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Stress-Induced Changes in Sensitivity to Thermal Nociceptive Stimulation in Normal Rats and Following Excitotoxic Spinal...

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STRESS-INDUCED CHANGES IN SENSITIVITY TO THERMAL NOCICEPTIVE STIMULATION IN NORMAL RATS AND FOLLOWING EXCI TOTOXIC SPINAL CORD INJURY By CHRISTOPHER DUNCAN KING A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Christopher Duncan King

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To my family and friends who supported me through these years. With your guidance and patience, I achieved a great accomplishment. I would have not been as successful without you. Thank you.

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iv ACKNOWLEDGMENTS I want to acknowledge family and friends who sustained me. In particular, I am extremely thankful for the guidance, love, and patience of my family. My father Richard, mother Sharren, stepmother Sally, sister Kels ey, and wife Natasha have been my rock. Each of them has played a special role in my life. With their inspiration and encouragement, I accomplished my goals and understood the importance of family especially during the tough times. I also w ould like to remember the individuals who have left our family including my mother Sharren, grandfather King, and grandmother Flint. They are loved and missed. I also thank the professors who helped me during my graduate studies. I am fortunate to have worked with them. Dr. Caudle provided me with an opportunity to work in his lab, visit foreign lands, and fu rther develop my research and academic proficiencies during a tough time in my life. Also, I appreciate Dr. Vierck’s guidance and wisdom over the past few years. I w ould also acknowledge help and advice from Drs. Darragh Devine and Andre Mauderli about stress and the poten tial pitfalls of behavioral testing. Last but not least, I express my gratitude to Dr. Yezierski as an honest and supportive mentor. In the process of de veloping my training and research program, he was able to convey his knowledge about pa in, and was very patie nt in educating me about writing. I am thankful to each of my professors for their involvement in my development as a scientist and a person.

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v In Drs. Yezierski’s and Vierk’s lab, I wo rked with several amazing individuals who also gave me technical and moral support. I would like to express my appreciation to my research backbone: my “rat ladies” Jackie Karen, and Jean. I would not have accomplished my research goals without their dedication and assistance. They educated me on many things related to my research, and also showed that you are only as good as the people around you. I also like to thank Dr. Cannon for his computer, histology, and perfusion expertise; and for listening to my unending questions about these issues. Also, I thank Victoria Gority for administrative assi stance and long talks about world problems. I also thank Sandra for her assistance, ev en though I have only known her for a short time. Finally, I would like to thank my friends; es pecially my old roommate and lab mate Federico. I also like to tha nk Sara (a fellow graduate student in Dr. Yezierski’s lab) for her support and friendship through our graduate training. Finally, I thank my God and my savior Jesus Christ for giving me a gr eat family, friends, and an opportunity to develop into a scientist.

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vi TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...........................................................................................................viii LIST OF FIGURES...........................................................................................................ix ABSTRACT.....................................................................................................................xi ii CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW....................................................1 The Pain Experience.....................................................................................................1 Animal Models of Pain.................................................................................................8 Chronic Pain...............................................................................................................13 Influence of Stress on Pain.........................................................................................21 Thermoregulation by Sympathetic Vasoconstriction.................................................25 Summary.....................................................................................................................27 2 EXPERIMENTAL METHODS AND DESIGN........................................................29 Experimental Animals................................................................................................30 Behavioral Tes ting Procedures...................................................................................31 Drug Administration...................................................................................................42 Surgical Procedures....................................................................................................42 Assessment of Core and Cutaneous Temperature......................................................44 Statistical Analysis......................................................................................................48 Study Design...............................................................................................................48 3 EFFECTS OF RESTRAINT STRESS ON NOCICEPTIVE RESPONSES IN NORMAL SUBJECTS...............................................................................................52 Effects of Restraint Stress on Refl ex Lick/Guard Responses at 44.0 C.....................53 Effects of Restraint Stress on Operant Escape Responses at 44.0 C.........................55 Time Course of Restraint Stress on Operant Escape Responses at 44.0 C................65 Effects of Restraint Stress on Core Temperature.......................................................68 Effects of Restraint St ress on Control Responses.......................................................76 Effects of Restraint Stress on Operant Thermal Preference.......................................80

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vii Effects of Endogenous Opioids on Stress -Induced Changes in Nociception.............86 Effects of Morphine on Stress-Induced Changes in Nociception...............................92 Summary and Discussion...........................................................................................98 Notes.........................................................................................................................1 06 4 EFFECTS OF RESTRAINT STRE SS ON NOCICEPTIVE RESPONSES FOLLOWING EXCITOTOXIC SPINAL CORD INJURY....................................107 Effects of Excitotoxic Spinal Cord Injury on Operant Escape.................................108 Overall Effect of Spinal Injury on Escape Responses.......................................108 Effects of Spinal Injury in Individual Groups...................................................113 Effects of Restraint Stress on Operan t Escape Following Excitotoxic Injury..........118 Overall Effects of Stress on Es cape Responses after Injury..............................118 Effects of Stress on Individual Groups..............................................................120 Effects of Excitotoxic Spinal Co rd Injury on Thermal Preference...........................125 Effects of Restraint Stress on Thermal Pr eference Following Excitotoxic Injury...131 Prediction of Behavioral Responses Based on Open Field Responses.....................138 Comparison between Normal and Spinally Injured Animals...................................139 Histology...................................................................................................................141 Summary...................................................................................................................150 5 EFFECT OF STRESS AND EXCITOTOXIC INJURY ON PERIPHERAL VASOCONSTRICTION..........................................................................................155 Effects of Restraint Stress on Peripheral Vasoconstriction......................................157 Effects of Excitotoxi c Injury on Periphera l Vasoconstriction..................................158 Summary...................................................................................................................167 6 CONCLUSIONS AND FUTURE STUDIES...........................................................173 Future Directions......................................................................................................177 Conclusions...............................................................................................................182 LIST OF REFERENCES.................................................................................................185 BIOGRAPHICAL SKETCH...........................................................................................209

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viii LIST OF TABLES Table page 3-1 Cumulative reflex lick/guard and operan t escape durations over two sessions of restraint stress...........................................................................................................65 3-2 Darkbox latencies for cont rol and restraint groups..................................................80 4-1 Number and duration of escape responses at 44.5 C before and during testing sessions in which animals we re tested fifteen minutes..........................................121 4-2 Effect of open field re sponses on operant responses fo r groups after excitotoxic injury......................................................................................................................138 4-3 Histological data for groups after ex citotoxic injury that were behavioral assessed in the operant escape and thermal preference tests..................................147 4-4 Histological data for groups after ex citotoxic injury that were behavioral assessed in the operant escape and thermal preference tests..................................148 4-5 Effects of histological variables on operant escape and thermal preference responses after ex citotoxic injury...........................................................................149

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ix LIST OF FIGURES Figure page 1-1 Hierarchical behavioral responses to nociceptive stimuli including spinal, supraspinal, and cortic al mediated responses.............................................................9 2-1 Reflex apparatus.......................................................................................................32 2-2 Operant escape apparatus.........................................................................................34 2-3 Thermal preference apparatus..................................................................................37 2-4 Open filed apparatus.................................................................................................40 2-5 Restraint tube............................................................................................................4 1 2-6 Behavioral testing sequence, stre ss exposure, and injection schedule for evaluation of operant and reflex lick/guard responses.............................................41 2-7 Skin temperature record ing in anesth etized rats.......................................................47 3-1 Behavioral testing sequen ce for the restraint group.................................................53 3-2 Reflex lick/guard latencies during testing trials at 44.0 C......................................56 3-3 Cumulative reflex lick/guard dur ations during testing trials at 44.0 C....................57 3-4 Escape latencies during testing trials at 44.0 C.......................................................58 3-5 Cumulative escape durations during testing trials at 44.0 C...................................60 3-6 Sequence analysis of successive esca pe plate and platform durations during testing trials at 44.0 C..............................................................................................63 3-7 Average escape duration of the first six plate and platform responses during testing trials at 44.0 C..............................................................................................64 3-8 Temporal profile of restraint stress on escape responses during trials at 44.0 C.....66 3-9 Escape latencies during testing trials at 44.0 C.......................................................69 3-10 Escape durations duri ng testing trials at 44.0 C......................................................70

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x 3-11 Core body temperatures dur ing testing trials at 44.0 C...........................................73 3-12 Core and cutaneous hindpaw temperat ures for control a nd restraint groups...........75 3-13 Escape latencies during testing trials at 36.0 C.......................................................77 3-14 Cumulative escape durations during testing trials at 36.0 C...................................78 3-15 Sequence analysis of successive esca pe plate and platform durations during testing trials at 36.0 C..............................................................................................81 3-16 Average escape duration of the first six plate and platform responses during testing trials at 36.0 C..............................................................................................82 3-17 Cumulative thermal preference durati ons during testing trials at 15.0 and 45.0 C.84 3-18 Average of the first six cold and heat durations.......................................................85 3-19 Reflexive lick/guard latencies at 44.5 C during testing sessions.............................88 3-20 Cumulative reflexive lic k/guard durations at 44.5 C...............................................89 3-21 Cumulative escape durations at 44.5 C....................................................................91 3-22 Reflexive lick/guard latencies at 44.5 C..................................................................94 3-23 Reflexive lick/guard durations at 44.5 C.................................................................95 3-24 Cumulative escape durations during testing trials at 44.5 C...................................97 4-1 The number of escape platform re sponses during testing trials at 44.5 C before and after excitotoxic injury....................................................................................109 4-2 Cumulative escape platform dura tions during testing trials at 44.5 C before and after excitotoxic injury...........................................................................................110 4-3 Sequence analysis of successive esca pe plate and platform durations during testing trials at 44.5 C before and after excitotoxic injury....................................111 4-4 Average duration of the first six plat e and platform responses during testing trials at 44.5 C before and after excitotoxic injury................................................112 4-5 Weekly postoperative platform respons es across several week s of testing during trials at 44.5 C before and after excitotoxic injury................................................114 4-6 Number of escape platform res ponses during testing trials at 44.5 C before and after excitotoxic injury...........................................................................................116

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xi 4-7 Cumulative escape platform dura tions during testing trials at 44.5 C before and after excitotoxic injury...........................................................................................117 4-8 Weekly postoperative platform respons es across several week s of testing during testing trials at 44.5 C before and after excitotoxic injury....................................119 4-9 The number of escape platform responses at 44.5 C.............................................122 4-10 Cumulative escape platform responses at 44.5 C..................................................126 4-11 Cumulative escape platform responses at 44.5 C..................................................127 4-12 Correlation between postope rative responses following QUIS and change in skin temperature regulation during sessions..................................................................128 4-13 The number of thermal preference res ponses during testing trials at 15.0-45.0 C before and after excitotoxic injury.........................................................................129 4-14 Cumulative durations of thermal prefer ence responses during testing trials at 15.0-45.0 C before and after excitotoxic injury.....................................................130 4-15 Sequence analysis of successive co ld and heat preference durations during testing trials at 15.0-45.0 C before and after excitotoxic injury............................132 4-16 Average durations of the first six co ld and heat preference responses during testing trials at 15.0-45.0 C before and after excitotoxic injury............................133 4-17 Weekly postoperative cold and heat preference responses across several weeks of testing during trials at 15.0-45.0 C before and after excitotoxic injury............134 4-18 Number of thermal preference res ponses during testing trials at 15.0-45.0 C......136 4-19 Cumulative cold and heat preference responses at 15.0-45.0 C............................137 4-20 Difference scores for plate and platfo rm durations during escape trials at 44.5 C in normal and after excitotoxic...............................................................................140 4-21 Difference scores for cold and heat preference durations during trial at 15.0-45.0 C in normal and after excitotoxic injury...............................................142 4-22 A comparison of in vitro MRI images...................................................................144 4-23 Summary of transverse and sagittal spinal co rd images obtained through in vitro MRI after excitotoxic injury...................................................................................145 5-1 Reduction of skin temperatures by symp athetically mediated vasoconstriction....156

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xii 5-2 Skin temperature measurements from the plantar surface of non-stimulated paws during and after thermal stimul ation of the left hindpaw.......................................159 5-3 Skin temperature measurements from the plantar surface of non-stimulated paws during and after thermal stimul ation of the left hindpaw.......................................163 5-4 Skin temperature measurements from the plantar surface of non-stimulated paws during and after thermal stimul ation of the left hindpaw.......................................164 5-5 Skin temperature measurements from the plantar surface of non-stimulated paws during and after thermal stim ulation of left hindpaw.............................................165 5-6 Skin temperature measurements from the plantar surface of non-stimulated paws during and after thermal stim ulation of left hindpaw.............................................166

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xiii Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy STRESS-INDUCED CHANGES IN SENSITIVITY TO THERMAL NOCICEPTIVE STIMULATION IN NORMAL RATS AND FOLLOWING EXCI TOTOXIC SPINAL CORD INJURY By Christopher Duncan King August 2006 Chair: Robert Yezierski Cochair: Charles Vierck Major Department: Medical Science-Neuroscience The sensation of pain is a complex e xperience that requires processing of nociceptive stimulation by cortical structures Various manipulations (including stress and injury to the nervous system) influen ce activity in these structures and thus influencing pain perception. To unders tand the effects of stress on nociceptive sensitivity, behavioral responses of normal (inj ury nave) and spinal injured animals were evaluated before and after a 15 minute expos ure to restraint stre ss. Two types of behavioral assessment strategies were used, including reflex (dependent on spino-bulbo-spinal processing) and operant (d ependent on cerebral processing) responses to low-intensity thermal stimulation (44.0 to 44.5 C) that activates C-nociceptors. Excitotoxic spinal cord injury was acco mplished by intraspina l injection of the AMPA/metabotropic receptor agonist quisqua lic acid (QUIS). Additional features of

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xiv stress-induced changes in nociception were al so investigated, incl uding the impact of opioids and sympathetic-mediated thermo regulation of skin temperature. Results suggest that restraint stress decreased thermal sensitivity of reflex responses by activating an endogenous opioi d system, supporting previous reports of stress-induced hyporeflexia. In terestingly, low-dose morphine enhanced reflex lick/guard responses and opposed inhibito ry effects of restraint stress on reflexes, suggesting a separate mechanism mediating these effects. In contrast, restraint stress increased thermal sensitivity to heat in the operant escape and thermal preference tests, which was opposed by tonic endogenous opioids and by e xogenous opioid administration. Results provide evidence for stress-i nduced hyperalgesia, which wa s not observed the following day or during sessions at neutral temperatures (36.0 C) suggesting that this effect is specific to activation of C-noci ceptors. Excitotoxic spinal cord injury also increased thermal sensitivity to heat in some animals, which was enhanced by stress in subsequent testing sessions. In summary, results suggest that exposure to acute restraint stress has a differential effect depending on the behavioral assessment strategy. Furthermore, stress was found to enhance thermal hyperalgesia after excitotoxic in jury. Finally, assessment of skin temperatures during thermal stimulation showed an association between the regulation of sympathetic vasoconstriction and enhanced sens itivity to heat on ope rant responses after stress and excitotoxic injury.

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1 CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW The purpose of my study is to advance our understanding of the behavioral and pharmacological mechanisms responsible fo r modulating nociceptiv e responses after acute stress. Pre-clinical a nd clinical studies of pain ha ve described changes to the psychological condition initiated by stressors that may lead to changes in nociceptive sensitivity and precipitate psychopathologies. Psychological stressor s are encountered on a daily basis and appear to correlate with conditions of increased pain sensitivity in individuals with a variety of pain conditions including chronic pain syndromes such as fibromyalgia, rheumatoid arthritis, and ir ritable bowel syndrome (Bennet et al., 1998; Blackburn-Munro and BlackburnMunro, 2001; Davis et al., 2001; Mayer et al., 2001). Because these studies are limited, additional research is needed to understand the negative effects of stress on acute and chronic pain conditio ns. However, pre-clinical research has been hampered by poorly defined behavioral assessment strategies that focus on reflex responses dependent on spinal and br ainstem processing of painful information. A well-defined, unambiguous animal model th at demonstrates the stress-induced enhancement of pain is therefore required. Fortunately, operant escape task, a recently developed behavioral assay, can address these issues. The Pain Experience Based on the original hypothe sis by Melzack and Casey (19 68), the pain experience may be thought of in terms of a sensory discriminative component in which precise anatomical mapping of stimulus intensity, location, and modality are maintained. The

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2 pain experience is also thought to have an affective motivational component (in which pain perception is modulated by the concurre nt overlay of an emotional component as well as previously learned asso ciations). This organization reflects the definition of the International Association for th e Study of Pain (IASP) that pain is “an unpleasant sensory and emotional experience associated with actual or potential tissue damage” (Merskey and Bogduck, 1994, page 210). Although complex, it is heuristic ally useful to consider that pain is a valuable response to potentially tissue damaging stimuli. Pain is detected in the periphery through the activation of primary A or C-nociceptors, transmitted to the dorsal horn of the spinal cord, and transmitted to supraspinal structures through ascending pathways. Painful information is processed in the brainstem a nd cerebrum and results in the activation of descending modulatory pathways th at inhibit or facilitate pain transmission in the spinal cord. Subsequent responses are organi zed through a complex interaction of neuroanatomical structures. These mechanis ms encompass primary afferent transduction to spinal encoding, and finally supraspinal s timulus-response relationships. Within the nervous system, numerous structures a nd pathways (e.g., ascending and descending) transmit, process, and modulate information associated with the pain experience. Ascending Pain Pathways The complexity of processing sensory input in the spinal cord shows that it is a critical conduit for transmitting sensory nocic eptive information. Studies have shown that nociceptive stimulation activates primary afferent nociceptors in the skin including fast conducting small diameter myelinated A mechanoreceptors and slow conducting unmyelinated C polymodal fibers (Fleisch er et al., 1983; Willis and Westlund, 1997).

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3 Depending on the nature of the stimulus, A and C-nociceptors are activated differently. High and low intensity stimula tion is required to excite A and C-nociceptors, respectively (Yeomans et al., 1996). The initial location of nociceptive informa tion processing occurs in the dorsal horn of the spinal cord. Nociceptive information is conveyed by afferent fibers that terminate on second order neurons located in the dorsal horn (e.g., superficial laminae I/II; Todd et al., 2000, 2002; Millan, 2002). Stud ies show the role of dorsal horn neurons in the rostral transmission of nociceptive informati on and descending modulation (Millan, 2002, 2003; Willis and Westlund, 1997). Several ascending nociceptive pathways have been identified in conveying nociceptive in formation including spinothalamic, spinomesencephalic, spinoreticular, spinocervi cal, and spinolimbic pathways (Burstein et al., 1987, 1990; Burstein and Giesler, 1989; Willis and Westlund, 1997; Yezierski, 1988). In addition to nociceptive transmission, dorsa l horn neurons modulate other nociceptive projection neurons and motor neurons (W illis and Westlund, 1997). Nociception does not indicate pain perception (Le Bars et al., 2001; Vierck, 2006). Rather, pain perception requires cerebral processing of the nociceptiv e stimulus (Mauderli et al., 2000; Vierck, 2006). Nociceptive input is transmitted alon g ascending pathways to supraspinal structures. Ascending pathways innervat e brainstem (e.g., PAG, Bulbar reticular formation) and cortical (e.g., thalamus, hypotha lamus, amygdala) structures involved in higher order processing of nociceptive info rmation (Giesler et al., 1994; Price, 2000; Willis and Westlund, 1997). These cortical systems are important for the affective component of pain (Price, 2000). Spinothalami c tract (STT) cells ar e implicated in the

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4 sensation of pain as a conse quence of anterolateral cordotom ies or spinal lesions (Price, 2000; Vierck and Light, 1999, 2002; Willis a nd Westlund, 1997; Yezierski, 1988). Spinothalamic tract (STT) cells are also activ ated in responses to thermal stimulation (Ferrington et al., 1987; Price et al., 1978). Descending Pain Pathways Previous studies show the presence of a complex endogenous inhibitory system that modulates spinal circuitry involved in nociceptive proces sing. Several supraspinal structures have been implicated in the modul ation of spinal processing of nociception and nociceptive behavior. Cortical structures im plicated in modulation include the amygdala, anterior cingulate cortex, insular corte x, and hypothalamus (Willis and Westlund, 1997; Price, 2000). In addition, brainstem structur es have been shown to impact nociception including the locus ceruleus (LC), A7 catecholamine cell group, periaqueductal gray (PAG), reticular formation, and rostroventrome dial medulla (RVM; Mitchell et al., 1998; Nuseir and Proudfit, 2000; Proudfit and Clark, 1991; Westlund and Coulter, 1980; Willis and Westlund, 1997). In addition, ascending pr ojections from neurons expressing NK-1R influence the activation of descending pathways (Suzuki et al., 2002). This suggests that nociceptive stimuli activate a spino-bulbo-spin al system in which ascending projections provide afferent input to supr aspinal loci. In turn, supras pinal neurons modulate spinal activity by descending projections. In c onditions of pain and stress, enhanced nociceptive sensitivity most likely invol ves components of both ascending and descending projections among spinal cor d, brainstem, and cortical regions. Activation of descending inhibitory pathways suppresses nociceptive reflex responses, evidence for stimulation-induced hypoalgesia. An important bulbo-spinal circuit mediating the expre ssion of hypoalgesia includes th e connection between the PAG

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5 and RVM. Other pathways include connect ions between the PAG and LC (Willis and Westlund, 1997). The PAG projects to nuclei of the RVM (Fields and Basbaum, 1999). From the RVM, descending pathways project to the spinal cord via the dorsolateral funiculus (DLF) and influence nociceptive ne urons in the dorsal horn (Basbaum and Fields, 1984; Millan, 2002). Activation of neurons in the PAG and RVM by electrical stimulation or microinjections of opioids produces a decrease in the activity of nociceptive neurons and nociceptive reflexes to thermal stimulation (Carstens et al., 1979, 1980, 1981; Fields and Basbaum, 1999; Fi elds et al., 1988, 1991; Peng et al., 1996). STT cells that are implicated in transmitting pain sensations are particularly inhibited after stimulation of th e PAG (Yezierski, et al., 1982). Based on anatomical, electrophysiological and pharmacological evidence, RVM is thought to have a substantial role in th e modulation of nociceptive responses and transmission of nociceptive input (Mason, 1999). In the RVM, cells have been characterized as “OFF”, “ON”, and neutra l cells based on responses to thermal stimulation (Fields et al., 1991; Heinrich er et al., 1989, 1997). The “OFF” cells are tonically active and pause in firing immediately before ta il withdrawal from a noxious thermal stimulus and are thus thought to be involved in inhibition of spinal nociceptive neurons. The “ON” cells accelerate firing imme diately before the nociceptive reflex and are directly inhibited by mu-opioid agoni sts; these cells ar e thought to produce facilitation of spinal nocicep tive neurons (Fields et al., 1983, 1991; Heinricher et al., 1994; Urban and Gebhart, 1999). Both cell types pr oject to dorsal horn (e.g., lamina I, II, and V) to modulate nociceptive transmission a nd responses to thermal stimulation (Fields et al., 1983; Morgan and Fields, 1994; Mitchell et al., 1998).

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6 While traditional studies focused on des cending inhibition from the RVM, several studies show that descending pathways exer t facilitatory influences on nociceptive processing and responses through activity of RVM neurons in chronic pain states (Porreca et al., 2001, 2002; Urban and Gebhart, 1999). Descending pathways appear to facilitate nocicepti on through activity of -opioid receptor expressing pronociceptive “ON” cells (Ossipov et al., 2000; Pertovaara et al., 1996). These observations led to hypothesis that spino-bulbo-sp inal loop could co ntribute to the development and maintenance of exaggerated pain behavi ors produced by noxious and non-noxious stimuli (Porreca et al., 2002; Urban a nd Gebhart, 1999). However, que stions have been raised concerning descending pathways in the cortical processing of pain because these studies are based on reflex-mediated responses not dependent on cortical processing. Descending systems are also involved in regulating other phy siological functions (autonomic, motor) especially to innocuous stimuli (Manson, 2005). Descending bulbo-spinal pathways origin ating from the RVM are critical for expression of exogenous opioid anti-nociception as assessed by reflex responses (Fields et al., 1983; Gilbert and Frank lin, 2002; Gebhart and Jones, 1988). Neurons responsible for descending pathways display high levels of opioid receptors and peptide expression (Marinelli et al., 2002). Microinjection of opi oid agonists into discrete brainstem sites (e.g. PAG and RVM) produces reduced activit y of dorsal horn neur ons and nociceptive tail and hindpaw withdrawal responses to noxious stimulation (Jenson and Yaksh, 1986a, 1986b, 1986c; Jones and Gebhart, 1988; Ya ksh, 1997, 1999; Yaksh et al., 1976). Furthermore, the hyporeflexic effects of system ic opioids appear to activate descending modulatory systems through these sites. Fo r example, microinjections of opioid

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7 antagonists into the PAG and RVM oppose the hy poalgesic effects of systemic morphine (Manning and Franklin, 1998; Yaksh and Rudy, 1978). In addition, neural pathways associated with stress-induced changes in nociceptive reflexes include supraspinal neurons that ex ert a descending inhib itory effect on dorsal horn neurons including descending pathways from the RVM. Activation of the RVM during times of stress was shown to be critic al for expression of mo rphine inhibition of reflex responses. In stressed rats, the e nhancement of morphine inhibition of reflex responses was reduced by injections of lidocai ne or muscimol into the RVM (Mitchell et al., 1998). Supraspinal structures mediate morphine -induced inhibition of reflex responses through descending projections that are blocked by RVM and DLF lesions or inactivation of the RVM by lidocaine (Abbott et al., 1996; Basbaum and Fields, 1984; Fields et al., 1988, 1991; Gilbert and Franklin, 2002; Mitc hell et al., 1998). The RVM is also implicated in the expression of stress-indu ced antinociception. In activation of the RVM by lidocaine attenuated reflexive behavior to heat in stressed rats (Mitchell et al., 1998). Damage to descending pathways in the DLF also reduced the development of stress-induced inhibition of reflex responses (Watkins and Mayer, 1982; Watkins et al., 1982). At the spinal level, bulbo-spinal terminals release several neurotransmitters that modulate dorsal horn activ ity and nociceptive responses, including catecholamines, and opioid peptides (Schmauss and Yaksh, 1984 ; Takano and Yaksh, 1992). Activation of the bulbo-spinal descending inhibitory pathwa ys are mimicked and enhanced by spinal application of 2 and receptor agonists (Nuseir and Proudfit, 2000; Schmauss and

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8 Yaksh, 1984 ; Takano and Yaksh, 1992). In contrast the effects of activation of bulbo-spinal projections are reve rsed by spinal application of 2 (e.g., phenotlamine) and (e.g., naloxone) receptor anta gonists (Camarata and Yaksh, 1986; Yaksh, 1979; Yaksh and Rudy, 1977). Animal Models of Pain The sensation of pain provides important information to an organism about its internal and external environment in order to maintain homeostasis. In the presence of a painful stimulus, various systems are activat ed to avoid the stimulus and limit damage (Le Bars et al., 2001). Assessment of pain sens itivity in animal studies is inferred from a variety of behavioral responses to nociceptive stimuli as illust rated in Figure 1-1 (adapted from C. Vierck). These responses can be cat egorized hierarchically within the neuroaxis including segmental reflexes, supraspinal reflexes, and lear ned escape responses (Le Bars et al., 2001; Vierck, 2006). As demonstrated in numerous studies, sp inally mediated reflex responses are demonstrated by simple limb or tail withdraw al from a nociceptive stimulus (Franklin and Abbott, 1989). Reflex responses mediat ed by spino-bulbo-spinal circuitry are revealed by more complex responses incl uding licking, guardi ng, vocalization, and jumping (Le Bars et al., 2001; Matthies a nd Franklin, 1992; Wool f, 1984). Finally, learned escape responses requires cerebral processing of nociceptive information and development of a proper strategy to terminate the stimulus (Mauderli et al., 2000). The concept of learning is not included in most te sts of nociception that utilize reflex assays (Le Bars et al., 2001).

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9 Figure 1-1. Hierarchical behavi oral responses to nociceptive stimuli including spinal, supraspinal, and cortical mediated responses. Nociceptive Responses Mediated by Spina l and Spino-Bulbo-Spinal Processing In pre-clinical models, evaluation of se nsory processing has ut ilized reflex based assessment strategies. These behavioral e ndpoints have been us ed to evaluate the presence of pain and assess alterations in nociception by various experimental manipulations. Segmentally orga nized spinal pathways regulat e withdrawal of a rodent’s tail or hindpaw from a nociceptive stimulus. Furthermore, segmentally-mediated tail or hindpaw withdrawal responses can be elicited in spinalized animals (Figure 1-1; Borszcz et al., 1992; Franklin and Abbott, 1989; Kauppila et al., 1998). In addition,

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10 bulbo-spinal-mediated responses including pawlicking and vocalization can be elicited in decerebrate animals (Figur e 1-1; Woolf, 1984; Matthies and Franklin, 1992). It is important to note that a majority of animal studies utilize brief high intensity thermal stimulation above 50.0 C that activates mylinated A nociceptors (Yeomans et al., 1996). Finally, reflex responses re present an important measur e of nociception and can be modulated by brainstem (e.g., LC, RVM) and cerebral structures (L e Bars et al., 2001; Vierck, 2006). Experimental manipulations such as stress and nerve injury ca n alter both types of reflex responses. A heightened or dimini shed sensitivity to a noxious stimulus after an experimental manipulation illustrates two concepts in the field of pain: hypereflexia and hyporeflexia, respectively. For example, acute stress reduces nociceptive reflex responses presumably by endogenous opioid m echanisms (Bodnar et al., 1978b; Gamaro et al., 1998; Watkins and Mayer, 1982), and injury to the central nervous system heightens sensitivity to thermal stimulation (A costa Rua, 2003; Yezierski et al., 1998). However, these end-points fail to account fo r the interactions be tween manipulations (stress) and higher order func tions that are responsible fo r the affective dimension of pain. Reflexive responses do not represent the c onscious or clinical aspect of pain perception, but rather spinal ly and supraspinally mediat ed nociceptive responses to thermal stimulation (Mauderli et al., 2000; Le Bars et al., 2001). Studies utilizing reflex-mediated responses assume that change s in reflex responses are a consequence of altered sensory processing at different leve ls of the neuroaxis (Le Bars et al., 2001; Vierck, 2006). However, these studies ma y fail to address other non-sensory factors

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11 affected by an experimental manipulation including changes in motor output, posture, motivation, attention, and cogni tion. Finally, pathways underl ying reflex responses are associated with other physiological functi ons unrelated to nociception (Mason, 2005; Le Bars et al., 2001). Therefore, assessment of reflexive responses can lead to deceptive conclusions about their importance in the overall sensation of pain. Nociceptive Responses Mediated by Cerebral Processing A main feature working against reflex ba sed assessment strategies is that these strategies do not take into account interactions between e xperimental manipulations and higher cortical activit y that are critical for the per ception of nociceptive stimuli. Therefore, in contrast to reflex responses, conscious and motivated responses to thermal stimulation are believed to characterize cl inically relevant aspects of nociceptive perception dependent on highe r order cerebral processi ng of nociceptive input. Consequently, operant responses are absent in decerebrate animals as they are dependent on cerebral processing of nociceptive input and environmental cues for the execution of appropriate escape responses (F igure 1-1; Mauderli et al., 200 0; Vierck, 2006; Vierck et al., 2003, 2004). Recently, an operant escape task was deve loped that evaluates thermal nociceptive sensitivity in awake, unrestrained, and consci ous rats (Mauderli et al., 2000). This test overcomes the limitations inherent with reflex withdrawal responses and offers a strategy to evaluate changes in the affective dimens ion of pain. The escape test provides an opportunity to evaluate: a) the consequen ces of experimental manipulations on a non-reflexive behavioral outcome measure; b) the mechanisms involved in hyporeflexia (e.g., decrease in nociceptive sensitivity) and hyperaglesia (e.g., incr ease in nociceptive sensitivity); and c) the effects of these ma nipulations on behaviors dependent on spinal

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12 and brainstem or cerebral pr ocessing of nociceptive input More importantly, an opportunity exists to directly compare reflex and escape responses to similar levels of thermal stimulation. Differences between reflex lick/guard a nd operant escape responses have been observed in several studies. Systemic inje ctions of low dose morphine (0.5 to1.5 mg/kg) attenuate escape responses (e .g., increase response latencie s and decreased duration) dependent on unmyelinated C-nociceptor activation (~44.0 C; Cooper et al., 1986; Vierck et al., 2002). By comparison, escape re sponses were not aff ected by morphine at temperatures activating A -nociceptors. In contrast, reflex responses were augmented (e.g., decreased response latencies and increase d duration) at the sa me temperature after morphine administration. Typically, suppres sion of reflex response is reported after injections of higher dose morphine (3 to 10 mg/kg; Holtman and Wala, 2005; O’Callaghan and Holtzman, 1975). Reflex re sponses were more sensitive to the hyporeflexic effects of morphine at temperatures lower than 50.0 C (Holtman and Wala, 2005). Based on these and other studies, the differe nce in sensitivity to morphine that depends on the activation of A or C-nociceptors illustrates an importance of the rate of cutaneous heating by a thermal stimulus. It has been shown that near threshold for nociceptor activation occurs at temp eratures ranging from 43.0 to 45.0 C (Le Bars et al., 2001; Treede, 1995; Vierck et al ., 2000). This temperature ra nge preferentia lly activates C-nociceptors as a result of a slow rate of skin heating a nd is involved in human pain sensations. C-nociceptors are essential for the affective sensation of a painful stimulus and, therefore, important to the elicitation of operant escape res ponses (Cooper and

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13 Vierck, 1986; Cooper et al., 1986; Vierck et al., 2000, 2004). In cont rast, temperatures above 45.0 C produce a rapid rate of heating that activates A and C–nociceptors (Cooper et al., 1986; Yeomans and Proudfit, 199 6; Yeomans et al., 1996). Clearly, the activation of C-nociceptors by gradual heating of the skin is important to overall pain sensation. The ability of low dose morphine to selectively suppress nociception mediated by C-nociceptors and not A -nociceptors supports the id ea that C-nociceptors are activated by sustained low inte nsity thermal stimulation. Other manipulations demonstrate a differen ce in reflex and operant responses to thermal stimulation. Operant escape responses appear to be mediated in part by NK-1R neurons. Vierck et al. (2003) demonstrat ed that lesioning of NK-1R neurons with substance-P saporin reduced escape responses to low intensity thermal stimulation while reflex responses were not affected. Also, Vierck et al. (2005) reported that chronic constriction (CCI) of the scia tic nerve produced increase in cold sensitivity. Finally, other operant test may offer unique opport unities to evaluated responses dependent on cortical processing (Neubert et al., 2005, 2006). Chronic Pain While acute pain serves a protective functi on to the organism, chronic pain persists beyond its intended purpose as a result of a bnormal activity in the central nervous system. In fact, chronic pain can last for a long period of time (> six months; Herr, 2004; Willis, 2002). Chronic pain is characterized as spontaneous, stimulus-independent, or evoked, stimulus dependent, pain sensati ons (Herr, 2004). Although the features of spontaneous and evoked pain will not be di scussed, it appears that several mechanisms mediate these pain etiologies including se nsitization (Willis, 2002; Willis and Westlund,

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14 1997). Furthermore, a prominent feature of a bnormal pain sensations is the presence of either allodynia (e.g., enha nced response to normally non-painful stimulus) or hyperalgesia (e.g., enhanced response to nor mally painful stimulus). Central pain conditions are initiated by injury to the cen tral nervous system without involvement of peripheral nociceptors (Willis and Westlund, 1997). Central pain was defined by the IASP as “pain initiated or caused by a prim ary lesion of dysfunction within the CNS” (Merskey and Bogduk, 1994, page 211). In support of the definition, studies have shown that lesions of the central ne rvous system (e.g., spinal cord, brainstem, and brain) may result in central pain presumably through alte red activity within nociceptive pathways. In particular, central pain after spinal cord injury will be discussed. Spinal Cord Injury Pain Spinal cord injury (SCI) is a chal lenging healthcare problem in terms of understanding the pathophysiology underlying th e condition and treatment strategies. Spinal cord injury pain can devel op immediately or ove r a period of time (Widertrom-Noga, 2002; Widertrom-Noga et al., 1999), and SCI pain can be either spontaneous or evoked (Siddall et al., 2002; Vierck et al., 2000; Yezierski, 2002). Several studies have reported between 60–90% of individuals with SCI experience pain of some type (Beric, 1997; Bonica, 1991; Kennedy et al., 1 997; Mariano, 1992; Siddall et al., 2002; Widertrom-Noga et al ., 1999). However, the development of chronic pain is higher in individuals with part ial interruption of gray and white matter (e.g., incomplete SCI) compared to complete spinal injuries (Beric et al., 1988). In most cases, SCI pain is a major obstacle and overshadows other physiolo gical consequences (e.g., impairment of motor functioning) based on the fact that SC I patients would forgo functional recovery for pain relief (Finnerup et al., 2001; Nepo muceno et al., 1979; Yezierski, 1996). In

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15 addition, subsequent treatments strategies to treat SCI pain are limited and mostly ineffective (Davidoff et al., 1987; Yezierski, 1996). Central pain after injury to the spinal cord is often characterized by abnormal sensations located in dermatomes at or be low the level of injury. An increase in nociception in dermatomes or segments at or adjacent to the injury location is defined as at-level pain (Siddall et al., 2002; Vierck et al ., 2000). In contrast, below-level pain after spinal cord injury is identified by an incr ease and spontaneous sens ations in nociception in dermatomes caudal to the in jury location (Siddall et al., 2002; Vierck et al., 2000). Another factor that distinguishes below-level pain is a delaye d onset of weeks, months, or years. Other sensations are also reported in individuals suffering with chronic pain conditions. Abnormal sensations such as ting ling, numbness, and itchi ng are identified as either dysesthesias or paraesthesias (Herr, 2004). Several potential mechanisms have been hypothesized to mediate alte red pain sensation after SCI (see blow). Some of these conditions include: a) abnormal activity (e.g., hy peractivity) of neurons associated with pain transmission in the spinal cord and lo ss of afferent input to rostral targets (e.g., deafferentation of thalamic and cortical areas), b) hypofunctio ning of the endogenous opioid system, c) hyperfunction of glutaminer gic excitatory systems, and d) loss of inhibitory mechanisms (Eide, 1998; Willis, 2002; Yezierski, 2002). Mechanism of SCI Pain Neuronal hyperexcitability afte r loss of inhibitory modula tion, in areas above or below the lesion site, may also influenced pa in sensation (evoked and/or spontaneous) in humans (Finnerup et al., 2003a 2003b; Milhorat et al., 1996 ) and rodents (Vierck and Light, 1999, 2000; Yezierski and Park, 1993; Yezier ski et al., 1998). In individuals with SCI, spinal and thalamic neurons show eviden ce of hyperexcitability that is characterized

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16 as an abnormal increase in activity (resting and evoked; Lenz et al., 1987, 1994; Loeser and Ward, 1967, 1968). Additionally, in animal s SCI models, the presence of neuronal hyperexcitability at spin al segments bordering the injury si te is associated with at-level pain (Christensen and Hulsebosch, 1997; Drew et al., 2001, 2004; Hao et al., 1992a; Yezierski and Park, 1993). Using electrophysio logical techniques, several studies have shown that neurons within pain pathwa ys display abnormal spontaneous activity, expansion of receptive field, a diminish thre shold for activation, an increased responses to stimulation, and extended afterdischarge (Eide 1998). Pharmacological investigations have demonstrated the role of neuronal hypere xcitability in altered pain sensitivity by administration of lidocaine (Loubser and Donvan, 1991) or NMDA antagonists (Hao and Xu, 1996; Hao et al., 19 91b; Liu et al., 1997). As mentioned previously, noc iceptive information is conveyed from the spinal cord to rostral targets via ascending pathways including the spinothalamic tract (Willis and Westlund, 1997). Abnormal activity of STT pathway has traditionally been thought as a critical feature of central pa in after SCI. Studies have supported this hyp othesis after interruption of STT pathways (Vierck and Light, 1999, 2000) or lesioning of its rostral sites in the cerebral cortex particularly in post-stroke pa in (Anderson et al., 1995; Boivie, 1994; Boivie et al., 1989). Although involvement of the STT pathway is important to the development of central pain, other factors app ear to be equally impor tant. In fact, some studies have reported similar damage to STT pathways in SCI patients with and without pain (Finnerup et al., 2003a, b). Usi ng MRI methods, Finnerup et al. (2003a) demonstrated that individuals with central below-level pain, compared to patients without central pain, displayed similar damage to the STT pathways, but patients with pain had a

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17 larger loss of gray matter. Damage to gr ay and white matter (e.g., interruption of the spinothalamic pathway) in the spinal cord appears to be crit ical factors in the development of below-level pain after SCI (Boivie et al., 198 9; Vierck and Light, 1999). Thus, in support of other clini cal studies implicating damage to STT pathways as critical factors in the development of central pain, damage to the sp inal gray matter is also a critical factor. Because the STT is the major ascending pa thway to supraspinal targets, rostral sites, such as the thalamus and cerebral corte x, lose critical input if the STT is damaged (Loeser and Ward, 1967, 1968). Lesions of the an terolateral spinal co rd after cordotomy produce spontaneous and evoked pain as a consequence of pathways originating from gray matter (Vierck and Light, 1999, 2000). Th is evidence supports the suggestion that central pain after interruption of the STT path way is a consequence of deafferentation. Altered activity patterns are detected in d eafferentated nuclei targeted by the STT pathways including the thalamus (Lenz et al., 1978, 1987; Weng et al., 2000) and cerebral cortex (Lenz et al., 1987, 1994). In addition, antero lateral cordotomies disrupt descending modulatory pathways, which also co ntributes to the enhancement of neuronal excitability in areas borderi ng the lesion (Vierck and Light, 2000). From these studies, it is clear that interruption of STT tract and changes to corresponding rostral targets are important for central pain. However, other factors will determine the expression of central pain including gray matter damage (Vierck and Light, 2000). The endogenous opioid system is implicated in the pathophysiology of neuropathic pain (Edie, 1998; Hao et al., 1998; Ossipov et al., 1997; Porrecca et al., 2001). Based on several studies in rodents, the opioid system (e.g., PPD, PPE) is activat ed after injury in

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18 spinal and supraspinal areas and appears to suppress abnormal pain sensation. But, dysfunctions of the opioid system lead to de velopment of hypersensitivity to thermal and mechanical stimulation (Abraham et al., 2000, 20 01; Xu et al., 1994). Finally, despite the unidentified pathophysiological mechanisms underlying SCI pa in, psychosocial factors contribute this condition. Studies have identified a relationship between several psychological factors and SCI pain includi ng depression, anxiety, fatigue, and stress (Kennedy et al., 1997; Mariano, 1992; Summe rs et al., 1991). Unfortunately, no pre-clinical studies have examined the impact of stress on SCI pain. Animal Models of SCI Pain Several pre-clinical models of SCI are used to examine pathophysiological mechanisms underlying alter se nsitivity to nocice ptive stimuli. Models such as hemisection (Christensen et al., 1997), phot ochemical lesions (Hao et al., 1991a, 1991b, 1992a, 1992b), contusion (Drew et al., 2004), an d anterior lateral spinal cordotomy (Vierck and Light, 1999, 2000) have been empl oyed to evaluate pathophysiological and behavioral changes occurring after SCI. A lthough these models will not be discussed, several reviews have compared and contra sted the models (Vierck et al., 2000). While mechanisms underlying SCI pain are still unclear, evidence from experimental studies have demonstrated a re lationship between abnormal pain sensitivity and several pathophysiological factors. Behavi orally, abnormal SCI pain in animals is evaluated by the presence of at-level or below-level change s in sensitivity. A common method to examine at-level pain sensations af ter SCI is assessing th e presence of caudally directed grooming in dermatomes adjacent to the injury level. In addition, changes in nociceptive responses after SC I provide evidence for a llodynia and hyperalgesia to

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19 thermal and mechanical stimulation in dermat omes adjacent to or below the level of injury. Excitotoxic Model of SCI Recent research into SCI has demonstrated that trauma to the spinal cord produces damage to the gray matter through mechanisms of cell death. The release of excitatory amino acids (EAA) is implicated in the de velopment of damage after SCI (Choi and Rothman, 1990). Subsequent release of glut amate after an insult activates AMPA and NMDA receptors initiating an excitotoxic cascade, which leads to neuronal cell loss within the gray matter of the dorsal horn (Berens et al., 2005 ; Gorman et al., 2001; Liu et al., 1991; Yezierski, 2002). The excitotoxic eff ect of EAAs is a cri tical initiating event for lesion progression and devel opment of SCI pain. Furtherm ore, protection of neurons from the excitotoxic effects of EAA release has been minimized by the administration of NMDA and AMPA antagonists (Choi and Rothma n, 1990; Liu et al., 1997) in addition to other treatments (agmatine; Yu et al., 2000, 2003). The excitotoxic model of spinal cord inju ry utilizes an intraspinal injection of quisqualic acid (QUIS), an mG luR and ionotropic GluR agonist, to produce lesions of the gray matter (Berens et al., 2005; Caudle et al., 2003; Gorman et al., 2001; Yezierski et al., 1993, 1998). An important feature of the excito toxic model is the o ccurrence of at-level and below-level pain, which is associated with neuronal loss (Yezierski et al., 1993, 1998; Berens et al., 2005). After an inj ection of QUIS, expression of spontaneous pain-like behaviors (e.g., overgrooming) is de monstrated at dermatomes corresponding to spinal segments near the lesion site. Mo re importantly, overgrooming was prominent after sparing of the superficial dorsal horn (Berens et al., 2005; Yezierski et al., 1998). Superficial dorsal horn neurons (e.g., lamina I) are implicated in chronic pain conditions

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20 (Ikeda at al., 2003). These cel ls also participate in the expression of injury induced overgrooming especially NK-1R expressi ng neurons (Khasabov et al., 2002). For example, Yezierski et al. (2004) reported that elimination of NK-1R neurons with a selective neurotoxin (e.g., subs tance-P saporin) reduced spon taneous pain-like behaviors after excitotoxic injury. Similar strategi es have been used to reduce nociceptive responses to capsaicin (Mantyh et al., 1997) and nerve injury (Nichols et al., 2001). By comparison, nociceptive responses to mechanical and thermal stimulation are augmented particularly in dermatomes ad jacent to and below the lesion epicenter (Yezierski and Park, 1993; Yezierski et al ., 1998). Evidence suggests a relationship between the enhancement of nociceptive res ponses and hyperexcitabil ity of neurons (e.g., increased spontaneous activity, increased res ponse to stimulation) bordering the area of neuronal loss (Yezierski and Park, 1993; Y ezierski et al., 1998). Based on these and other lesion studies, a critical component of below-level spinal cord injury pain appears to be gray and white matter damage. Additional factors impact the expression of heightened spontaneous and evoked nociceptive responses. In particular, these f actors include the longitu dinal progression, or the rostral-caudal distribution, of neuronal loss from the epicenter (~4.0 mm; Gorman et al., 2001; Yezierski, 1998). Furthermore, ar eas remote to the lesion epicenter also demonstrate changes after injury. Morrow et al., (2000) and Pa ulson et al. (2005) measured regional cerebral blood flow (rCBF), which indicates levels of neuronal activity in rodents. After excitotoxi c injury, several supr aspinal structures, which are targeted by rostral projecting pathways, were activated including forebrain (e.g., somatosensory cortex and thalamus). These ar eas are critical for the proce ssing of pain and demonstrated

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21 a remote effect of injury as a consequence of reorganization and/or deafferentation (Lenz et al., 1991). Other factors have already di scussed including genetic factors (Brewer et al., 2001), sex hormones (Gorman et al., 2001 ), and endogenous opioid mechanisms (Abraham et al., 2000, 2001) are important for the expression of pain-like behaviors after excitotoxic injury. Influence of Stress on Pain Types of Stress Stressors are characterized as either physical (systemic) or psychological (processive) and appear to activate different neural pathways. Systemic stressors (e.g., illness) primarily activate brainstem structures to restore homeostasis. By contrast, processive stressors (e.g. rest raint) are processed by limbic st ructures and elicit emotional responses. Limbic activation by stress acts through hypothalamic and brainstem systems to initiate physiological and hormonal responses, and may modulate motor output through higher cortical cente rs (Herman and Cullinan, 1997; Herman et al., 1996). For example, the hypothalamus may not directly m odulate a behavioral response, but rather modulates sensory input and the organization of learned responses driving the behavior. Biological responses to stress Several lines of research have suggested the ability of stress to modulate sensory perception in humans and reflex responses of la boratory animals. A stressor is defined as either an internal or external stimulus that presents an actual or perceived threat to the homeostasis of the organism (Herman a nd Cullinan, 1997). Exposure to a stressor induces a wide variety of adaptive stre ss responses including immune, hormonal, endocrine, physiological, and behavioral re sponses (Drolet et al., 2001; Herman and Cullinam, 1997). Ultimately, the stress response permits an individual to cope with the

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22 stressor and maintain homeostasis under norma l conditions, but after nerve injury, studies have suggested that stress can contribute to the development of psychopathologies and maintain the cycle of chronic pain (H erman and Cullinan, 1997; Melzack, 1999). Modulation of Nociceptive Responses by Stress Several studies have demonstrated that acute exposure to psychological stressors such as restraint produce attenuation of segm ental and bulbo-spinal reflexive withdrawal responses to high intensity thermal stimuli as measured by both tail-flick and hotplate tests (Amir and Amit, 1978; Bodnar et al., 1978a, 1978b, 1978c, 1979; Calcagnetti and Holtzman, 1992; Calcagnetti et al., 1990, 1992; Ga maro et al., 1998), an effect referred to as stress-induced analgesia (SIA; Lewis et al ., 1980). Reduction of re flexive responses to nociceptive stimuli is an adaptiv e response to acute stress exposure in order to cope with challenging situations. Transmitters regulating changes in nociceptive sensitivity on reflexive responses by stress include the endogenous opioid (Lewis et al., 1980; Porro and Carli, 1988), serotonine rgic (Quintero et al, 2000), and noradrenergic systems (Watkins and Mayer, 1982). Interestingly, chro nic stress exposure can increase sensitivity of reflex responses. For example, repeated exposure to an inescap able and uncontrollable stressor appears to induce sensi tization of sensory neurons in the spinal cord. In addition, repeated exposure to cold-water swims pr oduced a cutaneous thermal hyperalgesia as measured by reflex latencies (Quintero et al., 2000). Likewise, daily exposures to restraint stress over a forty-da y period resulted in cutaneous thermal hyperalgesia as assessed by tail flick res ponses (Gamaro et al., 1998). Modulation of Nociceptive Responses by Stress: Pharmacology The antinociceptive effects of endogenous opi oids, which are released after stress exposure, have been demonstrated after exposure to stressful stimuli. Opioid peptides are

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23 derived from three separate precursor pe ptides and include e nkephalin, endorphin, and dynorphin (Drolet et al., 2001; Yamada and Na besima, 1995). These peptides interact with receptors distributed throughout the cent ral and peripheral ne rvous system and are capable of modulating nociceptiv e sensations during stressful and painful stimuli (Kelley, 1982; Yamada and Nabesima, 1995). Threats to homeostasis induce the release of endogenous opioid peptides and ar e speculated to permit the or ganism to cope with the stressful situation (Amit and Galin a, 1988; Terman et al., 1984). Activation of the endogenous opioid system has been shown to parallel the induction of stress-induced hyporeflexia, or s uppression of nocifensive reflex responses, by various stressors (Bodnar et al., 1978a; Gama ro et al., 1998; Madden et al., 1977). For example, a single exposure to foot-shock produced hyporeflexia, as measured by increasing time to elicit ta il-flick responses to therma l stimulation. Stress-induced hyporeflexia also parallels incr eases in endogenous opioid leve ls in the central nervous system (Madden et al., 1977). Involvement of the opioid system in stress-induced changes in nocifensive responses was furthe r characterized by: 1) naloxone, an opioid antagonist, which reversed stress-induced hypor eflexia in rats; and, 2) cross-tolerance between stress-induced hyporef lexia and morphine after re peated exposure to stress (Bodnar et al., 1978b; Girardot and Holloway, 1984; Lewis et al., 1980). Restraint stress In relation to other stressful stimuli, restraint stress is considered to be a psychological stressor and has been used to induce stress-induced hyporeflexia in rats (Tusuda et al., 1989; Calcagnetti et al., 1992; Gamaro et al., 1998). For example, Gamaro et al. (1998) demonstrated that expos ure to a single session of restraint for one hour produced stress-induced hyporeflexia as assessed by tail-flick assay in both male

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24 and female rats. Studies have also dem onstrated the role of endogenous opioids in restraint-induced hyporeflexia on tail-flick and hindpaw withdr awal. The antinociceptive effects of -opioid agonists on reflexes (e.g., mor phine, DAMGO) were potentiated after acute exposure to restraint stress (Abb elbaum and Holtzman, 1984; Abbelbaum and Holtzman, 1985; Calcagnetti et al., 1990; Ca lcagnetti and Holtzman, 1992; Calcagnetti et al., 1992). For example, restraint stress enha nced the hyporeflexive effects of opioids, indicated by increases in re flexive withdrawal latencies to thermal stimulation after systemic (Abbelbaum and Holtzman, 1984, 1985, 1986; Fleetwood and Holtzman 1989; Calcagnetti and Holtzman, 1990, 1992), intrat hecal (Calcagnetti et al., 1992), and intracerebroventricular (Abbelbaum and Ho ltzman, 1985, 1986; Calcagnetti et al., 1990) administration compared to unstressed contro ls. These studies also suggest that both spinal and supraspinal opioid mechanisms contribute to stress-induced potentiation of opioids after i.t. and i.c.v. opioids on reflexive tests of nociception. Additional evidence for the involvement of endogenous opioids in stress-induced hyporeflexia is observed after sy stemic injections of opioi d antagonists. Administration of naloxone reverses the hyporeflexic eff ects of stress (Pilcher and Browne, 1983). Finally, evidence of endogenous opioids in stress-induced hyporeflexia is supported by the development of cross-tolerance between stress-induced hyporef lexia and morphine after repeated exposure to stress (e.g., hab ituation) or repeated exposure to morphine (e.g., tolerance). For example, the potentiation of the inhib itory effects of opioids by stress is reduced in habituated rats (Fleetwood and Holtzman, 1989) and morphine-tolerant rats (Torres et al., 2003). It is clear from these studies that restraint stress activates components of the endogenous opioid system and is involved in the

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25 modulation of responses to noc iceptive stimuli. While th e impact of stress on nociceptive reflex responses has been appreciated, no prev ious studies have examined the effects of stress on operant responses. Modulation of Nociceptive Responses by Stress: Chronic Pain Stress is reported to increase nociceptive sensitivity in individuals with chronic pain (Ditor et al., 2003; Galv in and Godfrey, 2001). This eff ect of stress is especially significant, as stress has been linked to th e onset and maintenance of numerous life threatening medical conditions, including those that severely compromise ones quality of life. Clinically, the presen ce of psychological stressors co rrelate with conditions of increased sensitivity in indivi duals with a variety of defi ned pain conditions, including fibromyalgia and those arising from nerve in juries (spinal cord injury). Furthermore, acute stress has been shown to increase pain se nsitivity in chronic pain patients, and has been suggested to contribute to the deve lopment of chronic pain syndromes like fibromyalgia, rheumatoid arthritis, and ir ritable bowel syndrome (Bennet et al., 1998; Blackburn-Munro and BlackburnMunro, 2001; Davis et al., 2001; Mayer et al., 2001). Even though patients with conditions such as sp inal cord injury deve lop chronic pain, the effect of stress in clinical settings has not been adequately addressed. Likewise, pre-clinical models of chr onic pain have not addressed the impact of stress on altered sensation after spinal injury. Thermoregulation by Sympathetic Vasoconstriction Numerous physiological mechanisms medi ate behavioral responding to nociceptive stimulation including the sympathetic component of the autonomic nervous system. The autonomic system plays an essential functi on in mediating physiological responses to internal or external stimuli (McDougall et al ., 2005). It also is implicated in pain

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26 perception and affective/motivational st ates (Thayer and Brosschot, 2005). The regulation of heat (thermoregul ation) is a consequence of sympathetic activity. Various manipulations can alter sympathetic tone, ultimately affecting the distribution of body heat and blood flow. For example, exposure to mental stress increases body temperature. In response to increase body temperatures sympathetic-mediated vasoconstriction reduced peripheral temperature (cooling) by restricting blood flow (Cooke et al., 1990; Larsson et al., 1995; Ni cotra et al., 2005). Sources of sympathetic regulat ion are localized in the intermediolateral column of the thoracolumbar spinal cord (Hofstette r et al., 2005). Vari ous neuroanatomical structures are involved in re gulating the outflow of sympat hetic preganglionic neuronal cell bodies including the hypothalamus, pr efrontal cortex, amalgdala, and RVM (Dampney, 1994; Korsak and Gilbey, 2004; McDougall and Widdop, 2005; Nalivaiko and Blessing, 2001). Activation of the sympathetic nervous system is also accomplished by the HPA axis after exposur e to nociceptive stimulation (J anig, 1995; Mage rl et al., 1996) or stress (Herman and Cullina n, 1997; McDougall et al., 2005). Activity of the sympathetic system can be evaluated indirectly by assessment of peripheral vasoconstriction during thermal s timulation (Shimodoa et al., 1998; Vierck, Unpublished Observations; Wakisaka et al., 1991; Willette et al., 1992). Overall, nociceptive stimulation decreases skin temperature ipsilaterally and contralaterally in non-stimulated areas. It app ears that activation of nocicepto rs by stimulation triggers a sympathetic response (Magerl et al., 1996). Ma nipulations have been shown to increase sympathetic-mediated vasoconstriction, includin g stress (Larsson et al., 1995), peripheral injury (CCI: Kurves et al., 1997; Wakisaka et al., 1991), and spinal injury (Acosta-Rau,

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27 2003). Furthermore, a relationship exists between the ability to demonstrate vasoconstriction and change in thermal se nsitivity. If a ma nipulation blunts the expression to vasoconstriction, it will also di splay an enhanced sensitivity to thermal stimulation (e.g., increase escape response to he at). Reduction of vasoconstriction in response to thermal stimulation has been dem onstrated after formalin (C. Vierck and R. Cannon, Unpublished Observations; C. Vierck and A. Light, Unpublished Observations) and excitotoxic injury to gr ay matter (Acosta-Rua, 2003). Thus, enhanced nociceptive responding is a consequence of peripheral and central injury that dramatically alters ability of the sympathetic nervous system to regulated cutaneous temperatures via vasoconstriction. Summary Efforts to study the effects of stress on se nsory processing in pr e-clinical models have frequently sought to employ reflex ive behaviors as e ndpoints for assessing stress-induced alterations in nociception. It is clear that such reflex functions are mediated by systems that respond to environmen tal cues and previous experience. These end-points fail to account for the interactions between stress and higher order functions initiated by a particular stimulus condition. In contrast, operant escape responses reflect a higher order organizational function, pres enting an approach by which we might establish a clinically relevant model of mo tivated behavioral responses to nociceptive stimuli that permits an evaluation of the affective component of pain. Though the importance of stressors on higher order functi on has been long appreciated, there are few studies that have examined the effects of stress on operant responses before and after injury to the central nervous system. The ultimate goal of the present proposal is to

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28 understand the apparent differential modulation of sensory processing by stress in normal and after spinal cord injury on several tests of nociception.

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29 CHAPTER 2 EXPERIMENTAL METHODS AND DESIGN The goal of this research is to increase our understanding of the effects of stress on sensory processing. In order to accomplish this goal, behavioral and physiological techniques were used to a ssess changes in nociceptive sensitivity in injury-nave and spinally injured rats. Each behavioral testing session cons isted of 2 consecutive testing trials in separate apparatuses that were constructed of plexiglass. Animals were exposed to a neutral temperature during the first trial (e.g., pre-test), which was used to normalize temperatures of the rodent’s hindpaw and acclim ate the animal to the apparatus. Thermal stimulation was delivered through a heated or cooled aluminum plate. During succeeding testing trials (e.g., test), animals were e xposed to a range of non-nociceptive and nociceptive temperatures. The responses coll ected during the second trial were recorded through customized computer software. Assessment of nociceptive responses was accomplished by comparing reflex lick/guard, operant escape, and thermal preference responses before and after exposure to restra int stress and following spinal injury. In order to produce stress-induced cha nges in nociception, restraint stress, a psychological stressor, was sele cted based on an extensive li terature demonstrating that restraint activates limbi c circuits and affects reflex re sponses to thermal stimulation. Restraint stress is a useful and convenient stressor, which can be delivered without difficulty, and does not present a direct th ermoregulatory challenge to the animal compared to other stressors (e.g., cold wate r swim). Likewise, several studies have concluded that the underlying mechanisms mediating restraint-induced changes in

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30 nociception is a result of activation of the endogenous opioid system. Based on these studies, pharmacological agents (e.g., naloxone and morphine) were used to determine the effect of the endogenous and exoge nous opioids on stress-induced changes in nociception. Furthermore, a common condition confronting in dividuals with spinal cord injury is chronic pain. In order to study the pathophys iology underlying SCI pain, the excitotoxic model of SCI that was developed by Dr. Y ezierski shares similar pathophysiological consequences common after traumatic and ischemic SCI. This model provides an excellent platform to study altered pain proc essing after spinal gray matter damage. Behavioral manifestations of altered nociception after excitotoxic injury include spontaneous (e.g., at level grooming) and evoked (mechanical allodynia and thermal hyperalgesia) pain sensations (Gorman et al ., 2001; Yezierski et al., 1998). Finally, experimental manipulations (e.g., stress and sp inal injury) affect pain sensations by various mechanisms including modulation of cutaneous skin temperature by the autonomic nervous system. Experimental Animals Female Long Evans rats were housed in pairs and maintained on a 12-12 hour light-dark cycle with free access to food and wate r. The reasons for using female rats for behavioral testing were based on observations that females were easier to handle, less aggressive, and maintained their body weight over time. Also, chronic pain is more common in females compared to males. The rats were adapted to the testing apparatus and handled prior to behavior al training and baseline tes ting. All experiments were carried out according to the Guide for the Ca re and Use of Laboratory Animals and were

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31 approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Florida (B193 and C013). Behavioral Testing Procedures Assessment of Reflex Lick-Guard Responses Reflex responses represent a supraspina lly-mediated behavior. Lick responses were recognized as stereotyped lifting of one hindlimb, then holding and licking the hindpaw. Guard responses were scored when a hindlimb was raised from the platform and flexed in an exaggerated fashion. Guar d responses were longer in duration than limb flexion that occurred during ambulation. Hindlimb reflex responses were measured during the second trial including frequency (number of responses during a trial), duration (total time spent licking or guarding during a trial), and latency to first lick-guard response. Reflex apparatus The apparatus used to evaluate lick-guard responses consisted of the reflex apparatus consists of a plexig lass box with a thermally regulated floor without an escape option (Figure 2-1). The enclosure was ventilated to permit airflow. Although no training is required for reflex responding, rats were familiarized with the apparatus and the testing procedure over 2-week period. Rats, which are not properly adapted to the testing environment, display stress-induced hypoalgesia due to the novel environment (Plone et al, 1996). Two consecutive trials were used to asse ss reflex responses. Similar to escape testing, a 15 minute trial at 36.0 C (pre-test trial) was used to standardize foot temperatures, which was followed by a second 10 minute trial at 44.5 C (test trial).

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32 Figure 2-1. Reflex apparatus.

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33 Because the animal cannot escape thermal stimulation, trial durations of 10 minutes were selected to prevent tissue dama ge. Reflex responses were exhibited during thermal stimulation at 44.0 C but not at 36.0 C. Assessment of Operant Thermal Escape Responses Operant escape apparatus The escape apparatus incorporates a shu ttle-box design, as described previously (Figure 2-2; Mauderli et al., 2000; Vierck et al., 2002). Th e escape test was carried out in a plexiglass box divided into two compartments by a hanging wall with an opening to permit rats to move freely between the compartments. The first compartment is dimly illuminated (0.5 foot candles) and includes a th ermally regulated floor, which can deliver either non-nociceptive or nocic eptive stimuli (43.0 to 47.0 C) to the paws during occupancy. Thermal stimulation was delivered by an aluminum plate regulated by a water bath (Neslab). The adjacent compartment contains a brightly illuminated (35-watt) halogen bulb above a thermally neutral escape platform. The platform provides animals an opportunity to escape noci ceptive thermal stimulation. The dual compartment set-up provides a conflict between aver sion to light and thermal noc iception. As a consequence, rats will proportion their time on the platform in relation to the intensity of stimulation. Operant escape training and assessment Rats were trained over 3 weeks to lear n to escape from thermal stimulation by climbing onto the neutral escape platform. During the training period, rats were familiarized with the testing procedure and trained to discriminate between gradually increasing floor temperatures (36.0, 40.0, 42.0, 44.0, 45.0, and 47.0 C) in the absence (first phase) and presence of bright light over the escape platform (second phase).

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34 Figure 2-2. Operant escape apparatus.

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35 Each training session consisted of two cons ecutive 15 minute trials. The first trial consisted of pre-te st condition at 36.0 C, and the second trial consisted of a range of gradually increasing temperatures over successi ve daily sessions. The pre-test was used to standardize foot temperatures prior to te sting, acclimate the rats to the apparatus, and extinguish avoidance behavior (e.g., occupancy of the escape platform unrelated to floor temperature). After operant training, base line escape responses were assessed over a 6 week period. Similar to training, rats were tested da ily with two consecutive 15 minute trials at 36.0 C (pre-test) and then at 36.0, 44.0, or 44.5 C (test trial). Es cape responses during the second (test) trial were assessed including frequency ( number of responses during a trial), duration (total time o ccupying escape platform during a trial), and latency to first escape response. Assessment of Operant Thermal Preference Responses An additional operant assessment strategy included the thermal preference test (Mauderli et al., 2000; Vierck et al., 2002). This test can de termine if an experimental manipulation (stress or injury) selectively affects cold ( first compartment ) or heat ( second compartment ) nociception. Preference of a thermal modality (cold or heat) will depend on the temperatures and experimental manipulation used. For example, if a manipulation affects sensitivity to heat nocicep tion, the animal will spend less time on the heated compartment and more time on the cold compartment. In cases when both modalities are affected, animals will increase their preference for a modality less affected by the manipulation

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36 Thermal preference apparatus Similar to the operant escape test, thermal preference apparatus (Figure 2-3) uses a shuttle-box design that requires an animal to choose between two distinct compartments. However, unlike the operant escape test with one thermally regulated floor, both floors of the thermal preference are thermally regulated at different temperat ures. The first and second compartments presented cold (0.3, 10.0, 15.0, or 36.0 C.) and heat (43.0, 44.0, 44.5, 45.0, 46.0 or 47.0 C.) nociceptive temperatures, re spectively. In addition, the preference test was precede d by a pre-test at 36.0 C to standardize foot temperatures prior to placement into the testing apparatus. Thermal preference training and assessment Following one week of preference training, ba seline responses were assessed over a 2 month depending on the stability of operant behavioral resp onses. It is important to note that only one cold and hot temperature, which are listed above were used during a single testing session (e .g., 15.0 paired with 45.0 C.). The duration of a single thermal preference was 12 minutes to avoid tissue da mage. Thermal preference responses were assessed by frequency (number of crossing dur ing a trial), duration (total time spent occupying the escape platform during a trial) and latency to first thermal preference response. Assessment of Darkbox Responses The darkbox test was used to assess motiv ation to escape the light and to evaluate whether motor deficits (e.g., freezing beha vior) were induced by experimental manipulations like restraint stress. The appa ratus consisted of two compartments with a 2 by 2 inch opening in the dividing wall.

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37 Figure 2-3. Thermal preference apparatus.

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38 Each testing session began with ten-seconds of acclimation in which a computer identified the location of the rat by weight Then, a 70-sec trial was initiated with presentation of light in both compartments. When the rat moved from the compartment it occupied at the start of the session to the adjacent compartment, the light was extinguished in the selected compartment for the remainder of the 70 second trial. At the end of the trial, both compartm ents were lit to in itiate the next trial. Darkbox latency was defined as the time required for the rat to move to the adjacent compartment. Each session consisted of seven light escape trials over fifteen minu tes. During stress testing, each rat was placed in the darkbox apparatus 15 minutes after termination of the stress exposure. Assessment of Open Field Responses The modified open field test consisted of a 90 cm x 90 cm square black Plexiglas container with an adjacent 20 cm x 20 cm st art-box which allowed the animal to either remain in the start-box or enter into the ope n field (Figure 2-4; picture provided by Dr. Darragh Devine). A light fixtur e illuminated the open field a bout (5 to 150 Lux). A door separated the two boxes, which was opened vi a a rope and pulley system. Upon opening of the door, the rope was secured with a hook until the next trial. A camera, located above the box, recorded the animal’s behavior. The trial duration for the open field was 5 minutes. After exposure to the field, the rat was returned to its homecage. The open field assesses anxiety-like responses in rats duri ng exposure to a novel environment. Restraint Stress Procedures After stabilization of behavi oral responses, rats were se lected to receive an acute exposure to restraint stress ( stress condition ) or remained in their home cage until behavioral testing ( control condition ). Rats were removed from their home cages, and

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39 rats were restrained for 15-minutes. The rest raint tube (Figure 2-5; D. Devine, Personal Communications) is composed of a soft flexible sheet of plastic 11” X 7 “ mounted to a rigid plexiglas cradle (8 ” X 3’ X 3”) by m eans of two small bolts with convex heads. There are ventilation holes at one end to allow unrestricte d breathing, and the other end has a vertical slot to allow co mfortable placement of the tail during the restraint process. The plastic sheet is then gently rolled around the animal and held s ecurely in place with two 12’ X 1” Velcro strips. Groups either remained in their home cage until testing or received a 15 minute exposure to restraint st ress (Figure 2-6). Then, each ra t was removed from the restraint tube and placed in the pre-test apparatus at 36.0 C for 15 minutes. Rats were then placed in the adjacent test apparatus at 36.0 C (thermally neutral control temperature) or 44.5 C (testing temperature) for an a dditional 10 to 15 minutes depe nding on the behavioral test. Control rats followed the same protocol and di d not receive stress on the day of testing. Both groups of rats remained in their home cages in a separate room until stress exposure or behavioral testing was complete. All temp eratures were held constant over the two days of testing. On successive testing weeks, exposure to restraint stress was switched to the group previously in the control condition. For exam ple, group 1 received restraint stress while group 2 was not be exposed to stress, servi ng as a control. The following week group 2 was exposed to restraint stress and group 1 se rved as a control. The experiment was designed to expose animals to stress every two weeks with the aim of 1) avoiding adaptation and 2) using each animal as their ow n control. This testing protocol has been shown not to cause carry-over effect s of stress (King et al., 2003).

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40 Figure 2-4. Open filed apparatus.

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41 Figure 2-5. Restraint tube. Figure 2-6. Behavioral testi ng sequence, stress exposure, and injection schedule for evaluation of operant and reflex lick/guard responses.

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42 Drug Administration In a separate group of animals, behavi oral responses in normal animals were evaluated after an injection of naloxone and morphine. Af ter stabilization of baseline responses, rats were randomly assigned to receive either stress or no stress before behavioral testing. After 15 minutes of restra int stress, rats were immediately injected with either an injection of morphine (opi oid agonist, 1 mg/kg, i .p.), naloxone (opioid antagonist, 3 mg/kg, i.p.), or sa line (1 mg/kg, i.p.). Then, rats were placed into the pre-test apparatus at 36.0 C for an additional 15 minutes. Behavioral responses were recorded during the second trial at 44.5 C. Control subjects remained in their home cage separate from stress subjects. Control subjects were injected and tested similarly to stress subjects, but control subjects did not receive stress on the day of testing. Surgical Procedures In this study, the effects of stress on operant responses were assessed after excitotoxic lesioning of mid-thorasic (T8) or lumbar (L2) segments of the spinal cord. Rats received an intraspinal injection of quisqualic acid (QUIS) which is a non-NMDA receptor agonist that interacts with AMPA a nd mGlu receptors. Previous studies have shown that nociceptive responses are enhanced after an intraspinal in jection (Acosta-Rua, 2003; Gorman et al., 2001; Yezierski et al., 1998). The escape and thermal preference responses were recorded before and after su rgery. Excitotoxic lesioning of the spinal cord was conducted after several weeks of ba seline testing. Beha vioral testing was resumed 2 weeks after surgery. At 8 weeks post-op, rats were removed from their home cages and placed in a restraint tube for 15 minutes. After stress exposure, rats were placed in the pre-exposure testing apparatus at 36.0 C. After fifteen minutes of

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43 pre-exposure, rats were placed in the adjacent testing apparatus at 44.5 C. Both control animals and animals waiting to receive st ress were kept separate from animals undergoing stress. Intraspinal Injection of Quisqualic Acid (QUIS) As mentioned previously, an imals underwent excitotoxic injury as mentioned in other studies (Gorman et al., 2001; Yezierski et al., 1998). Rats were anesthetized with a combination of ketamine (3 ml), acepromazine (1 ml), and xylazine (1 ml) at 0.65 ml/kg administered subcutaneously. Level of anes thesia was evaluated by noxious pinch of the hindpaw. Body temperature was maintained at normal levels (36.5 C) during QUIS surgery and post-operative period. Several pathological features occur after QUIS injections including neuronal loss, cavitations, demylinati on, and alteration of glia (Yezierski et al., 1993, 1998; Berens et al., 2005). Following injections muscles were closed in layers, the skin closed with w ound clips, and animals re turned to their home cages. Intraspinal Injection Procedures After placement into a sterotaxic frame, the dorsal surface of the spinal cord was exposed via laminectomy correspon ding between spinal segments T8 to L2. After removal of the dura and pia matter, QUIS was inj ected bilaterally into the spinal cord to target mid-thoracic and upper lumber spinal cord segments. Glass micropipettes (tip diameter 5 to 10m) attached to a Hamilton mi croliter syringe (volume 5l) are used for injections. The syringe was mounted on a micr oinjector attached to a micromanipulator. Injections were made between the dorsal ve in and dorsal root en try zone at depths ranging from 500 to 1200 m below the surfac e of the cord. To avoid white matter

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44 damage, all intraspinal injections were placed in the middle of the gray matter (lumbosacral cord: T8-L2). Stock solutions of 125 mM QUIS (Sigma) was made fresh daily using sterile saline and buffered to physio logical pH as needed. At each injection site 0.1-0.6 l of QUIS was injected (over a 60 second time interval). The total volume of QUIS injected/animal was 1.2 to 1.5 l per side. The standard injection consisted of three bi-lateral injection tracks separated by 0.5 mm. In Vitro MRI Analysis of Spinal Cord At the end of the study, animals were inje cted with sodium pentobarbital (1 ml, i.p.) and underwent transcardial perfusion with PBS followed by 10% formalin in PBS (Fischer Scientific). Spinal cord segmen ts containing QUIS lesions were removed and placed into an MRI tube. All excised cords were subjected to in vitro three-dimensional MR microscopy (3D MRM). Images were acquired with a three-dimensional (3D) gradient echo pulse sequence using a TR = 150 msecs, TE = 10 msecs with NA = 2. The image FOV was 2 cm x 0.5 cm x 0.5 cm in a matrix of 512 x 128 x 128 in a total data acquisition time of 1.5 hours. Therefore, MR images were acquired with a resolution of ~40 microns x 40 microns x 40 microns. A 3D Fourier transformation was applied to the acquired data matrix to produce the 3D image. General image processing and analysis was performed using custom software writte n in the Interactive Data Language (IDL, from Research Systems, Boulder, CO). Assessment of Core and Cutaneous Temperature The effects of experimental manipulati ons could be a consequence of altered temperature regulation (core and cu taneous). In order to evalua te the impact of stress and spinal injury on thermoregulation, core a nd cutaneous temperatures were evaluated

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45 before and after exposure to restraint stre ss. In addition, temperatures were also evaluated before and after excitotoxic spinal injury. Core Temperature An implantable thermal probe (IPTT-300; BMDS, Delaware) recorded core body temperature. After induction of anesthesia with isoflurane, the injection site for the probe was prepared by removing the hair on the animal’s back followed by aseptic preparation of the site with alcohol and iodine wipes. The probe was injected subcutaneously with a specialized injector. Temperatures were recorded by portable reader (BMDS, Delaware) without any restraint of the rat. Temperatures were recorded before and after exposure to the first testing apparatus. Data are expressed in degrees Celsius. Autonomic-Mediated Skin Temperatures The autonomic nervous system (ANS) regul ates skin temperature by changes in vasoconstriction through activ ity of the sympathetic nervous system. Experimental conditions, including stress, pain, and injury, ma y activate and potential ly alter the ANS. In order to determine if these manipulati ons could modulate an animal’s autonomic response to thermal stimulation, skin temperat ures were recorded in the absence (resting conditions) and presence of heat stimulation (44.5 C; Figure 2-7). In order to assess skin temperatures, all animals were injected with diazepam (10 mg/ kg, i.p.). Previous research has demonstrated that isoflurane negatively affected au tonomic activity, which is counteracted by diazepam (C. Vierck, Pers onal Communication). 1% Isoflurane is used to induce (5%) and mainta in (1%) anesthesia. A thermal heating blanket is used to maintain normal body temperature (36.0 C). Several sites were monitored to changes in temperatures including rectal core temperature, both fo repaws, and both hindpaws. For

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46 each paw, a thermocouple was applied to the skin with an adhesive foam pad. For the right forepaw, left forepaw, and right hindpaw, thermocouples session were applied to the plantar surface, and a thermocouple was applied to the top of the left hindpaw (stimulated paw). After stabilization of paw temp eratures, a pre-heated thermode (44.5 C.) was applied against the left hindpaw for 10 mi nutes. Cutaneous temperature of each (non-stimulated) paw was recorded for 20 minutes during (10 minutes) and after stimulation (10 minutes; resting period) to perm it skin and core temperatures to return to baseline. For each manipulation, pre-stress or pre-opera tive skin temperatures were collected several weeks prior to restraint or excitot oxic injury. For the stress condition, animals were stressed for 15 minutes followed by inducti on of anesthesia with isoflurane. Skin temperature was assessed 15 minutes after the termination of restraint, which permitted stabilization of skin temperatures before te sting and corresponding to 30 minutes after the onset of stress. Temporal Profile of Skin Temperature: Effects of Restraint Stress condition In order to evaluate the effects of a pre-exposure to 36.0 C (pre-test) on skin temperature after restraint st ress, a thermocouple was secu red to the left and right hindpaw (plantar surface) to the skin with an adhesive foam pad. Temperatures were recorded over a 30 second period. Then, animals were placed into a restraint tube for 15 minute. Skin temperature was continuously r ecorded during the entire trial. Animals were removed from the tube at the end of th e restraint period. Fo llowing removal of the thermocouples, animals were placed into a 36.0 C pre-test for 15 minutes. Then,

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47 thermocouples were reattached to both hi ndpaws for 1 minute. Core temperature was also recorded before restraint and at 5 mi nute intervals thereafter. The sequence of events paralleled the testing conditi ons for operant and reflex testing. Figure 2-7. Skin temperature reco rding in anesthetized rats.

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48 Control conditions In addition, non-stressed control animals were tested before and after exposure to 36.0 C. Skin temperature was recorded for 1 minute prior to placement into the pre-test and then removed. After a 15 minute pretest trial, animals were removed and thermocouples were reaffixed to both hindpaws. Statistical Analysis The frequency, latency, and duration of be havioral responses (escape, thermal preference, and licking/guarding, as appropria te) were collected by custom software (EVENTLOG, Autorat, Robot). The data ar e expressed in seconds, and values were represented as absolute group means S.E.M. Statistical analysis of behavioral responses between groups was performed by t-te sts. Analysis of behavioral responses between groups was performed by an one-w ay analysis of variance (ANOVA) or two-way analysis of variance (ANOVA) with or without repeated measures followed by Newman-Keuls post-tests. P-values less than 0.05 were considered significant. Analysis was performed using GraphPad Prism vers ion 4.00 for Windows, GraphPad Software, San Diego California USA ( www.graphpad.com ). Analysis of Covariate and correlations were performed by SSPS. Study Design The current experiments were based on previous literatur e and studies conducted in the labs of Drs. Vierck and Yezierski sugge sting that nociceptive re sponses are mediated through different neuroanatomical pathways. Previous research has suggested that stressors inhibit reflex mediated nociceptive responses particularly to high intensity thermal stimulation. Inhibition of refl ex responses has s upported evidence for stress-induced analgesia (SIA). For example, acute exposure to restraint stress has been

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49 shown to increase segmental (tail withdrawal ) and spino-bulbo-spina l reflexes (hindpaw withdrawal or licking) to thermal stimulation. However, despite limit anecdotal and clinical evidence that stress enhances pain sensations, the effects of stress on thermal sensitivity in rats have not been examined by operant tests of nociception. Because pain is a complex sensation, it requires cortical st ructures to process and elicit appropriate actions. Thus, in order to study pain, proper be havioral strategies ar e required to examine these structures. Thus, the current study will de termine effects of acute restraint stress on reflex lick/guard responses (a spino-bul bo-spinally mediated behavior) and operant escape responses (a cerebrally mediated beha vior) in rats. It is hypothesized that an exposure to acute restraint stress produces a differential effect on reflex lick/guard and operant escape responses evoke d by thermal stimulation. In order to characterize the effects of st ress on behavioral responses, the impact of various pharmacological agents and temporal profile were evaluated. First, the contribution of endogenous opioids to stress-induced changes in lick/guard and operant escape responses to thermal stimulation were evaluated by systemic administration of an opioid receptor agonist (morphine) or antagon ist (naloxone) before behavioral testing. Previous studies have implicated end ogenous opioid peptides as mediators of stress-induced hyporeflexia, as shown by the effects of either opioid agonists or antagonists on reflexive tests of noci ception (Calcagnetti and Holtzman, 1992; Calcagnetti et al., 1990). Furt hermore, stress has been show n to increase the release of endogenous opioids and modulate the physio logical and psychological response to stressful and painful stimulation (Drolet et al., 2000; Madden et al., 1978). The effects of endogenous opioid peptides, however, after st ress on responses dependent on cerebral

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50 processing have not been examined. The i nhibitory effects of stress on reflexive responses are modulated by the endogenous opioid system, but the system could oppose the facilitatory effects of stress on operan t responses. It is hypothesized that the endogenous opioid system contri butes to stress-induced re duction of spino-bulbo-spinal reflexes while opposing the excitatory effect s of stress on cerebrally mediated operant escape responses to thermal stimulation. Second, the temporal profile of stress on operant responses was assessed 15 minutes, 30 minutes, and 24 hours after the onset of stress. Previous stress literature has pointed out that magnitude of altered nocicep tive responses is dependent on the duration and intensity of the stressor. Typically, l ong exposure to a stressor or a single exposure an intense stressor resulted in extended behavioral or physio logical responses. Because the current stressor was only 15 minutes, it wa s hypothesized that the effects of restraint stress on operant responses would gradually disappear across time. In addition, the effects of stress were examined on a well-established model of spinal cord injury (SCI), which results in e nhanced expression of below-level behaviors. Stress triggers changes in several impor tant physiological systems including the autonomic nervous system (e.g., sympathetic-adrenal system) and the hypothalamic-pituitary-adrenal axis (HPA axis ). Clinically, psychological stress is associated with the progression and mainte nance of several chronic pain conditions including fibromyalgia, irritable bowel syndrome, nerve injury, and rheumatoid arthritis. Likewise, physiological systems are alte red in conditions of chronic pain. In light of these clinical observations only a limited number of studies have examined the effects of stress on altered noci ceptive responses in chronic pain models,

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51 particularly models of spinal cord injury. The current aim will use an excitotoxic model of SCI. In this model, animals underwent a bilateral injection of the AMPA/metabotropic receptor agonist quisqualic acid (QUIS) into the spinal cord. Operant responses were assessed before surgery (pre-op), after surg ery (post-op), and after an exposure to restraint stress. It was hypot hesized that excitoto xic injury of the spinal cord would produce an increase in thermal sensitivity a nd subsequent exposure to acute restraint stress will enhance injury-induced operant es cape responses. Finally, the study examined a potential mechanism (thermoregulation) me diating altered thermal sensitivity by stress and excitotoxic spinal injury.

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52 CHAPTER 3 EFFECTS OF RESTRAINT STRESS ON NO CICEPTIVE RESPONSES IN NORMAL SUBJECTS Various experimental manipulations can influence processing of input from nociceptive afferents. Exposure to a stressor has been associated with both suppression (Abbelbaum and Holtzman, 1984; Borszcz et al., 1992; Gamaro et al., 1998) and enhancement (Borszcz et al., 1992; Huang and Shyu, 1987; Illich et al ., 1995; King et al., 1996, 1999) of pain sensations. Based on severa l pre-clinical studies stress inhibits reflex mediated responses (w ithdrawal of the tail or hindpa w) to thermal stimulation. However, some questions have been raised pe rtaining to relevance of reflex responses in the perception of pain, which is dependent on higher cortical pro cessing. An important question can be raised concerni ng the effect of stress on pain sensations. Does stress affect responses dependent on operant proces sing of nociceptive information differently than reflex responses? Or does stress suppress reflex and operant responses similarly? In this chapter, the effects of restraint stre ss on reflex and operant (escape and thermal preference) responses to low intensity thermal stimulation, which activates C-nociceptor afferents by heat (44.0 to 44.5 C), were examined. To control for confounding effects of stre ss (e.g., avoidance), control tests were also examined including operant escape responses at a neutral temperature (36.0 C) and darkbox responses. In order to further char acterize the effects of stress on nociception, pharmacological conditions, which were based on previous research using reflex-based behavioral responses, were also investigated. Naloxone or morphine was administered to

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53 determine the contribution of endogenous and exogenous opioids on normal and stress-induced changes in thermal sensitivity, respectively. Due to study limitations, opioid pharmacology was limited to reflex lick-guard and operant escape. Effects of Restraint Stress on Re flex Lick/Guard Responses at 44.0 C Behavioral responses of fe male rats (n=11) were a ssessed during a 3-day period, with baseline testing on Day 1 (baseline), post-stress or control testing on Day 2 (15 minutes), and evaluation of long-term stress effects Day 3 (24 hours). On Day 2, half the animals received 15 minutes of restraint stre ss, followed by a 15 minut e pre-test and test trials as shown in Figure 3-1. Testing sessi ons included a 15 minute pre-test exposure to 36.0 C, followed by a test trial at 44.0 C. Reflex (Figure 3-1A) and operant escape (Figure 3-1B) responses were assessed fo r 10 and 15 minutes, respectively, during the testing trial. The control group remained in their homecage until behavioral testing. Figure 3-1. Behavioral testing sequence for th e restraint group. (A) Reflex hindpaw. (B) Operant escape.

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54 Reflex Lick-Guard Latency The latency of first lick/guard responses to 44.0 C during baseline sessions (Day 1), testing sessions in which rats received 15 mi nutes of stress (Day 2; restraint group) or no stress (control group), and sessions 24 hour s afterwards (Day 3) are presented in Figure 3-2. Latencies of lick/gua rd responses were significan tly greater on Day 2 for the restraint group than for th e control group (Figure 3-2A; F= 24.61, P< 0.001). Reflex response latencies were also greater for stre ssed rats on Day 2 than on days when the same rats were not stressed (Days 1 and 3; F= 10.08; P< 0.001). Difference scores between control and restraint groups revealed that reflex latencies were significantly higher after stress on Day 2 after stress (F igure 3-2B; F= 8.78, P< 0.001). Reflex Lick-Guard Duration The duration of lick/gua rd responses to 44.0 C were significantly lower for the restraint group than for the c ontrol group on Day 2 (Figure 3-3A; F= 39.18, P< 0.001). Reflex response durations were also signi ficantly lower for the restraint group on Day 2 than on days when the same rats were not stressed (Days 1 and 3; F= 13.61, P< 0.001). Reflex responses for the control and restraint groups were stable on testing Days 1 and 3, demonstrating no adaptation to daily testing at 44.0 C. Difference scores between control and restraint groups revealed that reflex duration were si gnificantly lower after stress on Day 2 after stress (Figure 3-3B; F= 11.361, P< 0.001). Base on these observations on reflex late ncies and durations, it can be concluded that exposure to restraint stre ss suppressed reflex responses. Stress-induced hyporeflexia was characterized by a longer latency to el icit a hindpaw response and a shorter time engaging in licking or guarding of the hindpaw. The effect was transient and did not

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55 persist the following day. Thus, this data supports previous research suggesting that stress inhibits spino-bulbo-spinal reflexes to thermal stimulation. Effects of Restraint Stress on Operant Escape Responses at 44.0 C Operant Escape Latencies To determine the effects of stress on opera nt responses, the same groups of animals (described above) was stressed on weeks interspe rsed between reflex testing (Figure 3-4). The first latency of operant escap e responses during trials at 44.0 C were observed during baseline sessions (Day 1), testing sessions in which rats received 15 minutes of restraint stress (restraint group; Day 2) or no stre ss (control group), and se ssion the following day (24 hours, Day 3). Previous studies have sugge sted that the first escape latencies are less dependable outcome measures compared to es cape durations (Vierc k et al., 2002; 2003). Consistent with these observations, no diffe rences in escape latencies were observed between the restraint and control groups on any day of testing (Figure 3-4A; F= 0.2570, P= 0.7740). Difference scores revealed no significant effects (Figure 3-4B, F= 0.818, P= 0.447). Operant Escape Durations The duration of escape responses was si gnificantly greater on Day 2 for the restraint group than for the c ontrol group on Day 2 (Figure 3-5A; F= 38.48, P< 0.001). Furthermore, the duration of escape was great er after stress exposur e on Day 2 than it was for the same rats on unstressed days (Days 1 and 3; F= 49.01, P< 0.001). Therefore, acute restraint stress did not produce a longterm (24 hour) effect on escape duration. Difference scores revealed that escape durat ions were significantly higher than controls after exposure to stress (Figure 3-5B; F= 11.305, P< 0.001).

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56 Figure 3-2. Reflex lick/guard late ncies during testing trials at 44.0 C for control (open bar) and restraint (closed ba r) groups during baseline se ssions (Day 1), testing sessions in which rats received 15 minutes of restraint stress (restraint group; Day 2) or no stress (control group), a nd sessions the following day (Day 3). (A) Exposure to restraint significantly in creased reflex latencies when tested fifteen minutes after stress (15 minutes; Day 2) compared to the control group and compared to unstressed trials on ba seline and 24 hours after stress. (B) Difference scores confirmed that stress in creased reflex latencies compared to controls (15 minutes; Day 2). Data are expressed in seconds and are represented as absolute group means S.E.M. Significant within-subject differences between trials 15 minutes af ter stress exposure and trials by the same rats on baseline and 24 hours are indicated as: *** P< 0.001. Significant between-subject differences between the control and restraint groups on Day 2 are indicated as: P< 0.001.

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57 Figure 3-3. Cumulative reflex lick/guard durations during tes ting trials at 44.0 C for control (open bar) and rest raint (closed bar) groups during baseline sessions (Day 1), testing sessions in which rats received 15 minutes of restraint stress (restraint group; Day 2) or no stress (control group), and sessions the following day (Day 3). (A) Exposure to restraint signi ficantly decreased reflex durations when tested fifteen minutes after stress (15 minutes; Day 2) compared to the control group and compar ed to unstressed trials on baseline and 24 hours after stress. (B) Differe nce scores confirmed that stress decreased reflex durations compared to controls (15 minutes; Day2). Data are expressed in seconds and are represente d as absolute group means S.E.M. Significant within-subject differences be tween trials 15 minutes after stress exposure and trials by the same rats on baseline and 24 hours are indicated as: *** P< 0.001. Significant between-subject di fferences between the control and restraint groups on Day 2 are indicated as: P< 0.001.

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58 Figure 3-4. Escape latencies dur ing testing trials at 44.0 C for control (open bar) and restraint (closed bar) groups during baseline sessions (Day 1), testing sessions in which rats received 15 minutes of re straint stress (restrai nt group; Day 2) or no stress (control group), and session s the following day (Day 3). (A) Exposure to restraint did not affect escape latencies compared to the control group and compared to unstressed tria ls on baseline (Day 1) and 24 hours (Day 3) after stress. (B) Difference scor es confirmed that stress did not alter escape latencies (15 minutes; Day 2). Da ta are expressed in seconds and are represented as absolute group means S.E.M.

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59 A comparison of escape durations for the co ntrol group did exhibit a slight increase across the three testing days. Statistical an alysis revealed no si gnificant change across testing Days ( F= 2.968; P= 0.0623). This effect has been obs erved previously in our lab as a consequence of repeated testi ng at the same thermal temperature. Unlike reflex responses, operant responses ar e enhanced (increased heat sensitivity) after an acute exposure to re straint stress when assessed by durations. This effect, stress-induced hyperalgesia was characterized by a longe r time spent on the escape platform during a trial, which were more re liable than latencies. Similar to reflex responses, this effect was transient and did not persist the following day. Thus, it can be concluded that stress produced a differential e ffect where reflexes were suppressed while nociceptive responses dependent on cortical processing of thermal stimulation were enhanced. Sequence Analysis of Successive Operant Escape Durations In addition to analysis of the total duration of escape, examinations of successive operant escape duration within trials presents an additional strategy to analyze the effect of experimental manipulations on behavioral responses. Successive operant escape plate (A, B) and platform (C, D) durations are show n in Figure 3-6 for control (left panel) and restraint (right panel) groups In general, the maximum nu mber of escape responses was twelve (12), but a majority of animals respond approximately 6 times. Plate durations for control (A) and re straint (B) groups do not change across baseline (Day 1), 15 minutes (Day 2), and 24 hours (Day 3). Plate durations peaked between the 2nd and 3rd responses.

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60 Figure 3-5. Cumulative escape durati ons during testing trials at 44.0 C for control (open bar) and restraint (closed ba r) groups during baseline se ssions (Day 1), testing sessions in which rats received 15 minutes of restraint stress (restraint group; Day 2) or no stress (control group), a nd sessions the following day (Day 3). (A) Exposure to restraint significantly in creased escape durations when tested fifteen minutes after stress (15 minutes; Day 2) compared to the control group and compared to unstressed trials on ba seline (Day 1) and 24 hours (Day 3) after stress. (B) Difference scores co nfirmed that stress enhanced escape durations compared to controls (15 mi nutes; Day 2). Data are expressed in seconds and are represented as absolu te group means S.E.M. Significant within-subject differences between tria ls 15 minutes after stress exposure and trials by the same rats on baseli ne and 24 hours are indicated as: *** P< 0.001. Significant between-subject differences between the control and restraint groups on Day 2 are indicated as: P< 0.001.

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61 In contrast, control and restraint groups displayed different patterns of responding during trials at 44.0 C on Day 2 (15 minutes) in which one group of animals was exposed to acute stress (restraint gr oup) while the other group remained in their home cage (control group). Platform durations for th e control (C) and rest raint (D) groups were shorter than plate times in this gr oup of animals and peak between the 3rd and 6th platform responses. On the day of stress, platform dur ations in the restraint group dramatically increased, which indicates an enhanced therma l sensitivity by restraint stress, compared to the control group. The peak of the effect occurs on the 3rd and persisted until the 9th platform response. Importantly, platform dura tions were comparable to baseline values when assessed the following Day (24 hours). In summary, the average duration across of the first 6 responses for the control and restraint groups are compared (Figure 3-7). This number was selected because majority of animals quit responding after the sixth res ponse. In the control group (Figure 3-7A), plate durations did not differ over the three consecutive testing sessions ( F= 0.793, P= 0.453), but platform durations did change significantly ( F= 7.368, P= 0.001). Platform durations were significantly higher th an baseline during sessions on Day 3 ( P< 0.05) but not during the 15 minute testing period on Day 2 ( P> 0.05). In contrast, the average dura tion for plate (Figure 3-7B; F= 3.727, P= 0.025) and platform ( F= 18.64, P< 0.001) durations were different over the three consecutive sessions for the restraint group. In particul ar, plate durations gr adually decreased over sessions (Days 1 through 3) with durations significantly lower than baseline during the Day 3 session only ( P< 0.05). Platform durations after re straint were significantly longer than baseline ( P< 0.001) but not 24 hours later ( P> 0.05). Furthermore, platform

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62 durations were higher after re straint than the control group ( F= 38.004, P< 0.001; Day 2). During sessions 24 hours after stress, platform durations were similar between the two groups ( F= 0.255, P= 0.594; Day 3). Thus, analysis of successive operant escape responses revealed a transient hyperalgesia (enhancement of h eat sensitivity) as indicated by increase in escape platform duration after restraint stress. Effects of Repeated Stress Exposures: Adaptation In order to avoid adaptation to repeated expos ures to restraint stress (De Boer et al., 1999; Gamaro et al., 1998) at l east 2 weeks elapsed between stre ss tests for each animal. The effectiveness of this strategy and the po ssibility that there might be carryover effects of repeated stress were evaluated by three t ypes of statistical comparisons related to escape and lick/guard durations at 44.0 C, as shown in Table 3-1. First, the effectiveness of stress wa s evaluated for the first and second administration of restraint prior to reflex or operant testing. Reflex durations were lower for stressed rats compared to unstr essed rats on Day 2 for the first ( F= 9.15, P< 0.01) and second stress sessions ( F= 5.06, P= 0.01). Also, escape durations were greater for stressed rats compared to unstresse d rats on Day 2 for the first (F =5.69, P< 0.01) and second stress sessions ( F= 5.89, P< 0.01). Second, performance on day 1, 2, and 3 was compared for the first and second reflex and operant testing sessions, in order to determine whether there were cumulative effects of repeated stress on performance. None of these 12 comparisons revealed significant effects: for the c ontrol and restraint groups during either reflex or operant testing on each of the three days.

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63 Figure 3-6. Sequence analysis of succe ssive escape plate and platform dura tions during testing trials at 44.0 C for control (left panel) and restraint (right panel) groups during ba seline sessions (asterisk; Day 1), testi ng sessions in which rats received 15 minutes of restraint stress (restraint gr oup, gray circle; Day 2) or no stress (contro l group, gray circle), and sessions the following day (closed square; Day 3). Da ta are expressed in seconds and are represented as absolute group means S.E.M.

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64 Figure 3-7. Average escape duration of the firs t six plate (open bar) and platform (closed bar) responses during te sting trials at 44.0 C for control and restraint groups during baseline sessions (D ay 1), testing sessions in which rats received 15 minutes of restraint stress (restraint gr oup; Day 2) or no stress (control group), and sessions the following day (Day 3). (A) In the control group, no differences were observed in plate, but platform durat ions were slightly higher over repeated sessions. (B) Exposure to acute restraint stress significantly increased escape platform durations when tested fifteen mi nutes after stress (15 minutes; Day 2) compared to th e control group and compared to unstressed trials on baseline (Day 1) and 24 hours (Day 3). Data are expressed in seconds and are represente d as absolute group means S.E.M. Significant within-subject differences between trials 15 minutes and 24 hours after stress exposure or no stress and tr ials by the same rats on baseline and 24 hours are indicated as: P< 0.05 and *** P< 0.001. Significant between-subject differences between th e control and rest raint groups during trials at 15 minutes and 24 hours are indicated as: P< 0.05 and P< 0.001.

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65 Finally, neither escape ( F= 0.306; P= 0.583) nor lick/guard ( F= 0.302, P= 0.585) durations during baselin e testing (Day 1) differed signifi cantly across 4 weeks of testing at 44oC (twice prior to the restrain t group and twice prior to th e control group). Overall, these data show that acute restraint stress re mained effective with repetition at two (2) week intervals and did not accumulate (ca rryover) from one exposure to the next. Table 3-1. Cumulative reflex lick/guard and operant escape durati ons over two sessions of restraint stress. Stress produced similar effects on reflex and escape durations to 44.0 C during the first and second exposures. The effects of stress were not diminished by repetition at 2-week intervals. The data are expressed in seconds, and values are represented as absolute group means S.E.M. 44.0 C Reflex L/G 44.0 C Operant Escape Baseline (Day 1) Baseline (Day 1) 1st Session 2nd Session 1st Session 2nd Session Control 91.1 13.3 93.0 12.1 314.5 55.1 262.5 41.9 Pre-Stress 102.8 14.8 93.0 7.0 271.4 61.9 358.4 50.6 Test (Day 2) Test (Day 2) 1st Session 2nd Session 1st Session 2nd Session Control 94.9 11.5 92.8 8.1 344.4 35.1 289.3 56.3 Stress 50.4 9.2 51.1 8.2 565.9 49.7 505.1 53.3 24 Hours (Day 3) 24 Hours (Day 3) 1st Session 2nd Session 1st Session 2nd Session Control 76.4 14.1 90.2 13.4 411.4 37.8 379.6 67.1 Post-Stress 75.2 13 81.1 8.9 499.9 37.3 383.6 23.4 Time Course of Restraint Stress on Operant Escape Responses at 44.0 C Previous studies have demons trated that the effects of stress are detected after termination of the stressor and may last for several hours or days la ter (Calcagnetti et al., 1992; Drolet et al., 2001; Gamaro et al., 1998; Quintero et al., 2000; Tusuda et al., 1989). The duration and magnitude of stress effect is dependent on several factors including stressor intensity and the le ngth of exposure. To examin e the temporal profile of

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66 stress-induced hyperalgesia, a separate group of female rats (n=12) were restrained for a period of 15 minutes followed by assessmen t of behavioral responses immediately (without a 15 minute pre-test at 36.0 C) after exposure to restraint stress as illustrated in Figure 3-8A. In addition, the testing protocol used in experiments described above is shown in Figure 3-8B (with a 15 minute pre-test at 36.0 C). Finally, operant escape responses were assessed the following day (24 hours). The control group did not receive stress, but were tested under similar circumstances without or with a pre-test at 36.0 C. Figure 3-8. Temporal profile of restraint st ress on escape responses during trials at 44.0 C. Responses were assessed 15 minut es (A), 30 minutes after the onset of stress (B), and 24 hours after restrain t stress (not shown) The control group followed the same testing schedule but without exposure to restraint stress.

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67 Operant Escape Latencies To determine the effects of stress on es cape latencies across multiple time points, animals received 15 minutes of restraint st ress (restraint group) or no stress (control group) followed by testing sessions at 44.0 C immediately or 15 minut es after stress, and sessions the following day (24 hour s; Figure 3-9). Escape late ncies in the control group did not differ across testi ng sessions (Figure 3-9A; F= 0.8555, P= 0.4387). However, latencies were affected by restraint stress ( F= 4.279; P= 0.0269). Escape latencies were shorter immediately and 15 minutes after restraint compared to the following day ( P< 0.05). Differences between the two groups (Fi gure 3-9B) revealed th at latencies were shorter than controls wh en assessed immediately ( F= 12.7, P= 0.002) and 15 minutes ( F= 6.358, P= 0.019) but not 24 hours ( F= 0.137, P= 0.714) after the termination of stress. Previous data suggested that escape late ncies were unreliable and a poor outcome measure. Unlike the first se t of experiments in a differe nt group of animals, escape latencies were sensitive to stress. A reduc tion of escape latencies, which indicates a lower threshold to elicit an escape response, supports the conclusion of stress-induced hyperalgesia. Operant Escape Durations To determine the effects of stress on es cape durations across multiple time points, responses in the same group of animals are pres ented in Figure 3-10. Similar to latencies, escape durations in Figure 3-10A did not di ffer across testing sessi ons in the control group ( F= 2.147, P= 0.1407), but durations were significan tly different in the restraint group ( F= 3.675, P= 0.0420). Escape durations were si gnificantly longer immediately and 15 minutes after restraint compared to the following day ( P< 0.05). Difference scores (Figure 3-10B) revealed that escape durations were longer th an controls when assessed

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68 immediately ( F= 16.035, P< 0.001) and 15 minutes ( F= 5.989, P= 0.023) but not 24 hours ( F= 0.518, P= 0.479) after stress. In agreement with previous data (Figure 3-5), escape durations were influenced by stress. Stress-induced hyperalgesia was displayed immediately (greatest effect) after the termination of restraint as well as when evaluated 15 minutes after stress exposure. The effect was transient based on the fact that both groups showed similar re sponding 24 hours later. Effects of Restraint Stress on Core Temperature Various experimental manipulations such as restraint stress influence thermoregulation, which can impact behavioral responses to thermal stimulation. For example, restraint stress produces hyporeflexia and is associated with increased core temperature (~1.0 C; Chen and Herbert, 1995; Keim and Sigg, 1976; Le Bars et al., 2001; Thompson et al., 2003; Tjolsen and Ho le, 1993). Furthermore, evidence suggests that changes in thermoregulation can infl uence the interpreta tion of behavioral responding (Le Bars et al., 2001; Tjolsen and Hole, 1993). In the context of the previous study, the fo llowing question can be raised. Is the expression of hyperalgesia a consequence of increased body temperature after exposure to stress? Thus, a possible underlying reas on for stress-induced changes in operant responses is altered body temperature. On th e other hand, an increase in core temperature may not be a critical factor in the expre ssion of stress-induced changes in nociception, but rather changes in skin temperature. In f act, skin temperature has been identified as a potential confounding variable (Tjolsen and Hole, 1993), and therefore, requires techniques to stabilize temper atures before behavioral asse ssment (e.g., pre-test trials at 36.0 C).

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69 Figure 3-9. Escape latencies dur ing testing trials at 44.0 C for control (open bar) and restraint (closed bar) groups when test ed immediately, fifteen minutes, and 24 hours after exposure to restraint. (A) Escape latencies were shorter immediately and fifteen minutes after restraint stress compared to control groups. However, responses did not carry over to the following day. (B) Difference scores confirmed that st ress reduced latencies compared to controls. Data are expre ssed in seconds and are repr esented as absolute group means S.E.M. Significant within-s ubject differences between 24 hours after stress exposure and trials by the same rats on baseline and 15 minutes are indicated as: P< 0.05. Significant between-subject differences between the control and restraint gr oups are indicated as: P< 0.05 and P< 0.001.

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70 Figure 3-10. Escape durations during testing trials at 44.0 C for control (open bar) and restraint (closed bar) groups when test ed immediately, fifteen minutes, and 24 hours after exposure to restraint. (A) Escape durations were immediately increased (stress-induced hyperalgesia) af ter restraint and when tested fifteen minutes after stress. However, response s did not carry over to the following day. (B) Difference scores confirmed that stress enhanced escape durations compared to controls. Data are expre ssed in seconds and are represented as absolute group means S.E.M. Si gnificant within-subject differences 24 hours after stress exposure and trials by the same rats immediately and 15 minutes are indicated as: P< 0.05. Significant between-subject differences between the control and restra int groups are indicated as: P< 0.05 and P< 0.001.

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71 Experiment 1 In order to determine the effect of re straint stress on thermoregulation, core temperatures of female rats (n=12) were recorded during separate sessions in which animals were exposed to no stress (control group ) or stress (restraint group; Figure 3-11). Temperature recordings were a ssessed prior to trials at 44.0 C either immediately (Figure 3-8A), 15 minutes (Figure 3-8B), or 24 hours (not shown) afte r restraint stress. Control animals were tested under identical conditi ons without exposure to restraint. In Figure 3-11A, body temperatures remained constant over the testing sessions in control group ( F= 0.047, P= 0.954), but temperatures were significantly affected by stress (restraint group; F= 18.06, P< 0.001). Core temperatures were significantly greater when evaluated immediately ( P< 0.001) but not 15 minutes ( P> 0.05) or the following day (24 hours; P> 0.05). Increased core temperatures could be a consequence of struggling during restraint, which consequently raises te mperature. A feature of restraint stress is the uncontrolled restrictions impos ed by the device that an animal initially tries to escape. Differences between the two groups revealed that temperatures were significantly higher when assessed immediately (Figure 3-11B; F= 30.807, P< 0.001) and 15 minutes ( F= 7.506, P= 0.043) but not twenty-four hours ( F= 2.012, P= 0.170) after restraint. Thus, while restraint temporarily incr eased core temperatures (0.86 0.09), they were slightly comparable to controls 15 minutes after the ons et of stress. Changes in core temperature therefore cannot account entirely for the obser vation of stress-induced hyperalgesia on operant responses.

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72 Experiment 2 An additional group of female rats (n=6) were used to examine the effects of stress on core (Figure 3-12A) and cutaneous temperat ures (Figure 3-12B). Core temperatures were assessed over five minute intervals duri ng restraint and exposure to a thermal plate at 36.0 C (similar to pre-test trials ). Recordings during contro l and restraint groups were obtained in different sessions. In addition to core temperature, cutaneous temperatures were measured in rats in which animals were not stressed (control group) or stressed for 15 minutes (restraint group). For the restra int group, recordings were obtained before and after restraint and after exposure to a 15 minute pre-test trial at 36.0 C. Control animals were assessed before and after the pre-test. Core body temperature In Figure 3-12A, stress increased core te mperature (restraint group, gray circle; F= 9.420, P< 0.001). Specifically, a significant increas e in core temperatures was observed at ten ( P< 0.001) and 15 ( P< 0.001) minutes during restraint. Temperatures peaked at 38.62 0.11OC. These results are in agreement w ith Figure 3-11. But, after placement into the pre-test at 36.0 C, core temperatures gradually decreased. Temperatures were greater than baseline at five ( P< 0.001) but not after ten ( P> 0.05) or 15 ( P> 0.05) minutes during pre-test conditions. In the control group (asterisk with dashed line), temperatures increased slightly after placement into the pr e-test, but this effect was not significant ( F= 0.48, P= 0.7). Differences between groups (not shown) suggested that temperatures were higher after stress than control before placement into the pre-test at 36.0 OC ( F= 14.511, P= 0.003) but did not differ five ( F= 2.207 P= 0.168), ten ( F= 0.000, P= 1.0), and 15 ( F= 0.009, P= 0.926) minutes into the trial.

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73 Figure 3-11. Core body temperatures during testing trials at 44.0 C for control (open bar) and restraint (closed bar) groups when tested immediately, fifteen minutes, and 24 hours after exposure to restrain t. (A) Core temperatures were immediately increased ( stress-induced hyperthermia ) compared to the control group. Core temperature was still higher than controls fifteen minutes after stress. Core temperature did not di ffer the following day. (B) Difference scores revealed that stress-induced increase in temperature was most prominent immediately after stress and sl owly returned to control values. Data are expressed in seconds and are represented as absolute group means S.E.M. Significant within-subject di fferences immediately after stress exposure or not stress are indicated as: *** P <0.001. Significant between-subject differences between the control and re straint groups are indicated as: P< 0.05 and P< 0.001.

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74 Thus, stress-induced increases in core temperature are most prevalent during exposure to stress. Temperatures quickly return to levels comparable to controls during a pre-test trial at 36.0 C. This data supports the idea that altered body temperature is not responsible for the expression of stress-induced hyperalgesia when animals are tested at 44.0 C (consistent with data shown in Figure 3-11). Peripheral cutaneous temperature In general, cutaneous temperatures were lower than body temperatures (27.97 0.48 vs. 37.53 0.27 C; F= 300.836, P< 0.001). Cutaneous temperatur es were significantly different across testing sessions (Figure 3-12B; F= 19.621, P< 0.001). Recordings were greater than baseline when assessed after termination of restraint ( P< 0.01) and exposure to 36.0 C ( P< 0.001). In the control group, skin temperatures also increased ( F= 90.886, P< 0.001). Temperatures increased af ter the pre-test trial at 36.0 C in the absence of stress because of increased activity of the an imal and exposure to thermal stimulation that was provided by the heated plate. No differenc es in temperatures were observed between control and restraint groups during baseline before stress ( F= 1.019, P= 0.337) or after exposure to 36.0 C ( F= 0.554, P= 0.474). However, recordings after restraint were higher than control before placement into the pre-test ( F= 15.689, P= 0.003). In summary, while stress increased skin temperatures ( stress-induced hyperthermia ), this effect does not pers ist, and quickly returned to levels comparable to controls. Because temperatur es in control and restrain t groups were equalized by 36.0 C before placement into the second trial at 44.0 C, core and cutaneous temperatures did not play an important role in the expres sion of stress-induced hyperalgesia.

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75 Figure 3-12. Core and cutaneous hindpaw temper atures for control (a sterisk with dashed lines) and restraint (gray circle) groups (A) Core temperature was increased during a fifteen-minute exposure to rest raint stress. After termination of restraint, animals were placed into a pre-test at 36.0 C in which temperatures progressively returned to levels comp arable to controls. (B) Cutaneous temperatures were greater after restraint, but exposure to pre-test normalized skin temperatures. Data are expressed in seconds and are represented as absolute group means S.E.M. Si gnificant within-subject differences between temperature assessed during and af ter restraint compared to pre-stress baseline values are indicated as: ** P< 0.01 and *** P< 0.001. Significant between-subject differences between te mperature assessed before and after a pre-test trial are indicated as: P< 0.01.

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76 Effects of Restraint Stress on Control Responses Operant Escape Responses at 36.0 C Behavioral responses during 36.0 C trials were obtained in female rats (n=11) during baseline sessions (Day 1), testing sessions in which rats received 15 minutes of restraint stress (restra int group; Day 2) or no stress (control group), and sessions the following Day (24 hours; Day 3). Asse ssment of escape responses during 36.0 C trials is an important strategy to determine if a pa rticular experimental manipulation produces avoidance, or increased platform durati on unrelated to thermal stimulation. Operant escape latencies As illustrated in Figure 3-13A, escape latenc ies were not affected by restraint stress ( F= 0.23, P= 0.795). Durations were not different between control a nd restraint groups (Figure 3-13B; F= 1.124, P= 0.33). Response latencies were highly variable at 36.0 C, a non-noxious temperature, due to explorator y behaviors and the inability of this temperature to elicit escape behavior. Operant escape durations Similar to latencies during 36.0 C trials, escape durations we re also not affected by restraint stress (Figure 3-14A; F= 2.599, P= 0.0869). Durations were not different between control and restra int groups (Figure 3-14B; F= 0.993, P= 0.382). At this neutral plate temperature, platform occupancy was short and the duration of operant escape response did not change signifi cantly after restraint stress. Because responses were not affected, stress does not induce av oidance during the escape test.

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77 Figure 3-13. Escape latencies during testing trials at 36.0 C for control (open bar) and restraint (closed bar) groups during baseline sessions (Day 1), testing sessions in which rats received 15 minutes of re straint stress (restrai nt group; Day 2) or no stress (control group), and session s the following day (Day 3). (A) Exposure to restraint did not affect escape latencies compared to the control groups and compared to unstressed trials on baseline and 24 hours after stress. (B) Difference scores confirmed that stre ss did not alter escape latencies (15 minutes; Day 2). The data are expresse d in seconds and are represented as absolute group means S.E.M.

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78 Figure 3-14. Cumulative escape durati ons during testing trials at 36.0 C for control (open bar) and restraint (closed ba r) groups during baseline se ssions (Day 1), testing sessions in which rats received 15 minutes of restraint stress (restraint group; Day 2) or no stress (control group), a nd sessions the following day (Day 3). (A) Exposure to acute stress produced no significant alteration of escape durations during 36.0OC trials. (B) Difference scores confirmed that stress did not significantly alter es cape durations (15 minutes; Day 2). Data are expressed in seconds, and values are represented as absolute group means S.E.M.

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79 Sequence analysis of successive operant escape durations In addition to analysis of the total du ration of escape within trials at 36.0 C, successive escape plate (A, B) and platform (C, D) durations were examined between control (left panel) and restrain t (right panel) groups (Figure 3-15). Overall, compared to plate responses at 44.0 C, plate times were longer at 36.0 C. For both groups, plate durations were higher during the beginning of the trial and gradually decreased. Plate durations for control (Figure 3-15A) and rest raint (Figure 3-15B) groups did not change across the baseline, test, or 24 hours periods. In contrast, platform durations remained stable throughout the trial and were substantia lly shorter than plate durations for control (Figure 3-15C) and restraint (Figure 3-15D) groups. Alt hough restraint stress did not influence platform durations, the number of re sponses was reduced compared to baseline. In support of the cumulative platform durati ons represented in Fi gure 3-16, restraint did not significantly affect escape responses at neutral temperatures. In summary, the average duration of the first 6 responses during trials at 36.0 C for the control and restra int groups are compared in Figur e 3-16. In the control group, neither plate (Figure 3-16A; F= 1.306, P= 0.2859) nor platform ( F= 2.382, P= 0.1096) durations changed over the three consecutive se ssions. Similar to controls, stress did not affect plate (Figure 3-16B; F= 2.256, P= 0.1233) or platform ( F= 1.112, P= 0.3422) durations over the three consecutive sessions. Thus, stress does not produce an incr ease in platform duration at neutral temperatures suggesting that the expre ssion of stress-induced hyperalgesia at 44.0 C, which activates C-nociceptors, is dependent on higher levels of sensory processing. Furthermore, stress-induced hyperalgesia is not a consequen ce of avoidance.

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80 Darkbox Responses In order to control from changes in motor functioning and motivation, darkbox responses were evaluated in female rats (n=11) for control and stress conditions (Table 3-2). Latencies of escape from bright light in the darkbox test were unaffected by prior stress ( F= 1.633, P= 0.2358). Therefore, acute restraint st ress did not sign ificantly alter aversion to light or produce motor effects (e.g. freezing) that inte rfered with escape performance. Table 3-2. Darkbox latencies for control and re straint groups. Responses were unaffected by stress. Data are expressed in se conds, and values are represented as absolute group means S.E.M. Baseline 15 Minutes 24 Hours Control 12.7 1.3 Control13.4 1.5 Control 15.3 1.6 Pre-Stress 16.4 1.8 Stress 15.3 1.8 Post-Stress 12.6 1.5 Effects of Restraint Stress on Operant Thermal Preference Based on the preceding results, restraint st ress produces a height ened sensitivity to heat (e.g., decreased plate durations; increase d escape platform durations). But, is stress-induced hyperalgesia spec ific to the escape paradigm? Or, does acute restraint stress produce a generalized heightened sens itivity to heat? To determine if this observation was limited to the escape paradigm or could be generali zed to other operant paradigms, an additional operant paradigm (the rmal preference) was used to clarify this issue. Enhanced heat sensitivity would be indicated by an increase in duration (preference) for the cold compartment. In these experiments, the floor was cooled to 15.0 C in one compartment and heated to 45.0 C in the adjacent compartment. Responses were recorded during a 12-minute trial preceded by a pre-test trial at 36.0 C (15 minutes).

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81 Figure 3-15. Sequence analysis of succe ssive escape plate and platform du rations during testing trials at 36.0 C for control (left panel) and restraint (right panel) groups during ba seline sessions (asterisk; Day 1), testi ng sessions in which rats received 15 minutes of restraint stress (restraint gr oup, gray circle; Day 2) or no stress (contro l group, gray circle), and sessions the following day (closed square; Day 3). Data are expressed in seconds and are repr esented as absolute group means S.E.M

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82 Figure 3-16. Average escape dur ation of the first six plat e (open bar) and platform (closed bar) responses duri ng testing trials at 36.0 C for control and restraint groups during baseline sessions (Day 1), testing sessions in which rats received 15 minutes of restraint stress (restraint group; Day 2) or no stress (control group), and sessions the followi ng day (Day 3). In both groups, plate durations were longer than platform durations. In addition, no differences were observed in plate and platform dur ations over sessions. The data are expressed in seconds and are represen ted as absolute group means S.E.M.

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83 Thermal Preference Durations To determine the effects of stress on the thermal preference test, baseline responses (n=20; Day 1) were compared to sessions in which all animals underw ent restraint stress for 15 minutes and testing 15 minutes (test; Day 2), 24 hours (Day 3) and 48 hours (Day 4) after restraint (Figure 3-17). Similar to the operant escape test, thermal preference responses were continuously assessed during th e testing trial. In Figure 3-17A, restraint stress influenced thermal preference responses ( F= 2.865, P= 0.041). Cold preference was significantly higher than baseline after stress (Day 2; P< 0.05) but not 24 (Day 3; P> 0.05) or 48 (Day 4; P> 0.05) hours after stress. Convers ely, heat preference was lower after stress (Day 2; P< 0.05) but not 24 (Day 3; P> 0.05) or 48 (Day 4; P> 0.05) hours later. Difference scores (Figure 3-17B) revealed that the effect of stress was most prominent when thermal preference was asse ssed 15 minutes after stress. Responses quickly returned to levels comparable to baseline during subsequent testing sessions. Thus, similar to escape (e.g., decreased time spent on the heat plate), restraint stress produced a heightened sensitivity to heat as indicated by an increase in cold preference and a decrease in heat preference. Sequence Analysis of Successive Thermal Preference Durations In addition to analysis of the cumulative preference responses, su ccessive cold and heat durations within trials were compared. The average duration of the first 6 cold and heat preference responses were analyzed to determine the effect of stress on successive preference responses (Figure 3-18).

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84 Figure 3-17. Cumulative thermal preference dura tions during testing tr ials at 15.0 (cold: open bar) and 45.0 C (heat: closed bar) during baseline sessions, testing session in which rats received 15 minutes of restraint stress, and sessions 24 and 48 hours after stress. (A) Under base line conditions (Day 1), preference for heat was greater than the prefer ence for cold. An acute exposure to restraint significantly decreased preference for heat on Day 2. However, preference for heat returned to pre-stre ss levels when assessed 24 (Day 3) or 48 hours (Day 4) hours after restraint st ress. (B) Based on difference scores, stress produced a substantial change in preference that dissipated the following testing sessions. Data are expressed in s econds and are represented as absolute group means S.E.M. Signi ficant differences between the control and restraint groups on Day 2 are indicated as: P <0.05.

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85 Restraint stress had an effect on cold ( F= 8.786, P= 0.01) and heat ( F= 3.632, P= 0.038) preference responses. The averag e duration of cold responses were significantly higher than baseline after exposure to stress ( P< 0.01) but not higher 24 ( P> 0.05) or 48 ( P> 0.05) hours later. Consequently, the average duration of heat responses were significantly lower than baseline after exposure to stress ( P< 0.05) but not higher 24 ( P> 0.05) or 48 ( P> 0.05) hours later. As show n on operant escape, stress produced a transient increase sensitivity to h eat as indicated by an increase in cold preference to 15.0 C (longer duration) and a decreas e in heat preference to 45.0 C (shorter duration). Figure 3-18. Average of the first six cold (open bar) and heat (c losed bar) durations during baseline sessions, testing sessions in which rats received 15 minutes of restraint stress (test), and sessions 24 and 48 hours after stress. Restraint stress affected the average cold a nd heat preference responses. Cold responses were increased while heat responses were reduced. This effect did not continue during sessions assessed the following days (24 and 48 hours). Data are expressed in seconds and are represented as absolute group means S.E.M. Significant differences between restraint for cold and heat preference are indicated as: P <0.05.

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86 Effects of Endogenous Opioids on St ress-Induced Changes in Nociception Previous studies suggest that restra int-induced changes in nociception are modulated by opioid mechanisms. Stress-induced changes in nociception are characterized as either op ioid-dependent or non-opioid dependent (Lewis, Cannon, and Liebeskind, 1980; Porro and Carli, 1988; Dr olet et al., 2001). Involvement of endogenous opioids, which are released duri ng stress (Madden et al. 1977), in the expression of stress-induced hyporeflexia is confirmed by admini stration of an opioid antagonist (naloxone; Pilcher and Browne, 1983). In the present study, injections of naloxone were used to dete rmine the role of endogenous opioids in modulating e ffects of stress on reflex ( hyporeflexia ) and operant ( hyperalgesia ). It was hypothesized that the endo genous opioid system contributes to stress-induced reduction of reflexes while opposing the excitatory effects of stress on cerebrally mediated operant escape re sponses to thermal stimulation. Reflex Lick/Guard Responses As mentioned previously, re straint stress suppressed reflex lick/guard responses (increased latencies; decr eased duration). To determine if the expression of stress-induced hyporeflexia was mediated by endogenous opioids, behavioral responses were assessed in a group of female rats duri ng sessions in which ra ts received 15 minutes of restraint stress (restraint group; n=19) or no stress (con trol group; n=19) followed by an injection of saline (1.0 mg/kg) or naloxone (3.0 mg/kg). Then, animals were tested during a trial at 44.5 C for 10 minutes that was preceded by a pre-test at 36.0 C (15 minutes).

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87 Reflex lick-guard latency Reflex latencies are presented in Figure 3-19. Reflex latencies were significantly longer in the restraint group af ter an injection of saline comp ared to non-stressed controls (Figure 3-19A; F= 10.065, P= 0.003). Stress-induced increases of reflex latencies were reduced after administration of naloxone at 3 mg/kg ( F= 4.475, P= 0.041). Naloxone did not affect reflex latencie s in the control groups ( F= 0.517, P= 0.477). Difference scores between control and restraint groups revealed th at the reduction of re flex latencies after stress, a characteristic of stress-induced hyporeflexia was reduced by naloxone (Figure 3-19B, F= 9.704, P= 0.004). Reflex lick-guard duration The duration of reflex responses were also assessed in this group of rats during trials at 44.5 C (Figure 3-20). After saline injection, durations were significantly shorter after restraint than the co ntrol group (Figure 3-20A, F= 7.526, P= 0.009). Naloxone did not affect reflex durations in the control groups ( F= 0.013, P= 0.723) but reversed stress-induced decreases of reflex durations ( F= 5.459, P= 0.025). Reduction of reflex durations to thermal stimulat ion by stress, a feature of stress-induced hyporeflexia was reversed by naloxone as reve aled by differences between both groups (Figure 3-20B, F= 11.403, P= 0.002). Thus, the ability of naloxone to reduce expression of stress-induced hyporeflexia as assessed by both reflex latenc ies and duration provides evidence for the involvement of an endogenous opioid system in mediating these effects.

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88 Figure 3-19. Reflexive lick /guard latencies at 44.5 C during testing sessions in which rats received 15 minutes of restraint stress (restraint group, closed bar) or no stress (control groups, open bar) followed by an injection of saline or naloxone. (A) Reflex latencies were longer after exposure to restraint stress than control group. Naloxone did not affect reflex latencies under control group, but naloxone reduced stress-induced inhibition of reflex latencies. (B) Difference scores between control a nd restraint groups revealed that naloxone reduced the increase in reflex latenc ies after stress. Data ar e expressed in seconds and are represented as absolute group means S.E.M. Significant within-subject differences after saline and naloxone injections are indicated as: P <0.05. Significant between-subject differences between control and restraint groups are indicated as: P< 0.01.

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89 Figure 3-20. Cumulative reflexiv e lick/guard durations at 44.5 C during testing sessions in which rats received 15 minutes of restraint stress (res traint group, closed bar) or no stress (control groups, open ba r) followed by an injection of saline or naloxone. (A) Reflex responses were reduced by restraint stress compared to control group. Similar to latencies, injection of naloxone did not affect reflex durations under control group, but naloxone reduced stress-induced inhibition of reflex durations. (B) Di fference scores between control and restraint groups revealed that naloxone reduced the decrease of reflex durations after stress. Data are expre ssed in seconds and are represented as absolute group means S.E.M. Signi ficant within-subject differences after saline and naloxone injecti ons are indicated as: P <0.05. Significant between-subject differences between cont rol and restraint groups are indicated as: P< 0.01.

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90 Operant Escape Responses The expression of stress-induced hyporeflexia is mediated by the endogenous opioid system. However, the role of this system in mediating enhanced sensitivity on cortically mediated responses to thermal stimulation is unknown. To determine if the expression of stress-induced hyperalges ia was mediated by endogenous opioids, behavioral responses were assessed in a separa te group of female rats during sessions in which rats received 15 minutes of restraint stress (restraint group; n=10) or no stress (control group; n=10) followed by an injecti on of saline (1.0 mg/kg) or naloxone (3.0 mg/kg). Then, animals were tested during a trial at 44.5 C for 15 minutes that was preceded by a pre-test at 36.0 C (15 minutes). Figure 3-21 presents the cumulative duration of reflex responses that were assessed in a group of rats during trials at 44.5 C. Restraint stress enhanced thermal sensitivity (Figure 3-21A stress-induced hyperalgesia ). Specifically, durations were significantly greater after stress (saline; F= 4.725, 0.043). Interestingly, na loxone increased escape durations in the control ( F= 4.080, P= 0.050) and restraint ( F= 8.325, P= 0.010) groups. Escape durations remained higher in the re straint group to the control group after injection of naloxone ( F= 5.209, P= 0.035). However, difference scores (from controls) revealed that naloxone produced similar eff ects (e.g., increased duration) in both groups (Figure 3-21B, F= 0.145, P= 0.707). The ability of naloxone to augment stre ss-induced hyperalgesia provides evidence for endogenous opioid system in mediating these e ffects. It appears that this system is important for mediating escape behavior and suppressing mechanisms underlying the expression of hyperalgesia.

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91 Figure 3-21. Cumulative escape durations at 44.5 C during testing sessions in which rats received 15 minutes of restraint stress (restraint group, closed bar) or no stress (control groups, open bar) followed by an injection of saline or naloxone. (A) Escape durations were significantly great er after an injection of naloxone when compared to control groups. Na loxone significantly increased escape durations after restraint stress. (B) Di fference scores revealed that stress significantly increased escape durations in saline treated animals, which were slightly longer after naloxone. Data are expressed in seconds and are represented as absolute group means S.E.M. Significant within-subject differences after saline and naloxone injections are indicated as: P <0.05 and ** P <0.01. Significant between-subject differences between control and restraint groups are indicated as: P< 0.05.

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92 Effects of Morphine on Stress-In duced Changes in Nociception Under non-stressful conditions, morphine pr oduces hyporeflexia at high doses (5 to 10 mg/kg), as indicated by a longer latency to elicit a reflex response to high intensity stimulation (>50 C; for reviews see Drolet et al., 2001; Yamada and Nabesima, 1995). Studies have shown that mor phine-induced hyporeflexia is en hanced by restraint stress (Abbelbaum and Holtzman, 1984, 1985; Calcagnetti and Holtzman, 1990, 1992; Fleetwood and Holtzman 1989). Paradoxically, low dose morphine (0.5 to 5.0 mg/kg) results in hypereflexia, which is demonstrat ed by a shorter latency and longer duration of reflex responses (Cooper and Vierck, 1986; Guirimand et al., 1995; Holtman and Wala, 2005; Vierck et al., 2002; Wiley, Personal Co mmunication) presumably from excitatory effects on motor activity (Lee et al., 1978; Le Bars et al., 2001 ). Hyperactivity after low dose morphine was observed particularly in stereotyped behaviors and is mediated by -opioid (Negus et al., 1993; Weinger et al., 1991) and 2 (Weinger et al., 1992, 1995) receptors. Mu-opioid receptors are widely distributed throughout the nervous system particularly in areas of the brainstem a nd cortex involved in sensory processing and motor output (Drolet et al., 2001; Yamada and Nabesima, 1995). Similar doses of morphine have been shown to reduce opera nt responses (morphine-induced hyporeflexia; Vierck et al., 2002). Reflex Lick/Guard Responses First response latency of refl ex lick/guard responses to 44.5 C thermal was evaluated to determine if exogenous opio ids could modulate the expression of stress-induced hyporeflexia. Behavioral re sponses were obtained during sessions in which rats received 15 minutes of restraint stress (restraint group) or no stress (control

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93 group) followed by an injection of morphine. The drug was administered at 1 (n=19), 5 (n=13), 8 (n=10), and 10 (n=8) mg/kg in addition to saline (n=19). Reflex lick-guard latency In the control group, morphine significantly affected reflex latencies (Figure 3-22A, F= 14.006, P< 0.001). No differences were seen with 1 mg/kg ( P> 0.05), but reflex latencies longer than salin e were observed after 5.0 ( P< 0.05), 8.0 ( P< 0.01) and 10.0 ( P< 0.01) mg/kg of morphine. In a similar protocol, morphine also significantly increased latencies after stress ( F= 26.600, P< 0.001). Specifically, latencies were significantly longer afte r morphine at 5.0 ( P< 0.05), 8.0 ( P< 0.01) and 10.0 ( P< 0.01) but not 1 mg/kg of morphine ( P> 0.05). Difference between control and restraint groups (Figure 3-22B) revealed that reflex latencies were si gnificantly longer after stress compared to the control groups for all doses of morphine: 1.0 ( F= 4.065, P= 0.04), 5.0 mg/kg ( F= 6.030, P= 0.02), 8.0 ( F= 7.947, P= 0.011), and 10 ( F= 5.224, P= 0.038) mg/kg. This supports previous data that stress enha nced the hyporeflexic effect of morphine. Reflex lick-guard durations The duration of reflex responses were also assessed in this group of rats during trials at 44.5 C after morphine administration (Fi gure 3-23). Reflex durations were significantly shorter after rest raint than the control group, but morphine increased reflex durations in stressed animals suggesting that morphine countered an inhibitory effect of stress (Figure 3-23A, B). For example, refl ex durations were shorter in the restraint group compared to the control group especia lly after injections of morphine at 1 ( F= 4.965, P= 0.032), 5 ( F= 4.965, P= 0.032) and 10 ( F= 5.395, P= 0.039) mg/kg, but no significant differences were observed at 8 mg/kg ( F= 1.260, P= 0.276) due to high degree of variability.

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94 Figure 3-22. Reflexive lick /guard latencies at 44.5 C during testing sessions in which rats received 15 minutes of restraint stress (restraint group, closed bar) or no stress (control groups, open bar) followed by an in jection of saline or morphine. (A, B) Reflex latencies were longer after e xposure to restraint stress compared to the control group. Injections of morphi ne dose-dependently increased reflex latencies in both groups. Morphine potentiated stre ss-induced inhibition of reflex latencies compared to the contro l group. Data are expressed in seconds and are represented as absolute group means S.E.M. Significant within-subject differences after saline and morphine injections are indicated as: P <0.05 and ** P <0.01. Significant between-subject differences between control and restrain t groups are indicated as: P< 0.05 and P< 0.01.

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95 Figure 3-23. Reflexive lick /guard durations at 44.5 C during testing sessions in which rats received 15 minutes of restraint stress (restraint group, closed bar) or no stress (control groups, open bar) fo llowed by an injection of saline or morphine. (A, B) Reflex durations were shorter af ter exposure to restraint stress compared to the control group. Injections of morphine produced a bi-directional effect depending on the dose of morphine. In the control group, reflex durations were longer after injections of 1.0 and 5.0, mg/kg of morphine compared to saline treated groups. Longer durations peaked at 5.0 mg/kg. Reflex durations were shorte r after injections of 10.0 mg/kg of morphine. In the restraint group, reflex durations were shorter than morphine treated groups in the control group. Ho wever, reflex durations were longer after injections of 1.0 a nd 5.0 mg/kg of morphine co mpared to saline treated groups in the stress group. Reflex durat ions were shorter after an 8.0 and 10.0 mg/kg dose of morphine. Data are expr essed in seconds and are represented as absolute group means S.E.M. Signi ficant within-subject differences after saline and morphine injections are indicated as: P <0.05. Significant between-subject differences between cont rol and restraint groups are indicated as: P< 0.05 and P< 0.01.

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96 In summary, morphine produced a bi-direc tional effect of reflex response duration depending on the dose. Hypereflexia and hyporeflexia was observed at lower and higher doses, respectively. Stress-induced h yporeflexia was reduced by morphine. Furthermore, a discrepancy between reflex la tencies and duration occurred. While reflex latencies were inhibited, durations were enhanced. It is impo rtant to note that a majority of studies use latencies as the behavioral outcome measure, but only a few groups use durations (Vierck et al., 2002). Studies that rely on reflex la tencies failed to recognize changes in duration within a trial. Becau se previous studies used high intensity stimulation, trial durations were short and ranged from 10 to 30 seconds (Holtman and Wala, 2005). In the present experiment, the discrepancy may be a result of an initial decrease in reflex responding fo llowed by a period of excitation. Operant Escape Responses Based on the ability of morphi ne to affect reflex respons es in control and stress conditions, the role of exogenous opioids on the expression of stress-induced hyperalgesia was examined. Figure 3-24 (A B) presents the effects of low dose morphine on operant esca pe durations during 44.5 C trials for control (n=10) and restraint (n=10) groups. In th e control group, while durations were shorter after injection of morphine, but this effect was not quite significant ( F= 3.426, P= 0.081). However, morphine significantly reduced dur ations in the stress groups ( F= 5.689, P= 0.028). The effect of morphine was similar be tween control and stress groups ( F= 0.397, P= 0.536). Morphine reduced the expression of stress -induced hyperalgesia (e.g. decreased escape platform duration; increased thermal plate duration).

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97 Figure 3-24. Cumulative escape durati ons during testing trials at 44.5 C during testing sessions in which rats received 15 minutes of restraint stress (restraint group, closed bar) or no stress (c ontrol groups, open bar) follo wed by an injection of saline or morphine. (A, B) Escape dura tions were significantly greater after restraint stress when compared to c ontrol groups. However, morphine reduced escape durations in control groups and reduced stress-induced excitation of escape durations. Data are expressed in seconds and are represented as absolute group means S.E.M. Significant within-subject differences after saline and morphine injections are indicated as: P <0.05. Significant between-subject differences between control and restraint groups are indicated as: P< 0.05.

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98 Summary and Discussion Brief (15 minutes) exposure to restrain t stress produced differential effects on reflex and operant responses to nociceptiv e thermal stimulation. Restraint stress depressed lick/guard responses, increasing response latencies and decreasing the time spent performing reflex responses during a tria l. In contrast, stress enhanced escape responding to thermal stimulation. The late ncy of initial escape responses was not affected by stress in one group (Figure 3-4) but was affected in another group (Figure 3-9). Escape latency is a highly variable measure of nociceptive responsivity, due to factors that include a strong i nnate tendency to explore the co nfines of a test environment upon initial entry. Escape latency has been show n to be consistently less sensitive than response duration when measured against variati ons in stimulus intensity or experimental treatments other than stress (Vierck et. al., 2002, 2003). In contrast to latency, rats consistently apportion their time on the escape platform according to the intensity of nociceptive stimul ation, indicating that escape duration most reliably reflects the aversive magnitude of a stimulus. In addition, restraint stress produced a transient hyperalgesia in another op erant escape test (the thermal preference) characterized by a decrease in tim e spent on a plate heated to 45.0 C. Based on these two operant behavioral assessment strategies, the primary c onsequence of stress is the enhancement of cortically mediated responses to heat (e.g., stress-induced heat hyperalgesia ). Role of the Endogenous Opioid System and Morphine The expression of stress-induced cha nges in nociception was affected by endogenous and exogenous opioids Studies have demonstrat ed exposure to restraint stress increases first response latency to e ngage in tail withdr awal, hindpaw licking, and

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99 hindpaw guarding (Amir and Amit, 1978; Calcagnetti and Holtzman, 1992; Calcagnetti et al., 1992; Gamaro et al., 1998; Terman et al ., 1986; Vidal and Jacobs, 1984). Reduction of reflex responses by stress has been interp reted as stress-induced hyporeflexia. Various mechanisms modulate the expression of stress-induced hyporefle xia including opioid mechanisms (Lewis et al., 1980; Porro and Carli, 1988; Yamada and Nabeshima, 1995). In addition to regulating th e stress response, opioid pep tides regulate sensory and affective components of pain processing during painful and st ressful situations through activity of -opioid receptors (Yamada and Nabe sima, 1995; Zubieta et al., 2001). Several lines of evidence support the hypot hesis that endogenous opioid peptides modulate stress-induced hyporefle xia. Release of opioid peptides and expression of stress-induced hyporeflexia share a simila r temporal profile (Bodnar et al., 1978a; Madden et al., 1977). The contribution of endogenous opioids on the expression of stress-induced hyporeflexia is illustrated by its cross-tole rance with morphine and reversibility by naloxone (Pilcher and Br owne, 1983; Terman et al., 1982). In the current study, endogenous opioids have no tonic inhibitory effect on reflexes response as indicated by the in ability of naloxone to infl uence reflex responses. However, cortical mediated responses (escape) are subjected to toni c inhibitory control by this system. In response to thermal stimulation, endogenous opioids are released (Cesselin et al., 1989; Kuraishi et al., 1984; Takagi, 1984) an d consequently may regulate escape responses in the absence of stress. Ad ministration of an opioid antagonist or other anti-opioid molecule (e. g., CCK) suppresses this system; endogenous opioids can no longer mediate responses, which should resu lt in an increase sensitivity to heat stimulation.

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100 Interestingly, the release of endogenous opioi ds under non-restraint conditions is an important factor in the expression of cer erbrally-mediated responses, but not the neuroanatomical mechanisms underlying refl ex responses. Under stress conditions, hyporeflexia was reversed by naloxone suggest ing that the hyporeflexia was mediated by endogenous opioids. In contrast stress-induced hyperalgesia was enhanced by naloxone suggesting that the expression of hyperalges ia was opposed by the same system. An increase in endogenous opioid release has been observed after stre ss (Larsen and Mau, 1994; Madden et al., 1977) part icularly in areas traditiona lly involved in movement and drug addition (mesocorticolimbic systems; Kalivas and Abhold, 1987; Kalivas and Duffy, 1995). In the current study, exogenous opioids (morphine) enhanced the expression of stress-induced hyporeflexia and hyperalgesia to low intensit y suprathreshold thermal stimulation. However, depending on the behavi oral outcome, differe nces between reflex responses were observed. In confirmation of previous studies, morphine and stress increased the latencies to elic it a reflex response. Previous studies have demonstrated that stress can enhance the hyporeflexic (e.g., increase reflex latencies) effects of -opioid agonists (e.g., morphine; Abbelb aum and Holtzman, 1984, 1985; Calcagnetti and Holtzman, 1990, 1992; Fleetwood and Holtzman 1989). These effects of stress are reduced in chronically stressed and morphine tolerant animals (Fleetwood and Holtzman 1989). In agreement with these studies, th e hyporeflexic effect of morphine was enhanced by stress when assessed by reflex latencies. However, low dose morphine enhanced reflex responses ( morphine-induced hypereflexia ) and higher doses were required (> 8 mg/kg) to reduce reflex responses ( morphine-induced hyporeflexia ). If

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101 latencies were only assessed, it would be falsely concluded that morphine produced hyporeflexia, which was stress enhanced, co mparable to previous studies. But, evaluation of response durations provides evidence that morphine has pro-nociceptive properties presumably through the activation of NMDA receptors (Heinricher, et al., 1997, 2001; Holtman and Wala, 2005; Manni ng et al., 1996; Pr ice et al., 2000). Furthermore, motor output is altered by opioids due to inhibition of GABAergic transmission in the cortex (e.g., substantia nigra and striatum; Turs ki et al., 1982) and brainstem (RVM; Weinger et al., 1991). Based on reflex durations, morphine oppos ed the expression of stress-induced hyporeflexia and hyperalgesia, when assesse d by response durations. These results suggest that morphine simultaneously activat ed descending inhibito ry and facilitatory pathways, but activity within these pathways affected spino-bulbo-spinal and cerebrally mediated responses differently. The e ffects on facilitation (morphine-induced hypereflexia) were more significant than those on inhibition (stress-induced hyporeflexia). The effects on inhibition (m orphine-induced hypoalgesia) were more significant than those on facilitati on (stress-induced hyperalgesia). Expression of Stress-induced Hyperalgesia The expression of hyperalgesia is most not iceable when assessed immediately after stress. Escape responses were highest comp ared to controls during this time point. Although the effects of stress remained signi ficant 15 minutes later, it was slightly smaller than responses assessed immediately after th e termination of stress. Even though behavioral responses were not evaluated afte r 30 minutes, future st udies could perform a more intensive assessment across multiple time points. Responses would be expected to

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102 diminish over time based on the fact that re sponses were greater immediate after stress and returned to pre-stress levels the following day. Evidence has suggested that the release of opioid peptides during exposure to a stressor regulates the stress response and allows an animal to cope with stressful situations (Amit and Galina, 1988; Curtis et al., 2001; Sumova and Jakoubek, 1989; Tanaka et al., 2000; Terman et al., 1984). But, the release of endogenous opioids does not explain the underlying mechanisms for stress-induced hyperalgesia Possible mechanisms may be related to changes in va rious physiological systems, which are most apparent immediately after stress. The release of excitatory or anti-opioid peptides could account for the increase in sensitivity. While the role of endogenous opioids was addressed, the current study did not evalua te additional neurochemicals previously identified to enhance nociceptive sens itivity such as cholecystokinin (CCK), noradrenaline (NE), and dopamine (DA). Evidence suggests that CCK possess anti-opioid properties. CCK mRNA is found in areas of opioid expression (Stengaard-Pedersen and Larsson, 1981, 1982). CCK is a contributing factor in the development of opioid tolerance after repeated morphine administra tion (Stanfa et al., 1994) and injury (Vanderah et al., 2001a, 2001b; Xie et al., 2005). Modulation of this system with agonists (CCK8) or antagonist (L365, 260; proglumide) can reduce or enhance the hyporeflexic effects of morphine, respectively (F aris et al., 1993; Hawranko and Smith, 1999; Pu et al., 1994; Xie et al ., 2005). Furthermore, stress-induced hyporeflexia is enhanced by CCK antagonist s (Hawranko and Smith, 1999; Watkins et al., 1984, 1985).

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103 Furthermore, the expression of hypera lgesia may be a consequence of hypervigilance or attentiveness to the environment. Stress has been known to increase vigilance in animals (Castilho et al., 1999; Hayes and Katayama, 1986; Maier et al., 1992) presumably through NE (Tanaka et al., 2000). Immobilizati on stress induces the release of NE (Glavin et al., 1983; Iimori et al., 1982; Keim and Sigg, 1976; Tanaka et al., 1982, 1983a, 1983b). Interestingly, exogenously administered opioids antagonists or agonists can increase (naloxone) or decrease (morphine) stress -induced release of NE in limbic and other cortical structures, respectivel y. It has been speculated that the release of NE by stress is a critical factor in the expression of anxiety (Tanaka et al., 2000). Opioid peptides, in turn, are responsible for the termination of the stress response (Valentino and Van Bockstaele, 2001), whic h also supports evidence that opioids regulate affective and motivational states dur ing pain and stress (Zubieta et al., 2001). Other possible mechanisms include alterations in the DA system (Altier and Stewart, 1996, 1999a, 1999b; Beaulieu et al., 1987; De S ouza and Van Loon GR, 1986; Kalivas and Duffy, 1995; Watanabe, 1984) and activity of the sympathetic nervous system (Elam et al., 1986), which will be addressed in later chapters. Role of Thermoregulation In support of other studies, body temperat ure was higher after restraint stress (e.g., stress-induced hyperthermia). Stress has b een shown to increase body temperature (Chen and Herbert, 1995; Keim and Sigg, 1976; Le Bars et al., 2001; Thompson et al., 2003; Tjolsen and Hole, 1993), but these studies did not properly evaluate the impact of altered thermoregulation on nociceptive responding. Cu taneous temperatures (tail or plantar surface of the hindpaw) were identified as a confounding variable in the interpretation of reflex responses (Tjolsen and Hole, 1993). A relationship was also observed between

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104 temperatures and tail (primary thermoregulat ory organ) of the ra t during withdrawal responses. If the tail was cooler, the late ncy to withdrawal was longer suggesting a reduced sensitivity. Temperatures can pr ofoundly impact nociceptive responses and subsequently provide erroneous interpretations of those responses. Thus, it is important to determine if changes in behavioral responding is a consequence of altered thermoregulation. In the current study, while restraint incr eased body temperature, animals quickly cooled down after termination of restra int and during pre-test trials at 36.0 C (neutral temperatures). The pre-test trial serves an important role to normalize temperatures in stressed animals with non-stressed animals pr ior to testing at low intensity thermal stimulation. In addition, cutane ous temperatures also were in creased after stress. But, the increase in temperature can be attributed to postural factors. Because the animal is confined within the tube, their hindpaws rema ined close to their body. As a consequence, an increase in hindpaw temperatures was obser ved. Even though tail temperature was not included in the present study, it underwent a peri od of cooling during restraint stress. While this can be attributed to the tail bei ng exposed to the environm ent, restraint stress reduced cutaneous temperature of the tail a nd may indicate activation of the sympathetic nervous system (Chapter 5). Similar to core temperatures, pr e-test trials at 36.0 C brought cutaneous temperatures of the control group to levels comparable to the restraint group. This provides evidence that pre-e xposure to a neutral temperature normalize hindpaw temperatures prior to pl acement into the testing appa ratus. Thus, the expression of stress-induced hyperalgesia is not influenced by change s in core body temperature.

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105 Future studies may compare the effects of restraint stress and exercise (e.g. wheel running), which is a not stressful, on changes to both body and thermal sensitivity. Control Procedures Several control procedures ensured that th e effects of stress on escape responding were not the result of changes in the aversi ve qualities of bright light, which discouraged occupancy of the escape platform. Latencies to escape light in the da rkbox test were not altered by prior stress, and ne ither latencies nor durations of platform occupancy were altered during trials at a neutral temperature (36.0 C). Long latencies and short durations of platform occupancy at 36.0 C also showed that animals had not learned to avoid thermal stimulation by occupying the escape pl atform regardless of plate temperature. The increase in escape duration produced by acute stress has important implications in relation to an extensive animal literature that has reported elevated latencies or thresholds for innate, unlearned nociceptive responses after exposure to a variety of stressors (Bodnar et al., 1980a). The present study confirmed the usual result for reflex responses, using the same animals and the same nociceptive stimulus for lick/guard responses as for operant escape. The attenua ting effects of acute st ress on lick/guard and other innate reflex responses have been c onsidered as evidence for stress induced analgesia (SIA), based on an assumption that stress acts at spinal le vels to suppress both segmental reflex and rostral projection system s receiving nociceptive input. However, in light of our finding that acute stress dimini shes reflex responses to nociceptive input while enhancing operant responding to the same stimulus, it appears that stress-induced hyporeflexia can coexist with stress induced hype ralgesia. This combination of effects may have adaptive significance. For example, in stressful environmental circumstances

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106 such as a predator/prey interaction, repeated elicitation of stereotyped reflexes such as limb flexion or licking would interfere with intended motor actions required of the situation (Brandao et al., 1994, 1999; Coimbra et al., 2006; Leo-Borges et al., 1988). Under the same circumstances, pain would be an important motivator and could initiate fighting or fleeing. These defensive respons es are independent of stress-induced hypoalgesia (Coimbra et al., 2006; Prado and R oberts, 1985) and appear to be modulated by different mechanisms (Brandao et al., 1999; Castilho et al., 1999; Maier, 1990). Notes 1. Parts of this chapter were previously published. 2. Reprinted from Brain Research, King, et al., Differential effects of stress on escape and reflex responses to nociceptiv e thermal stimuli in the rat, Volume 987(2), 214-222, Copyright 2003, with permission from Elsevier.

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107 CHAPTER 4 EFFECTS OF RESTRAINT STRESS ON NO CICEPTIVE RESPONSES FOLLOWING EXCITOTOXIC SPINAL CORD INJURY While other complications impact the daily activities in SCI patients, pain is a relatively common, serious health problem that negatively affects th e quality of life in these individuals. To better unders tand altered pain sensations after injury to the spinal cord, an animal model (excito toxic model of SCI) was devel oped by Dr. Robert Yezierski to identify central mechanisms underlying SC I pain and develop novel preventative and treatment strategies. Excitotoxic injury to the spinal cord is accomplished by an intraspinal injection of the AMPA/metabotr opic receptor agonist qui squalic acid (QUIS), which results in damage to the spinal gray matter. Histological examination of spinal inju red cords reveals a relationship between pain sensations (atand below-level) and se veral anatomical changes (regional neuronal loss; longitudinal extent of injury). Behaviorally, animals display a heightened sensitivity to mechanical and thermal sens itivity as assessed by reflex mediated responses. As discussed previously, reflex responses to nociceptive stimulation do not represent cortical mechanisms or pain sensat ion, but rather spinal or spino-bulbo-spinal mediated responses to nociceptive stimuli. Based on these observations, a question can be raised regarding the impact of spinal injury on pain sensati ons. First, are responses depe ndent on cortical processing of nociceptive thermal stimulation affected by spinal injury? If so, what potential mechanism(s) are involved? Therefore, the e ffects of excitotoxic le sions of the spinal

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108 gray matter, confined to the thoracic and lumbosacral cord, on operant responses were examined. Two types of operant tasks were utilized to examine these effects including operant escape and thermal preference. It was hypothesized that ope rant responses would be affected by excitotoxic injury. Injury-i nduced hyperalgesia (e.g., increased sensitivity to heat) after QUIS could be demonstrated by change in operant behavior following injury. Finally, while a positive relationship exists between clinical pain conditions and exposure to stress, limited studies have ex amined the effects of stress on nociceptive responses in chronic pain models, particularly models of spinal in jury. Therefore, the effects of restraint stress on operant responses were examined after excitotoxic lesions to the spinal gray matter. Seve ral weeks after inject ions of QUIS, animals were exposed to restraint stress for fifteen minutes followed by assessment of thermal sensitivity on the day of stress and several days afterward. Re straint stress was expected to further enhance operant responses afte r excitotoxic injury. Effects of Excitotoxic Spinal Cord Injury on Operant Escape Overall Effect of Spinal Injury on Escape Responses The effects of excitotoxic lesions of th e spinal gray matter on operant escape responses (n=15) during trials at 44.5 C are shown in Figures 4-1 through 4-5. Escape performances were evaluated by two differe nt response measures: a) the number of responses elicited during a tr ial (count); and, b) the total time spen t on the platform (duration). Preoperative responses were r ecorded over several months before spinal injury. After excitotoxic injury (QUIS), be havioral assessment resumed three weeks later and continued over 10 weeks.

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109 Escape counts The effects of QUIS on the number of escap e platform responses are presented in Figure 4-1. The number of postoperative res ponses (QUIS) was slightly higher than preoperative responses (baseline), but platform responses were not significantly different after QUIS ( F= 0.945, P= 0.329). Failure to reveal an injury-induced in crease in the number of responses suggests that spinal in jury did not alter th ermal sensitivity on the escape test in this group of rats. Figure 4-1. The number of escape platform responses during testing trials at 44.5 C before (baseline, open bar) and after ( QUIS, closed bar) excitotoxic injury. Escape responses did not di ffer after QUIS. Data are expressed in seconds and are represented as absolute group means S.E.M. Escape durations A better evaluation of an experimental ma nipulation on operant responses is escape duration. Previous evidence has shown that an imals display an enhanced sensitivity to heat after injury of the upper thoracic co rd (Acosta-Rua, 2003). Injury-induced hyperalgesia was indicated by an increase in escape duration and a decrease in plate duration. It was hypothesized that operant responses woul d increase afte r excitotoxic

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110 injury of the mid-thoracic and upper lumbar sp inal cord. Figure 4-2 shows the effects of excitotoxic lesions on the duration of escape responses (platform). In the current group of rats, an injury -induced hyperalgesia was not observed for platform duration ( F= 0.030, P= 0.863). Conversely, the total time spent on the thermal plate was not different before or after QUIS (data not shown; F= 0.029, P= 0.865). The inability to alter thermal sensitivity assesse d by operant responses (counts and duration) after QUIS suggests that cortically-mediated responses to thermal stimulation are not affected by QUIS injuries. Figure 4-2. Cumulative escape platform durations during tes ting trials at 44.5 C before (baseline, open bar) and after (QUIS, cl osed bar) excitotoxic injury. Platform durations were affected by QUIS. Da ta are expressed in seconds and are represented as absolute group means S.E.M. Sequence analysis of succe ssive escape durations To further examine the effect of QUIS on operant responses, successive escape plate and platform durati ons within a trial at 44.5 C are presented in Figure 4-3. A summary of the successive escape plate and plat form durations is presented in Figure 4-4.

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111 Figure 4-3. Sequence analysis of successive escape plate a nd platform durations during testing trials at 44.5 C before (baseline, asterisk) and after (QUIS, gray circle) excitotoxic injury. QUIS failed to infl uence successive operant escape plate (A) and platform (B) durations. Data are expressed in seconds and are represented as absolute group means S.E.M.

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112 Figure 4-4. Average duration of the first six plate and platform responses during testing trials at 44.5 C before (baseline, open bar) and after (QUIS, closed bar) excitotoxic injury. No differences we re observed in plate (A) and platform (B) durations between baseline and QUIS testing conditions. Data are expressed in seconds and are represen ted as absolute group means S.E.M.

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113 In general, plate durations (Figure 4-3A ) were initially higher than platform durations (corresponding to res ponses 1 through 6) and stabi lized after the 7th response for the remainder of the trial. Excitotoxic injury did not affect plate durations, which appeared to be similar to baseline respons es. In addition, platform durations (Figure 4-3B) appeared to increase slightly from the 1st response to the 5th response (peaking around 4th response). However, similar to pl ate responses, baseline platform durations were similar to these obtained following excitotoxic injuries. The average durations of the first 6 dura tions are compared between baseline (open bar) and QUIS (closed bar). In agreement with cumulative escape durations, QUIS did not affect the average durati ons of plate (Figure 4-4A, F= 0.106, P= 0745) or platform (Figure 4-4B, F= 0.064, P= 0.800) responses. The average ba seline plate and platform durations were similar between testing conditions. Weekly assessment of escape durations Assessment of weekly postoperative escap e responses is shown in Figure 4-5 before (asterisk) and after QUIS (gray ci rcle). Postoperative assessment of escape responses was reinstated three weeks after an injection of QUI S. Platform durations were variable across each week (peaking during the 7th week). Statistical analysis revealed that QUIS did not affect escape respon ses across weekly testing sessions ( F= 0.507, P= 0.851). Effects of Spinal Injury in Individual Groups In evaluating operant responses after inju ry, QUIS failed to enhance sensitivity to heat (no injury-induced hyperalg esia). However, two types of responses were identified in the population of animals with spinal injury.

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114 Figure 4-5. Weekly postoperative platform responses across several weeks of testing during trials at 44.5 C before (baseline, asterisk) and after (QUIS, gray circle) excitotoxic injury. Data are expresse d in seconds and are represented as absolute group means S.E.M. One group (QUIS hyperalgesia ; n=7) displayed an optimal change in responding after injury that was characterized by an in crease in platform escape duration. In the second group (QUIS hypoalgesia ; n=8), platform escape du rations were significantly reduced compared to baseline values. In order to characterize the groups, further analyses of platform durations as well as the number of platform responses were conducted and are presented in Figures 4-6 through 4-8. Based on previous analysis, the number of platform responses was not affected by QUIS. However, based on the revised grouping, an effect of QUIS was revealed for the first group (QUIS hyperalgesia ), which exhibited enhanced responding postoperatively, and the second group (QUIS hypoalgesia ) that postoperative re sponding was marginally reduced after injury.

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115 Escape counts The number of baseline platform responses for these two groups are presented in Figure 4-6. Interestingly, the number of platform responses was lower in the hyperalgesic group compared to the hypoalgesic group. After injection of QUIS, platform counts were significantly higher in the hyperalgesic group (Figure 4-6A, F= 19.627, P< 0.001). By contrast, QUIS did not si gnificantly affect the number of platform responses in the hypoalgesic group (Figure 4-6B, F= 2.057, P= 0.153). Escape durations The duration of escape responses did not di ffer before and after an injection of QUIS based on the original grouping. Howeve r, additional characterization revealed a difference in escape responses between the tw o groups (Figure 4-7). Platform durations (total time on the escape platform) were a ffected by QUIS. Platform durations in the hyperalgesic group were significantly higher duri ng sessions after QUIS (Figure 4-7A; F= 3.869, P= 0.05) compared to baseline sessions. Conversely, plate durations were significantly lower during sessions after QUIS (data not shown; F= 3.903, P= 0.05) compared to baseline sessions. In contrast to this group, QUIS pr oduced an opposite effect in the hypoalgesic group, with platform durations significantly lower during sessions after QUIS (Figure 4-7B; F= 8.066, P= 0.005) compared to baseline se ssions. Plate durations were significantly higher during sessions after QUIS (data not shown; F= 8.115, P= 0.005) compared to baseline sessions. Thus, QUI S produced either enhanced (decrease plate duration; increase platform dur ation) or reduced (increas e plate duration; decrease platform duration) sensitivity to heat as represented in these two groups.

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116 Figure 4-6. Number of escape platform responses during testing trials at 44.5 C before (baseline, open bar) and after (QUIS, closed bar) excitotoxic injury. While the number of responses in the hyperalgesic group (A) was increased, no effect was observed in the hypoalgesic group (B). Data are expressed in seconds and are represented as absolu te group means S.E.M. Significant differences between baseline and QUIS testing periods are indicated by: *** P< 0.001.

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117 Figure 4-7. Cumulative escape platform dur ations during testing trials at 44.5 C before (baseline, open bar) and after (QUIS, closed bar) excitotoxic injury. The duration of platform responses was increased for the hyperalgesic group (A) but the hypoalgesic group (B) spent less time on the platform. Data are expressed in seconds and are represente d as absolute group means S.E.M. Significant differences between base line and QUIS testing sessions are indicated by: P< 0.05 and ** P< 0.01.

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118 Weekly assessment of escape durations Assessment of weekly postope rative escape responses in hyperalgesia (A) and hypoalgesia (B) groups was compared before (ast erisk) and after QUIS (gray circle) excitotoxic injury (Figure 4-8). QUIS enhanced escape responses to 44.5 C across several weeks after excitotoxic injury in the hyperalgesic group (Figure 4-8A). This enhancement appears to be greater during ea rlier weeks followed by a progressive return of responses to pre-QUIS levels. While QUIS enhanced platform responses in the hyperalgesic group, it decreased escape respons es across several weeks in the hypoalgesic group (Figure 4-8B). Responses began to progressively return to baseline levels at the end of the te sting period. Thus, QUIS produ ced two different effects: increased or decreased sensitivity to heat Effects of Restraint Stress on Operant Escape Following Excitotoxic Injury Overall Effects of Stress on Escape Responses after Injury Behavioral responses dependent on cortical processing of thermal information are increased after a 15 minute exposure to rest raint stress in normal uninjured animals (Chapter 3). Based on previous results s uggesting that stress produces a heightened sensitivity to heat, how would animals with spinal cord injury respond to stress?While clinical evidence indicates that stress is an important factor in chr onic pain, these effects have not been examined in an animal mode l. The excitotoxic model of SCI offers a unique opportunity to evaluate the effects of stress on altered se nsation after spinal injury. The fact that animals have experienced injury to the spinal cord could be a critical factor in the development and/or enhancement of stress-induced increases in operant responding.

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119 Figure 4-8. Weekly postoperative platform responses across several weeks of testing during testing trials at 44.5 C before (baseline, asterisk) and after (QUIS, gray circle) excitotoxic injury. Platform durations remained higher for the hyperalgesic group (A) and lower for the hypoalgesic group (B). Data are expressed in seconds and are represen ted as absolute group means S.E.M.

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120 To examine if stress could enhance operant responses in spinally injured animals, animals were exposed restraint stress eight weeks after surgery, which was similar to the previous stress protocol in intact animals. Then, animals were placed into a pre-test for 15 minutes followed by a testing trial at 44.5 C. The effects of restraint stress were evaluated over three consecutive days includi ng the day of stress (re straint stress), the following day (24 hours), and two days (48 hours) af ter restraint. In th is initial analysis, animals were not subdivided into hyperalgesic or hypoalgesic groups. In order to simplify the results from a ll animals that underwent escape testing, a summary of operant responding (escape count s; durations) after restraint stress is presented in Table 4-1. Re straint stress significantly reduced platform counts ( F= 7.347, P= 0.007). On the following testing days, platfo rm counts were not significantly lower, 24 ( F= 0.214, P= 0.644) or 48 ( F= 0.789, P= 0.376) hours after stress. Platform escape durations were not significan tly affected by restraint stress fifteen minutes ( F= 1.293, P= 0.257), 24 hours ( F= 0.628, P= 0.429), and 48 hours ( F= 0.002, P= 0.967). Conversely, no significa nt differences were obser ved for plate durations fifteen minutes ( F= 1.284, P= 0.258), 24 hours ( F= 1.284, P= 0.258), and 48 hours ( F= 1.284, P= 0.258) after stress. Effects of Stress on Individual Groups After extensive evaluation of behavioral responses, two types of behavioral responses after spinal injury emerged in cluding animals displaying an increase or decrease to thermal stimulation. Based on th ese observations, analys is of stress-induced changes in operant responses was carried out on animals demonstrating an enhanced or a diminished response to thermal stimulation after QUIS. Based on the revised grouping,

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121 these testing sessions were evaluated between hyperalgesic (increased responding after QUIS; left panel) and hypoalgesic (reduced responding after QUI S; right panel) groups. Table 4-1. Number and duration of escape responses at 44.5 C before and during testing sessions in which animals were tested fi fteen minutes (restraint stress), the following day (24 hours), and two days ( 48 hours) after stress. The data are expressed in seconds and are represente d as absolute group means S.E.M. Count Thermal Plate Duration Escape Platform Duration QUIS 8.03 0.29 554.12 13.50 345.69 13.50 Restraint Stress 5.07 1.17 P <0.01 494.35 70.50ns 404.67 70.53ns 24 Hours 7.53 0.92ns 514.15 53.07ns 385.53 52.97ns 48 Hours 7.07 1.10ns 556.24 52.59ns 343.60 13.05ns Escape count in the hyperalg esic and hypoalgesic groups The number of postoperative escape platform responses is illustrated in Figure 4-9 during sessions in which animals were tested after restraint stress (restraint stress; top panel) and one (24 hours; middle panel) or two days (48 hours; bottom panel) after stress. Restraint stress significantly reduced th e number of platform responses in the hyperalgesic group (Figure 4-9A, F= 8.147, P= 0.005). However, the reduction of platform counts did not pe rsist during sessions assessed 24 hours (Figure 4-9C, F= 0.287, P= 0.593) or 48 hours (Figure 4-9E, F= 0.395, P= 0.531) after stress. In the hypoalgesic group, platform counts were no t significantly different during sessions fifteen minutes (Fi gure 4-9B, restraint stress: F= 1.42, P= 0.236), 24 hours (Figure 4-9D, F= 0.018, P= 0.894), or 48 hours (Figure 4-9F, F= 0.354, P= 0.553) after stress.

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122 Figure 4-9. The number of escape platform responses at 44.5 C before (QUIS, open bar) and during testing sessions (closed bar) in which animals were tested fifteen minutes (restraint stress), the followi ng day (24 hours), and two days (48 hours) after stress. Restraint stress decreased the number of platform responses for the hype ralgesic (A) and hypoalgesic (B) groups, but responses were only significantly reduced in the hyperalgesic group. In subsequent testing sessions, platform counts were comparable to pre-stress when assessed 24 (C, D) and 48 (E, F) hours after stress. Data are expressed in seconds and are represented as absolute group mean s S.E.M. Significant differences between baseline and QUIS testi ng periods are indicated by: ** P< 0.01.

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123 Escape durations in the hyperalgesic group In Figure 4-10, the effects of restraint st ress on the duration of plate and platform responses are compared during testing sessi ons, which animals were tested fifteen minutes (restraint stress), the following day (24 hours), and two da ys (48 hours) after stress. After excitotoxic injury, posto perative platform durations during 44.5 C trials for the hyperalgesic group were affected by stress. Rest raint stress significantly increased platform durations (Figure 4-10A, F= 5.981, P= 0.016). Conversely, plate durations were lower after stress (data not shown; F= 5.96, P= 0.016). Subsequent testing sessions that asse ssed the following day (24 hours) and two days (48 hours) after restraint rev ealed similar stress effect. For the hyperalgesic group, platform durations remained higher but were not significant at 24 (Figure 4-10B, F= 2.765, P= 0.099) or 48 (Figure 4-10C, F= 2.765, P= 0.099) hours. In addition, plate durations were not significantly lowe r during these sessions (24 hours, F= 3.782, P= 0.055; 48 hours, F= 3.756, P= 0.055). Escape duration in the hypoalgesic group In Figure 4-11, the effects of restraint st ress on the duration of plate and platform responses were evaluated during testing sessi ons. Animals were tested fifteen minutes (restraint stress), the following day (24 hours) and two days (48 hours) after stress. Unlike the hyperalgesic group, stress failed to alte r platform (Figure 4-11A, F= 0.027, P= 0.87) or plate (data not shown; F= 0.027, P= 0.87) durations during 44.5 C trials for the hypoalgesic group. Escape durations were simila r to pre-stress responses. Responses were comparable when assessed the following days. Platform durations did not differ from pre-stress respons es during sessions 24 (Figure 4-11B, F= 0.091,

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124 P= 0.764) or 48 (Figure 4-11C, F= 1.656, P= 0.201) hours after stress. Similar to platform responses, plate durations did not cha nge across sessions 24 (data not shown; F= 0.090, P= 0.765) or 48 (data not shown; F= 1.661, P= 0.2) hours after stress. Additional evaluation of escape responses As mentioned previously, no effects were revealed following excitotoxic injury when evaluations included th e entire group. Attempts were made to further characterize the response profile of these animals, which are discu ssed later in this chapter (e.g., histology, open field responses). However, no variables were able to further differentiate escape responses following injury. As a result, animals were characterized by changes in their escape responses. Animals showed either an enhanced ( hyperalgesic group: increased duration; decreased la tency: F=4.908, P=0.028) or reduced ( hypoalgesic group: decreased duration; increased latency: F=6.564, P=0.011) thermal sensitivity following injury. In addition, restrain t stress decreased and increase d escape latencies in the hyperalgesic (F=1.123, P=0.292) and hypoalgesic groups ( F= 4.609, P= 0.034), respectively. Latencies were not shown, but ar e presented here for comparison purposes. Therefore, excitotoxic injury increased th ermal sensitivity in some animals, which was also characterized by changes follo wing restraint stress and during thermal stimulation. Stress significantly affected the hyperalgesic group but not the hypoalgesic group. Finally, changes in temperature re gulation were more pronounced in the hypoalgesia group. While this differentiation may appear artifi cial, further statisti cal analysis supports the subdivision of animals. Based on the Shapiro-Wilk analysis, test for normality revealed a nearly unequal dist ribution of escape durations ( P =0.059) and latencies ( P =0.06). However, a relationship exists be tween postoperative responses and other

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125 conditions: responses following restraint stress and during baseline (preoperative) sessions. Further analysis revealed a signi ficant association betw een postoperative and stress sessions (Figure 4-12) when a ssessed by escape latency (Pearson: r = 0.696, P =0.004) but not escape duration (r = 0.405, P =0.135). In addition, evaluations of skin temperature in anesthetized rats (c hapter 5) revealed additional association between postoperative escape responding and va soconstriction (see Figure 5-4) when assessed after i mmediately (15 minute period; r = 0.5, P =0.05) or five minutes (20 minute period; r = 0.542, P =0.037) after the te rmination of 44.5 C (thermal stimulus). Effects of Excitotoxic Spinal Cord Injury on Thermal Preference In addition to operant escape, the effects of excitotoxic lesions (QUIS) on thermal preference (n=12) responses were evaluated preoperatively and postoperatively after the third week and continued ove r a 10-week period. Responses were obtained from a separate group of animals. The thermal pref erence test required an animal to decide between two thermal plates at 15.0 (cold) and 45.0 C (heat). Therefore, an increase in time spent in one thermal modality versus an other indicates a change in sensitivity. Thermal Preference Counts The number of thermal preference respons es (crossings between compartments) was not affected by QUIS (Figure 4-13). Following spinal injury, the number of preference responses did not ch ange compared to preoperativ e responses. Thus, as with operant escape counts, no diffe rences were seen with the number of responses before and after QUIS. While this may indicate the sp inal injury did not produce altered thermal sensations, other outcome measures showed the effect of injury more reliably.

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126 Figure 4-10. Cumulative escape platform responses at 44.5 C before (QUIS, open bar) and during testing sessions (closed bar) in animals tested fifteen minutes (restraint stress), the following day ( 24 hours), and two days (48 hours) after stress for the hyperalgesic group. Restraint significantly increased platform durations (A). While these effects were still present, platform durations were not significantly different from QUIS during sessions 24 hours (B) or 48 (C) hours after stress. Data are expresse d in seconds and are represented as absolute group means S.E.M. Significant differences between baseline and QUIS testing periods are indicated by: P< 0.05.

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127 Figure 4-11. Cumulative escape platform responses at 44.5 C before (QUIS, open bar) and during testing sessions (closed bar) in animals tested fifteen minutes (restraint stress), the following day ( 24 hours), and two days (48 hours) after stress for the hypoalgesic group. Platform durations were not affected by restraint stress (A) and du ring sessions 24 hours (B) or 48 (C) hours later. Data are expressed in seconds and are represented as absolute group means S.E.M.

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128 Figure 4-12. Correlation between postoperative responses foll owing QUIS and change in skin temperature regulation during sessi ons following thermal stimulation of the left hindpaw. Data are expresse d in seconds and are represented as absolute group means.

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129 Figure 4-13. The number of thermal prefer ence responses during testing trials at 15.0-45.0 C before (baseline, open bar) and after (QUIS, closed bar) excitotoxic injury. Thermal preference did not differ after QUIS. Data are expressed in seconds and are represen ted as absolute group means S.E.M. Thermal Preference Durations As shown in Figure 4-14, despite the nu mber of responses being unaffected, QUIS had an impact on the total time spent on the co ld versus heat plates QUIS increased the time spent on the cold plate. Increase d cold preference was significantly higher compared to baseline (Figure 4-14A; F= 6.754, P= 0.01). Conversely, QUIS reduced the duration spent on the heated plate, which wa s significantly different from baseline (Figure 4-14B; F= 6.67, P= 0.01). Thus, an increase in cold preference and decrease in heat preference indicates that spinal injury produced a heightened sensitivity to heat. Sequence Analysis of Successive Thermal Preference Durations To further examine the effect of QUIS on the thermal preference test, successive cold and heat durations are presented in Figur e 4-15. In general, cold and heat preference responses peaked around the 4th and 5th responses.

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130 Figure 4-14. Cumulative durations of thermal pr eference responses during testing trials at 15.0-45.0 C before (baseline, open bar) and after (QUIS, closed bar) excitotoxic injury. (A) Cold preferen ce was significantly higher while (B) heat preference was significantly lo wer after QUIS compared to baseline responses indicating a incr ease sensitivity to heat stimulation. Data are expressed in seconds and are represente d as absolute group means S.E.M. Significant differences between preopera tive and postoperative periods for cold and heat preference are indicated by: ** P< 0.01.

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131 As indicated by the cumulative preferen ce durations, cold preference (Figure 4-15A) was initially higher, particularly in the beginning of the testing trial, after QUIS compared to baseline responses. On th e other hand, QUIS lowered heat preference slightly compared to baseline values (F igure 4-15B). Figure 4-16 summarizes the average duration of the first six cold and heat preference responses that are compared between baseline (open bar) and QUIS (closed ba r). QUIS did not significantly affect the average duration for cold (Figure 4-16A, F= 1.025, P= 0335) or heat (Figure 4-16B, F= 0.010, P= 0.921). Weekly Assessment of Thermal Preference Durations Assessment of weekly postoperative therma l preference responses is shown before (asterisk) and after QUIS (gray circle; Figure 4-17). Similar to animals in the escape paradigm, postoperative assessment of therma l preference responses was initiated three weeks after an injection of QUIS. Cold a nd heat preference progressively changed over time. The trend continued and was most evident at the 9th and 10th week postoperatively. Statis tical analysis revealed that pr eference for cold (Figure 4-17A, F= 2.225, P= 0.025) and heat (Figure 4-17B, F= 2.225, P= 0.025) were significantly affected by QUIS across weekly testing sessions. Effects of Restraint Stress on Thermal Preference Following Excitotoxic Injury To examine if stress could enhance thermal preference responses in spinally injured animals, animals were exposed to acute rest raint stress eight week s after surgery, which was similar to the previous stress protocol in intact animals. Then, animals were placed into a pre-test for 15 minutes followed by testing trial at 15.0 and 45.0 C. Postoperative responses were examined over three consecutive days.

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132 Figure 4-15. Sequence analysis of successive cold and heat preference durations during testing trials at 15.0-45.0 C before (baseline: asterisk) and after (QUIS: gray circle) excitotoxic injury. (A) Cold preference was slight ly higher after QUIS throughout the trial especially at the beginning. (B) Heat preference was slightly decreased over successive response. Data are expressed in seconds and are represented as absolute group means S.E.M.

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133 Figure 4-16. Average durations of the first si x cold and heat pref erence responses during testing trials at 15.0-45.0 C before (baseline, open bar) and after (QUIS, closed bar) excitotoxic injury. Co ld (A) and heat (B) preference was increased and decreased after spinal inju ry, respectively. However, the effects were not significantly different from ba seline. Data are expressed in seconds and are represented as absolute group means S.E.M.

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134 Figure 4-17. Weekly postoperative cold and heat preference responses across several weeks of testing duri ng trials at 15.0-45.0 C before (baseline, asterisk) and after (QUIS, gray circle) excitotoxic in jury. Data are expressed in seconds and are represented as absolute group means S.E.M.

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135 Thermal Preference Counts In Figure 4-18, the number of postoperative thermal preference responses before (QUIS) and after sessions in which animals were stressed and tested fifteen minutes (restraint stress), the following day (24 hours) and two days (48 hours) are presented. Thermal preference counts were not signi ficantly different fifteen (Figure 4-18A, F= 1.230, P= 0. 269), 24 hour (Figure 4-18B, F= 0.198, P= 0.657), and 48-hour (Figure 4-18C, F= 0.013, P= 0. .908) hours after stress. Thermal Preference Durations In Figure 4-19, the duration of cold and heat preference were compared during sessions before (QUIS) and fifteen minutes (restraint stress), the following day (24 hours), and two days (48 hours) after stre ss. Preference for cold (Figure 4-19A, F= 5.381, P= 0.021) and heat (Figure 4-19B, F= 5.540, P= 0.019) was significantly affected by restraint. Specifically, cold durations were longer and, heat durations were shorter after exposure to restraint stress. During assessment of subsequent testi ng sessions, stress-induced changes in thermal preference responses remained si gnificantly different the following day (24 hours), but not two days (48 hours) after restraint. Statistical analysis revealed that cold preference during sessions the following day was significantly different compared to pre-stress responses (Figure 4-19C, F= 8.911, P= 0.003), but sessions two days later was not significant (Figure 4-19E, F= 0.475, P= 0.491). Conversely, preference for heat 24 hours afterwards was significantly different compared to pre-stress responses (Figure 4-19D, F= 9.213, P= 0.003), but sessions two days late r were not significant (Figure 4-19F, F= 0.507, P= 0.477). Thus, restraint stress fu rther enhanced injury-induced sensitivity to heat, which persisted the following day.

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136 Figure 4-18. Number of thermal preference responses during testing trials at 15.0-45.0 C before (QUIS, open bar) and during te sting sessions (closed bar) in which animals were tested fifteen minutes (re straint stress), the following day (24 hours), and two days (48 hours) after stress. Restraint stress did not significantly affect the number of res ponses 15 minutes (A), 24 hours (B), or 48 hours (C) afterwards. Data are expressed in seconds and are represented as absolute group means S.E.M.

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137 Figure 4-19. Cumulative cold (ri ght panel) and heat (left panel) preference responses at 15.0-45.0 C before (QUIS, open bar) and dur ing testing sessions (closed bar) in which animals were tested fifteen minutes (restraint stress), the following day (24 hours), and two days (48 hours) af ter stress. The dur ation of cold (A) and heat (B) preference were significantly higher and consequently lower after restraint stress indicating an increased sensitivity to heat. In subsequent testing sessions, preference remained hi gher for cold (C) and lower for heat (D) 24 hours after stress. However, pref erence for cold (E) and heat (F) were not different 48 hours after stress. Da ta are expressed in seconds and are represented as absolute group means S.E.M. Significant differences between baseline and QUIS testi ng periods are indicated by: P< 0.05 and ** P< 0.01.

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138 Prediction of Behavioral Response s Based on Open Field Responses Another variable that could affect the expr ession of altered sens ation is anxiety-like responses (assessed by the open field test). As mentioned previously, open field responses provide a measure of anxiet y-like responses a nd determine stress responsiveness (Fernandez et al., 2004; Van den Buuse et al., 2001), which may be a predictor of individual sensit ivity to thermal stimulation after stress (Jorum, 1989) or injury (Kontinen et al., 1999; Vatine et al., 2000). For example, stress sensitive animals are characterized by lower expl oration of the open field (Kab baj et al., 2000). During some preliminary sessions, a relationship was revealed in groups that displayed stress-induced hyperalgesia and anxiety-like response (e .g., shorter duration, longer latency). To determine if responses in the open field could differentia te the behavioral groups after injury, open fiel d responses were used as covariate including counts, durations, and latencies (Table 4-2). However, open fiel d responses did not reveal significant effects of excitotoxic injury on behavioral responses Table 4-2. Effect of open field response s on operant responses for groups after excitotoxic injury. Open Field Count Duration Latency Escape P=0.232 P=0.597 P=0.430 Thermal Preference P=0.809 P=0.984 P=0.849 Inner Field Count Duration Latency Escape P=0.877 P=0.631 P=0.441 Thermal Preference P=0.650 P=0.768 P=0.812

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139 Possible reasons for the inability of open fiel d-test to predict an injury or stress response is the time between assessment of anxiety responses and other conditions (injury, stress). From the time animals were tested in the open field, several months had passed before excitotoxic inju ry and restraint stress. During this time, animals were extensively, acclimated, and trained in the opera nt escape tests. Future studies could use the open field test several weeks before excitotoxic injury for evaluation of the relationship between open fiel d results and behavioral re sponse to stress and spinal injury. Comparison between Normal and Spinally Injured Animals The effects of stress on QUIS animals were compared to responses in normal, uninjured animals. Difference scores from pre-stress values were used to determine significant effects. These comparisons betw een groups are illustrated for the escape (Figure 4-20) and thermal preference (Figure 4-21) testing sessions. Differences in Escape Duration For the escape test, restraint decrease d plate (15 minutes; Figure 4-20A) and increased platform (15 minutes; Figure 4-20B) durations compared to pre-stress levels. In the QUIS condition, durations were sligh tly larger than responses in the normal condition but were similar between groups (plate and platform: F= 0.576, P= 0.458). The effects of stress, which were characteri zed by a decrease in plate time and an increase in platform time, were observed in subsequent testing sessions. Plate and platform durations remained lower and highe r than the normal condi tion, respectively, during sessions 24 (plate and platform: F= 6.437, P= 0.021) and 48 (plate and platform: F= 5.470, P= 0.032) hours after stress. In contrast, normal animals demonstrated no carry-over effects, and returned to pre-stress values 24 hours after stress.

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140 Figure 4-20. Difference scores for plate and platform durations during escape trials at 44.5 C in normal (open bar) and after excito toxic injury (QUIS: closed bar) in which animals were tested for fifteen mi nutes (restraint stress), the following day (24 hours), and two days (48 hours) after stress. Restraint stress reduced plate (A) and increased platform (B) dur ations in both conditions. While the normal condition recovered 24 and 48 hours later (spending less time on the platform), the QUIS condition spent mo re time on the platform during the same time points. Data are expresse d in seconds and are represented as difference scores from pre-stress va lues (means S.E.M). Significant differences between baseline and QUIS conditions are indicated by: P< 0.05.

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141 Differences in Thermal Preference Duration Restraint increased cold (15 minutes; Figure 4-21A) and decreased heat (15 minutes; Figure 4-21B) preference durations comp ared to pre-stress levels. In the QUIS condition, durations were slightly lower than responses in the normal condition but were similar between groups (cold and heat preference: F= 0.118, P= 0.732). In the normal condition, preference responses progressively decreased 24 and 48 hours after stress. In the QUIS condition, responses were signifi cantly higher than the normal condition 24 hours ( F= 5.732, P= 0.022) but only slightly higher 48 hours ( F= 0.024, P= 0.876) after stress. Thus, carry-over effect s were most prominent in spinally injured animals. These results show that stress further enhanced operant responding to low intensity thermal stimulation in spinally injured animals. Histology After the completion of behavioral testi ng, animals were euthanasized and purfused with PBS followed by 10% formalin. Spinal cords were removed and underwent further analysis by in vitro MRI microscopy (as men tioned in chapter 2). The use of this technique has been recently de scribed (Berens et al., 2005). Images of spinal cord sections obtained from in vitro MRI confir med the presence of pathological features (neuronal loss and cavitation) after QUIS. Intraspinal inje ction of QUIS has been shown to produce cavitation that is similar to c onditions found in patients with syringomyelia (Berens et al., 2005; Yezi erski, et al., 1993). It has been shown that the release of excitatory amino acids (EAA) is responsible for development of excitotoxic injury and pain (Choi and Rothman, 1990; Yezierski, 2000, 2001). Based on histological data, ex citotoxic injury re sulted in pathology characterized in previous studies (Ber ens, et al., 2005; Yezierski, 2002).

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142 Figure 4-21. Difference scores for cold and heat preference durati ons during trial at 15.0-45.0 C in normal (open bar) and after ex citotoxic injury (QUIS: closed bar) in which animals were tested for fifteen minutes (restraint stress), the following day (24 hours), and two days ( 48 hours) after stress. Similar to escape animals, restraint stress increa sed preference for cold (A) and reduced preference for heat (B) in both conditions. While the normal condition recovered 24 and 48 hours later (spending more time on the heated plate), the QUIS condition spent more time in the cold compartment during the same time points. Data are expressed in se conds and are represented as difference scores from pre-stress values (mean s S.E.M). Significant differences between baseline and QUIS conditions are indicated by: P< 0.05.

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143 In addition, in vitro MRI visualized the le ngth and epicenter of damage within the spinal cord. Representative transverse im ages, which provide a better evaluation of neuronal loss, were assessed in spinal cord se ctions above and below level of excitotoxic injury (Figure 4-22). Similar to findings from Berens et al (2005), white and gray matter are distinguishable. Two pathological features are observed in these images including evidence for hemorrhage (Figure 4-22; dark, hypotensive signal) and cavitation (Figure 4-22; light, hypertensive si gnal) particularly in the central canal. Based on these sections, spinal cords were evaluated for the presence of cavitations, the rost ral-caudal extent of injury along the cord, degree of gray matter da mage, and level of injury (Tables 4-3 and 4-4). To illustrate some features that occur afte r excitotoxic injury to the spinal; cord, representative images from three animals are presented in Figure 4-23. Gray matter damage varied among animals after excitotoxic injury, which was eith er localized to the dorsal horn (Figure 4-23A; section 2) or included the ventral horn (Figure 4-23B, C; section 2). In addition, the formation of cav ities after injury was variable. Cavitation was not present in some animals (Figure 4-23A ; section 2) but presen t in others (Figure 4-23B; light signal in section 2). Finally, longitudinal extent of damage following injuries was also variable (ranging from 4 mm to 10 mm). Re presentation of injury length s is illustrated in sagittal images in which injury length was small (F igure 4-23A), moderate (Figure 4-23B), or extensive (Figure 4-23C).

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144 Figure 4-22. A comparison of in vitro MRI images in spinal cord section rostral (A) and at the level (B) of excitotoxic injury. Features of excitotoxic injury are identified as the following: A, hemorrhage; B, cavitation, C, gray matter; and D, white matter.

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145 Figure 4-23. Summary of tran sverse and sagittal spinal cord images obtained through in vitro MRI after excitotoxic injury. Images are presented based on the rostral-caudal orient ation, with rostra l sections located at the t op. Transverse sections are represented as follows: rostral (top image), injury level (m iddle image), and caudal (botto m image). Sagittal sections illustrate the length or rostral-caudal extent of injury.

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146 In the present study, comparisons with be havioral responding were attempted. Tables 4-3 and 4-4 show the percentage of animals (each testing condition: escape, thermal preference) that exhibited the fo llowing pathology after excitotoxic injury: cavitation, longitudinal extent of damage, degree of gray matter damage, and level of injury. In regards to the frequency of cavit ation after QUIS, groups differed. A majority (66.7%) of the animals in the escape group faile d to develop cavities compared to a large number (41.7%) of thermal preference anim als exhibiting an extensive amount of cavitation. As for the extent of longitudinal damage, a majority of animals exhibited relatively moderate (4-7 mm) degree of damage along the spinal cord in both groups (escape: 60.0%; thermal preference: 41.0%). Interestingly, groups diffe red on the degree of gray matter damage. For the escape group, a major ity of animals (40.0%) exhibited complete damage to the dorsal horn with some sparing of the ventral horn. However, the thermal preference group (58.3%) had extensive damage to the dorsal and ventral horn with involvement of the white matter. Finally, the level of injury varied between areas within the thoracic level to lower levels of the lumb ar cord. A majority of animals in both groups possessed similar injury levels (escape: ~T13; thermal preference: T11-L1). To summarize the data in Tables 4-3 and 4-4, histological data was normalized into specific bins. For example, scores were assigned depending on the degree of cavitation (Table columns left to right: 1, cavitation; 2, minor expansion of the central canal; 3, major expansion of the central canal; 4, extens ive cavitation within gray matter). Based on these values, averages were obtained and analyzed.

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147Table 4-3. Histological data for groups af ter excitotoxic injury that were behavior al assessed in the ope rant escape and therma l preference tests. Data are re presented as percentage of an imals within each condition. Cavitation None Minor Moderate Severe Escape All Rats 66.7 13.3 13.3 6.7 Hyperalgesia 71.3 14.3 0 14.3 Hypoalgesia 62.5 12.5 25 0 Thermal Preference 16.7 25 16.7 41.7 Rostral-Caudal Extent of Injury 4-6 mm6-7 mm 7-8 mm 8-9 mm 9-10 mm >10 mm Escape All Rats 60 0 13.3 6.7 13.3 6.7 Hyperalgesia 71.4 14.3 0 0 14.3 0 Hypoalgesia 50 12.5 12.5 12.5 12.5 12.5 Thermal Preference 8.3 41.7 16.7 33.3 0 0

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148Table 4-4. Histological data for groups af ter excitotoxic injury that were behavior al assessed in the ope rant escape and therma l preference tests. Data are re presented as percentage of an imals within each condition. Gray Matter Damage DH DH/VH (Minor)DH/VH (Major)Com plete (Minor)Complete (Major) Escape All Rats 13.3 13.3 40 20 13.3 Hyperalgesia 14.3 28.6 42.9 14.3 0 Hypoalgesia 12.5 0 37.5 25 25 Thermal Preference 0 16.7 8.3 16.7 58.3 Level of Injury T7-8T9-10 T11-T12 T13-L1
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149 Overall, cavitation was relatively minor in the escape group regardless of the subgroup (hyperalgesia hypoalgesia) but was extensive in the thermal preference group. The average length of damage along the long itudinal axis was 7-8 mm for both groups. Finally, the average level of injury for the escape and thermal preference group was located around T11-T12 and T10-T11, respectively. In Table 45, operant responses were evaluated with the histological data as a cova riate. Cavitation, length of injury, amount of gray matter damage, or level of injury did not influence behavioral responses. Table 4-5. Effects of histological variable s on operant escape and thermal preference responses after excitotoxic injury. A summary of hist ological data for groups after excitotoxic injury that was behavi orally assessed in the operant escape and thermal preference tests. Four pathological features (cavitation, rostral-caudal extent of damage, the amount of gray matter damage, and the level of injury) were used a covariate. Cavitation Rostral-Caudal Extent of Injury Gray Matter Damage Level of Injury Escape P=0.224 P=0.352 P=0.812 P=0.323 Thermal Preference P=0.929 P=0.604 P=0.752 P=0.984 Overall, histological data did not assist in explaining the behavi oral responses from operant escape or thermal pr eference. One possible reason is that spinal cords were excised several months after ex citotoxic injury and behavioral testing (4 months). In previous studies (Berens et al., 2005), damage to the gray matter increased over 30 days (end of study). In the curre nt study, damage most likely pr ogressed over time. It is possible that the relationship between operant responding and histological variables could not be established because analysis of thes e cords was several w eeks after heightened sensitivity. In order to address these issu es in future studies; histol ogical analysis should be collected during heightened se nsitivity (e.g., within the firs t 10 weeks). A technique, in

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150 vivo MRI, used in Berens et al. (2005) offers unique opportunity to examine the progression of injury over multiple time points in the same animal. While labor intensive, animals can undergo both image sessions during the assessment of their behavioral responses, which would provide a better opportunity to re veal a relationship between pathology and altered sensation. Howe ver, this option may not be feasible due to time restraints (imaging requires 3 hours per rat) and financial resources ($100 per hour; each session is $300 per rat). Theref ore, spinal cords should be removed as mentioned in Chapter 2 within severa l weeks of heightened responding. Also, it would be beneficial to examin e the time course of damage over longer periods of time to determine the long-te rm progression regardless of behavioral responding. As mentioned previously, histologic al analysis of cords after QUIS has been limited to 30 days postoperatively (Ber ens et al., 2005). Furthermore, since the excitotoxic effect of EAA is critical for lesion progressi on and developmen t of altered sensitivity after SCI, these effects could be minimized by administration of NMDA and AMPA antagonists during behavioral anal ysis as a possible treatment opposing injury-induced hyperalgesia (Choi and Rothman, 1990; Goda et al., 2002). Summary In pre-clinical models, behavioral co nsequences of excitotoxic SCI include allodynia and hyperalgesia to mechanical and thermal stimulation (enhancement of heat sensitivity). Depending on the operant task, in jury-induced hyperalges ia is characterized by a reduction in the time spent on a heated plate. In the case of the operant escape test, increased platform durations were associated with reduction of thermal plate durations. Additionally, the thermal preference test re vealed an increased cold preference and decreased heat preference. Thus, excito toxic spinal cord injury produced an

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151 injury-induced hyperalgesia to heat. Furthermore, the abili ty of stress to enhance operant responses (e.g. further increase in heat sensitivity) for a peri od of several days provides evidence that stress can enhance thermal sensitivity in animals that displayed an injury-induced hyperalgesia af ter spinal cord injury. Several pathological features may contribute to heightened sensitivity to heat after damage to the gray matter For example, the longitudinal progression of neuronal loss from the epicenter (~4.0 mm) was found to be important in the development of at-level pain-like sensations (grooming; Vierck et al., 2000; Yezierski, 2000; Yezierski et al., 1998). But, based on the present histologica l data, no evidence was found to support this observation for the behavioral outcome measur es used. All animals had injury that expanded from 4 to 10 mm. It is also possibl e that areas remote to the lesion epicenter may play a role in enhanced sensitivity. In creased blood flow in supraspinal structures, which are involved in processing of somato sensation (e.g., somatosensory cortex and thalamus), are observed after QUIS (Morro w et al., 2000; Pauls on et al., 2005) One interesting observation was made in anim als tested in the ope rant escape test. Some animals exhibited an enhanced sens itivity to heat while a portion showed a reduction in sensitivity after excitotoxic inju ry. Possible explanations of these results include changes in the anxiety state, disr uption of nociceptive pathways (ascending STT tract and descending modulatory pathways ; Vierck and Light, 2000), and altered functioning of the endogenous opioid system. It has been shown that the opioid and anti-opioid (e.g., CCK) systems are activated af ter excitotoxic injury (Abraham et al., 2000, 2001).

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152 Considering that injury-insensitive animals were identified in the escape test, it is important to acknowledge the di fference between the two opera nt tests. While animals must decide between two nociceptive stimuli in the thermal preference test, the escape test presents only one nociceptive stimuli ba lanced by light. Under normal conditions, light is relatively unpleasant and an important factor in re gulating responding. In fact, when the light is turned off, platform durat ion will significantly increase. It has been demonstrated that anxious animals (innate or due to stress) will avoid open, lighted areas (Fernandez et al., 2004). Anxiety can be evaluated in several tests like the open field (Van den Buuse et al., 2001). It is possibl e that the injury insensitive group spent less time on the platform due to an increased aversion to light. Therefore, assessment of motivational (darkbox) or anxiety (open fi eld) would yield additional information regarding change in light sensitivity. In other models of SCI, dysfunction of the opioid system is associated with hypersensitivity to thermal and mechanical st imulation (Abraham et al., 2000; Hao et al., 1998; Xu et al., 1994). For example, Xu et al (1994) reported that 50% of animals developed mechanical allodynia after ischemic SCI. It was speculated that the opioid system was dysfunctional in injury-sensitive animals but hyperactive in injury-insensitive animals. In confirmation of this hypothesis, the nonallodynic animal s exhibited features of allodynia after administration of naloxone an opioid receptor antagonist. Similar effects were reported after intr athecal injection of naloxone (H ao et al., 1998). Thus, in the current study, the endogenous opioid syst em was compromised in animals that develop abnormal sensitivity after SCI injury, but functional in animals with reduced sensitivity. This, in part, may explain th e lack of stress-induced changes in the

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153 injury-insensitive group. Future studies s hould use opioid antagonists such as naloxone to determine if the opioid system is overac tive in animals that demonstrate a reduced sensitivity to heat. A potential mechanism modulating pain se nsation is cholecystokinin (CCK). Administration of a CCK antagonist (e.g., pr oglumide, L365,260) prevents morphine tolerance and enhances morphi ne anti-nocicep tion (Heinricher et al ., 2001; Millan, 2002; Stanfa, 1994; Valverde et al., 1994; Wiesnfield-H alilin et al., 1990). After injury, levels of CCK mRNA and peptides are elevated spinal and supraspi nal structures involved in pain processing (Abraham et al., 2000, 2001; Ossipov et al., 1997; Vanderah et al., 2001a, 2001b). CCK, also, is implicated in mediating abnormal sensation after SCI (Brewer et al., 2003; Wiesenfeld-Hallin et al., 2002; Xu et al., 1994). For example, CCK mRNA levels were increased in the cortex and brainstem after excitotoxic injury. However, CCK was higher in animals that e xhibited more spontaneous pain (grooming; Brewer et al., 2003). In anothe r study, administration of a CCKB antagonist reverses mechanical allodynia after ischemic SCI (X u et al., 1994). Thus, up-regulation of CCK after SCI may suppress endogenous opioid activit y that contributes to alterations in nociceptive responsiveness. The mechanisms may also contribute to the ineffectiveness of opioid therapy to treat SCI pain. While no pre-clinical studies have examined the impact of stress on SCI pain, this is the first documentation that stress can augmen t sensitivity to heat assessed by cortically dependent responses. The ability of stress to increase sens itivity was similar between normal and injury groups. Unlike normal animal s, the effects of st ress persisted over several days. These results are comparable to clinical observations that stress can

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154 exacerbate below-level pain (Ditor et al., 2003; Demirel et al., 1998; Galvin and Godfrey, 2001; Yezierski, 2002). Possible explanations for these observations include altered functioning of critical physio logical system (autonomic ne rvous system; HPA), which have been identified on other pain conditi ons (Okifuji and Turk, 2002). The functioning of the autonomic system will be addressed in Chapter 5.

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155 CHAPTER 5 EFFECT OF STRESS AND EXCITOTO XIC INJURY ON PERIPHERAL VASOCONSTRICTION A possible mechanism mediating behavioral responses to heat is thermoregulation. Based on extensive literature, thermoregulati on is regulated by the sympathetic nervous system (Willete et al., 1991). Various experi mental manipulations alter sympathetic tone including stress, injury, and nociceptive stimulation (Janig, 1995; Herman and Cullinan, 1997; Magerl et al., 1996; McLachlan et al., 1992; Vierck, Unpublished Observations). One consequence of sympathetic activation is an increase in peripheral vasoconstriction resulting in cooling of the skin (described in Figure 5-1). A rela tionship exists between alterations in cutaneous temperature during th ermal stimulation and enhanced sensitivity to thermal stimulation based on operant res ponses (Vierck, Unpublished Observations). Experimental manipulations (stress, formalin, excitotoxic injury) that affect an animal’s sensitivity to mild nociceptive thermal stimuli can alter autonomic responses. Thus, if a manipulation attenuates peripheral vasoconstric tion, the animal will lose the ability to counteract thermal stimulation. As a conseque nce, sensitivity to h eat will increase as indicated by an increase in operant responses (hyperalgesia). Recently, a method has been developed to assess peripheral vasoconstriction in rodents (Vierck, Unpublished Obse rvations). Briefly, temperat ures were recorded from the plantar surface of both forepaws and one hindpaw during sessions in which the remaining hindpaw is stimulated with low-intensity heat (44.5 C). During stimulation of

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156 the hindpaw, temperatures of the non-stimulated paws diminished significantly within the first five minutes followed by a progre ssive increase in temperature. Figure 5-1. Reduction of skin temperat ures by sympathetically mediated vasoconstriction.

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157 To determine if altered cutaneous th ermoregulation contributes to operant responding to heat, several groups of animals were tested during se veral sessions: stress only, QUIS only, and stress in spinally injured animals. Recordings were obtained during separate sessions: a) before and after stre ss in normal animals, b) before and after excitotoxic injury, and c) before and after stress in spinally injured animals. These manipulations can alter temperature regulat ion and subsequent responses to thermal simulation. Effects of Restraint Stress on Peripheral Vasoconstriction To determine the effects of stress on peri pheral vasoconstriction, skin temperatures were recorded during baseline sessions and sessions in which animals were stressed for fifteen minutes (Figure 5-2). Skin temperat ures during baseline and stress conditions were obtained during different sessions. Afte r induction of anesthes ia with isoflurane, skin temperatures were recorded exactly fift een minutes after the termination of stress, corresponding to the behavioral paradigm us ed in Chapters 3 and 4. The hindpaw was stimulated for ten minutes with a temperature of 44.5 C. As seen in figure 5-2A, skin temperatur es during baseline conditions were lower during stimulation of the left hindpaw. Coo ling of the skin suggests that stimulation increased sympathetic activity re sulting in vasoconstriction (Fi gure 5-1). After the first 5 minutes of stimulation, non-stimulated paws demonstrated a progressive increase in temperature, which continued for the remai nder of the recording period. On the other hand, exposure to restraint stress reduced th e cooling of the non-stimulated paws. Despite a transient drop in temperature with in the first three minutes of stimulation,

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158 temperatures increased over time (e.g., warmer; vasodilatation) suggest ing that a decrease in sympathetic activity. To evaluate changes in peripheral ther moregulation before and after stress, temperatures were collapsed over five minute periods (Figure 5-2B). Skin temperatures during baseline sessions we re significantly lower than the stress condition during stimulation (5 minutes: F= 452.72, P< 0.001; 10 minutes: F= 562.81, P< 0.001). At the termination of stimulation, baseline temperat ures remained significan tly cooler than the stress conditions (15 minutes: F= 545.67, P< 0.001; ten minutes: F= 11219.25, P< 0.001). Thus, the expression of stress-induced hype ralgesia is a cons equence of blunted peripheral vasoconstriction to thermal stimula tion. Behaviorally, stressed animals are sensitive to heat because they lack the ability to compensate for stimulation Effects of Excitotoxic Injury on Peripheral Vasoconstriction Operant Escape Test Overall vasoconstriction after spinal injury In the operant escape test, it was predicte d that animals would show an enhanced sensitivity to heat following excitotoxic spin al injury. But, as a group, no hyperalgesia was observed. Skin temperatures were record ed before (baseline) and after excitotoxic injury in order to determine the effects of QUIS on peripheral vasoconstriction (Figure 5-3). Similar to Figure 5-2A, stim ulation of the left hindpaw at 44.5 C reduced skin temperatures, which was followed by a progressi ve increase during the later stages of and in the absence stimulation (baseline; Figure 5-3A).

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159 Figure 5-2. Skin temperature measurements from the plantar surface of non-stimulated paws during and after thermal stim ulation of the left hindpaw at 44.5 C (thick red line) for 10 minutes for baseline (blue dotted line) and stress (black continuous line) conditions. (A) Base line temperatures in non-stimulated paws were reduced during stimulation of the left hindpaw and returned to pre-stimulation levels at the end of st imulation. Restraint stress attenuated the cooling of paw temperatures (no reduc tion of temperature in non-stimulated paws). (B) Baseline recording were cool er than temperatures after stress. Restraint stress prevented the decrease in paw temperature. In addition, restraint stress increased sk in temperatures after the first 5 minutes. Data are expressed as change from temperatures immediately prior to stimulation and subsequent temperatures during and af ter thermal stimulation. Significant differences between baseline a nd stress are indicated by: *** P< 0.001.

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160 Skin temperatures were not affected by exc itotoxic injury (QUIS). In Figure 5-3B, skin temperatures were equally lower in bo th conditions during the first 5 minutes of stimulation ( F= 0.006, P= 0.940) but recordings were si gnificantly lower in the QUIS condition at 10 minutes ( F= 349.782, P< 0.001). After stimulation ended, baseline temperatures rebounded while recordings in the QUIS group remained significantly cooler (15 minutes: F= 317.619, P< 0.001; 20 minutes: F= 12512.69, P< 0.001). Thus, QUIS failed to blunt peripheral vasoconstricti on in this group, which can be a primary reason why injury-induced hyperalgesia was not observed in this group of animals. QUIS, on the other hand, appeared to prolong th e cooling of paw temperatures even after termination of the stimulus. Individual vasoconstriction after spinal injury As a group, thermoregulation was not signi ficantly affected by QUIS. But, two distinct groups were identified in Chapter 4 (QUIS hyperalgesia vs. QUIS hypoalgesia ). These groups may have different sympathetic responses to thermal stimulation. Skin temperatures were further analyzed based on these categorizations (Figure 5-4). After excitotoxic injury, thermal st imulation of the left hindpaw lowered skin temperatures (e.g., cooling response) in the hyperalgesic (Figure 5-4A) and hypoalgesic (Figure 5-4B) groups. However, the hyperalgesic group showed a rebound (w arming of non-stimulated paws) that paralleled a similar re bound in non-injured animals. The hypoalgesic group, on the other hand, did not recover. Recordings were collapsed over 5-minute intervals for the hyperalgesic (Figure 5-4C) and hypoalgesic (Figure 5-4D) groups. During the first 5 minutes, skin temperatures were similar before and after QUIS for both groups ( hyperalgesic : F= 1.957, P= 0.164; hypoalgesic : F= 1.435, 0.233). But, temperatures between baseline and QUIS

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161 conditions diverge after the firs t five minutes. Skin temperatures remained lower for the hyperalgesic group (10 minutes: F= 576.720, P< 0.001; 15 minutes: F= 240.07, P< 0.001; 20 minutes: F= 5656.23, P< 0.001). Similar responses were obtained for the hypoalgesic group at the end of stimulation. Temperatures were lower than baseline at 15 minutes ( F= 307.72, P< 0.001), 20 minutes ( F= 21.092.7, P< 0.001) but not during the earlier stages of stimulation (warme r vs. baseline at 10 minutes: F= 108.58, P< 0.001). The groups differed on the rates of recovery af ter stimulation was terminated. Skin temperatures in the hyperalgesic group recovered but cool er temperatures in the hypoalgesic group were prolonged even af ter cessation of stimulation. Even after dividing animals into their re spective groups, vasoconstriction remained active and was enhanced after excitotoxic in jury. A difference was observed between the two groups. While temperatures of the hyperalgesic recovered, the hypoalgesic group failed to recover and remained cooler during the testing trial. Pr olonged vasoconstriction may explain the observation that this group was less sensitive to heat (e.g., decreased platform duration; increased plate duratio n). The absence of an injury-induced hyperalgesia could be due to the inability of QUIS lesions to alter thermoregulation. Also, similar to histological analysis, eval uation of vasoconstricti on was assessed several months after spinal injury. T hus, the effect of spinal injury on behavioral responses was no longer present. Thermal Preference Test In the thermal preference test, animals s howed an enhanced sensitivity to heat (lower preference for heat; in jury-induced hyperalgesia). To determine if QUIS altered thermoregulation, skin temperatures were record ed before (baseline) and after excitotoxic injury (Figure 5-5) similar to the escape group. While thermal stimulation of the left

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162 hindpaw at 44.5 C reduced skin temperatures, excito toxic injury (QUIS) diminished cooling of skin temperatures (Figure 5-5A). Recordings were hi gher during stimulation followed by a small drop at the end of stimulation (10 minutes). In Figure 5-5B, baseline skin temperatures were significantly lower than recording in the QUIS conditions during the initial (5 minutes: F= 91.148, P< 0.001) but not the later period (10 minutes: F= 3.588, P= 0.06) of stimulation. At the end of stimulation, temperature in both conditions began to re bound in which baseline temperatures were significantly higher than QUI S conditions (15 minutes: F= 435.18, P< 0.001; 20 minutes: F= 835.4, P< 0.001). After excitotoxic injury, periphe ral vasoconstriction was blunted in this group. The apparent consequence of alte red functioning of the sympathetic system is the expression of injury-induced hyperalgesia. Effects of Restraint Stress Skin temperatures were recorded during sess ions before and after restraint stress to determine the effects of stress on peripheral vasoconstriction after excitotoxic injury (Figure 5-6). Cooling of non-stimulated pa ws was significantly diminished by restraint stress (Figure 5-6A). Similar to normal c onditions (Figure 5-2), recordings revealed a transient drop in temperature followed by an increase in temperat ure over time. After restraint stress (Figure 5-6B), temperatures were higher than pre-stress levels in QUIS animals during (5 minutes: F= 196.883, P< 0.001; 10 minutes: F= 1123.85, P< 0.001) and after (15 minutes: F= 43.59, P< 0.001; 20 minutes: F= 68.427, P< 0.001) thermal stimulation. Even after ex citotoxic injury, a ltered peripheral vasoconstriction was affected by restraint stress. This supports behavioral data demons trating that restraint enhances sensitivity to heat in spinally injured animals.

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163 Figure 5-3. Skin temperature measurements from the plantar surface of non-stimulated paws during and after thermal stim ulation of the left hindpaw at 44.5 C (thick red line) for 10 minutes before (baselin e: blue dotted line) and after (QUIS: black continuous line) exc itotoxic injury. (A) Ba seline temperatures of non-stimulated paws were reduced duri ng stimulation of the left hindpaw and returned to pre-stimulation levels at the end of stimulation. QUIS failed to attenuate the cooling of skin temperatur es in the non-stimulated paws. (B) QUIS enhanced the cooling of paw te mperatures, which continued after stimulation was terminated. Data are expressed as change from temperatures immediately prior to stimulation and subs equent temperatures during and after thermal stimulation. Significant differ ences between baseline and stress are indicated by: *** P< 0.001.

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164 Figure 5-4. Skin temperature measurements from the plantar surface of non-stimulated paws during and after thermal stim ulation of the left hindpaw at 44.5 C (thick red line) for 10 minutes before (baselin e: blue dotted line) and after (QUIS: black continuous line) exc itotoxic injury. (A, B) Baseline temperatures of non-stimulated paws were reduced duri ng stimulation of the left hindpaw and returned to pre-stimulation levels at the end of stimulation. QUIS failed to attenuate the cooling of skin temperatures in the non-stimulated paws. (C, D) Cooling of paw temperatures was enha nced after QUIS. Reduction of paw temperatures continued in the hypoalgesic group but not the hyperalgesic group at the end of stimulation. Da ta are expressed as change from temperatures immediately prior to stim ulation and subsequent temperatures during and after thermal stimulation. Significant differences between baseline and stress are indicated by: *** P< 0.001.

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165 Figure 5-5. Skin temperature measurements from the plantar surface of non-stimulated paws during and after thermal s timulation of left hindpaw at 44.5 C (thick red line) for 10 minutes before (baseline: bl ue dotted line) and after (QUIS: black continuous line) excitoto xic lesioning. (A) Baseline temperatures of non-stimulated paws were reduced duri ng stimulation of the left hindpaw and returned to pre-stimulation levels at the end of stimulation. QUIS attenuated cooling of paw temperatures in non-stim ulated paws. (B) QUIS prevented the decrease in paw temperature within th e first 5 minutes. In addition, paws displayed a slight decrease in temperatur e at the end of stim ulation. Data are expressed as change from temperatures immediately prior to stimulation and subsequent temperatures during and af ter thermal stimulation. Significant differences between baseline a nd stress are indicated by: *** P< 0.001.

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166 Figure 5-6. Skin temperature measurements from the plantar surface of non-stimulated paws during and after thermal s timulation of left hindpaw at 44.5 C (thick red line) for 10 minutes for baseline (b lue dotted line) and stress (black continuous line) conditions after exci totoxic injury. (A) Restraint stress attenuated cooling of non-stimulated pa ws. (B) Restraint stress prevented the decrease in paw temperature. In ad dition, restraint stre ss increased skin temperatures after the first 7 minutes. Data are expressed as change from temperatures immediately prior to stim ulation and subsequent temperatures during and after thermal stimulation. Significant differences between baseline and stress are indicated by: *** P< 0.001.

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167 Summary Peripheral vasoconstriction is a potential mechanism that may mediate behavioral responses to thermal stimulation. A conseque nce of vasoconstriction due to a reduction in blood flow to the periphery is a coo ling of skin temperatures. Under normal conditions, vasoconstriction permits the anim al to compensate for heat (e.g., during exposure to a thermal plate). Af ter restraint stress or spinal injury, enhanced sensitivity to heat (hyperalgesia; increased platform dur ations and decrease plate durations) may be a result of compromised sympathetic tone that reduces cooling of skin temperatures (e.g., vasoconstriction). Thus, beha vioral hypersensitivity to heat (operant responses) appears to be a consequence of an impaired symp athetic vasoconstriction, which limits an animal’s ability to counteract th e effects of thermal stimulation. Sympathetic Vasoconstriction after Stress Some have speculated that the ANS is involved in the execution of context appropriate responses, goal-directed behavior s, and positive affectiv e states based on an integrated processing of information by prefrontal and limbic st ructures modulating “fight or flight” res ponses (Thayer and Brosschot, 2005). In fact, changes in blood flow have been associated with expression of defense responses (e.g., arousal, after an exposure to threatening environmental stimu li (Nalivaiko and Blessing, 1999; Vianna and Carrive, 2005). Similar effects are observed after electrical stimulation of selected brainstem structures (Nalivaiko and Ble ssing, 1999). Furthermore, a consequence following chronic activation of these systems by stress or injury results in a dysregulation that may be revealed by negative affective states, hypervigilance, impaired cognition, and a lower resistance to stress (Tha yer and Brosschot, 2005). Clear ly, the ANS is critical for the functional state of an animal dur ing exposure to thr eatening stimuli.

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168 The sympathetic nervous system has been shown to mediate temperature particularly in the peri phery (Janig and McLachlan, 1992; Owens et al., 2002). Sympathetic vasoconstriction, or reduction in skin temperatur e, is observed after several manipulations. In addition, skin cooling is observed during thermal stimulation in humans (Karlsson et al ., 1998, 2006; Krassioukov, 2005; Ni cotra et al., 2005, 2006; Shimdoa et al., 1998; Vierck, Unpublished Ob servations; Willette et al., 1991) and animals (Vierck, Unpublished Observations). It is also seen after an acute exposure to stress in humans (Cooke et al., 1990; La rsson et al., 1995) and animals (Vianna and Carrive, 2005). It has been known that activity within the sympat hetic nervous system is responsible for “fight or f light” responses to a stressor (Barron and Van Loon, 1989) and is characterized by arousal a nd increases in heart rate, bl ood pressure, body temperature, and the release of catecholamines (Appelmaum and Holtzman, 1986; Chen and Herbert, 1995; De Boer et al., 1999; Thompson et al., 2003). Interestingly, while the temperatures of extremities (hindpaws) were cooler after e xposure to a stressor, a warming of core temperature has been shown to occur si multaneously (Vianna and Carrive, 2005). Increased body temperature is a consequen ce of two factors: 1) increase metabolism (brain, muscles), and 2) decreasing vasoc onstriction (Gordon, 1990; Vianna and Carrive, 2005). During stressful situations, blood flow from non-essential areas (gastrointestinal tract; skin) is redirected to areas critical to “fight or flight” struct ures including skeletal muscles and the central nervous system (Apl er and Zink, 1994). It is hypothesized that reduction of skin temperatures through d ecreased blood flow to the extremities also serves as a protective mechanism to limit blood loss (Vianna and Carrive, 2005).

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169 In the current experiment, peripheral va soconstriction was reduced by restraint stress. A possible reason for this effect was illustrated by Cooke et al (1991) in which he used a mental stress paradigm (arithmetic) to stimulate sympathe tic vascular tone. Differences were seen between males and fema les. During these tasks, males exhibited vasoconstriction while female subjects dem onstrated vasodilatation (increase in blood flow) compared to baseline values. Females appear to possess higher resting levels of sympathetic vasoconstriction. During periods of sympathetic activation (mental stress), the additional release of adrenerg ic agents or vasoconstriction has a minimal effect due to a ceiling effect. In some cases, vasodilatati on, or an increase in peripheral temperature, can be revealed (Vanhyoutte, 2003). In the present experiment, restraint stre ss appeared to activate the sympathetic nervous system. Subsequently, further activatio n of this system resu lted in a reduction of vasoconstriction that is normally observe d under control conditions. Because stress heightened sympathetic vascul ar tone, thermal stimulation had a minimal effect and revealed an increase in skin temperature (e.g., vasodilatation). Thus, an inability to cool skin temperature during exposure to thermal stimulation appears to contribute to an enhancement of heat sensitivity. Sympathetic Vasoconstriction after Spinal Cord Injury Abnormalities of the autonomic (e.g., cardiovascular) nervous system are common after SCI caused by damage to descending sympathetic pathways. Especially, SCI patients exhibit abnormal sympathetic activity that can be characterized by a lack of cutaneous vasoconstriction (Karlsson, 2006; Krassioukov, 2005; Nicotr a et al., 2006). For example, Nicotra et al (2005) reported that skin bl ood flow, determined by laser Doppler, was reduced in SCI compared to nor mal subjects after nociceptive stimulation

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170 and mental stress. In addition, abnormal sympathetic tone was demonstrated by an overall decrease in sympathetic skin respons es (SRR; Cariga et al., 2002; Curt et al., 1996, 1997). Assessment of sympathetic nervous system is accomplished by sympathetic skin responses (SSR), an el ectrophysiological procedure in which responses are induced by electrical stimulation. L ack of a sympathetic skin res ponse (SSR) was most prominent below the lesion (hands vs. feet: Cariga et al., 2002) but not if th e descending afferent tracts were spared (Nair et al., 2001). Based these studies, it can be concluded that the autonomic system is functionally abnorma l after SCI particul arly if descending projections (lateral funiculus; Krassioukov, 2005) are damaged. Unlike the effects of restrain t stress, excitotoxic injury either increased (operant escape group) or decreased (thermal preferen ce group) peripheral va soconstriction. This discrepancy maybe related to the analysis of histological variables considering assessment of vasoconstriction was several m onths after spinal injury. Bravo et al. (2002) found evidence that the sympathetic ne rvous system was diminished during the early stage following spinal injury. Thus, fu ture studies should examine alterations in sympathetic vasoconstriction in parallel with behavioral testing esp ecially during the first few weeks following injury. However, based on these results, differences in cutaneous cooling may be related to behavioral responding. In the escape group, thermal stimulation produced a prolonged vasoconstriction. After spinal injury, lo ss of descending pathways and/or chronic activation of this system due to injury reduces resting sympathetic regulation of peripheral nerves. Consequently, nerves upregulate -adrenergic receptors. During phasic stimulation, vascular hypersensitivity to circulating noradrenaline is observed and

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171 results in an exaggerated vasoconstric tion (McLachan and Brock, 2006). A pronounced vasoconstriction could reduce behavioral res ponses (hypoalgesia) to thermal stimulation. Unlike the escape test, animals tested in th e thermal preference test demonstrated a diminished vasoconstriction after spinal in jury. Again, similar to restraint stress, abnormal vascular tone (e.g., hi gher resting level of sympat hetic vasoconstriction) may contribute to reduced vasoconstriction to ther mal stimulation. In this group of animals, spinal injury may have chronically activated the sympathetic nervous system. Additional stimulation had little effect. Interestingly, restraint stress diminished peripheral vasoconstriction after spinal injury. At first glance, the reduced cooling response of cutaneous temperatures appeared to be greater in normal animals (exhib iting an increase in temperature due to vasodilatation). However, this observati on is misleading because the QUIS group had a pronounced cooling response prior to stress. Clearly, additional research is needed to fully examine alteration in the sympathetic nervous system after spinal injury. Future Studies Additional research is require d to directly evaluate cha nges in sympathetic nervous system. To accomplish this task, seve ral techniques are available including radio-telemetric probes (measurement of h eart rate, body temperature, blood flow, and general activity) and infrared thermography (measurement of radiating heat from body and extremities). Both strategies are advantageous because of the accuracy of measurements and practicality of assessi ng responses in conscious, freely moving animals without interference (V ianna and Carrive, 2005). Furthermore, this system is also working in parallel with the HPA axis, as indicated by the secretion of gluocorticoids from the ad renal glands (Levine, 2000). Gluocorticoids

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172 mediate the responses to a stressor (Herman an d Cullinan, 1997) and indirectly affect the autonomic nervous system. Van Acker et al ( 2001) reported that rest raint stress induced an increase in heart rate (sympathetica lly regulated) that was reversed by an intracerebroventricular injecti on of glucocorticoid antagonist. These results suggest that classical stress hormones significantly influe nce cardiovascular activity after stress through interactions with the autonomic system However, no studies have examined the role of gluocorticoids in mediating vasocons triction during stimulation (stress, injury). Future studies could examine the modulation of skin temperatures by gluocorticoids and other stress related hormones.

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173 CHAPTER 6 CONCLUSIONS AND FUTURE STUDIES Given the differential effects of restraint stress on reflex and operant responses to thermal stimulation in the pr esent study, a variety of que stions arise concerning the generality and mechanisms for these influences. That is, what aspects of stress and which modulatory systems are related to the suppressi on of innate reflexes and/or enhancement of nociceptive sensations dependent on co rtical responses? For example, recent investigations indicate that an important de terminant of stress effects on innate reflex responses is the duration of st ress. Whereas acute stress at tenuates licking and guarding (e.g., increase latencies), repeated presentati on (chronic stress) is reported to enhance responsivity (e.g., decrease latencies; Gamaro et al., 1998; Quintero et al., 2000). Thus, suppression of innate reflexes may be unique to acute stress. In addition to the importance of stress duration, there appears to be significant differences between the effects of processi ve and systemic stressors, particularly on sensations of pain. Some forms of acut e systemic stress (e.g., vigorous exercise) attenuate pain sensitivity in humans (Vierck et al., 2001) but psychological stress can exacerbate experimental pain (Logan et al., 2001 ). In the present study, escape behavior may have been enhanced because restraint st ress preferentially activated forebrain and limbic circuits (Herman and Cullinan, 1997; Herman et al., 1996). This raises the possibility that psychological stressors have greater eff ects on cerebral components of pain transmission systems than do systemic stressors that act predominantly through brainstem systems (Cullinan et al., 1995; Herman and Cullinan, 1997). Psychological

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174 stress also stimulates forebrain catechol amine (e.g., noradrenalin; Tanaka et al., 2000) circuits, which project to supraspinal and spin al structures modulating innate reactions to nociceptive input (Milla n, 2002). Therefore, licking a nd guarding are likely suppressed after restraint because psychological stress indi rectly activates descending (brain stem to spinal) inhibitory systems (C alcagnetti et al., 1992; Gamaro et al., 1998; Tsuda et al., 1989). It is important to note that presumed activation of descending modulatory systems by restraint stress did not i nhibit nociceptive transmission fr om the spinal cord to the cerebrum, as assessed by escape duration. Not only was ascending nociceptive transmission spared, but it might have been accentuated at the spin al level by descending modulation. A potential mechanism for this co mbination of effects is that descending modulatory systems have both ex citatory and inhibitory actio ns (Millan, 2000) that could suppress spinal motoneuron output while differen tially activating cells of origin of pain transmission pathways. In addition to differences in the type or duration of stress, how important is the form of nociceptive stimulation to the effects of stress? Po st-stress effects have often been tested on behaviors elicited by intens e and brief stimuli that activate A-delta nociceptors – inputs which more effectively a nd reliably elicit reflexes than selective activation of C-nociceptors (Cooper and Vierc k, 1986; Cooper et al., 198 6; Vierck et al., 2000). However, it is possible that stress enhances sensitivity to tonic input from C-nociceptors. For example, average escape latencies for 44.0 C stimulation (as in the present study) occurred when foot temperatures reached 44.0 C (Vierck et al., 2004), which approximates thresholds for activati on of C-nociceptors (F leischer et al., 1983;

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175 Leem et al., 1993). Average lick/guard la tencies were more than twice the escape latencies, and these behaviors occurred when skin temperatures and durations sufficient to activate A-delta nociceptors might have been attained. This raises the possibility that acute stress has opposite effects on nociceptive input from A-de lta and C-nociceptors. In support of this hypothesis are studies show ing stress-induced de pression of reflex responses to intense thermal stimulation a nd demonstrations that operant escape from electrical stimulation is attenuated by acute stress (Bodnar et al., 1978c; 1979). Electrical stimulation provides an optimum model of A-delta nociception, because very high current levels are required to activate C-noc iceptors (Cooper and Vierck, 1986; Cooper et al., 1986). A key hypothesis of particular relevance to A-delta and C nocicep tion is that acute stress can attenuate nociceptive reflexes through opioid mechanisms (Amir and Amit, 1979; Bodnar et al., 1978c; Gamaro et al., 1998 ; Lewis et al., 1980). Involvement of opioid mechanisms is indicated by blockade of stress effects by opioid antagonists, enhancement of stress effects by opioid agoni sts, and development of cross-tolerance with morphine after repeated exposures (Bodnar et al., 1978b; Lewis et al., 1980; Calcagnetti et al., 1992; Willer et al., 1981). However, doses as high as 10 mg/kg of systemic morphine are required to produce a reflex suppression comparable to that produced by stress (Bodnar et al., 1978b). Doses in this range are likely to produce a generalized behavioral/motoric suppression an d are above threshold for attenuation of A-delta nociception (Cooper and Vierck, 1986, Mauderli et al., 2000). Higher dose morphine (3.0 to 5.0 mg/kg) attenuates A-delta nociception, whereas 0.5 mg/kg is sufficient to reduce operant responses to C-nociceptor input (Cooper and Vierck, 1986;

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176 Vierck et al., 2002; Yeomans et al., 1995). Because systemic mor phine preferentially attenuates C-nociception, the opioid hypothesis can be evaluated appropriately using low levels of nociceptive heat to selectively activates C-nociceptors (Yeomans and Proudfit, 1996; Yeomans et al., 1996), as in the pres ent study. Accordingly, low dose morphine (0.5 mg/kg) suppresses escape responding to 44.0 C stimulation, but lick/guard responding to the same stimulus is enhanced by this dose of morphine (Vierck et al., 2002). Restraint stress had the opposite eff ect: enhancement of escape responding to 44.0 C and depression of lick/guard responses. Therefore, the opioid hypothesis is contradictory to stress effects on responses to C-nociceptor input that are sensitive to physiological levels of opioid agonists. A probable reason for difficulties ascribing physiological levels of opioids or other transmitters to generalized effects of stress on nociceptive behaviors is that distinct responses are mediated by different central pa thways, and each of these neural systems is subject to different patterns of modulation at multiple levels of the neuraxis. Licking is present in decerebrate animals but absent in spinal animals (Matthie s and Franklin, 1992). Guarding, an elaboration of withdrawal reflexes that are present in spinal animals, is more difficult to interpret in terms of requis ite neural circuitry but can be elicited after decerebration. Thus, both licking and guard ing depend upon specialized spinal-brain stem-spinal loops that can be modulated at e ither of these levels. The tail flick response has characteristics of a strictly segmental refl ex that can be modulated directly at spinal levels or indirectly by descending connec tions from the brain stem. Acute stress attenuates each of these responses. In contrast, several other innate responses (vocalizations and jumping) ar e present in decerebrate animal s and have been reported to

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177 be increased by acute stress (King et al., 1996, 1999; Simone and Bodnar, 1982). Therefore, the distinction between behavior s that are accentuated or depressed by acute stress appears not to be ba sed entirely upon distinctions between operant and reflex effects or between actions at cerebral, brain stem or spinal levels. A possible commonality for rodent behaviors that are accentuated by stress is an adaptive significance for fight/flight situations in wh ich survival is optimized by a compatible combination of intentional reactions and i nnate responses. Vocalization alerts other animals, and jumping can be effectively integrated into an escape strategy. Future Directions Several possible studies could be evaluated from the current set of experiments. Future studies could examine the effects of chronic stress or expos ure of acute stress on lesioned animals with other chronic pain conditions (e.g., spinal nerve ligation, SNL; chronic constriction injury, CCI; formalin). In addition, the following studies were initially proposed for this dissertation. However, due to unforeseen limitations, the studies were not competed. These experime nts would examine the neural circuitry and transmitter systems involved in producing stress-induced changes in nociceptive sensitivity in rats. Specifically, the role of ascending and descending inhibitory/facilitatory pathways in stressinduced alterations in operant and reflex responses to thermal stimulation could be exam ined. The contributi on of these pathways to stress-induced changes in responses would be accomplished by: 1) intrathecal administration of adrenergic and opioid rece ptor agonists or anta gonists; and, 2) using receptor specific neurotoxic lesions.

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178 Spinal neurotransmitter systems in the ex pression of stress-induced changes in thermal stimulation These experiments would examine the effect s of intrathecal adrenergic and opioid agonists or antagonists on opera nt escape and reflexive respon ses to an acute exposure to restraint stress. In previous studies, spinally administer ed opioid or noradrenergic agonists reduced dorsal horn activ ity and reflex tail and hindpa w withdrawal responses to nociceptive thermal stimulation. The inhibito ry effects of intrat hecal agonists are mediated by spinal -opioid and 2 adrenergic receptors (C lark and Proudfit, 1991; Takano and Yaksh, 1992; Yaksh, 1999) It is hypothesized that spinal adrenergic and opioidergic systems contribute to stress-i nduced effects on responses evoked by thermal stimulation. These receptors mediate the in hibitory effects of stress on reflexive responses and oppose the facili tatory effects of stress on operant responses through activity of descending brainstem proj ections to the spinal cord. If the descending inhibitory effects of st ress are mediated by opioid and noradrenergic receptors, activation of spinal -opioid and 2-adrenergic will enhance the inhibitory effects of stress on reflex responses. In contrast, blockade of spinal -opioid and 2-adrenergic will decrease the inhibitory e ffects of stress on reflex responses. If the descending excitatory effects of st ress are opposed by opioid and noradrenergic receptors, activation of the spinal -opioid and 2-adrenergic receptors by i.t. DAMGO and clonidine, respectively, will decrease sensitivity of operant escape responses. In contrast, blockade of spinal -opioid and 2-adrenergic receptors by i.t. naloxone and yohimbine, respectively, will in crease the excitatory effe cts of stress on operant responses (see below for alternative results and future directions).

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179 The results of the proposed experiments may result in little or no effects of stress and drugs on reflex or operant escape responses. Fi rst, noredranaline has a differential effect on behavioral responses to thermal stimul ation depending on expression of adrenergic receptor subtypes. Due to the bi-directional activity of noredranaline in the spinal cord, the involvement of spinal 1(pronociceptive) and 2(antinociceptive) noradrenergic receptors in stress-induced changes in so matosensory processing can be examined by complementing 2 adrenergic antagonists with intrathecal injections of prazosin ( 1-adrenergic antagonist). Blockade of spinal 1-adrenergic (pronociceptive) receptors by i.t. prazosin will reduce the facilitatory effects of stress on escape and enhance the inhibitory effects of stress on reflex responses. The adrenergic 1-adrenergic antagonist, prazosin, is ideal for these initial studies due the extensive literature on stress and modulation of nociception (Camarata and Yaksh, 1986). The results of the proposed experiments may result in little or no effects of stress and drugs on reflex or operant escape responses Secondly, the opioid and adrenergic receptors may not be involved in stress eff ects on operant responses, and therefore, neither -opioid or 2-adrenergic receptor agonists (e.g., DAMGO, clonidine) nor antagonists (e.g., naloxone, yohimbin e) will affect increases in sensitivity observed on these responses following stress. This possibility will necessitate the use of other pharmacological agents targeting transmitter systems thought to have a role in the descending modulation of nocicep tive processing in the spin al cord (e.g., cholinergic, serotonergic, or cholecystokinin). Considering recent reports by Zeitz et al (2002) suggesting the pronociceptive properties of spinal 5-HT3 receptors, we could intrathecally administer a selective

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180 5-HT3R antagonist (ondansetron, 1-25 g) to reverse the facilitatory effects of these receptors on operant responses. Finally, it is possible that spinal neurotransmitter systems may not be critical in the stress-i nduced hypersensitivity. Future studies could examine the effects of intrace rebroventricular (i.c.v.) inject ions in order to examine neural structures involved in the supras pinal processing of noc iceptive information, which may produce a greater impact on es cape responses as mentioned below. Role neurokinin-1 receptor (NK-1R) expre ssing neurons on stress-induced changes in thermal responses These experiments would examine the eff ects of a specific population of spinal neurons on somatosensory processing in nonstressed and stressed animals. NK-1R expressing spinal neurons are implicated in nociceptive transmission, controlling behavioral responses, and activ ation of descending pathways from the brainstem (Dolye and Hunt, 1999; Wiley and Lappi, 1997). These neurons would be lesioned by intrathecal injection of substance P-saporin after training and baseli ne testing. It is hypothesize that ascending pathways from spin al neurons expressi ng NK-1R contribute to stress-induced hypersensitivity of operant responses and hyposensitivity of reflex responses evoked by thermal stimulation. If the excitatory effects of stress invo lve spinal neurons expressing NK-1R, we expect that ablation of these neurons will reduce operant escape responses in lesioned animals. If the inhibitory effects of stre ss involve spinal neur ons expressing NK-1R, we expect that ablation of these neurons will reduce reflex responses in lesioned animals. These results are based on th e hypothesis that an ascending pathway originating from NK-1R expressing neurons activates a descen ding excitatory as well as inhibitory feedback loop in the rostral medulla.

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181 The reliability and specificity of neurot oxins are a potential problem. However, recent collaborations with Dr. Wiley and ot her labs have insured the proper use and viability of the neurotoxin. Low-doses of the neurotoxin will prevent non-specific damage to non-receptor expressing cells. If the neurotoxin lesions fail to alter stress-induced changes in noci ception, moderate to high doses of the neurotoxin can be used. In addition, mechanical lesions to eliminate ascending (ALQ) and descending pathways (DLF) may be used instead. Elimin ation of these pathways will identify the location of critical pathways involved in producing stress effects. Depending on the intrathecal pharmacology, we could use speci fic lesions to eliminate influences by adrenergic and serotonergic sy stems in the spinal cord ac ting at nerve terminals (e.g., 5, 7-dihydroxytryptamine and 6-hydroxy DA). Descending pathways mediating stress-i nduced changes in thermal responses Finally, these experiments would examine the function of descending facilitatory and inhibitory pathways on somatosensory processing in non-stressed and stressed animals. Descending projections from the ro stroventral medulla (R VM) originating from -opioid receptor-expressing ne urons are implicated in the modulation of spinal nociceptive transmission and enha ncement of nociceptive behavior s in rats. The origin of descending facilitatory pathways will be le sioned by bilateral medu llary injection of dermorphin-saporin after training and baseline testing. It is hypothesized that descending pathways from the RVM contribute to stre ss-induced hypersensitivity of operant responses and hyposensitivity of reflex re sponses evoked by thermal stimulation. If the excitatory effects of stre ss are mediated by neurons expressing -opioid receptor in the RVM, we expect that ablati on of these neurons will reduce operant escape

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182 responses in lesioned animals as a result of eliminating facilitato ry pathways driving operant behavior. If the inhibitory effects of stress are mediated by neurons expressing -opioid receptor in the RVM, we expect that ablation of these neurons will reduce reflex responses in lesioned animals as a result of eliminating facilita tory pathways that normally antagonize descending inhibitory influences The reliability and specificity of neurotoxi ns are a potential problem. However, a recent collaboration with Dr. Wiley and ot her labs has insured the proper use and viability of the neurotoxin. If the neurotoxin lesions fail to alter stress-induced changes in nociception, we could use moderate to high dose of the neurotoxin or inject lidocaine into the RVM. Finally, mi croinjection of lidocaine could be used to reduce neuronal activity in the RVM. Mitchell et al (1998) reported that inactivation of RVM neurons by lidocaine reversed morphine and stress-i nduced inhibition of reflexive responses. Conclusions The present studies demonstrated that rest raint stress differentially affected innate reflex and operant escape responses to lo w intensity thermal stimulation, which is threshold activation for C-noci ceptors. Reflex lick/guard responses were reduced after restraint stress (stress-indu ced hyporeflexia), which confir ms previous studies of stress-induced hyporeflexia for A -activation. In contrast, op erant escape responses were enhanced in the same animals after st ress (stress-induced hyperalgesia). Additional characteristics of stress-induced changes in nociception were identified. First, control procedures showed that stress did not affect responding to non-nociceptive stimulation, did not influence aversion to light and did not enhance avoidance responding. Second, stress-induced changes in behavioral responses were greater when

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183 assessed immediately after rest raint. Third, the effect of stress on both responses was transient and did not undergo ad aptation with repetition at 2 w eek intervals. Fourth, core body and cutaneous temperatures re turned to levels similar to controls by fifteen minutes after stress termination. Finally, the expres sion of stress-induced changes in nociception was affected by endogenous and exogenous opioids. Stress-induced hyperalgesia (operant escape) is mediated by a non-opioi d mechanism, which is counteracted by tonic endogenous opioids and exogenous opioid admi nistration. In cont rast, stress-induced hypoalgesia is mediated by endogenous opioi d systems, which suppresses reflex lick/guard responses. Exogenous morphine enhances reflex responses and opposes effects of restraint stress on these reflexes through separate mechanisms. Taken together these observations suggest that acute expos ure to a psychological stressor activates forebrain circuits mediating pain percepti on, producing hyperalgesia Forebrain-limbic circuits in turn act ivate descending pathways to suppress nociceptive reflexes. In addition, pain sensations are affected by damage to the spinal cord gray matter. Assessment of operant escape responses revealed that heat sensitivity was enhanced after Excitotoxic injury of the spinal cord by altering sympathetic-mediated peripheral vasoconstriction. It is clear that damage to the gray matter is a cr itical factor in the development of heightened pain sensation in humans (Finnerup et al., 2003) and may underlie observations that the autonomic ner vous system is dysfunctional after spinal injury (Nicotra et al., 2005, 2006). Subse quent exposure to acute restraint stress enhanced injury-induced operant escape res ponses, which is consistent with anecdotal reports that stress increases clinical pain in humans especially after SCI (Ditor et al., 2003). Thus, the expression of stress-induced hy peralgesia is transient, dependent on the

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184 preferential activation of C-nociceptors not influence by changes in core body temperature but altered sympathetic regulati on of peripheral vasoconstriction, and more prominent in spinally injured conditions.

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209 BIOGRAPHICAL SKETCH Born in western Pennsylvania, I grew up outside of Pittsburgh with my father and mother. My childhood was like th at of other normal children: playing sports, wandering in the woods, and watching several Pittsburgh professional sports teams. I attended a Catholic elementary school and a public high sc hool. I was very activ e in sports during this time playing soccer and football. My parents divorced, but out of this unsettling situation I acquired a larger family after my parents remarried. I will never forget the day that my sister was born. At the end of my senior year in high sc hool, my mother passed away, leading to a rough period in my life. However, I found strength in my family, and I focused that strength into academic studies in college. I wanted to attend physical therapy school, but my attention shifted after taking Dr. Blustein ’s neuroscience class and working on several undergraduate projects at Beaver College. As my interest in neuroscience grew, I planned to attend graduate school, so I gained additional research experience in Dr. Caudle’s lab. At the University of Florida, I was given that opportunity to fulfill my goal and continue my research interests in neuros cience. During my time in graduate school, I met and married the most wonderful woman in the world: Natasha. As my graduate career ends, I look forward to the future.


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STRESS-INDUCED CHANGES IN SENSITIVITY TO THERMAL NOCICEPTIVE
STIMULATION IN NORMAL RATS AND FOLLOWING EXCITOTOXIC SPINAL
CORD INJURY
















By

CHRISTOPHER DUNCAN KING


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2006

































Copyright 2006

by

Christopher Duncan King

































To my family and friends who supported me through these years. With your guidance
and patience, I achieved a great accomplishment. I would have not been as successful
without you. Thank you.















ACKNOWLEDGMENTS

I want to acknowledge family and friends who sustained me. In particular, I am

extremely thankful for the guidance, love, and patience of my family. My father Richard,

mother Sharren, stepmother Sally, sister Kelsey, and wife Natasha have been my rock.

Each of them has played a special role in my life. With their inspiration and

encouragement, I accomplished my goals and understood the importance of family

especially during the tough times. I also would like to remember the individuals who

have left our family including my mother Sharren, grandfather King, and grandmother

Flint. They are loved and missed.

I also thank the professors who helped me during my graduate studies. I am

fortunate to have worked with them. Dr. Caudle provided me with an opportunity to

work in his lab, visit foreign lands, and further develop my research and academic

proficiencies during a tough time in my life. Also, I appreciate Dr. Vierck's guidance

and wisdom over the past few years. I would also acknowledge help and advice from

Drs. Darragh Devine and Andre Mauderli about stress and the potential pitfalls of

behavioral testing. Last but not least, I express my gratitude to Dr. Yezierski as an honest

and supportive mentor. In the process of developing my training and research program,

he was able to convey his knowledge about pain, and was very patient in educating me

about writing. I am thankful to each of my professors for their involvement in my

development as a scientist and a person.









In Drs. Yezierski's and Vierk's lab, I worked with several amazing individuals who

also gave me technical and moral support. I would like to express my appreciation to my

research backbone: my "rat ladies" Jackie, Karen, and Jean. I would not have

accomplished my research goals without their dedication and assistance. They educated

me on many things related to my research, and also showed that you are only as good as

the people around you. I also like to thank Dr. Cannon for his computer, histology, and

perfusion expertise; and for listening to my unending questions about these issues. Also,

I thank Victoria Gority for administrative assistance and long talks about world problems.

I also thank Sandra for her assistance, even though I have only known her for a short

time.

Finally, I would like to thank my friends; especially my old roommate and lab mate

Federico. I also like to thank Sara (a fellow graduate student in Dr. Yezierski's lab) for

her support and friendship through our graduate training. Finally, I thank my God and

my savior Jesus Christ for giving me a great family, friends, and an opportunity to

develop into a scientist.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TABLES ................................ ........... ....... .................. viii

LIST OF FIGURES ......... ........................................... ............ ix

A B S T R A C T .............................................. ..........................................x iii

CHAPTER

1 INTRODUCTION AND LITERATURE REVIEW ....................................................1

T h e P ain E x p erien ce .......................................................................... .....................
Anim al M models of Pain .............................................. ........ ................ .8
C h ro n ic P a in ...............................................................................................................1 3
Influence of Stress on Pain .................................................................................. 21
Thermoregulation by Sympathetic Vasoconstriction ...........................................25
Sum m ary ...................................... ................. ................. .......... 27

2 EXPERIMENTAL METHODS AND DESIGN................................29

E x p erim mental A nim als ..................................................................... .....................30
B ehavioral Testing Procedures ........................................................ ............. 31
D rug A dm inistration....... ........ .......................... .. .. .. ...... .... .... .. 42
Surgical Procedures ............ ....... .... .................................. .... ......... ........ 42
Assessment of Core and Cutaneous Temperature ............................................... 44
Statistical A n aly sis............................................................................. ............... 4 8
Stu dy D design ..................................................... ......... ...... 4 8

3 EFFECTS OF RESTRAINT STRESS ON NOCICEPTIVE RESPONSES IN
N O R M A L SU B JE C T S ............................................ .................... ...............52

Effects of Restraint Stress on Reflex Lick/Guard Responses at 44.00C...................53
Effects of Restraint Stress on Operant Escape Responses at 44.00C .........................55
Time Course of Restraint Stress on Operant Escape Responses at 44.00C ..............65
Effects of Restraint Stress on Core Temperature ....................................... .......... 68
Effects of Restraint Stress on Control Responses...........................................76
Effects of Restraint Stress on Operant Thermal Preference................. ..............80









Effects of Endogenous Opioids on Stress-Induced Changes in Nociception............86
Effects of Morphine on Stress-Induced Changes in Nociception .............................92
Sum m ary and D iscu ssion ........................................ .............................................98
N o te s ................................ ......................................................1 0 6

4 EFFECTS OF RESTRAINT STRESS ON NOCICEPTIVE RESPONSES
FOLLOWING EXCITOTOXIC SPINAL CORD INJURY ............... ............... 107

Effects of Excitotoxic Spinal Cord Injury on Operant Escape............................... 108
Overall Effect of Spinal Injury on Escape Responses ............... ...............108
Effects of Spinal Injury in Individual Groups.............................. ..................113
Effects of Restraint Stress on Operant Escape Following Excitotoxic Injury ..........118
Overall Effects of Stress on Escape Responses after Injury.............................118
Effects of Stress on Individual Groups................................... ................ ... 120
Effects of Excitotoxic Spinal Cord Injury on Thermal Preference.........................125
Effects of Restraint Stress on Thermal Preference Following Excitotoxic Injury ...131
Prediction of Behavioral Responses Based on Open Field Responses..................... 138
Comparison between Normal and Spinally Injured Animals................................139
H istology..................................... .................. ............... ........... 141
Su m m ary ...................................... ....................................................150

5 EFFECT OF STRESS AND EXCITOTOXIC INJURY ON PERIPHERAL
VASOCON STRICTION .............................. ............... .................. ............... 155

Effects of Restraint Stress on Peripheral Vasoconstriction.................................... 157
Effects of Excitotoxic Injury on Peripheral Vasoconstriction...............................158
Sum m ary ............................................................... ..... ..... ......... 167

6 CONCLUSIONS AND FUTURE STUDIES................. ....................................173

F u tu re D direction s ............................................................................ ................ .. 17 7
C o n clu sio n s.................................................... ................ 18 2

LIST OF REFEREN CES ........................................................... .. ............... 185

B IO G R A PH IC A L SK E T C H ........................................ ............................................209
















LIST OF TABLES


Table p

3-1 Cumulative reflex lick/guard and operant escape durations over two sessions of
re stra in t store ss .................................................. ............... ................ 6 5

3-2 Darkbox latencies for control and restraint groups ............................................80

4-1 Number and duration of escape responses at 44.50C before and during testing
sessions in which animals were tested fifteen minutes .......................................121

4-2 Effect of open field responses on operant responses for groups after excitotoxic
in ju ry ........................................................................... 1 3 8

4-3 Histological data for groups after excitotoxic injury that were behavioral
assessed in the operant escape and thermal preference tests................................147

4-4 Histological data for groups after excitotoxic injury that were behavioral
assessed in the operant escape and thermal preference tests............... ...............148

4-5 Effects of histological variables on operant escape and thermal preference
responses after excitotoxic injury..................................... .......................... 149
















LIST OF FIGURES


Figure page

1-1 Hierarchical behavioral responses to nociceptive stimuli including spinal,
supraspinal, and cortical mediated responses .......................................................9

2-1 Reflex apparatus ................................... ......................... ......... 32

2-2 O perant escape apparatu s ........................................ ...................... .....................34

2-3 Therm al preference apparatus ............................................................................ 37

2-4 O pen filed apparatus............................................ ................... ............... 40

2-5 Restraint tube.................................... ............................... .......... 41

2-6 Behavioral testing sequence, stress exposure, and injection schedule for
evaluation of operant and reflex lick/guard responses...........................................41

2-7 Skin temperature recording in anesthetized rats..................... ............................ 47

3-1 Behavioral testing sequence for the restraint group ...........................................53

3-2 Reflex lick/guard latencies during testing trials at 44.00C..................................56

3-3 Cumulative reflex lick/guard durations during testing trials at 44.00C....................57

3-4 Escape latencies during testing trials at 44.0 C ................... ................... .......... 58

3-5 Cumulative escape durations during testing trials at 44.00C .................................60

3-6 Sequence analysis of successive escape plate and platform durations during
testing trials at 44 .0 C ....................... .. ...................... .. ........ ........... 63

3-7 Average escape duration of the first six plate and platform responses during
testing trials at 44 .0 C ....................... .. ...................... .. ........ ........... 64

3-8 Temporal profile of restraint stress on escape responses during trials at 44.00C.....66

3-9 Escape latencies during testing trials at 44.0 C ................... ................... .......... 69

3-10 Escape durations during testing trials at 44.0C ............................................... 70









3-11 Core body temperatures during testing trials at 44.00C ........................................ 73

3-12 Core and cutaneous hindpaw temperatures for control and restraint groups ...........75

3-13 Escape latencies during testing trials at 36.0 C ................... ................... .......... 77

3-14 Cumulative escape durations during testing trials at 36.00C .................................78

3-15 Sequence analysis of successive escape plate and platform durations during
testing trials at 36.0 C ....................... .. .... ................ ........................ 8 1

3-16 Average escape duration of the first six plate and platform responses during
testing trials at 36.0 C ....................... .. ...................... .. ........ ........... 82

3-17 Cumulative thermal preference durations during testing trials at 15.0 and 45.00C .84

3-18 Average of the first six cold and heat durations.................................................... 85

3-19 Reflexive lick/guard latencies at 44.50C during testing sessions ..........................88

3-20 Cumulative reflexive lick/guard durations at 44.5C ......................... ............89

3-21 Cumulative escape durations at 44.50C............... ........................................ 91

3-22 Reflexive lick/guard latencies at 44.5 C .............................................................. 94

3-23 Reflexive lick/guard durations at 44.5 C ...................................... ............... 95

3-24 Cumulative escape durations during testing trials at 44.50C .................................97

4-1 The number of escape platform responses during testing trials at 44.50C before
and after excitotoxic injury ............................................................................. 109

4-2 Cumulative escape platform durations during testing trials at 44.50C before and
after excitotoxic injury. .................. .......................... .. .. .. .... .. ........ .... 110

4-3 Sequence analysis of successive escape plate and platform durations during
testing trials at 44.50C before and after excitotoxic injury ............... ............... 111

4-4 Average duration of the first six plate and platform responses during testing
trials at 44.50C before and after excitotoxic injury ..........................112

4-5 Weekly postoperative platform responses across several weeks of testing during
trials at 44.50C before and after excitotoxic injury ...............................................114

4-6 Number of escape platform responses during testing trials at 44.50C before and
after excitotoxic injury ............................................. ................. .. ........ .. .. 16









4-7 Cumulative escape platform durations during testing trials at 44.50C before and
after ex citotox ic inju ry ................................................................. .......... .... 117

4-8 Weekly postoperative platform responses across several weeks of testing during
testing trials at 44.50C before and after excitotoxic injury ...............................119

4-9 The number of escape platform responses at 44.5C .................. ....................122

4-10 Cumulative escape platform responses at 44.50C .............................................126

4-11 Cumulative escape platform responses at 44.5 C ........................................ ...... 127

4-12 Correlation between postoperative responses following QUIS and change in skin
temperature regulation during sessions .............................................................. 128

4-13 The number of thermal preference responses during testing trials at 15.0-45.00C
before and after excitotoxic injury .............................................. ............... 129

4-14 Cumulative durations of thermal preference responses during testing trials at
15.0-45.00C before and after excitotoxic injury................... ........... ............... 130

4-15 Sequence analysis of successive cold and heat preference durations during
testing trials at 15.0-45.00C before and after excitotoxic injury ..........................132

4-16 Average durations of the first six cold and heat preference responses during
testing trials at 15.0-45.00C before and after excitotoxic injury ..........................133

4-17 Weekly postoperative cold and heat preference responses across several weeks
of testing during trials at 15.0-45.00C before and after excitotoxic injury ............134

4-18 Number of thermal preference responses during testing trials at 15.0-45.00C ......136

4-19 Cumulative cold and heat preference responses at 15.0-45.0C ............................137

4-20 Difference scores for plate and platform durations during escape trials at 44.50C
in normal and after excitotoxic....................... .... ............................ 140

4-21 Difference scores for cold and heat preference durations during trial at
15.0-45.00C in normal and after excitotoxic injury ............................................ 142

4-22 A comparison of in vitro MRI images ........................................................144

4-23 Summary of transverse and sagittal spinal cord images obtained through in vitro
M R I after excitotoxic injury........................................................ ............... 145

5-1 Reduction of skin temperatures by sympathetically mediated vasoconstriction ....156









5-2 Skin temperature measurements from the plantar surface of non-stimulated paws
during and after thermal stimulation of the left hindpaw ................................159

5-3 Skin temperature measurements from the plantar surface of non-stimulated paws
during and after thermal stimulation of the left hindpaw ................................163

5-4 Skin temperature measurements from the plantar surface of non-stimulated paws
during and after thermal stimulation of the left hindpaw ................................164

5-5 Skin temperature measurements from the plantar surface of non-stimulated paws
during and after thermal stimulation of left hindpaw....................................... 165

5-6 Skin temperature measurements from the plantar surface of non-stimulated paws
during and after thermal stimulation of left hindpaw....................................... 166















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

STRESS-INDUCED CHANGES IN SENSITIVITY TO THERMAL NOCICEPTIVE
STIMULATION IN NORMAL RATS AND FOLLOWING EXCITOTOXIC SPINAL
CORD INJURY

By

Christopher Duncan King

August 2006

Chair: Robert Yezierski
Cochair: Charles Vierck
Major Department: Medical Science-Neuroscience

The sensation of pain is a complex experience that requires processing of

nociceptive stimulation by cortical structures. Various manipulations (including stress

and injury to the nervous system) influence activity in these structures and thus

influencing pain perception. To understand the effects of stress on nociceptive

sensitivity, behavioral responses of normal (injury naive) and spinal injured animals were

evaluated before and after a 15 minute exposure to restraint stress. Two types of

behavioral assessment strategies were used, including reflex (dependent on

spino-bulbo-spinal processing) and operant (dependent on cerebral processing) responses

to low-intensity thermal stimulation (44.0 to 44.50C) that activates C-nociceptors.

Excitotoxic spinal cord injury was accomplished by intraspinal injection of the

AMPA/metabotropic receptor agonist quisqualic acid (QUIS). Additional features of









stress-induced changes in nociception were also investigated, including the impact of

opioids and sympathetic-mediated thermoregulation of skin temperature.

Results suggest that restraint stress decreased thermal sensitivity of reflex

responses by activating an endogenous opioid system, supporting previous reports of

stress-induced hyporeflexia. Interestingly, low-dose morphine enhanced reflex lick/guard

responses and opposed inhibitory effects of restraint stress on reflexes, suggesting a

separate mechanism mediating these effects. In contrast, restraint stress increased

thermal sensitivity to heat in the operant escape and thermal preference tests, which was

opposed by tonic endogenous opioids and by exogenous opioid administration. Results

provide evidence for stress-induced hyperalgesia, which was not observed the following

day or during sessions at neutral temperatures (36.0C) suggesting that this effect is

specific to activation of C-nociceptors. Excitotoxic spinal cord injury also increased

thermal sensitivity to heat in some animals, which was enhanced by stress in subsequent

testing sessions.

In summary, results suggest that exposure to acute restraint stress has a differential

effect depending on the behavioral assessment strategy. Furthermore, stress was found to

enhance thermal hyperalgesia after excitotoxic injury. Finally, assessment of skin

temperatures during thermal stimulation showed an association between the regulation of

sympathetic vasoconstriction and enhanced sensitivity to heat on operant responses after

stress and excitotoxic injury.














CHAPTER 1
INTRODUCTION AND LITERATURE REVIEW

The purpose of my study is to advance our understanding of the behavioral and

pharmacological mechanisms responsible for modulating nociceptive responses after

acute stress. Pre-clinical and clinical studies of pain have described changes to the

psychological condition initiated by stressors that may lead to changes in nociceptive

sensitivity and precipitate psychopathologies. Psychological stressors are encountered on

a daily basis and appear to correlate with conditions of increased pain sensitivity in

individuals with a variety of pain conditions, including chronic pain syndromes such as

fibromyalgia, rheumatoid arthritis, and irritable bowel syndrome (Bennet et al., 1998;

Blackbum-Munro and Blackburn-Munro, 2001; Davis et al., 2001; Mayer et al., 2001).

Because these studies are limited, additional research is needed to understand the

negative effects of stress on acute and chronic pain conditions. However, pre-clinical

research has been hampered by poorly defined behavioral assessment strategies that focus

on reflex responses dependent on spinal and brainstem processing of painful information.

A well-defined, unambiguous animal model that demonstrates the stress-induced

enhancement of pain is therefore required. Fortunately, operant escape task, a recently

developed behavioral assay, can address these issues.

The Pain Experience

Based on the original hypothesis by Melzack and Casey (1968), the pain experience

may be thought of in terms of a sensory discriminative component in which precise

anatomical mapping of stimulus intensity, location, and modality are maintained. The









pain experience is also thought to have an affective motivational component (in which

pain perception is modulated by the concurrent overlay of an emotional component as

well as previously learned associations). This organization reflects the definition of the

International Association for the Study of Pain (IASP) that pain is "an unpleasant sensory

and emotional experience associated with actual or potential tissue damage" (Merskey

and Bogduck, 1994, page 210).

Although complex, it is heuristically useful to consider that pain is a valuable

response to potentially tissue damaging stimuli. Pain is detected in the periphery through

the activation of primary A6 or C-nociceptors, transmitted to the dorsal horn of the spinal

cord, and transmitted to supraspinal structures through ascending pathways. Painful

information is processed in the brainstem and cerebrum and results in the activation of

descending modulatory pathways that inhibit or facilitate pain transmission in the spinal

cord. Subsequent responses are organized through a complex interaction of

neuroanatomical structures. These mechanisms encompass primary afferent transduction

to spinal encoding, and finally supraspinal stimulus-response relationships. Within the

nervous system, numerous structures and pathways (e.g., ascending and descending)

transmit, process, and modulate information associated with the pain experience.

Ascending Pain Pathways

The complexity of processing sensory input in the spinal cord shows that it is a

critical conduit for transmitting sensory nociceptive information. Studies have shown

that nociceptive stimulation activates primary afferent nociceptors in the skin including

fast conducting small diameter myelinated A6 mechanoreceptors and slow conducting

unmyelinated C polymodal fibers (Fleischer et al., 1983; Willis and Westlund, 1997).









Depending on the nature of the stimulus, A6- and C-nociceptors are activated differently.

High and low intensity stimulation is required to excite A6- and C-nociceptors,

respectively (Yeomans et al., 1996).

The initial location of nociceptive information processing occurs in the dorsal horn

of the spinal cord. Nociceptive information is conveyed by afferent fibers that terminate

on second order neurons located in the dorsal horn (e.g., superficial laminae I/II; Todd et

al., 2000, 2002; Millan, 2002). Studies show the role of dorsal horn neurons in the rostral

transmission of nociceptive information and descending modulation (Millan, 2002, 2003;

Willis and Westlund, 1997). Several ascending nociceptive pathways have been

identified in conveying nociceptive information including spinothalamic,

spinomesencephalic, spinoreticular, spinocervical, and spinolimbic pathways (Burstein et

al., 1987, 1990; Burstein and Giesler, 1989; Willis and Westlund, 1997; Yezierski, 1988).

In addition to nociceptive transmission, dorsal horn neurons modulate other nociceptive

projection neurons and motor neurons (Willis and Westlund, 1997). Nociception does

not indicate pain perception (Le Bars et al., 2001; Vierck, 2006). Rather, pain perception

requires cerebral processing of the nociceptive stimulus (Mauderli et al., 2000; Vierck,

2006).

Nociceptive input is transmitted along ascending pathways to supraspinal

structures. Ascending pathways innervate brainstem (e.g., PAG, Bulbar reticular

formation) and cortical (e.g., thalamus, hypothalamus, amygdala) structures involved in

higher order processing of nociceptive information (Giesler et al., 1994; Price, 2000;

Willis and Westlund, 1997). These cortical systems are important for the affective

component of pain (Price, 2000). Spinothalamic tract (STT) cells are implicated in the









sensation of pain as a consequence of anterolateral cordotomies or spinal lesions (Price,

2000; Vierck and Light, 1999, 2002; Willis and Westlund, 1997; Yezierski, 1988).

Spinothalamic tract (STT) cells are also activated in responses to thermal stimulation

(Ferrington et al., 1987; Price et al., 1978).

Descending Pain Pathways

Previous studies show the presence of a complex endogenous inhibitory system that

modulates spinal circuitry involved in nociceptive processing. Several supraspinal

structures have been implicated in the modulation of spinal processing of nociception and

nociceptive behavior. Cortical structures implicated in modulation include the amygdala,

anterior cingulate cortex, insular cortex, and hypothalamus (Willis and Westlund, 1997;

Price, 2000). In addition, brainstem structures have been shown to impact nociception

including the locus ceruleus (LC), A7 catecholamine cell group, periaqueductal gray

(PAG), reticular formation, and rostroventromedial medulla (RVM; Mitchell et al., 1998;

Nuseir and Proudfit, 2000; Proudfit and Clark, 1991; Westlund and Coulter, 1980; Willis

and Westlund, 1997). In addition, ascending projections from neurons expressing NK-1R

influence the activation of descending pathways (Suzuki et al., 2002). This suggests that

nociceptive stimuli activate a spino-bulbo-spinal system in which ascending projections

provide afferent input to supraspinal loci. In turn, supraspinal neurons modulate spinal

activity by descending projections. In conditions of pain and stress, enhanced

nociceptive sensitivity most likely involves components of both ascending and

descending projections among spinal cord, brainstem, and cortical regions.

Activation of descending inhibitory pathways suppresses nociceptive reflex

responses, evidence for stimulation-induced hypoalgesia. An important bulbo-spinal

circuit mediating the expression of hypoalgesia includes the connection between the PAG









and RVM. Other pathways include connections between the PAG and LC (Willis and

Westlund, 1997). The PAG projects to nuclei of the RVM (Fields and Basbaum, 1999).

From the RVM, descending pathways project to the spinal cord via the dorsolateral

funiculus (DLF) and influence nociceptive neurons in the dorsal horn (Basbaum and

Fields, 1984; Millan, 2002). Activation of neurons in the PAG and RVM by electrical

stimulation or microinjections of opioids produces a decrease in the activity of

nociceptive neurons and nociceptive reflexes to thermal stimulation (Carstens et al.,

1979, 1980, 1981; Fields and Basbaum, 1999; Fields et al., 1988, 1991; Peng et al.,

1996). STT cells that are implicated in transmitting pain sensations are particularly

inhibited after stimulation of the PAG (Yezierski, et al., 1982).

Based on anatomical, electrophysiological and pharmacological evidence, RVM is

thought to have a substantial role in the modulation of nociceptive responses and

transmission of nociceptive input (Mason, 1999). In the RVM, cells have been

characterized as "OFF", "ON", and neutral cells based on responses to thermal

stimulation (Fields et al., 1991; Heinricher et al., 1989, 1997). The "OFF" cells are

tonically active and pause in firing immediately before tail withdrawal from a noxious

thermal stimulus and are thus thought to be involved in inhibition of spinal nociceptive

neurons. The "ON" cells accelerate firing immediately before the nociceptive reflex and

are directly inhibited by mu-opioid agonists; these cells are thought to produce

facilitation of spinal nociceptive neurons (Fields et al., 1983, 1991; Heinricher et al.,

1994; Urban and Gebhart, 1999). Both cell types project to dorsal horn (e.g., lamina I, II,

and V) to modulate nociceptive transmission and responses to thermal stimulation (Fields

et al., 1983; Morgan and Fields, 1994; Mitchell et al., 1998).









While traditional studies focused on descending inhibition from the RVM, several

studies show that descending pathways exert facilitatory influences on nociceptive

processing and responses through activity of RVM neurons in chronic pain states

(Porreca et al., 2001, 2002; Urban and Gebhart, 1999). Descending pathways appear to

facilitate nociception through activity of [t-opioid receptor expressing pronociceptive

"ON" cells (Ossipov et al., 2000; Pertovaara et al., 1996). These observations led to

hypothesis that spino-bulbo-spinal loop could contribute to the development and

maintenance of exaggerated pain behaviors produced by noxious and non-noxious stimuli

(Porreca et al., 2002; Urban and Gebhart, 1999). However, questions have been raised

concerning descending pathways in the cortical processing of pain because these studies

are based on reflex-mediated responses not dependent on cortical processing.

Descending systems are also involved in regulating other physiological functions

(autonomic, motor) especially to innocuous stimuli (Manson, 2005).

Descending bulbo-spinal pathways originating from the RVM are critical for

expression of exogenous opioid anti-nociception as assessed by reflex responses (Fields

et al., 1983; Gilbert and Franklin, 2002; Gebhart and Jones, 1988). Neurons responsible

for descending pathways display high levels of opioid receptors and peptide expression

(Marinelli et al., 2002). Microinjection of opioid agonists into discrete brainstem sites

(e.g. PAG and RVM) produces reduced activity of dorsal horn neurons and nociceptive

tail and hindpaw withdrawal responses to noxious stimulation (Jenson and Yaksh, 1986a,

1986b, 1986c; Jones and Gebhart, 1988; Yaksh, 1997, 1999; Yaksh et al., 1976).

Furthermore, the hyporeflexic effects of systemic opioids appear to activate descending

modulatory systems through these sites. For example, microinjections of opioid









antagonists into the PAG and RVM oppose the hypoalgesic effects of systemic morphine

(Manning and Franklin, 1998; Yaksh and Rudy, 1978).

In addition, neural pathways associated with stress-induced changes in nociceptive

reflexes include supraspinal neurons that exert a descending inhibitory effect on dorsal

horn neurons including descending pathways from the RVM. Activation of the RVM

during times of stress was shown to be critical for expression of morphine inhibition of

reflex responses. In stressed rats, the enhancement of morphine inhibition of reflex

responses was reduced by injections of lidocaine or muscimol into the RVM (Mitchell et

al., 1998).

Supraspinal structures mediate morphine-induced inhibition of reflex responses

through descending projections that are blocked by RVM and DLF lesions or inactivation

of the RVM by lidocaine (Abbott et al., 1996; Basbaum and Fields, 1984; Fields et al.,

1988, 1991; Gilbert and Franklin, 2002; Mitchell et al., 1998). The RVM is also

implicated in the expression of stress-induced antinociception. Inactivation of the RVM

by lidocaine attenuated reflexive behavior to heat in stressed rats (Mitchell et al., 1998).

Damage to descending pathways in the DLF also reduced the development of

stress-induced inhibition of reflex responses (Watkins and Mayer, 1982; Watkins et al.,

1982).

At the spinal level, bulbo-spinal terminals release several neurotransmitters that

modulate dorsal horn activity and nociceptive responses, including catecholamines, and

opioid peptides (Schmauss and Yaksh, 1984; Takano and Yaksh, 1992). Activation of

the bulbo-spinal descending inhibitory pathways are mimicked and enhanced by spinal

application of U2 and [t receptor agonists (Nuseir and Proudfit, 2000; Schmauss and









Yaksh, 1984; Takano and Yaksh, 1992). In contrast, the effects of activation of

bulbo-spinal projections are reversed by spinal application of U.2 (e.g., phenotlamine) and

[t (e.g., naloxone) receptor antagonists (Camarata and Yaksh, 1986; Yaksh, 1979; Yaksh

and Rudy, 1977).

Animal Models of Pain

The sensation of pain provides important information to an organism about its

internal and external environment in order to maintain homeostasis. In the presence of a

painful stimulus, various systems are activated to avoid the stimulus and limit damage

(Le Bars et al., 2001). Assessment of pain sensitivity in animal studies is inferred from a

variety of behavioral responses to nociceptive stimuli as illustrated in Figure 1-1 (adapted

from C. Vierck). These responses can be categorized hierarchically within the neuroaxis

including segmental reflexes, supraspinal reflexes, and learned escape responses (Le Bars

et al., 2001; Vierck, 2006).

As demonstrated in numerous studies, spinally mediated reflex responses are

demonstrated by simple limb or tail withdrawal from a nociceptive stimulus (Franklin

and Abbott, 1989). Reflex responses mediated by spino-bulbo-spinal circuitry are

revealed by more complex responses including licking, guarding, vocalization, and

jumping (Le Bars et al., 2001; Matthies and Franklin, 1992; Woolf, 1984). Finally,

learned escape responses requires cerebral processing of nociceptive information and

development of a proper strategy to terminate the stimulus (Mauderli et al., 2000). The

concept of learning is not included in most tests of nociception that utilize reflex assays

(Le Bars et al., 2001).











Cortex \ < Operant Escape
Operant Escape
............... ................. Thermal Preference

Brainstem. < Supraspinal Reflexes
Hindpaw Licking
Hindpaw Guarding





Spinal Cord c i Spinal Reflexes
Tail Withdrawal
Hindpaw Withdrawal






Figure 1-1. Hierarchical behavioral responses to nociceptive stimuli including spinal,
supraspinal, and cortical mediated responses.
Nociceptive Responses Mediated by Spinal and Spino-Bulbo-Spinal Processing
In pre-clinical models, evaluation of sensory processing has utilized reflex based

assessment strategies. These behavioral endpoints have been used to evaluate the

presence of pain and assess alterations in nociception by various experimental

manipulations. Segmentally organized spinal pathways regulate withdrawal of a rodent's

tail or hindpaw from a nociceptive stimulus. Furthermore, segmentally-mediated tail or

hindpaw withdrawal responses can be elicited in spinalized animals (Figure 1-1; Borszcz

et al., 1992; Franklin and Abbott, 1989; Kauppila et al., 1998). In addition,









bulbo-spinal-mediated responses including paw-licking and vocalization can be elicited

in decerebrate animals (Figure 1-1; Woolf, 1984; Matthies and Franklin, 1992). It is

important to note that a majority of animal studies utilize brief high intensity thermal

stimulation above 50.0C that activates mylinated A6 nociceptors (Yeomans et al., 1996).

Finally, reflex responses represent an important measure of nociception and can be

modulated by brainstem (e.g., LC, RVM) and cerebral structures (Le Bars et al., 2001;

Vierck, 2006).

Experimental manipulations such as stress and nerve injury can alter both types of

reflex responses. A heightened or diminished sensitivity to a noxious stimulus after an

experimental manipulation illustrates two concepts in the field of pain: hypereflexia and

hyporeflexia, respectively. For example, acute stress reduces nociceptive reflex

responses presumably by endogenous opioid mechanisms (Bodnar et al., 1978b; Gamaro

et al., 1998; Watkins and Mayer, 1982), and injury to the central nervous system

heightens sensitivity to thermal stimulation (Acosta Rua, 2003; Yezierski et al., 1998).

However, these end-points fail to account for the interactions between manipulations

(stress) and higher order functions that are responsible for the affective dimension of

pain.

Reflexive responses do not represent the conscious or clinical aspect of pain

perception, but rather spinally and supraspinally mediated nociceptive responses to

thermal stimulation (Mauderli et al., 2000; Le Bars et al., 2001). Studies utilizing

reflex-mediated responses assume that changes in reflex responses are a consequence of

altered sensory processing at different levels of the neuroaxis (Le Bars et al., 2001;

Vierck, 2006). However, these studies may fail to address other non-sensory factors









affected by an experimental manipulation including changes in motor output, posture,

motivation, attention, and cognition. Finally, pathways underlying reflex responses are

associated with other physiological functions unrelated to nociception (Mason, 2005; Le

Bars et al., 2001). Therefore, assessment of reflexive responses can lead to deceptive

conclusions about their importance in the overall sensation of pain.

Nociceptive Responses Mediated by Cerebral Processing

A main feature working against reflex based assessment strategies is that these

strategies do not take into account interactions between experimental manipulations and

higher cortical activity that are critical for the perception of nociceptive stimuli.

Therefore, in contrast to reflex responses, conscious and motivated responses to thermal

stimulation are believed to characterize clinically relevant aspects of nociceptive

perception dependent on higher order cerebral processing of nociceptive input.

Consequently, operant responses are absent in decerebrate animals as they are dependent

on cerebral processing of nociceptive input and environmental cues for the execution of

appropriate escape responses (Figure 1-1; Mauderli et al., 2000; Vierck, 2006; Vierck et

al., 2003, 2004).

Recently, an operant escape task was developed that evaluates thermal nociceptive

sensitivity in awake, unrestrained, and conscious rats (Mauderli et al., 2000). This test

overcomes the limitations inherent with reflex withdrawal responses and offers a strategy

to evaluate changes in the affective dimension of pain. The escape test provides an

opportunity to evaluate: a) the consequences of experimental manipulations on a

non-reflexive behavioral outcome measure; b) the mechanisms involved in hyporeflexia

(e.g., decrease in nociceptive sensitivity) and hyperaglesia (e.g., increase in nociceptive

sensitivity); and c) the effects of these manipulations on behaviors dependent on spinal









and brainstem or cerebral processing of nociceptive input. More importantly, an

opportunity exists to directly compare reflex and escape responses to similar levels of

thermal stimulation.

Differences between reflex lick/guard and operant escape responses have been

observed in several studies. Systemic injections of low dose morphine (0.5 tol.5 mg/kg)

attenuate escape responses (e.g., increase response latencies and decreased duration)

dependent on unmyelinated C-nociceptor activation (-44.00C; Cooper et al., 1986;

Vierck et al., 2002). By comparison, escape responses were not affected by morphine at

temperatures activating A6-nociceptors. In contrast, reflex responses were augmented

(e.g., decreased response latencies and increased duration) at the same temperature after

morphine administration. Typically, suppression of reflex response is reported after

injections of higher dose morphine (3 to 10 mg/kg; Holtman and Wala, 2005;

O'Callaghan and Holtzman, 1975). Reflex responses were more sensitive to the

hyporeflexic effects of morphine at temperatures lower than 50.0C (Holtman and Wala,

2005).

Based on these and other studies, the difference in sensitivity to morphine that

depends on the activation of A6 or C-nociceptors illustrates an importance of the rate of

cutaneous heating by a thermal stimulus. It has been shown that near threshold for

nociceptor activation occurs at temperatures ranging from 43.0 to 45.00C (Le Bars et al.,

2001; Treede, 1995; Vierck et al., 2000). This temperature range preferentially activates

C-nociceptors as a result of a slow rate of skin heating and is involved in human pain

sensations. C-nociceptors are essential for the affective sensation of a painful stimulus

and, therefore, important to the elicitation of operant escape responses (Cooper and









Vierck, 1986; Cooper et al., 1986; Vierck et al., 2000, 2004). In contrast, temperatures

above 45.0C produce a rapid rate of heating that activates A6- and C-nociceptors

(Cooper et al., 1986; Yeomans and Proudfit, 1996; Yeomans et al., 1996). Clearly, the

activation of C-nociceptors by gradual heating of the skin is important to overall pain

sensation. The ability of low dose morphine to selectively suppress nociception mediated

by C-nociceptors and not A6-nociceptors supports the idea that C-nociceptors are

activated by sustained low intensity thermal stimulation.

Other manipulations demonstrate a difference in reflex and operant responses to

thermal stimulation. Operant escape responses appear to be mediated in part by NK-1R

neurons. Vierck et al. (2003) demonstrated that lesioning of NK-1R neurons with

substance-P saporin reduced escape responses to low intensity thermal stimulation while

reflex responses were not affected. Also, Vierck et al. (2005) reported that chronic

constriction (CCI) of the sciatic nerve produced increase in cold sensitivity. Finally,

other operant test may offer unique opportunities to evaluated responses dependent on

cortical processing (Neubert et al., 2005, 2006).

Chronic Pain

While acute pain serves a protective function to the organism, chronic pain persists

beyond its intended purpose as a result of abnormal activity in the central nervous

system. In fact, chronic pain can last for a long period of time (> six months; Herr, 2004;

Willis, 2002). Chronic pain is characterized as spontaneous, stimulus-independent, or

evoked, stimulus dependent, pain sensations (Herr, 2004). Although the features of

spontaneous and evoked pain will not be discussed, it appears that several mechanisms

mediate these pain etiologies including sensitization (Willis, 2002; Willis and Westlund,









1997). Furthermore, a prominent feature of abnormal pain sensations is the presence of

either allodynia (e.g., enhanced response to normally non-painful stimulus) or

hyperalgesia (e.g., enhanced response to normally painful stimulus). Central pain

conditions are initiated by injury to the central nervous system without involvement of

peripheral nociceptors (Willis and Westlund, 1997). Central pain was defined by the

IASP as "pain initiated or caused by a primary lesion of dysfunction within the CNS"

(Merskey and Bogduk, 1994, page 211). In support of the definition, studies have shown

that lesions of the central nervous system (e.g., spinal cord, brainstem, and brain) may

result in central pain presumably through altered activity within nociceptive pathways. In

particular, central pain after spinal cord injury will be discussed.

Spinal Cord Injury Pain

Spinal cord injury (SCI) is a challenging healthcare problem in terms of

understanding the pathophysiology underlying the condition and treatment strategies.

Spinal cord injury pain can develop immediately or over a period of time

(Widertrom-Noga, 2002; Widertrom-Noga et al., 1999), and SCI pain can be either

spontaneous or evoked (Siddall et al., 2002; Vierck et al., 2000; Yezierski, 2002).

Several studies have reported between 60-90% of individuals with SCI experience pain

of some type (Beric, 1997; Bonica, 1991; Kennedy et al., 1997; Mariano, 1992; Siddall et

al., 2002; Widertrom-Noga et al., 1999). However, the development of chronic pain is

higher in individuals with partial interruption of gray and white matter (e.g., incomplete

SCI) compared to complete spinal injuries (Beric et al., 1988). In most cases, SCI pain is

a major obstacle and overshadows other physiological consequences (e.g., impairment of

motor functioning) based on the fact that SCI patients would forgo functional recovery

for pain relief (Finnerup et al., 2001; Nepomuceno et al., 1979; Yezierski, 1996). In









addition, subsequent treatments strategies to treat SCI pain are limited and mostly

ineffective (Davidoff et al., 1987; Yezierski, 1996).

Central pain after injury to the spinal cord is often characterized by abnormal

sensations located in dermatomes at or below the level of injury. An increase in

nociception in dermatomes or segments at or adjacent to the injury location is defined as

at-level pain (Siddall et al., 2002; Vierck et al., 2000). In contrast, below-level pain after

spinal cord injury is identified by an increase and spontaneous sensations in nociception

in dermatomes caudal to the injury location (Siddall et al., 2002; Vierck et al., 2000).

Another factor that distinguishes below-level pain is a delayed onset of weeks, months, or

years. Other sensations are also reported in individuals suffering with chronic pain

conditions. Abnormal sensations such as tingling, numbness, and itching are identified as

either dysesthesias or paraesthesias (Herr, 2004). Several potential mechanisms have

been hypothesized to mediate altered pain sensation after SCI (see blow). Some of these

conditions include: a) abnormal activity (e.g., hyperactivity) of neurons associated with

pain transmission in the spinal cord and loss of afferent input to rostral targets (e.g.,

deafferentation of thalamic and cortical areas), b) hypofunctioning of the endogenous

opioid system, c) hyperfunction of glutaminergic excitatory systems, and d) loss of

inhibitory mechanisms (Eide, 1998; Willis, 2002; Yezierski, 2002).

Mechanism of SCI Pain

Neuronal hyperexcitability after loss of inhibitory modulation, in areas above or

below the lesion site, may also influenced pain sensation (evoked and/or spontaneous) in

humans (Finnerup et al., 2003a, 2003b; Milhorat et al., 1996) and rodents (Vierck and

Light, 1999, 2000; Yezierski and Park, 1993; Yezierski et al., 1998). In individuals with

SCI, spinal and thalamic neurons show evidence of hyperexcitability that is characterized









as an abnormal increase in activity (resting and evoked; Lenz et al., 1987, 1994; Loeser

and Ward, 1967, 1968). Additionally, in animals SCI models, the presence of neuronal

hyperexcitability at spinal segments bordering the injury site is associated with at-level

pain (Christensen and Hulsebosch, 1997; Drew et al., 2001, 2004; Hao et al., 1992a;

Yezierski and Park, 1993). Using electrophysiological techniques, several studies have

shown that neurons within pain pathways display abnormal spontaneous activity,

expansion of receptive field, a diminish threshold for activation, an increased responses

to stimulation, and extended afterdischarge (Eide 1998). Pharmacological investigations

have demonstrated the role of neuronal hyperexcitability in altered pain sensitivity by

administration of lidocaine (Loubser and Donvan, 1991) or NMDA antagonists (Hao and

Xu, 1996; Hao et al., 1991b; Liu et al., 1997).

As mentioned previously, nociceptive information is conveyed from the spinal cord

to rostral targets via ascending pathways including the spinothalamic tract (Willis and

Westlund, 1997). Abnormal activity of STT pathway has traditionally been thought as a

critical feature of central pain after SCI. Studies have supported this hypothesis after

interruption of STT pathways (Vierck and Light, 1999, 2000) or lesioning of its rostral

sites in the cerebral cortex particularly in post-stroke pain (Anderson et al., 1995; Boivie,

1994; Boivie et al., 1989). Although involvement of the STT pathway is important to the

development of central pain, other factors appear to be equally important. In fact, some

studies have reported similar damage to STT pathways in SCI patients with and without

pain (Finnerup et al., 2003a, b). Using MRI methods, Finnerup et al. (2003a)

demonstrated that individuals with central below-level pain, compared to patients without

central pain, displayed similar damage to the STT pathways, but patients with pain had a









larger loss of gray matter. Damage to gray and white matter (e.g., interruption of the

spinothalamic pathway) in the spinal cord appears to be critical factors in the

development of below-level pain after SCI (Boivie et al., 1989; Vierck and Light, 1999).

Thus, in support of other clinical studies implicating damage to STT pathways as critical

factors in the development of central pain, damage to the spinal gray matter is also a

critical factor.

Because the STT is the major ascending pathway to supraspinal targets, rostral

sites, such as the thalamus and cerebral cortex, lose critical input if the STT is damaged

(Loeser and Ward, 1967, 1968). Lesions of the anterolateral spinal cord after cordotomy

produce spontaneous and evoked pain as a consequence of pathways originating from

gray matter (Vierck and Light, 1999, 2000). This evidence supports the suggestion that

central pain after interruption of the STT pathway is a consequence of deafferentation.

Altered activity patterns are detected in deafferentated nuclei targeted by the STT

pathways including the thalamus (Lenz et al., 1978, 1987; Weng et al., 2000) and

cerebral cortex (Lenz et al., 1987, 1994). In addition, anterolateral cordotomies disrupt

descending modulatory pathways, which also contributes to the enhancement of neuronal

excitability in areas bordering the lesion (Vierck and Light, 2000). From these studies, it

is clear that interruption of STT tract and changes to corresponding rostral targets are

important for central pain. However, other factors will determine the expression of

central pain including gray matter damage (Vierck and Light, 2000).

The endogenous opioid system is implicated in the pathophysiology of neuropathic

pain (Edie, 1998; Hao et al., 1998; Ossipov et al., 1997; Porrecca et al., 2001). Based on

several studies in rodents, the opioid system (e.g., PPD, PPE) is activated after injury in









spinal and supraspinal areas and appears to suppress abnormal pain sensation. But,

dysfunctions of the opioid system lead to development of hypersensitivity to thermal and

mechanical stimulation (Abraham et al., 2000, 2001; Xu et al., 1994). Finally, despite the

unidentified pathophysiological mechanisms underlying SCI pain, psychosocial factors

contribute this condition. Studies have identified a relationship between several

psychological factors and SCI pain including depression, anxiety, fatigue, and stress

(Kennedy et al., 1997; Mariano, 1992; Summers et al., 1991). Unfortunately, no

pre-clinical studies have examined the impact of stress on SCI pain.

Animal Models of SCI Pain

Several pre-clinical models of SCI are used to examine pathophysiological

mechanisms underlying alter sensitivity to nociceptive stimuli. Models such as

hemisection (Christensen et al., 1997), photochemical lesions (Hao et al., 1991a, 1991b,

1992a, 1992b), contusion (Drew et al., 2004), and anterior lateral spinal cordotomy

(Vierck and Light, 1999, 2000) have been employed to evaluate pathophysiological and

behavioral changes occurring after SCI. Although these models will not be discussed,

several reviews have compared and contrasted the models (Vierck et al., 2000).

While mechanisms underlying SCI pain are still unclear, evidence from

experimental studies have demonstrated a relationship between abnormal pain sensitivity

and several pathophysiological factors. Behaviorally, abnormal SCI pain in animals is

evaluated by the presence of at-level or below-level changes in sensitivity. A common

method to examine at-level pain sensations after SCI is assessing the presence of caudally

directed grooming in dermatomes adjacent to the injury level. In addition, changes in

nociceptive responses after SCI provide evidence for allodynia and hyperalgesia to









thermal and mechanical stimulation in dermatomes adjacent to or below the level of

injury.

Excitotoxic Model of SCI

Recent research into SCI has demonstrated that trauma to the spinal cord produces

damage to the gray matter through mechanisms of cell death. The release of excitatory

amino acids (EAA) is implicated in the development of damage after SCI (Choi and

Rothman, 1990). Subsequent release of glutamate after an insult activates AMPA and

NMDA receptors initiating an excitotoxic cascade, which leads to neuronal cell loss

within the gray matter of the dorsal horn (Berens et al., 2005; Gorman et al., 2001; Liu et

al., 1991; Yezierski, 2002). The excitotoxic effect of EAAs is a critical initiating event

for lesion progression and development of SCI pain. Furthermore, protection of neurons

from the excitotoxic effects of EAA release has been minimized by the administration of

NMDA and AMPA antagonists (Choi and Rothman, 1990; Liu et al., 1997) in addition to

other treatments (agmatine; Yu et al., 2000, 2003).

The excitotoxic model of spinal cord injury utilizes an intraspinal injection of

quisqualic acid (QUIS), an mGluR and ionotropic GluR agonist, to produce lesions of the

gray matter (Berens et al., 2005; Caudle et al., 2003; Gorman et al., 2001; Yezierski et al.,

1993, 1998). An important feature of the excitotoxic model is the occurrence of at-level

and below-level pain, which is associated with neuronal loss (Yezierski et al., 1993,

1998; Berens et al., 2005). After an injection of QUIS, expression of spontaneous

pain-like behaviors (e.g., overgrooming) is demonstrated at dermatomes corresponding to

spinal segments near the lesion site. More importantly, overgrooming was prominent

after sparing of the superficial dorsal horn (Berens et al., 2005; Yezierski et al., 1998).

Superficial dorsal horn neurons (e.g., lamina I) are implicated in chronic pain conditions









(Ikeda at al., 2003). These cells also participate in the expression of injury induced

overgrooming especially NK-1R expressing neurons (Khasabov et al., 2002). For

example, Yezierski et al. (2004) reported that elimination of NK-1R neurons with a

selective neurotoxin (e.g., substance-P saporin) reduced spontaneous pain-like behaviors

after excitotoxic injury. Similar strategies have been used to reduce nociceptive

responses to capsaicin (Mantyh et al., 1997) and nerve injury (Nichols et al., 2001).

By comparison, nociceptive responses to mechanical and thermal stimulation are

augmented particularly in dermatomes adjacent to and below the lesion epicenter

(Yezierski and Park, 1993; Yezierski et al., 1998). Evidence suggests a relationship

between the enhancement of nociceptive responses and hyperexcitability of neurons (e.g.,

increased spontaneous activity, increased response to stimulation) bordering the area of

neuronal loss (Yezierski and Park, 1993; Yezierski et al., 1998). Based on these and

other lesion studies, a critical component of below-level spinal cord injury pain appears

to be gray and white matter damage.

Additional factors impact the expression of heightened spontaneous and evoked

nociceptive responses. In particular, these factors include the longitudinal progression, or

the rostral-caudal distribution, of neuronal loss from the epicenter (-4.0 mm; Gorman et

al., 2001; Yezierski, 1998). Furthermore, areas remote to the lesion epicenter also

demonstrate changes after injury. Morrow et al., (2000) and Paulson et al. (2005)

measured regional cerebral blood flow (rCBF), which indicates levels of neuronal activity

in rodents. After excitotoxic injury, several supraspinal structures, which are targeted by

rostral projecting pathways, were activated including forebrain (e.g., somatosensory

cortex and thalamus). These areas are critical for the processing of pain and demonstrated









a remote effect of injury as a consequence of reorganization and/or deafferentation (Lenz

et al., 1991). Other factors have already discussed including genetic factors (Brewer et

al., 2001), sex hormones (Gorman et al., 2001), and endogenous opioid mechanisms

(Abraham et al., 2000, 2001) are important for the expression of pain-like behaviors after

excitotoxic injury.

Influence of Stress on Pain

Types of Stress

Stressors are characterized as either physical (systemic) or psychological

(processive) and appear to activate different neural pathways. Systemic stressors (e.g.,

illness) primarily activate brainstem structures to restore homeostasis. By contrast,

processive stressors (e.g. restraint) are processed by limbic structures and elicit emotional

responses. Limbic activation by stress acts through hypothalamic and brainstem systems

to initiate physiological and hormonal responses, and may modulate motor output

through higher cortical centers (Herman and Cullinan, 1997; Herman et al., 1996). For

example, the hypothalamus may not directly modulate a behavioral response, but rather

modulates sensory input and the organization of learned responses driving the behavior.

Biological responses to stress

Several lines of research have suggested the ability of stress to modulate sensory

perception in humans and reflex responses of laboratory animals. A stressor is defined as

either an internal or external stimulus that presents an actual or perceived threat to the

homeostasis of the organism (Herman and Cullinan, 1997). Exposure to a stressor

induces a wide variety of adaptive stress responses including immune, hormonal,

endocrine, physiological, and behavioral responses (Drolet et al., 2001; Herman and

Cullinam, 1997). Ultimately, the stress response permits an individual to cope with the









stressor and maintain homeostasis under normal conditions, but after nerve injury, studies

have suggested that stress can contribute to the development of psychopathologies and

maintain the cycle of chronic pain (Herman and Cullinan, 1997; Melzack, 1999).

Modulation of Nociceptive Responses by Stress

Several studies have demonstrated that acute exposure to psychological stressors

such as restraint produce attenuation of segmental and bulbo-spinal reflexive withdrawal

responses to high intensity thermal stimuli as measured by both tail-flick and hotplate

tests (Amir and Amit, 1978; Bodnar et al., 1978a, 1978b, 1978c, 1979; Calcagnetti and

Holtzman, 1992; Calcagnetti et al., 1990, 1992; Gamaro et al., 1998), an effect referred to

as stress-induced analgesia (SIA; Lewis et al., 1980). Reduction of reflexive responses to

nociceptive stimuli is an adaptive response to acute stress exposure in order to cope with

challenging situations. Transmitters regulating changes in nociceptive sensitivity on

reflexive responses by stress include the endogenous opioid (Lewis et al., 1980; Porro

and Carli, 1988), serotoninergic (Quintero et al, 2000), and noradrenergic systems

(Watkins and Mayer, 1982). Interestingly, chronic stress exposure can increase sensitivity

of reflex responses. For example, repeated exposure to an inescapable and uncontrollable

stressor appears to induce sensitization of sensory neurons in the spinal cord. In addition,

repeated exposure to cold-water swims produced a cutaneous thermal hyperalgesia as

measured by reflex latencies (Quintero et al., 2000). Likewise, daily exposures to

restraint stress over a forty-day period resulted in cutaneous thermal hyperalgesia as

assessed by tail flick responses (Gamaro et al., 1998).

Modulation of Nociceptive Responses by Stress: Pharmacology

The antinociceptive effects of endogenous opioids, which are released after stress

exposure, have been demonstrated after exposure to stressful stimuli. Opioid peptides are









derived from three separate precursor peptides and include enkephalin, endorphin, and

dynorphin (Drolet et al., 2001; Yamada and Nabesima, 1995). These peptides interact

with receptors distributed throughout the central and peripheral nervous system and are

capable of modulating nociceptive sensations during stressful and painful stimuli (Kelley,

1982; Yamada and Nabesima, 1995). Threats to homeostasis induce the release of

endogenous opioid peptides and are speculated to permit the organism to cope with the

stressful situation (Amit and Galina, 1988; Terman et al., 1984).

Activation of the endogenous opioid system has been shown to parallel the

induction of stress-induced hyporeflexia, or suppression of nocifensive reflex responses,

by various stressors (Bodnar et al., 1978a; Gamaro et al., 1998; Madden et al., 1977). For

example, a single exposure to foot-shock produced hyporeflexia, as measured by

increasing time to elicit tail-flick responses to thermal stimulation. Stress-induced

hyporeflexia also parallels increases in endogenous opioid levels in the central nervous

system (Madden et al., 1977). Involvement of the opioid system in stress-induced

changes in nocifensive responses was further characterized by: 1) naloxone, an opioid

antagonist, which reversed stress-induced hyporeflexia in rats; and, 2) cross-tolerance

between stress-induced hyporeflexia and morphine after repeated exposure to stress

(Bodnar et al., 1978b; Girardot and Holloway, 1984; Lewis et al., 1980).

Restraint stress

In relation to other stressful stimuli, restraint stress is considered to be a

psychological stressor and has been used to induce stress-induced hyporeflexia in rats

(Tusuda et al., 1989; Calcagnetti et al., 1992; Gamaro et al., 1998). For example,

Gamaro et al. (1998) demonstrated that exposure to a single session of restraint for one

hour produced stress-induced hyporeflexia as assessed by tail-flick assay in both male









and female rats. Studies have also demonstrated the role of endogenous opioids in

restraint-induced hyporeflexia on tail-flick and hindpaw withdrawal. The antinociceptive

effects of [t-opioid agonists on reflexes (e.g., morphine, DAMGO) were potentiated after

acute exposure to restraint stress (Abbelbaum and Holtzman, 1984; Abbelbaum and

Holtzman, 1985; Calcagnetti et al., 1990; Calcagnetti and Holtzman, 1992; Calcagnetti et

al., 1992). For example, restraint stress enhanced the hyporeflexive effects of opioids,

indicated by increases in reflexive withdrawal latencies to thermal stimulation after

systemic (Abbelbaum and Holtzman, 1984, 1985, 1986; Fleetwood and Holtzman 1989;

Calcagnetti and Holtzman, 1990, 1992), intrathecal (Calcagnetti et al., 1992), and

intracerebroventricular (Abbelbaum and Holtzman, 1985, 1986; Calcagnetti et al., 1990)

administration compared to unstressed controls. These studies also suggest that both

spinal and supraspinal opioid mechanisms contribute to stress-induced potentiation of

opioids after i.t. and i.c.v. opioids on reflexive tests of nociception.

Additional evidence for the involvement of endogenous opioids in stress-induced

hyporeflexia is observed after systemic injections of opioid antagonists. Administration

of naloxone reverses the hyporeflexic effects of stress (Pilcher and Browne, 1983).

Finally, evidence of endogenous opioids in stress-induced hyporeflexia is supported by

the development of cross-tolerance between stress-induced hyporeflexia and morphine

after repeated exposure to stress (e.g., habituation) or repeated exposure to morphine

(e.g., tolerance). For example, the potentiation of the inhibitory effects of opioids by

stress is reduced in habituated rats (Fleetwood and Holtzman, 1989) and

morphine-tolerant rats (Torres et al., 2003). It is clear from these studies that restraint

stress activates components of the endogenous opioid system and is involved in the









modulation of responses to nociceptive stimuli. While the impact of stress on nociceptive

reflex responses has been appreciated, no previous studies have examined the effects of

stress on operant responses.

Modulation of Nociceptive Responses by Stress: Chronic Pain

Stress is reported to increase nociceptive sensitivity in individuals with chronic

pain (Ditor et al., 2003; Galvin and Godfrey, 2001). This effect of stress is especially

significant, as stress has been linked to the onset and maintenance of numerous life

threatening medical conditions, including those that severely compromise ones quality of

life. Clinically, the presence of psychological stressors correlate with conditions of

increased sensitivity in individuals with a variety of defined pain conditions, including

fibromyalgia and those arising from nerve injuries (spinal cord injury). Furthermore,

acute stress has been shown to increase pain sensitivity in chronic pain patients, and has

been suggested to contribute to the development of chronic pain syndromes like

fibromyalgia, rheumatoid arthritis, and irritable bowel syndrome (Bennet et al., 1998;

Blackbum-Munro and Blackburn-Munro, 2001; Davis et al., 2001; Mayer et al., 2001).

Even though patients with conditions such as spinal cord injury develop chronic pain, the

effect of stress in clinical settings has not been adequately addressed. Likewise,

pre-clinical models of chronic pain have not addressed the impact of stress on altered

sensation after spinal injury.

Thermoregulation by Sympathetic Vasoconstriction

Numerous physiological mechanisms mediate behavioral responding to nociceptive

stimulation including the sympathetic component of the autonomic nervous system. The

autonomic system plays an essential function in mediating physiological responses to

internal or external stimuli (McDougall et al., 2005). It also is implicated in pain









perception and affective/motivational states (Thayer and Brosschot, 2005). The

regulation of heat (thermoregulation) is a consequence of sympathetic activity. Various

manipulations can alter sympathetic tone, ultimately affecting the distribution of body

heat and blood flow. For example, exposure to mental stress increases body temperature.

In response to increase body temperatures, sympathetic-mediated vasoconstriction

reduced peripheral temperature (cooling) by restricting blood flow (Cooke et al., 1990;

Larsson et al., 1995; Nicotra et al., 2005).

Sources of sympathetic regulation are localized in the intermediolateral column of

the thoracolumbar spinal cord (Hofstetter et al., 2005). Various neuroanatomical

structures are involved in regulating the outflow of sympathetic preganglionic neuronal

cell bodies including the hypothalamus, prefrontal cortex, amalgdala, and RVM

(Dampney, 1994; Korsak and Gilbey, 2004; McDougall and Widdop, 2005; Nalivaiko

and Blessing, 2001). Activation of the sympathetic nervous system is also accomplished

by the HPA axis after exposure to nociceptive stimulation (Janig, 1995; Magerl et al.,

1996) or stress (Herman and Cullinan, 1997; McDougall et al., 2005).

Activity of the sympathetic system can be evaluated indirectly by assessment of

peripheral vasoconstriction during thermal stimulation (Shimodoa et al., 1998; Vierck,

Unpublished Observations; Wakisaka et al., 1991; Willette et al., 1992). Overall,

nociceptive stimulation decreases skin temperature ipsilaterally and contralaterally in

non-stimulated areas. It appears that activation of nociceptors by stimulation triggers a

sympathetic response (Magerl et al., 1996). Manipulations have been shown to increase

sympathetic-mediated vasoconstriction, including stress (Larsson et al., 1995), peripheral

injury (CCI: Kurves et al., 1997; Wakisaka et al., 1991), and spinal injury (Acosta-Rau,









2003). Furthermore, a relationship exists between the ability to demonstrate

vasoconstriction and change in thermal sensitivity. If a manipulation blunts the

expression to vasoconstriction, it will also display an enhanced sensitivity to thermal

stimulation (e.g., increase escape response to heat). Reduction of vasoconstriction in

response to thermal stimulation has been demonstrated after formalin (C. Vierck and R.

Cannon, Unpublished Observations; C. Vierck and A. Light, Unpublished Observations)

and excitotoxic injury to gray matter (Acosta-Rua, 2003). Thus, enhanced nociceptive

responding is a consequence of peripheral and central injury that dramatically alters

ability of the sympathetic nervous system to regulated cutaneous temperatures via

vasoconstriction.

Summary

Efforts to study the effects of stress on sensory processing in pre-clinical models

have frequently sought to employ reflexive behaviors as endpoints for assessing

stress-induced alterations in nociception. It is clear that such reflex functions are

mediated by systems that respond to environmental cues and previous experience. These

end-points fail to account for the interactions between stress and higher order functions

initiated by a particular stimulus condition. In contrast, operant escape responses reflect

a higher order organizational function, presenting an approach by which we might

establish a clinically relevant model of motivated behavioral responses to nociceptive

stimuli that permits an evaluation of the affective component of pain. Though the

importance of stressors on higher order function has been long appreciated, there are few

studies that have examined the effects of stress on operant responses before and after

injury to the central nervous system. The ultimate goal of the present proposal is to






28


understand the apparent differential modulation of sensory processing by stress in normal

and after spinal cord injury on several tests of nociception.














CHAPTER 2
EXPERIMENTAL METHODS AND DESIGN

The goal of this research is to increase our understanding of the effects of stress on

sensory processing. In order to accomplish this goal, behavioral and physiological

techniques were used to assess changes in nociceptive sensitivity in injury-naive and

spinally injured rats. Each behavioral testing session consisted of 2 consecutive testing

trials in separate apparatuses that were constructed of plexiglass. Animals were exposed

to a neutral temperature during the first trial (e.g., pre-test), which was used to normalize

temperatures of the rodent's hindpaw and acclimate the animal to the apparatus. Thermal

stimulation was delivered through a heated or cooled aluminum plate. During succeeding

testing trials (e.g., test), animals were exposed to a range of non-nociceptive and

nociceptive temperatures. The responses collected during the second trial were recorded

through customized computer software. Assessment of nociceptive responses was

accomplished by comparing reflex lick/guard, operant escape, and thermal preference

responses before and after exposure to restraint stress and following spinal injury.

In order to produce stress-induced changes in nociception, restraint stress, a

psychological stressor, was selected based on an extensive literature demonstrating that

restraint activates limbic circuits and affects reflex responses to thermal stimulation.

Restraint stress is a useful and convenient stressor, which can be delivered without

difficulty, and does not present a direct thermoregulatory challenge to the animal

compared to other stressors (e.g., cold water swim). Likewise, several studies have

concluded that the underlying mechanisms mediating restraint-induced changes in









nociception is a result of activation of the endogenous opioid system. Based on these

studies, pharmacological agents (e.g., naloxone and morphine) were used to determine

the effect of the endogenous and exogenous opioids on stress-induced changes in

nociception.

Furthermore, a common condition confronting individuals with spinal cord injury is

chronic pain. In order to study the pathophysiology underlying SCI pain, the excitotoxic

model of SCI that was developed by Dr. Yezierski shares similar pathophysiological

consequences common after traumatic and ischemic SCI. This model provides an

excellent platform to study altered pain processing after spinal gray matter damage.

Behavioral manifestations of altered nociception after excitotoxic injury include

spontaneous (e.g., at level grooming) and evoked (mechanical allodynia and thermal

hyperalgesia) pain sensations (Gorman et al., 2001; Yezierski et al., 1998). Finally,

experimental manipulations (e.g., stress and spinal injury) affect pain sensations by

various mechanisms including modulation of cutaneous skin temperature by the

autonomic nervous system.

Experimental Animals

Female Long Evans rats were housed in pairs and maintained on a 12-12 hour

light-dark cycle with free access to food and water. The reasons for using female rats for

behavioral testing were based on observations that females were easier to handle, less

aggressive, and maintained their body weight over time. Also, chronic pain is more

common in females compared to males. The rats were adapted to the testing apparatus

and handled prior to behavioral training and baseline testing. All experiments were

carried out according to the Guide for the Care and Use of Laboratory Animals and were









approved by the Institutional Animal Care and Use Committee (IACUC) at the

University of Florida (B193 and C013).

Behavioral Testing Procedures

Assessment of Reflex Lick-Guard Responses

Reflex responses represent a supraspinally-mediated behavior. Lick responses

were recognized as stereotyped lifting of one hindlimb, then holding and licking the

hindpaw. Guard responses were scored when a hindlimb was raised from the platform

and flexed in an exaggerated fashion. Guard responses were longer in duration than limb

flexion that occurred during ambulation. Hindlimb reflex responses were measured

during the second trial including frequency (number of responses during a trial), duration

(total time spent licking or guarding during a trial), and latency to first lick-guard

response.

Reflex apparatus

The apparatus used to evaluate lick-guard responses consisted of the reflex

apparatus consists of a plexiglass box with a thermally regulated floor without an escape

option (Figure 2-1). The enclosure was ventilated to permit airflow. Although no

training is required for reflex responding, rats were familiarized with the apparatus and

the testing procedure over 2-week period. Rats, which are not properly adapted to the

testing environment, display stress-induced hypoalgesia due to the novel environment

(Plone et al, 1996).

Two consecutive trials were used to assess reflex responses. Similar to escape

testing, a 15 minute trial at 36.00C (pre-test trial) was used to standardize foot

temperatures, which was followed by a second 10 minute trial at 44.50C (test trial).















































Heat Plate
No Escape Platform


Figure 2-1. Reflex apparatus.









Because the animal cannot escape thermal stimulation, trial durations of 10

minutes were selected to prevent tissue damage. Reflex responses were exhibited during

thermal stimulation at 44.00C but not at 36.00C.

Assessment of Operant Thermal Escape Responses

Operant escape apparatus

The escape apparatus incorporates a shuttle-box design, as described previously

(Figure 2-2; Mauderli et al., 2000; Vierck et al., 2002). The escape test was carried out in

a plexiglass box divided into two compartments by a hanging wall with an opening to

permit rats to move freely between the compartments. The first compartment is dimly

illuminated (0.5 foot candles) and includes a thermally regulated floor, which can deliver

either non-nociceptive or nociceptive stimuli (43.0 to 47.00C) to the paws during

occupancy. Thermal stimulation was delivered by an aluminum plate regulated by a

water bath (Neslab). The adjacent compartment contains a brightly illuminated (35-watt)

halogen bulb above a thermally neutral escape platform. The platform provides animals

an opportunity to escape nociceptive thermal stimulation. The dual compartment set-up

provides a conflict between aversion to light and thermal nociception. As a consequence,

rats will proportion their time on the platform in relation to the intensity of stimulation.

Operant escape training and assessment

Rats were trained over 3 weeks to learn to escape from thermal stimulation by

climbing onto the neutral escape platform. During the training period, rats were

familiarized with the testing procedure and trained to discriminate between gradually

increasing floor temperatures (36.0, 40.0, 42.0, 44.0, 45.0, and 47.00C) in the absence

(first phase) and presence of bright light over the escape platform (second phase).




























U


/ \


C-


Heat Plate

Figure 2-2. Operant escape apparatus.


Escape Platform


,-2, -









Each training session consisted of two consecutive 15 minute trials. The first trial

consisted of pre-test condition at 36.00C, and the second trial consisted of a range of

gradually increasing temperatures over successive daily sessions. The pre-test was used

to standardize foot temperatures prior to testing, acclimate the rats to the apparatus, and

extinguish avoidance behavior (e.g., occupancy of the escape platform unrelated to floor

temperature).

After operant training, baseline escape responses were assessed over a 6 week

period. Similar to training, rats were tested daily with two consecutive 15 minute trials at

36.0C (pre-test) and then at 36.0, 44.0, or 44.50C (test trial). Escape responses during

the second (test) trial were assessed including frequency (number of responses during a

trial), duration (total time occupying escape platform during a trial), and latency to first

escape response.

Assessment of Operant Thermal Preference Responses

An additional operant assessment strategy included the thermal preference test

(Mauderli et al., 2000; Vierck et al., 2002). This test can determine if an experimental

manipulation (stress or injury) selectively affects cold (first compartment) or heat

(second compartment) nociception. Preference of a thermal modality (cold or heat) will

depend on the temperatures and experimental manipulation used. For example, if a

manipulation affects sensitivity to heat nociception, the animal will spend less time on the

heated compartment and more time on the cold compartment. In cases when both

modalities are affected, animals will increase their preference for a modality less affected

by the manipulation









Thermal preference apparatus

Similar to the operant escape test, thermal preference apparatus (Figure 2-3) uses a

shuttle-box design that requires an animal to choose between two distinct compartments.

However, unlike the operant escape test with one thermally regulated floor, both floors of

the thermal preference are thermally regulated at different temperatures. The first and

second compartments presented cold (0.3, 10.0, 15.0, or 36.00C.) and heat (43.0, 44.0,

44.5, 45.0, 46.0 or 47.00C.) nociceptive temperatures, respectively. In addition, the

preference test was preceded by a pre-test at 36.00C to standardize foot temperatures

prior to placement into the testing apparatus.

Thermal preference training and assessment

Following one week of preference training, baseline responses were assessed over a

2 month depending on the stability of operant behavioral responses. It is important to

note that only one cold and hot temperature, which are listed above, were used during a

single testing session (e.g., 15.0 paired with 45.00C.). The duration of a single thermal

preference was 12 minutes to avoid tissue damage. Thermal preference responses were

assessed by frequency (number of crossing during a trial), duration (total time spent

occupying the escape platform during a trial), and latency to first thermal preference

response.

Assessment of Darkbox Responses

The darkbox test was used to assess motivation to escape the light and to evaluate

whether motor deficits (e.g., freezing behavior) were induced by experimental

manipulations like restraint stress. The apparatus consisted of two compartments with a

212 by 212 inch opening in the dividing wall.








































Compartment 1 Compartment 2
Cold Plate Hot Plate
Figure 2-3. Thermal preference apparatus.









Each testing session began with ten-seconds of acclimation in which a computer

identified the location of the rat by weight. Then, a 70-sec trial was initiated with

presentation of light in both compartments. When the rat moved from the compartment

it occupied at the start of the session to the adjacent compartment, the light was

extinguished in the selected compartment for the remainder of the 70 second trial. At the

end of the trial, both compartments were lit to initiate the next trial. Darkbox latency was

defined as the time required for the rat to move to the adjacent compartment. Each

session consisted of seven light escape trials over fifteen minutes. During stress testing,

each rat was placed in the darkbox apparatus 15 minutes after termination of the stress

exposure.

Assessment of Open Field Responses

The modified open field test consisted of a 90 cm x 90 cm square black Plexiglas

container with an adjacent 20 cm x 20 cm start-box which allowed the animal to either

remain in the start-box or enter into the open field (Figure 2-4; picture provided by Dr.

Darragh Devine). A light fixture illuminated the open field about (5 to 150 Lux). A door

separated the two boxes, which was opened via a rope and pulley system. Upon opening

of the door, the rope was secured with a hook until the next trial. A camera, located

above the box, recorded the animal's behavior. The trial duration for the open field was 5

minutes. After exposure to the field, the rat was returned to its homecage. The open field

assesses anxiety-like responses in rats during exposure to a novel environment.

Restraint Stress Procedures

After stabilization of behavioral responses, rats were selected to receive an acute

exposure to restraint stress (stress condition) or remained in their home cage until

behavioral testing (control condition). Rats were removed from their home cages, and









rats were restrained for 15-minutes. The restraint tube (Figure 2-5; D. Devine, Personal

Communications) is composed of a soft flexible sheet of plastic 11" X 7 34" mounted to a

rigid plexiglas cradle (8 1/2" X 3' X 3") by means of two small bolts with convex heads.

There are ventilation holes at one end to allow unrestricted breathing, and the other end

has a vertical slot to allow comfortable placement of the tail during the restraint process.

The plastic sheet is then gently rolled around the animal and held securely in place with

two 12' X 1" Velcro strips.

Groups either remained in their home cage until testing or received a 15 minute

exposure to restraint stress (Figure 2-6). Then, each rat was removed from the restraint

tube and placed in the pre-test apparatus at 36.00C for 15 minutes. Rats were then placed

in the adjacent test apparatus at 36.00C (thermally neutral control temperature) or 44.5C

(testing temperature) for an additional 10 to 15 minutes depending on the behavioral test.

Control rats followed the same protocol and did not receive stress on the day of testing.

Both groups of rats remained in their home cages in a separate room until stress exposure

or behavioral testing was complete. All temperatures were held constant over the two

days of testing.

On successive testing weeks, exposure to restraint stress was switched to the group

previously in the control condition. For example, group 1 received restraint stress while

group 2 was not be exposed to stress, serving as a control. The following week group 2

was exposed to restraint stress and group 1 served as a control. The experiment was

designed to expose animals to stress every two weeks with the aim of 1) avoiding

adaptation and 2) using each animal as their own control. This testing protocol has been

shown not to cause carry-over effects of stress (King et al., 2003).











/~
id


Figure 2-4. Open filed apparatus.
Figure 2-4. Open filed apparatus.



































Figure 2-5. Restraint tube.


Operant Escape

Restraint Pre-test Test

Home Cage Pre-test Test
51 10 15 201 25 30 351 40 45


Reflex Lick/Guard


Restraint Pre-test Test

Home Cage Pre-test Test
51 101 15 201 25 30 351 40


S' I


Figure 2-6. Behavioral testing sequence, stress exposure, and injection schedule for
evaluation of operant and reflex lick/guard responses.









Drug Administration

In a separate group of animals, behavioral responses in normal animals were

evaluated after an injection of naloxone and morphine. After stabilization of baseline

responses, rats were randomly assigned to receive either stress or no stress before

behavioral testing. After 15 minutes of restraint stress, rats were immediately injected

with either an injection of morphine (opioid agonist, 1 mg/kg, i.p.), naloxone (opioid

antagonist, 3 mg/kg, i.p.), or saline (1 mg/kg, i.p.). Then, rats were placed into the

pre-test apparatus at 36.00C for an additional 15 minutes. Behavioral responses were

recorded during the second trial at 44.50C. Control subjects remained in their home cage

separate from stress subjects. Control subjects were injected and tested similarly to stress

subjects, but control subjects did not receive stress on the day of testing.

Surgical Procedures

In this study, the effects of stress on operant responses were assessed after

excitotoxic lesioning of mid-thorasic (Ts) or lumbar (L2) segments of the spinal cord.

Rats received an intraspinal injection of quisqualic acid (QUIS), which is a non-NMDA

receptor agonist that interacts with AMPA and mGlu receptors. Previous studies have

shown that nociceptive responses are enhanced after an intraspinal injection (Acosta-Rua,

2003; Gorman et al., 2001; Yezierski et al., 1998). The escape and thermal preference

responses were recorded before and after surgery. Excitotoxic lesioning of the spinal

cord was conducted after several weeks of baseline testing. Behavioral testing was

resumed 2 weeks after surgery. At 8 weeks post-op, rats were removed from their home

cages and placed in a restraint tube for 15 minutes. After stress exposure, rats were

placed in the pre-exposure testing apparatus at 36.00C. After fifteen minutes of









pre-exposure, rats were placed in the adjacent testing apparatus at 44.50C. Both control

animals and animals waiting to receive stress were kept separate from animals

undergoing stress.

Intraspinal Injection of Quisqualic Acid (QUIS)

As mentioned previously, animals underwent excitotoxic injury as mentioned in

other studies (Gorman et al., 2001; Yezierski et al., 1998). Rats were anesthetized with a

combination of ketamine (3 ml), acepromazine (1 ml), and xylazine (1 ml) at 0.65 ml/kg

administered subcutaneously. Level of anesthesia was evaluated by noxious pinch of the

hindpaw. Body temperature was maintained at normal levels (36.50C) during QUIS

surgery and post-operative period. Several pathological features occur after QUIS

injections including neuronal loss, cavitations, demylination, and alteration of glia

(Yezierski et al., 1993, 1998; Berens et al., 2005). Following injections muscles were

closed in layers, the skin closed with wound clips, and animals returned to their home

cages.

Intraspinal Injection Procedures

After placement into a sterotaxic frame, the dorsal surface of the spinal cord was

exposed via laminectomy corresponding between spinal segments T8 to L2. After

removal of the dura and pia matter, QUIS was injected bilaterally into the spinal cord to

target mid-thoracic and upper lumber spinal cord segments. Glass micropipettes (tip

diameter 5 to 10lm) attached to a Hamilton microliter syringe (volume 5 1) are used for

injections. The syringe was mounted on a microinjector attached to a micromanipulator.

Injections were made between the dorsal vein and dorsal root entry zone at depths

ranging from 500 to 1200 pm below the surface of the cord. To avoid white matter









damage, all intraspinal injections were placed in the middle of the gray matter

(lumbosacral cord: Ts-L2). Stock solutions of 125 mM QUIS (Sigma) was made fresh

daily using sterile saline and buffered to physiological pH as needed. At each injection

site 0.1-0.6 il of QUIS was injected (over a 60 second time interval). The total volume of

QUIS injected/animal was 1.2 to 1.5 il per side. The standard injection consisted of three

bi-lateral injection tracks separated by 0.5 mm.

In Vitro MRI Analysis of Spinal Cord

At the end of the study, animals were injected with sodium pentobarbital (1 ml,

i.p.) and underwent transcardial perfusion with PBS followed by 10% formalin in PBS

(Fischer Scientific). Spinal cord segments containing QUIS lesions were removed and

placed into an MRI tube. All excised cords were subjected to in vitro three-dimensional

MR microscopy (3D MRM). Images were acquired with a three-dimensional (3D)

gradient echo pulse sequence using a TR = 150 msecs, TE = 10 msecs with NA = 2. The

image FOV was 2 cm x 0.5 cm x 0.5 cm in a matrix of 512 x 128 x 128 in a total data

acquisition time of 1.5 hours. Therefore, MR images were acquired with a resolution of

-40 microns x 40 microns x 40 microns. A 3D Fourier transformation was applied to the

acquired data matrix to produce the 3D image. General image processing and analysis

was performed using custom software written in the Interactive Data Language (IDL,

from Research Systems, Boulder, CO).

Assessment of Core and Cutaneous Temperature

The effects of experimental manipulations could be a consequence of altered

temperature regulation (core and cutaneous). In order to evaluate the impact of stress and

spinal injury on thermoregulation, core and cutaneous temperatures were evaluated









before and after exposure to restraint stress. In addition, temperatures were also

evaluated before and after excitotoxic spinal injury.

Core Temperature

An implantable thermal probe (IPTT-300; BMDS, Delaware) recorded core body

temperature. After induction of anesthesia with isoflurane, the injection site for the probe

was prepared by removing the hair on the animal's back followed by aseptic preparation

of the site with alcohol and iodine wipes. The probe was injected subcutaneously with a

specialized injector. Temperatures were recorded by portable reader (BMDS, Delaware)

without any restraint of the rat. Temperatures were recorded before and after exposure to

the first testing apparatus. Data are expressed in degrees Celsius.

Autonomic-Mediated Skin Temperatures

The autonomic nervous system (ANS) regulates skin temperature by changes in

vasoconstriction through activity of the sympathetic nervous system. Experimental

conditions, including stress, pain, and injury, may activate and potentially alter the ANS.

In order to determine if these manipulations could modulate an animal's autonomic

response to thermal stimulation, skin temperatures were recorded in the absence (resting

conditions) and presence of heat stimulation (44.50C; Figure 2-7). In order to assess skin

temperatures, all animals were injected with diazepam (10 mg/kg, i.p.). Previous

research has demonstrated that isoflurane negatively affected autonomic activity, which

is counteracted by diazepam (C. Vierck, Personal Communication). 1% Isoflurane is

used to induce (5%) and maintain (1%) anesthesia. A thermal heating blanket is used to

maintain normal body temperature (36.0C). Several sites were monitored to changes in

temperatures including rectal core temperature, both forepaws, and both hindpaws. For









each paw, a thermocouple was applied to the skin with an adhesive foam pad. For the

right forepaw, left forepaw, and right hindpaw, thermocouples session were applied to the

plantar surface, and a thermocouple was applied to the top of the left hindpaw (stimulated

paw). After stabilization of paw temperatures, a pre-heated thermode (44.50C.) was

applied against the left hindpaw for 10 minutes. Cutaneous temperature of each

(non-stimulated) paw was recorded for 20 minutes during (10 minutes) and after

stimulation (10 minutes; resting period) to permit skin and core temperatures to return to

baseline.

For each manipulation, pre-stress or pre-operative skin temperatures were collected

several weeks prior to restraint or excitotoxic injury. For the stress condition, animals

were stressed for 15 minutes followed by induction of anesthesia with isoflurane. Skin

temperature was assessed 15 minutes after the termination of restraint, which permitted

stabilization of skin temperatures before testing and corresponding to 30 minutes after the

onset of stress.

Temporal Profile of Skin Temperature: Effects of Restraint

Stress condition

In order to evaluate the effects of a pre-exposure to 36.00C (pre-test) on skin

temperature after restraint stress, a thermocouple was secured to the left and right

hindpaw (plantar surface) to the skin with an adhesive foam pad. Temperatures were

recorded over a 30 second period. Then, animals were placed into a restraint tube for 15

minute. Skin temperature was continuously recorded during the entire trial. Animals

were removed from the tube at the end of the restraint period. Following removal of the

thermocouples, animals were placed into a 36.00C pre-test for 15 minutes. Then,









thermocouples were reattached to both hindpaws for 1 minute. Core temperature was

also recorded before restraint and at 5 minute intervals thereafter. The sequence of

events paralleled the testing conditions for operant and reflex testing.


Figure 2-7. Skin temperature recording in anesthetized rats.









Control conditions

In addition, non-stressed control animals were tested before and after exposure to

36.00C. Skin temperature was recorded for 1 minute prior to placement into the pre-test

and then removed. After a 15 minute pre-test trial, animals were removed and

thermocouples were reaffixed to both hindpaws.

Statistical Analysis

The frequency, latency, and duration of behavioral responses (escape, thermal

preference, and licking/guarding, as appropriate) were collected by custom software

(EVENTLOG, Autorat, Robot). The data are expressed in seconds, and values were

represented as absolute group means S.E.M. Statistical analysis of behavioral

responses between groups was performed by t-tests. Analysis of behavioral responses

between groups was performed by an one-way analysis of variance (ANOVA) or

two-way analysis of variance (ANOVA) with or without repeated measures followed by

Newman-Keuls post-tests. P-values less than 0.05 were considered significant. Analysis

was performed using GraphPad Prism version 4.00 for Windows, GraphPad Software,

San Diego California USA (www.graphpad.com). Analysis of Covariate and correlations

were performed by SSPS.

Study Design

The current experiments were based on previous literature and studies conducted in

the labs of Drs. Vierck and Yezierski suggesting that nociceptive responses are mediated

through different neuroanatomical pathways. Previous research has suggested that

stressors inhibit reflex mediated nociceptive responses particularly to high intensity

thermal stimulation. Inhibition of reflex responses has supported evidence for

stress-induced analgesia (SIA). For example, acute exposure to restraint stress has been









shown to increase segmental (tail withdrawal) and spino-bulbo-spinal reflexes (hindpaw

withdrawal or licking) to thermal stimulation. However, despite limit anecdotal and

clinical evidence that stress enhances pain sensations, the effects of stress on thermal

sensitivity in rats have not been examined by operant tests of nociception. Because pain

is a complex sensation, it requires cortical structures to process and elicit appropriate

actions. Thus, in order to study pain, proper behavioral strategies are required to examine

these structures. Thus, the current study will determine effects of acute restraint stress on

reflex lick/guard responses (a spino-bulbo-spinally mediated behavior) and operant

escape responses (a cerebrally mediated behavior) in rats. It is hypothesized that an

exposure to acute restraint stress produces a differential effect on reflex lick/guard and

operant escape responses evoked by thermal stimulation.

In order to characterize the effects of stress on behavioral responses, the impact of

various pharmacological agents and temporal profile were evaluated. First, the

contribution of endogenous opioids to stress-induced changes in lick/guard and operant

escape responses to thermal stimulation were evaluated by systemic administration of an

opioid receptor agonist (morphine) or antagonist (naloxone) before behavioral testing.

Previous studies have implicated endogenous opioid peptides as mediators of

stress-induced hyporeflexia, as shown by the effects of either opioid agonists or

antagonists on reflexive tests of nociception (Calcagnetti and Holtzman, 1992;

Calcagnetti et al., 1990). Furthermore, stress has been shown to increase the release of

endogenous opioids and modulate the physiological and psychological response to

stressful and painful stimulation (Drolet et al., 2000; Madden et al., 1978). The effects of

endogenous opioid peptides, however, after stress on responses dependent on cerebral









processing have not been examined. The inhibitory effects of stress on reflexive

responses are modulated by the endogenous opioid system, but the system could oppose

the facilitatory effects of stress on operant responses. It is hypothesized that the

endogenous opioid system contributes to stress-induced reduction of spino-bulbo-spinal

reflexes while opposing the excitatory effects of stress on cerebrally mediated operant

escape responses to thermal stimulation.

Second, the temporal profile of stress on operant responses was assessed 15

minutes, 30 minutes, and 24 hours after the onset of stress. Previous stress literature has

pointed out that magnitude of altered nociceptive responses is dependent on the duration

and intensity of the stressor. Typically, long exposure to a stressor or a single exposure

an intense stressor resulted in extended behavioral or physiological responses. Because

the current stressor was only 15 minutes, it was hypothesized that the effects of restraint

stress on operant responses would gradually disappear across time.

In addition, the effects of stress were examined on a well-established model of

spinal cord injury (SCI), which results in enhanced expression of below-level behaviors.

Stress triggers changes in several important physiological systems including the

autonomic nervous system (e.g., sympathetic-adrenal system) and the

hypothalamic-pituitary-adrenal axis (HPA axis). Clinically, psychological stress is

associated with the progression and maintenance of several chronic pain conditions

including fibromyalgia, irritable bowel syndrome, nerve injury, and rheumatoid arthritis.

Likewise, physiological systems are altered in conditions of chronic pain.

In light of these clinical observations, only a limited number of studies have

examined the effects of stress on altered nociceptive responses in chronic pain models,









particularly models of spinal cord injury. The current aim will use an excitotoxic model

of SCI. In this model, animals underwent a bilateral injection of the AMPA/metabotropic

receptor agonist quisqualic acid (QUIS) into the spinal cord. Operant responses were

assessed before surgery (pre-op), after surgery (post-op), and after an exposure to

restraint stress. It was hypothesized that excitotoxic injury of the spinal cord would

produce an increase in thermal sensitivity and subsequent exposure to acute restraint

stress will enhance injury-induced operant escape responses. Finally, the study examined

a potential mechanism (thermoregulation) mediating altered thermal sensitivity by stress

and excitotoxic spinal injury.














CHAPTER 3
EFFECTS OF RESTRAINT STRESS ON NOCICEPTIVE RESPONSES IN NORMAL
SUBJECTS

Various experimental manipulations can influence processing of input from

nociceptive afferents. Exposure to a stressor has been associated with both suppression

(Abbelbaum and Holtzman, 1984; Borszcz et al., 1992; Gamaro et al., 1998) and

enhancement (Borszcz et al., 1992; Huang and Shyu, 1987; Illich et al., 1995; King et al.,

1996, 1999) of pain sensations. Based on several pre-clinical studies, stress inhibits

reflex mediated responses (withdrawal of the tail or hindpaw) to thermal stimulation.

However, some questions have been raised pertaining to relevance of reflex responses in

the perception of pain, which is dependent on higher cortical processing. An important

question can be raised concerning the effect of stress on pain sensations. Does stress

affect responses dependent on operant processing of nociceptive information differently

than reflex responses? Or does stress suppress reflex and operant responses similarly? In

this chapter, the effects of restraint stress on reflex and operant (escape and thermal

preference) responses to low intensity thermal stimulation, which activates C-nociceptor

afferents by heat (44.0 to 44.50C), were examined.

To control for confounding effects of stress (e.g., avoidance), control tests were

also examined including operant escape responses at a neutral temperature (36.0C) and

darkbox responses. In order to further characterize the effects of stress on nociception,

pharmacological conditions, which were based on previous research using reflex-based

behavioral responses, were also investigated. Naloxone or morphine was administered to









determine the contribution of endogenous and exogenous opioids on normal and

stress-induced changes in thermal sensitivity, respectively. Due to study limitations,

opioid pharmacology was limited to reflex lick-guard and operant escape.

Effects of Restraint Stress on Reflex Lick/Guard Responses at 44.00C

Behavioral responses of female rats (n=l 1) were assessed during a 3-day period,

with baseline testing on Day 1 (baseline), post-stress or control testing on Day 2 (15

minutes), and evaluation of long-term stress effects Day 3 (24 hours). On Day 2, half the

animals received 15 minutes of restraint stress, followed by a 15 minute pre-test and test

trials as shown in Figure 3-1. Testing sessions included a 15 minute pre-test exposure to

36.0C, followed by a test trial at 44.00C. Reflex (Figure 3-1A) and operant escape

(Figure 3-1B) responses were assessed for 10 and 15 minutes, respectively, during the

testing trial. The control group remained in their homecage until behavioral testing.

(A) Reflex Hindpaw

Restraint Pre-Test HP Test
Stress 36.0C 44.0C


I I I I
0 15 30 40 (Minutes)


(B) Operant Escape

Restraint Pre-Test Escape Test
Stress 36.0C 44.0C


I I I I
0 15 30 45 (Minutes)

Figure 3-1. Behavioral testing sequence for the restraint group. (A) Reflex hindpaw. (B)
Operant escape.









Reflex Lick-Guard Latency

The latency of first lick/guard responses to 44.00C during baseline sessions (Day

1), testing sessions in which rats received 15 minutes of stress (Day 2; restraint group) or

no stress (control group), and sessions 24 hours afterwards (Day 3) are presented in

Figure 3-2. Latencies of lick/guard responses were significantly greater on Day 2 for the

restraint group than for the control group (Figure 3-2A; F=24.61, P<0.001). Reflex

response latencies were also greater for stressed rats on Day 2 than on days when the

same rats were not stressed (Days 1 and 3; F=10.08; P<0.001). Difference scores

between control and restraint groups revealed that reflex latencies were significantly

higher after stress on Day 2 after stress (Figure 3-2B; F 8.78, P<0.001).

Reflex Lick-Guard Duration

The duration of lick/guard responses to 44.00C were significantly lower for the

restraint group than for the control group on Day 2 (Figure 3-3A; F 39.18, P<0.001).

Reflex response durations were also significantly lower for the restraint group on Day 2

than on days when the same rats were not stressed (Days 1 and 3; F 13.61, P<0.001).

Reflex responses for the control and restraint groups were stable on testing Days 1 and 3,

demonstrating no adaptation to daily testing at 44.00C. Difference scores between

control and restraint groups revealed that reflex duration were significantly lower after

stress on Day 2 after stress (Figure 3-3B; F 11.361, P<0.001).

Base on these observations on reflex latencies and durations, it can be concluded

that exposure to restraint stress suppressed reflex responses. Stress-induced hyporeflexia

was characterized by a longer latency to elicit a hindpaw response and a shorter time

engaging in licking or guarding of the hindpaw. The effect was transient and did not









persist the following day. Thus, this data supports previous research suggesting that

stress inhibits spino-bulbo-spinal reflexes to thermal stimulation.

Effects of Restraint Stress on Operant Escape Responses at 44.00C

Operant Escape Latencies

To determine the effects of stress on operant responses, the same groups of animals

(described above) was stressed on weeks interspersed between reflex testing (Figure 3-4).

The first latency of operant escape responses during trials at 44.00C were observed during

baseline sessions (Day 1), testing sessions in which rats received 15 minutes of restraint

stress (restraint group; Day 2) or no stress (control group), and session the following day

(24 hours, Day 3). Previous studies have suggested that the first escape latencies are less

dependable outcome measures compared to escape durations (Vierck et al., 2002; 2003).

Consistent with these observations, no differences in escape latencies were observed

between the restraint and control groups on any day of testing (Figure 3-4A; F=0.2570,

P=0.7740). Difference scores revealed no significant effects (Figure 3-4B, F=0.818,

P=0.447).

Operant Escape Durations

The duration of escape responses was significantly greater on Day 2 for the

restraint group than for the control group on Day 2 (Figure 3-5A; F=38.48, P<0.001).

Furthermore, the duration of escape was greater after stress exposure on Day 2 than it

was for the same rats on unstressed days (Days 1 and 3; F=49.01, P<0.001). Therefore,

acute restraint stress did not produce a long-term (24 hour) effect on escape duration.

Difference scores revealed that escape durations were significantly higher than controls

after exposure to stress (Figure 3-5B; F 11.305, P<0.001).











A
S 250' *** EControl Group
2 00 *Restraint Group

100-





Baseline 15 Minutes 24 Hours
Day I Day 2 Day 3
Successive Testing Days


B
100-


50-






Baseline 15 Minutes 24 Hours
Day I Day 2 Day 3
Successive Testing Days


Figure 3-2. Reflex lick/guard latencies during testing trials at 44.00C for control (open
bar) and restraint (closed bar) groups during baseline sessions (Day 1), testing
sessions in which rats received 15 minutes of restraint stress (restraint group;
Day 2) or no stress (control group), and sessions the following day (Day 3).
(A) Exposure to restraint significantly increased reflex latencies when tested
fifteen minutes after stress (15 minutes; Day 2) compared to the control group
and compared to unstressed trials on baseline and 24 hours after stress. (B)
Difference scores confirmed that stress increased reflex latencies compared to
controls (15 minutes; Day 2). Data are expressed in seconds and are
represented as absolute group means + S.E.M. Significant within-subject
differences between trials 15 minutes after stress exposure and trials by the
same rats on baseline and 24 hours are indicated as: *** P<0.001.
Significant between-subject differences between the control and restraint
groups on Day 2 are indicated as: *** P<0.001.






57



A
S 120- EControl Group
: 100- _E T Restraint Group
go-
60-
| 0* -
40-
20-

Baseline 15 Minutes 24 Hours
Day 1 Day 2 Day 3
Successive Testing Days




B
20-
10-
0

r,-20
Q -30-
-40-
-50-
Baseline 15 Minutes 24 Hours
Day I Day 2 Day 3
Successive Testing Days


Figure 3-3. Cumulative reflex lick/guard durations during testing trials at 44.00C for
control (open bar) and restraint (closed bar) groups during baseline sessions
(Day 1), testing sessions in which rats received 15 minutes of restraint stress
(restraint group; Day 2) or no stress (control group), and sessions the
following day (Day 3). (A) Exposure to restraint significantly decreased
reflex durations when tested fifteen minutes after stress (15 minutes; Day 2)
compared to the control group and compared to unstressed trials on baseline
and 24 hours after stress. (B) Difference scores confirmed that stress
decreased reflex durations compared to controls (15 minutes; Day2). Data are
expressed in seconds and are represented as absolute group means + S.E.M.
Significant within-subject differences between trials 15 minutes after stress
exposure and trials by the same rats on baseline and 24 hours are indicated as:
*** P<0.001. Significant between-subject differences between the control
and restraint groups on Day 2 are indicated as: e** P<0.001.










A

5

bd
ouh
cU


ElControl Group
*Restraint Group


II1 01


Baseline 15 Minutes 24 Hours
Day I Day 2 Day 3
Successive Testing Days


I_


Baseline 15 Minutes 24 Hours
Day I Day 2 Day 3
Successive Testing Days

Figure 3-4. Escape latencies during testing trials at 44.00C for control (open bar) and
restraint (closed bar) groups during baseline sessions (Day 1), testing sessions
in which rats received 15 minutes of restraint stress (restraint group; Day 2) or
no stress (control group), and sessions the following day (Day 3). (A)
Exposure to restraint did not affect escape latencies compared to the control
group and compared to unstressed trials on baseline (Day 1) and 24 hours
(Day 3) after stress. (B) Difference scores confirmed that stress did not alter
escape latencies (15 minutes; Day 2). Data are expressed in seconds and are
represented as absolute group means S.E.M.









A comparison of escape durations for the control group did exhibit a slight increase

across the three testing days. Statistical analysis revealed no significant change across

testing Days (F=2.968; P=0.0623). This effect has been observed previously in our lab

as a consequence of repeated testing at the same thermal temperature.

Unlike reflex responses, operant responses are enhanced (increased heat sensitivity)

after an acute exposure to restraint stress when assessed by durations. This effect,

stress-induced hyperalgesia, was characterized by a longer time spent on the escape

platform during a trial, which were more reliable than latencies. Similar to reflex

responses, this effect was transient and did not persist the following day. Thus, it can be

concluded that stress produced a differential effect where reflexes were suppressed while

nociceptive responses dependent on cortical processing of thermal stimulation were

enhanced.

Sequence Analysis of Successive Operant Escape Durations

In addition to analysis of the total duration of escape, examinations of successive

operant escape duration within trials presents an additional strategy to analyze the effect

of experimental manipulations on behavioral responses. Successive operant escape plate

(A, B) and platform (C, D) durations are shown in Figure 3-6 for control (left panel) and

restraint (right panel) groups. In general, the maximum number of escape responses was

twelve (12), but a majority of animals respond approximately 6 times.

Plate durations for control (A) and restraint (B) groups do not change across

baseline (Day 1), 15 minutes (Day 2), and 24 hours (Day 3). Plate durations peaked

between the 2nd and 3rd responses.












A

2

P- C
OJ

u fi
uI-
rt
W


EDControl Group
*Restraint Group


B
300-
250-
200-
I ,-

t.150
o
100

01

Baseline 15 Minutes 24 Hours
Duy 1 Day 2 Day 3
Successive Testing Days


Figure 3-5. Cumulative escape durations during testing trials at 44.00C for control (open
bar) and restraint (closed bar) groups during baseline sessions (Day 1), testing
sessions in which rats received 15 minutes of restraint stress (restraint group;
Day 2) or no stress (control group), and sessions the following day (Day 3).
(A) Exposure to restraint significantly increased escape durations when tested
fifteen minutes after stress (15 minutes; Day 2) compared to the control group
and compared to unstressed trials on baseline (Day 1) and 24 hours (Day 3)
after stress. (B) Difference scores confirmed that stress enhanced escape
durations compared to controls (15 minutes; Day 2). Data are expressed in
seconds and are represented as absolute group means + S.E.M. Significant
within-subject differences between trials 15 minutes after stress exposure and
trials by the same rats on baseline and 24 hours are indicated as: *** P<0.001.
Significant between-subject differences between the control and restraint
groups on Day 2 are indicated as: *** P<0.001.


***










Baseline 15 Minutes 24 Hours
Day I Day 2 Day 3
Successive Testing Days









In contrast, control and restraint groups displayed different patterns of responding

during trials at 44.00C on Day 2 (15 minutes) in which one group of animals was exposed

to acute stress (restraint group) while the other group remained in their home cage

(control group). Platform durations for the control (C) and restraint (D) groups were

shorter than plate times in this group of animals and peak between the 3rd and 6th platform

responses. On the day of stress, platform durations in the restraint group dramatically

increased, which indicates an enhanced thermal sensitivity by restraint stress, compared

to the control group. The peak of the effect occurs on the 3rd and persisted until the 9th

platform response. Importantly, platform durations were comparable to baseline values

when assessed the following Day (24 hours).

In summary, the average duration across of the first 6 responses for the control and

restraint groups are compared (Figure 3-7). This number was selected because majority

of animals quit responding after the sixth response. In the control group (Figure 3-7A),

plate durations did not differ over the three consecutive testing sessions (F=0.793,

P=0.453), but platform durations did change significantly (F 7.368, P 0.001). Platform

durations were significantly higher than baseline during sessions on Day 3 (P<0.05) but

not during the 15 minute testing period on Day 2 (P>0.05).

In contrast, the average duration for plate (Figure 3-7B; F 3.727, P 0.025) and

platform (F=18.64, P<0.001) durations were different over the three consecutive

sessions for the restraint group. In particular, plate durations gradually decreased over

sessions (Days 1 through 3) with durations significantly lower than baseline during the

Day 3 session only (P<0.05). Platform durations after restraint were significantly longer

than baseline (P<0.001) but not 24 hours later (P>0.05). Furthermore, platform









durations were higher after restraint than the control group (F=38.004, P<0.001; Day 2).

During sessions 24 hours after stress, platform durations were similar between the two

groups (F=0.255, P=0.594; Day 3). Thus, analysis of successive operant escape

responses revealed a transient hyperalgesia (enhancement of heat sensitivity) as indicated

by increase in escape platform duration after restraint stress.

Effects of Repeated Stress Exposures: Adaptation

In order to avoid adaptation to repeated exposures to restraint stress (De Boer et al.,

1999; Gamaro et al., 1998) at least 2 weeks elapsed between stress tests for each animal.

The effectiveness of this strategy and the possibility that there might be carryover effects

of repeated stress were evaluated by three types of statistical comparisons related to

escape and lick/guard durations at 44.00C, as shown in Table 3-1.

First, the effectiveness of stress was evaluated for the first and second

administration of restraint prior to reflex or operant testing. Reflex durations were lower

for stressed rats compared to unstressed rats on Day 2 for the first (F=9.15, P<0.01) and

second stress sessions (F=5.06, P=0.01). Also, escape durations were greater for

stressed rats compared to unstressed rats on Day 2 for the first (F =5.69, P<0.01) and

second stress sessions (F=5.89, P<0.01).

Second, performance on day 1, 2, and 3 was compared for the first and second

reflex and operant testing sessions, in order to determine whether there were cumulative

effects of repeated stress on performance. None of these 12 comparisons revealed

significant effects: for the control and restraint groups during either reflex or operant

testing on each of the three days.
















Control Group


Restraint Group


P 1so-



0-


--Baseline u
-*- 15 Minutes
-0-24 Hours 150,

100
a* m


0 1 2 3 4 5 6 7 8 9 10 I'I12
Successive Escape Responses


0i 12 S 6 7 8 9 1 111213
Successive Escape Responses


2 150-
I-
Platform oo
50-

0-


-*-Baseline '
-*- 15 Minutes
--24 Hours 150s

100

50


0 1 2 i 4 5 6 7 8 9 10 11'123
Successive Escape Responses


0 i 2 4 5 6 7 8 9 10l 12 13
Successive Escape Responses


Figure 3-6. Sequence analysis of successive escape plate and platform durations during testing trials at 44.00C for control (left panel)
and restraint (right panel) groups during baseline sessions (asterisk; Day 1), testing sessions in which rats received 15
minutes of restraint stress (restraint group, gray circle; Day 2) or no stress (control group, gray circle), and sessions the
following day (closed square; Day 3). Data are expressed in seconds and are represented as absolute group means +
S.E.M.


- Baseline
-*- 15 Minutes
--24 Hours


--Baseline
-*- 15 Minutes
--24 Hours


~b~tj











A Control Group
100- ElPlate
*Platform


275


0 --

Baseline 15 Minutes 24 Hours
Successive Testing Days



B Restraint Group
100.*** Plate

S75
lPlatform



25-i



Baseline 15 Minutes 24 Hours
Successive Testing Days


Figure 3-7. Average escape duration of the first six plate (open bar) and platform (closed
bar) responses during testing trials at 44.00C for control and restraint groups
during baseline sessions (Day 1), testing sessions in which rats received 15
minutes of restraint stress (restraint group; Day 2) or no stress (control group),
and sessions the following day (Day 3). (A) In the control group, no
differences were observed in plate, but platform durations were slightly higher
over repeated sessions. (B) Exposure to acute restraint stress significantly
increased escape platform durations when tested fifteen minutes after stress
(15 minutes; Day 2) compared to the control group and compared to
unstressed trials on baseline (Day 1) and 24 hours (Day 3). Data are
expressed in seconds and are represented as absolute group means + S.E.M.
Significant within-subject differences between trials 15 minutes and 24 hours
after stress exposure or no stress and trials by the same rats on baseline and 24
hours are indicated as: P<0.05 and *** P<0.001. Significant
between-subject differences between the control and restraint groups during
trials at 15 minutes and 24 hours are indicated as: P<0.05 and *** P<0.001.









Finally, neither escape (F 0.306; P 0.583) nor lick/guard (F 0.302, P 0.585)

durations during baseline testing (Day 1) differed significantly across 4 weeks of testing

at 44C (twice prior to the restraint group and twice prior to the control group). Overall,

these data show that acute restraint stress remained effective with repetition at two (2)

week intervals and did not accumulate (carryover) from one exposure to the next.

Table 3-1. Cumulative reflex lick/guard and operant escape durations over two sessions
of restraint stress. Stress produced similar effects on reflex and escape
durations to 44.00C during the first and second exposures. The effects of
stress were not diminished by repetition at 2-week intervals. The data are
expressed in seconds, and values are represented as absolute group means +
S.E.M.
44.0C Reflex L/G 44.0C Operant Escape
Baseline (Day 1) Baseline (Day 1)
1't Session 2"d Session s't Session 2nd Session

Control 91.1 + 13.3 93.0 12.1 314.5 55.1 262.5 41.9
Pre-Stress 102.8 + 14.8 93.0 + 7.0 271.4 + 61.9 358.4 + 50.6


Test (Day 2)
1st Session 2nd Session
94.9 11.5 92.8 8.1
50.4 + 9.2 51.1 + 8.2


Test (Day 2)
1st Session 2nd Session
344.4 35.1 289.3 56.3
565.9 49.7 505.1 53.3


24 Hours (Day 3) 24 Hours (Day 3)
1st Session 2nd Session 1st Session 2nd Session
Control 76.4 14.1 90.2 13.4 411.4 37.8 379.6 67.1
Post-Stress 75.2 + 13 81.1 + 8.9 499.9 + 37.3 383.6 + 23.4

Time Course of Restraint Stress on Operant Escape Responses at 44.00C

Previous studies have demonstrated that the effects of stress are detected after

termination of the stressor and may last for several hours or days later (Calcagnetti et al.,

1992; Drolet et al., 2001; Gamaro et al., 1998; Quintero et al., 2000; Tusuda et al., 1989).

The duration and magnitude of stress effect is dependent on several factors including

stressor intensity and the length of exposure. To examine the temporal profile of


Control
Stress








stress-induced hyperalgesia, a separate group of female rats (n=12) were restrained for a

period of 15 minutes followed by assessment of behavioral responses immediately

(without a 15 minute pre-test at 36.00C) after exposure to restraint stress as illustrated in

Figure 3-8A.

In addition, the testing protocol used in experiments described above is shown in

Figure 3-8B (with a 15 minute pre-test at 36.00C). Finally, operant escape responses

were assessed the following day (24 hours). The control group did not receive stress, but

were tested under similar circumstances without or with a pre-test at 36.00C.

(A)

Restraint Escape Test
Stress 44.0C


I I I
0 15 30 (Minutes)


(B)

Restraint Pre-Test Escape Test
Stress 36.00C 44.0C


I I I I
0 15 30 45 (Minutes)

Figure 3-8. Temporal profile of restraint stress on escape responses during trials at
44.0C. Responses were assessed 15 minutes (A), 30 minutes after the onset
of stress (B), and 24 hours after restraint stress (not shown). The control group
followed the same testing schedule but without exposure to restraint stress.









Operant Escape Latencies

To determine the effects of stress on escape latencies across multiple time points,

animals received 15 minutes of restraint stress (restraint group) or no stress (control

group) followed by testing sessions at 44.00C immediately or 15 minutes after stress, and

sessions the following day (24 hours; Figure 3-9). Escape latencies in the control group

did not differ across testing sessions (Figure 3-9A; F=0.8555, P=0.4387). However,

latencies were affected by restraint stress (F 4.279; P=0.0269). Escape latencies were

shorter immediately and 15 minutes after restraint compared to the following day

(P<0.05). Differences between the two groups (Figure 3-9B) revealed that latencies were

shorter than controls when assessed immediately (F=12.7, P=0.002) and 15 minutes

(F 6.358, P 0.019) but not 24 hours (F=0.137, P 0.714) after the termination of stress.

Previous data suggested that escape latencies were unreliable and a poor outcome

measure. Unlike the first set of experiments in a different group of animals, escape

latencies were sensitive to stress. A reduction of escape latencies, which indicates a

lower threshold to elicit an escape response, supports the conclusion of stress-induced

hyperalgesia.

Operant Escape Durations

To determine the effects of stress on escape durations across multiple time points,

responses in the same group of animals are presented in Figure 3-10. Similar to latencies,

escape durations in Figure 3-10A did not differ across testing sessions in the control

group (F=2.147, P 0.1407), but durations were significantly different in the restraint

group (F=3.675, P=0.0420). Escape durations were significantly longer immediately

and 15 minutes after restraint compared to the following day (P<0.05). Difference scores

(Figure 3-10B) revealed that escape durations were longer than controls when assessed









immediately (F 16.035, P<0.001) and 15 minutes (F 5.989, P 0.023) but not 24 hours

(F=0.518, P=0.479) after stress. In agreement with previous data (Figure 3-5), escape

durations were influenced by stress. Stress-induced hyperalgesia was displayed

immediately (greatest effect) after the termination of restraint as well as when evaluated

15 minutes after stress exposure. The effect was transient based on the fact that both

groups showed similar responding 24 hours later.

Effects of Restraint Stress on Core Temperature

Various experimental manipulations such as restraint stress influence

thermoregulation, which can impact behavioral responses to thermal stimulation. For

example, restraint stress produces hyporeflexia and is associated with increased core

temperature (-1.0C; Chen and Herbert, 1995; Keim and Sigg, 1976; Le Bars et al.,

2001; Thompson et al., 2003; Tjolsen and Hole, 1993). Furthermore, evidence suggests

that changes in thermoregulation can influence the interpretation of behavioral

responding (Le Bars et al., 2001; Tjolsen and Hole, 1993).

In the context of the previous study, the following question can be raised. Is the

expression of hyperalgesia a consequence of increased body temperature after exposure

to stress? Thus, a possible underlying reason for stress-induced changes in operant

responses is altered body temperature. On the other hand, an increase in core temperature

may not be a critical factor in the expression of stress-induced changes in nociception,

but rather changes in skin temperature. In fact, skin temperature has been identified as a

potential confounding variable (Tjolsen and Hole, 1993), and therefore, requires

techniques to stabilize temperatures before behavioral assessment (e.g., pre-test trials at

36.0C).











A
7* ElControl Group
ERestraint Group
5 0-


a 301 ***
o 2o 01


Immediately 15 Minutes 24 Hours

Testing Session



B
20-
1- 10-


mt -10-
A U -20-
-30-
-40-
Immediately 15 Minutes 24 Hours

Testing Session



Figure 3-9. Escape latencies during testing trials at 44.00C for control (open bar) and
restraint (closed bar) groups when tested immediately, fifteen minutes, and 24
hours after exposure to restraint. (A) Escape latencies were shorter
immediately and fifteen minutes after restraint stress compared to control
groups. However, responses did not carry over to the following day. (B)
Difference scores confirmed that stress reduced latencies compared to
controls. Data are expressed in seconds and are represented as absolute group
means + S.E.M. Significant within-subject differences between 24 hours after
stress exposure and trials by the same rats on baseline and 15 minutes are
indicated as: P<0.05. Significant between-subject differences between the
control and restraint groups are indicated as: P<0.05 and *** P<0.001.












0



OSS


24 Hours


Testing Session


24 Hours


Testing Session


Figure 3-10. Escape durations during testing trials at 44.00C for control (open bar) and
restraint (closed bar) groups when tested immediately, fifteen minutes, and 24
hours after exposure to restraint. (A) Escape durations were immediately
increased (stress-induced hyperalgesia) after restraint and when tested fifteen
minutes after stress. However, responses did not carry over to the following
day. (B) Difference scores confirmed that stress enhanced escape durations
compared to controls. Data are expressed in seconds and are represented as
absolute group means + S.E.M. Significant within-subject differences 24
hours after stress exposure and trials by the same rats immediately and 15
minutes are indicated as: P<0.05. Significant between-subject differences
between the control and restraint groups are indicated as: P<0.05 and ***
P<0.001.


EControl Group
*Restraint Group


Immediately 15 Minutes


Immediately 15 Minutes









Experiment 1

In order to determine the effect of restraint stress on thermoregulation, core

temperatures of female rats (n=12) were recorded during separate sessions in which

animals were exposed to no stress (control group) or stress (restraint group; Figure 3-11).

Temperature recordings were assessed prior to trials at 44.00C either immediately (Figure

3-8A), 15 minutes (Figure 3-8B), or 24 hours (not shown) after restraint stress. Control

animals were tested under identical conditions without exposure to restraint.

In Figure 3-11A, body temperatures remained constant over the testing sessions in

control group (F=0.047, P=0.954), but temperatures were significantly affected by stress

(restraint group; F=18.06, P<0.001). Core temperatures were significantly greater when

evaluated immediately (P<0.001) but not 15 minutes (P>0.05) or the following day (24

hours; P>0.05). Increased core temperatures could be a consequence of struggling

during restraint, which consequently raises temperature. A feature of restraint stress is

the uncontrolled restrictions imposed by the device that an animal initially tries to escape.

Differences between the two groups revealed that temperatures were significantly

higher when assessed immediately (Figure 3-11B; F=30.807, P<0.001) and 15 minutes

(F 7.506, P 0.043) but not twenty-four hours (F 2.012, P=0.170) after restraint. Thus,

while restraint temporarily increased core temperatures (0.86 + 0.09), they were slightly

comparable to controls 15 minutes after the onset of stress. Changes in core temperature

therefore cannot account entirely for the observation of stress-induced hyperalgesia on

operant responses.









Experiment 2

An additional group of female rats (n=6) were used to examine the effects of stress

on core (Figure 3-12A) and cutaneous temperatures (Figure 3-12B). Core temperatures

were assessed over five minute intervals during restraint and exposure to a thermal plate

at 36.0C (similar to pre-test trials). Recordings during control and restraint groups were

obtained in different sessions. In addition to core temperature, cutaneous temperatures

were measured in rats in which animals were not stressed (control group) or stressed for

15 minutes (restraint group). For the restraint group, recordings were obtained before

and after restraint and after exposure to a 15 minute pre-test trial at 36.00C. Control

animals were assessed before and after the pre-test.

Core body temperature

In Figure 3-12A, stress increased core temperature (restraint group, gray circle; F=

9.420, P<0.001). Specifically, a significant increase in core temperatures was observed

at ten (P<0.001) and 15 (P<0.001) minutes during restraint. Temperatures peaked at

38.62+0.11 C. These results are in agreement with Figure 3-11. But, after placement

into the pre-test at 36.00C, core temperatures gradually decreased. Temperatures were

greater than baseline at five (P<0.001) but not after ten (P>0.05) or 15 (P>0.05) minutes

during pre-test conditions. In the control group (asterisk with dashed line), temperatures

increased slightly after placement into the pre-test, but this effect was not significant

(F 0.48, P=0.7). Differences between groups (not shown) suggested that temperatures

were higher after stress than control before placement into the pre-test at 36.0 C

(F 14.511, P 0.003) but did not differ five (F 2.207 P 0.168), ten (F 0.000, P 1.0),

and 15 (F 0.009, P 0.926) minutes into the trial.

















U
* 38.5

a 38.0,

S37.5,

37.0-


]jControl Group
*Restraint Group


**
t**









Immediately 15 Minutes 24 Hours


Testing Session


Immediately 15 Minutes


r-T-


24 Hours


Testing Session


Figure 3-11. Core body temperatures during testing trials at 44.00C for control (open bar)
and restraint (closed bar) groups when tested immediately, fifteen minutes,
and 24 hours after exposure to restraint. (A) Core temperatures were
immediately increased (stress-induced hyperthermia) compared to the control
group. Core temperature was still higher than controls fifteen minutes after
stress. Core temperature did not differ the following day. (B) Difference
scores revealed that stress-induced increase in temperature was most
prominent immediately after stress and slowly returned to control values.
Data are expressed in seconds and are represented as absolute group means +
S.E.M. Significant within-subject differences immediately after stress
exposure or not stress are indicated as: *** P<0.001. Significant
between-subject differences between the control and restraint groups are
indicated as: P<0.05 and *** P<0.001.












Thus, stress-induced increases in core temperature are most prevalent during

exposure to stress. Temperatures quickly return to levels comparable to controls during a

pre-test trial at 36.00C. This data supports the idea that altered body temperature is not

responsible for the expression of stress-induced hyperalgesia when animals are tested at

44.0C (consistent with data shown in Figure 3-11).

Peripheral cutaneous temperature

In general, cutaneous temperatures were lower than body temperatures (27.97+0.48

vs. 37.53+0.270C; F 300.836, P<0.001). Cutaneous temperatures were significantly

different across testing sessions (Figure 3-12B; F=19.621, P<0.001). Recordings were

greater than baseline when assessed after termination of restraint (P<0.01) and exposure

to 36.00C (P<0.001). In the control group, skin temperatures also increased (F 90.886,

P<0.001). Temperatures increased after the pre-test trial at 36.00C in the absence of

stress because of increased activity of the animal and exposure to thermal stimulation that

was provided by the heated plate. No differences in temperatures were observed between

control and restraint groups during baseline before stress (F=1.019, P=0.337) or after

exposure to 36.00C (F=0.554, P=0.474). However, recordings after restraint were higher

than control before placement into the pre-test (F=15.689, P=0.003).

In summary, while stress increased skin temperatures (stress-induced

hyperthermia), this effect does not persist, and quickly returned to levels comparable to

controls. Because temperatures in control and restraint groups were equalized by 36.0C

before placement into the second trial at 44.00C, core and cutaneous temperatures did not

play an important role in the expression of stress-induced hyperalgesia.












- Control
--Stress


A
39.0-

"-" 38.5-

m 38.0-

E 37.5-

37.0-


36.0-
0'
S33.5-
C) w
I ; 310-
sI
Ua
S28.5

26.0-


-*-Control
S-Stress


Restraint Pre-Test
Baseline Post-Stress Post Pre-Test


Figure 3-12. Core and cutaneous hindpaw temperatures for control (asterisk with dashed
lines) and restraint (gray circle) groups. (A) Core temperature was increased
during a fifteen-minute exposure to restraint stress. After termination of
restraint, animals were placed into a pre-test at 36.00C in which temperatures
progressively returned to levels comparable to controls. (B) Cutaneous
temperatures were greater after restraint, but exposure to pre-test normalized
skin temperatures. Data are expressed in seconds and are represented as
absolute group means + S.E.M. Significant within-subject differences
between temperature assessed during and after restraint compared to pre-stress
baseline values are indicated as: ** P<0.01 and *** P<0.001. Significant
between-subject differences between temperature assessed before and after a
pre-test trial are indicated as: ** P<0.01.


Restraint Pre-Test


4$ +! 1;^ 4 4 14
'b '% _1% e









Effects of Restraint Stress on Control Responses

Operant Escape Responses at 36.00C

Behavioral responses during 36.0C trials were obtained in female rats (n=l 1)

during baseline sessions (Day 1), testing sessions in which rats received 15 minutes of

restraint stress (restraint group; Day 2) or no stress (control group), and sessions the

following Day (24 hours; Day 3). Assessment of escape responses during 36.0C trials is

an important strategy to determine if a particular experimental manipulation produces

avoidance, or increased platform duration unrelated to thermal stimulation.

Operant escape latencies

As illustrated in Figure 3-13A, escape latencies were not affected by restraint stress

(F 0.23, P=0.795). Durations were not different between control and restraint groups

(Figure 3-13B; F 1.124, P 0.33). Response latencies were highly variable at 36.00C, a

non-noxious temperature, due to exploratory behaviors and the inability of this

temperature to elicit escape behavior.

Operant escape durations

Similar to latencies during 36.0C trials, escape durations were also not affected by

restraint stress (Figure 3-14A; F=2.599, P=0.0869). Durations were not different

between control and restraint groups (Figure 3-14B; F=0.993, P=0.382). At this neutral

plate temperature, platform occupancy was short and the duration of operant escape

response did not change significantly after restraint stress. Because responses were not

affected, stress does not induce avoidance during the escape test.











A

-
| 3:
SE
aeP
a-
2 W


EIControl Group
ERestraint Group


I 1 1


Baseline 15 Minutes 24 Hours
Day 1 Day 2 Day 3
Successive Testing Days


B

S-
vl
Pag
a o
s^


Baseline 15 Minutes 24 Hours
Day I Day 2 Day 3
Successive Testing Days


Figure 3-13. Escape latencies during testing trials at 36.00C for control (open bar) and
restraint (closed bar) groups during baseline sessions (Day 1), testing sessions
in which rats received 15 minutes of restraint stress (restraint group; Day 2) or
no stress (control group), and sessions the following day (Day 3). (A)
Exposure to restraint did not affect escape latencies compared to the control
groups and compared to unstressed trials on baseline and 24 hours after stress.
(B) Difference scores confirmed that stress did not alter escape latencies (15
minutes; Day 2). The data are expressed in seconds and are represented as
absolute group means + S.E.M.











A



2 E

0
~a 1
o 5
^ Q
Ed


B




Au

"U


ElControl Group
*Restraint Group


Baseline 15 Minutes 24 Hours
Day 1 Day 2 Day 3
Successive Testing Days


Baseline 15 Minutes 24 Hours
Day I Day 2 Day 3
Successive Testing Days


Figure 3-14. Cumulative escape durations during testing trials at 36.00C for control (open
bar) and restraint (closed bar) groups during baseline sessions (Day 1), testing
sessions in which rats received 15 minutes of restraint stress (restraint group;
Day 2) or no stress (control group), and sessions the following day (Day 3).
(A) Exposure to acute stress produced no significant alteration of escape
durations during 36.00C trials. (B) Difference scores confirmed that stress did
not significantly alter escape durations (15 minutes; Day 2). Data are
expressed in seconds, and values are represented as absolute group means +
S.E.M.









Sequence analysis of successive operant escape durations

In addition to analysis of the total duration of escape within trials at 36.00C,

successive escape plate (A, B) and platform (C, D) durations were examined between

control (left panel) and restraint (right panel) groups (Figure 3-15). Overall, compared to

plate responses at 44.00C, plate times were longer at 36.00C. For both groups, plate

durations were higher during the beginning of the trial and gradually decreased. Plate

durations for control (Figure 3-15A) and restraint (Figure 3-15B) groups did not change

across the baseline, test, or 24 hours periods. In contrast, platform durations remained

stable throughout the trial and were substantially shorter than plate durations for control

(Figure 3-15C) and restraint (Figure 3-15D) groups. Although restraint stress did not

influence platform durations, the number of responses was reduced compared to baseline.

In support of the cumulative platform durations represented in Figure 3-16, restraint did

not significantly affect escape responses at neutral temperatures. .

In summary, the average duration of the first 6 responses during trials at 36.00C for

the control and restraint groups are compared in Figure 3-16. In the control group,

neither plate (Figure 3-16A; F 1.306, P 0.2859) nor platform (F 2.382, P 0.1096)

durations changed over the three consecutive sessions. Similar to controls, stress did not

affect plate (Figure 3-16B; F 2.256, P 0.1233) or platform (F 1.112, P 0.3422)

durations over the three consecutive sessions.

Thus, stress does not produce an increase in platform duration at neutral

temperatures suggesting that the expression of stress-induced hyperalgesia at 44.00C,

which activates C-nociceptors, is dependent on higher levels of sensory processing.

Furthermore, stress-induced hyperalgesia is not a consequence of avoidance.









Darkbox Responses

In order to control from changes in motor functioning and motivation, darkbox

responses were evaluated in female rats (n=l 1) for control and stress conditions (Table

3-2). Latencies of escape from bright light in the darkbox test were unaffected by prior

stress (F=1.633, P=0.2358). Therefore, acute restraint stress did not significantly alter

aversion to light or produce motor effects (e.g. freezing) that interfered with escape

performance.

Table 3-2. Darkbox latencies for control and restraint groups. Responses were unaffected
by stress. Data are expressed in seconds, and values are represented as
absolute group means S.E.M.
Baseline 15 Minutes 24 Hours
Control 12.7 1.3 Control 13.4 1.5 Control 15.3 1.6
Pre-Stress 16.4 1.8 Stress 15.3 + 1.8 Post-Stress 12.6 1.5

Effects of Restraint Stress on Operant Thermal Preference

Based on the preceding results, restraint stress produces a heightened sensitivity to

heat (e.g., decreased plate durations; increased escape platform durations). But, is

stress-induced hyperalgesia specific to the escape paradigm? Or, does acute restraint

stress produce a generalized heightened sensitivity to heat? To determine if this

observation was limited to the escape paradigm or could be generalized to other operant

paradigms, an additional operant paradigm (thermal preference) was used to clarify this

issue. Enhanced heat sensitivity would be indicated by an increase in duration

(preference) for the cold compartment. In these experiments, the floor was cooled to

15.0C in one compartment and heated to 45.00C in the adjacent compartment.

Responses were recorded during a 12-minute trial preceded by a pre-test trial at 36.00C

(15 minutes).















Control Group







A-'


Restraint Group


--Baseline
-*- 15 Minutes
--24 Hours


0 1 2 4 5 6 7 8 9 10 11 12 13
Successive Escape Responses


0 2 3 4 S 6 7 9 10 II 12 13
Successive Escape Responses


C




Platform I
p


-*-Baseline
-- 15 Minutes
--24 Hours


ErI4-


0 1 2 3 4 5 6 7 8 9 10 13 1 3
Successive Escape Responses


0 1 2 3 4 5 6 7 8 9 10 II 12 13
Successive Escape Responses


Figure 3-15. Sequence analysis of successive escape plate and platform durations during testing trials at 36.00C for control (left panel)
and restraint (right panel) groups during baseline sessions (asterisk; Day 1), testing sessions in which rats received 15
minutes of restraint stress (restraint group, gray circle; Day 2) or no stress (control group, gray circle), and sessions the
following day (closed square; Day 3). Data are expressed in seconds and are represented as absolute group means + S.E.M


A




Plate J
g


-- Baseline
-- 15 Minutes
--24 Hours


--Baseline
-- 15 Minutes
-24 Hours










Control Group


E"Plate
mPlatform


Baseline 15 Minutes 24 Hours
Successive Testing Days


Restraint Group


E Plate
EPlatform


Baseline 15 Minutes 24 Hours
Successive Testing Days


Figure 3-16. Average escape duration of the first six plate (open bar) and platform
(closed bar) responses during testing trials at 36.00C for control and restraint
groups during baseline sessions (Day 1), testing sessions in which rats
received 15 minutes of restraint stress (restraint group; Day 2) or no stress
(control group), and sessions the following day (Day 3). In both groups, plate
durations were longer than platform durations. In addition, no differences
were observed in plate and platform durations over sessions. The data are
expressed in seconds and are represented as absolute group means + S.E.M.









Thermal Preference Durations

To determine the effects of stress on the thermal preference test, baseline responses

(n=20; Day 1) were compared to sessions in which all animals underwent restraint stress

for 15 minutes and testing 15 minutes (test; Day 2), 24 hours (Day 3) and 48 hours (Day

4) after restraint (Figure 3-17). Similar to the operant escape test, thermal preference

responses were continuously assessed during the testing trial. In Figure 3-17A, restraint

stress influenced thermal preference responses (F=2.865, P=0.041). Cold preference

was significantly higher than baseline after stress (Day 2; P<0.05) but not 24 (Day 3;

P>0.05) or 48 (Day 4; P>0.05) hours after stress. Conversely, heat preference was lower

after stress (Day 2; P<0.05) but not 24 (Day 3; P>0.05) or 48 (Day 4; P>0.05) hours

later.

Difference scores (Figure 3-17B) revealed that the effect of stress was most

prominent when thermal preference was assessed 15 minutes after stress. Responses

quickly returned to levels comparable to baseline during subsequent testing sessions.

Thus, similar to escape (e.g., decreased time spent on the heat plate), restraint stress

produced a heightened sensitivity to heat as indicated by an increase in cold preference

and a decrease in heat preference.

Sequence Analysis of Successive Thermal Preference Durations

In addition to analysis of the cumulative preference responses, successive cold and

heat durations within trials were compared. The average duration of the first 6 cold and

heat preference responses were analyzed to determine the effect of stress on successive

preference responses (Figure 3-18).











A
500-
509 ElCold Preference
400- Heat Preference


u 200'


0- --
rlol



Baseline Test 24 Hours 48 Hours
Day [ Day 2 Day 3 Day 4
Testing Session





B
S150Cold Preference
100- o Heat Preference
S~509
o 9

o-s

-1009
-150-
Test 24 Hours 48 Hours
Day 2 Day 3 Day 4
Testing Session


Figure 3-17. Cumulative thermal preference durations during testing trials at 15.0 (cold:
open bar) and 45.00C (heat: closed bar) during baseline sessions, testing
session in which rats received 15 minutes of restraint stress, and sessions 24
and 48 hours after stress. (A) Under baseline conditions (Day 1), preference
for heat was greater than the preference for cold. An acute exposure to
restraint significantly decreased preference for heat on Day 2. However,
preference for heat returned to pre-stress levels when assessed 24 (Day 3) or
48 hours (Day 4) hours after restraint stress. (B) Based on difference scores,
stress produced a substantial change in preference that dissipated the
following testing sessions. Data are expressed in seconds and are represented
as absolute group means + S.E.M. Significant differences between the control
and restraint groups on Day 2 are indicated as: P<0.05.









Restraint stress had an effect on cold (F=8.786, P 0.01) and heat (F=3.632,

P=0.038) preference responses. The average duration of cold responses were

significantly higher than baseline after exposure to stress (P<0.01) but not higher 24

(P>0.05) or 48 (P>0.05) hours later. Consequently, the average duration of heat

responses were significantly lower than baseline after exposure to stress (P<0.05) but not

higher 24 (P>0.05) or 48 (P>0.05) hours later. As shown on operant escape, stress

produced a transient increase sensitivity to heat as indicated by an increase in cold

preference to 15.00C (longer duration) and a decrease in heat preference to 45.00C

(shorter duration).


W 80-
70-
~ 60 -
50
S40-



1_ 10-
0

Baseline Test 24 Hours 48 Hours
Day 1 Day 2 Day 3 Day 4
Testing Session


ECold Preference
MHeat Preference


Figure 3-18. Average of the first six cold (open bar) and heat (closed bar) durations
during baseline sessions, testing sessions in which rats received 15 minutes of
restraint stress (test), and sessions 24 and 48 hours after stress. Restraint
stress affected the average cold and heat preference responses. Cold
responses were increased while heat responses were reduced. This effect did
not continue during sessions assessed the following days (24 and 48 hours).
Data are expressed in seconds and are represented as absolute group means +
S.E.M. Significant differences between restraint for cold and heat preference
are indicated as: P<0.05.









Effects of Endogenous Opioids on Stress-Induced Changes in Nociception

Previous studies suggest that restraint-induced changes in nociception are

modulated by opioid mechanisms. Stress-induced changes in nociception are

characterized as either opioid-dependent or non-opioid dependent (Lewis, Cannon, and

Liebeskind, 1980; Porro and Carli, 1988; Drolet et al., 2001). Involvement of

endogenous opioids, which are released during stress (Madden et al. 1977), in the

expression of stress-induced hyporeflexia is confirmed by administration of an opioid

antagonist (naloxone; Pilcher and Browne, 1983).

In the present study, injections of naloxone were used to determine the role of

endogenous opioids in modulating effects of stress on reflex (hyporeflexia) and operant

(hyperalgesia). It was hypothesized that the endogenous opioid system contributes to

stress-induced reduction of reflexes while opposing the excitatory effects of stress on

cerebrally mediated operant escape responses to thermal stimulation.

Reflex Lick/Guard Responses

As mentioned previously, restraint stress suppressed reflex lick/guard responses

(increased latencies; decreased duration). To determine if the expression of

stress-induced hyporeflexia was mediated by endogenous opioids, behavioral responses

were assessed in a group of female rats during sessions in which rats received 15 minutes

of restraint stress (restraint group; n=19) or no stress (control group; n=19) followed by

an injection of saline (1.0 mg/kg) or naloxone (3.0 mg/kg). Then, animals were tested

during a trial at 44.50C for 10 minutes that was preceded by a pre-test at 36.00C (15

minutes).