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Effects of Nociceptin/orphanin FQ Microinjections into Amygdala on Anxiety-Related Behaviors and Hypothalamic-Pituitary-...

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PAGE 1

EFFECTS OF NOCICEPTIN/ORPHANIN FQ MICROINJECTIONS INTO THE AMYGDALA ON ANXIETY-REL ATED BEHAVIORS AND HYPOTHALAMIC-PITUITARY-ADRENAL AXIS ACTIVATION By MEGAN K. GREEN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by MEGAN K. GREEN

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This document is dedicated to all who believe in the scientific pur suit of knowledge and truth.

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iv ACKNOWLEDGMENTS I thank my committee members Dr. Henr ietta Logan, Dr. John Petitto, and Dr. Margaret Bradley for their assistance in the preparation of this thesis. I also, and especially, thank my advisor, Dr. Darragh De vine, for his incredibly dedicated guidance and support of my graduate studies. I would also like to thank my mother, Lisa, my stepfather, Michael, and my brother, Damion, for guiding and supporting me on my journey, in life and in academics. I thank Terry and Karen for their support during my first 2 years of graduate training. I thank “my” Michael for motivating me during the writing of this thesis. And I thank Simon Poindexter, the brilliant Siamese, for keeping my lap warm and occasionally editing this manuscript while I wrote.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF FIGURES...........................................................................................................vi ABSTRACT......................................................................................................................v ii CHAPTER 1 INTRODUCTION............................................................................................................1 2 METHODS...................................................................................................................... .7 Animals........................................................................................................................ .7 Drugs.......................................................................................................................... ...7 Surgery........................................................................................................................ ..8 Equipment.....................................................................................................................8 Anxiety-Testing Procedure.........................................................................................10 Statistical Analyses.....................................................................................................11 3 RESULTS...................................................................................................................... .13 Intracerebroventricular Injections...............................................................................13 Intra-Amygdaloid Injections.......................................................................................16 Thymus, Adrenal, and Spleen Masses........................................................................24 4 DISCUSSION.................................................................................................................26 Anxiety-Related Behavior..........................................................................................26 Corticosterone.............................................................................................................30 Organ Masses..............................................................................................................32 Summary.....................................................................................................................33 LIST OF REFERENCES...................................................................................................34 BIOGRAPHICAL SKETCH.............................................................................................41

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vi LIST OF FIGURES Figure page 2-1. Photograph of the open field and a diagram of the zones used for scoring…………9 3-1. Anxiety-related behaviors follow ing ICV injections of N/OFQ. ............................14 3-2. Concentrations of circul ating corticosterone following IC V injections of N/OFQ .16 3-3. Anxiety-related behaviors following in tra-amygdaloid injections of N/OFQ. ........18 3-4. Anatomical map of amygdaloid placements showing latency to enter open field. .20 3-5. Anatomical map of amygdaloid placements showing time spent in the open field. 22 3-6. Concentrations of circ ulating corticosterone followi ng intra-amygdaloid injections of N/OFQ. ...............................................................................................................24 3-7. Analysis of glandular masses. .................................................................................25

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vii Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EFFECTS OF NOCICEPTIN/ORPHANIN FQ MICROINJECTIONS INTO THE AMYGDALA ON ANXIETY-REL ATED BEHAVIORS AND HYPOTHALAMIC-PITUITARY-ADRENAL AXIS ACTIVATION By Megan K. Green December 2005 Chair: Darragh P. Devine Major Department: Psychology Intracerebroventricular (ICV) mi croinjections of the opioid-like neurotransmitter nociceptin/orphanin FQ (N/OFQ ) produce elevations in hypo thalamic-pituitary-adrenal axis (HPA axis) activity and anxiety-relate d behaviors in rats. Furthermore, these increases in HPA axis activity can be produ ced by N/OFQ injections into a number of limbic structures. We examined the potential role of one limbic structure, the amygdala, in the N/OFQ-induced anxiogenic effects. Male Long Evans rats were each implanted with a guide cannula into the ri ght lateral ventricle or amygda la. Each rat received an injection of N/OFQ (0, 0.01, 0.1, or 1.0 nmole) prior to behavioral testing in a neophobic test of anxiety. In the anxiety test, each rat was placed in a start box connected to an open field, where the rats had fr ee access to the field for 5 mi nutes. Latency to enter the open field, number of entries into the open field, total time spent in the open field, and thigmotactic behaviors were used as measur es of anxiety. Follow ing testing, the rats

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viii were euthanized and plasma samples were obt ained for analysis of HPA axis activity. Additionally, each rat brain was removed fo r cannula verification, and thymus glands, adrenal glands, and spleens were dissected out fo r analysis of overall health of the rats. N/OFQ injections produced doseorderly increases in anxiety -related behaviors, and these effects were greater in the IC V-implanted rats than they were in the amygdala-implanted rats. The ICV N/OFQ-treated rats displayed longer latencies to enter the open field, fewer numbers of entries into the open field, and less total time spent in the open field than the ICV vehicle-treated rats did. Th e intra-amygdaloid N/OFQ-treated rats also exhibited longer latencies to enter the open field, but did not differ in the number of entries into the open field or the total time spent in the open field when compared with the behavior of the intra-amygdaloid vehicletreated rats. N/OFQ injections into the lateral ventricle also produced elevations in circulating cort icosterone, indicating that the HPA axis activity was greater in these rats However, amygdaloid injections did not affect corticosterone levels. In conclusi on, the amygdala appears to be involved in the anxiogenic behavioral effects of N/OFQ. However, the differences in potency of effects between ICV N/OFQ injections and intraamygdaloid injections on anxiety-related behaviors and circulating CORT in rats indicat e that the amygdala is not the primary site of drug action and that extraamygdaloid sites are involved.

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1 CHAPTER 1 INTRODUCTION Nociceptin/Orphanin FQ (N/OFQ) and its cognate receptor NOP constitute a highly conserved (Danielson and Dores, 1999) peptide neurotransmitter system that affects an interesting range of very important behavioral and physiological activities. N/OFQ is a 17 amino acid peptide that is st ructurally similar to the endogenous opioids, particularly dynorphin A (Meuni er et al., 1995; Reinscheid et al., 1995). However, N/OFQ does not bind to the , or opioid receptors with high affinity (Shimohigashi et al., 1996), but does bind with high affinity to the NOP receptor (Butour et al., 1997; Reinscheid et al., 1995; Shimohigashi et al., 1996). The NOP receptor is a 7-transmembrane, G-protein coupled receptor (Bunzow et al., 1994; Chen et al., 1994; Lachowicz et al., 1995; Reinsche id et al., 1996; Wang et al., 1994; Wick et al., 1994) that is negatively linked to adenylate cyclase (Lac howicz et al., 1995; Mollereau et al., 1994; Reinscheid et al., 1995; Reinscheid et al., 1996), increases inward rectifying K+ channel conductance (Connor et al., 1996a ; Vaughan and Christie, 1 996; Vaughan et al., 1997), and inhibits Ca2+ conductance (Connor et al, 1996b) The NOP receptor shows high structural homology with the opioid recepto rs (Bunzow et al., 1994; Chen et al., 1994; Lachowicz et al., 1995; Moller eau et al., 1994; Wang et al ., 1994; Wick et al., 1994), although it does not bind any of the opioids with high affinity (Bunzow et al., 1994; Butour et al., 1997; Lachowi cz et al., 1995; Wang et al., 1994). This low affinity between N/OFQ and opioid receptors and betw een NOP and opioid peptides suggests that the N/OFQ-NOP system is functionally distinct from the opioid system.

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2 N/OFQ, its precursor protein, and the NOP receptor are all found widely distributed throughout the brain, spinal cord, and periphery (Bunzow et al., 1994; Chen et al., 1994; Devine et al., 2003; L achowicz et al., 1995; Neal et al., 1999a&b; Nothacker et al., 1996; Mollereau et al., 1994; Wang et al., 19 94; Wick et al., 1994), consistent with a wide range of functions including pain modul ation (Meunier et al ., 1995; Reinscheid et al., 1995; Tian et al., 1997), motor performan ce (Devine et al., 1996; Reinscheid et al., 1995), spatial learning (Sandin et al., 1997; Sandin et al., 2004), and feeding (Nicholson et al., 2002; Pomonis et al., 1996) However, localization is particularly high in limbic regions including the hypothalamus, septum, be d nucleus of stria terminalis (BNST), and amygdala (Bunzow et al., 1994; Devine et al., 2003; Lachowicz et al., 1995; Neal et al., 1999a&b; Nothacker et al., 1996; Wang et al, 1994). This limbic localization is consistent with the known role of N/OFQ in stress responses (physiological, homeostatic responses to stimuli that represent a cha nge or potential change to the organism’s environment; e.g., Herman and Cullinan, 1997) and anxiety responses (behavioral responses to non-specific, potentially threaten ing stimuli; e.g., Walker et al., 2003). For example, N/OFQ is released from forebrain ne urons in rats following exposure to a mild stressor (Devine et al., 2003) Additionally, intracranial injections of N/OFQ alter hypothalamic-pituitary-adrenal axis (HPA axis ) activity and anxiety -related behaviors. Unfortunately, some studies have desc ribed increased HPA-axis activity and anxiogenesis (Devine et al., 2001; Fernand ez et al., 2004; Misilmeri and Devine, 2000; Misilmeri et al., 2002), while other studies have reported decreased HPA-axis activity and anxiolysis (Gavioli et al ., 2002; Griebel et al., 1999; Jenc k et al., 1997; Le Cudennec et al., 2002).

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3 To examine anxiety-related behaviors, standard neophobic tests are generally used, such as the open field test, elevated pl us maze, and light-dark test. These tests are based on the natural behaviors of rats, includ ing the tendency to explore during foraging activities and, on the other hand, the tende ncy to avoid open spaces due to their vulnerability to predation. For example, ra ts show thigmotaxis in the open field (for example see Simon et al., 1994), and they show a preference for the en closed arms of the elevated plus maze (Handley and Mithani, 1984; Pellow et al., 1985) and for the dark box of the light-dark test (Chaouloff et al., 1997; Costall et al., 1989; Crawley and Goodwin, 1980; Onaivi and Martin, 1989). The balance between exploration and avoidance can be manipulated in a highly reproducible manner by anxiolytic drugs (i.e., drugs that humans report to be anxiety-reducing, such as diazep am) and by anxiogenic drugs (i.e., drugs that humans report to be anxiety-inducing, such as FG 7142). Rats increa se their exploration of open or lit spaces following administration of anxiolytic compounds and decrease their exploration following administration of a nxiogenic compounds (Chaouloff et al., 1997; Costall et al., 1989; Crawle y, 1981; Crawley and Goodwin, 1980; Fernandez et al., 2004; Handley and Mithani, 1984; Hughes, 1972; Onaivi and Martin, 1989; Pellow and File, 1986; Pellow et al., 1985; Simon et al., 1994; Stefanski et al., 1992). In the present experiment, we use a modi fied version of the open field test, in which a start box was attached to one wall of the open field. This addition allows us to use latency to enter the open field and tim e spent in the open field as measures of exploratory behavior (in addi tion to thigmotactic behavior that has been reported in previous versions of the open field). Also, we divide the open field into 3 zones, a proximal peripheral zone, a distal periphera l zone, and an inner zone (see Methods

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4 section for a full description). The separate zones allow us to measure exploration near the “safety” of the start box versus exploration farther away from this “safe” zone, as well as the traditional measure of i nner zone exploration. Furtherm ore, we have calibrated the test so that our vehicle-treated rats spend approximately 25% of the test time in the open field, allowing us to easily observe both in creases and decreases in anxiety-related behaviors under one set of conditions (ligh ting, handling, etc.). Under these conditions, rats that have been treated with diazepam generally show shorter latencies to enter the open field, more total time spent in the open field, and more exploration away from the start box and in the middle of the open field, as compared to vehicletreated rats. On the other hand, rats treated with FG 7142 generally show longer latencie s to enter the open field, less total time spent in the open field, and less exploration away from the start box and in the center of the open field. These data provide evidence that the modified open field is a valid and sensitive tool for measur ing changes in expressi on of anxiety-related behaviors (Fernandez et al., 2002). Intracerebroventricular (ICV) injections of N/OFQ have been shown to increase anxiety-related behaviors in the modified open field test, the elevated plus maze, and the light-dark test (Devine et al., 2004; Fernandez et al., 2004) N/OFQ-treated rats, as compared to vehicle-treated ra ts, display longer latencies to enter the open arms of the elevated plus maze, the light box of the lig ht-dark test, and the open field of the open field test. Additionally, N/OFQ-treated rats spend less total time in the open arms, light box, and open field. These behaviors resemble the effects following injections of other anxiogenic drugs, such as FG 7142. Furthermore, injections of anxiol ytic drugs, such as diazepam, produce behavioral effects that ar e opposite to those produced by the N/OFQ

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5 injections. Specifically, injections of diazep am produce shorter latenc ies to enter open/lit areas and greater total time spent in these ar eas. These results suggest that N/OFQ has an anxiogenic action after ICV administration. In addition to these behavioral effects, ICV injections of N/OFQ increase HPA axis activity in rats. When rats are injected into the lateral ventricle under unstressed conditions (i.e., the rats are allowed to rec over from the stress of handling and cannula implantation prior to the delivery of the drug) they exhibit substa ntial elevations in circulating adrenocorticotropic hormone (ACTH) and corticosterone (CORT; Devine et al., 2001). These elevations of stress-rela ted hormones are also observed when ICV N/OFQ injections are administ ered without allowing rats to recover from the stress of handling (Nicholson et al., 2002). Additiona lly, elevations of ACTH and CORT are observed when rats are injected and then exposed to the mild stress of a novel environment or following testing in the open field (Devine et al., 2001; Fernandez et al., 2004). These N/OFQ-induced elevations in circulating hormone concentrations are mediated by limbic inputs, including the se ptum, BNST, and amygdala (Misilmeri and Devine, 2000; Misilmeri et al., 2002). Speci fically, unstressed inje ctions into these limbic structures produce elevations in circul ating ACTH and CORT. These data suggest that N/OFQ activates the HPA axis in unstr essed conditions and enhances its activity during exposure to mild stress. The behavioral and hormona l results described above c onflict with other reports that N/OFQ is anxiolytic and attenuates stre ss-induced CORT eleva tions (Gavioli et al., 2002; Griebel et al., 1999; Jenc k et al., 1997; Le Cudennec et al., 2002). The reasons for these discrepancies are currently unclear, but we have consistently observed dose-orderly

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6 anxiogenic and HPA axis-activating effect s under a variety of conditions and with experimenters who are blind to the treatment conditions. Anxiogenic actions have also been reported by Vitale and colleagues (2003), using an elevated plus maze. In light of these observations that N/ OFQ produces anxiogenic behavioral effects and activation of the HPA axis, and that th e hormonal alterations produced by N/OFQ are mediated by limbic structures, we were intere sted in whether the anxiogenic behavioral effects are also mediated by such limbic regions In particular, we examined the potential role of the amygdala, a limbic structure that is known to participat e in the regulation of behavioral and hormonal responses to fear-i nducing stimuli (for examples see Goldstein, 1965; Rogan et al., 1997; Walker and Davis, 1997).

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7 CHAPTER 2 METHODS Animals Male Long Evans rats (n = 85, Harla n, Indianapolis, IN) were housed in polycarbonate cages (43 x 21.5 x 25.5 cm) on a 1 2hr-12hr light-dark cycle (lights on at 7:00 am). The rats were pair-housed until surgery, then singly-housed in a climate-controlled vivarium (temperature 21-23o C, humidity 55-60%). Standard laboratory chow and tap water were available ad libitum All procedures were pre-approved by the University of Florid a’s Institutional Animal Care and Use Committee, and the experiments were conducte d in compliance with the NIH Guide for the Care and Use of Laboratory Animals. Drugs Ketamine and xylazine were both obtained from Henry Schein (Melville, NY) at concentrations of 100 mg/ml. Ketamine-xyl azine was mixed by adding 2 ml of xylazine to 10ml of ketamine yielding a 12ml solution of 83.3 mg/ml ketamine and 16.7 mg/ml xylazine. Ketorolac tromethamine (30 mg/m l) and AErrane (99.9 % isoflurane) were also purchased from Henry Schein. N/OFQ was obtained from Si gma-Aldrich (St. Louis, MO) and was dissolved in artificial extracellular flui d (aECF) composed of 2.0 mM Sorenson’s phosphate buffer (pH 7.4) containing 145 mM Na+, 2.7 mM K+, 1.0 mM Mg2+, 1.2 mM Ca2+, 150 mM Cl-, and 0.2 mM ascorbate. These ion concentra tions replicate the c oncentrations found in extracellular fluid in the brain (Moghaddam and Bunney, 1989) N/OFQ was prepared at

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8 concentrations of 0.01, 0.1, and 1.0 nmole per 1.0 l for ICV injections or per 0.5 l for amygdaloid injections. Surgery Each rat (260-355g) was implanted with a guide cannula under ketamine-xylazine anesthesia (62.5 mg/kg ketamine + 12.5 mg/kg xylazine, i.p. in a volume of 0.75 ml/kg). Ketorolac tromethamine (2 mg/kg, s.c.) was inje cted for analgesia at the time of surgery. During surgery, AErrane was administered as supplemental anesthesia as necessary. Once the rat was anesthetized, a stainle ss steel guide cannula (11mm, 22 gauge) was vertically implanted into the right lateral ventricle (ICV; 0.8 mm posterior to bregma, 1.4 mm lateral from the midline, a nd 2.7 mm ventral from dura; n = 35) or into the right amygdala (1.8 mm posterior to bregma 3.9 mm lateral, and 6.2 mm ventral; n = 50). The cannula was secured with dental cement anchored to stainless steel screws (0.80 x 3/32”, Plastics One Inc.). An obturator that ex tended 1.2 mm beyond th e guide cannula tip was inserted at the time of surgery and removed on the day of the experiment at the time that an intracranial injection was administered. Following surgery, each rat was injected with 1.0 ml warm 0.9% NaCl and placed in a warm, clean cage to recover from anesthesia. The rats were then returned to the vivarium where they were given 7-10 days to fully recover from surgery. Equipment Anxiety-related behavior was measured in the modified open fi eld test (see Fig. 2-1). The open field was composed of a 90 x 90 x 60 cm field with a 20 x 30 x 60 cm start box attached to the outsi de of the open field, at the midpoint of one side. The bottom and sides were constructed of black acrylic. Separating the start box and the open field was a black acrylic guillotine door attached to a rope and pulley system, which

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9 allowed the door to be opened from outside th e testing room. The tops of the start box and open field were open and a camera was mounted on the ceiling above the testing apparatus to record the rats’ behaviors. Illumination of the start box was approximately 14 lux, and illumination throughout the open fiel d was even at approximately 30 lux. Figure 2-1. Photograph of the open field and a diagram of the zones used for scoring. Zone 0 represents the start box where th e rat is placed for the first minute of the test. After the door opens and the ra t enters the open field, he may move between zone 0 and the open field free ly. Zone 1 represents the proximal periphery of the open field. Zone 2 repres ents the distal periphery, and zone 3 represents the central region of the open field. An observer who was blind to the treatm ent conditions scored the exploratory behavior of the rats from the videotapes, us ing a grid that was superimposed on the video monitor. This grid divided the open field in to 25 equal squares. The outer 16 squares defined an outer zone that was further subdivi ded such that the half of the outer zone proximal to the start box defined zone 1 and the half of the outer zone distal from the start box defined zone 2. Zone 3 was defined by the inner 9 squares. Additionally, for consistency, the start box was defined as zone 0. Latency to enter the open field (i.e. entry into zone 1), total time spent in the ope n field (sum of zones 1-3), latency to enter Zone 3 Zone1 Zone 0 Zone2

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10 and time spent in each of the 3 zones, and th e number of entries in to the open field and the inner zone 3 were used as measures of anxiety. An entry into the open field or movement from one zone to another was counted when all 4 paws of the rat left one zone and entered a new zone. Anxiety-Testing Procedure Beginning 7-10 days after surgery, each rat was handled for 5 minutes on each of 3 consecutive days, and then given one da y with no disturbance. On the 5th day, each rat was fitted with a 28-gauge stainless steel in jector connected with polyethylene (PE20) tubing to a Hamilton syringe (5 l syringe for ICV injections and 1 l syringe for amygdaloid injections) mounted in a syringe pump. Each rat then received a 1.0 l (ICV) or 0.5 l (amygdala) injection of aECF or aECF containing 0.01, 0.1, or 1.0 nmole N/OFQ by an experimenter blind to the dose. The injections were administered over a 2minute period and the injector was left in place for 3 additional minutes for diffusion. Each rat was freely moving in its home cage during the injection procedure. These injections and the subsequent behavioral tests were completed 90-210 minutes after the vivarium lights were turned on, the time during which the HPA axis is at its daily nadir (Ixart et al., 1977; Kwak et al., 1993). Five minut es after the injection, each rat was individually placed in the start box (zone 0) of the open field test, and the door to the testing room was cl osed, isolating the rat from th e experimenter and any other disruptive influences. The rat was then given 1 minut e to acclimate to the novel environment of the start box. After 1 minut e the guillotine door was opened remotely, and it remained open throughout the test period. The rat was given 5 minutes to explore the start box and open field. Each rat was then returned to its home cage until sacrifice by rapid decapitation 30 minutes afte r the start of the injection.

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11 Immediately after decapitation, 6 ml of trunk blood was collected into chilled polypropylene tubes containing 600 l of Na2EDTA at 20 g/ l. The tubes were centrifuged at 1000x gravity. The plasma fractio n was collected, aliquotted, and frozen at -80C until use. Each brain was rem oved, frozen in 2-methylbutane at -40oC, and stored at -80oC until use. Later each brain was sectioned at 30 m and stained with cresyl violet for cannula placement verification. Thymus gl ands, adrenal glands, and spleens were dissected out and weighed for verification of the health status of the rats. Radioimmunoassay (RIA) was performe d for quantification of plasma concentrations of CORT using a kit by Dia gnostic Products Corp. (Los Angeles, CA). The interassay variability for this kit increased with sample CORT concentrations, ranging from less than 5% for lower plasma CO RT concentrations to less than 15% for higher plasma CORT concentrations. Statistical Analyses All between groups differences (0, 0.01, 0.1, and 1.0 nmole N/OFQ) for latency to enter the open field and the individual zone s, total open field time and individual zone times, number of entries in to the open field and inner zone 3, plasma CORT concentrations, and organ masses were anal yzed by separate one-way ANOVAs for ICV and for amygdala placements. All signi ficant effects were further analyzed by Newman-Keuls post-tests. Standard deviations were calculated for circulating CORT concentrations for the ICV-injected rats and the amygdala-injected rats, separately. Outliers (values beyond 2 standard deviations from the mean) were re moved from further analysis. Two outlying CORT values from the ICV-implanted groups and 2 values from the amygdala-implanted groups were eliminated prior to further analysis.

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12 Anatomical misplacements occurred in 2 ICV-implanted rats and necrotic lesions at the site of injection were obs erved in 2 additional ICV-implan ted rats. These 4 rats were removed from the study. Anatomical misplacements also occurred in 9 amygdala-implanted rats. Five of the rats with extra-amygdaloid placements were treated with N/OFQ (1 rat at 1.0 nmole, 2 rats at 0.1 nmole, and 2 rats at 0.01 nmole) and are reported as anatomical controls. The remain ing 4 rats with misplacements were treated with aECF and were removed from the st udy. Additionally, 2 amygdala-implanted rats had necrotic lesions at the site of the inje ction and were also re moved from the study. Differences between anatomical controls and intra-amygdaloid vehicle controls in latency to enter the open field and the indi vidual zones, total open field time and individual zone times, number of entries in to the open field and inner zone 3, plasma CORT concentrations, and organ masses we re analyzed by individual T-tests.

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13 CHAPTER 3 RESULTS Intracerebroventricular Injections The N/OFQ-treated rats showed greater ex pression of anxiety-related behaviors than the aECF vehicle-treated rats did after ICV injections. These N/OFQ-treated rats displayed significantly longer latencies to enter the open field (i.e. zone 1; F(3,27) = 4.83, p < 0.01) than did the aECF-treated rats. The N/OFQ-treated rats also exhibited significantly longer latenc ies to enter zone 2 ( F(3,27) = 4.06, p < 0.05) and zone 3 ( F(3,27) = 3.62, p < 0.05) (Fig. 3-1A). Additionally, the N/ OFQ-treated rats spent significantly less time in the open field, at all N/OFQ doses (Fig 3-1B; F(3,27) = 6.44, p < 0.01), in comparison with the behaviors of the aECF-treat ed rats. This decrea se in open field time was also significant for zone 1 ( F(3,27) = 6.90, p < 0.01), zone 2 ( F(3,27) = 4.44, p < 0.05), and zone 3 ( F(3,27) = 4.18, p < 0.05) independently (Fig. 3-1C). The N/OFQ-treated rats also displayed significantly fewer entries in to the open field from the start box (Fig. 3-1D; F(3,27) = 8.24, p < 0.01) and significantly fewer entr ies into the inner zone 3 (Fig. 3-1E ; F(3,27) = 5.7, p < 0.01). Injections of N/OFQ into the right latera l ventricle produced significant elevations in circulating CORT at every dose administered as compared to the CORT concentrations in the aECF-treated rats (Fig. 3-2; F(3,25) = 5.6, p < 0.01).

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14 Figure 3-1. Anxiety-relate d behaviors following ICV injections of N/OFQ. N/OFQ-treated rats exhibited: (A) longer latencies to enter all 3 zones of the open field, (B) decreased total time in th e open field, (C) decr eased total time in each of the 3 zones, (D) fewer entrie s into the open fiel d from the start box and (E) fewer entries into zone 3 (the inner zone). All of these effects occurred in a manner that was generally dose-orderly. Values expressed are group means SEM ( n = 7-8 rats per group). At some doses, all rats in the group failed to enter zones 2 or 3. Thes e groups show a mean latency of 300s, a mean zone time of 0s, a mean numer of entries equal to 0, and a SEM of 0 for each of these measures. Signi ficant differences between the N/OFQ-treated rats and the aECF-treated controls (0.0 nmoles) are expressed as p < 0.05 and ** p < 0.01

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15 0 0 0. 0 1 0.1 1 0 0 .0 0 .01 0.1 1.0 0.0 0 .0 1 0 .1 1.0 0 100 200 300Zone 1 Zone 2Zone 3 ICV Latency to Enter ZonesDose N/OFQ (nmoles)* * * *Latency (seconds) 0 .0 0 .01 0 1 1.0 0 100 200 300 ICV Open Field Time ** ** *Dose N/OFQ (nmoles)Open Field Time (seconds) 0.0 0.01 0.1 1 0 0.0 0 .01 0.1 1.0 0 0 0.0 1 0 .1 1.0 0 100 200 300Zone 1Zone 2 Zone 3 ICV Time Spent in ZonesDose N/OFQ (nmoles)** ** * * *Zone Time (seconds) ICV Entries into Open Field 0 0 .0 1 0. 1 1 .0 0 1 2 3 4 5 6* ** **Dose N/OFQ (nmoles)Number of entries ICV Inner Zone Entries 0 0 01 0 .1 1.0 0 1 2 3 4 5 6* ** **Dose N/OFQ (nmoles)Number of entries A B C D E

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16 Figure 3-2. Concentrations of circulating co rticosterone following ICV injections of N/OFQ. Injections of N/OFQ into th e lateral ventricle produced dose-orderly elevations in circulating CORT at all doses administered. Values expressed are group means SEM ( n = 7-8 rats per group). Significant differences between the N/OFQ-treated rats and the aECF-treated controls are expressed as p < 0.05 and ** p < 0.01 Intra-Amygdaloid Injections The N/OFQ-treated rats with amygdaloi d implants exhibited elevations in anxiety-related behavior as i ndicated by significantly longer latencies to enter the open field (Fig. 3-3A; F(3,35) = 3.34, p < 0.05) and significantly longer latencies to enter zone 2 ( F(3,35) = 2.93, p < 0.05). However, the latencies to enter zone 3 were not significantly different from the latencies of the aECF-treated rats ( F(3,35) = 1.81, p > 0.05). The N/OFQ-treated rats did not exhibit significant di fferences from aECF-treated rats in total open field time (Fig 3-3B.; F(3,35) = 1.5, p > 0.05) or time spent in any of the 3 zones (Fig 3-3C.; F(3,35) = 0.98, p > 0.05 for zone 1; F(3,35) = 2.63, p > 0.05 for zone 2; F(3,35) = 0.3, p > 0.05 for zone 3). These rats also did not display significant differences in the number of entries into the open field (Fig. 3-3D; F(3,35) = 0.45, p > 0.05) or into the inner zone 3 (Fig. 3-3E; F(3,35) = 0.93, p > 0.05). Most of the amygdaloid placements were located in or around the basomedial (BMA) and the central (CeA) nuclei of the amygdala (see figures 3-4 and 3-5). The ICV Circulating Corticosterone 0 .0 0 .0 1 0 .1 1. 0 0 100 200 300 400 500** ** *Dose N/OFQ (nmoles)Corticosterone (ng/ml)

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17 effects of aECF injections in to these amygdaloid sites were compared against the effects of injections into extra-amygda loid sites (defined as anatom ical controls). Comparisons between the vehicle-treated rats and the N/OF Q-treated anatomical controls showed no significant differences on any m easure of latency (Fig. 3-3, A; t(12) = 0.87, p > 0.05 for open field latency, t(12) = 0.72, p > 0.05 for zone 2 latency, t(12) = 2.1, p > 0.05 for zone 3 latency). Similarly, the anatomical controls did not differ from the vehicle-treated rats on any measure of time (Fig. 3-3, B-C; t(12) = 0.46, p > 0.05 for open field time, t(12) = 0.29, p > 0.05 for zone 1 time, t(12) = 0.62, p > 0.05 for zone 2 time, t(12) = 0.49, p > 0.05 for zone 3 time), or on any measure of zone entries (Fig. 3-3, D-E; t(12) = 1.92, p > 0.05 for open field entries, and t(12) = 0.82, p > 0.05 for inner zone 3 entries). Injections of N/OFQ into the right amygda la did not produce significant changes in circulating CORT (Fig. 3-6; F(3,34) = 1.78; p > 0.05) as compared with the CORT concentrations in the aECF-treated rats. C oncentrations of circul ating CORT were not significantly different between N/OFQ-treated anatomical co ntrols and vehicle-treated rats (Fig. 3-6, t(12) = 2.10, p > 0.05).

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18 Figure 3-3. Anxiety-related be haviors following intra-amygdaloid injections of N/OFQ. N/OFQ-treated rats exhibited (A) longer latencies to enter zones 1 and 2 of the open field. However, injections of N/OFQ into the right amygdala did not significantly alter (B) total open field time, (C) time spent in any of the 3 zones, (D) the number of entries into the open field, (E) or the number of entries into the inner zone 3. Values expressed are group means SEM (n = 9-10 rats per group). Significant differ ences between the N/OFQ-treated rats and the aECF-treated controls (0.0 nmol es) are expressed as p < 0.05. AC = anatomical controls

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19 Amygdala Latency to Enter Zones 0.0 0.01 0.1 1.0 AC 0.0 0.01 0.1 1.0 AC 0.0 0.01 0.1 1.0 A C 0 100 200 300Zone 1Zone 2Zone 3Dose N/OFQ (nmoles)* *Latency (seconds) Amygdala Open Field Time 0.0 0.01 0.1 1.0 AC 0 100 200 300 Dose N/OFQ (nmoles)Open Field Time (seconds) Amygdala Time Spent in Zones 0.0 0 .01 0.1 1.0 AC 0.0 0.01 0.1 1.0 AC 0.0 0.01 0 .1 1.0 AC 0 100 200 300Zone 1Zone 2Zone 3Dose N/OFQ (nmoles)Zone Time (seconds) Amygdala Entries into Open Field 0 0.01 0.1 1.0 AC 0 1 2 3 4 5 6 Dose N/OFQ (nmoles)Number of entries Amygdala Inner Zone Entries 0 0 .01 0.1 1.0 AC 0 1 2 3 4 5 6 Dose N/OFQ (nmoles)Number of Entries A B C D E

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20 Figure 3-4. Anatomical map of amygdaloid placements showing latency to enter open field. Shown on the map are the placements for each rat, including anatomical controls. The shapes and colors of the symbols indicate the doses of N/OFQ and the latency to enter the open field for individual rats. The 1.0 nmole doses are marked by an upward facing triangle, 0.1 nmole doses are marked by a diamond, 0.01 nmole doses are marked by a downward facing triangle, and aECF controls are marked by a circ le. Additionally, anatomical controls are identified by a black dot placed within the marker. Latency to enter the open field is identified by color with red 131 seconds, orange = 45-130 seconds, yellow = 10-44 seconds, and green 9 seconds. (Atlas diagrams taken from Paxinos and Watson, 1998.)

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21

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22 Figure 3-5. Anatomical map of amygdaloid placements showing time spent in the open field. Shown on the map are the placements for each rat, including anatomical controls. The shapes and colors of the symbols indicate the doses of N/OFQ and the time spent in the open field fo r individual rats. 1.0 nmole doses are marked by an upward facing triangle, 0.1 nmole doses are marked by a diamond, 0.01 nmole doses are marked by a downward facing triangle, and aECF controls are marked by a circle. Additionally, anatomical controls are identified by a black dot placed within the marker. Amount of time spent in the open field is identified by color with red 29 seconds, orange = 30-99 seconds, yellow = 100-149 seconds, and green 150seconds. (Atlas diagrams taken from Paxinos and Watson, 1998.)

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23

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24 Figure 3-6. Concentrations of circulating corticosterone following intra-amygdaloid injections of N/OFQ. N/OFQ administ ration did not signifi cantly alter the levels of circulating corticosterone Values expressed are group means SEM ( n = 9-10 rats per group). AC = anatomical controls Thymus, Adrenal, and Spleen Masses There were no significant differences in adrenal masses (Fig. 3-7A.; ICV: F(3,26) = 0.23, p > 0.05; Amygdala: F(3,30) = 0.46, p > 0.05), thymus gland masses(Fig. 3-7B; ICV: F(3,27) = 0.30, p > 0.05; Amygdala: F(3,35) = 0.73, p > 0.05), or spleen masses (Fig. 3-7C; ICV: F(3,27) = 0.13, p > 0.05; Amygdala: F(3,35) = 0.49, p > 0.05) between the aECF and N/OFQ-treated rats for ICVor amygdala-implanted groups. Additionally, these organ masses were comparable to the masses that were found in previous experiments involving groups of rats that did or did not undergo surg ical procedures (data not shown; Fernandez et al., 2004). Amygdala Circulating Corticosterone 0.0 0 01 0.1 1 .0 AC 0 100 200 300 400 500 Dose N/OFQ ( nmoles ) Corticosterone (ng/ml)

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25 Figure 3-7. Analysis of gla ndular masses. (A) Adrenal gland masses, (B) thymus gland masses and (C) spleen masses showed no significant differences between groups, regardless of the injection site or N/OFQ treatment. Values expressed are group means the standard error of the mean (SEM) ( n = 7-9 rats per group for adrenal glands; n = 7-10 for thymus glands and spleens). Adrenal Gland Weights 0 0 0.01 0 1 1.0 0 0 0 0 1 0 1 1.0 A C 0 10 20 30 40 50 60ICV AmygdalaDose N/OFQ (nmoles)Weight (mg) Thymus Gland Weights 0 .0 0.01 0 1 1.0 0 .0 0 0 1 0 1 1.0 A C 0 100 200 300 400 500ICV AmygdalaDose N/OFQ (nmole)Weight (mg) Spleen Weights 0.0 0 01 0. 1 1 .0 0.0 0.0 1 0. 1 1 .0 AC 0 100 200 300 400 500 600 700 800 900ICV AmygdalaDose N/OFQ (nmoles)weight (mg) A B C

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26 CHAPTER 4 DISCUSSION Anxiety-Related Behavior Injections of N/OFQ into the right lateral ventricle were very effective at producing dose orderly elevations in all measured anxiety-related behavi ors. These results replicate previous findings from our lab (Fernandez et al., 2004). Injecti ons into the right amygdala also elevated anxiety -related behaviors as seen in measures of latency to enter the open field and zone 2. However, amygdaloid injections were less effective at altering the expression of these behavi ors than were the ICV inject ions, when equimolar amounts of N/OFQ were injected. There are multiple reasons why we might observe differences in potency between ICV and amygdaloid injections of N/OFQ. First, the amygdala may not be the primary site of drug action. Davis and colleagues (Lee and Davis, 1997; Walker et al., 2003) proposed that one condition for a structure to be considered a primary site of drug action is that the effects of direct injections into that structure mimic the effects of injections into the ventricle. In fact, in their stud ies of acoustic startle responses, Lee and Davis (1997) found that injections of corticotropi n releasing hormone (CRH) into the BNST, like injections of CRH into the lateral ve ntricle, produced an enhancement of rats’ acoustic startle reflexes. These increases in startle following intra-BNST injections occurred more rapidly than the enhancem ent produced by ICV inje ctions. Additionally, the increases in startle occurred at much lo wer concentrations than were required with ICV injections (40 and 80 ng intra-BNST versus 1 g ICV), although the ultimate

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27 behavioral change was not as great with th ese low concentration intra-BNST injections (70% enhancement following intra-BNST injections versus approximately 200% enhancement following ICV injections). This in addition to lesion and CRH antagonist data, demonstrates that the BNST is a prim ary site of action for the startle-enhancing effects of CRH. These results provide a model for localizing the effects of neurotransmitter action. In c ontrast, our results that equimo lar injections of N/OFQ into the amygdala produced less potent effects than in jections into the ve ntricle suggest that the amygdala may not be the primary site of action for the anxioge nic effects of NOFQ. It could be argued that the effects of our amygdaloid injections were less potent because of the fact that they were done unilaterally. However, we injected into the right amygdala, which is generally more dominantly involved in emotionally-relevant behavioral responses (Adamec et al., 2001; Andersen and Teicher, 1999; Coleman-Mesches and McGaugh, 1995a&b; Peper et al., 2001; Scicli et al., 2004), or is at least no less involved than the left amygdala (Good and West brook, 1995; Izquierdo and Murray, 2004; LaBar and LeDoux, 1996). In fact, bilateral injections may add little in terms of changes in emotionally-relevant behaviors when compared to the effects of unilateral injections into the right amygdala. For example, in one study, Coleman-Mesches and McGaugh (1995b) found impairments in the retention of inhibitory avoidance learning following in tra-amygdaloid injections of the GABA-A agonist, muscimol. Interestingly, unilateral right amygdaloid injec tions and bilateral amygdaloid injections produced equivalent degrees of learning impairment (and the impairment was minimal following unilateral in jections into the left amygdala). These data suggest that the right amygdala is domin ant in inhibitory avoidance learning tasks

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28 and the left amygdala plays little role, whic h is consistent with other studies showing right amygdaloid dominance in emotionally-rele vant behaviors. Accordingly, the fact that we did not get more potent effects with in jections into the righ t amygdala versus the ventricle further supports the contention that the amygdala is not a primary site for the anxiogenic actions of N/OFQ. Importantly, our ICV injections were also done unilaterally. Previous work in our lab with 125I-N/OFQ demonstrated that N/OFQ injected unilaterally into the lateral ventricle primarily reaches ipsilateral structures with little radioact ive label detection in the contralateral hemisphere (Devine, D.P ., unpublished). This provides further evidence that the potent effects seen following ICV inj ections in the present experiment were not likely due to bilateral actions at the amygdalae. ICV injectio ns are, however, expected to diffuse more widely than intra-amygdaloid inje ctions, and so, these injections are likely to have effects at multiple sites. Moreover, if there are multiple sites all contributing in an additive or synergistic manner to emotiona lly-relevant behavioral effects, then it is possible that no single site will meet the crit eria of a primary site of drug action. ICV injections, then, may actually produce a stronger result by affecting multiple primary sites concurrently. Another factor that could c ontribute to the apparently w eak behavioral effects of intra-amygdaloid injections of N/OFQ is that the amygdala is a very complex structure that consists of multiple inte rconnected nuclei. A number of these nuclei are involved in fear-related behaviors including the central amygdala (CeA), the basolateral nucleus (BLA), the basomedial nucleus (BMA), the lateral nucleus (LA), the medial nucleus (MeA), and the intercalated neurons (for examples s ee Bhatnagar and Dallman, 1998;

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29 Good and Westbrook, 1995; Goosen and Mare n, 2001; Par et al., 2003; Walker and Davis, 1997). However, among these nuclei, only the BLA and the MeA show high levels of NOP receptor mRNA expression and radiolabelled N/OFQ binding (Neal et al., 1999b). Additionally, the BMA and LA show some NOP mRNA expression and N/OFQ binding. However, the CeA and intercalat ed neurons show little to no NOP mRNA expression or N/OFQ binding. It is possible, then, that inje ctions of N/OFQ into these different nuclei could ha ve differing effects, introducing gr eater variability in the results. While there was some scatter in placement in the present study, most placements were in the CeA or the BMA. Although we did not statistically analyze differences between injections into these 2 sets of nuclei, there wa s no apparent difference in effect. This may be due, largely, to diffusion of N/OFQ following the injections, such that our injections affected multiple subnuclei. However, it is unclear how far our inj ections diffused within the amygdala. An alternative possibility is that another limbic site, such as the septum or BNST, is the primary site of drug action, mediati ng the behavioral effects of N/OFQ after ICV administration. In fact, the amygdala and BNST can be differentiated in terms of their roles in fear and anxiety (Walker and Davis, 1997; for review see Walker et al., 2003). Although the distinctions are not entirely clear, the amygdala appears to play a larger role in fear-related behaviors (such as startle responses to a specific, usually conditioned, stimulus), and the BNST appears to be mo re important in generalized anxiety (for example increases in startle reflexes that are not produced by a specific and immediate stimulus). The modified open field test used in the present experi ment more resembles tests of generalized anxiety, as there is no sp ecific, conditioned fear stimulus. In this

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30 respect, the BNST may be more involved in the behavioral res ponses during the open field test. In conclusion, it appears that the amy gdala plays a role in N/OFQ-induced increases in anxiety-related behaviors. Howe ver, because the behavioral effects observed in amygdala-implanted rats were not as great as those seen in ICVimplanted rats; it is evident that other structures must also be involved. The specific brain regions involved and the manner in which these regions interact to yield the anxioge nic effect of N/OFQ remain to be determined. Corticosterone ICV injections of N/OFQ enhanced HPA axis-activity following exposure of the rats to mild stress (i.e., handling and expos ure to the novel environment of the anxiety test). These data for ICV injections are cons istent with previous research (Fernandez et al., 2004). However, in this and the Ferna ndez experiment, the CORT concentrations appear to be higher after injec tion of N/OFQ at all the doses that were tested, than they were following ICV injections of equimolar N/OFQ doses in unstressed rats (Devine et al., 2001). This supports the assertion that th e handling and injecti on procedures, as well as the exposure to a novel environment (the open field), are mildly to moderately stressful for the rats. Nevertheless, the rats that were injected with N/OFQ still displayed higher levels of circulating CORT in re sponse to these stressors than did the vehicle-treated rats. These data provide ev idence that N/OFQ actions produce further enhancement of HPA axis activity beyond that produced by stressor exposure. In fact, Devine and colleagues (2001) demonstrated th at injections of N/OFQ into the lateral ventricle prolonged the CORT elevation produ ced by exposure to a mild stressor such that at 30 minutes following injection, the CO RT levels of vehicle-treated rats were

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31 returning to baseline while the CORT leve ls of N/OFQ-treated rats remained highly elevated. This suggests that N/OFQ has an enduring pharmacological effect or that it is interfering with negative feedback mechanis ms of the HPA axis (Devine et al., 2001). While we did not conduct a time course ex amination of CORT levels in the present experiment, the elevation in CORT levels observed at 30 minutes post-injection in the ICV N/OFQ-treated rats is compatible w ith the idea that ICV N/OFQ administration prolongs CORT elevation through this time point. Injections of N/OFQ into the amygdala did not enhance HPA axis activity following exposure to mild stress. This may se em inconsistent with the behavioral data; however, hormonal and behavioral responses to pharmacological manipulations can be dissociated. For example, diazepam, an anxiol ytic, does produce elevations in circulating corticosterone (Chabot et al., 1982; Ferna ndez et al., 2004; Marc and Morselli, 1969; Massoco and Palermo-Neto, 1999). Nevertheless, previous research in our lab found that intra-amygdaloid injections of N/OFQ in unstressed rats did produce elevations of circulating CORT, although the elevations we re relatively small (Misilmeri and Devine, 2000). It is possible, then, th at the experiences of handling, injection, and exposure to the open field produce stress effects in rats th at are great enough to obscure the modest effects of intra-amygdaloid N/OFQ injections on CORT concentrati ons. Additionally, if the hypothesis of Devine and colleagues ( 2001), that ICV injections of N/OFQ are reaching structures involved in negative feedba ck regulation, is correct, then it would be expected that intra-amygdaloid injecti ons of N/OFQ would not produce the same enhancement of HPA axis activity.

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32 It is important to note that the CORT data from 4 rats (2 ICV-implanted rats and 2 amygdala-implanted rats) were removed prior to analysis, as they were outlying values. Of major concern were the very low CORT concentrations (unde r 100 ng/ml) of the 2 ICV-implanted rats, consideri ng the level of stressor expos ure these rats experienced. This may have been due to high amounts of coagulation in the plasma tested, although this was not systematically recorded. For consistency, standard deviations were also calculated for the amygdala-implanted groups and outlying values were subsequently removed. The removal of the 2 outlying valu es from the 1.0 nmole ICV group did affect the final statistical analysis; however, rem oval of the 2 values from the 0.01 nmole and 1.0 nmole intra-amygdaloid groups did not affect further analysis. Organ Masses In the present study, we measured the t hymus, adrenal, and spleen masses to establish that there were no sy stematic differences in health status or stress exposure between the various groups of rats (especially since the rats underwent differing types of intracranial cannulation surgery) Thymus glands and spleens tend to decrease in mass and adrenal glands tend to increase in ma ss following exposure to physiological stressors (such as physical insults, poor diet, or expos ure to toxic chemical s) and psychological stressors (such as restraint, crowding, or predator exposure) particularly after chronic exposure to these stressors (for examples see Blanchard et al., 1998; Bryant et al., 1991; Dominguez-Gerpe and Rey-Mendez, 2001; Ha segawa and Saiki, 2002; Selye, 1936; Watzl et al., 1993). While we did not include any rats that did not undergo the stress of surgery and handling, the gland ma sses in this experiment were similar to those measured in other experiments where non-su rgical unstressed c ontrols were includ ed (Fernandez et al., 2004). Additionally, there were no significa nt differences in thymus gland masses,

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33 spleen masses, or adrenal gland masses between any of the groups test ed. Therefore, we can conclude that there were no apparent diffe rences in the health of the rats that may have affected the ultimate results of the experiment. Summary These data show that N/OFQ injections affect anxiety and HPA axis activity through actions in the amygdala (among other potential sites) suggesting the possibility that amygdaloid N/OFQ neurotransmission may be involved in regul ation of affect. However, because ICV injections of N/OFQ pr oduced greater, more potent effects than injections into the right amygda la did, additional structures must be involved. This may include actions in the contrala teral amygdala or other limbic st ructures such as the BNST or, potentially, in multiple limbic regions involving synergistic actions at these sites. To examine other structures involved, we are cu rrently conducting a number of studies. For example, we are examining the effects of inj ecting N/OFQ into the BNST to determine if the BNST contributes to, or is the primary site of action fo r, the anxiogenic behavioral effects of N/OFQ. Additionally, we are using other tools, such as in situ hybridization, to examine changes in N/OFQ and NOP expressi on throughout the brai n following social stress. These studies will help us to better understand the role of N/OFQ in the neurocircuitry of stress, anxi ety, and HPA axis functioning.

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41 BIOGRAPHICAL SKETCH Megan K. Green received her Associate of Arts in August 1998 at Okaloosa Walton College. In May 2000, she received a dual Bachelor of Arts in psychology and anthropology from the University of West Fl orida. Megan began her graduate studies in experimental psychology at the Universi ty of West Florida in August 2001. She continued graduate school at the Universi ty of Florida in August 2003, where she is currently pursuing studies in behavior al neuroscience through the psychology department.


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Title: Effects of Nociceptin/orphanin FQ Microinjections into Amygdala on Anxiety-Related Behaviors and Hypothalamic-Pituitary-Adrenal Axis Activation
Physical Description: Mixed Material
Copyright Date: 2008

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EFFECTS OF NOCICEPTIN/ORPHANIN FQ MICROINJECTIONS INTO THE
AMYGDALA ON ANXIETY-RELATED BEHAVIORS AND
HYPOTHALAMIC-PITUITARY-ADRENAL AXIS ACTIVATION















By

MEGAN K. GREEN


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2005

































Copyright 2005

by

MEGAN K. GREEN



































This document is dedicated to all who believe in the scientific pursuit of knowledge and
truth.















ACKNOWLEDGMENTS

I thank my committee members Dr. Henrietta Logan, Dr. John Petitto, and Dr.

Margaret Bradley for their assistance in the preparation of this thesis. I also, and

especially, thank my advisor, Dr. Darragh Devine, for his incredibly dedicated guidance

and support of my graduate studies.

I would also like to thank my mother, Lisa, my stepfather, Michael, and my

brother, Damion, for guiding and supporting me on my journey, in life and in academics.

I thank Terry and Karen for their support during my first 2 years of graduate training. I

thank "my" Michael for motivating me during the writing of this thesis. And I thank

Simon Poindexter, the brilliant Siamese, for keeping my lap warm and occasionally

editing this manuscript while I wrote.
















TABLE OF CONTENTS

page

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

L IST O F F IG U R E S ...... ...... .......................... ........................ .. .. ...... ............ vi

ABSTRACT .............. .............................................. vii

CHAPTER

1 IN TR O D U C TIO N ................................................ ......... .. ................ .

2 M E T H O D S ........................................................................... .7

A nim als .................................................. 7
D ru g s.................................................. 7
S u rg e ry ................................................................................. 8
E qu ipm ent .................................................................................... . 8
A nxiety-T testing P procedure .................................................................................... 10
S statistic al A n aly se s ................................................................................................ 1 1

3 R E S U L T S ...............................................................................13

Intracerebroventricular Inj sections ...................................................................... 13
Intra-Amygdaloid Injections ................. .............................. 16
Thymus, Adrenal, and Spleen Masses ............................ ......... ......... 24

4 D IS C U S S IO N ............. ......... .. .............. .. .....................................................2 6

A nxiety-Related Behavior ...................... ....................... .. ...........................26
C o rtic o ste ro n e ........................................................................................................ 3 0
O rgan M asses........................................................................................ 32
S u m m a ry ......................................................................................................3 3

L IST O F R E FE R E N C E S .............................................................................. 34

B IO G R A PH IC A L SK E T C H ........................................................................................ 4 1






v
















LIST OF FIGURES


Figure pge

2-1. Photograph of the open field and a diagram of the zones used for scoring............ 9

3-1. Anxiety-related behaviors following ICV injections of N/OFQ. .........................14

3-2. Concentrations of circulating corticosterone following ICV injections of N/OFQ .16

3-3. Anxiety-related behaviors following intra-amygdaloid injections ofN/OFQ. ........18

3-4. Anatomical map of amygdaloid placements showing latency to enter open field. .20

3-5. Anatomical map of amygdaloid placements showing time spent in the open field. 22

3-6. Concentrations of circulating corticosterone following intra-amygdaloid injections
ofN /O F Q ................................................................................24

3-7. A analysis of glandular m asses. ............................................................................ 25















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

EFFECTS OF NOCICEPTIN/ORPHANIN FQ MICROINJECTIONS INTO THE
AMYGDALA ON ANXIETY-RELATED BEHAVIORS AND
HYPOTHALAMIC-PITUITARY-ADRENAL AXIS ACTIVATION


By

Megan K. Green

December 2005

Chair: Darragh P. Devine
Major Department: Psychology

Intracerebroventricular (ICV) microinjections of the opioid-like neurotransmitter

nociceptin/orphanin FQ (N/OFQ) produce elevations in hypothalamic-pituitary-adrenal

axis (HPA axis) activity and anxiety-related behaviors in rats. Furthermore, these

increases in HPA axis activity can be produced by N/OFQ injections into a number of

limbic structures. We examined the potential role of one limbic structure, the amygdala,

in the N/OFQ-induced anxiogenic effects. Male Long Evans rats were each implanted

with a guide cannula into the right lateral ventricle or amygdala. Each rat received an

injection of N/OFQ (0, 0.01, 0.1, or 1.0 nmole) prior to behavioral testing in a neophobic

test of anxiety. In the anxiety test, each rat was placed in a start box connected to an

open field, where the rats had free access to the field for 5 minutes. Latency to enter the

open field, number of entries into the open field, total time spent in the open field, and

thigmotactic behaviors were used as measures of anxiety. Following testing, the rats









were euthanized and plasma samples were obtained for analysis of HPA axis activity.

Additionally, each rat brain was removed for cannula verification, and thymus glands,

adrenal glands, and spleens were dissected out for analysis of overall health of the rats.

N/OFQ injections produced dose-orderly increases in anxiety-related behaviors, and these

effects were greater in the ICV-implanted rats than they were in the amygdala-implanted

rats. The ICV N/OFQ-treated rats displayed longer latencies to enter the open field,

fewer numbers of entries into the open field, and less total time spent in the open field

than the ICV vehicle-treated rats did. The intra-amygdaloid N/OFQ-treated rats also

exhibited longer latencies to enter the open field, but did not differ in the number of

entries into the open field or the total time spent in the open field when compared with

the behavior of the intra-amygdaloid vehicle-treated rats. N/OFQ injections into the

lateral ventricle also produced elevations in circulating corticosterone, indicating that the

HPA axis activity was greater in these rats. However, amygdaloid injections did not

affect corticosterone levels. In conclusion, the amygdala appears to be involved in the

anxiogenic behavioral effects of N/OFQ. However, the differences in potency of effects

between ICV N/OFQ injections and intra-amygdaloid injections on anxiety-related

behaviors and circulating CORT in rats indicate that the amygdala is not the primary site

of drug action and that extra-amygdaloid sites are involved.














CHAPTER 1
INTRODUCTION

Nociceptin/Orphanin FQ (N/OFQ) and its cognate receptor NOP constitute a

highly conserved (Danielson and Dores, 1999) peptide neurotransmitter system that

affects an interesting range of very important behavioral and physiological activities.

N/OFQ is a 17 amino acid peptide that is structurally similar to the endogenous opioids,

particularly dynorphin A (Meunier et al., 1995; Reinscheid et al., 1995). However,

N/OFQ does not bind to the i, 6, or K opioid receptors with high affinity (Shimohigashi

et al., 1996), but does bind with high affinity to the NOP receptor (Butour et al., 1997;

Reinscheid et al., 1995; Shimohigashi et al., 1996). The NOP receptor is a

7-transmembrane, G-protein coupled receptor (Bunzow et al., 1994; Chen et al., 1994;

Lachowicz et al., 1995; Reinscheid et al., 1996; Wang et al., 1994; Wick et al., 1994) that

is negatively linked to adenylate cyclase (Lachowicz et al., 1995; Mollereau et al., 1994;

Reinscheid et al., 1995; Reinscheid et al., 1996), increases inward rectifying K+ channel

conductance (Connor et al., 1996a; Vaughan and Christie, 1996; Vaughan et al., 1997),

and inhibits Ca2+ conductance (Connor et al, 1996b). The NOP receptor shows high

structural homology with the opioid receptors (Bunzow et al., 1994; Chen et al., 1994;

Lachowicz et al., 1995; Mollereau et al., 1994; Wang et al., 1994; Wick et al., 1994),

although it does not bind any of the opioids with high affinity (Bunzow et al., 1994;

Butour et al., 1997; Lachowicz et al., 1995; Wang et al., 1994). This low affinity

between N/OFQ and opioid receptors and between NOP and opioid peptides suggests that

the N/OFQ-NOP system is functionally distinct from the opioid system.









N/OFQ, its precursor protein, and the NOP receptor are all found widely

distributed throughout the brain, spinal cord, and periphery (Bunzow et al., 1994; Chen et

al., 1994; Devine et al., 2003; Lachowicz et al., 1995; Neal et al., 1999a&b; Nothacker et

al., 1996; Mollereau et al., 1994; Wang et al., 1994; Wick et al., 1994), consistent with a

wide range of functions including pain modulation (Meunier et al., 1995; Reinscheid et

al., 1995; Tian et al., 1997), motor performance (Devine et al., 1996; Reinscheid et al.,

1995), spatial learning (Sandin et al., 1997; Sandin et al., 2004), and feeding (Nicholson

et al., 2002; Pomonis et al., 1996). However, localization is particularly high in limbic

regions including the hypothalamus, septum, bed nucleus of stria terminalis (BNST), and

amygdala (Bunzow et al., 1994; Devine et al., 2003; Lachowicz et al., 1995; Neal et al.,

1999a&b; Nothacker et al., 1996; Wang et al, 1994). This limbic localization is

consistent with the known role of N/OFQ in stress responses (physiological, homeostatic

responses to stimuli that represent a change or potential change to the organism's

environment; e.g., Herman and Cullinan, 1997) and anxiety responses (behavioral

responses to non-specific, potentially threatening stimuli; e.g., Walker et al., 2003). For

example, N/OFQ is released from forebrain neurons in rats following exposure to a mild

stressor (Devine et al., 2003). Additionally, intracranial injections of N/OFQ alter

hypothalamic-pituitary-adrenal axis (HPA axis) activity and anxiety-related behaviors.

Unfortunately, some studies have described increased HPA-axis activity and

anxiogenesis (Devine et al., 2001; Fernandez et al., 2004; Misilmeri and Devine, 2000;

Misilmeri et al., 2002), while other studies have reported decreased HPA-axis activity

and anxiolysis (Gavioli et al., 2002; Griebel et al., 1999; Jenck et al., 1997; Le Cudennec

et al., 2002).









To examine anxiety-related behaviors, standard neophobic tests are generally

used, such as the open field test, elevated plus maze, and light-dark test. These tests are

based on the natural behaviors of rats, including the tendency to explore during foraging

activities and, on the other hand, the tendency to avoid open spaces due to their

vulnerability to predation. For example, rats show thigmotaxis in the open field (for

example see Simon et al., 1994), and they show a preference for the enclosed arms of the

elevated plus maze (Handley and Mithani, 1984; Pellow et al., 1985) and for the dark box

of the light-dark test (Chaouloff et al., 1997; Costall et al., 1989; Crawley and Goodwin,

1980; Onaivi and Martin, 1989). The balance between exploration and avoidance can be

manipulated in a highly reproducible manner by anxiolytic drugs (i.e., drugs that humans

report to be anxiety-reducing, such as diazepam) and by anxiogenic drugs (i.e., drugs that

humans report to be anxiety-inducing, such as FG 7142). Rats increase their exploration

of open or lit spaces following administration of anxiolytic compounds and decrease their

exploration following administration of anxiogenic compounds (Chaouloff et al., 1997;

Costall et al., 1989; Crawley, 1981; Crawley and Goodwin, 1980; Fernandez et al., 2004;

Handley and Mithani, 1984; Hughes, 1972; Onaivi and Martin, 1989; Pellow and File,

1986; Pellow et al., 1985; Simon et al., 1994; Stefanski et al., 1992).

In the present experiment, we use a modified version of the open field test, in

which a start box was attached to one wall of the open field. This addition allows us to

use latency to enter the open field and time spent in the open field as measures of

exploratory behavior (in addition to thigmotactic behavior that has been reported in

previous versions of the open field). Also, we divide the open field into 3 zones, a

proximal peripheral zone, a distal peripheral zone, and an inner zone (see Methods









section for a full description). The separate zones allow us to measure exploration near

the "safety" of the start box versus exploration farther away from this "safe" zone, as well

as the traditional measure of inner zone exploration. Furthermore, we have calibrated the

test so that our vehicle-treated rats spend approximately 25% of the test time in the open

field, allowing us to easily observe both increases and decreases in anxiety-related

behaviors under one set of conditions (lighting, handling, etc.). Under these conditions,

rats that have been treated with diazepam generally show shorter latencies to enter the

open field, more total time spent in the open field, and more exploration away from the

start box and in the middle of the open field, as compared to vehicle-treated rats. On the

other hand, rats treated with FG 7142 generally show longer latencies to enter the open

field, less total time spent in the open field, and less exploration away from the start box

and in the center of the open field. These data provide evidence that the modified open

field is a valid and sensitive tool for measuring changes in expression of anxiety-related

behaviors (Fernandez et al., 2002).

Intracerebroventricular (ICV) injections of N/OFQ have been shown to increase

anxiety-related behaviors in the modified open field test, the elevated plus maze, and the

light-dark test (Devine et al., 2004; Fernandez et al., 2004). N/OFQ-treated rats, as

compared to vehicle-treated rats, display longer latencies to enter the open arms of the

elevated plus maze, the light box of the light-dark test, and the open field of the open

field test. Additionally, N/OFQ-treated rats spend less total time in the open arms, light

box, and open field. These behaviors resemble the effects following injections of other

anxiogenic drugs, such as FG 7142. Furthermore, injections of anxiolytic drugs, such as

diazepam, produce behavioral effects that are opposite to those produced by the N/OFQ









injections. Specifically, injections of diazepam produce shorter latencies to enter open/lit

areas and greater total time spent in these areas. These results suggest that N/OFQ has an

anxiogenic action after ICV administration.

In addition to these behavioral effects, ICV injections of N/OFQ increase HPA axis

activity in rats. When rats are injected into the lateral ventricle under unstressed

conditions (i.e., the rats are allowed to recover from the stress of handling and cannula

implantation prior to the delivery of the drug), they exhibit substantial elevations in

circulating adrenocorticotropic hormone (ACTH) and corticosterone (CORT; Devine et

al., 2001). These elevations of stress-related hormones are also observed when ICV

N/OFQ injections are administered without allowing rats to recover from the stress of

handling (Nicholson et al., 2002). Additionally, elevations of ACTH and CORT are

observed when rats are injected and then exposed to the mild stress of a novel

environment or following testing in the open field (Devine et al., 2001; Fernandez et al.,

2004). These N/OFQ-induced elevations in circulating hormone concentrations are

mediated by limbic inputs, including the septum, BNST, and amygdala (Misilmeri and

Devine, 2000; Misilmeri et al., 2002). Specifically, unstressed injections into these

limbic structures produce elevations in circulating ACTH and CORT. These data suggest

that N/OFQ activates the HPA axis in unstressed conditions and enhances its activity

during exposure to mild stress.

The behavioral and hormonal results described above conflict with other reports

that N/OFQ is anxiolytic and attenuates stress-induced CORT elevations (Gavioli et al.,

2002; Griebel et al., 1999; Jenck et al., 1997; Le Cudennec et al., 2002). The reasons for

these discrepancies are currently unclear, but we have consistently observed dose-orderly









anxiogenic and HPA axis-activating effects under a variety of conditions and with

experimenters who are blind to the treatment conditions. Anxiogenic actions have also

been reported by Vitale and colleagues (2003), using an elevated plus maze.

In light of these observations that N/OFQ produces anxiogenic behavioral effects

and activation of the HPA axis, and that the hormonal alterations produced by N/OFQ are

mediated by limbic structures, we were interested in whether the anxiogenic behavioral

effects are also mediated by such limbic regions. In particular, we examined the potential

role of the amygdala, a limbic structure that is known to participate in the regulation of

behavioral and hormonal responses to fear-inducing stimuli (for examples see Goldstein,

1965; Rogan et al., 1997; Walker and Davis, 1997).














CHAPTER 2
METHODS

Animals

Male Long Evans rats (n = 85, Harlan, Indianapolis, IN) were housed in

polycarbonate cages (43 x 21.5 x 25.5 cm) on a 12hr-12hr light-dark cycle (lights on at

7:00 am). The rats were pair-housed until surgery, then singly-housed in a

climate-controlled vivarium (temperature 21-23o C, humidity 55-60%). Standard

laboratory chow and tap water were available ad libitum. All procedures were

pre-approved by the University of Florida's Institutional Animal Care and Use

Committee, and the experiments were conducted in compliance with the NIH Guide for

the Care and Use of Laboratory Animals.

Drugs

Ketamine and xylazine were both obtained from Henry Schein (Melville, NY) at

concentrations of 100 mg/ml. Ketamine-xylazine was mixed by adding 2 ml ofxylazine

to 10ml of ketamine yielding a 12ml solution of 83.3 mg/ml ketamine and 16.7 mg/ml

xylazine. Ketorolac tromethamine (30 mg/ml) and AErrane (99.9 % isoflurane) were

also purchased from Henry Schein.

N/OFQ was obtained from Sigma-Aldrich (St. Louis, MO) and was dissolved in

artificial extracellular fluid (aECF) composed of 2.0 mM Sorenson's phosphate buffer

(pH 7.4) containing 145 mM Na+, 2.7 mM K+, 1.0 mM Mg2+, 1.2 mM Ca2+, 150 mM C1-,

and 0.2 mM ascorbate. These ion concentrations replicate the concentrations found in

extracellular fluid in the brain (Moghaddam and Bunney, 1989). N/OFQ was prepared at









concentrations of 0.01, 0.1, and 1.0 nmole per 1.0 pl for ICV injections or per 0.5 pl for

amygdaloid injections.

Surgery

Each rat (260-3 5 5g) was implanted with a guide cannula under ketamine-xylazine

anesthesia (62.5 mg/kg ketamine + 12.5 mg/kg xylazine, i.p. in a volume of 0.75 ml/kg).

Ketorolac tromethamine (2 mg/kg, s.c.) was injected for analgesia at the time of surgery.

During surgery, AErrane was administered as supplemental anesthesia as necessary.

Once the rat was anesthetized, a stainless steel guide cannula (11mm, 22 gauge) was

vertically implanted into the right lateral ventricle (ICV; 0.8 mm posterior to bregma, 1.4

mm lateral from the midline, and 2.7 mm ventral from dura; n = 35) or into the right

amygdala (1.8 mm posterior to bregma, 3.9 mm lateral, and 6.2 mm ventral; n = 50). The

cannula was secured with dental cement anchored to stainless steel screws (0.80 x 3/32",

Plastics One Inc.). An obturator that extended 1.2 mm beyond the guide cannula tip was

inserted at the time of surgery and removed on the day of the experiment at the time that

an intracranial injection was administered. Following surgery, each rat was injected with

1.0 ml warm 0.9% NaCl and placed in a warm, clean cage to recover from anesthesia.

The rats were then returned to the vivarium where they were given 7-10 days to fully

recover from surgery.

Equipment

Anxiety-related behavior was measured in the modified open field test (see Fig.

2-1). The open field was composed of a 90 x 90 x 60 cm field with a 20 x 30 x 60 cm

start box attached to the outside of the open field, at the midpoint of one side. The

bottom and sides were constructed of black acrylic. Separating the start box and the open

field was a black acrylic guillotine door attached to a rope and pulley system, which









allowed the door to be opened from outside the testing room. The tops of the start box

and open field were open and a camera was mounted on the ceiling above the testing

apparatus to record the rats' behaviors. Illumination of the start box was approximately

14 lux, and illumination throughout the open field was even at approximately 30 lux.


Figure 2-1.Photograph of the open field and a diagram of the zones used for scoring.
Zone 0 represents the start box where the rat is placed for the first minute of
the test. After the door opens and the rat enters the open field, he may move
between zone 0 and the open field freely. Zone 1 represents the proximal
periphery of the open field. Zone 2 represents the distal periphery, and zone 3
represents the central region of the open field.


An observer who was blind to the treatment conditions scored the exploratory

behavior of the rats from the videotapes, using a grid that was superimposed on the video

monitor. This grid divided the open field into 25 equal squares. The outer 16 squares

defined an outer zone that was further subdivided such that the half of the outer zone

proximal to the start box defined zone 1 and the half of the outer zone distal from the start

box defined zone 2. Zone 3 was defined by the inner 9 squares. Additionally, for

consistency, the start box was defined as zone 0. Latency to enter the open field (i.e.

entry into zone 1), total time spent in the open field (sum of zones 1-3), latency to enter









and time spent in each of the 3 zones, and the number of entries into the open field and

the inner zone 3 were used as measures of anxiety. An entry into the open field or

movement from one zone to another was counted when all 4 paws of the rat left one zone

and entered a new zone.

Anxiety-Testing Procedure

Beginning 7-10 days after surgery, each rat was handled for 5 minutes on each of 3

consecutive days, and then given one day with no disturbance. On the 5th day, each rat

was fitted with a 28-gauge stainless steel injector connected with polyethylene (PE20)

tubing to a Hamilton syringe (5 [l syringe for ICV injections and 1 [l syringe for

amygdaloid injections) mounted in a syringe pump. Each rat then received a 1.0 pl (ICV)

or 0.5 [l (amygdala) injection of aECF or aECF containing 0.01, 0.1, or 1.0 nmole

N/OFQ by an experimenter blind to the dose. The injections were administered over a 2-

minute period and the injector was left in place for 3 additional minutes for diffusion.

Each rat was freely moving in its home cage during the injection procedure.

These injections and the subsequent behavioral tests were completed 90-210

minutes after the vivarium lights were turned on, the time during which the HPA axis is

at its daily nadir (Ixart et al., 1977; Kwak et al., 1993). Five minutes after the injection,

each rat was individually placed in the start box (zone 0) of the open field test, and the

door to the testing room was closed, isolating the rat from the experimenter and any other

disruptive influences. The rat was then given 1 minute to acclimate to the novel

environment of the start box. After 1 minute the guillotine door was opened remotely,

and it remained open throughout the test period. The rat was given 5 minutes to explore

the start box and open field. Each rat was then returned to its home cage until sacrifice

by rapid decapitation 30 minutes after the start of the injection.









Immediately after decapitation, 6 ml of trunk blood was collected into chilled

polypropylene tubes containing 600 ul of Na2EDTA at 20 gg/dl. The tubes were

centrifuged at 1000x gravity. The plasma fraction was collected, aliquotted, and frozen at

-80C until use. Each brain was removed, frozen in 2-methylbutane at -400C, and stored

at -80C until use. Later each brain was sectioned at 30 tm and stained with cresyl violet

for cannula placement verification. Thymus glands, adrenal glands, and spleens were

dissected out and weighed for verification of the health status of the rats.

Radioimmunoassay (RIA) was performed for quantification of plasma

concentrations of CORT using a kit by Diagnostic Products Corp. (Los Angeles, CA).

The interassay variability for this kit increased with sample CORT concentrations,

ranging from less than 5% for lower plasma CORT concentrations to less than 15% for

higher plasma CORT concentrations.

Statistical Analyses

All between groups differences (0, 0.01, 0.1, and 1.0 nmole N/OFQ) for latency to

enter the open field and the individual zones, total open field time and individual zone

times, number of entries into the open field and inner zone 3, plasma CORT

concentrations, and organ masses were analyzed by separate one-way ANOVAs for ICV

and for amygdala placements. All significant effects were further analyzed by

Newman-Keuls post-tests.

Standard deviations were calculated for circulating CORT concentrations for the

ICV-injected rats and the amygdala-injected rats, separately. Outliers (values beyond 2

standard deviations from the mean) were removed from further analysis. Two outlying

CORT values from the ICV-implanted groups and 2 values from the amygdala-implanted

groups were eliminated prior to further analysis.









Anatomical misplacements occurred in 2 ICV-implanted rats and necrotic lesions at

the site of injection were observed in 2 additional ICV-implanted rats. These 4 rats were

removed from the study. Anatomical misplacements also occurred in 9

amygdala-implanted rats. Five of the rats with extra-amygdaloid placements were treated

with N/OFQ (1 rat at 1.0 nmole, 2 rats at 0.1 nmole, and 2 rats at 0.01 nmole) and are

reported as anatomical controls. The remaining 4 rats with misplacements were treated

with aECF and were removed from the study. Additionally, 2 amygdala-implanted rats

had necrotic lesions at the site of the injection and were also removed from the study.

Differences between anatomical controls and intra-amygdaloid vehicle controls in

latency to enter the open field and the individual zones, total open field time and

individual zone times, number of entries into the open field and inner zone 3, plasma

CORT concentrations, and organ masses were analyzed by individual T-tests.














CHAPTER 3
RESULTS

Intracerebroventricular Injections

The N/OFQ-treated rats showed greater expression of anxiety-related behaviors

than the aECF vehicle-treated rats did after ICV injections. These N/OFQ-treated rats

displayed significantly longer latencies to enter the open field (i.e. zone 1; F(3,2) = 4.83, p

< 0.01) than did the aECF-treated rats. The N/OFQ-treated rats also exhibited

significantly longer latencies to enter zone 2 (F(3,2) = 4.06, p < 0.05) and zone 3 (F(3,2) =

3.62, p < 0.05) (Fig. 3-1A). Additionally, the N/OFQ-treated rats spent significantly less

time in the open field, at all N/OFQ doses (Fig 3-1B; F(3,27) = 6.44,p < 0.01), in

comparison with the behaviors of the aECF-treated rats. This decrease in open field time

was also significant for zone 1 (F(3,27) = 6.90, p < 0.01), zone 2 (F(3,27) = 4.44, p < 0.05),

and zone 3 (F(3,27 = 4.18,p < 0.05) independently (Fig. 3-1C). The N/OFQ-treated rats

also displayed significantly fewer entries into the open field from the start box (Fig.

3-1D; F(3,27 = 8.24, p < 0.01) and significantly fewer entries into the inner zone 3 (Fig.

3-1E ; F(3,2 = 5.7,p < 0.01).

Injections of N/OFQ into the right lateral ventricle produced significant elevations

in circulating CORT at every dose administered as compared to the CORT concentrations

in the aECF-treated rats (Fig. 3-2; F(3,25) = 5.6, p < 0.01).






14


Figure 3-1. Anxiety-related behaviors following ICV injections of N/OFQ.
N/OFQ-treated rats exhibited: (A) longer latencies to enter all 3 zones of the
open field, (B) decreased total time in the open field, (C) decreased total time
in each of the 3 zones, (D) fewer entries into the open field from the start box,
and (E) fewer entries into zone 3 (the inner zone). All of these effects
occurred in a manner that was generally dose-orderly. Values expressed are
group means + SEM (n = 7-8 rats per group). At some doses, all rats in the
group failed to enter zones 2 or 3. These groups show a mean latency of 300s,
a mean zone time of Os, a mean number of entries equal to 0, and a SEM of 0
for each of these measures. Significant differences between the
N/OFQ-treated rats and the aECF-treated controls (0.0 nmoles) are expressed
as *p < 0.05 and **p < 0.01













A ICV
Latency to Enter Zones
** ** **


Zone 1 Zone 2 Zone 3
Dose NIOFQ (nmoles)


ICV
Open Field Time


** **


ICV
C Time Spent in Zones


* *


Dose NIOFQ (nmoles)




ICV
D Entries into Open Field


w 5-
5-

S4-
0 3-

.0 2
.-
E
z 1-


Dose NIOFQ (nmoles)


Zone 1 Zone 2 Zone 3

Dose NIOFQ (nmoles)


ICV
Inner Zone Entries


*


Dose OFQ (n
Dose NIOFQ (nmoles)


H


=m=A


1 1l- ) **










ICV
Circulating Corticosterone
500-
E .**
S400- ** *

300-


1 100-
0
0 .

Dose NIOFQ (nmoles)

Figure 3-2. Concentrations of circulating corticosterone following ICV injections of
N/OFQ. Injections of N/OFQ into the lateral ventricle produced dose-orderly
elevations in circulating CORT at all doses administered. Values expressed
are group means + SEM (n = 7-8 rats per group). Significant differences
between the N/OFQ-treated rats and the aECF-treated controls are expressed
as *p < 0.05 and **p < 0.01

Intra-Amygdaloid Injections

The N/OFQ-treated rats with amygdaloid implants exhibited elevations in

anxiety-related behavior as indicated by significantly longer latencies to enter the open

field (Fig. 3-3A; F(3,35) = 3.34, p < 0.05) and significantly longer latencies to enter zone 2

(F(3,35) = 2.93, p < 0.05). However, the latencies to enter zone 3 were not significantly

different from the latencies of the aECF-treated rats (F(3,3 = 1.81, p > 0.05). The

N/OFQ-treated rats did not exhibit significant differences from aECF-treated rats in total

open field time (Fig 3-3B.; F(3,3) = 1.5,p > 0.05) or time spent in any of the 3 zones (Fig

3-3C.; F(3,3s) = 0.98, p > 0.05 for zone 1; F(3,35) = 2.63, p > 0.05 for zone 2; F(3,35) = 0.3, p

> 0.05 for zone 3). These rats also did not display significant differences in the number

of entries into the open field (Fig. 3-3D; F(3,35) = 0.45, p > 0.05) or into the inner zone 3

(Fig. 3-3E; F(3,35) = 0.93,p > 0.05).

Most of the amygdaloid placements were located in or around the basomedial

(BMA) and the central (CeA) nuclei of the amygdala (see figures 3-4 and 3-5). The









effects of aECF injections into these amygdaloid sites were compared against the effects

of injections into extra-amygdaloid sites (defined as anatomical controls). Comparisons

between the vehicle-treated rats and the N/OFQ-treated anatomical controls showed no

significant differences on any measure of latency (Fig. 3-3, A; t(2) = 0.87,p > 0.05 for

open field latency, t(12) = 0.72, p > 0.05 for zone 2 latency, t(2) =2.1, p > 0.05 for zone 3

latency). Similarly, the anatomical controls did not differ from the vehicle-treated rats on

any measure of time (Fig. 3-3, B-C; t(2 = 0.46, p > 0.05 for open field time, t12) = 0.29,

p > 0.05 for zone 1 time, t2) = 0.62, p > 0.05 for zone 2 time, t12) 0.49, p > 0.05 for

zone 3 time), or on any measure of zone entries (Fig. 3-3, D-E; t( ) = 1.92,p > 0.05 for

open field entries, and t(2) = 0.82, p > 0.05 for inner zone 3 entries).

Injections of N/OFQ into the right amygdala did not produce significant changes in

circulating CORT (Fig. 3-6; F(3,34 = 1.78; p > 0.05) as compared with the CORT

concentrations in the aECF-treated rats. Concentrations of circulating CORT were not

significantly different between N/OFQ-treated anatomical controls and vehicle-treated

rats (Fig. 3-6, t() = 2.10, p > 0.05).






18


Figure 3-3. Anxiety-related behaviors following intra-amygdaloid injections of N/OFQ.
N/OFQ-treated rats exhibited (A) longer latencies to enter zones 1 and 2 of the
open field. However, injections ofN/OFQ into the right amygdala did not
significantly alter (B) total open field time, (C) time spent in any of the 3
zones, (D) the number of entries into the open field, (E) or the number of
entries into the inner zone 3. Values expressed are group means + SEM (n =
9-10 rats per group). Significant differences between the N/OFQ-treated rats
and the aECF-treated controls (0.0 nmoles) are expressed as p < 0.05. AC =
anatomical controls







19


A Amygdala
Latency to Enter Zones
300-
*
o
0 200.


S100


0
5 ,:j~," O ^La~~p obol,P i


Zone 1 Zone 2
Dose N/OFQ (nmoles)


Zone 3


B

- 300-
0
U
200-
E
S100-
I-

0 0-


Amygdala
Open Field Time


Amygdala
Time Spent in Zones


0
u 200-

E
F 100
0


nr


Dose N/OFQ (nmoles)


Amygdala
D Entries into Open Field
6-
5-
4-
o 3
S2-
E

0 1- 1 1 1

Dose N/OFQ (nmoles)


is is, i


Zone 1 Zone 2
Dose N/OFQ (nmoles)

Amygdala
Inner Zone Entries


Zone 3


Dose N/OFQ (nmoles)


-'--


I1 .ii


nhr~~ ~n.rh






20




Figure 3-4. Anatomical map of amygdaloid placements showing latency to enter open
field. Shown on the map are the placements for each rat, including anatomical
controls. The shapes and colors of the symbols indicate the doses of N/OFQ
and the latency to enter the open field for individual rats. The 1.0 nmole
doses are marked by an upward facing triangle, 0.1 nmole doses are marked
by a diamond, 0.01 nmole doses are marked by a downward facing triangle,
and aECF controls are marked by a circle. Additionally, anatomical controls
are identified by a black dot placed within the marker. Latency to enter the
open field is identified by color with red > 131 seconds, orange = 45-130
seconds, yellow = 10-44 seconds, and green < 9 seconds. (Atlas diagrams
taken from Paxinos and Watson, 1998.)













Amygdala Placements
Latency in sec Dose NOFQ
Bregma -0.80 m 131+ A 1.Onmole
45-130 -
10-44 < 0.1 nmole
0-9 0.01 nmole

0 0 nmole] aECF
Bregma -0.92 m Anatomical
Control






Bregma 1.30 tmm






Bregma -1.40 mm





Bregma -1.60 mm





Bregma -1.80 mm







Bregma -1.88 mm






Bregma 2.12 mm







Bregma -2 30 mm





\ Bregma -2.56 mm


-2.80 mm






22



Figure 3-5. Anatomical map of amygdaloid placements showing time spent in the open
field. Shown on the map are the placements for each rat, including anatomical
controls. The shapes and colors of the symbols indicate the doses of N/OFQ
and the time spent in the open field for individual rats. 1.0 nmole doses are
marked by an upward facing triangle, 0.1 nmole doses are marked by a
diamond, 0.01 nmole doses are marked by a downward facing triangle, and
aECF controls are marked by a circle. Additionally, anatomical controls are
identified by a black dot placed within the marker. Amount of time spent in
the open field is identified by color with red < 29 seconds, orange = 30-99
seconds, yellow = 100-149 seconds, and green > 150seconds. (Atlas diagrams
taken from Paxinos and Watson, 1998.)













Amygdala Placements
Open Field Time in sec Dose N/OFQ
Bregma -0.80mm 0-29 A 1 0 mole
30-99 -
100-149 0.l1 nole
150+ V 0.01 Onmole

0 0 mnolet aECF
Bregma -0.92 mnm n Anatomical
Control







Bregma -1 30 mm






Bregma -1.40 mm





Bregma -1.60 mria






Bregma -1.80 mm






Bregma -1 88 nns






Bregma -2.12 mm






Bregma -2.30 mm







\ Bregma -2.56 nn


-2.80 mm










Amygdala
Circulating Corticosterone
500-

S400-

S300-
0
200-

S100-
0



Dose NIOFQ (nmoles)

Figure 3-6. Concentrations of circulating corticosterone following intra-amygdaloid
injections of N/OFQ. N/OFQ administration did not significantly alter the
levels of circulating corticosterone. Values expressed are group means +
SEM (n = 9-10 rats per group). AC = anatomical controls




Thymus, Adrenal, and Spleen Masses

There were no significant differences in adrenal masses (Fig. 3-7A.; ICV: F(3,26) =

0.23, p > 0.05; Amygdala: F(3,3) = 0.46, p > 0.05), thymus gland masses(Fig. 3-7B; ICV:

F(3,27) = 0.30, p > 0.05; Amygdala: F(3,35) = 0.73,p > 0.05), or spleen masses (Fig. 3-7C;

ICV: F(3,27) = 0.13, p > 0.05; Amygdala: F(3,35) = 0.49, p > 0.05) between the aECF and

N/OFQ-treated rats for ICV- or amygdala-implanted groups. Additionally, these organ

masses were comparable to the masses that were found in previous experiments involving

groups of rats that did or did not undergo surgical procedures (data not shown; Fernandez

et al., 2004).










A
Adrenal Gland Weights
o60-
50-
40-
E
M30-
20-
10



ICV Amygdala
Dose N/OFQ (nmoles)

B
Thymus Gland Weights

400-
30-

._P 200
100-
I I II I

ICV Amygdala
Dose N/OFQ (nmole)

C
Spleen Weights
900-
800-
700-
S600-
E
4-00


100
1 1 1 1

ICV Amygdala
Dose N/OFQ (nmoles)




Figure 3-7. Analysis of glandular masses. (A) Adrenal gland masses, (B) thymus gland
masses and (C) spleen masses showed no significant differences between
groups, regardless of the injection site or N/OFQ treatment. Values expressed
are group means + the standard error of the mean (SEM) (n = 7-9 rats per
group for adrenal glands; n = 7-10 for thymus glands and spleens).














CHAPTER 4
DISCUSSION

Anxiety-Related Behavior

Injections of N/OFQ into the right lateral ventricle were very effective at producing

dose orderly elevations in all measured anxiety-related behaviors. These results replicate

previous findings from our lab (Fernandez et al., 2004). Injections into the right

amygdala also elevated anxiety-related behaviors as seen in measures of latency to enter

the open field and zone 2. However, amygdaloid injections were less effective at altering

the expression of these behaviors than were the ICV injections, when equimolar amounts

of N/OFQ were injected.

There are multiple reasons why we might observe differences in potency between

ICV and amygdaloid injections of N/OFQ. First, the amygdala may not be the primary

site of drug action. Davis and colleagues (Lee and Davis, 1997; Walker et al., 2003)

proposed that one condition for a structure to be considered a primary site of drug action

is that the effects of direct injections into that structure mimic the effects of injections

into the ventricle. In fact, in their studies of acoustic startle responses, Lee and Davis

(1997) found that injections of corticotropin releasing hormone (CRH) into the BNST,

like injections of CRH into the lateral ventricle, produced an enhancement of rats'

acoustic startle reflexes. These increases in startle following intra-BNST injections

occurred more rapidly than the enhancement produced by ICV injections. Additionally,

the increases in startle occurred at much lower concentrations than were required with

ICV injections (40 and 80 ng intra-BNST versus 1 tg ICV), although the ultimate









behavioral change was not as great with these low concentration intra-BNST injections

(70% enhancement following intra-BNST injections versus approximately 200%

enhancement following ICV injections). This, in addition to lesion and CRH antagonist

data, demonstrates that the BNST is a primary site of action for the startle-enhancing

effects of CRH. These results provide a model for localizing the effects of

neurotransmitter action. In contrast, our results that equimolar injections of N/OFQ into

the amygdala produced less potent effects than injections into the ventricle suggest that

the amygdala may not be the primary site of action for the anxiogenic effects of NOFQ.

It could be argued that the effects of our amygdaloid injections were less potent

because of the fact that they were done unilaterally. However, we injected into the right

amygdala, which is generally more dominantly involved in emotionally-relevant

behavioral responses (Adamec et al., 2001; Andersen and Teicher, 1999;

Coleman-Mesches and McGaugh, 1995a&b; Peper et al., 2001; Scicli et al., 2004), or is

at least no less involved than the left amygdala (Good and Westbrook, 1995; Izquierdo

and Murray, 2004; LaBar and LeDoux, 1996). In fact, bilateral injections may add little

in terms of changes in emotionally-relevant behaviors when compared to the effects of

unilateral injections into the right amygdala. For example, in one study,

Coleman-Mesches and McGaugh (1995b) found impairments in the retention of

inhibitory avoidance learning following intra-amygdaloid injections of the GABA-A

agonist, muscimol. Interestingly, unilateral right amygdaloid injections and bilateral

amygdaloid injections produced equivalent degrees of learning impairment (and the

impairment was minimal following unilateral injections into the left amygdala). These

data suggest that the right amygdala is dominant in inhibitory avoidance learning tasks









and the left amygdala plays little role, which is consistent with other studies showing

right amygdaloid dominance in emotionally-relevant behaviors. Accordingly, the fact

that we did not get more potent effects with injections into the right amygdala versus the

ventricle further supports the contention that the amygdala is not a primary site for the

anxiogenic actions of N/OFQ.

Importantly, our ICV injections were also done unilaterally. Previous work in our

lab with 125I-N/OFQ demonstrated that N/OFQ injected unilaterally into the lateral

ventricle primarily reaches ipsilateral structures, with little radioactive label detection in

the contralateral hemisphere (Devine, D.P., unpublished). This provides further evidence

that the potent effects seen following ICV injections in the present experiment were not

likely due to bilateral actions at the amygdalae. ICV injections are, however, expected to

diffuse more widely than intra-amygdaloid injections, and so, these injections are likely

to have effects at multiple sites. Moreover, if there are multiple sites all contributing in

an additive or synergistic manner to emotionally-relevant behavioral effects, then it is

possible that no single site will meet the criteria of a primary site of drug action. ICV

injections, then, may actually produce a stronger result by affecting multiple primary sites

concurrently.

Another factor that could contribute to the apparently weak behavioral effects of

intra-amygdaloid injections of N/OFQ is that the amygdala is a very complex structure

that consists of multiple interconnected nuclei. A number of these nuclei are involved in

fear-related behaviors including the central amygdala (CeA), the basolateral nucleus

(BLA), the basomedial nucleus (BMA), the lateral nucleus (LA), the medial nucleus

(MeA), and the intercalated neurons (for examples see Bhatnagar and Dallman, 1998;









Good and Westbrook, 1995; Goosen and Maren, 2001; Pare et al., 2003; Walker and

Davis, 1997). However, among these nuclei, only the BLA and the MeA show high

levels of NOP receptor mRNA expression and radiolabelled N/OFQ binding (Neal et al.,

1999b). Additionally, the BMA and LA show some NOP mRNA expression and N/OFQ

binding. However, the CeA and intercalated neurons show little to no NOP mRNA

expression or N/OFQ binding. It is possible, then, that injections of N/OFQ into these

different nuclei could have differing effects, introducing greater variability in the results.

While there was some scatter in placement in the present study, most placements were in

the CeA or the BMA. Although we did not statistically analyze differences between

injections into these 2 sets of nuclei, there was no apparent difference in effect. This may

be due, largely, to diffusion of N/OFQ following the injections, such that our injections

affected multiple subnuclei. However, it is unclear how far our injections diffused within

the amygdala.

An alternative possibility is that another limbic site, such as the septum or BNST,

is the primary site of drug action, mediating the behavioral effects of N/OFQ after ICV

administration. In fact, the amygdala and BNST can be differentiated in terms of their

roles in fear and anxiety (Walker and Davis, 1997; for review see Walker et al., 2003).

Although the distinctions are not entirely clear, the amygdala appears to play a larger role

in fear-related behaviors (such as startle responses to a specific, usually conditioned,

stimulus), and the BNST appears to be more important in generalized anxiety (for

example increases in startle reflexes that are not produced by a specific and immediate

stimulus). The modified open field test used in the present experiment more resembles

tests of generalized anxiety, as there is no specific, conditioned fear stimulus. In this









respect, the BNST may be more involved in the behavioral responses during the open

field test.

In conclusion, it appears that the amygdala plays a role in N/OFQ-induced

increases in anxiety-related behaviors. However, because the behavioral effects observed

in amygdala-implanted rats were not as great as those seen in ICV-implanted rats; it is

evident that other structures must also be involved. The specific brain regions involved

and the manner in which these regions interact to yield the anxiogenic effect of N/OFQ

remain to be determined.

Corticosterone

ICV injections of N/OFQ enhanced HPA axis-activity following exposure of the

rats to mild stress (i.e., handling and exposure to the novel environment of the anxiety

test). These data for ICV injections are consistent with previous research (Fernandez et

al., 2004). However, in this and the Fernandez experiment, the CORT concentrations

appear to be higher after injection of N/OFQ at all the doses that were tested, than they

were following ICV injections of equimolar N/OFQ doses in unstressed rats (Devine et

al., 2001). This supports the assertion that the handling and injection procedures, as well

as the exposure to a novel environment (the open field), are mildly to moderately

stressful for the rats. Nevertheless, the rats that were injected with N/OFQ still displayed

higher levels of circulating CORT in response to these stressors than did the

vehicle-treated rats. These data provide evidence that N/OFQ actions produce further

enhancement of HPA axis activity beyond that produced by stressor exposure. In fact,

Devine and colleagues (2001) demonstrated that injections of N/OFQ into the lateral

ventricle prolonged the CORT elevation produced by exposure to a mild stressor such

that at 30 minutes following injection, the CORT levels of vehicle-treated rats were









returning to baseline while the CORT levels of N/OFQ-treated rats remained highly

elevated. This suggests that N/OFQ has an enduring pharmacological effect or that it is

interfering with negative feedback mechanisms of the HPA axis (Devine et al., 2001).

While we did not conduct a time course examination of CORT levels in the present

experiment, the elevation in CORT levels observed at 30 minutes post-injection in the

ICV N/OFQ-treated rats is compatible with the idea that ICV N/OFQ administration

prolongs CORT elevation through this time point.

Injections of N/OFQ into the amygdala did not enhance HPA axis activity

following exposure to mild stress. This may seem inconsistent with the behavioral data;

however, hormonal and behavioral responses to pharmacological manipulations can be

dissociated. For example, diazepam, an anxiolytic, does produce elevations in circulating

corticosterone (Chabot et al., 1982; Fernandez et al., 2004; Marc and Morselli, 1969;

Massoco and Palermo-Neto, 1999). Nevertheless, previous research in our lab found that

intra-amygdaloid injections of N/OFQ in unstressed rats did produce elevations of

circulating CORT, although the elevations were relatively small (Misilmeri and Devine,

2000). It is possible, then, that the experiences of handling, injection, and exposure to the

open field produce stress effects in rats that are great enough to obscure the modest

effects of intra-amygdaloid N/OFQ injections on CORT concentrations. Additionally, if

the hypothesis of Devine and colleagues (2001), that ICV injections of N/OFQ are

reaching structures involved in negative feedback regulation, is correct, then it would be

expected that intra-amygdaloid injections of N/OFQ would not produce the same

enhancement of HPA axis activity.









It is important to note that the CORT data from 4 rats (2 ICV-implanted rats and 2

amygdala-implanted rats) were removed prior to analysis, as they were outlying values.

Of major concern were the very low CORT concentrations (under 100 ng/ml) of the 2

ICV-implanted rats, considering the level of stressor exposure these rats experienced.

This may have been due to high amounts of coagulation in the plasma tested, although

this was not systematically recorded. For consistency, standard deviations were also

calculated for the amygdala-implanted groups and outlying values were subsequently

removed. The removal of the 2 outlying values from the 1.0 nmole ICV group did affect

the final statistical analysis; however, removal of the 2 values from the 0.01 nmole and

1.0 nmole intra-amygdaloid groups did not affect further analysis.

Organ Masses

In the present study, we measured the thymus, adrenal, and spleen masses to

establish that there were no systematic differences in health status or stress exposure

between the various groups of rats (especially since the rats underwent differing types of

intracranial cannulation surgery). Thymus glands and spleens tend to decrease in mass

and adrenal glands tend to increase in mass following exposure to physiological stressors

(such as physical insults, poor diet, or exposure to toxic chemicals) and psychological

stressors (such as restraint, crowding, or predator exposure), particularly after chronic

exposure to these stressors (for examples see Blanchard et al., 1998; Bryant et al., 1991;

Dominguez-Gerpe and Rey-Mendez, 2001; Hasegawa and Saiki, 2002; Selye, 1936;

Watzl et al., 1993). While we did not include any rats that did not undergo the stress of

surgery and handling, the gland masses in this experiment were similar to those measured

in other experiments where non-surgical unstressed controls were included (Fernandez et

al., 2004). Additionally, there were no significant differences in thymus gland masses,









spleen masses, or adrenal gland masses between any of the groups tested. Therefore, we

can conclude that there were no apparent differences in the health of the rats that may

have affected the ultimate results of the experiment.

Summary

These data show that N/OFQ injections affect anxiety and HPA axis activity

through actions in the amygdala (among other potential sites), suggesting the possibility

that amygdaloid N/OFQ neurotransmission may be involved in regulation of affect.

However, because ICV injections of N/OFQ produced greater, more potent effects than

injections into the right amygdala did, additional structures must be involved. This may

include actions in the contralateral amygdala or other limbic structures such as the BNST

or, potentially, in multiple limbic regions involving synergistic actions at these sites. To

examine other structures involved, we are currently conducting a number of studies. For

example, we are examining the effects of injecting N/OFQ into the BNST to determine if

the BNST contributes to, or is the primary site of action for, the anxiogenic behavioral

effects of N/OFQ. Additionally, we are using other tools, such as in situ hybridization, to

examine changes in N/OFQ and NOP expression throughout the brain following social

stress. These studies will help us to better understand the role of N/OFQ in the

neurocircuitry of stress, anxiety, and HPA axis functioning.















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BIOGRAPHICAL SKETCH

Megan K. Green received her Associate of Arts in August 1998 at Okaloosa

Walton College. In May 2000, she received a dual Bachelor of Arts in psychology and

anthropology from the University of West Florida. Megan began her graduate studies in

experimental psychology at the University of West Florida in August 2001. She

continued graduate school at the University of Florida in August 2003, where she is

currently pursuing studies in behavioral neuroscience through the psychology

department.