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1 N METHYL D ASPARTATE RECEPTOR 1 SPLICING AND PHOSPHORYLATION CHANGES ARE ASSOCIATED WITH BEHAVIORAL ALTERATIONS DURING MORPHINE TOLERANCE AND WITHDRAWAL By ETHAN MICHAEL ANDERSON A DISSERTATION PRESENTED TO THE GRADUATE S CHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012
2 2012 Ethan Anderson
3 ACKNOWLEDGMENTS I would like to thank Dr. Robert Caudle for his mentorship over the last four years as well as my committee members Dr. John Neubert, Dr. Drake Morgan, Dr. J effrey Harrison, and Dr. Neil Rowland for their criticisms and suggestions for this work. I would also like to thank everyone who helped to teach me the methodolo gy I used in this work. They include Dr. Arseima Del Valle Pinero, Dr. Shelby Suckow Nahir, Dr. Todd Nolan, and Alan Jenkins. I would also like to thank the other members of the lab that helped produce the data for this work : Kathy Kapernaros and Turi Reev es I also thank the National Institute on Drug Abuse and National Institutes of Health as grant number DA030044 supported this research. Finally, I would like to thank my family, especially Keitha McCall and Penelope Anderson, for their support during thi s time.
4 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 3 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 LIST OF ABBREVIATIONS ................................ ................................ ........................... 10 ABSTRACT ................................ ................................ ................................ ................... 13 CH AP TE R 1 INTRODUCTION ................................ ................................ ................................ .... 15 Morphine Tolerance, Dependence, and Withdrawal in Humans ............................. 15 Neuronal Pathways Involved in Opioid Pain Relief and Reward ............................. 16 Pain Pathways ................................ ................................ ................................ .. 17 The ascending pain pathway ................................ ................................ ..... 17 The descending pain pathway ................................ ................................ ... 20 Limbic System and Reward Pathways ................................ ............................. 22 The Interconnections between the Pain and Reward Systems ........................ 23 Stages of Opioid Use ................................ ................................ .............................. 24 Acute Morphine ................................ ................................ ................................ 24 Morphine Tolerance ................................ ................................ ......................... 25 Acute Morphine Withdrawal ................................ ................................ .............. 26 Extended Morphine Withdrawal ................................ ................................ ........ 27 NMDARs and Plasticity ................................ ................................ ........................... 28 NR1 Splicing ................................ ................................ ................................ ..... 29 NR1 Phosphorylation ................................ ................................ ....................... 33 Interactions between NMDARs and Opioid Signa ling ................................ ............. 33 Co localization of NR1 and MOR in Distinct Brain Regions .............................. 34 Pain ................................ ................................ ................................ .................. 35 Reward ................................ ................................ ................................ ............. 35 Acute Morphine ................................ ................................ ................................ 36 Tolerance ................................ ................................ ................................ ......... 36 Acute Withd rawal ................................ ................................ ............................. 37 Extended Withdrawal ................................ ................................ ....................... 38 Intracellular Molecules Which Interact With Both NMDARs and MORs ........... 39 cAMP and PKA ................................ ................................ .......................... 39 CREB ................................ ................................ ................................ ......... 39 Calcineurin ................................ ................................ ................................ 41 Hypotheses ................................ ................................ ................................ ............. 42 Morphine will alter NR1 Splicing ................................ ................................ ....... 42 MK 801 Will Block Morphine Tolerance and Associated NR1 Splicing ............ 42
5 Altered Pain Behavior during the Extended Withdrawal Period will be Associated with NR1 Changes ................................ ................................ ...... 44 Altered NR1 Phosphorylation during Withdrawal ................................ .............. 44 2 EXPERIMENTAL PROCEDURES ................................ ................................ .......... 46 Animal Care ................................ ................................ ................................ ............ 46 B ehavioral Assessment of Morphine Tolerance and Withdrawal ............................ 46 Acute Morphine Administration ................................ ................................ ............... 47 Operant Orofacial Testing at Avers ive and Non aversive Temperatures during Morphine Tolerance ................................ ................................ ............................. 47 Assay ................................ ................................ ................................ ................... 49 Behavioral Testing during an Extended Withdrawal Period ................................ .... 49 Open Field Assay ................................ ................................ ................................ ... 51 Tissue Collection ................................ ................................ ................................ .... 51 Primary Neuronal Cultures ................................ ................................ ...................... 52 Western Blotting ................................ ................................ ................................ ..... 53 Double labeled Immunocyto chemistry ................................ ................................ .... 54 Quantitative Internalization Assay ................................ ................................ ........... 55 3 RESULTS ................................ ................................ ................................ ............... 59 Be havioral Assessment of Morphine Tolerance and Withdrawal ............................ 59 Morphine Tolerance and Withdrawal Alters NR1 Expression ................................ 59 Acute Mor phine Administration does not Alter NR1 Splice Variant Expression ...... 61 Morphine Tolerance Alters Pain and Motivational Reward Seeking Behavior on the Operant Orofacial Nociception Assay. ................................ ........................... 61 No Differences in an Acute Morphine Dose at Hot, Cold, or Neutral Temperatures ................................ ................................ ................................ ...... 63 NMDAR Antagonism Alters Morphine induced Behavior on the Operant Orofacial Assay ................................ ................................ ................................ ... 64 Effect of NMDAR Antagonism on Morphine Induced NR1 Expression ................... 65 Weight Change during an Exten ded Withdrawal Period ................................ ......... 66 Behavioral Testing with Escalating Morphine Doses ................................ .............. 67 Behavioral Testing during an Acute and Extended Wi thdrawal Period ................... 68 NR1 Expression Differences in the NACC and AMY after an Extended Withdrawal Period ................................ ................................ ............................... 69 C1 and C2 Expression in the AMY is Associated with Differences in Behavior during Aversive, Painful Conditions ................................ ................................ ..... 70 N1 Expression in the AMY and C1 Expression in the NACC are not Associated with Differences in Behavio r During Aversive, Painful Conditions ....................... 71 pNR1 and CAL levels in the NACC are Associated with Reward seeking Behavior ................................ ................................ ................................ .............. 73 CAL, pNR 1, and pCREB Levels Alter during Withdrawal in Culture and are Regulated by NMDAR and PKA signaling ................................ ........................... 74
6 NR1 Surface Expression alters with Morphine Withdrawal in Culture and is Regulated by NMD AR and PKA signaling ................................ ........................... 76 4 DISCUSSION ................................ ................................ ................................ ....... 105 Morphine Tolerance and Withdrawal Alters NR1 Expression ............................... 106 Morphine Tolerance Alters Pain and Motivational Reward Seeking Behavior on an Operant Orofacial Nociception Assay ................................ ........................... 107 NMDAR Antagonism Alters Morphine induce d Behavior on the Operant Orofacial Assay ................................ ................................ ................................ 109 Effect of NMDAR Antagonism on Morphine Induced NR1 Expression ................. 110 Altered NR1 Spli cing in the AMY ................................ ................................ .......... 113 Weight Change during an Extended Withdrawal Period ................................ ....... 113 Behavioral Testing with Escalating Morphine Doses ................................ ............ 113 Open Field Results ................................ ................................ ............................... 114 Behavioral Testing during an Acute and Extended Withdrawal Period ................. 114 NR1 Expression Differences in the NACC and AMY after an Extended Withdrawal Period ................................ ................................ ............................. 116 C1 and C2 Expression in the AMY is Associated with Differences in Behavior during Aversive, Painful Conditions ................................ ................................ ... 116 N1 Expression in the AMY and C1 Expression in the NACC are not Associated with Differences in Behavior during Aversive, Painful Conditions ...................... 118 pNR1 and CAL levels in the NACC are Associated with Reward seeking Behavior ................................ ................................ ................................ ............ 119 CAL Levels in the NACC Increase during Acute Withdrawal ................................ 120 CAL, pNR1, and pCREB Levels Alter during Withdrawal in Culture and are Regulated by NMDAR and PKA Signaling ................................ ......................... 121 NR1 Surface E xpression alters with Morphine Withdrawal in Culture and is Regulated by NMDAR and PKA Signaling ................................ ......................... 123 5 CONCLUSIONS ................................ ................................ ................................ ... 125 LIST OF REF ERENCES ................................ ................................ ............................. 129 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 148
7 LIST OF TABLES Table page 2 1 Escalating morphin e doses. These doses were administered twice daily to rats on days 1 10. Control animals received equivalent volumes of saline. ........ 57 2 2 Experimental outline for morphine induced NR1 splice vari ant expression changes. ................................ ................................ ................................ ............. 57 2 3 Experimental outline to study the effects of morphine tolerance on aversive and non aversive temperatures on the operant orofacial pain assay. ................ 57 2 4 Experimental outline to study the effects of NMDAR antagonism on morphine induced behavior on the operant orofacial pain assay and NR1 splice variant expression. ................................ ................................ ................... 57 3 1 Tolerance and withdrawal changes in total NR1 and splice cassette expressi on as measured by western blots ................................ .......................... 78 3 2 Two Way ANOVA values for Figure 3 7 ................................ ............................. 78 3 3 Repeated Measures Two Way ANOVA values for Figure 3 9 ............................ 79 3 4 T tests and F tests for NR1 total and all splice variants in the NACC and AMY after an extended withdrawal. ................................ ................................ .... 80
8 LIST OF FIGURES Figure page 1 1 NMDAR subunit 1 alternative splicing ................................ ................................ 45 2 1 Experimental timeline for extended withdrawal study ................................ ......... 58 2 2 Cell culture timeline ................................ ................................ ............................ 58 3 1 The effec ts of morphine on thermal sensitivity and weight ................................ 81 3 2 A representative blot of NR1 changes in the nu cleus accumbens due to morphine ................................ ................................ ................................ ............ 81 3 3 Quantification of NR1 splicing changes in the brain due to mo rphine tolerance and withdrawal ................................ ................................ .................... 82 3 4 Quantification of NR1 total protein changes in the brain due to mo rphine tolera nce and withdrawal ................................ ................................ ................... 83 3 5 An acute dose of morphine does not alter NR1 splice variation ......................... 84 3 6 A representative sample of the e ffects of heat aversion and morphine on the operant orofacial pain assay ................................ ................................ ............... 85 3 7 Morphine alters pain and reward seeking behavior at aversive and non aversive temperatures on the operant orofac ial nociception assay .................... 86 3 8 Morphine increases time per contact values, facial contact times, and time spent licking at hot, cold, and neutral temperatures equally. .............................. 88 3 9 Morphine and MK 801 alter pain and reward seeking behavior on the operant orofacial nociception assay at aversiv e and non aversive temperatures ............ 88 3 10 Morphine and MK 801 co administration alters NR1 splice varia tion in the nucleus accumbens ................................ ................................ ............................ 90 3 11 Weight change during morphine administration, acute, and extended withdrawal ................................ ................................ ................................ ........... 92 3 12 The effects of morphine tolerance on behavior at non aversive temperatures ove r an escalating dosing paradigm ................................ ................................ ... 92 3 13 Behavioral di fferences between acute versus extended withdrawal at 37C and 46C ................................ ................................ ................................ ............ 93 3 14 Naloxone decreases motivational behavior at non aversive tempe ratures during acute withdrawal ................................ ................................ ...................... 94
9 3 15 No differences were observed in locomotor or rearing behavior during morphine administration o r acute and extended withdrawal ............................... 94 3 16 AMY C1 c hanges are associated with long ter m responses to aversive stimuli ................................ ................................ ................................ ................ 95 3 17 AMY C2 changes are associated with long term motivation an d responses to aversive stimuli ................................ ................................ ................................ ... 96 3 18 AMY N1 changes are not associated with long term motivation an d responses to aversive stimuli ................................ ................................ .............. 97 3 19 NACC C1 changes are not associated with long term motivation an d responses to aversive stimuli ................................ ................................ .............. 98 3 20 Long term alterations in phosphorylated NR1 and CAL in the NACC are associated with altered motiva tion during extended withdrawal ........................ 99 3 21 CAL increases after three days of with drawal in the nucleus accumbens ........ 100 3 22 NMDARs and MORs are co localiz ed in primary neuronal cultures ................. 100 3 23 Naloxone induced withdrawal increases CAL pNR1, and pCREB in culture ... 101 3 24 NMDAR antagonism bloc ks some withdrawal induced alteratio ns in pNR1 and pCREB in culture ................................ ................................ ....................... 101 3 25 NMDAR anta gonism increases CAL in culture ................................ ................. 102 3 26 PKA inhibition alters CAL, pNR1, and pCREB during naloxone pre cipitated withdrawal in culture ................................ ................................ ......................... 102 3 27 PKA activation alters naloxone precipitated withdrawal induced alterations in CAL, pNR1, and pCREB in culture ................................ ................................ ... 103 3 28 Altered NR1 cell surface expression occurs with naloxone precipitated withdrawal, NMDAR antagonism, and PKA acti vation or inhibition in culture ... 104
10 LIST OF ABBREVIATIONS AC adenylate cyclase ACC anterior cingulate cortex AMY amygdala ANOVA analysis of variance APV 2 amino 5 phosphonopentanoic acid B37 baseline measures at 37C CAL calcineurin C AMP cyclic adenosine monophospha te CCI chronic constriction injury C E A central nucleus of the amygdala CNS central nervous system CPA conditioned place aversion CPP conditioned place preference CRE cAMP response element CREB cAMP response element binding protein DA dopamine ER endoplasmi c reticulum G guanine GABA gamma aminobutyric acid GAP glyceraldehyde 3 phosphate dehydrogenase GLU glutamate HIPP dorsal hippocampus LC locus coeruleus M morphine injected group
11 MK MK 801 injected group MM MK 801 and morphine injected group MOR mu opioid receptor M OR 37 M OR 46 NACC nucleus accumbens NAL naloxone NMDAR N methyl D aspartate receptor NR1 NMDAR1 subunit of the NMDA receptor NS non significant PAG periaq ueductal gray PFC prefrontal cortex PKA protein kinase A PKC protein kinase C P NR1 the NR1 subunit when phosphorylated at serine 897 PP1 protein phosphatase 1 PP2B protein phosphatase 2B PSD postsynaptic density RM repeated measures S saline injected group S AL 37 rats tes S AL 46 SC spinal cord SS1 secondary somatosensory cerebral cortex area 1 SS2 secondary somatosensory cerebral cortex area 2
12 STT spinothalamic tract VP ventral pallidum VPL ven tral posterolateral nucleus of the thalamus VPM ventral posteromedial nucleus of the thalamus VTA ventral tegmental area WDR wide dynamic range neurons
13 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy N METHYL D ASPARTATE RECEPTOR 1 SPLICING AND PHOSPHORYLATION CHANGES ARE ASSOCIATED WITH BEHAVIORAL ALTERATIONS DURING MORPHINE TOLERANCE AND WITHDRAWAL By Ethan Michael And erson December 2012 Chair: Robert M. Caudle Major: Medical Sciences Neuroscience Morphine is an opioid drug which is used clinically for the treatment of pain and recreationally for its rewarding effects. Repeated use of morphine can lead to tolerance a nd dependence A n aversive withdrawal state occurs once the drug is no longer taken. This withdrawal state often leads to reuse of the drug and may have long term negative effects on the individual. These states are believed to be dependent on drug induced plastic changes in the nervous system of which the N methyl D aspartate (NMDA) receptor is a likely component. The NR1 subunit of the NMDA receptor can be alternatively spliced and phosphorylated and these changes influence the plastic properties of the r eceptor. We examined the effects of acute and chronic morphine as well as an acute and long term withdrawal on the splicing of NR1 in many regions of the brain associated with morphine use including the nucleus accumbens and amygdala. Here we demonstrate t hat chronic morphine alters splicing of the NR1 subunit and this can be modulated by blocking the activity of the receptor with MK 801. Withdrawal also alters the NR1 subunit as e ven after two months of an extended withdrawal period reductions in the C1 ca ssette were still present in the amygdala of some rats. These low
14 levels were associated with increased sensitivity to aversive conditions. In the accumbens, phosphorylation of the C1 cassette was increased after extended withdrawal and this was associated with increased motivation for food rewards. A reduction in the expression of the phosphatase calcineurin was also observed which may be responsible for this finding. Finally, using a cell culture model of morphine withdrawal we observed an association bet ween the levels of NR1 phosphorylation with cell surface expression of the NR1 subunit suggesting a mechanism for altered plasticity during long term withdrawal. These data demonstrate that morphine can induce long term changes in NR1 expression which can alter synaptic function and behavior. Further research into the role of the NMDA receptor during withdrawal may provide a way to halt the negative long term changes in plasticity associated with opioid use and benefit patients around the world.
15 CHAPTER 1 1 INTRODUCTION Morphine Tolerance, Dependence, and Withdrawal in Humans Whether opioids like morphine are used to treat pain or for their euphoric effects, tolerance, dependence and withdrawal are important issues facing many humans today. Opioids like mor phine have been used for the treatment of pain for thousands of years (Levinthal, 2010) and c linically, they are potent analgesics that are e xcellent for relieving acute pain. Unfort unately, tolerance and dependence can develop when used repeatedly over time like in the treatment of chronic pain. Finding ways to attenuate tolerance would allow opioid treatments to be effective for much longer periods of time. On the other hand, opioid s are also used recreationally for their pleasurable and reinforcing effects. Extended use can lead to dependence and sometimes addiction. Ceasing opioid use can lead to a negative, aversive state termed withdrawal. This state can help propagate opioid use regardless of detrimental consequences (Koob and Le Moal, 2005) The development of tolerance, dependence, and withdrawal may involve ronic opioid use (Williams et al., 2001) Much research in the field has demonstrated the N methyl D aspartate receptor (NMDAR) to be a major player in these changes. Specific alterations in NMDARs correlate with these opioid induced states and halting these altera tions with antagonists can modulate tolerance and dependence in animal models (Ueda and Ueda, 2009) These long term effects of (Noda and Nabeshima, 1 Some elements of this dissertation are reprinted with permission from two publications (Anderson et al., 2012b;Anderson et al., 2012a)
16 2004) and are believed to be mechanisms partially responsible for the predicted 1.7% of people in the world who suffer from addiction (Nagy et al., 2005) The NR1 subunit of the NMDAR can be alternati vely spliced and phosphorylated and this can alter plasticity in the brain as well (Lau and Zukin, 2007;Scott et al., 2003) Therefore, NR1 modulation co uld be responsible in part for the alterations in the nervous system responsible for tolerance, dependence, and withdrawal. Neuronal Pathways Involved in Opioid Pain Relief and Reward Opioids exert their effects on pain and reward by the modulation of two somewhat separable neuronal pathways. In order to provide information necessary for the continuation of life as well as the motivation and desir e to obtain these various wants and needs, evolution has given rise to various systems that help to inform us ab out when we are doing something beneficial and when we are doing something harmful. Pain is a way that the body informs itself about the inherent dangers in the world. This evolutionarily ancient system informs the organism about harmful and deadly situati ons in the environment and trains them to stay away from these stimuli so that they are more likely to live longer and reproduce (Price, 2002;Williams et al., 2001) A second system is in place that informs us when a situation is good or beneficial to our wellbeing. Thi s reward system drives hedonistic pleasures like the love of energy rich palatable food, sexual drives for increased reproductive chances, and positive social interactions for increased cooperation within our species (Nocjar and Panksepp, 2007) These two systems are interrelated through shared anatomical connections in areas like the amygdala (AMY) and the dorsal hippocampus (HIPP). The nucleus accumbens (NA CC) can also modulate both pain and rewarding behaviors as it is important for the motivation to avoid the former and seek the latter (Fields, 2007;Simon et al., 2011)
17 They also share a common mode of modulation in the endogenous opioid peptides, which are released in response to activity in these areas (Koepp et al., 2009;Zubieta and Stohler, 2009) The effects of these endorphins are mimicked in part by exogenous opioids like morphine When morphine is taken acutely, it can both inhibit pain and facilitate reward, but chronic use can cause extreme dysregulation of these systems (Koob, 2009a) Pain Pathways Clinically, opioid drugs are usually administered to relieve pain by altering the transmission of neurons responsi ble for sending and interpreting nociceptive messages to the brain. These neurons form ascending pathway s that travel to the brain and d escending pathways that return to the periphery. The ascending pain pathway The ascending pain system is responsible for bringing noxious, tissue damaging stimuli to the attention of the brain. The classic pain pathway is called the spinothalamic tract (STT) and starts with nociceptive afferents in the periphery, travels up the spinal cord (SC), goes through the midbrain, i s relayed through the thalamus, and ends in the cortex. Although this traditional pathway is accurate, it is not the whole story. Several other pathways exist in the central nervous system (CNS) and their various afferents synapse onto a variety of nuclei. Taken together, the ascending pain system is a wide reaching system that sends direct messages to many different brain areas (Price, 2002) Pain generally begins in the periphery. Various types of potentially damaging stimuli are det ected by receptors in the skin, viscera, and muscles of the body. These nociceptive stimuli include heat, cold, chemical, and mechanical damage. These are not
18 pain specific receptors, but when they are highly activated, tissue damage is more likely. Change s in the frequency of action potential firings alert the brain as to how intense the damage could be. Once these stimuli are detected, two different types of afferent fibers carry this information to the SC and they have different types of pain associated with them. When someone touches a hot stove on accident, there is a rapid, this alerting pain. They are myelinated and send their messages very quickly t o the SC to cause protective reflexes. In rodent studies these reflexive responses are often meas ured as the pain itself. N ociceptive tests like the tail flick and paw withdrawal (also known as the plantar assay) typically measure this reflexive response by applying an aversive stimulus like heat to the tail or paw and measuring the time it takes the animal to withdraw. In humans as well as rodents, o nce the hand /paw/tail is retracted and the This is a longer lasting, throbbing, burning, and more unpleasant pain t hat keeps a person aware that recent damage has occurred. This type of pain may keep a person from using their burned hand as often, force them to seek treatment for the burn, and would generally help the recovery process by keeping a person aware of their recent injury. C fibers are responsible for this type of pain and their messages take longer due to the fact that they are unmyelinated and have much slower conduction velocities (Price, 1999) Both of these types of pain fibers synapse onto the dorsal horn of the SC as their first stop in the CNS. These primary afferents synapse onto a few distinct regions of the SC known as lamina I and deep layers V VI. In these layer s, the SC has two main types of neurons which carry these messages: nociceptive specific neurons
19 and wide dynamic range (WDR) neurons. Nociceptive specific neurons carry only nociceptive information from the periphery, whereas WDR neurons carry multiple se nsory dimensions all the way from gentle touch to intense pain. Pain is again encoded by more frequent action potentials (Price, 1999) Traveling through the SC to the b rain are many tracts which have specific types of information associated with them. The STT is the classic pain tract beca use it mostly consists of WDR neurons and therefore encodes many different types of pain. It receives afferents from the periphery in lamina I and layers V VI, then projects to the ventroposterior lateral nucleus of the thalamus (VPL). From here, afferents travel to the secondary somatosensory cerebral cortex areas 1 and 2 (SS1 and SS2). This tract is responsible for the actual sensory p erception of pain. Other tracts carry pain information to many other brain regions. The next two tracts discussed start only in lamina I and contain mostly nociceptive specific neurons, so their targets receive messages that only represent pain. The spino parabrachio amygdaloid sends messages to the central nucleus of the amygdala (CeA) through the parabrachial nucleus. The second nociceptive specific neuron tract is the spino parabrachio hypothalamic pathway. These pathways together are responsible for ale rting the autonomic nervous system (ANS) (Price, 1999) The former is also responsible for e motions surrounding pain too. Pain is heavily innervations of the AMY I n humans these negative emotions can increase reported pain, while positive emotions can reduce it (Villemure and Bushnell, 2002)
20 Once pain signals have been delivered to the thalamus, cortex, AMY, and hypothalamus, they begin to integr ate with other sensory messages in the cortex to give limbic somatosensory pathway starts in the SS1 and SS2 and projects to the posterior parietal cortical areas and the insular cortex (IC). These cortical a reas have projections to the AMY and HIPP. The AMY starts an emotional response to the pain as well as playing a role in learning from the pain. Its projections to the HIPP help integrate this memory of pain s o that one can learn to avoid it the next time. Cortical areas are activated as well in parallel so that necessary decisions can be made to lessen the pain through the most effective and appropriate actions (Price, 19 99) Disruption of this pathway interrupts normal associations involved in pain processing. Typically feelings of threat are associated with feelings of pain, but patients who have had strokes in the IC report pain feelings normally, but have no associate d feelings of being threatened by it (Berthier et al., 1988) The IC also projects to the anterior cingulate co rtex (ACC) which is responsible for attending to pain and being motivated to escape it. From the ACC, projections head to the prefrontal cortical areas (PFC) and supplementary motor areas. The former is involved in executive functions and decision making, while the latter is involved in selecting appropriate responses to deal with the pain (Price, 1999) The descending pain pathway Evidence suggests that morphine alleviat es pain through the activation of the descending pain pathway. Once pain has come to the attention of the cortex, it is sometimes necessary to ignore it for a time, or at least lessen its intensity. Pain alerts the body of tissue damage, but sometimes an i mmedi ate escape is not possible or beneficial for the individual. If two organisms are in a fight for food, territory, or a mate,
21 the one that can disregard short term pain for the long term benefit will be more likely to live longer and reproduce. Situati ons like this may have been responsible for the evolution of a natural pain inhibiting system in organisms. This is a response that occurs in certain situations where pain must be endured for a period of time to allow the animal more success. This is a top down process that starts in the higher cognitive areas of the cortex and modulates the targets of the ascending pain system (Price, 2002) Higher cortical functions can change the pain response. In the laboratory, it has been demonst rated that anticipating pain can activate the ascending pathways in the thalamus. Direct afferents exist from the cortical areas to the CeA as well. After these messages are delivered to the AMY, projections from there head to the midbrain (Heinricher et al., 1999) The main nuclei of the descending pathway are found here at or around the fourth ventricle. The per iaqueductal gray (PAG) receives afferents from the ACC and the frontal cortex. The ACC is responsible for motivational escape from example for this could be an inj ured prey running away on a hurt leg from a predator. It is more beneficial to the organism to continue damaging the leg while running than to b e eaten. The PAG exerts its analgesic effects through the GABAergic ( gamma aminobutyric acid releasing) neurons in the rostroventral medulla (RVM) (Bodnar, 2009) These GABAerg ic neurons inhibit the spinal nociceptive afferents to provide a relief from these painful signals. The PAG also sends projections to the NACC which causes the release of opioid peptides and antinociceptive behavior (Xuan et al., 1986) Therefore, cortical messages sent through the AMY and NACC a re capable of modulating pain.
22 Limbic System and Reward Pathways In addition to avoiding pain, evolution has given rise to brain systems which motivate animals to seek out rewarding stimuli as well. This system shares common areas with pain processing like the HIPP, AMY, and the NACC. Opioids are involved in this system too as they he lp to encode the positive benefits of natural rewards like food, sex, and social interest (Nocjar and Panksepp, 2007) The involvement of opioid signaling i n the consuming of palatable food rewards for instance, has been well studied (Doyle et al., 1993;Katsuura et al., 2011;Taha et al., 2009;Zhang and Kelley, 2002) The start of this reward pathway is generally considered to be the v entral t egmental a rea (VTA). This has dopaminergic projections to the NACC and other limbic areas like the HIPP and AMY (Swanson, 1982) which is why this pathway is often electrical self stimulation in rats (Olds and Mi lner, 1954) and although the most sen sitive sites for reinforcement is the medial forebrain bundle (Koob, 2009a) rats will self stimulate the VTA (Burgess et al., 1993;Singh et al., 1996) which can alter dopamine (DA) transmission in the NACC (Fiorino et al., 1993;Owesson White et al., 2008) Though it was thought for a time that DA release into the NACC meant reward, this idea has been brought into question more recently. The NACC is now viewed as having a role in (Berridge and Robinson, 1998;Wyvell and Berridge, 2001) linked to DA. DA can cause an increase in the motivation to obtain rewards, but does (Pecina et al., 2003) The rewarding aspects are currently believed to coded by interactions between the NACC and the ventral pallidum (VP), another component of the limbic system (Smith et
23 al., 2009;Smith et al., 2011) Still, the NACC thus forms a major structure in the motivatio nal reward pathway and demonstrates the importance for DA in the reward process. Due to its central role in the limbic system it is a major component of reward and reward based learning which can be influenced by emotional responses from the AMY and past e xperiences from the HIPP. The Interconnections between the Pain and Reward Systems These two pathways help humans learn how to respond to their environment by avoiding that which is harmful and seeking out things which help maintain life. The pain and rewa rd pathways are mostly separable when examining the anatomy but have common connection points like the NACC, AMY, and HIPP. The pain system starts peripherally; travels up the SC; goes to the midbrain; heads to the thalamus, hypothalamus, or AMY, and ends in the cortical regions. This caudally located system alerts the body of danger and gives it the motivation to escape it. The reward system is located more in the basal forebrain area in the rostral portion of the brain and alerts the body to positive expe riences that are beneficial for survival and reproduction. One way these areas are connected is that these pathways can both release endorphins (short bind to the mu (MOR), delta, and/or kappa o pioid receptors to perform a variety to modulatory tasks. Analgesia for instance, is thought to be mainly a function of the MOR and morphine is considered its prototypical agonist Many of these receptors are found in the descending pain pathways and exert their effect by inhibiting the GABAergic cells of the PAG (Ko et al., 2006) Inhibiting these inhibitory signals turn on the analgesic effects of the PAG and RVM. Many other areas of the brain have high densities of MORs too. In the mesolimbic reward system the VP, VTA, HIPP, and AMY are all altered by endorphins
24 (W illiams et al., 2001) The amygdala can also release endorphins in response to positive emotions (Koepp et al., 2009) So the mesolim bic reward system which ha s projections to the AMY can act on the endogenous opioid system. Both of these pathways are involved heavily in learning so it is not surprising that the AMY and H IPP are common structures to both as they have a role in conditi onal learning and emotional responses to both pain and reward. Stages of Opioid Use The responses of these pathways to morphi ne are different depending on whether the drug is used acutely or chronically. Also, c hronic use can result in a withdrawal state o nce the drug is no longer taken which can also alter these pathways. Acute Morphine Opioids are widely prescribed because they greatly reduce acute pain Analgesia occurs through the activati on of the descending pain pathways and the inhibiti o n of spinal nociception Morphine binds to the MOR and these receptors are found in high levels in many areas of the CNS like in the SC, caudate putamen, LC, PAG, s triatum, thalamus, and AMY as demonstrated by autoradiography binding studie s (Pert et al., 1976) The MOR is a guanine (G) nucleotide binding protein coupled receptor which has seven transmembrane domains and is coupled to pertussis toxin sensitive G proteins. by a number of different ways. When the MOR is coupled to a Gi protein (inhibitory regulative G protein) its uncoupling results in the inhibit ion of adenylate cyclase (A C). cyclic adenosine monophosphate (cAMP) which in turn decreases the activation of protein kinase A (PKA). Low PKA activation reduces neurotransmitter release in neurons by shifting the voltage dependence of
25 ca tion channels to more negative potentials. The MOR can also increase potassium (K+) conductance in the LC, PAG, HIPP, VTA, and AMY through the activation of G protein activated inwardly rectifying K+ channels (GIRKs). This channel activation is produced th rough the beta/gamma subunits of the MOR protein The se beta/gamma subunits can also inhibit calcium conductance. Through these intercellular conductance changes morphine is able to alter glutamate (GLU) release in the LC, SC, PAG, HIPP, VTA, NACC, str iatum, and hypothalamus and can also in hibit GABA release in the AMY, RV M, NACC, PAG, HIPP, and VTA (Williams et al., 2001) Acute doses of MOR agonists generally alter pain and reward by inhibiting the neurons in the systems in which they are found (Zhu and Pan, 2005;Williams et al., 2001) For instance, MOR agonists inhibit GLU and GABA release in the PAG, a major site of morphine induced pain relief (Williams et al., 2001) In additi on to pain relief, opioids produce euphoria by acutely inhibiting neurons in the mesolimbic reward pathways (Williams et al., 2001;Koob, 2009a) Morphine Tolerance The r epeated administration of morphine over time causes dysregulations in the nervous system which can counter the acute effects of the drug. These changes produ ce morphine tolerance. This is defined as 1) a diminished drug effect over time produced by an equivalent dose of drug, or 2) the need to increase a drug dose to maintain the same level of response (Cleary and Backonja, 1996) Tolerance is the result of counter adaptations in the cell in response t o the acute effects of opioid exposure. Some of these responses include acute desensitization and internalization for which phosphorylation pathways involving protein kinase C (PKC) and GRKs (G prot ein coupled receptor kinase) are partially responsible (Williams et al., 2001) Later long
26 term desensitization and down regulation can occur which reduces the amount of MORs found on the cell by down regulating their production. In addition to changes in the surface and cellular expression of the MOR, other cellular components also undergo counter adaptations in response to tolerance. The most well known example s are probably AC and PKA. As acute MOR activation reduces AC activity through Gi, the cellular response over time is to increase the levels of AC produced. Long term adaptations also occur in potassium and calcium channel conductance typically in order to return the neuron to normal functioning. For instance, in the LC and PAG morphine inhibits neuronal firing and these mechanisms are believed to be responsible for respectively After chronic exposure adaptations occur so that firing in these areas returns to normal and morphine h as less of or no effect on anxiety and/or pain. Therefore these effects lead to a tolerant and dependent state where opioids must be continually taken to maintain normal neuronal functioning (Williams et al., 2001) Acute Morphine Withdrawal Withdrawa l from opioids is a dysphoric state which generally has the opposite effects as acute administration of opioids. The body has counter adapted to constant MOR activation at this point and the continuation of this activation is now necessary for normal neuro nal function to remain. For instance, high levels of AC were necessary for normal functioning during the tolerance phase, but during withdrawal when the MOR is no longer inhibiting some of this AC a hyperactivation of cAMP and PKA can occur. Cation channe l hyperactivity occurs in the PAG and LC also which plays a role in the hyperalgesia and anxiety which mark opioid withdrawal (Williams et al., 2001) Another area responsible for withdrawal induced anxiety is the AMY. GABA release is
27 decreased in this area during withdrawal and causes a general overexcitability of its neurons. Laboratory measures of anxiety in rodents are reduced when GABA(A) receptor agonists and MOR agonists are administered during withdrawal (Cabral et al., 2009) Decreases in motivation during w ithdrawal have also been reported as measured by reduced responding for natural food rewards (Zhang et al., 2007) This effect is likely related to the decreased activity of the mesolimbic DA system, as less activity of GABA, opioid peptide, and GLU systems in NACC and AMY occurs during withdrawal (Koob, 2009a) Similar to the acute and chronic stages, major changes in activity occur during withdrawal. Fos related antigens are increased after neural activit y and in the NACC, their levels peak during the third day of withdrawal (Nye and Nestler, 1996) Thus the positive effects on pain, anxiety, and reward of opioids are all reversed during the withdrawal state. These opposite changes in neuronal activity can often lead to the reuse of opioids (Ueda and Ueda, 2009) Extended Morphine Wit hdrawal In humans, many symptoms occur long after acute withdrawal has ended and can include craving, anxiety, dysphoria, hypersensitivity to pain, and cue associated memories which can all lead to relapse in addicted humans (Koob and Le Moal, 2005;Spanagel and Weiss, 1999;Williams et al., 2001) M any o f these s igns wer e traced to specific circuit pathways which include the NACC, HIPP and the AMY in animal studies (Crombag et al., 2008;Gardner, 2011;Heinz et al., 2009;Koob, 2009b) Extended withdrawal has been less studied in the animal literature but both behavioral changes to natural rewards (Dalley et al., 2005) and altered neurotransmitter release in the NACC (Schoffelmeer et al., 2001) were demonstrated to occur in rodents. These long term changes in behavior and brain function may be influenced by changes in neural
28 plasticity. The long term effe cts of opioid withdrawal could result from stable neuroplastic changes in these areas which could perpetuate these behaviors (Duka et al., 2011) NMDARs and Plasticity NMDARs are proteins in the nervous system capable of inducing long term effects through the stabilization and reorganization of synapses (Nikonenko et al., 2002) and are therefore prime candidates for stable, drug induced neuroplastic changes caused by repeated o pioid use and/or withdrawal. These receptors are highly involved in many long term disease states: chronic pain, tissue damage from inflammation, disorders like schizophren ia, obsessive compulsive disorder, and addiction (Lau and Zukin, 2007) The classic NMDAR is an ionotropic cation channel which is a tetramer of two NMDAR1 (NR1) subunits and two NMDAR2 (NR2) subunits (Behe et al., 1995;Premkumar and Auerbach, 1997) These two subunits are structurally similar in that they both have an extracellular N terminal region, three hydrophobic transmem brane domains (TM), a fourth hydrophobic domain (TM2) which is believed to make a hairpin loop in the membrane, extracellular regions that contain the binding site for endogenous agonists, and an intracellular C terminal domain that contains sites for phos phorylation by different kinases. The NMDAR1 subunit (NR1) is essential fo r NMDAR function (Dingledine et al., 1999) and the function of the body in general as demonstrated by the fact that NR1 knockout mice die shortly after birth (Forrest et al., 1994) Activation requires the simultaneous binding of the neurotransmitter GLU to the NR2 and the binding of the co agonist glycine to the NR1 (Kuryatov et al., 1994) More
29 recently, a third subunit, the NR3 subunit, has also been discovered. This modulates the NMDAR by influencing surface expression and calcium permeability (Cull Candy and Leszkiewicz, 2004) but is not a necessary component for its function. In functional NMDAR s, the TM2 regions of different subunits come together to form an ion channel that selectively allows calcium ions through (Zukin and Bennett, 1995) Under normal physiological conditions, Mg 2 + acts as a voltage dependent blocker of this channel so that it is only open when the membrane is depolarized. When th e cell is depolarized the Mg 2+ comes out of the channel and allows other ions like calcium to flow in to the cell (Mayer et al., 1984) These receptors are usually highly permeable to calcium (Morgan and Curran, 1988) which, once in the cell, has many effects on plasticity through signaling pathways involving calmoduli n and PKC (Fukunaga et al., 1992) NMDARs are detectors of neuronal activity and are themselves altered by neuronal activity. In normal NMDAR trafficking, some activity promotes more receptors reaching the cell surface (Grosshans et al., 2002) which can increas e Ca2+ influx into the cell C hronic activity, however, (Lau and Zukin, 2007) These studies involving the role of NMDAR activity are usually performed by selectively antagonizing them with MK 801 (Wong et al., 1986) Since NMDARs can detect changes in activity and are altered by these c hanges as well, they are good candidates for the long term plastic effects caused by morphine NR1 Splicing The NMDAR was demonstrated to play a role in drug induced neural plasticity (Ueda and Ueda, 2009) ; however, the alternative splicing of the NR1 subunit is often overlooked. The NMDAR1 subunit has eight variants due to the alternative splicing of exons 5, 21, and 22 of its parent gene GRIN1 (Figure 1 1) Wh en expressed they are
30 known as the N1, C1, and C2 regions, respectively (Cull Candy and Leszkiewicz, 2004) phosphorylation, and cellular distribution (Dingledine et al., 1999) as e ach of these splice variants have different pharmacological and/or regulatory effects on the NMDAR (Zukin and Bennett, 1995) NR1 splice variants are differentially expressed developmentally (Hoffmann et al., 2000;Laurie and Seeburg, 1994) They also have region specific expression in the brain (Laurie et al., 1995) SC (Prybylowski e t al., 2001) and enteric nervous system (Del Valle Pinero et al., 2007) Each of these splice variants have differen t effects on the NMDAR and their splicing is based on different factors. The splicing of both the N1 and the C1 cassettes are regulated by calcium, calcium/calmodulin dependent protein kinase IV (CaMKIV) (Lee et al., 2007) and the depolarization of the neuronal membrane (Xie, 2008) They are also spliced in a regional dependent manner based on the splicing factor NAPOR (neuroblastoma apoptosis related RNA binding protein). This protein is expressed in a region spe cific manner and binds to splicing silencers responsible for keeping C1 from being expressed thereby allowing its inclusion (Zhang et al., 2002) are mutually exclusive exons. The C2 region contains a stop codon for translation However, i f C2 is spliced out a different C terminal end is inserted. T region and contains its own stop codon (Zukin and Bennett, 1995) Mu et al. (2003) demonstrated that the splicing of these mutually exclusive ends can be dependent on the activity of the receptor. Decreased neural activity (NMDAR antagonism) can lead to an increase in the C2 cassette and in creased neural activity leads to more C2 inclusion.
31 The pathway(s) and the splicing factors that regulate these changes are yet to be determined. The inclusion or removal of the four alternatively spliced exons can alter the plastic properties of the NMDAR N1 is a 21 amino acid sequence located near the N terminus of the subunit which contains six positively charged amino acids (Zheng et al., 1994) The N1 insert changes the pharmacological properties of the receptor by forming a surface loop which makes the NMDAR insensitive to protons. NMDARs which do not contain the N1 cassette are 50% inhibited at physiological pH (Traynelis et al., 1998) N1 inclusion also leads to faster deactivation times (Rumbaugh et al., 2000) and more sensitivity to NMDAR antagonists (Rodriguez Paz et al., 1995) The C1 insert is a 37 amino acid region that contains an endoplasmic reticulum (ER) retention signal which keeps C1 containing NR1 subunits in the ER for longer periods of time (Scott et al., 2001) This increased time in the ER could mean that this subunit has had sufficient time to fold to a more perfect state while the other subunits are exported more quickly and are not assembled as well (Horak and Wenthold, 2009) C1 has three serines which can be phosphorylated in vivo by PKC and PKA (Tingley et al., 1997) The C1 region also contains many binding areas for post synaptic density (PSD) associated proteins like neurofilament L, yotaio, and calmodulin (Cull Candy and Leszkiewicz, 2004) The C2 insert hinders maturation of proteins between the ER and Golgi facilitates rapid movement between these two organelles, likely due to the presence of a three amino acid (threonine valine valine or TVV ) (Horak and Wenthol d, 2009) This motif is an ER export signal found on proteins which allows them to rapidly leave this organelle by binding to coatomer protein complex II proteins
32 (Barlowe, 2002) Another hypothesis is that the TVV region acts as a PDZ domain binding motif which interacts with other proteins to bring the subunit to the cell surface at a faster rate. These motifs are three consecutive amino acids (AA) represented as (T/S)XV: threonine or serine, any AA and a valine (Mu et al., 2003) NR2 subunits have these (T/S)XV regions as well which help bring complete NMDARs to the surface, but containing NR1 subunits go to the surface unaccompanied by their NR2 counterpart (Holmes et al., 2002) contain ing subunits exhibit higher basal surface expression than C2 containing receptors (Okabe et al., 1999) In culture, C2 containing NMDAR clusters are located synaptically and NMDAR antagonism causes (Pauly et al., 2005) T he C2 cassette however, is important for spine stability in the long term (Alvarez et al., 2007) state of plasticity. Rapid chan ges in surface expression could be produced by containing NR1 subunits for long term stabilization. Therefore, any changes found in NR1 splicing could have long term plastic effects on the activation of ne urons. Since NR1 splice variants have an effect on synaptic plasticity it is no surprise that changes in their mRNA and protein levels were previously reported in diseases of long term synaptic plasticity like drug abuse and chronic pain (Darstein et al., 2000;Gaunitz et al., 2002;Loftis and Janowsky, 2002;Prybylowski et al., 2001;Winkler et al., 1999a;Zhou et al., 2007;Zhou et al., 2006;Zhou et al., 2009) These data together suggest that alternative splicing could be a mechanism for inducing long term drug induced neuroplastic effects.
33 NR1 Phosphorylation Phosphorylation is a mechanism for rapidly altering neuronal function and can have many e ffects on plasticity. The NR1 subunit can be phosphorylated on three serines all located in the C1 cassette. Ser890 and Ser896 are phosphorylated by PKC and Ser897 is phosphorylated by PKA (Tingley et al., 1997) Phosphorylation alters the binding of scaffolding proteins like yotiao (Westphal et al., 1999) to the C1 cassette of the NR1 subunit (Lin et al., 1998) PKA Ser897 phosphorylation increases the sensitivity of the NMDAR to GLU (Dudman et al., 2003) and increases NMDAR currents (Westphal et al., 1999) NR1 phosphorylation increases cell surface delivery (Lau and Zukin, 2007;Scott et al., 2003) and can be increased in pain states (Zhou et al., 2006) and associated with hyperalgesia and allodynia in m odels of SC injury (Caudle et al., 2003) Drugs of abuse like ethanol (Ferrani Kile et al., 2003) and cocaine (Scheggi et al., 2007) can also increase PKA phosphorylation of NR1. Several phosphatases reduce the phosphorylation of NR1. Calcineurin ( CAL ) can desensitize NMDARs in culture (Tong et al., 1995) through dephosphorylation of the C1 cassette (Choe et al., 2005) This can allow calmodulin to bind to the C1 subunit and reduce NMDAR activity (Ehlers et al., 1996) Ser897 can also be dephosphorylated by protein phosphatase 2A (PP2A) which can form stable complexes in vivo with NR3 subuni ts (Chan and Sucher, 2001) Since l ong term changes in the phosphorylation state of the NR1 are associated with increased or decreased function and surface expression of the NMDAR plasticity produced by opioids could be related to phosp horylation Interactions between NMDARs and Opioid Signaling Many studies have examined possible interactions between NMDARs and opioid receptors. NMDARs are co localized on many of the same cells as MORs and they alter
34 thways, modulate pain and reward, and are both changed over time during acute and chronic opioid use, as well as withdrawal. These shared properties make NMDARs good candidates for mediating the plastic effects of morphine administration. Co localization o f NR1 and MOR in Distinct Brain Regions and AMY as they are co splic e able exons are expressed in both these areas, but localization studies have not been performed for each of them. In the NACC shell, 66% of the NR1 labeled neurons co localized with MORs. Most of the other MORs were located on synapses that opposed or received input from NR1 containing neurons (Gracy et al., 1997) All exons for the NR1 splice variants are expressed in the NACC (Laurie et al., 1995) Most (Winkler et al., 1999b) Glass et al. (2009) revealed a high degree of overlap between MORs and NR1 subunits on the dendrites of GABAergic interneurons and mRNA for a ll NR1 exons are present in the adult AMY (Laurie et al., 1995) In the HIPP however, NR1s and MORs rarely co localize (Milner and Drake, 2001) suggesting that interactions between the MOR and the NR1 subunit in this area are intercellular in nature and not intracellular. All NR1 splice variants are expressed (Laurie et al., 1995) and mRNA for C2 similarly to the NACC (Winkler et al., 1999b) Like the NACC and AMY morphine exerts its effects on hippocampal MORs located mainly on GABAergic neurons (Drake and Milner, 1999)
35 Pain One way that NMDARs and MORs interact in the above mentioned areas is the altering of pain perception. Both MOR agonists like morphine a nd NMDAR antagonists like ketamine were demonstrated to reduce pain in humans and animals. NMDARs are involved in the expression of pain mechanisms like windup and central sensitization and NMDAR blockade can reduce pain by inhibiting this pain sensitizati on (Woolf and Thompson, 1991) NMDAR antagonists can also block pain and potentiate opi oid analgesia in animal models (Carlezon et al., 2000;Lutfy et al., 1999;Nishiyama, 2000;Quartaroli et al., 2001;Trujillo and Akil, 1991) One major area of MOR and NMDAR interaction is the AMY. NMDARs in the AMY contribute to the antinociceptive properties of morphine (Manning and Mayer, 1995) and in the CeA they are responsible in part for the sensitization of chronic pain (Li and Neugebauer, 2004) PKA phosphorylation of NR1 is important for synaptic plastic ity in pain in the CeA as well (Fu et al., 2008) Importantly, NR1 splicing differences have been observed in pain models of peripheral inflammation (Caudle et al., 2005) SC injury (Prybylowski et al., 2001) and colitis (Zhou et al., 2009) As NMDARs and MORs both modulate pain their interactions could be re sponsible for the alterations in pain perception that occur over time with tolerance and withdrawal. Reward NR1 and MOR interact in sites important for reward and motivation as well. For instance, NMDAR antagonism can alter the rewarding properties of opio ids. Morphine produces a conditioned place preference ( CPP ) which can be blocked by NMDAR antagonists (Tzschentke and Schmidt, 1995) Opioids can also influence NMDAR signaling as MOR agonists inhibit GLU signaling in the VTA which can alter motivation
36 and reinforcement (Margolis et al., 2005) The AMY region is responsible for the positive and negative emotional aspects of morphine use (Hou et al., 2009) and NMDARs in the AMY are necessary for morphine induced CPP (Rezayof et al., 2007) NMDAR blockade in the prelimbic cortex can also enhance opioid induced reward which can be prevented by inactivating the BLA (Bishop et al., 2011) So NMDARs antagonism in several d istinct brain regions can modulate morphine induced reward an d motivation. Acute Morphine Interactions between the MOR and the NMDAR can occur with an acute dose of morphine. A single dose of morphine was demonstrated to change NR1 mRNA levels in the hippocampus and hypothalamus (Le Greves et al., 1998) Acute morphine causes activity changes in regions of the brain like the NACC, LC, AMY, and in the dorsal horn of the SC (Martin et al., 1999;Martin et al., 2004;Zhu et al., 2003) which could alt er the surface expression of NMDARs (Grosshans et al., 2002) Tolerance While acute morphine can alter NMDARs, the interactions between NR1 and MOR have been examined most thoroughly with respect to morphine tolerance. This relationship was first examined with a study suggesting NMDAR antagonists can attenuat e morphine tolerance in the tail flick assay (Trujillo and Akil, 1991) More studies supported this role of NMDAR receptors in tolerance by demonstrating that anti sense oligonucleotides against NR1 can also attenuate morphine tolerance in reflex based measures of nociception (Shimoyama et al., 2005) Also, morphine tolerance was accelerated with the co administra tion of morphine and NMDA together (Kolesnikov et al., 1998) In contrast to much of the research howeve r, Carlezon et al. (2000) suggested that MK 801 an NMDAR antagonist, simply potentiated morphine induced
37 antinociception, but did not attenuate tolerance. Clinical studies have also demonstrated that NMDAR antagonists do not alter opioid tolerance in humans (Compton et al., 2008;Galer et al., 2005) Part of the confusion may stem from the fact that NMDAR antagonists like ketamine have been demonstrated to have an additive effect on opioid analgesia (Suzuki et al., 2005;Wadhwa et al., 2001;Weinbroum et al., 2002;Wiesenfeld Hallin, 1998) which may have an effect on antinociception in an imal studies (Carlezon et al., 2000) induced change s have been found w i th NR1 mRNA and/or protein. The areas of the brain which have been demonstrated to have morphine induced NR1 expression changes include the LC and the thalamus (Zhu et al., 2003) PAG, VTA, NACC (Inoue et al., 2003) AMY (Bajo et al., 2006) co rtex, HIPP (Murray et al., 2007) and the SC (Lim et al., 2005) These expression changes may be responsible for the morphine induced alterations of NMDARs. Thes e include decreases in: the affinity of glycine (Siggins et al., 2003) the Mg2+ block, synaptic transmission, NMDA induced current, and PKC activation of the NMDARs (Martin et al., 1999) Though NMDARs may not attenuate tolerance, there is a well established interaction between repeated mor phine administration and NMDARs. Acute Withdrawal As mentioned above, counter adaptations occur during the tolerance phase which are still present when the opioid is removed and can cause an aversive withdrawal condition. When morphine dependent rats are a dministered NMDAR antagonists or NR1 antisense oligonucleotides in addition to naloxone, they display reduced precipitated withdrawal symptoms (Manning et al., 1996;Trujillo and Akil, 1991;Zhu and Ho, 1998) suggesting that morphine induced changes in NMDARs during tolerance are
38 at least partially responsible for the withdraw al state. Withdrawal increases activity in the LC, AMY, NACC, and VTA (Shaw Lutchman et al., 2002) and NMDAR antagonists can block this increase in neuronal firing in the NACC and AMY (Rasmussen et al., 2005) This increased NMDAR activity is partially responsible for withdrawal as ket amine injected into the NACC reduces naloxone precipitated withdrawal behaviors (Ji et al., 2004) The AMY is associated with withd rawal induced conditioned place aversion (CPA) (Hou et al., 2009) and conditionally deleting NR1 in the AMY can reduce withdrawal induced CPA (Glass et al., 2008) So NMDAR activity is responsible for changes in behav ior during the withdrawal state. Extended Withdrawal Extended withdrawal is rarely studied in the l aboratory except in the context of addiction and relapse. NMDARs have a role in the extended withdrawal phase as ifenprodil (a NR2 B selective blocker) can inhibit morphine induced reinstatement of an extinguished CPP in rats two months after their last inj ection. Morphine primed reinstatement was also blocked by microinjection of ifenprodil into the NACC and HIPP (Ma et al., 2007) CPP protocols do not typically induce dependence and withdrawal by repeated exposures but these tests do measure long term alterations in morphine reward. The memories of morphine use are still present in the long term as demonstrated by the presence of a CPP weeks later. If NMDARs are antagonized with memantine during the extinction phase the morphine induced reinstatement of CPP is no longer present when tested three weeks later (Popik et al., 2006) Th is suggests long term effects of opioid withdrawal and/or memory could result from neuroplastic changes in these areas This may propagate add ictive, drug seeking behaviors (Duka et al., 2011) Much evidence suggests that the chronic nature of addiction is the result of
39 drug induced neuroplastic changes that remain in place long after substance use ceases and the acute withdrawal phase is over (Koob and Le Moal, 2005) Stable NR1 splicing changes in these areas could provide a mechanism as to why addicts are still prone to relapse long after acute withdrawal has ended. Intracellular Molecules Which Interact With Both NMDARs and MO Rs pathways. Many of the components in these interactions like cAMP, PKA, CREB and CAL, have been previously investigat ed but their ro les are not fully understood. cAMP and PKA As ment ioned previously, cAMP and PKA are intracellular components which both alter NMDARs and MORs. MOR agonists reduce cAMP and PKA activity acutely and then counter adaptations occur during the tolerance stage where these cAMP levels increase to higher levels. During withdrawal, these levels remain high and contribute to the hyperexcitability of neurons which occurs during this stage (Fan et al., 2009) cAMP and PKA can also alter the molecular properties of NMDARs so these components could be involved in opioid induced neural plasticity. For instance, PKA activ ation increases NMDAR currents (Westphal et al., 1999) phosphorylated by PKA (Tingley et al., 1997) which increases cell surface delivery (Lau and Zukin, 2007;Scott et al., 2003) .This increased surface expression could alter N MDAR activation in neurons and provide a mechanism for morphine induced plastic changes. CREB Another component that can be altered by NMDARs, MORs, and cAMP/PKA is the cAMP response element binding protein (CREB). CREB is a nuclear protein which
40 binds to the promoter regions of DNA with cAMP response element (CRE) consensus sequences. This binding can alter transcription of many genes related to synaptic plasticity (McPherson and Lawrence, 2007) including opioid peptides and glutamatergic receptor subunits (Carlezon, Jr. et al., 2005) The NR1 promoter has a CRE binding site on it (Zarain Herzberg et al., 2005) and NMDAR activation induces gene expression via a CRE dependent mecha nism (Bradley et al., 2006) meaning these two components xpression. The splicing and phosphorylation of NR1 may be involved with this activation as NR1s which lack C1 induce lower levels of the phosphorylated version of this nuclear transcription factor, pCREB, when activated by NMDA (Bradley et al., 2006) PKA can generate pCREB by phosphorylating CREB on serine 133 (Gonzalez and Montminy, 1989) so stimulation of AC activity and increased cAMP levels can lead to active PKA subunits translocating into the nucleus and phosphorylating CREB (Carlezon, Jr. et al., 2005) Opposite effects occ ur with acute morphine administration as Gi activation can decrease pCREB levels in the LC. As with some other cellular components chronic morphine exposure returns it to normal levels, and withdrawal increases its expression (Guitart et al., 1992) Similarly to NMDAR activation, the promot e r for the MOR gene also has a CRE element so pCREB can alter its transcription rate (Lee and Lee, 2003) making these two components co regulatory as well. Behaviorally, shRNA knockdown of CREB can attenuate morphine withdrawal behaviors and reduce AC, PKA, and CREB levels in vivo (Wu et al., 2012a) and CREB mutant mice have reduced morphine withdrawal behaviors (Maldonado et al., 1996) As MORs, NMDARs, an
41 interconnected neuronal components may have roles in long term opioid induced plasticity and opioid induced behaviors. Calcineurin CAL can often counteract the effects of PKA on NMDARs and CREB and is viewed as a negative modulator of synaptic plasticity (Biala et al., 2005) Therefore dysregulation of CAL is another potential method for opioid induced plasticity. CAL (also known as calcium/calmodulin dependent serine/threonine protein phosphatase, protein phosphatase 2B, and PP2B) is a calcium dependent protein phosph atase which can alter the strength of synapses through dephosphorylatin g various neuronal proteins. It s alpha subunit interacts with calmodulin while its beta subunit binds calcium (Klee et al., 1979) CAL can directly alter synaptic transmission as it is anchored in synaptic complexes along with PKA, NMDARs AMPARs, and PSD 95 (Ehlers, 2003) Due to this close association, inhibiting CAL can prolong NMDAR channel openings (Li et al., 2002;Lieberman and Mody, 1994;Liu et al., 1991) allowing increased calcium influx and the strengthening of synapses. CAL exerts its effects on the NR1 subunit by dephosphorylat ing S897 on C1 cassette (Choe et al., 2005) Once C1 is dephosphorylated, calmodulin can bind to this re gion and reduce NMDAR a ctivity (Ehlers et al., 1996) CAL can also reduce the phosphorylation of CREB on serine 133 (Lee et al., 2005) and modulate CREB mediated gene expression (Lam et al., 2009) CAL has also been demonstrated to alter the activation of protein phosphatase 1 (PP1) in hippocampal neur ons which leads to alter ed pCREB levels (Bito et al., 1996) Therefore, CAL can alter CREB phosphorylation in both direct and indirect ways. CAL can alter behavior as well as d ecreasing levels of CAL in the AMY produces increases in anxiety and depression as measured by the elevated plus maze and tail suspension
42 tests (Bahi et al., 2009) CAL can also alter opioid specific behaviors as its inhibition decreases naloxone induced withdrawal symptoms in mice (Dougherty et al., 1987;Dougherty and Dafny, 1988;Homayoun et al., 2003) and can block CPP for morphine in mice (Langroudi et al., 2005;Suzuki et al., 1993) As CAL appears to be able to alter NMDARs, MORs, and CREB activation in models of withdrawal and reward, it may be a component in long term opioid induced neural plasticity. Hypotheses Morphine will alter NR1 Splicing We hypothesized that morphine tolerance and withdrawal would produce changes reward seeking behavior. The changes in activity produced by opioids at eac h of the different stages of their use have been demonstrated to have effects on the expression of the NR1 subunit as a whole. Opioid induced alterations have been viewed as a disease of plasticity and NR1 splicing changes have been demonstrated previously in disease models of long term synaptic plasticity like chronic pain as well as both cocaine and alcohol abuse (Darstein et al., 2000;Gaunitz et al., 2002;Loftis and Janowsky, 2002;Prybylowski et al., 2001;Winkler et al., 1999a;Zhou et al., 2007;Zhou et al., 2006;Zhou et al., 2009;Raed er et al., 2008) The alternative splicing of NR1 could therefore be a mechanism for inducing long term opioid induced neuroplastic effects. MK 801 Will Block Morphine Tolerance and Associated NR1 Splicing Some reports suggest that NMDAR antagonism may be able to attenuate morphine tolerance (Shimoyama et al., 2005;Trujillo and Akil, 1991) while others disagree (Carlezon et al., 2000) All of these reports use reflex based measures of nociception however so we investigated the effects of the NMDAR antagonist MK 801
43 o n morphine tolerance in a non reflex based operant orofaci al thermal pain assay. Operant pain measures often give different results when compared to reflex based measures in terms of opioid dose effects and pain thresholds. While high doses of opioids are typically used for reflex based measures (Trujillo and Akil, 1991) several studies indicate that lower doses are needed for responses on operant assays (King et al., 2007;Morgan et al., 2008;Vincler et al., 2001) Other studies have also demonstrated that the thre sholds for escape from a painful stimulus are different for operant versus reflex based measures (Mauderli et al., 2000;Vierck et al., 2008;Vierck, Jr. et al., 2004) versus the speed of their spinal reflexes. A benefit of th is operant task is that the rodent can choose whether or not to perform the task, this allows the rodent to express escape or avoidance behavior. This complex behavior requires cortical decision making to control the amount of nociception the rodent feels (Dubner et al., 1976;Mauderli et al., 2000;Vierck, Jr. et al., 1971;Vierck, Jr. et al. 2004) While escape and avoidance behaviors can interfere with reflex based measures these pain behaviors are an integral component of our operant orofacial pain assay The differences in pain thresholds and the lower doses of opioids needed for operant assays suggest a higher sensitivity to pain and analgesia than traditional reflex based measures. We hypothesized that MK 801 would attenuate tolerance on our operant assay and would also alter the splicing of NR1, possibly blocking the morphine induced
44 Altered Pain Behavior during the Extended Withdrawal Period will be Associated with NR1 Changes Changes in NMDARs play a large role in many plastic conditions of the nervous sy stem like central sensitization (Ossipov et al., 2000) and chronic pain syndromes (Gaunitz et al., 2002;Prybylowski et al., 2001;Zhou et al., 2009) but sometimes only have an effect on a subset of the population. In a model of chronic gastrointestinal pain, rats expressed visceral and somatic hypersensitivity to stimuli and these ch anges correlated with increased NR1 phosphorylation and increased expression of N1, C1, and C2 splice variants (Zhou et al., 2009) In this case, long after the original insult occurred, a 25% subset of the population displayed changes in pain sensitivity. Since plasti c changes in brain pathways may mediate the long term effects of an extended withdrawal phase we hypothesized that if altered NR1 splicing is observed during the acute phase of withdrawal then perhaps these splice variants do not return to their baseline l evels long after the initial change. We investigated the effects of the extended withdrawal phase on operant measures of pain to determine if altered NR1 splicing and/or phosphorylation is associated with long term behavioral changes due to the extended wi thdrawal phase. Altered NR1 Phosphorylation during Withdrawal Finally, in a primary neuronal cell culture model of withdrawal, we examined the intracellular components which may be responsible for the transition between the acute and extended withdrawal ph ase to determine how NMDARs, CAL, and CREB may be altered during these stages. The phosphorylation state of NR1 and its expression can determine surface expression so modulation by CAL and CREB could be responsible for opioid induced changes in plasticity during withdrawal.
45 Figure 1 1. NMDAR subunit 1 alternative splicing. N1 and C1 are spliceable cassettes, boxes represent non spliceable exons.
46 CHAPTER 2 EXPERIMENTAL PROCEDU R ES Animal Care For all experiments, male Sprague Dawley rats (250 300 g, Charles River, Raleigh, NC) were housed in pairs in 22C temperature and 31% humidity controlled rooms with a 12 hour light/dark cycle (6am 6pm lights on) Rats had free access to foo d and water except when fasted for behavioral testing These facilities are a ccredited by the Association for Assessment and Accreditation of Laboratory Animal Care and all procedures were approved by the University of Florida Institutional Animal Care a nd Use Committee Behavioral Assessment of Morphine Tolerance and Withdrawal In order to test for morphine induced changes in NR1 splice variant expression, rats were randomly assigned to a saline (n=7), morphine (n=10), or withdrawal group (n=9). Nocicep tion was assessed using the Plantar Test (Ugo Basile, Collegeville, PA). Rats were placed in a clear chamber for thirty minutes, then an infrared heat stimulus (I.R. setting = 50) was placed under the hind paw and latency to withdraw was recorded. A cut of f time of 32.6 seconds prevented any tissue damage to the foot. Four trials (two per paw with a five minute intertrial interval) were averaged together for each animal as a daily score. Baseline latencies were taken for three days and then rats were given saline injections thirty minutes before testing for three additional days. This last injection day served as the baseline. Twice daily escalating morphine injections (Table 2 1) or equivalent volumes of subcutaneous saline were administered for the next te n days (1 10). Morphine sulfate (15mg/mL, Baxter, Deerfield, IL) was obtained from Webster Veterinary (Devens, MA). Four saline rats and the rats in the morphine group
47 were sacrificed as described below on day 10 within thirty minutes of their last injecti on. The remaining rats were tested for three more days (11 13) with no injectio ns before being placed in the chamber. After testing, rats were placed in individual empty cages and time spent grooming was recorded for fifteen minutes. These rats were euthan ized at the end of day 13. Rats were weighed every other day throughout these procedures. Rat weights and withdrawal latencies on the Plantar Test were analyzed using Repeated Measures Two hoc test using GraphPad Prism 4 so ftware (La Jolla, CA). For all analyses p values less than 0.05 were considered significant. See Table 2 2 for an abbreviated experimental design time course. Acute Morphine Administration Six rats were injected with saline and six were administered the ma ximal dose of morphine from the morphine tolerance experiment (60mg/kg). Thirty minutes later the rats were euthanized, tissue was harvested, and western blots were run as described below. Densitometry scores were analyzed with a two sample t test using Gr aphPad Prism 4 software. Operant Orofacial Testing at Aversive and Non aversive Temperatures during Morphine Tolerance Hairless Sprague Dawley rats (250 300 g, Charles River, Raleigh, NC) were used for this experiment as facial hair interferes with the tes ting procedure (Neubert et al., 2005) These rats were fasted for 20+/ 1hrs (from 5pm the previous night to12 2pm the next day) before each training and testing day then placed in a Plexiglas box for twenty minute periods and trained to press their faces between two aluminum tubes in order to receive diluted sweetened condensed milk. These tubes had water continuously flowing
48 through them from a circulating water bath which can be heated to aversive or non aversive temperatures. Mechanical sensors were attached to the tubes and the feeding bottle so that every time a rat touched its face to the tubes and licked the feeding bottle a recording was taken on DATAQ software (WinDa q Lite Data Acq DI 194, DATAQ Instruments, Inc. Akron, OH). After two weeks of baseline trai ning (three times a week) at a non 10mg/kg morphine or an equivalent volume of saline was adminis tered twice daily for ten days and testing continued every two to three days. Rats were randomly separated were tested on each day. The rats tested on day 1 were euthanized after testing and eight new rats were used for each subsequent day (eight for 3, eight for 5, etc.). These sensitivity was determined by taking the total amount of time spent making facial contacts and dividing it by the number of facial contacts at an aver defined as the time per contact. Analgesics cause the time per contact to increase from the facial contact for longer periods of time (Neubert et al., 2006;Neubert et al., 2007) We also measured two other variables, total facial contact time and total time spent licking, as measures of reward seeking behavior as they represent how much time the rats spent trying to obtain the food reward. Data on testing sessions were expressed as
49 performed. Significance was assessed with four separate Two Way ANOVAs with post hoc comparison (Sal37 vs. Sal46, Mor37 vs. Mor46, Sal37 vs. Mor37, and Sal46 vs. Mor46) to isolate the effects of morphine or temperature using GraphPad Prism 4 software. See Table 2 3 for an abbreviated experimental design time course. NMDAR Antagoni Assay Thirty two hairless rats were trained and baseline measures were established in the same way as above. Rats were then randomly split into four groups of eight. Each group received an intrape ritoneal injection of either saline or 0.1mg/kg MK 801 hydrogen maleate (Sigma, St. Louis, MO). Thirty minutes later they received a subcutaneous injection of either saline or 10mg/kg morphine. Doses of morphine and MK 801 were equivalent to those reported by Trujillo and Akil (1991) Forty minutes after the second injection rats were tested on the operant task for twenty minutes. Rats were tested fo euthanized and NACC and AMY tissue was harvested as described below forty minutes after a final morphine or saline injection on the evening of day 10. Behavioral data were analyzed with Repeated Measures (RM) Two hoc test using GraphPad Prism 4 software. See Table 2 4 for an abbreviated experimental design time course Behavioral Testing during an Extended Withdrawal Period To measure long term changes in motivational behavior and pain sensitivity during an extended withdrawal period we tested a new group of hairless rats as illustrated in
50 Figure 2 1. Forty rats were f asted for 17 +/ 1hrs (from 5pm the previous night to 9 11am the next day) then trained on the operant orofacial assay as described above. After six training sessions (three times a week) and one baseline at a non the temperature was changed separated into two groups while maintaining equal baselines on eight measures: weight, time per contact values at both temperatures, Eight rats were designated to be con trols and thirty two were placed in the morphine treatment group. Next, morphine or an equivalent volume of saline was administered twice daily for ten days in an escalating dosing paradigm as depicted in Table 2 1 and testing continued every two to three days. After the tenth day all injections were ceased and testing continued for two months. Typically rats were tested for two sessions at weights and behavioral outcomes by group were analyzed with Repeated Measures (RM) Two hoc comparison at several stages: During morphine injections, during an acute withdrawal stage of sixteen days and an extended withdrawal stage of days seventeen through fifty e ight. Unpaired t tests were used to compare expression levels between morphine and saline injected animals. F tests were used to examine variance in populations. Frequency distributions were used to place the western blot data into bins then best fit Gauss ian curves were used to determine if separate populations existed within the withdrawn rats. The morphine
51 splice variant being examined. RM Two Way ANOVAs with Bonferron p ost hoc comparison was used to examine differences i n behavioral outcomes for the separated groups. All tests were performed using GraphPad Prism 4 and 5 software La Jolla, CA). For all tests, p values less than 0.05 were considered significant. Open Field Assay Morphine can alter locomotor activity (Timar et al., 2005) so we examined locomotor and rearing effects during an extended withdrawal period. The forty rats (Med Associates, St. Albans, VT) for five minutes and locomotor and rearing behavior were analyzed. One attenuation and one baseline session were performed before the morphine injections began. After the baseline session, rats were divided into two groups which were not significantly diff erent with respect to three locomotor m easures in addition to the operant measures mentioned previously: total distance traveled, number of rears, and time spent rearing. Testing sessions began on day 2 of the morphine injections four hours after their thi rd 5mg/kg dose. One session was during the acute withdrawal stage on day 4, and three sessions occurred during the extended withdrawal period on days 18, 39, and 53. Tissue Collection At the end of each experiment rats were euthanized by CO 2 inhalation fo llowed by rapid decapitation. Brains were removed and placed in an ice cold Acrylic Rat Brain Slicer Matrix (Zivic Instruments, Pittsburg, PA). Bilateral areas of interest were removed using a 2mm Harris Uni Core puncher with the Paxinos & Watson rat brain atlas (1998) as a guide. Areas were removed from the slices cut using the following distances from
52 Bregma: NACC, 0 to 2mm; VPM, AMY, and HIPP 2 to 5mm; VTA and PAG, 5 to 8mm; (LC), 8 to 11mm. SC sect ions were taken from C1 to C4. Tissues were placed in 1.5ml tubes and immediately frozen in liquid N 2 T issues were later sonicated with a Sonics Vibra Cell Sonicator (Danbury, CT, USA) at 60 Amps for ten seconds in t issue disruption b uffer (0.3% SDS, 65mM DTT, 1mM EDTA, 20mM Tris, pH 8.0). Samples were centrifuged at 20,000g for ten minutes at 4C and protein quantification and western blots were performed as described below. Primary Neuronal Cultures Primary rat neuronal cell cultures were prepared with p rotocols based on Hilgenberg and Smith (2007) Ce ll culture vials (96 well plates, 12 well plates (Costar) with added 12mm round glass coverslips (Warner Instrument Corp, Hamden, CT), or 60x15mm plates (Falcon)) were coated with 0.001% Poly L ornithine (Sigma) and two hours later were rinsed twice with d dH20 then coated with 5ug/mL Laminin (Invitrogen) and kept in a 37C incubator in 5% CO 2 overnight. The next day an E17 dam was euthanized w ith CO 2 a nd decapitat ed Embryos were removed and placed into ice cold sterile dissecting solution (6.85mM NaCl 0.2 7mM KCl, 8.5 M Na2HPO4, 11 M KH2PO4, 0.27mM Hepes, 33.3mM D (+) Glucose, 43.8mM Sucrose, pH 7.4). Frontal areas containing the cortex, basal ganglia, AMY, and HIPP were separated, cut into pieces with razor blades, then placed in 37C dissociation solution (5mL TrypLE, and 500L 1M Hepes, Gibco) for 10mins. Cells were dissociated with a fire polished glass pipette, incubated for 5mins, then re dissociated and re incubated for 5mins. After a final dissociation, the solution was spun at 150g for 5mins at 4C. The supernatant was discarded and cells were re suspended in 5mL of 37C media (Neurobasal, 1mM N aPyruvate, 2mM L Glutamine (Cellgro), Pen Strep, B27 (Gibco), with 5% Fetal Bovine
53 Serum (Hyclone)). Cells were counted with a hemocytometer (Hausser Scientif ic, Horsham, PA) and plated with a density of 1x10 6 cells/mL. Starting the next day, the media was replaced with media without FBS for the duration of the experiment as needed. Drugs were added to these cultures as illustrated in Figure 2 2. On day in vitr o 21 (DIV 21), the media from non control plates was replaced with 0.1M morphine or 100M APV containing media as needed. On DIV28, 50M naloxone, 1 100 M MK 801, 10M H89, or 0.005 0.5M 8 Br cAMP (a membrane permeable cAMP analog ) was added to cultures as needed. All 60mm plates were harvested on DIV30. Plates were washed with phosphate buffered saline (PBS; 137 mM NaCl, 10 mM NaH2PO4, 2.7 mM KCl, pH 7.4), then 200uL of ice cold RIPA buffer (10% 10X RIPA buffer (Cell Signaling, Danvers, MA), 1% protease inhibitor cocktail (Thermo), 1% phosphatase inhibitor cocktail (Sigma), 1 M PMSF (dissolved in EtOH) (Sigma) in ddH20) was added and cells were loosened with a sterile scraper, pipetted into a 1.5mL tube, and sonicated with a Sonics Vibra Cell Sonicator ( Danbury, CT, USA) at 20 Amps for 10 seconds. Samples were centrifuged at 20,000g then resonicated, recentrifuged, and supernatant was retained. Protein quantification and western blot analysis were then performed as described below. Note: All western blot data for the primary neuronal cell culture experiments were performed on cells from two separate dissociations and their data was combined into a final statistical analysis. Western Blotting Protein concentration was determined by the Bicinchoninic Acid As say (Pierce Chemical Co., Rockford, IL) then a mixture of 20g of protein, ddH20, 5% 2 mercaptoethanol, and 50% 2X Sodium Dodecyl Sulfate buffer (Invitrogen, Carlsbad, CA, USA) was heated in a boiling water bath for five minutes, loaded into a 4 20 % Tris
54 g lycine gels (Invitrogen ) and run at 80V for fifteen minutes then 150V for forty five minutes. Gels were placed in transfer buffer (10% methanol, 48mM Tris, 39mM glycine, pH 9.2) for thirty minutes then transferred onto a Millipore (Bedford, MA, USA) Immob i lon P polyvinylidene fluoride membrane using a Biorad semi dry transfer device (Hercules, CA, USA). Membranes were blocked in 5% dry non fat milk TTBS buffer (20mM Tris HCL, 0.9% NaCl, 0.05% Tween 20, pH 7.4) for one hour. Primary antibodies for N1 and C1 (both 1:4000, rabbit, Iadarola, NIDCR/NIH), C2 (1:3000, phosphate dehydrogenase (GAP, 1:4000 15000, mouse, Pierce Thermo, Rockford, IL), CAL (1:1000, Mouse, BD Transdu ction Labs, San Jose, CA), pNR1 Ser897 (1:1000, rabbit, Millipore, Bedford, MA), PKA (1:1000, Mouse, BD Transduction Labs, San Jose, CA), pPKA C T197 (1:1000, rabbit, Cell Signaling, Danvers, MA), or pCREB pSer133 (1:1000, rabbit, Pierce Thermo, Rockford, IL) were placed on membranes on a rotator at 4C. The next day blots were washed in TTBS three times for ten minutes and secondary antibody was added (anti rabbit or anti mouse IgG, HRP linked, 1:4000, Cell Signaling, Danvers, MA) for one hour. Blots were washed three times for five minutes each and then detected using ECL Plus or ECL Prime (Amersham, Pittsburg, PA) and Biomax MR film (Kodak, Rochester, NY) or the Carestream Image Station 4000MM (Carestream Health, Rochester, NY). Band density was measured with ImageJ software (National Institutes of Health, Bethesda, MD) for the film or Carestream Molecular Imaging Software. Double labeled Immunocytochemistry Primary neuronal cell culture coverslips were removed from media and fixed in 4C 10% Buffered Form alin Phosphate (Fisher) for 10mins at RT (room temperature),
55 rinsed 3X for 10mins in rinse buffer (PBS with 0.1% TritonX (Fisher)), and blocked for 60mins with blocking buffer (5% Normal Donkey Serum (Sigma), 5% Bovine Serum Albumin (Sigma) in PBS with 0.1 % TritonX). Primary antibodies to the MOR (1:100, guinea pig, Abcam) and NR1 (1:100, rabbit, Epitomics) were added to new blocking buffer and left overnight at 4C. Slips were washed with rinse buffer 3X10mins, then fluorescent secondary antibodies (1:1000 Alexa Fluor 594 goat anti mouse and Alexa Fluor 488 goat anti rabbit (Invitrogen)) in new blocking buffer were added. After an hour of dark incubation at RT and 3X10mins washes in rinse buffer, cover slips were flipped onto a slide with Mowiol mounting m edia (0.1% Mowiol 4 88 (Millipore), 25% glycerol, 0.1M tris, in ddH20, pH 8.5). Images were obtained with a Photometrics cascade cooled EMCCD camera using the Open Source software package MicroManager connected to a spinning disk confocal system with a Lei cs DMIRB microscope with a 63X oil immersion objective according to the protocols of Brown et al. (2012) Images were analyzed and combined with ImageJ software. Quantitative Internalization Assay Neuronal cul tures were plated on DIV1 onto two 96 well plates and drugs (morphine, naloxone, H89, 8 Br cAMP, APV, and MK 801) were added as illustrated in Figure 2 2. On DIV30 the buffer was removed and cells were washed once in PBS before being fixed with 4% paraform aldehyde for 30 min at RT. Paraformaldehyde alone was demonstrated to not cause substantial permeablization of cell membranes in this assay which allows it to be used as a surface expression assay (Daigle et al., 2008) Following fixation, cells were washed 5X for 30 min in PBS (with no added detergent to avoid membrane permeabili zation), and blocked for 90 min in LI COR Odyssey Blocking Buffer (LI COR Biosciences, Lincoln, Nebraska) at room temperature with gentle
56 rocking. After blocking, cells were incubated overnight at 4C with an a ntiNR1 antibody which detects only the extrace llular portion of the NR1 subunit (1:250, BD Pharminogen, San Jose, CA). The cells were washed 5X the following day in Tris buffered saline containing 0.05% Tween 20 (TBST; 1 37 mM NaCl, 10 mM Tris, 0.05% Tween 20, pH 7.4) for 30 min. A fluorescent antibody (1:1000, Alexa Fluor 594 goat anti rabbit, Invitrogen) in blocking buffer was added for 90 min then plates were washed 5X in TBST and dried. The plates were then rewashed over several days with TBST and dried again to reduce background staining. Plates we re read on a Synergy HT plate reader (Biotek, Winooski, VT) with an excitation setting of 590 and an emission setting of 617. Note: This experiment was performed o n two separate 96 well plates.
57 Table 2 1. E scalating morphine doses. T hese doses were admin istered twice daily to rats on days 1 10. Control animals received equivalent volumes of saline. Day M orphine Dose 1 2 5 mg/kg 3 4 10 mg/kg 5 6 20 mg/kg 7 8 40 mg/kg 9 10 60 mg/kg Table 2 2 Experimental outline for morphine induced NR1 spli ce variant expression changes. Day (s) Procedures performed 0 Baseline hindpaw latencies 1 10 E scalating morphine inj ections (Table 2 1) 10 Harvest tissue from half of the saline and morphine treated groups 11 13 Withdrawal 13 14+ Harvest remaini ng tissue Western blot analysis Table 2 3 Experimental outline to study the effects of morphine tolerance on aversive and non aversive temperatures on the operant orofacial pain assay. Day (s) Procedures performed 1 4 2 Training ( t hree times a week for two weeks) 0 (B37) Baseline measures at 37 C for Sal37 and Mor37 0 (B46) Baseline measures at 46 C for Sal46 and Mor46 1 10 1, 3, 5, 8, 10 Twice daily 10mg/kg morphine (or saline) injections Operant testing at 37 C for Sal37 and Mor37 1, 3, 5, 8, 10 Operant testing at 46 C for Sal46 and Mor4 6 Table 2 4 Experimental outline to study the effects of NMDAR antagonism on morphine induced behavior on the operant orofacial pain assay and NR1 splice variant expression. Day (s) Procedures performed 19 5 Training ( t hree times a week for two wee ks) 3 (B37) Baseline measures at 37 C 0 (B46) Baseline measures at 46 C 1 10 3, 5 8, 10 Twice daily 0.1mg/kg MK 801 (or saline) injections, followed 30min later by 10mg/kg morphine (or saline) injections Operant testing at 37 C Operant testing at 46 C 10 11+ Harvest all tissue Wester n blot analysis
58 Figure 2 1. Experimental timeline for extended withdrawal study. Rats were trained on the operant task for two weeks, followed by two weeks of baseline testing. Ten days of morphine injections and acute withdrawal then took place follo wed by eight weeks of an extended withdrawal period where testing continued. Rats were then euthanized and tissue was harvested at the end of the 14th week. Figure 2 2. Cell culture timeline. Primary neuronal cell cultures were prepared with tissue from frontal areas containing the cortex, basal ganglia, amygdala, and hippocampus from E17 embryos on day in vitro 1 (DIV 1). On DIV 21, the media from non control plates was replaced with morphine or APV containing media as needed. On DIV 28, naloxone, MK 80 1, H89, or 8 Br cAMP was added to cultures as needed. All cultures were harvested on DIV30. In the case of the NR1 surface expression assay, cells were fixed with paraformaldehyde on DIV30.
59 CHAPTER 3 RESULTS Behavioral Assessment of Morphine Tolerance and Withdrawal Tolerance to the analgesic effects of morphine was measured using a thermal sensitivity assay. No significant differences were found between the groups at baseline. Rats were then injected with escalating doses of morphine (Table 2 1) or an equ ivalent volume of saline for ten days. A significant effect for morphine (F(1,150)=36.38, p<0.0001), time (F(10,150)=6.024, p<0.0001), and their interaction (F(10,150)=6.104, p<0.0001) were observed with RM Two Way ANOVA. Latencies returned to levels not s ignificantly different from saline controls during the three withdrawal days: 11 13 (RM Two Way ANOVA, treatment (F(1,12)=3.559), time (F(2,12)=0.1310), interaction (F(2,12)=1.921) (Figure 3 1A). Rats were weighed every other day throughout the procedure a nd during the ten days of morphine injections a significant effect for morphine was not observed with RM Two Way ANOVA (F(1,125)=1.865, p=ns) although a significant effect was observed for time (F(5,125)=12.21, p<0.0001), and interaction (F(5,125)=22.40, p <0.0001) likely due to morphine inhibiting weight gain. Morphine had a significant effect on weight during withdrawal (RM Two way ANOVA: morphine (F(1,12)=23.35, p=0.0004), time (F(1,12)=2.091, p=ns), interaction F(1,12)=6.794, p=0.0229). Post hoc tests we re significant for weight loss on withdrawal days 11 (p<0.05) and 13 (p<0.01) (Figure 3 1B). Morphine Tolerance and Withdrawal Alters NR1 Expression Brain regions were isolated from the rats mentioned above and western blots were run on their tissue. Weste rn blots were probed with antibodies specific for N1, C1, C2,
60 (four from each treatment group) but all rats were included in the analysis (Figure 3 2). Densitometry scores were normalized by GAP scores as a loading control and expressed as a percentage of control. All scores were analyzed with One Way ANOVA hoc test. Decreases in the N1 cassette occurred in the NACC and AMY during the tolerance phase a nd these levels stayed low during withdrawal (F(2,23)=5.825, p=0.0090 and F(2,23)=5.136, p=0.0143 respectively). A decrease in N1 was also observed between the morphine and withdrawn rats in the LC (F(2,23)=6.820, p=0.0047) (Figure 3 3A). A decrease in the C1 cassette was observed in the HIPP during the tolerance phase and these levels stayed low during withdrawal (F(2,23)=6.021, p=0.0079). C1 also decreased in the AMY during withdrawal (F(2,23)=3.451, p=0.0489) (Figure 3 3B). C2 containing NR1 subunits inc reased significantly in the HIPP during the tolerance phase and these levels were decreased when morphine rats were compared to withdrawn ones (F(2,23)=5.856, p=0.0085) (Figure 3 containing NR1 subunits decreased significantly during withd rawal in the NACC, AMY, and the HIPP (F(2,23)=6.564, p=0.0056; F(2,23)=7.655, withdrawal as well (F(2,23)=4.266, p=0.0266) (Figure 3 3D). No significant changes were found in th e VTA, PAG, or SC. Since all NR1 subunits must have either a C2 or due to their mutual exclusivity, their combined levels should reflect total NR1 levels therefore we also examined total NR1 levels whenever changes in these two splice variants were obs erved. All scores were analyzed with One hoc test. In the NACC, a significant decrease in total NR1 was observed in withdrawal when
61 compared to the morphine rats (F(2,23)=3.684, p=0.0409). In the AMY, a decrease in total NR1 was also observed in withdrawal when compared to the morphine rats (F(2,23)=3.827, p=0.0368). In the HIPP, a significant increase in total NR1 protein occurred in the morphine animals and returned to levels not different from the saline controls after the three days of withdrawal (F(2,23)=4.051, p=0.0311). No change in total NR1 was observed for the VPM (Figure 3 4). All significant western blot results for total NR1 and splice variant cassette expression are summarized in Table 3 1. Acute Morphine Adminis tration does not Alter NR1 Splice Variant Expression We examined the effects of an acute dose (60mg/kg) of morphine on NR1 splice variants in all the areas in which a change was observed during the morphine phase of experiment one. No significant changes w ere observed in the NACC, AMY, or HIPP for any of the variants tested (for all two sample t tests df=10 and p>0.05) (Figure 3 5A C). Morphine Tolerance Alters Pain and Motivational Reward Seeking Behavior on the O perant Orofacial Nociception Assay. The eff ects of morphine tolerance on an operant orofacial nociception assay were assessed at two different temperatures. Representative effects for contact behavior for Sal37, Sal46, Mor37, and Mor46 are displayed in Figure 3 6A D, respectively. The time per cont act is decreased when comparing Sal37 to Sal46, but morphine increases the time per contact at both temperatures. As observed in Figure 3 7A, an aversive temperature of 4 test, t=4.325, df=63, p<0.0001, n=64). Subsequent values in Figu re 3 7 B are expressed as a vidual rat.
62 Four separ ate Two hoc tests were run comparing Sal37 to Sal46, Mor37 to Mor46, Sal37 to Mor37, and Sal46 to Mor46. These r esults can be found in Table 3 2 The aversive temperature over the five testing sessions resulted in Sal46 ra ts having lower time per contacts compared to Sal37 rats (p=0.0248). This is likely an adaptive behavior to a constant aversive stimulus. The rats have lower time per contact values over time if they are constantly subjected to aversive stimuli each time t hey are placed in the operant box. A significant effect of temperature was not observed for the Mor37 versus Mor46 rats suggesting thermal pain was not having an effect with this dose of morphine. There was an effect for time (p<0.0003) demonstrating that over time tolerance occurred in both groups. Mor37 rats had significantly increased time per contact values compared to Sal37 rats (p=0.0077) especially on the first day of injections suggesting that morphine is having more than just an analgesic effect on this assay. Mor46 rats had greatly increased time per contact significant effects were observed for morphine (p<0.0001), time (p<0.0001), and an interaction (p<0.0001) s over time. Significant effects were observed on days 1 and 3 with post hoc tests (Figure 3 7B). Differences were also observed in reward seeking behavior as evidenced by differences in the time s pent making facial contacts to receive the reward. No differences were observed between Sal37 and Sal46 rats for facial contact times suggesting that over time neither of these groups changed from their baseline values. No differences were observed between Mor37 and Mor46 rats, suggesting that they
63 spent equivalent times responding for reward A significant effect of morphine on contact time was observed when comparing Sal37 to Mor37 (p<0.0001) suggesting that morphine treated rats spent more time obtain ing the r eward than saline controls at aversive temperatures. Mor46 rats also spent significantly more time making facial contacts than Sal46 rats did (p<0.0001). Differences were observed with post hoc tests on days 3, 5, and 8 suggesting that morphine has a larger effect at this aversive temperature. Time was not significant for any of the morphine treated rats suggesting that rats did not become tolerant to this reward seeking behavior over the course of the ten day treatment (Figure 3 7C). Similar results to contact time were observed for the time spent licking to receive the reward. Sal37 rats spent the same amount of time licking as Sal46 rats did and, over time, both increased their licking (p=0.0077). Mor46 and Mor37 rats spent equal amounts of time lic king and both increased their licking over time (p=0.0488). Although Mor37 rats had a trend of spending a larger amount of time licking than Sal37 rats this was not significant, but Mor46 rats spent a much larger amount of time licking than Sal46 rats (p<0 .0001). This effect changed over time (p<0.0045) likely due to the saline rats increasing their licking on day 10. Significant increases in licking for Mor46 rats were observed with post hoc tests on 3, 5, and 8 (Figure 3 7D). No Differences in an Acute Mo rphine Dose at Hot, Cold, or Neutral Temperatures Morphine increased time per contact behavior at hot, cold, and neutral temperatures equally ( One Way ANOVA, F(2,40 )= 0.9729 p=0. 3868, Figure 3 8 A) Similar results were observed with facial contact times ( O ne Way ANOVA, F(2,40 )= 2.231 p=0. 1207, Figure 3 8 B) and times spent licking ( One Way ANOVA,
64 F(2,40 )= 0.1381 p=0. 8714, Figure 3 8 C) at all temperatures effects on motivation are inhibited by neither hot nor cold temperatures. NMD AR Antagonism Alters Morphine induced Behavior on the Operant Orofacial Assay Hairless r ats were injected with either saline (S, n=8), MK 801 (MK, n=8), morphine (M, n=8), or morphine with an MK 801 pretreatment (MM, n=8) and tested on the operant orofacia l nociception assay. RM Two hoc test data for all of the following analyses can be foun d in Table 3 3 On days 3 and observed for the time (p=0.0197), likely due to the increases in the MK 801/morphine group (Figure 3 (p=0. 0411) and time (p=0.0036) but not an interaction on the time per contact values. On day 10 the MK 801/morphine group had a significantly higher time per contact values than the MK 801 rats (Figure 3 significant effect of treatment (p<0.0001), time (p<0.0001), and an interaction was observed (p=0.0002). Again, MK 801/morphine rats had the greatest change in behavior as they spent more time on the facial contact than any other group. On days 3 and 5 MK 801/morphine rats had higher contact times than saline, morphine, and MK 801 rats (Figure 3 9C). Morphine/MK 801 rats spent more time making facial contacts at (p<0.0001), and an interactio n (p<0.0001). On days 8 and 10 MK 801/morphine rats had higher contact times than the other three groups. Also, MK 801 rats had lower facial contact times on day 10 than morphine rats as well (Figure 3 9D). For licking behavior was observed for treatment group (p=0.0005), time
65 (p=0.0012), and an interaction between the two (p=0.0348). On day 3 the MK 801/morphine group spent more time licking than both saline and MK 801 treated rats. On day 5 MK 801/morphine rats spent more time obtaining reward than saline and morphine rats. Morphine rats also spent more time licking than MK 801 treated rats on day 5 (Figure 3 (p<0.0001), and an interaction (p=0.0023) on licki ng behavior. On day 8 the MK 801 rats licked significantly less than the other t hree groups. On day 10 the MK 801/morphine rats again had the largest increase in behavior from baseline as they licked more than saline, MK 801, and morphine rats. MK 801 rats also licked less than saline and morphine treated rats on day 10 (Figure 3 9F). Effect of NMDAR Antagonism on Morphine Induced NR1 Expression Western blot analysis of NACC, AMY, and HIPP extracts were conducted to determine if NR1 splicing was altered due to morphine and MK 801. Significance was assessed with One hoc test. In the NACC, the N1 cassette was significantly altered by treatment group (F(3,28)=5.718, p=0.0035) as N1 was slightly but not significantly, decreased i n the morphine and MK 801 groups and these appeared to have an additive effect in the combined group. The MK 801/morphine group had significantly decreased N1 as compared to the saline group (p<0.01) and the morphine group (p<0.05) (Figure 3 10A). Results were similar for the AMY (F(3,28)=6.386, p=0.0020) as N1 decreased compared to saline injected rats with both the MK 801/saline treatment (p<0.05) and the MK 801/morphine treated animals (p<0.01) (Figure 3 10B). No significant changes in N1 occurred in the HIPP (Figure 3 10C). In the NACC, C1 levels also decreased with drug treatment (F(3,28)=3.568, p=0.0265) and MK 801/morphine treated rats had significantly lower levels than saline
66 treated rats (p<0.05) (Figure 3 10D). Similar results again occurred in th e AMY with C1 (F(3,28)=27.80, p<0.0001). When compared to both saline injected and morphine injected rats C1 significantly decreased in the MK 801/saline rats (p<0.05, p<0.05) and the MK 801/morphine injected rats (p<0.001, p<0.001). Also, the MK 801/morph ine treated rats had lower C1 level s than the MK 801/saline treated rats (p<0.001) (Figure 3 10E). In the HIPP, C1 splicing also changed with drug treatment (F(3,28)=10.27, p<0.0001). The MK 801/saline treated rats had lower levels of C1 than the saline ra ts (p<0.001), morphine (p<0.05), and MK 801/morphine rats (p<0.001) (Figure 3 10F). No change was observed for C2 containing NR1 subunits in the NACC, AMY, or the HIPP (Figures 3 containing subunits significantly increased in the NACC ( F(3,28)=4.125, p=0.0153) when comparing the MK 801/morphine group to controls (p<0.05) (Figure 3 3 10K and L). Finally, NR1pan levels did not differ significantly due to any of the drug trea tments for the NACC, AMY, or HIPP (Figures 3 10M, N, and O). the NACC we would have expected NR1 total to increase as well but it did not. This could be due to NR1 subunits in the NACC contain ing more C2 (85%) 15% ) (Winkler et al., 1999b) levels of NR1 total to produce a detectable change in expression with our western blots. Weight Change during an Extended Withdrawal Period Rats were weighed three times a week throughout the course of the study. No group effect for a difference in weight w as observed during the baseline or injection periods between the saline and morphine treated groups (Figure 3 11A; F(1,38)=0.3613, p>0.05). There was a significant effect for time (F(6,38)=182.2, p<0.0001) and an interaction however (F(6,38)=12.83, p<0.0 001) as both groups
67 tended to gain weight through this period but morphine treated rats gained less. During the acute withdrawal stage morphine treated rats had significantly decreased weights compared to saline injected rats (Figure 3 11B; F(1,38)=12.77, p=0.0010). Significant effects were also observed for time (F(6,38)=498.0, p<0.0001) and an interaction (F(6,38)=32.17, p<0.0001) as both groups still tended to gain weight during this period. During the extended withdrawal phase, the morphine treated rats different than the saline treated rats as they had gained back most of the lost weight (Figure 3 11C; F(1,38)=0.7006, p>0.05). There was a significant effect for time (F(6,38)=272.8, p<0.0001) due to both groups gaining weight but no int erac tion (F(6,38)=0.8461, p>0.05). Behavioral Testing with Escalating Morphine Doses Escalating doses of morphine (Table 2 1) significantly increased motivational b 12A; RM Tw o Way, F(1,217)=30.31, p<0.0001). Post hoc tests were significant for the 10, 20, and 40 mg/kg doses (p<0.001, p<0.001, and p<0.05). No significant effect was observed for time (RM Two Way, F(5,217)=2.151, p>0.05) or an interaction (RM Two Way, F(5,217)=2. 013, p>0.05) as saline rats had consistent time per contact levels. by an increase in facial contact times (Figure 3 12B; RM Two Way ANOVA, F(1,217)=15.74, p<0.0001) at the 1 0 and 40 mg/kg doses (post hoc tests, p<0.05). A significant effect was also observed for time as the saline treated rats also increased their contact times each session (RM Two Way, F(5,217)=7.334, p<0.0001) albeit at a lesser amount than the morphine tre ated rats. No interaction was observed (RM Two Way, F(5,217)=1.263, p>0.05).
68 Behavioral Testing during an Acute and Extended Withdrawal Period Figure 3 13A illustrates that rats during the acute stage of morphine withdrawal had a higher time per contact a Way ANOVA revealed a significant effect for group (F(1,38)=6.904, p=0.0123) but not for time or an interaction (F(3,38)=2.153, p>0.05; F(3,38)=0.5802, p>0.05). During extended withdrawal this effect on the time per contact leve ls was no longer evident (RM Two Way A NOVA; group F(1,38)=1.587, p>0.05; time F(3,38)=3.719, p<0.0001; interaction F(3,38)=0.3613, p>0.05). This suggests that at non aversive temperatures withdrawing rats have a higher motivation to obtain the reward than saline controls in the acute stages of withdrawal. Figure 3 13B illustrates that both groups have similar total facial contact times throughout withdrawa observed for group (RM Two Way ANOVA, F(1,38)=1.008, p>0.05) or time (F(3,38)=1.252, p>0.05), but an effect is observed for an interaction (F(3,38)=4.831, p=0.0033). During the extended phase no effect is obse rved for group (F(1,38)= 0.1752, p>0.05) or an interaction (F(3,38)=1.691, p>0.05), but a significant effect for time was observed (F(3,38)=6.641, p<0.0001) suggesting both groups slightly increased their facial contact times over the course of extended wi thdrawal in non aversive conditions. No obvious differences were observed between groups during the acute withdrawal phase for the time per contact leve ly inhibited by t he aversive heat stimuli (Figure 3 13C; group F(1,38)=0.08164, p>0.05; time (3,38)=2.939, p>0.05; interaction F(3,38)=1.020, p>0.05) or the extended withdrawal p h ase (Figure 3 13 C, group F(1,38)=0.7164, p>0.05; time F(3,38)=0.1076, p>0.05; interaction F(3,38)=0.3139, p>0.05). Facial contact times between groups were similar during the acute phase and both changed over time (Figure 3 13D; group
69 F(1,38)=1.677, p>0.05; time F(3,38)=4.992, p=0.0092; interacti on F(3,38)=0.1988, p>0.05). During the extended phase however, an effect was observed for group as the (Figure 3 13 D; group F(1,38)=5.123, p=0.0294; time F(3,38)=0.3425, p>0.05; interaction F(3,38)=0.4801, p>0.05) demonstrating that alterations in sensitivity to aversive st imuli can be detected by this assay for up to two months after chronic morphine withdrawal. With a new set of six rats the escalating morphine doses from Table 2 1 were administered. On day 2 and 5 of withdrawal a 5mg/kg injection of naloxone was given 40 mins before testing. At an aversive temperature on day two day of withdrawal, naloxone had no effect on time per contact (Figure 3 14A) or facial contact behavior (Figure 3 14B). However, on day five of withdrawal while testing at non aversive temperatures naloxone did decrease motivational behavior (Figure 3 14C, One Way hoc test was significant for the withdrawal vs. withdrawal and naloxone group, p<0.05)). Naloxone also had a significant effect on facial con 14D, One Way ANOVA, F(2,43)=4.634, p=0.0150, no post hoc tests were significant). NR1 Expression Differences in the NACC and AMY after an Extended Withdrawal Period After euthanizing and harvesting tissue western blot analysis w as performed and t tests and F tests were used to determine if differences existed between the saline and withdrawn grou ps. As demonstrated in Table 3 4 no overall densitometry differences in the means were observed in the total NR1 subunit or any splice cassettes. F tests revealed that the variances were altered in the AMY (for the N1, C1, and C2 cassettes)
70 and the NACC (C1 cassette). We hypothesized that the differences in variances between these groups could be due to there being more than one populatio n of rats in the withdrawn group. One of these groups may be more sensitive to the aversive stimuli C1 and C2 Expression in the AMY is Associated with Differences in Behavior during Aversive, Painful Condi tions In the AMY, the C1 cassette of all withdrawn animals was not significantly different from controls (Figure 3 15A, unpaired t test, t(37)=1.084, p>0.05). Howev er, the withdrawn group did have a larger variance (F(30,7)=5.959, p=0.0203). Densitometry d ata was plotted into a frequency distribution with bins of five percentage points as compared to controls. A Gaussian best fit for one population has an R 2 of 0.6509 (Figure 3 15B), but fitting the data to two populations gives R 2 values of 0.8650 (Figure 3 15C). An extra sum of squares comparison of fits was also performed and a p<0.0001 was given in favor of the two population fit over the one population fit for the withdrawn animals (F(3,28)=14.80). This further suggests that two distinct populations of extended withdrawal period, the Low C1 than either the High C1 or control groups (Figure 3 15D). RM Two Way ANOVA was significant for a group effect (F(2,111)=9.92, p=0.0309) but not for time (F(3,111)=0.56, p>0.05) or an interaction (F(6,111)=0.61, p>0.05) throughout this phase. These data indicate that lower C1 levels in the AMY are associated with inhibited motivational behavior during aversive conditions in a subset of the withdrawn population.
71 Also in the AMY, the C2 cassette of all withdrawn ani mals was not significantly different from controls (Figure 3 16A, unpaired t test t(37)=1.029, p>0.05). However, the withdrawn group had a larger variance (F(30,7)=8.020, p=0.0082). Densitometry data was plotted into a frequency distribution with bins of t en percentage points as compared to controls. A Gaussian best fit for one population has an R 2 of 0.791 (Figure 3 16B), but fitting the data to two populations gives R 2 values of 0.9934 (Figure 3 16C). An extra sum of squares comparison of fits was perform ed and a p<0.0001 was given in favor of the two population fit over the one population fit for the withdrawn animals (F(3,6)=60.85). This strongly suggests that two distinct populations of withdrawn rats Throughout the extended of the other groups (Figure 3 16D). RM Two Way ANOVA was significant for a group effect (F(2,108)=3.717, p=0.0341) but not for time (F(3,108)=0 .5438, p>0.05) or an interaction (F(6,108)=0.3540, p>0.05) throughout this phase. These data indicate that lower C2 levels in the AMY are also associated with inhibited motivational behavior during aversive conditions in a subset of the withdrawn populatio n. Since both low C1 and lo correlation gave a p=0.0961 and an R 2 value of 0.09 (Figure 3 17). This indicates that the loss of either C1 or C2 alone could be N1 Expression in the AMY and C1 Expression in the NACC are not Associated with Differences in Behavior During Aversive, Painful Conditions Also in the AMY, the N1 cassette of all withdrawn animals was not sign ificantly different from controls (Figure 3 18A, unpaired t test t(37)=0.8959, p>0.05). However,
72 the withdrawn group did have a larger variance (F(30,7)=6.831, p=0.0135). Densitometry data was plotted into a frequency distribution with bins of ten percenta ge points as compared to controls. A Gaussian best fit for one population has an R 2 of 0.7709 (Figure 3 18B), but fitting the data to two populations gives R 2 values of 0.8731 (Figure 3 18C). An extra sum of squares comparison of fits was also performed an d a p=0.0470 was given in favor of the two population fit over the one population fit for the withdrawn animals s were observed between th 18D). (RM Two Way ANOVA, group (F(2,108)=2.540, p=n.s.) but not for time (F(3,108)=0.56, p>0.05) or an interaction (F(6,111)=0.61, p>0.05) throughout this phase. Finally, the C1 cassette of all withdraw n animals in the NACC was not significantly different from controls (Figure 3 19A, unpaired t test t(37)=0.3431, p>0.05). However, the withdrawn group did have a larger variance (F(30,7)=4.681, p=0.0408). Densitometry data was plotted into a frequency dist ribution with bins of five percentage points as compared to controls. A Gaussian best fit for one population has an R 2 of 0.7305 (Figure 3 19B), but fitting the data to two populations gives R 2 values of 0.8015 (Figure 3 19C). An extra sum of squares compa rison of fits was also performed and a p=0.0054 was given in favor of the two population fit over the one population fit for the withdrawn animals group based on the two pop ulation distribution. Similar to findings for N1 in the AMY, no (Figure 3 19D). (RM Two Way ANOVA, group (F(2,111)=2.610, p>0.05) but not for time
73 (F(3,111)=0.4979, p>0.05) or an interaction (F(6,111)=0.9481, p>0.05) throughout this phase. pNR1 and CAL levels in the NACC are Associated with Reward seeking Behavior Phosphorylation changes and as sociated alterations in phosphat ase pathways in the accumbens were also observed after extended withdrawal. Figure 3 20A illustrates that serine 897 o f the NR1 subunit (pNR1) increases in the withdrawn group of r ats (t(38)=2.365, p=0.0232). This level of phosphorylation correlated with increased on the operant orofacial assay (Figure 3 20B, Pearson p=0.0287). The withdrawn rats were then separated by a mean split into two groups ba sed on their pNR1 expression: Low pNR1 and High pNR1. The rats with the highest pNR1 levels had significantly higher time per contact value on the last day of testing (Figure 3 20C, One hoc test was significant when comparing the Low to High pNR1 group (p<0.05). Plotting these three groups of rats over time demonstrates that the High pNR1 rats had consistently greater motivational behavior throughout the extended withdrawal period (Figure 3 20D). A Tw o Way ANOVA was significant for a group (F(2,296)=3.680, p=0.0349) and time effect (F(8,296)=2.248, p=0.0241), but not significant for an interaction between the two during the extended withdrawal period (F(16,296)=0.9537, p=0.5081). A likely cause of the increase in phosphorylated pNR1 could be greater kinase activity or lower phosphatase activity in the NACC. Western blot analysis demonstrated that there was a de crease in the pNR1 S897 phosphat ase CAL in the accumbens (Figure 3 20E, t(38)=2.216, p=0.0327) No differences in PKA (Figure 3 20F, t(38)=0.2555, p=n.s.) or pPKA levels (Figure 3 20G, t(38)=1.338, p=n.s.) were
74 observed suggesting that kinase activity is at normal levels at the end of the extended withdrawal period. Representative blots for these d ata are illustrated in Figure 3 20H. Since CAL levels were low in the extended withdrawal period we wanted to examine their levels during the acute withdrawal period as well. Contrary to a long term withdrawal we observed an increase in CAL in the NACC aft er three days of withdrawal from the ten days of escalating morphine injections (Figure 3 21, One Way ANOVA, hoc test p<0.05 for the tolerant versus acute withdrawal group p>0.05 for all other comparisons ). CAL, pNR1, and pCREB Levels Alter during Withdrawal in Culture and are Regulated by NMDAR and PKA signaling In order to examine the components necessary for CAL levels to alter during withdrawal, we turned to a primary neuronal cell culture model. Dual labeled fluorescent immunohistochemistry demonstrated that NR1s and MORs were co localized in the same neurons (Figure 3 22) suggesting that this is a good model for examining the intracellular relationship between NR1 and morphine during withdrawal. In our primar y n euronal cell culture model, withdrawal had a significant effect on CAL protein levels (Figure 3 23A, One hoc was significant for an effect between the Mor and Mor+Nal treated cells (p<0.05). PKA activation was likely increased as pNR1 levels (Figure 3 23B, One Way ANOVA, Mor+Nal (p<0.01)) and pCREB levels were also increased during withdrawal (Figure 3 23C, One Way ANOVA, F(2,16)=8.648, p=0.0028) Mor vs. Mor+Nal (p<0.01) Control vs. Mor+Nal (p<0.05)).
75 To determine if these changes are due to NMDAR activation, new cultures were prepared and MK 801 was added in increasing doses during the withdrawal stage. The increa se in CAL during withdrawal was not altered with MK 801 (Figure 3 24A, One Way ANOVA, F(3,37)=0.6934, p=n.s.). The increase in pNR1 during withdrawal was dose dependently blocked by MK 801 (Figure 3 24B, One Way ANOVA, F(3,37)=21.71, s post hoc tests were significant when comparing 0 M to 1 M (p<0.01), 10 M (p<0.001), and 100 M (p<0.001). Post ho c tests were also significant between the highest and lowest doses (p<0.05). A similar effect was observed for pCREB as it was blocked by MK 8 01 as well (Figure 3 24C, One Way ANOVA, F(3,37)=9.488, p<0.0001). Post hoc tests were significant when comparing 0 M to both 10 M (p<0.001) and 100 M (p<0.01). To determine if these changes were due to NMDAR antagonism alone and withdrawal was not a neces sary prerequisite, new cultures were prepared and 100 M APV was added for nine days with no other drugs. CAL was increased by the APV treatment (Figure 3 25A, t(12)=3.236, p=0.0071), but neither pNR1 (Figure 3 25B, t(12)=1.319, p=n.s.) nor pCREB was altere d (Figure 3 25C, t(12)=0.5488, p=n.s.). As the alterations in pNR1 and pCREB are likely caused by PKA changes we decided to pharmacologically activate and inhibit its activity. PKA inhibition with 10 M H89 potentiated the increase in CAL levels during with drawal (Figure 3 26A, t(8)=2.751, p=0.0250), but decreased pNR1 (Figure 3 26B, t(8)=2.393, p=0.0436) and pCREB levels (Figure 3 26C, t(8)=3.884, p=0.0046). PKA activation with 8 Br cAMP had opposite effects. The CAL increase during withdrawal was dose dep endently reduced by PKA activation (Figure 3 27A, One Way ANOVA, F(3,24)=4.060, p=0.0182) as p ost
76 hoc tests were significant when comparing 0 M to 0.05 M (p<0.05). pNR1 was increased by 8 Br cAMP (Figure 3 27B, One Way ANOVA, F(3,23)=3.815, p=0.0235) and p ost hoc tests were significant between the 0 M and 0.5 M doses (p<0.05). pCREB levels also increased (Figure 3 27C, One Way ANOVA, F(3,23)=4.427, p=0.0135) as the 0 M and 0.05 M 8 Br cAMP groups were significantly different (p<0.0 5). NR1 Surface Expression alters with Morphine Withdrawal in Culture and is Regulated by NMDAR and PKA signaling Phosphorylating S897 was demonstrated previously to alter cell surface expression of NR1 subunits in culture (Scott et al., 2003) To determine if surface expression was altered in our cultures, two 96 well plates of primary neuronal cell cultures were prepared and went through similar drug treatments as the experiments above. Naloxone precipitated withdrawal increased NR1 surface expression (Figure 3 28A, One Way ANOVA, F(2,77)=7.322, p=0.0012) when compare d to cells with no drugs added (post hoc test, p<0.01) and morphine only added (post hoc test, p<0.05). No differences were observed when naloxone was added to cultures without prior morphine treatment (Figure 3 28B, t(46)=0.9054, p=n.s.). NMDAR antagonism with APV increased NR1 surface expression (Figure 3 28C, t(46)=3.178, p=0.0027) however MK 801 was able to partially block the withdrawal induced surface expression increase (Figure 3 28D, One Way ANOVA, F(3,92)=5.660, p=0.0013) as significant changes fro m control wells were observed for Mor+Nal (p<0.01) and the 1 M dose of MK 801 (p<0.01) but not at the 10 M dose. PKA inhibition reduced surface expression as well (Figure 3 28E, One Way ANOVA, F(2,77)=5.808, p=0.0045) as significant changes
77 from control we lls were observed for Mor+Nal (p<0.01) but not when H89 was added with naloxone. Unexpectedly, surface expression was also decreased when PKA was activated with 8 Br cAMP (Figure 3 28F, One Way ANOVA, F(3,92)=4.592, p=0.0048) as the 0.005 M and 0.05 M dos es appeared to block the withdrawal induced increase from control levels (p<0.01). Therefore, surface expression can be explained by the changes observed in earlier cell culture experiments for pNR1 in every case except when PKA is over activated.
78 Table 3 1. Tolerance and withdrawal changes in total NR1 and splice cassette expression as measured by western blots An represent s a significant down regulation, significant upr egulation, or no change respectively Tolerance Withdrawal NR1 N1 C1 C2 NR1 N1 C1 C2 NACC NACC AMY AMY HIPP HIPP VPM VPM LC LC VTA VTA PAG PAG SC SC Table 3 2 Two Way ANOVA values for Figure 3 7. Bonferroni post hoc tests results: an i ndicates a p value < 0.05, ** indicates a p value < 0.01, and *** indicates a p value < 0.001. Analysis Group Time Interaction Day 1 3 5 8 10 B S37 vs. S46 F(1,70)=4.591 p=0.0248 F(4,70)=1.149 p=ns F(4,70)=1.918 p=ns ns ns ns ns ns M37 vs. M46 F(1, 70)=2.553 p=ns F(4,70)=6.000 P=0.0003 F(4,70)=1.032 p=ns ns ns ns ns ns S37 vs. M37 F(1,70)=7.535 p=0.0077 F(4,70)=0.8588 p=ns F(4,70)=1.036 p=ns ns ns ns ns S46 vs. M46 F(1,70)=45.01 p<0.0001 F(4,70)=8.366 p<0.0001 F(4,70)=7.841 p<0.0001 *** ** ns n s ns C S37 vs. S46 F(1,70)=0.4016 p=ns F(4,70)=1.477 p=ns F(4,70)=1.147 p=ns ns ns ns ns ns M37 vs. M46 F(1,70)=0.7322 p=ns F(4,70)=1.519 p=ns F(4,70)=0.2285 p=ns ns ns ns ns ns S37 vs. M37 F(1,70)=18.48 p<0.0001 F(4,70)=1.413 p=ns F(4,70)=0.4721 p=ns ns ns ns ns ns S46 vs. M46 F(1,70)=60.63 p<0.0001 F(4,70)=0.5867 p=ns F(4,70)=1.325 p=ns ns *** ** ** D S37 vs. S46 F(1,70)=0.6081 p=ns F(4,70)=3.778 p=0.0077 F(4,70)=0.4857 p=ns ns ns ns ns ns M37 vs. M46 F(1,70)=3.878 p=ns F(4,70)=2.519 p=0.0488 F(4,70)=0.3475 p=ns ns ns ns ns ns S37 vs. M37 F(1,70)=2.097 p=ns F(4,70)=2.411 p=ns F(4,70)=0.3293 p=ns ns ns ns ns ns S46 vs. M46 F(1,70)=27.80 p<0.0001 F(4,70)=4.155 p=0.0045 F(4,70)=1.335 p=ns ns ** ns
79 Table 3 3 Repeated Measures Two Way AN OVA values for Figure 3 9 Bonferroni post hoc tests results: an indicates a p value < 0.05, ** indicates a p value < 0.01, and *** indicates a p value < 0.001. Analysis Group Time Interaction SvM SvMK SvMM MvMK MvMM MKvMM A Time Per Contact ns P=0.019 7 ns 3 ns 3 ns 3 ns 3 ns 3 ns 3 ns 37 C F(3,56)=1.55 F(2,56)=4.21 F(6,56)=1.60 5 ns 5 ns 5 ns 5 ns 5 ns 5 ns B Time Per Contact p=0.0411 p=0.0036 ns 8 ns 8 ns 8 ns 8 ns 8 ns 8 ns 46 C F(3,56)=3.14 F(2,56)=6.23 F(6,56)=1.98 10 ns 10 ns 10 ns 10 ns 10 ns 10 ** C Facial Contact Time p<0.0001 p<0.0001 p=0.0002 3 ns 3 ns 3 ** 3 ns 3 ** 3 *** 37 C F(3,56)=13.63 F(2,56)=17.32 F(6,56)=5.37 5 ns 5 ns 5 *** 5 ns 5 *** 5 D Facial Contact Time p<0.0001 p<0.0001 p<0.0001 8 ns 8 ns 8 8 ns 8 8 *** 46 C F(3,56)=18.50 F(2,56)=27.71 F(6,56)=7.97 10 ns 10 ns 10 *** 10 10 *** 10 *** E Time Spent Licking p=0.0005 p=0.0012 p=0.0348 3 ns 3 ns 3 ** 3 ns 3 ns 3 37 C F(3,56)=8.16 F(2,56)=7.61 F(6,56)=2.46 5 ns 5 ns 5 *** 5 5 *** 5 ns F Time Spent Licking p<0.0001 p<0.0001 P=0.0023 8 ns 8 ** 8 ns 8 ** 8 ns 8 *** 46 C F(3,56)=11.93 F(2,56)=19.32 F(6,56)=3.95 10 ns 10 10 10 ** 10 10 ***
80 Table 3 4 T tests and F tests for NR1 total and all splice variants in the NACC and AMY after an extended withdrawal. NACC AMY t test F test t test F test NR1 t(37)=1.333 p=n.s. F(30,7)=1.506 p=n.s. t(37)=0.3249 p=n.s. F(30,7)=2.649 p=n.s. N1 t(37)=0.1500 p=n.s. F(30,7)=2.817 p=n.s. t(37)=0.8959 p=n.s. F(30,7)=5.959 p=0.0135 C1 t(37)= 0.9601 p=n.s. F(30,7)=4.681 p=0.0408 t(37)=1.084 p=n.s. F(30,7)=5.959 p=0.0203 C2 t(37)=1.969 p=n.s. F(30,7)=3.517 p=n.s. t(37)=1.029 p=n.s. F(30,7)=8.020 p=0.0082 C2' t(37)=0.7447 p=n.s. F(30,7)=3.246 p=n.s. t(37)=0.5824 p=n.s. F(30,7)=2.499 p=n.s.
81 Figure 3 1. The effects of m orphine on thermal sensitivity and weight. Significance for each analysis was assessed with Repeated Measures Two Way ANOVA. A) The latency to withdraw from a heat stimulus increased from saline injected controls (n=6) on day s 1 10 when morphine was injected (n=11). Significant effects on hindpaw latency for morphine, time, and their interaction were observed. Latencies of withdrawn rats (n=5) returned to levels not significant from saline injected rats (n=3) during the three day withdrawal period (days 11 13). B) A significant effect for morphine was not observed during the injection period but a significant effect was observed for time and an interaction. Weight decreased significantly during morphine withdrawal. Significant effects on weight loss were observed for morphine, time, and their interaction. The data presented is the mean +/ SEM, an indicates a p value hoc test and ** indicates a p value < 0.01. See Table 2 2 for experiment design. Figure 3 2. A representative blot of NR1 changes in the nucleus accumbens due to morphine. Sample western blots illustrating results from 12 of the 26 animals used in this study. Each row contains data from one antibody (NR1pan, N1, See Table 2 2 for experiment design.
82 Figure 3 3. Quantification of NR1 splicing changes in the brain due to morphine tolerance and withdrawal. A) N1 decreased significantly in the morphine animals and did not return to baseline levels during withdrawal in both the NACC and the AMY. N1 also decreased in the LC during withdrawal. B) C1 decreased significantly in the HIPP during the morphine phase and these levels did not return to pre drug levels during withdrawal. C1 also decreased in the AMY during withd rawal. C) C2 increased significantly in the HIPP during the tolerance phase. These levels dropped significantly during increased in the VPM during withdrawal. No significant changes were found in the VTA, PAG, or SC for any cassettes. The data presented is the mean +/ SEM. An hoc test when compared to the saline group and a # represents significance between the morphine and withdrawn groups. Significance for all analyses was assessed with One Way hoc tests. See Table 2 2 for experiment design.
83 Figure 3 4. Quantification of NR1 total prot ein changes in the brain due to morphine tolerance and withdrawal. A significant decrease in total NR1 was observed in the NACC and the AMY in withdrawal when compared to the morphine rats. In the HIPP, a significant increase in total NR1 protein occurred in the morphine animals and returned to normal levels after the 3 days of withdrawal. No change in total NR1 was observed for the VPM. The data presented is the mean +/ SEM. An represents a significant result in hoc test when compared to the saline group and a # represents significance between the morphine and withdrawn groups. Significance for all analyses was assessed with One Way ANOVAs and hoc tests. See Table 2 2 for experiment design.
84 Figure 3 5. An acute dose of morphine does not alter NR1 splice variation. No significant changes were found in the A) NACC, B) AMY, or C) HIPP due to an acute dose of morphine. The data presented is the mean +/ SEM. Significance was assessed with two sample t tests (for all, df=1 0) for each cassette in each area with p<0.05 being considered significant.
85 Figure 3 6. A representative sample of the effects of heat aversion and morphine on the operant orofacial pain assay. This figure displays a typical reading for the contact therm odes during a 20 minute session for four different stages. On the Y axis is the amplitude of the signal which represents when a rat is pressing its face into the metal thermodes. Though it is not displayed here, during each of these bursts of contacts, the rat can reach the reward bottle and its licks are recorded by a separate channel. A. The Baseline 37C figure illustrates the normal eating bursts at a non aversive temperature. B. At aversive temperatures, the average time per contact decreases as the rat cannot hold its face on the thermode for as long a time period. C. When injected with 10mg/kg of morphine 40mins before a session, a bursting pattern with much longer bouts of eating is observed. D. This pattern of longer eating bouts is unaltered by the aversive 46C temperature acutely. See Table 2 3 for experiment design.
86 Figure 3 7. Morphine alters pain and reward seeking behavior at aversive and non aversive temperatures on the operant orofacial nociception assay. A) An increased temperature is aversi ve as demonstrated by the decrease in time baseline. B) Aversive temperatures decreased the time per contact values for saline treated rats. Morphine increased the time per contact val ues at both temperatures (Mor37 vs. Sal 37 on day 1 and Mor46 vs. Sal37 on days 1 and 3) and tolerance occurred to this over time. C) Saline treated rats had similar facial contact times. The two morphine treated groups also had similar facial contact time s. Morphine treated rats spent more time making facial contacts than saline controls at both temperatures. This behavior is most evident for times changed over time suggesting no tole rance to the morphine treated groups over the course of 10 days. D) Both saline treated groups spent the same amount of time licking and both increased their licking over time. Mor46 and Mor37 rats spent equal amounts of time licking and both increased the ir licking over time as well. Mor46 rats spent a much larger amount of time licking than Sal46 rats especially on days 3, 5, and 8. This effect changed over time likely due to the saline rats increasing their licking on day 10. Significance for each analys is was assessed with Two Way ANOVA and hoc test. N=8 for each time point for each group. These results can also be found in Table 3 2 The data presented is the mean +/ SEM, an indicates a p hoc test, ** indicates a p value < 0.01, and *** indicates a p value < 0.001. See Table 2 3 for experiment design.
88 Figure 3 8. Morphine increases time per contact values, facial contact times, and time spent licking at hot, cold, and neutral temperatures equally. Figure 3 9. Morphine and MK 801 alter pain and reward seeking behavior on the operant orofacial nociception assay at aversive and non aversiv e temperatures. A) On days 3 and 5 of injections all rats were and a significant difference was only observed for the time (p=0.0197), likely due to the increases in the morphine and the MK 801/morphine groups. B) At time per con tact values. On day 10 the MK 801/morphine group had a significantly higher time per contact values than the MK 801 rats. C) For total interaction was observed. MK 801/morphine ra ts had the greatest change in behavior as they spent more time on the facial contact than any other group. On days 3 and 5 MK 801/morphine rats had higher contact times than saline, morphine, and MK r treatment, time, and an interaction. Morphine/MK 801 rats spent more time making facial contacts than any other group on days 8 and 10. Also, MK 801 rats had lower facial contact times on day 10 than morphine rats as well. E) C a significant effect was observed for treatment group, time, and an interaction between the two. On day 3 the MK 801/morphine group spent more time licking than both saline and MK 801 treated rats. On day 5 MK 801/morphine rats spent more time obtaining reward than saline and morphine rats. Morphine rats also spent more time licking than MK effect of group, time, and an interaction between the two on licking behavior. On day 8 the MK 801 rats l icked significantly less than the other three groups. On day 10 the MK 801/morphine rats licked more than saline, MK 801, and morphine rats. MK 801 rats also licked less than saline and morphine treated rats on day 10. Results from all Repeated Measures Tw o Way ANOVAs and hoc test s can also be found in Table 3 3 The data presented is the mean +/ SEM, an indicates a p value < 0.05 for hoc test, ** indicates a p value < 0.01, and *** indicates a pvalue < 0 .001. Each gro up has an N=8. See Table 2 4 for experiment design.
90 Figure 3 10. Morphine and MK 801 co administration alters NR1 splice variation in the nucleus accumbens. A) N1 was significantly decreased in the morphine and MK 801 group as compared to the saline gro up and the morphine group. B) N1 decreased in both the MK 801 and the MK 801/morphine group compared to saline treated rats in the AMY. C) No significant changes in N1 occurred in the HIPP. D) C1 levels decreased in the morphine and MK 801 group in the NA CC. E) In the AMY, C1 significantly decreased in the MK 801 group compared to both the saline and morphine group. The MK 801/morphine group also had decreased C1 compared to the saline, morphine, and MK 801 groups. F) In the HIPP, C1 decreased in the MK 80 1 group when compared to all other groups. G) No change was observed for C2 containing NR1 subunits in the NACC. H) No change was observed for C2 containing NR1 subunits in the AMY. I) No change was observed for C2 containing NR1 subunits in the HIPP. J) C levels did not change with any treatment in the NACC. N) NR1pan levels did not change with any treatment in the AMY. O) NR1pan levels did not change with any treatment in the HIPP. The data presented is the mean +/ SEM, an *, **, *** indicates a p post hoc test wh en compared to the saline/saline group. A # and ### indicates a p hoc test when compared to the saline/morphine group. A @ and @@@ indicates a hoc tes t when compared to the MK 801/saline group. Significance was assessed with One Way ANOVAs. N=8 for each group. See Table 2 4 for experiment design.
92 Figure 3 11. Weight change during morphine administration, acute, and extended withdrawal. A. No grou p effect for a difference in weight w as observed at baseline or during morphine administration. There was an interaction and time effect though as both groups tended to gain weight, but the saline rats gained more. B. Morphine injected rats had significant ly lower weights during the acute withdrawal phase. C. No differences in weight were observed during extended withdrawal. For all graphs: saline N=8, morphine N=32. See Figure 2 1 for experiment design. Figure 3 12. The effects of m orphine tolerance on behavior at non aversive t emperatures over an escalating dosing paradigm A. Morphine treated rats had significantly higher time per c ontact values throughout the administration period. Post hoc tests were significant for the 10, 20, and 40 mg/kg doses (p <0.001, p<0.001, and p<0.05). B. Morphine also increased the facial contact times during the administration period especially at the 10 and 40 mg/kg doses (post hoc tests, p<0.05). For both graphs: saline N=8, morphine N=32. See Figure 2 1 for experiment d esign.
93 Figure 3 13. Behavioral differences between acute versus extended withdrawal at 37C and 46C. A. Morphine treated rats had significantly higher time per contact as compared to b aseline measures (Day 0) B. No differences were observed in differences were observed in the time per contact values for acute or observed for morphine extended withdrawal however, significantly lower facial contact times were observed for morphine treated rats. For all graphs: saline N=8, morphine N=32. Se e Figure 2 1 for experiment design.
94 Figure 3 14. Naloxone decreases motivational behavior at non aversive temperatures during acute withdrawal. A. A 5mg/kg injection of naloxone 40 mins before testing at an aversive temperature on the second day of with drawal does not alter time per contact behavior. B. Naloxone does not alter facial contact time at aversive temperatures on withdrawal day two. C. Naloxone does alter time per contact behavior at a non aversive temperature on the fifth day of withdrawal. D Naloxone had a significant effect on behavior at 37C for total facial contact time on day five of withdrawal. For all graphs: Saline N=8, Withdrawal N=32, and Withdrawal and Naloxone N=6. A represents a hoc test. Figure 3 15. No differences were observed in locomotor or rearing behavior during morphine administration or acute and extended withdrawal. A. No differences for total distance traveled were found with t tests during the morphine or acute morphine testing days. A Repeated Measures Two Way ANOVA was also insignificant for the extended withdrawal stage. B. and C. A similar pattern was observed for number of rearings and rearing time as no signi ficant differences were found. See Figure 2 1 for experiment design.
95 Figure 3 16. AMY C1 changes are associated with long term responses to aversive stimuli. A. No differences in the means were observed in the C1 cassette of withdrawn animals and controls but more variance is found in the withdrawn group. B. One Gaussian p opulation explains some of the data. C. Two Gaussian populations best explain the data. D. Significantly lower facial contact times were observed for the altered group during the extended f three saline, high C1, and low C1 rats in the AMY. All bands illustrated are from the same gel and blot. For A: Saline N=8, Morphine N=39. For E: Saline N=8, High C1 N=23, Low C 1 N=8. See Figure 2 1 for experiment design.
96 Figure 3 17. AMY C2 changes are associated with long term motivation and responses to aversive stimuli. A. C2 expression levels in the withdrawn animals were not significantly different from controls but more variance is found in the withdrawn group. B. One Gaussian population explai ns some of the data. C. Two Gaussian populations best explain the data. D. Low C2 rats had lower of three saline, high C2, and low C2 rats in the AMY. All bands illustrated are from the same gel and blot. For A: Saline N=8, Morphine N=31. For D: Saline N =8, Low C2 N=21, High C2 N=10. See Figure 2 1 for experiment design.
97 Figure 3 18. AMY N1 changes are not associated with long term motivation and responses to aversive stimul i. A. N1 expression levels in the withdrawn animals were not significantly different from controls but more variance is found in the withdrawn group. B. One Gaussian population explains some of the data. C. Two Gaussian populations best explain the data. D No between the groups. For A: Saline N=8, Morphine N=31. For D: Saline N=8, Low N1 N=23, High N1 N=8. See Figure 2 1 for experiment design.
98 Figure 3 19. NACC C1 changes are no t associated with long term motivation and responses to aversive stimuli. A. NACC C1 expression levels in the withdrawn animals were not significantly different from controls but more variance is found in the withdrawn group. B. One Gaussian population exp lains some of the data. C. Two Gaussian populations best explain the data. D. No differe between the groups. For A: Saline N=8, Morphine N=31. For D: Saline N=8, Low N1 N=22, High N1 N=9. See Figure 2 1 for experiment design.
99 Figure 3 20. Long term alterations in phosphory lated NR1 and CAL in the NACC are associated with altered motivation during extended withdrawal. A) pNR1 (S897) is increased in the NACC of morphine treated rats which underwent a two month withdrawal period. B) pNR1 levels have a significant positive cor relation with their time per contact behaviour on the last behavioral session at 37C on the operant orofacial assay. C) Withdrawn rats were divided into two groups based on a mean split of the pNR1 densitometry. Those with higher pNR1 levels had the highe st time per contact values on the last testing day. D) Those with higher pNR1 levels had significantly higher time per contact values throughout the extended withdrawal period. E) CAL levels were significantly lower in the withdrawn group of rats but F) PK A and G) pPKA levels were not significantly different from saline controls. H) Representative blots for pNR1, CAL, PKA, pPKA, and GAP are provided. For hoc test compared to the Low pNR1 group. For al l other graphs, an represents a p<0.05 for an unpaired t test. For all graphs: Saline (N=8), Withdrawn (N=32), Low pNR1 (N= 19), and High pNR1 (N=13). See Figure 2 1 for experiment design.
100 Figure 3 21. CAL increases after three days of withdrawal in the nucleus accumbens. CAL levels increase in the NACC after three days of withdrawal from the ten days of escalating morphine injections. Saline N=7, Morphine N=10, Withdrawn N=9 See Table 2 2 for experiment design. Figure 3 22. NMDARs and MORs a re co localized in primary neuronal cultures. Dual labeled immunohistochemistry with antibodies to the NR1 subunit of the NMDAR and the MOR and fluorescent secondary antibodies co localize in neuron s harvested from E17 embryos.
101 Figure 3 23. Naloxone in duced withdrawal increases CAL, pNR1, and pCREB in culture. Naloxone precipitated withdrawal in primary neuronal cultures increases A) CAL, B) pNR1 (S897), and C) pCREB (S133) as compared to control and/or morphine treated cultures. For all graphs: Control (N=8), Morphine (N=5), and Morphine + Naloxone (N=6). An represents a p<0.05 hoc test as compared to the Morphine+Naloxone group. Representative blots are provided below each graph. See Figure 2 2 for experiment design. Figure 3 24. NMDAR antagonism blocks some withdrawal induced alterations in pNR1 and pCREB in culture. Adding MK 801 to cultures during naloxone precipitated withdrawal has no effect on A) CAL, but dose dependently decreases B ) pNR1 (S897), and C) pCREB (S133) as compared to MOR+NAL treated cultures. For all graphs: 0 M MK 801 (N=13), 1 M (N=9), 10 M (N=9), and 100 M (N=10). An *, **, and *** represents a p<0.05, hoc test as com pared to the 0 M MK 801 group, respectively. A # represents a p<0.05 for a hoc test when comparing the 1 M to 100 M group. Representative blots a re provided below each graph. See Figure 2 2 for experiment design.
102 Figure 3 25. NMDAR ant agonism increases CAL in culture. APV treatment for nine days A) increases CAL, and has no effect on B) pNR1 or C) pCREB. For all graphs: Control (N=8), 100 M APV (N=6). A ** represents a p<0.01 value for an unpaired t test. Representative blots are provi ded below each graph. See Figure 2 2 for experiment design. Figure 3 26. PKA inhibition alters CAL, pNR1, and pCREB during n aloxone precipitated withdrawal in culture. When 10 M H89 is added to m orphine treated cultures with naloxone for two days A) CA L is increased, but B) pNR1 and C) pCREB are increased. For all graphs: Control (N=5), 10 M H89 (N=5). A and ** represent a p<0.05 and p<0.01 value for an unpaired t test, respectively. Representative blots a re provided below each graph. See Figure 2 2 f or experiment design.
103 Figure 3 27. PKA activation alters naloxone precipitated withdrawal induced alterations in CAL, pNR1, and pCREB in culture. Adding the PKA activator 8 Br cAMP to cultures during naloxone precipitated withdrawal dose dependently A) reduces CAL, but increases B) pNR1 (S897), and C) pCREB (S133) as compared to MOR+NAL treated cultures. For all graphs: 0 M 8 Br cAMP (N=7), 1 M (N=7), 10 M (N=8), and 100 M (N=6). An represents a p<0.05 hoc test as compare d to the 0 M group. Representative blots a re provided below each graph. See Figure 2 2 for experiment design.
104 Figure 3 28. Altered NR1 cell surface expression occurs with naloxone precipitated withdrawal, NMDAR antagonism, and PKA activation or inhibi tion in culture.A) Morphine treatment alone did not alter NR1 expression, but naloxone precipitated withdrawal did. B) Naloxone added to cultures without a previous morphine treatment had no effect but C) NMDAR antagonism with APV increased NR1 surface exp ression. D) 10 M MK 801 was able to partially block the withdrawal induced increase in NR1 surface expression. Also, PKA E) inhibition and F) activation were also able to partially block the withdrawal induced increase in NR1 surface expression. For all gr aphs: None (N=32), Mor (N=16), Mor+Nal (N=32), Nal (N=16), APV (N=16), Mor+Nal+1 M MK 801 (N=16), Mor+Nal+10 M MK 801 (N=16), Mor+Nal+H89 (N=16), Mor+Nal+0.005 M 8 Br cAMP (N=16), and Mor+Nal+0.05 M 8 Br cAMP (N=16). For C) an ** represents a p<0.01 value for an unpaired t test. For all other graphs and ** represents a p<0.05 and a p<0.01 value for a hoc test as compared to the None group, respectively. See Figure 2 2 for experiment design.
105 CHAPTER 4 DISCUSSION In the first experiment we demonstrated that chronic morphine administration and a subsequent acute withdrawal leads to an altered set of NMDARs in several areas of the limbic system. This was not found in acutely treated animals suggesting that the effects observed are due to an adaptation to morphine over time. We also demonstrated whether MK 801 could attenu ate tolerance i n this assay. Our findings were that MK 801 did not appear to attenuate tolerance. In addition, these two drugs had interesting effects on NR1 splicing in several brain areas, likely due to their ability to alter the excitability of neurons. The end result of these splicing changes will be altered NMDAR activity in the future which may be a mechanism for some of the detrimental effects of long term chronic opioid administration. We also observed tha t altered C1 and C2 in the AMY are associated with increased inhibition of reward seeking behavior during aversive conditions in an extended withdrawal period of sixty days. During non aversive conditions during e xtended withdrawal however, reward seeking behavior was correlated with increased levels of pNR1 in the NACC. As demonstrated by withdrawal studies in vivo and in vitro CAL expression levels are partially responsible for this increase in pNR1 and may lead to increased NR1 surface expression. This may result in altered plasticity in the NACC as the neuronal response of the NMDAR to GLU will be increased. This could provide a mechanism for the long term alterations in craving behavior seen in patients months afte r acute withdrawal has ended.
106 Morphine Tolerance and Withdrawal Alters NR1 Expression Escalating morphine doses did lead to tolerance and subsequent withdrawal in rats but there were no significant changes in NR1 in areas generally associated with pai n (PAG and SC), as determined by Western blot analysis. These results are similar to the findings of Kozela and Popik (2007) and suggest that changes in NR1 splice areas. These treatments were associated with large differences in NR1 splice varian t composition in frontal areas more related to higher cognitive functions like motivation, anxiety, and memory. Two common themes arose in the first experiment. First, it is of interest that N1 in the NACC and AMY and C1 in the HIPP decreased in the morphi ne rats and did not return to normal levels during withdrawal. This suggests that some morphine induced changes in splicing may last long after the drug is withdrawn and could be a mechanism for extended alterations in behavior after chronic drug administr ation. N1 and C1 can be regulated by calcium, membrane depolarization and neuronal activity (Lee et al., 2007;Xie, 2008) therefore these common results may be a compensating mechanism for the increased neuronal activity in these areas during morphine a dministration and withdrawal (Rasmussen et al., 1995) In the HIPP however, the morphine induced alteration did not continue through withdrawal. C2 levels rose during the tolerance phase and did return to pre drug levels during spontaneous withdrawal. The increas e in C2 containing NR1 subunits observed in the Increasing NMDAR activation in the HIPP reverses morphine induced memory loss (Zarrindast et al., 2011) and over the ten day injection period this cellular adaptation could have occ urred in an attempt to offset this morphine effect. Once the morphine is
107 no longer administ er ed and withdrawal ensues, C2 levels could then return to pre drug concomitant d a result of increased neuronal activity in culture (Mu et al., 2 003) Since the AMY, NACC, and HIPP are all very active during withdrawal (Rasmussen et al., 1995) this suggests that activity dependent changes in NR1 splicing are occurring in these areas. ivation (Cull Candy and Leszkiewicz, 2004) phosphorylation (Scott et al., 2001) and neuronal surface expression (Okabe et al., 19 99) therefore the likely end result is that after three days of withdrawal from morphine, NMDARs are going to be less responsive to GLU and less capable of future plastic events in the NACC, AMY, and HIPP. These results along with previously reported long term decreases in C2 containing NR1 subunits following cocaine withdrawal (Loftis and Janowsky, 2002) and long term increases in (Winkler et al., 1999a) suggest a commo n splicing mechanism for chronic drug induced changes in neuronal plasticity. Morphine Tolerance Alters Pain and Motivational Reward Seeking Behavior on an Operant Orofacial Nociception Assay NMDAR antagonism has been suggested to attenuate tolerance in s pinal reflex based pain assays like the thermal tail flick (Trujillo and Akil, 1991) and has also been demonstrated to alter NR1 splicing (Mu et al., 2003) We therefore hypothesized that NMDAR antagonism would lead to a different set of NR1 receptors which would be associated with attenuated morphin e tolerance. MK 801 leads to increased locomotor activity (Carlsson and Svensson, 1990) and studies in ou r lab suggest that this interferes with assessing nociception on the Plantar Test (data not shown).
108 Furthermore, spinal reflex based measures of pain may not be appropriate for measuring the alterations in morphine analgesia and tolerance we expected (King et al., 2007;Morgan et al., 2008;Vincler et al., 2001) with MK 801 pre administration (Carlezon et al., 2000) We therefore tested for NMDAR antagonist effects on morphine tolerance with an operant orofacial thermal sensi tivity assay. This assay is capable of measuring the effects of aversive thermal pain through a decrease in time per contact values and morphine antinociception which increases the time per contact (Neubert e t al., 2006;Neubert et al., 2007) When compared to baseline levels, morphine greatly increased the time per contact values versus Sal46 rats and this reduced over time to levels near baseline confirming that tolerance can be measured with this assay. Thi s effect is not entirely due to the reduction of heat pain by morphine as morphine also had the increased reward seeking behavior demonstrated by increased time spe nt making facial contacts and time spent licking. This increased reward seeking behavior is consistent with the fact that MOR agonists have previously been demonstrated to induce increases in food intake following inter accumbal infusion (Katsuura et al., 2011;Zhang and Kelley, 2002) possibly due to a ltered palatability of food rewards (Taha et al., 2009) This increased time per contact occurred regardless of temperature as it that morphine injected rats have an increased motivation to obtain the sweet reward and this is effects on the limbic system.
109 NMDAR Antagonism Alters Morphine induced Behavior on the Operant Orofacial Assay To further investigate this dual effect of morphine on motivational reward seeking injection of MK 801 on rats administered saline or morphine at both temperatures. Previous results therefore we decided to test for attenuated tolerance on these days. We also decided to period. Similar to the findings of Carlezon and Wise (1993) MK 801 potentiated onal reward morphine tolerance although studies by Carlezon et al. (2000) demonstrate that NMDAR antagonists likely do not attenuate morphine tolerance. Instead it could be a result of MK possibility is that this increase could be a continuation of the change in the motivational reward seeking properties o f morphine observed at non aversive temperatures. there is different effect when these two drugs are combined. There is a more complicated relationship than MK 801 simply attenuating morphine tolerance as suggested by previous studies (Shimoyama et al., 2005;Trujillo and Akil, 1991) This suggests that the time per co ntact values for MK some interaction between these factors. An unexpected result was that MK 801 injected rats remained at baseline levels of the time per contact values, total contact time, and time spent licking throu ghout testing.
110 This was not anticipated as MK 801 was demonstrated to increase the preference for food reward using the CPP test (Yonghui et al., 2006) and also increases food intake in rats (Burns and Ritter, 1997) On the other hand, MK 801 reduced motivation on progressive ratio schedules (Buffalo et al., 1994) therefore it could be interfering with the motivational factors necessary for reward seeking behavior on our task. Another possibility is that MK 801 may be interfering with cognition in this task. The nature of operant tasks is that a functioning cortex is needed. MK 801 alone may have interfered A dose response curve experiment in the future would shed light on this issue. Effect of NMDAR Antagonism on Morphine Induced NR1 Expression Since morphine injected into the NACC increases reward seeking behavior (Katsuura et al., 2011;Taha et al., 2009;Zhang and Kelley, 2002) we investigated this area for ch anges in NR1 splicing. Both the N1 and C1 cassettes appeared to decrease with MK 801 or morphine alone, but when both drugs were co administered statistical significance was achieved. No change in C2 expression was detected. NMDAR antagonism and morphine a mRNA (Winkler et al., 1999b) n increase in neurons without having a large measurable effect on NR1pan western blots. Together these changes suggest that the co administration of morphine and MK 801 leads to modulation of glutamatergic signaling in the NACC. MORs are most often found o n different neurons than NR1 subunits in co localization studies on NACC shell neurons (Gracy et al., 1997) Although we took tissue from the NACC shell and core,
111 t his suggests that separate groups of neurons in this motivational area would be inhibited with morphine and NMDAR antagonists. Thus, when the two drugs are combined the NACC would be highly activated. This increase in activity could initially be responsible for the potentiating effects of MK 801 on m orphine induced motivation al reward seeking. Over ten days of injections this increased activation o f the NACC would likely result in long term compensatory NR1 splice variant adaptations (Lee et al., 2007) Indeed, the neurons in MK 801/morphine rats have less responsive NR1 su bunits than the control NR1 subunits (Cull Candy and Leszkiewicz, 2004) supporting this hypothesis. These results demonstrate that the NACC may be less responsive to glutamatergic activation in the future and could play a role in d rug induced changes in behavior associated with this area. Differences in N1 and C1 splicing were also obser ved in the AMY in a similar pattern to the changes in the NACC. The MK 801 treated rats had lower levels of both splice variants and the MK 801/morphine treated rats had the lowest. These decreases in N1 and C1 in the AMY are likely due to changes in activ ity due to the drug treatments. Their similarity to the changes observed in the NACC are likely due the fact that MORs and NMDARs are found co localized on GABAergic neurons in the AMY (Glass et al., 2008) neurons (Gracy et al., 1997) Other similarities between these areas are that MK 801 was demonstrated to increase Fos like immunoreactivity in both the AMY and NACC (Carr and Kutchukhidze, 2000) Once again, this alteration in neuronal activity may lead to similar splicing patterns in both of these areas for the MK 801 treated rats. In the HIPP, splicing changes due to drug treatment were only observed in the C1 cassette
112 which decreased in both the morphine treated and MK 801 treated groups. The lowest levels of C1 were found in the MK 801 treated rats and these rats also likely had problems learning on our operant task This problem in learning is noticeable when comparing the saline treated rats to the MK 801 rats on day 8 and 10 of this experiment. The saline treated rats continued to have higher time per contacts over time, but the MK 801 treated group stayed at the same levels. This could be due to the saline rats being able to learn over time while the MK 801 rats are stalled. On days 8 and 10 every group except those treated with MK 801 alone had responding rates that were increased from baseline. This stagnant beh avior by the MK 801 rats could represent impaired learning over time. Lower C1 levels may reflect NMDARs that are less capable of synaptic plasticity (Scott et al., 2001) which could be a mechanism for the effects on learning on our operant testing. This behavior was not the case for the morphine treated rats that also had lowered C1 levels, however suggesting that other factors like an overshadowing role on the behavior in learning in this task. Interestingly, when MK 801 and morphine were co administered these changes appeared to be blocked and were at levels not significantly different from those of the saline controls. This blocki ng effect of MK 801 on morphine induced C1 changes could be a mechanism for MK acquisition of morphine induced CPP a behavior dependent on HIPP functioning (Zarrindast et al., 2007) It should be noted that some of the splice variant changes we report here may be dose dependent. The escalating dose paradigm used in a previous experiment result ed in much higher amounts of morphine being administered over time than the 1 0mg/kg
113 twice daily dose paradigm in latter experiments. As a result more subtle effects are observed in our western blots. The N1 changes in the NACC and AMY and the C2 changes in the HIPP did not obtain statistical significance though there were similar t rends between the two data sets. The C1 cassette changes observed in the HIPP did occur in both dosing paradigms however, so this change may be especially sensitive to morphine administration. Altered NR1 Splicing in the AMY Altered NR1 splice variants rem ained in the AMY for at least two months following repeated morphine administration. While these altered splice variants were not observed in all rats, the subsets that did retain them were more sensitive to aversive, painful conditions. These long term al terations in NR1 splice variants could play a role in the negative long term effects of morphine withdrawal like increased anxiety and stress. Weight Change during an Extended Withdrawal Period The effects of morphine administration and withdrawal were ana lyzed in two different ways: by total group changes and by examining individual differences based on observed in the entire group. As expected, total group effects were observ ed for weight loss during the acute, but not extended withdrawal phase. This demonstrates that the morphine treated rats were dependent (Goode, 1971) Behavioral Testing with Escalating Morphine Doses Group effects were observed on the operant orofacial assay during morphine administration. Increases in time per contact values and facial contact times to alter motivation for the reward at non aversive
114 temperatures similarly to previous results of taste palatability (Doyle et al., 1993;Katsuura et al., 2011;Taha et al., 2009;Zhang and Kelley, 2002) O pen Field Results Morphine can either increase or decrease locomotion depending on dose and time period tested. Unexpectantly no differences in locomotor activity were observed with morphine injections or any stage of withdrawal. We had hypothesized that testing four hours after the third 5mg/kg dose of morphine would be a highly variable time point in which some rats would still have increased activity due to morphine (Babbini and Davis, 1972) but others would have higher activity levels perhaps due to recovering from the drug effect more quickly. We had hoped to use this a s an indicator of sensitivity to morphine, but no differences were observed. This could be due to the relatively short duration of the test as perhaps more than a five minute sample is needed to fully o pioid induced locomotor activity. Also this was the third 5mg/kg dose so perhaps some tolerance had already occurred which interfered with this assay. During withdrawal from chronic morphine, reduced activity was reported (Timar et al., 2005) but we did not observe any differences in either the acute or extended with drawal period. Behavioral Testing during an Acute and Extended Withdrawal Period This group effect continued during acute withdrawal as morphine treated animals had higher facial contact times and significantly higher time per contact values. A similar inc rease in food reward seeking during withdrawal was reported (Nocjar and Panksepp, 2007) and this could be an attempt to compensate for the averseness of wit hdrawal or could be due to the associations made between morphine and the reward during
115 previous pairings. The subsequent decrease over time could be attributed to an extinction of this behavior (Hayes and Gardner, 2004;Marlatt, 1990;Schmidt et al., 2005) As the effects on motivational behavior on day five at non aversive temperatures was blocked by naloxo ne, it is possible that there was still MOR activation occurring. This could be due to the presence of metabolites at morphine which may be responsible for the increase in motivational behavior during the acute withdrawal phase. During e xtended withdrawal morphine treated rats no longer had significantly higher as compared to saline treated rats This suggests that these rats have an increased sensitivity to the aversive stimulus over time. Many animal and human studies suggest that a long lasting hypersensitivity to pain may exist after chronic opioid administration (Angst and Clark, 2006) so it is possible that this is reflected here. However, in the operant orofacial assay pain sensitivity is generally determined by mperatures (Neubert et al., 2005;Neubert et al., 2006) Hypersensitivity should be reflected as a decrease in the extended period so it may be that hypersensitivity is not the cause of this long term behavior. We did observe that treated rats. This indicates that the withdrawn rats spent less time seeking reward at more aversive temperatures, suggesting they may be more sensitive to aversive conditions than controls. One well characterized aspect of withdrawal is increased stress and anxiety which could be responsible for this increa sed sensitivity (Avila et al.,
116 2008;Castilho et al., 2008;Schulteis et al., 1998) Although hypersensitivity to pain is a possible explanation for the inhibition during aversive conditions over time, we suspect that a better explanation lies in the modulation of the emotional response to the stimulus, not the painfulness of it. NR1 Expression Differences in the NACC and AMY after an Extended Withdrawal Period As glutamatergic activity and NMDARs are both linked to emotional responses to pain, negative emotional states, and anxiety in the AMY, (Ansah et al., 2010;Cabral et al., 2009;Glass et al., 2008;Spuz and Borszcz, 2012) we examined this area for changes in NR1 subunits. We observed altered NR1 splicing in the AMY in a subpopulation of withdrawn rats that was associated w ith behavioral changes on our operant task. The AMY is a region responsible in part for pain processing as NMDARs in t he AMY contribute to the antinociceptive properties of morphine (Manning and Mayer, 1995) It is also important for the positive and negative emotional aspects of morphine use (Hou et al., 2009) as measured by morphine induced CPP (Rezayof et al., 2007) and withdrawal induced CPA place aversion (Glass et al., 2008;Stinus et al., 1990) Plastic effects in the negativit y of opioid withdrawal have been linked to glutamatergic activity in the extended amygdala and can remain in place for over a month using CPP tasks (Reti et al., 2008) so this area is a prime candidate for being responsible for inhibited rewar d seeking during extended withdrawal. C1 and C2 Expression in the AMY is Associated with Differences in Behavior during Aversive, Painful Conditions NR1 splicing in the AMY was predictive of motivational behaviors i n aversive, but not non aversive, conditi ons in our study. One subset of rats retained low levels of C1 in the AMY. A different subset had lower C2 levels. Both of these expression levels
117 predicted lower average facial contact times throughout the withdrawal period and both of these effects would result in less active NMDARs in the AMY. It should be noted that seven of the eight animals in the Low C1 group were also in the Low C2 group suggesting that these groups are not completely distinct. The C1 insert has three serines which can be phosphoryl ated in vivo by PKC and PKA (Tingley et al., 1997) The C1 region als o contains many binding areas for PSD associated proteins like neurofilament L, yotaio, and calmodulin which can alter the stability of NMDARs in the postsynaptic density (PSD) (Cull Candy and Leszkiewicz, 2004) It is likely that this subset of rats have fewer NMDARs anchored in the synapse or lower levels of phosphorylation. NR1 subuni ts with lower levels of C2 may allow less calcium into the cell (Rameau et al., 2000) Like the animals with lower C1 expre ssion, less active NMDARs would be expressed in the AMY. These morphine induced splicing changes in the NMDAR may be responsible for generating and/or propagating this long term sensitivity to aversive conditions. The C1 c hanges observed could be due to th e interactions between NMDARs and MORs in the CeA. About 40% of neurons in the CeA are hyperpolarized by MOR agonists like morphine (Zhu and Pan, 2004) and these agonists reduce the probability of GLU release presynaptically (Zhu and Pan, 2005) This relationship between NMDARs and MORs is likely responsible for the inhibition of the aversiveness of opioid withdrawal as demonstrated by the injection of NMDAR antagonists into the CeA (Watanabe et al., 2002) or the delet ion of NR1 within the CeA (Glass et al., 2008) In the CeA, as well as many other brain areas, an upregulation of the cAMP pathway and CRE dependent gene expression was demonstrated to occur during naltrexone precipitated withdrawal (Bie et al., 2005;Shaw Lutchma n et al., 2002)
118 expression via a CRE dependent mechanism (Bradley et al., 2006) While the presence or absence of C1 does not alter receptor inactivation, these lower C1 levels could be an adaptation in response to the activity of a subset of MOR and NMDAR c ontaining cells in the CeA during withdrawal (Glass et al., 2008;Glass et al., 2009;Glass, 2010;Zhu and Pan, 2004;Zhu and Pan, 2005) Our previous data suggests that the C1 changes found in the long term withdrawn subset occurred within three days of withdr awal. This was an effect for all withdrawn animals however. This posed the question of whether or not these splice variants return to normal or not. Interestingly, it looks as if it does recover to control levels in some animals after two months. The anima ls that are left with abnormal C1 levels also have behavioral side effects that could be related to anxiety and reactions to stress. N1 Expression in the AMY and C1 Expression in the NACC are not Associated with Differences in Behavior during Aversive, Pai nful Conditions F tests were also significant for C1 in the NACC and N1 in the AMY but we did not observe any associations between these two splice variant expression levels and any behavioral effect. This suggests that although C1 and C2 levels in the AMY may be alterations in the AMY. The NACC is responsible in part for aversive effects of opioid withdrawal (Williams et al., 2001) Therefore it is possible that altera tions of NMDARs in the NACC do not support this hypothesis as we observed no association between N1 levels in the NACC and any behaviors measured by our operant assay The inhibited
119 behavior at aversive temperatures is more clearly associated with C1 and C2 expression in the AMY. pNR1 and CAL levels in the NACC are Associated with Reward seeking Behavior Phosphorylation and phosphat ase pathways in the NACC were also al tered after the extended withdrawal period. Unlike the splice variant changes after the extended withdrawal phase in which only a subset appear to be altered, statistically significant increases in pNR1 were observed in the NACC for the withdrawn group as a whole. This increase was correlated with an increased motivation to obtain the sweet food reward as the rats with the highest levels of pNR1 in the NACC had the highest time per contact values at non aversive temperatures. It is likely that the NMDARs in the NACC had higher levels of surface expression as a result of these increased pNR1 levels (Scott et al., 2003) This could allow for an increa sed response to GLU release in NACC. This increased GLU response could encode that the milk reward is more palatable or just inherently more rewardin g. This predicted increase in the response to GLU by NMDARs in the NACC was recently reported after a ten day withdrawal (Wu et al., 2012b) Though these authors did not examine phosphorylation in this study, this in crease in pNR1 could be the mechanism responsible. Since pNR1 increased, the kinases and phosphatases which control its levels were investigated to determine what cellular mechanisms were responsible. No changes in kinase expression were observed as PKA o r pPKA levels were unchanged for the withdrawal group. A significant decrease in CAL was found though, suggesting that decreased phosphatase activity was responsible for the increased pNR1 in the NACC. Decreased CAL levels have been previously reported to occur with chronic DA receptor antagonism by antipsychotics. Interestingly, in this previous study the
120 phosphatase activity was actually higher despite lower protein levels. These authors suggested that lower protein levels may be an attempt to reduce the excess CAL activity overtime which could also be occurring here. CAL Levels in the NACC Increase d uring Acute Withdrawal Though extended withdrawal decreased CAL levels in the NACC, acute withdrawal had an opposite effect. The increase in CAL during withd rawal may be responsible for some of the withdrawal induced behavioral changes as CAL inhibition was demonstrated to decrease naloxone induced withdrawal symptoms in mice (Dougherty et al., 1987;Dougherty and Dafny, 1988;Homayoun et al., 2003) and naloxone induced contractions in guinea pig ileum (Mehr et al., 2003) CAL inhibition can a lso reduce reward seeking behavior as cyclosporine A attenuates m orphine induced CPP in mice (Langroudi et al., 2005;Suzuki et al., 1993) Furthermore, a morphine induced increase in CAL activity was reported previously. Morphine exposure to primary hippocampal neuronal cultures causes increased CAL activity which was associated with decreased amplitudes of miniature excitatory postsynaptic currents and internalized GLU receptors. Inhibiting CAL with FK506 in these cultures blocked the MOR induced structural rearrangement of spines, its effects on mEPSCs, and the redistribution of GLU receptors (Kam et al., 2010;Miller et al., 2012) These s tructural changes in synapses by CAL may have a role in opioid induced plasticity but it is not clear what cellular phenomena are responsible for the expression of this phosphatase in withdrawal.
121 CAL, pNR1, and pCREB Levels Alter during Withdrawal in Cult ure and are Regulated by NMDAR and PKA S ignaling In order to study the withdrawal induced increases in pNR1 and CAL during withdrawal in more depth, we turned to a primary neuronal cell culture model. Our cultures have neurons which express bot h NMDARs and MORs so they were an appropriate model for studying the intracellular pathway changes we detected in adult rat brain tissue. Opioid withdrawal was previously demonstrated to increase adenylate cyclase activity, cAMP levels, PKA activity, and pCREB in cult ure (Chartoff et al., 2003;Fan et al., 2009) as well as in brain regions like the NACC, AMY, HIPP, the striatum, and prefrontal cortex (Edwards et al., 2009;Yang and Pu, 2009 ) Considering this, our results which demonstrated increases in pNR1 and pCREB during morphine withdrawal are not surprising a nd are likely dependent on cAMP superactivation. Interestingly, the increase in CAL should have reduced these p hosphorylation le vels, but as the AC/cAMP/PKA pathway is hyperactive, this increase in CAL was not able to offset the increase phosphorylation. It appears that increased CAL and PKA levels are both a common element of opiate withdrawal. The subsequent return to baseline of PKA and the reduction in CAL observed in the extended withdrawal phase may then be a counter adaptation to their high levels in the acute phase. Just like counter adaptations occur during the transition from the acute to tolerant phase of morphine use, a similar transition occurs during the changes from acute to extended withdrawal. Determining how to block these changes in acute withdrawal may provide a way to halt the long term differences in motivational behavior, pNR1, and CAL observed during extended withdrawal. To determine what components are important for the changes in CAL we attempted to alter this increase with NMDAR antagonism and PKA inhibition
122 and activation. NMDAR antagonism under control conditions increased CAL but NMDAR a ntagonism during w ithdrawal had no effect. Since CAL levels were already high during withdrawal, a ceiling effect may have occurred which kept MK 801 from having a noticeable effect. There was no ceiling effect evident for PKA inhibition during withdrawal though, as H89 inc reased CAL protein An opposite effect was observed for PKA activation as 8 Br cAMP actually reduced expression. T he AC/cAMP/PKA pathway is already hyperact ivated during withdrawal but activating it even more was able to decrease CAL. This suggests t hat the increase in CAL is being constantly decreased by the PKA pathway and more activation is simply speeding up the process. One explanation is that a counter adaptation to CAL expression has formed during the tolerance phase and the addition of naloxon e releases this suppression. During acute withdrawal the PKA pathway begins to reduce CAL and continues this throug hout extended withdrawal. If this pattern continued into the extended withdrawal period it w ould result in an overshoot of PKA inhibition of CAL which would result in the decreased levels in the long term similar to those observed in the NACC. In future experiments CAL should be inhibited by either FK506 or cyclosporine A (Choe et al., 200 5;Liu et al., 1991) to determine the effects of CAL reduction earlier in the withdrawal stage. Behaviorally, inhibiting CAL in vivo in the hippocampus produces memory enhancement (Malleret et al., 2001 ) and its inhibition in the AMY propagates the memory of aversive events as demonstrated by deficits in the extinction of conditioned fear memory (Lin et al., 2003) If CAL in the NACC plays a role in motivational learning then blocking this protein during acute withdrawal may block the propagation of reward seeking behavior observed in extended withdrawal.
123 NR1 Surface Expression alters with Morphine Withdrawal in Culture and is Regulated by NMDAR and PKA S ignaling NR1 sur face expression can alter the plastic properties of neurons by increasing the probability of activation by GLU and allowing more calcium into the cell. PKA activation of NR1 increases the surface expression of the NMDAR (Scott et al., 2003) and since pNR1 correlates with altered motivational b ehavior in the extended withdrawal phase this may have to do with increased surface expression. In the cell culture experiments and surface expression assay, w ithdrawal increased both pNR1 levels and surface expression levels, NMDAR antagonism during withd rawal reduced both pNR1 and surface expression levels, and as expected PKA inhibition reduced both levels. Conflicting results were observed for the effects of NMDAR antagonism during normal, non withdrawal conditions. There was increased surface expressio n, but not increased pNR1 levels detected with western blots. The different results with NMDAR antagonism (Mu et al., 2003) which can increase surface expression of NR1 (Holmes et al., 2002) Indeed, p was reduced in NACC, HIPP, and AMY tissue from adult rats during acute withdrawal. If this is also occurring in our culture model it could be overpowering and neutralizing the NMDAR antagonis NR1 surface expression. Other conflicting results were observed with PKA activation. Increased pNR1 was observed with western blotting, but the surf ace expression was partially blocked at th e 10 M dose of 8 Br be responsible but it is currently unknown if PKA alters the expression of this exon as t
124 Nevertheless, CAL, PKA, and pNR1 alter NR1 surface expression and this could be a mechanism for the long term altered plasticity in the NACC observed during withdrawal. This could be responsible for the increased motivation for food rewards on our operant task so it would be interest ing to inhibit CAL and activate or inactivate PKA in the NACC in vivo to determine if similar increases in feeding behavior are observed. A next step would also be to block the changes in CAL in the NACC during the acute withdrawal stage to determine if it can halt these long term behaviors.
125 CHAPTER 5 CONCLUSIONS In these experiments, p rotein expression levels and the splicing of the NR1 subunit of the NMDAR were demonstrated to change both with repeated morphine injections and a subsequent three day withd rawal in the NACC, AMY, and HIPP This suggests that splice variation could be a mechanism for morphine tolerance. Blocking these changes could possibly attenuate tolerance and help provide analgesia for those humans that experience morphine tolerance. In an attempt to block these morphine induced changes in splicing, we used the NMDAR antagonist MK 801 and tested pain with a more clinically relevant operant orofacial pain assay. Morphine was demonstrated to increase motivational reward seeking behavior in rats as measured by the operant orofacial pain assay and this effect was potentiated by a pretreatment with MK 801 When these two drugs were combined, MK 801 and morphine alter ed NR1 splicing in the NACC demonstrating the activity dependent nature of thes e changes. Contrary to previous studies, NMDAR antagonism did not attenuate tolerance on this assay. tolerance have failed, this suggests that this operant assay may be a better predictor of clinical pain outcomes than traditional reflex based measures. As the expression changes observed during withdrawal were dissimilar from baseline levels, we investigated whether or not these changes persist for long periods of time in withdrawal. NR1 splice variant expression level changes were observed in the AMY after a two month withdrawal period. Lower levels of C1 and C2 cassette expression were demonstrated to occur in a subset of the population of withdrawn rats. T hese sub sets h ad associated changes in motivational behavior and/or hypersensitivity
126 to adverse conditions which may reflect long term alterations in the withdrawn population. We conclude that over time the splicing changes observed in the acute withdrawal stage had ret urned to baseline levels in most rats. However, since some rats still had altered levels there could be a permanent change in these animals which would reflect altered plasticity. Another possibility is that these alterations are indeed transient in nature but simply take months to return to normal levels of expression. For instance, perhaps if we had waited until three months had passed, all the rats may have returned to baseline levels of C1 and C2 expression and no differences in behavior would be detec table by our assay Nonetheless, a subset of the population did retain these altered NR1 splic e variants after two months of withdrawal and had associated alterations in motivational behavior during aversive, stressful si tuations. This study also demonstra te d that increase s in pNR1 levels in the NACC also occur red during an extended withdrawal from morphine. This increase wa s associated with an increased motivation to obtain sweet food rewards and lowered CAL levels in the NACC. CAL expression was high in a cute withdrawal however suggesting that different stages of withdrawal may be marked by different protein levels of this phosphatase. T he low CAL levels observed in the extended withdrawal phase are likely the result of altered plastic changes which occur red during the first three days of withdrawal Just like the transition from acute to chronic morphine administration is characterized by counter adaptations, the transition from the acute to extended withdrawal phase may also have counter adaptations. The drastic changes in activity and cAMP superactivation during acute withdrawal may have long lasting effects in extended withdrawal Some of these effects are responsible for the expression changes
127 in CAL and pNR1 in the NACC as well as the alter ed motivati onal reward seeking behavior. T h e increase in CAL in acute withdrawal was demonstrated to be modulated by PKA activity which suggest s a future means of blocking or modulating these alterations and their effects on pNR1 in the NACC. Since pNR1 is responsib le in part for NMDAR surface expression levels and surface expression alter s the strength of synapses, blocking these events in the NACC may reduce the negative plastic effects of the acute withdrawal stage. In the human population, changes in motivation, anxiety, and adverse reactions to stress are hallmarks o f the dependent population (Koob and Le Moal, 2005) Recovering human patients report that anxiety is a common component of extended abstinence (Shi et al., 2008) and s tress and anxiety can also increase the probability of drug use in animals (Shaham et al., 2000) These drug induced behaviors can be partially localized to the AMY (Crombag et al., 2008;Gardner, 2011;Heinz et al., 2009;Koob, 2009b) If these NR1 splicing changes in the AMY are similar in humans then they could contribute to the lon g term changes in anxiety found in many dependent patients. New non drug related behaviors would be more difficult to learn as the NMDARs in the AMY would not function at pre drug levels as a result of the plastic changes which have occurred during the tol erance and withdrawal phases of opioid use. Also, if the pNR1 levels of humans also increase during the extended withdrawal phase in the NACC then this may be a mechanism for the altered motivational changes like craving in abstinent addicts. Furthermore since motivational factors and stress are common causes of relapse in the human addicted population (Gardner, 2011;Koob,
128 2009b) altered NR1 splicing or phosphoryla tion could be a marker for an increased propensity to relapse. In conclusion, since NR1 splicing and phosphorylation are modulated by mor phine tolerance and withdrawal and are associated with changes in motivational behavior in both the presence and absenc e of aversive, stressful conditions they may prove to be good pharmacological target s for the treatment of opioid withdrawal and possibly even addiction. In humans, treatment during the acute withdrawal phase may provide a means to reduce the long term n egative effects of extended withdrawal like dysphoria, emotional dysregulation, and possible ev en cravings
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148 BIOGRAPHICAL SKETCH Ethan Michael Anderson received an associate of arts d egree from Lake City Community College in 2002 and took several classes at Santa Fe Community College before being accepted at the University of Florida where he majored in p sychology. He received a c um l aude Bachelor of Science d egree in thi s field in the spring of 2008. Ethan was accepted into the In terdisciplinary Program in Biomedical Sciences in the f all of 2008 and rotated in the labs of Thomas Yang and Christy Carter before obtaining a position in the l aboratory of Robert M. Caudle. He was admitted to candidacy in October of 2010 and his graduati on as a d octor of p hilosophy is set for December of 2012. He published two papers in peer review ed scientific journals during his time in the Caudle 801 administration leads to alternative N methyl d aspartate receptor 1 spl icing and associated changes in reward seeking behavior and 27 and term changes in reward seeking following morphine withdrawal are associated with altered N methyl D asparta in Neuroscience 223C:45 55 both in 2012