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Characterization of the Effects of Novel 5-Ht2c Receptor Agonists on Neurotransmission and Voluntary Alcohol Consumption...

Permanent Link: http://ufdc.ufl.edu/UFE0044635/00001

Material Information

Title: Characterization of the Effects of Novel 5-Ht2c Receptor Agonists on Neurotransmission and Voluntary Alcohol Consumption in Rats
Physical Description: 1 online resource (106 p.)
Language: english
Creator: Kasper, James M
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: addiction -- alcoholism -- operant -- serotonin
Pharmacodynamics -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: This dissertation project studies the ability of 5-HT2C receptor modulators to alter voluntary ethanol intake and to investigate the changes in neurotransmission that accompany 5-HT2C agonists in a neuronal pathway associated with reward, the mesolimbic pathway.  The 5-HT2C receptors are expressed throughout this pathway including in the nucleus accumbens, NAc.  Due to high transmembrane sequence homology of the 5-HT2 subfamily of receptors it is difficult to find selective agonists for the 5-HT2C receptor that do not activate 5-HT2A/B receptors as well.  This is problematic because while 5-HT2C agonists are predicted to be a pharmacotherapy for alcoholism, agonists for 5-HT2A cause psychotomimetic effects and agonists for 5-HT2B cause cardio-valvulopathy.  Recently, a promising series of phenylaminotetralin-based structures, PAT, have been discovered and certain PATs have been shown to have functional selectivity at the 5-HT2 subfamily of receptors.  Of special interest are compounds that can act as agonists at 5-HT2C while acting as antagonists/inverse agonists at 5-HT2A/B receptors.  The goal of this dissertation is to determine if these functionally selective PATs can alter voluntary ethanol consumption, both under  basal conditions  and in ethanol deprived animals, and examine changes in neurotransmission that might mediate this effect.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by James M Kasper.
Thesis: Thesis (Ph.D.)--University of Florida, 2012.
Local: Adviser: Peris, Joanna N.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044635:00001

Permanent Link: http://ufdc.ufl.edu/UFE0044635/00001

Material Information

Title: Characterization of the Effects of Novel 5-Ht2c Receptor Agonists on Neurotransmission and Voluntary Alcohol Consumption in Rats
Physical Description: 1 online resource (106 p.)
Language: english
Creator: Kasper, James M
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: addiction -- alcoholism -- operant -- serotonin
Pharmacodynamics -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: This dissertation project studies the ability of 5-HT2C receptor modulators to alter voluntary ethanol intake and to investigate the changes in neurotransmission that accompany 5-HT2C agonists in a neuronal pathway associated with reward, the mesolimbic pathway.  The 5-HT2C receptors are expressed throughout this pathway including in the nucleus accumbens, NAc.  Due to high transmembrane sequence homology of the 5-HT2 subfamily of receptors it is difficult to find selective agonists for the 5-HT2C receptor that do not activate 5-HT2A/B receptors as well.  This is problematic because while 5-HT2C agonists are predicted to be a pharmacotherapy for alcoholism, agonists for 5-HT2A cause psychotomimetic effects and agonists for 5-HT2B cause cardio-valvulopathy.  Recently, a promising series of phenylaminotetralin-based structures, PAT, have been discovered and certain PATs have been shown to have functional selectivity at the 5-HT2 subfamily of receptors.  Of special interest are compounds that can act as agonists at 5-HT2C while acting as antagonists/inverse agonists at 5-HT2A/B receptors.  The goal of this dissertation is to determine if these functionally selective PATs can alter voluntary ethanol consumption, both under  basal conditions  and in ethanol deprived animals, and examine changes in neurotransmission that might mediate this effect.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by James M Kasper.
Thesis: Thesis (Ph.D.)--University of Florida, 2012.
Local: Adviser: Peris, Joanna N.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044635:00001


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1 CHARACTERIZATION OF THE EFFECTS OF NOVEL 5 HT2C RECEPTOR AGONISTS ON NEUROTRANSMISSION AND VOLUNTARY ALCOHOL CONSUMPTION IN RATS By JAMES MICHAEL KASPER A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLO RIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012

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2 2012 James Michael Kasper

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3 To my wife Reyna and my parents: Dan and Peggy

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4 ACKNOWLEDGMENTS There are many wonderful people w ho have helped me reach this goal. I wish to thank my advisor, Joanna Peris, for her guidance, helpfulness and forceful encouragement when needed. She has shaped my views on scientific discovery and has shown me what true scientific passion looks like. I also wish to thank my committee for their knowledge and expertise. Learning how to see my work through the eyes of another has helped me gain new perspectives on my research. I also thank all the rotation had the pleasure to work with who help ed me accomplish these studies. Lastly, I give thanks to my family for nurturing my creativity and supporting me. Richard and Jo Ann Kasper and David and Suzann e Winter.

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5 TABLE OF CONTENTS P age ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 11 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 LITERATURE REVIEW ................................ ................................ .......................... 14 Substance Addiction ................................ ................................ ............................... 14 Alcoholism ................................ ................................ ................................ ........ 14 Mesolimbic Pathway ................................ ................................ ......................... 15 Neurotransmitter Implicated In Ethanol Addiction ................................ ................... 16 Dopamine ................................ ................................ ................................ ......... 16 Gamma Aminobutyric A cid ( GABA ) ................................ ................................ 17 Serotonin (5 HT) ................................ ................................ ............................... 19 Current Pharmacotherapies For Alcoholism ................................ ........................... 22 Phe nyl Aminotetranils ( PATs ) ................................ ................................ ................. 24 Tools to Study Ethanol Addiction ................................ ................................ ............ 25 Microdialysis And Capillary El ectrophoresis With Laser Induced F luorescence ( CE LIF ) ................................ ................................ .................. 25 Operant Conditioning ................................ ................................ ....................... 27 Summary ................................ ................................ ................................ ................ 30 Objectives ................................ ................................ ................................ ............... 30 AIM #1: Role Of 5 HT 2C Receptor Modulation On Voluntary Ethanol Intake Measured Using Operant Conditioning ................................ ......................... 31 AIM #2: PAT Effect On The Alcohol Deprivation Effect ................................ .... 31 AIM #3: Modulation Of Neurotransmission In The N ucleus Ac cumbens (NAc) By 5 HT 2C Receptor Agonists Using CE LIF ................................ ....... 31 2 METHODS ................................ ................................ ................................ .............. 34 Animals And Housing ................................ ................................ .............................. 34 Ethanol Self Administrat ion Training ................................ ................................ ....... 34 Microdialysis Experiments ................................ ................................ ...................... 36 Drugs ................................ ................................ ................................ ...................... 37 Statistica l Analysis ................................ ................................ ................................ .. 37

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6 3 ROLE OF 5HT 2C MODULATION ON VOLUNTARY ETHANOL INTAKE MEASURED USING OPERANT CONDITIONING ................................ ................. 39 Rational For These S tudies ................................ ................................ .................... 39 Animals And Housing ................................ ................................ ....................... 41 Ethanol Self Administration Training ................................ ................................ 41 Fixed Ratio Studies ................................ ................................ .......................... 41 Effects of Ro60 0175 and the selective 5 HT 2C receptor antagonist, SB242,084 on ethanol gel self administration and plain gel consumption ................................ ................................ ........................... 41 Effect of the 5 HT 2C agonist and 5 HT 2A antagonist, PAT on ethanol gel self administration and plain gel consumption ................................ ........ 42 Progressive Ratio Studies ................................ ................................ ................ 42 Drugs ................................ ................................ ................................ ................ 43 Statistical Analysis ................................ ................................ ............................ 43 Results ................................ ................................ ................................ .................... 43 Ro60 0175 Attenuates And SB242,084 Enhances Fixed Ratio Operant Responding For Ethanol Containing Gelatin ................................ ................. 43 ( ) trans PAT Attenuates Fixed Ratio Operant Responding For Ethanol Containing Gelatin ................................ ................................ ......................... 44 ( ) trans PAT Alters Consumption Pattern But Not Total Consumption During Progressive Ratio Schedule ................................ .............................. 45 Discussion ................................ ................................ ................................ .............. 46 4 PAT INHIBITS THE ALCOHOL DEPRIVATION EFFECT ................................ ...... 55 Rational For Experiment ................................ ................................ ......................... 55 Animals And Housing ................................ ................................ ....................... 55 Ethanol Self Administration Training ................................ ................................ 56 Drug Studi es ................................ ................................ ................................ ..... 56 Drugs ................................ ................................ ................................ ................ 58 Statistical Analysis ................................ ................................ ............................ 58 Results ................................ ................................ ................................ .................... 58 ( ) trans PAT Alters The ADE ................................ ................................ ........... 58 ( ) trans PAT decreases voluntary consumption of ethanol containing gel in non deprived rats ................................ ................................ .......... 59 ( ) trans PAT prevents the ethanol deprivation effect in deprived rats ....... 59 ( ) trans m Br PAT Does Not Alter Non deprived Or Deprived Ethanol Consumption ................................ ................................ ................................ 60 Discussion ................................ ................................ ................................ .............. 61 5 MODULATION OF NEUROTRANSMISSION IN THE NAC BY 5 HT 2C AGONISTS USING CAPILLARY ELECTROPHORESES WI TH LASER INDUCED FLUORESCENCE DETECTION ................................ ........................... 70 Methods ................................ ................................ ................................ .................. 72 Animals ................................ ................................ ................................ ............. 72

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7 Chemicals ................................ ................................ ................................ ......... 73 Microdialysis And Surgeries ................................ ................................ ............. 73 Experiment Procedures ................................ ................................ .................... 73 CE LIF ................................ ................................ ................................ .............. 74 Data Analysis And Histology ................................ ................................ ............ 74 Results ................................ ................................ ................................ .................... 75 Effect Of ( ) trans PAT Perfusion On Basal Neurotransmitter Release In NAc ................................ ................................ ................................ ............... 75 Effect Of ( ) trans PAT On K+ Induced Neurotransmitter Release In The NAc. ................................ ................................ ................................ .............. 75 Effect Of Additional 5 HT 2C Receptor Agonists On Potassium ( K+ ) Induced Neurotransmitter Release In The NAc. ................................ ......................... 76 Effect Of 5 HT 2 Receptor Modulation On K+ Induced Neurotra nsmitter Release In The NAc. ................................ ................................ ..................... 76 Effect Of Novel ( ) trans PAT Perfusion On K+ Induced Neurotransmitter Release In The Striatum. ................................ ................................ ............... 77 Discussion ................................ ................................ ................................ .............. 77 PATs Effect On K+ Stimulated Release In NAc And Striatum .......................... 78 H istomine 1 Receptors Do Not Alter GABA During High K+ Induced GABA Release. ................................ ................................ ................................ ........ 78 5 HT 2C Receptors Mediate Decreases In High K+ Induced GABA Release. .... 79 6 GENERAL DISCUSSION ................................ ................................ ....................... 91 Discussion ................................ ................................ ................................ .............. 91 ................................ ................................ ..... 91 Mesolimbic Pathway ................................ ................................ ......................... 92 PAT Analogs ................................ ................................ ................................ .... 94 Future Studies ................................ ................................ ................................ ........ 94 APPENDIX LIST OF REFERENCES ................................ ................................ ............................... 97 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 106

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8 LIST OF TABLES Table P age 1 1 List of all affinities a nd functional activit y of drugs in the current study ............... 33

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9 LIST OF FIGURES Figure P age 3 1 Ro60 0175 decreases and SB242,084 increases voluntary et hano l gel consumption in rats. ................................ ................................ ........................... 50 3 2 Ro60 0175 decreases voluntary plain gel consumption and SB242,084 has no significant effect. ................................ ................................ ................................ 50 3 3 ( ) trans PAT decreases voluntary ethanol containing gel consumption in a dose dependent manner and (+) trans PAT also decreases consumption at 10 mg/kg ................................ ................................ ................................ ............ 51 3 4 ( ) trans PAT at 5 mg/ kg reduced basal consumption patterns during a FR5 operant session. ................................ ................................ ................................ 51 3 5 ( ) trans PAT has no effect on voluntary plain gel consumption but (+) trans PAT decreases consumption at 10 mg/kg. ................................ ......................... 52 3 6 ( ) trans PAT has no effect on breakpoints for 0.15 g gelatin under a PR10 schedule. ................................ ................................ ................................ ............ 52 3 7 ( ) trans PAT at 10 mg/kg a lters gel deliveries between 5 and 10 minutes into the PR10 operant session for 0.15 g of ethanol containing gelatin. .................... 53 3 8 ( ) trans PAT at 5 mg/kg has no effect on consumption patterns dur ing a PR10 operant session for 0.15 g of ethanol containing gelatin. ................................ .... 53 3 9 Ro60 0157 and ( ) trans PAT at 1 and 10 mg/kg has decreased ethanol breakpoints during PR10 operant session respond ing for 0.28 g of gelatin reward. ................................ ................................ ................................ ............... 54 3 10 Ro60 0157 at 1 mg/kg but not ( ) trans PAT decreased plain gelatin breakpoints during PR10 operant session responding for 0.28 g of gelatin reward. ................................ ................................ ................................ ............... 54 4 1 ( ) trans PAT administration causes a temporary decrease in voluntary ethanol consumption. ................................ ................................ ................................ ...... 64 4 2 ( ) trans PAT administ ration prevents the ADE. Ethanol deprived rats demonstrate increased consumption of ethanol after reinstatement. ................. 65 4 3 ( ) trans m Br PAT administration had no effect on voluntary ethanol c onsumption. ................................ ................................ ................................ ...... 66 4 4 ( ) trans m Br PAT administration had no effect on ADE. ................................ ...... 67

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10 4 5 Ro60 0175 administration decreases experimen tal day voluntary ethanol consumption. ................................ ................................ ................................ ...... 68 4 6 Ro60 0175 administration does not alter the ADE. ................................ ................ 69 5 1 Coronal sections showing microdialysis probe placement within the NAc. ............ 81 5 2 ( ) trans PAT does not alter basal GABA concentrations in the NAc. .................... 82 5 3 ( ) trans PAT decreased K+ stimulated GABA release in the NAc. ....................... 82 5 4 ( ) trans PAT did not alter K+ stimulated taurine release in the NAc. .................... 83 5 5 Ketanserin had no effect on K+ stimu lated GABA release in the NAc. .................. 83 5 6 ( ) trans PAT had no effect on K+ stimulated GABA release in the NAc in the presence of ketans erin. ................................ ................................ ...................... 84 5 7 Mepyramine had no effect on K+ stimu lated GABA release in the NAc. ................ 84 5 8 ( ) trans PAT decreased K+ stimulated G ABA release in the NAc in the presence of mepyramine. ................................ ................................ ................... 85 5 9 ( ) trans p Cl PAT decreased K+ stimu lated GABA release in the NAc. ................ 85 5 10 ( ) trans p Cl PAT had no effect on K+ stimulated GABA release in the NAc in the presence of ketanserin. ................................ ................................ ................ 86 5 11 TOMCAT decreased K+ stimulated GABA release in the NAc. ........................... 86 5 12 TOMCAT had no effect on K+ stimulated GABA release in the NAc in the presence of ketanserin. ................................ ................................ ...................... 87 5 13 Ro60 0175 decreased K+ stimu lated GABA release in the NAc. ......................... 87 5 14 ( ) trans p Me PAT had no effect on K+ stimulated GABA release in the NAc. ... 88 5 15 (+) trans PAT had no effect on K+ stimulated GABA release in the NAc. ............ 88 5 16 ( ) trans CAT had no effect on K+ stimulated GABA release in the NAc. ............ 89 5 17 ( ) trans PAT decreased K+ stimulated GABA release in the striatum. ............... 89 5 18 ( ) trans PAT had no effect on K+ stimulated taurine release in the striatum. ...... 90

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11 LIST OF ABBREVIATIONS 5 HT Serotonin ADE Alcohol Deprivation Effect FR Fixed Ratio GABA Gamma Aminobutyric A cid IP Intraperitoneal K+ Potassium NAc Nucleus Accumbens PR Progressive Ratio SC S ubcutaneous VTA Ventral Tegmental Area

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12 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 CHARACTERIZATION OF THE EFFECTS OF NOVEL 5 HT2C RECEPTOR AGONISTS ON NEUROTRANSMISSION AND VOLUNTARY ALCOHO L CONSUMPTION IN RATS By James Michael Kasper August 2012 Chair: Joanna Peris Major: Pharmaceutical Sciences This dissertation project studies the ability of 5 HT 2C receptor modulators to alter voluntary ethanol intake and to i nvestigate the changes in neuro trans mission that accompany 5 HT 2C agonists in a neuronal pathway associated with reward, the mesolimbic pathway The 5 HT 2C receptors are expressed throughout this pathway including in the nucleus accumbens, NAc. Due to high trans membrane sequence homolog y of the 5 HT 2 subfamily of receptors it is difficult to find selective agonists for the 5 HT 2C receptor that do not activate 5 HT 2A/B receptors as well. This is problematic because while 5 HT 2C agonists are predicted to be a pharmacotherapy for alcoholism, agonists for 5 HT 2A cause psychotomimetic effects and agonists for 5 HT 2B cause cardio valvulopathy Recently, a promising series of phenylaminotetralin based structures PAT, have been discovered and cer tain PAT s have been shown to have functional selectivity at the 5 HT 2 subfamily of receptors. Of special inter est are compounds that can act as agonists at 5 HT 2C while acting as antagonists/inverse agonists at 5 HT 2A/B receptors. The goal of this disser tation is to determine if these functionally selective PAT s can alter voluntary ethanol consumption, both under basal

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13 conditions and in ethanol de prived animals, and examine changes in neuro trans mission that might mediate this effect.

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14 CHAPTER 1 LITERAT URE REVIEW Substance Addiction Substance addiction in humans is characterized by continued drug seeking and drug taking despite negative or dangerous consequences These drugs often initially induce euphoria or remove distress. Over time and repeated dru g use, homeostatic changes in the body occur which result in tolerance, sensitization, physical dependence, and /or craving (Cami and Farre, 2003). Many of these changes are mediated by altered receptor populations, synaptic turnover, and cellular protein expression in the central nervous system (Wolf, 2002). For this reason, key areas of the brain have been identi fied and studied to discover the process of addiction development and identify therapeutic targets. Alcoholism An estimated half of Americans over the age of 12 are reported to be ethanol drinkers in a 2006 study by the Department of Health and Human Services. This study also found that a fifth of Americans over 12 engaged in binge drinking (five or more drinks on one occasion). Ethanol mec hanism of action for reward is not fully understood but there is effects At low acute doses ethanol is perceived as a stimulant due to suppressing inhibitory drive in the CNS. As dose s increase, sedation and motor dis coordination can be observed (Holdstock and de Wit, 1998). Pharmacologically relevant doses of ethanol have been shown to act on a wide variety of proteins. GABA A receptors can be stimulated by ethanol while glu tamate NMDA receptors are inhibited (Melis et al., 2009).

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15 Neuronal calcium channels (Wang et al., 1992) an d potassium channels (Chu et al. 1992) are also altered by ethanol. But ethanol does not only affect extracellular proteins, it can also act direct ly on second m essenger pathways like adenylyl cyclase (Yoshimura and Tabakoff, 1999) and protein kinase C (Solem et al., 1997). many mechanisms of action make alcoholism difficult to study because there are numerous ethanol induced effects to tr eat or block by potential pharmacotherapy. Mesolimbic Pathway of the brain but the mesolimbic pathway has been shown to mediate the rewarding effects of ethanol (S derpalm and Ericson, 2009) and the acquisition of alc ohol consumption (Vengeliene et al., 2007). The mesolimbic pathway consists of dopaminergic neurons that project f rom the ventral tegmental area (VTA) to the nucleus accumbens (NAc) and is known for its role in reward. The neuronal cell bodies in the NAc are primarily GABAergic and project either back to the VTA or to oth er brain regions including the striatum and raphe nucleus. The raphe n ucleus has serotonergic cell bodies that can project to the VTA or NAc. These projections explain why dopamine, GABA and serotonin receptors in the mesolimbic pathway are expressed in those regions ( West erink et. al., 1996, Clemett et al., 1999) and are able to alter neuro trans mitter release (Alex and Phek, 2007) The impo rtance of the mesolimbic pathway in voluntary ethanol intake is demonstrated by studies that show that r ats will self administer ethanol directly to the VTA (Rodd et al., 2004) This indicates that the VTA is a site of action for ethanol and that ethanol in the VTA has reinforcing value. E thanol self administration directly to the V T A i s blocked by coinfusion of a D2/3 agonist, indicating the importance of dopamine

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16 ) It is clear that the m esolimbic pathway plays a critical role in ethanol reinforcement and therefore the development of alcoholism. However, there are probably more neurotransmitters involved in this circuitry besides just dopamine. Neuro trans mitter s Implicated I n Alcoholis m Dopamine Dopamine is a catecholamine neuro trans mitter that plays an important role in addiction (S derpalm and Ericson, 2011) This is because drugs of abuse often increase dopamine concentrations in the NAc (Sulzer, 2010) Dopamine receptors are sorte d into 5 subtypes, D 1 through D 5 Those subtypes are often grouped into D 1 like and D 2 like receptor families (Hurely and Jenner, 2006) D 1 like receptors include D 1 and D 5 receptors which cou 1 like receptors will cause activation of adenylyl cyclase and an increase in the second messenger cAMP in most brain regions This can lead to increase in neuro trans mitter release of the neuron. The D 2 like family include s D 2 D 3 and D 4 receptors that mostly this family will generally inhibit adenylyl cyclase and thus inhibit neuro trans mission (Neves et al., 2002) In the m esolimbic pathway, both D 1 and D 2 like receptor families have been show n to be important regulators of neuro trans mission. The D 1 and D 5 receptors are expressed in the NAc and can mediate both the effects of ethanol and ethanol self administration (S derpalm and Ericson, 2011) D 1 like receptor antagonists in the NAc can dec rease ethanol self administration (Hodge et al., 1997). D 2 and D 3 receptors are highly expressed in the NAc but their role is more complicated. D 2 like receptor

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17 a gonists have biphasic effects; with low doses increas ing and high doses decreasing ethanol s elf administration (Hodge et al., 1997). Ethanol causes dose dependent excitation of dopamine neurons in the VTA (Gessa et al. 1985 ). Ethanol directly administered to the VTA causes dopamine release in the NAc (Ding et al., 2009) and increased ethanol seeking behavior (Hauser et al., 2010) but the D 2 / 3 receptor agonist quinpirole reduced this seeking when microinjected alongside the ethanol. reinforcing and that reinforcement can be modulated by dopamine receptors. It is clear that ethanol induced changes in dopamine in the mesolimbic pathway are related to the rewarding value of ethanol. GABA Aminobutyric acid, is one of the main inhibitory neuro trans mitters. It is widely present throughout the CNS and its receptors have been implicated in addiction as therapeutic targets (Lingford Hughes, 2010) GABA receptors include the GABA A and GABA B receptor families The GABA A receptor is a ligand gated ion channel that will selecti vely allow chloride ions to enter the neuron In the majority of neurons, this hyperpolarizes the cell, preventing action potentials, and thus inhibiting neuro trans mission. Ethanol acts as a positive allosteric modulator at this receptor and increases the effect of GABA when present (We iner and Valenzuela, 2006) Antagonists for GABA A receptors administered directly to the NAc or VTA decrease ethanol self administration in rats (Chester and Cunningham, 2002). Interestingly, Agonism of the GABA A receptors by muscimol in the NAc also cau ses a decrease in ethanol self administration in rats (Hodge et al., 1995).

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18 The GABA B receptor is coupled to a G protein and can inhibit adenylyl cyclase and open potassium channels. As with the GABA A receptor, the flow of ions causes hyperpolarization and inhibits the neuron from firing (Kelm et al., 2011) Positive modulation of GABA B receptors inhibit s the reinforcing ability of numerous substances of abuse including ethanol (Vlachou and Markou, 2010). This decrease in reinforcing ability is thought to be mediated by GABA B receptors expressed in the VTA on do paminergic cell bodies (Boehm et al 2002). Activation of GABA B receptors on VTA dopamine neurons decreases the mesolimbic dopamine release induced by ethanol (Westerink et al., 1996). Ex viv o studies have demonstrated that bath applied ethanol causes a dose dependent decrease in VTA GABA neuron firing along with an increase in dopamine neuron firing (Xiao et al., 2007). The increase in dopamine firing was able to be blocked by a GABA A recept or antagonist. This suggests that ethanol causes a disinhibition of dopamine neurons through presynaptic effects on GABA neurons. In vivo acute ethanol administration (2g/kg ; large enough to cause loss of righting reflex ) given i.p. causes a decrease of VTA GABA neuron firing in the VTA (Gallegos et al., 1999). This decrease in GABA firing can be blocked by systemic administration of a D 2 receptor antagonist, eticlopride (Ludlow et al., 2009). However, when animals are given a lower ethanol dose (0.1 g /kg ; small enough to induce euphoria and stimulate locomotor activity ) i.v., there is an increase in VTA GABA neuron firing (Steffensen et al., 2009). In addition to these electrophysiology studies, microdialysis studies showed there were no significant changes in VTA GABA concentrations ( 2 g/kg ethanol i.p. ) observed

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19 using both alcohol preferri ng and non preferring rats (Kemppainen et al., 2010). Indeed, few microdialysis studies on ethanol induced changes in overall GABA concentrations show significant changes in any brain region (Klem et al., 2011). Two notable exceptions were in the NAc : GABA concentrations decrease after ethanol administration in ethanol tolerant (Piepponen et al., 2002) and ethanol dependent rats (Dahchour and De Witte, 2000). The se seemingly conflicting results from microdialysis and electrophysiology are currently unresolved. Even though the mesolimbic pathway is thought of as a dopaminergic pathway, the importance of GABAergic neurons is great. GABA cell bodies represent a la rge portion of the cell bodies in the NAc and form a major pathway back to the VTA (Conrad and Pfaff, 1976). Mesolimbic GABA provides important regulation of dopamine rgic neurons in the mesolimbic pathway and represents an interesting pharmacotherapeutic target for addictive substances ( Hodge et al., 1995) Serotonin Serotonin, 5 HT, is a monoamine neuro trans mitter. In the central nervous system serotonin can alter mood, desire, and sleep (Filip et al., 2005) Receptors for 5 HT are classified into s even families 5 HT 1 to 5 HT 7 Most of these receptor families couple to a G protein while one, 5 HT 3 is a ligand gated ion channel (Hoyer et al., 1994) Of particular interest to addiction and reward is the 5 HT 2 family. They include the 5 HT 2A 5 HT 2B and 5 HT 2C receptors. All members of this family are G protein coupled (Hoyer et al., 1994) When activated, these receptors activate phospholip ase C second messenger pathways, o ne outcome of which is neuronal excitati on and increased neuro trans mission (Eison and Mullins, 1996) This family has been implicated as a therapeutic target for regulating anxiety, appetite,

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20 memory, mood, and drug use (Bubar and Cunningham, 2006; Meltzer and Massey, 2011) These numerous diff erent effects make the 5 HT 2 family of receptors an important target of study. There are many similarities with a few important differences between the 5 HT 2C and 5 HT 2A VTA NAc prefrontal cortex a nd s triatum ( Pompeiano et al., 1994). All of these brain regions have been implicated in addiction. Both receptor subtypes are coupled to Gq, and they show a >80% sequence homology in the trans membrane region, which makes designing receptor specific drug s challenging (Bubar and Cunningham, 2006). There are several specific antagonists for both of these rec eptors, but there are few specific agonists. This has complicated the study of 5 HT 2C and 5 HT 2A receptor activation. To exacerbate the problem, the 5 HT 2C and 5 HT 2A receptors often have opposing behavioral effects, e.g. craving for psychostimulant s (Bubar and Cunningham, 2006) Specific drugs for this family of receptors are important because activation of 5 HT 2A receptor is known to cause psychoto mimetic effects (Hoyer et al., 2002) Activating 5 HT 2B in the periphery causes valvular heart disease ( Fitzgerald et al., 2000 ). A ctivation of 5 HT 2C receptors causes decreased appetite and ethanol consumption ( Bickerdike, 20 03 ; Tomkins et al., 2002) These results suggest that a 5 HT 2C agonist that does not activate 5 HT 2A/2B receptors would have therapeutic value. Unfortunately, the most popular commercially available 5 HT 2C receptor agonist, Ro60 0175, demonstrates a ten fold selectivity for the 5 H T 2C receptor over 5 HT 2A/2B making the literature difficult to interpret. Thankfully there are fairly selective antagonists for

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21 the receptor subtypes demonstrating 100 fold preference or more ( Di Matteo et al., 1999) In the mesolimbic pathway, 5 HT r eceptors are known for their ability to modify dopamine release. When 5 HT 2C receptor agonist is administered systemically, it causes a decrease in dopamine release in the NAc that can be blocked by a selective 5 HT 2C receptor antagonist (Dremencov et al. 2006). Indeed, when a 5 HT 2B/2C receptor antagonist is given systemically, there is an increase in basal dopamine neuron firing (Di Matteo et al., 2001). These studies strongly suggest that 5 HT 2C receptor agonists administered systemically leads to de creased dopamine in the NAc. When 5 HT 2C receptors agonists are administered directly to the VTA they d ecrease cocaine induced accumbal dopamine outflow Low doses of 5 HT 2C agonist, Ro60 0175, directly administered in to the NAc increase and higher doses decrease cocaine induced dopamine release (Navailles et al., 2008). This shows that there is a balance of 5 HT 2C receptor populations in the brain and that there are brain region dependent differences in the effect of 5 HT 2C receptors on accumbal dopamin e release. The 5 HT 2A receptor has limited effects on substance abuse 5 HT 2A antagonists block while agonists enhance increased locomotor effects of cocaine (Bubar and Cunningham, 2006). However the 5 HT 2A antagonist M100907 did not alter operant resp onding for cocaine (Fletcher et al., 2002). Agonism of the 5 HT 2A receptor is shown to be important in the initiation of events leading to hallucinogenic activity associated with LSD and related drugs (Egan et al., 1998). The studies of the role 5 HT 2A r eceptors play in other drugs of abuse provide direction to designing an alcoholism

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22 pharmacotherapy that does not activate 5 HT 2A receptors in order to avoid hallucinogenic like effects. The 5 HT 2C receptor has also been shown to be important for ethanol induced changes in neuro trans mission. Agonism of this receptor increases GABA release in the VTA and blocks the ethanol induced increases in VTA dopamine neuron firing (Theile et al., 2009). 5 HT 2C receptor agonism reduces while antagonism increases etha nol self administration in rats (Tomkins et al., 2002), suggesting that basal 5 HT 2C receptor activity can influence ethanol consumption. Current Pharmacotherapies For Alcoholism There are few drug treatments approved for human alcoholism and they have only been moderately effective (Edwards et al., 2011). Disulfiram, an acetaldehyde dehydrogenase inhibitor, results in large amounts of acetaldehyde produced after drinking which quickly produces headache and nausea. Disulfiram has been proven to be effe ctive on short term (less than 1 month) but not on long term (more than 6 months) alcohol consumption (Wright and Moore, 1990). The mechanism of action for n altrexone, a and opioid antagonist, is not fully understood but naltrexone has been proven to increase initial ethanol abstinence period and decrease ethanol consumption during relapse (Latt et al., 2002). In humans receiving additional cognitive behavioral therapy, naltrexone 50 mg/day demonstrates a 66.1% abstinence rate at 12 weeks (Feeney et. al., 2006). Acamprosate, an antagonist for glutaminergic N methyl D aspartate (NMDA) receptors and agonist for GABA A receptors, increases abstinence in both the short and long term when combined with psychosocial treatments (Mason, 2001). All three of these drugs have previously been shown to be effective at delaying relapse to ethanol consumption (Williams, 2005) but are only marginally effective at

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23 treating alcoholism over placebo. For example, a meta analysis shows that naltrexone causes an increase in desirable outcomes (decreased drinks per day, decreased number of drinking days, and increased length of abstinence periods) by 12% to 19% over placebo (Kransler and Van Kirk, 2001) There are also pharmacotherapies to treat alcoholism that are not a pproved for this use. They includ e t opiramate, b aclofen and o ndansetron. Topiramate is a GABA A receptor positive modulator and AMPA receptor antagonist. Topiramate is an anticonvulsant drug used to treat epilepsy but has been shown to have promise tre ating alcoholism. Topiramate increases the number of abstinent days decreases drinks per day, and decreases the number of heavy drinking (more than 4 drinks) days (Johnson et al., 2003). Baclofen, a GABA B receptor agonist, was originally approved for hu man use to treat spasticity It has been shown that baclofen increases the length of abstinence periods and the overall number of abstinent days (Leggio et al., 2010). Ondansetron, a 5 HT 3 receptor antagonist, is used as an antiemetic but has been shown to decrease ethanol craving and increase the number of abstinent days compared to placebo (Sellers et al., 1994). This ondansetron induced decrease in ethanol consumption changed the basal average drinks per day from 3.8 to 2.8 while placebos lowered drin ks per day to 3.3. Topiramate, b aclofen and o ndansetron all show promise in treating alcoholism but display similar overall marginal effectiveness as approved pharmacotherapies for alcoholism. The generally mild effectiveness of the current pharmacothe rapies for alcoholism could be explained in several ways. Some of the treatments have been shown to be mo re efficacious in different subtypes of alcoholics. For example, baclofen has more

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24 pronounced effects on alcoholics with liver cirrhosis, indicating severe alcohol dependence (Leggio et al., 2010). Another explanation for the lackluster results of the current pharmacotherapies is that the mechanism of action for ethanol addiction is not known. Without a clear target, it is d ifficult to intelligently design a pharmacotherapy for alcoholism. Modeling human alcoholism in animals and choosing a therapeutic target sites of action. These difficulties in studying alcoholis m carry over to the drug discovery process PATs Phenylaminotetralins, PATs, represent a novel scaffold for drug discovery. Some PATs have been shown to modulate 5 HT 2 subfamily of receptors (Booth et al., 2009). ( ) trans PAT, ( 2S,4R ) ( ) trans 1 phen yl 3 dimethylamino 1,2,3,4 tetrahydronaphthalene ( ) trans m Br PAT, ( ) trans (2S,4R) N,N dimethyl 4 (3 Brom ophenyl) 1,2,3,4 tetrahydro 2 naphthalene amine ( ) trans CAT, (2S,4R) ( ) trans cyclohexylaminotetralin, TOMCAT ( ) trans (2S,4R) N,N dimethyl 4 (3 chlorophenyl) 6 methoxy 1,2,3,4 tetrahydro 2 naphthalene amine and ( ) trans p Cl PAT ( ) trans (2S,4R ) N,N dimethyl 4 (4 Chlorophenyl) 1,2,3,4 tetrahydro 2 naphthalene amine have all been shown to act at 5 HT 2C and 5 HT 2A receptors. Previo us stu dies demonstrate that trit iated PAT binding sites include the hippocampus, N Ac, and s triatum (Booth et al., 1999). The current understanding of affinit y and function for ( ) trans PAT and many other PAT analogues is summarized in Table 1 1 along with othe r established 5 HT 2 receptor modulators The PAT compound s are a valuable tool and potential alcoholism pharmacotherapy because some have demonstrated agonis m at 5 HT 2C receptors and

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25 an inverse agonism at 5 HT 2A receptors (Booth et al., 2009). TOM CAT, ( ) trans p Cl PAT, and ( ) trans m Br PAT have a similar affinity and functional profile as ( ) trans PAT PAT administrat ion can separate the effect of 5 HT 2C receptor activation on neuro trans mission an d behavior from confounding influence by 5 HT 2A rece ptor activation. These receptor specific functions also make PAT a potential addiction therapy because activation of 5 HT 2C receptors and blockade of 5 HT 2A receptors has been shown to be important for cocaine (Bubar and Cunningham, 2006) and alcoholism ( Di Matteo et al., 2001) ( ) trans PAT also alter s behavior by decreasing consumption of palatable dessert in mice, indicating anorectic effects (Rowland et al., 2008). This is important to know when studying ethanol consumption because ethanol has calo ric value itself. It will be necessary to test for any non specific decreases in ethanol consumption that are due to anorectic effects and not specifically decreasing voluntary ethanol consumption. Together, some PAT s display functional selectivity for agonism of the 5 HT 2C receptor. The functional profiles of these PAT s suggest that they can modulate ethanol intake (Di Matteo et al., 2001). Although it will be necessary to control for non specific decreases in overall caloric intake, select PAT s repre sent a novel pharmacotherapy for alcoholism. Tools to Study Ethanol Addiction Microdialysis A nd CE LIF Microdialysis is a popular technique to measure unbound substances in any medium. Using passive diffusion, substances can be collected from a complex environment for identification or quantification. It is an invasive technique which requires a probe, subject, and sample collection method. Microdialysis samples can be

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26 taken from a living animal that is awake and freely moving. Microdialysis is a pop ular method to measure extracellular neuro trans mitters in the brain of because of the simplicity of the technique and the minimal damage to the surrounding tissue. There are many types of probes commercially available and they can be highly customized. A basic probe will trans port fluid from a pump into a small semipermeable membrane tip where passive diffusion can take place. The fluid is constantly mobile and pushed out of the probe tip and though a collection tube. The substance containing fluid, d ialysate, is then collected for analysis. The probe can be customized by altering the volume of the trans porting tubes, flow rate, and length and molecular cut off of the semipermeable membrane tip. Increasing tube size or flow rate will increase fluid t urnover in the membrane tip causing more dialysate to be collected but decreasing the concentration of substances collected due to decreased time for passive diffusion. Increasing the length of the membrane tip allows for a more concentrated sample to be collected. In the brain, it is common for researchers to use the maximum length of membrane that will fit into their area of interest. Altering the molecular cutoff of the membrane determines what substances can enter the probe and what is excluded. O nce the sample is gathered the most popular analysis method is to inject 10 to 30 L onto a HPLC. This allows for quantification of substance s with a very low limit of det ection. The biggest limitation to this technique is that it requires such a large volume of dialysate. This dialysate injected represents the average concentration of neuro trans mitters across 5 to 30 minutes of dialysis which results in poor temporal resolution. One way to overcome the poor temporal resolution of HPLC is to instead us e capillary electrophoresis coupled to laser induced fl uorescence ( CE LIF )

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27 CE LIF is a sensitive technique (Robert et al., 1998) and only requires 20 nL of dialysate to measure the quantities of multiple substances including amino acids and catecholam ines. The small sample size can be gathered from microdialysis in less than a second which gives high temporal resolution of multiple neuro trans mitters (Bowser and Kennedy, 2001). The accurate measuring of multiple neuro trans mitters with high temporal re solution p rovides a complete picture when observing how neuro trans mitters change in relation to each other and determining the onset and duration of changes in neuro trans mission. Operant Conditioning Operant conditioning is a form of learning during whic h the behavior of an animal is modified due to the consequences of that behavior. This includes drug self administration setups using an operant conditioning chamber. There are many potential setups for operant conditioning chambers and their customizati on has been the strength of the technique. Operant chambers allow the anima l s to bar press in order to receive a substance. The result of pressing the bar is considered to be a positive reinforcement if the animal increases the behavior that caused the r eward (Staddon and Ettenger, 1989) These rewards include substances of abuse, including ethanol. By giving the animal a novel alcoholism pharmacotherapy one can screen drugs that reduce an animal intake of ethanol In order to fully explore the eff ect of therapeutics on operant responding for abused substances, the operant chamber can be set up to determine the effect on dose titration or motivation alone. A fixed ratio, FR, schedule is used to examine how much of the reward the animal want s to hav e during the session. Typically this involves the animal pressing the bar a fixed amount of presses to receive the reward. The fixed

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28 amount of bar presses to receive the reward can vary with easier schedules requiring a lower number of bar presses per de livery. If pressing the bar a fixed amount is fairly easy (FR5) the animals will consume as much of the abused substance as they desire. If pharmacotherapy reduces the amount of rewards the animal receives, it has altered self titration. There are many explanations for why a potential alcoholism pharmacotherapy could result in decreases in FR responding. Self titration of ethanol will lower if the animal finds the ethanol more potent, if the animal is being punished for ethanol consumption, or the anim al finds the ethanol to be more or less rewarding. Motivation for the reward can be measured using a progressive ratio, PR, schedule ( Hoffmeister, 1979 ) This involves progressively more bar presses for each subsequent reward delivery. The ideal PR sch edule requires enough bar presses that the animal is unable to receive the same amount of the reward as they would under a FR5 schedule. T he point at which the animal stops responding for the reward is called the break point. This represents the maximum a mount of effort the animal will put in to receiving that reward. P harmacotherapeutic agents can alter breakpoints as well indicating a decreased wanting of the reinforcement Significant changes during PR responding can be explained with greater ease tha n FR significant changes because PR responding reflects animal desire for the reinforce ment Measuring the effect of a pharmacotherapy on basal daily low levels of ethanol consumption may not be as relevant to human alcoholism as measuring the effect of the pharmacotherapy on escalated ethanol intake such as during the Alcohol Deprivation Effect (ADE) The ADE model s alcoholic relapse Relapse in alcoholism is characterized by compulsive alcohol seeking and consumption. Alcohol deprivation

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29 induced cra ving is a tremendous difficulty for alcoholics (Martin Fardon and Weiss, 2012). When alcohol is reintroduced following a period of abstinence, consumption temporarily increases compared to previous basal consumption (Rodd, et al. 2009). This can be teste d using operant chambers by removing the ethanol from the reward for days or weeks depending on the animal strain. When the animals are once again allowed to respond for ethanol reward they display the increased ethanol consumption indicative of the ADE In rats both acamprosate and naltrexone decrease aspects of the ADE singularly and when administered together (Heyser et al., 2003). It is difficult to model voluntary ethanol consumption because rats tend not to consume pharmacologically relevant dos es of ethanol (Chester and Cunningham, 2002). Researchers have addressed the low ethanol consumption problem in multiple ways including: selective rat breeding for ethanol consumption, food and water restriction, and use of sweetener in the ethanol (Samso n and Czachowski, 2003). The use of sweetener in ethanol was advanced by the development of a gelatin vehicle that contains 10% ethanol and polycose This method sustains fairly high self administration levels in Sprague Dawley rats without the need for food or water restriction (Peris et al 2006). Consumption of ethanol containing gel atin results in dose related increases in brain ethanol levels. The jello shot procedure is easily applied to operant responding (Li et al ., 2008, 2010) w ith p lain gel serving as a control for drug effects on appetite, response rate and other confounding variables. The jello shot model of voluntary ethanol consumption allows for pharmacologically relevant doses of ethanol to be consumed without food or w ater depriving non genetically selected rats.

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30 Summa ry Substance addiction affects many people and key brain regions have been identified that mediate the rewarding aspects. The mesolimbic pathway is composed of dopaminergic projections from the VTA to t he NAc In turn, GABA cell bodies in the NAc project back to the VTA These projections make a loop where the VTA is sending signals to the NAc and the NAc sends signals back to the VTA. There are many other neuron projections to these brain regions whi ch play a role in regulating the pathway. This includes serotonergic input and the 5 HT 2 family of receptors. 5 HT 2A and 5 HT 2C receptors in particular have been shown to be key modulators of the mesolimbic pathway. To this end, the 5 HT 2A inverse agoni st and 5 HT 2C receptor agonist PAT and other novel analogs are exciting new tools to investigate the role of 5 HT 2 receptors in the mesolimbic pathway and represent potential alcoholism pharmacotherapeutics. Objectives It is hypothesized that the 5 HT 2C receptor represents a potential pharmacotherapy for alcoholism (Di Matteo et al., 2001) but the 5 HT 2C receptor has proven difficult to study due to a lack of selective agonists. Some PAT compounds represent a novel pharmacotherapy for a poorly studied a lcoholism target. ( ) trans PAT and select analogs have been shown to have functional selectivity for the 5 HT 2 family of receptors. PAT compounds are promising in their 5 HT 2C agonism and 5 HT 2A/B antagonism/inverse agonism. The goal of this thesis is to determine the effect of these PAT compounds on alcohol consumption models and establish the PAT induced change s in neuro trans mission that may underlie the changes in ethanol consumption behavior. Investigating the effect of these PAT s on rat voluntary ethanol consumption can illuminate the role that 5 HT 2C agonists play in self titration and motivation for

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31 ethanol. The PAT compounds will be tested on an ethanol deprivation model of alcoholism to examine their role in alcohol consumption relapse The m echanisms of action of PAT analogs will then be studied in the NAc to determine what neuronal systems are involved by this potential pharmacotherapy AIM #1: R ole Of 5 HT 2C M odulation On V oluntary E thanol I ntake Measured U sing O perant C onditioning This a im tests the hypothesis that 5 HT 2C agonists specifically reduce voluntary ethanol containing gel atin consumption on a FR schedule. ( ) trans PAT along with the known 5 HT 2 receptor agonist Ro60 0175 and specific 5 HT 2C antagonist SB242,080 will be admini stered before the daily operant session responding for plain or ethanol containing gelatin. These experiments will illuminate the role that 5 HT 2C receptors play in titration of ethanol intake AIM #2: PAT E ffect On The Alcohol Deprivation Effect The AD E represents a model of ethanol consumption that parallels the tendency to increase ethanol consumption after a period of abstinence (Martin Fardon and Weiss, 2012) This aim will both demonstrate that the ADE can be modeled using the jello shot model o f alcoholism and test the hypothesis that ( ) trans PAT and ( ) trans m Br PAT will remove the deprivation induced increase in voluntary ethanol consumption. Pharmacotherapies that reduce the number of drinks on the reinstatement day and the subsequent hi gh ethanol consumption days would represent a valuable treatment for alcoholism in humans AIM #3: M odulation Of Neuro trans mission In The NAc By 5 HT 2C Agonists Using CE LIF This aim tests the hypothesis that agonism of 5 HT 2C receptors in the NAc will r esult in modulation of GABA release. The 5 HT 2C receptor is expressed on both

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32 dopamine and GABA neurons in the mesolimbic pathway. The effect of systemic 5 HT 2C agonists on dopamine has been well studied but the effect of 5 HT 2C agonists on GABA is uncle ar. GABA release is viewed as a possible mechanism to alter ethanol induced dopamine release Establishing the mechanism of action for pharmacotherapies that alter ethanol consumption can yield new targets for future pharmacotherapies and drive forward t he understanding of alcoholism itself.

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33 5 HT 2A 5 HT 2C Ro60 0175 31 nM agonist 1 nM agonist SB242,084 160 nM antagonist 1 nM antagonist Ketanserin 2 nM antagonist 50 nM antagonist ( ) trans PAT 80 nM inverse agonist 20 nM agonist (+) trans P AT 520 nM inverse agonist 1300 nM agonist ( ) trans m Br PAT 1 5 nM antagonist 6 nM agonist ( ) trans p Cl PAT 240 nM inverse agonist 130 nM agonist TOMCAT 35 nM inverse agonist 17 nM agonist ( ) trans p Me PAT 210 nM inverse agonist 330 nM ag onist ( ) trans CAT 1.6 nM inverse agonist 14 nM inverse agonist Table 1 1. List of affinities and functional activity of serotonergic drugs in the current study.

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34 CHAPTER 2 METHODS Animals A nd Housing Female Sprague Dawley rats (90 days old) weighi ng approximately 275 g at the start of the studies, were individually housed in plastic cages with food and water available ad libitum throughout the study except for the time spent in the operant chambers. Female rats were used for operant experiments si nce they maintain stable body weight over several months. For microdialysis experiments, m ale Sprague Dawley rats, 300 400 grams, were used. All rats were maintained on a 12 h light/dark cycle in an environmentally controlled room (lights on at 6:00 am temperature: 233 C, humidity: 4525 %) and allowed a 1 week acclimatization period to the animal facilities prior to the start of the studies. Ethanol Self A dministration T raining Ethanol self administration was established by using sweetened gelatin ve hicle (jello shots) containing ethanol (10% Polycose, 10% ethanol, 0.25% gelatin, all by weight, in water). This method sustains fairly high self administration levels in Sprague Dawley rats without the need for food or water restriction (Peris et al 20 06). Consumption of ethanol containing gel atin results in dose related increases in brain ethanol levels (Peris et al 2006) responding (Li et al., 2008, 2010) with p lain gel serving as a control for drug effects on appetite, response rate and other confounding variables. Ethanol self administration tr aining consisted of two stages: free access training and operant conditioning. First during free access the animals had 24 h home cage access to 1 0% ethanol containing gel for 2 days followed by 6 h and 3 h access periods per day for 2 days each. After

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35 this animals had a daily one hour access period for 11 days. Next, operant training was initiated. Operant training was conducted in five operant chambers. Each chamber was housed in a sound attenuating box equipped with a house light that illuminates with each delivery. Each chamber contained a gel dispenser calibrated to deliver 0.15 g of gel into a recessed dish positioned between the two leve rs. The apparatus was controlled by a microcomputer interface linked to a DELL computer using Graphic state software to record and manage the data. The rats were placed daily in the operant boxes for 30 min utes and trained to press the active lever to ge t the reinforce ment To encourage the association of the active lever with gel delivery, 0.3 g of gel was placed gel coming from the spout. Initially, the animals were trained on a fixed ratio (FR) reinforcement schedules. First, FR 1 schedule was employed, where each response on the active lever resulted in a delivery of gel atin while responses on the inactive lever were recorded but had no scheduled consequences When reliable responding on the FR 1 schedule was achieved (10% day to day variation) the reinforcement schedule was increased to FR 5. When stable responding and pharmacologically significant consumption levels (0.8 g/kg) had been established, the dr ug studies were started. Each drug study was divided into four key days: b asal, experimental, first day post experimental and second day post experimental. The basal day represented the normal consumption of the rats. Experimental day showed the consum ption levels during drug administration The first and second post experimental days determine if there were any drug induced long lasting effects on consumption. This four day cycle was then repeated multiple times using a latin square design to test mu ltiple drug treatments.

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36 Before each new cycle of drug treatments we waited for the animals to reach baseline consumption. Microdialysis Experiments Each rat was anesthetized with isoflurane and placed in a stereotaxic instrument for implantation of a guide canulla. The guide canulla was anchored with two stainless steel screws and dental cement. The following coordinates from bregma were used for implantation: NAc +1.8 anteroposterior, +1 .3 lateral, 6.2 dorsoventral, s triatum +0.8 anteroposterior, + 3.0 lateral, 4.0 dorsoventral After implantation, animals were given at least 2 days to recover before either resuming operant sessions or undergoing microdialysis testing. The technique used to gather extracellular neurotransmitter concentrations wa s microdialysis coupled with capillary electrophoresis separation using laser induced fluorescence detection and has been described previously (Bowser and Kennedy 2001 ; Li et al., 2008 ). On the experiment day, a standard calibration curve (0 to 20 ) w as first performed using a microdialysis probe with outer diameter 270 13,000 molecular weight cutoff, and variable active length (2 to 4 mm) depending on the brain region being dialysed. The probe was placed in standard solutions kept at 37 degr ees Celsius. A standard curve was then produced relating the relative fluorescence to the known concentrations. After calibration, the probe was implanted in a non anesthetized and freely moving rat. The experiment b egan after neuro trans mitter concen trations reach ed steady state which was typically 2 hours after implantation. The substances measured include d glutamate, aspartate, serotonin, dopamine, ornathine, GABA, taurine, glutamine, serine, and glycine.

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37 The perfusion setup is described in Chap ter 5 It allow ed for determining the effect of drug reverse dialysis on both basal and stimulated neuro trans mitter release. Basal effects were discovered during the 3 0 minute pretreatment period before the second potassium stimulation. Changes in 50 m M K+ stimulated neuro trans mitter release were measured by comparing the first stimulation to the drug treated second stimulation. The third stimulation serves as a control for instrument sensitivity and should not be significantly different from the first stimulation. Drugs PAT compound s were D epartment of M edicinal C hemistry at College of Pharmacy, University of Florida. Ro60 0175 ((S) 2 (chloro 5 fluoro indol 1 yl) 1 methylethylamine 1:1 C4H4O4) and SB242,084 (6 ch loro 5 methyl 1 [2 (2 methylpyridyl 3 oxy) pyrid 5 yl carbomyl]indoline) were purchased from Tocris Bioscience. Ro60 0175 was dissolved in 0.9% saline. SB242,084 was prepared in 0.9% saline solution containing 8% hydroxypropyl b cyclodextrin and 25 mM cit ric acid. All drug doses are expressed as that of the salt. Gelatin and polycose were purchased from Knox brand, Kraft Foods and Abbott Laboratories respectively Statistical A nalysis Operant d ata w as analyzed by one or two way repeated measures ANOVA u sing SPSS software. Post hoc comparisons were carried out with Bonferroni test. In all cases, the accepted leve l of significance was taken at p<0 .05. Microdialysis d ata analysis was performed using Lab View to determine the peak height for each measured amino acid and neuro trans mitter. There was one of these data points for each analyte every 15 seconds during the experiment. The peak heights for each analyte were changed to and then graphed over time. Data

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38 points from specific time periods, such as during high K+ stimulations, were examined using area under the curve, AUC. Basal concentration of neuro trans mission was determined by finding the average concen tration for the 5 minutes before the time period of interest. AUC was determined by subtracting the basal value from the data points during the 10 minute long time period of interest. This results in the AUC value for the time period of interest. This A UC value can then be compared other AUC values in the same experiment using one way ANOVA to determine if the AUC values are different from each other.

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3 9 CHAPTER 3 ROLE OF 5HT 2C MODULATION ON VOLUNTARY ETHANOL INTAKE MEAS URED USING OPERANT CONDITIONING Rational F or T hese S tudies Alcoholism is a disorder that involves changes of neuro trans mitter release in the central nervous system. Ethanol induced changes in dopamine in the nucleus accumbens and related brain areas have been widely studied (Salamone and Correa, 2002) and it is clear that dopamine plays a significant role in alcoholism (Sulzer, 2011). Serotonergic drive, via 5 HT 2 receptor family, is able to modulate dopamine release in brain regions associated with addiction. Agonism of the 5 HT 2C r eceptor has been shown to decrease voluntary ethanol consumption in rats (Tomkinset al., 2002). However, agonism of the 5 HT 2A receptor causes psyc h otomimetic effects ( Fitzgerald et al., 2000 ). There are currently no specific agonists for the 5 HT 2C rece ptor available due to the high trans membrane sequence homology between the two receptor subtypes. However, the novel ( ) trans PAT series of compounds have several drugs that exhibit functional selectivity; they act as agonists at 5 HT 2C and antagonists a t 5 HT 2A These drugs represent a possible pharmacotherapy for alcoholism. self administration (Rowland et al., 2005) It models human alcohol intake through voluntary ora l consumption of ethanol. Rats will bar press for a reinforcement consisting of 0.15 grams of a 10% ethanol containing gelatin. This model results in physiologically relevant doses of ethanol self administered without the need for food/water restriction ( Peris et al 2006 ). The rats voluntarily consume the human equivalent of approximately 1.2 drinks or 0.8g/kg. This model is well suited for testing drugs that affect voluntary ethanol self administration because the large physiologically

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40 relevant doses of ethanol self administered allow for the visualization of pharmacotherapy induced decreases in ethanol consumption. Prior to the daily operant session, drug is administered and changes in daily consumption observed. An important strength of this mode l is that experiments can then be repeated using gelatin ethanol. If a drug decreased both ethanol containing and ethanol free gelatin consumption then it can be con cluded that the drug has nonspecific effects on overall ethanol intake is a good model to test if drugs alter voluntary, physiologically relevant, ethanol consumption t hat is specific for the ethanol itself. Thus, this model was used to assess the effects of PAT and its analogs on voluntary ethanol consumption. For comparison purposes, we also chose to test some commercially available 5 HT 2 ligands. Currently, the m ost specific 5 HT 2C receptor agonist commercially available is Ro60 0175. It has a modest 30 fold selectivity for activating the 5 HT 2C receptor over the 5 HT 2A receptor. This makes it difficult to attribute the behavioral effects of Ro60 0175 to just on e receptor. Addition of a specific 5 HT 2C antagonist will be required to ensure Ro60 0175 has its action solely via agonism of 5 HT 2C receptors. The 5 HT 2C receptor antagonist SB242,084 will be used to confirm the role that 5 HT 2C receptor agonists pla y. SB242,084 has 150 fold selectivity for the 5 HT 2C receptor over the related 5 HT 2A By administering this compound and blocking the 5 HT 2C receptors before an agonist is administered, it is possible to then determine if the effects of the 5 HT 2C agoni st are due to activity at the 5 HT 2C receptor. Methods

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41 Animals A nd H ousing Female Sprague Dawley rats (90 days old) weighing approximately 275 g at the start of the studies, were individually housed in plastic cages with food and water available ad libit um throughout the study except for the time spent in the operant chambers. Female rats were used since they maintain stable body weight and stable ethanol consumption over several months (Li et al., 2010) They were maintained on a 12 h light/dark cycle in an environmentally controlled room (lights on at 6:00 am, temperature: 233 C, humidity: 4525 %) and allowed a 1 week acclimatization period to the animal facilities prior to the start of the studies. Ethanol S elf A dministration T raining As described i n Chapter 2 General Methods. Fixed R atio S tudies Effects of Ro60 0175 and the selective 5 HT 2C receptor antagonist, SB242,084 on ethanol gel self administration and plain gel consumption The rats (n = 10) were initially trained to consume ethanol as outl ined above. Animals received once daily IP (30 min prior) and SC (50 min prior) vehicles injections prior to drug treatments to acclimate the rats to injections. When the total number of reinforcements response pattern had stabilized, the effects of the different drug treatments on ethanol self administration behavior were evaluated. Four treatments were implemented: Ro60 0175 at two doses (0.5 and 1 mg/kg sc, 30 min pretreatment), SB242,084 (0.5 mg/kg ip, 50 min pretreatment) in combination with Ro60 0 175 (0.5 mg/kg sc, 30 min pretreatment) and SB242,084 (0.5 mg/kg ip, 50 min pretreatment) alone. A Latin square design was employed such that each animal received each dose /dose combination in a balanced order. Each treatment day was

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42 separated from the n ext by at least 3 days. Following the data analysis of this study, a second experiment was conducted to determine the effects of the same four treatments on plain gel consumption as a control for the first experiment. When the response pattern for plain gel had stabilized, the effects of the different drug treatments on plain gel consumption were evaluated in a similar manner. Effect of the 5 HT 2C agonist and 5 HT 2A antagonist, PAT on ethanol gel self administration and plain gel consumption The rats (n=1 0) were switched back to ethanol gel and the consumption level was stabilized The effect of ( ) trans PAT (0.5, 1, 5 and 10 mg/kg ip, 2 0 min pretreatment) and (+) trans PAT (10 mg/kg ip, 20 min pretreatment) on ethanol self administration behavior was ev aluated. A Latin square design was employed such that each animal received each dose in a balanced order. Each treatment day was separated from the next by at least 3 days. Following the data analysis of this study, a second experiment was conducted to determ ine the effects of the same PAT doses on plain gel consumption as a control for the first experiment. When the response pattern had stabilized, the effects of the different drug treatments on plain gel consumption were evaluated in a similar manner. Progressive R atio S tudies Rats (n=10) were divided into two groups and placed on a PR 10 schedule of responding for ethanol containing gel. After a 2 week stabilization (three consecutive days of gel deliveries 1 from previous day) rats were adminis tered either 5 or 10 mg/kg ( ) trans PAT 20 minutes prior to the operant session. Breakpoint and consumption data were compared from the experimental day and the two following session days to determine the effect of ( ) trans PAT.

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43 Drugs PAT compound s we re synthesized in the laboratory of Dr. Raymond G. Booth in the D epartment of M edicinal C hemistry College of Pharmacy, University of Florida. Ro60 0175 ((S) 2 (chloro 5 fluoro indol 1 yl) 1 methylethylamine 1:1 C4H4O4) and SB242,084 (6 chloro 5 methyl 1 [ 2 (2 methylpyridyl 3 oxy) pyrid 5 yl carbomyl]indoline) were purchased from Tocris Bioscience. Ro60 0175 (+) trans PAT and ( ) trans PAT was dissolved in 0.9% saline. SB242,084 was prepared in 0.9% saline solution containing 8% hydroxypropyl b cyclodextri n and 25 mM citric acid. All drug doses are expressed as that of the salt. Gelatin an d polycose were purchased from Kraft Foods and Abbott Laboratories respectively Statistical A nalysis Data were analyzed by one or two way repeated measures ANOVA using S PSS software. Post hoc comparisons were carried out using t test with Bonferroni correction In all cases, the accepted leve l of significance was taken at p < 0 .05. Results Ro60 0175 A ttenuates A nd SB 242,084 E nhances F ixed R atio O perant R esponding F or E thanol C ontaining G elatin Average basal consumption was 8.5 2.1 (average standard error of the mean) grams of ethanol containing gelatin per kilogram of rat (equal to 0.85 g/kg ethanol). ANOVA testing indicated a significant effect of days (F (3, 36 ) = 6.6, p<0.01) drug treatment (F (3, 36 ) = 6.3, p<0.01) and interaction between days and drug treatment (F (9, 144 ) = 9.6, p<0.001) This indicates that the effect of the different drug treatments is changing over days of the experiment. Ro60 0175, the 5 HT 2 agonist caused a significant decrease of daily voluntary ethanol containing gel consumption at both 0.5

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44 and 1.0 mg/kg, changing ethanol containing gelatin consumption to 6.1 1.1 g/kg and 0.86 0.31 g/kg respectively (Figure 3 1). The 5 HT 2C anta gonist SB242,08 4 significantly increased ethanol containing gel consumption to 13 1.9 g/ k g. When both SB242,08 4 and Ro60 0175 are administered, there is no significant change, 11 1.7 g/kg, in ethanol containing gel atin consumption. Then Ro60 0175 and SB242,084 were tested on operant responding for plain gel and basal consumption was 18 2.6 g/kg. ANOVA testing indicated a significant effect of days (F (3, 36 ) = 5.0, p<0.01), drug treatment (F (3, 36 ) = 7.0, p<0.01) and interaction between days and dru g treatment (F (9,144) = 11.5, p<0.001) Ro60 0175 caused a significant decrease in plain gel consumption at both 0.5 and 1.0 mg/kg, to 10 2.2 g/kg and 7.5 1.5 g/kg (Figure 3 2). The 5 HT 2C antagonist SB242,08 4 had no effect on plain gel consumption with an average consumption of 23 3.5 g/Kg. When both SB242,08 4 and Ro60 0175 were administered in combination there was no significant change, 22 2.0 g/kg, in ethanol containing gel consumption. ( ) trans PAT A ttenuates F ixed R atio O perant R espondin g F or E thanol C ontaining G elatin The average ethanol containing gelatin consumption for this experiment was 12 2.2 g/kg. Three way ANOVA testing showed a significant effect of days (F (3,36) = 2.6, p<0.05) and an interaction between days and drug trea tment (F (3,144) = 2.6, p<0.05) but no significant effect of drug treatment alone (F (3,36) = 2.6, p=0.07). The 5 HT 2C agonist, 5 HT 2A/B antagonist/inverse agonist ( ) trans PAT caused a dose dependent decrease in ethanol containing gel consumption (Figur e 3 3). ( ) trans PAT at 5 mg/kg was the lowest dose to cause a significant decrease from basal to 8.1 1.5 g/Kg. In addition to overall ethanol consumption, the pattern of ethanol consumption was altered

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45 by 5 mg/kg ( ) trans PAT as well (Figure 3 4) The pattern of ethanol consumption demonstrated a significant main effect of bins (F (10,48) = 3.2, p<0.001) and a significant interaction between days and bins (F (20,144) = 1.1, p<0.001). Post hoc analysis determined ( ) trans PAT reduced ethanol consu mption during the 1 0 20 minute period s of the operant session when compared to basal Average plain gel consumption while testing ( ) trans PAT was 22 1.8 g/kg. ANOVA testing indicated no significant effects of days (F (3,36) = 1.6, p=0.22), drug treat ment (F (3,36) = 1.1, p=0.36), or interaction between days and drug treatment (F (9,144) = 0.38, p=0.94). ( ) trans PAT did not alter plain gel consumption at any dose (Figure 3 5 ). When looking at the effect of (+) trans PAT administered at 10 mg/kg on both ethanol and plain gelatin co nsumption o ne way ANOVA indicates there is a significant effect of days (F (3,28) = 7.4, p<0.001) during ethanol gelatin consumption while testing (+) trans PAT Likewise, there was a significant effect of days (F (3, 20) = 4.9, p<0.05) during plain gel consumption in the presence of (+) trans PAT. (+) trans PAT decreased both ethanol containing and plain gel consumption at 10 mg/kg (Figure 3 3, 3 5) ( ) trans PAT A lters C onsumption P attern And T otal C onsumption D uring P rogressive R atio S chedule s The average breakpoint of these rats responding for 0.15 g of ethanol gel on a PR10 schedule was 37 10. ANOVA testing on breakpoints for ( ) trans PAT at both doses indicate no significant effect of days (F (2,12) = 1.1, p=0 .32), drug treatment (F (2,12) = 0.8 2, p=0.34), or interaction between days and drug treatment (F (6,24) = 0.53). ( ) trans PAT did not alter overall consumption at either 5 or 10 mg/kg (Figure 3 6 ).

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46 However, when the deliveries were binned into 5 minute intervals across the 30 minute operant session, ANOVA of 10 mg/kg ( ) trans PAT on ethanol gelatin revealed a main effect of bins (F ( 5,24) = 7.8, p<0.05) and a significant in teraction between days and bins ( F (10,72) = 2.1, p<0.05) 10 mg/kg ( ) trans P AT decreased basal consumption between 5 and 10 minutes from 2.2 0.73 to 0.80 0.22 (Figure 3 7 ). In the ( ) trans PAT 5 mg/kg treated group (Figure 3 8 ), there was a significant main effect of bins ( F (5,24) = 8.5 p< 0.00 1) but no main effect or inter action involving treatment days. ( ) trans PAT administered at 5 mg/kg ha d no effect on consumption patterns during PR10 responding for ethanol gelatin. When the reward size was increased to 0.28 g on a PR10, a verage breakpoints for ethanol containing g elatin (65.3 11.6) and plain gelatin (67.1 16.3) were similar (Figure 3 9, 3 10) At 1.0 mg/kg, Ro 60 0175 decreased both ethanol containing (68.3 11.1% of basal) (Figure 3 9) and plain (79.2 4.8% of basal) (Figure 3 10) gelatin breakpoints, thoug h it did not have significant effects at a dose of 0.5 mg/kg. However, at 10 mg/kg, ( ) trans PAT decreased only ethanol containing gelatin (70.4 4.1% of basal) (Figure 3 9) but not plain gelatin (106.3 7.2% of basal) (Figure 3 10) ANOVA results dem onstrated significant effects of days (F(1,32) = 4.3, p = 0.048), days by drug treatment (F(3,13) = 19.0, p = 0.001), days by gelatin type (F(1,64) = 10.2, p = 0.004), and days by drug treatment by gelatin type (F(3,52) = 3.7, p = 0.025). Discussion Thes e results show that modulation of 5 HT 2 subfamily of receptors can alter voluntary consumption of both plain and ethanol containing gel. The 5 HT 2C agonist Ro60 0175 has been shown to decrease ethanol consumption in another model of alcohol consumption (T omkins et al., 2002) and the present experiments have

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47 replicated that result using the jello shot model of alcohol consumption on a FR5 schedule. The Ro60 0175 induced decrease in plain gelatin consumption was also expected because the effective dose fo r decreasing ethanol consumption is close to the effective dose for decreasing food intake (Higgins and Fletcher, 2003) Ro60 nonspecific decrease in both ethanol and plain gelatin consumption makes it difficult to determine the effect that Ro60 01 75 has specifically on ethanol consumption. Unfortunately, the 5 HT 2C antagonist SB242,084 increased ethanol gel consumption making the combination of SB242,084 and Ro60 0175 difficult to interpret. While the affinity and functional profile of Ro60 0175 and SB242,084 suggest that the decreased/increased ethanol consumption are 5 HT 2C mediated, this cannot be confirmed. It should be noted that while SB242,084 demonstrates >100 fold affinity preference for the 5 HT 2C receptor (Table 1 1), SB242,084 also ac ts on other 5 HT, dopamine and adrenergic receptors (Kennett et al., 1997). It is possible that the nonspecific decrease in gelatin consumption was due to Ro60 0175 acting on and SB242,084 blocking a different variant of the 5 HT 2C receptor This theory is supported by the fact that (+) trans PAT decreased both ethanol and plain gelatin. Comparing (+) trans PAT to ( ) trans PAT in terms of affinity and function at 5 HT 2A and 5 HT 2C receptors (Table 1 1) it is obvious that ( ) trans PAT should outperform (+) trans PAT at these receptors. Further tests are needed to confirm what mediates the (+) trans PAT nonspecific effects on gelatin consumption. However, ( ) trans PAT caused an ethanol specific decrease in consumption on a FR5 schedule that was dose d ependent. This was in agreement with previous studies observing that 5 HT 2C agonism leads to decreased ethanol consumption (Tomkins et

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48 al., 2002). It was unexpected that ( ) trans PAT did not alter plain gel consumption at any of the doses tested because of previous studies that showed ( ) trans PAT ting at 4.2 mg/kg (Rowland et a l., 2008). While it is unknown if higher doses of ( ) trans PAT will alter plain gel consumption, it is possible that the dose respons e curves for ( ) trans PAT on ethanol intake and food intake are farther apart than those dose response curves for Ro60 0175. This could be attributed to ( ) trans PAT activating second messenger systems, PLC vs PLA2, in a different manner than Ro60 0175. Regardless of the mechanism of action, ( ) trans PAT specificity to reduction of ethanol containing gelatin assures us that the treatment is specific to ethanol itself, and not due to nausea sedation or otherwise unable to press the bar ( ) trans P AT had no effect on overall breakpoints in the PR10 studies at either 5 or 10 mg/kg but did alter the consumption pattern with 0.15 g reward size With 0.28 g of gel as a reward, Ro60 0175 decreased both ethanol and plain gelatin breakpoints. However, ( ) trans PAT only decreased the breakpoints for ethanol gelatin and not plain gelatin. This specific change in ethanol breakpoints signifies that ( ) trans PAT alter s the value of the ethanol to the rat. ( ) trans e Ro60 0175 decreases the value of both plain and ethanol containing gelatin During PR10 with 0.15 g reward and 10 mg/kg ( ) trans PAT there was a decrease in consumption on the experimental day between 10 and 15 minutes. This change in consumption pat tern hints that ( ) trans PAT is having an effect but it was not great enough to cause changes in overall breakpoints.

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49 One aspect that stands out in th e PR10 experiment s is the relatively low basal breakpoints, 37 10, for 0.15 g of gelatin compared to breakpoints previously reported with this model, 52 3.2 using 0.28 g rewards (Li et al., 2010). As reinforcement size increases there is a tendency for breakpoints in increase as well ( Hoffmeister, 1979 ). The breakpoints using 0.15 g reward may be so low that there is a floor effect and they are unable to be decreased by ( ) trans PAT. Increasing the reward size allows the visualization of both Ro60 0175 and ( ) trans PAT decrease in breakpoints

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50 Figure 3 1. Ro60 0175 decreases and SB242,08 4 incr eases voluntary ethanol gel consumption in rats. Black columns represent the average consumption before experimental day, red represents consumption after drug administration, and grey represents consumption the day after drug administration. Shown are m ean values and SEMs for N = 10. indicates p<0.05 compared to basal. Figure 3 2. Ro60 0175 decreases voluntary plain gel consumption and SB242,08 4 has no significant effect. Black columns represent the average consumption before experimental day, r ed represents consumption after drug administration, and grey represents consumption the day after drug administration. Shown are mean values and SEMs for N = 10. indicates p<0.05 compared to basal.

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51 Figure 3 3. ( ) trans PAT decreases voluntary e thanol containing gel consumption in a dose dependent manner and (+) trans PAT also decreases consumption at 10 mg/kg. Black columns represent the average consumption before experimental day, red represents consumption after drug administration, and grey represents consumption the day after drug administration. Shown are mean values and SEMs for N = 10. indicates p<0.05 compared to basal. Figure 3 4. ( ) trans PAT at 5 mg/kg reduced basal consumption patterns during a FR5 operant session. Shown are mean values and SEMs for N = 10 indicates experimental day has p<0.05 compared to basal bin value.

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52 Figure 3 5 ( ) trans PAT has no effect on voluntary plain gel consumption but (+) trans PAT decreases consumption at 10 mg/kg. Black columns re present the average consumption before experimental day, red represents consumption after drug administration, and grey represents consumption the day after drug administration. Shown are mean values and SEMs for N = 10. indicates p<0.05 compared to ba sal. Figure 3 6 ( ) trans PAT has no effect on breakpoints for 0.15 g gelatin under a PR10 schedule. Shown are mean values and SEMs for N = 5. indicates p<0.05 compared to basal.

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53 Figure 3 7 ( ) trans PAT at 10 mg/kg alters gel deliveries between 5 and 10 minutes into the PR10 operant session for 0.15 g of ethanol containing gelatin Shown are mean values and SEMs for N = 5. indicates p<0.05 compared to basal bin value. Figure 3 8 ( ) trans PAT at 5 mg/kg has no effect on consumpt ion patterns during a PR10 operant session for 0.15 g of ethanol containing gelatin Shown are mean values and SEMs for N = 5. indicates p<0.05 compared to basal bin value.

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54 Figure 3 9. Ro60 0157 and ( ) trans PAT at 1 and 10 mg/kg has decreased ethanol breakpoints during PR10 operant session responding for 0.28 g of gelatin reward. Shown are breakpoints expressed as a percentage of the basal breakpoint responding with N = 4 5. indicates p<0.05 compared to basal. Figure 3 10. Ro60 0157 at 1 mg/kg but not ( ) trans PAT decreased plain gelatin breakpoints during PR10 operant session responding for 0.28 g of gelatin reward. Shown are breakpoints expressed as a percentage of the basal bre akpoint responding with N = 4 5. indicates p<0.05 compared to basal.

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55 CHAPTER 4 PAT INHIBITS THE ALCOHOL DEPRIVATION EFFECT Rational e F or E xperiment The Alcohol Deprivation Effect (ADE) is a model of relapse in recovering alcoholics. Relapse in alco holism is characterized by compulsive alcohol seeking and consumption. Alcohol deprivation induced craving is a tremendous difficulty for alcoholics (Martin Fardon and Weiss, 2012). In animals, when alcohol is reintroduced following a period of abstinenc e, consumption temporarily increases compared to previous basal consumption (Rodd, et al. 2009). This can be tested using operant chambers by removing the ethanol from the reward for days or weeks. When the animals are once again allowed to respond for e thanol reward, they display the increased ethanol consumption indicative of the ADE. In rats both acamprosate and naltrexone decrease aspects of the ADE singularly and when administered together (Heyser et al., 2003). The aim of this study was to test no vel 5 HT 2C agonists that could decrease the severity and duration of the ADE. Methods Animals A nd H ousing Female Sprague Dawley rats (90 days old) weighing approximately 275 g at the start of the studies, were individually housed in plastic cages with fo od and water available ad libitum throughout the study except for the time spent in the operant chambers. Female rats were used since they maintain stable body weight over several months. They were maintained on a 12 h light/dark cycle in an environmental ly controlled room (lights on at 6:00 am, temperature: 233 C, humidity: 4525 %) and allowed a 1 week acclimatization period to the animal facilities prior to the start of the studies.

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56 Ethanol S elf Administration T raining Ethanol self administration was e stablished by using sweetened gelatin vehicle (jello shots) containing ethanol (10% Polycose, 10% ethanol, 0.25% gelatin, all by weight, in water). This method sustains fairly high self administration levels in Sprague Dawley rats without the need for foo d or water restriction (Peris et al 2006). Ethanol self administration tr aining consisted of two stages: free access training and operant conditioning. First during free access the animals had 24 h home cage access to 10% ethanol containing gel for 2 days followed by 6 h and 3 h access periods per day for 2 days each. After this, animals had a daily one hour access period for 11 days. Next, operant training was initiated. Operant training was conducted in five operant chambers. Each chamber was hou sed in a sound attenuating box equipped with a house light that illuminates with each delivery. Each chamber contained a gel dispenser calibrated to deliver 0.15 g of gel into a recessed dish positioned between the two levers. The apparatus was controlle d by a microcomputer interface linked to a DELL computer using Graphic state software to record and manage the data. The rats were placed daily in the operant boxes for 30 min utes and trained to press the active lever to get the reinforce ment To encoura ge the association of the active lever with gel session along with a single delivery of gel coming from the spout. Initially, the animals were trained on a fixed ratio ( FR) reinforcement schedules. First, FR 1 schedule was employed, where each response on the active lever resulted in a delivery of gel atin while responses on the inactive lever were recorded but had no scheduled consequences. When reliable responding on t he FR 1 schedule was achieved (10% day to day variation) the reinforcement schedule was increased to FR 5. When stable

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57 responding and pharmacologically significant consumption levels (0.8 g/kg) had been established, the drug studies were started. Drug S tudies The jello shot model of inducing voluntary ethanol self administration (Peris, et al. 2006) was used to habituate alcohol nave female Sprague Dawley rats to freely tains 10% ethanol in gelatin with a glucose polymer caloric source (polycose). After tapering of free access sessions from 24 hr to 1 hr (Peris et al., 2006) animals were introduced to an operant responding environment and incrementally trained to bar pre ss for gel deliveries during a 30 min session. A fixed ratio schedule, increased from FR 1 to FR 5, regulated the amount of effort required for each delivery. This mechanism allowed for accurate determination of consumption levels surrounding periods of al cohol availability and deprivation. Animals were then divided into 4 groups (N = 6 for each group): the non deprived saline control group, the non deprived 5 mg/kg PAT group, the alcohol deprived saline control group and the alcohol deprived 5 mg/kg PAT g roup. Animals were habituated to once daily saline injections prior to operant responding to control for the effect of injections on operant responding. The experimental timeline contained 10 days of all groups responding for ethanol gelatin to establish baseline consumption for each group. Then the alcohol deprived animals responded for plain gelatin for 3 weeks while the non deprived animals continued to respond for ethanol gelatin. After the 3 week deprivation (or continued daily ethanol access) all animals responded for ethanol gelatin again and this day is called the reinstatement day. The reinstatement day also marks the day where two groups of animals received PAT injection 20 min prior to the

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58 start of the operant session while the other two gro ups continued to receive daily saline. All four groups continued responding for ethanol gel for 15 days after reinstatement to observe the ADE induced changes in ethanol consumption return to basal. After that the 5 week long experiment was repeated agai n with ( ) trans m Br PAT instead of ( ) trans PAT. Animals that were non deprived in the ( ) trans PAT experiment remained non deprived during the ( ) trans m Br PAT experiment and likewise for the deprived animals. However, to not bias our study to the drug history, half of all animals in each group had previous 5 mg/kg ( ) trans PAT administration and the other half were drug naive. Drugs PAT compound s were M edicinal C hemistry at College of Pharmacy, University of Florida All drug doses are expressed as that of the salt. Gelatin and polycose were purchased from Kraft Foods and Abbott Laboratories respectively Statistical A nalysis Data were analyzed by one or two way repeated measures ANO VA using SPSS software. Post hoc comparisons were carried out using T test with Bonferroni correction In all cases, the accepted leve l of significance was taken at p < 0 .05. Results ( ) trans PAT Alters The ADE The jello shot model of alcoholism produ ced a stable baseline of ethanol containing gelatin consumption 1.2 0.37 g/Kg. Animals were then divided into four groups, N = 6, and half were deprived of daily ethanol by switching the deprived animals to operant responding for plain gelatin. These a nimals were deprived for 3 weeks while

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59 the other two groups received daily ethanol gelatin operant sessions. After 3 weeks, the deprived animals were switched back to ethanol gelatin. On that day of reinstatement, one of the deprived and one of the non d eprived groups were administered 5 m/kg ( ) trans PAT. Both the ethanol deprivation and administration of ( ) trans PAT influenced the consumption of ethanol. Three way ANOVA indicates that t here were significant main effect s of deprivation (F (1,11 ) = 2 4, p<0.05), drug treatment (F (1,11) = 1800, p<0.001), days (F (4,25) = 36, p<0.05). All interactions were also significant, including deprivation and drug (F (3,20) = 34, p<0.05), deprivation and days (F ( 9, 110 ) = 21, p<0.05), drug treatment and days (F (9, 110 ) = 23, p<0.05), and deprivation and drug treatment and days (F (19, 230 ) 7.1, p<0.05). ( ) trans PAT decreases voluntary consumption of ethanol containing gel in non deprived rats Looking at the non deprived rats, ( ) trans PAT decreased ethanol co nsumption to 0.69 0.03 g/kg on the experimental day while saline control animals consumed 1.3 0.32 g/kg (Figure 4 1). Follow up post hoc comparisons indicated that among the non deprived animals, there was a significant effect of drug treatment (F (1, 5) = 190, p<0.001) and interaction between days and drug treatment (F ( 9 ,110) = 9.2, p<0.05), but not days alone (F (4,19) = 3.2, p=0.054). Bonferroni post hoc tests showed that the only significant difference between non deprived ( ) trans PAT and saline treated animals was on the experimental day. ( ) trans PAT prevents the ethanol deprivation effect in deprived rats The ADE is an increase in alcohol consumption after a period of abstinence. ANOVA testing of the ethanol deprived ethanol consumption sho wed a significant effect of drug treatment (F (1,5) = 100, p<0.001), days (F (4,19) = 30, p<0.001), and

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60 interaction between days and drug treatment (F (9,110) = 8.0, p<0.05). T he jello shot model o f alcoholism demonstrated an alcoho l deprivation effect that started one day after ethanol reinstatement and returns to basal levels, 1.2 0.37 g/kg, six days after reinstatement (Figure 4 2). When ( ) trans PAT is injected on the day of reinstatement, there is a decrease in experimental day consumption, 0.6 9 0.16 g/kg, similar to experimental day effect of non deprived ( ) trans PAT in Figure 4 1. The ethanol deprived ( ) trans PAT group then returns to basal consumption and does not display the increased consumption found in the ethanol deprived saline i njected rats. ( ) trans m Br PAT D oes N ot A lter N on deprived O r D eprived E thanol C onsumption Non deprived animals demonstrated a lower basal consumption, 0.93 0.28 g/kg, than deprived animals, 1.2 0.23 g/kg (Figure 4 3 and 4 4) Three way ANOVA com paring main effect of deprivation, drug treatment and days found that only deprivation was significant (F (1,11) = 35 p<0.05) No interactions between main variables were found to be significant. In the non deprived animals, there was no difference in consumption between control and ( ) trans m Br PAT (Figure 4 3). Deprived saline injected control animals displayed the ADE as indicated by their increasing consumption to 1.8 0.21 g/kg. Deprived ( ) m Br trans PAT treated animals were not significantl y different from control deprived animals throughout the experiment (Figure 4 4). Ro60 0175 Alters Non deprived But Not Deprived Ethanol Consumption When rats underwent a three week period of responding for plain gelatin, the level of ethanol self admini stration was higher upon reaccess to ethanol gelatin (51.1 5.2% increase on Post 1) compared to non deprived rats (Figure 4 5, 4 6), which lasted for

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61 three to five days. Systemic administration of the 5 HT 2C agonist, Ro60 0175, on the first day of ethan ol gelatin reaccess produced differing effects on consumption, depending on ethanol deprivation. In non deprived rats, Ro60 0175 reduced ethanol consumption by about 30% (Figure 4 5) compared to non deprived rats that received saline (0.69 0.12 g/kg vs 0.98 0.13 g/kg, respectively). Ethanol self administration returned to normal levels in drug treated rats on subsequent days. In the deprived rats, Ro60 0175 did not significantly alter ethanol consumption compared to saline control s on experimental day (0.93 0.17 g/kg vs 1.13 0.21 g/kg respectively) (Figure 4 6) Three way ANOVA showed a significant main effect of ethanol deprivation (F(1,5) = 10.9, p<0.05) but no other main effects or interactions were significant. Discussion This data shows th alcoholism and it produces a five day long increase in ethanol consumption after a three week deprivation There is evidence in the literature that the ADE can be observed after shorter deprivation s (2 days) using P rats, a strain of rat genetically selected to consume ethanol (Bell et al., 2006). Other models of the ADE use a 2 week deprivation to produce an ADE that lasts for only 24 hours (Theilen et al., 2004). The jello shot model of alcoho lism produced a 47% increase in ethanol consumption on the second day which is comparable to the increases observed in other models ( Martin Fardon and Weiss, 2012). These results also show that ( ) trans PAT reduces both daily basal consumption and ADE i nduced increases in ethanol consumption. This indicates that ( ) trans PAT is capable of preventing the changes that result in the ADE because ( ) trans PAT

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62 significantly decreases consumption in deprived animals for multiple days compared to the one day in non deprived rats. This suggests that functionally selective 5 HT 2C agonists both decrease basal consumption and decrease the elevated consumption seen after deprivation in human alcoholics. Surprisingly, ( ) trans m Br PAT had no effect on daily or ADE consumption. Previously we observed that strong 5 HT 2C receptor agonism causes a decrease in voluntary ethanol containing gelatin consumption. When comparing available affinity and functional data, ( ) trans m Br PAT is very similar to ( ) trans PAT ( Table 1 1). These unexpected results may be due to insufficient dose, 5 mg/kg, of the ( ) trans m Br PAT. Unlike ( ) trans PAT, a dose response curve was not established for ( ) trans m Br PAT. There may also be differences in the human 5 HT 2C receptor that is used for affinity studies and the rat 5 HT 2C receptor that mediates the drugs ability to alter voluntary ethanol consumption. Possible pharmacokinetic reasons for this seem unlikely because of the structural similarity of ( ) trans m Br PAT and ( ) trans PAT. ( ) trans m Br PAT remains a powerful potential drug to reduce alcohol intake that requires further testing to ascertain the full effects on voluntary ethanol consumption. Ro60 0175 decreased non deprived but not deprivation induced increas e in consumption. This was expected because of the overlapping dose effect curves for Ro60 0175 explained in the previous chapter. Briefly, Ro60 0175 decreases both plain and ethanol containing gelatin consumption on FR5 schedules. This makes Ro60 0175 seem to have a more potent effect on ethanol containing gelatin consumption (Ro60 0175 decreases consumption for the vehicle gelatin and ethanol together) than the

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63 specific effect of Ro60 0175 has on ethanol consumption. Therefore it was unsurprising that the dose of Ro60 0175 selected was too low to alter the ADE. Rats that were deprived of ethanol in the Ro60 0175 and ( ) trans m Br PAT experiment were the same rats deprived in the ( ) trans PAT experiment. While the basal values for the ( ) trans m B r PAT experiment of the deprived and non deprived rats were not significantly different from each other, there does seem to be variation in basal consumption between repeated deprivation s in this animal model. In summary the ADE is a model of alcoholism relapse that is important to study the pharmacotheraputic value of a novel drug (L and Shaham, 2002). The 5 HT 2C agonist ( ) trans PAT, administered once before reinstatement to ethanol consumption, completely prevents the ADE from occurring over the ne xt five days. Another 5 HT 2C agonist, ( ) trans m Br PAT however, did not alter ethanol consumption or prevent the ADE from occurring. This was most likely due to using too low of a dose of ( ) trans m Br PAT because of differences in ( ) trans m Br PAT affinity between the human and rat receptor. The 5 HT2 family agonist Ro60 0175, did not alter the ADE either due to difficulty in determining the proper dose from the confounding effects on plain gel consumption. Functionally selective 5 HT 2C receptor agonists show promise as a novel pharmacotherapy for alcoholism that decrease daily ethanol consumption and prevent ethanol deprivation induced increases in ethanol consumption.

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64 Figure 4 1. ( ) trans PAT administration causes a temporary decrease in voluntary ethanol consumption. Basal consumption values were taken 3 weeks before Experimental day with Post 1 3 showing the consumption the days immediately following the Experimental day. N = 6 indicates a significant difference between groups on t hat day.

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65 Figure 4 2. ( ) trans PAT administration prevents the ADE. Ethanol deprived rats demonstrate increased consumption of ethanol after reinstatement. Basal consumption values were taken 3 weeks before Experimental day with Post 1 3 showing th e consumption the days immediately following the Experimental day. N = 6 indicates a significant difference between groups on that day.

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66 Figure 4 3. ( ) trans m Br PAT administration had no effect on voluntary ethanol consumption. Ethanol non dep rived rats demonstrate stable baseline of ethanol consumption. Basal consumption values were taken 3 weeks before Experimental day with Post 1 3 showing the consumption the days immediately following the Experimental day. N = 6 indicates a significant difference between groups on that day.

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67 Figure 4 4. ( ) trans m Br PAT administration had no effect on ADE. Ethanol deprived rats demonstrate increased consumption of ethanol after reinstatement. Basal consumption values were taken 3 weeks before E xperimental day with Post 1 3 showing the consumption the days immediately following the Experimental day. N = 6 indicates a significant difference between groups on that day.

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68 Figure 4 5. Ro60 0175 administration decre ases experimental day voluntary ethanol consumption. Ethanol non deprived rats demonstrate stable baseline of ethanol consumption. Basal consumption values were taken 3 weeks before Experimental day with Post 1 3 showing the consumption the days immediat ely following the Experimental day. N = 6 indicates a significant difference between groups on that day.

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69 Figure 4 6. Ro60 0175 administration does not alter the ADE. Ethanol deprived rats demonstrate increased consumpt ion of ethanol after reinstatement. Basal consumption values were taken 3 weeks before Experimental day with Post 1 3 showing the consumption the days immediately following the Experimental day. N = 6 indicates a significant difference between groups on that day.

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70 CHAPTER 5 MODULATION OF NEUROTRANSMISSION IN THE NAC BY 5 HT 2C AGONISTS USING CAPILLARY ELECTROPHORESES WITH LASER INDUCED FLUORESCENCE DETECTION Alcoholism is a complex disorder that has a wide range of effects on neuro trans mitters, incl uding GABA (Koob et a l., 1998). While there are conflicting reports about ethanol induced changes of GABA concentrations in the mesolimbic pathway (Theile et a l., 2009; Xaio and Ye, 2008), it is clear that 5 HT 2C receptors can modulate both GABA and dopam ine release (Bubar et a l., 2011). This makes the 5 HT 2C receptor a promising therapeutic target for alcoholism. Unfortunately, the 5 HT 2C receptor is difficult to study because it shares a high trans membrane sequence homology with the rest of the 5 HT 2 family (Julius et al., 1990), making it difficult to find agonists that activate only one out of the three members of this family. This is problematic because 5 HT 2C receptor agonists are thought to have therapeutic properties while activating 5 HT 2A rece ptors causes psyc h o to mimetic effects. These opposing effects of 5 HT 2A/C agonists make it difficult to study the role these receptors play. The current most popular 5 HT 2C agonist, R o 60 0175, reports having good binding specificity for 5 HT 2C (pKi = 9.0) compared to 5 HT 2A (pKi = 7.5) (Damjanoska et al., 2003). T he EC50 values for Ro60 0175 have only a 10 fold favoring for 5 HT 2C over 5 HT 2A (Porter et al., 1999). Recently ( ) trans PAT has demonstrated functional selectivity for this receptor family. It acts as a high affinity 5 HT 2C agonist while being an inverse agonist at 5 HT 2A/B receptors (Table 1 1) This compound inspired the creation of PAT analogs some of which share this functional specificity. Using these compounds it is now possible to investigate the effects of 5 HT 2C agonists without confounding 5 HT 2A agonism.

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71 There are two conditions to test a drugs effect on neuro trans mitter release in microdialysis experiments, basal or stimulated. Administering the drug during basal conditions will determine the effect of the drug on basal neuro trans mission. This can prove difficult to detect decreases in neuro trans mitter concentrations if the neuro trans mitter is found in low nM range. Stimulated release results in an increase in extracellular neuro trans mitter concentrations and the effects of drugs can be detected. There are many chemicals to increase neuro trans mitter concentrations including cocaine, ethano l, and high potassium (K+). K+ stimulation results in all neurons being brought to th reshold and causes calcium dependent release of neuro trans mitters (Sellstr m and Hamberger, 1977). By testing PAT compounds during K+ stimulated conditions, it is possible to observe decreases in neuro trans mitters found in low nM concentrations (e.g. GABA ) using CE LIF. The 5 HT 2C receptor agonists are notorious for binding to multiple different receptor families and subtypes. The PAT analogs are no exception, binding with high affinity to 5 HT 2A 5 HT 2C and H 1 receptors. This makes it problematic to determine which action at which receptor causes PAT analog induced changes. Follow up studies must be performed to determine the site of action for PAT induced changes in neuro trans mission. Mepyramine is a high affinity antagonist that is specific for t he H 1 receptor and has been widely used in research for decades. T he PAT analogs have the highest affinity for H 1 receptors, so it is possible that their ability to alter K+ induced changes in neuro trans mission is mediated by H 1 receptor and not through 5 HT 2C H 1 receptors have been shown to alter K+ stimulated 5 HT release (Son et al., 2001) but the role of

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72 H1 receptors on K+ stimulated GABA release remains unclear. To this end, mepyramine was tested alone on K+ stimulation induced increases in neuro tr ans mitter release Mepyramine and ( ) trans PAT share similar affinities for the H1 receptor so equivalent doses can be tested. Once the effects of mepyramine on basal and K+ stimulated GABA release was determined its ability to block any effects of PAT analogs mediated via H 1 receptors was assessed Ketanserin is a high affinity antagonist for the 5 HT 2A 5 HT 2C H 1 and alpha 1 adrenergic receptors. It is commercially available and has been widely used in research for the last 25 years. Using this c ompound, it is possible to block the 5 HT 2C agonism from PAT analogs. When comparing the functional profile of these PAT analogs to ketanserin, there is overlapping antagonism/inverse agonism of 5 HT 2A and H 1 receptors. The largest difference between the se PATs and ketanserin is the function at the 5 HT 2C receptor. If k etanserin blocks PAT analogs from attenuating K+ induced GABA release, then this effect is mediated by the 5 HT 2C receptors. Methods Animals Male Sprague Dawley rats 60 days of age (Harl an, Indianapolis, IN), weighing 225 to 250 g, were singly housed in a temperature and humidity controlled environment with a 12 hour normal phase light/dark cycle (06:00 18:00). All tests were conducted during the light phase. Rats were acclimated to ou r housing facilities for at least 1 week prior to experimentation. The subjects had ad libitum access to food and water throughout the experiment. Rat use was approved by the Institutional Animal Care and Use Committee and was consistent with the NIH Guid e for the Care and Use of Laboratory Animals.

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73 Chemicals NaCl, CaCl2, KCl, MgSO4, monobasic borate, dibasic borate, o phthaldialdehyde mercaptoethanol (BME), hydroxypropyl cyclodextrin (HPBCD), sodium metaboisulfate, citric acid, RO60 0175, mepyr amine and ketanserin were supplied by Sigma, St. Louis (USA). PAT analogs were supplied by the laboratory of Dr. Raymond Booth, D epartment of M edicinal C hemistry at College of Pharmacy, University of Florida. Microdialysis A nd Surgeries As described i n Chapter 2. Each rat will be anesthetized with isoflurane and placed in a stereotaxic instrument for implantation of a guide canulla. The guide canulla will be anchored with two stainless steel screws and dental cement. The following coordinates from b regma will be used for implantation: Nucleus Accumbens +1.8 anteroposterior, +1.3 lateral, 6.2 dorsoventral, Striatum +0.8 anteroposterior, +3.0 lateral, 4.0 dorsoventral After implantation, animals are given at least 2 days to recover before microdial ysis testing. Experiment Procedures E xperiment al procedures are described as following. (1) When measuring the effect of novel PATs on bas al neuro trans mission aCSF was constantly perfused throughout the experiment. The animal was then switched from pl ain aCSF to drug in an aCSF vehicle for 15 minu tes Lastly the animal was switched back to plain aCSF to investigate if any dru g induced changes dissipated. ( 2 ) In the K+ stimulation experiment, aCSF was first perfused for 5 minutes. Then high K+ aCSF (95 mM NaCl, 50 mM KCl, 1.0 mM MgCl2, 1.2 mM CaCl2, 0.45 mM monobasic phosphate, 1.55 mM dibasic phosphate, pH 7.4) was perfused into the NAc

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74 or striatum for 10 minutes. The a nim al was then switched back to plain aCSF for 40 minutes. The perfusion and wa shing procedures were repeated three times with the second stimulation paired with drug perfusion (3) K+ stimulation experiments with constant ketanserin or mepyramine are performed similar to Experimental Procedure 2. This experiment includes three 10 minute high K+ stimulations with drug perfusion paired to the second stimulation. In addition to this, there is a constant perfusion throughout the entire experiment of either 50 M m epyramine or 50 M k etanserin. CE LIF As described in Chapter 2. The technique used to gather data will be microdialysis coupled with capillary electrophoresis with laser induced fluorescence detection and has been described previously (Bowser and Kennedy 2001). On the experiment day, a standard calibration curve (0 to 20 ) will first be performed using a microdialysis probe with outer diameter 270 13,000 molecular weight cutoff, and variable active length (2 to 4 mm) depending on the brain region being dialysed. After calibration, the probe will be implanted in the non anesthetized and free moving rat. The experiment begins after neuro trans mitter concentrations reach steady state which is typically 2 hours after implantation. The substances measured will include glutamate, aspartate, serotonin, dopamine, ornathine, GABA, taurine, glutamine, serine, and glycine. Data Analysis A nd Histology Data output was converted into concentrations of neuro trans mitters over time as described in Chapter 2. AUC was then determined for each K+ induced increase in neuro trans mitter release. The three AUC values were then converted to a percent of

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75 the first (control) AUC. One way ANOVA with Tukey post hoc tests with significance level p < 0.05 were performed using SPSS on the AUC values to determine significant changes. A Bright In struments cryostat was used to section the frozen brains after the experiment. Coronal sections 40 m thick were removed until probe tract is observed. Probe placement was determined by tract location in relation to brain arcetecture described with a bra in atlas (Paxinos and Watson, 2005). Only animals with the majority (over 50%) of the 2 or 4 mm long probe in the targeted brain region were included in the study. Results Effect O f ( ) trans PAT P erfusion O n B asal N euro trans mitter R elease I n NAc In ou r study the m icrodialysis experiment started with a 15 minute basal period. We then directly per fused 5 M of ( ) trans PAT directly into the NAc for 15 minutes. The GABA concentrations before, during, and after are plotted in Figure 5 2 The PAT compo unds had no measureable effect on basal accumbal neuro trans mitter concentration in rats (F (2,8) = 2.8, p>0.05) Effect O f ( ) trans PAT O n K+ I nduced Neuro trans mitter R elease I n T he NAc. The effects of ( ) trans PAT on K+ stimulated increases in accum bal GABA and taurine were examined using the procedure described in Experimental Procedure 2. ( ) trans PAT reduced K+ stimulated GABA release (F (2,8) = 180, p<0.0001) (Figure 5 3 ) but not taurine (t (2) = 0.69, p>0.05) (Figure 5 4 ). To determine what r eceptor is mediating this effect, ketanserin or m epyramine were constantly perfused as in Experimental Procedure 3 to block 5 HT 2C and H 1 receptors respectively. Ketanserin (Figure 5 5 ) and mepyramine (Figure 5 7 ) ha d no effect on stimulated GABA release (F

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76 (2,8) = 0.60, p>0.05) (F (2,8) = 3.7, p>0.05) However when ketanserin is perfused throughout the experiment, it blocks ( ) trans PAT ability to decrease stimulated GABA release (Figure 5 6 ) (F (2,8) = 1.6, p>0.05) Mepyramine however, was unable to alter the decrease in stimulated GABA levels that was caused by ( ) trans PAT (Figure 5 8 ) (F (2,8) = 180, p<0.0001) Effect O f A dditional 5 HT 2C R eceptor A gonists O n K+ I nduced N euro trans mitter R elease I n T he NAc. Other novel and established 5 HT 2C ag onists were then tested for the ability to reduce K+ stimulated GABA release. This includes ( ) trans p Cl PAT, TOMCAT, and Ro60 0175. All three compounds were able to reduce stimulated GABA release as shown in Figure 5 9 (F (2,8) = 220, p<0.0001) Figur e 5 1 1 (F (2,8) = 39, p<0.01) and Figure 5 1 3 (F (2,8) = 75, p<0.001) When ketanserin was perfused throughout the experiment to block 5 HT 2C agonism, both ( ) trans p Cl PAT (Figure 5 10 ) and TOMCAT (Figure 5 1 2 ) were unable to change stimulated GABA co ncentrations (F (2,8) = 0.72, p>0.05) (F (2,8) = 2.2, p>0.05) Effect O f 5 HT 2 R eceptor M odulation O n K+ I nduced N euro trans mitter R elease I n T he NAc. To fully explore the pharmacology behind the 5 HT 2C receptor effects described above, 5 HT 2C agonists with poor affinity partial efficacy, or 5 HT 2C antagonist were tested. The ( ) trans p Me PAT is a 5 HT 2C agonist that has 10 fold lower affinity for the 5 HT 2C receptor compared to ( ) trans PAT. When ( ) trans p Me PAT was administered during K+ stim ulation, there was no significant change in K+ stimulated GABA release ( Figure 5 1 4 ) (F (2,8) = 0.28, p>0.05) A partial agonist, (+) trans PAT was also tested and showed no effect on stimulated GABA release ( Figure 5 1 5 ) (F (2,8) = 2.4, p>0.05) Lastly the 5 HT 2C receptor antagonist, ( ) trans CAT was tested

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77 but caused no ch ange in stimulated GABA release ( Figure 5 1 6 ) (F (2,8) = 0.55, p>0.05) Together, this shows that high affinity full agonists for the 5 HT 2C receptor are required for reduction of K + stimulated GABA release in the NAc. Effect O f N ovel ( ) trans PAT P erfusion O n K+ I nduced N euro trans mitter R elease I n T he S triatum. ( ) trans PAT was then tested for its ability to alter stimulated GABA release in the striatum, another brain region receiving large amounts of dopaminergic input K+ stimulation caused a robust increase in GABA and taurine similar to what was observed in the NAc however when ( ) trans PAT was perfused during the second stimulation, K+ nificantly reduced (Figure 5 1 7 ) (F (2,11) = 120, p<0.001) Taurine increases remain unaffected by ( ) trans PAT (Figure 5 1 8 ) (t (2) = 076, p>0.05) Discussion This study examined the effect of multiple novel and established 5 HT 2 receptor modulators on both basal and stimulated neuro trans mission in the NAc and s triatum. Reverse dialysis of ( ) trans PAT had no observable effect on basal neuro trans mission in the NAc. However, novel PAT analogs with 5 HT 2C agonism reduced K+ stimulated GABA release K+ stimulated taurine concentrations were unaffected by the presence of PAT analogs Analogs that are 5 HT 2C antagonist/inverse agonists had no effect on extracellular neuro trans mitter concentrations Although we were able to reliably measure K+ stimulat ed GABA release, our ability to detect hypothesized decreases in basal GABA release were limited by our detection sensitivity.

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78 PATs E ffect O n K+ S timulated R elease I n NAc A nd S triatum This study replicated previous work with K+ stimulation (Sellstr m and Hamberger, 1977) and advanced it into an in vivo screening procedure for 5 HT 2 receptor modulators. Drugs with good affinity and agonism for the 5 HT 2C receptor e.g., ( ) trans PAT, ( ) trans p Cl PAT, TO MCAT, and Ro 60 0175, caused a decrease of K+ st imulated GABA release in the NAc. Taurine release remained unaffected. The only 5 HT 2C agonist tested that did not decrease GABA release was the ( ) trans p Me PAT and this can be attributed to its poor affinity for the 5 HT 2C receptor Drugs that are 5 HT 2C antagonists/inverse agonists did not alter K+ stimulated GABA or taurine release in the NAc Although it was originally hypothesized that the inverse agonists should have the opposite effect of the agonists, this was not observed. The K+ stimulati on might be so powerful that potentiating more release is impossible due to a ceiling effect. It is also possible there was not enough endogenous drive on the 5 HT 2C receptor for an antagonist to block. Either of these could explain why 5 HT 2C receptor a ntagonists/inverse agonists were unable to alter stimulated GABA release. In the striatum, 5 HT 2C agonism caused a decrease in K+ stimulated GABA release but not taurine The effects of both the K+ stimulation and 5 HT 2C agonism are the same that was ob served in the NAc. Both the striatum and NAc both have a large amount of dopamine neurons projecting to them and both express 5 HT receptors ( Alex and Pehek, 2007 ). H1 R eceptors D o N ot A lter GABA D uring H igh K+ I nduced GABA R elease. Mepyramine at 50 M directly perfused into the NAc had no effect on basal or high K+ to decrease high K+ stimulated GABA release is mediated by their action on 5 HT 2C

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79 receptors and not thro ugh their action on H 1 receptors. M epyramine locally perfused into the NAc did not influence basal or stimulated release of GABA, therefore it is fit to be used to block H 1 receptors such that PAT analogs cannot act on them. 5 HT 2C R eceptors M ediate D ec reases I n H igh K+ I nduced GABA R elease. The experiments in which k etanserin was constant ly perfused throughout demonstrated that ( ) trans PAT, TOMCAT, and ( ) trans p Cl PAT were mediating their effects through a receptor that is common to the binding p rofile of the PAT analogs and k etanserin. These receptors include the 5 HT 2A/C and H 1 receptors. From the results of the m epyramine studies, we can rule out H 1 receptors as h aving a role in attenuating K+ induced GABA release. This leaves only the 5 HT 2 A/C receptors. To determine if the ( ) trans PAT, TOMCAT, and ( ) trans p Cl PAT mediate their effect through 5 HT 2A or 5 HT 2C receptors, the function of these drugs at both receptors was examined These three PAT analogs are all 5 HT 2A antagonist/inver se agonist and 5 HT 2C agonists. From the drugs studied it is apparent that the only drugs that altered GABA release had strong affinity and agonism at 5 HT 2C receptors. 5 HT 2A receptor antagonism /inverse agonism by ( ) trans CAT or k etanserin did not de crease K+ stimulated GABA release Together, this indicates that 5 HT 2A antagonism/inverse agonism does not play a role in the attenuation of K+ stimulated GABA release, and 5 HT 2C agonism does. Previous electrophysiological studies have shown that 5 HT 2C agonism causes an increase in local GABA release onto dopaminergic neurons in the VTA (Theile et a l., 2009). T hese results conflict with the results of the present study where we observed 5 HT 2C agonists had no measurable effect on basal GABA levels an d decreased stimulated GABA release in both the striatum and NAc. However it should be pointed

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80 out that the electrophysiological studies were performed in different brain regions than the current dialysis experiments This apparent disagreement is rectif ied in light of previous studies (Navailles et al., 2008) where 5 HT 2C receptors have been shown to differentially regulate cocaine induced effects depending on brai n region ( VTA vs NAc ) Taken together, these findings demonstrate that 5 HT 2C receptor ago nists modulate GABAergic drive in the mesolimbic pathway differently in the NAc than in the VTA

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81 Figure 5 1. Coronal sections showing microdialysis probe placement withi n the NAc. Lines indicate the active dialysis regions. Numbers below the figure represent the position of the slice relative to bregma. Figure was adapted from Paxinos and Watson, 2005. 2.2mm 1.7mm 1.2mm

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82 Figure 5 2 ( ) trans PAT does not alter basal GABA concent rations in the NAc. ( A) The concentration of GABA over time. Red line indicates when 5 M ( ) trans PAT was added to the aCSF. (B) AUC values for the data presented in Panel A. Data shown are mean values SEMs for N = 3. indicates p < 0.05. (F (2 ,8) = 2.8, p>0.05) Figure 5 3 ( ) trans PAT decreased K+ stimulated GABA release in the NAc. ( A) The concentration of GABA over time. Blue shaded areas represent perfusion with 50 mM K+ containing aCSF. Red line indicates when 50 M ( ) trans PA T was added to the aCSF. (B) AUC values for the data presented in Panel A. Data shown are mean values SEMs for N = 3. indicates p < 0.05. (F (2,8) = 180, p<0.0001)

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83 Figure 5 4 ( ) trans PAT did not alter K+ stimulated taurine release in t he NAc. ( A) The concentration of taurine over time. Blue shaded areas represent perfusion with 50 mM K+ containing aCSF. Red line indicates when 50 M ( ) trans PAT was added to the aCSF. (B) AUC values for the data presented in Panel A. Data shown a re mean values SEMs for N = 3. indicates p < 0.05. (t (2) = 0.69, p>0.05) Figure 5 5 Ketanserin had no effect on K+ stimulated GABA release in the NAc. ( A) The concentration of GABA over time. Blue shaded areas represent perfusion with 50 mM K+ containing aCSF. Red line indicates when 50 M ketanserin was added to the aCSF. (B) AUC values for the data presented in Panel A. Data shown are mean values SEMs for N = 3. indicates p < 0.05. (F (2,8) = 0.60, p>0.05)

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84 Figure 5 6 ( ) tra ns PAT had no effect on K+ stimulated GABA release in the NAc in the presence of ketanserin ( A) The concentration of GABA over time. Blue shaded areas represent perfusion with 50 mM K+ containing aCSF. Red line indicates when 50 M ( ) trans PAT was a dded to the aCSF. Ketanserin was perfused throughout the whole experiment. (B) AUC values for the data presented in Panel A. Data shown are mean values SEMs for N = 3. indicates p < 0.05. (F (2,8) = 1.6, p>0.05) Figure 5 7 Mepyramine had no ef fect on K+ stimulated GABA release in the NAc. ( A) The concentration of GABA over time. Blue shaded areas represent perfusion with 50 mM K+ containing aCSF. Red line indicates when 50 M mepyramine was added to the aCSF. (B) AUC values for the data pr esented in Panel A. Data shown are mean values SEMs for N = 3. indicates p < 0.05. (F (2,8) = 3.7, p>0.05)

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85 Figure 5 8 ( ) trans PAT decreased K+ stimulated GABA release in the NAc in the presence of mepyramine ( A) The concentration of GAB A over time. Blue shaded areas represent perfusion with 50 mM K+ containing aCSF. Red line indicates when 50 M ( ) trans PAT was added to the aCSF. Mepyramine was perfused throughout the experiment. (B) AUC values for the data presented in Panel A. D ata shown are mean values SEMs for N = 3. indicates p < 0.05. (F (2,8) = 180, p<0.0001) Figure 5 9 ( ) trans p Cl PAT decreased K+ stimulated GABA release in the NAc. ( A) The concentration of GABA over time. Blue shaded areas represent perf usion with 50 mM K+ containing aCSF. Red line indicates when 50 M ( ) trans p Cl PAT was added to the aCSF. (B) AUC values for the data presented in Panel A. Data shown are mean values SEMs for N = 3. indicates p < 0.05. (F (2,8) = 220, p<0.0001)

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86 Figure 5 10 ( ) trans p Cl PAT had no effect on K+ stimulated GABA release in the NAc in the presence of ketanserin ( A) The concentration of GABA over time. Blue shaded areas represent perfusion with 50 mM K+ containing aCSF. Red line indicat es when 50 M ( ) trans p Cl PAT was added to the aCSF. Ketanserin was perfused throughout the whole experiment. (B) AUC values for the data presented in Panel A. Data shown are mean values SEMs for N = 3. indicates p < 0.05. (F (2,8) = 0.72, p>0. 05) Figure 5 1 1 TOMCAT decreased K+ stimulated GABA release in the NAc. ( A) The concentration of GABA over time. Blue shaded areas represent perfusion with 50 mM K+ containing aCSF. Red line indicates when 50 M TOMCAT was added to the aCSF. (B ) AUC values for the data presented in Panel A. Data shown are mean values SEMs for N = 3. indicates p < 0.05. (F (2,8) = 39, p<0.01)

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87 Figure 5 1 2 TOMCAT had no effect on K+ stimulated GABA release in the NAc in the presence of ketanserin ( A) The concentration of GABA over time. Blue shaded areas represent perfusion with 50 mM K+ containing aCSF. Red line indicates when 50 M TOMCAT was added to the aCSF. Ketanserin was perfused throughout the whole experiment. (B) AUC values for the da ta presented in Panel A. Data shown are mean values SEMs for N = 3. indicates p < 0.05. (F (2,8) = 2.2, p>0.05) Figure 5 1 3 Ro60 0175 decreased K+ stimulated GABA release in the NAc. ( A) The concentration of GABA over time. Blue shaded areas represent perfusion with 50 mM K+ containing aCSF. Red line indicates when 50 M Ro60 0175 was added to the aCSF. (B) AUC values for the data presented in Panel A. Data shown are mean values SEMs for N = 3. indicates p < 0.05. (F (2,8) = 75, p<0. 001)

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88 Figure 5 1 4 ( ) trans p Me PAT had no effect on K+ stimulated GABA release in the NAc. ( A) The concentration of GABA over time. Blue shaded areas represent perfusion with 50 mM K+ containing aCSF. Red line indicates when 50 M ( ) trans p Me PAT was added to the aCSF. (B) AUC values for the data presented in Panel A. Data shown are mean values SEMs for N = 3. indicates p < 0.05. (F (2,8) = 0.28, p>0.05) Figure 5 1 5 ( + ) trans PAT had no effect on K+ stimulated GABA release in th e NAc. ( A) The concentration of GABA over time. Blue shaded areas represent perfusion with 50 mM K+ containing aCSF. Red line indicates when 50 M ( + ) trans PAT was added to the aCSF. (B) AUC values for the data presented in Panel A. Data shown are m ean values SEMs for N = 3. indicates p < 0.05. (F (2,8) = 2.4, p>0.05)

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89 Figure 5 1 6 ( ) trans C AT had no effect on K+ stimulated GABA release in the NAc. ( A) The concentration of GABA over time. Blue shaded areas represent perfusion with 5 0 mM K+ containing aCSF. Red line indicates when 50 M ( ) trans C AT was added to the aCSF. (B) AUC values for the data presented in Panel A. Data shown are mean values SEMs for N = 3. indicates p < 0.05. (F (2,8) = 0.55, p>0.05) Figure 5 1 7 ( ) trans PAT decreased K+ stimulated GABA release in the striatum ( A) The concentration of GABA over time. Blue shaded areas represent perfusion with 50 mM K+ containing aCSF. Red line indicates when 50 M ( ) trans PAT was added to the aCSF. (B) AUC values for the data presented in Panel A. Data shown are mean values SEMs for N = 4 indicates p < 0.05. (F (2,11) = 120, p<0.001)

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90 Figure 5 1 8 ( ) trans PAT had no effect on K+ stimulated taurine release in the striatum ( A) The concen tration of taurine over time. Blue shaded areas represent perfusion with 50 mM K+ containing aCSF. Red line indicates when 50 M ( ) trans PAT was added to the aCSF. (B) AUC values for the data presented in Panel A. Data shown are mean values SEMs fo r N = 4 indicates p < 0.05. (t (2) = 076, p>0.05)

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91 CHAPTER 6 GENERAL DISCUSSION Discussion Alcoholism is a complex disorder in humans that lacks highly effective pharmacotherapies (Edwards et al., 2011) An estimated 17.8% of Americans have abused alcohol and 12.5% of Americans have demonstrated alcohol dependence sometime during their life (Hasin et al., 2007) In the current study, we investigated the pharmacotheraputic potential of 5 HT 2C agonists using the jello shot model of alcoholism and examined the mechanism of action of these drugs in reward centers of the brain. This dissertation in total provides encouraging evidence for functionally selective agonists of the 5 HT 2C receptor acting as pharmacotherapy for alcoholism. Jello Shot Mo del O f Alcoholism administration of ethanol and shows an ADE after 3 week deprivation. An ideal model of alcoholism should have the following 7 attributes (Cicero et. al., 197 1): oral self administration of ethanol ethanol consumed should result in pharmacologically relevant dose ethanol should be consumed for post ingestive pharmacological effects ethanol should be positively reinforcing chronic ethanol consumption should lead to metabolic and functional tolerance chronic ethanol consumption should lead to dependence animals should display characteristics associated with relapse of a n ideal model of alcoholism. The rats voluntarily self administer ethanol in a sweetened vehicle (Rowland et. al., 2005). Pharmacologically relevant dose of ethanol has been observed previously (Peris et. al., 2006), and the current study demonstrated p harmacologically relevant doses with 0.85 0.21 g/kg basal ethanol consumption

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92 during a 30 minute operant session. Additionally, for the first time using this model, the characteristics associated with relapse were explored when looking at the ADE. Ani mals deprived of ethanol for 3 weeks were shown to greatly increase consumption after reinstatement. The other 4 attributes are not covered in this dissertation though there are previous studies examining aspects of positive reinforcement such as motivati on for ethanol reinforcements (Li et. al., 2009). Overall, the ADE experiments add support to this animal model mirroring the beginning pattern of human alcohol consumption Mesolimbic Pathway The mesolimbic pathway is widely known to be important in th e development of addiction. Changes in neuro trans mission in this pathway mediate the rewarding effects of many substances of abuse including alcohol. One of the most prevalent modulators of neuro trans mission in the mesolimbic pathway is GABA. Modulating GABA release in the mesolimbic pathway represents a potential target to reduce the rewarding value of ethanol. Ethanol has complex effects on GABA release in the mesolimbic pathway. Microdialysis studies have shown that systemic ethanol administration does not change GABA concentrations in the NAc (Smith et. al, 2004) or VTA (Kemppainen et. al., 2010). This conflicts with the electrophysiology studies which demonstrate that ethanol increases spontaneous and evoked GABA release in the VTA and numerous o ther brain regions (Kelm et. al., 2011). Apparently, the net effect of ethanol on GABA concentration is negligible but ethanol causes significant effects on individual GABA neurons. This supports the hypothesis of addictive substances increasing GABA rel ease onto inhibitory neurons that then project onto dopaminergic neurons. To put it

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93 simply, ethanol causes GABA to inhibit neurons that would otherwise inhibit dopamine release. Thus an increase in DA concentrations is seen and the reward value of the dr ug is perceived. This explanation was first used to explain the addictive proper ties of benzodiazepines (Tan et al., 2010) and quickly applied to alcoholism (Kelm et. al., 2011). The above mechanism of action for ethanol is problematic in terms of findi ng a singular target for potential pharmacotherapy. This is because both of the receptors in the pathway are for the same ligand, GABA, and treatment would require antagonism of the first and agonism of the second. When looking at the current available p harmacotherapies, topiramate directly and naltrexone indirectly have the ability to modulate GABA A receptors. Naltrexone acts as an indirect GABA agonist (Johnson and for decreasing ethanol consumption is partially mediated by the chloride channel associated with GABA A receptors (Gewiss et al., 1994). Topiramate, a GABA A receptor positive modulator and AMPA receptor antagonist, also has therapeutic effects for alcoholics (Johnson et al., 2003). Baclofen, a GABA B agonist has demonstrated the ability to reduce ethanol withdrawal, craving, and intake (Colombo et al., 2004). GABA A/B receptors are expressed throughout the mesolimbic pathway (Chester et al., 2002; Li et al., 2004) and because both GABA a nd dop amine neurons express both GABA A/B receptors it is difficult to design a pharmacotherapy that inhibits the GABA receptors on GABA neurons but not GABA receptors on dopamine neurons. These three drugs provide strong evidence that modulating GABA in the mes olimbic pathway represents a pharmacotherapeutic target but improved results may be observed with the ability to selectively modulate the GABA

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94 neurons in the reward pathway. Specifically, an alcoholism treatment c ould decrease GABAergic drive onto GABA ne urons that then project onto DA neurons. 5 HT 2C receptors represent an elusive target for alcoholism pharmacotherapy because of their ability to modulate dopamine in the mesolimbic pathway (Di Matteo et al., 2001). Agonism of this receptor increases GAB A release in the VTA and blocks the ethanol induced increases in VTA dopamine neuron firing (Theile et al., 2009). The current study demonstrates that 5 HT 2C agonism decreases stimulated GABA release in the NAc. Together, 5 HT 2C rec eptor agonism decrease s NAc and increases VTA GABA concentrations. These brain region specific modulations of GABA in the mesolimbic pathway via 5 HT 2C agonists represent a powerful pharmacotherapy for alcoholism PAT Analogs Several novel PAT analogs have demonstrated the a bility to decrease K+ stimulated GABA release in the NAc. This decrease has been shown to be mediated specifically by 5 HT 2C receptors. Another 5 HT 2C receptor agonist, Ro60 0175 showed the same decrease in K+ stimulated GABA release. It is evident that agonists for the 5 HT 2C receptor are able to modulate GABA in the NAc. The 5 HT 2C agonists ( ) trans PAT and Ro60 0175 tested in both microdialysis and operant responding experiments, reduced K+ stimulated GABA in the NAc and consumption of ethanol conta ining gelatin. The importance of 5 HT 2C agonists in decreasing ethanol consumption is clear and there is strong evidence that the mechanism by which these drugs alter behavior is through GABA. Future Studies This dissertation clearly demonstrated the im portance of the 5 HT 2C receptor in treating alcoholism. There are a few aspects of this work that remain unclear or

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95 untested. This includes experiments surrounding the inability of ( ) m Br trans PAT to alter the ADE and the lack of effect of ( ) trans PAT on progressive ratio responding for ethanol gel. To clarify the results of testing ( ) m Br trans PAT in Chapter 4, the affinity and functional studies for both ( ) m Br trans PAT and ( ) trans PAT should be performed using rat 5 HT 2C receptors as op posed to human. This may clarify why these two compounds had similar human affinity values and functional profile but only ( ) trans PAT was able to reduce voluntary ethanol consumption and the ADE. To further explore ( ) m Br trans PAT, a full dose resp onse curve on voluntary FR5 consumption of both ethanol and plain gelatin similar to the experiments performed with ( ) trans PAT ( Figure 3 3 and Figure 3 5 ) should be performed Then, knowing the lowest effective dose, repeat the ADE experiment to determ ine if ( ) trans m Br PAT alters ethanol consumption after a deprivation. There was a significant difference in breakpoints observed during PR10 operant sessions using ( ) trans PAT responding for 0.28 g of ethanol containing gelatin but not with 0.15 g. Obviously if the reinforce r is larger, the animals will work harder for another administration resulting in a larger breakpoint. With the breakpoints so low responding for 0.15 g of gelatin there may have been a floor effect where the breakpoints couldn go lower. Using 0.28 g of gelatin as a reward solved this problem and allowed the visualization of ( ) trans PAT reducing the rewarding value of ethanol specifically. ( ) trans PAT decreases motivation for ethanol but not the plain gelatin vehicle The current work went into great detail about the role of 5 HT 2C receptor agonism and K+ stimulated GABA concentrations in the NAc. While this setup yields very

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96 consistent information about stimulated release of GABA and taurine, a more alcoholism focused project would be to use ethanol as the GABA stimulating agent. Ethanol (1 mol/L) reverse dialyzed into the central nucleus of the amygdala has been shown to stimulate GABA release measured by microdialysis (Roberto et. al., 2010). A literature search di d not show any experiments using this ethanol reverse dialyzation technique in the NAc. After a study to determine if reverse dialyzed ethanol will significantly stimulate GABA release in the NAc, it would then be possible to test novel PAT on ethanol sti mulated GABA release. The ethanol induced GABA release would be more directly related to alcoholism than the K+ stimulated GABA release. Lastly, there are other neuro trans mitters involved in alcoholism besides GABA and DA and they are currently understu died. For example, increases in glycine are associated with anticipation before an operant session responding for ethanol containing gelatin (Li et. al., 2008). Administering PAT before an operant session may decrease this anticipatory glycine and theref ore demonstrate that PAT decreases anticipation for ethanol. This would further bolster PAT as a pharmacotherapy that decreases voluntary ethanol consumption, prevents the ADE seen in relapsing alcoholics, and reduces daily anticipation for ethanol.

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106 BIOGRAPHICAL SKETCH James was born to D an and Peggy Kasper He grew up in Lisbon, Wisconsin and graduated from Hamilton High School in 2001. He then attended University of Wisconsin Eau Claire and graduated in 2006 with a After college he attended University of Florida unde r the guidance of Dr. Joanna Peris where he received his Ph.D in the s ummer of 2012