Relationship Between Nicotine and Ingestive Behaviors in Rats

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Title:
Relationship Between Nicotine and Ingestive Behaviors in Rats
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1 online resource (247 p.)
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english
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Grebenstein, Patricia E
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Degree:
Doctorate ( Ph.D.)
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University of Florida
Degree Disciplines:
Psychology
Committee Chair:
Rowland, Neil E
Committee Members:
Dallery, Jesse
Dotson, Cedrick Deshawn
Smith, David W
Morgan, Drake

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Subjects / Keywords:
addiction -- cues -- cytisine -- eating -- flavor -- meals -- nicotine -- psychopharmacology -- treatment
Psychology -- Dissertations, Academic -- UF
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Psychology thesis, Ph.D.
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theses   ( marcgt )
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Abstract:
The experiments described in this dissertation examine the interactions between nicotine and ingestive behaviors in rats. The first experiment examines the effect of nicotine on flavor preferences. In this experiment, rats were given noncontingent infusions of nicotine and ad libitum access to a flavored Kool-Aid solution. After two and three weeks, flavor preference tests were administered. Various parameters of interest included changes in nicotine dose, flavor novelty, pretest preferences, and an initial week without the flavor presentation. Animals preferred the flavor that they consumed while receiving the nicotine over a novel solution, though these preferences didn’t differ significantly from preferences for flavors paired with saline infusions. In light of evidence indicating that individuals who smoke cigarettes weigh less than those who do not, and smoking cessation results in increased weight gain compared to controls, the second experiment examined nicotine’s effects on body weight and meal patterns. Rats that received noncontingent infusions of nicotine over 23 hours weighed less and ate smaller meals in both the light and dark phases, with compensatory increases in meal number in the dark. In the third experiment, rats received noncontingent infusions of cytisine, a selective a4ß2 nicotinic acetylcholine receptor partial agonist. Administration of cytisine occurred over 23 hours and body weight and meal patterns were recorded. In this experiment, animals weighed less and ate fewer meals in the light and dark phases without any changes in meal size, and these effects on body weight persisted after administration of the drug was discontinued. The final experiment examined the ability of cytisine to substitute for nicotine in a self-administration protocol. Animals were allowed to self-administer nicotine and once stable self-administration was attained, the nicotine was substituted with cytisine. A dose response relationship was examined within animals and responses for the cytisine were compared to extinction with vehicle. Cytisine increased total pellet intake with respect to nicotine and animals self-administered cytisine significantly less than the nicotine. There was no effect of cytisine dose on meal patterns. Cytisine maintained decreased food intake and weight gain compared to baseline.
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In the series University of Florida Digital Collections.
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Includes vita.
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Statement of Responsibility:
by Patricia E Grebenstein.
Thesis:
Thesis (Ph.D.)--University of Florida, 2012.
Local:
Adviser: Rowland, Neil E.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-08-31

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1 RELATIONSHIP BETWEEN NICOTINE AND INGESTIVE BEHAVIORS IN RATS By PATRICIA E. GREBENSTEIN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSPHY UNIVERSITY OF FLORIDA 2012

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2 2012 Patricia E. Grebenstein

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3 To my parents for their unending love and support and to my advisor Dr. Neil E. Rowland for his guidance and patience

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4 ACKNOWLEDGMENTS I would like to thank my mentor Neil Rowland for his support throughout my time at the University of Florida. My graduate experience was greatly enriched because of him and his patience and willingness to teach. I would also like to thank our lab technician, Kim Robertson, for helping my experiments run smoothly and to our various undergraduate research assistants, particularly Joseph Harp, for their hard work and dedication. I would like to thank my diss ertation committee members Jesse Dallery, David Smith, Drake Morgan and Shawn Dotson for their priceless advice regarding experimental design and proper writing techniques. Lastly, I want to give tremendous thanks to my parents. They have provided limitl ess support throughout my graduate experience and I am blessed to have such a caring, generous family. Their belief in me and their unfailing encouragement provided me with the support I needed to accomplish my dreams.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ............... 4 LIST OF TABLES ................................ ................................ ................................ ........................... 8 LIST OF FIGURES ................................ ................................ ................................ ....................... 11 ABSTRACT ................................ ................................ ................................ ................................ ... 15 CHAPTER 1 BACKGROUND INFORMATION ON NICOTINE AND INGESTIVE BEHAVIORS ..... 17 Prevalence of Cigarette Smoking ................................ ................................ ........................... 17 The Role of Cues in Nicotine Self Administration ................................ ................................ 18 The Relationship between Nicotine Abuse and Overweight/Obesity ................................ .... 21 Factors Associated with Weight Loss during Smoking ................................ .......................... 24 Factors Associated with Weight Gain during Smoking Cessation ................................ ......... 25 ................................ ................ 36 Implications for Treatment ................................ ................................ ................................ ..... 41 Conclusions ................................ ................................ ................................ ............................. 45 2 THE RELATIONSHIP BETWEEN NICOTINE AND FLAVOR CUES ............................. 47 Introduction ................................ ................................ ................................ ............................. 47 Materials and Methods ................................ ................................ ................................ ........... 51 Animals and Housing ................................ ................................ ................................ ...... 51 Surgery ................................ ................................ ................................ ............................ 51 Apparatus ................................ ................................ ................................ ......................... 52 Nicotine ................................ ................................ ................................ ........................... 53 Procedure ................................ ................................ ................................ ......................... 54 Experiment one ................................ ................................ ................................ ........ 54 Experiment two ................................ ................................ ................................ ........ 55 Experiment three ................................ ................................ ................................ ...... 55 Experiment four ................................ ................................ ................................ ........ 56 Data Analysis ................................ ................................ ................................ ................... 56 Results ................................ ................................ ................................ ................................ ..... 57 Experiment One: High Dose Compared with Vehicle ................................ .................... 57 Experiment Two: High Dose Compared with Low Dose ................................ ............... 57 Experiment Three: Flavor Compared with No Flavor during Week One ....................... 58 Experiment Four: Initial Preference ................................ ................................ ................ 58 Discussion ................................ ................................ ................................ ............................... 58 3 THE EFFECTS OF NICOTINE ON BODY WEIGHT AND MEAL PATTERNS .............. 70

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6 Introduction ................................ ................................ ................................ ............................. 70 Materials and Methods ................................ ................................ ................................ ........... 75 Animal s and Housing ................................ ................................ ................................ ...... 75 Surgery ................................ ................................ ................................ ............................ 75 Apparatus ................................ ................................ ................................ ......................... 76 Nicotine ................................ ................................ ................................ ........................... 77 Procedure ................................ ................................ ................................ ......................... 77 Data Analysis ................................ ................................ ................................ ................... 78 Results ................................ ................................ ................................ ................................ ..... 79 Body Weight ................................ ................................ ................................ .................... 79 Meal Patterns .06 mg/kg/inf ................................ ................................ .......................... 79 Baseline ................................ ................................ ................................ .................... 79 Vehicle first two days of treatment ................................ ................................ ....... 80 Treatment number of pellets ................................ ................................ ................. 80 Treatment meal size ................................ ................................ ............................... 81 Trea tment number of meals ................................ ................................ ................... 82 Cessation ................................ ................................ ................................ .................. 83 Meal Patterns .03 mg/kg/inf ................................ ................................ .......................... 84 Total pellets ................................ ................................ ................................ .............. 84 Night pellets ................................ ................................ ................................ ............. 84 Day pellets ................................ ................................ ................................ ................ 85 Excess Pellets ................................ ................................ ................................ .................. 86 Day Only Meal Patterns ................................ ................................ ................................ .. 86 Total pellets ................................ ................................ ................................ .............. 86 Number of meals ................................ ................................ ................................ ...... 87 Meal size ................................ ................................ ................................ .................. 87 Discussion ................................ ................................ ................................ ............................... 88 4 THE EFFECTS OF CYTISINE ON BODY WEIGHT AND MEAL PATTERNS ............ 122 Introduction ................................ ................................ ................................ ........................... 122 Materials and Methods ................................ ................................ ................................ ......... 129 Animal s and Housing ................................ ................................ ................................ .... 129 Surgery ................................ ................................ ................................ .......................... 130 Apparatus ................................ ................................ ................................ ....................... 131 Cytisine ................................ ................................ ................................ .......................... 132 Procedure ................................ ................................ ................................ ....................... 132 Data Analysis ................................ ................................ ................................ ................. 133 Results ................................ ................................ ................................ ................................ ... 133 Body Weight ................................ ................................ ................................ .................. 133 Meal Patterns ................................ ................................ ................................ ................. 133 Baseline ................................ ................................ ................................ .................. 133 Vehicle first two days of treatment ................................ ................................ ..... 134 Treatment number of pellets ................................ ................................ ............... 134 Treatment number of meals ................................ ................................ ................. 135 Treatment meal size ................................ ................................ ............................. 136 Cessation ................................ ................................ ................................ ........................ 137

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7 Excess Pellets ................................ ................................ ................................ ................ 137 Discussion ................................ ................................ ................................ ............................. 138 5 SELF ADMINISTRATION OF NICOTINE AND CYTISINE: EFFECTS ON BODY WEIGHT AND MEAL PATTERNS ................................ ................................ ................... 165 Introduction ................................ ................................ ................................ ........................... 165 Materials and Methods ................................ ................................ ................................ ......... 167 Animal s and Housing ................................ ................................ ................................ .... 167 Surgery ................................ ................................ ................................ .......................... 168 Apparatus ................................ ................................ ................................ ....................... 168 Nicotine ................................ ................................ ................................ ......................... 170 Cytisine ................................ ................................ ................................ .......................... 170 Procedure ................................ ................................ ................................ ....................... 1 70 Data Analysis ................................ ................................ ................................ ................. 171 Results ................................ ................................ ................................ ................................ ... 172 Body Weight ................................ ................................ ................................ .................. 172 Infusions ................................ ................................ ................................ ........................ 172 Meal Patterns ................................ ................................ ................................ ................. 173 Number of pellets ................................ ................................ ................................ ... 173 Meal number ................................ ................................ ................................ .......... 174 Meal size ................................ ................................ ................................ ................ 176 Discussion ................................ ................................ ................................ ............................. 177 6 GENERAL DISCUSSION ................................ ................................ ................................ ... 219 The Re lationship between Nicotine and Flavor Cues ................................ ........................... 219 The Effects of Nicotine on Body Weight and Meal Patterns ................................ ............... 220 The Effects of Cytisine on Body Weight and Meal Patterns ................................ ................ 222 LIST OF REFERENCES ................................ ................................ ................................ ............. 225 BIOGRAPH ICAL SKETCH ................................ ................................ ................................ ....... 247

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8 LIST OF TABLES Table page 3 1 P and F values for the changes in body weight over each day of the baseline, treatment, and cessation phases ................................ ................................ ......................... 94 3 2 F and P values for all meal pattern variables of int erest, with days clustered into groups, throughout the baseline and cessation phases for the vehicle and high nicotine groups ................................ ................................ ................................ ................... 95 3 3 F and P values for all meal pattern variables of interest, with days clustered into groups, throughout the treatment phase for the vehicle and high nicotine groups ............ 95 3 4 P and F values for the mean number of pellets eaten per day, during the treatment phase ................................ ................................ ................................ ................................ .. 96 3 5 P and F values for the mean meal size eaten per day, during the treatment phase ............ 97 3 6 P and F values for the mean number of meals eaten per day, during the treatment phase ................................ ................................ ................................ ................................ .. 97 3 7 P and F values for the mean number of pellets eaten, with days clustered into groups, throughout the baseline, treatment and cessation phases for the vehicle and low nicotine groups ................................ ................................ ................................ ................... 98 3 8 P and F values for the mean number of excess pellets eaten, with days clustered into groups, throughout the baseline, treatment and cessation phases for the vehicle and high nicotine groups ................................ ................................ ................................ ........... 99 3 9 P and F values for all meal pattern variables of interest, with days clustered into groups, throughout the baseline, treatment and cessation phases for the nicotine g roup during the light cycle ................................ ................................ ............................... 99 4 1 P and F values for the changes in body weight over each day of the baseline, treatment, and cessation phases ................................ ................................ ....................... 142 4 2 F and P values for all meal pattern variables of interest, with days clustered into groups, throughout t he baseline and treatment phase ................................ ...................... 142 4 3 P and F values for the total number of pellets eaten over each day of the baseline and tre atment phases ................................ ................................ ................................ ............... 143 4 4 P and F values for the average total number of meals eaten over each day of the baseline and treatment phases ................................ ................................ .......................... 144 4 5 F and P values for the average meal size eaten over each day of the baseline and treatment phases ................................ ................................ ................................ ............... 144

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9 4 6 F and P values for the averages of all meal pattern variables of interest, with days clustered into groups, throughout the cessation phase ................................ .................... 145 4 7 F and P values for the average number of excess pellets eaten, with days clustered into groups, throughout the baseline, treatment, and cessation phases ............................ 145 5 1 F and P values for the effects of phase and order of treatment on changes on body weight number of infusions, and the meal pattern variables of interest ......................... 184 5 2 T and P values for the changes in body weight gain throu ghout each phase of treatment, compared with the first phase of nicotine and previous phase ....................... 184 5 3 T and P values for the changes in number of infusions throughout each phase of treatment, compared with the first phase of nicotine and the previous phase ................. 184 5 4 T and P values for the changes in total number of pellets throughout each phase of treatment, compared with the first phase of nicotine and the previous phase ................. 185 5 5 T and P values for the changes in the total number of pellets during the dark cycle throughout each phase of treatment, compared with the first phase of nicotine and the previous phase ................................ ................................ ................................ .................. 185 5 6 T and P values for the changes in the total number of pellets during the light cycle throughout each phase of treatment, compared with the first phase of nicotine and the previous phase ................................ ................................ ................................ .................. 185 5 7 T and P values for the changes in the total number of meals throughout each phase of t reatment, compared with the first phase of nicotine and the previous phase ................. 186 5 8 T and P values for the changes in the total nu mber of meals during the dark cycle throughout each phase of treatment, compared with the first phase of nicotine and the previous phase ................................ ................................ ................................ .................. 186 5 9 T and P values for the changes in the total number of meals during the light cycle throughout each phase of treatment, compared with the first phase of nicotine and the previous phase ................................ ................................ ................................ .................. 186 5 10 T and P values for the changes in meal size throughout each phase of treatment, compared with the first phase of nicotine and the previous phase ................................ .. 187 5 11 T and P values for the changes in meal size during the dark cycle throughout each phase of treatment, compared with the first phase o f nicotine and the previous phase ... 187 5 12 T and P values for the changes in meal size during the light cycle throughout each phase of treatment, compared with the first phase of nicotine and the previous phase ... 187 5 13 T and P values for the changes in b ody weight gained throughout each phase of treatment, throughout the baseline, 2.5 cytisine, 5 cytisine, and extinction periods ....... 188

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10 5 14 T and P values for the changes in total number of pellets throughout each phase of treatment, throughout the baseline, 2.5 cytisine, 5 cytisine, and extinction periods ....... 188 5 15 T and P values for the changes in total number of pellets during the dark cycle, throughout the baseline, 2.5 cytisine, 5 cytisine, and extinction periods ......................... 188 5 16 T and P values for the changes in total number of pellets during the light cycle, throughout the baseline, 2.5 cytisine, 5 cytisine, and extinction perio ds ......................... 188 5 17 T and P values for the changes in total number of meals throughout each phase of treatment, throughout the baseline, 2.5 cytisine, 5 cytisine, and extinction periods ....... 189 5 18 T and P values for the changes in total number of meals during the dark cycle, throughout the baseline, 2.5 cytisine, 5 cytisine, and extinction periods ......................... 189 5 19 T and P values for the changes in total number of meals during the light cycle, throughout the baseline, 2.5 cytisine, 5 cytisine, and extinction periods ......................... 189 5 20 T and P values for the changes in total meal size throughout each phase of treatment, throughout the baseline, 2.5 cytisine, 5 cytisine, and extinction periods ......................... 190 5 21 T and P values for the changes in total meal size during the dark cycle, throughout the baseline, 2.5 cytisine, 5 cytisine, and extinction periods ................................ ........... 190 5 22 T and P values for the changes in total meal size during the light cycle, throughout the baseline, 2.5 cytisine, 5 cytisine, and extinction periods ................................ ........... 190

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11 LIST OF FIGURES Figure page 2 1 Flavor preference test vehicle v. high dose ................................ ................................ ..... 65 2 2 Vehicle v. nicotine individual data. ................................ ................................ ................. 66 2 3 Flavor preference test low v. high dose. ................................ ................................ .......... 67 2 4 Week one with or without flavor. ................................ ................................ ...................... 68 2 5 Initial preference effects. ................................ ................................ ................................ ... 69 3 1 Nicotine: body weight change over treatment and cessation. ................................ .......... 100 3 2 Nicotine: total number of pellets. ................................ ................................ ..................... 101 3 3 Nicotine: total pellets per day. ................................ ................................ ......................... 102 3 4 Nicotine: total pellets dark cycle. ................................ ................................ .................. 103 3 5 Nicotine: total pellets light cycle. ................................ ................................ .................. 104 3 6 Nicotine: total pellets per day light cycle. ................................ ................................ ..... 105 3 7 Nicotine: total meal size. ................................ ................................ ................................ .. 106 3 8 Nicotine: total meal size dark cycle ................................ ................................ ............... 107 3 9 Nicotine: total meal size light cycle. ................................ ................................ ............. 108 3 10 Nicotine: total number of meals. ................................ ................................ ...................... 109 3 11 Nicotine: total number of meals dark cycle ................................ ................................ ... 110 3 12 Nicotine: total number of meals light cycle. ................................ ................................ .. 111 3 13 Nicotine: total pellets .03 mg/kg/inf. ................................ ................................ ............. 112 3 14 Nicotine: total pellets dark cycle .03 mg/kg/inf. ................................ ......................... 113 3 15 Nicotine: total number of pelle ts light cycle .03 mg/kg/inf ................................ ........ 114 3 16 Nicotine: total number of pellets light cycle all doses ................................ ................ 115 3 17 Nicotine: total excess pellets ................................ ................................ ............................ 116 3 18 Nicotine: total excess pellets dark cycle. ................................ ................................ ....... 117

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12 3 19 Nicotine: total excess pellets light cycle ................................ ................................ ........ 118 3 20 Nicotine: total pellets day only ................................ ................................ ...................... 119 3 21 Nicotine: total number of meals day only ................................ ................................ ..... 120 3 22 Nicotine: Total meal size day only. ................................ ................................ ............... 121 4 1 Cytisine: body weight over treatment and cessation. ................................ ....................... 146 4 2 Cytisine: total pellets ................................ ................................ ................................ ........ 147 4 3 Cytisine: total pellets per day ................................ ................................ ........................... 148 4 4 Cytisine: total pellets dark cycle ................................ ................................ ................... 149 4 5 Cytisine: total pellets per day dark cycle ................................ ................................ ...... 150 4 6 Cytisine: total pellets light cycle ................................ ................................ ................... 151 4 7 Cytisine: total pellets per day light cycle ................................ ................................ ...... 152 4 8 Cytisine: total number of meals ................................ ................................ ....................... 153 4 9 Cytisine: total meals per day ................................ ................................ ............................ 154 4 10 Cytisine: total meals dark cycle ................................ ................................ .................... 1 55 4 11 Cytisine: total meals per day dark cycle ................................ ................................ ....... 156 4 12 Cytisine: total meals light cycle ................................ ................................ .................... 157 4 13 Cytisine: total meals per day light cycle ................................ ................................ ....... 158 4 14 Cytisine: total meal size ................................ ................................ ................................ ... 159 4 15 Cytisine: total meal size dark cycle ................................ ................................ .............. 160 4 16 Cytisine: total meal size light cycle. ................................ ................................ ............. 161 4 17 Cytisine: total number of excess pellets ................................ ................................ ........... 162 4 18 Cytisine: total excess pellets dark cycle ................................ ................................ ........ 163 4 19 Cytisine: total excess pellets light cycle. ................................ ................................ ...... 164 5 1 Body weight: comparisons with nicotine t reatments ................................ ....................... 191 5 2 Body weight: comparisons between treatments ................................ ............................... 192

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13 5 3 Infusions: comparisons with nicotine treatment ................................ .............................. 193 5 4 Infusions: comparisons with the previous treatment ................................ ....................... 194 5 5 Total pellets: comparisons with nicotine treatment ................................ ......................... 195 5 6 Total pellets: comparisons between treatments ................................ ............................... 196 5 7 Night pellets: comparisons with nicotine treatment ................................ ......................... 197 5 8 Night pellets: comparisons between treatments ................................ ............................... 198 5 9 Day pellets: comparisons with nicotine treatment ................................ ........................... 199 5 10 Day pellets: comparisons between treatments ................................ ................................ 200 5 11 Total meal number: comparison with nicotine treatment ................................ ................ 201 5 12 Night meals: comparisons with nicotine treatment ................................ .......................... 202 5 13 Night meals: comparisons between treatments. ................................ ............................... 203 5 14 Day meals: comparisons with nicotine treatment ................................ ............................ 204 5 15 Total meal size: comparisons with nicotine treatment ................................ ..................... 205 5 16 Total meal size: comparisons between treatments ................................ ........................... 206 5 17 Night meal size: comparisons with nicotine treatment ................................ .................... 207 5 18 Night meal size: comparisons between treatments ................................ .......................... 208 5 19 Day meal size: comparison with nicotine treatment ................................ ........................ 209 5 20 Day meal size: comparisons between treatments. ................................ ............................ 210 5 21 Body weight comparisons of cytisine treatments. ................................ ........................... 211 5 22 Infusions: comparisons of cytisine treatments.. ................................ ............................... 212 5 23 Total pellets: comparisons of cytisine treatment ................................ ............................. 213 5:24 Night pellets: comparisons of cytisine treatment ................................ ............................. 214 5 25 Day pellets: comparisons of cytisine treatment ................................ ............................... 215 5 26 Total meal size: comparisons of cytisine treatment ................................ ......................... 216

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14 5 27 Night meal size: comparisons of cytisine treatment ................................ ........................ 217 5 28 Day meal size: comparison of cytisine treatment ................................ ............................ 218

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15 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 RELATIONSHIP BETWEEN NICOTINE AND INGESTIVE BEHAVIORS IN RATS By Patricia E. Grebenstein August 2012 Chair: Neil Rowland Major: Psychology The experiments described in this dissertation examine the interactions between nicotine an d ingestive behaviors in rats The first experiment examines the effect of nicotine on flavor preferences In this experiment, rats were given noncontingent infusions of nicotine and ad libitum access to a flavored Kool Aid solution After two and three weeks, flavor preference tests were administered Various parameters of interest included changes in nicotine dose, flavor novelty, pretest preferences, and an initial week without the flavor presentation Animals preferred the flavor that they consumed while receiving the nicotine over a novel solution, saline infusions In light of evidence indicating that individuals who smoke cigarettes weigh less than thos e who do not, and smoking cessation results in increased weight gain compared to Rats that received noncontingent infusions of nicotine over 23 hours weighed les s and ate smaller meals in both the light and dark phases, with compensatory increases in meal number in the dark In the third experiment, rats received noncontingent infusions of cytisine, a selective t Administration of cytisine occurred over 23 hours and body weight and meal patterns were recorded In this experiment, animals weighed

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16 less and ate fewer meals in the light and dark phases without any changes in meal size, and these effects on body we ight persisted after administration of the drug was discontinued The final experiment examined the ability of cytisine to substitute for nicotine in a self administration protocol Animals self administer ed nicotine and cytisine on alternate four day pe riods. Cytisine increased total pellet intake with respect to nicotine and animals self administered cytisine significantly less than the nicotine There were no differences between cytisine treatment and the cessation phase for meal patterns and body we ight change. Cytisine maintained decreased food intake and w eight gain compared to baseline through decreases in size from the second experiment were replicate d.

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17 CHAPTER 1 BACKGROUND INFORMATI ON ON NICOTINE AND I NGESTIVE BEHAVIORS Prevalence of Cigarette Smoking Cigarette smoking is the leading cause of preventable death among the 45 million adult smokers in the United States and harms nearly every organ in the body Approximately 1 in every 5 deaths is in the United States is smoking related, with around 440,000 people prematurely dying every year (CDC, 2002) While the number of adults who smoke has decreased over the past 2 3 decades in the United States the number of teens who engage in smoking has not Likewise smoking is becoming a much bigger problem in developing countries where over 40% of people smoke, particularly males (Benowitz 2008a) It is estimated that over 5 million deaths occur worldwide every year and that this number will increase to over 10 million annually in 30 40 years (Benowitz 2008a; Peto et al. 1996) Smoking in psychiatric populations has increased as well, particularly in people with schizophrenia of which around 90% u se tobacco (Dalack et al. 1998). Despite the health risks associated with smoking cigarettes, people continue to engage in this behavior Although over 70% of individuals who smoke express a desire to qu it, only 3% of them are successful over a long ter m period (Shiffman et al. 1996 ) Around half of smokers attempt to cut do wn on daily intake (West and Shiffman 2001), and approximately 80% of people who attempt to quit on their own, using various methods available, relapse within the first month (Hughes et al. 1992). The success rates of the different smoking interventions available range from 10 30% for the first 6 months to a y ear (Hughes 2009; Silagy et al. 2007; West and Shiffman 2001), with the aver age success rate for different nicotine replacement therapies at around 17% compared with 10% in control groups (Silagy et al 2007) Combining therapies

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18 ch added b enefit (Silagy et al. 2007). The Role of Cues in Nicotine Self Administration Nicotine itself can serve as a weak primary reinforcer, and causes the release of many neurotransmitters and long term changes in brain reward regions (Koob and Volkow 2009) N icotine is an agonist at nicotinic acetylcholine receptors (nAChRs), ligand gated ion channels located peripherally and centrally (Markou and Paterson 2009) Moderate to heavy smokers are estimated to have plasma levels of nicotine that result in complete saturation of the high affinity Though people smoke in part to avoid withdrawal symptoms perpetuated by long term receptor changes, this is only one of the reasons p eople smoke. Given the near complete nAChR occupancy from nicotine, a separate mechanism may be maintaini ng the behavior (Balfour et al. 2000; Benowitz et al. 2008b). The initial dopamine release that occurs is thought to influence communication between areas of the brain involved in encoding the rewarding prop erties of a stimulus (Di Chiara 2000), and there is mounting evidence to suggest that the stimuli associated with nicotine are a crucial component in maintaining the drug seeking behavior. The neur smoking and relapse has also been studied extensively For example, when presented with smoking cues (ashtrays, cigarettes, pictures of people smoking etc.) people smoke their firs t cigarette sooner and take longer puffs (Payne et al. 1991) The effect of cues on craving and relapse seem to be more important in women than men, particularly for olfactory and taste stimuli which seem to be more reinforcing for women Women are also less able than men to identify the changes in nicotine levels in cigarettes and place more emphasis on cues when rating smoki ng satisfaction (Perkins et al. 2001 a ) Brain imaging studies have been used to evaluate

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19 the effect of cues on brain activation in smokers These studies demonstrate enhanced activity in areas like the amygdala, ventral striatum, thalamus, hippocampus, orbitofrontal cortex, and dorsolateral prefrontal cortex, indicating a role for the emotional significance of the cues presented ( Fr anklin et al. 2007). Cues seem to have the greatest effect on brain activation when associated with an expectancy to smoke and dr ug availability (McBride et al. 2006). Rats acquire self administration behavior more quickly and move up in reinforcement sc hedules (from FR1 to FR5) when the nicotine self administration ( NSA ) is paired with some type of stimulus such as a light cue without which they self administer nicotine at much lower levels (Cohen et al. 2005) These cues are then able to maintain respo nding when nicotine is removed, and support high levels of responding during reinstatement after periods of withdraw al and extinction (Cohen et al. 2005; Liu et al. 2007) These effects have been blocked with drugs such as the cannabinoid receptor anta gon ist rimonabant (Cohen et al. 2005), the nonselective nAChR anta gonist mecamylamine (Liu et al. 2007), the GABA B receptor antag onist CGP44532 (Paterson et al. 2005), the D 3 receptor antag onist BP 897 (Le Foll et al. 2003), the mGlu5 receptor antagonist MEP and mGlu2/3 receptor agonists (Markou and Paterson 2009), indicating a role for a variety of systems in the integration of cues with nicotine administration. Recent research by several different groups has underlined the relative importance of stimuli as sociated with nicotine use For example, there seems to be an increase in an imuli (Caggiula et al. 2002) Similarly, people who are presented with environmental stimuli which are associated wi th smoking are more likely to relapse and experience increased craving for the drug (Conklin et al. 2002) When people smoke at a high enough level that the relevant nAChRs are desensitized, it is most likely the conditioned reinforcers within the cigaret te smoke that maintain the behavior

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20 Several studies have examined human smoking behavior in the presence of smoking cues without nicotine as well as in the presence of nicotine without smoking cues The presentation of cues to smokers while they are not allowed to smoke results in increased craving (Caggiula et al. 2001) Blocking cues on the other hand reduces satisfaction For example, anesthetizing the respiratory airway but allowing people to smoke leads to reduced liking and this effect can be see n when olfactory cues are blocked ( Westman et al. 1996). Research on nicotine and its relationship with different stimuli usually pair infusions of nicotine with a compound visual stimulus (CVS) and examine the effect of the CVS on the acquisition and mai ntenance of nicotine self administration The CVS is typically composed of a house light which shuts off and a dim cue light above the response device which turns on (i.e. Donny et al. 1995) understood or agreed upon The traditional view is that the pairing of the nicotine with various stimuli occurs through the typical Pavlovian method of conditioning The cue itself which is relatively neutral, becomes associated with the reinforcing effect of nicotine which then leads to an increase in responding for both the cue and nicotine (Caggiula et al. 2002; Chaudhri et al. 2006 b; Raiff et al. 2006) An alternate theory sug gests that nicotine has a reinforcement enhancing effect on other concurrent stimuli as well Thus, when nicotine is administered noncontingently, rats press at much higher levels for the slightly reinforcing nonpharmacological stimulus (a light cue) (Don ny et al. 2003). As re viewed in Chaudhri et al. (2006 a ), evidence seems to indicate that the strength of a stimulus and the degree that it is reinforcing helps to determine which property of nicotine dominates in the facilitation of self administration Thus, responding for nonpharmacological stimuli which are moderately to strongly reinforcing will be acquired at greater rates in the

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21 presence of nicotine because of the reinforcement enhancing properties of nicotine On the other hand, nonpharmacological stimuli which are weak primary reinforcers will be potentiated by the primary reinforcing actions of nicotine, enabling the weak stimulus to become a conditioned reinforcer According to this view, contingent nicotine increases responding for weaker non pharmacological stimuli, whereas non contingent nicotine can be seen to elevate responding for already reinf orcing stimuli (Chaudhri et al. 2006 b; Chaudhri et al. 2007 ) Similarly, increasing the reinforcing property of a stimulus (i.e. by previously pai ring it with sucrose) enhances the effect nicotine has on responding for that stimulus (Chaudhri et al. 200b ). Rats also show sex differences that corroborate the clinical observation s that women are more sensitive to nicotine associated cues (Perkins et al. 2001 a ) Female rats show a greater rate of responding when nicotine and a visual stimulus are paired together, indicating that female rats either show greater d effects of the nicotine plus v isual stimulus (Chaudhri et al. 2005). The Relationship between Nicotine Abuse and Overweight/Obesity In 2000, the two leading causes of mortality in the United States were smoking and obesity, with 435,000 deaths from tob acco and 400,000 deaths related to poor diet and physical inactivity (Mokdad et al. et al. 2004) Estimated rates of obesity in the United States from the National Health and Nutrition Examination Survey (NHANES) collected in 2007 2008, show a ~34% preval ence of age adjusted obesity and a prevalence of ~68% for obesity and overweight combined (Ogden & Carroll 2010) The prevalence of obesity and overweight in the United States has been steadily increasing since 1976 in nearly all age groups and across sex es (Flegal et al. 2010) Within the last 10 years, the per person medical costs for obesity have been estimated between $1700 and $2700; the aggregate national cost of overweight and obesity combined cost anywhere from $113.9 billion to 209.7 billion (Tsa i et al. 2011; Cawley and Meyerhoefer 2012)

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22 These astronomical health costs reflect both the growing trend in obesity and the associated medical and cost burdens, with somewhere between 5% and 10% of US healthcare spending delineated to obesity related h ealth issues (Tsai et al. 2011). One mechanism that may be behind the rise in obesity is the negative impact smoking has on general health Obese smokers have a lower life expectancy (<13 years) than normal weight nonsmokers, and have double the decline i n life expectancy compared with people who are either obese or are smokers, but not both (Peeters et al. 2003) People who smoke cigarettes are at increased risk for a number of disorders that are related to and/or are exacerbated by overweight and obesit y, including coronary heart disease ( Miyazaki et al. 2003 ), insulin resistance (Jensen et al. 1995; Chiolero et al. 2008), and type II diabetes (Eliasson 2003) Adiponectin, a protein secreted by fat cells that helps to mediate lipid and glucose metabolis m, is lower in people who have coronary artery disease, those who are obese, and people who smoke cigarettes, and is also associated with insulin resistance (Miyazaki et al. 2003 ) There is some evidence to suggest that heavier smokers might be more overw eight than light smokers, with the metabolic advantages of smoking being overwhelmed by other important factors (Albanes et al. 1987; Chiolero et al. 2008) who repeatedly try to quit, relapse, and subsequently gain and lose weight depending on their smoking status, which may then increase the risk for overweight or obesity (Ch iolero et al. 2008) Increased percentages of visceral fat in smokers may then subsequently confer an increased risk for cardiovascular disease via a differential release of hormones (Audrain McGovern and Benowitz 2011) There is also evidence to suggest that smoking decreases insulin sensitivity and that smokers have higher insulin levels than nonsmokers, ( as reviewed by Wack and Rodin 1982 and Audrain McGovern and Benowitz 2011), whereas smoking cessation

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23 improves insulin sensitivity ( as reviewed by Pis telli et al. 2009) Similarly, smoking increases insulin resistance in people with type II diabetes (as reviewed by Filozof et al. 2004) All of these factors are associated with the increased prevalence of overweight and obesity. Another mechanism that might contribute to the increased prevalence of obesity is the relative decline in cigarette smoking among the adult population From 1976 to 1991, while the prevalence of obesity was increasing, the rates of smoking among adults were steadily decreasing (Flegal et al. 1995) About 25% of the inc rease in overweight in men and 15% of the increase in overweight in women could be linked to smoking cessation (Flegal et al. 1995) Many studies have shown that nicotine results in decreased weight gain (Schec hter and Cook 1976; Grunberg and Bowen 1985; Albanes et al. 1987; Klesges and Meyers 1989; Flegal et al. 1995; Swan and Carmelli 1995) and that people who quit smoking (and relevant animal models) weigh more than current smokers (Schechter and Cook 1976; R odin 1987; Grunberg et al. 1985; Klesges and Meyers 1989; Flegal et al. 1995; Swan and Carmelli 1995; Pistelli et al. 2009) Overweight and obesity occurs more frequently between 45 64 years of age, during the period when people are most likely to quit sm oking as well In people over 50 years old, 27% of overweight women and 27% of obese women are former smokers, and 44% of overweight men and 48% of obese men are former smokers (Audrain McGovern and Benowitz 2011) A comprehensiv e literature review by Kl esges and Meyers (1989) examined the relative weight loss among smokers and the relative weight gain among quitters 83% of reviewed studies showed that smokers weigh on average 7.75lb less than nonsmokers, with moderate smokers weighing the least and hea vy smokers weighing approximately the same as nonsmokers Women have a greater weight loss advantage from smoking than do men In terms of weight gain following cessation, 76% of the reviewed articles provided evidence to suggest that those

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24 who quit smok ing weighed on average 6.40lb more than nonsmokers, with heavier smokers and women gaining the most weight after quitting Although some studies have not shown these effects (Albanes et al. 1987), many studies have replicated these weight changes and have sought to parse out what relative factors might predispose individuals to gain weight after cessation, which mechanisms might be involved in this weight gain, and how information about weight gain might be able to influence treatments for smoking cessatio n. Factors Associated with Weight Loss during Smoking There are several mechanisms that could account for the decrease in body weight observed in conjunction with nicotine use As an individual smokes larger quantities of cigarettes, there may also be a r elative decrease in appetite and food intake ( as reviewed in Chiolero et al. 2008; Grunberg et al. 1985; Grunberg et al. 1986) However, not all studies on humans have shown this effect ( as reviewed in Wack and Rodin 1982 and Klesges and Meyers 1989; Sche chter and Cook 1976; Rodin 1989) with some studies even showing elevated food consumption in smokers ( as reviewed in Klesges and Meyers 1989) There also seems to be an increase in the waist hip ratio in smokers, which may be accounted for by a decrease i n hip circumference in conjunction with the weight loss seen in smokers (Jensen et al. 1995; Filozof et al. 2004) In contrast, a large proportion of the studies in rats that have received chronic nicotine infusions have reported a decrease in food consum ption compared to those that received saline (McNair and Bryson 1983; Bowen et al. 1986; Miyata et al. 1999) This inverse dose response relationship between nicotine and food consumption is stronger in heavy smokers and/or high daily doses of nicotine (M cNair and Bryson 1983; Grunberg et al. 1986) though the effect has been seen in some experiments with lower doses (Bowen et al. 1986), and with moderate doses in some human studies (Albanes et al. 1987).

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25 Another possible mechanism for the weight loss seen during nicotine administration is a dose dependent change in physical activity Several studies have found increases in physical activity in animals (Grunberg and Bowen 1985; Bowen et al. 1986) whereas other have not seen an effect of nicotine on activit y levels ( as reviewed by Klesg es and Meyers 1989; Albanes 1987 ) Effects on physical activity have also been reported in some (Rodin 1987) but not all human studies, with subjects who stay the same weight or lose weight reporting more physical activity th an those who gain weight while smoking. Sex differences have also been reported in animal studies, with males but not females showing increased levels of physical activity during nicotine administration (Grunberg et al. 1986). Factors Associated with Wei ght Gain during Smoking Cessation The fact that weight loss occurs in many people who smoke leads to the notion that people who stop smoking should therefore gain weight There is a considerable amount of evidence showing that this is indeed the case (see section on The Relationship between Nicotine Abuse and Overweight/Obesity ), with approximately 80% of smokers who quit gaining weight ( as reviewed by Pistelli et al. 2009 ) and only about 25% of ex smokers able to maintain a healthy body weight ( as reviewed by Audrain McGovern and Benowitz 2011) Weight gain is typically the greatest in the first couple of months after cessation, but continues for up to two years ( as revie wed by Klesges and Meyers 1989; Flegal et al. 1995; Audrain McGovern and Benowitz 2011; Kamaura et al. 2011). Several studies have attempted to parse apart which factors and characteristics might explain the relative variability of weight gain in ex smoke rs Some of the most important variables that have an impact on post cessation weight gain are age, sex, and race Other factors that influence weight gain after smoking cessation include socioeconomic status ( as reviewed by Pistelli et al. 2009 and Filo zof et al. 2004; Swan and Carmelli 1995), and marital status, with

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26 increased weight gain associated with a single status ( as reviewed by Gritz and Jeor 1992; Swan and Carmelli 1995). Sex differences: Much evidence supports higher weight gain in female th an male ex smokers, an effect that seems at least partially mediated by actual biological differences in the effects of smoking on body weight (Grunberg et al. 1986) These differences may be due to how men and women differ in their metabolism of nicotine with female smokers showing decreased clearance of nicotine ( as reviewed by Grunberg et al. 1986) Some animal studies have found body weight changes in female rats but not in male rats after chronic administration of a high dose of nicotine (Grunberg e t al. 1986) Human studies have shown increases in weight gain among women who have quit smoking compared with men, with women also showing a larger percentage of extreme weight gain ( as reviewed by Pistelli et al. 2009 and Filozof et al. 2004). Beliefs about weight: As reviewed in Pistelli et al. (2009), there are several weight related smoking variables that characterize smokers who are concerned about weight, including: beliefs eight, withdrawal weight gain, weight gain as an obstacle to quitting, and a preoccupation with body image Many of these variables differ between men and w omen ( as reviewed by Perkins et al. 2001 b ; Gritz and Jeor 1992; Camp et al. 1993) Females are more likely to agree that smoking can help them control their body weight, and approximately 39% of women report smoking to control their weight compared to onl y 12% of men (Camp et al. 1993) ability to control post cessation weight gain increases the ability to successfully quit smoking, particularly in men (Jeffery et al. 2000) About 53% of women smokers worry about their we ight compared with 31% in nonsmokers Overweight women (20%) start smoking for weight

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27 related reasons more than normal weight women (2%; as reviewed by Klesges and Meyers 1989) Women (39%) are also more likely than men (25%) to recommend smoking as an a ctive weight loss strategy, less likely to endorse the health benefits of quitting, and are also more likely to relapse for weight related reasons than men (20% and 7% respectively; as reviewed by Klesges and Meyers 1989), making them less likely to attemp t to quit in the first place (Jeffery et al. 2000). Women (26%) report excessive concern about weight gain following quitting more often than men (7%), and conversely men (38%) report no concern for weight gain after cessation more often than women (14%) This lack of concern about post cessation weight gain is related to greater success with quitting smoking in both men and women (Jeffery et al. 2000). Age: Age is a factor in weight gain after smoking, with people younger than 55 at risk for gaining mor e weight than older adults ( as reviewed by Pistelli et al. 2009; Swan and Carmelli 1995) and aging smokers failing to gain as much weight as aging nonsmokers ( as reviewed by Klesges and Meyers 1989) Adolescents who report smoking initiation and maintenan ce with the purpose of weight control or loss and those who are trying to lose weight are 40% more likely to start smoking ( as reviewed by Audrain McGovern 2010) In a survey of 1600 adolescent smokers, the heaviest smokers were most likely to agree that smoking is an effective control of weight gain ( as reviewed by Klesges and Meyers 1989). Race: Race is a determinant of post cessation weight gain and smoking to l ose weight About 45% of Caucasian adolescents believe smoking can help control their weight, whereas African American girls (10%) are the least likely group to believe this is true; Caucasian boys (~30%) also believe smoking can help with weight control more than African American boys (~14%; Camp et al. 1993) Only Caucasian smokers reported smoking in order to control weight gain, with one study not finding a single African American person, regardless of sex, who

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28 reported smoking for this reason (Camp e t al. 1993) However, despite these racial differences in reasons for smoking initiation, African American people are at a higher risk for extreme weight gain as compared to Caucasians ( as reviewed by Filozof et al. 2004 and Gritz and Jeor 1992; Swan and Carmelli 1995 ) Weight control does not seem to be a factor in the initiation of smoking in Hispanic women or Asian Americans (Gritz and Jeor 1992) The rate of obesity is high in Native Americans, as is the prevalence of smoking in various tribes, putt ing this particular group at increased risk for post cessation weight gain (Gritz and Jeor 1992). Nicotine dose: The average daily dose of nicotine plays an important role in the magnitudes of weight loss while smoking and weight gain during cessation R ats given the highest doses of nicotine lose the most weight (Schechter and Cook 1976; Grunberg et al. 1985 and 1986) and subsequently gain the most weight after c essation (McNair and Bryson 1983 ; Bowen et al. 1986; Grunberg et al. 1986 and 1985; Flegal et al. 1995) Over 70% of reviewed studies reported increased weight gain after smoking cessation in the heaviest smokers ( as reviewed by Klesges and Meyers 1989 ) Many (73%) of the reviewed studies suggest a curvilinear relationship between nicotine dose and weight change, with moderate smoking producing the greatest relative weight loss while, paradoxically, heavy smokers behave more like nonsmokers ( as reviewed by Klesges and Meyers 1989; Albanes et al. 1987). This dose dependent effect may be mo dulated by two possible factors: differences in food intake and changes in energy expenditure, which highlight a differential level of physical activity or metabolism in moderate compared to heavy smokers This idea of energy balance, determined by caloric intak e, energy expenditure through physical activity, and metabolic effects, underlies the nicotine induced changes in weight gain Heavier smokers seem to adopt more unhealthy eating habits than do light smokers, eating fewer fruit s and vegetables and more ca ndy, drinking

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29 more alcohol and coffee, and engaging in less physical activity ( as reviewed by Chiolero et al. 2008; Swan and Carmelli 1995) The difference in activity level, if it continues post cessation, will then lead to increased weight gain (Filozof et al. 2004; Saules et al. 2004; Audrain McGovern and Benowitz 2011) Some evidence suggests that heavy smokers may also eat more than li ght smokers which might also may underlie their greater weigh t gain after cessation (Rodin 1987; Saules et al. 2004). Physical activity: In light of evidence that smokers may in fact be consuming equal or increased amounts of food as compared to nonsmokers, it is possible that the weight gain seen in ex smokers may be in part attributable to a change in physical activit y, similar to that seen in the weight loss of smokers (Rodin 1987; Swan and Carmelli 1995) In a 1987 study by Rodin et al., individuals who did not report weight gain after smoking were more active while smoking than those who ended up gaining weight and also reported no change in or increase of physical activity while smoking Those that gained weight showed either low physical activity or a decrease in physical activity, illustrating a role for physical activity in the relative weight gain seen by peop le who stop smoking This variability in physical activity may partially account for the inconsistencies in studies examining whether or not people gain weight after smoking However, a large number of studies have not found a difference in physical acti vity between smokers and ex smokers ( as reviewed by Klesges and Meyers 1989; Albanes et al. 1987; Jensen et al. 1995; Flegal et al. 1995) Some animal studies also report decreased physical activity during nicotine cessation and estimate the relative cont ribution of physical activity on weight gain to be around 9% (Grunberg and Bowen 1985) Food intake: Smoking cessation may be accompanied by a change in food intake The animal literature has been able to parse this effect out relatively well, with many studies showing

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30 a change in the ingestive behavior of animals after nicotine administration is over Compared to nave controls, animals that received infusions of ni cotine in the past gain more weight (Bowen et al. 1986; Miyata et al. 1999) Meal pattern analysis shows that rats increase meal size relative to controls after nicotine, suggesting an effect of nicotine removal on satiety (Miyata et al. 1999) This effe ct may however, be modulated by sex, food preference, and dose Some studies have found a change in food intake in female rats but not male rats (Bowen et al. 1986), while others have observed changes for males but not femal es (McNair and Bryson 1983 ) T he weight gain seen in both animals and people during cessation seems to be the greatest with the highest doses of nicotine, indicating a positive relationship between smoking rate and weight change ( as reviewed by Klesges and Meyers 1989 and Pistelli et a l. 2009; Schechter and Cook 1976; McNair and Bryson 1982; Grunberg et al. 1985; Rodin 1987) One study in people quitting smoking found an average increase in intake of 227 calories per day, which explains roughly 69% of the weight gain after 3 months of abstinence ( as reviewed by Audrain McGovern and Benowitz 2011) This weight gain after cessation brings ex of nonsmokers, indicating a return to age projected body weights ( as reviewed by Redington 1984 and Audrain McGo vern and Benowitz 2011; Albanes et al. 1987) This increase in food consumption supports the oral gratification hypothesis, which states that people may eat more when they quit smoking as a substitute for cigarettes (Wack and Rodin 1982) Here, one rewar ding stimulus is substituted with another Conversely, it is also possible that since smoking naturally excludes the possibility of eating at the same time, smokers may be replacing their old smoking behavior with the interchangeable option of eating, eff ectively serving as an activity which substitutes nicotine use throughout a period of time (Wack and Rodin 1982)

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31 Food preferences: Both human and animal studies have examined changes in food preference post nicotine There is mounting evidence that an inc rease in preference for sweet and high carbohydrate foods accompanies nicotine cessation, whereas smoking cigarettes is associated with a decrease in the consumption of sweet foods (Saules et al. 2004) Ex smokers that increase their carbohydrate intake a nd decrease their protein intake seem to be the ex smokers who are gaining the most weight, whereas those ex smokers that remain the same weight or show weight loss decrease the percent of their total calories that is attributed to carbohydrates, an effect that seems particularly relevant for females (Rodin 1987) In an effort to parse out the importance of sweet taste, calorie content, and nutritive content on this effect, one study examined the effects of chronic nicotine administration in rats and measu red a change in preference for various foods compared to standard chow during and after drug delivery Compared to controls, they found that animals that had previously received nicotine ate more of sweet high or low calorie foods than the regular chow, i ndicating an important role for sweetness in changes in dietary intake in ex smokers (Grunberg et al 1985) One possible mechanism behind decreased sweet consumption in the presence of nicotine and increased sweet consumption during cessation may be nico Nicotine may decrease sweet consumption because of increased glucose availability and cessation may result in increased sweet consumption due to decreased glucose availability (Redi ngton 1984 ; Grunberg et al. 1985), as decreased liking for sweet solutions in smokers follows a glucose load (Redington 198 4 ) This change in the relative pleasantness of sweet tasting foods has been seen in other studies as well (Bowen et al. 1991; Spring et al. 2003) Nicoti ne may also increase the in between snacking of meals, typically composed of

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32 Another possible mechanism behind changes in dietary intake may be a change in the perceived intensities of various foods, though research in support of this has been relatively inconsistent (Pursell et al. 1973; Redington 198 4 ; Rodin 1987 ) Hunger and food consumption have been negatively associated with increasing doses of nicotin e, whereas satiety and fullness are positively associated with nicotine ( as reviewed by Chiorelo 2008) Smokers often begin a smoking episode right after the completion of a meal, so it is possible when they quit smoking that a learned marker of meal ter mination has been lost, and as a result meals are larger (Wack and Rodin 1982) It is also possible that withdrawal from nicotine results in a reward deficiency that subsequently increases the desire for readily available rewards including snacks, without a change in the pleasantness or hunger for the particular food In this case, carbohydrates would substitute for the missing nicotine reward, which would then activate the reward circuits in the brain that were similarly activated by nicotine and bring p leasure to the ex smoker (Spring et al. 2003; Audrain McGovern and Benowitz 2011) A study in humans illustrated a change in the reward value of carbohydrates in female smokers withdrawing from nicotine In this study, females in withdrawal were willing to work harder for carbohydrate snacks relative to money, and earned an additional 5 6% increase in calories (100 300 kcal) per day from these snacks compared with nonsmokers, without a corresponding hedonic or intensity change (Spring et al. 2003). Change in carbohydrate intake: The increase in consumption of carbohydrate rich food in ex smokers may be in part related to serotonin High carbohydrate, low protein diets increase the synthesis of serotonin in the brain via an increase in tryptophan (Bo wen et al. 1991) Nicotine withdrawal is characterized by cravings, difficulty concentrating, restlessness and mood disturbances including depression, anxiety, anger, irritability and frustration ( as reviewed by

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33 DiFranza et al. 2000) There is some evide nce to suggest that in people who crave carbohydrates, consuming carbohydrates alleviates negative affect ( as reviewed by Bowen et al. 1991) During the first week of smoking cessation, people reported less withdrawal related anxiety when taking tryptopha n and consuming a high carbohydrate diet, providing additional support for the hypothesis that this effect is mediated by serotonin (Bowen et al. 1991). Disordered eating: The variability of weight gain among ex smokers is influenced by an oncern with body weight and eating habits People who express features of disordered eating, including binge eating and eating to reduce negative affect, are more likely to smoke for weight control reason ( as reviewed by Saules et al. 2004) Higher rates of weight gain and increased restrained eating scores are seen in bingeing rather than non bingeing overweight ex smokers (Camp et al. 1993; Saules et al. 2004; White et al. 2010). Individuals who reported two or more bingeing episodes per week were also more likely to report weight gain than those with fewer episodes (White et al. 2010) Over three quarters of the participants in the study reported that they began binge eating before they quit smoking, while about 20% reported the start of binge eating episodes after smoking cessation (White et al. 2010) Thus, the majority of ex smokers who are bingeing were doing so before cessation, but cessation can also induce binging in a minority and may exacerbate binging in those who have previously binged. A weight post cessation, defined as gaining 13kg or more over a 10 year period ( as reviewed by Pistelli et al. 2009) Approximately 10 21% of individuals who quit smoking fall in to this subgroup ( as reviewed by Pistelli et al. 2009; Swan and Carmelli 1995; Williamson et al. 1991; Flegal et al. 1995) These individuals are more likely to be African American <55 yrs old (Williamson et al. 1991), heavier smokers (Williamson et al. 1 991; Flegal et al. 1995) and of

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34 lower socioeconomic status They also exercise less strenuously and drink more coffee and liquor (Swan and Carmelli 1995) Additionally there appears to be a genetic component involved in whether or not someone gains exces sive weight after smoking cessation, with concordance rates for weight gain at 53% in monogygotic twins and 38% in dizygotic twins (Swan and Carmelli 1995). Metabolism: The relatively inconsistent evidence implicating either physical activity or food con sumption as factors in weight gain during smoking cessation suggest and additional role for metabolic changes due to nicotine Nicotine seems to effect metabolic pathways by causing food derived metabolites to stay in the blood stream longer, where they are then subsequently either used or excreted (Wack and Rodin 1982) This metabolically inefficient effect is supported by the mobilization of lipid deposits (Hersch et al. 1962) and the subsequent increase in free fatty acids in the bloodstream (Hersch et al. 1962; Wack and Rodin 1982; Hellerstein et al. 1994) Nicotine also seems to affect body weight by increasing the release of catecholamines and sympathetic activity, and increased thermogenesis ( as revi ewed by Hellerstein et al. 1994; Wack and Rodin 1982) Evidence for this thermogenic effect includes a 10% increase in oxygen consumption 45 minutes after smoking and oxygen consumption increases after smoking cessation ( as reviewed by Wack and Rodin 1982 and Hellerstein et al. 1994) This corresponds to the expenditure of approximately 200 kcal per 24 hours, which would result in the loss of 10 kg over the course of one year (Audrain McGovern and Benowitz 2011) Conversely, upon smoking cessation metabo lism becomes more efficient, more energy is stored, and weight is gained (Wack and Rodin 1982). At a population level, 75% of total energy expenditure is in the form of basal metabolism well established

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35 (Hellerstein et al. 1994). For example, female ex smokers have been shown to have a resting metabolic rate that is 16% lower than females who are currently smoking ( as reviewed by Chiolero et al. 2008) Further, animal studies have show n that nicotine increases resting metabolism in rodents receiving cigarette smoke ( as reviewed by Klesges and Meyers 1989) This measurement of basal metabolism has been criticized, however Since most people spend the majority of their time in casual ac tivity throughout the day, a measurement of metabolic rate while fasting and at rest may not accurately assess the metabolic effects of nicotine under everyday conditions In light of this, Perkins et al. (1994) examined energy expenditure after smoking d uring light physical activity, and found energy expenditure was greater in activity as opposed to rest after smoking. lipase, an enzyme important in the uptake and stor age of free fatty acids ( as reviewed by Klesges and Meyers 1989 and Chiolero et al. 2008). Increased lipolysis decreases the storage of triglycerides in adipose tissue ( as reviewed by Jo et al. 2002) and increases the thermogenic effects of nicotine ( as r eviewed by Audrain McGovern and Benowitz 2011) Gluteal and abdominal activities of lipoprotein lipase are increased in women who have quit smoking (Ferrara et al. 2001) which corresponds to evidence that smoking alters lipogenesis and might lead to incre ased weight in subcutaneous regions of the body (Jensen et al. 1995) Fat oxidation is positively correlated with plasma nicotine levels (Jensen et al. 1995) with smokers seeming to use more lipids to maintain fasting resting energy expenditure This may lead to disproportionate lipid intake when smokers stop smoking, and thus an increase in body weight (Jensen et al. 1995; Filozof et al. 2004).

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36 Another variable that affects metabolism is insulin. Acutely, nicotine lowers insulin levels and thus increase s blood levels of glucose, possibly impeding metabolism and making the metabolic system less efficient ( as reviewed by Klesges and Meyers 1989) However, chronic smoking is associated with increased plasma insulin levels and eventually insulin resistance, which may then account for weight gain and increased visceral adipose tissue ( as reviewed by Filozof et al. 2004 and Chiolero et al. 2008) These differences in acute and chronic nicotine use have been replicated ( as reviewed by Audrain McGovern and Beno witz 2011) Fasting plasma glucose seems to be higher in ex smokers than current smokers and nonsmokers, a change that may last for up to two years and parallels the changes in body mass index that accompany smoking cessation (Kamaura et al. 2011) Lastl y, plasma adiponectin may also contribute to Plasma adiponectin is typically lower in obese subjects and is associated with insulin resistance, and there is evidence to indicate that concentrations of adiponectin are low er in smokers than nonsmokers ( as reviewed by Filozof et al. 2004). Nicotine also increases leptin binding and receptor sensitivity ( as reviewed by Audrain McGovern and Benowitz 2011) There is some evidence to suggest that the decrease in food intake du ring nicotine use is caused by increased levels of leptin ( as reviewed by Jo at al. 2002 ) However, other evidence suggests that plasma leptin levels are lower in smokers compared to nonsmokers ( as reviewed by Filozof et al. 2004) Another peptide involv ed in ingestive behavior, NPY, may also be affected by nicotine There is evidence to suggest that nicotine decreases NPY expression in animals and therefore inhibits food intake, although support for this is mixed ( as reviewed by Filozof et al. 2004). T the limbic system which integrates and receives inputs from signals arising from the periphery

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37 including adipose tissue, the liver and pancreas, and the gastrointestinal tract ( as reviewed by Jo et al. 2002) Peripheral satiety signals from the liver and gut send information to the nucleus of the solitary tract (NTS) in the brainstem and from there are sent to the hypothalamus, while the information about fat storage acts directly on the hypothalamus ( as reviewed by Jo et al. 2002) The hypothalamus is made up of many different nuclei, including areas specific to ingestive behavior. The arcuate nucleus is a major area for the production of NPY, galanin, and orexigenic peptides There are two populations of cells within the arcuate nucleus, one that increases food intake and another that decreases food intake As reviewed by Jo et al. ( 2002 ) and Meister et al. ( 2006 ) the agouti related peptide (AgRP) is co expressed with NPY mRNA in this area, and both of these neuropeptides are involved in the initiation of food intake Melanocortins, including alpha melanocyte stimulating hormone ( MSH), are cleaved fro m the pro opiomelanocortin (POMC) precursor molecule, bind to melanocortin receptors, and are involved in suppression of food intake (Bellinger et al. 2003 a ) Not only are the pro feeding peptides responsible for promoting food intake, they also interact with the satiety signals by inhibiting those messages, and vice versa The cocaine and amphetamine regulated transcript (CART) has an opposing action to NPY induced feeding in rodents, and is co localized with POMC neurons in an area of the arcuate nucle us ( as reviewed by Jo et al. 2002 and Meister et al. 2006) AgRP is an antagonist for alpha MSH, and therefore initiates food intake by inhibiting the inhibitory role of alpha MSH brain barrier and so can receive blood borne chemical signals d irectly, including leptin and insulin A large proportion of NPY/AgRP and POMC/CART neurons express leptin receptors ( as reviewed by Jo et al. 2002 and Meister et

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38 al. 2006). The arcuate nucleus therefore is structured specif ically to regulate food intake via opposing mechanisms (Meister et al. 2006). The lateral hypothalamus (LH) is another area within the hypothalamus that is important for the regulation of food intake, and as reviewed by Jo et al. 2002, expresses orexin (a lso known as hypocretin) and melanin concentrating hormone (MCH) A subset of cells in this area produces protein prepro orexin for o rexins A and B (Kane et al. 2001 ) The orexin neurons from this area synapse onto NPY neurons in the arcuate nucleus and stimulate feeding, whereas leptin has the opposite effect on NPY neurons and decreases feeding MCH is normally a signal which initiates food intake, and increased CART/alpha MSH and increased GABAergic input can cause a decrease in MCH, and therefore dec reased food intake. In rats, measurements of daily food intake are further analyzed by looking at meal size and meal number (total food intake = meal size x meal number) It follows that some of the neurotransmitters in the brain are involved in the dete rmination of either meal size or meal number through the regulation of feeding related peptides Meguid et al. ( 2000 ) and Miyata et al. ( 1999 & 2001 ) examined the role of dopamine and serotonin in meal patterns, and found that dopamine effects meal number by acting on alpha MSH/CART neurons and meal size via actions on NPY/AgRP. Nicotine may therefore be exerting its effects on food intake in the various areas of the hypothalamus through changes in neurotransmission and levels of neuropeptides Nicotine h as specific effects on meal patterns, effecting changes in both meal number and meal size ( as reviewed by Miyata et al. 1999 and Meguid et al. 2000; Bellinger et al. 2003 b ). After nicotine administration, dopamine and serotonin levels are increased within the LH during the infusion ( as reviewed by Meguid et al. 2000; Miyata et al. 1999) and are significantly lower after the

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39 nicotine infusion (Miyata et al. 1999) When food intake is examined after nicotine withdrawal, dopamine levels remain higher after a meal and are associated with increased meal size (Miyata et al. 1999). Acute nicotine administration reduces food intake, and decreases NPY and NPY mRNA levels in the arcuate nucleus ( as reviewed by Meguid et al. 2000) Chronic nicotine administration h owever, increases levels of NPY mRNA (Li et al. 2000), indicating a change in sensitivity and perhaps a desensitization of these receptors in the arcuate nucleus ( as reviewed by Jo at al. 2002) It is also possible that this effect is seen as an indirect result of nicotine influencing leptin secretion or biosynthesis, which would then affect levels of NPY (Li et al. 2000) which has been seen to facilitate increase s in meal size but not meal number (Leibowitz and Alexander 1991) Nicotine administration also affects levels of MCH most likely by either decreasing the various inputs onto MCH or by reducing the inhibition of MCH neurons ( as reviewed by Jo at al. 2002) There are markers for cholinergic transmission on POMC/CART neurons in the arcuate nucleus of the hypothalamus, indicating another possible target for nicotine to modulate food intake (Meister et al. 2006), as nicotine has been found to increase the sec retion and circulating levels of POMC ( as reviewed by Li et al. 2001) When CART is injected directly into the third ventricle near the hypothalamus, a subsequent decrease in food intake and meal size is seen ( as reviewed by Kramer et al. 2007) Nicotine also seems to effect mRNA levels of CART in the hypothalamus, and this may be yet another mechanism responsible for the changes in meal patterns that are observed with nicotine administration (Kramer et al. 2007), seen acutely, chronically, and during wit hdrawal (Dandekar et al. 2011).

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40 In addition to affecting dopamine levels, nicotine also increases GABA release within the hypothalamus may inhibit MCH neurons which receive input from GABA neurons, thereby reducing food intake ( as reviewed by Jo at al. 2002) Decreased food intake results from direct nicotine injection into the LH, and nicotine administration results in changes in neurotransmission in the LH as well as the expres sion of the previously mentioned neuropeptides involved in food intake ( as reviewed by Jo at al. 2002 and Meguid et al. 2000) Nicotine regulates the monoamines dopamine and serotonin in areas which project to the LH and GABA and glutamate within the LH i tself ( as reviewed by Jo at al. 2002) Chronic nicotine administration increases prepro orexin mRNA expression (Kane et al. 2000) which might lead to increases in food intake This increase in mRNA may decrease orexin receptor expression or change orexin signaling downstream, and this might partially explain the hypophagia seen with nicotine ( as reviewed by Jo at al. 2002; Kane et al. 2000) Other studies have indicated a change in the sensitivity of orexin receptors, such that nicotine administration si gnificantly decreases the affinity of orexin A binding while simultaneously decreasing NPY receptors, might the n result in subsequent decreases in food intake (Kane et al. 2001). Nicotinic acetylcholine receptors are located throughout enteric, sensory, a nd autonomic neurons, and it is likely that smoking activates receptors in the periphery that are involved in food intake ( as reviewed by Jo at al. 2002) Systemic injections of nicotine activate brain stem areas involved in the regulation of feeding, dec rease food intake, and increase sympathetic activity in the peripheral systems of food intake such as the liver and adipose tissue ( as reviewed by Jo at al. 2002) There are also nAChRs located throughout the hypothalamus, particularly within the arcuate nucleus and the LH ( as reviewed by Jo at al. 2002).

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41 Implications for Treatment The high prevalence of post cessation weight gain presents a major problem for many people wanting to quit smoking In light of this, many studies have focused on behavioral t herapies in conjunction with drug therapy to help people both lose weight and succeed in quitting smoking Concurrently, there has been an effort to develop pharmacotherapies to reduce post cessation weight gain, with a secondary goal of increasing abstin ence. Evidence suggests that smokers who quit successfully gain more weight than those who relapse, indicating that individuals who are able to handle some post cessation weight gain will remain abstinent in the long term as compared to those with weight concerns (Gritz and Jeor 1992) rather than the actual weight gain itself Some of the most successful behavioral interventions have been those that have focused on overall healthy eating and lifestyle, as opposed to focusing on calories and weight loss ( as reviewed by Gritz and Jeor 1992) Hall et al (1992) used an innovative approach to weight loss which included a group with an individualized behavioral self mana gement aspect which was compared to a group given nonspecific therapy and a no intervention control The participants who were in the weight management groups were more likely to smoke than subjects in the control group, with men gaining more weight in th at condition, and neither of the treatments resulted in any differences in weight gain compared to controls This study shed light on the relative contribution that the complexity of treatment might play in adherence to that treatment, with intensive weight loss therapies paradoxically detracting from nicotine abstinence (Hall et al. 1992). In contrast, a study which provided a weight loss intervention to only those women who explicitly expressed concern with weight gain had more success in achieving prolonged abstinence, although there was still a lack of a weight attenuating effect (Pirie et al. 1992).

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42 Target ing the beliefs underlying the concern for weight gain may result in increased abstinence ( as reviewed by Pistelli et al. 2009; Gritz and Jeor 1992) There is increasing evidence to suggest that there are higher rates for smoking cessation when weight los s and smoking cessation strategies are offered in conjunction with one another ( as reviewed by Filozof et al. 2004) Cognitive behavioral therapy (CBT) combines both, with the goal of reducing the weight related concerns and facilitating a healthy body im age (Perkins et al. 2001 b ) CBT was more successful at maintaining sustained abstinence and reducing relapse compared to a standard aimed at actually reducing w eight Those in both the weight control group and the CBT group showed increased weight loss compared to the standard treatment, indicating both a weight attenuating effect and an increased rate of long term abstinence for those receiving CBT Another po ssible reason for the relative success seen with CBT may be its effects on mood, as the participants in that study also showed decreased negative affect in the first week after they quit smoking compared to the standard group (Perkins et al. 2001 b ). Many drugs that are currently approved for smoking cessation, as well as other popularly investigated drugs used to treat depression and obesity, have been examined for their weight attenuating properties as well as their ability to help people quit smoking A cross the board, one of the biggest problems seen with using these methods to reduce post cessation weight gain is their inability to foster weight loss after the treatment stops According to Borrelli et al. (1999) the post cessation weight loss from ano rectic drugs is divided into two phases The first phase is where the initial weight loss occurs, and as time progresses eventual tolerance to drug occurs and patients slowly regain the weight lost This phase is followed by a second phase where patients

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43 rapidly gain weight once treatment is stopped This last phase is one of the biggest obstacles to finding a treatment to achieve permanent weight loss. There are currently two drugs approved by the FDA for smoking cessation in the United States Buprop ion (Wellbutrin ) affects the neurotransmitters dopamine and norepinephrine and was initially prescribed for the treatment for depression Varenicline (Chantix ), a drug specific for treating nicotine addiction, serves as a partial agonist at the high aff inity nicotinic acetylcholine receptors in the brain Though there is a lot of support for these two drugs in the effects on weight loss are sparse Several stu cessation weight gain in humans (Stoops et al. 2008; Parsons et al. 2009) whereas a recent animal study found attenuated responding for food in animals given a high dose of varenicline onnor et al. 2010) The studies on buproprion seem more promising and consistent Patients receiving bupropion gained less weight at the end of treatment than patients receiving placebo, and bupropion plus the nicotine patch seems to increase weight loss compared with bupropion alone ( as reviewed by Filofoz 2004) In a population of obese nonsmokers, bupropion was found to facilitate weight loss (Gadde et al. 2001; Anderson et al. 2002) In addition to its effects on weight loss, bupropion also delays r elapse and increases long term abstinence ( as reviewed by Filofoz 2004) Unfortunately, the effects of bupropion on weight loss do not seem to extend past the treatment phase, with subjects eventually gaining weight similar to other ex smokers ( as reviewe d by Filofoz 2004) The relative success with bupropion on smoking cessation and weight loss has drawn receptors Fluoxetine (Prozac ) is an antidepressant as is bupropion, and has been investigated

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44 by itself and in conjunction with other nicotine cessation therapies for an improved outcome and increased weight loss Compared to placebo, ex smokers taking fluoxetine gained less weight over 10 weeks with bo th a high and low dose of the drug (Borrelli et al. 1999), and these decreases in body weight seem to be mediated by decreases in food consumption (Gritz and Jeor 1992) However, toward the end of the experiment, subjects began gaining weight, and 6 month s after patients stopped taking the drug, the group receiving the highest dose of bupropion gained the most weight, though the lower dose maintained a lower weight than those on placebo (Borrelli et al. 1999) ty to attenuate weight gain compared to placebo in the first month after quitting (Spring et al. 1995) However, once the drug was stopped, those taking fluoxetine gained weight and fluoxetine did not seem to influence success at quitting smoking any more than a placebo (Spring et al. 1995) These results indicate that fluoxetine may help buy time for smokers who are afraid to gain weight, giving them an opportunity to quit smoking without worrying about preventing weight gain Another serotonergic agent dexfenfluramine, has also been examined for post cessation weight gain prevention Similar to fluoxetine, dexfenfluramine reduces body weight in the short term but tolerance to the drug results in eventual weight gain and rebound back up to baseline lev els (Spring et al. 1995), an effect that also seems to be moderated by reductions in caloric and carbohydrate intakes ( as reviewed by Gritz and Jeor 1992). Two other drugs that have been examined for effects on post cessation weight gain are naltrexone and rimonabant Despite the fact that rimonabant has been prohibited for use in the United States and Europe because of negative side effects, many researchers have continued to study its effects on weight loss, both outside of and in conjunction with nicoti ne cessation A study which examined both smoking cessation and weight gain found that rimonabant combined

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45 with the nicotine patch resulted in increased rates of abstinence compared to rimonabant alone, and both treatments resulted in very limited weight gain (Rigotti et al. 2009) This is consistent with previous studies indicating the rimonabant alone has positive effects on smoking cessation and helps prevent weight gain (Cahill and Ussher 2011) Naltrexone is an FDA approved drug for treatment of drug and alcohol abuse, and has been examined in conjunction with other pharmacotherapies for the prevention of post cessation weight gain Several studies have found that naltrexone in combination with the nicotine patch suppresses weight gain and helps maintain abstinence, particularly in women (Krishnan Sarin et al. 2003; King et al. 2006), although others have not found this effect (Toll et al. 2008 and 2010). Nicotine replacement therapies (NRTs) by themselves without the addi tion of another pharmacotherapy do not have a large impact on post cessation weight gain Frequent nicotine gum users seem to gain slightly less weight 6 months post treatment compared to infrequent gum users though other stu dies have found a weight rebound effect after people stop using the gum ( as reviewed by Klesges and Meyers 198 9) This ability of nicotine gum to attenuate post cessation weight gain may only be significant for the heaviest smokers ( as reviewed by Gritz a nd Jeor 1992) cessation weight gain compared to a placebo patch ( as reviewed by Rigotti et al. 2009) and this may be due to differences in delivery between the two NRTs (Gritz a nd Jeor 1992). Conclusions The relationship between nicotine addiction and ingestive behaviors are important to understand and yet not very well known or widely studied Ingestive behaviors may h ave implications on all levels of the addiction cycle contributing to the craving for the drug, the intoxication that comes from the using drug and the withdrawal and negative affect that comes when the drug is no longer available (Koob and Volkow 2009) The next two chapters focus on

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46 noncontingent nicotine body weight Chapter 4 examines the relationship between noncontingent cytisine administration (a non nicotine pharmacotherapy) and food intake, while Chapter 5 examines nicotine an d cytisine self administration and the effects this protocol has on food intake The last chapter ties in all of these chapters and summarizes many of the effects nicotine has on ingestive behaviors.

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47 CHAPTER 2 THE RELATIONSHIP BET WEEN NICOTINE AND FL AVO R CUES Introduction As will be discussed in more detail in Chapter 3, smoking cessation aids, including nicotine replacement therapies (NRTs) (Silagy et al. 200 7 ) and non nicotine pharmacotherapies (such as buproprion and varenicline) increase the odds of quitting as high as two fold relative to vehicle ( Silagy et al. 2007 ; Tonstad et al. 2011; Covey et al. 2007) However, the long term abstinence rates are still extremely low (Benowitz 2009; Etter and Stapleton 20 06 ; Hughes et al 2003; Kralikova et al. 2009; Epstein et al. 2006) The limited success of smoking cessation aids highlights the complexity of nicotine addiction and hints at the involvement of other mechanisms beyond the direct effects of nicotine. Research into the success of smoking cessati on therapies has illustrated the relative inability of many of these methods to attenuate relapse brought on by the presence of smoking related cues (Shiffman et al. 1996) Cue induced responding, a method of reinstatement considered relatively valid for emulating the role of external stimuli in nicotine addiction (Lerman et al. 2007; Epstein et al. 2006), has been tested against various smoking cessation therapies These studies have provided evidence for the role of drug associated cues in nicotine depe ndence and relapse, and have demonstrated only limited success for pharmacotherapies in preventing cue et al. 2010; Lerman et al. 2007; Caggiula et al., 2001) Therefore, there seems to be an important but often overlooked relationship between the success of a particular therapy and its ability to prevent cue induced reinstatement. agreed upon Nicotine seems to serve as both a weak prim ary reinforce r whereby cues become associated with the drug in a Pavlovian manner (Caggiula et al. 2002; Chaudhri et al. 2006 b;

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48 Raiff et al., 2006), and as a reinforcement enhancer of other stimuli, such that noncontingent nicotine increases responding for other cues (Donny et al., 2003) For more detail on this topic, refer back to Chapter 1. The majority of studies examining the role of cues in nicotine self administration in animal models have focused on external stimuli, such as light and sound cues ( as referenced by Foll and Goldberg 2005) However, one of the key attributes of cigarettes valued by smokers is their Recognizing this, in 2009 the FDA banned flavored cigarettes in a move to discourage smoking by younger populations that have been shown to smoke flavored cigarettes more t han other groups (Mitka 2009) Unfortunately, basic research on the contribution of flavors to nicotine addiction has been scant. In contrast, the role of flavor cues in eating behavior has been well establi shed For example, Ackroff and Sclafani ( 2001 ) pioneered a method of infusing nutrients such as glucose through a gastric catheter contingent upon a lick operant by a rat Responding for a non caloric, saccharin flavored Kool Aid solution paired with int ragastic delivery of glucose established a robust flavor preference for the nutrient paired flavor This flavor preference indicated that the post ingestive reinforcing effects of the glucose could be associated with a flavor Previous research in our la b has shown that in contrast to the robust flavor preference seen for a flavor paired with a nutrient, flavor solutions paired with nicotine and cocaine show a lack of preference and an aversion, respectively These results illustrate a possible fundament al difference between the interoceptive cues from drugs and those from food. Other studies which have examined the relationship between psychoactive substances and flavor cues have similarly found avoidance and aversive responses to the solution Many st udies have shown the ability of cocaine to induce a weak CTA with varying results due to differences

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49 in dose and route of administration, as well as the strain, sex, and age of the animals (Goudie et al. 1977, Ferrari et al. 1991 Fenu et al. 2010) In these studies, the drug was administered intraperitoneally or subcutaneously either directly before or after being allowed to consume the flavor solution examined the pres ence of a CTA in association with intravenous drug administration The literature surrounding taste preference learning for nicotine is somewhat less comprehensive than the cocaine literature Most studies have found reductions in the percentage consumed for the drug paired flavor compared to the unpaired flavor (Iwamoto and Williamson 1984, Ossenkopp and Giugno 1990, Rinker et al. 2007, Fenu et al. 2010), indications of weak CTAs similar to those seen in cocaine studies, with variations in the CTA due to similar variables. The acute aversion produced by nicotine has been relatively well characterized (Pomerlaeu et al. 1993) and is most likely mediated by dopamine activity in the VTA, as illustrated by a reversal of nicotine induced CTAs when dopamine is blocked The role of dopamine then is to initially mediate the aversive effe cts of the drug (Laviolette et al. 2002 Tan et al. 2009) It is possible that the previous experiments examining this relationship were only investigating the acute effects of n icotine, which are predominantly aversive If this is the case, then the decreased consumption of the drug paired flavor may be mediated by the aversive effects upon initial exposure, and protocols with much longer exposures may be needed to reveal any pr eference effects of nicotine on flavor cues. Considerable research has been done showing that the longer the daily self administration or exposure session, the higher the potential for development of addiction and measureable signs e t al. 2007) Extended access models of nicotine self administration for example, allow rats the ability to self administer nicotine seven days a week for 6 23 hours a

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50 day These longer sessions result in increased addiction as measured by increased withd rawal symptoms It is possible, therefore, that the manifestation of a flavor preference may be related to the development of an addicted state, and this simply has not been achieved in previous studies of taste cues and nicotine If the flavor solution were to become associated with the administration of nicotine and contribute to the addictive potential of the drug via classical condition ing (Caggiula et al., 2002), tha n one would expect the drug paired flavor to be consumed more on the test day If ra ts were addicted to nicotine on the test day, consumption of the drug paired flavor would result in the amelioration of withdrawal symptoms Since this has lon g enough to produce dependence and thus a flavor preference In Wilmouth and Spear (2006), when animals were given a flavor solution during the dark phase only and then allowed to drink water during the light phase, they restricted the majority of their f luid consump tion to the period where water wa s available In contrast, when water was not given at all, the suppression of intake was no longer present and animals consumed the flavored solution. In order to understand the role of session length and tast e conditioning with nicotine as the unconditioned stimulus, the present studies examined flavor preference conditioning in animals receiving noncontingent nicotine in an extended access protocol Animals were placed in the operant chambers for 23 hours a day and were given a flavored Kool Aid solution as their only source of fluid Thirty noncontingent infusions of nicotine were administered every 30 minutes of the dark cycle and the last 3 hours of the light cycle After two weeks, animals were given a two bottle preference test with the nicotine paired flavor and a novel flavor; this was repeated after a third week of exposure We also examined a variety of parameters within this protocol including changes in nico tine dose, flavor novelty, pretest preferences, and an initial week

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51 without the flavor presentation We hypothesized that animals would prefer the nicotine paired flavor over the novel flavor and that this preference would increase with time, as the init ial aversive effects of nicotine were bypassed and animals became more dependent on the drug. Materials and Methods Animals and Housing 16 male Sprague Dawley rats initially weighing ~275 g were used in the present study. All rats were purchased from Har lan (Indianapolis IN). The principles embraced in the Guide for the Care and Use of Laboratory Animals were followed throughout, and the project was approved by the UF IACUC. Upon arrival, rats were housed individually in a conventional vivarium that w as maintained at 22 26 o C and 40 80% relative humidity, on a reverse 12:12 hr light:dark cycle (lights off at 10:00AM). Home cages were of polycarbonate, with Sani Chips contact bedding Purina 5001 Chow pellets available ad libitum for the portion of the experiment where animals were in their home cages. When animals were housed in operant chambers, food was removed from the cages. Autoclaved tap water was available at all times from a standard bottle and sipper tube, except during the course of the expe riment when animals received all fluid in the chambers. A reverse light cycle (on: 2200 1000 h) was in effect in both the vivarium and the operant chambers. Surgery Twenty r ats were anaesthetized with isofluorane and implanted with a ~9cm Micro Renathane catheter (.0 37" O.D. x .023" I.D, Micro Renathane tubing #037 ; Braintree Scientific, Braintree, MA) sterilized in ethylene oxide Two collars, ~.2cm, made from Silastic tubing (.51mm ID x .94mm OD, Silastic #508 002; Dow Corning, Midland, MI) were fitted onto one end of the catheter and 3.2 cm from the opposite end of the catheter The catheter was advanced 3.2 cm into the right jugular vein and secured with a suture around the collar The distal end was

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52 tunneled subcutaneously to an incision in the scap ular region and attached to a port made of 21 ga stainless steel tubing with surgical mesh attached ( 313 000BM 15; Plastics One Inc., Roanoke, VA): the second collar served as a reinforcer at this union. The incision was then closed with non wicking sutur e around the port. The exterior of the port was fitted with a small (~1.5cm) piece of polyvinyl tubing (.51mm ID x 1.52mm OD; Norton Performance Plastics, Akron, OH) and closed with a pin. Immediately after surgery, rats were given of an analgesic and an ti i nflammatory medication (5 mg/kg, Ketorolac tromethamine; Henry Schein, Melville NY). After one day had passed, catheters were flushed with heparinized saline daily and the antibiotics enrofloxacin, 1.5mg per animal per day (trade name: Baytril; Sigma Chemicals, St. Louis MO) and Streptokinase, 200 units per animal per day (Sigma Chemicals, St Louis, MO) were administered via the catheter on alternating days Most rats recovered operative weights within 2 3 days. Apparatus Operant chambers (30.5 cm x 24.1cm x 21.0 cm, ENV 008CT; Med Associates St. Albans, VT) were used throughout the experiment. Chambers were located individually in sound attenuating boxes One side of the chamber had two symmetrically placed holes, one of which provided access to a sipper tube containing the test solutions and was available at all times The sipper tubes were mounted on a retractable motorized stage (ENV 252M, Med Associates) which controlled access to the tubes During the experiment, except on test days, the bot tles remained in the advanced position, allowing the animals I access to the test solutions On test days, animals were given tw o bottles to drink from in a 50 minute session; licking on these tubes was recorded by an electronic dual contact lickometer (E NV 250B, Med Associates). Nicotine or the vehicle (sodium phosphate buffer) were delivered to rats via polyethylene tubing (PE60), protected by a stainless steel spring, connected to an infusion pump (PHM 100

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53 set at 3.33RPM; Med Associates ) via a fluid swi vel ( 375/22PS; Instech laboratories, Plymouth Meeting, PA) to allow almost unlimited movement within the chamber. A hole was located in the center of the ceiling of the chamber which allowed passage of the polyethylene tubing out of the chamber Another length of PE tubing connected the swivel to a syringe pump (PHM 100, 3.33RPM; Med Associates ) located on a shelf mounted outsid e the cage. The pump held a 30 mL syringe containing either sterile nicotine tartrate solution or the vehicle and fitted with an in line nitrocellulose filter (0.22m; Cameo #25ES; Osmonics). The timing and duration of the infusions were controlled by a computer program (MedPC IV). The chambers were fitted with a single fixed lever on the wall opposite the water tube next to a fo od receptacle also controlled by a computer A single press on the lever caused a feeder to release one 45 mg nutritionally complete food pellet (Purified Rodent Tablet, 5TUL, Test Diet, Richmond, IN; energy content 12.7% fat, 20.5% protein, 66.8% carbohy drate) into a trough located next to the feeder. Each apparatus was interfaced to a computer through an input/output module (DIG 716; Med Associates) Rats lived in these chambers for 23 h/day, and were removed only for chamber cleaning Chambers were enclosed in ventilated sound attenuating boxes A 15 W house light (ENV 215M; Med Associates) was located ~2cm below the ceiling on the wall above the openings for the sipper tubes, running the same 12:12 lighting cycle as the vivarium. The max/min tempe rature inside the chambers was recorded every 4 th day and typically was ~1 o C above room temperature. Nicotine Nicotine hydrogen tartrate (Sigma Chemicals, St Louis MO) was dissolved in a sodium phosphate buffer (pH~7.35; 0.1 M ) containing 17mg heparin per 100 mL s. Vehicle was the buffer heparin solution The concentrations of the nicotine solution were adjusted to deliver doses of 0.01 or 0.06 mg/kg body wt per injection, calculated as the free base.

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54 Procedure Experiment one The purpose of experiment one was to determine if there is a difference in flavor preferences between animals given nicotine in conjunction with a Kool Aid solution and control animals who receive the Kool Aid solution without receiving nicotine A week after surgery, animals were placed into the operant chambers, where they were tested using a FR1 schedule to obtain pellets No specific lever training was conducted: all rats acquired the operant task to obtain food within the first night For t he first week, animals were give ad libitum access to autoclaved water in sipper tubes After the first week, they were then given ad libitum access to the test solution (.1% saccharin flavored with .05% Kool Aid; Kraft Foods Global Inc., Northfield, IL) in the sipper tubes in place of the water Cherry and grape were the flavors that were used; half of the rats in each group received cherry as a CS+ and the other half grape as a CS+. Rats were tested for three weeks During this time, they were given p rogrammed injections of nicotine The progra mmed infusions (0.04 mL ) were 1 s in duration every 30 mi n for the entire dark phase (12 h) and for the last 3 h of the light phase This resulted in a total of 30 injectio ns over a 15h period of the 23 h test sessi on that coincided with the maximal feeding and active times of rats Animals were randomly divided; the control group received infusions of the buffer/heparin solution and the second group received infusions of nicotine The nicotine group initially rece ived 0.01 mg/kg/infusion for two days and then had their drug dose increased to avoid possible aversive effects. Terminal doses of nicotine maintained for the rest of the treatment phase, was 0.06 mg/kg per injection (1.8 mg/kg total daily dose) After t wo weeks, and again after three weeks, animals were given a two bottle preference test in which two bottles were filled with cherry and grape Kool Aid and placed into the retractable motorized stages

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55 Approximately 16 hours before the test, animals were w ater restricted to increase responding for the test solution. At the start of the session, both bottles were advanced and licks on each spout were recorded over a 50 minute period. Experiment two A second experiment was performed to determine if there is a nicotine dose dependency in flavor preference Experimentally nave animals were given ad libitum access to the Kool Aid solution from the start of the experiment The groups initially received 0.01 mg/kg/infusion for two days to avoid possible aversiv e effects, then had their drug dose increased. Terminal doses of nicotine for the two groups, maintained for the rest of the treatment phase, were 0.06 or 0.03 mg/kg per injection (1.8 mg/kg or 0.9 mg/kg total daily dose) Two preference tests were given one after 2 weeks and another after 3 weeks The Kool Aid solution, access to food, and the preference test procedure were all identical to the first experiment. Experiment three The purpose of experiment three was to determine whether pre exposure to n icotine during the first week without access to the Kool Aid solution would affect the outcome The procedure was generally similar to experiment one, but the two groups of rats differed Similar to Experiments 1 and 2, animals in group one were given ad libitum access to water in the chambers for the first week in conjunction with the nicotine and then received the Kool Aid solution for the last two weeks Animals in group two were given ad libitum access to Kool Aid from the first day of nicotine and f or the duration of the experiment The Kool Aid solution, access to food, and the preference test procedure were all identical to the first experiment, and the high dose of nicotine (.06 mg/kg/inf) was used.

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56 Experiment four T he purpose of experiment fou r was to determine if pairing nicotine with the Kool Aid solution over a three week period would reverse initial flavor avoidance or enhance an initial flavor preference Rats in this experiment were given an initial preference test for the grape and cherry test solutions before they were put into the chambers in the 23h experiment They were then divided into two groups that were matched on the basis of results from the two bottle preference test Animals in group one were given the flavor they consumed most in the preference test for the duration of the experiment; animals in group two were given the flavor they consumed least for the three weeks of the experiment One additional preference test was given after 3 weeks, and the number of licks for each flavor was recorded again. To avoid possible aversive effect of the drug, both groups initially received 0.01 mg/kg/infusion of nicotine for two days and then had their drug dose increased to 0.03 mg/kg per injection (0.9 mg/kg total daily dose), and this was maintained for the rest of the study. The Kool Aid solution, access to food, and the preference test procedure were all identical to the first experiment. Data Anal ysis For experiment 1, independent samples t tests were used to examine differences in paired and unpaired flavor preferences between animals that received nicotine and those that received the vehicle Interactions between the dose and preferences for the paired flavor over the two sessions of interest were examined using a repeated measures ANOVA, with paired flavor preferences over the two sessions as the within subjects factor and dose as the between subjects factor Independent samples t tests were us ed in experiment 2, to examine differences in paired and unpaired flavor preferences between animals that received the high compared with the low dose of nicotine Likewise, differences in preference for the paired and unpaired flavors between

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57 animals tha t received a flavor the first week compared with those that did not, were examined using independent samples t tests Lastly, a univariate ANOVA was used in experiment 3 to determine if there were differences in preferences for the paired and unpaired fla vors for animals that were given the flavor they initial preferred or avoided, and to determine if there was an effect of dose on these preferences. Results Experiment One: High Dose Compared with Vehicle On the first test day after 2 weeks of exposure, there were no statistically significant differences in preferences for the paired flavors between animals that received the high dose of nicotine (M = .60; SE = .14) compared with those that received the vehicle (M = .68; SE =.09) during treatment [t (9) = .517, ns], (Figure 2 1) On the second test day after a third week of exposure, there were again no statistically significant differences in preferences between animals that received the high dose of nicotine (M = .73; SE = .17) compared with those that received the vehicle (M = .49; SE = .19), [t (7) = .895, ns] There was also no significant interaction between the preference for the paired flavor during the first session compared to the second session for animals receiving nicotine or vehicle [F (1, 7 ) = .958, ns] Individual data for this experiment are presented in Figure 2 2. Experiment Two: High Dose Compared with Low Dose On the first test day after 2 weeks of exposure, there were no statistically significant differences in preferences for the pa ired versus unpaired flavors between animals that received the high dose of nicotine (M = .53; SE = .10) compared with those that received the low dose (M = .41; SE = .05) during treatment [t (10) = .321, ns] (Figure 2 3). On the second test day after a t hird week of exposure, there were again no statistically significant differences between animals

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58 that received the high dose of nicotine (M = .65; SE = .16) compared with those that received the vehicle (M = .66; SE = .06) during the second test session [t (8) = 1.060, ns]. Experiment Three: Flavor Compared with No Flavor during Week One On the first test day after 2 weeks of exposure, there were no statistically significant differences in preferences for the paired versus unpaired flavors on the first test day between animals that received the flavor solution (M = .54; SE = .13) compared with those that did not (M = .60; SE = .14) during the first week [t (10) = .321, ns] (Figure 2 4) On the second test day after a third week of exposure, there were no st atistically significant differences between animals that received the flavor solution the first week (M = .50; SE = .13) compared with those that did not (M = .73; SE = .17), [t (8) = 1.060, ns]. Experiment Four: Initial Preference There was no main effect of initial preference on flavor preferences on the test day: the animals for whom nicotine was paired with the flavor solution they preferred initially did not significantly differ from those for whom nicotine was paired with the initially less preferred flavor [F (1, 29) = .012, ns] (Figure 2 5) Similarly, there was no significant main effect of dose for the paired and unpaired flavors on the test day between animals that received the vehicle compared with those that received the low dose of nicotine [F (1, 29) = .518, ns]. There was likewise no significant interaction between dose and initial preference [F (1,29) = 1.478]. Discussion In the present study, infusions of noncontingent nicotine in the presence of a Kool Aid flavored solution resulted in a modest preference for this flavor over a novel flavor in several conditions Interestingly, this preference appeared to increase throughout time, from the first preference test given after two weeks of nicotine exposure to the second preference test at we ek 3.

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59 In experiment 1, animals that received a high dose of nicotine showed a small 60% preference for the flavor during the first test which then increased to a 73% preference after an additional week In contrast, the flavored Kool Aid solution that wa s available while animals received infusions of the vehicle (essentially, a mere exposure control group) was preferred 68% over the novel flavor during the first test and then decreased to 48% after the second test To the first study to date that has not reported an aversion for a flavor that was consumed in the presence of nicotine Though the differences between vehicle and nicotine groups were not statistically significant, most likely due to a small number of animals a nd individual variability, the trends seen in this experiment are important to consider For individual data, reference Figure 2 2. The pattern of results seen in animals who received the vehicle aligns nicely with previous research examining the influen ce of mere exposure on the development of flavor preferences. In a classic study, Domjan (1976) first explored the contribution of prior exposure on the intake of a saccharin solution In a series of experiments, he demonstrated that exposure to a sacchar in solution for 30 minutes preceding water availability for a subsequent 30 minutes resulted in increased consumption of the saccharin solution over time This increase in saccharin intake was primarily a function of the number of exposures to the solutio n and was also related to the duration of the exposures, such that animals that received access to the saccharin solutions for increased amounts of time consumed more of the flavor during the test session The mere exposure hypothesis operates under the a ssumption that humans and animals are initially neophobic to new foods and flavors until they acquire enough experience with the new substance to learn that it is not harmful, such that the intake of the substance increases over time (Domjan 1976; Hill 197 8; Meyer and Sclafani 2006) Not only does prior exposure to a beneficial

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60 substance result in increased consumption, it also results in elevated likeness ratings Pliner (1982) gave human subjects different amounts of exposure to a variety of novel foods and measured affective response to the stimuli and found that liking ratings increased with increasing experience with the food Taken together, the results from previous studies on exposure provide an explanation for why animals would initially prefer a substance they had experienced in the past over a novel flavor Perhaps unexpectedly, we observed a decline in preference in the vehicle group between the second and third weeks It is possible that once a food or drink is no longer novel, people or ani mals may adaptively sample other available items to introduce variety in their diet (Hill 1978) A second possibility is that the increased preference for the less experienced flavor is due to the negative recency effect, which states that preferences will emerge for tastes which have not been experienced in a while ( as reviewed by Hill 1978) such th at one or two exposures to the substance will increase the intake of that substance (Domjan 1976) hypothesis on which the expected result of mere exposure depends. Compare d with this control group, the nicotine group exhibited some differences in their preferences. Again, there was not a significant interaction between the vehicle and nicotine groups for preferences during the first and second t est sessions but, rather tha n a decline in preference between the tests at the ends of weeks 2 and 3, preference for the nicotine associated flavor increased between these tests This trend was also present in experiment 2 in which the nicotine dose was varied, suggesting that alth ough small, there is a consistently different trend between vehicle and nicotine paired animals in the direction that preference for a nicotine paired flavor is increasing This result stands in marked contrast to all previous published studies in which n icotine was found to produce robust conditioned taste aversions, not preferences or even

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61 indifference As noted in the Introduction of this chapter all of these previous studies used short term nicotine exposures The present experiments using long term exposure have yielded quite a different result of persistent (albeit weak) preferences by week three in animals receiving both low and high doses of nicotine One possible mechanism underlying these effects might be the increased opportunities for the ni cotine and flavor solution to become associated with one another If acute administration of nicotine paired with a flavored solution results in conditioned taste aversions, and chronic administration results in preferences which increase over time, than the duration of exposure must be taken into consideration. We would therefore predict that even longer exposures might lead to even stronger preferences, although for technical and other reasons we did not pursue this hypothesis. Though few studies have been conducted on the relationship between nicotine and flavor (CPPs) Similar to conditioned taste learning, CPP can reveal either reinforcing or aversive pro perties of drugs Similar to traditional conditioned taste protocols, CPPs are evaluated by giving the animal acute injection(s) of a drug in one environment and saline in a different environment, after which the preference for one environment over the ot her is examined on a drug free test day (Fudala et al. 1985; Belluzzi et al. 2004; Grabus et al. 2006) CPP testing measures the time spent in each environment and functions under the assumption that the unique drug paired environment and all related cues become associated with the effects of the drug (van der Kooy 1987) It might be expected that results with nicotine from CPP studies might generalize to conditioned taste protocols, since within classical associative learning theory the nature or modalit y of the CS+ should be of little importance CPPs in animals receiving nicotine

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62 increasing CPP as dose increases, but then an aversion at still higher doses ( a s reviewed by Foll and Goldberg 2005; Fudala et al. 1985; Grabus et al. 2006) In the present study, we were unable to find any clear dose effects. In contrast, in self administration studies, to be reviewed more fully in Chapters 3 and 5, the high dose used in this study produces a higher rate of self administration than the lower dose though both doses are readily self administered by rats and so are inferred to be reinforcing Both the low and high doses used in this study are able to support respondi ng for nicotine and visual cues and the low dose has been frequently use to demonstrate et al. 2003; Chaudhri et al. 2007) The increased preferences and consis tent trends seen in the current study support the idea that both of these nicotine doses are reinforcing, and become associated with stimuli in the environment. In experiment 3, animals were given noncontingent nicotine for the first week without the flavor solution to allow for any acute aversive effects of nicotine to dissipate, the preference also tended to increase over time compared to the preferences seen for animals that received a high dose of nicotine from the start The results indicate that eliminating the associative pairings between potentially aversive consequences like nausea (Benowitz 1986) with an ingestible solution may increase the percentage of the flavor consumed on the test day, whereas the animals that received the flavor from th e start did not show a preference during the second session It is possible therefore, that not presenting the flavor in conjunction with the nicotine for the first week increased the preferences for the flavor because the acute side effects of nicotine w ere bypassed. The first three studies did not present the non paired solution prior to the preference test, and so did not assess initial or pre exposure preference. It is possible however, that initial

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63 preference modulates the manifestation of a taste preference or aversion (Hill 1978). For example, it is well known that it is easy to condition an aversion with a drug to a neutral flavor, but very hard to obtain a robust aversion using a highly preferred tastant like sucrose. Experiment 4 examined init ability to become positively associated with the flavor We expected that if nicotine were able to condition a robust flavor preference after three weeks, as seen in experiment 1, that the se preferences would be augmented by giving a flavor that was more reinforcing Studies have shown that noncontingent infusions of nicotine increase responding for other reinforcing visual stimuli (Donny et al. 2003; Chaudhri et al. 2007), such that anima ls demonstrate increased responding on a lever for the presentation of a visual stimulus when in the context of nicotine In the current study, there was no significant difference between animals that received the nicotine in conjunction with the flavor t hey initially preferred compared to those that received the flavor they did not prefer In contrast, animals that received the flavor they preferred paired with the vehicle consumed virtually equal amounts of both the two flavors, while those given the fl avor they least preferred continued to consume less of this flavor on the test day Although not significant, these results would indicate that instead of continuing to avoid the flavor they least preferred like animals that received the vehicle, animals given nicotine no longer showed an avoidance of this flavor after three weeks. There are a few limitations of the current study which should be addressed First, the relatively small number of animals completing this study definitely limits the statistica l analyses. Secondly, b ecause we did not give a preference test after the first week in any of the studies, it is difficult to determine whether or not there would have been an initial aversion seen due to the acute negative effects of nicotine, similar to that seen in week long preference testing Similarly,

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64 it would have been interesting to see how long the flavor preference would last, either by examining extinction or continuing the experiment for an additional week Previous studies examining flavor preferences associated with post ingestive consequences like nutrient infusions have demonstrated that these preferences are strong (typically >90%) and are resistant to extinction persisting for weeks after the flavor is no longer paired with nutrient infusions (Drucker et al. 1994) Similarly, other studies have shown that flavor preferences under these conditions persist and remain constant over time while the pairing still occurs (Lucas and Sclafani 1989) Though dru gs of abuse and ingestive behaviors are quite different, these studies provide some evidence that flavor preferences might develop under some conditions when paired with drugs of abuse and, by analogy with the nutrient studies, might be resistant to extinc tion. The current study evaluated the relationship between noncontingent nicotine and flavor preferences Because we did not examine concurrent self administration and flavor consumption, results from these studies cannot be extrapolated to provide evide nce for the role of flavor cues in enhancing cigarette smoking throughout time However, they do lend support to the idea that flavor cues might contribute to the reinforcing properties of smoking.

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65 Figure 2 1. Fla vor preference test vehicle v. high dose Shown are mean +/ SE percent licks of the paired and unpaired flavors, over two separate sessions, for animals in Experiment 1 that received the vehicle (n = 6) or the high dose of nicotine (n = 5)

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66 F igure 2 2. Vehicle v. nicotine individual data S hown are individual data, medians, and percentiles for percent licks of the paired flavors, over two separate sessions, for animals in Experiment 1 that received the vehicl e (n = 6) or the high dose (n = 5) of nicotine Symbols correspond to individual animals.

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67 Figure 2 3. Flavor p reference test low v. high d ose S hown are mean +/ SE percent licks of the paired and unpaired flavors, over two separate sessions, for animals in Experiment 2 that received the low dose (n = 7) or the high dose of nicotine (n = 7)

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68 Figure 2 4. Week on e with or without flavor S hown are mean +/ SE percent licks of the paired and unpaired flavors, over two separate sessions, for animals in Experiment 3 that received either water (n = 5) or the flavor solution (n = 7) during the first week of nicotine administration

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69 Figure 2 5. Initial preference effects S hown are mean +/ SE percent licks of the paired and unpaired flavors, over two separate sessions, for animals in Experiment 4 that received the flavor they initially preferred (n = 14) or avoi ded (n = 16) in conjunction with either nicotine or the vehicle

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70 CHAPTER 3 THE EFFECTS OF NICOT INE ON BODY WEIGHT A ND MEAL PATTERNS Introduction In Chapter 1, data were reviewed on the relationship between cigarette smoking and obesity, both of which are the two major causes of morbidity and mortality in the United States T he relationship between these disorders has been examined in humans through self report and in animals via passive nicotine administration. Weight gain and ultimately obesit y is due to positive energy balance, achieved either through increased food intake and/or decreased physical activity, or metabolism N icotine has been shown to affect all three of these factors, and these observations were summarized in Chapter 1. A stan dard method for examining the effects of nicotine on food intake is to analyze meal patterns in rodents T his technique measures total food intake, total number of meals and average meal size. Total food intake is normally the product of meal number and meal size although, depending upon the criterion applied to define a meal, animals may take some portion of their daily intake as small episodes or snacks P revious studies have administered nicotine via osmotic minipumps or multiple intraperitoneal (IP) injections T he advantages of minipumps are that they allow nicotine to be administered continuously without having to repeatedly handle and inject the animals ( Bishop et al. 2004; Grunberg and Bowen 1985; Grunberg et al. 1985; Bow en et al. 1986; Miyata et al. 199 9; Miyata et al. 2001) I n contrast, IP injections more closely mimic the episodic nature of smoking and are typical ly administered 4 5 times throughout the dark cycle (Bellinger et al. 2003 a & b ; Guan et al. 2004; Wellman et al. 2005) Studies, u sing different modes of administration, have reported diverse effects on weight, food intake and meal patterns I n general, animals given nicotine through the osmotic minipumps have shown dose dependent reductions in body weight (Grunberg and Bowen 1985;

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71 Grunberg et al. 1985; Grunberg et al. 1986; Miyata 2001), although some studies have only reported this effect for the first few days of administration (Miyata et al. 1999) C hanges in food consumption in these experiments were relatively inconsistent: so me studies reported that only the highest dose of nicotine (12mg/kg/day) decreased total food intake (Grunberg and Bowen 1985; Grunberg et al. 1986) or no significant change, while other studies reported a decrease of intake that was specific for sweet or high calorie foods and in male but not female rats (Grunberg et al. 1985; Grunberg et al. 1986) T he pattern of decreased food consumption with the administration of nicotine and increased food consumption after the drug is stopped have been reported in s ome (Grunberg et al. 1986; Miyata et al. 1999 & 2001) but not all (Grunberg et al. 1985; Bowen et al. 1986) experiments D espite some evidence of changes in food intake, studies using osmotic minipumps typically do not find that such changes persist for m ore than the first couple of days of nicotine administration (Miyata et al.1999 & 2001). Specific changes in meal patterns have been more readily seen in experiments using IP injections of nicotine, indicating more specific effects when nicotine is admini stered intermittently I P injections of nicotine result in relatively consistent decreases in total food intake (Li et al. 2000; Bellinger et al. 2002; Guan et al. 2004) I n these studies, nicotine decreased meal size in the dark phase of a 12:12 cycle ( Bellinger et al. 2003 b ; Guan et al. 2004), sometimes with an increase in meal number also during the dark (Bellinger et al. 2003 b ), and without any effects during the light (Bellinger et al. 2003 b ; Guan et al. 2004) A dministration of nicotine via IP inje ctions caused a dose dependent decrease in body weight (Kane et al. 2000; Li et al. 2000; Bellinger et al. 2003 a & b; Guan et al. 2004), an effect that was sustained across days compared with the relatively transient body weight changes using osmotic mini pump infusions (Kane et al. 2000; Li et al. 2000; Bellinger et al. 2003 b ; Guan et al. 2004)

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72 S urprisingly, none of these experiments has shown a gain or rebound effects in body weight after the nicotine regimen is stopped, and instead show a persistent, l ong lasting reduction in body weight in nicotine treated animals compared with drug nave controls T his effect is largely attributable to persistent differences in meal size and meal number after nicotine removal (Bellinger et al. 2003 b ) T his finding i n rats contrasts sharply with the human literature, and illustrates a critical shortcoming of these methods in relation to modeling relative weight gain that humans so often experience after quitting smoking. One potentially critical difference between IP injections or minipump infusions of nicotine in animals is that neither accurately emulates the episodic changes in plasma concentrations of nicotine associated with cigarette smoking (Russell and Feyerabend 1978; Sanderson et al. 1993; Winders 1998) T h ere are only a few studies which have investigated plasma nicotine levels during the administration of nicotine via a mimipump W inder et al. (1998) reported an average plasma concentration of 257 ng/ mL in rats that received 12 mg/kg/day of nicotine throu gh a minipump S anderson et al. (1993) found a similarly high level of plasma nicotine (~300 ng/ mL ) at the 12 mg/kg/day dose but only for the first day, after which levels dropped and were maintained at a consistent level around 40 ng/ mL H uman plasma ni cotine levels are typically 10 40 ng/ mL (Russell et al. 1976; Henningfield et al. 1993; Henningfield and Keenan 1993) T he reason for the discrepancy in the rat plasma levels above may include procedural and/or metabolic changes, but in any event they may account for the relatively temporary changes in body weight and food intake seen in experiments where nicotine is administered via a minipump (i.e. Miyata et al. 1999). Perhaps a more critical difference between minipump administration and smoking is tha t the constant infusion of nicotine through the minipump does not produce the rapid fluctuations in

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73 plasma levels of nicotine that occur while smoking cigarettes (Benowitz 1983; Henningfield et al. 1993; Henningfield and Keenan 1993; Sanderson et al. 1993) T he bolus infusions of nicotine from each puff may be associated with repeated and intense subjective reinforcement (Russell and Feyerabend 1978; Henningfield et al. 1993) T he reinforcement of smoking is associated with the activation and desensitization of nAChRs throughout the mesocorticolimbic system within the brain T which is located in large part on GABAergic terminals (Wooltorton et al. 2003) A s reviewed in Mansvelder et al. (2002), as an individual smokes, these receptors become desensitized and are stimulated T hese effects in turn enhance t he firing of dopamine neurons and long term potentiation T he relative activation of these subtypes occurs during the first minute of smoking when the blood arterial level rises dramatically, and is quickly followed by the desensitization of ptors I t takes anywhere from 13 33 minutes for this system to reset so that the receptors are no longer desensitized and can be activated again T he protocol of repeated IP injections of nicotine to rats (Russell and Feyerabend 1978) was designed in par t to emulate these episodic infusions of nicotine H owever, these studies typically administer nicotine 4 5 times during the night resulting in far fewer (but probably larger) spikes in plasma levels than are seen in heavy smokers who are puffing on cigar ettes several hundred times a day (Russell and Feyerabend 1978). In order to better define the effects of nicotine, and nicotine cessation, on food intake, there is a need to develop animal protocols that approximate the relative frequency and quantity of nicotine administered by smokers I ntravenous administration provides a route to produce rapid increases in blood nicotine levels that will directly impact the brain that stands in marked

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74 contrast to the IP route which results in a slower absorption and extensive first pass metabolism to cotinine in the liver (Russel and Feyerabend 1978). Because the plasma half life of nicotine is relatively short, epi sodic IV infusions of nicotine will result in peaks and troughs of blood nicotine levels (Be nowitz 1983 ; Rose et al. 1999) and, depe nding on the dose, with levels (40 120 ng/ mL ) that are relative ly comparable to those seen in smokers (Shoaib and Stolerman 1999). Indeed, such rapid spikes in plasma nicotine seem to be essential to demonstrate a reinforcing role of nicotine in rodents through self administration T hus, we have chosen the IV patterns of rats. Further, because food intake is mainly nocturnal, it is impo rtant to administer nicotine throughout the normal eating period W e hypothesized that the administration of nicotine would result in decreased weight gain and decreased food intake, followed by a rapid increase in weight gain and food intake after the re moval of the nicotine P revious studies using extended or continuous access to nicotine have shown that rats will reliably self administer 7 ). Unpublished studies from our lab have found that rats self administering in this protocol do so during the last ~3 hours of the light and throughout the dark cycle, and mostly in conjunction with spontaneous meals H owever, because the amounts of self administered nicotine varies considerabl y between individual rats, we elected to give programmed infusions of nicotine every 30 minutes throughout the 15 hours when they are most active, spanning the dark cycle and for the last three hours of the light cycle. This emulates modal self administra tion patterns (~30/day) but in a manner that the experimenter controls the dose and timing. We hypothesized that nicotine will decrease food intake in the dark cycle when animals are receiving nicotine every 30 minutes, and further hypothesize d that there may be a compensatory increase in food intake in the 8 hours of

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75 the light cycle when nicotine was not being administered I n light of previous results suggesting an effect of nicotine on satiety (i.e. Miyata et al. 1999 ), we hypothesized that programmed infusions of nicotine will decrease food intake in the night through specific decreases in meal size. Materials and Methods Animals and Housing Twenty two male Sprague Dawley rats initially weighing ~275 g were used in the present study. All rats were pur chased from Harlan ( Indianapolis, IN ). The principles described in the Guide for the Care and Use of Laboratory Animals were followed throughout, and the project was approved by the UF IACUC. Upon arrival, rats were housed individually in a conventional vivarium that was maintained at 22 26 o C and 40 80% relative humidity, on a reverse 12:12 hr light:dark cycle (lights off at 10:00AM). Home cages were of polycarbonate, with Sani Chips contact bedding. Purina 5001 Chow pellets were available ad libitum f or the initial portion of the experiment when animals were housed in their home cages D uring the test phase, animals were housed in operant chambers A utoclaved tap water was available at all times from a standard bottle and sipper tube. A reverse light cycle (on: 2200 1000 h) was in effect in both the vivarium and the operant chambers. Surgery Rats were anaesthetized with isofluorane and implanted wit h a ~9cm Micro Renathane catheter ( .037 O.D. x .023" I.D, Micro Renathane tubing #037; Braintree Scientific, Braintree, MA) sterilized in ethylene oxide T wo collars, ~.2cm, made from Silastic tubing (.51mm ID x .94mm OD, Silastic #508 002; Dow Corning, Midland, MI) were fitted onto one end of the catheter and 3.2 cm from the opposite end of the catheter T he catheter was advanced 3.2 cm into the right jugular vein and secured with a suture around the collar T he distal end was

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76 tunneled subcutaneously t o an incision in the scapular region and attached to a port made of 21 ga stainless steel tubing with surgical mesh attached ( 313 000BM 15; Plastics One Inc., Roanoke, VA): the second collar served as a reinforcer at this union. The incision was then clos ed with non wicking suture around the port. The exterior of the port was fitted with a small (~1.5cm) piece of polyvinyl tubing (.51mm ID x 1.52mm OD; Norton Performance Plastics, Akron, OH) and closed with a pin. Immediately after surgery, rats were giv en of an analgesic and anti inflammatory medication ( 5 mg/kg, Ketorolac tromethamine; Henry Schein, Melville NY). After 1 day had passed, catheters were flushed with heparinized saline daily and the antibiotics enrofloxacin, 1.5mg per animal per day (trad e name: Baytril; Sigma Chemicals, St. Louis MO) and Streptokinase, 200 units per animal per day (Sigma Chemicals, St Louis, MO) were administered via the catheter on alternating days M ost rats recovered operative weights within 2 3 days. Apparatus Op erant chambers (30.5 cm x 24.1cm x 21.0 cm, ENV 008CT; Med Associates St. Albans, VT) were used throughout the experiment C hambers were located individually in sound attenuating boxes. One side of the chamber had two symmetrically placed holes, one of which provided access to a sipper tube containing autoclaved water available at all times N icotine and vehicle (sodium phosphate buffer; pH ~ 7.35; 0.1 M ) were delivered to rats via polyethylene tubing (PE60), protected by a stainless steel spring, connec ted to an infusion pump (PHM 100 set at 3.33RPM; Med Associates ) via a fluid swivel ( 375/22PS; Instech laboratories, Plymouth Meeting, PA) to allow almost unlimited movement within the chamber A hole was located in the center of the ceiling of the chamb er which allowed passage of the polyethylene tubing out of the chamber A nother length of PE tubing connected the swivel to the infusion pump which was located on a shelf mounted outside the cage T he pump held a 30mL syringe

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77 containing either sterile ni cotine tartrate solution or the vehicle and fitted with an in line nitrocellulose filter (0.22m; Cameo #25ES; Osmonics) T he timing and duration of the infusions were controlled by a computer program (MedPC IV) T he chambers were fitted with a single fi xed lever on the wall opposite the water tube and next to a foo d receptacle also controlled by a computer A single press on the lever caused a feeder to release one 45 mg nutritionally complete food pellet (Purified Rodent Tablet, 5TUL, Test Diet, Richmo nd, IN; energy content 12.7% fat, 20.5% protein, 66.8% carbohydrate) into a trough located next to the feeder. Each apparatus was interfaced to a computer through an input/output module ( DIG 716; Med Associates ) R ats lived in these chambers for 23 h/da y, and were removed only for chamber cleaning C hambers were enclosed in ventilated sound attenuating boxes A 15 W house light ( ENV 215M; Med Associates ) was located ~2cm below the ceiling on the wall above the openings for the sipper tubes, running the same 12:12 lighting cycle as the vivarium T he max/min temperature inside the chambers was recorded periodically and typically was ~1 o C above room temperature. Nicotine Nicotine hydrogen tartrate (Sigma Chemicals, St Louis MO) was dissolved in a sodium phosphate buffer (pH~7.35; 0.1 M ) containing 17mg heparin per 100 mL s V ehicle was the buffer heparin solution T he concentrations of the nicotine solution were adjusted to deliver doses of approximately 0.01, 0.03 or 0.06 mg/kg body wt per injection, calc ulated as the free base. Procedure Two days after surgery, animals were placed into the operant chambers and baseline measures of body weight, pellets consumed and meal patterns were measured for 5 days. No specific lever training was conducted: all rats acquired the operant task within the first night

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78 A fter the baseline period, rats were divided into three groups matched for body weight T hey were then returned to the chambers and their catheters attached to the infusion line. For the first 2 days the y received programmed injections of vehicle T he programmed infusions (0.04mL ) were 1 s in duration every 30 min for the entire dark phase (12 h) and for the last 3 h of the light phase T his resulted in a total of 30 injections over a 15 h period of the 23 h test session that coincided with the maximal feeding and active times of rats. During the treatment phase, which lasted for 12 days, one group of rats received vehicle and two groups received nicotine B oth nicotine groups initially received 0.01 m g/kg/injection then had their drug dose increased gradually over 3 days to avoid possible aversive effects. Terminal doses of nicotine for the two groups, maintained for the last 9 days of the treatment phase, were either 0.06 or 0.03 mg/kg per injection (1.8 and 0.9 mg/kg total daily dose). After the treatment phase, rats entered a cessation phase during which nicotine infusions were replaced by vehicle T he cessation phase lasted 12 days. Body weight was recorded daily and meal parameters were recorde d continuously throughout the experiment. In addition, the e xcess number of pellets, defined as the number of pellets consumed that were not within a defined meal (< 4 pellets every 10 minutes) were also calculated This study was run in 2 replications, with all 3 groups represented approximately equally in each replication. There were no marked differences between these replications so the data were combined as planned for analysis. Data Analysis Because we expected the group data would be normally di stributed, parametric statistical tests were chosen. One way ANOVAs were used to examine differences in body weight and meal patterns (food intake, meal number, meal size and excess pellets) between animals that received the vehicle or the .06 mg/kg dose o f nicotine O ne way ANOVAs were also used to

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79 examine differences between the vehicle and the .03 mg/kg dose of nicotine S ignificance levels were set at p < .05. Results Body Weight The results of all statistical calculations are located in Table 3 1 D uring baseline, the groups of rats did not differ in the amount of weight gained over the five day period D uring the treatment phase, the animals that received nicotine gained less weight than those that received the vehicle (Figure 3 1) T his effect w as significant on days 8, 9, 10, 11, 12, and 13 D uring the cessation phase, the animals that received nicotine continued to gain less weight for a period of time than those that received the vehicle T his effect was significant on days 14, 15, 16, 17, 1 8, 19, 20 and 21 T he body weight gained for animals that received nicotine no longer differed from those that received the vehicle after the 21 st day. Meal Patterns .06 mg/kg/inf Baseline The results of all statistical calculations for means over the entire period are located in Table 2 and the means for each individual day can be found in Table 3 4 T here were no significant differences throughout baseline between the two groups in total pellet intake (Figures 3 2, 3 4, and 3 5) T his lack of difference was seen for the mean daily number of pellets eaten, the mean number of pellets eaten during the dark cycle, and the mean number of pellets eaten in the light cycle T he mean meal size also did not differ between the two groups (Figures 3 7, 3 8, and 3 9) for the total mean meal size, the mean meal size in the dark cycle, and the mean meal size during the light cycle S imilarly, there were no significant differences between the two groups in total meal intake (Figures 3 10, 3 11 and 3 12) T hi s similarity between groups was

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80 seen for the total mean number of meals, the mean number of meals in the dark cycle, and the mean number of meals in the light cycle. Vehicle first two days of treatment The results of all statistical calculations for mean s over this entire period are located in Table 3 3 (Days 6 7) T here were no differences across groups during the first two days of treatment, when all animals received the vehicle (not shown in Figures) T his lack of difference was seen for the mean dai ly number of pellets eaten, the mean number of pellets eaten during the dark cycle, and the mean number of pellets eaten in the light cycle S imilarly, there were no significant differences between the two groups in total meal intake (not shown in Figures ) T his similarity between groups was seen for the total mean number of meals, the mean number of meals in the dark cycle, and the mean number of meals in the light cycle T he mean meal size did not differ between the two groups (not shown in Figures) fo r the total mean meal size, and the mean meal size in the dark cycle, but did significantly differ for the mean meal size during the light cycle, with the nicotine group eating bigger meals during these two days than the vehicle group (not shown in Figures ). Treatment number of pellets The results of all statistical calculations for means over the entire period are located in Table 3 3 and the means for each individual day can be found in Table 3 4 A nimals that received the nicotine ate fewer pellets o verall than those that received the vehicle (Figures 3 2, 3 4, and 3 5) T he treatment phase was separated into three groups following the two days of vehicle administration (days 6 7), days 8 10 when animals received either .01 mg/kg/inf or the vehicle a nd days 11 14 and days 15 19 where the animals received their terminal dose of nicotine or the vehicle T he mean number of total pellets eaten by rats in the nicotine group was significantly less than animals in the vehicle group during days 8 10, 11 14 a nd 15 19 of the

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81 experiment F urther analysis revealed that animals receiving nicotine ate fewer pellets compared to vehicle animals daily (Figure 3 3), and this was significant on days 10, 11, 12, 13, 15, 16, and 19 and approached significance on days 14 and 17 T he mean number of total pellets eaten in the dark cycle by rats in the nicotine group did not significantly differ from animals in the vehicle group during days 8 10, 11 14 and 15 19 of the experiment F urther analysis revealed that animals rece iving nicotine ate moderately fewer pellets compared to vehicle animals daily during the dark cycle (not shown in Figures) on days 10 and 12. The mean number of total pellets eaten in the light cycle by rats in the nicotine group was not significant from t he vehicle group during days 8 10 and but was significantly less during days 11 14 and 15 19 of the experiment F urther analysis revealed that animals receiving nicotine ate fewer pellets than vehicle animals daily in the light cycle (Figure 3 6) and this was significant on days 10, 11, 12, 13, 14, 15, 16, 17 and 19. Treatment meal size The results of all statistical calculations for means over the entire period are located in Table 3 3 and the means for each individual day can be found in Table 3 5 A n imals that received the nicotine ate smaller sized meals overall than those that received the vehicle (Figures 3 7, 3 8 and 3 9) T he treatment phase was separated into three groups following the two days of vehicle administration T he mean meal size eat en by rats in the nicotine group did not significantly differ from animals in the vehicle group during days 8 10 but did significantly differ during days 11 14 and 15 19 of the experiment F urther analysis revealed that animals receiving nicotine ate sign ificantly smaller meals than those that received the vehicle on days 10, 12, 15 and 19 and approached significance on days 14 and 18 T he mean meal size eaten in the dark cycle by rats in the nicotine group was significantly different from animals in the vehicle group during days 11 14 and 15 19 of the experiment, and approached significance during days

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82 8 10 F urther analysis revealed that animals receiving nicotine ate significantly smaller meals compared to nicotine animals daily during the dark cycle on day 10, 12, 14, 15, 18, and 19 and approached significance on day 11 T he mean meal size eaten in the light cycle by rats in the nicotine group was smaller than animals in the vehicle group during days 11 14 but did not differ during days 8 10 and 15 1 9 of the experiment F urther analysis revealed that animals receiving nicotine ate fewer meals compared to nicotine animals daily during the dark cycle and this was significant on days 11 and 19. Treatment number of meals The results of all statistica l calculations for means over the entire period are located in Table 3 3 and the means for each individual day can be found in Table 3 6 A nimals that received the nicotine did not differ in the number of meals eaten overall compared with those that recei ved the vehicle (Figures 3 10, 3 11, and 3 12) T he treatment phase was separated into three groups following the two days of vehicle administration T he mean number of meals eaten by rats in the nicotine group did not differ from animals in the vehicle group during days 8 10, 11 14, and 15 19 of the experiment F urther analysis revealed that animals receiving nicotine ate the same number of meals as animals receiving the vehicle for all days of the treatment phase T he mean number of meals eaten in the dark cycle by rats in the nicotine group was greater than animals in the vehicle group and this approached significance on days 11 14 and was significant during days 15 19 but not during days 8 10 of the experiment F urther analysis revealed that animals receiving nicotine ate more meals compared to nicotine animals daily during the dark cycle, and this was significant on days 14, 15 and 19 and approached significance on days 10 and 7 T he mean number of meals eaten in the light cycle by rats in the nico tine group was moderately less than animals in the vehicle group and approached significance during days 11 14 and 15 19, but not during days 8 10 of the experiment F urther analysis revealed that animals

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83 receiving nicotine ate fewer meals compared to nic otine animals daily during the dark cycle, and this was significant on days 13, 14, 16 and 17. Cessation The results of all statistical calculations for means over the entire period are located in Table 3 2 T he cessation phase was separated into three g roups following the treatment phase T here were no significant differences throughout cessation between the two groups in total pellet intake (Figures 3 2, 3 4 and 3 5) T his lack of difference was seen for the mean daily number of pellets eaten during d ays 24 27 and 28 31 of the experiment though there was a moderate difference on days 20 23 T he mean number of pellets eaten during the dark cycle did not differ between groups during days 20 23, 24 27, and 28 31 of the experiment The mean number of pell ets eaten during the light cycle did not significantly differ between groups during days 20 23, 24 27, and 28 31 of the experiment L ikewise, the mean meal size did not significantly differ between the two groups (Figures 3 7, 3 8, and 3 9) for the total mean meal size during days 17 20, 20 13, and 25 28 of the experiment T he mean meal size in the dark cycle did not significantly differ between groups during days 20 23, 24 27, and 28 31 of the experiment T he mean meal size in the light cycle did not si gnificantly differ between groups during days 20 23, 24 27, and 28 31 of the experiment S imilarly, there were no significant differences between the two groups in total meal intake (Figures 3 10, 3 11 and 3 12) T his similarity between groups was seen f or the total mean number of meals during days 17 20, 20 13 and 25 28 of the experiment T he mean number of meals in the dark cycle did not differ between groups during days 20 23, 24 27, and 28 31 of the experiment T he mean number of meals in the light cycle did not differ between groups during days 20 23, 24 27, and 28 31 of the experiment.

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84 Meal Patterns .03 mg/kg/inf Total pellets The results for all statistical calculations for means over the entire period are located in Table 3 7 T here was no si gnificant difference throughout baseline between animals that received the vehicle and those that received the .03 mg/kg/inf dose of nicotine in total pellet intake (Figure 3 13) T he treatment phase was separated into three groups following the two days of vehicle administration (days 6 7), days 8 10 when animals received.01 mg/kg/inf and days 11 14 and days 15 19 when the animals received their terminal dose of nicotine T he mean number of total pellets eaten by rats in the nicotine group was significan tly less than animals that received the vehicle during days 11 14 and 15 19 of the experiment, but not during days 8 10 T here was no significant difference throughout cessation for the mean number of pellets consumed by animals receiving the low dose of nicotine compared with those that received the vehicle. Night pellets The results for all statistical calculations for means over the entire period are located in Table 3 7 T here was no significant difference throughout baseline between animals that re ceived the vehicle and those that received the .03 mg/kg/inf dose of nicotine in total pellet intake during the dark cycle (Figure 3 14) T he treatment phase was separated into three groups following the two days of vehicle administration (days 6 7), days 8 10 when animals received.01 mg/kg/inf and days 11 14 and days 15 19 when the animals were receiving their terminal dose of nicotine T he mean number of total pellets eaten in the dark cycle by rats in the nicotine group was not significantly different than animals that received the vehicle during days 8 10 and 15 19 of the experiment, but did approach significance on days 11 14 T here was no significant

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85 difference throughout cessation for the mean number of pellets consumed in the dark cycle by animals receiving the low dose of nicotine compared with those that received the vehicle. Day pellets The results for all statistical calculations for means over the entire period are located in Table 3 7 T here was no significant difference throughout baselin e between animals that received the vehicle and those that received the .03 mg/kg/inf dose of nicotine in total pellet intake during the light cycle (Figure 3 15) T he treatment phase was separated into three groups following the two days of vehicle admin istration (days 6 7), days 8 10 when animals received.01 mg/kg/inf and days 11 14 and days 15 19 when the animals received their terminal dose of nicotine T he mean number of total pellets eaten in the light cycle by rats in the nicotine group was moderat ely lower than animals that received the vehicle on days 11 14, but not on days 8 10 and days 15 19 T here was no significant difference throughout cessation for the mean number of pellets consumed in the dark cycle by animals receiving the low dose of ni cotine compared with those that received the vehicle. Comparisons were also made between the animals that received the high or lose dose of nicotine and the vehicle (Figure 3 16) T here was no significant difference throughout baseline for animals that r eceived the vehicle or the high or low dose of nicotine T he treatment phase was separated into three groups following the two days of vehicle administration (days 6 7), days 8 10 where animals received.01 mg/kg/inf and days 11 14 and days 15 19 where the animals received their terminal dose of nicotine T he mean number of total pellets eaten in the light cycle by rats the received the high dose of nicotine was significantly lower than animals that received the vehicle on days 11 14, but not on days 8 10 and days 15 19 when all three groups were compared T here was no significant difference throughout cessation for the mean number of

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86 pellets consumed in the dark cycle by animals receiving the low or high dose of nicotine compared with those that received the vehicle. Excess Pellets The results of all statistical calculations for means over the entire period are located in Table 3 8 T here was no significant difference throughout baseline between the two group s in total excess pellet intake (Figure 3 17), e xcess pellet intake during the dark cycle (Figure 3 18) and excess pellet intake during the dark cycle (Figure 3 19) with animals in the nicotine and vehicle groups eating the same number of excess pellets T here was a significant difference during the tr eatment phase between the nicotine and vehicle groups overall and during the dark cycle, with nicotine animals consuming more excess pellets than the vehicle group, but there was no difference during the light cycle T hese differences disappeared during t he cessation phase, when animals in both groups consumed similar amounts of excess pellets. Day Only Meal Patterns Total pellets The results of all statistical calculations for means over the entire period are located in Table 3 9 T here was no significan t difference throughout baseline for the mean number of pellets consumed, for animals receiving the high dose of nicotine during the first part of the light 3 20) T he treatment phase was separated into three groups following the two days of vehicle administration (days 6 7), days 8 10 when animals received.01 mg/kg/inf and days 11 14 and days 15 19 when the animals received their terminal dose of nicotine T h e mean number of total pellets eaten by rats in the nicotine group was significantly less during the time of day when the animals did not receive nicotine than when the animals received nicotine during days 11 14 and 15 19 of the experiment but not during days 8 10 T here was no significant difference

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87 throughout cessation for the mean number of pellets consumed by animals receiving the high nicotine and the latt er part when they did. Number of meals The results of all statistical calculations for means over the entire period are located in Table 3 9 T here was no significant difference throughout baseline for the mean number of meals consumed, for animals receiving the high dose of nicotine during the first part of the light they did (Figure 3 21) T he treatment phase was separated into three groups following the two days of vehicle administration (days 6 7), days 8 10 where animals received .01 mg/kg/inf and days 11 14 and days 15 19 where the animals received their termina l dose of nicotine T he mean number of total meals eaten by rats in the nicotine group was not significantly different during the time of day when the animals did not receive nicotine than when the animals received nicotine during days 9 11, 11 14 and 15 19 of the experiment T here was no significant difference throughout cessation for the mean number of meals consumed, for animals receiving the high dose of the latter part wher e they did. Meal size The results of all statistical calculations for means over the entire period are located in Table 3 9 T here was no significant difference throughout baseline for the mean meal size consumed, for animals receiving the high dose of n icotine during the first part of the light cycle 3 21) T he treatment phase was separated into three groups following the two days of vehicle administration (days 6 7), days 8 10 where animals received.01 mg/kg/inf and days 11 14 and

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88 days 15 19 where the animals received their terminal dose of nicotine T he mean meal size eaten by rats in the nicotine group was significantly less during the time of day when the anim als did not receive nicotine than when the animals received nicotine during days 8 10, 11 14 and 15 19 of the experiment. Discussion The current study was able to successfully replicate some of the major findings of past research on the relationship betwee n nicotine and food intake as well as uncover new and important findings S imilar to past studies that administered nicotine to animals via osmotic minipumps (Grunberg and Bowen 1985; Grunberg et al. 1985; Grunberg et al. 1986; Miyata 2001), animals in th e pres ent study showed a dose dependent decrease in weight gain T his decreased weight gain was persistent throughout the nicotine treatment phase in a manner similar to that reported in animals receiving IP injections of nicotine (Kane et al. 2000; Li et al. 2000; Bellinger et al. 2003 a & b; Guan et al. 2004) I n contrast to some studies using the IP injections (i.e Bellinger et al. 2003 b ) animals in the present study demonstrated a rebound in weight gain that occurred about a week after nicotine was re moved H owever, it should be noted that the weight gain observed was not as dramatic as we expected, nor was the change in weight gain immediate as seen in Dandekar et al. ( 2011 ) I nvestigations into the time course of post cessation weight gain in human s have yielded somewhat inconsistent results, with weight gain occurring anytime from a couple of weeks to 6 months later ( as reviewed by Perkins 1993) T seen du ring smoking in humans and most likely the pattern of weight gain after quitting as well F continue to monitor weight gain for longer than the 12 days in the current study.

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89 meal patterns I n the present study, animals showed decreases in total food intake during the entire treatment phase and then increases in food intake bac k to control levels post cessation T hese results are in contrast to those obtained from studies using osmotic minipumps which found only temporary decreases in food intake which then returned back to control levels, despite continuous infusions of nicoti ne (Miyata et al.1999 & 2001) T he present study also demonstrated an eventual increase in food intake at the end of the 12 days of monitoring T hese increases in total food intake did not occur right away, and this effect most likely accounts for the pe rsistent difference in weight gain between nicotine and control animals that occurred in the first week after cessation S tudies using minipumps or IP injections in contrast, have reported increases in food intake post cessation which occur immediately an d are significantly greater than intake by controls (Miyata et al. 2001; Dandekar et al. 2011) I nvestigations into weight gain after smoking in humans have typically revealed a sharp and sudden increase in food intake in the weeks following cessation, al though how immediate this effect is remains unclear ( as reviewed by Perkins 1993) T aken together, the weight gain and total food intake post cessation observed in the present study may more likely replicate the evidence for increased eating and weight ga in in humans, rather than the gain and intake seen in other animal experiments which are well above control and baseline levels. In line with studies which administered nicotine via IP injections, the current study found specific effects which occurred in the dark cycle T hough pellet intake during the dark cycle was not significantly different from controls, it did trend towards being lower S imilar to the previous studies using IP injections, the current study showed a decrease in meal size and a compe nsatory increase in meal number during nicotine administration, which was confined to

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90 the dark cycle T his effect seems to occur as a way for the animals to maintain consistent total food intake in light of the increased satiety produced by nicotine I n addition to these effects which replicate work from other studies, we also found an increase in pellet intake outside of the meal structure which was confined to the dark cycle T reflect increased motor activity that is often seen in conjunction with nicotine administration (Clark and Kumar 1983; Benwell and Balfour 1992; Corrigall and Coen 1994) B ecause we sure S ince we did not look to see if there were increases in excess pellets during the 3 hours of the light cycle where nicotine was administered compared to the 8 hours it was not, we cannot rule out that this effect was due to nicot I t is possible instead that this increase in pellets in the dark cycle is another compensatory mechanism similar to account for the s L astly, this effect may simply be an artifact of our meal criteria; many other studies define a meal as 3 or more pellets in a given period of time (i.e. Guan et al. 2004), whereas our lab uses the more stringent criteria of 4 pellets every 10 minutes I be translated into meals which would then simply contribute to the increase in meal number that we already observe d Importantly howev er, the current study also found effects of nicotine on meal size and total pellet intake in the light cycle that have not been previously reported A nimals receiving nicotine ate smaller meals in the light cycle, and therefore their total pellet intake i n the light cycle was also lower I n contrast with meal pattern changes in the dark cycle, there was no compensatory increase in meal number seen in the light cycle.

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91 The effects seen in the light cycle may be caused by a variety of factors A s opposed t o which administered nicotine to animals in a portion of the light cycle E ffects of constant examined light v. dark effects, and experiments in which IP injections were used only administered nicotine a few times throughout the dark cycle H uman smokers do not confine their smoking to the daylight, and therefore an animal model should not restri ct administration to the dark cycle, but should instead administer nicotine during the period of greatest activity I n light of the present results, this protocol seems crucial in parsing out some important effects which may be missed in other studies: na mely, the persistent decrease in food intake in the light cycle without any compensatory mechanism W hereas total pellet intake in the dark cycle was not significant due to the increase in meal number meant to compensate for the decrease in meal size, ani mals significantly reduced their intake in the light cycle and this effect largely accounted for the decrease in body weight that was also observed. Previous work from our lab indicates that when nicotine and food are simultaneously available, rats will c luster nicotine self administration and food intake together I n the present study we examined total pellet intake, meal number, and average meal size in the light cycle during the first 8 hours when animals were not receiving infusions of nicotine and th e last 3 hours when they received infusions every 30 minutes B ecause the last 3 hours of the light cycle are when animals are most active, they seem to eat a larger proportion of their food during that time T his is evidenced by the fact that there is n o significant difference between the first 8 hours and the last 3 hours in total food intake, meal number, and meal size D uring nicotine administration, food intake during both periods exhibit a downward trend, but food intake

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92 decreased less in the perio d of time when animals receive d nicotine T hese results, combined with other studies in our lab, show that animals prefer to eat around the time they also receive nicotine T herefore, when animals are not receiving nicotine infusions, they are eating les s, and this at least partially explains why we see an effect of nicotine on food intake in the light cycle that has not be previously reported by other groups. From an evolutionary standpoint, the effects observed in the current study in the light cycle m ake sense A s summarized by Strubbe and Woods (2004) in an article on the timing of meals, nocturnal fee d ing in rats occurs as a response to an aversion to the light itself as well as for the purpose of avoiding predators E ating is reduced in the light cycle in nocturnal animals for the purpose of minimizing interaction with predators and maximizing the availability of food I t stands to reason then, that any changes in meal patterns precipitated by nicotine would occur in the light cycle, where it is m ost advantageous to eat less, rather than in the dark cycle where food availability is greater and risk is lessened. In light of the results in the current study which show increased eating while animals receive nicotine and increased excess pellet intake in the dark cycle, it is possible that animals eat less in the light cycle simply because they are sleeping during that time N icotine functions as a stimulant, and if animals are receiving nicotine every 30 minutes, and eating more frequently, there is most likely little time for them to sleep I t is known that acetylcholine plays a role in sleep and that nicotine administration alters sleep S pecifically, nicotine dose dependently decreases REM and slow wave sleep and increases wakefulness in rats, ef fects which are mediated by nAChRs (Salin Pascual et al. 1999) I n light of the ability of nicotine infusions to decrease total sleep time, it is realistic to suppose that animals are sleeping during the period of time when they are no longer receiving in jections of the drug L evels of activity

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93 and sleep patterns were not measured in this study however, so we cannot be sure if this is the case. As discussed in Chapter 1, nicotine may be affecting food intake and meal patterns via changes in lipolysis, th e mobilization of previously stored f at. Extensive work by l e Magnen (1985) has shown that lipolysis is increased during the light cycle in rodents, such that weight gained during the nighttime is lost during the day L ipogenesis, the synthesis of fats, an increased respiratory quotient, and increased feeding rates mediated by increased sensitivity to glucose, all occur in the dark cycle T herefore, if nicotine is increasing lipolysis via changes in glucose or insulin, and lipolysis occurs during the day then rats should eat less during the light cycle which is exactly what we observed in the current study. In the present study, we gave animals intravenous nicotine every 30 minutes throughout the dark cycle and the last 3 hours of the light cycle and r ecorded changes in body weight and food intake T new results that have not otherwise been detected T hese effects illustrate the validity and sensitivity of the model to detect new effects as well as replicate the most important and consistent effects from previous experiments.

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94 Table 3 1. P and F values for the changes in body weight over each day of the baseline, treatment, and cessation phases Day Body Weight Day Body Weight Baseline F P 1 5 .763 ns Treatment Cessation F P 1 .029 ns 14 5.724 **P < .01 2 .138 ns 15 4.861 *P < .05 3 .151 ns 16 5.634 *P < .05 4 .175 ns 17 5.925 **P < .01 5 .717 ns 18 5.426 *P < .05 6 1.651 ns 19 4.620 *P < .05 7 2.990 ns 20 3.654 *P < .05 8 3.863 *P < .05 21 4.603 *P < .05 9 4.060 *P < .05 22 .627 ns 10 4.481 *P < .05 23 .479 ns 11 4.021 *P < .05 24 .528 ns 12 4.161 *P < .05 25 .545 ns 13 4.309 *P < .05

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95 Table 3 2. F and P values for all meal pattern variables of interest, with days clustered into groups, throughout the baseline and cessation phases for the vehicle and high nicotine groups Baseline Cessation Variables Days 1 5 Days 20 23 Days 24 27 Days 28 31 F P F P F P F P Total Pellets .272 ns 3.260 ^P = .09 2.023 ns 2.672 ns Night Pellets .300 ns .242 ns .514 ns 2.089 ns Day Pellets .025 ns 1.775 ns 2.312 ns .134 ns Total Meals .045 ns .467 ns 1.483 ns .327 ns Night Meals .058 ns .071 ns .048 ns .007 ns Day Meals .354 ns .345 ns 3.107 ns .633 ns Total Meal Size .222 ns .327 ns .001 ns 2.308 ns Night Meal Size .000 ns .042 ns .066 ns 2.965 ns Day Meal Size .102 ns 1.407 ns .077 ns .000 ns Table 3 3. F and P values for all meal pattern variables of interest, with days clustered into groups, throughout the treatment phase for the vehicle and high nicotine groups Treatment Variables Days 6 7 Days 8 10 Days 11 14 Days 15 19 F P F P F P F P Total Pellets .380 ns 5.071 *P<.05 13.310 *P < .05 11.672 *P < .05 Night Pellets .885 ns 2.226 ns 1.993 ns .693 ns Day Pellets .015 ns .175 ns 19.695 **P<.01 9.281 **P< .01 Total Meals .382 ns .271 ns .021 ns .009 ns Night Meals 1.432 ns 1.232 ns 3.703 ^P = .07 6.647 *P < .05 Day Meals .092 ns .001 ns 3.545 ^P = .08 3.905 ^P = .07 Total Meal Size .025 ns 1.493 ns 7.679 *P < .05 5.319 *P < .05 Night Meal Size .002 ns 4.300 ^P=.06 7.226 *P < .05 8.485 *P < .05 Day Meal Size .004 ns .008 ns 7.707 *P < .05 1.793 ns

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96 Table 3 4. P and F values for the mean number of pellets eaten per day, during the treatment phase Day Total Pellets Night Pellets Day Pellets F P F P F P 6 .102 ns .210 ns .001 ns 7 .814 ns 1.180 ns .044 ns 8 .096 ns .033 ns .000 ns 9 1.211 ns 1.865 ns .252 ns 10 9.804 **P < .01 3.300 ^P = .09 4.011 ^P = .06 11 4.523 ^P = .05 3.163 ns 6.437 ^P = .05 12 13.710 **P < .01 3.344 ^P = .09 15.132 **P < .01 13 10.037 **P < .01 .600 ns 25.496 **P < .01 14 4.009 ^P = .06 .249 ns 10.823 **P < .01 15 6.596 *P < .05 .589 ns 8.036 *P < .05 16 5.201 *P < .05 .015 ns 13.607 **P < .01 17 4.094 ^P = .06 .011 ns 9.226 *P < .05 18 3.111 ns 1.144 ns 1.162 ns 19 15.136 **P < .01 1.845 ns 5.106 ^P = .05

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97 Table 3 5. P and F values for the mean meal size eaten per day, during the treatment phase Day Total Meal Size Night Meal Size Day Meal Size Treatment F P F P F P 6 .083 ns .464 ns .185 ns 7 .703 ns 1.030 ns .359 ns 8 .883 ns .709 ns 1.359 ns 9 .166 ns .776 ns 1.100 ns 10 4.815 *P < .05 8.910 **P < .01 .065 ns 11 2.466 ns 4.453 ^P = .05 .953 *P < .05 12 5.182 *P < .05 4.767 *P < .05 4.500 ns 13 3.071 ns 3.062 ns 1.275 ns 14 3.914 ^P = .07 4.873 *P < .05 1.617 ns 15 5.388 *P < .05 8.476 *P < .05 .027 ns 16 .175 ns .518 ns .183 ns 17 .812 ns 1.838 ns .045 ns 18 4.329 ^P = .06 8.247 *P < .05 .095 ns 19 15.233 **P < .01 10.056 **P < .01 7.108 **P < .01 Table 3 6. P and F values for the mean number of meals eaten per day, during the treatment phase Day Total Meals Night Meals Day Meals Treatment F P F P F P 6 1.142 ns 3.204 ^P = .09 .166 ns 7 .007 ns .000 ns .017 ns 8 1.057 ns .497 ns .735 ns 9 .000 ns .067 ns .031 ns 10 .178 ns 4.486 ^P = .05 1.132 ns 11 .055 ns .700 ns 1.126 ns 12 .089 ns .966 ns 1.579 ns 13 .196 ns 1.889 ns 4.940 ^P = .05 14 .669 ns 9.942 **P < .01 5.027 ^P = .05 15 1.585 ns 15.467 **P < .01 2.883 ns 16 .361 ns .790 ns 3.278 ^P = .08 17 .027 ns 2.058 ns 6.669 ^P = .05 18 .356 ns 3.814 ^P = .07 1.450 ns 19 .423 ns 4.835 *P < .05 .929 ns

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98 Table 3 7. P and F values for the mean number of pellets eaten, with days clustered into groups, throughout the baseline, treatment and cessation phases for the vehicle and low nicotine groups Days Total Pellets Night Pellets Day Pellets Baseline F P F P F P 1 5 .003 ns .012 ns .126 ns Treatment 6 7 .435 ns 1.834 ns .298 ns 8 10 2.891 ns 2.283 ns .006 ns 11 14 18.972 **P < .01 3.985 ^P = .06 5.563 *P < .05 15 19 17.671 **P < .01 1.124 ns 2.353 ns Cessation 20 23 1.637 ns .872 ns .182 ns 24 27 .331 ns .741 ns .329 ns 28 31 .495 ns .341 ns .464 ns

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99 Table 3 8. P and F values for the mean number of excess pellets eaten, with days clustered into groups, throughout the baseline, treatment and cessation phases for the vehicle and high nicotine groups Time Point Experimental Phase/Day Total Night Day Baseline F P F P F P 1 5 2.450 ns .541 ns 1.406 ns Treatment 6 7 .436 ns .001 ns 2.576 ns 8 10 1.567 ns 1.086 ns .925 ns 11 14 8.679 **P < .01 10.423 **P < .01 2.546 ns 15 19 4.467 ^P = .05 6.197 *P < .05 .490 ns Cessation 20 23 .001 ns .307 ns .100 ns 24 27 .134 ns .022 ns .386 ns 28 31 .222 ns 2.776 ns .015 ns Table 3 9. P and F values for all meal pattern variables of interest, with days clustered into groups, throughout the baseline, treatment and cessation phases for the nicotine group during the light cycle when they received either the high dose of nicotine or no nicotine Day Data Experimental Phase/Day Pellets Meals Meal S ize Baseline F P F P F P 1 5 2.209 ns 1.045 ns 1.220 ns Treatment 6 7 .691 ns 2.108 ns 1.052 ns 8 10 2.167 ns .004 ns 5.557 *P < .05 11 14 4.283 ^P = .06 2.750 ns 7.081 *P < .05 15 19 6.445 *P < .05 3.131 ns 6.141 *P < .05 Cessation 20 23 .007 ns .148 ns .045 ns 24 27 .806 ns .172 ns .250 ns 28 31 .046 ns .033 ns .000 ns

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100 Figure 3 1. Nicotine: body weight change over treatment and cessation Shown are mean +/ SE cumulative change in body weight (g) on consecutive days of treatment and cessation phases, in animals that received either vehicle (n = 9), 0.03 mg/kg/inf of nicotine (n = 8), or 0.06 mg/kg/inf of nicotine (n = 8). ^P = .07, *P < 0.05, *P < 0.01 difference between vehicle and 0.06 m g/kg/inf nicotine

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101 Figure 3 2. Nicotine: total number of pellets Shown are mean +/ SE total lever presses for pellets, averaged over the ranges of days indicated, i n animals that received either vehicle (n = 9) or 0.06 mg/kg/inf of nicotine (n = 8) during the treatment phase. ^P = .07, *P < 0.05, **P < 0.01 difference between vehicle and nicotine

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102 Figure 3 3. Nicotine : total pellets per day S hown are number of lever presses on consecutive days of baseline and treatment phases, by individual rats that received either vehicle (n = 9) or .06 mg/kg/inf nicotine (n = 8) during the treatment phase L ines show the group me ans T hese means were significant on days 10, 11, 12, 13, 15, 16, and 19, *P < .05, ** P < .01 and approached significance on days 14 and 17, ^P = .06, difference between vehicle and nicotine

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103 Figure 3 4. Nicot ine: total pellets dark cycle S hown are mean +/ SE lever presses for pellets during the 12 hours of the dark cycle, averaged over the ranges of days indicated, in animals that received either vehicle (n = 9) or .06 mg/kg/inf nicotine (n = 8) during th e treatment phase

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104 Figure 3 5. Nicotine: total pellets light cycle S hown are mean +/ SE lever presses for pellets during the 11 hours of the light cycle, averaged over the ranges of days indicated, in animals that received either vehicle (n = 9) or .06 mg/kg/inf nicotine (n = 8) during the treatment phase. **P < 0.01 difference b etween vehicle and nicotine

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105 Figure 3 6. Nicotine: total pellets per day light cycle S hown are number of lever presses during the 11 hours of the light cycle, on consecutive days of baseline and treatment phases, by individual rats that received either vehicle (n = 9) or .06 mg/kg/inf (n = 8) during the treatment phase L ines show the group means T hese means were significant on days 11, 12, 13, 14, 15, 16, 17 and 19, *P < .05, ** P < .01 and approached significance on day 10, ^P = .06, difference between vehicle and nicotine

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106 Figure 3 7. Nicotine: total meal size S hown are mean +/ SE total number of pellets per meal, averaged over the ranges of days indicated, in animals that received either vehicle (n = 9) or .06 mg/kg/inf nicotine (n = 8) during the treatment phase. *P < 0.05 difference between vehicle and nicotine

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107 Figure 3 8. Nicotine: total meal size dark cycle S hown are mean +/ SE total number of pellets per meal during the 12 hours of the dark cycle, averaged over the ranges of days indicated, in animals that received either vehicle (n = 9) or .06 mg/k g/inf nicotine (n = 8) during the treatment phase. ^ P = .06, *P < 0.05 difference between vehicle and nicotine

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108 Figure 3 9. Nicotine: total meal size light cycle S hown are mean +/ SE total number of pel lets per meal during the 11 hours of the light cycle, averaged over the ranges of days indicated, in animals that received either vehicle (n = 9) or .06 mg/kg/inf nicotine (n = 8) during the treatment phase. **P < 0.01 difference between vehicle and n icotine

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109 Figure 3 10. Nicotine: total number of meals S hown are mean +/ SE total number of meals, averaged over the ranges of days indicated, in animals that received either vehicle (n = 9) or .06 mg/kg/inf ni cotine (n = 8) during the treatment phase

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110 Figure 3 11. Nicotine: total number of meals dark cycle S hown are mean +/ SE number of meals during the 12 hours of the dark cycle, averaged over the ranges of days indicated, in animals that received either vehicle (n = 9) or .06 mg/kg/inf nicotine (n = 8) during the treatment phase. ^P = .07, **P < 0.01 differen ce between vehicle and nicotine

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111 Figure 3 12. Nicotine: total number of meals light cycle S hown are mean +/ SE number of meals during the 11 hours of the light cycle, averaged over the ranges of days indicated, in animals that received either vehicle (n = 9) or .06 mg/kg/inf nicotine (n = 8) during the treatment phase. ^P = 0.08 for days 12 15 and ^P = 0.07 for days 16 20 difference between vehicle and nicotine

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112 Fi gure 3 13. Nicotine: total pellets .03 mg/kg/inf S hown are mean +/ SE total lever presses for pellets, averaged over the ranges of days indicated, in animals that received either vehicle (n = 9) or 0.03 mg/kg/inf of nicotine (n = 8) during the treatme nt phase. **P < 0.01 difference between vehicle and nicotine

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113 Figure 3 14. Nicotine: total pellets dark cycle .03 mg/kg/inf S hown are mean +/ SE lever presses for pellets during the 12 hours of the dark cycle, averaged over the ranges of days indicated, in animals that received either vehicle (n = 9) or .03 mg/kg/inf nicotine (n = 8) during the treatment phase. ^P = 0.06 difference bet ween vehicle and nicotine

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114 Figure 3 15. Nicotine: total number of pellets light cycle .03 mg/kg/inf S hown are mean +/ SE lever presses for pellets during the 11 hours of the light cycle, averaged over the ra nges of days indicated, in animals that rece ived either vehicle (n = 9) or 03 mg/kg/inf nicotine (n = 8) during the treatment phase. *P < 0.05 difference between vehicle and nicotine

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115 Figure 3 16. Nicotine: total number of pellets light cycle all doses S hown are mean +/ SE lever presses for pellets during the 11 hours of the light cycle, averaged over the ranges of days indicated, in animals that received either vehicle (n = 9), .03 mg/kg/inf nicotine (n = 8), or .06 mg/kg/inf nicotine (n = 8) during the treatment phase. **P < 0.01 difference between vehicle and nicotine

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116 Figure 3 17. Nicotine: total excess pellets S hown are mean +/ SE number of lever presses (for excess pellets) averaged over the ranges of days indicated, in animals that received either vehicle (n = 9) or .06 mg/kg/inf nicotine (n = 8) during the treatment phase. *P < 0.05, **P < 0.01 difference between vehicle and nicotine

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117 Figure 3 18. Nicotine: total excess pellets dark cycle S hown are mean +/ SE number of lever presses (for excess pellets) during the 12 hours of the dark cycle, averaged over the ranges of days indicated, in animals that r eceived either vehicle (n = 9) or .06 mg/kg/inf nicotine (n = 8) during the treatment phase. *P < 0.05, **P < 0.01 difference between vehicle and nicotine

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118 Figure 3 19. Nicotine: total excess pellets light cycle S hown are mean +/ SE number of lever presses (for excess pellets) during the 11 hours of the light cycle, averaged over the ranges of days indicated, in animals that received either vehicle (ns = 9) or .06 mg/kg/inf nicotine ( n = 8) during the treatment phase

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119 Figure 3 20. Nicotine: total pellets light only S hown are mean +/ SE total lever presses for pellets during the first 8 hours of the light cycle (without nicotine) and the last 3 hours of the light cycle (with nicotine), averaged over the ranges of days indicated, in animals that received .06 mg/kg/inf nicotine (n = 8) during the treatment phase. *P < 0.05, ^P = .06, difference between the time with nicotine and without

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120 Figure 3 21. Nicotine: t otal number of meals light only S hown are mean +/ SE number of meals during the first 8 hours of the light cycle (without nicotine) and the last 3 hours of the light cycle (with nicotine), averaged over the ranges of days indicated, in animals that received .06 mg/kg/inf nicotine (n = 8) during the treatment phase

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121 Figure 3 22. Nicotine: Total meal size light only S hown are mean +/ SE number of pellets per meal during the first 8 hours of the light cycle (without nicotine) and the last 3 hours of th e light cycle (with nicotine), averaged over the ranges of days indicated, in animals that received .06 mg/kg/inf nicotine (n = 8) during the treatment phase. *P < 0.05 difference between the time with nicotine and without

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122 CHAPTER 4 THE EFFECTS OF CYTISINE ON BODY WEIGHT AND MEAL PATT ERNS Introduction As previously mentioned in Chapter 1, a majority of smokers express a desire to quit smoking (>70%), yet very few are successful long term (~5%) M any individuals who attempt to quit do so using one of the approved smoking cessation drugs T his so group of medications includes nicotine replacement therapies (NRTs), brupropion, and varenicline (Polosa and Benowitz 2011) T nortript yline and clonidine which are not approved for smoking cessation, but are given when the first line drugs are either unsuccessful or not well tolerated (Polosa and Benowitz 2011) C urrently, researchers are also working to develop and test new medications (i.e M cRobbie et al. 2010; Tonstad et al. 2010), and vaccines (i.e C ornuz et al. 2008; deVilliers et al. 2010). Nicotine is the primary psychoactive ingredient in cigarettes and serves as a reinforcer for both humans and animals (Henningfield and Keen an 1993) T he rationale behind the development of NRTs therefore, was that the administration of nicotine would alleviate withdrawal symptoms and craving without the negative health effects of cigarette smoking, thereby helping people remain abstinent ( as reviewed by Stead et al. 2008) A ccording to the five approved NRTs for use in smoking cessation: skin patches, chewing gum, nasal spray, inhalers, and lozenges/ tablets, all of which increase the rates of quitting long term by 50 70% in people with a strong desire to quit T hough these methods differ in ease of use, cost, and adherence to treatment, there is no NRT that stands out as being better than the rest (K ralikove et al. 2009) S ome smokers however, express an inability and unwillingness to quit A new

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123 over an extended period of time by allowing them to use NRTs wh ile they smoke, which results in a moderate improvement in long term cessation (Wang et a. 2008; Kralikova et al. 2009) T ypical adverse events associated with NRT use include heart palpitations and chest pains, nausea and vomiting, gastrointestinal compl aints, and insomnia (Mills et al. 2010). The speed with which an NRT can satisfy nicotine cravings depends largely on its pharmacokinetics (Henningfield et al. 1993; McRobbie et al. 2010) T hose that deliver nicotine more rapidly and are absorbed more qu ickly provide the fastest relief (McRobbie et al. 2010) T he skin patch and gum both have slower kinetics whereas the nasal spray more closely mimics the kinetics of cigarette smoking (Henningfield and Keenan 1993) T here are two new NRTs that are curren tly undergoing randomized trials: a mouth spray and a Sweetmint lozenge M cRobbie et al. (2010) found that although all the NRTs tested provided relief from craving within an hour, the mouth spray reduced cravings within the first 5 minutes and was rated as being more helpful than the gum T hese benefits of the mouth spray are most likely due to the fast buccal absorption of nicotine, as opposed to the slower absorption rates of other NRTs B ecause of these differences in drug kinetics, research is curre ntly being conducted on the effectiveness of combining NRTs to result in long term abstinence C oncurrent administration of a long acting NRT such as the patch with the short term impromptu use of NRTs like the nasal spray or inhaler may increase blood ni cotine levels and promote abstinence (Ebbert et al. 2010). In light of the concern many smokers have with weight gain, it is important to look for a medication for smoking cessation which will not only mitigate craving and withdrawal symptoms, but will al so prevent weight gain A s summarized in Chapter 1, NRTs are relatively

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124 unsuccessful at preventing weight gain in ex smokers, with only long term gum users showing a modest decrease in weight gain ( as reviewed by Klesges and Meyers 1989). There are currently two non nicotine pharmocotherapies approved by the FDA for smoking cessation: bupropion and varenicline B upropion is the only antidepressant approved as I t acts as a dopamine a nd norepinephrine reuptake inhibitor and an antagonist for nicotinic acetylcholine receptors and decreases withdrawal symptoms via these mechanisms (Polosa and Benowitz 2011) S ome studies have found a moderate improvement of bupropion over the patch (i.e Jorenby et al. 1999), whereas others have found equal efficacy for both NRTs and bupropion (Hughes et al. 2003) T hough bupropion doubles the odds of quitting, individuals who take the drug are no more likely to remain abstinent after 1 year than those who took nothing ( as reviewed by Hughes et al. 2003 and Tong et al. 2006) T he most common side effects of bupropion are dry mouth, insomnia and nausea and it works equally well for people with or without a history of depression ( as reviewed by Hughes et al. 2003 and Tong et al. 2006). Research investigating the combination of bupropion and NRTs has yielded moderately significant results J orenby et al. (1999) found that the co administration of bupropion and the skin patch were higher than the patch or placebo alone and trended toward being higher than bupropion alone, although this was not significant S imon et al. (2004) reported that the skin patch did not increase quit rates when combined with bupropion A n additional benefit to the co administrat ion of bupropion and NRT may be decreased weight gain post cessation J orenby et al. ( 1999 ) found that individuals trying to quit who used the combination therapy gained less weight (1.1kg) than individuals who received the placebo (2.1kg) I ndependently bupropion may also cause a slight decrease in food intake (~15%) in rodents ( Bruijnzeel and Markou 2003)

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125 F Chapter 1. The second FDA approved non nicotine pha rmacotherapy offered is varenicline, a partial primary reinforcing effects in the brain (Polosa and Benowitz 2011) V arenicline also binds to 4 as reviewed by Cunningham and McMahon 2011) V a both agonist and antagonist properties: varenicline partially stimulates nAChRs and al so blocks these receptors via competition with nicotine ( as reviewed by LeSage et al. 2009) T hese properties allow varenicline to reduce craving and withdrawal symptoms as well as reduce smoking satisfaction by minimally activating the receptors and kee ping them occupied so as to prevent nicotine from binding to them ( as reviewed by Polosa and Benowitz 2011) I ndividuals taking varenicline are allowed to continue smoking, and this combination of nicotine and varenicline results in a 60 80% reduction of cigarettes smoked in the first 2 4 days ( as reviewed by Ebbert et al. 2010). Unlike NRTs and bupropion which demonstrate equal effectiveness, varenicline seems to be superior in helping people quit smoking, with healthy smokers having a greater chance of quitting on varenicline than placebo (~2.5 times greater) or bupropion ( ~ 1.7 times greater; as reviewed by Polosa and Benowitz 2011) L ikewise, the long term abstinence rates of individuals who have taken varenicline appear to be better than those given p lacebo or bupropion (Sofuoglu et al. 2011) A nimal studies support the effectiveness of varenicline as well A nimals given varenicline show decreased nicotine self 2011), partial substitution for nicotine in drug discrimination tasks (LeSage et al. 2009)

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126 attenuation of nicotine enhanced brain stimulation (Spiller et al. 2009), and decreased nicotine T he most common side effects of varenicline are nausea (Sofuoglu et al. 2011), insomnia, gastrointestinal complains, and headaches ( as reviewed by Polosa and Benowitz 2011). The aforementioned decreases in nicotine self administration and reinstatement in animals given varenicline were also accompanied by decreases in food intake at the highest dose (3.0 mg/kg), though this effect gradually decreased over the course 2010) H owever, additional evidence that varenicline effects food intake is sparse F or example, a study examining the behavioral effects of varenicline in humans did not find a change in caloric intake or number of items c onsumed in those given an acute dose of the drug (Stoops et al. 2008) S ood et al. (2009) examined weight gain in individuals taking a combination of varenicline and the weight loss drug sibutramine, and found an attenuation of weight gain in many of thei r subjects that was less than reported levels of weight gain in studies with varenicline only. dianicline and cytisine U nlike varenicline, dianicline appears to have onl y minimal clinical efficacy (Tonstadt et al. 2011), at least in part due to its limited ability to desensitize these receptors (Rollema et al. 2010). Cytisine on the other hand, has been used as a treatment for smoking cessation in east and central Europe an countries for the last 50 years (Etter et al. 2008) L ack of published clinical trials in English has recently prompted a slew of experiments in the United States and other English speaking countries examining the efficacy of cytisine in smoking cessat ion C ytisine originally comes from the golden rain acacia (cytisus laburnum) plant, and is relatively inexpensive A course of treatment in Poland costs approximately $15 and is roughly

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127 $6 over the counter in Russia (West et al. 2011) I n contrast, an 8 week course of NRT is approximately $230, an 8 week course of bupropion is $123 and a 12 week course of varenicline is $327 in China, whereas a pack of cigarettes only costs about 73 cents (West et al. 2011) C onsidering approximately 67% of smokers liv e in countries where the monthly income is less than $800 a month, the opportunity for an inexpensive treatment for cigarette smoking would have the potential to reach millions (Etter et al. 2008; West et al. 2011). Similar to varenicline, cytisine binds as reviewed by Tutka et al. 2006 ) C ytisine binds as reviewed by Tutka et al. 2006 ) H owever, the amount of cytisine that actually enters the brain is relat ively small when compared to varenicline, for example (Reavill et al. 1990; Rollema et al. 2010), so treatment with cytisine involves frequent dosing, around 1.5 mg six times a day ( as reviewed by Etter et al. 2008) W hereas varenicline concentrations are sufficient to result in low, sustained possible differential effects of the drugs on craving and alleviation of withdrawal symptoms ( as reviewed by Tutka et al. 200 6 ; Rollema et al. 2010) I n light o f the different pharmacodynamic and pharmacokinetic profiles between the two drugs, this analog to varenicline results in lower abstinence rates compared with varenicline H owever, cytisine has been found to h ave comparable quit rates to NRTs and bupropion (Walker et al. 2011; West et al. 2011), making it T he most

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12 8 common side effects associated with cytisine are gastrointestinal complaints, such as stomachaches, nausea, and dry mouth (West et al. 2011). Many of the experiments examining the relative effectiveness of cytisine on smoking ce, and many of them lack proper placebo controls and have other methodological issues ( as reviewed by Walker et al. 2011) A meta analysis of three placebo controlled studies conducted during this time revealed that cytisine doubled the odds of quitting compared to placebo (Etter et al. 2008) I n addition to the study mentioned previously comparing cytisine to NRT and the experiments conducted over 40 years ago, only one other double blind, placebo controlled experiment has been conducted V innikov et a l. (2008) found more abstinent individuals in the cytisine group (10.6%) at six months compared to those that had received the placebo (1.2%), with no changes in weight gain in either group. Behavioral studies in animals support the idea that cytisine is similar in action to nicotine, though there are a lack of studies in animals examining the relative reinforcing and rewarding properties of the drug ( as reviewed by Etter et al. 2008) L imited evidence indicates that mice will acutely self administer cyti sine (Rassmussen and Swedberg 1998) and cytisine injections into the ventral tegmental area result in a conditioned place preference (Museo and Wise 1994) D rug discrimination tests indicate that cytisine can substitute for nicotine from 20% (LeSage et al 2009) to 65% (Reavill et al. 1990). sparse A summary of commonly reported metabolic complaints found slight increases in reported weight gain and appetite in those taking cytisine, however these changes could be attributable to smoking cessation rather than cy tisine itself (Tutka et al. 2006 ) M ineur et al.

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129 in both during the course of cytisine administration T hese changes in food intake were not types were accompanied by increases in cFos expression of POMC neurons in the arcuate nucleus of the hypothalamus T hese findings led the authors to conclude that cytisine may exert ht gain and meal patterns in rats using a similar procedure described in Chapter 3 B ecause of the associations between socioeconomic status, obesity, and smoking (Swinburn et al. 2004; West et al. 2011), rug that is inexpensive, promotes abstinence, and also attenuates post cessation weight gain I n light of the results described in Mineur et al. (2011), we hypothesized that if cytisine is in fact exerting a direct effect on POMC neurons in the hypothalamus, it should affect satiety mechanisms (see Chapter 1 for details on POMC and satiety) T herefore we hypothesized that noncontingent infusions of the drug would result in decreased food intake via decreases in meal size, a hallmark of satiety, in a manner similar to what we observed with nicotine. Materials and Methods Animals and Housing Fifteen male Sprague Dawley rats initially weighing ~275 g were used in the present study. All rats were purchased from Harlan ( Indianapolis, IN ). The prin ciples described in the Guide for the Care and Use of Laboratory Animals were followed throughout, and the project was approved by the UF IACUC. Upon arrival, rats were housed individually in a conventional vivarium that was maintained at 22 26 o C and 40 8 0% relative humidity, on a reverse 12:12 hr light:dark cycle (lights off at 10:00AM). Home cages were of polycarbonate, with Sani Chips

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130 contact bedding. Purina 5001 Chow pellets were available ad libitum for the initial portion of the experiment when ani mals were housed in their home cages D uring the test phase, animals were housed in operant chambers A utoclaved tap water was available at all times from a standard bottle and sipper tube. A reverse light cycle (on: 2200 1000 h) was in effect in both t he vivarium and the operant chambers. Surgery Rats were anaesthetized with isofluorane and implanted with a ~9cm Micro Renathane catheter ( .037 O.D. x .023" I.D, Micro Renathane tubing #037; Braintree Scientific, Braintree, MA) sterilized in ethylene oxid e T wo collars, ~.2cm, made from Silastic tubing (.51mm ID x .94mm OD, Silastic #508 002; Dow Corning, Midland, MI) were fitted onto one end of the catheter and 3.2 cm from the opposite end of the catheter T he catheter was advanced 3.2 cm into the right jugular vein and secured with a suture around the collar T he distal end was tunneled subcutaneously to an incision in the scapular region and attached to a port made of 21 ga stainless steel tubing with surgical mesh attached ( 313 000BM 15; Plastics One Inc., Roanoke, VA): the second collar served as a reinforcer at this union. The incision was then closed with non wicking suture around the port. The exterior of the port was fitted with a small (~1.5cm) piece of polyvinyl tubing (.51mm ID x 1.52mm OD; Norton Performance Plastics, Akron, OH) and closed with a pin. Immediately after surgery, rats were given of an analgesic and anti inflammatory medication ( 5 mg/kg, Ketorolac tromethamine; Henry Schein, Melville NY). After 1 day had passed, catheters wer e flushed with heparinized saline daily and the antibiotics enrofloxacin, 1.5mg per animal per day (trade name: Baytril; Sigma Chemicals, St. Louis MO) and Streptokinase, 200 units per animal per day (Sigma Chemicals, St Louis, MO) were administered via t he catheter on alternating days M ost rats recovered operative weights within 2 3 days.

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131 Apparatus Operant chambers (30.5 cm x 24.1cm x 21.0 cm, ENV 008CT; Med Associates St. Albans, VT) were used throughout the experiment. Chambers were located indiv idually in sound attenuating boxes. One side of the chamber had two symmetrically placed holes, one of which provided access to a sipper tube containing autoclaved water available at all times C ytisine and vehicle (sodium phosphate buffer were delivered to rats via polyethylene tubing (PE60), protected by a stainless steel spring, connected to an infusion pump (PHM 100 set at 3.33RPM; Med Associates ) via a fluid swivel ( 375/22PS; Instech laboratories, Plymouth Meeting, PA) to allow almost unlimited move ment within the chamber. A hole was located in the center of the ceiling of the chamber which allowed passage of the polyethylene tubing out of the chamber A nother length of PE tubing connected the swivel to the infusion pump which was located on a shel f mounted outsid e the cage. The pump held a 30mL syringe containing either sterile cytisine solution or the vehicle and fitted with an in line nitrocellulose filter (0.22m; Cameo #25ES; Osmonics). The timing and duration of the infusions were controlled by a computer program (MedPC IV). The chambers were fitted with a single fixed lever on the wall opposite the water tube and next to a food receptacle also controlled by a computer A single press on the lever caused a feeder to release one 45 mg nutritionally complete food pellet (Purified Rodent Tab let, 5TUL, Test Diet, Richmond, IN; energy content 12.7% fat, 20.5% protein, 66.8% carbohydrate) into a trough located next to the feeder. Each apparatus was interfaced to a computer through an input/output module ( DIG 716; Med Associates ) R ats lived i n these chambers for 23 h/day, and were removed only for chamber cleaning C hambers were enclosed in ventilated sound attenuating boxes A 15 W house light ( ENV 215M; Med Associates ) was located ~2cm below the ceiling on the wall above the openings for t he sipper

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132 tubes, running the same 12:12 lighting cycle as the vivarium. The max/min temperature inside the chambers was recorded periodically and typically was ~1 o C above room temperature. Cytisine Cytisine, (Sigma Chemicals, St L ouis MO) was dissolved in a sodium phosphate buffer (pH~7.35; 0.1 M ). Vehicle was the buffer heparin solution containing 17mg heparin per 100 mL s T he concentrations of the Cytisine solution were adjusted to deliver doses of approximately 1.25mg/ mL (5mg/kg per tot al daily dose). Procedure Two days after surgery, animals were placed into the operant chambers and baseline measures of body weight, pellets consumed and meal patterns were measured for 5 days. No specific lever training was conducted: all rats acquired the operant task within the first night A fter the baseline period, rats were divided into two groups matched for body weight T hey were then returned to the chambers and their catheters attached to the infusion line. For the first 2 days they received programmed injections of vehicle T he programmed in fusions (0.04mL ) were 1 s in duration every 30 min for the entire dark phase (12 h) and for the last 3 h of the light phase T his resulted in a total of 30 injections over a 15 h period of the 23 h test session that coincided with the maximal feeding and active times of rats. During the treatment phase, one group of rats received vehicle and the other group received cytisine T he dose of cytisine given to the treatment group, maintained for the 9 days o f the treatment phase, was 5mg/kg. After the treatment phase, rats entered a cessation phase during which nicotine infusions were replaced by vehicle T he cessation phase lasted 12 days. Body weight was recorded daily and meal parameters were recorded c ontinuously throughout the experiment as defined in Chapter 3 This study was run in 2 replications, with

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133 both groups represented approximately equally in each replication. There were no marked differences between these replications so the data were com bined as planned for analysis. Data Analysis One way ANOVAs were used to examine differences in body weight and meal patterns (food intake, meal number, meal size and excess pellets) between animals that received the vehicle or cytisine S ignificance le vels were set at p < .05. Results Body Weight The results of all statistical calculations are located in Table 4 1 D uring baseline, the two groups of rats did not significantly differ in the amount of weight gained over the five day period D uring the treatment phase, the animals that received cytisine gained less weight than those that received the vehicle (Figure 4 1) T his effect was significant on days 4, 5, 6, 7, 8, 9, and 10 D uring the cessation phase, the animals that received cytisine continued to weigh less than those that received the vehicle T his effect was significant on days 11, 12, 14, 15, 16, 17, 18, 19, 20 and 21 T he decreased body weight gained for animals that r eceived cytisine compared to those that received the vehicle approached significance for days 13, and 22. Meal Patterns Baseline The results of all statistical calculations for means over the entire period are located in Table 2 and the means for each in dividual day can be found in Table 4 3 T here were no significant differences throughout baseline between the two groups for the mean daily number of pellets eaten, the mean number of pellets eaten during the dark cycle, and the mean number of pellets eat en in the light cycle (Figures 4 2, 4 4 and 4 6) S imilarly, there were no significant differences between the two groups in total mean number of meals, the mean number of meals in

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134 the dark cycle, and the mean number of meals in the light cycle (Figures 4 8, 4 10 and 4 12) T he mean meal size did not significantly differ between the two groups (Figures 4 14 and 4 15) for the total mean meal size, and the mean meal size in the dark cycle, but did significantly differ between the two groups during the light cycle, with the cytisine group eating bigger meals at baseline than the vehicle group (Figure 4 16). Vehicle first two days of treatment The results of all statistical calculations for means over this entire period are located in Table 4 2 (Days 6 7) T here were no significant differences across groups during the first two days of treatment, when all animals received the vehicle (not shown in Figures) T his lack of statistical significance was seen for the mean daily number of pellets eaten, the mean number of pellets eaten during the dark cycle, and the mean number of pellets eaten in the light cycle S imilarly, there were no significant differences between the two groups in total meal intake (not shown in Figures) T his similarity between groups wa s seen for the total mean number of meals, the mean number of meals in the dark cycle, and the mean number of meals in the light cycle T he mean meal size did not significantly differ between the two groups (not shown in Figures) for the total mean meal s ize, and the mean meal size in the dark cycle, but did significantly differ for the mean meal size during the light cycle, with the cytisine group eating bigger meals during these two days than the vehicle group (not shown in Figures). Treatment number of pellets The results of all statistical calculations for means over the entire period are located in Table 4 2 and the means for each individual day can be found in Table 4 3 A nimals that received cytisine ate significantly fewer pellets overall than t hose that received the vehicle (Figures 4 2, 4 4, and 4 6) T he treatment phase was separated into two periods following the two days of vehicle administration T he mean number of total pellets eaten by rats in the cytisine

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135 group was significantly less t han animals in the vehicle group during days 8 11 and 12 16 of the experiment F urther analysis revealed that animals receiving cytisine ate fewer pellets compared to vehicle animals daily (Figure 4 3), and this was significant on days 8, 9, 10, 12, 13, 1 4, and 15 and approached significance on day 16 T he mean number of total pellets eaten in the dark cycle by rats in the cytisine group was significantly less than animals in the vehicle group during days 8 11 and during days 12 16 of the experiment F urther analysis revealed that animals receiving cytisine ate fewer pellets compared to vehicle treated animals daily during the dark cycle (Figure 4 5), and this was significant on days 8, 9 and 15 and approached significance on days 12 and 16 T he mean n umber of total pellets eaten in the light cycle by rats in the cytisine group was not significant from the vehicle group during days 8 11 and only approached being significantly less during days 12 16 of the experiment F urther analysis revealed that anim als receiving cytisine ate fewer pellets than vehicle animals daily in the light cycle (Figure 4 7) and this was significant on day 13 and approached significance on days 12 and 16. Treatment number of meals The results of all statistical calculations for means over the entire period are located in Table 4 2 and the means for each individual day can be found in Table 4 A nimals that received the cytisine ate significantly fewer meals overall than those that received the vehicle (Figures 4 8, 4 10 and 4 12) T he treatment phase was separated into two periods following the two days of vehicle administration T he mean number of meals eaten by rats in the cytisine group was less than animals in the vehicle group, and this approached significance during da ys 8 11 and was significant during days 12 16 of the experiment F urther analysis revealed that animals receiving cytisine ate fewer meals compared to nicotine animals daily (Figure 4 9), and this was significant on days 8, 9, 10, 12, 14, 15 and 16 and ap proached significance on day 13 T he mean number of meals eaten in the dark cycle by rats in the cytisine group was less than animals in the vehicle

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136 group and was significant during days 12 16, but not during days 8 11 of the experiment F urther analysis revealed that animals receiving cytisine ate fewer meals compared to nicotine animals daily during the dark cycle (Figure 4 9), and this was significant on days 10 and 12 15 and approached significance on day 13 T he mean number of meals eaten in the lig ht cycle by rats in the cytisine group was less than animals in the vehicle group and was significant during days 8 11, and days 12 16 of the experiment F urther analysis revealed that animals receiving cytisine ate fewer meals compared to nicotine animal s daily during the dark cycle (Figure 4 9), and this was significant on days 10, 12, 13, 15, and 16 and approached significance on day 13. Treatment meal size The results of all statistical calculations for means over the entire period are located in Tab le 4 2 and the means for each individual day can be found in Table 4 5 A nimals that received the cytisine ate the same size meals overall than those that received the vehicle (Figures 4 8, 4 10 and 4 12) T he treatment phase was separated into two group s following the two days of vehicle administration T he mean meal size eaten by rats in the cytisine group did not significantly differ from animals in the vehicle group during days 8 11 and days 12 16 of the experiment T he mean meal size eaten in the d ark cycle by rats in the cytisine group did not differ from animals in the vehicle group during days 12 16 and days 8 11 of the experiment F urther analysis revealed that animals receiving cytisine ate significantly smaller meals compared to nicotine anim als during the dark cycle on day 8 T he mean meal size eaten in the light cycle by rats in the cytisine group was moderately greater than animals in the vehicle group and approached significance during days 8 11 and days 12 16 of the experiment F urther analysis revealed that animals receiving cytisine ate smaller meals compared to nicotine animals daily during the dark cycle (Figure 4 9), and this was significant on days 7, 10 and 15.

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137 Cessation The results of all statistical calculations for means over the entire period are located in Table 4 6 T he cessation phase was separated into three periods following the treatment phase T here were no significant differences throughout cessation between the two groups in total pellet intake (Figures 4 2, 4 4 and 4 6) T his lack of statistical significance was seen for the mean daily number of pellets eaten during days 17 20, 21 24, and 25 28 of the experiment T he mean number of pellets eaten during the dark cycle did not significantly differ between groups on days 17 20, 21 24, and 25 28 of the experiment The mean number of pellets eaten during the light cycle did not significantly differ between groups on days 17 20, 21 24, and 25 28 of the experiment S imilarly, there were no significant differences between the two groups in total meal intake (Figures 4 8, 4 10 and 4 12) T his similarity between groups was seen for the total mean number of meals during days 17 20 and 21 24, although the animals in the c ytisine group ate fewer meals during days 25 28 of the experiment T he mean number of meals in the dark cycle did not significantly differ between groups on days 17 20, 21 24, and 25 28 of the experiment T he mean number of meals in the light cycle did n ot significantly differ between groups on days 17 20, 21 24, and 25 28 of the experiment L ikewise, the mean meal size did not significantly differ between the two groups (Figures 4 14 and 4 15) for the total mean meal size during days 17 20, 21 24, and 2 5 28 of the experiment T he mean meal size in the dark cycle did not significantly differ between groups on days 17 20, 21 24, and 25 28 of the experiment T he mean meal size in the light cycle did not significantly differ between groups on days 17 20, 2 1 24, and 25 28 of the experiment. Excess Pellets The results of all statistical calculations for means over the entire period are located in Table 4 7 T here was a significant difference throughout baseline between the two groups in

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138 total excess pellet intake (Figure 4 17) and excess pellet intake during the dark cycle (Figure 4 18) with the vehicle group consuming more excess pellets than the cytisine group T here was no significant difference between the groups in excess pellet intake during the ligh t cycle throughout baseline (Figure 4 19) D uring the treatment period, there were no significant differences between the two groups in total excess pellet intake (Figure 4 17), excess pellet intake in the dark cycle (Figure 4 18) and excess pellet intake in the light cycle (Figure 4 19) T hese similarities persisted into the cessation period (Figures 4 17, 4 18, and 4 19). Discussion The current study examined the effects of intravenous noncontingent cytisine on body weight and meal patterns T he model used was sensitive enough to detect effects of cytisine that were unique and independent from the results obtained and outlined in chapter 3 with nicotine I n line with the results reported by Mineur et al. (2011), administration of cytisine resulted in decreased weight gain I nterestingly, this difference in weight gain between animals that received the vehicle and those that received cytisine persisted for the entire cessation period T his result was somewhat unexpected in light of how difficult it ca n be to find a drug that will both help people quit smoking and prevent weight gain (for a review on this topic refer to chapter 1) E vidence in the human literature on the effects of cytisine on body weight is inconclusive (Tutka et al. 2005) and not oft en reported T herefore the current study provides additional evidence and support for cytisine as an option for people who are concerned about the possible weight gain that can occur after quitting smoking I n contrast to other available pharmacotherapie s where small weight gain occurs gradually while taking the drug or a large weight gain occurs after the drug is stopped ( as reviewed by Filofoz 2004; Borrelli et al. 1999), cytisine appears to have lasting effects on body weight throughout treatment and p ost cessation.

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139 Drugs that are manufactured to promote weight loss usually fall into one of two categories, those that increase energy expenditure by influencing metabolism and those that decrease energy intake by reducing food intake or the absorption of food, with some drugs affecting both processes (Halford 2006) T he results from the present study provide evidence that in rodents, cytisine acts to reduce energy intake and thus inhibit weight gain I t is possible that cytisine is also having a long term effect on metabolism which would account for the persistent differences in body weight gain post cessation, although the mechanisms behind this effect are unknown I t is important note however, that many drugs which decrease weight gain and food intake in animals, do not show similar results in humans, so the results from the current study on body weight would need to be investigated in humans. In the present study, we found that cytisine administration reduced total food intake which was confin ed to a decrease in pellet intake during the dark cycle F urther meal pattern analysis revealed a decrease in meal number in the dark cycle during the last few days of treatment and a decrease in meal number that was significant all throughout the light c ycle C ytisine had no discernible effect on meal size; animals in the cytisine group consumed more meals during baseline than animals that received the vehicle and this difference persisted throughout the treatment phase T he larger effect of cytisine on meal number in the light cycle was most likely due to the small number of meals typically consumed during this period such that any change at all in meal number would be significant T he primary reduction of food intake in the experiment occurred during the dark cycle, when proportionally fewer pellets were consumed via a decrease in meal frequency T here was no compensatory mechanism observed in the current study; a decrease in meal number did not result in increased meal size to maintain caloric intak e T here was also no difference among the phases of the experiment for the number of excess pellets (as

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140 defined in the discussion for chapter 3), indicating that cytisine did not significantly decrease meal size to below 4 pellets or increase the number o f small meals, as was seen with nicotine (and detailed in the discussion of chapter 3). It is known that POMC neurons communicate via acetylcholine ( as reviewed by Adan et al. 2008), and evidence from Mineur et al. (2011) indicates that this system is imp ortant in H owever, changes in POMC signaling typically manifest as changes in meal size rather than meal number ( as reviewed by Adan et al. 2008) L eptin stimulation causes an increase in POMC signaling, which then releases melanocortins and stimulates the MC3 and 4 receptors P OMC output can therefore be evaluated via melanocortin receptor activity S tudies examining meal patterns in MC4 knockouts and animals given MC4 antagonists shown changes in meal size but not meal frequency ( as reviewed by Adan et al. 2009; Zhang et al. 2005) S imilarly, leptin signaling can also be evaluated for its effects on POMC neurons, and studies examining the effects of leptin on meal patterns also demonstrate a change in meal size (Morton et al. 2005) T he results from the present study do not provide support for the action of cytisine on POMC neurons S ince some success has been found in individuals taking bupropion, it would be useful in the future to examine the effects of that drug in rodents using the current model B upropion is known to affect the POMC system within the hypothalamus ( as reviewed by Cook and Bloom 2006), and if cytisine is also affecting that system, we would expect to see similar changes in meal pattern s between the two drugs. One of the main side effects of orally administered cytisine in humans is nausea (West et al. 2011). It rats given iv administration of cytosine may be experiencing some form of malaise and so eating less. While our animals, unl ike those given a sickness inducing agent such as LiCl, were alert and in good appearance, we cannot rule this out simply on the basis of meal pattern

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141 data. S tudies examining the effects of lithium chloride on meal patterns indicate that animals consume smaller meals when given injections of the drug and it is sometimes reported that animals also show increased latency to the next meal (Dess and Vanderweele 1994) T hese results indicate that an animal that is sick as a result of a drug will terminate t he meal sooner and this change in meal patterns is reflective specifically of malaise and differs quantitatively from changes in meal patterns due to satiety mechanisms T herefore it is unlikely that the changes in meal patterns in the current study are d ue to the gastrointest inal side effects of cytisine, but future studies should be conducted to verify this. The change in meal number observed in the current study reflects a reduction in the drive to initiate meals and may be mediated by a variety of dif ferent systems within the hypothalamus C ytisine may be exerting its effects via dopamine signaling in the ventromedial hypothalamus (VMH) for example, which is a hypothesized regulator of meal number (Meguid et al. 2000) A nother possible mechanism may be through changes in agouti related peptide (AgRP), an antagonist and inverse agonist for the MC3 and 4 receptors T he administration of a drug known as C75, a fatty acid synthase inhibitor, causes decreases in meal number that are accompanied by a reduc tion in AgRP mRNA expression (Aha et al. 2006) H owever, the evidence for a specific brain responsible for regulating meal number are poorly understood. The pre F urther experiments should be done to evaluate the ability of cytisine to prevent weight gain after nicotine administration in order to better predict whether or not cytisine may b e a superior alternative to help those individuals who want to quit smoking but are worried about post

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142 cessation weight gain T he relative inexpensiveness of the drug makes it an especially good candidate for people who cannot afford the other approved me dications for smoking cessation. Table 4 1. P and F values for the changes in body weight over each day of the baseline, treatment, and cessation phases Day Body Weight Day Body Weight Baseline F P Cessation F P 1 5 .537 ns 10 16.809 **P < .01 Treatment 11 15.087 **P < .01 1 .433 ns 12 8.940 *P < .05 2 .046 ns 13 3.650 **P < .01 3 3.837 ns 14 4.933 *P < .05 4 5.883 *P < .05 15 6.353 *P < .05 5 7.690 *P < .05 16 7.129 *P < .05 6 7.598 *P < .05 17 8.564 *P < .05 7 18.969 **P < .01 18 8.570 *P < .05 8 18.683 **P < .01 19 6.270 *P < .05 9 15.911 **P < .01 20 6.667 *P < .05 21 6.053 *P < .05 22 4.802 ^P = .05 Table 4 2. F and P values for all meal pattern variables of interest, with days clustered into groups, throughout the baseline and treatment phase Baseline Treatment Variables Days 1 5 Days 6 7 Days 8 11 Days 12 16 F P F P F P F P Total Pellets 1.352 ns .004 ns 23.459 **P<.01 20.330 **P<.01 Night Pellets .611 ns .385 ns 9.392 **P<.01 8.004 *P<.05 Day Pellets .273 ns 1.750 ns 6.42 ns 3.654 ^P=.08 Total Meals 3.756 ns 2.584 ns 3.622 ^P=.08 18.661 **P<.01 Night Meals .226 ns 1.518 ns .505 ns 5.990 *P<.05 Day Meals 1.791 ns .402 ns 5.246 *P<.05 12.959 **P<.01 Total Meal Size 4.826 *P<.05 1.573 ns .341 ns .370 ns Night Meal Size 2.135 ns .515 ns 1.673 ns .010 ns Day Meal Size 5.437 ns 6.161 ^P=.08 4.401 ^P=.06 3.506 ^P=.08

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143 Table 4 3. P and F values for the total number of pellets eaten over each day of the baseline and treatment phases Day Total Pellets Night Pellets Day Pellets Baseline F P F P F P 1 .173 ns .098 ns .944 ns 2 3.449 ^P = .08 3.373 ns .220 ns 3 1.431 ns .441 ns 1.416 ns 4 .882 ns .009 ns 2.191 ns 5 1.972 ns .741 ns .328 ns Treatment 6 .214 ns .894 ns 1.305 ns 7 .625 ns .001 ns 2.172 ns 8 12.839 **P < .01 13.849 **P < .01 .290 ns 9 9.655 **P < .01 5.743 *P < .05 .108 ns 10 6.106 *P < .05 2.410 ns .897 ns 11 1.838 ns 4.563 ^P = .05 .043 ns 12 10.970 *P < .05 4.050 ^P = .06 4.007 ^P = .07 13 10.983 **P < .01 2.645 ns 5.814 *P < .05 14 6.117 *P < .05 3.112 ns .000 ns 15 6.563 *P < .05 6.350 *P < .05 .010 ns 16 4.408 ^P = .05 4.040 ^P = .07 4.410 ^P = .06

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144 Table 4 4. P and F values for the average total number of meals eaten over each day of the baseline and treatment phases Day Total Meals Night Meals Day Meals Baseline F P F P F P 1 .011 ns .354 ns 1.178 ns 2 1.082 ns .280 ns .747 ns 3 .028 ns .084 ns .033 ns 4 6.760 *P < .05 2.333 ns 1.389 ns 5 6.720 *P < .05 .429 ns 2.296 ns Treatment 6 3.361 ns 2.552 ns .000 ns 7 .237 ns .015 ns 1.068 ns 8 1.352 ns .038 ns 2.240 ns 9 1.969 ns .517 ns 2.372 ns 10 7.097 *P < .05 .120 ns 12.064 **P < .01 11 .622 ns .309 ns .294 ns 12 6.333 *P < .05 .627 ns 5.765 *P < .05 13 4.256 ^P = .06 .517 ns 9.906 **P < .01 14 5.780 *P < .05 2.425 ns 3.866 ^P = .07 15 8.246 *P < .05 3.698 ^P = .08 6.760 *P < .05 16 20.067 **P < .01 12.287 **P < .01 4.825 *P < .05 Table 4 5. F and P values for the average meal size eaten over each day of the baseline and treatment phases Day Total Meal Size Night Meal Size Day Meal Size Baseline F P F P F P 1 .100 ns .565 ns .111 ns 2 4.138 ns .066 ns 1.859 ns 3 .930 ns .365 ns 1.343 ns 4 6.730 *P < .05 .205 ns 2.146 ns 5 7.576 *P < .05 .276 ns 9.076 ns Treatment 6 .499 ns .241 ns 1.043 ns 7 .925 ns .150 ns 6.969 *P < .05 8 1.727 ns 4.632 *P < .05 1.304 ns 9 .027 ns .017 ns .553 ns 10 .059 ns 1.679 ns 9.105 **P < .01 11 .022 ns .019 ns .619 ns 12 .314 ns 3.240 ns .038 ns 13 .113 ns .240 ns 1.04 ns 14 .038 ns .100 ns 1.795 ns 15 1.178 ns .352 ns 6.017 *P < .05 16 2.272 ns .606 ns 2.144 ns

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145 Table 4 6. F and P values for the averages of all meal pattern variables of interest, with days clustered into groups, throughout the cessation phase. Cessation Variables of Interest Days 17 20 Days 21 24 Days 25 28 F P F P F P Total Pellets .004 ns 2.570 ns .029 ns Night Pellets .003 ns 1.669 ns .636 ns Day Pellets .335 ns .337 ns 2.189 ns Total Meals 3.137 ns 1.834 ns 4.803 ns Night Meals 2.629 ns .490 ns 1.043 ns Day Meals 1.943 ns 1.766 ns 3.184 ns Total Meal Size 2.716 ns .060 ns 1.572 ns Night Meal Size 2.149 ns .195 ns 2.445 ns Day Meal Size 1.746 ns .000 ns .226 ns Table 4 7. F and P values for the average number of excess pellets eaten, with days clustered into groups, throughout the baseline, treatment, and cessation phases Time Point Experimental Phase/Day Total Night Day Baseline F P F P F P 1 5 4.945 *P < .05 6.185 *P < .05 .264 ns Treatment 6 7 2.38 ns 2.005 ns .665 ns 8 11 .017 ns 3.290 ^P = .09 1.761 ns 12 16 1.724 ns .627 ns .147 ns Cessation 17 20 .752 ns .257 ns .137 ns 21 24 .051 ns 1.196 ns .391 ns 25 28 .772 ns 1.646 ns .000 ns

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146 Figure 4 1. Cytisine: b ody weight over treatment and cessation S hown are mean +/ SE cumulative change in body weight (g) on consecutive days of treatment and cessation phases, in animals that received either cytisine (n = 8) or vehicle (n = 8). ^P < .06, *P < 0.05, **P < 0.01 difference between vehicle and cytisine

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147 Figure 4 2. Cytisine: total pellets S hown are mean +/ SE total lever presses for pellets, averaged over the ranges of days indicated, in animals that received either cytisine (n = 8) or vehicle (n = 8) during the t reatment phase. *P < 0.05, **P < 0.01 difference between vehicle and cytisine

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148 Figure 4 3. Cytisine: total pellets per day S hown are number of lever presses on consecutive days of baseline and treatment phases, by individual rats that received either cytisine (n = 8) or vehicle (n = 8) during the treatment phase L ines show the group means T he means were significant on days 8 10 and 11 15, P <0.05 and approached significance on day 16 (p < 0.06) difference between vehicle and cytisine

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149 Figure 4 4. Cytisine: total pellets dark cycle S hown are mean +/ SE lever presses for pellets during the 12 hours of the dark cycle, averaged over the ranges of days indicated, in animals that received either cytisine (n = 8) or vehicle (n = 8) during the treatment phase. *P < 0.05, **P < 0.01 difference betwe en vehicle and cytisine

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150 Figure 4 5. Cytisine: total pellets per day dark cycle S hown are number of lever presses during the 12 hours of the dark cycle, on consecutive days of baseline and treatment phases, by individual rats that received either cytisine (n = 8) or vehicle (n = 8) during the treatment phase L ines show the group means T hese means were significant on days 8 9 and 15, P <0.05 and approached significance on days 11 (p = 0.05), 12 (p = 0.06) and 16 (p = .07) difference betw een vehicle and cytisine

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151 Figure 4 6. Cytisi ne: total pellets light cycle S hown are mean +/ SE lever presses for pellets during the 11 hours of the light cycle, averaged over the ranges of days indicated, in animals that received either cytisine (n = 8) or vehicle (n = 8) during the treatment phase. ^P = 0.08 difference between vehicle and cytisine

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152 Figure 4 7. Cytisine: tota l pellets per day light cycle S hown are number of lever presses during the 11 hours of the light cycle, on consecutive days of baseline and treatment phases, by individual rats that received either cytisine (n = 8) or vehicle (n = 8) during the treatment phase L ines show the group means T he se means were significant on day 13, P <0.05 and approached significance on days 12 (p = .07) and 16 (p = .06) difference between vehicle and cytisine

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153 Figure 4 8. Cytisine: total number of meals S hown are mean +/ SE total number of meals, averaged over the ranges of days indicated, in animals that received either cytisine (n = 8) or vehicle (n = 8) during the treatment phase. ^P = 0.08, **P < 0.01 difference between vehicle and cytisine

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154 Figure 4 9. Cytisine: total meals per day S hown are total number of meals on consecutive days of baseline and treatment phases, by individual rats that received either cytisine (n = 8) or vehicle (n = 8) during the treatment phase L ines show the group means T hese means were significant on days 4 5, 10, 12, and 14 16, p < 0.05 and approached significance on day 13 (p = .06) difference between vehicle and cytisine

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155 Figure 4 10. Cytisine: total meals dark cycle S hown are mean +/ SE number of meals during the 12 hours of the dark cycle, averaged over the ranges of days indicated, in animals that received either cytisine (n = 8) or vehicle (n = 8) during the treatment phase. *P < 0.05 difference between vehicle and cytisine

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156 Figure 4 11. Cytisine: total meals per day dark cycle S hown are number of meals during the 12 hours of the dark cycle on consecutive days of baseline and treatment phases, by individual rats that received either c ytisine (n = 8) or vehicle (n = 8) during the treatment phase L ines show the group means T hese means were significant on day 16, p < .01 and approached significance on day 15, p = .08, difference between vehicle and cytisine

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157 Figure 4 12. Cytisine: total meals light cycle S hown are mean +/ SE number of meals during the 11 hours of the light cycle, averaged over the ranges of days indicated, in animals that received either cytisine (n = 8) or vehicle (n = 8 ) during the treatment phase. *P < 0.05, **P < 0.01 difference between vehicle and cytisine

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158 Figure 4 13. Cytisine: to tal meals per day light cycle S hown are number of meals during the 11 hours of the dark cycle, on consecutive days of baseline and treatment phases, by individual rats that received either cytisine (n = 8) or vehicle (n = 8) during the treatment phase L ines show the group means T hese means were significant on days 10, 12, 13, and 15 16, p < .05 and approached significance on day 14 (p = .07) difference between vehicle and cytisine

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159 Figure 4 14. Cytisine: total meal size S hown are mean +/ SE total number of pellets per meal, averaged over the ranges of days indicated, in animals that received either cytisine (n = 8) or vehicle (n = 8) during the treatment phase. *P < 0.05 difference between vehicle and cytisine

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160 Figure 4 15. Cytisin e: total meal size dark cycle S hown are mean +/ SE total number of pellets per meal during the 12 hours of the dark cycle, averaged over the ranges of days indicated, in animals that received either cytisine (n = 8) or ve hicle (n = 8) during the treatment phase

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161 Figure 4 16. Cytisine: total meal size light cycle S hown are mean +/ SE total number of pellets per meal during the 11 hours of the light cycle, averaged over the ranges of days indicated, in animals that received either cytisine (n = 8) or vehicle (n = 8) during the treatment phase. *P < 0.05, ^P = 0.06 for days 8 11 and p = .08 for days 12 16 difference between vehicle and cytisine

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162 Figure 4 17. Cytisine: tot al number of excess pellets S hown are mean +/ SE number of lever presses (for excess pellets) averaged over the range s of days indicated, in animals that received either cytisine (n = 8) or vehicle (n = 8) during the treatment phase

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163 Figure 4 18. Cytisine: to tal excess pellets dark cycle S hown are mean +/ SE number of lever presses (for excess pellets) durin g the 12 hours of the dark cycle, averaged over the ranges of days indicated, in animals that received either cytisine (n = 8) or vehicle (n = 8) during the treatment phase

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164 Figure 4 19. Cytisine: tot al excess pellets light cycle S hown are mean +/ SE number of lever presses (for excess pellets) during the 11 hours of the light cycle, in animals that received either cytisine (n = 8) or vehicle (n = 8) during the treatment phase

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165 CHAPTER 5 SELF ADMI NISTRATION OF NICOTI NE AND CYTISINE: EFF ECTS ON BODY WEIGHT AND MEAL PATTERNS Introduction There are a variety of behavioral models for studying the reinforcing properties of drugs including conditioned place preference testing, electrical brain stimulat ion, and self and Gardner 2005) T he self administration procedure, which is the focus of this chapter, is used most often in comparison with human drug seeking and drug taking and Gardner 2005) and its ability to assess the addictive potential and abuse liability of drugs (Donny et al. 1995) A and Gardener (2005), the most commonly used schedule of reinforcement in a self administration paradigm is the fixed ratio sc hedule, which seems to reflect the rewarding properties of the drug I n contrast, progressive ratio schedules which are also used in self administration paradigms, more accurately examine the reinforcing efficacy of and craving for the drug (Richardson an d Roberts 1996). Intravenous nicotine self administration has been demonstrated in rats (Corrigall and Coen 1989), dogs (Risner and Goldberg 1983), primates (Goldberg et al. 1981), and humans (Henningfield et al. 1983) T hough stable self administration was initially hard to come by in rodents without food deprivation or pretreatment, Corrigall and Coen (1989) were able to achieve nicotine self administration in rats on an FR5 schedule of reinforcement in combination with a light/tone cue, and these resul ts have since been replicated (Donny et al. 1995) R ats readily self administer nicotine in doses of .01, .03, and .06 mg/kg/inf (Corrigall and Coen 1989; Donny et al. 1995; Shram et al. 2008). Until recently, the majority of nicotine self administration experiments were limited access studies, with animals receiving nicotine for 1 2 hours a day for 5 days a week (Paterson and

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166 Markou 2004) O ver the last decade, extended access protocols have been increasingly used in order to more closely mimic patterns of human smoking T hese studies provide access to nicotine anywhere from 6 to 23 hours a day (Paterson and administration in extended access protocols (Patterson and Markou 2004), many have found an increase in number of infusions and total nicotine intake when nicotine is available for longer periods of time (Kenny and D espite these inconsistent findings, it is clear that nicotine self administration in extended access protocols results in increased signs of withdrawal (Patterson and Markou 2004; Kenny and The self administration procedu re has been extensively used to evaluate treatments for nicotine dependence (Rose and Corrigall 1997) T he evaluation of NRTs in rodents has been examined through the concurrent administration of nicotine via an osmotic minipump as a model for the patch a longside self administered nicotine R esults from these studies lent support to the human literature, such that continuous nicotine infusions suppressed nicotine self administration (LeSage et al. 2002) D rugs like varenicline and bupropion have also bee n evaluated in the context of self administration V arenicline attenuates nicotine self administration in both limited and extended access protocols (Rollema et al 2007; George et al. 2011), while bupropion exhibits a biphasic dose response curve by incr easing self administration at low doses and decreasing it at high doses (Rauhut et al. 2003) T olerance to non nicotine pharmacotherapies has also been examined using the self administration procedure (Rauhut et al. 2005), highlighting the ability of this method to evaluate the long term benefits and efficacy of these treatments.

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167 The abuse liability and reinforcing properties of potential pharmacotherapies can also be evaluated using the self administration procedure T he ability of a drug to substitute for nicotine in this protocol is indicative of its ability to both serve as a treatment for nicotine addiction and serve as a reinforcer on its own R esults indicate the varenicline for example, is similar to saline in that it causes a reduction of nicoti ne self administration by approximately 50% when it is substituted for nicotine in this protocol (Rollema et al. 2007) T he purpose of the current study A fter the establishment of stable self administration, cytisine was substituted for nicotine and the number of infusions was recorded A dose response relationship was examined within animals and responses for the cytisine were compared to extinction W e hypothesized that similar to the Rolle ma et al. (2007) study with varenicline, cytisine would cause a reduction in self administration comparable to that seen with the vehicle. Materials and Methods Animals and Housing Fifteen male Sprague Dawley rats initially weighing ~275 g were used in th e present study. All rats were purchased from Harlan ( Indianapolis, IN ). The principles described in the Guide for the Care and Use of Laboratory Animals were followed throughout, and the project was approved by the UF IACUC. Upon arrival, rats were ho used individually in a conventional vivarium that was maintained at 22 26 o C and 40 80% relative humidity, on a reverse 12:12 hr light:dark cycle (lights off at 10:00AM). Home cages were of polycarbonate, with Sani Chips contact bedding. Purina 5001 Chow pellets were available ad libitum for the initial portion of the experiment when animals were housed in their home cages D uring the test phase, animals were housed in operant chambers A utoclaved tap water was available at all times from a

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168 standard bott le and sipper tube. A reverse light cycle (on: 2200 1000 h) was in effect in both the vivarium and the operant chambers. Surgery Rats were anaesthetized with isofluorane and implanted with a ~9cm Micro Renathane catheter ( .037 O.D. x .023" I.D, Micro Renathane tubing #037; Braintree Scientific, Braintree, MA) sterilized in ethylene oxide T wo collars, ~.2cm, made from Silastic tubing (.51mm ID x .94mm OD, Silastic #508 002; Dow Corning, Midland, MI) were fitted onto one end o f the catheter and 3.2 cm from the opposite end of the catheter T he catheter was advanced 3.2 cm into the right jugular vein and secured with a suture around the collar T he distal end was tunneled subcutaneously to an incision in the scapular region an d attached to a port made of 21 ga stainless steel tubing with surgical mesh attached ( 313 000BM 15; Plastics One Inc., Roanoke, VA): the second collar served as a reinforcer at this union. The incision was then closed with non wicking suture around the p ort. The exterior of the port was fitted with a small (~1.5cm) piece of polyvinyl tubing (.51mm ID x 1.52mm OD; Norton Performance Plastics, Akron, OH) and closed with a pin. Immediately after surgery, rats were given of an analgesic and anti inflammator y medication ( 5 mg/kg, Ketorolac tromethamine; Henry Schein, Melville NY). After 1 day had passed, catheters were flushed with heparinized saline daily and the antibiotics enrofloxacin, 1.5mg per animal per day (trade name: Baytril; Sigma Chemicals, St. Louis MO) and Streptokinase, 200 units per animal per day (Sigma Chemicals, St Louis, MO) were administered via the catheter on alternating days M ost rats recovered operative weights within 2 3 days. Apparatus Operant chambers (30.5 cm x 24.1cm x 21.0 cm, ENV 008CT; Med Associates St. Albans, VT) were used throughout the experiment. Chambers were located individually in

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169 sound attenuating boxes. One side of the chamber had two symmetrically placed holes, one of which provided access to a sipper tube containing autoclaved water available at all times N icotine, cytisine, and vehicle (sodium phosphate buffer) were delivered to rats via polyethylene tubing (PE60), protected by a stainless steel spring, connected to an infusion pump (PHM 1 00 set at 3.33RPM; Med Associates ) via a fluid swivel ( 375/22PS; Instech laboratories, Plymouth Meeting, PA) to allow almost unlimited movement within the chamber. A hole was located in the center of the ceiling of the chamber which allowed passage of th e polyethylene tubing out of the chamber A nother length of PE tubing connected the swivel to the infusion pump which was located on a shelf mounted outsid e the cage. The pump held a 30mL syringe containing either sterile nicotine tartrate solution, cyti sine solution, or the vehicle and fitted with an in line nitrocellulose filter (0.22m; Cameo #25ES; Osmonics). The chambers were fitted with two fixed levers on the wall opposite the water tube on either side of a food receptacle. A single press on the inside lever caused a feeder to release one 45 mg nutritionally complete food pellet (Purified Rodent Tablet, 5TUL, Test Diet, Richmond, IN; energy content 12.7% fat, 20.5% protein, 66.8% carbohydrate) into a trough located next to the feeder. A single p ress on the outside lever, during the treatment phase, resulted in a 1sec infusion of nicotine accompanied by the initiation of a cue light directly above the outside lever. After a press on the outside lever, the cue light turned on and remained on for a 20 second period, during which the lever was inactive and presses on the lever would not result in an infusion of the drug T he duration of the infusions and the time the cue light was on were controlled by a computer program (MedPC IV) E ach apparatus was interfaced to a computer through an input/output module ( DIG 716; Med Associates ) R ats lived in these chambers for 23 h/day, and were removed only for chamber cleaning C hambers were enclosed in ventilated sound attenuating boxes A 15 W house ligh t

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170 ( ENV 215M; Med Associates ) was located ~2cm below the ceiling on the wall above the openings for the sipper tubes, running the same 12:12 lighting cycle as the vivarium. The max/min temperature inside the chambers was recorded periodically and typically was ~1 o C above room temperature. Nicotine Nicotine hydrogen tartrate (Sigma Chemicals, St Louis MO) was dissolved in a sodium phosphate buffer (pH~7.35; 0.1 M ) containing 17mg heparin per 100 mL s. Vehicle was the buffer heparin solution T he concentratio ns of the nicotine solution were adjusted to deliver doses of approximately 0.01 or 0.06 mg/kg body wt per injection, calculated as the free base. Cytisine Cytisine, (Sigma Chemicals, St. Louis MO) was dissolved in a sodium phosphate buffer (pH~7.35; 0.1 M ) Vehicle was the buffer heparin solution containing 17mg heparin per 100 mL s T he concentrations of the Cytisine solution were adjusted to deliver doses of approximately 1.25mg/ mL (5mg/kg per total daily dose). Procedure Two days after surgery, animals were placed into the operant chambers and baseline measures of body weight, pellets consumed and meal patterns were measured for 5 days. No specific lever training was conducted: all rats acquired the operant task within th e first night by pressing the inside lever on an FR1 schedule of reinforcement for food pellets. After the baseline period, rats were divided into two groups matched for body weight T hey were then returned to the chambers and their catheters attached to the infusion lines, which were filled with the vehicle, for two days D uring this period, the outside lever remained retracted and the animals were not allowed to respond on the lever for the drug T he following two days the vehicle was replaced with .0 1 mg/kg/inf of nicotine, the outside lever was protracted, and

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171 animals were allowed to respond for the nicotine on an FR1 schedule of reinforcement A ll subsequent drug self administration was on this FR1 schedule of reinforcement T he .01 mg/kg/inf dose of nicotine was then replaced with the terminal dose of nicotine, .06 mg/kg/inf, and animals were allowed to self administered this dose O nce the animal reached stable self administration, defined as 30 +/ 6 infusions of the nicotine for 3 days, the ni cotine was substituted with cytisine for 4 days D uring this period, the animal was able to respond on the outside lever to receive infusions of cytisine H alf of the animals received a low dose of cytisine (2.5mg/kg) while the other half received a high dose of cytisine (5mg/kg) A fter four days of cytisine self administration, the cytisine was replaced with the terminal dose of nicotine and animals were again allowed to self administer the nicotine T his continued until the animal reached stable self administration again, upon which the cytisine was substituted for the nicotine A nimals that had received the high dose of cytisine were given the low dose during this period, while animals who had initially received the low dose were then given the high dose D uring this period which lasted for four days, animals were again allowed to self administer the cytisine, after which they were then given nicotine for a final time A fter the animals again attained stable self administration, the nicotine was rem oved and replaced with vehicle, and extinction was measured for 4 days. Body weight was recorded daily and meal parameters were recorded continuously throughout the experiment. Data Analysis Repeated measures ANOVAs were used to examine differences in b ody weight gain, infusion number, and the meal pattern variables of interest, with the phase of the experiment as the within subjects factor and the order of treatment as a between subjects factor S ubsequent paired sample t tests were used to parse apart the differences between these variables throughout

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172 different phases of the experiment: all phases were compared to the initial nicotine 1 phase as well as to the phase of the experiment that preceded any given particular phase. Results Body Weight The res ults of all statistical calcula tions are located in Tables 5 1, 5 2 and 5 13. There was an overall main effect of phase on changes in body weight gain throughout the experiment E ach phase of the experiment was compared against the first nicotine treatment (from here on known 1) M ean body weight gain throughout nic 1 was significantly less than during the baseline phase T here was no difference between nic 1 and any other phase, although body weight gain in the extinction phase w as moderately greater E ach phase of the experiment was also compared against the previous phase (Figure 5 2) O verall, animals gained less weight during the nicotine phases of the experiment as compared to the cytisine phases S pecifically, animals in the nic 2 phase gained less weight than they did in the 2.5 mg/kg cytisine phase, animals in the nic 3 phase gained less weight than in the 5 mg/kg cytisine phase, and animals in the extinction phase gained more weight than they did during the final nicoti ne phase (nic 3) T here was no effect of the order of cytisine treatment on body weight gained throughout the different phases T he cytisine treatments were also compared against the baseline and extinction phases M ean body weight gain throughout the b aseline period was significantly greater than the 2.5 mg/kg cytisine phase, the 5 mg/kg cytisine phase, and the extinction phase (Figure 5 21) T here were no significant differences between the two cytisine phases and the extinction phase. Infusions The results of all statistical calculations are located in Tables 5 1 and 5 3 T here was an overall main effect of phase on the number of lever presses for infusions of the available drug throughout the experiment E ach phase of the experiment was compared a gainst the nic 1

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173 (Figure 5 3) T he mean number of infusions during nic 1 was significantly higher than the number of infusions received during all other treatments, except for during nic 3 where there was no significant difference between the two phases E ach phase of the experiment was also compared against the previous phase (Figure 5 4) O verall, there were no significant differences between these phases for the number of infusions received, except for between nic1 and 2.5 mg/kg cytisine T here was n o effect of the order of cytisine treatment on body weight gained throughout the different phases T he cytisine treatments were also compared against the extinction phase T here were no significant differences between the mean number of infusions throughout the cytisine treatments compared to the number of infusions in the extinction phase (Figure 5 22). Meal Patterns Number of pellets The results of all statistical calculations are located in Tables 5 1 and 5 4 5 6 T here was an overall main effect of phase on the total number of lever presses for pellets throughout the experiment E ach phase of the experiment was compared against the nic 1 (Figure 5 5) A nimals ate fewer pellets during nic 1 phase than they did at baseline and more pellets during the 2.5 mg/kg cyt phase, the nic 3 phase, and the extinction phase as well as moderately more pellets during the 5 mg/kg cyt phase as compared to the nic 1 phase W hen pellet intake in the dark cycle was examined, there were no signifi cant differences between any of the phases with night pellet intake during the nic 1 phase (Figure 5 7) W hen day pellet intake was examined, there were only moderate increases in daytime pellet intake in the nic 3 and extinction phases compared with the nic 1 phase (Figure 5 9). Each phase of the experiment was also compared against the previous phase (Figure 5 6) O verall, animals ate fewer pellets when they received the nicotine compared with the difference

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174 doses of cytisine S pecifically, they ate m ore pellets during 2.5 mg/kg cyt than at nic 1 and ate less during nic 2 than at 2.5 mg/kg cyt T hey also ate moderately more during 5 mg/kg cyt than during nic 2 and during extinction as well W hen pellet intake in the dark cycle was examined, animals a te significantly fewer pellets during nic 1 in the dark cycle compared with baseline, and fewer pellets during nic 2 compared with the 2.5 mg/kg cyt phase (Figure 5 8) D uring the light cycle however, the only significant difference in pellet intake betwe en phases was between baseline and nic 1 T here was no effect of the order of cytisine treatment on body weight gained throughout the different phases. The cytisine treatments were also compared against the baseline and extinction phases T he mean total number of pellets consumed throughout the baseline period was significantly greater than the 2.5 mg/kg cytisine phase, the 5 mg/kg cytisine phase, and the extinction phase (Figure 5 23) T here were no significant differences between the two cytisine phase s and the extinction phase L ikewise, the mean number of pellets during the dark cycle was significantly greater than the 2.5 mg/kg cytisine phase, the 5 mg/kg cytisine phase, and the extinction phase (Figure 5 24) T here were no significant differences between the two cytisine phases and the extinction phase T here was also a significant difference for the mean number of pellets consumed during the light cycle between the baseline phase and the 5 mg/kg cytisine phase but not between the baseline phase and the 2.5 mg/kg cytisine phase or the extinction phase. (Figure 5 25) T here were no significant differences between the two cytisine phases and the extinction phase Meal number The results of all statistical calculations are located in Tables 5 1 and 5 7 5 9 T here was not an overall main effect of phase on the total number meals throughout the experiment E ach phase of the experiment was compared against nic 1, and there were similarly no differences

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175 between any of the phases compared with the in itial nicotine phase W hen meal number was examined in the dark (Fig ure 5 12) and light (Figure 5 14 ) cycles, there were also no significant differences between the phases and nic 1. Each phase of the experiment was also compared against the previous pha se T here were no significant differences in total meals consumed between any of the phases W hen meal number was examine d in the dark phase (Figure 5 13 ), animals in the nic 1 phase ate a greater number of meals compared to the number of meals consumed during baseline A nimals also ate more meals during the nic 3 phase than during the 5 mg/kg cyt phase T here were no significant differences in number of meals consumed between any of the other phases during the dark cycle T here were also no differences in total meal number between any of the phases dur ing the light cycle (figure not shown ) T here was no effect of the order of cytisine treatment on body weight gained throughout the different phases. The cytisine treatments were also compared against the baseline and extinction phases (figures not shown) T he mean total number of meals consumed throughout the baseline period was not significantly different from the 2.5 mg/kg cytisine phase, the 5 mg/kg cytisine phase, and the extinction phase T here were no significant differences between the two cytisine phases and the extinction phase L ikewise, the mean number of pellets during the dark cycle did not differ significantly from the 2.5 mg/kg cytisine phase, the 5 mg/kg cytisine phase, and th e extinction phase T here were no significant differences between the two cytisine phases and the extinction phase T here was also no significant difference for the mean number of pellets consumed during the light cycle between the baseline phase and the 2.5 mg/kg cytisine phase, the 5 mg/kg cytisine phase, and the extinction phase T here were no significant differences between the two cytisine phases and the extinction phase.

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176 Meal size The results of all statistical calculations are located in Tables 5 1 and 5 10 5 12 T here was an overall main effect of phase on the total number of pellets per meal (meal size) throughout the experiment E ach phase of the experiment was compared against nic 1 (Figure 5 15) T here was a moderate increase in the ave rage meal size for animals during the 5 mg/kg cyt group compared to the nic 1 group T here were no other significant differences between the other phases compared with the nic 1 phase for average meal size W hen the average meal size in the dark cycle wa s examined (Figure 5 17), there was a significant increase in meal size for animals in the 2.5 mg/kg cyt phase compared with the nic 1 phase T here were no other differences detected between the nic 1 phase and the other phases for average meal size T he re were also no significant differences between meal size in the light cycle during nic 1 compared to meal size during the other phases (Figure 5 19). Each phase of the experiment was also compared against the previous phase O ther than a moderate increas e in total meal size between extinction and nic 3, there were no significant differences in meal size between any of the other phases (Figure 5 16) W hen the average meal size in the dark cycle was examined (Figure 5 18), there was a significant decrease in meal size between baseline and nic 1 and a moderate decrease between 2.5 mg/kg cyt and nic 2 T here were no significant differences in average meal size in the dark cycle between the other phases of the study W hen the average meal size in the light c ycle was examined (Figure 5 20), the only significant difference was between nic 1 and baseline, with animals in nic 1 eating smaller meals A ll other phases did not differ from one another on meal size in the light cycle. There was no effect of the orde r of cytisine treatment on body weight gained throughout the different phases. The cytisine treatments were also compared against the baseline and extinction phases T he mean total meal size consumed throughout the baseline period was significantly great er than

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177 the 2.5 mg/kg cytisine phase, the 5 mg/kg cytisine phase, and the extinction phase (Figure 5.26) T here were no significant differences between the two cytisine phases and the extinction phase L ikewise, the mean meal size during the dark cycle w as significantly greater than the than the 2.5 mg/kg cytisine phase, the 5 mg/kg cytisine phase, and the extinction phase (Figure 5 27) T here were no significant differences between the two cytisine phases and the extinction phase T here was a significa nt difference for the mean meal size consumed during the light cycle between the baseline phase and the 2.5 mg/kg cytisine phase, the 5 mg/kg cytisine phase, and the extinction phase (Figure 5 28) T here were no significant differences between the two cyt isine phases and the extinction phase. Discussion The current study examined the ability of cytisine to substitute for nicotine in a self administration procedure C hanges in body weight and meal patterns were also examined as animals self administered the different drugs throughout the study. This discussion will focus solely on the present experiment. The relationship between this study and the previous study exam will be discussed in Chapter 6. As expected, there was a significant decrease in body weight between the baseline period and the first several days of nicotine administration T here was also a sma ll but not significant increase in body weight gained from the last nicotine administration to the extinction phase T hese results support previous research in animals and humans (as outlined in Chapter 1) which demonstrates that nicotine administration r esults in decreased weight gain T here was a small but not significant increase in body weight gain during the extinction phase H owever, b ecause the extinction period only lasted for four days, we cannot address whether the animals would gain more or le ss weight over a more extended period S imilarly, there were fewer animals that completed the study and these results might be resolved with more animals.

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178 The present study examined two doses of cytisine for differential effects on body weight and meal p atterns, but no significant differences were found T herefore we will consider the two doses together for the purposes of this discussion T he current study also examined changes in weight gain between periods of nicotine administration and subsequent pe riods of cytisine administration, and found that animals gained less weight while receiving nicotine than they did while they received cytisine S tatistical calculations (data not presented) were done after the experiment to determine whether or not this effect was due to the number of self administrations the animals received for each drug W hile the number of nicotine administrations in the first nicotine period was negatively correlated with body weight gain, there were no significant correlations in o ther periods between number of administrations and weight gain. As mentioned above, the extinction phase was analyzed against the final nicotine phase that preceded it T herefore, the current extinction results only demonstrate d a small weight gain immed iately following nicotine administration, and thus it is unknown whether weight loss would have been observed had cytisine been given as the final administered substance I t is possible that cytisine may result in decreased weight gain in the long term I t is also unknown whether or not the change in body weight observed during the extinction phase is at all related to the administration of cytisine, or if cytisine administration interspersed with nicotine administration has any effect on body weight gain F uture studies need to examine whether or not cytisine administration can prevent weight gain after nicotine self administration, for example by allowing animals to self administer nicotine for a longer period of time and then allowing animals to self a dminister cytisine in a manner similar to individuals taking the drug. Despite a lack of correlation between number of infusions and weight gain among the various phases, there were significant differences in the number of infusions animals received

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179 betw een the phases A nimals self administered less of the available drug in every phase following the initial nicotine phase with the exception of the third nicotine phase T his effect may be due to the slight decrease in responding for nicotine that has bee n observed after the first 7 ). This problem might have been eliminated had we allowed rats to self administer for a longer period of time before substituting for cytisine T herefore, it cannot be deter mined unequivocally from this study whether the decrease in infusion number throughout the treatments is due to either the passage of time or to the change from nicotine to cytisine administration P eriods of nicotine administration and subsequent periods of cytisine administration were also compared A nimals self administered significantly less cytisine than they did nicotine during the first phase, such that the number of cytisine administrations was ~57% of the number of nicotine infusions T hese resu lts are supported by previous research examining the ability of cytisine to substitute for nicotine in drug discrimination tasks anywhere from 20 65% (Reavill et al. 1990; LeSage et al. 2009) A lthough there were no other significant differences in the nu mber of infusions, there was a distinct trend demonstrating that animals self administer cytisine less than they do nicotine. Meal patterns were also examined in the present study A nimals ate significantly fewer pellets overall during the first nicotine phase compared to the baseline phase A nimals also ate a small amount more during extinction compared to the last nicotine phase, which explains the moderate increase in weight gain observed in this phase as well A nimals also ate significantly less during the first phase compared to baseline in both the light and dark cycles T his decrease in total pellet intake overall and in the dark cycle is in line with previous studies examining Guan et al. 2 00 4 ; Guan et al. 2004) and is the first report of such effects during IV nicotine self administration.

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180 The effects of nicotine on meal number and meal size were also consistent with previous research T here were increases in meal number in the first nico tine phase compared to baseline and in the third phase compared to the second cytisine phase during the dark cycle T hese increases in meal number served a compensatory mechanism to account for the decreases in meal size that were also observed during the dark cycle A s opposed to other studies which have not found an effect of nicotine on meal patterns in the light cycle ( Guan et al. 200 4 ; Guan et al. 2004), the present study was able to detect these effects T here was a decrease in meal size between ba seline and the first nicotine phase but no change or compensatory increase in meal number in the light cycle F or explanations on these results, please see the discussions to Chapters 3 and 6. The changes in meal patterns between nicotine and cytisine ad ministration were examined as well D uring the cytisine periods, animals consumed more pellets overall than they did during the first phase of nicotine administration as well as compared to the preceding nicotine phases H owever, there were no difference s in total pellet intake during the dark and light cycle throughout the different phases compared to the first nicotine phase T here were no differences in meal number overall or throughout the dark and light phases between the initial nicotine phase and all others T here was a small increase in overall meal size during the second cytisine treatment, but no effects of cytisine on meal size during the dark or light periods when analyzed separately T nt effects are not as great as A nimals in this study never at e less when they received cyt i sine, than they did while receiving nicotine W e have previously found that animals tend to eat durin g the same 1 0 minute bins that they self administer nicotine (data not published); we do not know whether this temporal coincidence is also true of cytisine self

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181 administration and food intake I specific circadian effects I t may also be that animals are self administering cytisine less often and less regularly than they do nicotine, and thus their food intake is greater in general. We also compared meal patterns in the baseline and extinction phases with the cytisine phases O verall, these results indicate that cytisine results in decreased weight gain over time, decreased total pellet intake in the dark and light periods (5mg/kg only) and decreased average meal size during the dark and light periods T hese findings lend support to the idea that cytisine decreases body weight and food intake, similar to what was found in Mineur et al. (2011) W e also compared the baseline and extinction phases with each other and found persistent decreases in bod y weight gain and food intake T here were no differences between the cytisine phases and the extinction phase for any of the meal pattern variables of interest, indicating that the effects of cytisine may extend past the time the drug stopped T hough the extinction period did not last longer than 4 days, these results are encouraging and lend support to the notion that cytisine may not just delay weight gain, but prevent it even after the drug is no longer administered. This is the first study to exam ine whether or not rats will self administer cytisine after establishing nicotine self administration C ytisine was able to maintain stable self administration at levels that were just over 50% of the nicotine self administration levels T he lack of a do se effect on infusions was somewhat surprising B ecause the cytisine doses were counterbalanced within animals, we tested to see if there were effects of treatment order on the variables of interest and none were found T herefore, the order in which the animals received the high dose of cytisine compared with the low dose did not have an effect on weight gain, infusions, or meal patterns T hough side by side comparisons were not made between the two doses themselves for the variables of interest, it is c lear from the data that these groups did not

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182 differ T his difference is not due to a titration of intake between the doses either, because the self administration rates were comparable for both doses. One possibility for the lack of a dose effect is that animals were pressing for the light cues that became associated with nicotine infusions rather than for the cytisine itself T he level of infusions during the extinction period did not significantly differ from those seen when cytisine was administered, and this lends increased support to the notion that stimuli associated with nicotine contribute to self administration behaviors (Donn y et al. 1999; 2003) T he proper control to test for this effect was not implemented in the current study T he lack of a n inactive lever prevents us from concluding that animals were pressing the lever to receive infusions of cytisine rather than to turn the cue light off W e could have used yoked controls that would have received cytisine or nicotine and been allowed to l ever press to turn the light off, but did not because we chose to examin e a dose effect instead, given time constraints and a limited number of animals to study. As mentioned previously, we did not examine the temporal pattern of infusions in this study H ad we examined the distribution of infusions throughout each session, we could have compared the cytisine phases with the extinction phases to determine if the pattern of responding differed between the two H owever, previous research indicates that in c ontrast to the extinction pattern seen with other substances I nstead, extinction from nicotine results in either a small increase in responding for the first day decline in responding the moment the drug is replaced by saline (Shoiab et al. 1997 ; Harris et al. 2007 ) in a manner which is nearly identical to self administration observed throughout the dark and l ight cycles during the treatment phase (Harris et al. 2007) S ince nicotine extinction lacks a

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183 administration with cytisine, as there would be no reason to assume t hat cytisine and extinction responding would differ from one another M ost likely, responding in both phases would be spread out throughout the entire period and would look quite similar E vidence from studies on nicotine extinction also highlights the c ontribution of cues, such that responding during this A nimals continue to press for the compound visual stimulus that accompanies the nicotine administration U nfortunately we cannot asses s th e trajectory of responding throughout extinction in the current study because this phase only lasted a few days. The meal pattern effects in this study indicate that in the short term, cytisine can to some extent prevent the kind of weight gain and incre ased food intake seen after nicotine cessation T hough animals receiving cytisine showed less of a decrease in weight gain and food intake compared to when they received nicotine these variables never returned to baseline levels during cytisine administr ation I ndividuals who are trying to quit smoking with cytisine take the drug frequently throughout the day and it is possible that this more consistent, frequent dosing would yield changes in body weight and food intake that were not detected by our self administration procedure N evertheless, this study provides additional evidence that cytisine may limit post cessation weight gain and may be a suitable alternative for people who want to quit smoking but are afraid to gain weight.

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184 Table 5 1. F and P va lues for the effects of phase and order of treatment on changes on body weight, number of infusions, and the meal pattern variables of interest Variables Phase Order F P F P Body Weight 5.221 **P < .01 .645 Infusions 4.606 *P < .05 6.089 ^ P = .09 Total Pellets 7.750 **P < .01 .203 ns Night Pellets 3.073 *P < .05 .445 ns Day Pellets 4.717 **P < .01 .023 ns Total Meals 2.143 ns .152 ns Night Meals 2.781 *P < .05 .018 ns Day Meals .842 ns 7.639 ^P = .07 Total Meal Size 4.102 **P < .01 1.972 ns Night Meal Size 4.100 **P < .01 1.487 ns Day Meal Size 2.775 *P < .05 .360 ns Table 5 2. T and P values for the changes in body weight gain throughout each phase of treatment, compared with the first phase of nicotine and previous phase Body Weight Gain Nic Comparison t P Phase Comparison t P Baseline Nic 1 6.862 **P < .01 Nic 1 2.5 Cyt 1.146 ns Nic 1 2.5 Cyt 1.146 ns Nic 1 Nic 2 .770 ns 2.5 Cyt Nic 2 3.015 *P < .05 Nic 1 5 Cyt 1.351 ns Nic 2 5 Cyt 1.681 ns Nic 1 Nic 3 1.308 ns 5 Cyt Nic 3 2.356 ^P = .07 Nic 1 Extinction 1.897 ^P = .09 Nic 3 Extinction 4.028 *P < .05 Table 5 3. T and P values for the changes in number of infusions throughout each phase of treatment, compared with the first phase of nicotine and the previous phase Number of Infusions Nic Comparison t P Phase Comparison t P Nic 1 2.5 Cyt 4.366 **P < .01 Nic 1 2.5 Cyt 4.366 **P < .01 Nic 1 Nic 2 5.485 **P < .01 2.5 Cyt Nic 2 1.790 ns Nic 1 5 Cyt 3.924 **P < .01 Nic 2 5 Cyt 1.297 ns Nic 1 Nic 3 1.563 ns 5 Cyt Nic 3 .899 ns Nic 1 Extinction 3.262 *P < .05 Nic 3 Extinction 1.821 ns

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185 Table 5 4. T and P values for the changes in total number of pellets throughout each phase of treatment, compared with the first phase of nicotine and the previous phase Total Pellets Nic Comparison t P Phase Comparison t P Baseline Nic 1 7.347 **P < .01 Nic 1 2.5 Cyt 2.630 *P < .05 Nic 1 2.5 Cyt 2.630 *P < .05 Nic 1 Nic 2 .775 ns 2.5 Cyt Nic 2 3.726 **P < .01 Nic 1 5 Cyt 2.064 ^P = .07 Nic 2 5 Cyt 1.950 ^P = .09 Nic 1 Nic 3 2.660 *P < .05 5 Cyt Nic 3 1.679 ns Nic 1 Extinction 2.516 *P < .05 Nic 3 Extinction 2.314 ^P = .07 Table 5 5. T and P values for the changes in the total number of pellets during the dark cycle throughout each phase of treatment, compared with the first phase of nicotine and the previous phase Night Pellets Nic Comparison t P Phase Comparison t P Baseline Nic 1 4.311 **P < .01 Nic 1 2.5 Cyt .577 ns Nic 1 2.5 Cyt .577 ns Nic 1 Nic 2 1.152 ns 2.5 Cyt Nic 2 3.294 *P < .05 Nic 1 5 Cyt .714 ns Nic 2 5 Cyt 1.298 ns Nic 1 Nic 3 .185 ns 5 Cyt Nic 3 1.218 ns Nic 1 Extinction 1.174 ns Nic 3 Extinction 1.370 ns Table 5 6. T and P values for the changes in the total number of pellets during the light cycle throughout each phase of treatment, compared with the first phase of nicotine and the previous phase Day Pellets Nic Comparison t P Phase Comparison t P Baseline Nic 1 5.801 **P < .01 Nic 1 2.5 Cyt 1.436 ns Nic 1 2.5 Cyt 1.436 ns Nic 1 Nic 2 1.402 ns 2.5 Cyt Nic 2 1.105 ns Nic 1 5 Cyt 1.670 ns Nic 2 5 Cyt .255 ns Nic 1 Nic 3 2.051 ns 5 Cyt Nic 3 .192 ns Nic 1 Extinction 2.028 ^P = .07 Nic 3 Extinction 1.392 ns

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186 Table 5 7. T and P values for the changes in the total number of meals throughout each phase of treatment, compared with the first phase of nicotine and the previous phase Total Meal Number Nic Comparison t P Phase Comparison t P Baseline Nic 1 1.571 ns Nic 1 2.5 Cyt .393 ns Nic 1 2.5 Cyt .393 ns Nic 1 Nic 2 .335 ns 2.5 Cyt Nic 2 .083 ns Nic 1 5 Cyt .039 ns Nic 2 5 Cyt .362 ns Nic 1 Nic 3 1.008 ns 5 Cyt Nic 3 1.707 ns Nic 1 Extinction .548 ns Nic 3 Extinction .045 ns Table 5 8. T and P values for the changes in the total number of meals during the dark cycle throughout each phase of treatment, compared with the first phase of nicotine and the previous phase Night Meal Number Nic Comparison t P Phase Comparison t P Baseline Nic 1 2.789 *P < .05 Nic 1 2.5 Cyt 1.112 ns Nic 1 2.5 Cyt 1.112 ns Nic 1 Nic 2 1.598 ns 2.5 Cyt Nic 2 .805 ns Nic 1 5 Cyt 1.441 ns Nic 2 5 Cyt .158 ns Nic 1 Nic 3 .177 ns 5 Cyt Nic 3 2.747 *P < .05 Nic 1 Extinction .615 ns Nic 3 Extinction .317 ns Table 5 9. T and P values for the changes in the total number of meals during the light cycle throughout each phase of treatment, compared with the first phase of nicotine and the previous phase Day Meal Number Nic Comparison t P Phase Comparison t P Baseline Nic 1 .978 ns Nic 1 2.5 Cyt .259 ns Nic 1 2.5 Cyt .259 ns Nic 1 Nic 2 .111 ns 2.5 Cyt Nic 2 .505 ns Nic 1 5 Cyt .213 ns Nic 2 5 Cyt .717 ns Nic 1 Nic 3 .665 ns 5 Cyt Nic 3 .295 ns Nic 1 Extinction .445 ns Nic 3 Extinction .989 ns

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187 Table 5 10. T and P values for the changes in meal size throughout each phase of treatment, compared with the first phase of nicotine and the previous phase Total Meal Size Nic Comparison t P Phase Comparison t P Baseline Nic 1 4.668 **P < .01 Nic 1 2.5 Cyt 1.602 ns Nic 1 2.5 Cyt 1.602 ns Nic 1 Nic 2 .781 ns 2.5 Cyt Nic 2 1.657 ns Nic 1 5 Cyt 1.939 ^P = .08 Nic 2 5 Cyt 1.844 ns Nic 1 Nic 3 .110 ns 5 Cyt Nic 3 2.441 ^P = .06 Nic 1 Extinction 1.857 ns Nic 3 Extinction 1.552 ns Table 5 11. T and P values for the changes in meal size during the dark cycle throughout each phase of treatment, compared with the first phase of nicotine and the previous phase Night Meal Size Nic Comparison t P Phase Comparison t P Baseline Nic 1 4.942 **P < .01 Nic 1 2.5 Cyt 1.744 ns Nic 1 2.5 Cyt 1.744 ns Nic 1 Nic 2 .080 ns 2.5 Cyt Nic 2 2.169 ^ P = .06 Nic 1 5 Cyt 1.677 ns Nic 2 5 Cyt 1.443 ns Nic 1 Nic 3 .381 ns 5 Cyt Nic 3 1.823 ns Nic 1 Extinction 1.693 ns Nic 3 Extinction 1.322 ns Table 5 12. T and P values for the changes in meal size during the light cycle throughout each phase of treatment, compared with the first phase of nicotine and the previous phase Day Meal Size Nic Comparison t P Phase Comparison t P Baseline Nic 1 5.241 **P < .01 Nic 1 2.5 Cyt 1.599 ns Nic 1 2.5 Cyt 1.599 ns Nic 1 Nic 2 1.229 ns 2.5 Cyt Nic 2 .941 ns Nic 1 5 Cyt 1.146 ns Nic 2 5 Cyt .628 ns Nic 1 Nic 3 1.461 ns 5 Cyt Nic 3 .347 ns Nic 1 Extinction 1.276 ns Nic 3 Extinction .585 ns

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188 Table 5 13. T and P values for the changes in body weight gained throughout each phase of treatment, throughout the baseline, 2.5 cytisine, 5 cytisine, and extinction periods Body Weight Base line Comparison t P Ext inction Comparison t P Base 2.5 Cyt 4.986 **P < .01 Ext. 2.5 Cyt .229 ns Base 5 Cyt 4.370 **P < .01 Ext. 5 Cyt .830 ns Base Ext 2.866 *P < .05 Table 5 1 4 T and P values for the changes in total number of pellets throughout each phase of treatment, throughout the baseline, 2.5 cytisine, 5 cytisine, and extinction periods Total Pellets Base line Comparison t P Ext inction Comparison t P Base 2.5 Cyt 7.160 **P < .01 Ext. 2.5 Cyt .426 ns Base 5 Cyt 3.930 **P < .01 Ext. 5 Cyt 1.906 ns Base Ext 3.509 P < .0 1 Table 5 1 5 T and P values for the changes in total number of pellets during the dark cycle, throughout the baseline, 2.5 cytisine, 5 cytisine, and extinction periods Night Pellets Base line Comparison t P Ext inction Comparison t P Base 2.5 Cyt 2.832 *P < .05 Ext. 2.5 Cyt .479 ns Base 5 Cyt 2.261 *P < .05 Ext. 5 Cyt .533 ns Base Ext 2.773 *P < .05 Table 5 1 6 T and P values for the changes in total number of pellets during the light cycle, throughout the baseline, 2.5 cytisine, 5 cytisine, and extinction periods Day Pellets Base line Comparison t P Ext inction Comparison t P Base 2.5 Cyt 1.521 ns Ext. 2.5 Cyt .233 ns Base 5 Cyt 2.637 *P < .05 Ext. 5 Cyt 1.006 ns Base Ext 1.659 ns

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189 Table 5 1 7 T and P values for the changes in total number of meals throughout each phase of treatment, throughout the baseline, 2.5 cytisine, 5 cytisine, and extinction periods Total Meals Base line Comparison t P p Ext inction Comparison t P Base 2.5 Cyt 1.869 ^P = .09 Ext. 2.5 Cyt .795 ns Base 5 Cyt 1.442 ns Ext. 5 Cyt .875 ns Base Ext 2.088 ^P = .07 Table 5 1 8 T and P values for the changes in total number of meals during the dark cycle, throughout the baseline, 2.5 cytisine, 5 cytisine, and extinction periods Night Meals Base line Comparison t P Ext inction Comparison t P Base 2.5 Cyt .709 ns Ext. 2.5 Cyt .580 ns Base 5 Cyt 1.825 ns Ext. 5 Cyt .585 ns Base Ext 1.685 ns Table 5 1 9 T and P values for the changes in total number of meals during the light cycle, throughout the baseline, 2.5 cytisine, 5 cytisine, and extinction periods Day Meals Baseline Comparison t P Extinction Comparison t P Base 2.5 Cyt 1.825 ns Ext. 2.5 Cyt .612 ns Base 5 Cyt .549 ns Ext. 5 Cyt 1.000 ns Base Ext 1.710 ns

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190 Table: 5 20 T and P values for the changes in total meal size throughout each phase of treatment, throughout the baseline, 2.5 cytisine, 5 cytisine, and extinction periods Total Meal Size Base line Comparison t P Ext inction Comparison t P Base 2.5 Cyt 4.393 **P < .01 Ext. 2.5 Cyt .227 ns Base 5 Cyt 2.782 *P < .05 Ext. 5 Cyt .275 ns Base Ext 3.093 *P < .05 Table: 5 2 1 T and P values for the changes in total meal size during the dark cycle, throughout the baseline, 2.5 cytisine, 5 cytisine, and extinction periods Night Meal Size Baseline Comparison t P Extinction Comparison t P Base 2.5 Cyt 3.633 **P < .01 Ext. 2.5 Cyt .502 ns Base 5 Cyt 2.849 *P < .05 Ext. 5 Cyt .255 ns Base Ext 3.439 **P < .01 Table: 5 2 2 T and P values for the changes in total meal size during the light cycle, throughout the baseline, 2.5 cytisine, 5 cytisine, and extinction periods Day Meal Size Baseline Comparison t P Extinction Comparison t P Base 2.5 Cyt 3.166 *P < .05 Ext. 2.5 Cyt .649 ns Base 5 Cyt 3.804 **P < .01 Ext. 5 Cyt .408 ns Base Ext 2.398 *P < .05

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191 Figure 5 1. Body weight: comparisons with nicotine treatments S hown are mean +/ SE body weight gained for animals that received the vehicle during baseline, the first session of nico tine (n = 12), 2.5 mg/kg cytisine (n = 11), the second session of nicotine (n = 9), 5 mg/kg cytisine (n = 10), the third session of nicotine (n = 6), and extinction (n = 10). **P < .01 difference between nicotine 1 and other treatments (Note: The nicotine 1 individual data are th e same for each phase, but the plotted values differ slightly because numbers of animals contributed to the paired comparison used in each phase)

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192 Figure 5 2. Body weight: comparisons between treatments S hown are mean +/ SE body weight gained, for a nimals that were monitored during baseline (n = 12) and received the first session of nicotine (n = 12), 2.5 mg/kg cytisine (n = 11), the second session of nicotine (n = 9), 5 mg/kg cytisine (n = 10), the third session of n icotine (n = 6), and extinction ( n = 10). *P < .05, **P < .01, ^P = .07, difference between nicotine 1 and other treatments

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193 Figure 5 3. Infusions: comparisons with nicotine treatment S hown are mean +/ SE number of lever presses for infusions, for animals that received the first session of nicotine (n = 12), 2.5 mg/kg cytisine (n = 11), the second session of nicotine (n = 9), 5 mg/kg cytisine (n = 10), the third session of nicotine (n = 6), and extinction (n = 10). *P < .05, **P < .01 difference between nicotine 1 and other treatments (Note: The Nicotine 1 individual data are the same for each phase, but the plotted values differ slightly because numbers of animals contributed to the paired comparison used in each phase)

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194 Figure 5 4. Infusions: comparisons with the previous treatment S hown are mean +/ SE number of lever presses for infusions, for animals that were monitored during baseline (n = 12) and received the first session of nicotine (n = 12), 2.5 mg/kg cytisine (n = 11), the second session of nicotine (n = 9) 5 mg/kg cytisine (n = 10), the third session of n icotine (n = 6), and extinction (n = 10). **P < .01, difference between nicotine 1 all other treatments

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195 Figure 5 5. Total pellets: comparisons with nicotine trea tment S hown are mean +/ SE total lever presses for pellets for animals that received the first session of nicotine (n = 12), 2.5 mg/kg cytisine (n = 11), the second session of nicotine (n = 9), 5 mg/kg cytisine (n = 10), the third session of nicotine (n = 6), and extinction (n = 10). ^P = .07, *P < .01, difference between nicotine 1 and other treatments (Note: The Nicotine 1 individual data are the same for each phase, but the plotted values differ slightly because numbers of animals contributed t o the paired comparison used in each phase)

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196 Figure 5 6. Total pellets: comparisons between treatments S hown are mean +/ SE total lever presses for pellets for animals that were monitored during baseline (n = 12) and received the first session of nicotine (n = 12), 2.5 mg/kg cytisine (n = 11), the second session of nicotine (n = 9), 5 mg/kg cytisine (n = 10), the third session of nicotine (n = 6), and extinction (n = 10). *P < .05, **P < .01, ^P = .09 (5 Cyt), ^P = .07 (Extinction) difference between nicotine 1 and other treatments

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197 Figure 5 7. Night pellets: comparisons with nicotine treatment S hown are mean +/ SE lever presses for pellets during the 12 hours of the dark cycle, for animals that rec eived the first session of nicotine (n = 12), 2.5 mg/kg cytisine (n = 11), the second session of nicotine (n = 9), 5 mg/kg cytisine (n = 10), the third session of nicotine (n = 6), and extinction (n = 10) (Note: The Nicotine 1 individual data are the same for each phase, but the plotted values differ slightly because numbers of animals contributed to the paired comparison used in each phase)

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198 Figure 5 8. Night pellets: comparisons between treatments S hown are mean +/ SE total lever presses for pellets during the 12 hours of the dark cycle, for animals that were monitored during baseline (n = 12) and received the first session of nicotine (n = 12), 2.5 mg/kg cytisine (n = 11), the second session of nicotine (n = 9), 5 mg/kg cytisine (n = 10), the third session of nicotine (n = 6), and extinction (n = 10). *P < .05, ** P < .01, difference between nicotine 1 and other treatments

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199 Figure 5 9. Day pellets: comparis ons with nicotine treatment S hown are mean +/ SE lever presses for pellets during the 11 hours of the light cycle, for animals that received the first session of nicotine (n = 12), 2.5 mg/kg cytisine (n = 11), the second session of nicotine (n = 9), 5 m g/kg cytisine (n = 10), the third session of nicotine (n = 6), and extinction (n = 10). ^P = .09 (Nic 3), ^P = .07 (Extinction) difference between nicotine 1 and other treatments (Note: The Nicotine 1 individual data are the same for each phase, but the pl otted values differ slightly because numbers of animals contributed to the paired comparison used in each phase)

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200 Figure 5 10. Day pellets: comparisons between treatments S hown are mean +/ SE total lever presses for pellets during the 11 hours of the light cycle, for animals that were monitored during baseline (n = 12) and received the first session of nicotine (n = 12), 2.5 mg/kg cytisine (n = 11), the second session of nicotine (n = 9), 5 mg/kg cytisine (n = 10) the third session of nicotine (n = 6), and extinction (n = 10). ** P < .01, difference between nicotine 1 and other treatments

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201 Figure 5 11. Total meal number: comparison with nicotine treatment S hown are mean +/ SE total number of meals, for animals that received the first session of nicotine (n = 12), 2.5 mg/kg cytisine (n = 11), the second session of nicotine (n = 9), 5 mg/kg cytisine (n = 10), the third session of nicotine (n = 6), and extinction (n = 10) (Note: The Nicotine 1 individual data are the same for each phase, but the plotted values differ slightly because numbers of animals contributed to the paired comparison used in each phase)

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202 Figure 5 12. Nigh t meals: comparisons with nicotine treatment S hown are mean +/ SE total number of meals during the 12 hours of the dark cycle, for animals that received the first session of nicotine (n = 12), 2.5 mg/kg cytisine (n = 11), the second session of nicotine (n = 9), 5 mg/kg cytisine (n = 10), the third session of nicotine (n = 6), and extinction (n = 10) (Note: The Nicotine 1 individual data are the same for each phase, but the plotted values differ slightly because numbers of animals contributed to the paire d comparison used in each phase)

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203 Figure 5 13. Night meals: comparisons between treatments S hown are mean +/ SE number of meals during the 12 hours of the dark cycle, for animals that were monitored during baseli ne (n = 12) and received the first session of nicotine (n = 12), 2.5 mg/kg cytisine (n = 11), the second session of nicotine (n = 9), 5 mg/kg cytisine (n = 10), the third session of nicotine (n = 6), and extinction (n = 10). *P < .05, difference between ni cotine 1 and other treatments

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204 Figure 5 14. Day meals: comparisons with nicotine treatment S hown are mean +/ SE total number of meals during the 11 hours of the light cycle, for animals that received the first session of nicotine (n = 12), 2.5 mg/kg cytisine (n = 11), the second session of nicotine (n = 9), 5 mg/kg cytisine (n = 10), the third session o f nicotine (n = 6), and extinction (n = 10) (Note: The Nicotine 1 individual data are the same for each phase, but the plotted values differ slightly because numbers of animals contributed to the paired comparison used in each phase)

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205 Figure 5 15. T otal meal size: comparisons with nicotine treatment S hown are mean +/ SE total number of pellets per meal, for animals that received the first session of nicotine (n = 12), 2.5 mg/kg cytisine (n = 11), the second session of nicotine (n = 9), 5 mg /kg cytisine (n = 10), the third session of nicotine (n = 6), and extinction (n = 10). ^P = .08 (5 Cyt), difference between nicotine 1 and other treatments (Note: The Nicotine 1 individual data are the same for each phase, but the plotted value s differ slightly because numbers of animals contributed to the paired comparison used in each phase)

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206 Figure 5 16. Total meal size: comparisons between treatments S hown are mean +/ SE total number of pellets per me al, for animals that were monitored during baseline (n = 12) and received the first session of nicotine (n = 12), 2.5 mg/kg cytisine (n = 11), the second session of nicotine (n = 9), 5 mg/kg cytisine (n = 10), the third session of nicotine (n = 6), and ext inction (n = 10). ^P = .06, difference between nicotine 1 and other treatments

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207 Figure 5 17. Night meal size: comparisons with nicotine treatment S hown are mean +/ SE total number of pellets per meal during the 12 hours of the dark cycle, for animals that received the first session of nicotine (n = 12), 2.5 mg/kg cytisine (n = 11), the second session of nicotine (n = 9), 5 mg/kg cytisine (n = 10), the third session of nicotine (n = 6), and extinction (n = 10). **P < .01, difference between nicotine 1 and other treatments (Note: The Nicotine 1 individual data are the same for each phase, but the plotted values differ slightly because numbers of animals c ontributed to the paired comparison used in each phase)

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208 Figure 5 18. Night meal size: comparisons between treatments S hown are mean +/ SE number of pellets per meal during the 12 hours of the dark cycle, for animals that were monitored during baseline (n = 12) and received the first session of nicotine (n = 12), 2.5 mg/kg cytisine (n = 11), the second session of nicotine (n = 9) 5 mg/kg cytisine (n = 10), the third session of nicotine (n = 6), and extinction (n = 10). ^P = .06, **P < .01, difference between nicotine 1 and other treatments.

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209 Figure 5 19. Day meal size: comparison with nicotine treatment S hown are me an +/ SE total number of pellets per meal during the 11 hours of the light cycle, for animals that received the first session of nicotine (n = 12), 2.5 mg/kg cytisine (n = 11), the second session of nicotine (n = 9), 5 mg/kg cytisine (n = 10), the third s ession of nicotine (n = 6), and extinction (n = 10) (Note: The Nicotine 1 individual data are the same for each phase, but the plotted values differ slightly because numbers of animals contributed to the paired comparison used in each phase)

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210 Figure 5 20 Day meal size: comparisons between treatments S hown are mean +/ SE number of pellets per meal during the 11 hours of the light cycle, for animals that were monitored during baseline (n = 12) and received th e first session of nicotine (n = 12), 2.5 mg/kg cytisine (n = 11), the second session of nicotine (n = 9), 5 mg/kg cytisine (n = 10), the third session of nicotine (n = 6), and extinction (n = 10). **P < .01, difference between nicotine 1 and other treatme nts.

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211 Figure 5 21. Body weight comparisons of cytisine treatment s S hown are mean +/ SE body weight gained, for animals that were monitored during baseline (n = 12) and received 2.5 mg/kg cytisine (n = 11), 5 mg/kg cytisine (n = 10), and extinction (n = 10). **P < .01, difference between treatments

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212 Figure 5 22. Infusions: comparisons of cytisine treatments S hown are mean +/ SE number of lever presses for infusions, for animals that were monitored during baseline (n = 12) and received 2.5 mg/kg cytisine (n = 11), 5 mg/kg cytisine (n = 10), and extinction (n = 10).

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213 Figure 5 23. Total pellets: comparisons of cytisine treatment S hown are mean +/ SE total lever presses for pellets, for animals that were monitored during baseline (n = 12) and received 2.5 mg/kg cytisine (n = 11), 5 mg/kg cytisine (n = 10), and extinction (n = 10). **P < .01, difference between treatments

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214 Figure 5:24. Night pellets: comparisons of cytisine treatment S hown are mean +/ SE total lever presses for pellets during the 12 hours of the dark cycle, for animals that were monitored during baseline (n = 12) and received 2.5 mg/kg cytisine (n = 11), 5 mg/kg cytisine (n = 10), and extinction (n = 10). *P < .05, difference between treatments

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215 Figure 5 25. Day pellets: comparisons of cytisine treatment S hown are mean +/ SE lever presses for pellets during the 11 hours of the light cycle, for animals that were monitored during baseline (n = 12) and received 2.5 mg/kg cytisine (n = 11), 5 mg/kg cytisine (n = 10), and extinction (n = 10). *P < .05, difference between treat ments

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216 Figure 5 26. Total meal size: comparisons of cytisine treatment S hown are mean +/ SE total number of pellets per meal, for animals that were monitored during baseline (n = 12) and received 2.5 mg/kg cytisine (n = 11), 5 mg/kg cytisine (n = 10), and extinction (n = 10). **P < .01, *P < .05, difference between treatments

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217 Figure 5 27. Night meal size: comparisons of cytisine treatment S hown are mean +/ SE total number of pellets per meal during the 12 hours of the dark cycle, mean +/ SE total number of pellets per meal, for animals that were monitored during baseline (n = 12) and received 2.5 mg/kg cytisine (n = 11), 5 mg/kg cytisine (n = 10 ), and extinction (n = 10). **P < .01, *P < .05, difference between treatments

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218 Figure 5 28. Day meal size: comparison of cytisine treatment S hown are mean +/ SE total number of pellets per meal during the 11 hours of the light cycle, mean +/ SE tota l number of pellets per meal, for animals that were monitored during baseline (n = 12) and received 2.5 mg/kg cytisine (n = 11), 5 mg/kg cytisine (n = 10), and extinction (n = 10). **P < .01, *P < .05, differe nce between treatments

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219 CHAPTER 6 GENERAL DISCUSSION The Relationship between Nicotine and Flavor Cues The relationship between drugs of abuse and flavor cues has been studied in a variety of ways T ypically, an injection of the drug is given and the animal is then allowed to consume a flavored solution ( Goudie 1977, Ferrari 1991, Fenu 2010) W hen nicotine is the drug of choice, a conditioned taste aversion (CTA) is typically found, and it is generally accepted that CTAs for nicotine demonstrate the a versive properties of the drug H owever, in light of the results presented in Chapter 2, it is possible that the methods used to study the interaction between flavor cues and nicotine are only examining the acute effects of nicotine B y providing the fla vored solution as the only source of fluid alongside noncontingent infusions of nicotine, we were able to measure preferences beyond the first few days where the aversion was occurring and look instead at the reinforcing properties of nicotine T he acquis ition of a nicotine associated flavor preference seems partially influenced by dose of nicotine and not by the initial or unconditional preference for the paired flavor, suggesting that flavor preference, similar to cue response learning, is influenced by how reinforcing the drug itself is B ecause animals were given preference tests in drug free conditions, it is likely that increased licking for the nicotine associated flavor is ameliorating withdrawal symptoms, which would account for the increased pref erence for the flavor over time T his is the first report of successful conditioning of preference for a flavor that was associated with presence of nicotine, and underscores the importance of the extended access protocol which both fosters nicotine depen dence and prevents the flavor from being avoided. The implications for these results on human smoking are large T obacco companies especially target new (young) smokers, and the creation of flavored cigarettes, though banned

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220 now, is just one example of t his strategy A dditionally, water pipe tobacco smoking (also known as hookah) is showing increased prevalence among college students across the nation W ater pipe tobacco typically uses sheesha tobacco, a mixture of tobacco, flavorings, and sweetener ( as reviewed by Cobb et al. 2010) A pproximately 20% of students reported smoking in the last 30 days (Eissenberg et al. 2008) and 34 41% of students have tried it at least once (Jackson and Aveyard 2008) A s reviewed by Eissenberg et al. (2009) water pipe tobacco smoking is typically perceived as being less harmful than cigarette smoking despite evidence to the contrary which suggests it may be more harmful because of increased exposure to toxicants via increased inhalation I n addition, individuals who s moke water pipe tobacco also typically smoke I ndividuals that use water pipe tobacco also report some symptoms of addiction including cravings (Jackson and Aveyard 2008 ) T here are no studies to date that have investigated the influence the flavor of water pipe tobacco has on the reinforcing properties of the tobacco, and the current study may provide some evidence that the flavor is contributing to the addictive potent ial of smoking hookah. The Effects of Nicotine on Body Weight and Meal Patterns Nicotine use influences body weight via metabolic mechanisms, changes in physical activity, and alterations in total food intake T he experiments described in Chapters 3 and 5 lend support to the notion that changes in body weight from nicotine use are at least partially mediated by changes in food intake P revious studies investigating the effects of nicotine on food intake in rats have administered the drug either subcutan eously (Grunberg et al. 1985; Grunberg et al. 1986; Miyata 2001) or intraperitoneally (Li et al. 2000; Bellinger et al. 2003a ; Guan et al. 2004) T he two studies described in this dissertation are the first to use intravenous administratio n to address this issue T he procedure used in Chapter 3 involved giving

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221 noncontingent administration of nicotine to rats every 30 minutes throughout the dark cycle and the last 3 hours of the light cycle, when the animals are typically most active I n this study, we replicated effects from other experiments which used IP injections, and discovered some new effects A s seen in previous studies, nicotine decreases total food intake via decreases in intake during the dark cycle (Bellinger et al. 2003 b ; Gu an et al. 2004) T hese decreases in total food intake are due to changes in both meal size and meal number N icotine decreases meal size in the dark cycle and, apparently in order to compensate for this decrease in calor ic intake, rats consume more frequ ent meals throughout the dark cycle as well T hough this compensatory mechanis m has been characterized before accompanies these changes in food intake T he novel finding in our study is a decrease in fo od intake and meal size in the light cycle without a compensatory increase in meal number T his decrease explains the decrease in body weight gain much more completely than previous studies. It is possible that the effects seen in the light cycle were si mply due to the experimental manipulation of giving nicotine to the animals during a portion (3 hours) the 11 hour light cycle H owever, animals in this study ate more food during the part of the light cycle during which they received nicotine and decreas ed their food intake during the 8 hours when nicotine was not given A s a result, the proportion of total intake during the daytime nicotine administration period was much greater than that seen during the period without nicotine T hese results indicate that because animals were given nicotine in the light cycle, they tailored their eating to when administered T he second study in Chapter 5 supports the validity of our m odel and the results that were obtained, by showing similar meal pattern changes to those observed during noncontingent administration W hen animals were allowed to self administer nicotine, the same

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222 changes in meal patterns were observed T here was a de crease in meal size in the light and dark cycles and a compensatory increase in meal number only in the dark cycle T hese changes in meal patterns which occur within the first four days of stable nicotine self administration demonstrate the validity of us ing noncontingent IV nicotine administration in a 23h protocol with infusions of the drug in both the light and dark cycles. food intake occur through changes in sat iety mechanisms, as evidenced by decreased meal size A t this point, it is unclear which neuropeptides in the brain are affected by nicotine, and possible candidates were reviewed in Chapter 1 I n addition to those already discussed, cholecystokinin (CCK ) is a possible target for nicotine E vidence indicates that chronic administration of nicotine via oral gavage in rats is associated with elevated plasma levels of CCK which is accompanied a decrease in body weight, although not a decrease in food intake (Chowdhury et al. 1989) H owever, other direct evidence linking nicotine use to differential levels of CCK is sparse I nterestingly, CCK administration produces changes in meal patterns similar to those we observed in nicotine treated animals A s a neu ropeptide involved in satiety, CCK administration results in decreases in meal size but also increases in meal number ( as reviewed by Adan et al. 2008) T hese specific changes in meal patterns seem somewhat unique to nicotine and CCK, and thus more resear ch needs to be done examining the effects of nicotine on CCK as well as any other neuropeptides that might influence this system. The Effects of Cytisine on Body Weight and Meal Patterns The evidence we found supporting our model of noncontingent nicotine administration in a 23h protocol suggested that this would be a good model to use to examine the effects of other drugs on meal patterns I n light of the fact that many individuals who smoke cite a fear of weight gain as a reason for not quitting ( as rev iewed by Pistelli et al. 2009) our model seems a

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223 good way to test pharmacotherapies for nicotine cessation and their potential to prevent weight gain Much of the current research being conducted on non nicotine pharmacotherapies surrounds the drug cytisi ne (Vinnikov et al. 2008; Rollema et al. 2010; Walker et al. 2011), which has been approved in Eastern Europe for smoking cessation C ytisine is similar in chemical composition to varenicline, the most successful smoking cessation aid to date and one of t wo drugs approved by the FDA to help people quit smoking ( as reviewed by Polosa and Benowitz 2011) T he second approved drug is bupropion which is approximately as effective as nicotine replacement therapies and has shown the most promise in delaying post cessation weight gain (Jorenby et al. 1999) D espite the availability of these two drugs, the quit rates among individuals that use them are still relatively low and the drugs are extremely expensive (West et al. 2011) O n the other hand, cytisine is much less expensive and seems at least as effective as bupropion (Walker et al. 2011; West et al. 2011) B ecause of the health concerns either help delay or prevent weight gain after smoking cessation. The results presented within Chapter 4 indicate that cytisine may be a good alternative for people who want to quit smoking but are concerned about gaining weight W hen given noncontingently, cytisine reduced body weight and food intake T he reductions in total food consumed manifested through changes in meal number in both the light and dark cycles, indicating that cytisine may not have effects on short term satiety signals as previously discussed I t is important to note that because of th e persistent difference in body weight gain between controls and those that received cytisine, there may be a long term change in metabolism that is also at work T he current model was sensitive enough to detect different

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224 changes in meal patterns from wha t was seen in the nicotine experiment, lending credence to the notion that cytisine differentially affects meal patterns and body weight. The results from the self administration study in Chapter 5 also support the notion that cytisine causes a reduction in body weight and food intake T hough self administration of cytisine without previous exposure to nicotine was not examined in this study, the administration of cytisine still resulted in decreased body weight gain and decreased food intake I nterestin gly, the meal patterns during the cytisine phase in this experiment differed from the previous study where cytisine was administered noncontingently T he self administration of cytisine resulted in decreases in meal size rather than meal number B ecause we did not examine cytisine I f nicotine administration is acting on receptors for which cytisine has different affinities, and is fundamentally altering these receptor s (through desensitization for example), it is possible that cytisine would have a different effect than if nicotine were never administered F uture studies should examine the self administration capabilities of cytisine alone and the meal patterns that c oincide with cytisine self administration to see if this might be the case T he interaction between these drugs is important to understand; at no point will cytisine be given without a previous history of nicotine R egardless of these questions however, the studies discussed within this chapter indicate that cytisine, in the presence of nicotine or by itself, results in decreased weight gain and food intake which persists for a period of time after administration of the drug is stopped.

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247 BIOGRAPHICAL SKETCH Patricia E. Grebenstein graduated cum laude from Wittenberg University i n Springfield, OH in 2007 with a Bachelor of Arts Degree in p sychology with a minor in E nglish Her interest in behavioral neuroscience began after taking several classes on physiological psychology, sensation and perception, and psychophysiology. An int erest in drug addiction arose during a summer internship where she assisted in writing a literature review on the neurobiology and neuropsychological effects of MDMA (Ecstacy). In the fall of 2007, she was accepted to the l Neuroscience PhD program in the Department of Psychology under the mentorship of Dr. Neil E. Rowland whose research interest included ingestive behavior and nicotine addiction. gaining a better understanding of the role of sensory reinforcers in maintaining and sustaining nicotine self administration, particularly a flavor stimulus. She received her Master of Science Degree from the University of Florida in the fall of 2009. Her inter ests then evolved into investigating the effects of nicotine on body weight and meal patterns and the implications these effects have on treatments for smoking cessation. Patricia then examined the non nicotine pharmacotherapy, cytisine, for effects on bo dy weight and meal patterns, along with its reinforcing potential in a self administration model. She received her Degree of Philosophy from the University of Florida in the summer of 2012.