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Murine Models of Overconsumption and Binge Eating

Permanent Link: http://ufdc.ufl.edu/UFE0022463/00001

Material Information

Title: Murine Models of Overconsumption and Binge Eating Effect of Melanocortin-4 and Cannabinoid CB1 Receptor Activity on Caloric Intake and Body Weight in Female C57Bl/6J Mice
Physical Description: 1 online resource (113 p.)
Language: english
Creator: Mathes, Clare
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: am251, choice, diet, feeding, mtii, shu9119
Psychology -- Dissertations, Academic -- UF
Genre: Psychology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Incidence of obesity and overweight in America has grown to epidemic proportions. Regulatory systems in the brain that modulate eating behavior are well understood; however, the extent to which the neurochemical components of these systems affect need-free eating behavior in environments similar to those that promote overconsumption in humans has not been thoroughly explored. We have proposed models of overconsumption and binge eating in rats that have allowed us to explore the effectiveness of drugs that have possible therapeutic value in treating obesity and the role of specific receptor systems in the control of eating. In this dissertation, we attempt to generalize these models to mice and specifically examine the role of the melanocortin-4 receptor (MC4R) and the cannabinoid CB1 receptor (CB1R) on diet selection, caloric intake, and body weight change, and to assess if these receptor systems differentially affect hedonic versus regulatory eating. Mice were given (in conjunction with ad libitum moist chow) access to a sugary and fat dessert (sugar fat whip, SFW) on one of two protocols: either an overconsumption protocol, which consisted of either 8 or 24 h access to SFW, or a binge eating protocol, which consisted of 2 h access to SFW in either a low restricted or high restricted fashion. Mice on the overconsumption and binge protocols were centrally injected daily for 24 days with MC4R agonist MTII or MC4R antagonist SHU9119. Mice on the overconsumption protocol only were peripherally injected daily for 21 days with CB1R antagonist AM251. Food intake and body weight were measured daily. We conclude that mice do not overconsume or binge eat on these protocols, suggesting species differences in regulation of food intake between rats and mice when these animals are presented with choices and/or limited access to commodities. MTII decreased and SHU9119 increased caloric intake at some time points, but did not affect total caloric intake in mice at the dose level and frequency used in these studies. The extent to which these modulations affected diet selection was unclear. Similar to its effect in rats, AM251 decreased total caloric intake in mice, but different from rats, this was not due to a selective decrease in SFW intake. These studies raise interesting questions regarding species differences and protocol functionality that must be reconciled in order for a valid model of human obesity to be properly explored.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Clare Mathes.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Rowland, Neil E.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022463:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022463/00001

Material Information

Title: Murine Models of Overconsumption and Binge Eating Effect of Melanocortin-4 and Cannabinoid CB1 Receptor Activity on Caloric Intake and Body Weight in Female C57Bl/6J Mice
Physical Description: 1 online resource (113 p.)
Language: english
Creator: Mathes, Clare
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: am251, choice, diet, feeding, mtii, shu9119
Psychology -- Dissertations, Academic -- UF
Genre: Psychology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Incidence of obesity and overweight in America has grown to epidemic proportions. Regulatory systems in the brain that modulate eating behavior are well understood; however, the extent to which the neurochemical components of these systems affect need-free eating behavior in environments similar to those that promote overconsumption in humans has not been thoroughly explored. We have proposed models of overconsumption and binge eating in rats that have allowed us to explore the effectiveness of drugs that have possible therapeutic value in treating obesity and the role of specific receptor systems in the control of eating. In this dissertation, we attempt to generalize these models to mice and specifically examine the role of the melanocortin-4 receptor (MC4R) and the cannabinoid CB1 receptor (CB1R) on diet selection, caloric intake, and body weight change, and to assess if these receptor systems differentially affect hedonic versus regulatory eating. Mice were given (in conjunction with ad libitum moist chow) access to a sugary and fat dessert (sugar fat whip, SFW) on one of two protocols: either an overconsumption protocol, which consisted of either 8 or 24 h access to SFW, or a binge eating protocol, which consisted of 2 h access to SFW in either a low restricted or high restricted fashion. Mice on the overconsumption and binge protocols were centrally injected daily for 24 days with MC4R agonist MTII or MC4R antagonist SHU9119. Mice on the overconsumption protocol only were peripherally injected daily for 21 days with CB1R antagonist AM251. Food intake and body weight were measured daily. We conclude that mice do not overconsume or binge eat on these protocols, suggesting species differences in regulation of food intake between rats and mice when these animals are presented with choices and/or limited access to commodities. MTII decreased and SHU9119 increased caloric intake at some time points, but did not affect total caloric intake in mice at the dose level and frequency used in these studies. The extent to which these modulations affected diet selection was unclear. Similar to its effect in rats, AM251 decreased total caloric intake in mice, but different from rats, this was not due to a selective decrease in SFW intake. These studies raise interesting questions regarding species differences and protocol functionality that must be reconciled in order for a valid model of human obesity to be properly explored.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Clare Mathes.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Rowland, Neil E.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022463:00001


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1 MURINE MODELS OF OVERCONS UMPTION AND BINGE EATING: EFFECT OF MELANOCORTIN-4 AND CAN NABINOID CB1 RECEPTOR ACTIVITY ON CALORIC INTAKE AND BODY WEIGHT IN FEMALE C57BL/6J MICE By CLARE M. MATHES A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2008 Clare M. Mathes

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3 ACKNOWLEDGMENTS I thank m y mentor, Dr. Neil Rowland, for his support and patience. I thank my committee members, Drs. Alan Spector, Jesse Dallery, Joanna Peris, and Carrie Haskell-Luevano, for their time and insightful aid. I thank research scient ist, Kim Robertson, and undergraduate assistants, Melissa Chaney, Marco Ferrara, and Deepak Sure sh, who provided time and energy. I thank my friends and lab members for their professional an d personal support, especially Anaya Mitra and Katherine Gamble. Finally, I thank my family for their loyalty.

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4 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................3LIST OF TABLES................................................................................................................. ..........6LIST OF FIGURES.........................................................................................................................7ABSTRACT.....................................................................................................................................9CHAPTER 1 GENERAL INTRODUCTION.............................................................................................. 11Obesity and Society............................................................................................................ ....11The Role of the Environment.................................................................................................12Physiological Regulation of Food Intake...............................................................................14Reward Mechanisms Affecting Food Intake.......................................................................... 16Melanocortin-4 Receptor and Food Intake............................................................................. 18Cannabinoid CB1 Receptor and Food Intake......................................................................... 21Summary.................................................................................................................................232 GENERAL METHODS......................................................................................................... 25Introduction and Previous Studies..........................................................................................25Diet Protocols..................................................................................................................25Mouse Models.................................................................................................................27Pharmacological Models................................................................................................. 29General Methods.....................................................................................................................29Animals............................................................................................................................29Diets.................................................................................................................................303 EFFECT OF A MELANOCORTIN RECEPTOR AGONIST AND ANTAGONIST ON OVERCONSUMPTION AND BINGE EATING IN FEMAL E MICE................................. 43Introduction................................................................................................................... ..........43Review of Diet Protocols.................................................................................................43Review of Mouse and Ph armacological Models............................................................. 43Methods..................................................................................................................................44Animals and Diets...........................................................................................................44Surgery............................................................................................................................44Experimental Design....................................................................................................... 45Experiment 1: Overconsumption..................................................................................... 45Experiment 2: Binge Eating............................................................................................ 46Experiment 3: Effect of SHU 9119 and MTII on Overconsumption............................... 46

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5 Experiment 4: Effect of S HU9119 a nd MTII on Binge Eating.......................................47Drugs...............................................................................................................................47Statistics...........................................................................................................................47Results.....................................................................................................................................48Experiment 1: Overconsumption..................................................................................... 48Caloric intake fr om moist chow............................................................................... 49Caloric intake from Su gar Fat Whip (SFW)............................................................ 50Change in body weight.............................................................................................51Experiment 2: Binge Eating............................................................................................ 51Total caloric intake...................................................................................................51Caloric intake fr om moist chow............................................................................... 52Caloric intake from SFW......................................................................................... 53Change in body weight.............................................................................................53Experiment 3: Effect of SHU 9119 and MTII on Overconsumption............................... 53Mice given no access to SFW.................................................................................. 53Mice given 24 h access to SFW...............................................................................55Mice given 8 h access to SFW................................................................................. 58Experiment 4: Effect of S HU9119 and MTII on Binge Eating.......................................62Mice given Low Restriction (LR) 2 h access to SFW.............................................. 62Mice given High Restriction (HR) 2 h Access to SFW........................................... 65Discussion...............................................................................................................................68Experiment 1: Overconsumption..................................................................................... 68Experiment 2: Binge Eating............................................................................................ 70Experiment 3: Effect of SHU 9119 and MTII on Overconsumption............................... 70Experiment 4: Effect of S HU9119 and MTII on Binge Eating.......................................73Summary..........................................................................................................................744 EFFECT OF CANNABINOID CB1 RECEPTOR ANTAGONIST ON OVERCONSUMPTION IN FEMALE MICE .......................................................................87Introduction................................................................................................................... ..........87Methods..................................................................................................................................88Animals and Housing......................................................................................................88Experimental Design....................................................................................................... 88Drugs...............................................................................................................................88Statistics...........................................................................................................................89Results.....................................................................................................................................89Discussion...............................................................................................................................915 GENERAL DISCUSSION..................................................................................................... 95LIST OF REFERENCES.............................................................................................................101BIOGRAPHICAL SKETCH.......................................................................................................113

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6 LIST OF TABLES Table page 3-1 Caloric intakes, reported as mean + standard error kilocalories (M + SE kcal), across replications of mice inject ed daily with vehicle................................................................ 833-2 Caloric intakes (M + SE kcal) across replications of mice in jected daily with SHU9119............................................................................................................................843-3 Caloric intakes (M + SE kcal) across replications of mice inject ed daily with MTII.......853-4 Cumulative body weight change (M + SE g) across feeding and dose groups..................86

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7 LIST OF FIGURES Figure page 2-1 Caloric intake (M + SE kcal) of rats given ad libitum access to m oist chow and 8 h access to either sugar gel (SG) (n=8) or sugar fat whip (SFW) (n=8) or no additional dessert (n=8) daily for 15 days.......................................................................................... 312-2 Cumulative change in body weight (M + SE g) from baseline of rats fed the desserts described in the caption for Figure 2-1.............................................................................. 322-3 Caloric intake (M + SE kcal ) of rats given 8 h access to SFW and injected with either vehicle or Rimonabant (1 mg/kg) daily for 7 days............................................................ 332-4 Cumulative change in body weight (M + SE g) from baseline of rats on the dessert protocol and drug regimen describe d in the caption for Figure 2-3.................................. 342-5 Caloric intake (M + SE kcal ) of rats given 8 h access to SFW and injected with either vehicle or one of three doses of AM 251 (0.3, 1.0, 3.0 mg/kg) daily for 15 days.............. 352-6 Cumulative change in body weight (M + SE g) from baseline of rats on the dessert protocol and drug regimen describe d in the caption for Figure 2-5.................................. 362-7 Caloric intake (M + SE k cal) of either young (ie, 45 days of age) or aged (retired breeders, approximately 9 months of age) rats given 2 h access to SFW either every day (low restriction, LR) or every othe r day (high restric tion, HR) for 50 days............... 372-8 Cumulative change in body weight (M + SE g) from baseline of rats on the dessert protocol described in th e caption for Figure 2-7................................................................ 382-9 Caloric intake (M + SE kcal) of mice w ith either full genetic expression (wild type, WT), heterozygous expression (HET), or deletion (knock out, KO) of the melanocortin-4 receptor (MC4R) that we re given 8 h access to SFW for 14 days...........392-10 Cumulative change in body weight (M + SE g) from baseline of mice of the genotypes and on the dessert protocol desc ribed in the caption for Figure 2-9................. 402-11 Caloric intake (M + SE kcal) of WT, MC4RHET, and MC4RKO mice either LR or HR 2 h access to SFW for 14 days.................................................................................... 412-12 Cumulative change in body weight (M + SE g) from baseline of mice of the genotypes and on the dessert protocol desc ribed in the caption for Figure 2-11............... 423-1 Caloric intake (M + SE kcal) of mice in jected with vehicle (V) and given no SFW or either 8 h or 24 h access to SFW for 24 days.....................................................................753-2 Cumulative change in body weight (M + SE g) from baseline of mice on the diet and drug regimen described in Figure 3-1................................................................................76

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8 3-3 Caloric intake (M + SE kcal) of mice inje cted with vehicle and given either HR or LR 2 h access to SFW for 24 days .................................................................................... 773-4 Caloric intake (M + SE kcal) of mi ce given no SFW and injected with either melanocortin-4 receptor (MC4R) agonist melanotan-II (MTII) or MC4R antagonist SHU9119 for 24 days......................................................................................................... 783-5 Caloric intake (M + SE kcal) of mice given 24 h access to SFW and injected with either MTII or SHU9119 for 24 days................................................................................ 793-6 Caloric intake (M + SE kcal) of mice given 8 h access to SFW and injected with either MTII or SHU9119 for 24 days ............................................................................... 803-7 Caloric intake (M + SE kcal) of mice gi ven LR 2 h access to SFW and injected with either MTII or SHU9119 for 24 days. Da ta were taken from binge days only................ 813-8 Caloric intake (M + SE kcal) of mice gi ven HR 2 h access to SFW and injected with either MTII or SHU9119 for 24 days. Da ta were taken from binge days only................ 824-1 Caloric intake (M + SE kcal) of mice given 8 h access to SFW and injected with either vehicle or one of three doses of AM251 (1, 5, 10 mg/kg) daily for 15 days........... 934-2 Cumulative change in body weight (M + SE g) from baseline of rats on the dessert protocol and drug regimen describe d in the caption for Figure 4-1.................................. 94

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9 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy MURINE MODELS OF OVERCONS UMPTION AND BINGE EATING: EFFECT OF MELANOCORTIN-4 AND CAN NABINOID CB1 RECEPTOR ACTIVITY ON CALORIC INTAKE AND BODY WEIGHT IN FEMALE C57BL/6J MICE By Clare M. Mathes August 2008 Chair: Neil Rowland Major: Psychology Incidence of obesity and overweight in Am erica has grown to epidemic proportions. Regulatory systems in the brain that modulate eating behavior ar e well understood; however, the extent to which the neurochemical components of these systems affect need-free eating behavior in environments similar to those that pr omote overconsumption in humans has not been thoroughly explored. We have proposed models of overconsumption and binge eating in rats that have allowed us to explore the effectiveness of drugs that have possi ble therapeutic value in treating obesity and the role of specific receptor systems in th e control of eating. In this dissertation, we attempt to generalize these models to mice and specifically examine the role of the melanocortin-4 receptor (MC4R) and th e cannabinoid CB1 receptor (CB1R) on diet selection, caloric intake, and body weight change, and to assess if these receptor systems differentially affect hedoni c versus regulatory eating. Mice were given (in conjunction with ad libitum moist chow) access to a sugary and fat dessert (sugar fat whip, SFW) on one of two protocols: either an overconsumption protocol, which consisted of either 8 or 24 h access to SFW, or a binge eating protocol, which consisted of 2 h access to SFW in either a low restricted or high restricted fashion. Mice on the

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10 overconsumption and binge protocols were centra lly injected daily for 24 days with MC4R agonist MTII or MC4R antagonist SHU9119. Mice on the overconsumption protocol only were peripherally injected daily for 21 days w ith CB1R antagonist AM251. Food intake and body weight were measured daily. We conclude th at mice do not overconsume or binge eat on these protocols, suggesting species differences in regulation of food intake between rats and mice when these animals are presented with c hoices and/or limited access to commodities. MTII decreased and SHU9119 increa sed caloric intake at some time points, but did not affect total caloric intake in mice at the dose le vel and frequency used in these studies. The extent to which these modulations affected diet selection was unclear. Similar to its effect in rats, AM251 decreased total caloric intake in mice, but different from rats, this was not due to a selective decrease in SFW intake. These studie s raise interesting questions regarding species differences and protocol functiona lity that must be reconciled in order for a valid model of human obesity to be properly explored.

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11 CHAPTER 1 GENERAL INTRODUCTION Obesity and Society Prevalence of obesity in the United States has risen at an alarming rate, and the trend does not seem to be slowing. Incidence of be ing overweight, clinica lly defined as a body mass index (BMI, weight in kilograms divided by the s quare of height in meters) greater than 25, and of being obese, defined as BMI greater than 30, has increased by 40 and 110%, respectively, since 1980 (Stein and Colditz, 2004). Data from 1999 to 2002 collected vi a the National Health and Nutrition Examination Survey (NHANES) show that nearly 1/3 of adults are obese and 1/6 of children and adolescents are overweight (Baskin et al. 2005; Ogden et al. 2002), suggesting this trend will continue in to the next generation. Obesity has a dramatic impact on society and individuals with and without these conditions. It is has been s uggested that 300,000 deaths each year in the United States are related to excess body weight and th at obesity is the second cause of preventable death in this country (Allison et al. 1999). Analysis of the NHANES links high BMI with cardiovascular-, kidney-, and cancer-related deaths (Flegal et al. 2007). Excess body weight and fat deposition are associated with increased in cidence of heart disease (Rimm et al. 1995), hypertension (Witteman et al. 1989), type-2 diabetes (Colditz et al. 1995), and some cancers (Calle et al. 2003). In addition to increased health risks, obesity results in an economic burden shouldered by overweight and normal weight indi viduals alike. The United Stat es Department of Health and Human Services estimated that $117 billion a ye ar is lost with obesity-associated loss of productivity and health care costs, and meta-analysis of empirical literature mi rrors this estimate at $70 billion (Thomspon and Wolf, 2001).

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12 Obesity is also associated with the preval ence of psychiatric di sorders including anxiety and depression (Scott et al. 2008; Simon et al. 2006; Britz et al. 2000; but see Lamertz et al. 2002). This is most often seen in women, wh ich also report a highe r incidence of eating disorders, such as binge eating (Presnell et al. 2008). Binge eating disorder (BED) is not an independent eating disorder as defined by the Di agnostic and Statistical Manual IV, but criteria for research include behaviors such as eating la rge portions of food in a short time and reporting a lack of control over this be havior. Lack of an independent diagnosis for BE D may contribute to its seemingly low prevalence (Grucza et al. 2007; Hudson et al. 2007), but BED is correlated with the incidence of obesity, and obese individua ls that binge eat report a lower quality of life compared to obese individuals that do not binge eat (Rieger et al. 2005; Masheb et al. 2004). The Role of the Environment There has been much debate whether obes ity, its associated metabolic syndrome, and maladaptive eating patterns like binge eating should be considered physiological and/or psychological disease (Heshka and Allison, 2001). It has been suggested that defining these disorders as disease sates is not a necessary path to successful treatment because the development of obesity is most related to environmental conditions, and that the environment is what must be changed to prevent and trea t obesity (Jefferey and Utter, 2003; Popkin et al. 2005; Cope and Allison, 2006; Faith et al. 2007). Accumulation of excess body weight and ultimately obesity occur when energy is taken in at a rate higher than it is expended. In the past 50 years, caloric intake has increased (J effery and Harnack, 2007; Nielsen et al. 2002) and level of activity has decreased (Gortmaker et al. 1990). Ecological review s suggest that energy availability has increased by 15% since 1970, and along with an increased incidence of eating meals outside the home, consuming convenience m eals, and consuming larger portions (despite also more often choosing decrease d-fat alternative foods), this increase in availability is

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13 contributing to the epidemic of obesity (Harnack et al. 2000). Clinical re search shows that increased food availability, ener gy density, portion size, and pa latability all contribute to increased caloric intake, and that these influe nces are not compensated for by a reduction in caloric intake across time, thus l eading to weight gain (Ledikwe et al. 2006; Ello-Martin et al. 2005; Young and Nestle, 2002; Rolls et al. 2007; Rolls et al. 2006; Ello-Martin et al. 2007; Ard et al. 2007). Environmental factors that play a role in the development of obesity are mirrored in animal models of obesity. When rodents are given sole acce ss to a preferred diet that is high in fat, they become temporarily hyperphagic and gain we ight over time (Levin and Dunn-Meynell, 2002; Levin, 2005). Animals given ad libitum access to a variety of foods, as seen in cafeteria diets (Rothwell and Stock, 1988), also sh ow increased caloric intake co mpared to animals maintained on a single standard maintenance diet. Rats given time-limited access to shortening in a highly restricted manner consume more shortening than rats given shortening in a less restricted manner; although this protocol does not result in overconsump tion and weight gain, it does model some attributes of binge eating (Dimitriou et al. 2000; Corwin, 2006). Some data suggest that this combination of a maintenance diet a nd a palatable supplement is the most effective means to induce overconsumption in rodents (Archer and Mercer, 2007). Our laboratory combined factors of these protocols into a dessert protocol in which rats were given ad libitum access to a standard maintenance diet as well as daily time-limited access to one of two palatable, but nutritionally incomplete, desserts that varied in macronutrient composition and caloric density (Mathes et al. 2008). Rats given a dessert that contained fat and sugar, and had a high caloric density, consumed more calories than rats given a dessert that contained only sugar, and had a low caloric density, or rats given no dessert ; these latter two groups of rats had

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14 equivalent caloric intakes and did not gain weight from baseline. This dessert protocol provides an animal model of the effect of clinically re levant environmental factors (ie, caloric density, availability, palatability) on ea ting behavior and weight gain, and allows analysis of diet selection and compensation. It also provides a way to expand the aforementioned binge eating model by using a sweet and fatty dessert, which is the type of food for which women often report craving and overconsumption (Raymond et al. 2003). Physiological Regulation of Food Intake Animal models have helped not only to identify the impact of the environment on feeding, but also the physiological circuitry that regulates feedi ng. Food intake is a behavior necessary for the maintenance of life, and so it would follow that regulation of the consumption of adequate quantities of food would be tightly regulated by the brain. Much research has focused on the hypothalamus, the brain region cl assically associated with homeostasis and maintenance of drives (Williams et al. 2001). Lesions of the lateral hypothalamus result in hypophagia or aphagia and lesions of the ventro medial hypothalamus resulted in hyperphagia (Keesey et al. 1979). Identification of s pontaneously obese mouse stra ins and analysis of their humeral signaling via parabiotic studies, in whic h the circulatory system of an obese mouse was linked to that of a normal weight mouse, showed that peripheral signals in the blood relay to the brain the nutritional status of th e body (Harris 1997; Harris 1999). Leptin was isolated as one of these important signals. Leptin is secreted from adipose tissue su ch that its levels in the blood correlate to the amount of fat in the body (Aja and Moran, 2006). This was seen as consistent with the lipostatic theory of weight regulation, which hypothe sized that the amount of fat accumulated in the body is sensed in the br ain and regulated (Mayer, 1955; Le Mangen et al. 1973). Leptin is actively transported into the br ain, and also crosses at the circumventricular organs, including the arcuate nucleus (ARC) of the hypothalamus (Cone et al. 2001). There are

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15 two classes of neurons in the ARC that express the Ob leptin receptor, and both sets project to the paraventricular nucleus (PVN) of the hypot halamus. One set pr oduces nueorpeptide-Y (NPY) and agouti-related protein (AgRP). When NPY is exogenously administered to animals, it results in robust hyperphagia (Beck, 2006); AgRP also increases food intake, though to a lesser extent. These neurons fire tonically and their firi ng rate is reduced when le ptin is present (Kalra et al. 1999). This default-setting towards feeding seems ecologica lly valid in an evolutionary sense, since ancestral human behavior that prom oted early satiety and cessation of eating would have been selected against in times of famine Another set of neurons found in the ARC produce pro-opiomelanocortin (POMC) and cocaine and amphetamine related transcript (CART). melanocyte stimulating hormone ( -MSH) is a product of POMC that when exogenously administered reduces food intake. CART mimics the appetite-suppressing effect of the drugs for which it was named (Vicentic and Jones, 2007). When leptin activates these neurons, their activity increases (Kalra et al. 1999). -MSH activates melanocortin-4 receptors (MC4R) in the PVN; it is noteworthy that AgRP serves as an antagonist at the MC4R (Ellacott and Cone, 2006). Mice that have been genetically altered such that they do not expre ss MC4R (knockout, KO), and thus do not have the satiet y brakes this system provi des, are hyperphagic and obese (Huszar et al. 1997). This arrangement of NPY/Ag RP and CART/POMC neurones in the hypothalamus seems as though the dual center theory of motivation propose d by Stellar seems to be accomplished at the receptor leve l (Stellar and Corbit, 1973). Humans with genetic mutations of the leptin and POMC systems resulting in obesity have also been identified (Farooqi S and O'Rahilly S, 2006). However, these cases are rare, and thus do not reflect the prevalence of obesity seen in the United States. Also, although it is a potent anorexic in normal weight humans, repeated exogenous administration of leptin does not

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16 decrease caloric intake or body weight in obese humans; indeed, leptin-resistance is one of the factors of the metabolic syndrome. Pharmaco therapy impinging on the MC4R is still in a preclinical phase, but shows some promise in an imal models and will be discussed in a later section. Reward Mechanisms Affecting Food Intake It has been proposed that the trend toward overconsumption is more closely related to addictive behavior than to a regulatory dysfunction (Corwin and Hajnal, 2005), and that drug addiction is simply an expression of ingestiv e behavior gone haywire (Volkow and Wise, 2005). Indeed, eating disorders and substance use di sorders in humans ofte n occur concurrently (Gadalla and Piran, 2007). Highly palatable foods, which often have a high caloric density, may, in a fashion similar to non-natural rewards like drugs of abuse, bypass natural inhibitory processes associated with ingestion and overactivate brain areas associated with the perseveration of rewarding behavior (S imansky, 2005). One hypothesis concerning the motivation underlying eating in the absence of ne ed is that it is mediated by dopamine (DA) activity in limbic and cortical ar eas. Specifically, dopaminergic ne urons project from the ventral tegmental area (VTA) and terminate in the nucle us accumbens (NuAc) and prefrontal cortex (PFC); these structures have reciprocal projections to the VTA via -amniobutyric acid (GABA) and glutamate (Pierce and Kumaresan, 2006). It has been suggested that this mesolimbic dopamine system (MLDAS) is responsible for the rewarding nature of commodities. This is based on early work showing that stimulation of ne urons in the medial fo rebrain bundle resulted in feeding or feeding-like behavi or (as reviewed in Wise, 2002). Animals will also work to selfadminister electrical stimulation into this area, and food deprivation increases the amount of work animals would perform to receive this stim ulation. It was hypothesized that feeding results in a release of DA, and this resulted in a ple asurable state that promoted the continuation or

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17 future probability of the behavior toward the food and stimuli with which it was associated. This hypothesis is supported by electrophysiological studies showi ng that VTA DA neurons, which normally fire in a slow irregular pattern that resu lts in a tonic release of DA, fire rapidly and in bursts when hungry rats are presented with f ood or cues predicting food (Palmiter, 2007). However, paradigms that distinguish between 'w anting', or the incentive salience, versus the 'liking', or hedonic value, of food suggest that DA is necessary for the former but not the latter (Berridge, 2006). Although DA may be involved more in attenti on to and sensorimotor activation in the presence of relevant environmental cues than in hedonic evaluation, ac tivation of DA neurons interact with other systems that may be dire ctly involved in the pe rception of pleasure. Consumption of food, which resu lts in DA release from the VTA to NuAc, also results in stimulation of opioid receptors located on GABA neurons in the NuAc; release of endogenous opioids disinhibits DA neurons in a feed-for ward manner (Volkow and Wise, 2005). Opioid activity has been shown to increase food intake especially of high-fat and high-carbohydrate foods (Olszewski and Levine, 2007; Naleid et al. 2007). Opioid and DA transmission has also been linked to endogenous cannabinoid (CB) sign aling, which promotes feeding, especially of palatable foods; this will be discussed in a later section. Opioid and CB antagonists reduce feeding, as well as reduce craving and taking of drugs of abuse, suggesting similarities at the behavioral level, although food and drug reinfo rcers may not act produce the same electrical profile of activity in the MLDAS (Carelli et al. 2000). Not only do physiologic regulation, the perceptio n of reward, and environmental factors individually impact behavior asso ciated with feeding, they also interact. Certain environments impinge to different degrees on certain genetic and physiologic variants, leading to a phenotypic

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18 expression of obesity; for example, strains of rats have been bred for preference for and development of obesity via access to high fa t diets (Levin and Dunn-Meynell, 2002; Levin, 2005) and the C57B6J mouse strain is more suscep tible to diet-induced ob esity (DIO) than other strains (Collins et al. 2004). Humans with heterozygous expression of MC4R only become morbidly obese in environments that promote overfeeding (Ma et al. 2004), and this is mirrored in animal research (Vaughan et al. 2005). Receptors for peripheral signals such as leptin and appetite-stimulating ghrelin are found in the VTA, and hypothalamic neurons containing peptides such as appetite-stimulating orexin project to limbic struct ures (Palmiter, 2007; Berthoud, 2007). Neurochemicals and receptors responsible for CB signaling are found in the hypothalamus where leptin facilit ates their levels (DiMarzo et al. 2001), and interactions between the CB system and MC4R system are coming to light (Verty et al. 2004; but see Sinnayah et al. 2008). This complex interaction requi res appropriate protocols that mimic aspects of the environment in which humans feed to explore and identify behavior patterns and the associated brain circuitry a ssociated with disorders of feed ing that lead to overconsumption, binge eating, and ultimately, obesity and the meta bolic disorder. Studies in this dissertation present a pharmacological approach to understanding the circuitry of the MC4R system, which is thought of as a mediator of regulation in feeding, and the CB system, which is characterized as a mediator of food reward and 'liking'. Aspects of pharmacology and clinical relevancy of these systems are discussed below. Melanocortin-4 Receptor and Food Intake The m elanocortin system plays a critical role in the control of energy balance and has ties to clinical obesity. Reportedly, 4-7% of seve rely obese humans have defects in this system, making it the single-most prevalent monogene tic cause of obesity in humans (Yeo et al., 2000; Farooqui et al. 2003; Lubrano-Berthelier et al. 2003; Ma et al. 2004; Valli-Jaakola et al. 2004)

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19 and MC4RKO mice exhibit hyperphagia and become obese compared to their WT littermates (Huszar et al. 1997; Butler and Cone, 2003; Yang and Harmn, 2003). Emphasis has been placed on the MC4R in the homeostatic re gulation of feeding since, as detailed above, it is located in the PVN and central administration of its endogenous agonist, -MSH, and endogenous antagonist, AgRP, results in decrease and incr ease in food intake, respectively (Zimanyi and Pelleymounter, 2003; Seeley et al. 2004; Jonsson et al. 2002; Pierroz et al. 2002). The MC4R, when activated, couples to Gs, activating adenylyl cyclase and increasing cAMP production (Pattern et al. 2007; Proneth et al. 2006). The extent to which the other me lanocortin receptor subtypes affect energy regulation has been debated (Irani et al. 2004). All five melanocorti n receptors are G-protein coupled receptors, distributed profusely thr oughout the brain and peri phery, and activated by derivatives of POMC. The MC1R, which is implicated in pigmentation of the skin and immune response, MC2R, which mediates glucocorticoid responses, and MC5R, which is involved in exocrine function, are unlikely to impact en ergy regulation. The MC3R may play a role, suggested by their presence in the ARC and the development of obesity related to defective functioning (Schalin-Jantti et al. 2003). However, obesity du e to MC3R dysfunction occurs independent of hyperphagia and seems to be metabol ic in nature with a role distinct from MC4R (Abbot et al. 2000). Obesity related to MC4R dysfunction has been attributed to and maintained by hyperphagia (Huszar et al. 1997) and differences in metabolic rate and feeding efficiency (Chen et al. 2000 ; Butler et al. 2001 ) Few studies have examined if this dysfunctional interaction with food is regulatory or hedonic in nature and those that do conflict, though using many different paradigms. One study shows that MC4RKO mice have normal responses to nutrient pre-loading and gut sa tiety hormones (Vaughan et al. 2006A), suggesting that hyperphagia may

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20 be driven by availability or palatability, but another study sh ows that MC4RKO mice also have normal affective gustatory responses to the major tastes (Eylam et al. 2005), suggesting that increased caloric intake is not due to an altered perception of palatabilit y. Studies using operant techniques, which assess motivation and the e ffect of food availabil ity, show that MC4RKO mice do not overconsume when work is required for food and have feeding patterns similar to WT mice (Vaughan et al. 2005), but will work harder and for food under a progressive ratio schedule of reinforcement (ie, ha ve a higher break point) (Vaughan et al. 2006B). Differences have also been assessed when animals are given free access a nd choice between food commodities. When allowed to regulate selection from each macronutrient group, MC4RKO mice select more calories from fat than WT mice and mice injected with MC4R agonist melanotan-II (MTII) select fewer calories from fat than mice injected with saline (Samama et al. 2003). One study shows that MTII is less effective in decreasing caloric in take when a high fat diet is presented as the sole diet option to rats (Clegg et al. 2003), whereas another study reports that caloric intake is decreased similarly by MTII in rats given either a high calorie cafeteria diet (ie, chocolate + chow) or just chow (Hamilton and Doods, 2002). In experiments assessing the effect of MC4R dysfunction on the mediation of reward associated with stimuli other than food, neither MC4R antagonist SHU9119 nor agonist MTII altered the threshold for lateral hypothlamic self-stimulation, although MTII poten tiated the threshold-lowering effect of amphetamine (Cabeza de Vaca et al. 2002). In contrast, MTII decr eased alcohol consumption in alcohol-preferring rats (Navarro et al. 2003). Hyperphagia associated with MC4R dysfunction has also been connected with incidence of binge eating in humans, but the data conflict across studies (Branson et al. 2003; Hebebrand et al. 2004; Lubrano-Berthelier et al. 2006), and this has not been explored in animal models. Thus, it is unclear whether the hyperphagia associated

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21 with MC4R dysfunction stems from mechanisms mediating the reward value of commodities or from dysfunctional interactions w ith the environment and economics associated with feeding. Cannabinoid CB1 Receptor and Food Intake Reports of the appetite-stimul ating properties of the plant Cannabis sativa have been documented anecdotally for centuries and empirically in human residential laboratories (Foltin et al. 1988). 9-tetrahydrocannabinol (THC) has been characterized as the principal agent responsible for this effect (Mechoulam et al. 1970). Originally, exogenous CB were thought to act by modulating the permeability and responsiveness of cell membrane signaling, but characterization of CB1 and CB2 receptors in the mammalian system reconciled the basis of their physiological effects (Matsuda et al. 1990; Munro et al. 1993). CB1 receptors (CB1R) will be discussed here as they are located in the brain and considered more relevant to feeding as opposed to the more peripherally-located CB2 recepto rs that play a role in immune responses. CB1R are G-protein coupled receptors that, wh en activated, inhibit adenylate cyclase and decrease Ca2+ channel function (Howlett et al. 2002). CB1R are extremely prevalent throughout the brain, specifically in the hippocampus and neocortex, and are located predominantly on neuron axon terminals. CB1R distribution and presynaptic location allows its activity to modulate signaling of nearly all the major neurotransmitters, including DA, in regions of the brain associated with behaviors responsible for reward, atte ntion, learning, and regulatory processes (Freund et al. 2003). Along with THC, CB1R are activated by e ndogenous chemicals found in the brain, called endocannabinoids, which are derived from arach idonic acid. First to be identified was arachidonoylethanolamine, known as anandamide (Devane et al. 1992). Anandamide is only a partial agonist at CB1R, with low receptor affinity (Ki estimates range from 50-100 nM). It is found throughout the brain, with highest conc entrations found in the brainstem and

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22 hippocampus. 2-Arachidonoylglycerol (2-AG) was isolated first in the canine gut, but has been shown to be distributed in the brain in a pattern similar to anandamide (Mechoulam et al. 1995). It is a full agonist at CB1R with a high affinity (Ki estimates range from 1-10 M). Both chemicals are made and released on demand fo llowing membrane depolarization and increased Ca2+ levels, but are not stored in or released from vesicles. Reuptake mechanisms have been proposed, though not characterized. Both anadamid e and 2-AG are degraded by fatty acid amide hydrolase (FAAH) (Cota et al. 2006). Both endogenous CB and THC stimulate food inta ke in animals and levels of endogenous CB are modulated by food deprivation and f eeding (Williams and Kirkham, 2002; Kirkham et al. 2002). CB1RKO mice eat less and maintain a leaner body composition than wild type (WT) littermates (Ravinet-Trillou et al. 2004). These effects on f ood intake paved the way for development of CB1R antagonists that may be useful for weight management (Kirkham and Williams, 2004). SR141716A (now Rimonabant) (Rinaldi-Carmona et al. 1994) and AM251 (Gatley et al. 1996) are potent and selectiv e antagonists with inverse agonist proper ties at the CB1R. These agents acutely decrease food cons umption and behaviors associated with feeding in many rodent models (reviewed in Salamone et al. 2007), including moderately obese Lewis rats prefed with Ensure (Chambers et al. 2004), obese and lean Zucker rats fed laboratory chow ad libitum (Vickers et al. 2003), and mice on standard DIO protocols (Ravinet Trillou et al. 2003). Rimonabant is in phase III clinical trials and shows potential as an effective weight loss agent in humans (Cleland et al. 2004). Similar to opioids and benzodiazepines (Cooper, 2004), activity at CB1R may affect feeding by modulating the hedonic evaluation of f oods, especially sweet solutions. Reports are inconsistent: some studies show a CB1R agoni st-dependent increase and antagonist-dependent

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23 decrease in appetitive behavior via taste reactivity and brief-access tests (Higgs et al. 2003; Jarrett et al. 2007; Mahler et al. 2007) and others report behavior akin to an effect on satiation or motivation (Jarrett et al. 2005; Thornton-Jones et al. 2007). This inconsistency is mirrored by the effect of CB1R agonists and antagonists on preference for more complete food types in rodents; some studies report a selective decrease in the intake of palatable substances (Arnone et al. 1997; Freedland et al. 2000; Koch, 2003; Miller et al. 2004; Simiand et al. 1998; Ward and Dykstra, 2005) and other studie s report equal suppression between diets of varying palatability (Foltin and Haney, 2007; Gessa et al. 2006; McLaughlin et al. 2003; Verty et al. 2004; though see McLaughlin et al. 2006). The activity of CB1R has also been implicated in other ingestive behavior that associated with drug-taking. Rimonabant decreas es nicotine and alcohol taking in humans and animals (Columbo et al. 2007; Cahill and Ussher, 2007). It would make anatomical sense that CB1R and endogenous CB would be involved in the rewarding and pleasurable aspects of ingestive behavior be cause they are found in limbic brain areas and mediate DA signaling (Gardner and Vorel, 1998), but this has not been consistently displayed across multiple behavioral paradigms. In fact, one study reported the lack of a hypophagic effect of CB1R antagonists on food and alcohol and attributed it to the within-s ubjects nature of the experiment, suggesting that the effect of CB1R antagonists may be sensitive to the experimental design (Ginsburg and Lamb, 2006). Protocols th at examine the effect of CB1R antagonistmediated depression of appetite may require analysis of diet selection across multiple meals. Summary Both the MC4R and CB1R are im plicated in the control of food intake, although MC4R seems better situated to impinge on homeostati c and economic regulation whereas CB1R has the potential to impact the hedonic a nd learning aspects of food intake. Pharmacotherapy at each receptor type, and perhaps at both simultaneously, ma y be beneficial in the treatment of obesity

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24 in humans. However, the impact of their activity on behavior needs to be explored in rodent protocols that more accurately represent the human eating environment. Studies in this dissertation explore in female mice the effect of a MC4R agonist a nd antagonist and a CB1R antagonist on diet selection in protocols that model clinical aspects of overconsumption and binge eating.

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25 CHAPTER 2 GENERAL METHODS Introduction and Previous Studies Diet Protocols Overconsumption has been modeled via DIO in rodents by providing them ad libitum access to a palatable high calorie diet, which resu lts in increased energy in take and weight gain over time (Levin and Dunn-Meynell, 2002). In contrast, humans are bombarded by diverse food choices, and an element of choice in diet selectio n may be a key to the exploration of the limits of caloric compensation in anim al models. Cafeteria style diets (Rothwell and Stock, 1988) provide rodents with access to multiple foods, but there are substantial individual differences in dietary preferences that often complicate the analys is of these results. A dessert protocol avoids these confounds while still allowing a choice as well as promoting overconsumption. Furthermore, dessert protocols are utilized during rather than after the development of obesity, thus allowing examination of manipulations on di et selection prior to a lterations resulting from the obese state. In dessert protocols, rodents ar e given time restricted access to a preferred, often nutritionally incomplete opti onal calorie source along with ad libitum access to bland, but nutritionally complete standard diet (Corwin and Buda-Levin, 2004; Dimitriou et al. 2000). We established a dessert protocol to indu ce hyperphagia and body weight gain in female retired breeder Sprague Dawley rats (Mathes et al. 2008). In these studies, caloric compensation referred to a decrease in standard diet consumption comparable to the caloric intake from dessert so that daily total caloric intake remained st atistically unchanged; in contrast, overconsumption was defined as the lack of caloric compensation th at occurred when caloric intake of the dessert was not accompanied by an equivalent decrease in standard diet intake. Our dessert protocol consisted of daily 8 h nocturnal access to a desser t that was high in calories from fat and sugar

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26 (sugar fat whip, SFW; 7.35 kcal/g)), as well as moist chow, which was presented ad libitum to 8 rats. Other rats were given no dessert (n=8) or 8 h access to a sugar gel (0.32 kcal/g). As shown in figures 2-1 and 2-2 (pages 34 and 35), access to SFW for 15 days increa sed caloric intake and body weight by disrupting caloric compensation, whereas access to a lower calorie option, like sugar gel, did not increase caloric intake or body weight gain, because rats maintain caloric balance in its presence. We have used this protocol to explore th e effect of CB1R antagonists Rimonabant and AM251 on consumption and selection of dessert and chow (Mathes et al. 2008). In the first experiment, 24 rats were divided into two groups based on SFW consumption. One group was injected daily intraperitoneally (IP) with vehicle (equal parts pol yethylene glycol and saline, 1 ml/kg) and the other was injected with Rimonaba nt (1 mg/kg IP). In the second experiment, 32 rats were divided into four groups and injected da ily with either vehicle or one of three doses of AM251 (0.3, 1.0, 3.0 mg/kg IP). Total caloric inta kes and intakes from each commodity were measured for 7 days in the first experiment and 15 days in the second. As shown in figures 2-3 through 2-6 (pages 36-39), both CB antagonists decreased caloric intake and body weight gain compared to vehicle-injected controls. Intere stingly, the difference in caloric intake came primarily from a reduction in intake from SFW in the CB1R antagonist-injected groups. These findings support the hypothesis th at CB1R antagonists reduce caloric intake by affecting intake of palatable commodities. We used this same SFW dessert to extend a protocol modeling binge eating in female rats. Corwin and colleagues have shown that rats gi ven highly restricted (HR) access to shortening, defined as 2 h nocturnal access every other day, consumed more shortening in the 2 h binge period than rats given less restricted (LR) acce ss to shortening, defined as 2 h nocturnal access

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27 every day (Dimitriou et al. 2000). However, the weight gain that accompanies binge eating disorder in humans is not seen in this model. Women who binge eat repo rt that they tend to do so with snack items that are sweet and fatty (Raymond et al. 2003), and so SFW may be a more relevant commodity for use in animal models. In our first experiment, we presented twelve retired breeder Sprague Dawley rats with eith er HR or LR access to SFW as described in Corwin's shortening protocol. After 14 days, we had not replicated the binge results reported by Corwin (data not shown); this ma y have been due to a ceiling e ffect of SFW consumption or the age of the rats. Increased age has been shown to exacerbate and elongate consumption in rodent models of binge eating (Thomas et al. 2002), and so we compared the binge eating of young (approximately 42 days of age) a nd aged rats (retired breeders, approximately 9 months of age) using the SFW binge protocol (Mathes et al. 2007A). Twelve rats of both age groups were divided into restriction groups and their intake was measured for 50 days. As presented in figures 2-7 and 2-8 (pages 40 and 41), both young and aged rats given HR SFW access showed evidence of binge eating and overconsumption accompanied by increased body weight. Similar to Corwin's findings, binge eating was more eviden t in aged rats, but body weight gain was more evident in young rats. This may have been due to increases in muscle mass in young, growing animals, rather than accumulation of fat, which wa s seen to a greater extent in aged animals in Corwins study; however, we did not analyze body fat. Although in neither group did body weight differ as a result of LR and HR access, th at restricted access resulted in not only binge eating, but also overconsumption an d weight gain, suggests that us ing SFW instead of shortening may make this binge eating protoc ol more clinically relevant. Mouse Models Because of the genetic malleability availab l e in murine models, we extended the SFW overconsumption and binge protocols to female mice (Mathes et al. 2007B). We used female

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28 mice because previous experiments were conducte d using female rats, which were originally chosen since body weight is more stable in fema le than male rodents and because women seek treatment for overconsumption and binge eating more often than men. As a pilot, we presented a small group of MC4RKO mice, mice heterozygous for expre ssion of MC4R (MC4RHET), and WT mice with SFW either using the overconsumption (i e, 8 h) or binge eating (ie, LR or HR 2 h) protocol for 14 days or 21 days, respectively. As shown in figures 2-9 and 2-10 (pages 42 and 43), although normally hyperphagic, caloric balance and body weight were further disrupted in MC4RKO mice by presentation of 8 h SFW, but not by selectively exacerbating consumption of the palatable dessert (eg, in c ontrast to CB1R antagonists in ra ts). Unlike rats, WT and MC4RHET mice displayed caloric balance when given 8 h access to SFW, although they did readily consume SFW. This was a surprising fi nding since mice are susceptible to most DIO protocols. It was especially surprising that MC4RHET mice did not display behavior in between that of WT and MC4RKO mice. As shown in figures 2-11 and 2-12 (pages 44 and 45), although MC4RKO mice were the only genotype of mice to exhibit binge eating at 2 h after dessert presentation, they did not exhibit a higher level of binge eating at 24 h after dessert presentation compared to WT and MC4RHET mice. In fact MC4RKO mice seemed less likely to continue to binge over time; this may be the result of a ceiling effect relate d to the propensity of MC4RKO mice to overconsume and be hyperphagic re gardless of the situation. WT mice did display binge-like eating, and similar to our overconsumption data, so did MC4RHET mice. However, these studies were conduc ted in leftover mice that were not littermates or even the same age; since my data in female rats suggest that age may be a factor in the development of binge-like eating, these data need to be replicated in same-age li ttermates before conclusions are made.

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29 Pharmacological Models Since a larg e quantity of MC4RKO, MC4RH ET, and WT littermate mice would be necessary for a complete analysis of overcons umption and binge eating, we decided to use normal C57Bl/6J mice and a MC4R agonist and anta gonist to extend these data. We chose to inject these agents centrally (intracerebroventricu larly, icv) in an attempt to limit effect to the brain, and in particular to permeable regions including the hypothalamus and brain stem. We planned to repeat our analysis of the effect of CB1R anta gonism on overconsumption and diet selection in mice to assess if the CB1R-medi ated hypophagia and decrease in palatable food extended to situations that did not promote overconsump tion. We administered CB1R antagonists peripherally to keep it as similar to our corresponding rat experiments as possible. General Methods Animals All experiments were conducte d using 6 week old fe male C57Bl/6J mice (Jackson Labs, Bar Harbor, ME) that weighed 16-20 g at the start of the experime nt. All mice were individually housed in either standard polycarbonate tubs c ontaining a 2-3 cm layer of bedding (SaniChips, Teklad, Madison, WI) placed on standard racks, or in polycarbonate tubs containing 1-2 cm conventional corncob bedding that were hung on a ve ntilated rack (HepaPleat II, Lab Products Inc., Seaford, DE). All mice were provided with material for nest building (Nestlet, AnCare, Bellmore, NY). All mice were provided with ad libitum access to tap water and to a standard maintenance diet as described below. The viva rium was temperature and humidity controlled (23+ 2 C, 45-55%) and on a 12 h revers e light cycle. All measures were taken during the dark cycle, which is the time when mice are most act ive and consume most of their daily food. All animal procedures were approved by the University of Florida IACUC.

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30 Diets Moist chow was used as the ad libitum m aintenance diet in the study because it is less easily spilled than pellets or powder and because the moist texture reduces some differences between the maintenance diet and dessert (see below). Moist chow (1.67 kcal/g) was made by mixing powdered standard chow (Purina 5001) with an equal amount of tap water; this was allowed to come to room temper ature and was spooned into 10 ml glass beakers. A beaker of moist chow was attached to a metal stirrup and su spended in the left corner of each cage. Fresh jars of chow were provided daily. Food consump tion was measured for a period before initiation of the experimental phase of the study to ensure stability of intake. Mice were assigned to groups that were matched for range and am ounts of intakes during the baseline period. Some mice were also presented with a de ssert throughout the study. The palatable food source used was a sugar fat whip (SFW, 7.35 kcal/g ) that was made by mixing two parts softened vegetable shortening with one part white suga r (both commodities were standard brands purchased from a local supermarket); SFW was allowed to come to room temperature and was spooned into 10 ml glass beakers. The beakers were attached to a metal stirrup and hung in the right corner of each cage. Mice were provided with 24 h access to SFW before the start of the experiment; this acclimation period was to redu ce neophobia. Mice were assigned to groups that were matched for range and amounts of intake s during the baseline period. Fresh SFW was provided daily.

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31 Intake (kcal) 0 20 40 60 80 100 120 Intake (kcal) 0 20 40 60 80 100 No DessertSugar Gel SFW Intake (kcal) 0 20 40 60 80 100 b b a a b c b a Figure 2-1. Caloric intake (M + SE kcal) of rats given ad libitum access to moist chow and 8 h access to either sugar gel (SG) (n=8) or sugar fat whip (SFW) (n=8) or no additional dessert (n=8) daily for 15 days. Panel A) Total caloric intake. Panel B) Caloric intake from moist chow. Panel C) Caloric intake from dessert. The letters above the bars denote a significant difference between groups: groups that ha ve different letters above them are statistically different (p< 0.05) and the letter 'a' represents the group consuming the most calories. Rats give n SFW consumed significantly more total calories than the othe r groups and significantly fewer calories from moist chow than the other groups. Rats given SG consumed fewer calories from moist chow than rats given no dessert. Rats given SFW consumed significantly more calories from SFW than rats given SG consumed from SG. A B C

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32 No DessertSugar Gel SFW Cumulative Body Weight Gain (g) -40 -20 0 20 40 b b a Figure 2-2. Cumulative change in body weight (M + SE g) from baseline of rats fed the desserts described in the caption for Figure 2-1. Ra ts given SFW gained significantly more weight than rats given no de ssert or rats given SG. Ra ts given no dessert and rats given SG did not significantly change in body weight from baseline.

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33 V e h i c l e R i m o n a b a n t Intake (kcal) 0 20 40 60 80 100 120 140 AM251 Vehicle Rimonabant Intake (kcal) 0 20 40 60 80 100 120 Vehicle Rimonabant Intake (kcal) 0 20 40 60 80 100 120 a b a a a b Figure 2-3. Caloric intake (M + SE kcal) of rats given 8 h ac cess to SFW and injected with either vehicle or Rimonabant (1 mg/kg) daily for 7 days. Panel A) Total caloric intake. Panel B) Caloric intake from moist chow. Panel C) Caloric intake from SFW. The letters above the bars denote a signifi cant difference between groups: groups that have different letters above them are statistically differe nt (p<0.05) and the letter 'a' represents the group consuming the most calories. Rats injected with Rimonabant consumed significantly fewer total calori es than vehicle controls by consuming significantly fewer cal ories from SFW. A B C

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34 Vehicle Rimonabant Cumulative Body Weight Change (g) -20 -10 0 10 20 a b Figure 2-4. Cumulative change in body weight (M + SE g) from baseline of rats on the dessert protocol and drug regimen described in the caption for Figure 2-3. Rats injected with Rimonabant gained significantly less weight than vehicle controls.

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35 0 0 m g / k g 0 3 m g / k g 1 0 m g / k g 3 0 m g / k g Intake (kcal) 0 20 40 60 80 100 120 140 AM251 0.0 mg/kg0.3 mg/kg1.0 mg/kg3.0 mg/kg Intake (kcal) 0 20 40 60 80 100 120 AM215 0.0 mg/kg0.3 mg/kg1.0 mg/kg3.0 mg/kg Intake (kcal) 0 20 40 60 80 100 120 a ab abc c a ab abc bc a a b a Figure 2-5. Caloric intake (M + SE kcal) of rats given 8 h ac cess to SFW and injected with either vehicle or one of three doses of AM251 (0.3, 1.0, 3.0 mg/kg) daily for 15 days. Panel A) Total caloric intake. Panel B) Ca loric intake from moist chow. Panel C) Caloric intake from SFW. The letters above the bars denote a significant difference between groups: groups that have different letters above them are statistically different (p<0.05) and the letter 'a' represen ts the group consuming the most calories. Rats injected with 3.0 mg/kg AM251 consumed significantly fewer total calories than vehicle controls by consuming signi ficantly fewer calories from SFW. A B C

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36 AM251 0.0 mg/kg0.3mg/kg1.0mg/kg3.0 mg/kg Cumulative Body Weight Change (g) 0 10 20 30 40 a b bc c Figure 2-6. Cumulative change in body weight (M + SE g) from baseline of rats on the dessert protocol and drug regimen described in the caption for Figure 2-5. Rats injected with any dose of AM251 gained significantly le ss weight than vehicle controls.

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37 Intake (kcal) 0 20 40 60 80 100 No SFW LR 2 h SFW HR 2 h SFW Young Aged Intake (kcal) 0 20 40 60 80 No SFW LR 2 h SFW HR 2 h SFW c b c a d b c a Figure 2-7. Caloric intake (M + SE kcal) of either young (ie, 45 days of age) or aged (retired breeders, approximately 9 months of age) rats given 2 h access to SFW either every day (low restriction, LR) or every other day (high restriction, HR) for 50 days. Data are shown only from days when all rats we re given SFW (ie, binge days). Panel A) Caloric intake from moist chow and SFW 2 h after diet presentation. Panel B) Total caloric intake 24 h after presen tation of both diets. The letters above the bars denote a significant difference between groups: groups that have differen t letters above them are statistically different (p<0.05) and the letter 'a' represents the group consuming the most calories. Differences are shown between age and restriction groups; caloric intakes from moist chow of the rats prior to the experiment are shown for comparison, but were not included in statistica l analysis. Rats of either age given HR 2 h access to SFW consumed more calories in 2 h and 24 h than rats given LR 2 h access to SFW; this was potentiated in aged rats, which consumed more calories than young rats. A B

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38 LR AgedLR YoungHR AgedHR Young Cumulative Body Weight Change (g) 0 10 20 30 40 50 60 ab a b ab Figure 2-8. Cumulative change in body weight (M + SE g) from baseline of rats on the dessert protocol described in the cap tion for Figure 2-7. Data are shown from all days of the experiment, not just binge days. Young ra ts given LR access to SFW ate more total calories than old rats given HR 2 h access to SFW.

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39 Intake (kcal) 0 5 10 15 20 25 No SFW 8 h SFW Intake (kcal) 0 5 10 15 20 No SFW 8 h SFW Genotype WT HET KO Intake (kcal) 0 5 10 15 20 No SFW 8 h SFW b a a a a b a b a b a a a a b Figure 2-9. Caloric intake (M + SE kcal) of mice with either fu ll genetic expression (wild type, WT), heterozygous expression (HET), or deletion (knock out, KO) of the melanocortin-4 receptor (MC4R) that we re given 8 h access to SFW for 14 days. Shown and analyzed for comparison are the total caloric intakes from moist chow only of the mice prior to experimentation. Panel A) Total caloric intake. Panel B) Caloric intake from moist chow. Panel C) Caloric intake from dessert. The letters above the bars denote a significant diffe rence between groups: groups that have different letters above them are statistically different (p<0.05) and the letter 'a' represents the group consuming the most calories. Mice of all three genotypes reduced their caloric intake from mois t chow when given 8 h access to SFW, but compensation was not complete in MC4R KO mice. MC4RKO mice given 8 h SFW consumed more total calories when given 8 h access to SFW compared to when given moist chow only. A B C

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40 Genotype WT HET KO Cumulative Body Weight Change (g) 0 2 4 6 8 10 12 14 b b a Figure 2-10. Cumulative change in body weight (M + SE g) from baseline of mice of the genotypes and on the dessert protocol desc ribed in the caption for Figure 2-9. MC4RKO mice given 8 h access to SFW gained more weight than WT or MC4RHET mice given 8 h access to SFW, which did not significantly change in body weight from baseline measures.

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41 Intake (kcal) 0 5 10 15 20 25 30 No SFW LR 2 h SFW HR 2 h SFW Genotype WT HET KO Intake (kcal) 0 5 10 15 20 25 No SFW LR 2 h SFW HR 2 h SFW a a a a a b a b a b a b Figure 2-11. Caloric intake (M + SE kcal) of WT, MC4RHET, and MC4RKO mice either LR or HR 2 h access to SFW for 14 days. Data ar e shown only from binge days when all mice were given SFW. Panel A shows calor ic intake from moist chow and SFW 2 h after diet presentation; panel B shows total caloric intake 24 h after diet presentation. The letters above the bars denote a signifi cant difference between groups: groups that have different letters above them are statistically differe nt (p<0.05) and the letter 'a' represents the group consuming the most calories. Differences are shown between genotype and restriction groups; caloric intakes from moist chow of the mice prior to the experiment are shown for comparison, but were not included in statistical analysis. Mice of any genotype given HR 2 h access to SFW consumed more calories in 24 h than mice given LR 2 h access to SFW; this was potentiated in MC4RKO mice given HR 2 h access to SFW, which consumed more calories than MC4RKO mice given LR 2 h access to SFW at the 2 h time point as well. A B

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42 Genotype WT HET KO Cumulative Body Weight Change (g) -8 -6 -4 -2 0 2 4 6 8 LR 2 h SFW HR 2 h SFW a a a a b b Figure 2-12. Cumulative change in body weight (M + SE g) from baseline of mice of the genotypes and on the dessert protocol descri bed in the caption for Figure 2-11. Data are shown across all days of the experime nt, not just binge days. MC4RKO mice given either LR or HR 2 h access to SF W gained more weight than WT or MC4RHET mice given LR or HR 2 h access to SFW, which did not significantly change in body weight from baseline meas ures. There were no differences seen between restriction gr oups in any genotype.

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43 CHAPTER 3 EFFECT OF A MELANOCORTIN RECEPTOR AGONIST AND ANTAGONIST ON OVER CONSUMPTION AND BINGE EATING IN FEMALE MICE Introduction Review of Diet Protocols We have described som e of the benefits and limitations of rodent models of dysfunctional eating and obesity and proposed the use of a desser t model as an efficient and effective means of inducing overconsumption and obesity, while allowing assessment of diet selection (Mathes et al. 2008). We have established a model of overc onsumption in which female rats presented daily with access to a high calorie dessert that was both sweet and fatty c onsumed more calories and gained more weight than rats given no dessert or rats given a low calorie dessert that was only sugary. We (Mathes et al. 2007A) and others (Dimitriou et al. 2000) have also described a model of binge eating in which female rats presented every other day with time-limited access to a high fat preferred diet consume more of the pref erred diet in at that time compared to rats presented every day with the preferred diet. Review of Mouse and Pharmacological Models Since obes ity is a polygenic disorder, and murine models allow examination of multiple gene families in a manner unavailable in rats, we sought to generalize these two dessert protocols to female mice. We hypothesized that these protocols would induce ove rconsumption and binge eating in mice in a manner similar to rats. Both dessert protocols will also allow exploration of the role of intermittent presentation on selection of complete diets in mice. We focus on the role of MC4R in these protocols sinc e it is linked to human obesity, which is mirrored in our mouse pilot work, and binge eating, which is not seen in our pilot work (Mathes et al. 2007B). In the present study, we explore in female mice the ef fect of repeated central MC4R agonist and antagonist administration on overconsumption and binge eating using th e dessert protocols

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44 described in the general methods. We hypothesi ze that a MC4R agonist will decrease and a MC4R antagonist will increase calor ic intake in our dessert mode l that induces overconsumption. We also hypothesize that although th ese agents may decrease caloric intake in our dessert model of binge eating, they will not eliminate feeding differences due to restriction. These protocols will also allow exploration of the impact of MC4R agents on selection of complete diets in mice. Methods Animals and Diets Fem ale C57Bl/J6 mice were maintained in ventilated cages as described in Chapter 2. After a week of acclimation to th e housing conditions and diets, th ey were divided into fifteen groups, to be described below. Surgery All m ice were implanted with an indwelling can nula aimed at the right lateral ventricle. The mice were anesthetized via inhalation of a mixture of 2% isoflura ne in oxygen (SurgiVet, Waukesha, WI; 2 l/min flow rate). Mice were then secured in a stereotaxic surgical instrument (Kopf, Tujunga, CA) with a mouse adapter (Stoe lting, Wood Dale, IL) that included a nose cone to provide constant administration of anesth esia during surgery. The skull was exposed by midline incision, and a guide cannula was lo wered using standard stereotaxic procedures. Cannulae (Plastics One, Roanoke, VA) were made of 28-gauge stainless stee l, cut to a 2-mm length below a Teflon screw cap. Skull coordinates wi th respect to bregma were .05 cm caudal and .10 cm lateral. The cannula was secured to the skull with cya noacrylate and dental acrylic, and 5-7 days were allowed for recovery. Af ter the experiments were completed, placement and patency of the cannulae were verified both physiologically and anatomically. Mice were injected with the dipsogenic agent, angi otensin II (50 ng / 2 l icv) using an injector needle that extended 1 mm beyond the implanted guide. Then the mice were injected icv with

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45 blue food coloring, killed by injection of sodi um pentobarbital (100 mg/ kg), and decapitated. The cannulae were removed and brains were sl iced coronally by hand at the site of cannula placement. Mice that consumed <0.3 ml of water within 30 min and for which the dyed track of the cannula did not reach the ventricle were excluded from analysis. Experimental Design We perfor med experiments 1-4 simultaneously, but in three replications because of the large numbers of mice used. Experimental groups were equally represented in the first two replications; the third replica tion was performed to increase th e sample size of specific groups since some mice in the first two replications were excluded due to excessive chow spillage, health problems following surgery or chronic dosi ng, or the inability to verify cannula placement following the experiments. Some differences were seen in intakes between replications with the general trend of intakes in replication two bei ng largest and those in replication three being smaller than intakes in replication one. Visual inspection of the data suggested that variability was due mostly to differences in intakes from individual mice between replications rather than from intakes of mice across days within replicati ons. These differences ar e presented in Table 31a-c (pages 86 and 87), using 24 h total intakes as the representative sample. Experiment 1: Overconsumption Experiment 1 sought to generalize to fema le mice a protocol modeling diet selection leading to caloric overconsumption that we have previously established in female rats (Mathes et al. 2008). Two groups of mice were presented daily for 24 days with SFW for either an 8 h nocturnal (0900-1700 h) session (n=9) or with 24 h ad libitum access (n=7). These groups allowed us to compare the effect of uninterrupt ed versus interrupted daily access to SFW on total consumption. Another group of mice were given only moist chow and did not have access to SFW at any time (n=11); these mice were used as comparison controls. Immediately prior to

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46 food presentation, all mice were gently restrained by hand to remove the cannula stylet and were injected with vehicle (distilled water, 2 l icv) using an injector needle that extended 1 mm beyond the implanted guide. Vehicle wa s delivered at a rate of 1 l per minute using a handdriven 50 l Hamilton syringe that was attached to a microinjector via polyethylene tubing (Intramedic PE50, Parsippany, NJ). SFW and chow intakes were measured by subtracting the remaining weight of the diet from that origina lly presented; measures were taken 2 h, 4 h, 8 h, and 24 h post-dosing. Total caloric intakes, as well as individual calori c intakes from moist chow and SFW, and changes in body weight we re calculated daily for the 24 days of the experiment. Experiment 2: Binge Eating Experiment 2 sought to generalize to mice a protocol modeling binge eating that has previously been established by Corwin's group (Dimitriou et al. 2000) and extended by our lab to use SFW. In the current experiment using mi ce, two groups of mice we re presented daily for 24 days with 2 h nocturnal acce ss (0900-1100 h) to SFW either in a low restriction (LR) manner (n=6), defined as access every day, or in a high restriction (HR) manner (n =7), defined as every other day. All mice were dosed daily with vehicl e as described in experiment 1. Food intake and changes in body weight were assesse d as described in experiment 1. Experiment 3: Effect of SHU9119 and MTII on Overconsumption Experiment 3 assessed in mice the effect of drugs that modify activity of the MC4R on overconsumption and diet selecti on using the SFW protocol detail ed in experiment 1. In the current experiment, 3 groups of mice were presen ted daily for 24 days with no SFW (n=17), 8 h nocturnal (0900-1700 h) access to SFW (n=15), or 24 h access to SFW (n=14). Approximately half the mice from each of these groups were dosed with MC4R agonist MTII (1.0 nmol / 2 l) and the other half dosed with MC 4R antagonist SHU9119 (1.0 nmol / 2 l). The mice from

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47 experiment 1 were used as comparison controls. Drugs were administered icv as described in experiment 1. Food intake and changes in body we ight changes were assessed as described in experiment 1. Experiment 4: Effect of SHU9119 and MTII on Binge Eating Experiment 4 assessed in mice the effect of MTII and SHU9119 on caloric selection using the SFW binge protocol detailed in expe riment 2. Two groups of mice were presented daily for 24 days with 2 h nocturnal access (09001100 h) to SFW either every day (LR) (n=12) or every other day (HR) (n=14). Approximately half the mice from each of these groups were dosed with MTII (1.0 nmol / 2 l) and the other half dosed with SHU9119 (1.0 nmol / 2 l). The mice from experiment 2 were used as comparison controls. Drugs were administered icv as described in experiment 1. Food intake and ch anges in body weight were assessed as described in experiment 1. Drugs MTII and S HU9119 were purchased from Bachem (King of Prussia, PA). The drugs were individually dissolved in distilled water and aliquots containing enough solution for each daily dose for all replications were frozen and stor ed at -60 degrees C. The dose of 1.0 nmol was chosen for both drugs because it was not expected to have maximal effects on feeding and would also be unlikely to produce cumulati ve effects with chronic dosing (Li et al. 2004; Pierroz et al. 2002). Statistics The daily in dividual total caloric intakes at each time point from all replications, as well as the component intakes from moist chow and de ssert, were analyzed via two-way ANOVA with groups and days as main factors. One-wa y ANOVA and Tukey post hoc tests were used to further assess significant differences between groups. Resu lts for experiments exploring binge

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48 eating (experiments 2 and 4) were analyzed on ly from days during which both LR and HR groups were given SFW (ie, binge days). Changes in body weight were assessed by subtracting the body weights of mice on the final day of the e xperiment from that at the beginning of the experiment and analyzed via one-way ANOVA. Results Experiment 1: Overconsumption Total calor ic intake The overall average daily total caloric intakes of mice injected daily with vehicle and given no access to SFW or either 24 h or 8 h access to SFW are presen ted in panel A of Figure 3-1 (page 78). Intakes are shown at 2 h, 4 h, 8 h, and 24 h after diet presentation and drug administration and are from mice from all replications of the experiment. Two-way ANOVA comparing th e effect of no SFW access to either 8 h or 24 h SFW access revealed a significant effect of group at the 2 h (F[2,621]=29.8, p<0.001), 4 h (F[2,537]=29.7, p<0.001), 8 h (F[2,622]=60.0, p<0.001), and 24 h (F[2,620]=9.9, p<0.001) time points. Post hoc analysis revealed that at 2 and 8 h after diet presentation mice given 8 h access to SFW consumed significantly more calories than mice given either no SFW access or mice given 24 h access to SFW. At 4 h after di et presentation, mice given 8 h access to SFW consumed more total calories than mice given 24 h access to SFW. Post hoc analysis also revealed that at 2 and 8 h after diet presen tation mice given no access to SFW consumed more calories than mice given 24 h access to SFW. At 24 h after diet presentation, mice given no access to SFW consumed significantly more calorie s than mice given either 24 h or 8 h access to SFW. Thus, for the first 8 h, SFW access for 8 h promoted a high total caloric intake, and SFW access for 24 h promoted a low caloric intake; however, this did not last 24 h after diet presentation at which time no access to SFW promoted the greatest intakes.

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49 While the data presented are 24 day averages, analysis of trends across the days of the experiment revealed sporadic differences between days. There was an effect of days at the 4 h (F[23,537]=2.2, p=0.001) and 8 h (F[23,622]=3.5, p<0.001) time points. At 4 h after diet presentation, mice consumed more total calories on days 8-10, 12, and 15 than on day 3. At 8 h after diet presentation, mice consumed more tota l calories on day 16 than on day 1. There was no group x day interaction at any of the time point s. Thus, although there are some day-to-day fluctuations, the only consistent trend seen was that intakes on days early in the experiment seemed lower than days later in the experiment. Caloric intake from moist chow The overall average d aily caloric intakes from moist chow of mice injected daily with vehicle and given no access to SFW or either 24 h or 8 h access to SFW are presented in panel B of Figure 3-1 (page 78). Intakes are shown at 2 h, 4 h, 8 h, and 24 h after diet presentation and drug administration and are from mice from all replications of the experiment. Two-way ANOVA revealed a significant e ffect of group at the 2 h (F[2,622]=109.8, p<0.001), 4 h (F[2,537]=185.8, p<0.001), 8 h (F[2,622]=443.7, p<0.001), and 24 h (F[2,621]=1087.6, p<0.001) time points. Post hoc analys is revealed that at 2 h, 4 h, 8 h, and 24 h after diet presentation mice give n no access to SFW consumed significantly more calories from moist chow than mice given either 8 h SFW acce ss or 24 h access to SFW. Post hoc analysis revealed that at 24 h after diet presentation mice given 8 h access to SFW consumed significantly more calories from moist chow than mice give n 24 h access to SFW. Thus, any access to SFW promoted low intakes from moist chow, though 8 h access to SFW promoted higher 24 h intakes from moist chow than 24 h access to SFW. There was an effect of days at the 8 h (F[2,622]=443.7, p<0.001) time point, during which mice consumed more calories from moist chow on days 8, 9, and 14 than on day 2. There was

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50 no group x day interaction at any of the time point s. Thus, day-to-day differences were very small, again resulting from low intakes early in the experiment. Caloric intake from Sugar Fat Whip (SFW) The overall average d aily caloric intakes from SFW of mice injected da ily with vehicle and given either 24 h or 8 h access to SFW are presen ted in panel C of Figure 3-1 (page 78). Intakes are shown at 2 h, 4 h, 8 h, and 24 h after diet pr esentation and drug administration and are from mice from all replications of the experiment. Two-way ANOVA revealed a significant e ffect of group at the 2 h (F[2,369]=99.1, p<0.001), 4 h (F[2,318]=98.3, p<0.001), 8 h (F[2,370]=230.9, p<0.001), and 24 h (F[2,369]=17.8, p<0.001) time points. At 2 h, 4 h, a nd 8 h after diet presentation, mice given 8 h access to SFW consumed significantly more calor ies from SFW than mice given 24 h access to SFW. Mice given 24 h access to SFW consumed significantly more to tal calories from SFW than mice given 8 h access to SFW. Thus, alt hough for the fist 8 h access to SFW for 8 h promoted higher intakes of SFW than access to SFW for 24 h, access to SFW for 24 h promoted higher total intake of SFW. There was an effect of days at the 2 h (F[23,369]=4.6, p<0.001) and 4 h (F[23,318]=1.8, p=0.013) time points. At 2 after diet presenta tion, mice consumed more SFW on day 1 than any of the other days. At 4 h after diet presenta tion, mice consumed more calories from SFW on day 1 than days 3 and 4. There was a group x da y interaction at the 24 h (F[23,322]=1.9, p=0.009) time point. The intakes from SFW of mice given 24 h access to SFW were more stable than the intake from SFW of mice given 8 h access to SF W, which increased slowly across days. Thus, large intakes on day 1 resulted in a difference between days.

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51 Change in body weight The overall cumulative change in body weight of mice injected daily with vehicle and given no access to SFW or either 24 h or 8 h access to SFW are presented in Figure 3-2 (page 79). One-way ANOVA revealed a difference be tween groups (F[2,26]-6.7, p=0.005). Post hoc analysis revealed that mice given 8 h or 24 h ac cess to SFW gained more weight than mice given no access to SFW, which did not change in weight from baseline measures. Experiment 2: Binge Eating Total caloric intake The overall average d aily total caloric intakes of mice injected daily with vehicle and given either LR or HR 2 h access to SFW are presented in panel A of Figure 3-3 (page 80). Intakes are shown at 2 h, 4 h, 8 h, and 24 h after diet presen tation and drug administration and are from mice from all replications of the experiment. Two-way ANOVA comparing th e effect of LR to HR 2 h SFW access revealed a significant effect of group at the 2 h (F [1,166]=14.5, p<0.001) and 4 h (F[1,123]=7.3, p=0.008) time points. At 2 and 4 h after diet presen tation, mice given LR 2 h access to SFW consumed significantly more calories than mice given HR 2h acce ss to SFW. Thus, in the first 4 h of each experimental day low restriction promot ed higher intakes than high restriction. There was an effect of days at th e 2 h (F[12,166]=2.6, p=0.004), 4 h (F[12,123]=4.6, p<0.001), 8 h (F[12,166]=1.3, p<0.001), and 24 h (F[12,166]=2.9, p<0.001) time points. At 2 h after diet presentation, mice consumed more total calories on day 11 than on day 3. At 4 h after diet presentation, mice consumed fe wer total calories on day 3 compared to all other binge days. At 8 and 24 h after diet presentation, mice consumed more total calories on day 9 than on days 1 and 3. There was a group x day interaction at the 4 h (F[12,123]=2.1, p=0.022) time point, due to high intakes of mice given LR 2 h access to SFW on days 11 and 23. Thus, these differences

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52 between days are due mostly to low intakes during the first days of the experiment and high intakes during the later da ys of the experiment. Caloric intake from moist chow The overall average d aily caloric intakes from moist chow of mice injected daily with vehicle and given either LR or HR 2 h access to SFW are presen ted in panel B of Figure 3-3 (page 80). Intakes are shown at 2 h, 4 h, 8 h, and 24 h after diet presentation and drug administration and are from mice from a ll replications of the experiment. Two-way ANOVA revealed a significant e ffect of group at the 2 h (F[1,166]=5.3, p=0.023) and 24 h (F[1,166]=8.3, p=0.005) time point s. At 2 h after diet presentation, mice given LR 2 h access to SFW consumed significantly more calories from moist chow than mice given HR 2 h access to SFW. At 24 h after diet presentation, mice given HR 2 h access to SFW consumed significantly more calories from moist chow than mice given LR 2h access to SFW. Thus, low restriction to SFW promoted higher in takes of moist chow than high restriction to SFW in the first 2 h of each experimental day, bu t this was compensated for by the end of each experimental day. There was an effect of days at th e 2 h (F[12,166]=2.0, p=0.023), 4 h (F[12,123]=4.2, p<0.001), 8 h (F[12,166]=3.5, p<0.001), and 24 h (F[12,166]=3.4, p<0.001) time points. At the 2 h, 4 h, and 8 h time points, mice consumed fewer ca lories in the first three days than the during the rest of the experiment. At 24 h after diet presentation, mice consumed more calories from moist chow on day 9 than on days 1, 15, 21, and 23. There was no group x day interaction at any time point. Thus, these differences between days are due mostly to low intakes during the first days of the experiment and high intakes during the later days of the experiment.

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53 Caloric intake from SFW The overall average d aily caloric intakes from SFW of mice injected da ily with vehicle and given either LR or HR 2 h access to SFW are presented in pane l C of Figure 3-3 (page 80). Intakes are shown at 2 h after diet presentation and drug administration and are from mice from all replications of the experiment. Two-way ANOVA revealed a significant eff ect of group (F[1,166]=4.3, p=0.039). At 2 h after diet presentation, mice given LR 2 h access to SFW consumed significantly more calories from SFW than mice given HR 2h access to SFW. Thus, low restriction to SFW promoted greater intakes of SFW than high restriction to SFW. There was an effect of days (F[12,166]= 2.6, p=0.004), which was due to mice consuming more calories from SFW on day 23 than on days 1 and 3. There was no group x day interaction. Thus, caloric intake from SFW was lower during th e first days of the experiment than during the later days of the experiment. Change in body weight One-way ANOVA revealed that mice injected with vehicle and given either LR or HR access to SFW ate similar amounts of calorie s (F[1,13]=0.5, p=0.517). Since there were no differences in caloric intake between groups, a change in body weight would not be expected, and so the data are not shown. Experiment 3: Effect of SHU9119 and MTII on Overconsumption Mice given no access to SFW Total calor ic intake: The overall average daily total caloric intakes of mice given no access to SFW and injected daily with either SHU9119 or MTII are pres ented in Figure 3-4 (page 81). Also redrawn from Figure 3-1 are the total in takes of mice given no access to SFW and dosed

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54 daily with vehicle. Intakes are shown at 2 h, 4 h, 8 h, and 24 h after diet presentation and drug administration and are from mice from a ll replications of the experiment. Two-way ANOVA comparing the effect of MTII and SHU91 19 to vehicle on intakes of mice given no access to SFW revealed a signific ant effect of group at the 2 h (F[2,657]=32.0, p<0.001), 4 h (F[2,559]=30.5, p<0.001), 8 h (F[2,656]=5.6, p=0.003), and 24 h (F[2,656]=19.6, p<0.001) time points. Post hoc anal ysis revealed that at the 2 h and 4 h time points mice given no access to SFW and injected da ily with SHU9119 consumed mo re total calories than mice given no access to SFW and injected daily with either vehicle or MTII. Post hoc analysis revealed that at the 2 h and 4 h time points mice given no access to SFW and injected daily with vehicle consumed more total calories than mi ce given no access to SFW and injected daily with MTII. Post hoc analysis revealed that at the 8 h time point mice given no access to SFW and injected daily with MTII or S HU9119 consumed more total calori es than mice given no access to SFW and injected daily with either vehicle. Post hoc analysis reve aled that at the 24 h time point mice given no access to SFW and injected with MT II consumed more total calories than mice given no access to SFW and injected with either SHU9119 or vehicle. Post hoc analysis revealed that at the 24 h time point mice given no SFW a nd injected daily with SHU9119 consumed more total calories than mice given no SFW and inj ected daily with vehicle. Thus, SHU9119 increased total caloric intake at every time point. MTII decreased total caloric intake at the 2 h and 4 h time points, but increased it at 8 h and 24 h after diet presentation and drug administration. There was an effect of days at th e 4 h (F[23,559]=3.6, p<0.001), 8 h (F[23,656]=4.7, p<0.001), and 24 h (F[23,656]=4.4, p<0.001) time point s. At all time points, mice consumed fewer calories in the first three days than the re st of the experiment. There was no group x day

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55 interaction at any of the time points. Thus, th ese small differences between days seem due to low intakes during the early days of the experiment. Changes in body weight: Changes in body weight in mice given no access to SFW and injected with either MTII or SHU9119 are pr esented in Table 3-2 (page 87). Body weight changes of mice given no access to SFW and injected with vehicle are presented for comparison. One-way ANOVA revealed a significant effect of group (F[2,27]=4.0, p=0.030). Post hoc analysis revealed that mice given no access to SFW and injected with SHU9119 gained more weight than mice given no SFW and injected with MTII. Mice given 24 h access to SFW Total calor ic intake: The overall average daily total calor ic intakes of mice given 24 h access to SFW and injected daily with either SHU9119 or MTII are presented in panel A of Figure 3-5 (page 82). Also redrawn from Figur e 3-1 are the total inta kes of mice given 24 h access to SFW and dosed daily with vehicle. Intakes are shown at 2 h, 4 h, 8 h, and 24 h after diet presentation and drug administration and are from mice from all replications of the experiment. Two-way ANOVA comparing the effect of MTII and SHU9119 to vehicle on total intakes of mice given 24 h access to SFW revealed a significant effect of group at the 2 h (F[2,519]=359.6, p<0.001), 4 h (F[2,438]=45.4, p< 0.001), 8 h (F[2,522]=21.5, p<0.001), and 24 h (F[2,519]=5.8, p=0.003) time points. Post hoc analys is revealed that at the 2 h, 4 h, and 8 h time points mice given 24 h access to SFW and in jected daily with SHU9119 consumed more total calories than mice given 24 h access to SFW and injected daily with either vehicle or MTII. At the 8 h time point, mice given 24 h access to SFW and injected daily with SHU9119 consumed more total calories than mice given 24 h access to SFW and injected daily with MTII. Post hoc analysis revealed that at the 2 h a nd 4 h time points mice given 24 h access to SFW and

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56 injected daily with vehicle consumed more to tal calories than mice given 24 h access to SFW and injected daily with MTII. Post hoc analysis revealed that at the 24 h time point mice given 24 h access to SFW and injected daily with ei ther SHU9119 or MTII consumed more total calories than mice given 24 h acce ss to SFW and injected daily with vehicle. Thus, SHU9119 increased total caloric intake at the 2 h, 4 h, and 24 h time points. MTII decreased total caloric intake at the 2 h and 4 h time points, but in creased it 24 h after diet presentation and drug administration. There was an effect of days at the 2 h (F[23,519]=5.3, p<0.001) and 4 h (F[23,438]=1.6, p=0.036) time points. At 2 h after diet presenta tion, mice consumed fewer calories on day 1 than on any other day. At 4 h after diet presentation, mice consumed more calories on day 1 than on day 4. There was no group x day interaction at a ny of the time points. Thus, these small differences between days seem due to low inta kes during the early days of the experiment. Caloric intake from moist chow: The overall average daily caloric intakes from moist chow of mice given 24 h access to SFW and inj ected daily with either SHU9119 or MTII are presented in panel B of Figure 35 (page 82). Also redrawn from Figure 3-1are the intakes from moist chow of mice given 24 h access to SFW and dos ed daily with vehicle. Intakes are shown at 2 h, 4 h, 8 h, and 24 h after diet presentation and drug administration and are from mice from all replications of the experiment. Two-way ANOVA comparing the effect of MTII and SHU91 19 to vehicle on intakes of moist chow of mice given 24 h access to SFW reveal ed a significant effect of group at the 2 h (F[2,521]=3.9, p=0.021), 4 h (F[2,438]=38.6, p<0.001), 8 h (F[2,521]=30.1, p<0.001), and 24 h (F[2,521]=27.5, p<0.001) time points. Post hoc analys is revealed that at the 2 h, 4 h, and 8 h time points mice given 24 h access to SFW and in jected daily with SHU9119 consumed more

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57 calories from moist chow than mice given 24 h access to SFW and injected daily with either vehicle or MTII. Post hoc analysis revealed that at the 8 h time point mice given 24 h access to SFW and injected daily with MTII consumed more calories from moist chow than mice given 24 h access to SFW and injected daily w ith vehicle. Post hoc analysis revealed that at the 24 h time point mice given 24 h access to SFW and injected daily with either SHU9119 or MTII consumed more calories from moist chow than mice give n 24 h access to SFW and injected daily with vehicle. Thus, SHU9119 increased caloric intake from moist chow at all time points. MTII did not decrease caloric intake from moist chow at any time point, but increased it at 8 h and 24 h after diet presentation and drug administration. There was no effect of days or group x day interaction at any time point. Caloric intake from SFW: The overall average daily calori c intakes from SFW of mice given 24 h access to SFW and injected daily w ith either SHU9119 or MTII are presented in panel C of Figure 3-5 (page 82). Also redraw n from Figure 3-1are the intakes from SFW of mice given 24 h access to SFW and dosed daily with vehicle. Intakes are shown at 2 h, 4 h, 8 h, and 24 h after diet presentation and drug administ ration and are from mice from all replications of the experiment. Two-way ANOVA comparing the effect of MTII and SHU91 19 to vehicle on intakes of SFW of mice given 24 h access to SFW revealed a significant effect of group at the 2 h (F[2,519]=11.3, p<0.001), 4 h (F[2,438]=38.0, p<0.001), and 8 h (F[2,521]=21.3, p<0.001) time points. Post hoc analysis revealed that at the 4 h time point mice give n 24 h access to SFW and injected daily with SHU9119 consumed more cal ories from SFW than mice given 24 h access to SFW and injected daily with eith er vehicle or MTII; also at 4 h after diet presentation, mice given 24 h access to SFW and injected daily w ith vehicle consumed mo re calories from SFW

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58 than mice given 24 h access to SFW and injected with MTII. Post hoc analys is revealed that at the 2 and 8 h time points mice given 24 h access to SFW and injected dail y with either SHU9119 or vehicle consumed more calories from SFW than mice given 24 h access to SFW and injected daily with MTII. Thus, SHU9119 in creased caloric intake from SFW only at the 4 h time point. MTII decreased caloric intake from SFW at ever y time point except 24 h after diet presentation and drug administration. There was an effect of days at th e 2 h (F[2,519]=12.4, p<0.001), 4 h (F[2,438]=3.3, p<0.001), and 24 h (F[2,518]=1.7, p=0.030) time points. At 2 h after diet presentation, mice consumed more calories from SFW on day 1 than on any other day. At 4 h after diet presentation, mice ate fewer calories from SF W on day 1 than on days 2-5, 8, 9, 11-14, 18, 20, 21, 23, and 24. At 24 h after diet presentation, mi ce ate more calories from SFW on day 1 than on days 9, 11, 20, and 21. There was no group x day inte raction at any of the time points. Thus, there was a higher intake of SFW early day compared to la ter days of the experiment. Changes in body weight: Changes in body weight in mice given 24 h access to SFW and injected with either MTII or SHU9119 are pr esented in Table 3-2 (page 87). Body weight changes of mice given 24 h access to SFW and injected with vehicle are presented for comparison. One-way ANOVA revealed no effect of group (F[2,19]=2.5, p=0.110). Mice given 8 h access to SFW Total calor ic intake: The overall average daily total cal oric intakes of mice given 8 h access to SFW and injected daily with either SHU9119 or MTII are presented in panel A of Figure 3-6 (page 83). Also redrawn from Figur e 3-1 are the total inta kes of mice given 8 h access to SFW and dosed daily with vehicle. Intakes are shown at 2 h, 4 h, 8 h, and 24 h after diet presentation and drug administration are from mice from all re plications of the experiment.

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59 Two-way ANOVA comparing the effect of MTII and SHU9119 to vehicle on total intakes of mice given 8 h access to SFW revealed a signi ficant effect of group at the 2 h (F[2,586]=5.4, p=0.005), 4 h (F[2,478]=43.0, p<0.001), 8 h (F[2,567]=5.4, p=0.005), and 24 h (F[2,567]=5.3, p=0.005) time points. Post hoc anal ysis revealed that at the 4 h and 8 h time points mice given 8 h access to SFW and injected daily with SHU9119 consumed more total calories than mice given 8 h access to SFW and injected daily with MTII. Post hoc analysis revealed that at the 2 h and 4 h time points mice given 8 h access to SFW and inject ed daily with vehicle consumed more total calories than mice given 8 h access to SFW and injected daily with MTII. Post hoc analysis revealed that at the 24 h time point mice given 8 h access to SFW and inj ected daily with MTII consumed more total calories than mice given 8 h access to SFW and injected daily with vehicle. Thus, SHU9119 increased total caloric intake only at the 4 h time point. MTII decreased total caloric intake at 2h and 4 h time point, but in creased it 24 h after diet presentation and drug administration. There was an effect of days at the 4 h (F[23,478]=2.6, p<0.001) and 8 h (F[23,567]=3.7, p<0.001) time points. At 4 h after diet presenta tion, mice consumed more total calories on day 5 than on days 3 and 4; at 8 h after diet presen tation, mice consumed mo re calories on days 4-24 than on day 1. There was a group x day inte raction at the 4 h (F[46,407]=1.9, p<0.001) time point. At 4 h after diet pres entation, mice injected daily with vehicle had stable intakes across days, whereas mice dosed with either MTII or SHU9119 had initially low intakes and increased steadily across days. Thus, intake s were higher during early days co mpared to later days of the experiment. Caloric intake from moist chow: The overall average daily caloric intakes from moist chow of mice given 8 h access to SFW and in jected daily with either SHU9119 or MTII are

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60 presented in panel B of Figure 36 (page 83). Also redrawn from Figure 3-1 are the intakes from moist chow of mice given 8 h access to SFW and dosed daily with vehicle. Intakes are shown at 2 h, 4 h, 8 h, and 24 h after diet presentation and drug administration and are from mice from all replications of the experiment. Two-way ANOVA comparing the effect of MTII and SHU91 19 to vehicle on intakes of moist chow of mice given 8 h access to SFW rev ealed a significant effect of group at the 4 h (F[2,479]=29.4, p<0.001), 8 h (F[2,568]=11.3, p<0.001), and 24 h (F[2,568]=38.8, p<0.001) time points. Post hoc analysis revealed that at the 4 h time poi nt mice given 8 h access to SFW and injected daily with SHU9119 consumed more calories from moist chow than mice given 8 h access to SFW and injected daily with either vehi cle or MTII. At the 8 h and 24 h time points, mice given 8 h access to SFW and injected daily with SHU9119 consumed more calories from moist chow than mice given 8 h access to SFW a nd injected daily with vehicle. Post hoc analysis revealed that at the 24 h time point mice given 8 h access to SFW and injected daily with MTII consumed more calo ries from moist chow than mice given 8 h access to SFW and injected daily with either SHU9119 or vehicle. At the 8 h time point, mice given 8 h access to SFW and injected daily with MTII consumed more calories from moist chow than mice given 8 h access to SFW and injected daily with vehicle. Thus, SHU9119 increased caloric intake from moist chow at the 4 h, 8 h, and 24 h time points. MTII did not decrease caloric intake from moist chow at any time point, but increased it at 8 h and 24 h after diet presentation and drug administration. There was an effect of days at the 4 h (F[23,479]=1.8, p=0.016) time point. Post hoc analysis revealed no differences between days at the 4 h time point, but generally mice consumed

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61 more calories from moist chow dur ing the first days of the experi ment than in the later days. There was no group x day interaction at any of the time points. Caloric intake from SFW: The overall average daily calo ric intakes from SFW of mice given 8 h access to SFW and inject ed daily with either SHU9119 or MTII are presented in panel C of Figure 3-6 (page 83). Also redrawn from Figure 3-1 are the intakes from SFW of mice given 8 h access to SFW and dosed daily with vehi cle. Intakes are shown at 2 h, 4 h, 8 h after diet presentation and drug administration and are from mice from all replications of the experiment. Two-way ANOVA comparing the effect of MTII and SHU91 19 to vehicle on intakes of SFW of mice given 8 h access to SFW revealed a significant effect of group at the 2 h (F[2,568]=9.8, p<0.001), 4 h (F[2,478]=43.9, p<0.001), and 8 h (F[2,567]=7.0, p=0.001) time points. Post hoc analysis revealed that at th e 4 and 8 h time points mice given 8 h access to SFW and injected daily with either SHU9119 or vehicl e consumed more calories from SFW than mice given 8 h access to SFW and injected daily with MTII. Post hoc anal ysis revealed that at the 2 h time point mice given 8 h access to SFW and injected daily with ve hicle consumed more calories from SFW than mice given 8 h access to SFW and in jected daily with either SHU9119 or MTII. Thus, SHU9119 did not increase calor ic intake from SFW at any tim e point, but rather decreased it at 2 h after diet presentation and drug administra tion. MTII decreased intake at all time points after diet presentation an d drug administration. There was an effect of days at th e 2 h (F[23,568]=1.9, p=0.006), 4 h (F[23,478]=2.9, p<0.001), and 8 h (F[23,567]=1.2, p<0.001) time points. At 2 h after diet presentation, mice ate more calories from SFW on day 5 than on day 11. At 4 h after diet presentation, mice ate more calories from SFW on days 5 and 22 than on days 3 and 4. At 8 h afte r diet presentation, mice

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62 ate more calories from SFW on days 4-24 than on day 1. There was a grou p x day interaction at the 4 h (F[46,478]=2.4, p<0.001) time point. At 4 h after diet presentati on, mice injected daily with vehicle had stable intakes across days, whereas mice dosed with either MTII or SHU9119 had initially low intakes and increased steadily across days. Thus, intakes tended to be higher early in the experiment compared to later. Change in body weight: Changes in body weight in mice given 8 h access to SFW and injected with either MTII or SHU9119 are pr esented in Table 3-2 (page 87). Body weight changes of mice given 8 h access to SFW and injected with vehicle are presented for comparison. One-way ANOVA revealed no effect of group (F[2,24]=2.2, p=0.136). Experiment 4: Effect of SHU9119 and MTII on Binge Eating Mice given Low Restriction (LR) 2 h access to SFW Total calor ic intake: The overall average daily total calor ic intakes of mice given LR 2 h access to SFW and injected daily with either SHU9119 or MTII are presented in panel A of Figure 3-7 (page 84). Also redrawn from Figure 3-3 are the total intake s of mice given LR 2 h access to SFW and dosed daily with vehicle. Intakes are shown at 2 h, 4 h, 8 h, and 24 h after diet presentation and drug administration and are from mice from all replications of the experiment. Two-way ANOVA comparing the effect of MTII and SHU9119 to vehicle on total intakes of mice given LR 2 h access to SFW revealed a significant effect of group at the 2 h (F[2,243]=35.2, p<0.001), 4 h (F[2,183]=28.9, p< 0.001), 8 h (F[2,243]=4.7, p=0.010), and 24 h (F[2,243]=3.7, p=0.026) time points. Po st hoc analysis revealed that at the 2 and 4 h time points mice given LR 2 h access to SFW and injected da ily with either SHU9119 or vehicle consumed more total calories than mice given LR 2 h access to SFW and injected daily with MTII. At the 8 h time point, mice given LR 2 h access to SFW and injected daily with SHU9119 consumed

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63 more calories than mice given LR 2 h access to SF W and injected daily with MTII. Post hoc analysis revealed that at the 24 h time point mice given LR 2 h access to SFW and injected daily with MTII consumed more total calories than mice given LR 2 h access to SFW and injected daily with vehicle. Thus, SHU9119 did not in crease caloric intake at any time point. MTII decreased caloric intake at the 2 h and 4 h time points, but increas ed caloric intake at 24 h after diet presentation and drug administration. There was an effect of days at th e 2 h (F[12,243]=2.5, p=0.005), 4 h (F[12,183]=3.8, p<0.001), 8 h (F[12,243]=5.3, p<0.001), and 24 h (F[12,243]=3.7, p=0.002) time points. At 2 h after diet presentation, mice ate more total calor ies on day 23 than on day 1 and 3. At 4 h after diet presentation, mice ate more total calories on day 21 than on days 3, 11, and 13. At 8 h after diet presentation, mice ate fewer to tal calories on day 1 compared to all other binge days. At 24 h after diet presentation, mice ate more total calo ries on days 9 and 13 than on day 1. There was a group x day interaction at th e 2 h (F[24,243]=1.8, p=0.012) time point. At 2 h after diet presentation, the intakes of mice injected daily with vehicle slightly increa sed and then stabilized across days, compared to the in takes of mice injected with S HU9119 or MTII, which increased across all days. Thus, intakes tended to be highe r early in the experiment compared to later. Caloric intake from moist chow: The overall average daily caloric intakes from moist chow of mice given LR 2 h access to SFW and in jected daily with eith er SHU9119 or MTII are presented in panel B of Figure 37 (page 84). Also redrawn from Figure 3-3 are the intakes from moist chow of mice given LR 2 h access to SFW and dosed daily with vehicle. Intakes are shown at 2 h, 4 h, 8 h, and 24 h after diet presen tation and drug administration and are from mice from all replications of the experiment.

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64 Two-way ANOVA comparing the effect of MTII and SHU91 19 to vehicle on intakes of moist chow of mice given LR 2 h access to SFW re vealed a significant effect of group at the 2 h (F[2,243]=6.4, p=0.002), 4 h (F[2,184]=6.7, p=0.002), 8 h (F[2,243]=4.9, p=0.008), and 24 h (F[2,243]=17.8, p<0.001) time points. Post hoc analys is revealed that at the 2 h and 4 h time points mice given LR 2 h access to SFW and inj ected daily with SHU9119 consumed more calories from moist chow than mice given LR 2 h access to SFW and inject ed daily with MTII. Post hoc analysis revealed that at the 8 h and 24 h time points mice given LR 2 h access to SFW and injected daily with MTII consumed more calor ies from moist chow than mice given LR 2 h access to SFW and injected daily with vehicle. At the 24 h time point, mice given LR 2 h access to SFW and injected daily with MTII consumed more calories fr om moist chow than mice given LR 2 h access to SFW and injected daily with SHU9119. Thus SHU9119 did not increase caloric intake from moist chow at any time poi nt. MTII did not decrease caloric intake from moist chow at any time point, and increased it at 8 h and 24 h after diet presentation and drug administration. There was an effect of days at the 8 h (F[12,244]=3.5, p<0.000) time point, during which mice consumed more total calories on days 9 an d 13 than on days 1 and 3. There was no group x day interaction at any of the time points. T hus, intakes tended to be higher early in the experiment compared to later. Caloric intake from SFW: The overall average daily calo ric intakes from SFW of mice given LR 2 h access to SFW and injected daily with either SHU9119 or MTII are presented in panel C of Figure 3-7 (page 84). Also redraw n from Figure 3-3 are the intakes from SFW of mice given LR 2 access to SFW and dosed daily with vehicle. Intakes are s hown at 2 h after diet presentation and drug administration and are from mice from all re plications of the experiment.

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65 Two-way ANOVA comparing the effect of MTII and SHU9119 to vehicle on intakes of SFW of mice given LR 2 h access to SFW revealed a significant eff ect of group (F[2,243]=30.1, p<0.001). Post hoc analysis revealed that mice given LR 2 h access to SFW and injected daily with either SHU9119 or vehicle consumed more calories from SFW than mice given LR 2 h access to SFW and injected daily with MTII. Thus, SHU9119 did not increase caloric intake from SFW, whereas MTII decreased caloric intake from SFW. There was no effect of days or group x day interaction. Change in body weight: Changes in body weight in mice given LR 2 h access to SFW and injected with either MTII or SHU9119 are presented in Tabl e 3-2 (page 87). Body weight changes of mice given 8 h access to SFW and injected with vehicle are presented for comparison. One-way ANOVA revealed no effect of group (F[2,19]=0.4, p=0.695). Mice given High Restriction (HR) 2 h Access to SFW Total calor ic intake: The overall average daily total calor ic intakes of mice given HR 2 h access to SFW and injected daily with either SHU9119 or MTII are presented in panel A of Figure 3-8 (page 85). Also redrawn from Figure 3-3 are the total intake s of mice given HR 2 h access to SFW and dosed daily with vehicle. Intakes are shown at 2 h, 4 h, 8 h, and 24 h after diet presentation and drug administration and are from mice from all replications of the experiment. Two-way ANOVA comparing the effect of MTII and SHU9119 to vehicle on total intakes of mice given HR 2 h access to SFW revealed a significant effect of group at the 2 h (F[2,251]=32.2, p<0.001) and 4 h (F[2,189]=22.5, p<0.001) time points. Post hoc analysis revealed that at the 2 and 4 h time points mice given HR 2 h access to SFW and injected daily with either SHU9119 or vehicle consumed more total calories than mice given HR 2 h access to SFW and injected daily with MT II. Thus, SHU9119 did not increase total caloric intake at any

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66 time point. MTII decreased caloric intake 2 h and 4 h after diet presentation and drug administration. There was an effect of days at th e 2 h (F[12,251]=2.4, p=0.006), 4 h (F[12,189]=4.2, p<0.001), 8 h (F[12,251]=4.7, p<0.001), and 24 h (F[12,251]=5.6, p<0.001) time points. At 2 h after diet presentation, mice ate fe wer calories on day 1 than all ot her binge days. At 4 h after diet presentation, mice ate more calories on day 11 than on days 1 and 3. At 8 h after diet presentation, mice ate more calories on days 7, 9, 11, 17, and 19 than on day 23. There was no group x day interaction at any of the time points. Thus, intakes tended to be higher early in the experiment compared to later, except at 8 h after diet presentation and drug administration. Caloric intake from moist chow: The overall average daily caloric intakes from moist chow of mice given HR 2 h access to SFW a nd injected daily with SHU9119 or MTII are presented in panel B of Figure 38 (page 85). Also redrawn from Figure 3-3are the intakes from moist chow of mice given HR 2 h access to SFW and dosed daily with vehicle. Intakes are shown at 2 h, 4 h, 8 h, and 24 h after diet presen tation and drug administration and are from mice from all replications of the experiment. Two-way ANOVA comparing the effect of MTII and SHU91 19 to vehicle on intakes of moist chow of mice given HR 2 h access to SFW re vealed a significant effect of group at the 2 h (F[2,251]=4.1, p=0.019), 4 h (F[2,189]=4.6, p=0.012), 8 h (F[2,251]=11.9, p<0.001), and 24 h (F[2,251]=17.8, p<0.001) time points. Post hoc analys is revealed that at the 2 h time point mice given HR 2 h access to SFW and in jected daily with either SHU 9119 or vehicle consumed more calories from moist chow than mice given HR 2 h access to SFW and injected daily with MTII. Post hoc analysis revealed th at at the 4 h time point mice given HR 2 h access to SFW and injected daily with vehicle consumed more cal ories from moist chow than mice given HR 2 h

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67 access to SFW and injected daily with MTII. Post hoc analysis revealed that at the 8 h and 24 h time points mice given HR 2 h access to SFW and injected daily with MTII consumed more calories from moist chow than mice given HR 2 h access to SFW and inject ed daily with either SHU9119 or vehicle. Thus, SHU9119 did not incr ease caloric intake from moist chow at any time point. MTII decreased caloric intake from moist chow at the 2 h and 4 h time point, but increased it at 8 h and 24 h after di et presentation and drug administration. There was an effect of days at th e 4 h (F[12,189]=3.6, p<0.001), 8 h (F[12,251]=3.7, p<0.001), and 24 h (F[12,251]=4.9, p<0.001) time points. At 4 h after diet presentation, mice consumed more calories from moist chow on day 11 than on days 1, 3, and 23. At 8 h after dessert presentation, mice consumed more total calories from moist chow on days 7, 9, 11, and 19 than on day 1. At 24 h after dessert presenta tion, mice ate more calorie s from moist chow on day 7 than on days 1, 21, and 23. There was no group x day interaction at any of the time points. Thus intakes toward the middle of the experiment tended to be larger than those at the beginning or the end. Caloric intake from SFW: The overall average daily calori c intakes from SFW of mice given LR 2 h access to SFW and injected daily with either SHU9119 or MTII are presented in panel C of Figure 3-8 (page 85). Also redraw n from Figure 3-3 are the intakes from SFW of mice given HR 2 h access to SFW and dosed daily w ith vehicle. Intakes are shown at 2 h after diet presentation and drug administration and are from mice from all replications of the experiment. Two-way ANOVA comparing the effect of MTII and SHU91 19 to vehicle on intakes of SFW of mice given HR 2 h access to SFW reveal ed a significant eff ect of group (F[2,251]=42.8, p<0.001). Post hoc analysis revealed that mice given HR 2 h access to SFW and injected daily

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68 with either SHU9119 or vehicle consumed more calories from SFW than mice given HR 2 h access to SFW and injected daily with MTII. Thus, SHU9119 did not increase caloric intake from SFW, whereas MTII decreased it. There was no effect of days or group x day interaction. Change in body weight: Changes in body weight in mice given HR 2 h access to SFW and injected with either MTII or SHU9119 are presented in Tabl e 3-2 (page 87). Body weight changes of mice given 8 h access to SFW and injected with vehicle are presented for comparison. One-way ANOVA revealed no effect of group (F[2,20]=0.7, p=0.492). Discussion Experiment 1: Overconsumption In this study, we sought to generalize to fe m ale mice a dessert protocol that we have previously reported as a model of overconsump tion in female rats. Generalization of the protocols to mice would have allowed analysis of monogenetic correlates of these behaviors. However, overconsumption was not seen in fema le C57Bl/6J mice provided with daily 8 h or 24 h access to SFW. Mice avidly consumed the SFW, consuming over twice the calories from SFW that they ate from moist chow; how ever, they adequately reduced th eir intake of moist chow such that their total daily intakes were not different from the intakes of mice that were provided with moist chow only. In fact, mice given no access to SFW consumed significantly more total calories than mice given either 8 h or 24 h access to SFW. This is congruent with our pilot work in which female mice of a wild type strain did not exhibit overconsumption while on the SFW dessert protocol, and suggests th at the age of the rodent may not be as influential in the overconsumption protocol as in the binge eating protocol. These findings that mice do not overconsume when subjected to this protocol differ from reports that C57Bl/6J mice are especially suscep tible to DIO compared to other mouse strain

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69 (Collins et al. 2004) and that female mice may be more susceptible to body weight gain in models of DIO than male mice (Matyskova, 2007). The differences among these findings may be due to the provision of a choice between diet s compared to access to a single nutritionallycomplete high energy diet, or the time-limited availability of the dessert compared to ad libitum access. These results in mice differ from our previ ous report in female rats in which the total daily caloric intakes and body weights of female rats robustly increased as a result of large caloric intakes from SFW that were not ade quately compensated for by a reduction in moist chow intake. This may suggest that SFW may not be as salient or palatable to mice compared to rats, or that mice may prefer moist chow to SFW, whereas SFW seems almost irresistible to rats and is highly preferred to moist chow. It ma y also suggest that mice have more complete mechanisms than rats of compensating for calorie s in an environment that presents choices and contrasts among commodities. Although this calori c balancing apparently negates use of the SFW dessert protocol as a model of overconsumpti on in mice, it provides a pure analysis of diet selection in mice unencumbered by differen ces in caloric inta ke between groups. This protocol also allowed expl oration of the effect of inte rmittent versus uninterrupted presentation of SFW on caloric intake. Although as mentioned above, mice given SFW for any duration consumed less total calories than mice not given SFW, mice given less frequent access to SFW (ie, 8 h) consumed more total cal ories and calories from SFW than mice given continuous access to SFW (ie, 24 h) 2 h, 4 h, a nd 8 h after diet presentation. However, mice given 24 h access to SFW consumed more calor ies from SFW than mice given 8 h access to SFW 24 h after diet presentation. Thus, intermittent presentation of SFW s eems to initiate eating larger amounts of SFW than what would be consum ed if SFW were always present, but this does not in mice translate into ove rconsumption across a 24 h period.

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70 Experiment 2: Binge Eating Surpris ingly, the effect of intermittent presentation seen with 8 h versus 24 h presentation is not seen when the intermittent presentation is between days. Although in the first 2 h of SFW presentation mice given 2 h access to SFW ever y other day (ie, HR) consumed more total calories and calories from SFW than mice given either 8 h or 24 h daily access to SFW, they did not consume more calories in 24 h than mice give n 2 h access to SFW daily (ie, LR). In fact, mice given LR 2 h access to SFW consumed more total calories and calories from SFW than mice given HR 2 h access to SFW 2 h after diet presentation. Thus, the binge protocol we established in rats did not genera lize to mice. This is surprisi ng since evidence of binge like eating was seen in our preliminary studies with WT and MC4RHET mice (Mathes et al. 2007B). Its possible that this could be due to surgery or repeated centra l dosing, and so in our second and third replications, we ran this pr otocol on intact mice. The results were similar (data not shown): 2 h after diet presentation, mice given LR 2 h access to SFW ate more calories than mice given HR access to SFW (5.61 + 0.17 > 4.53 + 0.17, F[1,143]=21.0, p<0.001). Although at 24 h after diet presentation, mice given HR 2 h access to SFW consumed more to tal calories than mice given LR 2 h access to SFW (12.70 + 0.26 > 11.75 + 0.17, F[1,143]=6.4, p=0.013), this doesnt seem definable as binge eating si nce SFW was no longer available. It is possible that we saw binge eating in WT mice but not C57Bl/6J mice due to differences in housi ng conditions prior to and during the experiments, age, strain differences, or some comb ination of these factors. Experiment 3: Effect of SHU9119 and MTII on Overconsumption As was hypothesized, mice giv en either no access to SFW or 24 h access to SFW and injected daily with MC4R antagonist SHU9119 at e more 24 h total calories than mice given either no access to SFW or 24 h access to SFW and injected daily with vehicle; there was a trend for mice given 8 h access to SFW to do the same. This difference was most apparent at the 2 h

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71 and 4 h time points. It seems that these differences between mice given 24 h access to SFW and injected with either SHU9119 or vehicle were due to specific reductions in moist chow, as opposed to SFW, intake. At 2 h and 8 h after diet presentation and drug administration, mice given 24 h access to SFW and injected with S HU9119 consumed more calories from moist chow than and the same amount of calories from SFW as mice given 24 h access to SFW and injected with vehicle. Only at the 4 h time point did mice given 24 h access to SFW and injected with SHU9119 consume more calories from SFW than mice given 24 h access to SFW and injected with vehicle. Mice given 8 h access to SFW and injected with SHU9119 ate more calories from moist chow than and the same amount of calories from SFW as mice given 8 h access to SFW and injected with vehicle at 4 h and 8 h after diet presentation and dr ug administration. The effect of SHU9119 administration on caloric intake, specifically on moist chow rather than SFW, supports our hypothesis that antagonism of the MC 4R system increases consumption, and that this increase is more likely to be via a comp lete diet than a palatable diet. In contrast to the hyperphagi c effect of SHU9119 and the ex pected hypophagic effect of a MC4R agonist, mice given no access to SFW or e ither 8 h or 24 h access to SFW and injected daily with MC4R agonist MTII ate more 24 h to tal calories than mice given no access to SFW or either 8 h or 24 h access to SFW and injected daily with vehicle. This seems due to compensation for reduced intakes at 2 h and 4 h after diet presentation and drug administration, at which time mice given no access to SFW or either 8 h or 24 h access to SFW and injected daily with MTII ate fewer total ca lories than mice given no access to SFW or either 8 h or 24 h access to SFW and injected daily with vehicle. It is possible that MTII was mostly or only effective for the first 4 h after dosing. It seems these differen ces between mice given 24 h access to SFW and injected with either MTII or vehicle were specific to reductions in SFW, as opposed

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72 to moist chow, intake. At 2 h, 4 h, and 8 h after diet presentation and drug administration, mice given 24 h access to SFW and injected with MTII ate fewer calories from SFW than and as many calories from moist chow as mice given 24 h access to SFW and injected with vehicle. At 2 h and 4 h after diet presentation and drug admi nistration, mice given 8 h access to SFW and injected with MTII ate fewer calories from SFW than and as many calories from moist chow as mice given 8 h access to SFW and injected with vehi cle. At 8 h after diet presentation and drug administration, mice given 8 h access to SFW and injected with MTII seemed to begin to compensate and ate as many calories as mice give n 8 h access to SFW and injected with vehicle, and they did so by eating more calories from mo ist chow. The effect of MTII administration on caloric intake, specifically on SFW rather than mo ist chow, does not support our hypotheses that agonism of the MC4R system decreases consump tion, and that this decr ease would be via a complete diet more so than a palatable diet. The effect of SHU9119 and MTII on total caloric intake and se lection of complete versus palatable diets in the SFW overconsumption pr otocol appear contra dictory: if SHU9119 specifically increases moist chow intake, as it does, MTII should specifi cally reduce moist chow intake, but instead it specifically decreases SFW in take. This discrepancy could be interpreted as support to the suggestion that SFW is not very sali ent to mice, a point suggested above to explain why mice did not overconsume SFW when injected with vehicle. This may be even more evident when analyzing a system that doesn't sp ecifically promote diet selection based on the palatability of the commodity. If SFW does not serve as a potent reinfo rcer or is not very rewarding to mice, drugs that increase hunger would not affect SFW intake as much as they would moist chow intake. Congruently, drugs that decrease hunger woul d make mice disregard SFW even more. However, the fact that in a ny drug or diet access situation mice consume more

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73 calories from SFW than from moist chow doe s not support the assumption that SFW is not preferred to moist chow by mice; in fact, it coul d be argued that this s upports the opposite. Also, intermittent presentation (within-days) promotes increased consumption at initial time periods, which could be interpreted as craving-like behavi or. It would be interesting to see if this occurred to a greater extent if moist chow access was restricted. Experiment 4: Effect of SHU9119 and MTII on Binge Eating Contrary to our hypothesis, m ice given either LR or HR 2 h access to SFW and injected daily with SHU9119 ate the same number of total calories as mice given either LR or HR 2 h access to SFW and injected daily with vehicle at all time points. This may be due to a ceiling effect driven by time-limited acce ss to SFW mice injected with vehicle may have consumed the maximum number of calories possible when gi ven only 2 h access to SFW. Consistent with our hypothesis, mice given either LR or HR 2 h access to SFW and injected daily with MTII ate fewer total calories than mice given either LR or HR 2 h access to SFW and injected daily with vehicle in the first 4 h after diet presentation a nd drug administration. However, contrary to our hypothesis, mice given either LR or HR 2 h access to SFW and injected daily with MTII ate the same number of or more 24 h to tal calories than mice given eith er LR or HR 2 h access to SFW and injected daily with vehicle. This appeared to be compensatory in na ture and occurred most obviously 8 h after diet presentation and drug admi nistration, and was due mostly to moist chow, since SFW was no longer available. Regardless of the interpretation, ne ither SHU9119 nor MTII prompted or diminished any evidence of binge ea ting in this protocol. This does not agree with our preliminary data suggesting that MC4RKO mice seemed less like ly to binge than WT mice. This could be due to compensatory mechanisms possible in genetic deletion models or due to differences in age, housing conditions, strain, or a combination of these factors. It would be

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74 interesting to repeat this experiment in rats, si nce this protocol instigates binge eating in this species and has been shown to be suscep tible to modification by drugs (Buda-Levin et al. 2005). Summary Neither SF W protocol resulted in overconsum ption or binge eating in female C57Bl/6J mice. The reasons are unclear as to why thes e protocols do not produce overconsumption in a species and strain susceptible to DIO and when they have been so effective in rat models. It may be that mice developed a better strategy than rats by which to balance calor ic intake within days rather than across days, and so are not as affected by diet choices and time-limited access across days. It would be interesting to explore the ecological rational e and neurological correlates of this behavior. However, it is first important to assess the utility of these protocols between species. We have shown that CB1R antagonist s decrease intake in rats on the SFW overconsumption protocol, and that this is selective to SFW. We now explore this in mice, a species which may not perceive SFW as reward ing as rats, and assess if our hypothesis that CB1R specifically decreases selection of palatable is similar in this species.

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75 Cumulative Intake (kcal) 0 2 4 6 8 10 12 14 No SFW / V 24 h SFW / V 8 h SFW / V Cumulative Intake (kcal) 0 2 4 6 8 10 12 14 No SFW / V 24 h SFW / V 8 h SFW / V Time (h) Since Diet Presentation 2 4 8 24 Cumulative Intake (kcal) 0 2 4 6 8 10 12 14 24 h SFW / V 8 h SFW / V b c a b c a a b a a b b a b b a b b a b b a c b b a b a b a Figure 3-1. Caloric intake (M + SE kcal) of mice injected with vehicle (V) and given no sugar fat whip desert (SFW) or either 8 h or 24 h access to SFW for 24 days. Panel A) Total caloric intake. Panel B) Caloric intake from moist chow. Panel C) Caloric intake from SFW. Differences are shown be tween diet groups at 2 h, 4 h, 8 h, and 24 h after diet presentation and drug administ ration. Also shown in panel C is the difference between total daily calories from SFW consumed by mice given 8 h or 24 h access to SFW. The letters above the ba rs denote a significant difference between groups at each time point: groups that ha ve different letters above them are statistically different (p<0.05) and the letter 'a' represents the group consuming the most calories. Although for the first 8 h access to SFW resulted in increased caloric intake compared to no access, at 24 h afte r diet presentation and drug administration, mice given no access to SFW consumed more tota l calories than mice given either 8 h or 24 h access to SFW. The longer the dur ation of access to SFW, the less moist chow and the more SFW was consumed. A B C

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76 No SFW 8 h SFW24 h SFW Cumulative Body Weight Change (g) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 b a a Figure 3-2. Cumulative change in body weight (M + SE g) from baseline of mice on the diet and drug regimen described in Figure 3-1. Di fferences are shown between diet groups. Mice given no access to SFW gained less weig ht than mice given either 8 h or 24 h access to SFW, which gained comparable amounts of weight.

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77 Cumulative Intake (kcal) 0 2 4 6 8 10 12 14 LR 2 h SFW / V HR 2 h SFW / V Cumulative Intake (kcal) 0 2 4 6 8 10 12 14 LR 2 h SFW / V HR 2 h SFW / V Time (h) Since Diet Presentation 2 4 8 24 Cumulative Intake (kcal) 0 2 4 6 8 10 12 14 LR 2 h SFW / V HR 2 h SFW / V a b a b a a a a a b a a a a a b b a Figure 3-3 Caloric intake (M + SE kcal) of mice injected with vehicle and given either HR or LR 2 h access to SFW for 24 days. Data were taken from binge days only when both groups had access to SFW. Panel A) Total caloric intake. Panel B) Caloric intake from moist chow. Panel C) Caloric inta ke from SFW. Di fferences are shown between diet groups at 2 h, 4 h, 8 h, a nd 24 h after diet presentation and drug administration. Panel C shows SFW at the 2 h time point only because the dessert was removed after 2 h. The letters above the bars denote a significant difference between groups at each time point: groups th at have different letters above them are statistically different (p<0.05) and the letter 'a' represents the group consuming the most calories. Mice given LR 2 h access to SFW consumed less moist chow and more SFW than mice given HR 2 h access to SFW, but no differences in caloric intake were seen between groups 24 h after diet presen tation and drug administration. A C B

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78 Total Caloric IntakeTime (h) Since Diet Presentation 2 4 8 24 Cumulative Intake (kcal) 0 2 4 6 8 10 12 14 No SFW / SHU9119 No SFW / V No SFW / MTII a b c a b c a b a b c a Figure 3-4. Caloric intake (M + SE kcal) of mice given no SF W and injected with either melanocortin-4 receptor (MC4R) agonist melanotan-II (MTII) or MC4R antagonist SHU9119 for 24 days. Intakes of mice give n no SFW and injected with vehicle are redrawn from Figure 3-1. Differences are shown between diet groups at 2 h, 4 h, 8 h, and 24 h after diet presentation and drug ad ministration. The letters above the bars denote a significant difference between groups at each time point: groups that have different letters above them are statistically different (p<0.05) and the letter 'a' represents the group consuming the most calories. SHU9119 increased and MTII decreased caloric intake compared to vehi cle at the 2 h and 4 h time points; however, mice given no access to SFW and injected wi th either MTII or SHU9119 consumed more calories than mice given no access to SFW and injected with vehicle 24 h after diet presentation and drug administration.

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79 Cumulative Intake (kcal) 0 2 4 6 8 10 12 14 24 h SFW / SHU9119 24 h SFW / V 24 h SFW / MTII Time (h) Since Diet Presentation 2 4 8 24 Cumulative Intake (kcal) 0 2 4 6 8 10 12 14 24 h SFW / SHU9119 24 h SFW / V 24 h SFW / MTII Cumulative Intake (kcal) 0 2 4 6 8 10 12 14 24 h SFW / SHU9119 24 h SFW / V 24 h SFW / MTII a b c a b c a a,b b a b a a b b a b b a c b a b a a a b a b c a a b a a a Figure 3-5. Caloric intake (M + SE kcal) of mice given 24 h acce ss to SFW and injected with either MTII or SHU9119 for 24 days. In takes of mice given 24 h access to SFW and injected with vehicle are redrawn from Fi gure 3-1. Panel A) Total caloric intake. Panel B) Caloric intake from moist chow. Panel C) Caloric intake from SFW. Differences are shown between diet groups at 2 h, 4 h, 8 h, and 24 h after diet presentation and drug administration. The letters above the bars denote a significant difference between groups at each timepoin t: groups that have different letters above them are statistically different (p<0.05) and the letter 'a' re presents the group consuming the most calories. SHU9119 incr eased and MTII decrea sed caloric intake compared to vehicle at the 2 h and 4 h time points; however, mice given 24 h access to SFW and injected with either MTII or SHU9119 consumed more calories than mice given 24 h access to SFW and injected w ith vehicle 24 h after diet presentation and drug administration, primarily due to lo wer intakes of moist chow in the vehicle group. A B C

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80 Cumulative Intake (kcal) 0 2 4 6 8 10 12 14 8 h SFW / SHU9119 8 h SFW / V 8 h SFW / MTII Time (h) Since Diet Presentation 2 4 8 24 Cumulative Intake (kcal) 0 2 4 6 8 10 12 14 8 h SFW / SHU9119 8 h SFW / V 8 h SFW / MTII Cumulative Intake (kcal) 0 2 4 6 8 10 12 14 8 h SFW / SHU9119 8 h SFW / V 8 h SFW / MTII a,b a b a b c a a,b b a,b b a a a a a b b a b a b c a b a b a a b a a b Figure 3-6. Caloric intake (M + SE kcal) of mice given 8 h access to SFW and injected with either MTII or SHU9119 for 24 days. In takes of mice given 8 h access to SFW and injected with vehicle are redrawn from Figure 3-1. Panel A shows total caloric intake; panel B shows caloric intake from moist chow; panel C shows caloric intake from dessert. Differences are shown betw een diet groups at 2 h, 4 h, 8 h, and 24 h after diet presentation and drug administ ration. Panel C shows SFW at the 2 h, 4h, and 8 h time points only because the dessert was removed after 8 h. The letters above the bars denote a significant difference be tween groups at each time point: groups that have different letters above them are statistically differe nt (p<0.05) and the letter 'a' represents the group consuming the most calories. SHU9119 increased and MTII decreased caloric intake compared to ve hicle at the 4 h time point; however, mice given 8 h access to SFW and injected with MTII consumed more calories than mice given 8 h access to SFW and injected with vehicle 24 h after diet presentation and drug administration. A B C

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81 A: Total Caloric Intake Cumulative Intake (kcal) 0 2 4 6 8 10 12 14 LR 2 h SFW / SHU9119 LR 2 h SFW / V LR 2 h SFW / MTII C: Caloric Intake from SFWTime (h) Since Diet Presentation 2 4 8 24 Cumulative Intake (kcal) 0 2 4 6 8 10 12 14 LR 2 h SFW / SHU9119 LR 2 h SFW / V LR 2 h SFW / MTII B: Caloric Intake from Moist Chow Cumulative Intake (kcal) 0 2 4 6 8 10 12 14 LR 2 h SFW / SHU9119 LR 2 h SFW / V LR 2 h SFW / MTII a a b a a b a a,b b a,b b a a a,b b a a,b b a,b b a b b a a a b Figure 3-7. Caloric intake (M + SE kcal) of mice given LR 2 h access to SFW and injected with either MTII or SHU9119 for 24 days. In takes of mice given LR 2 h access to SFW and injected with vehicle are redrawn from Figure 3-3. Data were taken from binge days only. Panel A) Total caloric intake. Panel B) Caloric intake from moist chow. Panel C) Caloric intake from SFW. Differe nces are shown between diet groups at 2 h, 4 h, 8 h, and 24 h after diet presentati on and drug administration. Panel C shows SFW at the 2 h time point only because the de ssert was removed after 2 h. The letters above the bars denote a significant diffe rence between groups at each time point: groups that have different le tters above them are statistically different (p<0.05) and the letter 'a' represents th e group consuming the most calories. MTII decreased caloric intake compared to vehicle at the 2 h and 4 h time points; however, mice given LR 2 h access to SFW and injected with MTII consumed more calories than mice given LR 2 h access to SFW and inje cted with vehicle 24 h after diet presentation and drug administration. A B C

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82 Cumulative Intake (kcal) 0 2 4 6 8 10 12 14 HR 2 h SFW / SHU9119 HR 2 h SFW / V HR 2 h SFW / MTII Time (h) Since Diet Presentation 2 4 8 24 Cumulative Intake (kcal) 0 2 4 6 8 10 12 14 HR 2 h SFW / SHU9119 HR 2 h SFW / V HR 2 h SFW / MTII Cumulative Intake (kcal) 0 2 4 6 8 10 12 14 HR 2 h SFW / SHU9119 HR 2 h SFW / V HR 2 h SFW / MTII a a b a a b a a a a a a a a b a,b a b b b a b b a a a b Figure 3-8. Caloric intake (M + SE kcal) of mice given HR 2 h access to SFW and injected with either MTII or SHU9119 for 24 days. In takes of mice given HR 2 h access to SFW and injected with vehicle are redrawn from Figure 3-3. Data were taken from binge days only. Panel A) Total caloric intake. Panel B) Caloric intake from moist chow. Panel C) Caloric intake from dessert. Differences are shown between diet groups at 2 h, 4 h, 8 h, and 24 h after diet presentati on and drug administration. Panel C shows SFW at the 2 h time point only because the de ssert was removed after 2 h. The letters above the bars denote a significant diffe rence between groups at each timepoint: groups that have different le tters above them are statistically different (p<0.05) and the letter 'a' represents th e group consuming the most calories. MTII decreased caloric intake compared to vehicle at the 2 h and 4 h time points; however, mice given LR 2 h access to SFW and injected with either drug consumed an equivalent number of calories as mice given LR 2 h access to SFW and injected with vehicle 24 h after diet presentation and drug administration. A C B

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83 Table 3-1. Daily caloric intakes (M + SE kcal) across replications of mi ce injected daily with vehicle Replication 1 Replica tion 2 Replication 3 2-way ANOVA (factors = batch and day ) No SFW 12.30 + 0.35b n=3 14.57 + 0.32a n=4 11.42 + 0.30c n=4 Group: F(2,185)=25.8 p<0.001 Day: F(23,185)=2.1 p=0.002 GxD: F(44,185)=1.5 p=0.039 24 h SFW 11.76 + 0.48a n=2 12.18 + 0.43a n=3 11.19 + 0.53a n=2 Group: F(2,97)=1.0 p=0.356 Day: F(23,97)=0.4 p=0.998 GxD: F(44,97)=2.0 p=0.002 8 h SFW 12.30 + 0.32a n=3 11.06 + 0.35b n=3 10.57 + 0.34b n=3 Group: F(2,141)=8.3 p<0.001 Day: F(23,141)=1.2 p=0.238 GxD: F(44,141)=1.3 p=0.103 LR 2 h SFW 13.12 + 0.51a n=3 12.12 + 0.49a n=3 NA Group: F(1,63)=1.7 p=0.199 Day: F(11,63)=1.5 p=0.151 GxD: F(10,63)=1.7 p=0.101 HR 2 h SFW 12.79 + 0.53a n=3 13.75 + 0.50a n=4 NA Group: F(1,64)=1.7 p=0.202 Day: F(11,64)=2.3 p=0.012 GxD: F(10,64)=1.2 p=0.287

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84 Table 3-2. Daily caloric intakes (M + SE kcal) across replications of mi ce injected daily with SHU9119 Replication 1 Replica tion 2 Replication 3 2-way ANOVA (factors = batch and day ) No SFW 14.04 + 0.28a n=4 13.67 + 0.31a n=4 NA Group: F(1,145)=3.0 p=0.084 Day: F(23,145)=2.2 p=0.002 GxD: F(21,185)=3.2 p<0.001 24 h SFW 13.39 + 0.30a n=4 12.51 + 0.47a n=2 NA Group: F(1,110)=2.0 p=0.160 Day: F(23,110)=0.7 p=0.892 GxD: F(21,110)=0.9 p=0.539 8 h SFW 12.62 + 0.39a n=3 11.97 + 0.39a n=4 NA Group: F(1,141)=1.0 p=0.318 Day: F(23,141)=1.1 p=0.336 GxD: F(21,141)=0.7 p=0.790 LR 2 h SFW 13.28 + 0.51a n=3 13.60 + 0.49a n=3 NA Group: F(1,68)=0.4 p=0.512 Day: F(11,68)=1.0 p=0.436 GxD: F(10,68)=1.3 p=0.249 HR 2 h SFW 13.15 + 0.52a n=4 13.19 + 0.65a n=3 NA Group: F(1,66)=0.1 p=0.713 Day: F(11,66)=2.1 p=0.025 GxD: F(10,66)=0.9 p=0.515

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85 Table 3-3 Daily caloric intakes (M + SE kcal) ac ross replications of mi ce injected daily with MTII Replication 1 Replica tion 2 Replication 3 2-way ANOVA (factors = batch and day ) No SFW 15.13 + 0.30a n=4 15.59 + 0.35a n=3 12.62 + 0.51b n=2 Group: F(2,165)=11.9 p<0.001 Day: F(23,165)=1.6 p=0.044 GxD: F(44,165)=1.2 p=0.262 24 h SFW 12.84 + 0.34a n=3 13.91 + 0.38a n=3 11.20 + 0.49b n=2 Group: F(2,151)=8.9 p<0.001 Day: F(23,151)=1.0 p=0.515 GxD: F(44,151)=1.1 p=0.374 8 h SFW 13.25 + 0.39a n=4 12.09 + 0.44a,b n=3 10.34 + 0.82b n=1 Group: F(2,144)=7.6 p=0.001 Day: F(23,144)=0.6 p=0.908 GxD: F(44,144)=1.2 p=0.185 LR 2 h SFW 13.92 + 0.53a n=4 14.43 + 0.79a n=2 NA Group: F(1,55)=0.2 p=0.664 Day: F(11,55)=0.8 p=0.664 GxD: F(10,55)=1.0 p=0.482 HR 2 h SFW 12.95 + 0.42b n=4 15.12 + 0.53a n=3 NA Group: F(1,66)=10.7 p=0.002 Day: F(11,66)=3.1 p=0.001 GxD: F(10,66)=2.3 p=0.023

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86 Table 3-4. Cumulative body weight change (M + SE g) across feed ing and dose groups SHU9119 Vehicle MTII No SFW 1.54+ 0.41a 0.45 + 0.33a,b 0.05 + 0.35b 24 h SFW 3.86 + 0.45a 2.44 + 0.53a 2.05 + 0.53a 8 h SFW 3.26 + 0.65a 1.98 + 0.42a 1.71 + 0.55a LR 2 h SFW 1.47 + 0.60a 1.07 + 0.59a 1.8 + 0.33a HR 2 h SFW 1.46 + 0.87a 0.56 + 0.49a 1.63 + 0.38a

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87 CHAPTER 4 EFFECT OF CANNABINOID CB1 RECEPTOR ANTAGONIST ON OVERCONSUMPTION IN FEMALE MICE Introduction Since the overconsumption SFW dessert protoc ol that we have used successfully in female rats (Mathes et al. 2008) provides animals with a choice between two diet types, we have suggested that this model may have benefits diffe rent from similar models that induce obesity by providing access to a single high fa t diet (Levin, 2005). We have also used this protocol in female rats to show that daily administrati on of CB1R antagonists decreased caloric intake specifically by reducing SFW intake (Mathes et al. 2008), supporting the hypot hesis that the CB system impinges on food intake by modulating the pa latability of foods. The previous chapter described experiments that demonstrated that th e protocol does not promote overconsumption in mice, but it does still provide a basis for the analys is of diet selection an d preference. We sought in the present study to explore th e effect of CB1R antagonism on diet selection in female mice to assess if CB1R antagonism would have a similar effect on a species that does not overconsume and to which SFW may not be as pa latable as it is to rats. The few studies that have examined the effect of repeated CB1R antagonist admini stration in mice provided with a high-fat diet designed to induce obesity showed a sustained de crease in caloric intake and body weight gain (Ravinet et al. 2003; Hidebrant et al. 2003); however, these were in mice given access to only one diet. Only one study that we know of has explored diet selection within a single group of mice (South et al. 2007). They reported that male mi ce injected with AM251 showed reduced preference for a nutritionally complete high fat di et over a nutritionally complete low fat diet, which were both provided ad libitum We hypothesize that the resu lts of our study with female C57Bl/6J mice will be similar to these and to our results seen in rats using a choice between a

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88 nutritionally-complete diet and time-limited access to SFW. Comparisons between this and our previous study in rats will allow assessment of species differences using this dessert protocol. Methods Animals and Housing Fe male C57Bl/J6 mice were maintained in standard polycarbonate tubs as described in Chapter 2. After a week of acclimation to the h ousing conditions and diet s, they were divided into four groups, to be described below. Experimental Design Four groups of 8 mice received 8 h noctur nal access (1130 1930 h) to SFW daily in addition to ad libitum (24 h) access to moist chow. Mice rece ived injections of either one dose of AM251 (1, 5, or 10 mg/kg IP) or vehicle (equal parts polyethylene glycol and saline, 2 ml/kg IP) daily 30 min prior to access to a beaker of SFW and a fresh beaker of moist chow. SFW and moist chow intakes were measured by subtracting the remaining weight of the diet from that originally presented. The body weight of each mouse was measured daily prior to AM251 injection. Total caloric intake, in dividual caloric intakes from chow and SFW, and body weight change from baseline were calculated daily for 21 days. Drugs AM251 was purchased f rom Tocris (Ellisville, MO). AM251 was suspended in polyethylene glycol (Sigma Chem ical Co., St. Louis, MO; molecular weight = 400), which was then mixed with an equal volume of saline. The drug precipitated slowly when saline was added, so was sonicated immediately prior to injection to provide a suitable suspension. This vehicle was used in our previous work with rats and shown to have little to no effect on food intake. Injections were given in volumes of 2 ml/kg since the syringes us ed had gradations accurate to

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89 0.02 ml. AM251 has been shown to possibly have inverse agonist properti es, but for simplicity it will be considered an antagonist here (Maclennan et al. 1998; Pertwee, 2005). Statistics The daily individual total calor ic intakes, as well as the com ponent intakes from moist chow and dessert were analyzed via two-way AN OVA with groups and days as main factors. When the analysis revealed a si gnificant (p<0.05) eff ect of days and/or a significant group x day interaction, the data were analyzed furthe r with one-way ANOVA followed by Tukey post hoc comparisons to examine daily differences betw een groups and within-gro up differences across days. The cumulative body weight changes between the first and last day of each experiment were analyzed with two-way ANOVA and significan t differences between or within groups were further analyzed with one-way ANOVA followed by Tukey post hoc comparisons of each day's average cumulative body weight change. Results The average daily total caloric intakes across the days of the experi ment are presented in panel A of Figure 4-1 (page 94). Two-way ANO VA revealed a significa nt effect of group (F[3,649]=44.5, p<0.001) and days (F[20,649]=3.7, p<0.001), but no group x day interaction (F[60,649]=0.6, p=0.992). One-way ANOVA and Tuke y post hoc analysis revealed mice injected with any dose of AM251 ate significantly fewer total calories than mice injected with vehicle. Mice injected with 5 and 10 mg/kg AM 251 ate fewer total calori es than mice injected with 1 mg/kg AM251. Mice ate more total calories on day 4 than on day 15, and so the effect of days seemed due to low intakes during the earl y days of the experiment. Thus, AM251 reduced total caloric intake, and there was some evidence of this being dose-dependent. The average daily caloric intakes from moist chow across the days of the experiment are presented in panel B of Figure 4-1 (page 94). Two-way ANOVA rev ealed a significant effect of

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90 group (F[3,650]=45.2, p<0.001) and day (F[ 20,650]=2.3, p=0.001), but no group x day interaction (F[60,650]=0.6, p=0.995). One-way ANO VA and Tukey post hoc analysis revealed mice injected with any dose of AM251 ate signi ficantly fewer calories from moist chow than mice injected with vehicle. Mice injected wi th 5 and 10 mg/kg AM251 ate fewer calories from moist chow than mice injected with 1 mg/kg AM251. Mice ate more calories from moist chow on days 1 and 2 than on day 15, and so the effect of days seemed due to low intakes during the early days of the experiment. Thus, AM251 redu ced caloric intake from moist chow, and there was some evidence of this being dose-dependent. The average daily caloric intakes from SFW across the days of the experiment are presented in panel C of Figure 4-1 (page 94). Two-way ANOVA rev ealed a significant effect of group (F[3,649]=7.8, p<0.001) and day (F[20,649]= 3.0, p<0.001), but no group x day interaction (F[60,649]=.7, p=0.923). One-way ANOVA and Tukey pos t hoc analysis revealed mice injected with 5 or 10 mg/kg AM251 ate si gnificantly fewer calories from SFW than mice injected with vehicle. Mice ate more calories from SFW on day 4 than on days 15, 19, and 20, and so the effect of days seemed due to low intakes during the early days of the experiment. Thus, the higher doses of AM251 reduced caloric intake from SFW. The cumulative average body weight changes fr om baseline across days are presented in Figure 4-2 (page 95). Two-way ANOVA revealed a significant effect of group (F[3,619]=142.6, p<0.001) and day (F[3,619]=7.5, p<0.001), and no group x day interaction (F[60,619]=0.5, p>0.9). One-way ANOVA and Tukey post hoc analysis revealed mice injected with any dose of AM251 weighed less than mice inject ed with vehicle. Mice inje cted with 5 or 10 mg/kg AM251 weighed less than mice injected with 1 mg/ kg AM251. Mice weighed less on day 2-4 than on days 15-20, and so the effect of days seem ed due to increasing body weight across the

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91 experiment. Thus, AM251 reduced body weight gain, and there was some evidence of this being dose-dependent. Discussion Studies in a wide array of species and usi ng m any techniques show that CB1R antagonists decrease caloric intake by selectively reduci ng consumption of palatable diets, while other studies show suppression of bland diets and between diets of varyi ng palatability. In the present study, mice injected with AM251 consumed fewer calo ries than mice injected with vehicle, but this decrease resulted from reductions in both moist chow and SFW consumption. This differs from a study in which AM251 reduced the total cal oric intakes of mice given a choice between ad libitum access to a nutritionally complete high fat diet and a nutritionally complete low fat diet by specifically reducing consum ption of the high fat diet (South et al. 2007). This may be due to the provision of a choice between one low calorie nutritionally comp lete diet and one high calorie dessert compared to access to two nutritiona lly complete diets of similar caloric densities or the time-limited availability of the dessert compared to ad libitum access. Also, we used female mice that had been fed only moist chow prior to AM251 administration compared to South's study in which male mice had been prov ided with diet choice for 20 days prior to injection with AM251, and so experience with high energy diets and an obese state may impact the results. This also differs from our results in which fe male rats on the SFW dessert protocol injected with CB1R antagonists consumed fe wer total calories than rats on the SFW protocol injected with vehicle, and this decrease was specific to the consumption of SFW. In fact, more differences between groups were seen in daily intakes from moist chow than from SFW. This may suggest that some of the inconsistency in the literature exploring the action of CB1R

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92 antagonists may be due to species differences, as well as protocol differences in di et provision or selection between experiments. This experiment replicates our findings from the previous chapter that female C57Bl/6J mice do not overconsume on the SFW dessert protocol. It seems that mice ar e able to accurately compensate for calories when choices are presented; it is unclear as to why this differs from rats. If, as some studies suggest, the CB system does pr imarily modulate the inta ke of palatable foods, especially in situations in wh ich there is a choice between commodities, the failure of AM251 to consistently reduce intake of SFW supports the id ea presented in the previous chapter than mice do not find SFW as appealing as rats. However, many other studi es suggest that the CB system affects satiety or the amount of work an anim al will perform to obtain a commodity. Other studies using brief access tests or methods that bypass taste via gastric catheters, as well as operant techniques in economic paradigms could be used to further explore these species differences.

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93 0 m g / k g 1 m g / k g 5 m g / k g 1 0 m g / k g Intake (kcal) 0 5 10 15 20 AM251 0 mg/kg1 mg/kg5 mg/kg10 mg/kg Intake (kcal) 0 5 10 15 AM215 0 mg/kg1 mg/kg5 mg/kg10 mg/kg Intake (kcal) 0 5 10 15 a b c c a ab b b a b c c Figure 4-1. Daily caloric intake (M + SE kcal ) of mice given 8 h access to SFW and injected with either vehicle or one of three doses of AM251 (1, 5, 10 mg/kg) daily for 15 days. Panel A) Total caloric intake. Panel B) Ca loric intake from moist chow. Panel C) Caloric intake from dessert. The letters a bove the bars denote a significant difference between groups: groups that have different letters above them are statistically different (p<0.05) and the letter 'a' represen ts the group consuming the most calories. Mice injected with any dose of AM251 cons umed significantly fewer total calories than vehicle controls by consuming signifi cantly fewer calories from SFW and moist chow. A B C

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94 AM251 0 mg/kg1 mg/kg5 mg/kg10 mg/kg Cumulative Body Weight Change (g) 0.0 0.5 1.0 1.5 2.0 a b c c Figure 4-2. Cumulative change in body weight (M + SE g) from baseline of rats on the dessert protocol and drug regimen described in the caption for Figure 4-1. Mice injected with any dose of AM251 gained significan tly less weight than vehicle controls.

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95 CHAPTER 5 GENERAL DISCUSSION Anim al and human studies using a variety of techniques have shown that organisms will eat when they have energy or nutrient needs, but also when they are in an environment that promotes eating, despite having excess energy stores Attributes of an obesigenic environment include an abundance and variety of foods that ar e easy to access, that are high in calories, and that are considered palatable. It makes sense in the scope of evolutiona ry history that animals that would eat whenever the opportunity presente d itself would have succeeded during times of famine. That the probability of engaging in f eeding behavior in times of abundance and that food would be considered reward ing would also make sense, a nd would recruit activity from parts of the brain that process re ward. However, this adaptive behavior is detrimental to the health of humans since the present food condition is one of ubiquitous availabi lity. In light of an epidemic of obesity, it is important for the scientific and medical community to identify behavioral and pharmacological solutions to prev ent and treat the metabolic syndrome. This begins with animal models that include attrib utes of the environment in which humans eat and develop obesity. In this dissertation, we attempted to generalize to mice a model that promotes overconsumption and binge eating in rats. In contra st to our results with rats, mice given daily 8 h or 24 h access to a sweet and fa tty dessert in conjunction with ad libitum access to moist chow did not eat more calories than mice given only moist chow; in fact, mice given SFW consumed slightly, but significantly less than mice given moist chow only. Mice given interrupted access to SFW initially ate more calories than mice given continuous access to SFW, but by 24 h, both groups of mice ate similarly and mice given 24 h access to SFW ate more calories from SFW than mice given 8 h access to SFW. Similarly, mice given 2 h access to SFW every other day

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96 did not consume more calories during those 2 h of SFW access than mice given 2 h access to SFW every day; in fact, mice given LR 2 h SF W ate more calories than those given HR 2 h SFW. It is unclear as to why mice behave differe ntly than rats in thes e dessert protocols when they behave similarly in other DIO protocols in which only one high fat diet is available. As discussed in the previous chapte rs, it is possible that mice do not find SFW as palatable or find moist chow more palatable than do rats; this is unlikely since both species avidly consume SFW and eat more calories (though not higher intakes by volume) from SFW than moist chow. Operant protocols using progressive ratios would be necessary to compare differences between the species in motivation for the two commodities. Another explanation is that SFW is more satiating in mice than in rats, perhaps due to differences in gastric emptying. This could be explored by analyzing stomach contents over tim e between species or by infusing SFW into the stomach via a gastric catheter a nd assessing the effect on caloric intake and diet selection. In this dissertation, we also assessed the effect of orexigenic MC4R antagonist SHU9119 and anorexigenic MC4R agonist MTII on caloric intake and diet selection in mice. SHU9119 consistently increased caloric intake regardle ss of any duration of access to SFW (though there was a possible ceiling effect during 2 h SFW access as discussed in chapter 4), and this increase seemed predominantly from moist chow intake. This supports our hypothesis that antagonism of the MC4R system may promote increases in feed ing focused toward nutritionally complete foods regardless of other foods available that may be relatively more pala table. However, this is not supported by our results with MTII. MTII reliably decreas ed caloric intake in the first 4 h after diet presentation and drug administration (alt hough by 24 h, it often incr eased caloric intake relative to controls, possibly due to compensation following decreasing effect of the drug), but this decrease seemed predominantly from SFW intake. However, the argument could be made

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97 that MTII decreased SFW intake so as to pres erve consumption of the nutritionally complete food, which endogenous agonist AgRP, or other co mpensatory systems (including CB) may have been promoting. It would be informative to a ssess where in the brain these drugs are exerting their most potent effect: for example, if S HU9119 is binding optimally to MC4R in the brainstem, it may be selectively influencing consumption of moist chow because the lower calorie commodity is the one which meal size w ould be the most malleable. On the other hand, if MTII administration results in activity in th e NuAc, that may explain its effect on SFW. Localized injections of the drugs or analysis of activity via mi crodialysis would be useful in addressing these questions. Activity of compensatory systems may also ex plain why differences were seen between the data from pharmacological models and genetic knockout models Since MC4RKO mice overconsume and seem less likely to binge on SFW, it would follow that mice injected with SHU9119 would behave similarly. This was not the case, though dose-response curves should be established and continuous dosing (ie, via a mini pump) should be explored before this is ruled out. It would also be informative to assess via autoradiography if other systems are upregulated in MC4RKO mice for example, if CB1R are expressed to a greater extent in MC4RKO mice compared to their WT littermates as well as assessing the interaction between genotype and pharmacological manipulations. We also assessed the effect of anorexigen ic CB1R antagonist AM251 on caloric intake and diet selection in mice using the SFW protocol th at promoted overconsumpti on in rats. In rats, CB1R antagonism resulted in a decrease in caloric intake compared to vehi cle injected controls, and this decrease was specific to SFW. This was not the case in mice, which did not overconsume on the protocol. AM251 did reduce cal oric intake in mice compared to vehicle

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98 injected controls and compared to baseline intake measures of only moist chow, but the reduction was due to decreases in moist chow intake more often than SFW intake. This suggests that either mice do not find SFW as palatable as rats, or that CB1R antagonism affects diet selection differently in mice than in rats. It would be important to assess the impact of CB1R agonism in both rats and mice to as sess if it is consistent with th e antagonist results. Again, it would also be interesting to assess the imp act of MC4R drugs on CB1RKO mice, especially since multidrug therapy for obesity is blossoming. These species differences beg the question as to the functionality and operational definition of the term palatability. Many reviews have de bated this definition, and the consensus is that palatability is a hedonic evalua tion of food under particular ci rcumstances (Yeomans, 1998; p 609). A related question is to whether drugs that arguably affect the evalua tion of palatability of foods such as opioids, benodiazepines, and cannabi noids do so by acting at forebrain areas, such as the NuAc, brain stem regions, or if they impi nge on sensory receptors so as to alter taste or odor detection or discrimination. This has not been systematically explored. The term palatability is often used incorrectly in a circul ar sense, in which sensor y factors of the food are said to increase food intake and thus increased food intake is said to result from the level of palatability of the food. However, this is no t always an accurate assessment: for example, animals given a choice between one bottle of a lo w concentration of sucrose and one bottle of a high concentration of sucrose wi ll consume a larger volume of the lower concentration of sucrose, suggesting that the lower concentration of sucrose is more palatable. However, brief access tests such as Davis rigs, which present sm all volumes of different concentrations of sucrose to animals and measure their licking av idity, and taste reactivity measures, in which fluids are passively infused in to the mouth and species-specifi c mouth movements are observed

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99 and quantified, suggest that higher concentrations of sucrose evoke greate r responsivity, as thus are more palatable. This rela tes to the studies in this diss ertation since, although mice ate a larger volume of moist chow compared to SFW, it could be argued that the SFW was still more palatable than moist chow because there was no n eed for the mice to ingest it and they ate more calories from SFW than moist chow. Which test type more accurately assesses palatability? Two-bottle preference tests or a ch oice between two diets as is pres ented in this dissertation are arguably more ecologically valid, but do not elim inate post-ingestive eff ects that have been shown to alter perception of palatability (ie, sens ory-specific satiety) as do Davis rigs or taste reactivity tests. With these considerations, is it functional or even po ssible to use a choice between complete diets to assess pharmaco logical impingement on palatability? Similarly, is it possible to dis tinguish between eating for need and eating for the rewarding properties inherent in it? Thes e two aspects of feeding are not mutually exclusive: when food deprived, animals will avidly consume less prefe rred (arguably, palatable) foods, and when in a state associated with reward, such as while se lf-administering electrical stimulation to the median forebrain bundle or following cocaine admi nistration, animals are less like to engage in feeding (note: it is debatable as to if drugs produce the same pattern of neuronal activation associated with reward as does food, but that is beyond the scope of this dissertation). The SFW dessert protocols were designed to mimic attributes of availability and choice inherent in human feeding situations in a non-re stricted state. Although the protocols promote overconsumption and binge eating in rats, it doe s not translate to mice, limiting it use for pharmacological and genetic explorations in this species. However, results from these studies should be combined with results from studies using other techniques, such as brief access tests, operant contingencies, and real-time assessment of neurochemical parameters, before conclusions are drawn.

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100 Finally, how does eating in the absence of need relate to addiction, and should it be considered a behavioral disorder or disease? Are overconsump tion and drug addiction separate or overlapping behavior sets with distinct or reciprocally asso ciated brain pathways? Although this is an important question for health care policy, it seems difficult to resolve until functional models are available and operational definitions are agreed upon.

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113 BIOGRAPHICAL SKETCH Clare Ma thes lived in the Detroit area until attending Western Michigan University in fall 1998. Upon graduating with honors in 2002, Clare work ed as a research associate in safety pharmacology at a research toxicology facility in Mattewan, MI. In 2003, Clare left MI to begin a PhD program in behavioral neuroscience expl oring food intake and drug addiction at the University of Florida under Dr. Neil Rowland. She received her MS in fall 2005 and continued for her PhD, which she will receive in the su mmer of 2008. From there, Clare will do her postdoctoral training in the fiel d of taste psychophysics at Flor ida State University under Dr. Alan Spector.