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2012 Glucagon-like 1 Receptors in the Nucleus Accumbens Affect Food Intake Amanda M. Dossat

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THE FLORIDA STATE UNIVERSITY

COLLEGE OF ARTS AND SCIENCES

GLUCAGON-LIKE PEPTIDE 1 RECEPTORS IN THE NUCLEUS ACCUMBENS AFFECT

FOOD INTAKE

By

AMANDA M. DOSSAT

A Thesis submitted to the College of Arts and Sciences in partial fulfillment of the requirements for the degree of Master of Science

Degree Awarded: Fall Semester, 2012

Amanda M. Dossat defended this thesis on August 30th, 2012. The members of the supervisory committee were:

Diana L. Williams Professor Directing Thesis

Lisa A. Eckel Committee Member

Pamela K. Keel Committee Member

The Graduate School has verified and approved the above-named committee members, and certifies that the thesis has been approved in accordance with university requirements.

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TABLE OF CONTENTS

List of Figures ...... v Abstract ...... vi 1. INTRODUCTION ...... 1

2. RETROGRADE TRACING AND IMMUNOHISTOCHEMICAL METHODS ...... 5 2.1 ANIMALS ...... 5 2.2 RETROGRADE TRACING ...... 5 2.3 INJECTIONS ...... 5 2.4 PERFUSIONS ...... 6 2.5 IMMUNOHISTOCHEMISTRY ...... 6 2.6 ANALYSIS ...... 7 3. FOOD INTAKE EXPERIMENTS ...... 8 3.1 CANNULATION ...... 8 3.2 LV ADMINISTRATION STUDIES ...... 8 3.3 NAC ADMINISTRATION STUDIES ...... 9

4. SPECIFICITY OF INTRA-NAC GLP-1 EFFECTS ON FEEDING BEHAVIOR ...... 10 4.1 CONDITIONED TASTE AVERSION ...... 10

5. INTRA-NAC GLP-1R STIMULATION ...... 11 5.1 GLP-1-INDUCED C-FOS ...... 11 5.2 STATISTICAL ANALYSIS ...... 11

6. RESULTS ...... 12 6.1 COLOCALIZATION OF RETROGRADE TRACER AND GLP-1 NEURONS ...... 12 6.2 LV GLP-1 AND EX9 EFFECTS ON FOOD INTAKE ...... 13 6.3 EFFECTS OF INTRA-NAC TREATMENT...... 13 6.4 CONDITIONED TASTE AVERSION ...... 15 6.5 LV C-FOS INDUCTION ...... 15

7. DISCUSSION ...... 17 APPENDIX ...... 22

A. IACUC APPROVAL OF PROTOCOL ...... 22

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REFERENCES ...... 25

BIOGRAPHICAL SKETCH ...... 30

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LIST OF FIGURES

6.1 Representative images of fluorescent immunostaining and retrograde tracing of the projection from NTS neurons to NAc ...... 12

6.2 Dose–response function for the food intake effects of LV-injected GLP-1 (A) and Ex9 (B)...... 13

6.3 A. NAc core, but not shell, injection of GLP-1 reduced food intake...... 14

6.4 Percentage of saccharin out of total saccharin plus water consumed in a 24 h test of CTA...... 15

6.5 A-D, Representative images of c-Fos immunoreactivity (dark nuclear staining) in the NAc of a rat that received intra-NAc core saline (A, core region; B, shell region) and a rat that received intra-NAc core GLP-1 (C, core region; D, shell region)...... 15

6.6 Diagram of representative NAc core and shell injection placements based on the atlas of Paxinos and Watson (2007)...... 16

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ABSTRACT

Central glucagon-like peptide 1 receptor (GLP-1R) stimulation suppresses food intake, and hindbrain GLP-1 neurons project to numerous feeding-relevant brain regions. One such region is the nucleus accumbens (NAc), which plays a role in reward and motivated behavior. Using immunohistochemical and retrograde tracing techniques in rats, we identified a robust projection from GLP-1 neurons in the nucleus of the solitary tract to the NAc.We hypothesized that activation of NAc GLP-1Rs suppresses feeding.When injected into the NAc core of rats at doses subthreshold for effect when administered to the lateral ventricle, GLP-1 significantly reduced food intake relative to vehicle at 1, 2, and 24 h posttreatment. The same doses had no effect\ when injected into the NAc shell. NAc core treatment with ventricle-subthreshold doses of the GLP-1R antagonist exendin (9 –39) caused significant hyperphagia at 2 h posttreatment, suggesting that endogenous stimulation of NAc core GLP-1Rs plays a role in limiting food intake. It has been suggested that GLP-1 can cause nausea, but we found that NAc core administration of GLP-1 did not cause a conditioned taste aversion to saccharin, suggesting that the anorexic effect of NAc core GLP-1 is not caused by malaise. Finally, we observed that NAc core injection of GLP-1 significantly increased c-Fos expression in the NAc core. We conclude that that GLP-1Rs in the NAc play a physiologic role in food intake control, and suggest that the GLP-1 projection to NAc core may link satiation signal processing in the hindbrain with forebrain processing of food reward.

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CHAPTER 1

INTRODUCTION

Obesity has become a widespread health problem in recent decades, and it has been suggested that overweight and obese individuals may be more sensitive to the rewarding aspects of food stimuli (Appelhans et al., 2011). A large body of research has focused on the physiologic mechanisms that contribute to maintenance of energy , a balance between energy intake and energy expenditure (Smith GP, 1996; Moran TH, 2009). However, it is clear that we eat for non-homeostatic reasons, as well (Lowe and Butryn, 2007). Humans and rats often consume preferred, palatable food in the absence of homeostatic need, and this is referred to as ―hedonic eating‖ (Berridge et al., 2010). Because overconsumption of this nature is thought to contribute to obesity and other related metabolic disorders (Swinburn et al., 2009), investigation of the neuroendocrine mechanisms for this behavior may provide useful insight into the pathophysiology of these disorders. Here, we examined brain glucagon-like peptide 1 (GLP-1) as a possible link between homeostatic and hedonic mechanisms for food intake control. GLP-1 and its receptors are expressed in the brain as well as the periphery, and appear to influence food intake through its actions at both sites. Within the periphery, GLP-1 is secreted by intestinal L-cells in response to nutrients in the gastrointestinal tract (Holst JJ, 2007). Centrally, GLP-1-producing neurons are restricted to a small region of the caudal nucleus of the solitary tract (NTS) (Larsen et al., 1997). These neurons receive vagal afferent input, so they are in the position to respond indirectly to vagally-mediated meal-related signals such as the satiation cholecystokinin (Wright et al., 2011) and gastric distention (Vrang et al., 2003). Because GLP-1 readily crosses the blood-brain barrier, there has been some question of whether the endogenous GLP-1 that binds to neuronal GLP-1 receptors (GLP-1R) derives primarily from GLP-1 neurons or from the circulation. We suggest that intestinal GLP-1 is not likely to reach brain receptors because of the very short half-life of the active form of GLP-1 in the blood (< 2 min) (Vilsbøll et al., 2003). Even pharmacologically administered peripheral GLP-1 appears to affect food intake exclusively through peripheral receptors, because centrally administered GLP- R antagonist fails to blunt the anorexic effect of peripheral GLP-1 injection. Thus, we focus here on NTS GLP-1 neurons as the sole relevant source of endogenous ligand for brain GLP-1R.

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GLP-1 neurons send projections to many nuclei throughout the CNS (Rinaman L, 2010) where GLP-1R are expressed (Merchenthaler et al., 1999). Numerous studies have shown that an injection of GLP-1 into the cerebral ventricles can reduce food intake (e.g., Turton et al., 1996; Asarian et al., 1998; Chelikani et al., 2005; Williams et al., 2009), and blockade of central GLP-1R not only increases food intake, but body weight as well (Barrera et al., 2011). Similarly, RNA interference-mediated knockdown of mRNA for preproglucagon (the precursor to GLP-1) in the NTS causes hyperphagia and weight gain (Barrera et al., 2011). Together, these loss-of-function studies lend support to the idea that neuronal GLP-1 plays a physiologic role in the maintenance of energy balance. GLP-1R are now used clinically in humans for treatment of type 2 , and in addition to their effects on glucose homeostasis, they also function to reduce appetite and body weight (Bradley et al., 2010). For these reasons, it is critical to determine which GLP-1R expressing neuronal populations mediate these effects. Experiments in which GLP-1R agonists and antagonists are administered intracerebroventricularly (icv) suggest that GLP-1R populations in the brain play a role in mediating GLP-1’s effects, but such studies do not allow for the identification of the specific nuclei that mediate this effect. Thus far, the paraventricular nucleus of the hypothalamus (PVN) (McMahon and Wellman, 1998) and the lateral hypothalamus (LH) (Schick et al., 2003) are the only identified sites at which direct injection of low doses of GLP-1 reduces food intake. Both the PVN and the LH have a high density of GLP-1 fibers and express GLP-1R (Tauchi et al., 2008; Merchenthaler et al., 1999). However, several other feeding-relevant brain regions also receive projections from GLP-1 neurons and contain GLP-1R, including the focus of the present study, the nucleus accumbens (NAc). In the rat, both the core and shell subregions of the NAc contain both GLP-1 fibers and receptors for GLP-1 (Merchenthaler et al., 1999; Rinaman, 2010). Previous studies have identified this structure as being involved in drug addiction and reward-motivated behavior, and it is established that manipulations of NAc can affect intake of palatable food (Berridge et al., 2010). Perhaps the most well studied example is the increase in high fat food intake elicited by intra-NAc injection of the μ-opioid receptor (D-Ala2, N-MePhe4, Gly-ol)-enkephalin (DAMGO). Stimulation of NAc opioid receptors increases intake of highly preferred foods, but has no effect on the consumption of regular chow (Will et al., 2003). NAc core injection of

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DAMGO also increases breakpoint for rats lever-pressing for sucrose pellets on a progressive ratio (PR) schedule (Zhang et al., 2003). These studies underline the critical role of the NAc in the control of ―hedonic eating‖. In the present studies, we focused on the NAc as a site of action for central GLP-1. We hypothesized that NAc GLP-1R activation reduces food intake, and that endogenous GLP-1 action in the NAc plays a physiologic role in the control of ingestion. First, we characterized the GLP-1 neuron projection to NAc using retrograde tracing and immunohistochemistry. Next, we determined whether pharmacologic stimulation of NAc core or shell GLP-1R reduces food intake. To determine whether endogenous GLP-1 affects food intake by acting on NAc GLP-1R, we examined the effect of intra-NAc GLP-1R antagonist. We also determined that intra-NAc GLP-1 induces expression of c-Fos, an immediate early gene that can be used as a marker of neuronal activity. With any drug treatment that reduces food intake, we must consider the possibility that it does so by producing general illness or gastrointestinal distress as opposed to a physiologic effect on satiation or satiety. This possibility is commonly investigated using the conditioned taste aversion (CTA) paradigm. CTA is a form of Pavlovian conditioning in which a flavor (the conditioned stimulus) is paired with a nausea-causing agent (the unconditioned stimulus), causing future aversion to that flavor. Typically, subjects are given access to a novel, palatable food (usually sucrose or saccharin solution) as the conditioned stimulus and are then injected with the drug treatment of interest. Following this pairing, preference vs. aversion for the palatable substance is assessed in a two-bottle preference test in which one bottle contains the conditioned stimulus while the other contains water. Based in part on such CTA studies, it has been suggested that central GLP-1 reduces food intake due to visceral malaise and not necessarily satiation or satiety. Thiele and colleagues (1997) first showed that 3RD icv delivery of GLP-1 supported a CTA. Moreover, GLP-1 neurons are activated by the nauseating stimuli lithium chloride (Thiele et al., 1998) and lipopolysaccharide (Rinaman L., 1999), and can mediate some of their effects on feeding (Grill et al., 2004). It seems that the anorexic and nausea-related effects of GLP-1 can be dissociated, because intra-amygdalar GLP-1 injection caused CTA without anorexia, whereas 4th-ventricular GLP-1 injection caused only anorexia and no CTA (Kinzig et al., 2002). Given this previous research, any newly identified site of GLP-1 action must be evaluated for the possibility that it plays a role in viscerosensory stress. To

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address this issue, we conducted a CTA study for GLP-1 delivered to the NAc.

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CHAPTER 2

RETROGRADE TRACING AND IMMUNOHISTOCHEMICAL METHODS

This section discusses the retrograde tracing and immunohistochemical methods used to examine the presence and extent of the GLP-1 projection to the NAc.

2.1 Animals

Male Wistar rats were obtained from Charles River Laboratories (Wilmington, MA), with a mean body weight 325 g at the start of experiments. Rats were housed individually in a temperature-controlled vivarium on a 12 h light: 12 h dark cycle in standard plastic cages with food hoppers. Distilled water (DiH2O) and rat chow (Purina, St. Louis, MO) was available ad libitum except where otherwise noted. Rats were handled daily and habituated to experimental procedures before the onset of experimentation. All experimental procedures were approved by the Florida State University Institutional Animal Care and Use Committee and conform to the standards of the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996).

2.2 Retrograde tracing

We used retrograde neuronal tracing and double-label immunohistochemistry in order to identify NAc-projecting GLP-1 neurons.

2. 3 Injections

Animals received unilateral 0.5 μl microinjections of retrograde tracer, either 4% Fluorogold (Fluorochrome, Denver, CO) (n=3) or Red RetroBeads (Lumaflor, Durham, NC) (n=3) into the NAc core under 2-4% isolflurane in 1 liter oxygen/minute inhaled continuously throughout the procedure. Fluorogold is a fluorescent retrograde tracer that is taken up by axon terminals located in and around the injection site and is retrogradely transported to the cell bodies of those projection neurons. Fluorogold was chosen because it is a reliable and commonly used tracer, however, it can be picked up by fibers of passage in the area of the site of injection, and may therefore cause over-estimation of projection neurons. Red RetroBeads are rhodamine-coated latex microspheres that also act as a retrograde tracer, but are resistant to photobleaching and are

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taken up only at synaptic terminals. Because the NAc is quite long in the rostro-caudal dimension, each rat was injected in two rostro-caudal locations with a 30G Hamilton microsyringe (VWR, Radnor, PA) and pump (World Precision Instruments, Sarasota, FL) at a rate of 100 nl/min for Fluorogold and 300 nl/min for RetroBeads. The different rates are due to the fact that RetroBeads are more viscous and prone to clogging at slower rates of delivery. Stereotaxic coordinates for the first injection are 1.5 mm lateral to midline, 1.2 or 1.5 mm anterior to bregma and 7.4 mm ventral to the skull surface. The second injection was made at 2.0 mm anterior to bregma at the same lateral and ventral coordinates. Carprofen (5 mg/kg) (Butler Schein Animal Health Supply, Columbus, OH) was administered subcutaneously prior to surgery. Body weight was monitored daily during recovery.

2.4 Perfusions

Rats were anesthetized (180 mg/kg ketamine and 30 mg/kg xylazine, intraperitoneally administered) and transcardially perfused with 10 mM phosphate buffered saline followed by 4% paraformaldehyde, at 2 (for RetroBead-injected rats) or 5 (for Fluorogold-injected rats) days post-injection. The difference in incubation time is to allow for complete pick up, transport, and filling of distal process by each tracer. Brains were removed, sunk in 15% followed by 30% sucrose, and subsequently frozen in isopentane on dry ice. Coronal cryostat sections (20 μm) through the NAc were collected, slide-mounted for injection placement verification and stored at -80°C. Coronal microtome sections (40 μm) through the NTS were collected on a freezing stage microtome and placed in 0.02 M tris-buffered saline with 0.1% sodium azide and stored at 4°C.

2.5 Immunohistochemistry

At the onset of immunohistochemical procedures, sections were removed from storage solution, rinsed in PBS, treated for 1 min in 1% sodium borohydride (Sigma Aldrich), and rinsed again in PBS. Antibodies were diluted in 0.3% Triton-X, 1% bovine serum albumin (BSA), and 1% normal donkey serum in 10 mM PBS. We used a rabbit anti-GLP-1 primary antibody (Bachem, Torrance, CA), diluted at 1:5000 and the secondary for RetroBead-containing sections is donkey anti-rabbit alexa-488 (Invitrogen, Carlsbad, CA) diluted 1:1000. Fluorogold-containing sections were processed with the secondary antibody, donkey anti-rabbit Cy3 (Jackson Immunoresearch, West Grove, PA) at a 1:500 concentration. RetroBead-containing sections were slide-mounted

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and cover slipped with Fluoro-Gel (Electron Microscopy Sciences, Hatfield, PA). Fluorogold- containing sections were double-labeled for Fluorogold for enhanced visual detection. Because both primary antibodies were rabbit, these sections were incubated in 10% normal rabbit serum in 3% Triton-X and 1% BSA in PBS for 2 h followed by overnight incubation with 1:100 donkey anti-rabbit IgG Fab fragment (Jackson Immunoresearch, West Grove, PA) before incubation with the secondary antibody rabbit anti-Fluorogold (Millipore, Billerica, MA) used at a concentration of 1:3000. The donkey anti-rabbit alexa-488 secondary was diluted 1:1000 (Invitrogen, Carlsbad, CA), and these sections were slide-mounted and cover slipped with Aqua Polymount (Polysciences, Warrington, PA). Control sections were incubated without primary or secondary antibodies and showed no staining. Control sections from Fluorogold-injected rats were stained for GLP-1 and subjected to all subsequent steps noted above, except for incubation with the anti-Fluorogold primary antibody.

2.6 Analysis

Tracer injection placement was verified by visual examination of the sections collected through the NAc. From each rat, a series of 16 alternating sections through the caudal NTS, approximately 14.16 mm through 15.24 mm posterior to bregma (Paxinos and Watson, 2007) were examined. Sections were viewed with an Olympus BX41 fluorescence microscope and monochromatic digital images acquired with a Retiga EXI Aqua camera and Q-Capture software (Hunt Optics, Pittsburgh, PA). We used Adobe Photoshop CS4 (Adobe, San Jose, CA) to adjust contrast, add color and merge images of tracer and GLP-1 immunoreactivity. GLP-1-labeled cells, RetroBead-containing cells, and cells containing both were counted by eye. Fluorogold- containing sections were not counted because of the potential for that tracer to label cells that do not actually have synaptic terminals at the injection site. We believe quantification based on RetroBeads provided a more conservative estimate of the number of NAc-projecting GLP-1 neurons.

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CHAPTER 3

FOOD INTAKE EXPERIMENTS

This section discusses the methods used to examine the behavioral consequences following GLP- 1 and Exendin (9-39) delivery into the NAc core and shell subregions.

Cerebrospinal fluid (CSF) in the cerebral ventricles flows constantly in a rostro-caudal direction. The NAc is located in close proximity to the rostral lateral ventricle (LV), so any drug injected into the NAc could potentially diffuse into the LV and ultimately stimulate receptors in other brain locations. To control for this possibility, we used doses of GLP-1 and the GLP-1R antagonist Exendin (9-39) (Ex9) that are subthreshold for effect when delivered into the LV. This increased our confidence that any effect we observe following intra-NAc delivery is due to local action within the NAc and not possibly the result of diffusion of the drug to other areas accessible by the LV. Therefore, our first experiments were dose-response functions for GLP-1 and Ex9 delivered to the LV.

3.1 Cannulation

Rats were implanted with a 26G guide cannula (Plastics One, Roanoke, VA) targeting the lateral ventricle (LV), NAc core or shell. Coordinates for the LV cannulation are: 1.5 mm lateral to midline, 0.9 mm posterior to bregma, and 2.7 mm ventral to skull surface. NAc core coordinates are: 1.5 mm lateral to midline, 1.2 mm anterior to bregma, and 4.9 mm ventral to skull surface. NAc shell coordinates are: 0.8 mm lateral to midline, 1.5 mm anterior to bregma, and 5.9 mm ventral to skull surface. Injectors (30G; Plastics One) extended 2.0 mm (LV) or 2.5 mm (NAc) below the end of the guide cannula. LV cannulation was verified through observation of the water drinking response induced by Angiotensin II (Sigma-Aldrich, St. Louis, MO), no rat failed to drink at least 5 ml of DH20 45 minutes following injection of 4 ng/2 μl and no animals were excluded from the study (Bryant et al., 1980). NAc placements were verified histologically at the conclusion of behavioral experiments. (Figure 5)

3.2 LV administration studies

Within-subjects counterbalanced designs (n=10) were used to determine the dose-response for

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the food intake effects of LV-injected GLP-1 and Ex9 (American , Sunnyvale, CA). On experiment days, food was removed 2 h before the onset of the dark cycle. One h prior to dark, rats received an LV injection of saline, 0.033, 0.15, 0.3, or 3 μg in 2 μl 0.9% sterile saline. We returned pre-weighed chow immediately before dark onset and intake was measured 1, 2, and 24 h later. Body weight was also recorded daily, with injections separated by at least 48 h. We examined feeding responses to saline, 2.5, 5, 10, or 20 μg Ex9 in 2 μl 0.9% sterile saline using the same protocol as above with the same animals.

3.3 NAc administration studies

After completion of the LV dose-response studies delivered subthreshold doses of GLP-1 and Ex9 directly into the NAc. Injections and food intake measurements were performed as described above, except injection volume was reduced to 0.5 μl for intraparenchymal infusions.

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CHAPTER 4

SPECIFICITY OF INTRA-NAC GLP-1 EFFECTS ON FEEDING BEHAVIOR

4.1 Conditioned taste aversion

Rats were placed on a water-restriction schedule under which they received daily water access in a single drinking session. An empty bottle was presented simultaneously with the water bottle to acclimate animals to a 2-bottle choice; with bottle position switched each day to eliminate any potential location preference. The initial drinking session was 3 h long and the session times reduced each day so that rats were conditioned to receiving water access in a 20 min session. After stable intakes were achieved, there was a conditioning day. On the conditioning day rats were treated with intra-NAc saline, a dose of GLP-1 that reduces intake in our behavioral studies, or intraperitoneal injection of 1 ml of 0.6 M Lithium Chloride (LiCl) (n=5/group) paired with 0.2% saccharin. Rats were given ad lib access to a 0.2% saccharin solution and water on the day following conditioning; intakes were measured at 30 m, 1 h, and 24 h after the two bottles are made available.

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CHAPTER 5

INTRA-NAC GLP-1R STIMULATION

5.1 GLP-1-induced c-Fos

GLP-1R are excitatory G-protein coupled receptors; stimulation of these receptors results in an increase in neuronal activity (Gromada et al., 1997). To determine whether GLP-1R stimulation in NAcC activates neurons in that nucleus, we used the NAc core-cannulated rats to examine intra-NAc GLP-1-induced c-Fos. As in the food intake experiments, food was removed 2 h prior to treatment and rats divided into 2 groups: intra-NAc core injection of 0.9% sterile saline (n=5) or a LV-subthreshold dose of GLP-1 (n=7). Ninety minutes post-injection, rats were anesthetized and transcardially perfused as described above. Coronal cryostat sections (20 μm) were collected, slide-mounted and stained for c-Fos, using anatomically matched sections through the NAc core. We used a rabbit anti-c-Fos primary (EMD Biosciences, Gibbstown, NJ) diluted 1:5000 in 0.1% BSA in 10 mM PBS, and the secondary antibody donkey anti-rabbit alexa-488 (Invitrogen, Carlsbad, CA) used at a concentration of 1:200 in 0.1% BSA in 10 mM PBS. Photographs were taken as described above and Image J (NIH, Bethesda, MD) used to count c-Fos-like immunoreactivity bilaterally on 4-5 sections through the NAc surrounding the site of injection. Sections through the center of the injection were excluded so that tissue damage-related artifact was not mistakenly counted as c-Fos.

5.2 Statistical analysis

Effects were evaluated by Student’s t-tests or within- or between subjects one-way ANOVA where appropriate. Post hoc comparisons were made with Tukey’s Honestly Significant Difference test. P-values of <0.05 are considered significant.

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CHAPTER 6

RESULTS

6.1 Colocalization of retrograde tracer and GLP-1 neurons

Neurons positive for GLP-1 were observed throughout the caudal NTS with the highest density near the level of the obex. Few were observed at the caudal-most level of the area postrema and none were observed anterior to that point. Retrograde tracer was found throughout the caudal NTS and colocalized with GLP-1in many cells (Fig. 1). We identified 251 ± 2.45 GLP-1 neurons and 180 ± 23.79 RetroBead-containing neurons. Among these, 65.67 ± 8.04 were both GLP-1- and RetroBead-positive. Thus, 26.21 ± 3.4% of NTS GLP-1 neurons were retrogradely labeled. Among all retrogradely labeled caudal NTS neurons, 37.8 ± 8.1% were GLP-1 positive.

Figure 6.1: Representative images of fluorescent immunostaining and retrograde tracing of the projection from NTS neurons to NAc. A, NAc image showing the location and minimal spread of RetroBead injection (ac, anterior commissure). B, C, A low-magnification image of the caudal NTS (B) and a corresponding diagram (C) based on Paxinos and Watson (2007) are provided for orientation (Cu, cuneate nucleus; Gr, gracile nucleus; st, solitary tract; 10N, dorsal motor nucleus of the vagus; c, central canal). Higher-magnification images are taken from the area inside the white box in B. B, D, F, G, I, GLP-1 neurons (green cytoplasmic fluorescence) are visible in the NTS. B, E, F, H, I, Magenta cytoplasmic fluorescence identifies neurons that are retrogradely labeled by RetroBeads (B, E, F ) or Fluorogold (H, I ). B, F, I, Colocalization of GLP-1 and tracer is shown by merging images of GLP-1 and tracer. Arrows indicate several double-labeled cells. (Note: asterisks represent statistical significance compared to vehicle control).

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6.2 LV GLP-1 and Ex9 effects on food intake

LV injection of 1 and 3 g of GLP-1 reduced food intake relative to saline at 1 and 2 h post-dark onset (p<0.01), while lower doses did not differ from saline (Fig. 2A, 1 h data not shown). There were no significant effects on 24 h intake or body weight. LV treatment with 20 g of Ex9 increased food intake at 1 and 2 h post-dark onset (p<0.01), with no effect of lower doses (Fig. 2B; 1 h data not shown). At 24 h, 20 g of Ex9 was still effective (saline, 24.61 ± 1.33 g; 20 g of Ex9, 29.4 ± 1.11; p<0.01).

Figure 6.2: Dose–response function for the food intake effects of LV-injected GLP-1 (A) and Ex9 (B). (Note: asterisks represent statistical significance compared to vehicle control).

6.3 Effects of intra-NAc treatment

Intra-NAc core injection of 0.025 and 0.1 g of GLP-1 reduced food intake relative to saline at 1 and 2 h post-dark onset (p<0.05)(Fig. 3A; 1 h data not shown). A significant effect was observed at 24 h (saline, 25.1 ± 0.9 g; 0.025 g, 23.0 ± 1.0 g; 0.1 g, 23.3 ± 0.8 g; p<0.05 for each dose vs. saline). Body weight was not affected. NAc shell injection of the same doses had no effect on food intake or body weight at any point measured (Fig. 3A). As shown in Figure 3B, NAc core injection of 3 g Ex9 (p<0.001) effectively increased food intake at 2 h post-treatment.

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There was a main effect of Ex9 at 24 h (F(3-18) = 3.54, p<0.05) with a trend toward increased intake after 3 g of Ex9 (saline, 26.1 ± 1.37 g; 3 g, 29.06 ± 1.17; p=0.08), but no effect on body weight.

Figure 6.3: A. NAc core, but not shell, injection of GLP-1 reduced food intake. B. NAc core Ex9 reduced 2-h chow intake. (Note: asterisks represent statistical significance compared to vehicle control).

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6.4 Conditioned taste aversion

Rats preferred saccharin after it was paired with intra-NAc core saline injections, and rats in the NAc core 0.1 g of GLP-1 group were not significantly different (Fig. 4). Rats in the control group given LiCl displayed an aversion to saccharin (LiCl vs. saline and GLP-1; p’s<0.001).

Figure 6.4: Percentage of saccharin out of total saccharin plus water consumed in a 24 h test of CTA. Rats that previously experienced saccharin paired with intra-NAc core saline or GLP-1 strongly preferred to drink saccharin over water, but rats that received a saccharin-LiCl pairing showed aversion to saccharin. *p<0.05 relative to saline.

6.5 c-Fos induction

Intra-NAc core injection of 0.1 g of GLP-1 substantially increased the number of c-Fos-positive nuclei in the NAc core region (mean number of c-Fos-positive cells per section, counted bilaterally: saline, 112.2 ± 32.7; GLP-1, 544.5 ± 37.6; p<0.001; Fig. 5). Although the injections were unilateral, c-Fos expression was similar on the injected and non-injected sides. The boundary between core and shell is difficult to delineate without counterstaining, but few shells in the shell region contained c-Fos immunoreactivity.

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Figure 6.5: A-D, Representative images of c-Fos immunoreactivity (dark nuclear staining) in the NAc of a rat that received intra-NAc core saline (A, core region; B, shell region) and a rat that received intra-NAc core GLP-1 (C, core region; D, shell region). ac, Anterior commissure.

Figure 6.6: Diagram of representative NAc core and shell injection placements based on the atlas of Paxinos and Watson (2007). Additional rats’ injections were identified in similar locations at points between the anterior-posterior levels shown here.

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CHAPTER 7

DISCUSSION

Our results support the hypothesis that the GLP-1 neuronal projection to the NAc plays a physiologic role in the control of food intake. Retrograde tracer delivered into the medial NAc core identified a strong projection from GLP-1-producing neurons of the hindbrain to this forebrain structure. GLP-1 injection directly into the NAc core suppressed intake at doses that were subthreshold for effect when delivered to the LV. Our findings suggest that the core is more relevant for GLP-1 action than the shell, because intra-NAc shell injection of the same doses of GLP-1 had no effect. We observed significant c-Fos induction by intra-NAc core GLP-1 as well, adding confidence to the conclusion that NAc core neurons are GLP-1-responsive. These effects demonstrate that feeding responses can be driven by pharmacologic GLP-1R activation in NAc core, but do not necessarily mean that endogenous GLP-1 release in NAc has any impact on feeding. Our demonstration that intra-NAc core Ex9 treatment increased food intake addresses this issue and supports the conclusion that neuronal GLP-1 release at this site plays a role in limiting food consumption. Central GLP-1Rs are diverse in function and have been implicated in the response to visceral illness in several studies. For example, LV or 3rd-icv injection of GLP-1 supports a CTA (Thiele et al., 1997; Kinzig et al., 2002), and blockade of GLP-1R impairs LiCl-induced anorexia and formation of CTA (Seeley et al., 2000). Therefore, it is important to consider the possibility that the effects of intra-NAc GLP-1 observed here are related to illness responses and not to the control of food intake under normal physiologic circumstances. The fact that NAc core Ex9 treatment increased food intake goes some way to address this issue. None of the manipulations in this experiment are known to cause malaise, so it seems unlikely that this hyperphagic response could be due to relief from visceral illness. Rather, we suggest that this Ex9-induced hyperphagia reveals a role of endogenous GLP-1 in the physiologic control of food intake. To further address this issue, we asked whether intra-NAc core GLP-1, at a dose that significantly reduced food intake, could also produce a CTA. Rats treated with LiCl showed a robust CTA, but intra-NAc core GLP-1 produced no aversion. Together, our data support the suggestion that the anorexia we observed after NAc GLP-1 treatment is not mediated by illness or viscerosensory distress.

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There is now evidence that GLP-1R in several brain regions play a physiologic role in food intake regulation, but some GLP-1R populations in brain areas that are known to affect feeding seem to serve other functions instead. For example, previous studies have identified the paraventricular nucleus as a site in which GLP-1 affects food intake, but has no effect on glucose homeostasis and fails to produce a CTA (McMahon and Wellman, 1998). Alternatively, GLP- 1R stimulation within the arcuate nucleus has been shown to regulate glucose homeostasis while having no effect on feeding (Sandoval et al., 2008). Furthermore, stimulation of GLP1-R within the central nucleus of the amygdala produces a CTA while having no effect on food intake (Kinzig et al., 2002). The present study adds the NAc core as a second site for GLP-1’s food intake effects without induction of a CTA. Further support for the diversity of GLP-1R function comes from our finding that NAc shell injections of GLP-1 failed to produce changes in chow intake, despite previous findings that showed this region plays a role in certain kinds of feeding behavior. A recent report from Alhadeff and colleagues (2012) supports the idea that NAc core GLP-1R contribute to food intake control. In contrast to our findings, they found that stimulation of NAc shell GLP-1R with Exendin-4 significantly reduced food intake, however the magnitude of the effect was smaller than that observed following intra-NAc core injections. It remains to be seen what other effects may be obtained following NAc shell GLP-1R stimulation. What aspect of feeding behavior does this NTS to NAc pathway influence? One possibility is that this NAc GLP-1R population receives meal-related information, and we could assess this possibility using a few approaches. We can determine whether this GLP-1 projection is activated by meal-related signals through the use of immunohistochemistry in combination with gut stimulation. Following nutrient delivery to the stomach, we can identify cells that are activated by this ―stimulation‖ (cells that are c-Fos-positive). Additionally, we can identify the NAc-projecting population of GLP-1 neurons by delivering retrograde tracer into the NAc. Finally, to identify the GLP-1 neurons that project to the NAc and are activated by meal-related signals we can look for cells that: 1) produce GLP-1 (are GLP-1-positive), 2) send projections to the NAc core (are retrograde tracer positive), and 3) are activated in response to nutrient stimulation of the gut (are c-Fos-positive). This allowed us to determine if this GLP-1 projection is activated by meal-related signals. A powerful method to demonstrate that a particular pathway (or neurotransmitter) is involved in satiation (signals generated during a meal that lead to meal termination) or satiety

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(post-meal period when animals are not interested in consuming food) is to examine whether the can block nutrient-induced satiation and/or satiety. This can be resolved through the use of meal pattern analysis. If NAc GLP-1R blockade increases meal size and/or meal frequency, this will provide the first evidence that this projection is involved in satiation and/or satiety, respectively. It is well established that manipulations of NAc neurons can affect palatability and motivation to obtain food, and it is possible that GLP-1R activity within this nucleus could affect food intake through either of these mechanisms. In order to examine the role of this receptor population in modulating palatability, we could conduct a taste reactivity test. In this test, a taste stimulus (such as sucrose solution) is delivered directly into the oral cavity. Examination of stereotypical orofacial responses (such as rhythmic mouth movements, rhythmic tongue protrusions, and lateral tongue movements) produced following intra-oral delivery of a taste stimulus is a sensitive measure of the rat’s evaluation of the taste stimulus in the absence of post- ingestive feedback (only 50 μl sample provided) (Grill and Norgren, 1978). Grill and Norgren (1978) demonstrated that different tastants (such as: hydrogen chloride, sodium chloride, quinine, and sucrose) produced distinct pattern and duration of these orofacial responses. Furthermore, as the concentration of the tastants increased they observed significant increases in the duration of the orofacial response. Pharmacologic stimulation of NAc GLP-1R immediately prior to intra-oral sucrose delivery will allow us to examine orofacial responses to sucrose and identify whether this receptor population contributes to palatability. Following intra-NAc GLP- 1R stimulation, we hypothesize that rats will perceive the taste stimulus as less palatable. In order to assess GLP-1R activity-induced changes in motivation we could examine the effects of GLP-1 on PR responding. PR responding is a model often used to determine rats’ motivation to obtain drug or food reinforcers (Richardson & Roberts, 1996; Zhang et al., 2003; Finger et al., 2010). In this paradigm, a rat must perform increasing numbers of operant responses for each successive reinforcer. Dickson and colleagues (2012) recently examined the effect of intra-NAc Exendin 4 (potent GLP-1R agonist) treatment on PR responding for sucrose. They found this treatment suppressed PR responding, indicating that pharmacologic GLP-1R activation can suppress food-motivated behavior. It is possible that GLP-1R activation within NAc both promotes satiety and reduces food reward, which could be a two-pronged effect to control food intake.

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For several decades, research has focused on NAc influence on motivated behavior with most NAc food intake research examining dopaminergic and -opioid activity. There are many neurotransmitters that act within NAc but these are the two most studied and well understood, and it is possible that GLP-1 interacts with either or both of these neurotransmitter systems. Ingestion of palatable food stimulates (DA) inputs (Kenny PJ, 2011) and DA activity within the NAc promotes eating, particularly motivation for food (Zhang et al., 2003). Brown and colleagues (2011) observed an increase in DA release within the NAc core following an unpredicted food reward, and this change was not observed in the NAc shell. Because GLP-1R stimulation within the NAc reduces food intake and motivation for food (Dickson et al., 2012), it is possible that these two neurotransmitter systems interact within this nucleus to contribute to food intake control. Perhaps DA and GLP-1 have opposing effects on motivation, with DA influencing motivation earlier in the meal and GLP-1R working to suppress motivation by reducing DA input later in the meal. One way to test this idea is to measure DA release utilizing in vivo microdialysis. If GLP-1 suppresses dopaminergic transmission, then we would expect that delivery of GLP-1 to the NAc will reduce DA release. To further examine this interaction, we can deliver a DA receptor agonist into and examine whether this can blunt the reduction in DA release produced by GLP-1R stimulation. Such experiments allow us to examine the contribution and the extent of interaction between the DA and GLP-1 neurotransmitter systems underlying motivated behavior. In addition to its interaction with DA, GLP-1 may interact with the opioid system to control food intake. Intra-NAc μ-opioid receptor (MOR) stimulation via delivery of the μ-opioid receptor agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) increases food consumption (Zhang et al., 1998) and augments palatability of food stimuli (Kelley et al., 2002). MORs are presynaptic inhibitory receptors that can reduce synaptic transmission to neurons in the NAc (Hakan and Henriksen, 1987). GLP-1R are excitatory and may work in opposition to MORs within the NAc to promote excitation of the neurons. If this is the case, stimulation of GLP-1R within the NAc may blunt the behavioral effects of MOR stimulation. We have some preliminary evidence of such an interaction, with activation of GLP-1R in combination with MOR blockade producing reductions in food intake beyond that which is observed when the same dose of either compound is delivered in isolation. Ongoing studies are examining the degree of interaction between these neurotransmitter systems in the control of palatable food intake.

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To determine whether NAcC GLP-1R stimulation activates neurons locally, we used NAc core-cannulated rats to examine intra-NAc GLP-1-induced c-Fos. Following the delivery of a food intake-reducing dose of GLP-1, we observed a significant induction of c-Fos within the NAcC. Because 95% of NAcC neurons are GABAergic, it is most likely that GLP-1R stimulation activates a significant number of GABA neurons. In order to develop a detailed understanding of how NAc GLP-1R stimulation reduces food intake, further study is needed to determine what feeding-relevant areas may be targeted by GLP-1 activated GABAergic efferent projections. One candidate nucleus that may receive GABAergic signals following NAc GLP-1R stimulation is the lateral hypothalamus (Heimer et al, 1991; Stratford and Kelley, 1999). Support for anatomical connectivity between the NAc core and the rostral lateral hypothalamus, an area that is critical for promoting food intake, comes from a tracing study published by Thompson and Swanson (2010). Furthermore, Maldonado and colleagues (1995) demonstrated functional connectivity between the medial NAc and the LH, showing that inactivation of the tonic inhibitory input from the NAc to LH reduced food intake. Because these GABAergic neurons may contain GLP-1R, the activity produced by these neurons may impinge on the LH ultimately mediating NAc GLP-1R effects on food intake. Research on the role of the NAc in food intake control has focused in particular on hedonic intake—ingestion in the absence of homeostatic need, due to factors such as or reward obtained by eating. Although the NAc has been implicated in hedonic eating (Kelley et al., 2005), it has not been considered to play a major role in the homeostatic control of food intake. Conversely, GLP-1 has been considered a player in homeostatic feeding, with mediatory roles in meal-related satiety (Grill and Hayes, 2009) and the central effects of the adiposity hormone leptin (Goldstone et al., 1997). NTS GLP-1 neurons receive direct vagal afferent input (Hisadome et al., 2010) and are activated by gastric distention (Vrang et al., 2003) and ingestion of palatable, energy-dense food (Gaykema et al., 2009). Based on the data presented here, we hypothesize that meal-related signals from the gastrointestinal tract promote the release of GLP-1 in the NAc, where stimulation of GLP-1R act to reduce food intake. This GLP-1 projection to NAc may be the first identified behaviorally relevant connection between hindbrain satiety- processing neurons and a rostral forebrain region involved in reward.

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APPENDIX A

IACUC APPROVAL OF PROTOCOL

1.

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23

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BIOGRAPHICAL SKETCH

AMANDA MARIE DOSSAT Florida State University Department of

EDUCATION

Ph.D., anticipated 2014 (entered program in 2009) Program in Neuroscience, Williams Laboratory Florida State University

B.A., 2008, Cum Laude Psychology Florida State University

PROFESSIONAL EXPERIENCE

May 2008- October 2008 Laboratory Technician University of Florida, Gainesville, Florida Principal Investigator: Dr. David Smith

October 2008- July 2009 Laboratory Technician University of Florida, Gainesville, Florida Principal Investigator: Dr. Colleen Garbe Le Prell

AWARDS AND HONORS

2012 Leslie N. Wilson-Delores Auzenne Assistantship, Department of Psychology, Florida State University

2012 New Investigator Travel Award, Society for the Study of Ingestive Behavior;

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Travel Award based on abstract submission to annual meeting

2012 Jane West Award; For the ―best first author publication‖ by a graduate student in FSU Program in Neuroscience

2011 Gerard P. Smith Award, Society for the Study of Ingestive Behavior; Awarded for best oral presentation by a graduate student at the SSIB annual meeting

PEER-REVIEWED ORIGINAL RESEARCH PUBLICATIONS

1. Dossat AM, Lilly N, Kay K, Williams DL. Glucagon-like peptide 1 receptors in nucleus accumbens affect food intake. J Neurosci,31(41):14453-7, 2011.

2. Parise EM, Lilly N, Kay K, Dossat AM, Seth R, Overton JM, Williams DL. Evidence for the role of hindbrain orexin-1 receptors in the control of meal size. Am J Physiol Regul Integr Comp Physiol, 301(6):R1692-9, 2011.

3. Williams DL, Hyvarinen N, Lilly N, Kay K, Dossat A, Parise E, Torregrossa AM. Maintenance on a high-fat diet impairs the anorexic response to glucagon-like-peptide-1 receptor activation. Physiol Behav, 103(5):557-64, 2011.

ABSTRACTS

1. Dossat AM, Williams DL. Nucleus Accumbens GLP-1 receptors contribute to nutrient- induced satiety. Society for the Study of Ingestive Behavior, 21st Annual Meeting, 2012.

2. Williams DL, Dossat AM. Nucleus Accumbens Glucagon-Like Peptide-1 Receptor Activation Suppresses Food Intake. Annual Meeting, Orlando, FL: The Obesity Society, 2011.

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4. Dossat AM, Williams DL. Nucleus accumbens glucagon-like peptide 1 receptor blockade dampens gastric preload stimulation. Society for the Study of Ingestive Behavior, 20th Annual Meeting, 2012.

5. Dossat AM, Williams DL. Nucleus accumbens glucagon-like peptide 1 receptor activation suppresses food intake. Society for the Study of Ingestive Behavior, 19th Annual Meeting, 2011.

6. Dossat AM, Houpt TA, Williams DL. Leptin pre-treatment does not enhance a cholecystokinin-induced conditioned taste aversion. Society for the Study of Ingestive Behavior, 18th Annual Meeting, 2010.

TEACHING EXPERIENCE

Introduction to Brain and Behavior, Instructor

Florida State University College of Arts and Sciences, Fall 2011, Spring 2012, Summer 2012

Neuroanatomy, Graduate Laboratory Instructor for Professor Orenda Lyons-Johnson

Florida State University College of Arts and Sciences, Spring 2011

Physiological Psychology, Laboratory Instructor for Professor Elaine Hull and Joshua Rodefer

Florida State University College of Arts and Sciences, Fall 2010, Spring 2011

Physiological Psychology, Guest Lecturer: ―Ingestive behaviors and eating disorders‖ for Professor Joshua Rodefer, Florida State University College of Arts and Sciences, Fall 2010

Introduction to Brain and Behavior, Guest Lecturer: ―Reproductive Behavior‖ for Professor Alan Spector, Florida State University College of Arts and Sciences, Spring 2010

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Introduction to Brain and Behavior, Teaching Assistant for Professor Alan Spector Florida State University College of Arts and Sciences, Fall 2009, Spring 2010

Introduction to Brain and Behavior, Teaching Assistant for Professor Orenda Lyons-Johnson Florida State University College of Arts and Sciences, Fall 2009, Spring 2010, Summer 2010

Conditioning and Learning, Teaching Assistant for Professor Diana Williams Florida State University College of Arts and Sciences, Fall 2009

PROFESSIONAL AFFILIATIONS

Society for the Study of Ingestive Behavior, student member

Society for Neuroscience, student member, 2011

SERVICE Brain Awareness Week (Secondary School Outreach), Fundraising Florida State University College of Arts and Sciences, 2009-2011

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