Peripheral administration of GLP-1 reduces alcohol reward.

<p>Mice treated with vehicle on the testing day spent significantly more time in the compartment previously (during the conditioning sessions) paired with alcohol as compared to the compartment paired with saline. In contrast, mice treated with 0.02 mg of GLP-1 spent an equal amount of time in both the saline- and alcohol-paired compartments (A). Alcohol induced a significant preference for the compartment it was paired with over the compartment paired with saline during the conditioning sessions in NMRI mice injected with vehicle (n = 48) but not those treated with GLP-1 (n = 31) (B). %CPP was determined with the following formula ((test-pretest)/(total time- pretest))×100 to indicate the % preference above a neutral response (i.e. equal preference for each compartment). All values represent mean ± SEM. *p<0.05, **p<0.01.</p

Peripheral administration of GLP-1 or EX4 reduces voluntary alcohol intake. In an intermittent-access 20% ethanol drinking paradigm, Wistar rats peripherally injected with GLP-1 (0.1 mg/kg) drank less alcohol than those injected with vehicle at 1 h (n = 12 per treatment group (A)).

<p>The reduced alcohol drinking response was primarily exhibited by high alcohol consuming rats (HIGH C, top 30% consumers) and was not detected in low alcohol consuming rats (LOW C; bottom 30%) (B). Rats that received an IP injection of EX4 at a dose of either 0.3 µg (C) or 1.0 µg/kg (D) reduced their 20% ethanol intake at 1 h after alcohol exposure n = 13−25. All values represent mean ± SEM. VEH, vehicle for GLP-1 (glucagon-like-peptide-1); EtOH, ethanol. *p<0.05, **p<0.01.</p

Identification of the mesolimbic VTA as the neuroanatomical substrate for GLP-1R-linked effects on alcohol consumption.

<p>VTA-selective unilateral microinjections of GLP-1 (vehicle n = 11; GLP-1 1 µg n = 7, A−B) and EX4 (vehicle n = 9; EX4 0.1 µg n = 9, C−D) reduced 20% ethanol consumption during a 16 h drinking session. A diagram based on Paxinos and Watson at the level of bregma −5.40 mm shows a representative VTA injection site (E). Additionally, schematics illustrate the injection site for each rat from the GLP-1 (F) and the EX4 (G) study. Black circles represent vehicle-injected rats, grey drug-injected and white missed placement. All values represent mean ± SEM. Aq; aqueduct, SNR; <i>substantia nigra pars reticulata</i>. ^#p<0.1,*p<0.05, ***p<0.005.</p

Activation of GLP-1R in the NTS alters gene expression in the mesolimbic reward system.

<p>GLP-1R activation by Ex4 in the NTS increased the mRNA expression of the gene that encodes tyrosine hydroxylase (<i>TH</i>), and dopamine 2 receptor (<i>Drd2</i>) without significantly changing the mRNA expression of other dopamine receptors in the VTA (A). The expression of several other genes previously associated with changes in reward behavior: <i>FosB</i>, <i>Creb1</i> and <i>Gad1</i> remained unchanged after intra-NTS Ex4 treatment (B). Intra-NTS Ex4 treatment did not alter the expression of dopamine receptors, dopamine transporter (DAT), <i>FosB</i>, <i>Gad1</i> or <i>Creb1</i> in the nucleus accumbens (Fig C-D). Data are expressed as mean ±SEM. n = 6 (vehicle group) and n = 5 (Ex4 group). ** p<0.01, *** p<0.005. Dopamine receptor 1 (<i>Drd1a</i>), dopamine receptor 3 (<i>Drd3</i>), dopamine receptor 5 (<i>Drd5</i>), glutamate decarboxylase 1 (<i>Gad1</i>), cAMP responsive element binding protein 1 (<i>Creb1</i>), FBJ osteosarcoma viral oncogene B (<i>FosB</i>).</p

GLP-1R stimulation in the NTS decreased food-motivated behavior.

<p>The effect of intra-NTS injection of Ex4 on progressive ratio operant responding for sucrose was tested. Ex4 potently decreased the number of sucrose rewards earned (A) and the number of active lever presses (B) in an operant lever-pressing paradigm. Importantly this suppression in food-motivated behavior was not associated with a reduction in locomotor activity (C). n = 11 per each treatment group. ** p<0.01, ***P < 0.0005.</p

GLP-1R stimulation by Ex4 in the NTS reduces chow intake and body weight.

<p>Intra-NTS delivery of Ex4 reduced the consumption of chow over the 22h period of data collection (A-B). Body weight (g) was also reduced 22h after injections (C). In a second group of rats intake of kaolin (PICA response) was measured simultaneously with chow intake. While the chow intake was significantly reduced after intra NTS Ex4 administration (D), intake of kaolin was not altered by Ex4 (E). Data are expressed as mean ±SEM. n = 11 per each treatment group (A-C), n = 8 per each treatment group (D-E). * p<0.05, ** p<0.01, ***P < 0.005, **** p<0.0005.</p

NTS GLP-1R activation preferentially affects intake of palatable food but not chow.

<p>Direct NTS GLP-1R stimulation with Ex4 suppressed the intake of palatable food (peanut butter) but not chow when both were offered simultaneously. The effect of Ex4 had a short latency (noted 1h after injection), and lasted throughout the 6h of measurements. The food intake data are represented as grams eaten (A), as calories consumed, since the two foods differ in their caloric density (B), and as the fraction of total intake (by calories) that was represented by the palatable peanut butter intake(C). Data are expressed as mean ±SEM. n = 12 per each treatment group. * p<0.05, ** p<0.01, *** p<0.005.</p

GLP-1R stimulation in the NTS decreased food reward behavior.

<p>The effect of intra-NTS injection of Ex4 on the ability of palatable food to condition a place preference was tested. Preference for the chamber paired to palatable food was abolished by Ex4 treatment. The preference [% conditioned place preference (CPP)] was calculated using the following formula: ((test − pre-test)/(total time − pre-test)) × 100. n = 11 (vehicle group) and n = 8 (Ex4 group). ****p < 0.0005. Data represent mean ±SEM.</p

Many YFP-immunoreactive axons (green) closely apposed the TH-positive neurons (red) of the NTS.

<p>Fluorescent YFP– preproglucagon neurons (green) and DAPI (nuclear stain, blue) in coronal sections through the NTS of YFP–PPG mice. Micrographs showing the caudal NTS (A-B), the NTS at the level of the area postrema (C-D) and the NTS at the level of the 4^th ventricle (E-F). Cell bodies of YFP-immunoreactive preproglucagon neurons (green) were detected at the level of the area postrema and just caudally to the area postrema (A-D). Many green YFP-immunoreactive axons closely appose blue DAPI-labeled cell bodies in the NTS. White arrows indicate NTS TH-positive neuronal cell bodies closely apposed by the GLP-1 fibers. Insets in panels B,D and F show the interaction at a single neuron level. Area postrema (AP), central canal (cc), dorsal motor nucleus of the vagus (DMV), gracile nucleus (Gr), 4^th ventricle (4thV). B,D and F show higher magnification of areas in A,C and D, respectively.</p

Representative photomicrograph of a coronal section of the rat brain at the level of the NTS illustrating the microinjection site (encircled area) for the behavioral experiments (right panel) and a schematic representation of the NTS from the rat atlas (Paxinos and Watson, 1998) (left panel).

<p>Area postrema (AP), central canal (cc), gracile nucleus (Gr).</p