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

Impact of intra-VTA ghrelin on dietary choice.

<p>Impact of acute intra-VTA (ventral tegmental area) injection of ghrelin on total energy intake and dietary choice during (A) 3 hr, (B) 6 hr and (C) 24 hr, in rats (n = 17) that had been <i>ad libitum</i> fed a choice diet comprising normal chow, 1 g sucrose pellets and lard (saturated animal fat) over the previous 14 days. All rats received ghrelin and vehicle solution in a cross over design with one day washout between injections. **P<0.01, ***P<0.001.</p

Stability of daily food selection.

<p>Stability of daily (24 hr) food selection in rats (n = 18) offered an <i>ad libitum</i> free choice diet of normal chow, 1 g sucrose pellets and lard (a saturated animal fat). Data are shown for the last 4 baseline days (day 11–14) prior to ICV injection, and for the injection days in which 2 μg ghrelin or aCSF were administered. The data are expressed as mean ± SEM kcal consumed as % of daily total kcal intake.</p

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

Impact of ICV ghrelin on dietary choice.

<p>Impact of acute intracerebroventricular (ICV) injection of ghrelin on total energy intake and dietary choice during (A) 3 hr, (B) 6 hr and (C) 24 hr, in rats (n = 18) that had been <i>ad libitum</i> fed a choice diet comprising normal chow, 1 g sucrose pellets and lard (saturated animal fat) over the previous 14 days. All rats received ghrelin and vehicle solution in a cross over design with one day washout between injections. *P<0.05, **P<0.01, ***P<0.001.</p

Impact of ghrelin receptor knockout on dietary choice.

<p>Total energy intake and dietary choice were measured during (A) 3 hr, (B) 6 hr and (C) 24 hr after the end of an overnight fast, in ghrelin receptor knockout mice (GHSR-KO) and wild-type (WT) controls that had been <i>ad libitum</i> fed a choice diet comprising normal chow, 1 g sucrose pellets and lard (saturated animal fat) for 14 days prior to the overnight fast. **P<0.01 (one-way ANOVA).</p

Impact of fasting and peripherally administered ghrelin receptor antagonist on dietary choice.

<p>Impact of acute intraperitoneal injection of a ghrelin receptor antagonist on fasting-induced changes in total energy intake and dietary choice measured during (A) 3 hr, (B) 6 hr and (C) 24 hr after the end of an overnight fast, in rats (n = 24) that had been <i>ad libitum</i> fed a choice diet comprising normal chow, 1 g sucrose pellets and lard (saturated animal fat) for 14 days prior to fast. All rats received the antagonist and saline solution in a cross over design with one day washout between injections. In panels A and B; **P<0.01. In panel C, the letters indicate significant differences (rANOVA, P<0.01).</p

Histological verification of the location of the injection cannula in the lateral amygdaloid nucleus.

<p>A: Photomicrograph of a 40 µm counterstained coronal section of rat brain at level Bregma −3.3, illustrating the injection site. B: Schematic representation of the amygdala according to the rat brain atlas <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046321#pone.0046321-Paxinos1" target="_blank">[37]</a>. The shadow area outlines the region defined as the lateral amygdaloid nucleus. Scale bar = 1 mm. Abbreviations: BLA (basolateral amygdaloid nucleus, anterior), BLP (basolateral amygdaloid nucleus, posterior), BMP (basomedial amygdaloid nucleus, posterior), BMA (basomedial amygdaloid nucleus, anterior), CeC (central amygdaloid nucleus, central), CeL (central amygdaloid nucleus, lateral), LaDL (lateral amygdaloid nucleus, lateral), LaVL (lateral amygdaloid nucleus, ventrolateral), LaVM (lateral amygdaloid nucleus, ventromedial), OT (optic tract).</p

Effects of intra-amygdala administration of ghrelin on anxiety-like behavior in rats given access to food.

<p>In rats given access to food during the first hour after intra-amygdala injection (FOOD ACCESS), ghrelin increased food intake relative to saline controls (g of chow), both during this hour and during the 1 hr measurement taken after the anxiety tests (A). In this paradigm there was no effect of ghrelin (relative to saline controls) on anxiety-like behavior in either the EPM test (time spent in the open arm; B) or the open field test (central activity or central rearing; C, D respectively). *P<0.05 **P<0.01, vs. saline. Independent samples t-test, SPSS.</p