Exendin-4 blocks the nicotine-induced locomotor sensitization in mice.

<p>In the present experiment nicotine or vehicle for five days. 72 hours following this sub-chronic treatment Ex4 or vehicle was administered. Sub chronic nicotine treatment induced (0.5 mg/kg) a significant sensitization and this effect was attenuated by a single injection of Ex4 (2.4 μg/kg) (n=8; * ***<i>P</i><0.001, two-way ANOVA followed by a Bonferroni post-hoc test). All values represent mean ± SEM. </p

Exendin-4 attenuates nicotine-induced locomotor stimulation, accumbal dopamine release and conditioned place preference in mice.

<p>(A) Nicotine-induced (0.5 mg/kg IP) locomotor stimulation was attenuated by a single injection of Ex4 (2.4 μg/kg IP) (n=8 in each group; **<i>P</i><0.01 and ***<i>P</i><0.001, one-way ANOVA followed by a Bonferroni post-hoc test). (B) First we demonstrated a significant effect of nicotine (0.5 mg/kg IP) to increase dopamine release in comparison to vehicle treatment at time intervals 40-180 minutes (**P<0.01, ***P<0.01, veh-nic compared to veh-veh treatment). Secondly we showed that pre-treatment with Ex4 (2.4 μg/kg IP) attenuated the nicotine-induced increase in dopamine release compared to vehicle pre-treatment at time interval 40-60 and 100-180 minutes (+<0.05, ++P<0.01, +++P<0.01, Ex4-nic compared to veh-nic treatment). There was no difference in response between the veh-veh and Ex4-nic groups at a dose of Ex4 that had no effect <i>per </i><i>se</i>. Arrows represent time points of injection of Ex4, vehicle and nicotine. Data analyzed with a Two-way ANOVA followed by a Bonferroni post-hoc test (n=8 in each group) (C) The nicotine-induced (0.5 m/kg IP) condition place preference (CPP) was attenuated by an acute single IP injection of Ex4 (2.4 μg/kg IP) in mice (n=8 in each group, *<i>P</i><0.05, unpaired t-test). All values represent mean ± SEM. Arrow shows time for injections. </p

A coronal mouse brain section showing probe placements (illustrated by vertical lines) in the nucleus of mice used in the present study.

<p>The number given indicates millimeters anterior (+) from bregma.</p

Exendin-4 attenuates amphetamine-induced locomotor stimulation, accumbal dopamine release and conditioned place preference in mice.

<div><p>(A) Amphetamine-induced (2 mg/kg IP) locomotor stimulation was attenuated by a single injection of Ex4 (2.4 µg/kg IP) (n=8 in each group; **<i>P</i><0.01, one-way ANOVA followed by a Bonferroni post-hoc test). (B) First we demonstrated a significant effect of amphetamine (2 mg/kg IP) to increase dopamine release in comparison to vehicle treatment (time interval 40-120 minutes (P<0.001) and 140-160 minutes (P<0.05), Veh-Veh vs Veh-Amph). As shown in (B) pre-treatment with Ex4 (2.4 µg/kg IP) attenuated the amphetamine-induced increase in dopamine release compared to vehicle pre-treatment at time interval 40-8 minutes (*** < 0.001, Ex4-Amph compared to Veh-Amph treatment). The selected dose had no significant effect on accumbal dopamine release compared to vehicle treatment at any time interval (<i>P</i>>0.05, Veh-Veh vs Ex4-Veh). There was a significant difference in accumbal dopamine response in vehicle treated mice and Ex4-Amph treated mice at time interval 40-100 minutes (<i>P</i>>0.001). Arrows represent time points of injection of Ex4, vehicle and amphetamine. Data analyzed with a Two-way ANOVA followed by a Bonferroni post-hoc test (n=8 in each group) (C) The amphetamine-induced (2 mg/kg IP) condition place preference (CPP) was attenuated by an acute single IP injection of Ex4 (2.4 µg/kg IP) in mice (n=8 in each group, *<i>P</i><0.05, unpaired t-test).</p> <p>All values represent mean ± SEM.</p></div

Protocol for anxiety-related studies.

<p>Fed rats were injected with saline or ghrelin directly into the amygdala at time zero. In the FOOD ACCESS paradigm, rats were allowed access to food during the first hour after injection whereas food access was denied in the FOOD WITHHELD paradigm. After this, all rats underwent tests exploring anxiety-like behaviour, first in the EPM test (5 min) and then in the open field test (40 min). Afterwards all the rats were returned to their home cages and post-test food intake measured for 1 hr (corresponding to time 2–3 hr after injection).</p

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

<p>In this “FOOD WITHHELD" paradigm rats were denied access to food during the first hour after intra-amygdala injection. (A) An orexigenic response to intra-amygdala ghrelin injection was detected when animals were returned to their home cages after the anxiety testing. Intra-amygdala ghrelin injection decreased anxiety-like behavior relative to saline controls, reflected by an increase in the amount of time spent in the open arms in the EPM test (B) and by the increase in central activity (C) and central rearings (D) in the open field test. *P<0.05 **P<0.01, ***P<0.001, vs. saline. Independent samples t-test, SPSS.</p

Effects of intra-amygdala administration of ghrelin or a ghrelin antagonist (JMV2959) on cumulative food intake.

<p>Normal chow intake was measured at 1, 2, 4, and 24 hr following unilateral intra-amygdala injection of either (A) ghrelin (0.3 and 1 µg) to freely-fed rats or (B) JMV2959 (2 or 10 µg) to overnight fasted rats. Data are expressed as mean ± S.E.M. *P<0.05 **P<0.01 ***P<0.001, vs. saline. Paired sample t-test, SPSS.</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

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

<i>In situ</i> hybridization detection of GHSR mRNA in the rat amygdala.

<p>The autoradiographic detection of the isotopic <i>in situ</i> hybridization probe to GHSR mRNA revealed GHSR mRNA expression in distinct neurons of the amygdala. Note in A that the highest levels of expression could be observed in the ventrolateral (LaVL) and ventromedial (LaVM) parts of the lateral amygdaloid nucleus and in the posteroventral part of the medial amygdaloid nucleus (MePV). The parcellation and nuclear structure of the amygdala is shown in B. Also note the heavy GHSR mRNA signal in the arcuate (ARC) and ventromedial (VMH) nuclei. High-power photomicrographs (C–E) were amplified from various regions of the amygdala shown in A (cresyl violet counterstaining). Scale bars = 500 µm in A and B and 13 µm in C–E.</p