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

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

<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

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