Expression of GHS-R mRNA in GnRH-GFP neurons.

<p>Expression of GHS-R mRNA in GnRH-GFP neurons was demonstrated by single cell real-time PCR. The amplification plot shows the expression of <i>Gnrh</i> (Ct: 20.4), <i>Kiss1r</i> (Ct: 27) and <i>Gapdh</i> (Ct: 22.4) genes. The regular amplification curve of <i>Ghsr</i> with Ct values of 29.6 proves the expression of GHS-R mRNA.</p

Effect of ghrelin on the intracellular free Ca²⁺-content of the GT1-7 neurons.

<p><b>a</b>) Ghrelin (1 µM) administration in an estrogen-free medium resulted in an increase in the intracellular Ca²⁺-content. <b>b</b>) The GHS-R antagonist JMV2959 (10 µM, 10 min) abolished the action of ghrelin. <b>c</b>) Estradiol (E2, 1 nM, 24 h) treatment eliminated the effect of ghrelin on the intracellular Ca²⁺-concentration. <b>d</b>) Bar graph of the area-under-curve data representing the net change in the free Ca²⁺-content. *=p<0.0001. Arrow shows the onset of ghrelin administration.</p

Effect of ghrelin on the firing of the GnRH neurons from the brain slice of the female (metestrus and proestrus) and male mice.

<p><b>a</b> and <b>b</b>) Ghrelin (40 nM N=4 and 4 µM N=14) decreased the firing rate in the metestrus with no change in the shape of the individual spikes (insets). Time course of the instantaneous frequency is figured under the firing recordings. <b>c</b>) In proestrus, firing rate showed no change (N=11). <b>d</b>) Ghrelin administration in the male mouse resulted in a decrease in the firing rate (N=15). <b>e</b>) Bar graph shows the significant changes in the firing rate in the metestrous female (40nM-4 µM but not at 4 nM) and male, whereas ghrelin exerted no effect in the proestrous female mice. <b>f</b>) Dose-response curve of effect of ghrelin on the firing in metestrous mice (0%=full effect, 100%=no effect). Met=metestrus; Pro=proestrus. Arrow shows onset of ghrelin administration whereas double arrow marks wash-out. *=p<0.05.</p

Schematic illustration of E2-dependent effects of ghrelin in GnRH neuron.

<div><p>E2: 17β-estradiol; ER: estrogen receptor; GHS-R: ghrelin receptor; G_βγ and G_αq: G-protein subunits; DAG: diacylglycerol; DGL: DAG-lipase; CB1: cannabinoid receptor type-1; AM251: CB1 antagonist; 2-AG: 2-arachidonoylglycerol; THL: tetrahydrolipstatin (DAG-lipase inhibitor); PIP₂: phosphatidylinositol 4,5-bisphosphate; PLC: phospholipase-C; GABA_A-R: GABA_A receptor; [Ca²⁺]_i: intracellular free calcium; VGCC: voltage-gated calcium channel; JMV2959: GHS-R antagonist. Dashed arrow denotes putative indirect action.</p> <p>Binding of ghrelin to GHS-R increases intracellular free Ca²⁺-content E2-dependently, that in turn activates synthesis and release of 2-AG in the postsynaptic GnRH neuron. The released endocannabinoid then binds to the CB1 located in the presynaptic terminal and eventually causes suppression of GABA release into the synaptic cleft.</p></div

Recordings of firing of GnRH neurons in the brain slice of the female (a,b) and male (c) mice.

<p><b>d</b>) Firing rate of GnRH neurons is higher in proestrous than in metestrous female and male mice. Met=metestrus; Pro=proestrus; *=p<0.05.</p

Effect of ghrelin (4 µM) on the mPSCs in the GnRH neurons of the female metestrous mice.

<p><b>a</b>) Ghrelin decreased the frequency of the mPSCs (N=14) with no change in the shape of the individual mPSCs (insets). Time course of instantaneous frequency is depicted under the mPSC-recording. <b>b</b>) Effect of ghrelin on the mPSCs was abolished by the pretreatment with AM251 (N=11). <b>c</b>) Intracellularly applied THL eliminated the ghrelin-evoked changes on the mPSCs (N=10). <b>d</b>) Bar graph reveals that ghrelin significantly diminished the frequency of mPSCs. Arrow shows the onset of ghrelin administration. *=p<0.05.</p

Effect of antagonists on the ghrelin-modulated firing activity and resting potential of GnRH neurons in metestrous mice.

<p><b>a</b>) Block of fast neurotransmission by kynurenic acid (kyn) and picrotoxin (pic) eliminated the action of ghrelin (N=4). <b>b</b>) The GHS-R antagonist JMV2959 also abolished effect of ghrelin (N=11). <b>c</b>) Antagonizing the endocannabinoid CB1 receptor by AM251 abolished effect of ghrelin (N=10). <b>d</b>) Depletion of the intracellular Ca²⁺-pools by thapsigargin (thap) showed no effect on the ghrelin-induced decrease in the firing activity (N=4). <b>e</b>) Bar graph shows elimination of effect of ghrelin by inhibition of fast neurotransmission, by antagonizing ghrelin receptor or by block of CB1 but not by depleting the intracellular Ca²⁺-sources. <b>f</b>) Current clamp measurement showed slight depolarization upon ghrelin administration. <b>g</b>) Depolarizing effect of ghrelin was eliminated by nifedipine. *=p<0.05.</p

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

<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