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 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

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 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

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

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

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

Ovariectomy-evoked changes in the expression of phagocytic and recognition receptors were responsive to ER agonists.

<p>mRNA expression of Cd11b (A), Cd18 (B), Tlr3 (C) and Tlr9 (D) in the hippocampus was determined by real-time PCR. Error bars show SD of five samples for each group. ANOVA identified statistically significant differences in gene expression among treatment groups for all genes (p<0.001 for Cd11b, p = 0.012 for Cd18, p<0.001 for Tlr3, p = 0.009 for Tlr9). Turquise, black and red asterisks mark statistically significant group differences compared to M, M/OVX and M/OVX+E2 animals, respectively, by Newman-Keuls post hoc test. * corresponds to 0.01</p

The impact of gonadectomy and ER agonist treatments on mRNA expression of aromatase and ER genes in the hippocampus of middle-aged OVX rats.

<p>Expression of Cyp19a1 (A), Esr1 (B) and Esr2 (C) was measured by real-time PCR. ANOVA revealed statistically significant treatment effects for each gene (p<0.001 for Cyp19a1, p = 0.006 for Esr1 and p = 0.028 for Esr2). Turquoise, black and red asterisks indicate statistically significant group differences compared to M, M/OVX and M/OVX+E2 animals, respectively, by Newman-Keuls post hoc test. * corresponds to 0.01</p

Expression of macrophage-associated genes and inhibitory neuronal ligands in the hippocampus of middle-aged female rats.

<p>mRNA expression was studied by real-time PCR using TaqMan chemistry. ΔCt represents the difference between the Ct of a given gene and of the endogenous control (geometric mean of the Ct values of the selected housekeeping genes glyceraldehyde-3-phosphate dehydrogenase and hypoxanthine guanine phosphoribosyl-transferase, 21.887±0.296). Ct, cycle threshold.</p