Figure 1.
Effect of ghrelin on the intracellular free Ca2+-content of the GT1-7 neurons.
a) Ghrelin (1 µM) administration in an estrogen-free medium resulted in an increase in the intracellular Ca2+-content. b) The GHS-R antagonist JMV2959 (10 µM, 10 min) abolished the action of ghrelin. c) Estradiol (E2, 1 nM, 24 h) treatment eliminated the effect of ghrelin on the intracellular Ca2+-concentration. d) 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.
Figure 2.
Expression of GHS-R mRNA in GnRH-GFP neurons.
Expression of GHS-R mRNA in GnRH-GFP neurons was demonstrated by single cell real-time PCR. The amplification plot shows the expression of Gnrh (Ct: 20.4), Kiss1r (Ct: 27) and Gapdh (Ct: 22.4) genes. The regular amplification curve of Ghsr with Ct values of 29.6 proves the expression of GHS-R mRNA.
Figure 3.
Recordings of firing of GnRH neurons in the brain slice of the female (a,b) and male (c) mice.
d) Firing rate of GnRH neurons is higher in proestrous than in metestrous female and male mice. Met=metestrus; Pro=proestrus; *=p<0.05.
Figure 4.
Effect of ghrelin on the firing of the GnRH neurons from the brain slice of the female (metestrus and proestrus) and male mice.
a and 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. c) In proestrus, firing rate showed no change (N=11). d) Ghrelin administration in the male mouse resulted in a decrease in the firing rate (N=15). e) 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. f) 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.
Figure 5.
Effect of antagonists on the ghrelin-modulated firing activity and resting potential of GnRH neurons in metestrous mice.
a) Block of fast neurotransmission by kynurenic acid (kyn) and picrotoxin (pic) eliminated the action of ghrelin (N=4). b) The GHS-R antagonist JMV2959 also abolished effect of ghrelin (N=11). c) Antagonizing the endocannabinoid CB1 receptor by AM251 abolished effect of ghrelin (N=10). d) Depletion of the intracellular Ca2+-pools by thapsigargin (thap) showed no effect on the ghrelin-induced decrease in the firing activity (N=4). e) 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. f) Current clamp measurement showed slight depolarization upon ghrelin administration. g) Depolarizing effect of ghrelin was eliminated by nifedipine. *=p<0.05.
Figure 6.
Effect of ghrelin (4 µM) on the mPSCs in the GnRH neurons of the female metestrous mice.
a) 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) Effect of ghrelin on the mPSCs was abolished by the pretreatment with AM251 (N=11). c) Intracellularly applied THL eliminated the ghrelin-evoked changes on the mPSCs (N=10). d) Bar graph reveals that ghrelin significantly diminished the frequency of mPSCs. Arrow shows the onset of ghrelin administration. *=p<0.05.
Figure 7.
Schematic illustration of E2-dependent effects of ghrelin in GnRH neuron.
E2: 17β-estradiol; ER: estrogen receptor; GHS-R: ghrelin receptor; Gβγ and Gαq: 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; GABAA-R: GABAA receptor; [Ca2+]i: intracellular free calcium; VGCC: voltage-gated calcium channel; JMV2959: GHS-R antagonist. Dashed arrow denotes putative indirect action.
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.