The majority of recorded GnRH neurons show irregular AP firing with transition between slow, high spike amplitude tonic firing, and intervals of fast, lower spike-amplitude, burst-like AP firing. Transition from slow to fast tonic AP firing is associated with switching APs that are characterized by fAHP, threshold afterdepolarization, and initiation of fast AP firing. Treatment of hypothalamic GnRH neurons with nanomolar GnRH concentrations increased the occurrence of fast tonic APs, and reduced the duration of mAHP current. In contrast, treatment of GnRH neurons with micromolar GnRH concentrations abolished the appearance of fast tonic AP firing and did not affect the occurrence of slow tonic AP firing. The inhibitory action of high GnRH concentrations on AP firing was prevented by PTX and the underlying currents were identified as GIRK currents. These responses indicate that agonist-stimulation of endogenous GnRH receptors expressed in GnRH neurons activates GIRK channels, leading to suppression of membrane excitability and inhibition of AP firing. These data indicate that AP- and GnRH-driven Ca2+influx, and coupling of GnRH-R to Gi/o in GnRH neurons, determine the profile of after-hyperpolarization currents and consequently mediate firing frequency and the spike-profile. GnRH-induced modulation of Ca2+ influx and the consequent changes in AHP current suggest that the GnRH receptors expressed in hypothalamic GnRH neurons are important modulators of their neuronal excitability. The binding properties, calcium signaling, and pulsatile GnRH release of GT1-7 neurons expressing GnRH-R-GFP are comparable to these of GT1-7 neurons expressing the native GnRH-R. GnRH-R-GFP is localized to cell bodies and processes, and in fully differentiated bipolar neurons is confined to a thin rim of cytosol at the plasma membrane and in neuronal processes. GnRH stimulation causes redistribution of GnRH-R-GFP, with movement in close proximity to their bipolar processes and in the area of apparent synaptic connections. GnRH-Rs expressed in native and GT1-7 neurons form homo-oligomers and activate diverse signaling pathways by coupling to at least three G proteins. Such coupling is time- and dose-dependent, and switches between Gq, Gs, and Gi. GnRH-induced activation of GnRH-R in both GT1-7 neurons and alphaT3 gonadotrophs caused stimulation of PKD. GnRH-induced PKD phosphorylated both Ser742/Ser744 and Ser 916 in a time- and dose-dependent manner. This activation was abolished by a GnRH-R antagonist, consistent with GnRH-R-mediated activation. The PKC inhibitor Go 6983 abolishes phorbol 12-myristate 13-acetate (PMA)-induced PKD phosphorylation, but only partly inhibits GnRH-induced PKD Ser742/744 activation. In contrast, GnRH-induced PKD activation was not affected by the PKC-specific antagonist Go 6976. These findings indicate that in addition to PKC, the agonist-stimulated GnRH-R also activates PKD, providing a mechanism of signal integration and amplification. These data demonstrate that GnRH-GnRH-R-induced activation of PKD in immortalized GnRH neurons and pituitary gonadotrophs causes complex molecular interactions that maintain GnRH and LH secretion from the hypothalamo-pituitary axis.
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