The episodic secretory activity of the hypothalamic GnRH-producing neuronal network, which is essential for normal mammalian reproduction, is dependent on an agonist concentration-dependent switch in coupling of the GnRHR between Gs/Gq stimulatory and Gi inhibitory mechanisms. The latter may also suppress episodic GnRH secretion by regulating the secretory process at the cell membrane, and by inhibiting membrane ion currents. This autocrine mechanism determines the frequency of pulsatile GnRH release, and is also influenced by cell membrane-associated and nuclear estrogen receptor (ER) alpha and beta isoforms expressed in hypothalamic GnRH neurons and immortalized GT1-7 cells. An analysis performed on immunocytochemically identified, laser-captured fetal rat hypothalamic GnRH neurons, and olfactory placode-derived GnRH neurons identified by differential interference contrast microscopy and RT-PCR, demonstrated the coexpression of mRNAs encoding GnRH and its type I receptor. Both placode-derived neurons and GT1-7 cells exhibited spontaneous electrical activity that was stimulated by GnRH agonist treatment. This evoked response, as well as basal neuronal firing, was abolished by treatment with a GnRH antagonist. GnRH stimulation elicited biphasic intracellular calcium ([Ca2+]i) responses, and both basal and GnRH-stimulated [Ca2+]i levels were reduced by antagonist treatment. Perifused cultures released GnRH in a pulsatile manner that was highly dependent on extracellular Ca2+. The amplitude of GnRH pulses was increased by GnRH agonist stimulation and was diminished during GnRH antagonist treatment. These findings demonstrate that expression of GnRH receptor, GnRH-dependent activation of Ca2+ signaling, and autocrine regulation of GnRH release are operative in early fetal GnRH neurons. In addition to providing regulated GnRH secretion, this process could serve as a mechanism for activating gene expression during embryonic migration. Both ERalpha and ERbeta estrogen receptors were found to be expressed in normal and immortalized GnRH neurons, and mediate the differential actions of estradiol on neuronal signaling and function. The cell-surface ERalpha receptor identifieded in GT1-7 neurons undergoes high-affinity interactions with adenylyl cyclase inhibitory G proteins, modulates intracellular cAMP signaling, and regulates the GnRH secretory profile. The sensitivity of these interactions to picomolar estradiol concentrations suggests that this process represents a physiological negative feedback action of estrogen on the GnRH neuron. This is supported by the finding that exposure of perifused GT1-7 cells and hypothalamic neurons to picomolar estradiol levels increases the GnRH peak interval, shortens peak duration, and increases peak amplitude. These findings demonstrate that occupancy of the plasma membrane-associated ERs expressed in GT1-7 neurons by physiological estradiol levels causes activation of a Gi protein and modulates cAMP signaling and neuropeptide secretion. Current studies are investigating the individual and distinct actions of estradiol that are mediated by the two receptor subtypes in the regulation of GnRH neuronal function. GnRH utilizes multiple signaling pathways to activate extracellularly regulated MAP kinases, such as ERK1/2, in normal and immortalized pituitary gonadotrophs and GT1-7 neurons, which express receptors for GnRH and epidermal EGF. An analysis of the processes involved in such EGFR transactivation showed that GnRH stimulation of GT1-7 cells causes release/shedding of the soluble ligand, HB-EGF, as a consequence of metalloprotease activation. GnRH-induced phosphorylation of the EGFR and subsequently of Shc, ERK1/2, and its dependent protein, RSK-1, was abolished by metalloprotease inhibition. Similarly, blockade of the effect of HB-EGF with a neutralizing antibody, or the selective inhibitor CRM197, attenuated signals generated by GnRH and PMA but not those stimulated by EGF. In contrast, phosphorylation of the EGFR, Shc, and ERK1/2 by EGF and HB-EGF was independent of PKC and metalloprotease activity. The signaling characteristics of HB-EGF closely resemble those of GnRH and EGF in terms of the phosphorylation of the EGFR, Shc, ERK1/2, and RSK-1, as well as the nuclear translocation of RSK-1. However, neither the selective Src kinase inhibitor PP2 nor overexpression of Csk and dominant negative Pyk2, impaired HB-EGF-induced responses. In contrast, HEK293 cells expressing GnRH-R did not exhibit metalloprotease induction and EGF-R transactivation during GnRH stimulation. These findings demonstrate that GnRH-induced transactivation of the EGF-R, and the subsequent ERK1/2 phosphorylation, result from ectodomain shedding of HB-EGF through PKC-dependent activation of metalloprotease(s) in neuronal GT1-7 cells. Serotonin (5-HT), the endogenous non-selective 5-HT receptor agonist, activates the inositol 1,4,5-triphosphate/calcium [InsP3/Ca(2+)] signaling pathway and exerts both stimulatory and inhibitory actions on cAMP production and GnRH release in immortalized GnRH neurons. The high degree of similarity between the signaling and secretory responses elicited by GnRH and serotonin prompted studies on specific 5-HT receptor subtypes to deconvolute the complex actions of these agonists on signal transduction and GnRH release. Specific mRNA transcripts for 5-HT1A, 5-HT2C, 5-HT4, and 5-HT7 were identified in immortalized GnRH neurons. The rate of firing of spontaneous APs by identified hypothalamic GnRH neurons, and cAMP production and pulsatile GnRH release in GT1-7 cells, were profoundly inhibited during activation of the Gi-coupled 5-HT1A receptor. Treatment with a selective agonist to activate the Gq-coupled 5-HT2C receptor increased the rate of firing of spontaneous action potentials (APs), stimulated InsP3 production and caused a delayed increase in GnRH release. Selective activation of the Gs-coupled 5-HT4 receptor also increased the rate of firing of APs, stimulated cAMP production, and caused a sustained and robust increase in GnRH release. The ability of 5-HT receptor subtypes expressed in GnRH neurons to activate single or multiple G proteins in a time- and dose-dependent manner differentially regulates the phospholipase C (PLC)/InsP3/Ca(2+), and adenylyl cyclase (AC)/cAMP signaling pathways, and thereby determines the frequency and amplitude of pulsatile GnRH release. This process, in conjunction with the modulation of spontaneous electrical activity of the GnRH neuron, contributes to the control of the pulsatile mode of neuropeptide secretion that is characteristic of GnRH neuronal function in vivo and in vitro. These studies have demonstrated the manner in which the actions of GnRH through one receptor coupled to three G proteins in the GnRH neuron are similar to those of serotonin acting through three individual 5HT receptors, each specifically coupled to one of the same three G proteins