In VivoRecordings of GnRH Neuron Firing Reveal Heterogeneity and Dependence upon GABA_AReceptor Signaling

Abstract

The gonadotropin-releasing hormone (GnRH) neurons are the key cells regulating fertility in all mammalian species. The scattered distribution of these neurons has made investigation of their properties extremely difficult and the key goal of recording their electrical activity in vivo near impossible. The caudal-most extension of the GnRH neuron continuum brings some cells very close to the base of the brain at the level of the anterior hypothalamic area. Taking insight from this, we developed an experimental procedure in anesthetized GnRH-GFP mice that allows the electrical activity of these GnRH neurons to be recorded in vivo. On-cell recordings revealed that the majority of GnRH neurons (86%) were spontaneously active, exhibiting a range of firing patterns, although only a minority (15%) exhibited burst firing. Mean firing frequencies ranged from 0.06 to 3.65 Hz, with the most common interspike interval being ϳ500 ms. All GnRH neurons tested were activated by AMPA and kisspeptin. Whereas the GABA A receptor agonist muscimol evoked excitatory, inhibitory, or mixed effects on GnRH neuron firing, the GABA A receptor antagonist picrotoxin resulted in a consistent suppression of firing. These observations represent the first electrical recordings of GnRH neurons in vivo. They reveal that GnRH neurons in vivo exhibit considerable heterogeneity in their firing patterns with both similarities and differences to firing in vitro. These variable patterns of firing in vivo are found to be critically dependent upon ongoing GABA A receptor signaling.

Optogenetic activation of GnRH neurons reveals minimal requirements for pulsatile luteinizing hormone secretion

“…We have addressed here directly whether synchronizing GnRH neurons to fire in a burst pattern can generate an LH pulse. To ensure that the burst firing was relevant, we activated GnRH neurons for 5 min in precisely the same manner as they are found to exhibit burst firing in adult mice in vivo (10). Against our expectations, this pattern of activation had no effect at all on LH secretion.…”

“…In the third stimulation paradigm, we sought to entrain silent GnRH neurons to exhibit their endogenous burst firing pattern as identified recently in vivo (10). This involved giving 5-ms light pulses at 1.9 Hz for 10 s followed by a silent period of 15 s and then 1.9 Hz for 10 s and so on for 5 min.…”

“…Disappointingly, however, direct electrical recordings of adult GnRH neurons in acute brain slices in vitro have provided no clear correlate of pulsatile hormone secretion (7,9). Recent investigations into GnRH neuron firing in vivo in anesthetized GnRH-green fluorescent protein (GFP) mice have similarly been unable to shed light on the pulse-generating properties of these cells (10). The most promising insights into the nature of GnRH pulsatility have come from studies of embryonic GnRH neurons in vitro where episodes of burst firing, represented by calcium transients, are found to synchronize occasionally in subpopulations of GnRH neurons in a time frame similar to that of pulsatile GnRH/LH secretion (11,12).…”

Neonatal testosterone suppresses a neuroendocrine pulse generator required for reproduction

“…It is therefore difficult to predict how the presently unveiled mechanisms apply to the other, parvicellular, neuroendocrine systems and their individual-specific ultradian rhythms. Another limitation is the paucity of available information concerning the in vivo activity of identified parvicellular neurons 29 and how this activity changes along with the occurrence of a secretory pulse of anterior pituitary hormones.…”

Cell-Based Models to Study GnRH Neuron Physiology

“…Primary cultures of GnRH neurons have also obtained by dispersion of fetal olfactory tissue derived from of GFP-GnRH mice [48,49]. The presence of GnRH neurons expressing GFP in tissue explant or mixed cell cultures allows to identify living GnRH neurons in tissue slice preparations before and during the experiments; this offers significant experimental advantages for biochemical or electrophysiological experiments [50,51].…”