The Effects of GABA on Embryonic Gonadotropin-Releasing Hormone Neurons in Rat Hypothalamic Primary Culture

Abstract

Abstract. Gonadotropin-releasing hormone (GnRH) neurons arise in the olfactory placode, migrate into the preoptic area (POA), and then extend axons to the median eminence during embryogenesis. Little information is available concerning the properties of GnRH neurons during the late gestational period when GnRH neurons reach the POA and form neuronal networks, although many studies have examined such properties during earlier developmental stages or the postnatal period. The present study was performed to elucidate the involvement of γ-aminobutyric acid (GABA), one of the major neurotransmitters modifying GnRH neural activity, in regulation of GnRH gene expression on embryonic day 18.5 (E18.5) using transgenic rats expressing enhanced green fluorescence protein (EGFP) under the control of GnRH promoter. First, using RT-PCR, the mRNA of two isoforms of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD), GAD65 and GAD67 was detected in E18.5 embryonic POA-containing tissues. GAD67-positive cells were also demonstrated in close vicinity to GnRH-positive cells by immunohistochemistry, and immunoreactivity for both the GABA-A and GABA-B receptor subunits was detected in GnRH neurons. Next, primary cultures derived from anterior hypothalamic tissue of E18.5 embryos were prepared, and the effects of GABA and its agonists on GnRH promoter activity were evaluated using EGFP expression as a marker. GABA and the GABA-A receptor agonist muscimol, but not the GABA-B receptor agonist baclofen, significantly increased the EGFP-positive/GnRH-positive cell ratio. These results suggest that GABA plays a role in stimulating GnRH gene expression through GABA-A receptors in embryonic GnRH neurons in late gestational stages.

Lack of functional GABAB receptors alters GnRH physiology and sexual dimorphic expression of GnRH and GAD-67 in the brain

“…Interestingly, increases of GnRH levels were detected in the cortex when migration of GnRH neurons toward their natural location was impaired, as in mice overexpressing GAD-67 in GnRH neurons (43) or in netrin 1 knockout mice (81). GABA B Rs are expressed in GnRH neurons early in development; they were demonstrated in several immortalized GnRH neuron cell lines (30,63,91), in green fluorescence protein-expressing embryonic GnRH neurons (26), and in in situ hypothalamic neurons (86). Intriguingly, GABA B Rs were recently shown to control cell migration and cell positioning within the ventromedial nucleus of the hypothalamus (64) and in the POA-AH (99) in mice.…”

“…Regulation of the gonadotropin axis is provided by both stimulatory and inhibitory inputs conveyed by neurotransmitters, neurohormones, and peripheral peptide and steroid hormones. Among the neurotransmitters involved in this regulation, GABA has been proposed to be of prime importance (16,26,43,65). GABA can regulate the hypothalamic-pituitary-gonadal axis by acting at ionotropic GABA A/C and/or metabotropic GABA B receptors (GABA B Rs).…”

Lack of Functional GABA_B Receptors Alters <b><i>Kiss1</i></b>, <b><i>Gnrh1 </i></b>and <b><i>Gad1</i></b> mRNA Expression in the Medial Basal Hypothalamus at Postnatal Day 4

“…GABA A R stimulation also modulates Gnrh1 mRNA expression in a species-, age-, and model-specific manner [9, 10]. Despite this, knockdown of GABA A Rs in GnRH neurons has minimal effects on fertility [11].…”

“…In contrast, pharmacological reports suggested that GABA B Rs do not participate in this event [4, 22]. However, because GABA B Rs: a) are present in migrating GnRH neurons [10, 22], b) participate in neuron migration and differentiation [23, 24], c) are present in neural progenitors and in embryonic stem cells [25, 26], and d) participate in E 2 -induced sexual differentiation of several hypothalamic nuclei [27, 28], their participation on GnRH neuron migration and subsequent Gnrh1 expression warrants further investigation.…”

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].…”