Enhancement of a robust arcuate GABAergic input to gonadotropin-releasing hormone neurons in a model of polycystic ovarian syndrome

Abstract

Polycystic ovarian syndrome (PCOS), the leading cause of female infertility, is associated with an increase in luteinizing hormone (LH) pulse frequency, implicating abnormal steroid hormone feedback to gonadotropin-releasing hormone (GnRH) neurons. This study investigated whether modifications in the synaptically connected neuronal network of GnRH neurons could account for this pathology. The PCOS phenotype was induced in mice following prenatal androgen (PNA) exposure. Serial blood sampling confirmed that PNA elicits increased LH pulse frequency and impaired progesterone negative feedback in adult females, mimicking the neuroendocrine abnormalities of the clinical syndrome. Imaging of GnRH neurons revealed greater dendritic spine density that correlated with increased putative GABAergic but not glutamatergic inputs in PNA mice. Mapping of steroid hormone receptor expression revealed that PNA mice had 59% fewer progesterone receptor-expressing cells in the arcuate nucleus of the hypothalamus (ARN). To address whether increased GABA innervation to GnRH neurons originates in the ARN, a viral-mediated Cre-lox approach was taken to trace the projections of ARN GABA neurons in vivo. Remarkably, projections from ARN GABAergic neurons heavily contacted and even bundled with GnRH neuron dendrites, and the density of fibers apposing GnRH neurons was even greater in PNA mice (56%). Additionally, this ARN GABA population showed significantly less colocalization with progesterone receptor in PNA animals compared with controls. Together, these data describe a robust GABAergic circuit originating in the ARN that is enhanced in a model of PCOS and may underpin the neuroendocrine pathophysiology of the syndrome.

Keywords: GABA; GnRH; PCOS; luteinizing hormone; progesterone receptor.

Fig. 1.

LH concentrations from representative control (A, n = 14) and PNA (B, n = 19) mice. Stars indicate LH pulses. (C) PNA treatment significantly increases LH pulse frequency. (D) LH concentrations from control (n = 6) and PNA (n = 8) mice sampled at three time points: intact, 3 d after OVX, and 48 h postprogesterone pellet implantation.

Fig. 2.

PR immunoreactivity in representative unilateral sections containing the AVPV (outlined, A), PeN (outlined, B), and ARN (outlined, C) in control (n = 7) and PNA (n = 7) mice. (Scale bar, 250 μm.) (D) The number of PR-labeled cells is significantly decreased in PNA mice compared with controls.

Fig. 3.

Projected confocal images (8-μm optical thickness) of GnRH neurons (green) closely apposed by vGaT-ir puncta (red) from control (A, n = 5) and PNA (B, n = 5) mice. (i and ii) Projected confocal images (1.35-μm optical thickness) of the GnRH neuron dendrite from corresponding white boxes. Arrowheads indicate points where vGaT-ir puncta are considered to contact the GnRH neuron. (Scale bar, 5 μm.) (C) The density of GnRH neuron spines (i), vGluT2-ir puncta closely apposed to GnRH neurons (ii), and vGaT-ir puncta closely apposed to GnRH neurons (iii) from control and PNA mice.

Fig. 4.

(A) A single injection of Ad-iZ/EGFPf induces EGFPf expression in GABAergic neurons of the rostral (i), middle (ii), and caudal (iii) regions of the ARN. (Scale bar, 0.5 mm.) GnRH neurons (red) from control (B, n = 6) and PNA (C, n = 6) mice closely apposed by ARN GABAergic fibers (green). (Scale bar, 5 μm.) 3V, third ventricle.

Fig. 5.

(A) Reconstructions using AMIRA software illustrating the robust degree of contact between ARN GABAergic fibers (green) and the lengthy dendrites of GnRH neurons (red). The density (B) and total number (C) of closely apposed fibers from ARN GABAergic neurons to GnRH neurons is significantly increased at the dendrite of PNA (n = 6) mice compared with controls (n = 6). (D) The number of ARN GABAergic fibers closely apposed to GnRH neurons is significantly increased along the first 30 μm of the GnRH neuron dendrite. (E) The percentage of GnRH neurons that are contacted by ARN GABAergic fibers at a single point is significantly decreased in PNA mice compared with controls.

Fig. 6.

Projected confocal images of EGFPf-positive GABA neurons and PR-positive nuclei in the ARN of control (A, n = 4) and PNA (B, n = 5) mice. High-magnification images of EGFPf-positive GABAergic neurons (i), PR-positive nuclei (ii), and merged images (iii) from corresponding white boxes. Arrows indicate ARN GABAergic cells that are colocalized with nuclear PRs. (Scale bars, 50 μm.) (C) The percentage of GABAergic cells colocalized with PRs is significantly decreased in the rostral, middle, and caudal ARN of PNA mice compared with controls. 3V, third ventricle.