Activation of arcuate nucleus GABA neurons promotes luteinizing hormone secretion and reproductive dysfunction: Implications for polycystic ovary syndrome

Abstract

Background: Enhanced GABA activity in the brain and a hyperactive hypothalamic-pituitary-gonadal axis are associated with polycystic ovary syndrome (PCOS), the most common form of anovulatory infertility. Women with PCOS exhibit elevated cerebrospinal fluid GABA levels and preclinical models of PCOS exhibit increased GABAergic input to GnRH neurons, the central regulators of reproduction. The arcuate nucleus (ARN) is postulated as the anatomical origin of elevated GABAergic innervation; however, the functional role of this circuit is undefined.

Methods: We employed a combination of targeted optogenetic and chemogenetic approaches to assess the impact of acute and chronic ARN GABA neuron activation. Selective acute activation of ARN GABA neurons and their fiber projections was coupled with serial blood sampling for luteinizing hormone secretion in anesthetized male, female and prenatally androgenised (PNA) mice modelling PCOS. In addition, GnRH neuron responses to ARN GABA fiber stimulation were recorded in ex vivo brain slices. Chronic activation of ARN GABA neurons in healthy female mice was coupled with reproductive phenotyping for PCOS-like features.

Findings: Acute stimulation of ARN GABA fibers adjacent to GnRH neurons resulted in a significant and long-lasting increase in LH secretion in male and female mice. The amplitude of this response was blunted in PNA mice, which also exhibited a blunted LH response to GnRH administration. Infrequent and variable GABA_A-dependent changes in GnRH neuron firing were observed in brain slices. Chronic activation of ARN GABA neurons in healthy females impaired estrous cyclicity, decreased corpora lutea number and increased circulating testosterone levels.

Interpretation: ARN GABA neurons can stimulate the hypothalamic-pituitary axis and chronic activation of ARN GABA neurons can mimic the reproductive deficits of PCOS in healthy females. Unexpectedly blunted HPG axis responses in PNA mice may reflect a history of high frequency GnRH/LH secretion and reduced LH stores, but also raise questions about impaired function within the ARN GABA population and the involvement of other circuits.

Keywords: Chemogenetics; GnRH neurons; Luteinizing hormone; Mouse; Optogenetics; PCOS.

Fig. 1

Selective targeting and activation of ARN GABA neurons with ChETA. (A,B) Schematic shows a sagittal and coronal view of the mouse brain, and the ARN location of the bilateral AAV9-ChETA-eYFP injection. (C-E) Representative confocal photomicrographs of VGAT-Cre;tdTomato cells in the ARN (magenta) transfected with AAV9-ChETA-eYFP (green) and their co-localization (white). (F—H) Representative images of viral transfection from the rostral to caudal extension of the ARN (rARN↔cARN). Dashed white lines delineate the lateral borders of the ARN (3 V = third ventricle; scale bar = 100 μm). (i) Schematic of cell-attached voltage-clamp recordings from ChETA-eYFP-transfected ARN neurons in ex vivo brain slices. Representative traces of the 20 Hz optogenetic activation protocol run 5–10 times per cell (black traces) and the averaged trace (red traces) and spontaneous ARN GABA activity. (J) Mean ± SEM evoked spike fidelity at various optogenetic activation frequencies in ARN ChETA-expressing neurons from VGAT-Cre male mice (N = 6). ***p < .001; Kruskal-Wallis H test with Dunn's post hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

High frequency optogenetic activation of ARN GABA neuron fibers in the rPOA elicits LH secretion in vivo. (A) ChETA-assisted circuit mapping is achieved by injecting AAV9-ChETA-eYFP into the ARN of VGAT-Cre mice and subsequently activating ARN GABA neuron fibers projecting into the rPOA, where many GnRH neurons are located. Serial tail-tip blood sampling for LH serves as a proxy for in vivo GnRH neuron stimulation. (B) Representative confocal images demonstrating optical fiber placement (white dashed lines) adjacent to GnRH neurons (blue) and AAV9-ChETA-eYFP transfected GABA fibers projecting from the ARN (green, scale bar = 100 μm). (C-G) Projected confocal image and rotated 3D reconstruction of rPOA GnRH neuron (blue) contacted by ChETA-expressing fibers (magenta arrowheads, scale bar = 10 μm in C and D, scale bar = 1.5 μm in E-G.). (H-J) Representative examples of evoked LH secretion following 10 min of 2- and 20-Hz photo-stimulation (blue bars) in the rPOA of VGAT-Cre^+/− male (blue symbols), diestrous female (red symbols) and in PNA female mice (black symbols) and VGAT-Cre^−/− controls (grey symbols). (K—P) Mean ± SEM LH secretion in VGAT-Cre^+/− male (N = 6), diestrous female (N = 10), PNA female (N = 5) and VGAT-Cre^−/− controls (Cre^−/− male, N = 5; diestrous female, N = 10; and diestrous PNA, N = 5 mice). Magenta triangles indicate LH levels significantly higher than baseline levels. Two-way ANOVA with Dunnett's post hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Optogenetic activation of ARN GABA neuron fibers elicits distinct LH release in normal and PCOS-like mice. (A) Histograms show optogenetic-evoked changes in LH levels following 2 Hz and 20 Hz photostimulation of ARN GABA neurons in VGAT-Cre male (N = 6), diestrous female (N = 10) and diestrous PNA (N = 5) mice. *p < .05; one-way ANOVA with Tukey's post hoc test was applied for each frequency. (B) Dynamics of increased LH release following the injection of s.c. GnRH (200 ng/kg) in Cre^−/− mice. (C) GnRH-evoked changes in LH release in Cre^−/− male (M) (N = 5), diestrous female (F) (N = 10), and diestrous PNA (N = 5) mice. **p < .01; ***p < .001; ****p < .0001; one-way ANOVA with Tukey's post hoc test.

Fig. 4

Selective targeting and activation of ARN GABA neurons with the chemogenetic activator hM3Dq. (A,B) Schematic of the coronal mouse brain depicting the ARN location of bilateral AAV5-hM3Dq-mCherry injection. (C-G) Representative confocal images showing τGFP reporter expression of VGAT cells (green), AAV5-hM3Dq-mCherry transfected cells (magenta) and co-localization (white) in the ARN at low and high magnification (scale bar in B-D = 100 μm, in E-G = 25 μm). (H) Cell-attached recordings from hM3Dq-mCherry-expressing neurons in the ARN. Yellow bar indicates the time of 1 μM CNO administration to the bath. (I) Mean ± SEM firing frequencies before and after CNO administration to hM3Dq-mCherry-expressing neurons in brain slices from VGAT-Cre;τGFP male mice (n = 6 cells from 3 animals). *p < .05; Wilcoxon matched pairs signed rank test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

CNO administration to hM3Dq-expressing ARN GABA neuron fibers in the rPOA elicits LH secretion in male and female mice. (A) Schematic of the regions targeted in a sagittal mouse brain map for transfection and CNO delivery. (B,C) Representative confocal images showing ARN GABA projecting fibers transfected with AAV5-hM3Dq-mCherry (red) nearby GnRH neurons (blue) in the rPOA at low and high magnification. Scale bar in B = 100 μm and in C = 5 μm. (D) Protocol timeline. (E,F) Representative LH secretion pattern from ARN GABA^{OFF Target} male and female controls in response to CNO delivery and peripheral GnRH administration. (G,H) Representative examples of circulating LH secretion patterns in response to CNO delivery into the rPOA of correctly targeted ARN GABA^hM3Dq+/rPOA^ON male (blue traces) and female (red traces) mice. (I-J) Mean ± SEM LH responses in ARN GABA^hM3Dq+/rPOA^ON males (blue) and females (red) and in corresponding control animals (grey). Purple triangles indicate LH levels significantly higher (p < .05) than baseline levels. Two-way ANOVA with Dunnett's post hoc test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Chronic exposure to CNO activation of ARN GABA neurons disrupts estrus cyclicity in female mice. (A) Schematic of the protocol to chronically activate hM3Dq-expressing ARN GABA neurons with CNO administration through the drinking water. (B,C) Representative examples of estrous cyclicity before and during CNO (yellow box) exposure in an ARN GABA^hM3Dq+ female and in a ARN GABA^{OFF Target} control animal. (D,E) Mean ± SEM frequency of each estrous cycle phase before (water; grey bars) and during CNO exposure (CNO; yellow bars) in ARN GABA^hM3Dq+ females (N = 13) and in control animals (N = 13, consisting of ARN GABA^{OFF Target} and ARN GABA^mCherry female mice). (F,G) Mean ± SEM cycle length in days before and during CNO exposure. (H—K) Representative images of ARN GABA^mCherry (H) and ARN GABA^hM3Dq+ (I) ovarian histology; corpora lutea marked with asterisks (*). Mean ± SEM number of preovulatory follicles (POF; J) and corpora lutea (CL; K) from control (ARN GABA^mCherry and ARN GABA^OFFTarget; N = 13) and ARN GABA^hM3Dq+ (N = 9) mice following CNO administration. * p < .05; ***p < .001; (D,E) Multiple Student's t-tests; (F,G) Mann-Whitney U test; (J,K) Student's t-tests. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

The effect of chronic ARN GABA neuron activation on testosterone and LH pulse frequency in female mice. (A,B) Individual levels of circulating testosterone levels before (black dots) and during treatment with CNO in the drinking water (orange squares) in ARN GABA ^hM3Dq+ (N = 13) and controls (consisting of ARN GABA^{OFF Target} (N = 5) and ARN GABA^mCherry (N = 6) mice. (C) Percentage change in testosterone in response to CNO compared between control and ARN GABA^hM3Dq+ mice. (D-F) Representative examples of LH release profiles over 2 h in individual ARN GABA ^hM3Dq+ and ARN GABA^{OFF Target} animals at the end of 2 weeks of water only (black line and dots) and then following 2 weeks of CNO administration through the drinking water (orange line and square). (G,H) LH pulse frequency before and after CNO treatment period, and (I,J) cumulative LH secretion (area under the curve) before and after CNO treatment period in ARN GABA ^hM3Dq+ (N = 11) and ARN GABA^{OFF Target} (N = 5) groups. (A,B,G-J) Paired Student's t-tests; (C) Mann-Whitney U test; *** p < .001.