Knockdown of GABA(A) receptor signaling in GnRH neurons has minimal effects upon fertility

Abstract

The amino acid gamma-aminobutyric acid (GABA) is thought to play a key role in shaping the activity of the GnRH neurons throughout embryonic and postnatal life. However, the physiological roles of direct GABA inputs to GnRH neurons remain unknown. Using a Cre-LoxP strategy, we generated a targeted mouse line, in which all (98 +/- 1%) GnRH neurons had the gamma2-subunit of the GABA(A) receptor deleted. Electrophysiological recordings of GABA(A)-mediated postsynaptic currents from green fluorescent protein-tagged GnRH neurons with the gamma2-subunit knocked out (GnRH gamma2 KO) showed that the amplitude and frequency of GABA(A) postsynaptic currents were reduced by 70% (P < 0.01) and 77% (P < 0.05), respectively, and that the response to exogenous GABA was reduced by 90% (P < 0.01). Evaluation of male and female GnRH gamma2 KO mice revealed completely normal fecundity, estrous cycles, and puberty onset. Further investigation with gonadectomy and different steroid replacement regimens showed normal basal levels of LH in both sexes, and a normal estradiol-evoked positive feedback mechanism in females. However, the increment in LH after gonadectomy in GnRH gamma2 KO female mice was double that of controls (P < 0.05) and also more potently suppressed by 17-beta-estradiol (P < 0.05). A similar but nonsignificant trend was observed in GnRH gamma2 KO male mice. Together, these findings show that 70-90% reductions in the normal levels of GABA(A) receptor activity at the GnRH neuron appear to impact upon the estrogen negative feedback mechanism but are, nevertheless, compatible with normal fertility in mice.

Fig. 1.

Xgal histochemistry in the brain of an adult female GnRH-Cre;GTRosa26 mouse showing the locations of cells where Cre has been effective in recombining LoxP sites to enable expression of β-galactosidase. A, A low-power view of a coronal section showing dense Xgal expression in the medial septum (MS) and a small number of scattered cells in the striatum (S). Inset shows higher power view of two Xgal-expressing GnRH neurons in the plane of focus from Xgal-GnRH dual labeling. Scale bar, 30 μm. B, Low-power view of the thalamus showing dense Xgal expression in several midline nuclei, including the paraventricular (PVT), mediodorsal (MDT), and reunions (Re) thalamic nuclei, as well as a smaller population of cells in the ventroposterior thalamic nuclei (VPT). AC, Anterior commisure; CC, corpus callosum; Hb, habenular; LV, lateral ventrical; MT, mammilothalamic tract. Scale bar, 200 μm.

Fig. 2.

Diminished GABA_A receptor signaling in GnRH γ2 KO. A, Representative voltage-clamp trace showing the effect of 50 μm GABA on two EGFP-tagged GnRH neurons, one with intact (fg2/fg2) and one with deleted (GnRH-Cre,fg2/fg2) γ2-subunit. B, Comparison of GABA-evoked inward currents in GnRH neurons of γ2-intact (n = 12) and γ2-deleted mice (n = 6) and in unidentified neurons located in the striatum (n = 6 and 7). C and D, Representative traces showing mPSCs from GnRH neurons in which the γ2-subunit is intact (fg2/fg2) or deleted (GnRH-Cre,fg2/fg2). E, Comparison of the shape of averaged mPSC traces from GnRH neurons in which the γ2-subunit is intact (fg2/fg2) or deleted (GnRH-Cre,fg2/fg2). F, Cumulative probability plot of mPSCs amplitude. G–I, Comparison of mPSC amplitude, frequency, and decay time constant in GnRH neurons of γ2-intact (n = 14) and γ2-deleted (n = 6) mice. *, P < 0.05; **, P < 0.01; Mann-Whitney U test.

Fig. 3.

Male fertility and negative feedback in GnRH γ2 KO mice. A, Histograms showing the mean + sem numbers of litters and pups per litter born to breeding pairs in which wild-type females were mated with either GnRH γ2 KO (GnRH-Cre, fg2/fg2; n = 5) or control (GnRH-Cre, n = 5) mice. B, Histograms showing mean + sem. LH levels of intact and goandectomized (GDX) GnRH γ2 KO (n = 5) and control (fg2/fg2; n = 5) mice. *, P < 0.05; ***, P < 0.001.

Fig. 4.

Female puberty and estrous cyclicity in GnRH γ2 KO mice. A, Histograms showing the mean + sem postnatal day of vaginal opening and first estrus for GnRH γ2 KO (n = 10) and control (fg2/fg2, n = 5) mice. B, Histograms showing the mean + sem number of days GnRH γ2 KO (n = 6) and control (fg2/fg2, n = 6) female mice were in diestrus, proestrus, and estrus over a 17 d vaginal smearing period.

Fig. 5.

Estrogen positive and negative feedback in female GnRH γ2 KO mice. A, Negative feedback. Histograms showing mean + sem. LH levels in mice before (intact) and 2 wk after ovariectomy (OVX) and then 3 h after treatment with 17-β-estradiol (E2) in GnRH γ2 KO (n = 10) and control (fg2/fg2 or wild type, n = 10) mice. The increment from basal in LH after OVX is shown to the right. *, P < 0.05; **, P < 0.01; ***, P < 0.001. B–D, Estrogen positive feedback. Histogram showing (B) the mean + sem number of GnRH-immunoreactive neurons detected per section at the level of the rPOA, (C) mean + sem % GnRH neurons expressing c-Fos, and (D) the mean + sem surge levels of LH in GnRH γ2 KO (n = 5) and control (fg2/fg2; n = 6) mice.