Changes in GABAergic Transmission to and Intrinsic Excitability of Gonadotropin-Releasing Hormone (GnRH) Neurons during the Estrous Cycle in Mice

Abstract

Gonadotropin-releasing hormone (GnRH) neurons form the final common central output pathway controlling fertility and are regulated by steroid feedback. In females, estradiol feedback action varies between negative and positive; negative feedback typically regulates episodic GnRH release whereas positive feedback initiates a surge of GnRH, and subsequently luteinizing hormone (LH) release ultimately triggering ovulation. During the estrous cycle, changes between estradiol negative and positive feedback occur with cycle stage and time of day, with positive feedback in the late afternoon of proestrus in nocturnal species. To test the hypotheses that synaptic and intrinsic properties of GnRH neurons are regulated by cycle stage and time of day, we performed whole-cell patch-clamp studies of GnRH neurons in brain slices from mice at two times considered negative feedback (diestrous PM and proestrous AM) and during positive feedback (proestrous PM). GABAergic transmission can excite GnRH neurons and was higher in cells from proestrous PM mice than cells from proestrous AM mice and approached traditional significance levels relative to cells from diestrous PM mice. Action potential response to current injection was also greater in cells from proestrous PM mice than the other two groups. Interestingly, the hormonal milieu of proestrous AM provided stronger negative feedback on both GnRH neuron excitability and GABAergic postsynaptic current (PSC) amplitude than diestrous PM. These observations demonstrate elements of both synaptic and intrinsic properties of GnRH neurons are regulated in a cycle-dependent manner and provide insight into the neurobiological mechanisms underlying cyclic changes in neuroendocrine function among states of estradiol negative and positive feedback.

Keywords: GABA; GnRH; action potential; estradiol; excitability; feedback.

Figure 1.

GABAergic sPSC frequency is highest on proestrous PM. A, Representative sPSC recording from a neuron in each group. B, Individual values and mean ± SEM of spontaneous GABAergic PSC frequency for cells recorded on diestrous (di) PM, proestrous (pro) AM and pro PM (Kruskal–Wallis, KW = 14.4, *p < 0.05 Dunn’s). C, Mean by-cell cumulative probability distribution of interevent interval (IEI) for each group (Kruskal–Wallis, KW = 191, *p < 0.0001, Dunn’s). D, By-cell average of all sPSC from all cells in each group. E, Individual values and mean ± SEM of sPSC amplitude (ANOVA F_(2,33) = 6.69, *p < 0.05, **p < 0.005 Tukey). F, Histogram of mean by-cell sPSC amplitude distribution (Kruskal–Wallis, KW = 23.9, proestrous AM vs both diestrous PM and proestrous PM, *p < 0.001, Dunn’s). G, Individual values and mean ± SEM of sPSC time decay time between 90% and 10% of the maximum event amplitude (ANOVA F_(2,33) = 1.34).

Figure 2.

Blocking action potentials does not affect GABAergic PSC frequency or amplitude in diestrous or proestrous mice. A, Representative recordings from a representative neuron in each group before (control or con, top) and during (bottom) TTX treatment (from n = 6 cells diestrous PM, n = 5 cells proestrous PM). B, Individual values and mean ± SEM of GABAergic PSC frequency. C, Average of all PSC traces for control or ttx periods from all cells in each group. D, E, Individual values and mean ± SEM for: (D) PSC amplitude, (E) decay time between 90% and 10% of the maximum current amplitude. No statistical differences were detected using two-way repeated-measures ANOVA/Bonferroni test (B, cycle stage: F_(1,9) = 1.3; TTX: F_(1,9) = 1.6; cycle stage × TTX: F_(1,9) = 0.0; D, cycle stage: F_(1,9) = 0.3; TTX: F_(1,9) = 0.6; cycle stage × TTX: F_(1,9) = 0.5; E, cycle stage: F_(1,9) = 0.5; TTX: F_(1,9) = 6.4 (p = 0.01); cycle stage × TTX: F_(1,9) = 0.9).

Figure 3.

GnRH neuron excitability is increased on proestrus versus diestrus. A, Representative traces from a neuron in each group during 500-ms current injections of 12 and 24 pA (current injection protocol below). B, Mean ± SEM spikes elicited for each current injection step (two-way repeated-measures ANOVA cycle stage: F_(2,22) = 10.2, current: F_(15,330) = 93.03, interaction: F_(30,330) = 9.503, *p < 0.05 diestrous PM vs proestrous PM and p < 0.05 proestrous AM vs proestrous PM; *p < 0.05 among all three groups, Fisher’s LSD). C–H, Individual values and mean ± SEM for: (C) rheobase current (ANOVA F_(2,22) = 12.8, *p < 0.05, **p < 0.0001), (D) latency to first spike (ANOVA F_(2,22) = 2.85, p = 0.0792), (E) action potential threshold (ANOVA F_(2,22) = 6.18, *p < 0.01 Tukey), (F) action potential amplitude (ANOVA, F_(2,22) = 0.676), (G) FWHM (ANOVA F_(2,22) = 26.2, **p < 0.0001 Tukey), (H) action potential rate of rise (Kruskal–Wallis, KW = 6.69, *p < 0.05 Dunn’s), (I) AHP amplitude (ANOVA F_(2,22) = 0.252), and (J) AHP time (Kruskal–Wallis, KW = 7.03, *p < 0.05 Dunn’s).