Estradiol induces diurnal shifts in GABA transmission to gonadotropin-releasing hormone neurons to provide a neural signal for ovulation

Abstract

Ovulation is initiated by a surge of gonadotropin-releasing hormone (GnRH) secretion by the brain. GnRH is normally under negative feedback control by ovarian steroids. During sustained exposure to estradiol in the late follicular phase of the reproductive cycle, however, the feedback action of this steroid switches to positive, inducing the surge. Here, we used an established ovariectomized, estradiol-treated (OVX+E) mouse model exhibiting daily surges to investigate the neurobiological mechanisms underlying this switch. Specifically, we examined changes in GABA transmission to GnRH neurons, which can be excited by GABA(A) receptor activation. Spontaneous GABAergic postsynaptic currents (PSCs) were recorded in GnRH neurons from OVX+E and OVX mice in coronal and sagittal slices. There were no diurnal changes in PSC frequency in cells from OVX mice in either slice orientation. In OVX+E cells in both orientations, PSC frequency was low during negative feedback but increased at surge onset. During the surge peak, this increase subsided in coronal slices but persisted in sagittal slices. Comparison of PSCs before and during tetrodotoxin (TTX) treatment showed TTX decreased PSC frequency in OVX+E cells in sagittal slices, but not coronal slices. This indicates estradiol acts on multiple GABAergic afferent populations to increase transmission through both activity-dependent and -independent mechanisms. Estradiol also increased PSC amplitude during the surge. Estradiol and the diurnal cycle thus interact to induce shifts in both GABA transmission and postsynaptic response that would produce appropriate changes in GnRH neuron firing activity and hormone release.

Figure 1.

Schematic of experimental design showing times of brain slice preparation (arrows) and recording (bars) relative to LH surge induction in OVX+E mice. No surges occur in OVX mice; for OVX groups, times of slice preparation and recording were matched to times of negative feedback (red), surge onset (blue), and surge peak (green) states in OVX+E mice for comparison. The LH surge profile is adapted from mean values reported by Christian et al. (2005).

Figure 2.

GABA transmission to GnRH neurons does not change in a diurnal manner in OVX mice. A, B, Representative sPSC recordings in coronal (A) and sagittal (B) slices during negative feedback (top; n = 13 cells, coronal; n = 12 sagittal), surge onset (middle; n = 13 coronal; n = 12 sagittal), and surge peak (bottom; n = 13 coronal; n = 14 sagittal).

Figure 3.

GABA transmission to GnRH neurons increases during the GnRH/LH surge in an estradiol-dependent manner. A, B, Representative sPSC recordings from cells in coronal (A) and sagittal (B) slices from OVX+E mice during negative feedback (top; n = 18 cells coronal; n = 15 sagittal), surge onset (middle; n = 38 coronal; n = 28 sagittal), and surge peak (bottom; n = 18 coronal; n = 26 sagittal). C, D, Mean + SEM sPSC frequency in OVX+E cells in coronal (C) and sagittal (D) slices. Negative feedback (−FB) values in each slice orientation serve as control. *p < 0.05.

Figure 4.

Estradiol decreases GABA transmission to GnRH neurons during negative feedback but increases it during surge onset and peak. A, Mean + SEM of sPSC frequency in cells from OVX (open bars) and OVX+E (filled bars) mice during negative feedback (−FB), surge onset, and surge peak (coronal and sagittal values combined). B–D, Estradiol lengthens sPSC interevent interval during negative feedback but shortens it during surge onset and peak. Cumulative probability distributions for interevent interval during negative feedback (OVX+E, n = 1232 events; OVX, n = 1671) (B), surge onset (OVX+E, n = 4985; OVX, n = 1513) (C), and surge peak (OVX+E, n = 3198; OVX, n = 1488) (D). *p < 0.05.

Figure 5.

Approximately one in three GnRH neurons from OVX+E mice receive estradiol-induced increased GABA transmission during the surge, and this effect correlates with cell location. A, B, PSC frequency in all individual cells from OVX+E mice plotted versus the time of recording in coronal (A) and sagittal (B) slices. C, D, Approximate location of all GnRH neurons from OVX+E mice recorded during surge onset (coronal and sagittal) and surge peak (sagittal only). Slice images adapted from Paxinos and Franklin (2001). Coronal slices are displayed from rostral (left) to caudal (right), and sagittal slices are displayed medial (left) to lateral (right). The filled circles indicate cells with PSC frequency >0.5 Hz during surge onset (coronal and sagittal) and surge peak (sagittal only). The blue circles are cells recorded during surge onset, and the green circles are cells recorded during surge peak. E, F, Percentage of cells in each slice position that showed PSC frequency >0.5 Hz in coronal (E) and sagittal (F) slices.

Figure 6.

Estradiol increases activity of some GABAergic afferents during the GnRH/LH surge. A–D, Representative traces of PSCs before (top) and during (bottom) 0.5 μm TTX treatment from an OVX+E cell with baseline PSC frequency >0.5 Hz (A, sagittal; B, coronal), an OVX+E cell with baseline <0.5 Hz (C), and an OVX cell (D). E, PSC frequency in individual GnRH neurons before (con) and during (TTX) in vitro TTX treatment. Values from OVX+E cells in sagittal slices (n = 12) are on the left, OVX+E cells in coronal slices (n = 8) are in the middle, and OVX cells (n = 11) are on the right. The filled circles indicate cells that showed >30% decrease in PSC frequency in TTX. F, Percentage change in PSC frequency from control after in vitro TTX treatment. *p < 0.05 versus control in respective hormone milieu and slice orientation.

Figure 7.

Razor blade cut in between SCN and AVPV alters GABA transmission to GnRH neurons during surge onset-peak. A, Location of razor blade cut (orange line) on a midsagittal section in relation to approximate locations of the SCN, AVPV (arrows), and the area containing GnRH neurons (green dashed circle). B, C, SCN cut lowers GABAergic sPSC frequency (B) and percentage of cells with sPSC frequency >0.5 Hz (C) in midsagittal slices (n = 9 cells) but has no effect in parasagittal slices (n = 17). D, PSC frequency in individual GnRH neurons before (con) and during (TTX) in vitro TTX treatment. No cells exhibiting high control frequency (n = 5 of 15) responded to TTX. *p < 0.05 versus intact slices in respective slice position.

Figure 8.

Estradiol modulates amplitude of GABAergic PSCs. A, B, Averaged sPSC (A) and mPSC (B) traces from representative OVX+E (left) and OVX (right) cells during negative feedback (red), surge onset (blue), and surge peak (green). C, Cumulative probability distribution for mPSC amplitude comparing events in OVX+E cells during negative feedback (n = 205 events) and combined surge onset and peak times (n = 1104). D, Distributions comparing OVX and OVX+E events during negative feedback (top; OVX, n = 333) and combined surge onset and surge peak times (bottom; OVX, n = 758). *p < 0.05 versus −FB (C) and OVX (D).