Diurnal in vivo and rapid in vitro effects of estradiol on voltage-gated calcium channels in gonadotropin-releasing hormone neurons

Abstract

A robust surge of gonadotropin-releasing hormone (GnRH) release triggers the luteinizing hormone surge that induces ovulation. The GnRH surge is attributable to estradiol feedback, but the mechanisms are incompletely understood. Voltage-gated calcium channels (VGCCs) regulate hormone release and neuronal excitability, and may be part of the surge-generating mechanism. We examined VGCCs of GnRH neurons in brain slices from a model exhibiting daily luteinizing hormone surges. Mice were ovariectomized (OVX), and a subset was treated with estradiol implants (OVX+E). OVX+E mice exhibit negative feedback in the A.M. and positive feedback in the P.M. GnRH neurons express prominent high-voltage-activated (HVA) and small low-voltage-activated (LVA) macroscopic (whole-cell) Ca currents (I(Ca)). LVA-mediated currents were not altered by estradiol or time of day. In contrast, in OVX+E mice, HVA-mediated currents varied with time of day; HVA currents in cells from OVX+E mice were lower than those in cells from OVX mice in the A.M. but were higher in the P.M. These changes were attributable to diurnal alternations in L- and N-type components. There were no diurnal changes in any aspect of HVA-mediated I(Ca) in OVX mice. Acute in vitro treatment of cells from OVX and OVX+E mice with estradiol rapidly increased HVA currents primarily through L- and R-type VGCCs by activating estrogen receptor beta and GPR30, respectively. These results suggest multiple mechanisms contribute to the overall feedback regulation of HVA-mediated I(Ca) by estradiol. In combination with changes in synaptic inputs to GnRH neurons, these intrinsic changes in GnRH neurons may play critical roles in estradiol feedback.

Figure 1.

Adult GnRH neurons exhibit limited LVA calcium current. A, B, Representative of calcium current recorded at different membrane potentials in GnRH and non-GnRH neuron, respectively. C, D, Average current–voltage curves of peak and sustained current from GnRH and non-GnRH neurons. E, Subtraction of calcium current recorded with a −50 mV prepulse from current recorded with a −100 mV prepulse does not reveal LVA-mediated current. F, Representative tail current and time course of tail current (τ₁ and τ₂) from GnRH neuron (top) and non-GnRH neuron (middle), and mean ± SEM time constants (bottom). *p < 0.05; **p < 0.01.

Figure 2.

LVA-mediated currents function in GnRH neurons but are not altered in a diurnal model of estradiol positive and negative feedback. A, Representative average of 50 repeats of a voltage protocol to reveal LVA current (top) and its blockade by Ni²⁺. B, LVA-mediated current does not change with estradiol or time of day in this model. Error bars indicate SEM. C–G, Differentiation of fast and slow rebound potential and current in GnRH neurons. C, D, Representative current-clamp recordings of the slow (C) and fast (D) rebound potential generated by termination of a 50 pA hyperpolarizing current injection. E, ZD7288 (right) inhibits the slow but not the fast rebound potential (p < 0.05). F, Voltage-clamp recording of a cell exhibiting both fast and slow rebound current. Cell was stepped from a holding potential of −50 mV to the potentials indicated on the left for 1.2 s, and then returned to −50 mV. G, Voltage dependence of the slow (open symbols) and fast (closed symbols) rebound current. H, Representative current-clamp recording of GnRH neuron with a resting potential near the average and that did not exhibit rebound depolarization. I, Representative example of a GnRH neuron with a depolarized resting potential that did not exhibit rebound depolarization. J, Left, Representative current-clamp recording of a GnRH neuron with a depolarized resting potential that exhibited a rebound potential after termination of hyperpolarizing current injections. In this example, the rebound potential contributes to action potential generation, which is blocked by TTX (right). K, Nickel (100 μm) completely blocked the rebound potential in GnRH neurons. L, Depolarizing GnRH neurons (right) that exhibit hyperpolarized resting membrane potential does not induce rebound potential.

Figure 3.

HVA-mediated I_Ca in GnRH neurons is modulated in an estradiol-dependent diurnal manner. A, B, Current–voltage plots of peak and sustained current density from OVX and OVX+E mice in the A.M. and P.M. [*p < 0.05, OVX vs OVX+E in the A.M.; ^#p < 0.05, OVX vs OVX+E in the P.M.; **p < 0.01, OVX+E A.M. vs OVX+E P.M. (not marked in A as all values are significant)]. C, D, Activation and steady-state inactivation curves from OVX and OVX+E mice in the A.M. and P.M. E–H, Mean ± SEM V_1/2act (E), V_1/2inact (F), rise time (10–90%) (G), and decay time (90–50%) (H) from OVX and OVX+E mice in the A.M. and P.M.

Figure 4.

Temperature alters decay time and peak current but does not alter in vivo estradiol-dependent diurnal modulation of calcium current. A–C, Average maximal current (at +10 mV) curve of cells from OVX mice, OVX+E mice in A.M. and OVX+E mice in P.M., respectively, at 25 and 32°C (gray) and 25°C scaled to compare kinetics (scaled 25). D, The amplitude of I_Ca is greater at 32°C than 25°C in cells from both OVX and OVX+E mice, but estradiol-dependent effects persist at both temperatures. E, F, Mean ± SEM effects of temperature on V_1/2act and V_1/2inact in cells from OVX and OVX+E mice in the A.M. and (*p < 0.05, OVX vs OVX+E; ^#p < 0.05, OVX+E A.M. vs OVX+E P.M.). G, H, Mean ± SEM effects of temperature on rise time (10–90%) and decay time (90–50%) in cells from OVX and OVX+E mice in the A.M. and P.M. (*p < 0.05, OVX vs OVX+E; ^#p < 0.05, OVX+E A.M. vs OVX+E P.M.).

Figure 5.

In vivo treatment with estradiol modulates specific subtypes of HVA calcium channels. A, Example of rundown of I_Ca under control conditions. B, SNX-482 was locally applied for 10 min after a 5 min control period. The proportion of HVA subtype blocked by specific agents was assessed by the linear fit of the rundown during the first 5 min of control recording. C, D, The proportions of peak and sustained current of different subtype HVA from OVX and OVX+E mice in the A.M. and P.M. (*p < 0.05 A.M. vs P.M. in OVX+E mice). Error bars indicate SEM.

Figure 6.

In vitro treatment with estradiol rapidly increases I_Ca through classical receptor ERβ and GPR30. A, Representative traces show estradiol (1 nm; gray) rapidly increases I_Ca in GnRH neurons from OVX mice in both the A.M. and P.M. B, Time course of the rapid effect of estradiol (1 nm) on I_Ca. C, The percentage of cells responding to estradiol with a rapid change in I_Ca. Black bars, Cells from OVX mice treated with vehicle or estradiol 17β; white bar, cells from OVX+E mice treated with 1 nm estradiol 17β; gray bars, cells from OVX mice treated with specific receptor agonists and antagonists.

Figure 7.

In vitro treatment with estradiol potentiates different subtypes of HVA through different receptors. A, Nitrendipine (50 μm) decreased the percentage of cells responding to estradiol (1 nm). Additional treatment with conotoxin GVIA (700 nm) did not change percentage of cells responding. Nitrendipine (50 μm) blocked the effect of DPN (10 nm). B, Nitrendipine (50 μm), SNX (1 μm), or agatoxin IVA (166 nm) did not block the effect of G1 (100 nm). SNX-482 (1 μm) did not change the percentage of cells responding to G1 (100 nm). C, SNX-482 decreased the percentage increase in I_Ca in response to G1 (*p < 0.05; 0% indicates no difference from control conditions). D, The combined application of nitrendipine and SNX-482 blocked the effect of 17β-estradiol in both OVX and OVX+E mice. Error bars indicate SEM.