17β-Estradiol increases persistent Na(+) current and excitability of AVPV/PeN Kiss1 neurons in female mice

Abstract

In vitro slice studies have revealed that there are significant differences in the spontaneous firing activity between anteroventral periventricular/periventricular preoptic nucleus (AVPV/PeN) and arcuate nucleus (ARC) kisspeptin (Kiss1) neurons in females. Although both populations express similar endogenous conductances, we have discovered that AVPV/PeN Kiss1 neurons express a subthreshold, persistent sodium current (INaP) that dramatically alters their firing activity. Based on whole-cell recording of Kiss1-Cre-green fluorescent protein (GFP) neurons, INaP was 4-fold greater in AVPV/PeN vs ARC Kiss1 neurons. An LH surge-producing dose of 17β-estradiol (E2) that increased Kiss1 mRNA expression in the AVPV/PeN, also augmented INaP in AVPV/PeN neurons by 2-fold. Because the activation threshold for INaP was close to the resting membrane potential (RMP) of AVPV/PeN Kiss1 neurons (-54 mV), it rendered them much more excitable and spontaneously active vs ARC Kiss1 neurons (RMP = -66 mV). Single-cell RT-PCR revealed that AVPV/PeN Kiss1 neurons expressed the requisite sodium channel α-subunit transcripts, NaV1.1, NaV1.2, and NaV1.6 and β subunits, β2 and β4. Importantly, NaV1.1α and -β2 transcripts in AVPV/PeN, but not ARC, were up-regulated 2- to 3-fold by a surge-producing dose of E2, similar to the transient calcium current channel subunit Cav3.1. The transient calcium current collaborates with INaP to generate burst firing, and selective blockade of INaP by riluzole significantly attenuated rebound burst firing and spontaneous activity. Therefore, INaP appears to play a prominent role in AVPV/PeN Kiss1 neurons to generate spontaneous, repetitive burst firing, which is required for the high-frequency-stimulated release of kisspeptin for exciting GnRH neurons and potentially generating the GnRH surge.

Figure 1.

I_NaP in AVPV/PeN and ARC Kiss1 neurons. A, Representative recordings showing the relationship between I_NaP and voltage ramps (speeds from 20 mV/sec to 100 mV/sec) from an AVPV/PeN Kiss1 neuron. B and C, Voltage clamp recordings showing I_NaP generated by a voltage ramp from −80 to −20 mV at 20 mV/sec before (black trace; control) and after (red trace) application of TTX (1 μM) in representative AVPV/PeN (B) and ARC (C) Kiss1 neurons. The black straight line indicates the linear part of the whole-cell current in the presence of TTX. The horizontal stippled line indicates the zero current level. The vertical stippled line marks the resting membrane potential, and the arrow points to activation threshold for the I_NaP. D, Comparison of I_NaP density measured at −45 mV in AVPV/PeN Kiss1 neurons and ARC Kiss1 neurons from high-E2 animals. **, P < .01, n = 9–14; Student's t test.

Figure 2.

High estradiol treatment increased I_NaP in AVPV/PeN Kiss1 neurons but not in ARC Kiss1 neurons. A, I_NaP current in AVPV/PeN Kiss1 neurons is increased in high-E2 as compared with low-E2 animals. **, P < .01, Student's t test. B, qPCR measurements of Na_V1.1α and Na_Vβ2 mRNAs in AVPV/PeN Kiss1 neuronal pools (three pools per animal) from low-E2 and high-E2 OVX mice (n = 5 animals in each group). **, P < .01, n = 10–14; Student's t test. The gel figures in panels A and B illustrate that AVPV/PeN GFP neurons from low-E2 Kiss1 animals harvested during recording in the slice (A) or after dispersion (B) are Kiss1 positive. C, There was no difference in the magnitude of I_NaP in ARC Kiss1 neurons between high-E2 and low-E2 animals. D, In ARC Kiss1 neurons, Kiss1 mRNA expression was decreased with high-E2 treatment vs vehicle treatment; however, Na_V1.1α and Na_Vβ2 mRNA expression from high-E2 groups were not different from vehicle-treated OVX females. ***, P < .001, n = 5; Student's t test.

Figure 3.

Riluzole blocked I_NaP and inhibited firing in AVPV/PeN Kiss1 neurons. A, Representative recordings showing that treatment with riluzole (10 μM) for 10 minutes inhibited the I_NaP in an AVPV/PeN neuron. The inset shows that the I_NaT was only slightly inhibited (<10%) by riluzole. B, Representative recording showing that riluzole inhibited the spontaneous firing of an AVPV/PeN neuron by 90%. C, The current-induced firing before and after exposure to riluzole. The current injection protocol (20 pA, 1 sec duration) is illustrated at the bottom. D, Summary of the inhibitory effects of riluzole (illustrated in panel C) on the mean firing rate. **, P < .01, n = 6; Student's t test.

Figure 4.

I_NaP modulates T-type calcium channel-mediated rebound burst firing. A, qPCR measurements of Ca_V3.1 mRNA in AVPV/PeN Kiss1 neuronal pools (three pools per animal) from low-E2 and high-E2 mice (n = 5 animals in each group). **, P < .01, Student's t test. B, Representative recordings showing that in control conditions hyperpolarizing a cell to −95 mV for 1 second from −60 mV induced rebound burst firing with four action potentials (upper trace); however, in the presence of riluzole (10 μM), the same hyperpolarization from −60 mV induced rebound burst firing with only two action potentials, and the interspike interval from the first to the second action potential increased from 12 milliseconds to 18 milliseconds. The inset (middle trace) shows that riluzole had no effect on the T-type calcium current amplitude. C and D, Summary of the effects of riluzole on the number of action potentials (spikes) in the rebound burst and interspike intervals. **, P < .01, n = 6; Student's t test.

Figure 5.

E2 increases the excitability of AVPV/PeN Kiss1 neurons. A and B, The spontaneous firing rate of Kiss1 neurons was increased in high-E2 vs low-E2 animals in the presence of ionic glutamatergic and GABAergic receptor blockers (10 μM CNQX, 50 μM AP-5, and 100 μM picrotoxin). C, A representative recording showing that E2-treated ARC Kiss1 neurons are silent in the presence of ionic glutamatergic and GABAergic receptor blockers. D, Summary of the effects E2 treatment on the spontaneous firing of AVPV/PeN and ARC Kiss1 neurons. *, P < .05; ***, P < .001, Student's t test.