Estradiol attenuates multiple tetrodotoxin-sensitive sodium currents in isolated gonadotropin-releasing hormone neurons

Abstract

Secretion from gonadotropin-releasing hormone (GnRH) neurons is necessary for the production of gametes and hormones from the gonads. Subsequently, GnRH release is regulated by steroid feedback. However, the mechanisms by which steroids, specifically estradiol, modulate GnRH secretion are poorly understood. We have previously shown that estradiol administered to the female mouse decreases inward currents in fluorescently labeled GnRH neurons. The purpose of this study was to examine the contribution of sodium currents in the negative feedback action of estradiol. Electrophysiology was performed on GnRH neurons dissociated from young, middle-aged, or old female mice. All mice were ovariectomized; half were estradiol replaced. The amplitude of the sodium current underlying the action potential was significantly decreased in GnRH neurons from young estradiol-treated animals. In addition, in vivo estradiol significantly decreased the transient sodium current amplitude, but prolonged the sodium current inactivation time constant. Estradiol decreased the persistent sodium current amplitude, and induced a significant negative shift in peak current potential. In contrast to results obtained from cells from young reproductive animals, estradiol did not significantly attenuate the sodium current underlying the action potential in cells isolated from middle-aged or old mice. Sodium channels can modulate cell threshold, latency of firing, and action potential characteristics. The reduction of sodium current amplitude by estradiol suggests a negative feedback on GnRH neurons, which could lead to a downregulation of cell excitability and hormone release. The attenuation of estradiol regulation in peripostreproductive and postreproductive animals could lead to dysregulated hormone release with advancing age.

Figure 1. Estradiol reduces the inward ionic current underlying the action potential

(A) Action potential command waveform, consisting of multiple previously recorded action potentials in GnRH neurons. The first spike is evoked, the following spikes are spontaneous. (B) Representative current response to action potential voltage command. The current demarked by the rectangle was used for all analyses. (C) Representative current traces after TTX application (upper panel) and subtracted TTX-sensitive sodium currents (lower panel). (D) Representative traces (average of 10 records) from cells isolated from an ovariectomized (OVX) or estradiol-replaced (OVX+E) animal (D1); Cells were of similar capacitance (9–10 pF). The traces are overlapped in D2; note the attenuated current amplitude in the OVX+E animal. Traces are normalized in D3 to demonstrate similar kinetics. The scale bar in D2 applies to all traces. (E) The sodium current underlying the action potential is significantly reduced in GnRH neurons from estradiol-treated animals (OVX+E; *, p<0.002, n = 18–20). In vivo estradiol does not significantly decrease sodium current density in non-green (non-GnRH) neurons isolated from the same dissociation (p = 0.36, n = 11–13). Sodium currents in non-green neurons are significantly smaller than currents in GnRH neurons (a, p<0.001).

Figure 2. Estradiol decreases transient sodium current amplitude, but prolongs the inactivation time constant

(A) Representative current traces from cells isolated from an ovariectomized (OVX) or estradiol replaced (OVX+E) animal at step voltages from −60 mV to 60 mV for 30 ms. Cells had similar capacitances (~9 pF). (B) Current-voltage curves demonstrate that the transient sodium current amplitude is attenuated at all voltages in neurons from OVX+E animals (*, p<0.05; **, p<0.005; n = 31–36). (C) Representative normalized traces recorded at 0 mV suggest that sodium current inactivation is prolonged in neurons from estradiol-treated animals when compared to neurons from OVX animals. (D) The inactivation time constant Tau is significantly prolonged in neurons from estradiol-treated animals (*, p<0.05; **, p<0.001; n = 31–36).

Figure 3. Estradiol does not affect steady-state activation or inactivation

(A) Steady-state activation curves do not vary significantly between GnRH neurons from control (OVX) and estradiol-treated (OVX+E) animals (p>0.05, n = 31–36). (B) Representative current traces, evoked by the inactivation protocol, recorded from a cell isolated from an OVX animal. The 230-ms traces have been shortened (hash lines) to better demonstrate the current responses. The box illustrates the tail current from which inactivation data is calculated. The inactivation protocol is inset; every other step is included in both the protocol and the responses for clarity. (C) Steady-state inactivation curves do not vary significantly between GnRH neurons from control (OVX) and estradiol-treated (OVX+E) animals (p>0.05, n = 27–29).

Figure 4. Estradiol modulates the persistent sodium current

(A) Ramp protocol and corresponding average current traces in GnRH neurons from control (OVX) and estradiol-treated (OVX+E) animals (n = 10–16). Traces have been normalized. (B) Current voltage relationship of the TTX-subtracted persistent current in neurons from control and estradiol-treated animals. The current in OVX+E animals peaks at a significantly more negative voltage (p = 0.03), resulting in an attenuated peak current (p = 0.03; n=7–9).

Figure 5. GnRH neurons do not express a resurgent sodium current

Representative current responses (B) to a protocol (A) designed to evoke a resurgent sodium current in a GnRH neuron isolated from a young control (OVX) mouse. Currents were elicited by a 2-ms conditioning step to +30 mV, followed by step repolarizations (A). Under this protocol, a resurgent sodium current would be expected to peak approximately 5 ms following the conditioning step (arrow), and last tens of milliseconds (Raman and Bean, 2001). No resurgent current was evoked in GnRH neurons. The current response to the conditioning step (3.5 nA) is truncated.

Figure 6. The effect of estradiol on sodium currents is attenuated with age

(A) Representative traces (average of 10 records) from cells isolated from a 15–18 month old oavariectomized (OVX) or estradiol-replaced (OVX+E) animal. The two traces are overlapped to demonstrate the similar sodium current amplitude. The capacitance of both cells was similar (~11 pF). (B) In vivo estradiol significantly decreases sodium current density only in neurons isolated from young animals. *: p = 0.002 compared to young OVX, n = 18–20. Differences in sodium current amplitude between GnRH neurons isolated from OVX and OVX+E animals were not significant in middle-aged (MA; p = 0.60, n = 15–17) or old (p = 0.27, n = 27) animals.