Physiologic regulation of a tetrodotoxin-sensitive sodium influx that mediates a slow afterdepolarization potential in gonadotropin-releasing hormone neurons: possible implications for the central regulation of fertility

Abstract

The brain controls fertility through release of gonadotropin-releasing hormone (GnRH), but the mechanisms underlying action potential patterning and GnRH release are not understood. We investigated whether GnRH neurons exhibit afterdepolarizing potentials (ADPs) and whether these are modified by reproductive state. Whole-cell current-clamp recordings of GnRH neurons in brain slices from ovariectomized mice revealed a slow ADP (sADP) after action potentials generated by brief current injection. Generating two or four spikes enhanced sADP amplitude and duration. sADP amplitude was not affected by blocking selected neurotransmitter/neuromodulator receptors, delayed-rectifier potassium channels, calcium-dependent cation channels, or hyperpolarization-activated cation channels but was halved by the calcium channel blocker cadmium and abolished by tetrodotoxin. Cadmium also reduced peak latency. Intrinsic mechanisms underlying the sADP were investigated using voltage-clamp protocols simulating action potential waveforms. A single action potential produced an inward current, which increased after double and quadruple stimulation. Cadmium did not affect current amplitude but reduced peak latency. Pretreatment with blockers of calcium-activated potassium currents (I(KCa)) reproduced this shift and blocked subsequent cadmium-induced changes, suggesting cadmium changes latency indirectly by blocking I(KCa). Tetrodotoxin abolished the inward current, suggesting that it is carried by sodium. In contrast, I(KCa) blockers increased the inward current, indicating that I(KCa) may oppose generation of the sADP. Strong sADPs were suprathreshold, generating repetitive spontaneous firing. I(ADP), sADP, and excitability were enhanced by in vivo estradiol, which triggers a preovulatory surge of GnRH release. Physiological feedback modification of this inward current and resulting sADP may modulate action potential firing and subsequent GnRH release.

Figure 1.

Firing properties and afterdepolarization in GnRH neurons. A, GFP signal (left) and infrared differential interference contrast image (right) of a GFP-identified GnRH neuron. B, Stereotypical response of a GnRH neuron to current injection between pulse and surge modes of secretion that is critical to reproductive success. C, Spontaneous action potentials are followed by ADPs (below, spikes truncated). Dashed line indicates prespike baseline. D, Brief current injections are terminated before the evoked spike begins. Bottom, Current injection protocol; top, membrane potential response. E, Brief current injection triggers action potentials followed by an sADP; inset shows expanded scale (spikes truncated in inset).

Figure 2.

sADPs are dependent on the number of preceding spikes and membrane potential. A, Representative example of sADP after one (top), two (middle), or four (bottom) preceding spikes. Data are aligned by last induced spike. Dashed line shows prespike baseline. B, Expansion of sADP from A; spikes are truncated. The number near the trace indicates number of preceding spikes. C–E, Mean ± SEM sADP amplitude, duration, and latency, respectively, as a function of spike number. *p < 0.05. F, Representative example of sADP after two spikes in an individual cell in which membrane potential was adjusted to the values shown on the left by current injection. Spikes are truncated; dashed lines show prespike baseline. G, Plot of sADP amplitude versus membrane potential for all 10 cells tested.

Figure 3.

sADPS are not altered by selected neurotransmitter and neuromodulator receptor blockers. A–C, Representative examples of sADP before and after treatment with receptor blockers (A, 20 μm APV plus 20 μm CNQX; B, 20 μm bicuculline; C, 20–40 nm d-P-Glu). Spikes are truncated; dashed lines show prespike baseline. D, Mean ± SEM change in sADP amplitude in response to receptor blockade. Con, Control.

Figure 4.

Effect of channel blockers on the sADP. A, sADP recorded before (black) and after (gray) blocking I_h with ZD7288 (40 μm). B, sADP recorded before (black) and after (gray) blocking I_K with TEA (10 μm). C, D, Absolute amplitude of the sADP is decreased by application of cadmium (Cd²⁺, 200 μm) to block calcium channels. E, F, Cadmium reduces ADP amplitude (E) and latency (F); each individual value is shown with horizontal line indicating mean). G, Cadmium also increases amplitude of the AHP relative to prespike baseline. Spikes are truncated; dashed lines show prespike baseline. *p < 0.05; **p < 0.001. con, Control.

Figure 5.

Blocking specific sodium conductances eliminates sADP. A, Two brief depolarizing current pulses (300 pA, 3 ms) evokes two spikes followed by an sADP. B, TTX (0.5 μm) eliminates the sADP. C, Higher-amplitude current pulses (2500 pA, 3 ms) restore membrane potential changes but not sADP. D, Overlap of expanded and truncated traces from A–C. E, The persistent sodium current blocker riluzole (5–10 μm) had no effect on sADP amplitude. F, Summary of pretreatment (black) and posttreatment (hatched) sADP amplitudes by treatment. Dashed lines show prespike baseline. ZD, ZD7288; Ril, riluzole; Flu, flufenamic acid (200 μm). *p < 0.001.

Figure 6.

An inward current underlies the sADP. A, Square pulses (left) or simulated action potentials (middle) were used as voltage commands to elicit the current underlying the sADP. Right shows comparison of simulated action potential (black) versus actual action potential (gray). B, Inward current (I_ADP) is elicited by voltage commands shown in A. B, C, Increasing the number of simulated spikes increases the current. D, Top, Voltage protocol to estimate input resistance before, during, and after induced I_ADP. Bottom, Corresponding current trace. E, Overlap of current trace from before and during I_ADP showing increased steady-state conductance during I_ADP. F, Changes in I_steady-state in all cells tested illustrating the increase in current and return to precommand levels. *p < 0.001.

Figure 7.

Effect of channel blockers on I_ADP. A, TEA (10 μm) does not alter I_ADP. B–D, Cadmium (200 μm) does not affect amplitude of I_ADP (B, C) but does reduce latency. C and D show each individual cell with horizontal line indicating the mean. *p < 0.05. sADP and I_ADP are TTX sensitive. con, Control. E, F, Current-clamp (E) and voltage-clamp (F) recordings of the same cell before (left) and after (right) application of TTX. G, H, Summary of effect of channel blockers on I_ADP latency (G) and amplitude (H). IbTx, 100 μm iberiotoxin; apa, 200 μm apamin; Rx, treatment. *p < 0.05; **p < 0.001.

Figure 8.

sADPs contribute to repeat firing in GnRH neurons, and both sADPs and the resulting firing are enhanced by estradiol. A, Spontaneous action potentials can occur during the sADP. B, Superimposition of sADP in representative cells (V_m of −58 to −62 mV) from OVX and OVX plus estradiol mice showing the increased magnitude induced by estradiol (measured at 200 ms). C, Current-clamp recordings showing that induction of a pair of spikes by brief current injection elicits spontaneous spikes during the subsequent sADP and that this is enhanced by estradiol treatment in both the morning and evening. Ten traces from each cell are superimposed. D, Effect of estradiol on spikes generated during the sADP. E, Summary of estradiol effects on sADP amplitude, latency, and duration and I_ADP amplitude. *p < 0.05; ^#p < 0.01, unpaired analyses. E, Estradiol; AM, morning; PM, evening.