Slow Inactivation of Sodium Channels Contributes to Short-Term Adaptation in Vomeronasal Sensory Neurons

Spike frequency adaptation to repeated urine stimulations. A, Representative whole-cell current-clamp recordings from a VSN repetitively stimulated with diluted urine for 5 s with increasing intervals between pulses of 5, 10, 20, or 60 s, as indicated. Black bars at the top indicate the time of urine application. B, The number of spikes during the first and the second stimulation at the corresponding IPI is shown in the same row in A. The paired-pulse protocol was repeated three times in the same neuron. C, Scatter dot plot with the average ± SD of normalized spike frequency of the second stimulation with respect to the first stimulation for each IPI (n =10, Demsar’s test after Friedman test, p = 0.00029).

The duration of urine stimulation affects the extent of spike frequency adaptation. A, B, Representative whole-cell current-clamp recordings obtained stimulating VSNs with diluted urine for 2 s (A) or 10 s (B), with increasing intervals between pulses of 2, 5, 10, 20, or 60 s, as indicated. Black bars indicate the time of urine application. C, D, Scatter dot plots with the average ± SD of normalized spike frequencies for each IPI for urine pulses of 2 s (C) or 10 s (D; for C: n = 9, Demsar’s test after Friedman test (p = 0.045 for IPI 2 s; for D: n = 6; paired t test with Bonferroni correction after ANOVA for repeated measurements: p = 0.018 for IPI 2 s; p = 0.008 for IPI 5 s; and p = 0.036 for IPI 10 s).

VSNs show spike frequency adaptation in response to repeated current steps. A, Representative whole-cell current-clamp recordings of a VSN repetitively stimulated with a 5 pA current step for 5 s with increasing intervals between steps of 2, 5, 10, 20, or 60 s, as indicated. Steps at the top indicate the time of current injection. B, Number of spikes during the first and the second stimulation at the corresponding IPI shown in the same row in A. C, Scatter dot plot with the average ± SD of the normalized spike frequency of the second with respect to the first stimulation for each IPI (n = 15; Demsar’s test after Friedman test: p = 7.5 * 10^–5 for 2 s; p = 0.006 for 5 s).

The duration of current step stimulation affects the extent of spike frequency adaptation. A, B, Representative whole-cell current-clamp recordings obtained stimulating VSNs with a 5 pA current step for 2 s (A) or 10 s (B), with increasing intervals between steps of 2, 5, 10, 20, or 60 s, as indicated. Steps at the top indicate the time of current injection. C, D, Scatter dot plots with the average ± SD of normalized spike frequencies for each IPI for current steps of 2 s (C) or 10 s (D; for C: n = 13; Demsar’s test after Friedman test, p = 0.005 for IPI 2 s; for D: n = 10; paired t test with Bonferroni correction after ANOVA for repeated measurements: p = 0.0019 for IPI 2 s; p = 0.01 for IPI 5 s; p = 0.01 for IPI 10 s).

Phase–plane plot analysis of action potentials from repeated stimulations. A, Phase–plane plots of the first action potential in response to a repeated urine stimulation of 10 s duration with increasing IPIs ranging from 2 to 60 s, as indicated. Continuous and dashed lines were calculated from the first action potential of the first and second urine pulses, respectively. B–D, Scatter dot plots with the average ± SD of the ratio between the maximal dV/dt values of the first action potentials at each IPI. Urine pulse durations of 10 s (B), 5 s (C), and 2 s (D; for B: n = 6; Demsar’s test after Friedman test: p = 0.0021 for IPI 2 s; p = 0.0041 for IPI 5 s; for C: n = 6; Friedman test, p = 0.89; for D: n = 9; Friedman test, p = 0.051). E, Phase–plane plots of the first action potential in response to a repeated 5 pA current step of 10 s duration with increasing IPIs ranging from 2 to 60 s, as indicated. Continuous and dashed lines were calculated from the first action potential of the first and second current steps, respectively. F–H, Scatter dot plots with the average ± SD of the ratio between the maximal dV/dt values of the first action potentials at each IPI. Current step durations of 10 s (F), 5 s (G), and 2 s (H; for F: n = 8; Demsar’s test after Friedman test: p = 0.00054 for IPI 2 s; p = 0.018 for IPI 5 s; for H: n = 14; Friedman test, p = 0.126).

Inactivation of Na⁺ currents in VSNs. A, Families of whole-cell voltage-gated inward currents recorded from a VSN elicited by voltage steps from −80 to +60 mV with 10 mV increments from a holding potential of −100 mV. The patch pipette contained a Cs⁺-based intracellular solution. The neuron was exposed successively to bath solutions containing 100 μm Cd²⁺ and 100 μm Cd²⁺ plus 2 μm TTX, as indicated. B, Current–voltage relations for the peak inward currents from the recordings in A. C, E, Current recordings from the same VSN shown in A in response to stimulation protocols to measure inactivation in the presence of 100 μm Cd²⁺. A test pulse was preceded by a prepulse at the indicated voltages of 30 ms (C) or 30 s (E) duration to measure fast and slow inactivation, respectively. In C, one every two traces is shown. D, F, Normalized currents versus membrane potential from the experiments shown in C and E were fitted with a Boltzmann equation with V_half = −46.7 ± 1.6 mV and k = 4.9 ± 1.3 mV (n = 8) for fast inactivation (D) and V_half = −50.6 ± 2.4 mV and k = 8.7 ± 1.3 mV (n = 6) and asymptotic value of A = 0.18 for slow inactivation (F).