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. 2004 Sep 29;24(39):8480-4.
doi: 10.1523/JNEUROSCI.1598-04.2004.

Role of T-type calcium current in identified D-hair mechanoreceptor neurons studied in vitro

Anne-Sophie Dubreuil  1 , Hassan Boukhaddaoui, Gilles Desmadryl, Carlos Martinez-Salgado, Rabih Moshourab, Gary R Lewin, Patrick Carroll, Jean Valmier, Frédérique Scamps
Affiliations

Role of T-type calcium current in identified D-hair mechanoreceptor neurons studied in vitro

Anne-Sophie Dubreuil et al. J Neurosci. .

Abstract

Different subsets of dorsal root ganglion (DRG) mechanoreceptors transduce low- and high-intensity mechanical stimuli. It was shown recently that, in vivo, neurotrophin-4 (NT-4)-dependent D-hair mechanoreceptors specifically express a voltage-activated T-type calcium channel (Ca(v)3.2) that may be required for their mechanoreceptive function. Here we show that D-hair mechanoreceptors can be identified in vitro by a rosette-like morphology in the presence of NT-4 and that these rosette neurons are almost all absent in DRG cultures taken from NT-4 knock-out mice. In vitro identification of the D-hair mechanoreceptor allowed us to explore the electrophysiological properties of these cells. We demonstrate that the T-type Ca(v)3.2 channel induced slow membrane depolarization that contributes to lower the voltage threshold for action potential generation and controls spike latency after stimulation of D-hair mechanoreceptors. Indeed, the properties of the T-type amplifier are particularly well suited to explain the high sensitivity of D-hair mechanoreceptors to slowly moving stimuli.

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Figures

Figure 1.
Figure 1.
Morphological and functional identification of D-hair neurons in vitro. A, Neurofilament staining of a neuron with a rosette-like morphology induced by incubation with 10 ng/ml NT-4. Scale bar, 40 μm. B, The percentage of rosette neurons was calculated relative to the total neuronal population and was significantly reduced in NT-4-/- mutant mice. WT, Wild type. C, A ramp protocol applied to a rosette neuron from -80 to +40 mV, 500 msec duration with the whole-cell patch-clamp technique showed the presence of a high-amplitude low-voltage-activated ICa compared with high-voltage-activated ICa. This calcium current profile was observed exclusively in the rosette-like neurons. D, Dose-response curve of NiCl2 inhibition of low-voltage-activated ICa. The inhibitory effect of Ni was evaluated on calcium currents elicited with ramp protocols. Dose-response curve was fitted with a Hill equation: y = Vmax × {Ni}n/(K1/2 + {Ni})n (dashed line). From fit of the data, the Ni2+ concentration for half-maximal effect K1/2 was 2.9 ± 0.2 μm.
Figure 2.
Figure 2.
T-type ICa increases D-hair mechanoreceptor excitability. A, Depolarizing currents at 2 msec (I) of increasing amplitudes were applied to a rosette neuron to trigger an action potential (current amplitudes are given in brackets on voltage traces) under control conditions and after application of NiCl2 (b). Action potential of rosette neurons is characterized by a pronounced afterdepolarization (a) inhibited by the application of 30 μm NiCl2 (b). B, Threshold current for an action potential generation in individual rosette neurons is significantly increased after inhibition of T-type ICa with 30 μm NiCl2 (n = 6; paired t test; p = 0.01) or 10 μm mibefradil (n = 6; paired t test; p = 0.02). In non-rosette neurons, threshold current was not changed after application of 30 μm NiCl2 (n = 6; paired t test; p = 0.1). C, In rosette neurons, the latency for action potential generation was measured as the time between peak voltage induced by current injection and fast spike potential. Under control conditions, spike latency was dependent on current amplitude, with longer latency observed with smallest current amplitudes. In 30 μm NiCl2-treated rosette neurons, latency for spike generation was short (<1 msec) and unrelated to current amplitude (see voltage traces in A).
Figure 3.
Figure 3.
Velocity sensitivity of RA and D-hair mechanoreceptors. Recordings from 11 D-hair and 10 RA mechanoreceptors (n = 4 mice) indicate that D-hair mechanoreceptors are exquisitely sensitive to movement, in particular, very slow movements (1.5-100 μm/sec). In A, the mean firing rate and proportion of neurons responding (inset) to a series of 100 μm ramps ranging from 1.5 to 3000 μm/sec is shown for D-hair (black filled circles) and RA (black filled squares) mechanoreceptors. The latency for the first mechanically evoked spike plotted against velocity is plotted separately for D-hair (C) and RA mechanoreceptors (D). In B, a schematic diagram is shown indicating how a theoretical threshold latency was calculated for each neuron. The fastest latency, which was obtained using the fastest movement, was used to extrapolate a threshold value with slower-moving stimuli with the assumption that the indentation needed to evoke the first spike is constant at different ramp on velocities. The threshold latency (open circles or squares) is therefore plotted together with the measured latency to determine whether the first spike is evoked faster or slower than the theoretical value (C, D).
Figure 4.
Figure 4.
Slow action potential induced by T-type ICa accounts for latency in D-hair neurons. A, Application of 1 μm TTX inhibited the fast spike of D-hair cell action potential (a); addition of 30 μm NiCl2 in the presence of TTX inhibited the slow depolarization (b). B, Rise time, but not maximal amplitude, of slow depolarization is voltage dependent, decreasing with increasing depolarizing current amplitude.
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