Characteristics of sodium currents in rat geniculate ganglion neurons

Abstract

Geniculate ganglion (GG) cell bodies of chorda tympani (CT), greater superficial petrosal (GSP), and posterior auricular (PA) nerves transmit orofacial sensory information to the rostral nucleus of the solitary tract. We have used whole cell recording to investigate the characteristics of the Na(+) channels in isolated Fluorogold-labeled GG neurons that innervate different peripheral receptive fields. GG neurons expressed two classes of Na(+) channels, TTX sensitive (TTX-S) and TTX resistant (TTX-R). The majority of GG neurons expressed TTX-R currents of different amplitudes. TTX-R currents were relatively small in 60% of the neurons but were large in 12% of the sampled population. In a further 28% of the neurons, TTX completely abolished all Na(+) currents. Application of TTX completely inhibited action potential generation in all CT and PA neurons but had little effect on the generation of action potentials in 40% of GSP neurons. Most CT, GSP, and PA neurons stained positively with IB(4), and 27% of the GSP neurons were capsaicin sensitive. The majority of IB(4)-positive GSP neurons with large TTX-R Na(+) currents responded to capsaicin, whereas IB(4)-positive GSP neurons with small TTX-R Na(+) currents were capsaicin insensitive. These data demonstrate the heterogeneity of GG neurons and indicate the existence of a subset of GSP neurons sensitive to capsaicin, usually associated with nociceptors. Since there are no reports of nociceptors in the GSP receptive field, the role of these capsaicin-sensitive neurons is not clear.

Fig. 1.

Whole cell Na⁺ currents in geniculate ganglion (GG) neurons. A–C: representative examples of total (left), tetrodotoxin (TTX)-resistant (TTX-R; middle), and TTX-sensitive (TTX-S; right) Na⁺ current traces activated by depolarizing step pulses (50 ms) from a holding potential of −90 mV to test potentials from −70 to 60 mV in 10-mV increments as shown at bottom left in C. TTX-S Na⁺ currents were obtained by digitally subtracting the TTX-R Na⁺ currents from the total Na⁺ currents before TTX (0.3 μM) application. A: in 60% of GG neurons, TTX caused a large reduction of Na⁺ currents, but small TTX-R currents still remained. B: 12% of GG neurons expressed relatively large TTX-R currents. C: TTX completely abolished the Na⁺ currents in 28% of GG neurons.

Fig. 2.

Mean whole cell Na⁺ currents at different test potentials in different subpopulations of GG neurons. Peak current densities of total (A), TTX-R (B), and TTX-S (C) Na⁺ currents were plotted against membrane potentials. The total and TTX-S Na⁺ current densities for posterior auricular (PA) neurons displayed a prominent enhancement at test potentials of near −20 mV compared with chorda tympani (CT) and greater superficial petrosal (GSP) neurons, whereas the TTX-R Na⁺ current density was significantly larger in GSP neurons than in other subpopulations of GG neurons at test potentials greater than −10 mV. Data are means ± SE. *P < 0.05; **P < 0.01 indicate significant values by ANOVA. V_t, test potential.

Fig. 3.

Separation of TTX-R Na⁺ current into low- and high-threshold components. A: total TTX-R Na⁺ currents evoked with 50-ms voltage steps from a holding potential of −90 mV to test potentials from −70 to 60 mV in 10-mV increments in the presence of 0.3 μM TTX. Fluoride was the primary anion in the internal solution in this set of experiments. B: the high-threshold components yielded by a 500-ms prepulse to −60 mV before voltage steps. C: the low-threshold components revealed by digital subtraction of the high-threshold components from the total TTX-R currents. Voltage protocols used to activate Na⁺ currents are shown below the current traces in A and B, respectively.

Fig. 4.

Frequency distributions of TTX-R and TTX-S Na⁺ current densities in CT, GSP, and PA neurons. TTX-R (left) and TTX-S (right) Na⁺ current densities were calculated by dividing the current amplitude (pA) at a test potential of −10 mV by the cell capacitance (pF) in 24 CT (A), 24 GSP (B), and 28 PA neurons (C). Whereas 38% (9 of 24 neurons) of GSP neurons expressed large TTX-R currents of >60 pA/pF, all CT and 96% (27 of 28 neurons) of PA neurons displayed small TTX-R currents of <60 pA/pF.

Fig. 5.

Effect of TTX on action potentials in different subpopulations of GG neurons. Membrane potentials were recorded by injection of hyperpolarizing and depolarizing current pulses from −0.4 nA for 500 ms with 0.1-nA increments before (left) and after (middle) 0.3 μM TTX application. A: TTX eliminated the generation of action potentials in all CT (n = 19) and PA neurons (n = 15). TTX also completely abolished the spike firing in the majority of GSP neurons (12/20, 60%). The effect of TTX was reversed after washout (right). B: TTX had little effect on the generation of action potentials in 40% (8/20) of GSP neurons but increased the threshold of the spike firing. The firing returned partially after washout (right).

Fig. 6.

TTX-R Na⁺ current expression and capsaicin sensitivity in isolectin B₄ (IB₄)-positive and -negative GG neurons. A: identification of IB₄-positive and IB₄-negative GG neurons: a, dissociated GG neurons viewed with bright-field illumination; b, same-field fluorescence image of Fluorogold-labeled neurons with ultraviolet excitation filter; c: same field of IB₄-labeled neurons with FITC. The Fluorogold-labeled, IB₄-positive neuron is indicated by the arrowheads. Scale bar, 50 μm. B–D: total (left) and TTX-R Na⁺ currents (middle) were obtained from IB₄-positive and IB₄-negative GG neurons using the same protocols as shown in Fig. 1. At completion of Na⁺ current recordings, 10 μM capsaicin was applied in voltage-clamp mode (right). B: example of an IB₄-positive GSP neuron with small TTX-R Na⁺ currents of <60 pA/pF at a test potential of 0 mV. This type of neuron did not respond to focal application of 10 μM capsaicin. C: example of an IB₄-positive GSP neuron with large TTX-R Na⁺ currents of >60 pA/pF. This type of neuron was capsaicin sensitive, and an inward current was elicited. D: example of an IB₄-negative GSP neuron. In this type of neuron, TTX caused a large reduction of total Na⁺ currents. These neurons were capsaicin insensitive. All CT and PA neurons with and without TTX-R currents also did not respond to capsaicin.

Fig. 7.

Comparison of peak TTX-R Na⁺ current density among 3 subgroups of GSP neurons categorized by IB₄ immunoreactivity and capsaicin sensitivity. GSP neurons were subdivided into 3 groups: IB₄ positive and capsaicin sensitive [IB₄(+) Cap(+)], IB₄ positive and capsaicin insensitive [IB₄(+) Cap(−)], and IB₄ negative and capsaicin insensitive [IB₄(−) Cap(−)]. There were significant differences in TTX-R and TTX-S Na⁺ current densities between subgroups of GSP neurons (ANOVA: P < 0.01). Data are means ± SE. *P < 0.05; **P < 0.01 indicate significant values by ANOVA, followed by Scheffé's host hoc tests.