Serotonergic inhibition of the T-type and high voltage-activated Ca2+ currents in the primary sensory neurons of Xenopus larvae

Abstract

The primary sensory Rohon-Beard (R-B) neurons of Xenopus larvae are highly analogous to the C fibers of the mammalian pain pathway. We explored the actions of 5-HT by studying the modulation of Ca2+ currents. In approximately 80% of the acutely isolated R-B neurons, 5-HT inhibited the high voltage-activated (HVA) currents by 16% (n = 29) and the T-type currents by 24% (n = 41). The modulation of the T-type and the HVA currents was mimicked by selective 5-HT1A and 5-HT1D agonists: 8-OH-DPAT and L-694,247. The effects of the agonists were blocked by their respective 5-HT1A or 5-HT1D antagonists: p-MPPI and GR127935, suggesting that both 5-HT1A and 5-HT1D receptors were involved. Approximately 70% of the actions of 5-HT on HVA currents was occluded by omega-conotoxin-GVIA (N-type channel blocker), whereas the rest of the modulation ( approximately 30%) was occluded by <100 nM omega-agatoxin-TK (P/Q-type channel blocker). This suggests that 5-HT acts on N- and P/Q-type Ca2+ channels. Neither the modulation of the T-type nor that of the HVA currents was accompanied by changes in their voltage-dependent kinetics. Cell-attached patch-clamp recordings suggest that the modulation of the T-type channel occurs through a membrane-delimited second messenger. We have studied the functional consequences of the modulation of T-type Ca2+ channels and have found that these channels play a role in spike initiation in R-B neurons. Modulation of T-type channels by 5-HT therefore could modulate the sensitivity of this sensory pathway by increasing the thresholds of R-B neurons. This is a new and potentially important locus for modulation of sensory pathways in vertebrates.

Fig. 1.

Both the HVA and the T-type Ca²⁺ currents were reduced by 5-HT in acutely isolated R–B neurons. A, R–B neurons possess both the HVA and the T-type Ca²⁺ currents.A₁, Whole-cell Ca²⁺ currents were recorded by using steps from a holding potential of −80 mV to test potentials between −60 and +30 mV in a stage 42 Xenopus R–B neuron.A₂, I/V curve measured from the peak (showing the T-type and HVA currents) and end (showing only the HVA currents) of the Ca²⁺ currents (symbols correspond to measurements inA₁).B₁, T-type and HVA currents were elicited in the same neuron by test steps of −30 and +10 mV, respectively, from a holding potential of −90 mV. Both were reduced by 1 μm 5-HT (shown by asterisk); unlabeled traces are control and wash. B₂, Courses of the effects of 5-HT showing that they were totally reversible on both currents in the same neuron (symbolscorrespond to measurements inB₁).

Fig. 2.

Voltage-independent inhibition of the HVA Ca²⁺ currents by 5-HT.A₁, Ca²⁺currents elicited by steps from a holding potential of −80 mV (asterisk = 1 μm 5-HT).A₂, The inhibition of HVA currents by 5-HT (1 μm) was independent of membrane potential (n = 5);I_5-HT, current in presence of 5-HT;I_Con, current in the control.B₁, Example showing that the inhibition of the HVA currents in RB neurons was not changed by applying a +120 mV prepulse (asterisk = trace elicited by test pulse with 120 mV prepulse in 1 μm5-HT). B₂, Summary showing no significant difference between the mean inhibition of the HVA currents before and after applying a 120 mV prepulse in five R–B neurons.

Fig. 3.

The inhibition of HVA Ca²⁺currents by 5-HT is dose-dependent and mediated via 5-HT_1Aand 5-HT_1D receptors.A₁, The selective 5-HT_1D agonist L-694,247 (100 nm) inhibited HVA currents. The effects of L-694,247 (100 nm) were blocked by the selective 5-HT_1D antagonist GR127935 (100 nm). A₂, The selective 5-HT_1A agonist 8-OH-DPAT (100 nm) also inhibited the HVA currents. The effects of 8-OH-DPAT (100 nm) were totally blocked by the selective 5-HT_1A antagonist p-MPPI (100 nm). B, Summary of the antagonist effects on the agonists of 8-OH-DPAT and L-694,247 (*p < 0.05 vs agonists alone). C, The inhibition of HVA currents by 5-HT was dose-dependent (n = 7–39 for each dose). The smooth line is the best-fitting Hill equation.

Fig. 4.

The identity of the calcium channels inhibited by 5-HT in RB neurons. A₁, Time courses of the effects of calcium channel blockers ω-agatoxin-TK (100 nm), ω-CgTx (1 μm), nifedipine (10 μm), and Cd²⁺ (100 μm) on the HVA currents. A₂, Calcium currents elicited by steps to 10 mV from a holding potential of −50 mV during the application of the blockers (same neuron as inA₁).A₃, Summary of the effects of calcium channel blockers on the HVA currents in R–B neurons.B₁, Recording showing that the inhibition of HVA currents by 5-HT (1 μm) was mostly occluded by ω-conotoxin (1 μm) in a R–B neuron.B₂, Recording showing that the inhibition of HVA currents by 5-HT (1 μm) was partially occluded by ω-agatoxin-TK (100 nm), whereas the remaining inhibition was totally occluded by 1 μm ω-conotoxin (asterisk = HVA currents recorded in 5-HT on top of Ca²⁺ channel blockers).B₃, Summary of the inhibition of the HVA currents by 5-HT alone and additional inhibition on top of calcium channel blockers (*p < 0.05 and **p < 0.01 vs 5-HT alone).

Fig. 5.

Inhibition of the T-type calcium currents by serotonergic agonists. A, Representative traces (asterisk represents recordings in 100 nmselective agonists; unlabeled traces are control and wash) and dose–response relations showing the block of T-type currents by selective serotonergic agonists: 5-HT (A₁), 8-OH-DPAT (A₂), and L-694,247 (A₃); n = 7–41 for each point. The solid line is the best fit of the Hill equation.A₄, The inhibition of T-type currents by L-694,247 (100 nm) and 8-OH-DPAT (100 nm) was additive. B, Summary of the mean inhibition of T-type currents by selective serotonergic agonists at maximum dose in R–B neurons: 5-HT (1 μm,n = 41), 8-OH-DPAT (100 nm,n = 23), L-694,247 (100 nm,n = 22), CGS-12066B (1 μm,n = 8), α-M-5-HT (1 μm,n = 7), and 5-CT (100 nm,n = 5) (**p < 0.01 vs control).

Fig. 6.

Effects of selective antagonists on the inhibition of T-type calcium currents by selective agonists inXenopus R–B neurons. Both p-MPPI (100 nm, A₁) and GR127935 (100 nm, A₂) partially blocked the effect of 5-HT (1 μm).A₃, p-MPPI (100 nm) and GR127935 (100 nm) produced an additive block of the effect of 5-HT (1 μm). p-MPPI blocked the effect of 8-OH-DPAT (100 nm,A₄), whereas GR127935 (100 nm) blocked the effect of L-694,247 (100 nm,A₅).B₁, Summary of the block by the selective antagonists on 8-OH-DPAT (100 nm) (**p < 0.01 vs 8-OH-DPAT).B₂, Summary of the block by antagonists on L-694,247 (100 nm) (**p< 0.01 vs L-694,247). B₃, Summary of the block by selective antagonists on 5-HT (1 μm) (**p < 0.01 vs 5-HT).

Fig. 7.

Effects of 5-HT on T-type unitary channel recordings. A₁, Consecutive unitary T-type Ba²⁺ currents were elicited by steps from potentials equivalent to the membrane potential of approximately −90 to −30 mV (after allowing for the screening effect of high Ba²⁺ levels on membrane surface charge) in a cell-attached patch. All traces are leak-subtracted.A₂, Average unitary T-type Ba²⁺ current record obtained by averaging of 50 consecutive recordings in the same patch. The solid lineis the best fit of the single exponential equation; time constant (τ) = 23.5 msec. B₁, 5-HT (1 μm) did not modulate the average (50–100 consecutive traces) unitary T-type Ba²⁺ currents recorded in cell-attached patches. In Cell A there were no changes in the T-type current. In Cell B, however, the averaged currents were reduced, but this did not reverse on washing, suggesting that the reduction may result from a “run down” of channel activity. B₂, Summary of the effects of 5-HT (1 μm) on T-type unitary channel recordings in six R–B neurons.

Fig. 8.

5-HT did not alter the kinetic characteristics of the whole-cell T-type calcium currents.A₁, Leak-subtracted T-type tail currents (shown by arrow) were elicited by a series of test pulses (−80 to −20 mV in 5 mV steps) from a holding potential of −100 mV in control. A₂, Summary showing that 5-HT (1 μm) had no effect on the activation of the T-type currents in five R–B neurons. The data were fit with the Boltzmann relation (solid line):I/I_max = {1 + exp[(V +V_½)/K]}⁻¹.Squares, Control (V_½= −37.2 mV; K = −7.6); filled circles, 5-HT (1 μm) (V_½ = −38.8 mV;K = −7.3).B₁, The T-type currents, measured at the peak, undergo steady-state inactivation.B₂, 5-HT (1 μm) had no effect on the steady-state inactivation of the T-type currents in four R–B neurons. The data were fit with the Boltzmann relation (solid line). Squares, Control (V_½ = −74.7 mV;K = 5.2); filled circles, 5-HT (1 μm) (V_½ = −75.2 mV;K = 5.4). C₁,Top left, T-type currents recorded in control and 1 μm 5-HT (shown by asterisk). Top right, The T-type currents recorded in 5-HT were scaled up to the same peak magnitude as control to show that the two traces overlap almost completely. The time-dependent inactivation of the T-type currents was measured by fitting with a single exponential (solid line). C₂, 5-HT (1 μm) had no effect on the time-dependent inactivation in three R–B neurons. Squares, Control;filled circles, 5-HT (1 μm).

Fig. 9.

Differential block of the T-type and HVA Ca²⁺ currents by Y³⁺ in R–B neurons. A, Time series measurements in a R–B neuron showing the dose–response for Y³⁺ on T-type currents (A₁) and HVA currents (A₂) recorded in the same neuron.A₃, Example of the HVA and T-type currents recorded simultaneously in the same neuron, as shown inA₁ andA₂ (asterisks= 10 nm Y³⁺). B, Summary of the effects of Y³⁺ (10 nm) on the T-type currents and HVA currents (**p < 0.01 vs control).

Fig. 10.

Effects of the selective T-type Ca²⁺ channel blockers and 5-HT on R–B neuron firing. A₁, Current-clamp recording in a R–B neuron, using steps of current injection to detect the threshold current that evoked an action potential.A₂, Y³⁺ (10 nm; asterisk), which selectively blocked T-type currents, blocked the action potential evoked at threshold current injection. B₁, Current-clamp recording from a R–B neuron showing that injection of repeated current pulses just above the threshold caused reliable firing. At resting membrane potentials more positive than −50 mV, neither Y³⁺ (10 nm) nor 5-HT (10 nm) had any effects on neuron firing.B₂, At more negative resting membrane potentials (−90 mV), both Y³⁺ (10 nm) and 5-HT (10 nm) reversibly reduced the probability of firing in the neuron. C, Summary of the effects of Y³⁺ (10 nm) and 5-HT (10 nm) on the probability of R–B neuron firing in response to repetitive threshold current injection at a resting membrane potential of −90 mV, where the probability was measured as the number of pulses that evoked a spike/total number of pulses during control, drug treatment, and wash (**p < 0.01 vs control or wash).