Neurokinins enhance excitability in capsaicin-responsive DRG neurons

Abstract

Neurokinins released by capsaicin-responsive (C-R) dorsal root ganglia neurons (DRG) may control firing in these neurons by an autofeedback mechanism. Here we used patch clamp techniques to examine the effects of neurokinins on firing properties of dissociated DRG neurons of male rats. In C-R neurons that generated only a few action potentials (APs, termed phasic) in response to long depolarizing current pulses (600 ms), substance P (SP, 0.5 microM) lowered the AP threshold by 11.0+/-0.3 mV and increased firing from 1.1+/-0.7 APs to 5.2+/-0.6 APs. In C-R tonic neurons that fire multiple APs, SP elicited smaller changes in AP threshold (6.0+/-0.1 mV reduction) and the number of APs (11+/-1 vs. 9+/-1 in control). The effects of SP were similar to the effect of heteropodatoxin II (0.05 microM) or low concentrations of 4-aminopyridine (50 microM) that block A-type K(+) currents. A selective NK(2) agonist, [betaAla(8)]-neurokinin A (4-10) (0.5 microM), mimicked the effects of SP. The effects of SP in C-R phasic neurons were fully reversed by an NK(2) receptor antagonist (MEN10376, 0.5 microM) but only partially by a protein kinase C (PKC) inhibitor (bisindolylmaleimide, 0.5 microM). An NK(3)-selective agonist ([MePhe(7)]-neurokinin B, 0.5 microM), an NK(1)-selective agonist ([Sar(9), Met(11)]-substance P, 0.5 microM) or activation of PKC with phorbol 12,13-dibutyrate (0.5 microM) did not change firing. Our data suggest that the excitability of C-R phasic afferent neurons is increased by activation of NK(2) receptors and intracellular signaling mediated only in part by PKC.

Fig 1

Effect of substance P (SP, A–F) in phasically firing neurons (A–C) and tonically firing (D–F) adult rat DRG neurons. A, SP (0.5 μM) increases firing in response to a current injection 600 ms long and 250 pA in intensity in a phasic neuron from 1 action potential (AP, control trace, con) to 5 AP. B, Average increase in APs in response to SP in 5 neurons. C, Dependence of firing on stimulus intensity for experiment in B, before (Control, empty circles) and after SP (0.5 μM, filled squares). D, SP (0.5 μM) increases firing in response to a current injection 600 ms long and 250 pA in intensity in a tonic firing neuron from 8 APs (control trace, con) to 11 APs. E, Average increase in APs in response to SP in 7 neurons. C, Dependence of firing on stimulus intensity for experiment in E, before (Control, empty circles) and after SP (0.5 μM, filled squares).

Fig 2

Effect of A-type K⁺ channel blockers in phasically firing neurons (A–E) adult rat DRG neurons. A, The Kv4 K⁺ channel blocker heteropodatoxin (HPTx, 0.05 μM) increases firing in response to a current injection 600 ms long and 250 pA in intensity in a phasic neuron. B, Average increase in firing for experiment as shown in A in 6 neurons. C, The A-type K⁺ channel blocker 4-aminiopyridine (4AP, 50 μM, middle trace) increases firing in response to a current injection 600 ms long and 250 pA in intensity in a phasic neuron. Further increasing 4AP to 1 mM (lower trace), prolongs the action potentials and inhibits the increase in firing in response to lower concentrations of the drug, D, Average increase in firing for experiment as shown in C in 8 neurons. Dependence of firing on stimulus intensity for experiment in C and D before (Control, empty circles) and after HPTx (0.05 μM, filled circles), and 4AP (50 μM, filled squares, 1 mM, filled triangles).

Fig. 3

The neurokinin-mediated increase in firing in phasic DRG neurons requires activation of NK₂ receptors. A, Substance P (0.5 μM, middle trace) increased firing from 3 APs (top trace, control) to 8 APs. Application of an NK₂ antagonist, MEN10376 (0.5μM, bottom trace) reduced the increase in firing to near control rates. Firing in response to a current injection 600 ms long and 250 pA in intensity. B, Average increase in APs in response to SP and MEN10376 after SP in 4 neurons. C, A NK₂-selective agonist, [βAla⁸]-neurokinin A (4–10) (NKA, 0.5 μM, bottom trace) increased firing from 1 APs (top trace, control) to 5 APs. Firing in response to a current injection 600 ms long and 250 pA in intensity. D, Average increase in APs in response to NKA in 6 neurons. E, Dependence of firing on stimulus intensity for experiment in D, before (Control, empty circles) and after NKA (0.5 μM, filled triangles).

Fig. 4

The NK₁ and NK₃ agonists do not increase firing in phasic DRG neurons. A, A NK₃-selective agonist, [MePhe⁷]-neurokinin B (NKB, 0.5 μM, bottom trace) did not change firing (top trace, control). Firing in response to a current injection 600 ms long and 250 pA in intensity. B, Average firing before and after NKB in 5 neurons. C, An NK₁-selective agonist, [Sar⁹, Met¹¹]-substance P (SarMetSP, 0.5 μM, bottom trace) did not change firing (top trace, control). Firing in response to a current injection 600 ms long and 250 pA in intensity. D, Average firing before and after SarMetSP in 5 neurons.

Fig. 5

Neurokinin mediated increase in firing is not reversed by inhibition of PKC or mimicked by direct activation of PKC with phorbol ester. A, In a phasic firing neuron SP (0.5 μM, middle trace) increased firing from 1 AP (top trace, control) to 5 APs. Application of a PKC inhibitor, bisindolylmaleimide I HCl (BIM, 0.5μM, bottom trace), partially reduced the increase in firing to 3APs. Firing in response to a current injection 600 ms long and 250 pA in intensity. B, Average increase in APs in response to SP and BIM after SP in 5 neurons. C, Direct activation of PKC with phorbol 12,13-dibutyrate (PDBu, 0.5 μM, bottom trace) did not alter significantly (top trace, control) firing. Firing in response to a current injection 600 ms long and 250 pA in intensity. D, Average firing before and after PDBu in 5 neurons. E, Dependence of firing on stimulus intensity for experiment in D, before (Control, empty circles) and after PDBu (0.5 μM, filled circles).

Fig. 6

The phorbol ester PDBu reduces firing in tonic neurons. A, Direct activation of PKC with phorbol 12,13-dibutyrate (PDBu, 0.5 μM, bottom trace) prolonged the APs and reduced firing (top trace, control) in tonic firing neurons. Firing in response to a current injection 600 ms long and 250 pA in intensity. B, Average firing before and after PDBu in 4 neurons. C, Dependence of firing on stimulus intensity for experiment in B, before (Control, empty circles) and after PDBu (0.5 μM, filled circles).