Primary afferents with TRPM8 and TRPA1 profiles target distinct subpopulations of rat superficial dorsal horn neurones

Abstract

Background and purpose: The transient receptor potential (TRP) channels, transient receptor potential melastatin-1 (TRPM8) and transient receptor potential ankyrin-1 (TRPA1), are expressed in subpopulations of sensory neurones and have been proposed to mediate innocuous and noxious cold sensation respectively. The aim of this study was to compare TRPM8 and TRPA1 modulation of glutamatergic afferent transmission within the spinal dorsal horn.

Experimental approach: Whole cell patch clamp recordings were made from rat spinal cord slices in vitro to examine the effect of TRP agonists and temperature on glutamatergic excitatory postsynaptic currents (EPSCs).

Key results: Icilin (3 or 100 micromol.L(-1)), menthol (200 micromol.L(-1)) and capsaicin (1 micromol.L(-1)) reduced the amplitude of primary afferent evoked EPSCs in subpopulations of lamina I and II neurones. In a subpopulation of superficial neurones, innocuous cold (threshold 29 degrees C), 3 micromol.L(-1) icilin (EC50 1.5 micromol.L(-1)) and menthol (EC50 263 micromol.L(-1)) increased the rate of spontaneous miniature EPSCs. In the majority of lamina I and II neurones, 100 micromol.L(-1) icilin (EC50 79 micromol.L(-1)), allyl isothiocyanate (EC50 226 micromol.L(-1)), cinnamaldehyde (EC50 38 micromol.L(-1)) and capsaicin (1 micromol.L(-1)) increased miniature EPSC rate. The response to 100 micromol.L(-1), but not 3 micromol.L(-1) icilin, was abolished by ruthenium red, while neither was affected by iodoresiniferatoxin. Responsiveness to 3 micromol.L(-1), but not to 100 micromol.L(-1) icilin, was highly predictive of innocuous cold responsiveness. Neurones responding to 3 micromol.L(-1) icilin and innocuous cold were located more superficially than those responding to 100 micromol.L(-1) icilin.

Conclusions and implications: Activation of TRPM8 and TRPA1 presynaptically modulated glutamatergic transmission onto partially overlapping but distinct populations of superficial dorsal horn neurones. Spinal TRPM8 and TRPA1 channels may therefore provide therapeutic targets in cold hyperesthesia.

Figure 1

Icilin modulates primary afferent evoked synaptic transmission in the superficial dorsal horn. (A) Time plot of the amplitude of evoked excitatory postsynaptic currents (eEPSC) during superfusion of icilin (3 µmol·L⁻¹), plus the spontaneous EPSC (sEPSC) rate averaged over a 5 s epoch preceding each eEPSC. Raw current traces of (B) evoked EPSCs and (C) sEPSCs taken prior to (control) and during icilin (3 µmol·L⁻¹). Bar charts depicting the mean (D) eEPSC amplitude (as a percentage of pre-drug control) and (E) sEPSC rate (expressed as the absolute increase above pre-drug control) for icilin (3, 100 µmol·L⁻¹), menthol (200 µmol·L⁻¹) and capsaicin (1 µmol·L⁻¹). EPSCs were obtained by electrical stimulation of the dorsal rootlet in the presence of strychnine (3 µmol·L⁻¹) and picrotoxin (100 µmol·L⁻¹). (A–C) are from one neurone.

Figure 2

Icilin enhances glutamatergic transmission via a presynaptic mechanism. (A) Time plot of miniature excitatory postsynaptic current (mEPSC) rate during superfusion of icilin (3 µmol·L⁻¹) and capsaicin (1 µmol·L⁻¹). (B) Raw current traces and (C) superimposed averaged mEPSCs prior to (control) and during icilin (3 µmol·L⁻¹) and capsaicin (1 µmol·L⁻¹). Cumulative probability (cum prob) distribution plots of mEPSC (D) inter-event interval and (E) amplitude for the epochs averaged in (C). Bar charts depicting (F) mEPSC rate (expressed as the absolute increase above pre-drug control) and (G) mEPSC amplitude (as a percentage of pre-drug control) for neurones that responded to icilin (3, 100 µmol·L⁻¹), menthol (200 µmol·L⁻¹) and capsaicin (1 µmol·L⁻¹). (A–E) are taken from one neurone in the presence of TTX (500 nmol·L⁻¹), strychnine (3 µmol·L⁻¹) and picrotoxin (100 µmol·L⁻¹).

Figure 3

Icilin responsiveness predicts two presynaptic TRP ion channel types. Concentration–response curves for the increase in miniature excitatory postsynaptic current (EPSC) rate produced by (A) icilin, (B) menthol, (C) allyl isothiocyanate (AIT) and (D) cinnamaldehyde. In (A) neurones were divided into low-concentration icilin responders (LC, responded at 1–10 µmol·L⁻¹) and high-concentration icilin responders (HC, only responded >30 µmol·L⁻¹). (E) Bar chart depicting the mean increase in miniature EPSC (mEPSC) rate (expressed as the absolute increase above pre-drug level) for icilin (3, 100 µmol·L⁻¹) and capsaicin (1 µmol·L⁻¹) in the absence (control) and presence of iodoresiniferatoxin (IRTX, 300 nmol·L⁻¹) or ruthenium red (RR, 10 µmol·L⁻¹). In (A–D) a logistic function was fitted to each curve to determine the EC₅₀. In (A–E) change in mEPSC rate is expressed as the absolute increase above the pre-drug level. In (E) * denotes P < 0.05 compared with control.

Figure 4

Temperature reductions enhance glutamatergic transmission via a presynaptic mechanism. (A) Time plot of miniature excitatory postsynaptic current (mEPSC) rate during successive temperature decreases to 25°C and 15°C. (B) Raw current traces and (C) superimposed averaged mEPSCs at the different temperatures indicated. Cumulative probability (Cum Prob) distribution plots of mEPSC (D) inter-event interval and (E) amplitude for the epochs averaged in (C). (A–E) are taken from one neurone in the presence of tetrodotoxin (500 nmol·L⁻¹), strychnine (3 µmol·L⁻¹) and picrotoxin (100 µmol·L⁻¹).

Figure 5

Cool and icilin sensitive glutamatergic inputs target a subpopulation of superficial dorsal horn neurones. Location plots of neurones that did (Resp) or did not (Non Resp) respond with an increase in miniature excitatory postsynaptic current rate to (A) a temperature reduction to 25°C, and (B) icilin (3 µmol·L⁻¹). (C) Bar chart showing the percentage of temperature reduction (25°C), icilin (3, 100 µmol·L⁻¹) and capsaicin (1 µmol·L⁻¹) responsive neurones in laminae I, II_o, II_i and III. (D) Bar chart showing the percentage of 25°C responsive (open bars) and non-responsive (filled bars) neurones, which were also responsive to icilin (3, 100 µmol·L⁻¹) and capsaicin (1 µmol·L⁻¹).

Figure 6

Postsynaptic effects of icilin in the superficial dorsal horn. Raw membrane current traces in the presence of (A) tetrodotoxin (TTX) (500 nmol·L⁻¹), or (B) TTX (500 nmol·L⁻¹), 6-cyano-2,3-dihdroxy-7-nitro-quinoxaline (CNQX) (5 µmol·L⁻¹), strychnine (Strych, 3 µmol·L⁻¹) and picrotoxin (Picrotx, 100 µmol·L⁻¹) during superfusion of icilin (100 µmol·L⁻¹). (C) Bar charts depicting the mean currents produced by icilin in the presence of TTX, or TTX, CNQX, strychnine and picrotoxin. (A) and (B) are taken from two different neurones.

Figure 7

Effect of icilin on GABAergic/glycinergic transmission in the superficial dorsal horn. (A) Time plot of miniature inhibitory postsynaptic current (mIPSC) rate during superfusion of icilin (100 µmol·L⁻¹). (B) Raw current traces and (C) superimposed averaged mIPSCs prior to (control) and during icilin. Bar charts depicting (D) mIPSC rate (expressed as the absolute increase above pre-drug control) and (E) mIPSC amplitude (expressed as a percentage of pre-drug control) for all neurones tested. (A)–(C) are taken from one neurone in the presence of tetrodotoxin (500nmol·L⁻¹) and 6-cyano-2,3-dihdroxy-7-nitro-quinoxaline (5 µmol·L⁻¹).