TRPA1-expressing primary afferents synapse with a morphologically identified subclass of substantia gelatinosa neurons in the adult rat spinal cord

Abstract

The TRPA1 channel has been proposed to be a molecular transducer of cold and inflammatory nociceptive signals. It is expressed on a subset of small primary afferent neurons both in the peripheral terminals, where it serves as a sensor, and on the central nerve endings in the dorsal horn. The substantia gelatinosa (SG) of the spinal cord is a key site for integration of noxious inputs. The SG neurons are morphologically and functionally heterogeneous and the precise synaptic circuits of the SG are poorly understood. We examined how activation of TRPA1 channels affects synaptic transmission onto SG neurons using whole-cell patch-clamp recordings and morphological analyses in adult rat spinal cord slices. Cinnamaldehyde (TRPA1 agonist) elicited a barrage of excitatory postsynaptic currents (EPSCs) in a subset of the SG neurons that responded to allyl isothiocyanate (less specific TRPA1 agonist) and capsaicin (TRPV1 agonist). Cinnamaldehyde evoked EPSCs in vertical and radial but not islet or central SG cells. Notably, cinnamaldehyde produced no change in inhibitory postsynaptic currents and nor did it produce direct postsynaptic effects. In the presence of tetrodotoxin, cinnamaldehyde increased the frequency but not amplitude of miniature EPSCs. Intriguingly, cinnamaldehyde had a selective inhibitory action on monosynaptic C- (but not Adelta-) fiber-evoked EPSCs. These results indicate that activation of spinal TRPA1 presynaptically facilitates miniature excitatory synaptic transmission from primary afferents onto vertical and radial cells to initiate action potentials. The presence of TRPA1 channels on the central terminals raises the possibility of bidirectional modulatory action in morphologically identified subclasses of SG neurons.

FIG. 1. Dose-dependent effects of cinnamaldehyde (CA) on spontaneous EPSCs (sEPSCs)

Voltage clamp recordings (holding potential −70mV) of sEPSCs. (A) Representative effects of TRPA1 agonist CA (300 and 500 μM) on sEPSCs in the same SG neuron. CA dose-dependently elicited barrages of EPSCs. (B) Histograms of the amplitude distribution of sEPSCs in this neuron during the control period and in the presence of CA (500 μM). Each histogram was constructed from 60 s of continuous recording. Note that the incidence of large amplitude EPSCs was greatly enhanced by CA. (C) Summary data of CA actions on sEPSC frequency and amplitude (**P < 0.01). Note that the significant increase in EPSC frequency was blocked in the presence of RR, in Ca²⁺ free solution and in the presence of HC-030031, however, the Ca²⁺ channel blocker Cd²⁺ was without significant effect. In this and subsequent figures, numbers in parentheses indicate the number of neurons tested, vertical bars show SD.

FIG. 2. Actions of capsaicin (Cap) and CA on sEPSCs in the same SG neurons

(A) Representative SG neuron showing responses to TRPV1 agonist, Cap (1 μM) and CA (300 μM) both of which elicited barrages of EPSCs. The lower records are on an expanded time base showing sEPSCs during the control period and in the presence of each agonist. (B) An example of a Cap-sensitive but CA-insensitive SG neuron. (C) SG neuron which did not respond to either Cap or CA. (D) Percentage of neurons which were sensitive to Cap and CA (29%, n = 14), sensitive to Cap but insensitive to CA (45%, n = 22), and insensitive to both Cap and CA (26%, n = 13).

FIG. 3. Firing patterns of SG neurons and action of CA on each firing group

In response to the injection of a depolarizing current pulse the SG neurons exhibited distinctive firing patterns: (A) Delayed firing type. (B) Sustained repetitive firing type. (C) Phasic firing type. (D) Initial firing type. Pooled results under the firing traces in A-D showing the Effect of CA on sEPSC frequency relative to that of control. CA had an excitatory action on firing in all types of neurons.

FIG. 4. Examples of the morphology of neurobiotin-stained SG neurons and action of CA on sEPSCs in each cell type

(A) Vertical cells. (B) Radial cells. (C) Islet cells. (D) Central cells. (E) Unclassified cells. Lower drawings are the camera lucida representations of each neuron. Pooled results on the right hand side in A-E showing the frequency of sEPSCs in the presence of CA relative to that of control. Note that CA had an excitatory action on the majority of vertical, redial, and unclassified, but not on islet or central cells.

FIG.5. Actions of CA on monosynaptic Aδ and C-fiber evoked EPSCs

(A) This example neuron showed one monosynaptic Aδ fiber-evoked EPSC and two monosynaptic C fiber-evoked monosynaptic EPSCs which had distinct threshold stimulus intensities (TSI, shown overlaid in left panel) and conduction velocities (CV). As the stimulus intensity was increased, first an Aδ fiber EPSC was evoked (TSI: 0.04 mA, CV: 4.3 m/s) followed by a first C fiber response (TSI: 0.27 mA, CV: 0.51 m/s) and then a second C-fiber EPSC at higher stimulus intensity (TSI: 0.68 mA, CV: 0.42 m/s). Application of CA (300 μM) completely and selectively blocked the second monosynaptic C fiber-evoked EPSCs (right panel). (B) Pooled data from all recordings showing the percentage of evoked EPSCs inhibited by CA showing that its actions were largely mediated on a subgroup of C fiber-afferents. (C) Summary showing proportional decrease in the amplitude of monosynaptic C fiber-evoked EPSCs by CA. (D) CA inhibited the amplitude of monosynaptic C fiber-evoked EPSCs and enhanced the frequency of sEPSCs in the same neuron with remarkably similar time courses. The CA action on C fiber-evoked EPSCs was first examined then after complete recovery the CA was reapplied and the effect on sEPSCs was examined.

FIG. 6. Effects of CA on GABAergic and glycinergic spontaneous IPSCs (sIPSCs)

(A, B) GABAergic and glycinergic sIPSCs recorded in the presence of strychnine (2 μM) and bicuculline (10 μM), respectively. CA (300 μM) did not change the frequency or amplitude of GABAergic and glycinergic sIPSCs. These GABAergic and glycinergic IPSCs were inhibited by the supplemental application of bicuculline (10 μM) and strychnine (2 μM), respectively at the end of each recording session. (C) Summary showing effects of CA on the frequency and amplitude of GABAergic and glycinergic sIPSCs.

FIG. 7. CA evokes action potential discharge in SG neurons

(A) A continuous current clamp recording of the membrane potential showing the effect of CA (300 μM) which evoked a barrage of EPSPs and resulted in the generation of action potentials (AP). Arrowhead shows an AP. (B) Time course of the frequency of APs elicited by CA. CA reversibly increased the frequency of APs (n = 5).