The modality-specific contribution of peptidergic and non-peptidergic nociceptors is manifest at the level of dorsal horn nociresponsive neurons

Abstract

We previously demonstrated that genetic and/or pharmacological ablation of the TRPV1+/peptidergic or the MrgprD+/non-peptidergic subset of nociceptors produced selective, modality-specific deficits in the behavioural responses to heat and mechanical stimuli, respectively. To assess whether this modality-specific contribution is also manifest at the level of spinal cord neuron responsiveness, here we made extracellular recordings from lumbar dorsal horn neurons of the mouse in response to graded thermal and mechanical stimulation. We found that, following intrathecal injection of capsaicin to eliminate the central terminals of TRPV1+ nociceptors, neurons in the region of laminae I and V of the spinal cord lost responsiveness to noxious heat (whether generated by a contact heat probe or diode laser), with no change in their response to noxious mechanical stimulation. In contrast, ablation of MrgprD+ afferents did not alter the response to noxious heat, but reduced the firing of superficial dorsal horn nociceptive-specific neurons in response to graded mechanical stimulation and decreased the relative number of wide dynamic range neurons that were exclusively mechanosensitive. Neither ablation procedure reduced the number of dorsal horn neurons that responded to noxious cold. These findings support the conclusion that TRPV1+ nociceptors are necessary and probably sufficient for noxious heat activation of dorsal horn neurons and that, despite their polymodal properties, TRPV1+ and MrgprD+ nociceptors provide modality-specific contributions to the response properties of spinal cord neurons.

Figures

Figure 1. Nociresponsive neurons recorded from superficial and deep dorsal horn of the spinal cord

A, example of a superficial dorsal horn nociceptive specific (NS) neuron that could be activated by graded mechanical (upper three traces) and heat stimulation (lower three traces) of the hindpaw. Each record contains three traces: firing rate (top); raw data (middle); and stimulus intensities (bottom; 130, 230, 330 and 430 mN for mechanical and 40, 45 and 49°C for heat). The x-axis defines the temporal stimulus sequence (in seconds). Note that the neuron did not discharge in response to the innocuous 130 mN stimulus. B, example of a lamina I wide dynamic range (WDR) neuron that responded only to mechanical stimulation, including the 130 mN stimulus. C, example of a deep dorsal horn neuron that responded to innocuous (20°C) and noxious (10°C) cold stimuli. This particular neuron also discharged when the temperature of the receptive field returned to baseline (36°C). D, recording sites (*) in the superficial (left panel) and in the deep dorsal horn (right panel). Scale bars represent 250 μm.

Figure 2. Categorization of mechanically responsive superficial and deep dorsal horn neurons in vehicle- and capsaicin-treated mice

A, these multilevel pie charts schematize the relative abundance of the different categories of lamina I neurons in vehicle- and capsaicin-treated mice. The percentage relative to the total number of neurons recorded is listed in the adjacent table. Note the near-complete loss of heat-responsive neurons following capsaicin treatment (****P < 0.0001, Fisher's exact test). B, these multilevel pie charts schematize the relative abundance of the different categories of deep dorsal horn neurons, with their percentage relative to the total number of neurons recorded listed in the adjacent table. As for superficial dorsal horn neurons, capsaicin treatment eliminated heat responses of lamina V neurons (***P < 0.001, Fisher's exact test). Percentages marked with a filled triangle refer to the relative number of NS/M or NS/HM in the NS group, or of WDR/M or WDR/HM in the WDR group. Abbreviations: HM, heat- and mechanosensitive; LTM, low-threshold mechanosensitive; M, selectively mechanosensitive; NS, nociceptive specific; and WDR, wide dynamic range.

Figure 3. Ablation of transient receptor potential vanilloid-1-positive (TRPV1+) afferents does not alter the mechanical responsiveness of spinal cord nociresponsive neurons

Peak firing (top panels) and total spikes (bottom panels) elicited by mechanical stimulation at the indicated intensities, for superficial dorsal horn NS neurons (A), superficial dorsal horn WDR neurons (B) and deep dorsal horn WDR neurons (C) in vehicle-treated (filled columns) and capsaicin-treated mice (open columns). No significant differences were observed for the mechanical responsiveness between vehicle- and capsaicin-treated animals in any of these cell subtypes (P > 0.05, two-way ANOVA). For all neuronal types, both the peak firing rate and the total number of spikes significantly increased in response to increased mechanical stimulus intensity (one-way ANOVA). Data are presented as means + SEM.

Figure 4. Responsiveness of superficial dorsal horn neurons to C fibre and Aδ fibre diode laser stimulation in vehicle-treated mice

A, coding (peak firing) using the C fibre stimulation protocol. First, the threshold intensity (T) to evoke firing was determined and then two incremental steps of suprathreshold intensity (S1 and S2) were studied (see Methods). There was a significant increase in firing as the intensity increased (P < 0.001). B, the latency in response to the C fibre protocol also decreased significantly at the higher laser intensities (P < 0.01). C, coding (peak firing) using the Aδ fibre stimulation protocol. As for the C fibre protocol, there was a significant increase in firing as laser intensity increased (P < 0.05). D, latency to firing decreased at higher stimulation intensities (P < 0.01). Data are presented as means + SEM. *P < 0.05, **P < 0.01 and ***P < 0.001, Bonferroni post hoc tests and one-way ANOVA.

Figure 5. Categorization of mechanically responsive superficial and deep dorsal horn neurons in vehicle- and diphtheria toxin (DTX)-treated mice

A, these multilevel pie charts schematize the relative abundance of the different categories of presumptive lamina I neurons in vehicle- and DTX-treated mice, with their percentage relative to the total number of neurons recorded listed in the adjacent table. Note the loss of WDR/M units following DTX treatment (*P < 0.05, Z-test). B, multilevel pie charts schematize the relative abundance of the different categories of deep dorsal horn neurons, with their percentage relative to the total number of neurons recorded listed in the adjacent table. Note the reduction in WDR/M units, and the relative increase in heat-responsive units, following DTX treatment. Percentages marked with a filled triangle refer to the relative number of NS/M or NS/HM in the NS group, or of WDR/M or WDR/HM in the WDR group. Abbreviations are as for Fig. 2.

Figure 6. Ablation of MrgprD+ afferents reduces the mechanical responsiveness of superficial dorsal horn NS neurons

Peak firing (top panels) and total spikes (bottom panels) elicited by mechanical stimulation at the indicated intensities in superficial dorsal horn NS neurons (A), superficial dorsal horn WDR neurons (B) and deep dorsal horn WDR neurons (C) in vehicle-treated (filled columns) and DTX-treated mice (open columns). Two-way ANOVA showed significant reduction of both peak firing and total spikes in response to the graded mechanical stimuli in superficial dorsal horn NS neurons following DTX treatment compared with vehicle-treated animals (*P < 0.05 and **P < 0.01). Data are presented as means + SEM.

Figure 7. Ablation of MrgprD+ afferents does not alter heat responsiveness of spinal cord nociresponsive neurons

Peak firing (top panels) and total spikes (bottom panels) elicited by heat stimulation at the indicated intensities in superficial dorsal horn NS neurons (A), superficial dorsal horn WDR neurons (B) and deep dorsal horn WDR neurons (C) of vehicle-treated (filled columns) and DTX-treated mice (open columns). All groups encoded stimulus intensity with increased firing (one-way ANOVA), and there were no significant differences in the heat responsiveness between vehicle- and DTX-treated mice (two-way ANOVA). Data are presented as means + SEM.