We found that capsaicin (10 μM; pressure ejected using a picospritzer for 10 s) caused a 2-fold increase in EPSC frequency in a subset of spinal neurons from saline- and DTX-treated mice (Figures 7A, 7C, and 7E; Table S1). However, the tonic (baseline) and selleck evoked EPSC frequency of capsaicin-responsive neurons was significantly lower in DTX-treated
mice than in saline-treated animals (lower by 41.4% at baseline, 44.7% evoked; Figure 7E). This reduction in capsaicin responsiveness at the spinal level was consistent with our observation that DTX-treated mice had ∼50% fewer TRPV1+ DRG neurons (Figure 1H) and were ∼50% less responsive to capsaicin injection (Table 1). In addition, significantly fewer total neurons responded to capsaicin in slices from DTX-treated mice (Table S1). Intriguingly, there were no capsaicin-responsive transient neurons in DTX-treated mice (Table S1), consistent with the fact that capsaicin-responsive primary afferents are monosynaptically connected to transient neurons (Zheng et al., 2010). We then pressure ejected icilin (40 μM) onto slices from saline- and DTX-treated mice to identify TRPM8/cold-responsive BMS-387032 supplier spinal neurons. We found that the total number of spinal neurons that responded to icilin did not differ between saline- and DTX-treated mice (approximately 14% of all lamina II neurons responded in both groups; Table S1). However, icilin-responsive spinal neurons from DTX-treated mice had significantly
higher tonic and evoked EPSC activity (270% increase at baseline, 157% increase evoked,
relative to saline-treated mice) (Figures 7B, 7D, and 7F). Collectively, these experiments suggest that CGRPα DRG neurons tonically cross-inhibit TRPM8/cold-sensing circuits all at the spinal level (see Figures 7G and 7H for mechanism). CGRP-IR has long served as a classic molecular marker of peptidergic nociceptive neurons. However, whether CGRP-IR DRG neurons were actually required to sense painful stimuli was never directly tested. Using a genetically precise ablation strategy, we found that CGRPα DRG neurons are required to sense noxious heat, capsaicin, and some pruritogens. In contrast, CGRPα DRG neurons were not required to sense cold, innocuous, or noxious mechanical or β-alanine itch stimuli. Our findings were remarkably consistent across a large number of mice, sexes, and across multiple behavioral assays, conclusively revealing that CGRPα DRG neurons contribute directly to noxious heat and itch sensations. CGRPα neurons are clearly not the only DRG neurons that sense these stimuli, as some noxious heat, capsaicin, and itch responses remained after ablation. This remaining sensitivity probably originates from TRPV1+/CGRPα− (nonpeptidergic) neurons that were not ablated. Indeed, ablation of TRPV1+ DRG neurons or spinal afferents completely eliminated responses to capsaicin, heat, and some pruritogens (Cavanaugh et al., 2009; Karai et al., 2004; Mishra and Hoon, 2010; Mishra et al., 2011).
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