, 2012; Zylka et al , 2005) To determine whether peptidergic end

, 2012; Zylka et al., 2005). To determine whether peptidergic endings were missing in DTX-treated CGRPα-DTR+/− mice, we immunostained hindpaw sections with antibodies to CGRP and the pannerve fiber marker PGP9.5. We found that DTX treatment eliminated CGRP-IR terminals from the glabrous skin and hairy skin (epidermis and dermis) and from guard hairs (Figures 3A–3F, Figure S2). In contrast, DTX treatment did not eliminate CGRP-IR−, PGP9.5+ terminals, including terminals in the epidermis, hair follicles, and sweat glands (Figures 3A–3F, Figure S2, data not shown). Since ∼50% of all TRPV1+ DRG neurons were ablated in DTX-treated CGRPα-DTR+/− mice

(Figure 1H), we hypothesized that peripheral nerve responses to noxious heat might be impaired. To test this hypothesis, we utilized a skin-nerve preparation to quantify hot, cold, and mechanical responses of isolated C-fibers in the hindpaw of saline- and DTX-treated this website CGRPα-DTR+/− mice (Koltzenburg et al., 1997; Pribisko and Perl,

2011). We also mapped the distribution of noxious heat- and cold-receptive fields in this preparation by recording from the entire sural nerve (Figures 3G–3K). A near-infrared diode laser was used to control the intensity and location of heat stimulation (Pribisko and Perl, 2011). In saline-treated mice, laser heat stimulation (using an intensity that is in excess GDC-0941 mouse of the threshold of most C-fibers) activated multiple units in all of the spots (Figures 3G, 3I, and 3K). However, in DTX-treated mice, activity was detected in only 38.1% ± 2.4% of the spots (Figures 3G, 3I, and 3K). This is a profound reduction, particularly given that a response was scored as positive if as few as one action potential was detected when recording from the entire sural nerve. When averaged over all 40 spots, significantly fewer heat-evoked action potentials were

generated over the 2 s stimulation period in DTX-treated mice (Figure 3K). Furthermore, Dipeptidyl peptidase the laser heat threshold to activate isolated C-fibers was ∼2-fold higher in DTX-treated mice (Figure 3K). In contrast, there was no statistically significant change in the number of cold-responsive spots when recording from the entire sural nerve and no change in the cold threshold of activation in isolated C-fibers between groups (Figures 3H, 3J, and 3K). There was also no change in the mechanical thresholds of isolated C-fibers between saline- and DTX-treated CGRPα-DTR+/− mice (Figure 3K). Taken together, these data demonstrate that ablation of CGRPα+ afferents causes a profound loss of noxious heat sensitivity in skin with no change in cold or mechanical sensitivity. To determine whether this profound physiological loss of heat sensitivity also affected behavioral responses to heat, we tested saline- and DTX-treated CGRPα-DTR+/− mice using multiple heat-related behavioral assays (Table 1). For all of these experiments, we studied mice pre- and postsaline/DTX treatment and separately tested males and females.

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