Trpv1 mediates spontaneous firing and heat sensitization of cutaneous primary afferents after plantar incision

Abstract

TrpV1, the receptor for capsaicin, contributes to nociception in animals but appears to be much more important for signaling increased behavioral sensitivity in the injured state. The current study examined the relationship between the marked reduction in heat hyperalgesia after incision in TrpV1 knockout (KO) mice and the activity of the nociceptors in these same mice. Also, the role of TrpV1 in spontaneous activity (SA) of afferents after incision was examined. Standard teased-fiber techniques were used to record from glabrous skin afferents from incised and control TrpV1 KO and C57Bl6 mice. The loss of TrpV1 had minimal effect on the responses of mechano-heat-sensitive C-fiber afferents in the normal and incised states. However, a different group of heat sensitive afferents, termed unclassified afferents, was sensitized to heat by incision and had markedly reduced sensitization in the TrpV1 KO mice. These unclassified afferents also developed SA after incision, and generally had a lower threshold temperature compared to unclassified afferents without SA. The rate of SA was inversely correlated to the threshold temperature for heat; afferents that exhibited a higher rate of SA had a lower heat threshold. The proportion of unclassified afferents with SA was also reduced in incised TrpV1 KO mice compared to incised C57Bl6 mice. We conclude that a distinct class of afferents outside the mechano-heat-sensitive afferent population likely contributes to heat hypersensitivity after plantar incision. KO of TrpV1 influences SA in these unclassified afferents in incised skin. SA in these afferents is perhaps a manifestation of heat sensitization.

Figures

Figure 1

The effects on TrpV1 gene deletion on guarding behavior, heat and mechanical sensitivity in mice before and after plantar incision. Before or after incision, no differences are observed in the guarding behaviors (a) and paw withdrawal frequencies to von Frey hair stimulation (b–e). In a, note that ‘Y’ axis is extended up to the maximal guarding behaviors reported in the rat after incision. f) In C57BL6 mice, incision produced significant reductions in the withdrawal latencies to a heat stimulus on the incised paw. The decrease was significantly different from the paw withdrawal latencies in the TrpV1 knock out (KO) mice. Asterisks indicate a significant difference on the incised side between C57BL6 and KO mice (*P<0.05, Repeated measures ANOVA followed by posthoc t test). mN=milli-Newtons.

Figure 2

Heat responses in mechanosensitive afferents from TrpV1 KO and C57Bl6 mice. a) The percentage of heat responsive C-fibers in C57BL6 and KO mice. The threshold temperature for heat response (b), total spikes evoked during heat stimuli (c), and the peak discharge frequency (d) were not different between genotypes. Threshold was the temperature that elicited the first action potential if background activity was absent or at least 2 standard deviations greater than SA (For details, see materials and methods). e) Stimulus-response function for heat response in C57BL6 and KO mice (bin width 1-s, mean number of spikes±SE). f: Digitized oscilloscope trace of two typical C-fiber activities during heat stimulus in C57BL6 (left) and KO mice (right). Below, real-time record of skin temperature, and above is the peri-stimulus time histogram.

Figure 3

Loss of TrpV1 had a small effect on heat responses of mechanosensitive afferents after incision. a) the percentage of heat responsive C-fibers was not different between incised C57BL6 and KO mice. b) Threshold temperatures for activation of mechanosensitive C-fiber afferents from incised mice. The horizontal line represents mean±SE, p

Figure 4

The total activity of all afferents during heat stimulation. a) Left: Example recording from incised C57BL6 mice. In addition to a mechanically identified C-fiber afferent (spikes with large amplitude, filled arrow), small amplitude action potentials from another fiber are noted immediately during the onset of the heat stimulus (open arrow). The small amplitude action potentials are presumably from another class of afferents that are not readily identified by mechanical stimulation. In this study we named these ‘unclassified’ since they could not be further characterized (for details, see Material and Methods). Analyses of all afferent activity during heat stimulation are shown in b–f. Stimulus response function for heat response of all afferents in unincised (b) and incised (c) C57BL6 and KO mice (bin width 1-s, mean number of spikes±SE, p

Figure 5

Spontaneous activity (SA) of afferents from incised mice. The proportion of mechanosensitive C-fibers with SA was not different in the incised C57BL6 and TrpV1KO mice (a). b) the rate of mechanosensitive C-fibers with SA was similar in the KO and C57BL6. c) The percentage of the unclassified heat responsive afferents that had SA was higher in the incised C57BL6 mice compared to incised KO mice (b, p

Figure 6

Relationships between SA and heat sensitivity of unclassified afferents from incised C57BL6 mice. a: During this recording, a mechanically identified C-fiber afferent had no spontaneous activity and was not heat responsive. An unclassified afferent with SA could not be identified using a mechanical stimulus, had spontaneous activity and responded to heating at 35°C. b: The heat responses of an unclassified afferent without any spontaneous activity. The afferent responded at 41°C. c: Unclassified, heat responsive, spontaneously active afferents had a significantly lower heat threshold when compared with unclassified, heat responsive afferents without SA in the incised C57BL6 mice (p

Figure 7

Effects of cooling on SA. a: Example recording of an unclassified afferent with SA from incised WT mouse. Cooling the receptive field to 12–16°C completely and reversibly eliminated SA from this afferent. b: The effects of cooling on the SA of afferents from incised WT mice. The schematic shows time points chosen for counting before, during and after cooling. Before, 60s before the start of cooling; during, last 60s of cooling; and after, 60s from the point when the dermal temperature returned to the normal bath temperature. The difference between before and during cooling is significant (p

Figure 8

Mechano-sensitivity of cutaneous primary afferents from unincised and incised C57BL6 (a–d) and KO (e–h) mice. The mechanosensitivity is measured by computer operated feedback controlled force stimuli (tip diameter 0.7 mm). Each mechanical stimulus was 5s in duration and started from zero to 5, 10, 20, 40, 80, 120 mN. The inter-stimuli intervals were one minute. When an afferent produced a response to a particular force controlled ramp, it received three more ascending series of stimuli. To avoid fatigue, in no cases, more than three suprathreshold stimuli were applied; for example, if an afferent produced a response during the 20mN ramp, it received the 40, 80 and 120mN ramp. The total number of spikes was calculated over the entire 5s of the stimulus. The number of fibers is shown in the parenthesis. Statistical significance is assessed by a two-way ANOVA for KO vs C57BL6.