Three functionally distinct classes of C-fibre nociceptors in primates

Abstract

In primates, C-fibre polymodal nociceptors are broadly classified into two groups based on mechanosensitivity. Here we demonstrate that mechanically sensitive polymodal nociceptors that respond either quickly (QC) or slowly (SC) to a heat stimulus differ in responses to a mild burn, heat sensitization, conductive properties and chemosensitivity. Superficially applied capsaicin and intradermal injection of β-alanine, an MrgprD agonist, excite vigorously all QCs. Only 40% of SCs respond to β-alanine, and their response is only half that of QCs. Mechanically insensitive C-fibres (C-MIAs) are β-alanine insensitive but vigorously respond to capsaicin and histamine with distinct discharge patterns. Calcium imaging reveals that β-alanine and histamine activate distinct populations of capsaicin-responsive neurons in primate dorsal root ganglion. We suggest that histamine itch and capsaicin pain are peripherally encoded in C-MIAs, and that primate polymodal nociceptive afferents form three functionally distinct subpopulations with β-alanine responsive QC fibres likely corresponding to murine MrgprD-expressing, non-peptidergic nociceptive afferents.

Figure 1. The response to a 49°C, 3 s stepped heat stimulus separates two classes of mechano-heat sensitive C fibers

A: Temperature waveforms of the heat stimulus. The skin was first heated to a 38°C, 3 s baseline temperature and then stepped (rise time < 500 ms) to a 49°C, 3 s. stimulus. The starting time of the temperature waveform was shifted by the conduction latency of each fiber to align it with the fiber’s response. B: Specimen recording showing the response of a QC fiber (red circles) and an SC fiber (blue circles) to the stimulus depicted in A. Instantaneous discharge frequency is plotted vs time with each dot representing an action potential. In quick C-fibers (QC), the evoked response starts during the rise in temperature, shows an early high frequency discharge (as indicated by the red dashed line), but quickly adapts during the stimulus duration. In contrast, the response of slow C-fibers (SC) reaches a peak during the plateau phase of the stimulus (as indicated by the blue dashed line). Start of the stimulus is indicated by the black dashed line. C: The time of peak discharge for each fiber is plotted as a function of the stimulus rise time plus the conduction time for that fiber. Data points below the line of equality (solid line) correspond to those fibers whose peak discharge occurred during the rising phase of the heat stimulus. The two highlighted symbols show the data points for the examples AF71.02C and AF67.02C whose responses were plotted in B and supplemental figure 2. D: Average response to heat staircase stimulus (36°C or 38°C to 49°C in 1°C, 1 s increments) for SC- and QC fibers. Mean number of action potentials (± SEM) recorded at a given temperature was plotted against applied temperature. Responses at given temperatures were compared between QC- and SC fibers using Mann-Whitney U test (***p<0.001). E: Histogram of heat thresholds for QC and SC fibers. No QC had a heat threshold greater than 45°C, whereas no SC had a heat threshold less than 41°C. All data were collected in cynomolgus monkeys

Figure 2. SC afferents show greater sensitization to heat after a mild burn injury than QC afferents

A, B: Averaged stimulus-response functions to the heat staircase before and after the mild burn injury. The increase in response after the burn for the QC afferents (A) reached a plateau at the higher stimulus temperatures. In contrast, the SC afferents (B) showed an enhanced response for all temperatures. Pre- and post burn data were first analyzed with Friedman ANOVA (pre QC: χ²_(12,12)=138.2, p<0.001; post QC: χ²_(12,12)=123.7, p<0.001; pre SC: χ²_(9,12)=90.0, p<0.001; post SC: χ²_(9,12)=101.3, p<0.001). Within each fiber class, pre- and post burn data at a given temperature were compared with Wilcoxon matched pairs test (* p<0.05; ** p<0.01; *** p<0.001) C: The increase in heat response in QC- and SC fibers is temperature dependent (QCs: Friedman ANOVA χ²_(12,12)=81.6, p<0.001; SCs: Friedman ANOVA χ²_(9,12)=99.1, p<0.001). In QCs, the largest change in response was observed at temperatures around 45°C, whereas in SCs, the largest increase was observed at the higher temperatures. At low temperatures (37 - 39 °C) and high temperatures (47-49 °C), the change in response was significantly different between the groups (*p<0.05; ** p<0.01; Mann Whitney U test). D: Time course of the averaged neuronal response of QC- and SC fibers to the 48°C, 120s mild burn injury (bin size = 5s). The discharge in the QC afferents adapted quickly, whereas the average response of the SC afferents increased during the burn. Error bars indicate SEM. Data are exclusively from cynomolgus monkeys.

Figure 3. SC afferents exhibit greater activity-dependent slowing of conduction than QC afferents

The overall decrease in conduction velocity induced by suprathreshold electrical stimulation (2Hz for 3 min) at the receptive field differed significantly between fiber classes (F_(2,73)= 29.7, p<0.001). In addition, slowing over the course of the stimulation differed significantly between fiber classes (mixed model ANOVA with pulse number and fiber class as factors (F_(20,730)= 9.26, p< 0.001)). SC afferent slowed significantly more than QC fibers (* p<0.05, t-test with Bonferroni correction for multiple testing). C-MIAs slowed significantly more than QC- and SC- afferent fibers (###, p<0.001; t-test, with Bonferroni correction for multiple testing). Error bars indicate SEM. Data are from pigtail and cynomolgus monkeys.

Figure 4. QC- and SC fibers differ in their responses to chemical stimuli

A: QC afferents responded more vigorously than SC afferents to heat-inactivated cowhage spicules coated either with histamine (10 mg/ml) or capsaicin (200 mg/ml) that were inserted into the superficial layers of the skin. For each fiber class, a Friedman ANOVA was performed (QC: χ²_(15,2)=26.1, p<0.001; SC: χ²_(15,2)=25.0, p<0.001). Pairwise comparisons of responses to different chemical stimuli within each fiber class were then performed using Wilcoxon matched pairs test (^## and ^&& p<0.01, with Bonferroni correction for multiple testing), comparisons across different stimuli are indicated by red and blue lines for QC- and SC- fibers, respectively. Responses of QC and SC fibers were compared with Mann-Whitney U test (**p<0.01,*** p<0.001) and comparisons are indicated by grey bars. Medians are indicated by horizontal lines, boxes represent 25^th and 75^th percentiles, and whiskers represent 10^th and 90^th percentiles. B: Responses to intradermal injection of β-alanine (90 μg/ 10 μl) were significantly greater in QC- than in SC afferents (*** p<0.001, Mann-Whitney U test). For each fiber class a Friedman ANOVA was performed (QC: χ²_(27,2)=47.4, p<0.001; SC: χ²_(32,2)=33.5, p<0.001). Pairwise comparisons within each fiber class were then conducted using using Wilcoxon matched pairs test (^### and ^&&& p<0.001; ^## and ^&& p<0.01, with Bonferroni correction for multiple testing). Responses between QC and SC classes were compared with Mann-Whitney U test (**p<0.01,*** p<0.001). Data representation is identical to panel A. C: All 27 QC fibers responded to β-alanine, but only 12/ 32 SC fibers were responsive (χ²₍₁₎=23.65, p<0.001, χ² test), and none of the MIAs responded. Net responses to β-alanine were significantly larger in responsive QC- than responsive SC fibers (** p<0.01, Mann-Whitney U test). Open symbols correspond to fibers that did not fulfill response criteria. Dashed line indicates net response of 10 action potentials in 5 min. Data in A are from cynomolgus monkeys only. Data in B and C are from cynomolgus and pigtail monkeys.

Figure 5. C-MIAs are vigorously activated by histamine and capsaicin

A: Intradermal injection of histamine produced a significantly greater net response in C-MIAs than in CMHs (* p<0.05, Mann-Whitney U test). Dashed line indicates net response of 10 action potentials in 5 min. B: In C-MIAs tested with both histamine and capsaicin (filled circles, n=11), capsaicin responses were larger than the responses to histamine. Open circles indicate C-MIAs only tested with one agent. Inset: When only fibers that fulfill the response criteria were included, the average histamine- and capsaicin-evoked response did not differ. C: Histograms of instantaneous frequencies of histamine responses in CMHs (blue columns) and C-MIAs (grey columns) and capsaicin responses in C-MIAs (red columns). All trials resulting in a response were included in the analysis. Bins are in log increments (factor $\sqrt{2}$). Error bars indicate SEM. Data are from cynomolgus and pigtail monkeys.

Figure 6. Histamine and β-alanine activate different populations of DRG neurons

A: Specimen fluorescent picture of cultured DRG neurons investigated in calcium imaging studies. The intracellular calcium levels of the neurons numbered are shown in the traces in B. (Scale bar: 100 μm). B: Traces of intracellular calcium responses from representative cells in A. Cells were tested with β-alanine (1 mM, 60 s), histamine (100 μM, 40 s), and capsaicin (5 μM) with at least a 3 min wash out period between applications. Cells with an increase above 20% in the 340/380 ratio were classified as responders. Cell #1 and #2 are responsive to β-alanine but not histamine. In contrast, cell #3 is responsive to histamine but not β-alanine and whereas cell #4 only responds to capsaicin. C: Venn diagram shows the incidence of response (percent) to different reagents. β-alanine responsive cells are separate from histamine-responsive cells. The majority of the β-alanine and histamine responsive cells also respond to capsaicin. A total of 484 cells from 3 monkeys was analyzed: 8% responded to β-alanine, 9% to histamine, and 27% to capsaicin. Less than 1% of the cells responded to both β-alanine and histamine, suggesting that β-alanine and histamine-responsive cells are separate subpopulations in DRG. Data are from cynomolgus monkeys only.