Cowhage and histamine produce scratching behavior in monkey
Studies were performed in three monkeys to investigate the behavioral responses induced by cowhage spicules, histamine, and capsaicin. In humans, histamine and cowhage mainly cause the sensation of itch, i.e., the desire to scratch. In contrast, capsaicin injection produces a burning pain sensation in humans. To quantify the behavior in monkey, the number of scratches over the observation period (30 min) was counted. A high number of scratches was observed after the application of histamine and cowhage (). The monkeys typically used the contralateral hand to scratch the site of active cowhage application unless the cowhage was delivered, as it was in a few cases, to the lateral side of the leg, in which case the ipsilateral hand was used. The animals did not use the leg to scratch. Scratching, consisted of a series of flexing motions of the fingernails against the pruritic site. In contrast, a much lower number of scratches occurred after the application of the appropriate controls (saline, inactive spicules) and after the application of capsaicin or its vehicle (). We conclude that cowhage and histamine produce the sensation of itch in monkeys, and that nonhuman primates are therefore a suitable model to investigate the neuronal mechanisms underlying the sensation of cowhage- and histamine-induced itch.
Figure 1 Cutaneous administration of histamine and active cowhage spicules induces scratching behavior in monkey (n = 3). Scratching was not observed after the application of saline, inactive cowhage spicules, or capsaicin and its vehicle. The number of scratches (more ...)
We recorded from 43 CMHs that were responsive to mechanical (threshold, 1.9 ± 0.1 bar) and heat stimuli (threshold, 42.9 ± 0.4°C), and they had an average conduction velocity of 0.83 ± 0.02 m/s. The majority of units (38 of 43) were also cold responsive, displaying a response to application of a cold brass rod, but little or no response to the blunt pressure of a plastic-covered brass rod of the same weight.
CMHs show two types of responses to a 3 s 49°C heat stimulus (). Based on their quickly adapting response to heat, 22 units were characterized as QC units. In contrast, 21 units had slowly adapting, sustained responses to heat and were classified as SC units (). Distributions of the heat thresholds in QC fibers and SC fibers were almost nonoverlapping (), but such clear separation was not apparent for mechanical thresholds. However, QC fibers had significantly lower thresholds than SC fibers to both heat and mechanical stimuli (). QC fibers also tended to have a larger response to the 49°C, 3 s heat stimulus.
Comparison of physiological parameters between QC and SC fibers and their responses to pruritic stimuli (cowhage and histamine)
CMHs respond to cowhage
All 43 CMHs were tested with both inactive and active cowhage. shows a typical cowhage response. The response during the 15 s cowhage application interval (shaded area in figure) was caused by the mechanical activation elicited by spicule insertion and did not differ between inactive versus active cowhage application (31 ± 3 vs 32 ± 3 APs, respectively, n = 43). Minimal activity occurred after completion of the application of heat-inactivated cowhage spicules ().
Figure 3 Active cowhage causes bursting discharge in CMH afferent. A, Response induced by inactive cowhage (4 spicules) in unit AB59.3C. Inset, Superimposed action potential waveforms (n = 10). B, Response of the same afferent to active cowhage (8 spicules). Instantaneous (more ...)
Active cowhage elicited a delayed, but vigorous response (121 AP/5 min) (). The afferent discharged with high-frequency bursts followed by ~10 s long intervals of no activity. An instantaneous frequency histogram for this fiber () revealed two peaks, the peak at 0.1 Hz reflects the 10 s interburst intervals, and the peak at 3 Hz corresponds to the most often observed intraburst frequency. Interestingly, in response to the 49°C noxious heat stimulus, the same afferent showed a relatively constant discharge at ~10 Hz (), and was classified as an SC fiber.
A single cowhage spicule can vigorously activate CMHs
In five units, we attempted to elicit a response from insertion of a single active cowhage spicule in a mechanosensitive spot in the receptive field. Of 12 single spicule trials in these five units (at least two trials/unit), a response was elicited in only four trials (in three different units). Two responses to the single spicule were quite large and were remarkably similar to those generated by the multispicule insertion (). In one fiber, labeled as AC46.3C, the response to 18 spicules (83 APs) () was less than the activity induced by a single spicule (97 APs) (). A mechanical response during spicule insertion was elicited in all trials for which a response to cowhage was obtained and also in seven of eight trials with no cowhage response. Therefore, the occurrence of a mechanical response during spicule insertion did not predict the response to a single spicule.
Figure 4 The response to a single cowhage spicule can be quite vigorous. A, C, Response elicited after insertion of multiple active cowhage spicules into the receptive field of two different units (AC46.3C, AC49.1C). B, D, The activity elicited after insertion (more ...)
Responses and response patterns to cowhage are variable
The total response to active cowhage could be quite variable across trials for a given fiber (). This variability was observed in most fibers that were tested multiple times with active cowhage. Of 33 fibers that were tested two to three times with cowhage, 21 fibers had at least one cowhage trial that would be classified as a “no-response” trial (<10 APs/5 min), and three units were nonresponders throughout multiple trials. “No response” does not appear to be the result of tachyphylaxis or desensitization of the cowhage response because no-response trials occurred with an equal frequency between trials 1, 2, and 3 across fibers. Furthermore, the average response did not differ between the three trials ().
Figure 5 Responses to cowhage varied within fibers. For each fiber tested with multiple applications of cowhage, the number of action potentials in each 5 min trial was plotted. Responses were corrected for ongoing spontaneous activity observed during the baseline (more ...)
Variability in the magnitude of the maximal cowhage response was also observed across fibers. Of the 43 CMHs tested, 39 were classified as cowhage responders. Cowhage responders had a mean response to inactive cowhage of 5.7 ± 1.1 APs/5 min, whereas the average response to active cowhage spicules was 72.5 ± 8.7 APs/5 min.
Examples of the three different discharge patterns that were observed are shown in (see also , ). Approximately two-thirds of the fibers exhibited a bursting response where the discharge frequencies during the burst were around 0.5–5 Hz and the quiet interval between bursts was around 10 s (, , ). Three fibers (all QC fibers) exhibited very high instantaneous frequencies during the burst (, note different scale). For approximately one-third of the fibers, the bursting was not as obvious and the discharge pattern was relatively irregular (). In a given fiber, bursting could be observed in response to one application of cowhage, and an irregular discharge in response to another.
Figure 6 Three different types of discharge patterns were seen in response to active cowhage. A, Bursting discharge at intermediate frequency in unit AC48.2C. B, High-frequency bursting discharge in unit AB61.1C. Note that the scale is changed. C, Slow irregular (more ...)
To obtain a first estimate of the population response pattern in C-fibers to cowhage, we generated the instantaneous frequency histogram (similar to ) for all trials in cowhage responsive units. We then averaged across all trials to obtain the averaged histogram of instantaneous frequency shown in . This histogram has three apparent peaks: the peak at ~0.1 Hz corresponds to the average interburst interval (of around 10 s), the peak at ~1 Hz reflects the average intraburst frequency, and the peak at 40 Hz corresponds to the high intraburst discharge seen in three QC fibers.
QC fibers respond more vigorously to cowhage than SC fibers
Of the 39 cowhage responders, 21 were classified as SC type units, whereas 18 were QC type units. The time course of the cowhage induced neuronal excitation was similar in both populations (). The highest activity occurred 50–60 s after administration of cowhage, and the discharge declined linearly with time thereafter. However, the total discharge in QC fibers was greater than in SC fibers (97.1 ± 13.7 vs 49.0 ± 8.3 APs/5 min, respectively; p < 0.01, t test) ().
Figure 7 QC and SC fibers respond differently to cowhage application. A, Time course of cowhage-induced excitation in QC and SC fibers looks similar, but the discharge in QC fibers is larger (bin size, 10 s). B, Parameters extracted from the discharge pattern (more ...)
We wished to determine whether the bursting discharge pattern in response to cowhage differed between the SC and QC populations. A defining feature for the onset of a burst is the occurrence of a long interspike interval followed by a short interspike interval. As shown in , we computed the ratio of t1/t2 for all action potentials in the response and defined the onset of a burst as the time when t1/t2 > 5. For the trial with the maximum response, we then determined the number of bursts during the 5 min after application, the average interval between bursts (t1), and the average instantaneous frequency of the first two action potentials in the bursts (1/t2). The number of bursts during the response to cowhage was not significantly different between QC and SC fibers (). In contrast, the interval between bursts (t1) was significantly smaller in QC fibers than SC fibers, and the peak frequency (1/t2) in QC fibers was significantly higher than in SC fibers (, ). A plot of the average intraburst frequencies reveals that there were 3 QC units with considerably higher intraburst peak discharge frequencies (). When these three fibers are excluded from the analysis, the QC fibers still have significantly higher intraburst frequencies than the SC fibers (2.1 ± 0.3 Hz vs 1.3 ±.0.2 Hz; p < 0.02, t test).
CMHs respond to histamine
The responses of a typical CMH to saline and histamine (1 μg) are shown in (same fiber as in ). The needle insertion and injection of the 10 μl volume into the receptive field produced a response during both the saline and histamine trials, presumably because of the mechanical stimulation of the afferent. In contrast to the injection of saline, however, histamine caused lasting excitation. Notably, histamine led to a bursting discharge that was similar to that seen for cowhage.
Figure 8 CMH response to histamine. A, The fiber (AB59.3C) responded to insertion of the needle and injection of the volume of saline (10 μl), but responded only weakly (5 APs) during the 5 min after the injection. B, Histamine injection (1 μg) (more ...)
Similar to the activity caused by cowhage, different discharge patterns were observed after the histamine injection. About half of the units showed a high-frequency bursting discharge, whereas the remainder showed an irregular discharge with intermittent bursts. Seven units that were responsive to cowhage were completely unresponsive to histamine and were excited only during needle insertion and injection.
Histamine was tested in 38 CMHs, and 76% (29 of 38) were classified as responders. All of these 29 histamine-responsive units were also responsive to cowhage. Of the nine remaining units tested with histamine and found to be nonresponsive, seven responded only to cowhage, and two units had no response to cowhage or histamine. Unlike cowhage, the bursting discharge patterns in response to histamine were similar for the QC and SC fibers; the number of bursts, the interburst interval (t1), and the intraburst frequency (1/t2) were not significantly different ().
The response of CMHs to cowhage is larger than to histamine
The responses of units classified as responders were used to construct time-course plots for the activity evoked by histamine and cowhage (). The time courses of the cowhage and histamine responses were similar. The peak discharge occurred within the first 60 s, and the response decayed substantially in the first 5 min. Importantly, time courses of the responses to 1 or 10 μg of histamine were similar. For the 38 fibers tested with both cowhage and histamine, there was no correlation between the magnitudes of the cowhage response and the histamine response (), suggesting that different activation mechanisms are involved. The total response to cowhage was significantly larger than the response to histamine (67 ± 8.4 vs 27.4 ± 3.4 APs/5 min; ANOVA followed by Bonferroni test, p < 0.001) ().
Figure 9 A, Time course of the neuronal activity after the administration of cowhage or histamine (1 μg, 10 μg). Time courses of cowhage (n = 39) and histamine (1 μg, n = 18; 10 μg, n = 11) excitation were similar. Time courses (more ...)
Capsaicin does not cause lasting excitation in the majority of CMH afferents
Although most CMH units responded during the injection of 10 μg capsaicin, this response was not substantially greater than the response to vehicle injection, and the units did not show activity beyond the injection period. Therefore, most CMH units were classified as nonresponders. Only three units showed lasting responses to capsaicin, and these units were also classified as cowhage and histamine responders. The one fiber that responded robustly to capsaicin (80 APs/5 min) had smaller responses to histamine and cowhage (31 and 15 APs/5 min, respectively). The other two capsaicin-responsive units just met our criteria, having exactly 10 APs/5 min. In these two units, the histamine response was at least two times greater than the capsaicin response, and the cowhage response was at least two times greater than the histamine response. Although most CMHs did not exhibit a prolonged response to capsaicin and were classified as nonresponders, capsaicin had an effect on them, because most CMHs (17 of 28 tested) were desensitized to subsequent mechanical stimuli applied to the capsaicin injection site.
Of the 34 CMHs that were tested with all three compounds, 32 responded to cowhage, 27 responded to histamine, and only 3 units with lasting activation after capsaicin fulfilled the criteria of responders. The majority of the units (27) responded to both histamine and cowhage. Two CMHs did not respond to any of the chemical stimuli presented.
Mechanically insensitive afferent fibers
Five CMi afferents were tested with cowhage and none responded. In contrast, a large histamine and capsaicin response was observed in one CMi, and two CMi afferents responded only to capsaicin. Finally, two CMi fibers did not respond to any of the stimuli presented.