Site-dependent and state-dependent inhibition of pruritogen-responsive spinal neurons by scratching

Abstract

The relief of itch by scratching is thought to involve inhibition of pruritogen-responsive neurons in the spinal cord. We recorded the responses of superficial dorsal horn neurons in mice to intradermal injection of the pruritogens chloroquine and histamine. Scratching within an area 5-17 mm distant from the injection site, outside of the units' mechanoreceptive fields (off-site), significantly inhibited chloroquine-evoked and histamine-evoked responses without affecting capsaicin-evoked firing. This is consistent with observations that scratching at a distance from a site of itch is antipruritic. In contrast, scratching directly at the injection site (within the receptive field; on-site) had no effect on chloroquine-evoked neuronal firing, but enhanced the same neurons' responses to intradermal injection of the algogen capsaicin. Moreover, neuronal responses to histamine were enhanced during on-site scratching, and this was followed by suppression of firing below baseline levels after termination of scratching. Scratching thus inhibits pruritogen-responsive neurons in a manner that depends on the input modality (i.e. pain vs. histamine-dependent or histamine-independent itch) and skin location.

Fig. 1

Increased firing of spinal neurons following chloroquine, histamine and capsaicin. Bar graphs show, from left to right, responses to id microinjection of vehicle saline (vehicle for chloroquine; n=8), chloroquine (CQ, 100 µg/µl, n=10), histamine (His, 50 µg/µl, n =6), 0.7% Tween 80 (vehicle for capsaicin; n=8), and capsaicin (Cap, 30 µg/µl, n=14). Mean firing rate was calculated over a 1 min period pre- and again 1 min immediately post-injection. Error bars: SEM. Mean firing post-chloroquine and - capsaicin injections was significantly greater compared to mean firing pre-injection as well as mean firing following vehicle injections (*p < 0.01 for both; paired t-test and unpaired t-test, respectively).

Fig. 2

Individual example. Upper left peristimulus-time histogram (PSTH; bin: 1 sec) shows response of superficial dorsal horn unit to id injection of chloroquine (CQ; at arrow; 100 µg/µl), and effects of off-site and on-site scratching. Gray area shows expanded portions of the PSTHs above; duration of scratching is indicated by large horizontal bars above lower PSTHs. Figurine drawings of hind paw show injection site (X) and location of scratching (doublt-headed aroows). Upper and lower middle PSTHs shows unit’s response to brush, pinch and scratching within (on-site) and outside the receptive field (off-site), after CQ-evoked firing had waned. In middle figurine drawings of hind paw, location of mechanosensitive receptive field indicated by gray area. Upper and lower right PSTHs shows same unit’s response to id capsaicin (30 µg/µl) and scratch stimuli.

Fig. 3

State- and site-dependent inhibition of activity of chloroquine-responsive superficial dorsal horn neurons. A: Averaged PSTH (bins: 1 sec) of activity following id chloroquine (CQ) in 10 superficial dorsal horn units, before, during and after scratching on- or off-site. Horizontal arrows indicate duration of scratching. Error bars: SEM. B: Graph plots individual (thin lines) and mean (thick line +/− SEM) responses of superficial dorsal horn units following id chloroquine, before (pre), during, and after (post) off-site scratching. Responses normalized to pre-scratch firing rate. *: significantly different compared to the pre-scratch baseline (p < 0.05 during; p< 0.05 post, Dunnett’s test). #: significantly different compared to during (p < 0.05; paired t-test). Inset figurine shows perimeters of units’ mechanical receptive fields (dashed lines) on drawing of ipsilateral hindpaw. C: PSTH of same units in A, in which scratching was delivered on-site during the unit’s response to chloroquine. D: Graph as in B showing chloroquine-evoked activity, pre-, during, and post- on-site scratching. One-half of the units exhibited increased firing during scratching, but the overall effect (124.3%) was not statistically significant. There was no significant correlation between the chloroquine-evoked firing rate and the degree of on-site scratch-evoked inhibition (r= 0.545, p=0.103, Pearson’s product moment correlation). E: PSTH as in A showing lack of effect of off-site scratching on mean response to capsaicin. F: Graph as in B showing capsaicin-evoked activity, pre-, during, and post- off-site scratching. Inset shows histologically-recovered recording sites in superficial dorsal horn of units tested with CQ. G: PSTH as in C showing facilitatory effect of on-site scratching during the response to capsaicin. H: Graph as in D showing capsaicin-evoked activity, pre-, during, and post- on-site scratching. *: significantly different compared to pre (p < 0.05; Dunnett’s test).

Fig. 4

State- and site-dependent inhibition of activity of histamine-responsive superficial dorsal horn neurons. A: Averaged PSTH (bins: 1 sec) of activity following id histamine (His) in 6 superficial dorsal horn units, before, during and after scratching on- or off-site. Horizontal arrows indicate duration of scratching. Error bars: SEM. B: Graph plots individual (thin lines) and mean (thick line +/− SEM) responses of superficial dorsal horn units following id histamine, before (pre), during, and after (post) off-site scratching. Responses normalized to pre-scratch firing rate. *: significantly different compared to the pre-scratch baseline (p < 0.05 during; Dunnett’s test). Inset figurine shows perimeters of units’ mechanical receptive fields (dashed lines) on drawing of ipsilateral hindpaw (two receptive fields spanned the entire hindpaw). C: PSTH of same units in A, in which scratching was delivered on-site during the unit’s response to histamine. D: Graph as in B showing histamine-evoked activity, pre-, during, and post- on-site scratching. *: significantly different compared to the pre-scratch baseline (p < 0.05 post, Dunnett’s test). E: PSTH as in A showing lack of effect of off-site scratching on mean response to capsaicin. F: Graph as in B showing capsaicin-evoked activity, pre-, during, and post- off-site scratching. Inset shows histologically-recovered recording sites in superficial dorsal horn of units tested with histamine. G: PSTH as in C showing facilitatory effect of on-site scratching during the response to capsaicin. H: Graph as in D showing capsaicin-evoked activity, pre-, during, and post- on-site scratching. *: significantly different compared to pre (p < 0.05; Dunnett’s test).