Behavioral model of itch, alloknesis, pain and allodynia in the lower hindlimb and correlative responses of lumbar dorsal horn neurons in the mouse

Abstract

We have further developed a behavioral model of itch and pain in the lower hindlimb (calf) originally reported by LaMotte et al. (2011) that allows comparisons with responses of lumbar dorsal horn neurons to pruritic and noxious stimuli. Intradermal (id) microinjection of the pruritogens histamine, SLIGRL-NH2 (agonist of PAR-2 and MrgprC11) and chloroquine (agonist of MrgprA3) into the calf of the lower limb elicited significant biting and a small amount of licking directed to the injection site, over a 30-min time course. Following id injection of histamine, low-threshold mechanical stimuli reliably elicited discrete episodes of biting (alloknesis) over a longer time course; significantly less alloknesis was observed following id injection of SLIGRL-NH2. Capsaicin injections elicited licking but little biting. Following id injection of capsaicin, low-threshold mechanical stimuli elicited discrete hindlimb flinches (allodynia) over a prolonged (>2h) time course. In single-unit recordings from superficial lumbar dorsal horn neurons, low-threshold mechanically evoked responses were significantly enhanced, accompanied by receptive field expansion, following id injection of histamine in histamine-responsive neurons. This was not observed in histamine-insensitive neurons, or following id injection of saline or SLIGRL-NH2, regardless of whether the latter activated the neuron or not. These results suggest that itch-responsive neurons are selectively sensitized by histamine but not SLIGRL-NH2 to account for alloknesis. The presently described "calf" model appears to distinguish between itch- and pain-related behavioral responses, and provides a basis to investigate lumbar spinal neural mechanisms underlying itch, alloknesis, pain and allodynia.

Keywords: allodynia; alloknesis; dorsal horn neuron; itch; pain; scratching.

Fig. 1

Time course of biting and licking elicited by histamine, SLIGRL and capsaicin. A. Cumulative duration of biting (■) or licking (∆) behaviors vs. time following id injection of histamine (50 µg/10 µl) in the calf skin of the hindlimb. Error bars: S.E.M. (n = 6). B. Same as in A for SLIGRL-NH2 (50 µg/10 µl). C. Capsaicin (10 µg/10 µl). D. Saline. E. 7% Tween 80. F. Mean cumulative duration of biting and licking elicited by each agent. *: significant difference between bite and lick durations (p<0.05, paired t-test; n=6/group).

Fig. 2

Time courses for alloknesis elicited by histamine and SLIGRL, and for allodynia elicited by capsaicin. A. At 5-min intervals after id injection of histamine (50 µg/10 µl) in calf skin, 3 von Frey stimuli (bending force 0.7 mN) were sequentially applied at separate sites 1–2 mm from the histamine injection. Presence or absence of biting (■), licking (∆) or flinching (●) immediately after each stimulus was used to calculate a behavior score (maximum value=3). Error bars: S.E.M. (n = 6). B. As in A for SLIGRL-NH2 (50 µg/10 µl). C: Capsaicin (10 µg/10 µl). D. Saline. E. 7% Tween 80. F. Cumulative behavior scores for von Frey stimulus-evoked biting, licking and flinching following id injections. *, #: significantly different from saline-evoked biting (p< 0.05, unpaired t-tests, n=6/group). $: significantly different from 7% Tween-80-evoked flinching (p< 0.05, unpaired t-test, n=6/group).

Fig. 3

Enhancement of mechanically-evoked response of dorsal horn neuron after histamine. Peristimulus-time histogram (bins: 1 sec) of unit’s responses to application of von Frey filament (VF, ↓, 0.7 mN),) before and after id injection of histamine (↓). Raw spike traces are shown above for portions of the response (dashed lines); dots show time of VF stimuli. Upper left inset: mechanosensitive receptive field (blue) on hindpaw. Lower left inset: lamina I recording site (●) on spinal section. Upper right inset: mechanosensitive receptive field expanded following histamine.

Fig. 4

Enhancement of mechanosensitivity in histamine-sensitive dorsal horn neurons. A. Histamine-sensitive units (n=8). Graphs plots mean peak responses to innocuous mechnanical von Frey stimulus (0.7 mN; ■, solid line), and mean unit firing rate over a 20 sec just prior to the von Frey stimulus (O, dashed line), vs. time following id injection of histamine (at time 0). Pre-histamine responses are shown at the −5 min timepoint. Error bars: S.E.M. *: significant difference compared to peak response before histamine (p < 0.05; paired t-test). B: graph as in A for 14 histamine-insensitive units. C: As in A for 10 units following id injection of saline (vehicle control). D: as in A for 6 units responsive to id injection of SLIGRL-NH2. E: as in A for 18 units insensitive to SLIGRL-NH2. F: 29 histologically recovered recording sites (dots) compiled on representative lumbar section.