Enhanced excitability of MRGPRA3- and MRGPRD-positive nociceptors in a model of inflammatory itch and pain

Abstract

Itch is a common symptom of diseases of the skin but can also accompany diseases of other tissues including the nervous system. Acute itch from chemicals experimentally applied to the skin is initiated and maintained by action potential activity in a subset of nociceptive neurons. But whether these pruriceptive neurons are active or might become intrinsically more excitable under the pathological conditions that produce persistent itch and nociceptive sensations in humans is largely unexplored. Recently, two distinct types of cutaneous nociceptive dorsal root ganglion neurons were identified as responding to pruritic chemicals and playing a role in itch sensation. One expressed the mas-related G-coupled protein receptor MRGPRA3 and the other MRGPRD (MRGPRA3+ and MRGPRD+ neurons, respectively). Here we tested whether these two distinct pruriceptive nociceptors exhibited an enhanced excitability after the development of contact hypersensitivity, an animal model of allergic contact dermatitis, a common pruritic disorder in humans. The characteristics of increased excitability of pruriceptive neurons during this disorder may also pertain to the same types of neurons active in other pruritic diseases or pathologies that affect the nervous system and other tissues or organs. We found that challenging the skin of the calf of the hind paw or the cheek of previously sensitized mice with the hapten, squaric acid dibutyl ester, produced symptoms of contact hypersensitivity including an increase in skin thickness and site-directed spontaneous pain-like (licking or wiping) and itch-like (biting or scratching) behaviours. Ablation of MRGPRA3+ neurons led to a significant reduction in spontaneous scratching of the hapten-challenged nape of the neck of previously sensitized mice. In vivo, electrophysiological recordings revealed that MRGPRA3+ and MRGPRD+ neurons innervating the hapten-challenged skin exhibited a greater incidence of spontaneous activity and/or abnormal after-discharges in response to mechanical and heat stimuli applied to their receptive fields compared with neurons from the vehicle-treated control animals. Whole-cell recordings in vitro showed that both MRGPRA3+ and MRGPRD+ neurons from hapten-challenged mice displayed a significantly more depolarized resting membrane potential, decreased rheobase, and greater number of action potentials at twice rheobase compared with neurons from vehicle controls. These signs of neuronal hyperexcitability were associated with a significant increase in the peak amplitude of tetrodotoxin-sensitive and resistant sodium currents. Thus, the hyperexcitability of MRGPRA3+ and MRGPRD+ neurons, brought about in part by enhanced sodium currents, may contribute to the spontaneous itch- and pain-related behaviours accompanying contact hypersensitivity and/or other inflammatory diseases in humans.

Keywords: MRGPRA3; MRGPRD; allergic contact dermatitis; itch; pain.

Figure 1

SADBE challenge induced CHS and spontaneous site-directed behaviours in previously sensitized mice. (A) In comparison with pre-challenge values, the thickness of calf skin was significantly increased 24 h after the second SADBE challenge (n = 13 mice) whereas applications of acetone alone (vehicle) had no significant effect (n = 6) (paired t-test). (B) After SADBE challenge of the calf, cumulative durations of spontaneous biting and licking of the calf were each significantly increased over pre-challenge (Pre) values (n = 20) but not after applications of acetone (n = 6) (repeated measures analysis of variance with Bonferroni adjustments). (C) After SADBE challenge of the cheek, the number of bouts of spontaneous scratching and wiping were each significantly increased over pre-challenge values (n = 10) whereas the acetone vehicle itself had no significant effect (n = 6) (repeated measures analysis of variance with Bonferroni adjustments). (D) SADBE challenge to the nape of the neck in previously sensitized mice evoked significantly fewer bouts of site-directed scratching in MRGPRA3^GFP-Cre;ROSA26^DTR mice with selective ablation of MRGPRA3⁺ neurons (n = 7) than control ROSA26^DTR mice (n = 8) (unpaired t-test).*P < 0.05.

Figure 2

CHS produced spontaneous activity and enhanced stimulus-evoked responses in MRGPRA3⁺ and MRGPRD⁺ neurons. (A) Bright-field image of a neuron under recording with the extracellular electrode (outlined with dashed blue lines). (B) Fluorescent microscopy revealed the expression of GFP (i.e. MRGPRA3) in this neuron. (C) Original extracellular recording trace (Ie) and action potential markers (AP) indicated the presence of abnormal spontaneous activity (SA) of this neuron, without any external stimulation. (D) Responses of this MRGPRA3-GFP⁺ neuron to 160 mN force through a 200 μm diameter probe (1 s). (E) Response to heat stimulation (38 to 51°C, 5 s), in the presence of ongoing spontaneous activity. (F) Location of cutaneous receptive field (red dot) of this neuron on the hairy skin of hind paw (the region challenged with SADBE), and conduction velocity (0.67 m/s, lower trace) obtained with electrical stimulation (arrow) to the peripheral receptive field. (G) Responses of the MRGPRA3⁺ neuron innervating the vehicle control or SADBE-challenged skin to mechanical stimulus of 160 mN force through a 200 μm probe for 1 s, and to heat stimuli (38 to 51°C, 5 s). The neuron innervating SADBE-challenged skin was initially silent but showed prolonged after-discharge following the 160 mN mechanical stimulation and heating. No stimulus-evoked after-discharges were observed in MRGPRA3 neurons from control animals. (H) Bright-field image of a MRGPRD⁺ neuron under recording with the extracellular electrode (outlined with dashed blue lines). (I) Fluorescent microscopy revealed the expression of GFP (or MRGPRD) in this neuron. (J) Location of cutaneous peripheral receptive field (red dot) of this neuron on the hairy skin of hind paw (the region challenged with SADBE), and conduction velocity (0.57 m/s, lower trace) obtained with electrical stimulation (arrow) to the peripheral receptive field. (K and L) Responses of MRGPRD-GFP⁺ neuron to 80 mN force through a 200 μm diameter probe (1 s), and to heat stimulation (38 to 51°C, 5 s). This neuron did not exhibit spontaneous activity. (M) Spontaneous activity was observed in a subset of MRGPRD⁺ neurons from CHS mice, but not from controls. (N) MRGPRD⁺ neurons from CHS mice exhibited prolonged after-discharges in response to noxious mechanical stimulus (80 mN, 1 s) but not heat (38 to 51°C, 5 s). No prolonged after-discharges were evoked by noxious mechanical and heat stimuli in MRGPRD⁺ neurons from control animals.

Figure 3

SADBE challenge increased the excitability of MRGPRA3⁺ and MRGPRD⁺ neurons. (A) Representative traces of action potentials elicited at rheobase and twice rheobase in MRGPRA3⁺ neurons from vehicle control and SADBE-treated mice. SADBE challenge results in a reduction of rheobase while increasing the number of action potentials evoked by a 2× rheobase current injection. (B) MRGPRA3⁺ and MRGPRD⁺ neurons from SADBE-challenged mice each exhibited a more depolarized resting membrane potential (RMP), lower mean rheobase, and greater number of action potentials at twice rheobase, as compared to control group. Cell numbers tested are indicated in the parentheses. *P < 0.05 versus vehicle control, unpaired t-test.

Figure 4

Voltage-gated Na⁺ currents of MRGPR neurons were increased after the second SADBE challenge. (A) Representative traces of voltage-gated Na⁺ currents in two MRGPRA3⁺ neurons, one from an acetone-treated control mouse (left) and the other from a SADBE-treated mouse. Total Na⁺ currents (I_total; top) were generated by stepwise 30-ms test pulses in 5-mV steps from −100 to +50 mV, preceded by a 500-ms prepulse of −100 mV (inset). TTX-resistant (TTX-R) currents (middle) were generated using the same series of test pulses but preceded by a 500-ms prepulse of −50 mV. TTX-sensitive (TTX-S) currents (bottom) were obtained by digitally subtracting TTX-resistant currents from total Na⁺ currents. (B) The peak current densities of both TTX-sensitive (left) and TTX-resistant (right) Na⁺ currents was significantly larger in SADBE-treated mice (closed circles, n = 27 neurons), as compared with controls (open circles, n = 24 neurons). Data for MRGPRA3⁺ and MRGPRD⁺ neurons were similar and thus combined (see main text). *P < 0.05, SADBE versus vehicle control, one-way repeated measures analysis of variance with Tukey post hoc comparisons.

Figure 5

Comparison of voltage-dependent activation and steady-state inactivation curves of TTX-sensitive and TTX-resistant Na⁺ currents in MRGPR neurons between vehicle-treated and SADBE-challenged mice. Data were pooled for MRGPRA3⁺ and MRGPRD⁺ neurons. (A) For activation curves, normalized conductance (G / Gmax) was plotted against test pulse voltage and fitted to a Boltzmann function. (B) For inactivation curves, normalized current (I / Imax) was plotted against prepulse voltage and fitted to a negative Boltzmann function. Line represents the average of the individual curve fits. There were no significant differences in the levels of activation or inactivation for TTX-sensitive (A) and TTX-resistant (B) currents between the neurons from control (open circles) and SADBE-treated mice (closed circles).