Mrgprd enhances excitability in specific populations of cutaneous murine polymodal nociceptors

Abstract

The Mas-related G protein-coupled receptor D (Mrgprd) is selectively expressed in nonpeptidergic nociceptors that innervate the outer layers of mammalian skin. The function of Mrgprd in nociceptive neurons and the physiologically relevant somatosensory stimuli that activate Mrgprd-expressing (Mrgprd(+)) neurons are currently unknown. To address these issues, we studied three Mrgprd knock-in mouse lines using an ex vivo somatosensory preparation to examine the role of the Mrgprd receptor and Mrgprd(+) afferents in cutaneous somatosensation. In mouse hairy skin, Mrgprd, as marked by expression of green fluorescent protein reporters, was expressed predominantly in the population of nonpeptidergic, TRPV1-negative, C-polymodal nociceptors. In mice lacking Mrgprd, this population of nociceptors exhibited decreased sensitivity to cold, heat, and mechanical stimuli. Additionally, in vitro patch-clamp studies were performed on cultured dorsal root ganglion neurons from Mrgprd(-/-) and Mrgprd(+/-) mice. These studies revealed a higher rheobase in neurons from Mrgprd(-/-) mice than from Mrgprd(+/-) mice. Furthermore, the application of the Mrgprd ligand beta-alanine significantly reduced the rheobase and increased the firing rate in neurons from Mrgprd(+/-) mice but was without effect in neurons from Mrgprd(-/-) mice. Our results demonstrate that Mrgprd influences the excitability of polymodal nonpeptidergic nociceptors to mechanical and thermal stimuli.

Figure 1.

CPMs in Mrgprd knock-in and WT mice bind IB4 but do not express either TRPV1 or CGRP. A–C, Sample immunohistochemistry of recorded CPM cells in Mrgpr ^+/+ (A), Mrgprd ^−/− (EGFPf) (B), and Mrgprd ^−/− (Cre) (C), as indicated by biotin labeling (left panels, green). Labeling of TRPV1, CGRP, and IB4 are shown for Mrgprd–IRES–EGFPf ^+/+, Mrgprd ^−/− (EGFPf), and Mrgprd ^−/− (Cre), respectively (middle panels, blue). GFP labeling is shown in the right panels (red). Arrows indicate recorded cell. Scale bar, 40 μm.

Figure 2.

CPMs in Mrgprd ^−/− mice exhibit decreased mechanical sensitivity. Sharp electrode recordings were made from WT (black), Mrgprd ^+/+ (white), and Mrgprd ^−/− (gray) CPM cells that were tested for their response to mechanical stimuli. A–C, No significant difference was observed between the mice in either the average mechanical threshold (A; millinewtons) or in the mean peak instantaneous frequency (B; hertz); however, the mean rate for multiple force stimuli (C; spikes per second; 5, 10, 25, 50, and 100 mN) were decreased in the Mrgprd ^−/− mice. Significance (p < 0.05) is indicated below Mrgprd ^−/− in relation to either just WT (*) or to both WT and Mrgprd ^+/+ (**).

Figure 3.

CPMs in Mrgprd ^−/− mice exhibit decreased cold sensitivity. Sharp electrode recordings were made from WT (black), Mrgprd ^+/+ (white), and Mrgprd ^−/− (gray) CPM cells that were tested for their response to cold stimuli. Mrgprd ^−/− CPM cells had lower cold thresholds (A; degrees Celsius) but maintained a similar peak instantaneous frequency (B; hertz) compared with Mrgprd ^+/+ mice. *p < 0.05.

Figure 4.

CPMs in Mrgprd ^−/− mice exhibit decreased heat sensitivity. Sharp electrode recordings were made from WT (black), Mrgprd ^+/+ (white), and Mrgprd ^−/− (gray) CPM cells that were tested for their response to heat stimuli. A, B, Compared with WT and Mrgprd ^+/+ strains, the average thermal threshold of CPMs in Mrgprd ^−/− mice showed decreased excitability in terms of a higher heat threshold (A; degrees Celsius) and a lower peak instantaneous frequency (B; hertz). C, A heat ramp from 31 to 52°C showed a significant reduction in the average spikes per second in Mrgprd ^−/− mice between the temperatures of 43 and 52°C. Significance (*p < 0.05) is indicated below Mrgprd ^−/− in relation to both WT and Mrgprd ^+/+.

Figure 5.

β-Alanine increases the firing rate of Mrgprd ^+/− but not Mrgprd ^−/− DRG neurons. A–D, Whole-cell patch-clamp recordings were made from cultured GFP⁺ DRG neurons from Mrgprd ^+/− (A, B) and Mrgprd ^−/− (C, D) mice. A, C, The threshold current (1 s pulse duration; bottom trace) required to evoke one AP (top trace) was identified for each neuron. B, D, β-Alanine (1 mm) was then bath applied for 5 min, and the same amount of threshold current was injected into each neuron. B, Neurons expressing one functional copy of Mrgprd increased their firing rate, whereas firing rate was not increased in Mrgprd ^−/− neurons (D). A–D, In these cells, the threshold current was 100 pA. Calibration: 40 mV, 200 ms.