This study examined the function of Mrgprd-containing sensory afferents and the role of Mrgprd as a modulator of cell excitability. Using an ex vivo preparation, we assessed the functional role of Mrgprd in cutaneous sensory neurons by examining three knockin mouse lines and comparing them to WT (C57BL/6) mice. Based on GFP and Neurobiotin co-labeling of recorded cells, we found that the Mrgprd receptor was almost exclusively localized in a large subset of C polymodal nociceptors (CPMs) that respond to mechanical stimuli, as well as to heat and sometimes cold stimuli. In the transgenic animals that we examined in this study, 76% and 92% of the CPM cells recorded from Mrgprd+/+ and Mrgprd−/− strains, respectively, exhibit expression of GFP driven by the Mrgprd promoter. The disparity in percentages could reflect the fact that in Mrgprd+/+ cells, EGFP (the surrogate marker for Mrgprd expression) is expressed after an IRES, while in Mrgprd−/− cells it is inserted as part of the coding sequence. Based on unpublished observations (M.J. Zylka and D. J. Anderson), the level of EGFP expression in the former line is about 10-fold lower than in the latter line. So the value of 76% could be an under-estimate, due to the lower expression of EGFP.
Deletion of Mrgprd significantly decreased sensitivity to mechanical and thermal stimuli. Specifically, a decreased firing rate was observed in cells lacking Mrgprd in response to lower mechanical forces and noxious heat. Additionally, thresholds of activation were remarkably lower in response to cold, and higher in response to hot stimuli. The decreased firing rates in response to mechanical and thermal stimuli, as well as the greater deviation from normal body temperatures that was required for CPMs to start firing suggests that Mrgprd may be necessary to maintain normal excitability. In support of this, in vitro patch clamp studies on cultured DRG neurons from mice lacking Mrgprd had a higher rheobase than heterozygous mice. The application of a known Mrgprd ligand, β-alanine, significantly reduced the rheobase and increased the firing rate in the neurons of heterozygous mice, but not in Mrgprd−/− mice.
Sensory neurons that express the Mrgprd receptor share multiple characteristics of unmyelinated nociceptors. Immunohistochemically these afferents bind IB4, and express P2X3, but not SP, CGRP or TRPV1 (Dong et al., 2001
; Zylka et al., 2003
; Zylka et al., 2005
). Similarly, in this study as well as in previous studies in our laboratory using C57BL/6, Swiss Webster and C3H/BL6 WT mice, the vast majority of CPM fibers innervating mouse hairy skin characteristically bind IB4 and express P2X3, rarely express CGRP, but always lack TRPV1 (Albers et al., 2006
; Lawson et al., 2008
). Additionally, patch clamp of dissociated mouse DRG by Dussor and colleagues (2008)
, indicated that Mrgprd+ cells have somas with small diameters, long-duration APs, TTX-resistant Na+
currents, and Ca2+
currents inhibited by opioids. Finally, immunocytochemical analysis in this study confirmed that the majority of Neurobiotin-labeled CPM cells were in fact GFP+ (and hence normally Mrgprd+).
All of the CPM cells that contained Mrgprd in this study responded to cold and/or heat. It is well established that some of the key proteins involved in thermal transduction belong to the TRP family, including TRPV1 (Caterina et al., 1997
), TRPV2 (Caterina et al., 1999
), TRPA1 (Story et al., 2003
), TRPM8 (Peier et al., 2002
), and others. Previous work from our laboratory indicates that CPM cells are the largest population of heat-responding sensory afferents found in mouse hairy skin, but they lack the heat, capsaicin, and proton-sensitive TRPV1 channel (Zwick et al., 2002
; Woodbury et al., 2004
; Lawson et al., 2008
). Instead, this protein appears to be isolated to a small population of mechanically-insensitive C-fibers that respond to heat (Lawson et al., 2008
). In agreement with this data, Dussor and colleagues (2008)
showed that dissociated cells containing Mrgprd fail to respond to either capsaicin or protons. Similarly, heat threshold and immunohistochemical evidence argue against the presence of heat-activated TRPV2 in the CPMs of WT animals (Woodbury et al., 2004
; Lawson et al., 2008
). As for the ionic currents evoked by cold stimuli in some Mrgprd+ CPMs, a previous report suggests that there is a lack of functional TRPM8 (≤25°C) and TRPA1 (≤17°C) as well, judging by the lack of a response to either the TRPM8 agonist menthol or the TRPA1 agonist cinnamaldehyde (Dussor et al., 2008
). Thus how these sensory afferents transduce thermal signals remains to be determined.
While direct activation of known TRP channels by temperature seems unlikely in Mrgprd+ neurons, temperature could be sensed indirectly in Mrgprd+ afferents via the release of ATP from keratinocytes (Dussor et al., 2008
). Mrgprd+ neurons express the ATP-gated receptor P2X3 (Zylka et al., 2005
) and respond robustly to ATP with P2X3-like current kinetics (Dussor et al., 2008
). In this way, changes in temperature (and perhaps other stimuli) are detected by skin cells, which then propagate signals to Mrgprd+ fibers in the stratum granulosum through the release of ATP. Such a role for keratinocytes in the sensation of the external environmental stimuli has been previously suggested (see reviews, Denda et al., 2007
; Lumpkin and Caterina, 2007
; and Peier et al., 2002
). However, support for such a signaling mechanism is mixed. For example, P2X3−/− mice exhibit a reduced response of wide dynamic range neurons in the spinal cord dorsal horn to hindpaw heating (Souslova et al., 2000
; Shimizu et al., 2005
); however, behavioral testing indicated normal withdrawal from a hot plate and paradoxically indicated an enhanced avoidance of hot and cold temperatures in a thermal gradient (Shimizu et al., 2005
). While these findings may reflect some form of compensatory mechanism exists, they also suggest that the mechanism of transduction is more complex than ATP release from kerotinocytes and activation of P2X3 receptors in this subset of nociceptive fibers. Although we tested for mechanical and thermal responsiveness in CPM fibers, we did not test these cells for responses to chemical stimuli using the ex vivo
preparation. It is likely that these Mrgprd+ afferents are activated by molecules other than ATP, including the Mrgprd agonist, β-alanine (Shinohara et al., 2004
). In this study, using a dissociated cell preparation, we demonstrated for the first time that Mrgprd+ neurons are directly sensitized by β-alanine. β-alanine, and the related dipeptide carnosine (β-alanyl-L-histidine), are present in high concentrations in vertebrate muscles and skin (Crush 1970
; Nagai et al., 1986
; Kohen et al., 1988
) as well as rat sciatic nerve (Marks et al., 1970
). In addition, β-alanine can be generated from carnosine by carnosinase enzymes. Taken together, this suggests β-alanine could be present and/or produced in skin and tonically activate Mrgprd on sensory afferents. Tonic activation of Mrgprd could in turn increase the excitability of Mrgprd+ neurons via inhibition of KCNQ/M-currents (Crozier, et al., 2007
). The loss of this tonic activation could explain why Mrgprd+ neurons are less sensitive to thermal, mechanical, cold and electrical stimulation when the Mrgprd receptor is deleted. However, it should be noted that we have made repeated attempts, using the same the voltage step protocol used in rat sensory neurons (Crozier et al.
2005), but have been unable to reliably record M-currents in Mrgprd-GFP neurons. In addition, our colleague, Dr. Gregg Dussor, has also experienced the same difficulties in recording the M-current in these cells (G. Dussor, personal communication). Therefore, we were unable to directly test this hypothesis.
Numerous studies have shown that the C fiber population consists of multiple subsets of cells having a diverse pattern of histochemical markers, including neuropeptides, growth factor receptors, purinergic receptors, heat/cold-sensing receptors, proton-sensing receptors, IB4-binding, and others. The majority of these markers, however, are expressed in overlapping patterns in a wide range of cells types and/or tissue types. In this study, Mrgprd+ neurons accounted for 76-92% of the total CPM cells recorded from knockin animals. Considering work done by this laboratory and by others, the overwhelming localization of Mrgprd in CPM cells of the skin and its absence in other C fiber groups and tissue types, makes Mrgprd useful for targeting a major population of non-peptidergic C-polymodal nociceptors that innervate the skin.
The results presented in this study verify two key aspects of Mrgprd+ fibers. First, this receptor is localized in sensory afferents of the skin that specifically function as C-polymodal nociceptors. Secondly, it directly supports the idea that Mrgprd can influence cell excitability, at least in response to mechanical and thermal stimuli, as well as to its ligand β–alanine.