These data show that mouse sensory neurons that express the Mrgprd receptor have classic properties of unmyelinated nociceptors: small-diameter cell bodies, long-duration action potentials, TTX-resistant Na+ currents, and Ca2+ currents inhibited by opioids. Unlike other small sensory neurons, nearly all Mrgprd+ neurons fail to respond to capsaicin, acid, and menthol yet uniformly exhibit currents evoked by extracellular ATP. These results suggest two conclusions: the sensory neurons that innervate the outermost layer of mouse skin are nociceptors and they are specialized to detect extracellular ATP. Strict dependence on ATP implies that the neurons themselves are not the primary sensor of local noxious events. Rather skin cells, such as keratinocytes, must sense the event and release the ATP.
Although the data presented here demonstrate that Mrgprd+ neurons display “nociceptor-like” properties, they cannot conclusively determine that these neurons are nociceptors. Because it is not yet known whether nonnoxious stimuli can cause the release of ATP from keratinocytes, these neurons may sense other stimuli. However, recent evidence using an ex vivo
preparation supports the conclusions presented here by demonstrating that these neurons are polymodal nociceptors (Lawson et al. 2007
). This finding is intriguing because it suggests that there may be more than one type of stimulus that can initiate signaling between keratinocytes and sensory neurons using ATP. Recent reports have demonstrated that both mechanical stimulation and cell damage can cause the release of ATP from keratinocytes (Cook and McCleskey 2002
; Koizumi et al. 2004
). The released ATP was shown to activate P2 receptors on neighboring DRG neurons in culture. Keratinocytes can also respond to changes in temperature as they express temperature-sensitive TRP channels (Lee and Caterina 2005
; Lumpkin and Caterina 2007
). However, it is unknown at this point whether keratinocytes actually release ATP in response to changes in temperature. Also unknown is whether keratinocytes and sensory neurons can in fact communicate in vivo, but they are known to be in direct contact (Chateau and Misery 2004
), which provides anatomical support for this concept.
What is surprising about the results presented here is that most Mrgprd+ neurons did not respond with ionic currents to ligands other than ATP. The ATP sensitivity of the Mrgprd+ population corresponds well with histological data demonstrating that all Mrgprd+ neurons express the P2X3 receptor (Zylka et al. 2005
). What the present data show is that this population appears to be selectively excited by ATP through this single ATP-gated channel. Although all Mrgprd+ neurons did respond to the GABA agonist muscimol, and GABA synthesis may occur in fibroblasts in the epidermis (Ito et al. 2007
), it is unclear whether GABA can be released in the epidermis. So GABA receptors may not participate in peripheral activation of these neurons but may be involved in central modulation. Additionally, we did not examine responsiveness to the proposed ligand for Mrgprd, β
-alanine (Shinohara et al. 2004
). If this ligand is present in the skin, it would represent an additional stimulus that Mrgprd+ neurons are capable of detecting. Based on the lack of response to the other ligands tested, direct sensation of temperature through TRPV1 and TRPM8 or the presence of chemical mediators of pain appear not to be important for Mrgprd+ neurons. The reasons for this are unclear, but because Mrgprd+ fibers make up 60% of epidermal innervation (Zylka et al. 2005
), it may be left to the other 40% of epidermal afferents that terminate in deeper epidermal layers to sense these stimuli. Further, Zylka et al. found that 10% of Mrgprd-containing fibers were intertwined with CGRP-containing fibers. The functional significance of this is unknown, but it may allow the nervous system to respond to stimuli in the stratum granulosum that Mrgprd+ neurons cannot detect.
Additionally, ATP may not be the only substance released from keratinocytes that Mrgprd+ neurons are capable of detecting. A recent study detailed the ability of CB2
cannabinoid receptor agonists to produce antinociception by causing the release of β
-endorphin from keratinocytes in the stratum granulosum of the epidermis (Ibrahim et al. 2005
). A similar study showed that activation of the ETB
endothelin receptor, which is present on keratinocytes in both the stratum granulosum and stratum spinosum, can also cause the release of β
-endorphin from keratinocytes producing antinociception (Khodorova et al. 2003
). Because Mrgprd+ neurons are the majority of the innervation of the stratum granulosum (Zylka et al. 2005
), these neurons may be responsive to β
-endorphin released from keratinocytes; this may explain the high percentage of opioid-sensitive Mrgprd+ neurons. Thus keratinocytes may be capable of both stimulating and inhibiting the activity of cutaneous afferents using ATP and β
-endorphin, respectively. Future studies will determine whether activators of other G-protein-coupled receptors in addition to μ
-opioid agonists, which might also be released in the skin, can modulate the activity of these neurons. Ultimately it may be the case that for certain sensations keratinocytes are the actual “sensors” in the skin and sensory neurons merely relay the signal by responding to one or more mediators released by keratinocytes (Denda et al. 2007
; Lumpkin and Caterina 2007
Labeling for Mrgprd allows the in vitro study of sensory neurons the target innervation of which, the stratum granulosum of the epidermis, is clearly defined. Using this method, the experiments described here and those performed previously have only begun to uncover properties of these cutaneous afferents that are some of the closest neuronal terminals to the outside world. It may not be surprising if their phenotype is in fact nociceptive as responses to potentially damaging stimuli would be initiated before any other. These data show that Mrgprd+ neurons are activated selectively by the presence of ATP. This suggests that an intermediate (i.e., keratinocytes) may initiate signaling into the CNS by releasing ATP onto Mrgprd+ nociceptors. Although it is possible that dependence on an intermediate can slow down the initiation of signaling, this may be outweighed by the additional level of stimulus filtering provided by the intermediate. This filtering ensures that stimuli that produce a response are those that are truly capable of causing damage, a determination made by the epidermis itself and not the nervous system.