Stroking of the skin produces pleasant sensations that can occur during social interactions with conspecifics, such as grooming1. Despite numerous physiological studies (reviewed in ref. 2), molecularly defined sensory neurons that detect pleasant stroking of hairy skin3,4 in vivo have not been reported. Previously, we identified a rare population of unmyelinated sensory neurons that express the G protein-coupled receptor (GPCR) MrgprB45,6. These neurons exclusively innervate hairy skin with large terminal arborizations7 that resemble the receptive fields of C-tactile (CT) afferents in humans8. Unlike other molecularly defined mechanosensory C-fiber subtypes9,10, MrgprB4+ neurons could not be detectably activated by sensory stimulation of the skin ex vivo. Therefore, we developed a preparation for calcium imaging in their spinal projections during stimulation of the periphery in intact animals. MrgprB4+ neurons were activated by massage-like stroking of hairy skin, but not by noxious punctate mechanical stimulation. By contrast, a different population of C-fibers expressing MrgprD11 was activated by pinching but not by stroking, consistent with previous physiological and behavioral data10,12. Pharmacogenetic activation of MrgprB4- expressing neurons in freely behaving animals promoted conditioned place preference13, suggesting that such activation is positively reinforcing and/or anxiolytic. These data open the way to understanding the function of MrgprB4 neurons during natural behaviors, and provide a general approach to functionally characterizing genetically identified subsets of somatosensory neurons in vivo.

Damage to peripheral nerves is known to contribute to chronic pain states, including mechanical and thermal hyperalgesia and allodynia. It is unknown whether the establishment of these states is attributable to peripheral changes, central modifications, or both. In this study, we used several different approaches to assess the changes in myelinated (A) and unmyelinated (C) cutaneous nociceptors after transection and regeneration of the saphenous nerve. An ex vivo recording preparation was used to examine response characteristics and neurochemical phenotype of different types of functionally defined neurons. We found that myelinated nociceptors had significantly lower mechanical and thermal thresholds after regeneration, whereas C-polymodal nociceptors (CPMs) had lower heat thresholds. There was a significant increase in the percentage of mechanically insensitive C-fibers that responded to heat (CHs) after regeneration. Immunocytochemical analysis of identified afferents revealed that most CPMs were isolectin B4 (IB4) positive and transient receptor potential vanilloid 1 (TRPV1) negative, whereas CHs were always TRPV1 positive and IB4 negative in naive animals (Lawson et al., 2008). However, after regeneration, some identified CPMs and CHs stained positively for both markers, which was apparently attributable to an increase in the total number of IB4-positive neurons. Real-time PCR analysis of L2/L3 DRGs and hairy hindpaw skin at various times after saphenous nerve axotomy suggested multiple changes in neurotrophic factor signaling that correlated with either denervation or reinnervation of the cutaneous target. These changes may underlie the functional alterations observed after nerve regeneration and may explain how nerve damage leads to chronic pain conditions.

While much is known about the functional properties of cutaneous nociceptors, relatively little is known about the comprehensive functional properties of group III and IV muscle afferents. We have developed a mouse ex vivo forepaw muscle, median and ulnar nerve, dorsal root ganglion (DRG), spinal cord recording preparation to examine the functional response properties, neurochemical phenotypes, and spinal projections of individual muscle afferents. We found that the majority of group III and IV muscle afferents were chemosensitive (52%) while only 34% responded to mechanical stimulation and fewer (32%) responded to thermal stimuli. The chemosensitive afferents could be grouped into those that responded to a "low"-metabolite mixture containing amounts of lactate and ATP at pH 7.0 simulating levels observed in muscle during exercise (metaboreceptors) and a "high"-metabolite mixture containing lactic acid concentrations and ATP at pH 6.6 mimicking levels observed during ischemic contractions (metabo-nociceptors). While the majority of the metabo-nociceptive fibers responding to the higher concentration levels were found to contain acid-sensing ion channel 3 (ASIC3) and/or transient receptor potential vanilloid type 1 (TRPV1), metaboreceptors responding to the lower concentration levels lacked these receptors. Anatomically, group III muscle afferents were found to have projections into laminae I and IIo, and deeper laminae in the spinal cord, while all functional types of group IV muscle afferents projected primarily into both laminae I and II. These results provide novel information about the variety of sensory afferents innervating the muscle and provide insight into the types of fibers that may exhibit plasticity after injuries.

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