Comprehensive phenotyping of group III and IV muscle afferents in mouse.

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.

Dynamic changes in heat transducing channel TRPV1 expression regulate mechanically insensitive, heat sensitive C-fiber recruitment after axotomy and regeneration.

Peripheral injury leads to a significant increase in the prevalence of mechanically insensitive, heat-sensitive C-fibers (CH) that contain the heat transducing TRPV1 (transient receptor potential vanilloid type I) channel in mice. We have recently shown that this recruitment of CH fibers is associated with increased expression of the receptor for GDNF (glial cell line-derived neurotrophic factor) family neurotrophic factor artemin (GFRα3), and that in vivo inhibition of GFRα3 prevented the increase in TRPV1 expression normally observed following axotomy. Here we have directly tested the hypothesis that the recruitment of functional CH fibers following nerve regeneration requires enhanced TRPV1 levels. We used in vivo siRNA-mediated knockdown to inhibit the injury-induced expression of TRPV1 coupled with ex vivo recording to examine response characteristics and neurochemical phenotypes of different functionally defined cutaneous sensory neurons after regeneration. We confirmed that inhibition of TRPV1 did not affect the axotomy-induced decrease in polymodal C-fiber (CPM) heat threshold, but transiently prevented the recruitment of CH neurons. Moreover, a recovery of TRPV1 protein was observed following resolution of siRNA-mediated inhibition that was correlated with a concomitant rebound in CH neuron recruitment. Thus dynamic changes in TRPV1 expression, not absolute levels, may underlie the functional alterations observed in CH neurons and may contribute to the development of heat hyperalgesia after nerve injury.

Purinergic receptor P2Y1 regulates polymodal C-fiber thermal thresholds and sensory neuron phenotypic switching during peripheral inflammation.

We have recently found that, following complete Freund's adjuvant (CFA)-induced inflammation, cutaneous polymodal nociceptors (CPM) lacking the transient receptor potential vanilloid 1 (TRPV1) are sensitized to heat stimuli. In order to determine possible mechanisms playing a role in this change, we examined gene expression in the L2/L3 sensory ganglia following CFA injection into the hairy hind paw skin and found that G-protein-coupled purinoreceptor P2Y1 expression was increased. This receptor is of particular interest, as most CPMs innervating mouse hairy skin bind isolectin B4, which co-localizes with P2Y1. Additionally, our recent findings have shown that cutaneous CPMs in P2Y1-/- mice displayed significantly reduced thermal sensitivity. Together, these findings suggested a possible role for P2Y1 in inflammation-induced heat sensitization in these fibers. To test this hypothesis, we utilized our in vivo small interfering RNA technique to knock down the inflammation-induced increase in P2Y1 expression and then examined the functional effects using ex vivo recording. We found that the normal reduction of heat thresholds in CPM fibers induced by CFA was completely blocked by inhibition of P2Y1. Surprisingly, inhibition of P2Y1 during inflammation also significantly increased the number of CPM neurons expressing TRPV1 without a change in the total number of TRPV1-positive cells in the L2 and L3 dorsal root ganglia. These results show that the inflammation-induced enhanced expression of P2Y1 is required for normal heat sensitization of cutaneous CPM fibers. They also suggest that P2Y1 plays a role in the maintenance of phenotype in cutaneous afferent fibers containing TRPV1.