An argon laser was used for excitation of GFP and YFP and HeNe1 was used for rhodamine

entially sensitive to temperature. The cross-cluster expression of genes encoding mechanoreceptive molecules such as Trpcs, Kcnks, Asic3 and Piezo2 suggests that mechanical stimuli are sensed by neurons in multiple clusters. The predicted sensitivity of clusters to chemicals depends on the differential expression of chemical receptors. The differential expression of pruritogen receptors and NPB encoded by Nppb suggests that C2, C4 and C5 selectively transduce itch signals generated by pruritogens. For example, C4 contains mRNAs encoding the histamine H1 receptor, Mrgprc11 and Mrgpra3, and therefore could be specific for histamine-, BAM822- and chloroquine-induced itch. To evaluate the functional phenotypes of neuron clusters, we carried out in vivo whole-cell patch clamp recording of single neurons selected at random in the L5 DRG of mice upon application of a noxious mechanical stimulus, heat, cold or innocuous mechanical stimulus to the plantar skin of the hindpaw. The appropriate peripheral stimulus induced an inward Na+ current and generated an action potential in the responsive neuron. mRNA diffusing into the internal solution of the recording pipette was collected and processed for single-cell quantitative real-time PCR to evaluate the expression levels of neuron-type markers, including Gal, Zcchc12, Cldn9, Nppb, Nts, Sst, Th, Mrgpra3, Mrgprb4, Mrgprd, Nefh, Pvalb, Nxph1 and Baiap2l1. This analysis was successfully carried out for 69 responsive neurons. Cluster sorting showed that C1, C2, C4, C5, C6 and C7 are involved in sensing heat and pinch but have different preferred stimuli. Most recorded C1-2 neurons responded to heat and pinch. Over half of the neurons in C6 and C7 were activated by Y-27632 dihydrochloride chemical information pressure and pinch, whereas most neurons in C4 responded to pinch. Furthermore, these results suggest the sensory selectivity of neuron clusters. C2 neurons responded to heat and pinch but not to pressure. All recorded C3 neurons were stimulated by brush but not heat, and these neurons were also responsive to pressure and pinch. C9 neurons primarily responded to pressure and pinch. Neurons responding to cold are scattered in C2, C4-1 C5 and C6, but none of these neurons were found to respond only to cold. These results suggest that the transcriptional heterogeneity among neuronal clusters contributes to the functional specificity and probability of neurons to respond to cutaneous stimuli. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19822627 The sensory thresholds of neurons in the same cluster could be different due to intra-cluster npg Types of primary sensory neurons 94 Chang-Lin Li et al. npg 95 PR B4-signaling network, whereas C4-1 neurons are low-threshold mechanoreceptive. C2-2 neurons may be A-fiber MHN. Cold-sensitive neurons are scattered among C2-1, C4-1 and C5, which can also act like MHNs. This classification suggests a new framework for classifying somatosensory neuron types. Discussion Tissue-specific approach incorporating single-cell RNAseq for neuron-typing The present study presents a DRG-specific approach to neuron sampling for neuron typing that incorporates single-cell RNA-seq with other methods. First, neuron sizes are recorded because the differences between small and large neurons correlate with the projections of afferent fibers in the spinal cord and their functions. The correlation between neuron size and neuron type was proven by transcriptome analysis. Second, the principal neuron subsets classified according to IB4 labeling and their proportions are incorporated int