, 2004 and Ruscheweyh and Sandkühler, 2002). We found that the majority (29 out of 34) of B5-I neurons showed tonic firing ( Figures 7B and S5A) and may therefore function as integrators. Neurons can be classified based on morphology and previous studies have described several types including vertical, islet, central, and radial, although many cells cannot be classified according to this scheme ( Grudt and Perl, 2002, Yasaka et al., 2007 and Yasaka et al., 2010). To determine whether B5-I neurons buy ZD6474 belonged to any of these

subsets, we reconstructed B5-I neurons. Though B5-I neurons did not fit strictly into a single class, the majority were either central or unclassified, with axons and dendrites mainly restricted to lamina II ( Figure 7C). Thus, B5-I neurons are likely to be involved in integrating sensory input within the substantia gelatinosa. One of the hallmarks of itch is that it is relieved by a variety of counterstimuli, such as see more scratching, noxious chemicals,

or menthol (Bromm et al., 1995, Ward et al., 1996 and Yosipovitch et al., 2007). While the neural basis for this phenomenon is unknown, it has been suggested that counterstimuli reduce itch through activation of spinal inhibitory interneurons (Akiyama et al., 2011, Ma, 2010, Patel and Dong, 2010, Ross, 2011 and Bautista et al., 2014). Based on our findings, B5-I neurons seemed well positioned to mediate the inhibition of itch by counterstimuli. If so, we reasoned that they would receive input (either directly

or indirectly) from primary afferents that mediate the counterstimuli. Capsaicin, mustard oil, and menthol activate discrete subsets of primary afferents (those that express TrpV1, TrpA1, and TrpM8, respectively). Since topical treatment with any of these substances can inhibit itch, we tested whether B5-I neurons receive input from primary afferents that express TrpV1, TrpA1, or TrpM8 (Figure 7D). Upon application of capsaicin Catechol oxidase to depolarize TrpV1-expressing afferents, we saw a significant increase in the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in 80% (4 of 5) of B5-I neurons, with an average 7.8-fold increase in EPSC frequency (Figures 7E and 7F). Moreover, a significant increase in mEPSC frequency was likewise observed in the presence of tetrodotoxin (TTX) to block action potential propagation, suggesting that B5-I neurons receive direct input from capsaicin-sensitive sensory neurons (Figure S5B). Similarly, allyl isothiocyanate, a key component of mustard oil, resulted in increased sEPSC frequency in 86% (6 out of 7) B5-I neurons, with an average increase of 3.3-fold (Figures 7G and 7H). Finally we observed that sEPSC frequency was also significantly increased 2.5-fold by menthol in 90% (9 out of 10) of B5-I neurons (Figures 7I and 7J).