This immunostaining showed that the vast majority of B5-I neurons (∼90%) coexpressed the somatostatin receptor sst2A (Figures 1A and S1A available online). Furthermore, when we recorded from spinal interneurons genetically

labeled with the Bhlhb5-cre allele ( Ross et al., 2010), half showed strong hyperpolarization in response to somatostatin ( Figure 1B), confirming that B5-I neurons express functional KPT-330 concentration sst2A receptors. Given the loss of B5-I neurons in Bhlhb5−/− mice, we reasoned that there would be a corresponding decrease in the number of sst2A-expressing neurons in these animals. As predicted, the number of sst2A-expressing neurons was reduced by two-thirds in Bhlhb5−/− mice, with no significant change in the number of sst2A-negative inhibitory neurons ( Figures 1C and 1D). Thus, the vast majority of B5-I neurons belong to the subset of inhibitory spinal interneurons that express sst2A, and a large proportion of the sst2A-expressing population is missing in Bhlhb5−/− mice. Since somatostatin inhibits

neuronal activity and sst2A is the only somatostatin receptor that is expressed by dorsal horn neurons (see http://www.brain-map.org), the finding that B5-I neurons express sst2A allowed us to directly test the FG-4592 purchase idea that B5-I neurons normally function to inhibit itch. This experiment was important because, although we had previously shown that loss of B5-I neurons during development is associated with abnormally elevated itch (Ross et al., 2010), the evidence was merely correlative. Specifically, it was not clear whether B5-I neurons function in the adult to inhibit itch, or whether B5-I neurons play a key developmental role in the formation of proper itch circuits. We hypothesized that if B5-I neurons normally function to inhibit itch, then acute inhibition of these neurons by somatostatin would increase itch sensitivity (Figure 1E). Indeed, upon intrathecal injection of the somatostatin analog octreotide, we observed vigorous scratching, biting, and licking behavior that was suggestive of itch (Figure 1F),

consistent with previous reports (Seybold et al., 1982). This spontaneous behavior was dose dependent, with an immediate onset and a duration of approximately half an hour. Because B5-I neurons account for Florfenicol the majority (two-thirds) of sst2A-expressing cells, the finding that acute treatment with octreotide results in elevated scratching behavior is consistent with the hypothesis that B5-I neurons inhibit itch. Nevertheless, it remained possible that the observed scratching behavior was due instead to the effect of octreotide on the one-third of sst2A-expressing neurons that are not B5-I neurons. We therefore tested the effect of octreotide on Bhlhb5−/− mice, which lack B5-I neurons. Specifically, we reasoned that, if octreotide-induced scratching is due to inhibition of B5-I neurons, this treatment would have no effect in mice that lack these cells.