This finding strongly suggests that octreotide-induced itch behavior is due to inhibition of B5-I neurons. To further assess whether the octreotide-induced scratching was due to elevated itch (rather than a nociceptive response or a grooming behavior), Androgen Receptor Antagonist ic50 we tested the effect of octreotide on pruritogen-evoked itch. For these experiments, we selected a very low dose of octreotide that had no significant effect on its own (3 ng) and tested its effect on chloroquine-induced itch. We found that intrathecal octreotide significantly increased the amount of time that mice spent biting at the injection site in

response to intradermal chloroquine (Figure 1G). In contrast, this dose of intrathecal octreotide had no effect on acute nociceptive reflexes, as measured by hindpaw withdrawal latency on a hot plate (Figure S1B). Furthermore, the effect of intrathecal octreotide was very likely mediated by spinal neurons (rather than the central terminals of primary afferents) since intradermal octreotide caused no itch-like behavior (Figure S1C). Together, these findings suggest that acute inhibition of B5-I neurons results in elevated itch. Sst2A-expressing inhibitory neurons in laminae I-II can be further subdivided based on the presence or absence of galanin and neuronal nitric oxide synthase (nNOS), which are expressed in mostly nonoverlapping subsets

(Figure 2B; Iwagaki et al., 2013 and Tiong et al., 2011). To investigate whether B5-I neurons constitute one or more of these subsets, we Farnesyltransferase performed PF-01367338 mw immunostaining experiments. These experiments revealed that virtually all (∼95%) of the galanin-expressing cells coexpress Bhlhb5 and that these account for 78% of the B5-I neurons (Figures 2A, left, and S2A). Likewise, many nNOS-expressing neurons coexpress Bhlhb5 (though the number is difficult to assess since nNOS is beginning to be expressed just as Bhlhb5 is being downregulated; Figures 2A, middle, and S2B). In contrast, Bhlhb5 was very seldom coexpressed with neuropeptide Y (NPY), a marker for a distinct inhibitory subpopulation (2% of NPY cells; Figures 2A, right,

and S2C). These findings suggest that B5-I neurons correspond to two, mostly nonoverlapping subpopulations of inhibitory interneurons: those that express galanin and those that express nNOS. We next investigated what happens to these populations in the Bhlhb5−/− mouse. Mice lacking Bhlhb5 showed a dramatic loss of galanin- and nNOS-expressing populations, but there was no difference in the distribution of two other populations of inhibitory interneurons marked by NPY and parvalbumin, respectively ( Figure S2D). To investigate this finding in more detail, we performed a quantitative analysis with the optical disector method ( Polgár et al., 2004) on sections reacted for sst2A, nNOS, galanin, and NeuN and stained with a nuclear marker ( Figures 2B–2D and S2E).