In the Bhlhb5−/− mice, galanin-expressing cells were almost completely absent, while the number of sst2A-expressing cells that contained nNOS but not galanin PI3K inhibitor was substantially reduced ( Figures 2C and 2D). In contrast, the number of sst2A-expressing cells that contained neither galanin

nor nNOS did not differ significantly between wild-type and Bhlhb5−/− mice. This analysis of sst2A-expressing interneurons, together with the previous quantification of inhibitory neurons that lack sst2A ( Figure 1D), suggest that the galanin and nNOS populations of inhibitory neurons are severely depleted in Bhlhb5−/− mice, whereas all other inhibitory populations are unchanged. The loss of galanin cells in the Bhlhb5−/− mice was of particular interest because these cells also express the BMS-754807 order kappa opioid dynorphin ( Bröhl et al., 2008 and Sardella et al., 2011), and there is precedent for the idea that kappa opioids inhibit itch ( Inan and Cowan, 2004, Ko et al., 2003 and Togashi et al., 2002). We therefore confirmed that dynorphin is expressed in

B5-I neurons by reacting spinal cords from P4 mice with antibodies against Bhlhb5 and the dynorphin precursor, preprodynorphin (PPD; Figure 3A). As expected, we found that virtually all dynorphin-expressing neurons in laminae I-II were Bhlhb5 immunoreactive. We also assessed the number of dynorphin-expressing inhibitory interneurons in adult Bhlhb5−/− animals

with antibodies against either PPD or dynorphin B, a cleavage product of the full-length dynorphin peptide. These experiments revealed an almost complete loss of dynorphin-expressing inhibitory interneurons in Bhlhb5−/− mice ( Figures 3B, 3C, S3B, S3A and S3C), consistent with the finding that galanin-expressing neurons are largely absent in these mice. The finding that Bhlhb5−/− mice lack spinal inhibitory neurons that release dynorphin raised the possibility that B5-I neurons normally inhibit itch in part through activation of the kappa opioid receptor (KOR). As a first step to ADP ribosylation factor test this idea, we investigated the effect of kappa agonists nalfurafine and U-50,488 ( Figure 4A; Morgan and Christie, 2011, Wikström et al., 2005 and Williams et al., 2013) on acute pruritogen-evoked itch behavior. To investigate whether kappa agonists inhibit itch mediated by MrgprA3/C11-expressing afferents, we quantified scratch bouts following intradermal injection of chloroquine into the nape of the neck of mice that had been pretreated with either nalfurafine (20 μg/kg) or vehicle. We found that nalfurafine significantly reduced chloroquine-induced itch behavior ( Figures 4B and 4C), consistent with previous findings ( Inan and Cowan, 2004). Likewise, nalfurafine significantly attenuated SLIGRL-mediated itch ( Figure 4D).