Third across all three signaling molecules examined the

Third, across all three signaling molecules examined, the baseline abnormalities seen in Hdc-KO mice resemble the effects seen after H3R activation in wild-type animals. Specifically, MAPK activity (indexed by MSK1 phosphorylation) is increased at baseline in dMSNs of Hdc-KO mice to a similar degree as it is after RAMH challenge in WT animals (Fig. 1). Similar parallel effects are seen in both dMSNs and iMSNs in the regulation of P-S235/236 rpS6 (Fig. 2), and in iMSNs in the dephosphorylation of Akt at T308 (Fig. 4). H3R is constitutively upregulated in Hdc-KO mice (Rapanelli et al., 2014), perhaps as a compensation for chronically deficient histamine. H3R has been shown in at least some contexts to have high basal activity, even in the absence of ligand (Morisset et al., 2000). This raises the intriguing possibility, to which we return below, that many of the basal signaling abnormalities seen in Hdc-KO mice are attributable to the baseline effects of elevated H3R expression, despite the absence of HA. Fourth, H3R activation leads to augmentation of these signaling abnormalities, in most instances. In Hdc-KO mice, RAMH elevated phosphorylation of rpS6 in both dMSNs and iMSNs (though the effect was attenuated in dMSNs, possibly due to a ceiling effect) and further decreased phosphorylation of Akt in iMSNs. This accentuation of activity-dependent striatal signaling abnormalities may underlie the behavioral effects of RAMH documented previously in these mice (Rapanelli et al., 2017). The exception to this pattern was in MAPK activity, as reflected in MSK phosphorylation at T581. MSK1 phosphorylation was elevated at baseline in dMSNs of Hdc-KO mice, but this elevation was attenuated by RAMH challenge; as a result there was a statistically significant interaction between Famprofazone and drug condition in this analysis (Fig. 1B). The cause of this interactive effect is unclear, but it suggests that H3R signaling may be qualitatively altered, not just amplified, in dMSNs. Alternatively, the activation of MAPK signaling by H3R activation may exhibit an inverted-U dose–response relationship, with phosphatases or other counterregulatory mechanisms being recruited only at higher levels of receptor activation. Further study is needed to elucidate these mechanistic details. H3R activation reduces Akt phosphorylation in iMSNs in both genotypes (Fig. 4), an effect similar to that produced by D2R activation in these cells via a β-arrestin-mediated mechanism (Beaulieu et al., 2004, Beaulieu et al., 2005, Beaulieu et al., 2011). Regulation of β-arrestin-mediated signaling by H3R has not been demonstrated to date, but it is possible that H3R regulates Akt via direct interaction with D2Rs, which has been described ex vivo (Ferrada et al., 2008). Akt phosphorylation is reduced at baseline in iMSNs of the Hdc-KO mice. This may be because of constitutive activity of upregulated H3R; however, an alternative explanation is that tonically elevated DA in the Hdc-KO mouse striatum (Castellan Baldan et al., 2014, Rapanelli et al., 2014) provides sufficient D2R tone to reduce Akt phosphorylation via the D2-β-arrestin-Akt pathway, without the direct involvement of H3R. Multiple signaling cascades converge on rpS6, including MAPK, S6 kinase, PKA, and mTOR (Meyuhas, 2008, Knight et al., 2012, Bonito-Oliva et al., 2013, Biever et al., 2015). We see alterations in rpS6 phosphorylation at S235/236 with both genotype and RAMH treatment, in both dMSNs and iMSNs. This site is regulated by the MAPK cascade; however, the dissociation between the effects seen on rpS6 and those seen on MSK1 suggest that other, convergent signaling pathways likely contribute. One possibility is the mTor pathway, although the lack of any modulation of the mTor-regulated S440/444 site on rpS6 argues against any central role for mTor. Another possibility is the PKA pathway; however, our previous work (Rapanelli et al., 2016) has revealed, perhaps surprisingly, that RAMH does not produce any detectable change in phosphorylation of DARPP-32, a major PKA target, in vivo. Further work is required to elucidate the complex interplay of different signaling pathways downstream of H3R in the striatum.