aru function is needed in the circadian PDF-expressing neurons for normal ethanol sensitivity, a feature shared with PI3K but not Egfr. In addition, aru mutants 17-AAG in vivo show increased synapse number in both larval and adult neurons, a phenotype also observed upon activation of PI3K, but not Egfr ( Martín-Peña et al., 2006). We propose that aru regulates

ethanol sensitivity of adult Drosophila by two distinct mechanisms, one involving the Egfr/Erk pathway and the other involving regulation of synapse number in conjunction with the PI3K/Akt pathway. Finally, we show that social isolation, which reduces the number of synaptic terminals in PDF neurons ( Donlea et al., 2009), causes a dramatic decrease in ethanol sensitivity in wild-type flies. This environmental manipulation also restores normal ethanol sensitivity and PDF synapse number to aru mutants. In summary, our results Galunisertib in vitro suggest that the regulation of synapse number is a mechanism of central importance in the regulation of

ethanol sensitivity. aru is a predicted adaptor protein containing PTB and SH3 domains ( Tocchetti et al., 2003) and probably forms protein complexes that mediate signal transduction. aru is orthologous to vertebrate Eps8L3, the atypical member of the Eps8 family ( Tocchetti et al., 2003). Interestingly, a mouse knockout (KO) of the founding family member, Eps8, shows reduced ethanol sensitivity and enhanced consumption, which is mediated in part by direct regulation of actin dynamics by Eps8 ( Offenhäuser et al., 2006). Since normal ethanol sensitivity ( Offenhäuser et al., 2006 and Rothenfluh et al., 2006) and synapse formation ( Hotulainen and Hoogenraad, 2010) require complex actin remodeling, aru may also affect ethanol sensitivity of and synapse number by regulating actin dynamics. However, this effect is probably indirect, since Aru, like Eps8L3,

lacks the predicted actin-binding and -capping domains found in Eps8 ( Offenhäuser et al., 2004). We uncover several differences in the ways the Egfr/Erk and PI3K/Akt pathways regulate ethanol sensitivity. Neuronal activation of the Egfr/Erk pathway reduces, while inhibition enhances ethanol sensitivity (Corl et al., 2009). Conversely, neuronal perturbations of the PI3K/Akt pathway alter ethanol sensitivity such that activation of the pathway enhances, whereas inhibition reduces ethanol sensitivity. In addition, both pathways have different temporal requirements: the Egfr/Erk pathway is required continuously (in development and adulthood), whereas the PI3K/Akt pathway is only required during development (which includes metamorphosis). The continuous requirement of Egfr function in neurons to affect ethanol sensitivity suggests that the previously described acute effects of Egfr inhibitors on adult ethanol behaviors ( Corl et al.