After Autophagy inhibitor order pretreatment with saline or nicotine, there was no difference in the basal DA concentrations prior to ethanol exposure: 1.0 ± 0.2 nM after nicotine pretreatment and 1.0 ± 0.1 nM after saline pretreatment. To avoid handling-related stress, we administered

ethanol intravenously over a 5 min period (Figures 1A–1C, shaded columns). Ethanol induced a sustained increase in DA release in the saline control group (Figures 1A–1C, black circles). Nicotine pretreatment (0.4 mg/kg, intraperitoneally [i.p.], 3 hr prior) significantly attenuated the ethanol-induced increase in DA release (Figure 1A, red circles) (group × time: F(10,100) = 2.37, p < 0.05). The administered ethanol dose falls within the typical range tested in rodents ( Gonzales et al., 2004) and produces brain ethanol concentrations in rodents

that humans commonly achieve ( Howard et al., 2008). Brain ethanol concentrations peaked 10 min after the ethanol infusion and then decreased to a relatively stable concentration just above 30 mM for more than 30 min ( Figure S2). Blood ethanol concentrations were 26 ± 4 mM when measured from blood samples taken 95 min after ethanol administration. To determine the duration of nicotine’s effect on ethanol-induced DA release, we increased the interval between the nicotine pretreatment and the ethanol exposure to 15 hr and 40 hr, respectively. Remarkably, the DA release induced by ethanol remained significantly (group × time: F(10,250) = 6.16, p < 0.01) blunted 15 hr after nicotine pretreatment Galunisertib solubility dmso (0.4 mg/kg, i.p.) ( Figure 1B, red circles) compared to the saline control ( Figure 1B, black circles). This effect was less evident 40 hr after nicotine pretreatment (group × time: F(10,150) = 1.31, p > 0.05). However, a post hoc analysis of the first three postethanol dialysate samples (plus baseline) revealed a statistical difference between the nicotine and saline pretreatments (group

× time: F(5,75) = 2.63, p < 0.05), suggesting at least some influence of nicotine 40 hr after administration ( Figure 1C). The distribution of the Carnitine dehydrogenase microdialysis probe placements within the NAc was similar between the cohort of animals pretreated with nicotine and those pretreated with saline ( Figure 1D), indicating that regional differences in DA release do not account for these results. Although the animals were habituated to needle injections, we further controlled for the stimulus effects of the intraperitoneal injection of nicotine, which could potentially contribute to a stress response. We pretreated a separate experimental group with nicotine administered intravenously by cannula 15 hr prior to ethanol administration. This group displayed the same attenuated DA response to ethanol compared to the group pretreated with intravenous saline (group × time: F(10,210) = 5.35, p < 0.01).