Atement Pan-RAS-IN-1 web testing and the percentage ofEur J Neurosci. Author manuscript; available in PMC 2016 March 08.Moorman et al.PageFos+ ORX neurons in the DMH (r=0.867, p<0.01; Figure 2C) and LH (r=0.720, p<0.05; Figure 2E) subregions. No such relationship was observed in the PF area (r=0.012, p>0.05; Figure 2D). We also examined the relationship between the percentage of Fos+ ORX neurons and the number of non-rewarded head entries the animals made into the reward well during the reinstatement test as a measure of general locomotor activity. Importantly, we did not observe a significant relationship between Fos activation and non-rewarded well-entries in any of the hypothalamic subregions (p>0.05; Figure 2 F ), indicating that Fos induction in DMH and LH ORX neurons is not related to non-specific locomotor behavior. Experiment 2: Cue-induced reinstatement of ethanol seeking Animals exhibited robust cue-induced reinstatement of alcohol seeking– Animals were trained to respond for EtOH, the delivery of which was paired with light-tone cues. Over the final three days of FR3 self-administration training, animals exhibited an average of 91.0 (?.1 SEM) active lever responses and 3.0 (?.8 SEM) inactive lever responses, and earned 28 (?.4 SEM) EtOH rewards (Figure 3A). Lever pressing was extinguished in the same context, but in the absence of light-tone cues and EtOH rewards. On the extinction day prior to reinstatement testing, animals exhibited an average of 5.0 (?.8 SEM) active lever presses and 2.9 (?.7 SEM) inactive lever responses. On test day, active lever presses resulted in the delivery of light-tone cues previously paired with EtOH, but no delivery of EtOH. Animals exhibited significant cue-induced reinstatement of EtOH seeking on test day, as evidenced by a significant increase in active lever responding (32.6?.5 SEM) compared to extinction levels (t(18)=6.048, p<0.001; Figure 3A, B). We did not observe a significant increase in inactive lever responding on test day (p>0.05; Figure 3A). Cue-induced reinstatement of EtOH seeking is not correlated with activation of ORX neurons–As in Experiment 1, we assessed whether there was a relationship between cueinduced reinstatement behavior and activation of ORX neurons. Interestingly, there were no significant correlations between active lever responding on test day and the percentage of Fos+ ORX neurons in all hypothalamic subregions (Figure 3 C ). There also was no UNC0642 msds correlation between ORX neuron activation and general locomotor activity, as assessed by non-rewarded entries the animals made into the reward well during testing (p>. 05). Experiment 3: Home cage conditioned response testing Animals exhibited significant variability in EtOH preference on 2-bottle choice and test day–Animals received 1 week of 2-bottle choice testing to characterize preference for EtOH. During this period, animals on average consumed 4.6 ml (?.3 SEM) of EtOH (1.8?.11 g/kg) and 2.1 ml (?.3 SEM) of water in each 2-hour session. On the final day of testing, animals received a conditioned response test, whereby the number of licks on each bottle was measured using a junction potential detection method (see Methods and Hayar et al., 2006). Examples of junction potential recordings of EtOH-preferring versus water-preferring rats are shown in Figure 4 A,B. Preference for the EtOH vs. waterAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptEur J Neurosci. Author manuscript; available in PMC 2016 March 08.Moo.Atement testing and the percentage ofEur J Neurosci. Author manuscript; available in PMC 2016 March 08.Moorman et al.PageFos+ ORX neurons in the DMH (r=0.867, p<0.01; Figure 2C) and LH (r=0.720, p<0.05; Figure 2E) subregions. No such relationship was observed in the PF area (r=0.012, p>0.05; Figure 2D). We also examined the relationship between the percentage of Fos+ ORX neurons and the number of non-rewarded head entries the animals made into the reward well during the reinstatement test as a measure of general locomotor activity. Importantly, we did not observe a significant relationship between Fos activation and non-rewarded well-entries in any of the hypothalamic subregions (p>0.05; Figure 2 F ), indicating that Fos induction in DMH and LH ORX neurons is not related to non-specific locomotor behavior. Experiment 2: Cue-induced reinstatement of ethanol seeking Animals exhibited robust cue-induced reinstatement of alcohol seeking– Animals were trained to respond for EtOH, the delivery of which was paired with light-tone cues. Over the final three days of FR3 self-administration training, animals exhibited an average of 91.0 (?.1 SEM) active lever responses and 3.0 (?.8 SEM) inactive lever responses, and earned 28 (?.4 SEM) EtOH rewards (Figure 3A). Lever pressing was extinguished in the same context, but in the absence of light-tone cues and EtOH rewards. On the extinction day prior to reinstatement testing, animals exhibited an average of 5.0 (?.8 SEM) active lever presses and 2.9 (?.7 SEM) inactive lever responses. On test day, active lever presses resulted in the delivery of light-tone cues previously paired with EtOH, but no delivery of EtOH. Animals exhibited significant cue-induced reinstatement of EtOH seeking on test day, as evidenced by a significant increase in active lever responding (32.6?.5 SEM) compared to extinction levels (t(18)=6.048, p<0.001; Figure 3A, B). We did not observe a significant increase in inactive lever responding on test day (p>0.05; Figure 3A). Cue-induced reinstatement of EtOH seeking is not correlated with activation of ORX neurons–As in Experiment 1, we assessed whether there was a relationship between cueinduced reinstatement behavior and activation of ORX neurons. Interestingly, there were no significant correlations between active lever responding on test day and the percentage of Fos+ ORX neurons in all hypothalamic subregions (Figure 3 C ). There also was no correlation between ORX neuron activation and general locomotor activity, as assessed by non-rewarded entries the animals made into the reward well during testing (p>. 05). Experiment 3: Home cage conditioned response testing Animals exhibited significant variability in EtOH preference on 2-bottle choice and test day–Animals received 1 week of 2-bottle choice testing to characterize preference for EtOH. During this period, animals on average consumed 4.6 ml (?.3 SEM) of EtOH (1.8?.11 g/kg) and 2.1 ml (?.3 SEM) of water in each 2-hour session. On the final day of testing, animals received a conditioned response test, whereby the number of licks on each bottle was measured using a junction potential detection method (see Methods and Hayar et al., 2006). Examples of junction potential recordings of EtOH-preferring versus water-preferring rats are shown in Figure 4 A,B. Preference for the EtOH vs. waterAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptEur J Neurosci. Author manuscript; available in PMC 2016 March 08.Moo.