Taken together, these circuit-activity mapping experiments reveal the functional significance of

the inhibitory THVTA-LHb pathway in regulating midbrain activity. In vivo, pharmacological inhibition of the LHb increases dopamine in forebrain regions such as the striatum (Lecourtier et al., Selumetinib cost 2008). Likewise, we observed that in vivo activation of the THVTA-LHb pathway increased the firing rate of midbrain dopaminergic neurons ( Figure 6). Therefore, we hypothesized that in vivo activation of the THVTA-LHb::ChR2 pathway would result in a reward-related phenotype. To test this hypothesis, we implanted bilateral optical fibers ( Sparta et al., 2012) aimed directly above the LHb in THVTA-LHb::ChR2 mice ( Figure S6) and determined the behavioral ramifications of selectively activating the THVTA-LHb::ChR2 pathway. Using a real-time place preference assay, as previously described ( Stamatakis and Stuber, 2012), THVTA-LHb::ChR2 mice exhibited a significant preference for the side of the chamber that was paired with optical stimulation. In contrast, littermate controls (THVTA-LHb::Control) displayed no preference, demonstrating that activation ABT-199 purchase of the THVTA-LHb::ChR2 pathway produces reward-related behaviors ( Figures 7A–7C). This preference was dependent on GABAA signaling within the LHb, Ketanserin as intra-LHb microinjections of a GABAA

receptor antagonist (gabazine) through guide cannulas

interfaced with the optical fibers ( Jennings et al., 2013) blocked the preference for the stimulation-paired side ( Figures 7D and S7). In contrast, intra-LHb microinjection of a dopamine receptor antagonist (D1 and D2) cocktail did not block the rewarding phenotype ( Figures 7D and S7), whereas a systemic injection of the dopamine antagonist cocktail did disrupt the preference ( Figures 7E and S7). These data suggest that the observed reward-related phenotype induced by optical stimulation of the THVTA-LHb::ChR2 pathway does not depend on dopamine signaling within the LHb, but rather on downstream dopamine signaling in brain regions such as the NAc. Finally, to determine if activation of the THVTA-LHb::ChR2 pathway is reinforcing, we trained mice to nose-poke for optical stimulation of the THVTA-LHb::ChR2 pathway ( Figures 7F–7H). THVTA-LHb::ChR2 mice made significantly more nose-pokes to receive optical stimulation than THVTA-LHb::control mice ( Figure 7F). Taken together, these data demonstrate that although activation of THVTA-LHb::ChR2 terminals does not result in detectable dopamine release in the LHb, selective activation of this pathway promotes reward-related behavior by suppressing LHb activity through the release of GABA, leading to disinhibition of VTA dopaminergic neurons.