Figure 2. Viral CRISPR/Cas9 ablation of GABABR and D2R in VTA DA neurons. A, Viral targeting in a DATCre(+):Cas9GFP(+) mouse treated with intra-VTA AAV8-U6-gRNA-hSyn-NLSmCherry; nucleus-localized mCherry fluorescence highlights the anatomic scope of viral targeting, and GFP fluorescence denotes the Cre-dependent expression of Cas9GFP in midbrain DA neurons of the VTA and substantia nigra. MT, medial terminal nucleus of the accessory optic tract; PBP, parabrachial pigmented nucleus of the VTA; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; VTAR, rostral part of the VTA. Scale bar: 500 μm. B, Somatodendritic inhibitory currents (Vhold = −60 mV) evoked by sequential bath application of quinpirole (quin; 20 μm) and then baclofen (bac; 200 μm) in VTA DA neurons from DATCre(+):Cas9GFP(+) mice treated with AAV8-U6-gD2R-hSyn-NLSmCherry or AAV8-U6-gLacZ-hSyn-NLSmCherry control. Currents were reversed by the D2/3R antagonist sulpiride (sulp; 5 μm) and GABABR antagonist CGP54626 (CGP; 2 μm). C, Summary of currents evoked by quinpirole (applied first) in VTA DA neurons from DATCre(+):Cas9GFP(+) mice treated with AAV8-U6-gD2R-hSyn-NLSmCherry or AAV8-U6-gLacZ-hSyn-NLSmCherry control. Main effects of sex (F(1,27) = 6.391, p = 0.0176) and viral treatment (F(1,27) = 104.6, p < 0.0001) were detected, along with an interaction between sex and viral treatment (F(1,27) = 5.590, p = 0.0255); #p < 0.05; ***p < 0.001 versus LacZ (within sex). D, Summary of currents evoked by baclofen, measured after quinpirole/sulpiride application (C), in VTA DA neurons from DATCre(+):Cas9GFP(+) mice treated with AAV8-U6-gD2R-hSyn-NLSmCherry or AAV8-U6-gLacZ-hSyn-NLSmCherry control. There was no main effect of sex (F(1,27) = 1.741, p = 0.1981) or viral treatment (F(1,27) = 1.183, p = 0.2864), nor was there an interaction between sex and viral treatment (F(1,27) = 0.0047, p = 0.9461). E, Somatodendritic inhibitory currents (Vhold = −60 mV) evoked by sequential bath application of baclofen (bac; 200 μm) and then quinpirole (quin; 20 μm) in VTA DA neurons from DATCre(+):Cas9GFP(+) mice treated with AAV8-U6-gGBR1-hSyn-NLSmCherry or AAV8-U6-gLacZ-hSyn-NLSmCherry control. Currents were reversed by CGP54626 (CGP; 2 μm) and sulpiride (sulp; 5 μm). F, Summary of currents evoked by baclofen (applied first) in VTA DA neurons from DATCre(+):Cas9GFP(+) mice treated with AAV8-U6-gGBR1-hSyn-NLSmCherry or AAV8-U6-gLacZ-hSyn-NLSmCherry control. A main effect of viral treatment was observed (F(1,27) = 120.9, p < 0.0001), but there was no main effect of sex (F(1,27) = 0.1910, p = 0.6655) or interaction between sex and viral treatment (F(1,27) = 0.2216, p = 0.6416); +++p < 0.001 (main effect of viral treatment). G, Summary of currents evoked by quinpirole, measured after baclofen/CGP54626 application (F) in VTA DA neurons from DATCre(+):Cas9GFP(+) mice treated with AAV8-U6-gGBR1-hSyn-NLSmCherry or AAV8-U6-gLacZ-hSyn-NLSmCherry control. A main effect of viral treatment was observed (F(1,24) = 8.182, p = 0.0086), but there was no main effect of sex (F(1,24) = 3.429, p = 0.0764) or interaction between sex and viral treatment (F(1,24) = 1.970, p = 0.1733); ++p < 0.001 (main effect of viral treatment). H, Summary of currents evoked by quinpirole in VTA DA neurons from a separate cohort of DATCre(+):Cas9GFP(+) mice treated with AAV8-U6-gGBR1-hSyn-NLSmCherry or AAV8-U6-gLacZ-hSyn-NLSmCherry control. A main effect of sex was observed (F(1,26) = 13.41, p = 0.0011), but there was no main effect of viral treatment (F(1,26) = 1.766, p = 0.1954) or interaction between sex and viral treatment (F(1,26) = 0.0129, p = 0.9105); ++p < 0.01 (main effect of sex). I, Impact of D2R or GABABR ablation on spontaneous activity in VTA DA neurons. No main effect of sex (F(1,56) = 0.7079, p = 0.4037) or viral treatment (F(2,56) = 0.6571, p = 0.5223) was detected, nor was there an interaction between sex and viral treatment (F(2,56) = 1.276, p = 0.2870). J, Impact of D2R or GABABR ablation on rheobase in VTA DA neurons. No main effect of sex (F(1,56) = 0.8080, p = 0.3725) or viral treatment (F(2,56) = 1.495, p = 0.2332) was detected, nor was there an interaction between sex and viral treatment (F(2,56) = 0.1573, p = 0.8548).