(a) Cre-inducible AAV-DIO-ChR2-eYFP vector injected into VTA of VGluT2::Cre mice. Light stimulation of ChR2-eYFP-fibers and LHb whole-cell recordings. (b) Recorded biocytin-filled LHb neuron intermingled with ChR2-eYFP-fibers. (c) Light pulses (473 nm; 5 ms) evoked inward and outward currents at varying holding potentials. Top: Light evoked both fast inward (dotted line) and slower outward currents (solid line). Light-evoked inward currents at hyperpolarized holding potentials (≈−70 mV), outward currents at depolarized holding potentials (≈−50 mV), and both inward and outward currents at intermediate holding potentials (≈−60 mV). Outward, but not inward, currents were blocked by Picrotoxin (PcTx, 50 µM; GABAA receptor antagonist). (d) Inward currents were glutamatergic. Inward currents were abolished by AMPA receptor antagonist NBQX (5 µM) at −70 mV [e, summary; mean ± s.e.m. orange circles; paired t-test, t(3)=4.1; p = 0.026, n = 4 neurons from 4 VGluT2-ChR2 mice], but not at −50 mV (middle) or −45 mV (bottom). (e) Summary responses at −50 mV; purple circles, paired t-test, t(4)=0.64, p = 0.56, n = 5 neurons from 5 VGluT2-ChR2 mice. (f) GABAergic outward currents shunt glutamatergic inward currents. Top: both, light-evoked small inward and larger outward currents (left) in cells at −60 mV. PcTx eliminated the outward current and enhanced the inward (middle), which was eliminated by NBQX (right). Bottom: PcTx eliminated outward currents in all cells held at −50 mV [mean ± s.e.m.; purple circles; paired t-test, t(9)=6.2; p = 0.0002, n = 10 neurons from 8 VGluT2-ChR2 mice]. PcTx enhanced inward currents in cells held at −70 mV [mean ± s.e.m; orange circles; paired t-test, t(4)=2.9; p = 0.0437, n = 5 neurons from 5 VGluT2-ChR2 mice]. (g) Confirmation of glutamatergic-GABAergic co-transmission by mesohabenular AT, Cre-inducible AAV-DIO-ChR2-eYFP vector injected into VTA of VGaT::Cre mice. (h) Top left: Light-evoked inward current (control) at −70 mV was abolished by NBQX (1 µM) [summary in bottom; mean ± s.e.m. orange circles; paired t-test, t(5)=3.4; p = 0.0189, n = 6 neurons from 3 VGaT-ChR2 mice]. Top right: Outward current at −50 mV in the same cell before (control) and after PcTx [summary in bottom; mean ± s.e.m. purple circles; paired t-test, t(5)=3.0; p = 0.03, n = 6 neurons from 3 VGaT-ChR2 mice].