Figure 3. Photostimulation of ChR2-expressing MCH terminals evoked glutamate release in the LS. A, Experimental design schematic showing patch-clamp recording while photostimulating ChR2-expressing MCH neurons. B, Merged epifluorescence and IR-DIC photomicrographs of a coronal Pmch–cre brain slice injected with AAV8–EF1α–DIO–hChR2(H134R)–mCherry showing whole-cell recording from a mCherry-labeled MCH neuron (inset, outlined region). Scale bars: 200 μm; inset, 50 μm. C, Sample traces of action potential firing from a ChR2-expressing MCH neuron evoked by 5 s light trains (5 ms pulses) at 1, 5, 20, and 100 Hz light frequency (dashed line, 0 mV; left). Percentage change in spike fidelity (black, left axis) and MCH neuronal firing (gray, right axis) showing effective entrainment up to 20 Hz photostimulation (n = 12). D, Experimental design schematic showing whole-cell recordings from LS neurons while photostimulating ChR2-expressing MCH projections. E, Merged low-magnification (i) and high-magnification (ii) epifluorescence and IR-DIC photomicrographs of a coronal brain slice showing ChR2-expressing MCH axon terminals in the LS (i) and mCherry-fluorescent puncta outlining LS cells (ii). Scale bars: i, 200 μm; ii, 100 μm. F–I, Overlay of individual (gray) and averaged (black) oIPSCs (Vh of −5 mV; F) and oEPSCs (Vh of −60 mV; H) after photostimulation (3 5-ms light pulses in 1 s, repeated every 5 s for 20 trials). Averaged responses (F, H) magnified in insets (top) and raster plots (bottom; 50 ms bins) showing event synchronization during photostimulation. Probability plots (50 ms bins) showing that photostimulation increased IPSC and EPSC probability in control (con; n = 14), which is abolished by 20 μm BIC (n = 4; G) and 1 mm KYN (n = 6; I), respectively. J, K, Range (line, median; box, 25th to 75th percentiles; whisker, minimum to maximum) of absolute amplitudes (J) and delay latency (K) of oIPSCs and oEPSCs averaged from 20 trials. ***p < 0.001. L, Overlay of individual (gray) and averaged (black) oIPSCs (top) and oEPSCs (bottom) in control (i), BIC (ii), KYN (iii), 500 nm TTX (iv), TTX and KYN (v), and washout (vi). M, Effect of KYN and BIC on oIPSC and oEPSC amplitude. 3V, Third ventricle; cc, corpus callosum; con, control; CPu, caudate putamen; f, fornix; LHA, lateral hypothalamic area; LV, lateral ventricle.