(A) Design of the experimental setup for concurrent recording and photocontrol of midbrain DA neurons in vivo. A micro-drive system (Mini Matrix) is mounted with a stereotaxic frame on the head of an anesthetized mouse, and enables to position in the VTA one cannula for photoswitch injection, up to three tetrodes for electrophysiological recordings, and one optic fiber connected to a beam combiner for optical stimulation. The photoswitch is injected at least an hour prior to the recordings. (B) Representative multi-unit recordings of transduced neurons on two channels of a tetrode while alternating illumination between 390 and 520 nm light. (C) Representative electrophysiological response of a MAHoCh-treated control neuron, while alternating illumination conditions between 390 (purple) and 520 nm light (green) every 5 s. Top, raster plot (n = 19 transitions) centered on the 390 nm light stimuli, and showing both the 390 to 520 and the 520 to 390 nm light transitions. Bottom, peri-stimulus time histogram (PSTH) of firing frequency using a 250 ms bin. (D) Change in firing frequency (expressed in absolute photoswitching) between 390 and 520 nm light for MAHoCh-treated control (Ctrl, light grey, n = 28) and β2E61C-transduced neurons (LinAChR, dark grey, n = 93). Photoswitching is calculated as ((Freq520 – Freq390)/Freq390) and represented in percent. Cumulative distribution indicates that virally transduced neurons significantly photoswitch compared to controls (p=0.0055, Kolmogorov-Smirnov test). Inset, absolute photoswitching for control neurons (1.87 ± 1.60%) is lower than that for transduced neurons (21.35 ± 5.90%). The threshold set at 15% absolute photoswitching (red) was used to determine the fraction of responding neurons in transduced animals (33/93, 35.5%). (E) Left, representative electrophysiological response of a virally transduced, MAHoCh-treated type 1 pDA neuron, represented as in (C). Right: Average firing rate of all type 1 pDA neurons (n = 24), under 520 (green) and 390 nm (purple) light. Firing frequency is significantly lower in 390 nm (1.85 Hz) compared to 520 nm light (3.41 Hz, p=1.19e−07). (F) Top left, raster plot (n = 20 transitions) for the spikes contained within bursts (SWB) under 390 nm and 520 nm light, for the same neuron as in (E). Bottom left, PSTH of instantaneous SWB frequency using a 250 ms bin. Right, average SWB frequency of all type 1 pDA neurons (n = 24), under both wavelengths of light. SWB frequency is significantly lower in 390 compared to 520 nm light (p=0.043). (G) Left, representative electrophysiological response of a virally transduced, MAHoCh-treated type 2 pDA neuron, represented as in (C). Right: Average firing rate of all type 2 pDA neurons (n = 9), under 520 (green) and 390 nm (purple) light. Firing frequency is significantly higher in 390 nm (5.25 Hz) compared to 520 nm light (3.48 Hz, p=0.0039). All values represent mean ± SEM.