Supplementary Figure 7: In vitro and in vivo experimental validation for optogenetic stimulation of the LC NE fibers in the VTA.

(a) Photomicrograph of the LC of DBHcre-TdTomato mice transduced with the virus AAV1-ChR2-eYFP showing co-localization of DBH positive neurons (Tomato red staining) and ChR2 expressing cells (eYFP green staining; scale bars: upper 50 µm, lower 20 µm). 89.9% of DBH positive cells co-expressed the ChR2-eYFP in the LC and DBH-Tdtomato cells could be detected in 97.2% of the ChR2-eYFP expressing cells (n=3 mice). (b) Photomicrograph of the VTA of DBHcre-TdTomato mice transduced with the virus AAV1-ChR2-eYFP showing co-localization of DBH positive fibers (Tomato red staining) and ChR2 expressing fibers (eYFP green staining). The triple labelling has been reproduced on 13 mice. TH immunostaining (blue) showed DA neurons contacting the NE fibers from the LC expressing the ChR2. Scale bars: left 100 µm, right 10 µm. (c) Example electrophysiological characterization of a TdTomato- and eYFP-positive noradrenergic neuron in the LC. NE neurons fired on average at 15.1 ± 1.1 Hz in response to a 180 pA depolarizing step. A large majority of NE neurons were spontaneously active (n = 3 mice, 10 out of 11 neurons) and fired on average at 3.1 ± 0.5 Hz. (d) Example of photocurrents recorded in voltage-clamp (holding = -70 mV) during a 500 ms continuous blue-light stimulation in a LC-NE ChR2-positive neuron. (e) Steady-state photocurrent size quantification. Photocurrents were on average 271.0 ± 89.2 pA (n = 3 mice, n = 12 neurons). (f) Example traces of blue-light stimulations of a LC-NE ChR2-positive neuron. In these examples, square pulses of 5 ms duration were delivered at 5 and 20 Hz. (g) Action potential fidelity when stimulating LC-NE ChR2-positive neurons with 5 ms blue-light pulses at frequencies 1-50 Hz. As expected considering NE neuron electrophysiological properties, reliable firing (> 75%) was observed for stimulation frequencies between 1 and 10 Hz (n = 3 mice, n = 8 neurons). (h) Photomicrograph of activated LC-NE neurons (c-fos, blue) of DBHcre-TdTomato (red) mice transduced with the virus AAV1-ChR2-eYFP or AAV1- eYFP (green) which received blue-light optical stimulation (10-ms at 10 Hz blue light pulses over 500 ms every 20 seconds) in the LC for 20 minutes. This experiment has been reproduced successfully on 6 mice per condition. Scale bars: 50 µm.