Fig. 4: Optogenetic silencing of the DR^Sert neurons.

The experimental strategy for acute silencing of the DR^Sert neurons (a) involved expressing the inhibitory opsin ArchT selectively in the DR^Sert neurons, by injecting AAV-Flex-ArchT-GFP in the DR of Sert-Cre mice. Photomicrographs in b show the histological validation of ArchT transduction (n = 6) in the DR^Sert neurons at two rostral-to-caudal levels, and their relationship with the tip of the optical fiber targeting the ArchT-DR^Sert cells (the flat fiber tip rested on the floor of the cerebral aqueduct; its position is marked by a dotted line and an arrow). To validate that ArchT is expressed only in the serotonin cells (n = 6), c1 and c2 show photomicrographs from a brain section labeled for both GFP (ArchT- green) and serotonin (red); and merged (double labeling is yellow) showing that nearly all the ArchT-expressing cells were serotoninergic. The fourth panel in both c1 and c2 are the same sections represented at higher magnification. Panel a shows a drawing of the mouse brain at the level of the mid-DR (equivalent to plane −4.6 mm from bregma, of the mouse brain atlas)⁵³. d–e In vitro brain slice recordings from a DRN ArchT-GFP neuron (d) photographed under IR-DIC (top) and under fluorescence optics (bottom; scale bar: 25 µm). e1 Exposure to 593 nm light (60 s duration) hyperpolarized and silenced action potential firing of DR^Sert neurons expressing ArchT. e2 Bar histogram graph representing the mean firing frequencies before (crtl), during (on) and after (10–70 s; 10 s bins) photoinhibition. One-way ANOVA, F = 7.11; p < 0.0001 n = 6; Holm–Sidak’s multiple comparisons post hoc test; *P = 0.038 ctrl vs light-on; *P = 0.014, ctrl vs 10 s Light-OFF; ^##P < 0.01 and ^#p < 0.05 Light-ON vs Light-OFF. Data in e2 are presented as mean values ± SEM. e3 Post hoc intracellular biocytin labeling (red with streptavidin-AF-555 in right top panel) of a recorded DRN neuron that expresses ArchT-GFP (green), at low magnification (top left panel; scale bar: 100 µm) and at higher magnification (bottom; scale bar: 20 µm). The results shown in d and e3 were reproduced in n = 6 cells that were recorded from two mice.