Fig. 2: LHb Astrocyte dynamics during active coping states and input organization.

a Schematic of experimental design for photometry experiments. b Left panel, schematic depicting LHb viral injection and optic fiber placement (top) and representative image of rAAV9-GfaABC1D-GCaMP8s expression and fiber track in LHb (bottom). 3 V: Third ventricle; MHb: medial habenula. Middle panel: Sample motion index trace from single mouse and aligned raw photometric signal in green. Light-red squares indicate AC. Right panel: Sample heatmap related to photometry signal across trials. Data are presented as a heatmap of Z-score for each AC bout, sorted from shortest to longest, dashed line shows onset of AC while thick black lines show offset. c Top: heatmap of normalized Z-score (per animal) for each AC bout (n = 14 animals, 256 trials), sorted and represented as (b). Bottom: aligned averaged and normalized Z-score trace for all AC bouts. d Left: average Z-score for the last second of AC and PC bout. Data are presented as box plot, error bars indicate min to max, median and scatter. Two-tailed Wilcoxon signed rank test, ****p < 0.0001. Right: calcium rising time in function of AC bout duration, red dashed line shows regression line, R² = 0.67 ****p < 0.0001. e Left panel, Illustration of a head-restrained, awake, behaving juvenile zebrafish under a two-photon microscope (top) and a raw image of a Tg(GFAP:Gal4;UAS;GCaMP6s) zebrafish expressing GCaMP6s in astroglial cells of the ventral habenula (bottom). Middle panel, top: Example trace of zebrafish locomotion measured as tail beat angle. Red shading indicates the duration of locomotor AC bouts. Bottom, raw calcium signal from astroglial cells in the ventral habenula of the zebrafish. Each red line marks the onset of a locomotor AC bout. Right panel, top: Astroglial calcium traces [relative change in calcium signals (ΔF/F)] during locomotor AC bouts from an example fish. Warm colors represent increased activity, cold colors represent decreased activity. The dashed line indicates AC bout onset, and the thick black line represents AC bout offset. f Astroglial calcium traces during locomotor AC bouts from all recorded fish (n = 5). Bottom, Comparison of average astroglial activity between active and passive coping periods. g Left, Mean light-evoked astroglial calcium signals from all animals after consecutive exposure to neutral light (top, n = 5 animals, 30 trials) or aversive vibrations (bottom, n = 5 animals, 25 trials). Right, distribution of average light-, and vibration-evoked activity in trials from all animals Wilcoxon signed-rank test, ***p < 0.001. Data are presented as box plot, error bars indicate min to max, median and scatter. h Schematic of experimental design for retrograde rabies strategy. i Representative images depicting LHb injection site for rAAV5-GFAP-TVA-oG counterstained with an anti-Glutamine synthetase (GS) antibody, after ΔG-Rabies-mCherry injections and brain regions with positive rabies positive cells. Scale bar, 200 μm. 3 V, third ventricle; ac anterior commissure, BNST bed nucleus stria terminalis, EPN entopeduncular nucleus, IPN interpeduncular nucleus, LH lateral hypothalamus, LPO lateral preoptic area, LV lateral ventricle, MHb medial habenula; pc posterior commissure, VP ventral pallidum, VTA, ventral tegmental area. j Rabies positive brain region cell count. Brain regions were considered when exceeding >5 rabies positive cells (n = 7 animals). Data are presented as box plot, error bars indicate min to max, and median. k Quantification of neuronal and non-neuronal rabies positive cells in EPN, MHb and LH. Data are presented as pie charts representing RV⁺NeuN⁺ cells in magenta and RV⁺NeuN⁻ cells in black over total RV⁺ cells (n = 7 animals).