Animal husbandry
The animal handling and experiments were performed according to protocols approved by the animal welfare officer at Institüt für Neurowissenschaften, Technische Universität München (TUM) and the relevant department at the regional government (Regierung von Oberbayern, Sachgebiet 55.2; animal protocol number 55-2-1-54-2532 10112 and 55.2-2532.Vet_02-24-5). Adult zebrafish (Danio rerio) were housed in the facility at the Institute for Neuronal Cell Biology at TUM. The adult fish were maintained in water temperature of 27.5–28.0 °C on the 14–10 h light–dark cycle. All experiments were performed on 6–9 days post-fertilization larvae of undetermined sex. The eggs were kept in 0.3× Danieau solution, and in the water from the fish facility upon hatching. The larvae were maintained at 28.0 °C and under the 14–10 h light–dark cycle.
Animal strains
All imaging experiments of the HD neurons were performed on fish carrying Tg(gad1b–Gal4)mpn155 (ref. 18) and Tg(UAS–GCaMP6s)mpn101 (ref. 46). To record the activity of habenula neurons (Extended Data Fig. 8), either Tg(vglut2a–Gal4)nns20 (ref. 26) (n = 6 fish) or a previously uncharacterized enhancer trap line Tg(18107–Gal4) was used (n = 2 fish) with UAS–GCaMP6s. The expression pattern of the 18107–Gal4 line can be browed on Z Brain Atlas (https://zebrafishexplorer.zib.de/home/). For labelling habenula for the ablation (Fig. 5 and Extended Data Fig. 9), Tg(18107–Gal4) was used. A subset of fish in the ablation experiment possessed Tg(UAS–nfsB–mCherry)47 for a logistical reason. This does not affect the results of the ablation experiments as they were evenly distributed across the conditions, and the fish were not treated with relevant chemogenetic reagents. The experiment to search for putative AHV cells (Extended Data Fig. 10e–j) was performed on fish carrying Tg(HuC–H2B–jGCaMP7c). All fish were mitfa−/− (that is, nacre) mutants lacking melanophores to allow optical access to the brain.
Two-photon microscopy experiments
Animal preparation and the stimulus presentation setup
Animals were embedded in 2% low-melting point agarose in 30-mm petri dishes. The agarose around the tail was carefully removed with a scalpel to allow tail movements. The dish was mounted on a 3D-printed pedestal and placed in a cube-shaped acrylic tank with the outer edge length of 51 mm. The height of the pedestal was designed so that the head of the animal came to the centre of the tank, taking the thickness of the dish and the typical amount of agarose into account. The tank was then filled with fish facility water to minimize the refraction due to the petri dish wall. The three sides of the tank (except for the one facing the back of the fish) were made of single-side frosted acrylic (PLEXIGLAS Satinice 0M033 SC), which functioned as projection screens. The diffusive side faced inwards to minimize the reflections between the walls. The visual stimuli were projected onto the three walls through two sets of mirrors with a previously described geometry16 (Supplementary Video 1), subtending 270° horizontally and 90° vertically. The larvae were lit with an infrared LED array through the transparent back wall of the tank. Their tail movements were monitored from below with a high-speed camera (Allied Vision Pike F032) at 200 Hz, through a hot mirror and a short pass filter to reject the excitation beam.
Microscope
Functional imaging was performed with a custom-built two-photon microscope. The excitation was provided by a femtosecond pulsed laser with 920-nm wavelength, the repetition rate of 80 MHz and the average source power of 1.8 W (Spark ALCOR 920-2). The average power at the sample was approximately 10 mW. The scan head consisted of a horizontally scanning 12-kHz resonant mirror and a vertically scanning galvo mirror, controlled by a FPGA running a custom LabView code (LabView 2015)48. Pixels were acquired at 20 MHz and averaged eightfold, resulting in the frame rate of 5 Hz. The typical dimension of the image was about 100 µm × 100 µm, with the resolution of about 0.2 µm per pixel. Only pixels corresponding to the middle 80% of the horizontal scanning range were acquired to avoid image distortion, and the area outside was not excited to minimize photo-damage. The fast power modulation was achieved with the acousto-optic modulator built in to the laser.
Stimulus protocols
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