Upright microscope

Vestibulospinal photoablations were performed on Tg(nefma::EGFP) larvae at either 4 dpf for use in behavioral experiments from 4–6 dpf, or at 6 or 7 dpf for use in behavioral experiments from 7–9 dpf. Additional experiments were performed on Tg(α-tubulin:C3PA-GFP) larvae after spinal photoconversion to target a larger number of vestibulospinal neurons. Larvae were anesthetized in 0.2 mg/ml MESAB and then mounted in 2% low-melting point agarose. Photoablations were performed on an upright microscope (ThorLabs) using a 80 MHz Ti:Sapphire oscillator-based laser at 920 nm for cell visualization (SpectraPhysics MaiTai HP) and a second, high-power pulsed infrared laser for twophoton mediated photoablation (SpectraPhysics Spirit 8W) at 1040 nm (200 kHz repetition rate, 500 pulse picker, 400 fs pulse duration, 4 pulses per neuron over 10 ms) at 25–75 nJ per pulse, depending on tissue depth. Sibling controls were anesthetized for matched durations to lesioned fish. Lesioned and control sibling larvae were allowed to recover for 4–24 hours post-procedure and were confirmed to be swimming spontaneously and responsive to acoustic stimuli before behavioral measurements.

otogelin mutants were screened at 2 dpf for bilateral loss of utricular otoliths. Photoablations of ascending neurons of the tangential nucleus were performed in Tg(−6.7Tru.Hcrtr2:GAL4-VP16; UAS:EGFP) larvae at 4 dpf. Lesions of the vestibulospinal neurons and the nMLF were performed in Tg(nefma::EGFP) on day 6–7 and 5 dpf, respectively.
All lesions were done using a 2-photon laser as previously described³⁴ . Briefly, larvae were anesthetized in 0.2 mg/ml MESAB and then mounted in 2% low-melting point agarose. Neurons of interest were identified and imaged using an upright microscope (ThorLabs Bergamo) with an 80 MHz Ti:Sapphire oscillator-based laser at 920 nm (SpectraPhysics MaiTai HP). A separate high-power pulsed infrared laser (SpectraPhysics Spirit 8W) was used for photoablation (1040 nm, 200 kHz repetition rate, 400 fs pulse duration, 1–4 pulses per neuron over 10 ms at 25–75 nJ per pulse). Lesion controls were sibling fish and were anesthetized for comparable durations to lesioned larvae. Lesioned and control sibling larvae were allowed to recover at 28.5°C until behavioral measurements.