FingR Synapse Imaging in Zebrafish

Tg(UAS:FingR(PSD95)-GFP-CCR5TC-KRAB(A)-P2A-mKate2f) larvae that had been electroporated with FoxP2.A:Gal4FF (see the ‘Single-cell FingR(PSD95) expression using electroporation’ section) were kept under a 14 h–10 h light–dark cycle until 7 d.p.f., then imaged at ZT4–ZT5 (see the ‘Repeated imaging of FingR-labelled synapses’ section). Larvae were transferred to individual wells of a six-well plate containing 10 ml of sleep-promoting drugs, alone or in combination, as follows: 30 µM melatonin (M5250, Sigma-Aldrich) in 0.02% DMSO; 30 µM of clonidine hydrochloride (C7897, Sigma-Aldrich) in 0.02% DMSO; 45 µM 2-chloroadenosine (C5134, Sigma-Aldrich) in 0.02% DMSO; and 0.02% DMSO in fish water as controls^{45 (link),52 (link),60 (link),61 (link)}. Combinations of drugs were applied at the same concentrations as the single-dose conditions, maintaining the final DMSO concentration of 0.02%. Sleep induction was monitored with video-tracking (see the ‘Locomotor activity assay’ section) for 5 h, after which the drugs were removed by 2–3 careful replacements of the fish water using a transfer pipette followed by transferring the larvae individually to a new six-well plate with fresh water. The larvae were then reimaged using the Airyscan system (see the ‘Repeated imaging of FingR-labelled synapses’ section).