Tricaine

For all experiments, we used a concentration of 60–100 µg/mL tricaine in filtered fish water (MS-222 Sigma-Aldrich) buffered to pH 7-8. We came to this concentration through extensive testing of a wide range of concentrations. We optimized for a solution that kept fish fully anesthetized for the maximum treatment duration of 4 days, while minimizing physiological effects and mortality. We applied 60 µg/mL tricaine up until day 5, and utilized 80 µg/mL on day 5 and 100 µg/mL on day 6, raising the concentration to combat any indication of habituation to the solution. We note that different fish lines tolerate different levels of anesthesia, although all tested lines fell into the 60–100 µg/mL range. The tricaine bath was exchanged every 12 h because we found that tricaine started to lose its potency after around 18 h, presumably because it was metabolized by the animals kept in the dish (Supplemental Fig. 2). We assessed whether larvae were indeed fully anesthetized through shaking the plate and tapping fish, as well as a long-term quantification of movement response, or lack thereof, under a light-on and light-off stimulus (Supplemental Fig. 2).
We discarded all fish in a Petri dish if we found any animal that responded to visual or physical stimulus.
Zebrafish embryos were placed in petri dishes (9 cm diameter) filled with tricaine solution and kept in the dark until experimental testing, because tricaine forms toxic byproducts under extended exposure to light. Since the onset of spontaneous neuronal activity in larval zebrafish happens prior to hatching, we used a pronase solution (50 mg/ml in fish water for 5 min, Sigma-Aldrich) to dissolve the chorion prior to 24 h post fertilization (hpf). This allowed us to anesthetize animals at 36 hpf and to have animals develop fully in the absence of their ability to hatch themselves. The same pronase-treatment was also performed for all “tricaine-control” fish used in the study.
For tricaine washout, we transfered the fish to a petri dish filled with standard fish water, and placed the plate in darkness for the duration of washout, thereby preventing visually-guided “entrainment” of neural network during this period.

Zebrafish brood stocks were collected from approved vendors, from Kolathur farms, Chennai, TamilNadu, India and maintained in the lab. The fish were anesthetised using Tricaine Methanesulfonate (MS-222; Sigma) during the experimental procedures as recommended by Zfin.org. Stocks of Tricaine solution (0.4% in 1 M Tris, pH 9) stored at 4 °C was used to prepare working solution by diluting 4.2 mL in a 100 mL clean tank water just before use. The fish lost movements in Tricaine in about 1.5 min, sustained for about 2–3 min and were moved to a recovery tank (fresh tank water) and monitored for revival of movements, which took about 3–5 min. The fish were wounded between these time periods. The fish in the recovery tank were observed for restoration of active movements and returned to the experimental tanks.
The fish were euthanised by exposing to overdose of Tricaine Methanesulfonate (200–300 mg/L); held immersed for about ten minutes leading to subsequent death by hypoxia [31 (link)]. The procedures were approved by the Institutional Animal Ethics Committee (XXXXII/24th January 2015).