Efficacy of Tricaine (MS-222) and Hypothermia as Anesthetic Agents for Blocking Sensorimotor Responses in Larval Zebrafish

Abstract

Tricaine, or MS-222, is the most commonly used chemical anesthetic in zebrafish research. It is thought to act via blocking voltage-gated sodium channels, though its mechanism of action, particularly at the neuronal level, is not yet fully understood. Here, we first characterized the effects of tricaine on both body balance and touch responses in freely swimming animals, before determining its effect on the neural activity underlying the optokinetic response at the level of motion perception, sensorimotor signaling and the generation of behavior in immobilized animals. We found that the standard dose for larvae (168 mg/L) induced loss of righting reflex within 30 seconds, which then recovered within 3 minutes. Optokinetic behavior recovered within 15 minutes. Calcium imaging showed that tricaine interferes with optokinetic behavior by interruption of the signals between the pretectum and hindbrain. The motion sensitivity indices of identified sensory neurons were unchanged in larvae exposed to tricaine, though fewer such neurons were detected, leaving a small population of active sensory neurons. We then compared tricaine with gradual cooling, a potential non-chemical alternative method of anesthesia. While neuronal tuning appeared to be affected in a similar manner during gradual cooling, gradual cooling induced a surge in calcium levels in both the pretectum and hindbrain. This calcium surge, alongside a drop in heartrate, is potentially associated with harmful changes in physiology and suggests that tricaine is a better anesthetic agent than gradual cooling for zebrafish laboratory research.

Nature over Nurture: Functional neuronal circuits emerge in the absence of developmental activity

“…To test to what extent spontaneous neuronal activity, across any and all circuit elements, is necessary for the maturation of functional neuronal circuits in larval zebrafish, we developed a protocol to raise zebrafish under complete anesthesia. Amongst a series of potential candidates that are used as anesthetics in fish and amphibia, we settled on tricaine, a sodium-channel blocker known to reversibly silence neuronal action potential firing (Popovic et al 2012; Stanley et al 2020; Ramlochansingh et al 2014; Leyden et al 2022).…”

Apoptotic mechanism of propofol-induced developmental toxicity in zebrafish embryos

“…A camera phone (Vivo Z5x, Shenzhen, China) captured images of the embryos from the microscopic ocular, and after slow playback, the auxiliary counter counted the number of heartbeats in one minute, to provide the heart rate. Tricaine was added to the petri dish [ 38 ], and without response to touch in the tail with a needle, the larva body length was adjusted under a microscope to the lateral position so that their eyes overlapped. Zebrafish in propofol group did not need tricaine, but all embryos without hatching needed manual stripping (discarding the chorion from the embryos using ophthalmic forceps under a stereo microscope).…”

Behavioral and Neurophysiological Effects of Electrical Stunning on Zebrafish Larvae

“…Tricaine (MS-222) is known to work relatively slowly in this respect, as it takes between 25 and 34 seconds for adult zebra sh to loose equilibrium at a concentration of 250 mg/l 13,39,40 . Even at 336 mg/l it takes about 15 s for larvae until the loss of the righting re ex 17 . Though, for rapid cooling (2-4°C) the time window to onset decreases to 5-9 s for adult zebra sh 13,40 .…”

“…This highlights how little is known about the exact time death results from this method, and prolongs the time window in which suffering is possible, if e.g. the hypothermic larvae brie y warm up to 11° before being placed in the freezer 17 .…”