Determination of anaesthesia effectiveness in zebrafish larvae

Zebrafish larvae are renowned for their utility in toxicology and neurology studies due to their absence of a skull and low brain lipid content. However, behavioural observation criteria alone are inadequate for determining appropriate anaesthetics to induce unconsciousness in these models. This study aims to assess Lidocaine and Tricaine at the neuronal activity level as a potential indicator of unconsciousness. A behavioural assay using WT/TU larvae at five days post-fertilization (dpf) determined adequate concentrations for the microscopy phase. Survival curve analysis showed Lidocaine's slower onset effect on spontaneous movements and touch response compared to Tricaine at 500-, 200- and 100 mg/l concentrations. We observed a significant impact on behaviour assay for both Lidocaine and Tricaine at 200 mg/l  , consistent with the reported standard anaesthesia dose of 200 mg/l. Conversely, 500- and 100 mg/l doses are known, respectively, for euthanisa and possibly only sedation in zebrafish.  Therefore, the 200 mg/l dose was chosen for confocal microscopy. Utilizing the pan-neuronal calcium sensor (Tg(elavl3:H2B-GCaMP6s) line), we employed confocal microscopy to measure calcium influx as a biomarker of neuronal activity. Fluorescent intensities in the pallium, habenula, tectum and cerebellum were quantified using the IMARIS software. Calcium imaging of these regions was recorded in time-lapse imaging over 30 minutes. Corresponding with the pronounced behavioural effects, Tricaine showed a faster downward trend in neuronal activity in the habenula than Lidocaine. Additional physiological and molecular markers are needed alongside this approach to assess this model's unconsciousness comprehensively. These findings suggest Lidocaine to be somewhat less effective in inducing unconsciousness and unreliable as an anaesthetic for invasive procedures requiring immediate anaesthesia. Conversely, the study supports Tricaine as the preferred immersion anaesthetic in this model.