Ethanol withdrawal affects behavior in the light/dark test and decreases convulsive thresholds in zebrafish

Zebrafish is gaining traction as a behavioral model to study the neurobehavioral effects of acute and chronic alcohol (EtOH) use, including withdrawal syndrome. The aim of this work was to evaluate the applicability of anxiety-like responses and pilocarpine-induced seizures in behavioral models for EtOH withdrawal in zebrafish. Twenty-four adult zebrafish from the longfin phenotype were used in this study. Fourteen animals were exposed to increasing EtOH concentrations in the water for 16 days and kept in a final concentration of 0.5% v/v, and ten specimens were used as control (no exposition to etanol) After treatment, fish were subjected to a 60 min withdrawal period and exposed to the light/dark tank (LDT). Immediately after the LDT, animals were injected with a sub-convulsive dose of pilocarpine (150 mg/kg). LDT data were analyzed via approximatively two-sample Fisher-Pitman permutation tests with 10.000 Monte-Carlo re-samplings, and seizure data (latency to reach Score IV) were analyzed by fitting a Kaplan-Meier model to survival curves. Withdrawal increased time in the white compartment (Z = 2.1207. p = 0.0261; g = -0.995), risk assessment (Z = 1.9895. p = 0.0405; g = 0.918), and number of squares crossed (Z = 2.5965. p = 0.0034; g = 1.31), without affecting erratic swimming, freezing, thigmotaxis, entry duration, or transitions to white. Withdrawal decreased latencies to Score IV seizure-like behavior (χ2[df=1]=8.8. p = 0.00308). These results suggest increased anxiety, psychomotor agitation, and seizure susceptibility after EtOH withdrawal in zebrafish, and call attention to analyzing additional endpoints in the LDT after withdrawal.