Response of zebrafish larvae to mild electrical stimuli: A 96-well setup for behavioural screening.

BACKGROUND: Zebrafish larvae have a high potential as model system to replace rodents, especially in screening and drug discovery applications. However, an experimental setup to deliver mild electrical stimuli with simultaneous high throughput behavioural tracking has not yet been described.

NEW METHOD: A new tool was designed, making the delivery of electrical stimuli in a 96-well plate format possible. Using custom made electrode clips that can be slid over the walls of a square 96-well plate, 80 larvae could be tested simultaneously and behavioural responses recorded.

RESULTS: As proof of principle, two applications were tested: 1) The behavioural response after a single stimulus and the effect of buprenorphine on this response. 2) Habituation of locomotor activity to multiple stimuli and the involvement of the NMDA receptor. Reduced locomotor activity was observed after a single 5 V stimulus, however not with lower intensity stimuli. Pre-treatment with the analgesic buprenorphine prevented this response. Specificity of buprenorphine was confirmed using the antagonist naloxone. Habituation of locomotor activity was seen in response to multiple stimuli, depending on the inter stimulus interval. Treatment with the NMDA receptor antagonist memantine disrupted behavioural habituation.

COMPARISON WITH EXISTING METHODS: The equipment and setup described here are the first of its kind using a 96-well plate format, thereby increasing the potential throughput in screening applications using zebrafish larvae.

CONCLUSION: The combination of the described electrode clips for stimulus delivery and behavioural tracking allows for the use of zebrafish larvae in a new array of medium to high throughput applications.