Non-Associative Learning in Larval Zebrafish

Habituation is a form of non-associative learning and can be measured in larval zebrafish. The commonest behaviour studied is contraction of body muscles (‘startle’) in response to sudden acoustic, tactile or visual stimulation. This behaviour is evolutionarily conserved as a protection against potential danger and predators.

Zebrafish larvae can be placed in a common laboratory well-plate containing embryo medium with one larva per well (n.b. zebrafish larvae develop the startle response and in general it is most reliably seen in 6-9 dpf). Currently the MWP measures the startle behaviour by measuring the distance moved in 500ms, or 1s, and the unit cannot distinguish between C-start and O-bend.

Response to different compounds on such a behaviour can also be tested in a similiar fashion.

Differing vibratory cues can be provided by the motor located in the unit beneath the screen. The cues are given at different intervals (refered to as the intertrial interval, ITI). Following each auditory cue a recovery period (generally a few minutes) should be provided between each auditory stimulation. The acoustic startle response (ASR) for each individual larva is analysed by measuring the distance moved following each stimulation, providing a quantitative read out of the startle response.

Habituation, or a non-associative form of learning, can also be tested using light, or absence of light (“dark flash”). In a light dark assay, performed in the MWP unit, you may use the built-in stimuli – the overhead lighting or the screen.


Jonathan D. Best, Stephane Berghmans, Julia J.F.G. Hunt, Samantha C. Clarke, Angeleen Fleming, Paul Goldsmith and Alan G. Roach (2008), Non-Associative Learning in Larval Zebrafish, Neuropsychopharmacology 33, 1206-1215

See also review article: Adam C Roerts, Brent R. Bill and David L Glanzman, Learning and memory in zebrafish larvae,  Frontiers in Neural Circuits, 2013