A model-based quantification of startle reflex habituation in larval zebrafish

Zebrafish is an established animal model for the reproduction and study of neurobiological pathogenesis of human neurological conditions. The ‘startle reflex’ in zebrafish larvae is an evolutionarily preserved defence response, manifesting as a quick body-bend in reaction to sudden sensory stimuli. Changes in startle reflex habituation characterise several neuropsychiatric disorders and hence represent an informative index of neurophysiological health. This study aimed at establishing a simple and reliable experimental protocol for the quantification of startle reflex response and habituation. The fish were stimulated with 20 repeated pulses of specific vibratory frequency, acoustic intensity/power, light-intensity and interstimulus-interval, in three separate studies. The cumulative distance travelled, namely the sum of the distance travelled (mm) during all 20 stimuli, was computed as a group-level description for all the experimental conditions in each study. Additionally, by the use of bootstrapping, the data was fitted to a model of habituation with a first-order exponential representing the decay of locomotor distance travelled over repeated stimulation. Our results suggest that startle habituation is a stereotypic first-order process with a decay constant ranging from 1 to 2 stimuli. Habituation memory lasts no more than 5 min, as manifested by the locomotor activity recovering to baseline levels. We further observed significant effects of vibratory frequency, acoustic intensity/power and interstimulus-interval on the amplitude, offset, decay constant and cumulative distance travelled. Instead, the intensity of the flashed light did not contribute to significant behavioural variations. The findings provide novel insights as to the influence of different stimuli parameters on the startle reflex habituation and constitute a helpful reference framework for further investigation.