Figure 1

Figure 1

High-throughput quantification of neural activity in freely-swimming zebrafish larvae. (a) CaMPARI photoconverts from green to red fluorescing versions in the neuron only in the presence of both high intracellular calcium concentrations and a bright 405 nm light source. (b) Lateral view of green fluorescing CaMPARI merged with brightfield image shows exclusive expression in the CNS due to a pan-neural promoter (elavl3). (c) Larvae were transferred to 48 wells in the centre of a 96-well plate to ensure that the 405 nm LED Flood Array covers all larvae entirely. To minimize overheating, the plate was floating in a waterbath at 10 cm distant to the LED while CaMPARI was photoconverted (PC) by the LED Flood Array. (d) Lateral view of zebrafish with exemplar “CaMPARI Activity” heat maps. Heat maps show ratio of red/green (R/G) fluorescent output as indicated by the calibration bar and can be interpreted as relative neural activity. CNS regions with higher levels of neural activity translate to “hotter” pixels. Images were acquired using an automated INCell 2000 high-content microscope and reveal a reduction in neural activity when the larvae were anesthetized with MS-222 or an increase with convulsants, PTZ and 4-AP. Top, Enlarged lateral view from freely swimming larvae illustrating the optic tectum and hindbrain areas (dashed line) from which the R/G ratio was obtained. (e) Neural activity, inferred from the mean R/G ratio in optic tectum and hindbrain. CaMPARI activity is reduced to baseline in MS222-anaesthetized fish and photoconversion is undetectable when CaMPARI photoconverting light is omitted. Dashed green line represents mean for MS-222-anesthetized samples, which show a clear reduction in signal compared to freely swimming larvae and provides a baseline “near-zero” activity level for reference in subsequent experiments. Drivers of neural activity, PTZ and 4-AP induce a significant increase in CaMPARI activity. (f) Locomotor activity was measured using behavioural tracking software. PC light from the day prior at 4 days post-fertilization (dpf) did not affect locomotion at 5 dpf. MS-222 treatment abolished any swimbouts. Biological replicates are individual larvae = n. **Significantly different from freely swimming samples, p < 0.01.