Automated measurement of zebrafish larval movement

Abstract

The zebrafish is a powerful vertebrate model that is readily amenable to genetic, pharmacological and environmental manipulations to elucidate the molecular and cellular basis of movement and behaviour. We report software enabling automated analysis of zebrafish movement from video recordings captured with cameras ranging from a basic camcorder to more specialized equipment. The software, which is provided as open-source MATLAB functions, can be freely modified and distributed, and is compatible with multiwell plates under a wide range of experimental conditions. Automated measurement of zebrafish movement using this technique will be useful for multiple applications in neuroscience, pharmacology and neuropsychiatry.

Figure 2. Three example applications of LSRtrack and LSRanalyze

A, LSRtrack was used to determine the activity of a population of 24 zebrafish larvae, housed in a 96-well plate, in response to transitions between light and dark at 7 dpf. Video was recorded using a modified camcorder under infrared illumination at a frame rate of 2 s⁻¹. The graph shows mean larval displacement in each consecutive frame of the video, the axes are scaled to show the equivalent mean velocity in mm s⁻¹ and time in minutes. Ambient illumination is indicated above the graph. B, LSRanalyze was used to determine V_M, V_A and T% (see Fig. 1E for calculations) of the population shown in panel A, and to determine how these parameters changed under different illumination conditions. Error bars show the standard error of the mean (n = 24). C, the individual movement profiles of 24 larvae at 5 dpf are shown before and after application of PTZ at the concentrations shown. The displacement in each consecutive 0.5 s time bin is shown using a colour scale (the scale is shown to the right of chart), illustrating alterations in movement patterns following PTZ exposure. D, LSRanalyze was used to determine how active velocity (V_A) and proportion of time moving (T%– see Fig. 1E for calculations) changed following PTZ exposure. Error bars show the standard error of the mean (n = 8). E, following a mechanical stimulus, the escape response of two different larvae at 9 dpf was recorded using a high speed camera at a frame rate of 400 s⁻¹. The main graphs show larval displacement in each consecutive video frame as black squares. The axes are scaled to show the equivalent instantaneous velocity in mm s⁻¹ and time in ms; the trend line was calculated as a moving mean. The inset panels show how the position of the larvae (yellow circles) changed in each successive frame of the video; the axes show distance in mm and the arrows indicate the direction of movement.

Figure 1. Software functions for measuring and analysing zebrafish movement

A, the flowchart provides an overview of the algorithm used by LSRtrack to track zebrafish movement and report errors. The source code is shown in the online Supplemental material. B, two series of consecutive video frames at 30 frames s⁻¹ are shown, illustrating zebrafish larval movements in individual wells of a 96-well plate. LSRtrack identified the area surrounding the well (red), the larval object (blue) and the location of the object centroid (yellow circle). Supplemental Video 1 shows a movie segment of the tracking process in operation, using the same colour scheme. C, the locus traversed by each zebrafish larva in a 96-well plate over a 10 min period is shown as a black line; detected movements are confined to individual wells. D, the flowchart summarizes manual verification of tracking errors reported by LSRtrack. E, simple calculations were performed by LSRanalyze, using larval tracking data, in order to determine displacement and to derive values for mean velocity (V_M), active velocity (V_A) and the proportion of frames during which a movement occurred (T%).