Fig. 1. The four-fish microfluidic device and setup for screening the electric-induced response of semi mobile 6 dpf zebrafish larvae. (A) The labelled device identifying the screening pools and valve channels. (B) A close-up view of the screening pool with a larva trapped in the TR with its tail free to move in the screening pool. (C) Exploded view drawing of the device layers. (D) Experimental setup for behavioural screening of zebrafish larvae including two syringe pumps, a microscope, an electrical sourcemeter, and a computer.
Fig. 1D displays the experimental setup including a Leica upright microscope (Stereomicroscope Leica MZ10F, Singapore), a digital C-mount camera (GS3-U3-23S6M-C, Point Grey Research Inc., Canada), two syringe pumps (LEGATO 111, KD Scientific Inc., USA), an electronic sourcemeter (Model 2410, Keithley, USA), and necessary syringes, tubing and wires to connect to the microfluidic device to control the flow and electric stimulus.
Prior to conducting an experiment, larvae were divided into groups and exposed to the desired chemicals as reported in Table 1. A total of 12 groups of larvae were tested, i.e., 1 control group with no electric and chemical exposure, 1 control group with no chemical exposure, 3 groups for DA non-selective antagonists and agonists, 6 groups for D1- and D2-like selective antagonists and agonists, and 1 group for DMSO (as Haloperidol solvent) control.
The first step in the experiment was loading four larvae into the inlet channel in Fig. 1A. The flow velocity was set on the syringe pump to 1 ml/min so that rheotaxis ensured the fish to be directed towards the main channel in tail-first orientation for the majority of the trials[11]. An indirect flow channel was connected to the main channel through a series of short horizontal channels, through which water was pumped to the main channel to increase the hydrodynamic flow focusing on each of the TRs. As soon as the first fish reached the main channel, the bulk stream carried it into the first TR with lower flow resistance. Then, the indirect flow channel was switched on to provide a volumetric flow rate of 0.8 ml/min to push the larvae into the empty TRs and position them with their head immobilized in the narrow TR and their tails free to move within the screening pools as shown in Fig. 1B and Supplementary video1. Following loading of the TRs on each side of the device, the corresponding valves were activated to prevent the fish from swimming out of the TRs.
A recovery period of 60 s was given to ensure that the fish could adapt to the new environment following the loading process. A 20 s direct current (DC) electric stimulus was then applied via the electrodes to induce tail movement. The larval response was recorded at a speed of 160 frames per second (fps) using the camera mounted on the upright stereomicroscope for later analysis. The fish were washed out from the device and the process was repeated until the desired sample size of 45 larvae for each group was attained.