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.