2.3. Behavioral Screening with Microfluidics
The behavioral experiments were performed with a three-layer
polydimethylsiloxane (PDMS) microfluidics device demonstrated
previously[20], complemented with key auxiliary components such as
syringe pumps (LEGATO 111, KD Scientific Inc., USA), a sourcemeter
(Model 2410, Keithley, USA) and an upright Leica stereomicroscope
(Stereomicroscope Leica MDG41, Singapore) with a camera (GS3-U3-23S6M-C,
Point Grey Research Inc., Canada) to enable manipulation, stimulation,
and imaging of zebrafish larvae (Fig. 1A).
The microfluidic device consisted of a series of microchannels (Fig.
1B). 3D master molds for the top and bottom layers were first designed
using SolidWorks (SolidWorks Corp., USA) and printed (Objet260 Connex3
printer (Stratasys Ltd., USA)) to allow for PDMS casting. The top mold
contained the inlet, outlet, main channel, electrodes, larva head trap,
and larva tail screening pool. The bottom mold contained a L-shaped
valving channel. The PDMS base and curing agent were mixed at a ratio of
10:1, degassed for half an hour and poured into the molds and with the
necessary tubes added. After curing for approximately 6 hours on a
hotplate at a temperature of 60°C, the molds were removed, and a 0.2mm
thick PDMS middle layer membrane was bonded between the top and bottom
PDMS layers.
During an experiment, a larva was transferred into the device via the
inlet in the top layer and moved along the main channel using the
syringe pump with a controlled flow rate of 2 mL/min until reaching the
trapping region (TR) (Fig. 1C). The narrowed section of the TR adjoined
to the open screening pool acted to immobilize the upper body and head
of the larvae while allowing the tail to move freely in the screening
pool. The valve, situated in the bottom layer, was then pressurized,
causing the middle membrane to deflect and create a physical barrier
that prevented the larva from swimming out of the TR. Following a
one-minute recovery period, shown to be sufficient to return cardiac
activity to baseline, the electric stimulus of 3μA was applied for 20 s
using the sourcemeter[39]. The larva’s locomotor response could be
recorded with a camera at 2x magnification on the Leica stereomicroscope
(Fig. 1B). The tested larva was removed from the device via the outlet
before repeating the experiment to reach the designated sample sizes for
each condition.