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\begin{document}

\title{Zebrafish Larvae's Response to Electricity is Mediated by Dopaminergic
Agonists and Antagonists}



\author[1]{Arezoo Khalili}%
\author[1]{Ellen  van Wijngaarden}%
\author[1]{Georg Zoidl}%
\author[1]{Pouya Rezai}%
\affil[1]{York University}%


\vspace{-1em}



  
  \date{June 14, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
The signaling molecular mechanisms in zebrafish response to electricity
are unknown, so here we asked if changes to dopaminergic signaling
pathways can affect their electrically-evoked locomotion. To answer this
question, the effects of multiple selective and non-selective dopamine
compounds on the electric response of zebrafish larvae is investigated.
A microfluidic device with enhanced control of experimentation with
multiple larvae is used, which features a novel design to immobilize
four zebrafish larvae in parallel and expose them to electric current
that induces tail locomotion. In 6 days post-fertilization zebrafish
larvae, the electric induced locomotor response is quantified in terms
of the tail movement duration and beating frequency to discern the
effect of non-lethal concentrations of dopaminergic agonists
(apomorphine, SKF-81297, and quinpirole), and antagonists (butaclamol,
SCH-23390, and haloperidol). All dopamine antagonists decrease locomotor
activity, while dopamine agonists do not induce similar behaviours in
larvae. The D2- like selective dopamine agonist quinpirole enhances
movement. However, exposure to non-selective and D1-selective dopamine
agonists apomorphine and SKF-81297 cause no significant change in the
electric response. Exposing larvae that were pre-treated with butaclamol
and haloperidol to apomorphine and quinpirole, respectively, restores
electric locomotion. The results demonstrate a correlation between
electric response and the dopamine signalling pathway. We propose that
the electrofluidic assay has profound application potential as a
chemical screening method when investigating biological pathways,
behaviors, and brain disorders.%
\end{abstract}%



\sloppy


\textbf{Zebrafish Larvae's Response to Electricity is Mediated by
Dopaminergic Agonists and Antagonists}

Arezoo Khalili\textsuperscript{1}, Ellen van
Wijngaarden\textsuperscript{1}, Georg R. Zoidl\textsuperscript{2}, Pouya
Rezai\textsuperscript{1,*}

\textsuperscript{1} Department of Mechanical Engineering, York
University, Toronto, ON, CANADA

\textsuperscript{2} Department of Biology, York University, Toronto, ON,
CANADA

* Corresponding Author: BRG 433B, 4700 Keele St, Toronto, ON, M3J 1P3,
Canada; Tel: 416-736-2100 ext. 44703; Email: prezai@yorku.ca

\textbf{Abbreviations}

DA: Dopamine

DAergic: Dopaminergic

DC: Direct current

DI: Deionized

DMSO: Dimethyl sulfoxide

dpf: Days post fertilization

FOV: Field of view

fps: Frame rate per second

PDMS: Polydimethylsiloxane

RD: Response duration

SEM: Standard error of the mean

TBF: Tail beat frequency

TL: Tupfel Long fin

TR: Trapping region

\textbf{Abstract}

The signaling molecular mechanisms in zebrafish response to electricity
are unknown, so here we asked if changes to dopaminergic signaling
pathways can affect their electrically-evoked locomotion. To answer this
question, the effects of multiple selective and non-selective dopamine
compounds on the electric response of zebrafish larvae is investigated.
A microfluidic device with enhanced control of experimentation with
multiple larvae is used, which features a novel design to immobilize
four zebrafish larvae in parallel and expose them to electric current
that induces tail locomotion. In 6 days post-fertilization zebrafish
larvae, the electric induced locomotor response is quantified in terms
of the tail movement duration and beating frequency to discern the
effect of non-lethal concentrations of dopaminergic agonists
(apomorphine, SKF-81297, and quinpirole), and antagonists (butaclamol,
SCH-23390, and haloperidol). All dopamine antagonists decrease locomotor
activity, while dopamine agonists do not induce similar behaviours in
larvae. The D2- like selective dopamine agonist quinpirole enhances
movement. However, exposure to non-selective and D1-selective dopamine
agonists apomorphine and SKF-81297 cause no significant change in the
electric response. Exposing larvae that were pre-treated with butaclamol
and haloperidol to apomorphine and quinpirole, respectively, restores
electric locomotion. The results demonstrate a correlation between
electric response and the dopamine signalling pathway. We propose that
the electrofluidic assay has profound application potential as a
chemical screening method when investigating biological pathways,
behaviors, and brain disorders.

\textbf{Keywords}

Zebrafish, Electric response, Microfluidic, Dopamine, Locomotion

\textbf{Introduction}

Zebrafish larvae offer several advantages for behavioural studies
applicable in examining and creating treatments to combat
neurodegenerative and psychiatric disorders. Many microfluidic devices
have been designed for manipulation of zebrafish embryos and
larvae\textsuperscript{{[}1--8{]}} and stimulating
them\textsuperscript{{[}9--16{]}} to gather information about their
neural\textsuperscript{{[}15,17{]}} and
behavioral\textsuperscript{{[}13--16,18,19{]}} activities in controlled
microenvironments. Various stimuli such as
thermal\textsuperscript{{[}9{]}}, chemical\textsuperscript{{[}20{]}} and
fluid flow\textsuperscript{{[}11{]}} have been tested. Yet, electrical
stimulation comes with a high degree of versatility for studying
zebrafish movement. Its properties such as signal magnitude, direction
and duration can be modulated with convenience.

Studies have observed no significant impact on model organisms such as
nematodes and zebrafish due to small electric
signals\textsuperscript{{[}12,21{]}}. Some studies have been performed
on zebrafish response to electric signal by our lab and
others\textsuperscript{{[}16,22--24{]}}. While insightful, these
experiments were either time consuming with fish tested individually on
single-fish microfluidic platforms or uncontrollable and difficult to
quantify on conventional multi-well plates and chambers. In this paper,
a quadruple-fish microfluidic device was employed to load, partially
immobilize, electrically stimulate and monitor behavioral responses of
four larvae, simultaneously. The design principles of this device were
previously reported by us\textsuperscript{{[}6{]}}, while in this paper,
we focus on the first time application of this multi-fish device to
screen the novel effect of various dopaminergic chemicals on the
electric behaviour. The electric response of the semi-mobile larvae was
analyzed using response duration (RD) and tail beat frequency (TBF) as
quantitative phenotypes\textsuperscript{{[}16{]}}. The new device
reduced the time required for behavioral screening and enabled an
increased sample size.

The molecular pathway involved in zebrafish response to electricity is
unknown, but dopaminergic (DAergic) pathway is likely involved in this
locomotion\textsuperscript{{[}24{]}}. A few studies have identified
variations in different behaviours of zebrafish when exposed to dopamine
(DA) compounds\textsuperscript{{[}25--32{]}}. However, not much is known
about the behavioural impacts of DA drugs on the electric response of
zebrafish larvae. Specifically, the effects of non-selective and
selective DA agonists and antagonists on the electric response are
unknown.

Our quadruple-fish device was utilized to characterize the acute effects
of various DAergic receptor drugs on the electric-induced locomotion of
zebrafish larvae. The response of larvae exposed to DA antagonists were
compared to those treated with DA agonists. We also examined whether the
observed impairment in electric-induced movement due to DA antagonist
exposure could be recovered through subsequent treatment with a DA
agonist. Our findings elevate the present knowledge about the electric
induced behavior of zebrafish larvae and its potential regulation by the
DAergic system in the context of a customizable assay platform for
on-demand and quantitative behavioral studies.

\textbf{2. Materials and Methods}

\textbf{2.1. Zebrafish husbandry and chemical exposure}

The fish were maintained at 28\selectlanguage{ngerman}°C with a light to dark cycle of 14:10
hours. Egg water was used as the swimming solution with a 60 mg/ml
concentration of instant ocean sea salt (Instant Ocean, Blacksburg, USA)
and 0.1\% methylene blue (Fisher Scientific, Ottawa, Canada). Relevant
guidelines were followed to ensure proper care, under the Canadian
Council for Animal Care (CCAC) guidelines, Animal Care Committee (ACC)
protocol GZ 2020-7 R3, and York University Biosafety Permit PR 02-19.

All experiments were conducted using 6 dpf Tupfel long fin (TL)
zebrafish. Fish were divided into several groups to generate sample
sizes of 45 fish per condition to study the effects of DA agonists and
antagonists presented in Table 1. All exposures were done off-chip, with
all larvae in each category exposed to a specific test chemical and
washed off at the same time. The concentrations and exposure times
presented by Iron et al. \textsuperscript{{[}33{]}} were used for each
drug and the desired compounds were administered in the swimming media.
A total of 15 embryos per well were placed in a 12-well plate, with each
well containing 3 ml of the prepared solution.

All chemical compounds were obtained from Sigma-Aldrich (St. Louis, MO,
USA) and mixed with deionized (DI) water to produce the desired stock
concentration with one exception. Haloperidol was the only drug that
required dimethyl sulfoxide (DMSO) as a solvent due to its low
solubility. The solutions were then diluted with egg water to reach the
desired concentrations ranging between 0.2--50 \selectlanguage{greek}μ\selectlanguage{english}M. In contrast,
Haloperidol was diluted with 0.4\% DMSO to make the stock solution and
further diluted with egg water to form the desired concentration.

Table 1. DA agonists and antagonists used in our chemical screening
assays along with their concentrations and exposure times which were
selected based on the literature\textsuperscript{{[}33{]}}.\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
& \textbf{Non-selective} & \textbf{Non-selective} & \textbf{D1-like
selective} & \textbf{D1-like selective} & \textbf{D2-like selective} &
\textbf{D2-like selective}\tabularnewline
\midrule
\endhead
& \textbf{Agonist} & \textbf{Antagonist} & \textbf{Agonist} &
\textbf{Antagonist} & \textbf{Agonist} &
\textbf{Antagonist}\tabularnewline
\textbf{Chemical} & Apomorphine & Butaclamol & SKF-81297 & SCH-23390 &
Quinpirole & Haloperidol\tabularnewline
\textbf{Concentration} & 0.2 \selectlanguage{greek}μ\selectlanguage{english}M & 16.7 \selectlanguage{greek}μ\selectlanguage{english}M & 50 \selectlanguage{greek}μ\selectlanguage{english}M & 0.6 \selectlanguage{greek}μ\selectlanguage{english}M & 16.7 \selectlanguage{greek}μ\selectlanguage{english}M &
16.7 \selectlanguage{greek}μ\selectlanguage{english}M\tabularnewline
\textbf{Exposure time} & 20 min & 20 min & 140 min & 260 min & 80 min &
200 min\tabularnewline
\bottomrule
\end{longtable}

\textbf{2.2. Microfluidic device and experimental procedures}

The polydimethylsiloxane (PDMS) microfluidic device used for zebrafish
electrical response studies, under exposure to chemicals in Table 1, is
depicted with key features labelled in Fig. 1A,1B and 1C. It consisted
of three layers shown in Fig. 1C with various microchannels to enable
loading and screening of four larvae simultaneously. The top section of
the three-layer device included the main inlet and outlets from which
the fish entered and exited the device, the indirect flow inlet and
outlet to help direct the fish into the trapping regions (TRs) and the
reverse flow to aid in positioning and orienting the fish. The screening
pools were designed to provide enough space allowing complete tail
strokes without any collision with the device walls. This, in
combination with limitations associated with our microscope field of
view (FOV), restricted the number of larvae that could be tested at the
same time. The bottom layer of the device included valve channels
containing air. The middle layer consisted of a thin membrane,
approximately 200 \selectlanguage{greek}μ\selectlanguage{english}m thick, separating the top and bottom layers. When
the bottom valve was pressurized, the membrane deflected into the upper
layer channel to prevent the fish from swimming out of the TR. Two
anodic copper wire electrodes were attached to the outlets and a copper
wire was run through the indirect flow channel (Fig. 1A), at the middle
of the device, to function as the cathode.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

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\textsuperscript{{[}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.

\textbf{2.3. Numerical Model}

The model of the final design, created in SolidWorks (SolidWorks Corp.,
USA), was imported into COMSOL Multiphysics (COMSOL Inc., Sweden) for
three-dimensional simulations. Generally, the design consisted of four
traps connected through the inlet and outlet reservoirs, where the
cathode and anode electrodes were inserted.

The electric current and laminar flow modules in COMSOL were applied for
electric and flow field simulation in the device, respectively. Using
the electric current simulation, we determined the required total
electric current at the electrodes in the quadruple-fish device so that
each fish was exposed to the electric current of 3 \selectlanguage{greek}µ\selectlanguage{english}A, consistent with
our previous single-fish device\textsuperscript{{[}16{]}}. The electric
field within a conductive media was modeled by solving Ohm's law
equation using the steady-state DC electric module. Flow field
simulation with the relevant boundary, material and initial conditions
was also used to understand the fluid behavior and its correlation with
the experiment in terms of flow patterns and fish loading sequence in
the traps. Mesh independency was checked for both electric and flow
simulations to ensure accuracy.

\textbf{2.4. Video and image analysis}

Videos of the larvae's locomotor response were analyzed using Kinovea,
an open-source software (www.kinovea.org, France) to determine the RD
and TBF, as reported in Eq. 1 and 2. The automatic point tracking
feature within Kinovea facilitated tail tracking, producing an Excel
file (Microsoft Corp., WA, USA) of the tail tip coordinates. Every video
frame was double checked to ensure that the software accurately tracked
the tail position. Manual adjustment was applied as necessary if
low-resolution images due to fast tail motion hindered the automatic
tail tip detection process. Any small tail flicks were not included to
be consistent with the methods reported in the
literature\textsuperscript{{[}16,34{]}}.

\(Response\ Duration\ (RD)=Movement\ End\ Time-Movement\ Start\ Time\ \)

\emph{(1)}

\(Tail\ Beat\ Frequency\ (TBF)=\frac{\text{Number\ of\ full\ tail\ cycles\ }}{\text{RD}}\ \)

\emph{(2)}

To meet the requirement of a tail cycle, the tail tip coordinate had to
surpass a value of \(y=\pm 0.25mm\) from the centerline, as shown in
Fig. 1B, generating a quarter cycle for each pass. A complete tail cycle
was, therefore, comprised of four quarter cycles.

\textbf{2.5. Data analysis}

Data in this paper is represented using box plots so the 25\% and 75\%
percentiles, the median values, and the minimum and maximum data points
could be provided. All errors are described using the Standard Error of
the Mean (SEM). The sample sizes required were first determined using
power analysis with an upper limit of 0.05 and a significance level of
80\%. Following the collection of data, the Shapiro-Wilk test method was
applied to check if the data followed a normal distribution. Considering
the non-normal distribution of data, Mann-Whitney test was used to
discern the presence of significant differences in the data.

\textbf{3. Results}

In this paper, we demonstrated a novel application of our quadruple
zebrafish microfluidic screening device and investigated the effects of
several DAergic receptor compounds (Table 1) on zebrafish
electric-induced locomotor response.

\textbf{3.1. Performance of the quadruple zebrafish microfluidic device}

Compared to our single-fish device reported
previously\textsuperscript{{[}16{]}}, the design modifications to the
quadrupole-fish device in this paper had an impact on the electric
signal distribution and the fluid flow through the device, meriting
characterization.

The indirect flow offered a convenient way of steering the fish from the
main channel to fill the open TRs. An example of how each fish was
trapped in place is seen in trap A in Fig. 2. The quadruple-fish device
functionality and success rate were assessed based on the testing time
per fish, loading and orientation efficiencies defined by Eq. (3), (4)
and (5), respectively.

\(Testing\ time\ per\ fish=\frac{Loading\ time+60\ s\ recovery\ period+20\ s\ stimulation\ interval}{Number\ of\ fish\ oriented\ tail-first\ in\ the\ device}\ \)

\emph{(3)}

\(Loading\ efficiency=\frac{\text{Number\ of\ larvae\ trapped\ in\ the\ TRs\ }}{\text{Number\ of\ traps\ in\ the\ device}}\)

\emph{(4)}

\(\text{Orientation\ efficiency}=\frac{Number\ of\ fish\ correctly\ positioned\ in\ the\ TR\ (with\ tails\ in\ the\ screening\ pool)\ }{\text{Total\ number\ of\ larvae\ trapped}}\)

\emph{(5)}

Considering an average loading time of 20.4\selectlanguage{ngerman}±4.1 s, we achieved a testing
per fish time of 44.4±4.8 s for the proposed quadruple-fish device which
was 60\% faster than the time spent for testing in the single-fish
device\textsuperscript{{[}16{]}}. Our quadruple-fish device also offered
loading and orientation efficiencies of 87.5±5.6\% and 90±7.1\%,
respectively. These data clearly demonstrate the advantage of our
quadruple-fish device when aiming to facilitate larger sample sizes in a
shorter period of time.

Electric current and flow field analyses were conducted using COMSOL to
ensure their uniformity throughout the device. According to the electric
simulation, applying a total electric current of 12 \selectlanguage{greek}μ\selectlanguage{english}A between the anode
and cathode electrodes (shown in Fig. 1A) resulted in a uniform voltage
drop of 1.1 V (as seen in trap B in Fig. 2) and electric current of 3 \selectlanguage{greek}μ\selectlanguage{english}A
across each trap, consistent with the current used in our previous
single-fish device\textsuperscript{{[}6,16{]}}.

The flow in the indirect flow channel ran opposite to that in the main
channel to generate comparable pressure drops across each TR, ensuring
uniform loading conditions. The analysis of the flow dynamics within the
chip showed a pressure drop and therefore a hydrodynamic force pointing
from the main channel towards the TRs enabling loading and
immobilization within the TRs. The pressure contour plot across each
trap closely resembled the contour shown in Fig. 2 for trap C. The shear
stress in the TRs was also obtained by multiplying the water viscosity
by the velocity gradient at the wall, as shown in trap D in Fig. 2. The
maximum shear stress experienced by zebrafish during the loading process
was 10 Pa which was less than the 45 Pa threshold to avoid injury
according to the studies done by Ulanowicz and
Morgan\textsuperscript{{[}35,36{]}}.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\emph{Fig. 2. The fabricated device showing a fish loaded in trap A and
simulation overlays of the electric voltage, pressure drop and shear
stress for traps B, C, and D respectively. The wire for electric
stimulation running through the indirect flow channel is highlighted in
red}

In all experiments with zebrafish larvae presented in the following
sections, the control groups with no off-chip chemical exposure and no
on-chip electric stimulation demonstrated no movement in the device with
zero RD and zero TBF. The responses observed in the test groups was
therefore associated with the effect of electric stimulation and the
assayed chemicals in Table 1 (Supplementary video2).

\textbf{3.2. Effect of DA non-selective antagonists and agonists on
zebrafish response to electricity}

Here, we investigated the effect of butaclamol and apomorphine
(non-selective DA antagonists and agonists, respectively) on the
electric-induced locomotor activity of larvae in our microfluidic device
(Fig. 3A). Then, the larvae treated with butaclamol was post treated
with apomorphine to examine the combined effect of the non-selective DA
antagonist and agonist on zebrafish locomotor response, for the first
time. As shown in Fig. 3B and 3C, the RD and TBF of zebrafish larvae
obtained for the control and apomorphine-exposed groups were
statistically similar (P-value\textgreater{}0.05), showing that
apomorphine treatment did not affect the electrically induced response
of zebrafish larvae. However, zebrafish receiving butaclamol exhibited
51\% and 27\% reduction in the average RD and TBF, respectively, which
were both statistically significant. Post treatment with apomorphine
rescued the electric-induced locomotor responses and resulted in a 71\%
increase in RD and 33\% increase in TBF. There was no statistical
difference between the control group and the post-treated larvae
(P-value\textgreater{}0.05), demonstrating an improvement of locomotor
function under treatment of butaclamol-exposed zebrafish larvae with
apomorphine due to an increase in DA concentration.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\selectlanguage{greek}\emph{Fig. 3. Effects of apomorphine and butaclamol on 6 dpf zebrafish
larvae's electric response. (A) Exposure timeline of zebrafish larvae.
Electric-induced (B) RD and (C) TBF of zebrafish larvae exposed to no
chemical (control), 0.2 μM apomorphine, 16.7 μM butaclamol, and 0.2 μM
apomorphine after exposure to 16.7 μM butaclamol. Lines within the box
mark the median data points. Upper and lower box boundaries are the
75\textsuperscript{th} and the 25\textsuperscript{th} percentiles,
respectively. Whiskers are the highest and lowest data points obtained.
*: P\textless{}0.05, **: P\textless{}0.01, ***: P\textless{}0.001. n=15
larvae per experimental condition in three independent trials, for a
total of N = 45 larvae tested per group.}\selectlanguage{english}

\textbf{3.3. Effect of D1- and D2-like selective antagonists and
agonists on zebrafish response to electricity}

After showing the effect of non-selective DA compounds on the
electric-induced response of zebrafish larvae, we assessed potential
roles of specific DA receptors in regulating this response.

We first assessed the effect of SKF-81297 (a D1-like selective agonist)
alone and observed no significant effect on the electric-induced RD and
TBF of zebrafish larvae (Fig. 4). In contrast, the response of larvae
exposed to SCH-23390 (a D1-like selective antagonist) was marked by a
shorter RD and TBF with values decreasing by 48\% and 27\%, respectively
(P-value\textless{}0.001 and 0.05, respectively). In zebrafish larvae
exposed to SCH-23390, we observed no significant change upon post
treatment with SKF-81297.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\selectlanguage{greek}\emph{Fig. 4. Effect of SCH-23390 and SKF-81297 on 6 dpf zebrafish
larvae's electric response. (A) Exposure timeline of zebrafish larvae.
Electric-induced (B) RD and (C) TBF of zebrafish larvae exposed to no
chemical (control), 50 μM SKF-81297, 0.6 μM SCH-23390, and SKF-81297
after exposure to 0.6 μM SCH-23390. Lines within the box mark the median
data points. Upper and lower box boundaries are the
75\textsuperscript{th} and the 25\textsuperscript{th} percentiles,
respectively. Whiskers are the highest and lowest data points obtained.
*: P\textless{}0.05, **: P\textless{}0.01, ***: P\textless{}0.001. n=15
larvae per experimental condition in three independent trials, for a
total of N = 45 larvae tested per group.}\selectlanguage{english}

We then determined the effects of two D2-like selective compounds on
zebrafish electric response (Fig. 4). When larval electric-induced
locomotor response was recorded after exposure to quinpirole (a D2-like
selective agonist), increased activity was observed
(P-value\textless{}0.05). Treatment with quinpirole raised RD and TBF by
49\% and 24\%, respectively. DMSO vehicle did not alter zebrafish larvae
locomotor activity from the control level, ensuring that DMSO can be
used as solvent for haloperidol in our assay. Haloperidol exposure
decreased the RD and TBF by 49\% and 33\%, respectively and completely
abolished the locomotor activities at 50 \selectlanguage{greek}μ\selectlanguage{english}M (Data not shown).
Posttreatment with quinpirole caused a 55\% increase in electric-induced
RD and a 70\% rise in TBF. No statistical difference was observed
between the control group and the post-treated larvae
(P-value\textgreater{}0.05), verifying an improvement of locomotor
activity under treatment of haloperidol-exposed zebrafish larvae with
quinpirole due to an increase in DA concentration.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\par\null

\selectlanguage{greek}\emph{Fig. 5. Effect of DMSO, haloperidol and quinpirole on 6 dpf
zebrafish larvae's electric response. (A) Exposure timeline for
zebrafish larvae. Electric-induced (B) RD and (C) TBF of zebrafish
larvae exposed to no chemical (control), 16.7 μM quinpirole, DMSO
(0.4\%), 16.7 μM haloperidol and quinpirole after exposure to 16.7 μM
haloperidol. Lines within the box mark the median data points. Upper and
lower box boundaries are the 75\textsuperscript{th} and the
25\textsuperscript{th} percentiles, respectively. Whiskers are the
highest and lowest data points obtained. *: P\textless{}0.05, **:
P\textless{}0.01, ***: P\textless{}0.001. n=15 larvae per experimental
condition in three independent trials, for a total of N = 45 larvae
tested per group.}\selectlanguage{english}

\textbf{4. Discussion}

Previous reports have established that zebrafish larvae display changes
in their natural behavior (i.e., behaviour without external stimulation)
following exposure to drugs that are known to act on DA receptors in
mammals\textsuperscript{{[}29,30,32,33,37,38{]}}. In our earlier works,
we reported that the acute administration of neuroactive drugs that
alter DAergic signaling (e.g., apomorphine, SKF-38393, quinpirole,
6-OHDA and levodopa) caused similar effects on locomotor activity in
zebrafish larvae as in mammals\textsuperscript{{[}12,16{]}}. The aim of
this study was to advance the knowledge about zebrafish larvae's
response to electric field. Using our quadruple fish microfluidic
device, the sensitivity of zebrafish larvae's electric-induced response
to different chemicals that target DAergic receptors in mammals was
evaluated for the first time, based on two quantitative behavioral
readouts of RD and TBF.

Based on the data presented for general locomotion assay, both
apomorphine and butaclamol (non-selective DAergic agents) produce their
maximum effect after 20 minutes exposure\textsuperscript{{[}33{]}}.
These nonselective compounds, acting on both D1- and D2- receptors, are
commonly described as rapid-acting as the other agonists and antagonists
used in our study required longer exposure times. The selective agonists
had shorter exposure times than the selective antagonists, with the
D2-like drugs taking effect faster than those targeting the D1
receptors.

We benefited from our novel quadruple fish device to examine a possible
link between zebrafish's electric induced response and the DA receptors
activities. Exposure to apomorphine, a non-selective DA agonist, had no
impact on the electric-induced response (Fig. 3). This may be either due
to the drug affecting multiple different
receptors\textsuperscript{{[}39{]}} or possible saturation of the DA
receptors after treatment with this specific
dose\textsuperscript{{[}40,41{]}}. In contrast, exposure to the
non-selective antagonist butaclamol, resulted in a decrease in
electric-induced movement (Fig. 3), possibly because of suppression of
the DAergic signalling\textsuperscript{{[}30{]}}, matching observations
from the literature for 6 dpf zebrafish
larvae\textsuperscript{{[}33{]}}and
mammals\textsuperscript{{[}42--44{]}}, suggesting the induction of
analogous actions in zebrafish and mammals caused by DA receptor agents.
For the first time, we showed that the hypoactivity caused by butaclamol
was reversed through subsequent treatment with apomorphine, restoring
the locomotor response to match that observed in the control group (Fig.
3).

Exposure to D1 and D2-like selective DA antagonists of SCH-23390 and
haloperidol caused lower general locomotor activity in 6 dpf zebrafish
larvae\textsuperscript{{[}33{]}} and
rodents\textsuperscript{{[}44--47{]}}. King et al also reported
haloperidol having sedative effects on humans\textsuperscript{{[}48{]}}.
These results suggest a similar drug action mechanism for mammals and
zebrafish triggered by the mentioned selective antagonists, possibly due
to blockage of either D1- or D2-like receptors. SCH-23390 and
haloperidol, reduced electric induced movement of zebrafish larvae in
our microfluidic device (Fig. 4 and Fig. 5), consistent with previous
findings reported in the literature\textsuperscript{{[}33,44--48{]}}.

SKF-81297 (a D1-like selective DA agonist) induced no significant
effects on general locomotor response when freely swimming 6 dpf
zebrafish larvae were treated with the drug\textsuperscript{{[}49{]}}.
However, the resulting phenotypes for the D1 receptor-deficient mice
were not uniform for all studies and did not align with expected
observations of D1 receptor ligand treatment\textsuperscript{{[}50{]}}.
Some researchers reported an increase in general locomotor activity in
rodents\textsuperscript{{[}51,52{]}}. The effect was less prevalent in
younger organisms such as 5-10 days old compared to weaned
rats\textsuperscript{{[}53{]}}. In contrast other researchers observed
no significant difference in general movement of WT weaned
mice\textsuperscript{{[}54{]}} or Swiss Webster
mice\textsuperscript{{[}55{]}} when exposed to D1-like receptor
agonists. Treatment with SKF-81297 resulted in no significant change in
electric-induced locomotor response of zebrafish larvae (Fig. 4),
suggesting that D1- DA receptors might not be heavily involved in
modulating zebrafish larvae's electric induced response. The results
obtained in our experiments match the previously reported results,
showing no significant change in electric induced locomotor response
upon exposure to SKF-81297\textsuperscript{{[}49,54,55{]}}.

Researchers also reported that treatment with quinpirole (a D2-like
selective DA agonist), not only stimulated general locomotor activity in
zebrafish larvae\textsuperscript{{[}31,38{]}}, but in rodents as
well\textsuperscript{{[}44,56{]}}. Similarly, exposure to quinpirole
significantly increased the electric induced movement of larvae (Fig.
5), revealing a dominant involvement of D2-like DA receptors in electric
induced response.

As another novel aspect of our project, SKF-81297 and quinpirole
(DA-selective agonists) were employed to ascertain whether they would
restore the disrupted electric induced RD and TBF of larvae pretreated
with SCH-23390 and haloperidol (DA-selective antagonists), respectively
(Fig. 4 and Fig. 5).

To the best of our knowledge, we demonstrate for the first time that
posttreatment with quinpirole restores the electric induced locomotor
response, while posttreatment with SKF-81297 did not fully improve
activity. We propose a mechanism of electric-induced behavioral response
which requires D2-DA receptors and the advanced microfluidic platform as
a powerful investigative tool.

\textbf{5. Conclusion}

The presented screening technique provides researchers with a valuable
investigative tool for studying the biological pathways in sensory-motor
systems. This can be particularly useful in future work regarding
systems that may be involved in zebrafish response to electricity along
with examining larvae's locomotor response, on-demand genetic testing
and in both toxicology and drug screening applications. Employing a
microscope with a larger FOV, along with further modifications to the
device, could increase the number of fish tested at the same time to
improve the throughput of behavioral assays with the presented
technology.

The functional assay presented in this research enabled us to
distinguish the actions of receptor antagonists from those of agonists.
We assessed alteration in electric-induced locomotor activity of
zebrafish larvae following acute exposure to a variety of selective or
non-selective DA antagonists and agonists. Behavioral profiles varied
according to each DA receptor drug in terms of RD and TBF. Exposure to
quinpirole was found to significantly increase the RD and TBF of the
larval response to electric stimulus, suggesting the involvement of
D2-like DA receptors in modulating zebrafish larvae electric induced
response. In contrast, no significant difference resulted due to
exposure to apomorphine and SKF-81297. Treatment to any of the three DA
antagonists decreased RD and TBF, however, only apomorphine and
quinpirole restored the response, reversing the impact of the
antagonists. These results may support the claim that there are similar
actions in mammals and zebrafish triggered by DA receptor drugs.
Additional studies are required to pharmacologically clarify DAergic
receptors' role and involvement in the zebrafish nervous system.
Although present reports typically match mammalian reports of impacted
movement as a result of induced changes to the DAergic system, further
work is needed to eliminate the possibility of effects on other
signaling pathways. The drugs tested have higher affinities for DAergic
targets. However, they cannot be solely DAergic receptor ligands.
Previous research has shown that these chemicals can also bind to
adrenergic, cholinergic, histaminergic, and serotonergic receptors but
with lower affinity\textsuperscript{{[}57--59{]}}. Additional research
is needed to determine the relative affinities for these receptors to
fill this knowledge gap. Internal concentrations would also need to be
determined to eliminate all ambiguity regarding the amount of a drug
successfully passing biological membranes such as the skin or the
blood-brain barrier to be able to interact with the receptors in the
brain. The proposed studies will advance the understanding of
electric-induced behaviors in a lower vertebrate model recognized for
high-throughput and high-content analysis.

\textbf{Data Availability}

Data available on request from the authors.

\textbf{Conflict of Interest}

The authors do not claim any conflict of interest.

\textbf{Author's Contributions}

Arezoo Khalili:~Methodology, Investigation, Formal analysis, Validation,
Data curation, Visualization, Writing - original draft.

Ellen van Wijngaarden:~Investigation, Numerical Analysis, Writing -
original draft.

Georg R. Zoidl:~Conceptualization, Resources, Writing - review and
editing.

Pouya Rezai:~Conceptualization, Methodology, Validation, Resources,
Writing - review and editing, Supervision, Funding acquisition.

\textbf{Acknowledgments}

The authors would like to acknowledge the funding sources that enabled
this research: the Ontario Early Researcher Award (ER18-14-042) to PR,
the Ontario Graduate Scholarship to AK and the National Sciences and
Engineering research Council (NSERC) support to EW, PR
(RGPIN-2020-06140) and GRZ (RGPIN-418142-2012). We thank Janet Fleites
Medina and Veronica Scavo for zebrafish husbandry.

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