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Characterization of the locomotor activities of zebrafish larvae under the influence of various neuroactive drugs

Authors:
© Annals of Translational Medicine. All rights reserved. Ann Transl Med 2018;6(10):173atm.amegroups.com
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Characterization of the locomotor activities of zebrafish larvae
under the influence of various neuroactive drugs
Fei Li1, Jia Lin1, Xiuyun Liu1, Wenhui Li2, Yifeng Ding2, Yunjian Zhang2, Shuizhen Zhou2, Ning Guo3,
Qiang Li1
1Translational Medical Center for Development and Disease, Institute of Pediatrics, Shanghai Key Laboratory of Birth Defect, 2Department of
Neurology, Children’s Hospital of Fudan University, Shanghai 201102, China; 3Center for Chinese Medical Therapy and Systems Biology, Shanghai
University of Traditional Chinese Medicine, Shanghai 201203, China
Contributions: (I) Conception and design: F Li, J Lin; (II) Administrative support: Q Li; (III) Provision of study materials or patients: Q Li; (IV)
Collection and assembly of data: J Lin, X Liu; (V) Data analysis and interpretation: All authors; (VI) Manuscript writing: All authors; (VII) Final
approval of manuscript: All authors.
Correspondence to: Qiang Li. Translational Medical Center for Development and Disease, Shanghai Key Laboratory of Birth Defect, Institute of
Pediatrics, Children’s Hospital of Fudan University, 399 Wanyuan Road, Shanghai 201102, China. Email: liq@fudan.edu.cn.
Background: Behavioral changes in animals reflect functional changes in their central nervous system.
Neuroactive drugs that act on different neural pathways can induce specific behavioral responses; therefore,
it is possible to infer the activities of neuroactive drugs by studying the behavioral changes induced by drugs
of interest in animals.
Methods: In this study, AB strain zebrafish larvae at 7 days post fertilization (dpf) were treated with
different concentrations of drugs that act on different neural pathways. Changes in the swimming distances
of zebrafish larvae under different illumination conditions and the differences in locomotor activities between
light and dark conditions (lighting motor index) were analyzed.
Results: Among the drugs studied, different concentrations of sulpiride had no effect on larval locomotor
activity either under light or dark conditions. Progressively decreased spontaneous movements were
observed in zebrafish larvae treated with increasing doses of MK-801 and valproic acid. With increasing
concentrations of pentylenetetrazole and yohimbine, the spontaneous movement of larval zebrafish presented
a bell-shaped response. When the illumination changed from light to dark, zebrafish larvae not treated with
drugs demonstrated increased locomotor activities. However, high levels of yohimbine, pentylenetetrazole
decreased the degree of change in the lighting motor index.
Conclusions: In conclusion, drugs that affect different neural pathways exert different influences on the
locomotor activities of zebrafish larvae. This study presents an initial effort to establish a framework that
correlates the drug activities and the behavioral responses of zebrafish larvae under drug treatments, which
may provide a potential identification of the pathways of novel drugs with neurological activities through
their behavioral influences.
Keywords: Zebrafish larvae; neuroactive drugs; locomotion; pathway
Submitted Mar 15, 2018. Accepted for publication Apr 09, 2018.
doi: 10.21037/atm.2018.04.25
View this article at: http://dx.doi.org/10.21037/atm.2018.04.25
173
Original Article on Translational Neurodegeneration
Li et al. Locomotion of zebrash larvae under various neuroactive drugs
© Annals of Translational Medicine. All rights reserved. Ann Transl Med 2018;6(10):173atm.amegroups.com
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Introduction
The zebrash (Danio rerio) is a popular model organism for
behavioral genetics and developmental studies, especially
forward genetics (1). Because the nervous system of the
zebrash exhibits developmentally, structurally, functionally
and pharmacologically conserved similarities with the
mammalian nervous system, zebrash are also widely used
as experimental animals in neural and neuropharmacological
studies (2,3). As early as 4–5 days post-fertilization (dpf),
zebrafish larvae develop different behaviors, including
emotions (anxiety and fear), learning and memory, goal-
directed behavior, optokinetic responses, and prepulse
inhibition (4-6).
Neuroactive drugs that interact with different receptors
of the central nervous system result in diverse behavioral
manifestations in zebrafish. It has been shown that acute
exposure to dopamine receptor agonists induced increases
in the locomotor activities of 6 dpf zebrash larvae, whereas
dopamine receptor antagonists demonstrated opposite
effects (7). This phenomenon is consistent with the
findings in mammals (8,9). In humans and other animals,
γ-aminobutyrate (GABA) receptor antagonists have dis-
inhibitory effects on the central nervous system, which
can cause clonic or tonic-clonic seizures (10,11). Low
concentrations of alcohol, d-amphetamine and cocaine were
found to increase the spontaneous movement of 6 dpf wild-
type zebrash larvae, whereas high concentrations of these
drugs inhibited their locomotor activities (12).
For zebrash, changes in illumination conditions usually
result in changes in the levels of locomotor activities. It has
been shown that the locomotor activities of larval zebrash
were signicantly enhanced when the illumination changed
from light to dark (12), and some neuroactive drugs were
able to modify or even reverse this phenomenon (7).
A visual motor response (VMR) assay that focused on
the instantaneous changes in the swimming activities of
zebrash in response to changes in lighting conditions was
previously described (13,14). To characterize drug effects
on locomotor activities, instead of focusing on the transient
instantaneous locomotor changes caused by changes in
the lighting conditions, we aimed to examine the more
sustained changes in the swimming activities that are caused
by differences in lighting, i.e., light and dark conditions.
However, studies thus far have primarily focused on
the qualitative aspect of this phenomenon; therefore, a
new parameter—the lighting motor index—was devised
in this work to focus on the quantitative aspect of this
phenomenon.
In this study, we examined the influences of different
concentrations of various neuroactive drugs on the
locomotor activities and the lighting motor indexes of the
zebrash larvae under light and dark conditions. With this
comparative study, we attempted to speculate a correlation
between the drug activities and their behavioral inuences
to promote the identication of the activities of novel drugs
through their effects on zebrash locomotion.
Methods
Drugs
The drugs included MK-801; pentylenetetrazole;
valproic acid sodium salt; yohimbine hydrochloride;
5,5-diphenylhydantoin sodium salt and sulpiride, which
were bought from Sigma-Aldrich. Drugs were dissolved in
sterile water to prepare the indicated stock solutions. Before
experiment, stock solutions were diluted to appropriate
concentrations.
Zebrash husbandry
AB strain wild-type zebrash were acquired from Zebrash
Facility of Children’s Hospital of Fudan University, and were
maintained at 28.5 according to standard protocols (15).
The sh were kept on a 14-h light: 10-h dark cycle (lights
on at 8:00 AM; lights off at 10:00 PM). Eggs were obtained
by natural spawning and were raised in groups of 50 in an
incubator at 28.5 from birth to 7 dpf, which was staged
according to a previously published method (16). Eggs and
larvae were kept under the same lighting schedule as adult
zebrafish. All animal experimental procedures complied
with local and international regulations. All protocols were
approved by the institutional animal care committee.
Behavior tests
Twenty-four-well plates were used to carry out the behavior
tests in order to provide adequate swimming space for
the zebrafish larvae. We use ZebraBox (ViewPoint Life
Sciences) to record videos of zebrash larvae activities. The
experiments consisted of 60 min of continuous illumination
(light period) followed by 5 min of darkness (dark period).
The activities of zebrash larvae during the last 10 min of
the light period and the 5 min dark period were analyzed.
The quantication of zebrash larvae locomotion activities
Annals of Translational Medicine, Vol 6, No 10 May 2018
© Annals of Translational Medicine. All rights reserved. Ann Transl Med 2018;6(10):173atm.amegroups.com
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was achieved using the tracking mode of ZebraLab software
with recorded videos.
In this study, the swimming distances of zebrash larvae
were employed as indicators of their locomotor activities.
To evaluate the inuence of the illumination change from
light to dark on the locomotor activity, a lighting motor
index was devised as indicated below.
lighting motor index = (the swimming distances of the
zebrafish larvae under dark conditions)/(the swimming
distances of the zebrash larvae under light conditions)
Data collection and statistical analyses
All data are presented as the mean ± standard error of the
mean (SEM). Statistical analyses were performed using
GraphPad Prism software. The data for each drug were rst
assessed using repeated-measure two-way ANOVA, and a P
value less than 0.05 was considered statistically signicant
(P<0.05).
Results
For sulpiride-treated zebrafish larvae, two-way ANOVA
indicated that the change in illumination exerted a
significant influence on their locomotor activities {F
[1, 372] =30.18, P<0.0001} but that the change in drug
concentrations did not signicantly affect their locomotion
{F [5, 372] =1.574, P=0.1666} (Figure 1). The locomotor
activities of the zebrash larvae had no obviously change no
matter in the light and dark conditions (Figure 1A). When
the lighting motor index was considered, the zebrafish
larvae treated with 500 μM sulpiride demonstrated a
statistically signicant increase in the lighting motor index
compare with the control group (Figure 1B).
For diphenylhydantoin-treated zebrafish larvae, two-
way ANOVA revealed that the change in illumination
significantly affected their locomotor activities {F [1,
372] =37.40, P<0.0001}. Drug treatments at different
concentrations also signicantly inuenced their locomotor
activities {F [5, 372] =4.854, P=0.0003} (Figure 2). Although
multiple comparison tests did not identify significant
influences of diphenylhydantoin treatment at different
concentrations under light conditions on the locomotor
activity of the zebrash larvae, high concentrations (500 μM)
of diphenylhydantoin significantly reduced the locomotor
activities of the larvae under dark conditions. No difference
in the lighting motor index was observed between the
control group and the drug-treated groups (Figure 2B).
For yohimbine-treated zebrafish larvae, two-way
ANOVA analysis indicated that the change in illumination
had significant influences on their locomotor activities
{F [1, 372] =109.7, P<0.0001}. In addition, drug treatment
at different concentrations exerted significant influences
on the locomotor activities of these zebrash larvae {F [5,
372] =217.9, P<0.0001} (Figure 3). Under light conditions,
signicantly reduced locomotor activities were observed in
groups treated with 25, 50, 100, or 200 mg/L yohimbine
compared with the control group. Under dark conditions,
10 mg/L yohimbine treatment resulted in a significant
Figure 1 Effects of sulpiride on zebrash locomotor activities (A)
and the lighting motor index (B). (A) Average distances moved
by zebrafish larvae within a 1-min time bin are plotted. The
horizontal axis denotes the sulpiride treatment concentration that
the larvae received. The vertical axis denotes the distance moved
by the zebrafish larvae in millimeters; (B) lighting motor index.
The horizontal axis denotes the sulpiride treatment concentration
that the larvae received. The vertical axis denotes the fold changes
in the zebrash locomotor activities under different illumination
conditions. The data are presented as the mean ± SEM; n=32
animals per group. *, P<0.05, signicantly differs from the control
group. SEM, standard error of the mean.
Locomotor activity
Lighting motor index
Average distance moved
per minute (mm)
Fold change
[Sulpiride]
Control 4 μM 20 μM 100 μM 500 μM 1000 μM
Control 4 μM 20 μM 100 μM 500 μM 1000 μM
[Sulpiride]
250
200
150
100
50
0
2.5
2.0
1.5
1.0
0.5
0.0
Light
Dark
A
B
Li et al. Locomotion of zebrash larvae under various neuroactive drugs
© Annals of Translational Medicine. All rights reserved. Ann Transl Med 2018;6(10):173atm.amegroups.com
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increase in the locomotor activities of the zebrash larvae,
whereas further increases in the yohimbine concentration
significantly inhibited the locomotor activities of the
zebrafish larvae compared with the control group. When
the illumination condition changed from light to dark,
the zebrash larvae treated with 25, 50, 100, or 200 mg/L
yohimbine presented significantly reduced lighting motor
indexes (Figure 3B).
For MK-801-treated zebrash larvae, two-way ANOVA
analysis revealed significant influences of illumination
conditions on their locomotor activities {F [1, 372] =35.88,
P<0.0001}. The changes in drug concentrations also had
signicant impacts {F [5, 372] =19.45, P<0.0001} (Figure 4).
With increasing drug concentrations, the average distance
moved by the zebrafish larvae per minute demonstrated
a progressive decrease compared with the control group
under light conditions. Under dark conditions, increasing
drug concentrations also caused a downward trend in the
locomotor activities of the zebrafish larvae. The lighting
motor indexes of most drug-treated groups were increased
compared with that of the control group; however, no
statistical signicance was found (Figure 4B).
Figure 2 Effects of diphenylhydantoin on zebrafish locomotor
activities (A) and the lighting motor index (B). (A) Average
distances moved by the zebrafish larvae within a 1-min time bin
are plotted. The horizontal axis denotes the diphenylhydantoin
treatment concentration that the larvae received. The vertical
axis denotes the distance moved by the zebrafish larvae in
millimeters; (B) lighting motor index. The horizontal axis denotes
the diphenylhydantoin treatment concentration that the larvae
received. The vertical axis denotes the fold changes in the zebrash
locomotor activities under different illumination conditions. The
data are presented as the mean ± SEM; n=32 animals per group. *,
P<0.05, signicantly differs from the control group. SEM, standard
error of the mean.
Figure 3 Effects of yohimbine on zebrafish locomotor activities
(A) and the lighting motor index (B). (A) Average distances moved
by the zebrash larvae within a 1-min time bin are plotted. The
horizontal axis denotes the yohimbine treatment concentration that
the larvae received. The vertical axis denotes the distance moved by
the zebrash larvae in millimeters; (B) lighting motor index. The
horizontal axis denotes the yohimbine treatment concentration
that the larvae received. The vertical axis denotes the fold changes
in the zebrash locomotor activities under different illumination
conditions. The data are presented as the mean ± SEM; n=32
animals per group. *, P<0.05, signicantly differs from the control
group. SEM, standard error of the mean.
Light
Dark
Locomotor activity
Lighting motor index
Average distance moved
per minute (mm)
Fold change
[Diphenylhydantoin]
[Diphenylhydantoin]
Control 1 μM 4 μM 20 μM 100 μM 500 μM
Control 1 μM 4 μM 20 μM 100 μM 500 μM
100
80
60
40
20
0
3
2
1
0
A
B
Locomotor activity
Lighting motor index
Average distance moved
per minute (mm)
Fold change
[Yohimbine]
[Yohimbine]
Control 10 mg/L 25 mg/L 50 mg/L 100 mg/L 200 mg/L
Control 10 mg/L 25 mg/L 50 mg/L 100 mg/L 200 mg/L
200
150
100
50
0
3
2
1
0
Light
Dark
A
B
Annals of Translational Medicine, Vol 6, No 10 May 2018
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For valproic acid-treated zebrafish larvae, two-way
ANOVA analysis indicated that the change in illumination
conditions exerted significant effects on their locomotor
activity {F [1, 372] =42.43, P<0.0001}. The differences in
drug concentrations also had significant influences on the
locomotor activities of these zebrafish larvae {F [5, 372]
=12.71, P<0.0001} (Figure 5). Under both light and dark
conditions, the average distance moved by the zebrafish
larvae per minute was signicantly reduced compared with
the control groups when treated with 500 μM valproic acid.
However, the lighting motor indexes did not differ between
the control group and the drug-treated groups (Figure 5B).
For pentylenetetrazole-treated zebrafish larvae,
two-way ANOVA analysis revealed that the change in
illumination conditions had no effect on their locomotor
activities {F [1, 372] =0.08475, P=0.7711}, but the change
in drug concentrations had signicant effects on zebrash
locomotion {F [5, 372] =16.61, P<0.0001} (Figure 6). Under
light conditions, a progressive increase in locomotor
activities was observed in the groups treated with 1, 2, 4,
or 8 mM pentylenetetrazole, although only the locomotor
activities of the 4 and 8 mM pentylenetetrazole-treated
groups significantly differed from those of the control
group. When the concentration reached 16 mM, the
Locomotor activity
Lighting motor index
Average distance moved
per minute (mm)
Fold change
[MK-801]
[MK-801]
Control 10 μM 20 μM 50 μM 100 μM 200 μM
Control 10 μM 20 μM 50 μM 100 μM 200 μM
150
100
50
0
3
2
1
0
Light
Dark
A
B
Figure 4 Effects of MK-801 on zebrash locomotor activities (A)
and the lighting motor index (B). (A) Average distances moved
by the zebrash larvae within a 1-min time bin are plotted. The
horizontal axis denotes the MK-801 treatment concentration that
the larvae received. The vertical axis denotes the distance moved
by the zebrafish larvae in millimeters; (B) lighting motor index.
The horizontal axis denotes the MK-801 treatment concentration
that the larvae received. The vertical axis denotes the fold changes
in the zebrash locomotor activities under different illumination
conditions. The data are presented as the mean ± SEM; n=32
animals per group. *, P<0.05, signicantly differs from the control
group. SEM, standard error of the mean.
Figure 5 Effects of valproic acid on zebrash locomotor activities
(A) and the lighting motor index (B). (A) Average distances moved
by the zebrash larvae within a 1-min time bin are plotted. The
horizontal axis denotes the valproic acid treatment concentration
that the larvae received. The vertical axis denotes the distance
moved by the zebrafish larvae in millimeters; (B) lighting motor
index. The horizontal axis denotes the valproic acid treatment
concentration that the larvae received. The vertical axis denotes the
fold changes in the zebrash locomotor activities under different
illumination conditions. The data are presented as the mean ±
SEM; n=32 animals per group. *, P<0.05, signicantly differs from
the control group. SEM, standard error of the mean.
Locomotor activity
Lighting motor index
Average distance moved
per minute (mm)
Fold change
[Valproic acid]
[Valproic acid]
Control 1 μM 4 μM 20 μM 100 μM 500 μM
Control 1 μM 4 μM 20 μM 100 μM 200 μM
150
100
50
0
4
3
2
1
0
Light
Dark
A
B
Li et al. Locomotion of zebrash larvae under various neuroactive drugs
© Annals of Translational Medicine. All rights reserved. Ann Transl Med 2018;6(10):173atm.amegroups.com
Page 6 of 9
locomotor activities of the zebrafish larvae dropped to a
level that was comparable to that of the control group.
Under dark conditions, only the 16 mM pentylenetetrazole-
treated zebrash larvae demonstrated signicantly reduced
locomotor activities, whereas all other drug-treated groups
exhibited similar levels of locomotor activities compared
to the control group. However, zebrafish larvae in the
drug-treated groups (4, 8, 16 mM) presented significantly
decreased lighting motor indexes compared with the control
group (Figure 6B).
Discussion
Not only did the larval zebrafish react differently to
different drugs, but also their responses were affected by
the drug concentration. At the same time, illumination also
had an influence. Therefore, Table 1 was constructed to
illustrate the relationships among the drugs, the targeted
pathways, and the behavioral manifestations of zebrafish
under different drug treatments. The aim of this study was
to present an initial effort to establish a framework that
correlates the drug activities and the behavioral responses of
zebrash larvae under drug treatments, which may provide a
potential identication of the pathways of novel drugs with
neurological activities through their behavioral inuences.
With increasing concentrations of yohimbine, the
spontaneous movement of zebrafish larvae was markedly
decreased, regardless of illumination. These observations
were similar to other studies with rodents. When Male
hooded Lister rats were acutely treated with yohimbine
at 2.5 and 5 mg/kg, the rats showed decreased locomotor
activities (17). Male Sprague-Dawley rats with yohimbine
treatment below 2 mg/kg demonstrated increased
locomotor activities, whereas yohimbine treatment at
higher doses resulted in decreased locomotion (18). One
possible explanation is that yohimbine inhibits the activity
of α2-adrenaline receptors, which leads to inhibition of
these neurons. Sulpiride, a specic D2 dopamine receptor
antagonist, is another drug tested in the current study,
and we found that sulpiride did not affect zebrafish larval
locomotor activities under the current protocol, irrespective
of whether the light or dark condition was imposed. Few
studies have examined the influence of sulpiride on the
locomotor activities of model organisms. However, a
study examining the effects of haloperidol, a drug that
functions on the same neurological pathway as sulpiride,
found no effect on the locomotor activity of zebrafish
larvae in the rst 80 minutes when haloperidol was applied
at different concentrations. This finding was consistent
with our observation. However, after 80 minutes, different
concentrations of haloperidol had different effects on the
spontaneous movement of zebrafish larvae (7), with high
concentrations exerting an inhibitory effect on locomotor
activity (7). As our purpose was to establish an effective
platform of drug screening, a longer protocol was not the
optimal choice. However, it would be advisable to include a
second round of screening with longer protocols for drugs
that did not show any observable effects during the first
round of rapid screening.
Figure 6 Effects of pentylenetetrazole on zebrafish locomotor
activities (A) and the lighting motor index (B). (A) Average
distances moved by the zebrafish larvae within a 1-min time bin
are plotted. The horizontal axis denotes the pentylenetetrazole
treatment concentration that the larvae received. The vertical
axis denotes the distance moved by the zebrafish larvae in
millimeters; (B) lighting motor index. The horizontal axis denotes
the pentylenetetrazole treatment concentration that the larvae
received. The vertical axis denotes the fold changes in the zebrash
locomotor activities under different illumination conditions. The
data are presented as the mean ± SEM; n=32 animals per group. *,
P<0.05, signicantly differs from the control group. SEM, standard
error of the mean.
Locomotor activity
Lighting motor index
Average distance moved
per minute (mm)
Fold change
[Pentylenetertrazole]
[Pentylenetertrazole]
Control 1 mM 2 mM 4 mM 8 mM 16 mM
Control 1 mM 2 mM 4 mM 8 mM 16 mM
150
100
50
0
2.5
2.0
1.5
1.0
0.5
0.0
Light
Dark
A
B
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GABA is an inhibitory neurotransmitter of the central
nervous system, and pentylenetetrazole, a GABA receptor
antagonist, has been shown in our previous work to increase
the locomotion of larval zebrafish under continuous
illumination (19). In addition, progressively increased bodily
movements were observed in adult zebrafish treated with
increasing concentrations of pentylenetetrazole (5, 7.5, 10
and 15 mM) (11), and seizure-like behaviors were observed
in zebrafish treated with 15 mM pentylenetetrazole (11).
These observations share great similarities with our current
results. In this present study, progressively increased
locomotor activities were observed in zebrafish larvae
treated with increasing concentrations of pentylenetetrazole,
regardless of illumination, though an obvious but non-
significant decrease in the spontaneous movement of the
larvae was observed at 16 mM pentylenetetrazole. This
could possibly be due to the toxicity to the nervous system
of the drug at high concentrations (7,19). The alterations
in startle response patterns immediately following light-
dark transitions reflects changes in stress/anxiety levels
of animals (20,21). Pentylenetetrazole-treated zebrafish
larvae exhibited signicantly decreased anxiety levels during
the light-dark transition, as indicated by decreases in the
lighting motor index. This is in accordance with a previous
study (20), in which 5 dpf zebrafish larvae were treated
with 5 mM pentylenetetrazole. Picrotoxin is another drug
that acts on the same neural pathway as pentylenetetrazole.
In a different work by our group, 5 dpf zebrafish larvae
under picrotoxin treatment showed progressively
enhanced locomotor activities with increasing picrotoxin
concentrations under the light condition; in contrast, an
inhibitory effect of picrotoxin treatment on locomotor
activities was observed under the highest picrotoxin
concentration tested under the light condition. However,
no effect on spontaneous movement by zebrafish larvae
under the dark condition was observed (22). Male albino
rats that were acutely treated with 1.5 mg/kg picrotoxin
intraperitoneally demonstrated epileptic seizures after 20–
30 minutes (23). Hyper-synchronous (HYP) activities were
observed in electroencephalography recordings in male
Sprague-Dawley rats receiving intraperitoneal injections of
picrotoxin at 3–5 mg/kg (24). These observations are the
result of a dis-inhibited nervous system caused by GABA
receptor antagonists.
Discrepancies between our results and previously publish
works were also noted. In the current study, decreased
locomotor activities were observed in 7 dpf larvae with acute
MK-801 treatment (20, 50, 100 and 200 μM) under both
light and dark conditions. Mice treated with 0.05 mg/kg
MK-801 from 7 to 49 days postpartum demonstrated
decreased locomotor activities (25). However, in other
studies, acute MK-801 treatments resulted in increased
locomotor activities in both zebrash and rodents. Increases
in locomotor activities were observed in Male C57BL/6
mice with 0.3 and 0.6 mg/kg MK-801 treatment (26), and
5 dpf zebrafish larvae demonstrated increased swimming
activities when treated with 50 μM MK-801 (27). Increases
in locomotor activities were also observed in adult zebrash
with 20 μM MK-801 treatment (28). These differences
between our results and the results of others indicate the
influences of species, genetic background, and age on the
neurological effects of various drugs.
In our study, valproic acid and 5,5-diphenylhydantoin
were the two drugs showing acute inuences on spontaneous
locomotor activities but not well documented in previously
published works. It is well known that prenatal exposure to
valproic acid could result in neurodevelopmental disorder
during adolescence (29). The offspring of pregnant Wistar
Table 1 Summary of drug effects on the locomotor activity, including the activity and the possible target
Compound name Activities Influence under light* Influence under dark** Target
Sulpiride Dopamine receptor antagonist No difference No difference Excitatory neuron
5,5-Diphenylhydantoin Sodium channel antagonist No difference Downward trend Excitatory neuron
MK-801 NMDA receptor antagonist Downward trend Downward trend Excitatory neuron
Valproic acid Histone acetylation enzyme inhibitor Downward trend Downward trend
Yohimbine α2-adrenaline receptor antagonist Downward trend Downward trend
Pentylenetetrazole γ-aminobutyric acid receptor antagonist Upward trend Upward trend Inhibitory neuron
*, The influence of locomotion under the light condition; **, the influence of locomotion under the dark condition; –, there is no clear
definition of the types of neurons.
Li et al. Locomotion of zebrash larvae under various neuroactive drugs
© Annals of Translational Medicine. All rights reserved. Ann Transl Med 2018;6(10):173atm.amegroups.com
Page 8 of 9
rats that received an intraperitoneal injection of 600 mg/kg
valproic acid demonstrated declined spontaneous
movements during puberty and maturity compared with rat
offspring not treated with valproic acid (30). In our work,
high concentration (500 μM) of valproic acid signicantly
reduced the spontaneous movement of zebrafish larvae
under both light and dark conditions. This observation is in
agreement with the role of valproic acid as an anti-epileptic
drug. Our study found that 500 μM 5,5-diphenylhydantoin
decreased the spontaneous movement of the zebrash larvae
under both light and dark conditions. A previous study
demonstrated that 5 and 10 mg/kg diphenylhydantoin could
inhibit methylphenidate-induced hyper-locomotor activities
in male Swiss albino mice (31). Thus far, few studies have
examined the locomotor influences of diphenylhydantoin
on model organisms. Therefore, our work provides a
foundation for further studies on the activities of these two
drugs.
Conclusions
Various zebrafish behavioral paradigms have been devised
to explore the influences of neuroactive drugs on the
behavioral manifestations of a spectrum of neurological
processes. Our purpose was to establish a correlation
between drug actions and their behavioral inuences using
a high-throughput screening protocol to facilitate as well
as to expedite the identification of the actions of novel
drugs through their behavioral manifestations. Therefore,
zebrash larvae at 7 dpf, an acute drug treatment regimen,
a short 65 min experimental duration, simple behavioral
tests, and an automated data acquisition system were used
to maximize the turnover of the experiments. This study
presents an initial effort to establish a framework that
correlates the drug activities and the behavioral responses
of zebrafish larvae under drug treatments, which could
facilitate the identification of the mechanisms of novel
drugs with neurological activities through their behavioral
inuences.
Acknowledgements
Funding: This work was supported by grants from the
National Natural Science Foundation of China [81771632]
and the National Key Research and Development Program
[2016YFC1000500] to Q Li; and by Shanghai Key
Laboratory of Birth Defect [13DZ2260600] to N Guo.
This work was also supported by grants to X Liu from the
National Natural Science Foundation of China [81601329].
Footnote
Conicts of Interest: The authors have no conicts of interest
to declare.
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Cite this article as: Li F, Lin J, Liu X, Li W, Ding Y, Zhang
Y, Zhou S, Guo N, Li Q. Characterization of the locomotor
activities of zebrafish larvae under the influence of various
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atm.2018.04.25
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