Forensic Toxicology

https://doi.org/10.1007/s11419-021-00577-8

ORIGINAL ARTICLE

Caffeine‑induced bradycardia, death, and anxiety‑like behavior

in zebrafish larvae
Hideyuki Maeda1 · Akihiro Hasumi1 · Ken‑ichi Yoshida1

Received: 29 November 2020 / Accepted: 10 April 2021

© Japanese Association of Forensic Toxicology 2021

Abstract
Purpose Caffeine, a major ingredient of coffee and other drinks, causes minimal health problems. However, excessive caf-
feine intake may induce emotional and cardiac events. Studies on zebrafish larvae are considered useful for understanding
drug poisoning because they can be used for multiple simulations.
Methods The locomotion (swim distance) of zebrafish larvae exposed to varying concentrations of caffeine was measured
using a light–dark test and an automatic video-tracking system. Mortality and heart rates were assessed 4 and 24 h after
caffeine exposure and 24 h after caffeine removal.
Results The heart and survival rates were reduced by exposure to caffeine of  > 300 mg/L after 24 h in a dose-dependent man-
ner; this was attenuated by caffeine removal at 4 h. Caffeine reduced the heart rate and survival considerably at 1000 mg/L,
supporting the notion of caffeine-induced cardiac arrest resulting from bradycardia. The locomotion of zebrafish larvae during
six alternating cycles of light–dark exposure was slower during light periods and faster during dark periods; 100–300 mg/L
caffeine increased and decreased locomotion during light and dark cycles, respectively, with high inter-cycle reproducibility.
Conclusions Given the anxiogenic effect of caffeine and light preference of zebrafish larvae, caffeine-induced changes in
light–dark cycles could provide a new reliable marker for anxiety. Therefore, the “light–dark locomotion test” employed in
this study is valuable for research on anxiogenic or anxiolytic substances. The test may allow simultaneous and automatic
analysis of the locomotion of many fish under multiple conditions, thereby enabling evaluation of dose–response effects of
caffeine.

Keywords Caffeine overdose · Toxicology · Zebrafish larvae · Light–dark test · Behavior analysis · Arrhythmia

Introduction or beverages; in these cases, blood caffeine concentrations

Caffeine is a major ingredient of coffee, soft drinks, green
tea, black tea, and nutritional drinks. Because they allevi-
ate drowsiness and enhance concentration, these drinks
have become very popular. However, in addition to neuro-
logical symptoms such as excitement, insomnia, delusions,
and panic attacks, excessive caffeine may induce cardiac
symptoms including tachycardia and (rarely lethal) arrhyth-
mias. At plasma concentrations ≥ 15 mg/L, caffeine usually
stimulates the central nervous system and heart [1]. Life-
threatening caffeine overdoses entail the ingestion of caf-
feine-containing medications, rather than caffeinated foods
* Hideyuki Maeda
hmaeda@tokyo-med.ac.jp
1
Department of Forensic Medicine, Tokyo Medical
University, 6‑1‑1 Shinjuku,, Tokyo 160‑8402, Japan
can reach > 80 mg/L [1]. Blood caffeine concentrations in
cases of overdose are typically 190 mg/L [2], and in two
cases of lethal caffeine intoxication, they were reported to
be 350 mg/L [3] and 567 mg/L [4]. Intravenous injection of
a solution containing 192 mg/L of caffeine has been reported
to result in death of an individual [4].
Zebrafish offer various advantages to investigate the
toxic effects of neuroactive substances. Drugs can be
administered non-invasively to zebrafish by immersing
them in a solution of the drug. Basic cardiovascular mor-
phology of and functions in zebrafish larvae, including the
heart, blood vessels, heart rate and rhythm, vasoconstric-
tion, and blood flow, can be monitored using microscopy
and imaging, taking advantage of the fish’s transparency
[5]. Additionally, zebrafish and humans share common
mechanisms for the regulation of the heart rate and car-
diac ion channels [6]. Rana et al. reported that caffeine at

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1–25 mM suppressed the heartbeat, leading to death in
zebrafish embryos in approximately 2–3 days postfertili-
zation (DPF) [2]. Zebrafish may thus contribute consider-
ably to the analysis of pathophysiology and the large-scale
screening of drugs and toxic substances that may result in
fatalities, organ injuries, and abnormal behavior.
In adult zebrafish, a natural preference for darkness
(scototaxis) is a useful measure of anxiety, which is
attenuated by anxiolytic drugs and enhanced by anxio-
genic agents [7]. In the “light–dark preference test,” adult
zebrafish are treated with a substance in the central com-
partment and allowed to select either white or black com-
partments on opposite sides. The selection and avoidance
of the black compartment reflect the anxiogenic and anxio-
lytic effects of the substance, respectively (and vice versa
for the white compartment); conversely, a shuttle between
the two compartments indicates psychostimulant effects
[7, 8]. Using this test, Maximino et al. demonstrated that
caffeine induces anxiogenic and psychostimulant effects
at different doses [7, 8].
The “light–dark locomotion test” is also based on the
dark preference of adult zebrafish; it has been proposed
as a useful screening method for psychoactive substances
using high-throughput video-tracking analysis systems,
which can automatically record locomotion and other
swim behaviors. In the test, zebrafish larvae are placed in
multi-well plates within a closed chamber and exposed to
alternating light and dark conditions while recording the
swimming tracts automatically.
In zebrafish larvae, light transition has been found
to reduce locomotive activity, whereas dark transition
increases the same; this reflects stress and anxiety levels
[9]. However, there have been no studies on the effect of
caffeine on zebrafish behavior, using the light–dark loco-
motion test. Additionally, the effects of caffeine on the
heart rate and survival have not been studied and com-
pared systematically in zebrafish.
This study aimed to investigate whether caffeine alters
locomotive activity in zebrafish using the light–dark loco-
motion test; it also evaluated whether caffeine affects their
heart rate and survival rate, using microscopy.
Fig. 1  Experimental protocol. The black areas indicate periods when
light is off, and the white areas indicate those when the light is on;
time is expressed in min. The fish were exposed to six alternating
cycles of light and dark for 15 min each, while their swimming was
13
Forensic Toxicology
Materials and methods
Animal model and zebrafish husbandry
The experiments were performed according to the Guide
for the Care and Use of Laboratory Animals published
by the US National Institutes of Health (NIH publication
85–23, revised 1996). The protocol was approved by the
corresponding Experimental Animal Committee (approval
number: R1-117 and R2-0038).
Adult zebrafish (Danio rerio; undefined, outbred stocks
obtained from aquatic research organisms) were reared and
maintained in an automated small aquarium fish housing
system under standard conditions in aquarium water, which
is defined as reverse osmosis water with controlled pH,
water temperature, and conductivity at 7.5, 27 ± 1.0 °C, and
400 ± 50 mS, respectively. Lighting was artificial, with 14 h
of light (8 AM–10 PM) and 10 h of darkness.
Two adult male and female zebrafish were placed in
a breeding tank with a partition. In the afternoon and at
night, the tank was placed in an incubator set at 28 °C.
The fish were left undisturbed overnight and the partition
was removed the next morning. As soon as embryos were
observed, the adults were returned to their colony tank;
all embryos from the same strain were then pooled from
the breeding tanks and maintained at 28 °C in plastic petri
dishes. The water was replaced by fresh aquarium water, and
dead eggs and larvae were removed on days 1, 3, 4, and 5.
Experimental protocol
The outline of the experiments is presented in Fig. 1. Each
zebrafish larva (6–7 DPF) was placed in 1 of 96 wells of
plastic plates along with 0.3 mL of water solution contain-
ing 0, 0.1, 1, 10, 100, 200, 300, 500, or 1000 mg/L of caf-
feine (C750, SIGMA-ALDRICH, St. Louis, MO, USA) and
acclimatized to the video-tracking apparatus (Danio-vision,
Noldus, Wageningen, The Netherlands) for 60 min under
dark condition. Subsequently, the fish were exposed to six
alternating cycles of light and dark for 15 min each, while
their swimming was monitored. The light intensity used dur-
ing the experiments was 5000 lx; during darkness, infrared
monitored, before removing caffeine by immersing the retrieved fish
in water. Twenty-four hours later, the fish underwent six more light–
dark cycles under monitoring
Forensic Toxicology

illumination had a light intensity of 0 lx. Shortly thereafter,

caffeine was removed from selected wells and the retrieved
fish were immersed in 100 mL water, twice. Twenty-four
hours later, fish underwent six more light–dark cycles under
monitoring. Heart rate and survival rate were examined
under anesthesia with 0.32% tricaine in water, using a ster-
eomicroscope apparatus Reica EZ4W (Reica Microsystems
GmbH, Wetzlar, Germany) at 4 and 24 h. The survival rate
was assessed morphologically, as described in the results
section.

Statistical analysis

Group data are expressed as means ± standard error

of the mean. The data were analyzed using Dunnett’s,
Tukey–Kramer, or Student’s t tests, at a confidence level
of 95%. Statistical analysis was performed using GraphPad
Prism 6 for Windows v6.05 (GraphPad Software, San Diego,
CA, USA).

Results

Effects of caffeine on heart rate

The effects of caffeine on the heart rate are presented in

Fig. 2. After the swimming experiments (4 h), the heart
rate of larvae in the lower caffeine concentration groups
(0.1 mg/L and 1 mg/L) was higher than that of control
larvae (107.1% and 108.1%, respectively). However, at
concentrations ≥ 300 mg/L, the heart rate decreased in a
dose-dependent manner (80.2%, 61.0%, and 41.3% at 300,
500, and 1000 mg/L of caffeine, respectively). After 24 h,
bradycardia progressed in a dose-dependent manner at caf-
feine concentrations ≥ 300 mg/L (72.0%, 41.6%, and 11.8%,
with caffeine concentrations of 300, 500, and 1000 mg/L,
respectively). However, when caffeine was removed at 4 h,
the heart rate recovered to the pretreatment level in 24 h
at caffeine concentrations ≤ 500 mg/L and to 76.1% at the
concentration of 1000 mg/L.
Judgment of death
Figure 3 shows representative images of live and dead
fish. Lee et al. listed edema of the periphery and/or abdo-
men, hemorrhage, abnormal body shape (scoliosis, tail
bent), and motility as morphological signs of death in
zebrafish [10]. These findings could hardly be identified
in our larvae. However, as we could assess cardiac arrest
and extravasation of red blood cells from the heart (Fig. 3,
arrow) in transparent fish, we used these morphologi-
cal characteristics to discriminate between live and dead
Fig. 2  Effects of caffeine concentrations on the heart rate. Heart rate
of larvae after immersion for (a) 4 h in caffeine solutions at the indi-
cated concentration, (b) 24 h after washing the caffeine, and c 24 h
without washing the caffeine. bpm: beats per minute. *P < 0.05 vs.
Control, **P < 0.01 vs. Control (Dunnett’s test). Values are presented
as mean ± standard error, n = 7–49 (n, number of zebrafish larvae)
fish. Live fish demonstrated integrity and clarity of con-
tours and patterns. In contrast, dead fish showed black-
ish eyes without glitter, necrotic appearance of tissues,
deranged patterns of the lateral pigments, and blurred
contours and patterns with eventual breakdown of the
body. We present data on the live and dead fish 24 h after
immersion in water (live) or caffeine solution (500 mg/L,
dead) (Fig. 3a), live and dead fish in the same well con-
taining caffeine solution (Fig. 3b), and the same fish in
caffeine solution being alive at 4 h (Fig. 3c) and dead
at 24 h (Fig. 3d). We trained ourselves to discriminate
dead from live fish, by evaluating the aforementioned
characteristics.

13

Fig. 3  Representative live and
dead fish. Images of larvae (a)
in two different caffeine concen-
trations in the same experiment
(0, 500 mg/L), b in the same
field for the same caffeine
concentration (500 mg/L), and
c, d of the same fish at different
time points (4 h and 24 h with
500 mg/L caffeine). Arrows
indicate blood in the heart
chamber. L living zebrafish, D
dead zebrafish
Effects of caffeine concentrations on survival rate
After 4 h of immersion in caffeine, nearly 100% of fish
survived. However, those immersed in 1000 mg/L caffeine
showed a lower survival rate (Fig. 4a, approximately 80%
survival). After 24 h of caffeine immersion at concentrations
of 300, 500, and 1000 mg/L, the survival rate was reduced
to 54.1%, 39.4%, and 12.9%, respectively (Fig. 4c). Removal
of caffeine at 4 h restored the survival rate of fish that were
immersed in 300, 500, and 1000 mg/L caffeine to 94.4%,
65.5%, and 51.6%, respectively (Fig. 4b).
Collectively, the survival rate at 24 h was reduced at
caffeine concentrations > 300 mg/L; at caffeine concentra-
tions < 300 mg/L, it was restored to the basal level when
caffeine was removed at 4 h. The effects of drug removal on
the survival rate 24 h after immersion in caffeine at concen-
trations of 500 and 1000 mg/L for 4 h were partial.
Effects of caffeine on locomotion (swim distance)
After acclimatization for 1 h in the dark, zebrafish larvae
were exposed to six cycles of light–dark (15 min–15 min)
conditions, and the distance swum was examined. After
the session was completed (at 4 h), fish were washed and
immersed in caffeine-free water. At 24 h, the same test
was repeated (Fig. 5). Each time point in Fig. 5 shows the
distance traveled over time on exposure to the light and
dark stimuli. The solid line represents the distance trave-
led during the first six light–dark stimuli, and the dashed
line represents the distance traveled during the light–dark
stimuli after returning to caffeine-free water for 24 h. The
bar graph shows the distance traveled during the sixth
light–dark stimuli. All graphs include the distance traveled
during the light–dark stimulus in caffeine-free water, as a
13
Forensic Toxicology
reference. The represented graph shows the data pertaining
to the light–dark tests; the distance traveled is indicated by
the dashed line in the line graph. (Fig. 5).
In control larvae, during the six comparative cycles of
light and dark stimulation (Fig. 5a), the locomotive activ-
ity represented by the swim distance was significantly
higher on dark transitions than on light transitions, with
reproducible cycles. During the light cycles (slower loco-
motion) of larvae exposed to caffeine, locomotive activ-
ity was significantly enhanced at 100–300 mg/L, but it
was reduced at 500–1000 mg/L. In contrast, during the
dark cycles (faster locomotion), locomotion was reduced
by caffeine at 100–1000 mg/L (Fig. 5e–i). After washing,
the test was repeated 24 h later. At this time, the inhibitory
effects induced by caffeine during the dark transition were
not observed (Fig. 5e–g), except in those larvae exposed to
500–1000 mg/L caffeine for 4 h (Fig. 5h,i).
Discussion
Heart rate
The heart rate was reduced at caffeine concentra-
tions > 300 mg/L in a dose-dependent manner, and the reduc-
tion progressed distinctly at a concentration of 1000 mg/L
(41.3% of control at 4 h and 11.8% at 24 h) (Fig. 2). On
removal of caffeine at 4 h, the heart rate returned to that
of the control larvae at 300 and 500 mg/L caffeine at 24 h;
however, it only returned to 76.1% when using 1000 mg/L
caffeine.
Caffeine may induce bradycardia either through physi-
ological mechanisms or by toxicological effects. It is gener-
ally accepted that tachycardia and bradycardia are caused
Forensic Toxicology
Fig. 4  Survival rates at different caffeine concentrations. a At 4 h of
caffeine immersion, b at 24 h after washing the caffeine solution, and
c after 24 h of caffeine immersion. n = 16–59 (n, number of zebrafish
larvae)
by activation of the sympathetic and vagal nervous systems,
respectively. The dose–effect relationship in this study con-
tradicts a study in dogs, in which low-dose caffeine gener-
ated benign bradycardic arrhythmias via vagal stimulation
and severe ventricular arrhythmias or atrial flutter/fibrilla-
tion via sympathetic activation [11]. Willson argued that
toxicological concentrations of caffeine paradoxically induce
cholinergic activation via inhibition of acetylcholinesterase
[12]; this may explain the caffeine-mediated bradycardia
observed in this study. However, as the cholinergic antago-
nist atropine does not affect the heart rate in zebrafish before
12 DPF [13], caffeine is unlikely to have induced brady-
cardia in this study through vagal stimulation in our 6–7
DPF-old zebrafish.
The ether-a-go-go (hERG) potassium channel may have
been involved in the caffeine-induced bradycardia. hERG
plays a pivotal role in cardiac repolarization, as genetic
defects in hERG are known to induce QT prolongation on
electrocardiograms and pleiomorphic ventricular tachycar-
dia that can be fatal [2]. It is known that bradycardia and
mortality in zebrafish are useful for the screening of drugs
that cause QT prolongation and carry potential risks of sud-
den death [14]. Reports suggest that caffeine can inhibit
hERG at 1–5 mM in vitro, thereby inducing QT prolongation
that can lead to sudden death following pleiomorphic ven-
tricular tachycardia [15]. Rana et al. speculated that caffeine
induces bradycardia and cardiac arrest in zebrafish embryos
via hERG inhibition [2]. Thus, it is likely that caffeine at
1000 mg/L induced cardiac arrest via hERG inhibition.
Case reports or experimental studies provide no solid
evidence on the vascular effects of caffeine, and no human
studies have consistently demonstrated that caffeine in cof-
fee and caffeinated drinks contributes to sympathetic activa-
tion and increased blood pressure [16]. Sudden deaths after
the ingestion of caffeinated drinks cannot be explained by
blood caffeine concentrations and may be related to non-
caffeine ingredients. However, taurine neither exerts behav-
ioral effects by itself nor influences the effects of caffeine in
zebrafish [17].
The biological and pathological effects and mortality of
caffeine cannot be explained solely by blood caffeine con-
centrations because various confounding factors and inter-
personal diversities intervene. This discrepancy may be
explained by (1) substances interacting with caffeine (e.g.,
psychiatric medications) or influencing caffeine metabo-
lism, (2) preexisting diseases in organs that act as targets
of caffeine (e.g., heart) or its metabolizer (e.g., liver), or (3)
genetic polymorphisms in caffeine-metabolizing enzymes
[1]. In particular, caffeine competitively inhibits its metabo-
lizing liver enzyme, CYP1A2.
Death and survival
As the fish were considerably small, morphological markers
for death were required for the quick judgment of death. In
addition to cardiac standstill and extravasation in the trans-
parent zebrafish, which provided solid evidence for death,
we identified morphologic characteristics of dead and live
fish (Fig. 3); after self-training, this enabled a rapid assess-
ment of whether the fish were dead or alive at a glance.
The survival rate was reduced in a dose-dependent man-
ner to a similar extent as the reduction in heart rate 24 h
after caffeine immersion, with extreme reduction at concen-
trations of 1000 mg/L (Figs. 2, 4). These findings support
the notion that severe and prolonged bradycardia contrib-
uted to the death of the zebrafish. However, the recovery of
the heart rate (76.1%) was greater than that of the survival
rate (51.6%) after removal of 1000 mg/L caffeine from the
solution.
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 Forensic Toxicology

C -o ff
C -o n
distance (mm)
2000
2000
n.s. n.s.

d is ta n c e (m m )
d is ta n c e (m m )
1500
1500

1000
1000

500
500

0
0

C C" C C"

0 .1 - o n 0 .1 - o ff
distance (mm)

2000 n.s.
2000
n.s.

d is ta n c e (m m )
d is ta n c e (m m )
1500 1500

1000 1000

500 500

0 0
C 0 .1 0 .1 " C 0 .1 " 0 .1 "
distance (mm)

1 -o n 1 - o ff
2000 2000
d is ta n c e (m m ) n.s. n.s.

d is ta n c e (m m )
1500 1500

1000 1000

500 500

0 0

C 1 1" C 1 1"

Fig. 5  Effects of caffeine on locomotion (swim distance). a Distance
traveled by control larvae during six light–dark cycles. b–i) Dis-
tance traveled by larvae immersed in the indicated concentration of
caffeine. The solid line connects the distances swum within 4 h and
the dashed line, those after 24 h. The bar graph labeled “-on” indi-
cates the distance traveled when the light was turned on for the sixth
time, and the bar graph labeled “-off” indicates the distance traveled
when the light was turned off for the sixth time, according to the indi-
cated caffeine concentration (“indicates the distance swum 24 h after
washing the indicated concentration of caffeine). All graphs include
the distance traveled by control larvae (C) as reference. * P < 0.05
vs. C, **P < 0.01 vs. C (Dunnett’s test). Values are presented as
mean ± standard error, n = 7–63 (n, numbers of zebrafish larvae)

Chen et al. reported that in some cardiac mutants,
zebrafish can survive through percutaneous oxygen diffu-
sion but not through cardiovascular circulation during the
first 10 DPF [18]. The findings suggest the contribution of
extra-cardiac factors to the death of zebrafish at high caf-
feine doses.
Assuming the survival rate 24 h after caffeine immersion
to be an indicator of death, the 300 mg/L and 1000 mg/L
concentrations would approximately be LD50 and LD100,
respectively. These values are comparable to the blood con-
centrations found (350 and 567 mg/L) in the cases of lethal
caffeine intoxication in humans [3, 4].
Effects on locomotion
The locomotive activity of zebrafish larvae increased dur-
ing dark periods and decreased during light periods in the
light–dark locomotion test (Fig. 5); this is consistent with the
findings of a previous report on a single light–dark transition
in zebrafish larvae [9]. Previous studies interpreted that the
dark-induced increase in locomotion reflects stress/anxiety
levels. For instance, a report suggested that thigmotaxis
(staying close to walls), a well-validated index of anxiety,
was enhanced by a sudden light-to-dark transition and sup-
pressed by reduced darkness in zebrafish larvae [19]. Addi-
tionally, in the light–dark preference test, caffeine increased
the duration of stay of adult zebrafish in the black compart-
ment (dark preference); this has been believed to indicate the
anxiogenic effect of caffeine [7, 8].
Notably, we found that caffeine at concentrations of
100–300 mg/L induced a remarkable increase and decrease
in locomotion in the background of slow and fast basal loco-
motion during light and dark periods, respectively (Fig. 5).
The bidirectional changes induced by caffeine in the light
and dark periods were different from the enhanced locomo-
tion with caffeine in the dark, observed in previous studies
using different methods [7, 8, 20]. The difference may be
derived from the light preference of zebrafish larvae in this

13
Forensic Toxicology
Fig. 5  (continued)
study and the dark preference of zebrafish adult in previous
studies [7, 8, 20]. The bidirectional changes in the light–dark
locomotion test would be a reliable indicator for anxiety,
if further studies had confirmed the findings for other sub-
stances. However, it should be noted that caffeine causes
both, anxiety-like behavior and psychomotor stimulation,
depending on the dose in the light–dark preference test [8].
Indeed, caffeine exerts a psychostimulant effect through
inhibition of the striatal adenosine receptor, independent of
the anxiogenic effects [21]. However, the “light–dark pref-
erence” behavior reflects the active (selective) behavior of
zebrafish, whereas the “light–dark locomotion” behavior
indicates the passive (non-selective) behavior in response
to light–dark stimuli [8, 20]. Burgess et al. reported that in a
high-throughput system for locomotive analysis in zebrafish
larvae, reduction in illumination elicited large angle turns
distinct from startle responses, which oriented the larvae
toward the light source [22]. In sustained darkness, larvae
are transiently hyperactive before adopting a quiescent
state [22]. They highlighted that the locomotive activity of
zebrafish is controlled by the state of light or dark adapta-
tion, similar to the masking phenomena in higher vertebrae
where light directly regulates motor activity independent of
the emotion [22]. In any event, further studies are needed
to validate the changes in the light–dark locomotion test as
solid indications for anxiety or psychostimulation.
Movement was greatly reduced by 1000 mg/L caffeine
and nearly stopped at 24 h without reversal on drug removal
(Fig. 2). Given the low survival rate, the lack in locomotion
at 1000 mg/L concentrations of caffeine may indicate death.
However, to validate locomotion as a measure of death, it is
essential to exclude the possibility that fish immobility can
also reflect circumstances other than death.
Freezing is the complete cessation of movement in fish
at the bottom of the tank; this is induced by severe stress or
anxiety [20]. While freezing, they show active movements
in the gills and eyes; this could not be confirmed in the pre-
sent study. As we have identified morphological markers
for death (Fig. 3), it is essential to confirm such markers in
preliminary experiments before the introduction of absent
motility as a marker of death in full-scale studies.
Merits of the light–dark locomotion test
Rodent studies require labor for injection/handling and
considerations of differences in drug metabolism; how-
ever, this study used video-tracking of the distance swum
by zebrafish larvae after treatment with a neuroactive
13

Fig. 5  (continued)
substance. This enabled simultaneous and rapid monitor-
ing of the behavior and mortality of many animals under
various conditions, while maintaining the drug concentra-
tion in the bathing solution. Few studies have evaluated
the effect of caffeine on behavior using the “preference
test” [7, 8] and novel tank test [20]; this is the first study
to use the video-tracking system for evaluating the effects
of caffeine using the “locomotion test.”
Maximino et al. demonstrated the anxiogenic effects
of caffeine in the light–dark “preference test” on adult
zebrafish [7, 8]. In contrast, this study used the light–dark
“locomotion test,” taking advantage of the light preference
of zebrafish larvae [20]. Table 1 highlights the merits of
the “locomotion test,” as compared with the “preference
test.”
Table 1  Comparison between
light–dark preference and light–
dark locomotion tests Require amount of drug
Conditions, analyzable fishes
Labor for observation
Confirming reproducibility
Previous studies on caffeine
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Forensic Toxicology
In the preference test, 4.5 L of water was used in one
aquarium, and zebrafish were placed in it for the entire
experiment. [7]. In the “locomotion test,” each zebrafish
was placed in each of the 96 wells containing 0.3 mL of
drug solution. Accordingly, the “locomotion test” requires
considerably smaller amounts of the drug than the “prefer-
ence test.”
Many more conditions can be examined in an experiment
using the “locomotion test” instead of the “preference test,”
as the former utilizes a small 96-well plate and the latter
uses one set of the large apparatus for each condition. This
study presented data on nine concentrations of caffeine in
the “locomotion test,” whereas the study by Maximino et al.
study showed only three conditions in the “preference test”
[7, 23]. These advantages of the “locomotion test” enabled
Preference test Locomotion test(this study)
Large Small
Few Many
Much(watch, calculation) Minimum(automatic)
Difficult(repeat experiments) Easy(inter-cycle difference)
Plural None(novel)
Forensic Toxicology
us to demonstrate the dose-dependent effects of caffeine on
locomotion; they can also promote toxicology research by
elucidating the toxic and lethal concentrations of substances.
The “locomotion test” is considerably more efficient
than the “preference test.” The recordings and analyses are
performed manually in the latter and are fully automatic in
the former. In the “preference test,” it is necessary to watch
and record movements of fish in the two compartments; in
the “locomotion test,” the video-tracking system automati-
cally records fish behavior and can present plural behavio-
ral markers as digital data and graphs almost immediately
using built-in software; it can also analyze data after batch
output. The “locomotion test” can visualize the effects of
drugs in each light and dark transition cycle at a glance
and confirm the reproducibility at once. On the contrary,
with the “preference test,” it is difficult to confirm repro-
ducibility, as it requires repetition of the experiments, and
manual analyses of the results for each experiment.
As the automatic video-tracking system will be widely
adopted for pharmacological and toxicological studies on
zebrafish larvae, the results of this study provide valuable
information for future studies.
Conclusions
Caffeine-induced bradycardia and death in zebrafish larvae
in a dose-dependent manner. Most fish died with 1000 mg/L
caffeine, with sustained bradycardia; however, removal of
caffeine 4 h post-administration greatly improved the sur-
vival rate at 24 h. The high-throughput analysis of locomo-
tion (swim distance) using a video-tracking auto-analysis
system revealed fast and slow locomotion during dark and
light periods, as well as a decrease and increase in loco-
motion with 100–300 mg/L caffeine during dark and light
periods, respectively; this served as a novel indicator for
anxiogenic or psychoactive substances, including caffeine.
Caffeine at a concentration of 1000 mg/L greatly reduced
survival, heart rate, and locomotion, reflecting toxicity. The
results of this study demonstrate that zebrafish larvae are
useful for evaluating the effects of neuroactive substances on
behavior, heart rate, and death. Additionally, the light–dark
locomotion test may contribute to toxicological and patho-
physiological studies on anxiogenic, anxiolytic, psychoac-
tive, and antipsychotic substances.
Compliance with ethical standards
Conflict of interest All authors certify that they have no affiliations
with or involvement in any organization or entity with any financial
interest or non-financial interest in the subject matter or materials dis-
cussed in this manuscript.
Ethical approval The use of zebrafish for this study was approved by
the Experimental Animal Committee of Tokyo Medical University
(approval number: R2-0038). This article does not contain the results
of any studies on human participants, performed by any of the authors.
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