The Effect of Caffeine towards Zebrafish (Danio rerio) Juvenile
Working Memory Exposed by Unpredictable Chronic Stress (UCS)
Alya Ayu Tazkia
1
a
, and Zainuri Sabta Nugraha
1
b
1
Departement of Anatomy, Faculty of Medicine Universitas Islam, Yogyakarta Indonesia
Keywords: Unpredictable Chronic Stress (UCS), Caffeine, Zebrafish (Danio rerio), Memory
Abstract: The changes in brain structure are caused by dependent and independent factors. Early Life Stress (ELS) is
one of the dependent factors that affect the brain’s structure and volume. ELS exposure increases synaptic
pruning in the neuron in which it disturbs the cognitive function, memory loss, emotion, and risk-taking.
Researchers strive to perceive the effect of caffeine in coffee, which can increase memory. Caffeine in coffee
has an active compound which increases memory. This study aimed to identify the effect of caffeine in coffee
to memory with exposure to stress. The method used in this research was experimental using a post-test with
controlled group design. The group were divided into four groups comprising of Negative Control (K1),
Positive Control (K2), and group in which exposed by caffeine using different doses (P1 and P2). The exposed
group used 20mg/dL and 50mg/dL doses. Hence, the exposure was accorded by the protocol of toxicity for
three days. After three days of exposure, group K2, P and P2 were given Unpredictable Chronic Stress (UCS)
intervention for seven days. All of the groups were tested using T-maze for three days to see the Zebrafish’s
memory. Data analysis were collected from Zebrafish time and selection of colour on every side of T-maze.
This study shows that there are differences in each group according to time, the average group of K1 (27,11
s), K2 (41,01 s), P1 (25,62 s), P2 (49,22 s). The fastest time was shown in P1. Meanwhile, the slowest time
was shown in P2. The test using Shapiro-Wilk showed (P=0,000) p>0,005 in which the data were not well
distributed. The data normality using Kruskal-Wallis showed (P=0,000) p<0,005. Hence, H1 can be accepted.
Post-hoc test for every group according to time showed group P1-K1, P1-P2, K1-K2, K1-P2 (p<0,005),
whereas K1-P1 and K2-P2 (0,063) (p>0,005). Otherwise, compared group according to colour using Chi-
Square showed a significant difference (p<0,005). As a conclusion, study report the exposure of caffeine and
intervention of UCS in Zebrafish affect memory in the T-maze test.
1 INTRODUCTION
Changes in brain structure are essential in adaptation
for the positive or negative external stimulation. A
baby’s brain structure develops very complexly and
matures depending on the environment, education,
social interaction, and experience (Elston, 2014). One
of the factors in brain development is influenced by a
positive environment which leads to resilience.
Several kinds of research presented discrepancy in
maternal, leading to a cognitive abnormality in
children (Weinstock, 2008). It Is necessary to concern
parents’ education because parents play an essential
role in stimulating the children’s brain development
regarding social and behaviour (Mychasiuk, 2012).
a
https://orcid.org/0000-0002-0453-3408
b
https://orcid.org/0000-0001-6506-9656
Embryologically, the brain develops in week
three. It happens when telencephalon becomes the
frontal cortex. The primitive cell is located in the
subventricular zone, including axon and the dendritic
cell. It proliferates and migrates to the target cell to
produce a new layer of the cortex: the afterbirth, post-
natal neuron forms sensory motoric and cognitive-
based on the stimulation provided. The neuron which
is connected well may increase dendritic sprouting
and synaptogenesis. Meanwhile, neurons are not
connected and used to synaptic pruning and depletion
in the cortex (Mychasiuk, 2012).
The stimulation factor in post-natal should be
considered as an effect in changing the brain structure
by neurogenesis. First, the independent factor is
208
Tazkia, A. and Nugraha, Z.
The Effect of Caffeine towards Zebrafish (Danio rerio) Juvenile Working Memory Exposed by Unpredictable Chronic Stress (UCS).
DOI: 10.5220/0010490202080213
In Proceedings of the 1st Jenderal Soedirman International Medical Conference in conjunction with the 5th Annual Scientific Meeting (Temilnas) Consortium of Biomedical Science Indonesia
(JIMC 2020), pages 208-213
ISBN: 978-989-758-499-2
Copyright
c
2021 by SCITEPRESS – Science and Technology Publications, Lda. All rights reserved
caused by genetics in which the neuron is formed
based on the image of each other’s gene. Second, the
dependent factor is created by externals providing
stimulation or enrichment of experience after birth
(Mundkur, 2005). An environment was rich in
cognitive, social and motoric increases brain volume,
especially in the prefrontal, hippocampus, and
striatum neurons. However, in the form of stress, a
hostile environment decreases brain volume with low
cognitive function.
Several research pieces have examined using mice
by exposing Early Life Stress (ELS) in juveniles as a
result of a brain volume decrease. These changes are
involved in neuron restriction in ELS exposure which
produces neuron restriction through synaptic pruning.
The restrictions reduce emotion, cognitive, memory,
and decision making (Rocher, 2004; Mundkur, 2005).
Another research which concerns on stress pre-natal
and post-natal restrict in neuron and reduce cognitive
function. PFC has an essential role in stress, especially
in the Pyramidal neuron (Kolb, 2017).
The effect of ELS in the restriction on pyramidal
neuron PFC correlates in Major Depressive Disorder
(MDD), Post-Traumatic Disorder (PTSD), verbal,
decision making, loneliness, and anxiety (Rocher,
2004) (Frodl, 2010). The longer the neuron’s stress
exposure, the easier the neuron restriction in
pyramidal apical neuron PFC, presented in several
studies (Radley, 2005; Anderson, 2019). It is indicated
by evaluating the pyramidal neurons in stress
cognitive used in the Y-maze trial. The result showed
that there was a difference in the first trial and second.
Stress method used other problems, for instance,
forced-swim test, elevated plus maze, etc. (Anderson,
2019).
Zebrafish has an analogy brain structure similar
to the human brain, especially in memory and
cognitive. Human has PFC, which functions as
cognitive and long-term memory which is located in
the pallium. The Zebrafish analogue brain’s research
in the pallium is activated when mental plays a role.
Stress affects motoric and behaviour to respond in
Zebrafish (Parker, 2013; Anderson, 2019). In neuronal
histology, there are changes in mitochondria optic
tectum following stress exposure in Zebrafish. In
stress, neuron situation restricts where it can be
applied with the neuroprotectant use. Coffee is one of
the choices for natural neuroprotectant because coffee
has caffeine content. The function of caffeine is to
reduce the apoptosis in the neuron cells. Research has
revealed that coffee contains caffeine used to reduce
neuron apoptosis with hampering calcium release and
decrease Reactive Oxygen Species (ROS)
(Camandola, 2018).
A thing differentiating this research from another
research is this research focused on zebrafish
memory. This research aims to identify caffeine’s
effect on Unconditional Chronic Stress (UCS) in
zebrafish memory using T-maze. Furthermore, the
objective of this research is also to gain a preventive
against neuron restriction significantly. How early
life stress can affect the development memory.
2 MATERIAL AND METHODS
2.1 Animals and Housing
Juvenile Zebrafish with the age of 30 days (wild type-
both sexes) was obtained from Institut Pertanian
Bogor (IPB) and was sent to the animal laboratory
(pharmacology department–Universitas Islam
Indonesia). The aquarium maintained in water quality
was based on the instructed protocol. Fish were fed
two times a day with micro fish food. The fish were
kept in a 1L tank with 40 x 20 x 10 cm
3
. The water
quality of fish was maintained in 28
o
C, pH 7.4 and
with water mineral below 800. Biological time for
fish was preserved at 7.00 AM 7.00 PM, and the
light was dark. The experimental procedure was
approved by ethical clearance in Universitas Gadjah
Mada.
2.2 Caffeine Exposure
The caffeine group was exposed by 20mg/dL caffein
in group 1(P1), and 50mg/dL caffein in group 2 (P2).
A group with exposure 20 mg/dL and 50 mg/dL
caffeine extract administered the protocol toxicity in
a range of 96 hours. The fish were initially held in a
tank with two fish. After 96 hours exposure of
caffeine group, P1 dan P2 was given a UCS. We were
trying to modify the method from several studies as
by (Kim, 2017). The other group was not assigned
caffeine, the negative control group (K1) which is
only tested with T-maze trial. Then, a positive control
group (K2) was only given Unconditional Chronic
Stress (UCS) for seven without caffein.
2.3 Stress Exposure
Stress exposure was given to the K2, P1 and P2 group
for seven days. UCS was given to the fish which were
different in a day. There were restrain stress which the
fish are in centrifuge tube 4ml in 90 minutes. Predator
exposure, this method was administered by putting
the fish beside the predator with a divider. Low water
level housing tank, this method decreased water until
The Effect of Caffeine towards Zebrafish (Danio rerio) Juvenile Working Memory Exposed by Unpredictable Chronic Stress (UCS)
209
dorsal fin of the fish in two minutes. Chasing animals
were applied for eight minutes using chaser.
Moreover, the tank water replacement method was
performed in changing zebrafish tank with new water
three times when the zebra must adapt in freshwater.
UCS exposure is the same in every group to prevent
the different outcome of stress.
2.4 T-Maze
The T-maze test was applied three times in 96 hours
where training was modified by the study (Hieu,
2020; Kim, 2017) research by (Hieu, 2020). Fishes
were individually transferred to T-maze and observed
in one minute. Then, every group was noted for time
and chosen arm colour in T-maze as statistical
analysis. There were two red and green arms. When
fish got into the red component, caught by a net for
30 seconds got punishment. However, in the green
arm, the Zebrafish got a reward of fish food.
Furthermore, isolated in a green tank, the fish
remembered the colour. The training which was
provided for the fish aims to increase neuroplasticity
memory.
2.5 Statistical Analysis
Data were evaluated using IBM SPSS 24 in searching
for homogeneity, normality. The data normality using
Shapiro-Wilk analysis (p=0,00) could not be used; the
data were not normal. Meanwhile, using Kruskal-
Wallis analysis (p=0,00), the hypothesis was
accepted. With this average of time, there were
significant differences in every group.
3 RESULTS
3.1 The Difference with an Average
Time
Table 1: difference with average time according to reaching
the target arm using T-maze
no. Groups Time average SD p
1
a
K1 27,11 21,88 0, 000
2
b
K2 41,01 19,61
3
c
P1 25,62 18,76
4
d
P2 49,22 15,24
Notes
a
K1: Negative control group
b
K2: Positive control group
c
P1: Caffeine Exposure 20mg/dL
d
P2 Caffeine Exposure 50mg/dL
Table 1 shows the average time with the slowest
time to reach the T-maze arm, which is P2.
Meanwhile, the average time of the fastest one is P1.
As for time average of training from the beginning
until last, the average of time is K1 (27,11±21,88
second, K2 (41,01±19,61s), P1 (25,62±18,76s), and
P2 (49,22 ±15,24 s). The fastest to slowest in reaching
the T-maze arm are P1, K1, K2, and P2.
Table 2: Kruskal-Wallis group according to time
Relation between groups p
a
K1 K2 0,001
P1 0,855
d
P2 0,000
b
K2 P1 0,001
P2 0,063
c
P1 P2 0,000
Notes
a
K1: Negative control group
b
K2: Positive control group
c
P1: Caffeine Exposure 20mg/dL
d
P2: Caffeine Exposure 50mg/dL
The values of average time are presented in table
2. The post-doc Mann-Whitney test showed
significance within the group in K1 with K2 (p=0,00)
p<0,005. Group K1 with P1 is not statistically
significant (p=0,85). Group K1 with P2 has statistical
significance (p=0,00). Meanwhile, K2 with P2 was
not statistically significant (p=0,06). Group P1 with
K2 is of statistical importance (p=0,00). P1 with P2
shows statistical significance (p=0.00). The
significance result shows in the group are P1 with P2
and K1 with P2. Mann-Whitney Test indicated group
with exposure of 50mg/dl caffeine which possesses
an effect higher time average across the group.
However, the group with 20mg/dL is the best time
average compared to another group. Therefore,
caffeine dose impacts UCS.
3.2 Colour Difference
Table 3. T-maze arm colour is chosen difference in every
group using Pearson Chi-Square
Arm Chosen
K1
a
K2
b
P1
c
P2
d
Total p
Colour Not choosing 9 18 5 25 57 0,000
Green 28 16 30 12 86
Red 8 11 10 8 37
Total 45 45 45 45 180
a
K1: Negative control group
b
K2: Positive control group
c
P1: Caffeine Exposure 20mg/dL
d
P2: Caffeine Exposure 50mg/dL
JIMC 2020 - 1’s t Jenderal Soedirman International Medical Conference (JIMC) in conjunction with the Annual Scientific Meeting
(Temilnas) Consortium of Biomedical Science Indonesia (KIBI )
210
The analysis of colour difference across the group
using the chi-square method with two variables was
nominal categorical. The values of colour difference
presented in table 3 show K1 group 9 Zebrafish
preferred not to choose red arm colour; 28 Zebrafish
decided green arm colour and 8 Zebrafish chose a red
arm colour. K2 group 18 Zebrafish picked not to
choose arm; 16 Zebrafish chose green arm colour, and
11 Zebrafish preferred to select a red arm colour. P1
group 5 Zebrafish liked not to choose arm; 30
Zebrafish chose green arm colour, and 10 Zebrafish
chose a red arm colour. However, the P2 group 25
Zebrafish decided not to select arm; 12 Zebrafish
chose green arm colour, and 8 Zebrafish chose a red
arm colour. The comprehensive training for T-maze
is three.
Table 3 presents a group for P2 as the highest for
not choosing any arm. The tallest red arm colour is
the K2 group, although the highest green arm colour
is the P1 group compared to the other groups. The
analysis result shows a significant difference between
the groups in choosing the arm colour (p=0,00).
Clinical analysis indicated for memory in Zebrafish is
based on the colour selected for each group. Zebrafish
which chose green colour received a reward.
Otherwise, Zebrafish which chose red colour received
a punishment of stress exposure.
4 DISCUSSION
The effect of caffeine on zebrafish behaviour has
been extensively studied using toxicity. Stress and
caffeine which have been applied to the fish, have a
variation on motoric and memory. There is a
difference in the behaviour given with high dose
(50mg/dL) and low dose caffeine (20mg/dL). This
study shows the use of low dose caffeine which can
control stress and increase the memory. Based on
Zebrafish’s research (Adlioglu, 2012; Ruiz-Oliveira,
2019), the low dose of caffeine (5-140 mg/dL) may
increase memory motility cognitive.
Furthermore, a study in mice using 20mg/dL
caffeine dose reveals an optimal result in the forced
swim test. Therefore, in this study, we used 20mg/dl
dose to Zebrafish to reach the optimal memory. The
increased memory performance by caffeine is related
to its effect in increasing Acetylcholine (Ach) which
can inhibit the oxidation in a neuron. Ach affects in
expanding the work of GABA-Benzodiazepine,
where GABA works to inhibit glutamate.
Furthermore, caffeine occurs 1-Methyl-4-Phenyl-
1,2,3,6-Tetrahydro-Pyridine (MPTP) functioning as a
glutamate inhibitor.
Otherwise, the fish which was not given with
caffein reduced movement, memory and aggressive
cognitive. It was also followed by UCS cognitive
change in Zebrafish. Chronic stress affects restriction
in the pyramidal neuron. Restriction in pyramidal
neuron reduces regulation on top-down control in
working memory dominated in emotion. When
Zebrafish were exposed by UCS (K2) and had been
tested, it did not improve for choosing T-maze arm
(Arnstern, 2015; Goldwater, 2009; Mizoguchi, 2003).
Anatomically, PFC in Zebrafish is related to pallium.
It controls the cognitive in the Zebrafish, and sub-
pallium works as memory similar to the amygdala
and hippocampus in humans.
Stress activates HPA-Axis stimulated by
catecholamine and corticosteroid in the amygdala. In
this situation, stress inhibits frontal cortex in which
PFC functions as cognitive and working memory.
Based on this study, the group exposed with UCS
(K2) reduced in movement and was inhibited in
choosing arm colour, in which time to reach the T-
maze arm was also slower presented in table 1 and
table 3. However, a group which was not exposed to
stress exposure (K1) was faster in choosing a T-maze
arm. This study has shown that memory and decision
depend on stress stimulation from catecholamine and
glutamate. Meanwhile, it affects the working memory
in Zebrafish (Haight, 2011).
Caffeine is a substance that reduces neuron
restriction in the enhancement of memory and
decision. Low dose caffeine is a more effective
substance which is shown in table 3 with the T-maze
test. This study indicates a similar result with study in
(Adiloglu, 2012; Ruiz-Oliveira, 2019). In Ruiz-
Oliveira et al. (2019), Zebrafish with low dose
reached arm target faster than the higher amount. The
P1 group has a similar average time in choosing a T-
maze arm compare to K1. Low caffeine substance
proved that stress exposure did not affect Zebrafish,
which were given caffeine in low dose. The
consumption of low caffeine dose decreases in
anxiety by inhibiting the work of A2a adenosine
receptors. Otherwise, the P2 group had a disruption
in memory. This response induced zebrafish freezing
and continued exploring the tank without choosing a
T-maze arm (table 3). It can be interpreted that a high
dose of caffeine may escalate anxiety and stress.
Based on the study (Ruiz-Oliveira, 2019; De
Carvalho, 2019) in Zebrafish, the psychoactive and
dependent fish were given 50mg/dL caffeine dose.
Based on the FDA, the consumption on caffeine
400mg/dL disrupts adults’ health.
The training was conducted to increase the
memory in neuroplasticity and a new memory.
The Effect of Caffeine towards Zebrafish (Danio rerio) Juvenile Working Memory Exposed by Unpredictable Chronic Stress (UCS)
211
However, the more Zebrafish are trained, the more
neuroplasticity makes recent memory. In this case,
pyramidal neurons function as working memory and
higher cognitive function (Arnstern, 2019).
Cognitive and memory may decrease when the
neuron’s restriction of repeated stress and a higher
dose of caffeine cause anxiety. The limitation in the
neuron is caused by glutamate exposure in the apical
neuron. It affects restriction dominated in the apical
neurons. The layer in the apical neurons becomes
dominant due to Excitatory Post Synaptic Currents
(EPSP). Neuron Restriction is caused by how long
neurotransmitter exposure in glutamate stands
towards the other neuron. It is called Long Term
Potentiation (LTP). LTP affects Spike timing-
dependent (SDPT) by glutamate exposure and
increases in calcium for cell apoptosis. The longer the
LTP is, the more neuron restriction happens.
Therefore, the relation between time and choice of T-
maze arm is related to the exposure of UCS and
caffein exposure presented in table 2 and table 1.
Stress situation, the neurotransmitter focuses on the
bottom-down in the amygdala in which pyramidal
neuron restricts. Meanwhile, K1 and P2 group have
better working memory.
5 CONCLUSION
Caffeine affects memory and stress. Caffeine may
reduce stress at a low dose. Moreover, caffeine affects
the memory in Zebrafish.
ACKNOWLEDGEMENTS
We are indebted for our supervisor dr. Zainuri Sabta
Nugraha M, Sc. Department of anatomy faculty of
medicine Universitas Islam Indonesia has taught us a
wide range of neuroscience in Zebrafish.
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