The Effect of Caffeine towards Zebrafish (Danio rerio) Juvenile

Working Memory Exposed by Unpredictable Chronic Stress (UCS)

Alya Ayu Tazkia

, and Zainuri Sabta Nugraha

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).

https://orcid.org/0000-0002-0453-3408

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

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

. The water

quality of fish was maintained in 28

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

K1 27,11 21,88 0, 000

K2 41,01 19,61

P1 25,62 18,76

P2 49,22 15,24

Notes

K1: Negative control group

K2: Positive control group

P1: Caffeine Exposure 20mg/dL

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

K1 K2 0,001

P1 0,855

P2 0,000

K2 P1 0,001

P2 0,063

P1 P2 0,000

Notes

K1: Negative control group

K2: Positive control group

P1: Caffeine Exposure 20mg/dL

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

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

K1: Negative control group

K2: Positive control group

P1: Caffeine Exposure 20mg/dL

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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