American University of Beirut

Faculty of Medicine
Department of Anatomy, Cell biology and Physiological
Sciences
Masters in Biomedical Sciences – Neuroscience program

Course: HUMR 310 Module C

Assignment: Zebrafish as an emerging model for

neuroscience research

Prepared by: Sara Fares and Habib Rammal

Term: Fall 2023 - 2024
Question:
You are interested in conducting research on Angelman Syndrome (AS). You read the article
“Circadian Rhythms and Sleep Are Dependent Upon Expression Levels of Key Ubiquitin Ligase
Ube3a” and would like to verify the results in zebrafish, an animal model that you have in your
lab.
1. How would you create a similar Ube3a-deficient AS model in zebrafish?

Morpholinos is an antisense oligonucleotide that is used to inhibit the expression of the Ube3a as
in a model in creating a Ube3a-deficient Angelman Syndrome (AS) model in zebrafish.
Binding to specific RNA sequences, knocking down gene expression and blocking translation or
splicing are all functions that morpholinos is responsible for as a synthetic molecule.
How do morpholinos play a role in creating a Ube3a-deficient zebrafish model, here are the
steps:

Designing Morpholinos:
1. Identify Target Sequence: Using a sequence that is involved in translation or splicing in a
form that it identifies specific target sequences within the zebrafish Ube3a gene that we're
targeting to knock down.
2. Morpholinos Design: Design antisense morpholinos that are complementary to the identified
target sequences in zebrafish Ube3a mRNA. Ube3a exon 6 deletion in the Ud6-m-/p+ mouse
model can be used as a design that reflects genetic alterations.

Preparation and Injection of Morpholinos:

1. Prepare Morpholinos: Order or synthesize the designed morpholinos for Ube3a knockdown
in zebrafish, To enable comparison, it is crucial to include a control morpholino.
Prepare Zebrafish Embryos: Obtain fertilized zebrafish eggs and maintain them under standard
conditions.

2. Microinjection: Inject the Ube3a-targeting morpholino and the control morpholino into
zebrafish embryos at the one-cell stage using the microinjection technique. Take into account the
appropriate dosage and injection volume. Fine-tune the experimental conditions to ensure
effective knockdown of the target gene.

Phenotype Analysis Validation:

1. Knockdown Confirmation: After the injected embryo, RNA is extracted to validate the
knockdown efficiency followed by performing RT-qPCR to measure Ube3a mRNA levels.
To confirm the reduction in Ube3a protein levels, western blotting is done to perform protein
analysis.

2. Phenotypic Analysis: Examine the morpholino-injected embryos for any alterations in their
physical characteristics, with particular attention to behavioral factors associated with circadian
rhythms and sleep patterns. Take into account tests that imitate the traits seen in Ube3a-deficient
mouse models, such as locomotor activity, sleep-wake cycles, and reactions to sleep deprivation.
Further characterization as long-term observation, comparison with mouse models, and adjusting
experimental design can act as a valuable addition to the models when examining the
involvement of Ube3a in circadian rhythms and sleep patterns linked to Angelman Syndrome.

2. Propose two different behavioral tests using your AS zebrafish model; describe the
experimental procedure of each test and give the expected results with the hypothetical
figures (curve or histogram...) that you could get.
1. T-maze test for assessing memory
Norepinephrine and corticosteroid expression have been shown to be reduced in AS models (Shi
et al., 2022). Knowing that norepinephrine plays an important role in arousal and attentiveness, a
downregulation of these hormones should correlate with an impairment in memory and cognitive
functions. Therefore behavioral tests that involve emotionally stressful tasks can be used to
assess memory and cognitive deficits in Ube3a-deficient AS zebrafish.
A T-maze test can be conducted, where the zebrafish is placed in a T shaped tank, with
removable colored sleeves on each extremity, and a removable door to create a start box.
The both wild type control and Ube3a-deficient zebrafish should be placed in the tank for 3-10
minutes and allowed to acclimate, and consistent water temperature, water quality and lighting
should be maintained.
After the zebrafish is introduced into the start box, the door is removed and the fish is allowed to
explore the tank and chose an arm. The data can be analyzed using a program (ex Ethovision
XT) that allows the measurement of the different parameters, including the latency, velocity, and
time spent in each arm.

https://conductscience.com/maze/portfolio/zebrafish-t-maze/
To assess memory, the right arm is labelled in green and the left arm in red. Food is placed at the
green arm. The zebrafish is rewarded with food every time it enters the green arm, and the
experiment is repeated until the fish consistently choses the right arm. After the reward is
temporarily removed, the response of the fish is recorded.
The experiment is repeated after 24 hours, and the fish’s spatial memory is recorded again.
In order to further test the fish’s learning ability, the reward is switched from the green to the red
arm, and the latency period (the period until the fish adapts to the new condition) is assessed.
Finally, the spatial memory performance results are compared between the wild-type and the
Ube3a-deficient zebrafish.
The Ube3a-deficient model should exhibit a smaller percentage of choice accuracy after 24
hours, and a longer learning latency period, where a longer adaptation time is needed after the
reversal of the reward side.

Choice Accuracy of Ube3a-deficient vs

Wild-Type Zebrafish
100%
90%
% Choice Accuracy

80%
70%
60%
50%
40%
30%
20%
10%
0%

Ube3a-deficient WT

Results show Ube3a-deficient AS zebrafish exhibit a decline in choice accuracy that Wild-Type
zebrafish, suggesting impaired special memory in Angelman syndrome.

Difference in Latency period (seconds) between

Ube3a-Deficient and Wild Type Zebrafish
400
300
200
100
Latency Period (s)

0
s) s) s)
y( y( y(
enc enc e nc
t t at
La La d
l
g ng
s in hi erio
e
Te itc n
p
lin sw tio
e d za
as ar ili
B w b
Re st
a

Ube3a-deficient WT

- Baseline latency: when reward location is consistent

 - Reward switching latency: when reward location is switched

- Stabilization period latency: time needed for each group to consistently chose the right
arm

Results show Ube3a-deficient zebrafish require a significantly longer time to adjust, and a longer
learning time compared to the wild type control, suggesting impaired adaptive behavior in
Angelman Syndrome.
2. Shoaling test for assessing behavior
Ube3a-deficient mouse models exhibit reduced anxiety and compulsiveness compared to wild-
type models, shown by a reduced marble-burying behavior (Shi et al., 2022). To verify these
results in zebrafish, and to test anxiety levels in Angelman Syndrome zebrafish models, a
shoaling test can be performed, where reduced shoaling patterns can be attributed to a decrease
in anxiety levels.
Usually, zebrafish aggregate together to form group, or “shoals”.
In this test, zebrafish are divided into a control group (wild type) and a Ube3a-deficient group.
The behavior of the fish can be assessed by studying their spatial behavior and movement
patterns. This can be attributed to anxiety levels, where higher anxiety levels are attributed with a
smaller inter-fish distance, tighter shoal cohesion and a longer shoaling time.
After ensuring consistent conditions for both groups, including consistent lighting and water
temperature, and after allowing the zebrafish to acclimate in the slanted tank for 3-10 minutes,
the zebrafish behavior is recorded. First, the distance between the fish is recorded, as well as the
time spent in close proximity to each other.

Ube3a-deficient zebrafish should exhibit reduced anxiety levels, hence a shorter shoaling time,
and a loose shoal cohesion (a larger distance between shoal mates) compared to their wild-type
counterparts.
 60

Average distance between shoal mates 50

40
(cm)

30

20

10

Ube3a-deficient WT

Results show a larger average distance between shoal mates of Ube3a-deficient zebrafish
compared to wild type zebrafish, suggesting reduced anxiety in Angelman Syndrome.

https://www.researchgate.net/publication/313408982_Social_Phenotypes_in_Zebrafish