Running Header: EFFECTS OF VITAMIN D3 ON THE -AMYLOID CONCENTRATION OF BRAINS

The Effects of Vitamin D3 on the Protein Concentration of Sleep-Deprived Danio rerio Brains
and Its Implications Regarding Aggregation-Associated Neurodegenerative Diseases
Kar Shin Lin, Mateo Quiroga, Prit Shah, and Isaiah Swann
15 December 2014

EFFECTS OF VITAMIN D3 ON THE -AMYLOID CONCENTRATION OF BRAINS

Abstract:
This experiment aimed toward
treating sleep-deprived Danio rerio, with
Vitamin D3, and investigating whether it
would decrease the -amyloid concentration
within the brain. From there, implications
for treating aggregation-associated
neurodegenerative diseases were made. The
hypothesis that Vitamin D3 will decrease
levels of -amyloid despite being sleepdeprived was tested by increasing the
amount of light exposure in a day while also
providing a daily dosage of Vitamin D3. The
fish were separated into four tanks; two
tanks were put in a group. Each group had a
different sleep cycle; one tank in each group
was given Vitamin D3. The Danio rerio

the hypothesis. Lower concentrations of the

protein were noticed in fish that presided in
tanks that received the Vitamin D3
treatment.
Introduction:
Sleep is a multifaceted biological
state in which the nervous system is
inactive: the eyes are closed, a majority of
the muscles are relaxed, and consciousness
is altered. Despite certain misconceptions
that may lead one to believe that sleep is a
passive cycle, sleep is an active cycle in
which many functions are occurring
simultaneously, such as energy conservation,
memory conservation, and -- most
importantly-- restoration and recovery of
damaged parts and systems.

brains were dissected and analyzed with

SDS-PAGE. Based on the results of the
protein gel electrophoresis, the molecular
weights of the protein bands revealed a
correlation between Vitamin D3 and the amyloid concentration that coincided with

When the brain is active, nerves are

vigorously and indefatigably processing
external and internal stimuli. Thousands of
synapses throughout the brain are
constructed between neurons as information
is transmitted and interpreted via proteins

EFFECTS OF VITAMIN D3 ON THE -AMYLOID CONCENTRATION OF BRAINS

and neurotransmitters. As the day

The recurring mechanism of sleep is

progresses, proteins agglomerate within the

described by the two-part model; the model

within the brains extracellular space. When

serves to show the interaction between the

there exists an excessive agglomeration of

two processes and the balance they serve to

proteins, tangles between neurons, called

maintain. The first component of the two-

plaques, form, which may lead to

part model is the circadian rhythm, which

aggregation-associated neurodegenerative

describes how the bodys circadian clock

diseases, such as Alzheimers Disease and

regulates its internal processes. This

Parkinsons Disease (Hamilton, 2013). The

mechanism is a natural time clock that

organism, however, is able to flush these

regulates body patterns such as sleeping,

proteins out of the extracellular space in the

eating, hormone production, and other

brain during its sleep cycle by releasing a

important biological processes, sometimes

clear liquid, called Cerebrospinal Fluid

through coordination with the light and dark

(CSF), that collects the proteins and carries

cycle. The second component is the sleep-

them out of the brain where they can be

wake homeostasis module; this biological

processed, digested, and recycled (Kang,

mechanism is credited with inducing sleep

2009). High amounts of energy are used up

throughout the day through the buildup of

in the process of restoration and recovery;

hypogenic substances. Once an organism is

therefore this process cannot be conducted

aroused, this component will start building

while the organism is fully awake, which is

up substances that contribute to sleepiness;

why a lack of sleep is so detrimental to the

this component, however, works in

health of an organism.

cooperation with the circadian rhythm. This

cooperation takes place when the circadian

EFFECTS OF VITAMIN D3 ON THE -AMYLOID CONCENTRATION OF BRAINS

rhythm regulates the sleep-wake

procures affects the efficiency of that

homeostasis component. Once the body has

organisms glymphatic system, which is a

built up enough hypogenic substances and

sleep-driven system responsible for

the circadian cycle dictates that it is time to

cleansing the brain of excess proteins.

sleep, it will commence in lowering

(Thomas, 2013). Danio rerio have become

alertness levels and begin hypogenic

an increasingly key model organism for

hormone production. These actions, in turn,

sleep studies due to similarities between its

decrease the activity levels of neurons. After

nervous system and circadian rhythm and

a certain threshold has been reached, the

the human nervous system and circadian

organism will fall asleep. Once the organism

rhythm (Singh, 2013).

has fallen asleep, restoration of the body

commences. While Rapid Eye Movement
Sleep (REM Sleep) is in progress, the body
is restoring the brain through the
neutralization of neurotoxins, which is
usually done through the release of
cleansing liquids that rinse the brain of
impurities.
Sleep deprivation is a practice that is

Vitamin D3 plays a vital role in an

organism, due to its diverse supportive
characteristics and properties. The pseudosteroid vitamin is rich in its capacities, being
able to enhance the absorption of chemical
elements, regulate cell growth, improve
immune function, and reduce inflammation
(Vitamin D, 2014). Perhaps one of the
most important characteristics of the

crucial to augmenting the number of

Vitamin D3 family is its startling anti-

proteins present in the brain (Brain Basics,

oxidative ability. More specifically, Vitamin

2014). It is believed that a reduction in the

D3 has been linked with the beneficial

amount of sleep that a mammalian organism

ability to improve cognition in organisms. In

EFFECTS OF VITAMIN D3 ON THE -AMYLOID CONCENTRATION OF BRAINS

a recent research study, deceased

which causes cell death, and consequently,

Alzheimers patients who underwent an

tissue degeneration (Alzheimers

autopsy were found to have lower Vitamin

Association, 2011). Vitamin D3 has been

D3 levels present in the brain, while

linked with the ability to digest -amyloid

deceased patients who were not diagnosed

plaques and improve the brains capacity in

with Alzheimers, or any similar

clearing these toxins in the restoration cycle

neurodegenerative disease, experienced

of sleep (Champeau, 2013). It was

normal Vitamin D3 levels. This difference

hypothesized that if a model organism, such

led researchers to make the connection that

as the Danio rerio, is administered Vitamin

Vitamin D3 improves cognition and its anti-

D3, then the overall concentration of -

oxidative properties are beneficial in treating

amyloid would decrease, despite

neurodegenerative diseases (Kulie, 2009).

manipulating the sleep cycle of the fish. The

Without Vitamin D3 treatments, it would

purpose of this lab was to analyze the

have been expected for the fish to

overall protein concentration in Danio rerio

experience protein plaque agglomerations

after being administered Vitamin D3 for

since it was not undergoing its brain

three days during a five-day period. It was

restoration and reparation cycle.

expected that the Danio rerio would show

Aggregation-associated
neurodegenerative diseases are characterized
by an assemblage of tangled proteins, which
form plaques, in the extracellular space
between neurons. This accumulation
obstructs the neuronal uptake of nutrients,

less protein concentration after the drug

administration. The model organism, Danio
rerio, was chosen based on its ability to
grow faster and display the effects of
administered drugs more quickly than any
other organism.

EFFECTS OF VITAMIN D3 ON THE -AMYLOID CONCENTRATION OF BRAINS

Methods:
This experiment required the
separate treatment of Danio rerio with
varying sleep cycles in addition to Vitamin
D3. Four groups of Danio rerio were kept in
separate 5.5-gallon fish tanks (Tanks 1-4).
These tanks were cleaned with a sponge and
rinsed with tap water. Moreover, three
quarters of each tank were filled with tap
water. In order to dechlorinate the water and
add beneficial bacteria, twelve drops of
API Tap Water Conditioner and five mL of

filter with the appropriate filter cartridge.

Each fish feeder was filled with TetraColor
Plus tropical flakes and set at the second
smallest dosage. Thirty-four Danio rerio
were purchased from Petsmart and the bag
was placed in one of the four tanks to
acclimate the fish to the temperature of the
water in the tanks. After the fish were
acclimated in the tank for fifteen minutes,
the fish in the bag were separated into two
tanks which utilized the 10i water filters.
The fish were allowed to acclimate

Seachem Stability Water Conditioner were

into their new environments for five days, at

added to each tank. Tanks 1 and 2 each

which time all the fish were transferred Tank

included a Marineland aquarium heater set

3. Tanks 1 and 2 were drained of water, and

at 27C, a Tetra Whisper 10i (up to ten

cleaned with a sponge. Each component of

gallons) water filter with a filter cartridge

the tanks was cleaned with tap water (heater,

and a bio scrubber, a Top Fin automatic

filter, filter cartridge, etc.) was rinsed with

fish feeder, and a Top Fin air pump. Tanks

tap water. Afterwards, three quarters of the

3 and 4 each included the same water heater,

Tanks 1 and 2 were filled with tap water and

fish feeder, and air pump (with a Top Fin

treated as previously stated. White aquarium

aquarium air stone), but utilized a Tetra

gravel was added to cover the bottoms, and

Whisper 3i (up to three gallons) water

fake plants were placed in each tank. Each

EFFECTS OF VITAMIN D3 ON THE -AMYLOID CONCENTRATION OF BRAINS

tanks pH was measured with pH testing

The water heater in Tank 3 was replaced.

strips to ensure that it was between six and

Five hours later, eight fish were placed into

seven. To record temperature, floating glass

Tanks 1 and 3; nine fish were placed into

thermometers were placed inside each tank.

Tanks 2 and 4. Tanks 1 and 2 were placed

Because the newly added tap water was not

on a cart and kept in a completely dark

at the ideal temperature for the Danio rerio,

closet; Tanks 3 and 4 were placed in dark

the fish were divided between the Tanks 3

cabinets inside a classroom. All the tanks

and 4 (with 3i filters) and placed on a

had a sixty-watt incandescent light clamp

fourteen hour lightten hour dark sleep

attached to the side and placed on a

cycle using a sixty-watt incandescent light

fourteen-hour lightten-hour dark sleep

clamp. Only one light clamp was used,

cycle. The sleep cycle during this

positioned between the tanks.

experiment was arranged so that every 24-

hour period would reset at 2:00 pm. The

final individual tank set can be seen in
Picture 1, with all the components of the
tank present and functioning.
Treatment of the Danio rerio with
Vitamin D3 was started forty-eight hours
Picture 1. Tank Setup. Consisting of an air source, a
fish feeder, a water heater, a water filter, a plastic fish
plant, a light source, and white gravel rocks.

later. Tanks 1 and 4 were treated, whereas

Tanks 2 and 3 would serve as controls. The
filter for Tank 4 was turned off at 1:30 pm,

Eighteen hours later, due to a

malfunctioning water heater in Tank 3, all
the fish in that tank were moved to Tank 1.

and the filter for Tank 1 was turned off at

1:50 pm. Tank 4 was treated at 2:00 pm, and

EFFECTS OF VITAMIN D3 ON THE -AMYLOID CONCENTRATION OF BRAINS

Tank 1 was treated at 2:20: using a

twenty-one hour lightthree-hour dark. The

micropipette, one mL of liquid Vitamin D3

sleep cycles for Tanks 3 and 4 were not

(from the Vitamin Shoppe, item number:

changed throughout the course of the

VS-2714/1725688) was injected below the

experiment. No treatment occurred for forty-

surface of the water. The filters were turned

eight hours after the second treatment. After

back on thirty minutes after their respective

this time period, there was a fluctuation in

treatment times. After the first treatment, the

the water levels of the tanks, and therefore

sleep cycles for Tanks 1 and 2 were changed

the tanks were reverted back to their

to eighteen-hour lightsix-hour dark.

adequate water levels. The filters of Tanks 1

Therefore, treatment was performed during

and 4 were cleaned, and the filters of Tanks

the light-portion of the sleep cycle. This

2 and 3 were replaced. The final treatment

ensured that the treatment had minimal

was given after the water levels were

impact on the sleep cycle of the fish. The

restored.

next day, the air pump in Tank 1 was

malfunctioning and therefore replaced with
a Petco air pump. The day after the first
treatment, Tanks 1 and 4 were treated again
with the same procedure. Tank 4s filter was
turned off at 2:00 pm, and Tank 1s filter
was turned off at 2:10 pm. Tank 4 was
treated at 2:30 pm, and Tank 1 was treated at
2:40 pm. After this treatment, the sleep
cycles for Tanks 1 and 2 were changed to

Twenty-four hours after the final

treatment, the fish in Tank 1 were
transferred to a 500 mL beaker filled with
100 mL of tap water at 2:15 pm. At 2:50 pm,
the fish in this tank were euthanized; the
beaker was placed into a Tupperware
container with four cups of ice and water.
The fish were kept in the ice apparatus until
3:30, when all the fish were confirmed dead.
Confirmation of death was judged by a lack

EFFECTS OF VITAMIN D3 ON THE -AMYLOID CONCENTRATION OF BRAINS

of any movement upon contact with a probe.

pm. Two fish from each Tank were chosen

At this time, the fish were sealed in a plastic

to be dissected. All tools were sterilized with

bag and placed in the refrigerator. Forty-

ethanol and a flame prior to dissection. A

eight hours after the final treatment, the fish

basic light microscope and wax tray covered

in Tank 2, 3, and 4 were transferred to

with plastic wrap was used. First, the head

different 500 mL beakers filled with 100 mL

of the Danio rerio was removed with a

of tap water. The fish in Tank 2 were placed

scalpel. As shown in Picture 2, the head was

inside the ice apparatus at 2:20 pm and

positioned supine on the wax tray.

confirmed dead at 3:10 pm. The fish in Tank

3 were placed inside the ice apparatus at
3:05 pm and confirmed dead at 3:30 pm.
The fish in Tank 4 were placed inside the ice
apparatus at 2:35 pm and confirmed dead at
3:05 pm. All the fish in each tank were
placed in separate plastic bags, labeled, and
placed inside the refrigerator (10C) for

Picture 2. Dissection. The fish is situated in the

supine position for brain sample collection.

twenty-four hours before they were

The gills were pinned to the tray. The soft

dissected.

tissue was removed from the head with

The dissection procedure of the

Danio rerio was adapted from Tripti Gupta
and Mary Mullins Dissection of Organs
from Adult Zebrafish (Gupta, Mullins 2010).
The dissection process was started at 2:00

probes. The eyes were also removed without

puncturing them. Once the skull was
isolated, caution was taken in removing the
bone. The brain was located on the bottom
side of the skull (closest to the wax tray).
After the skull was removed, the soft tissue

EFFECTS OF VITAMIN D3 ON THE -AMYLOID CONCENTRATION OF BRAINS

10

surrounding the brain was removed with

temperature for five minutes. The tubes

probes and isolated as shown in Picture 3.

were then heated for five minutes at 95C in

Each brain was placed inside a two mL

a heat block. During this time, the PAGE

micro-centrifuge tube, which was labeled

apparatus was prepared, and a running

with the tank number and fish (A or B). All

buffer solution of 1x Tris-glycine-SDS

the brains were dissected in a ninety-minute

(TGS) was prepared by mixing 100 mL of

period and stored in the fridge at 3:30 pm.

10x Tris-glycine-SDS buffer with 900 mL of

deionized water. The Ready Gel precast gel
was placed inside the module and filled
appropriately with the running buffer.
Because only one gel was used for this
experiment, a blank gel acted as a buffer
dam. The protein samples were taken from

Picture 3. Brain Sample. The matter shown above is

the brain sample extracted from a Danio rerio fish.

the heat block, and ten L of each sample

was loaded into the gel using protein

A day after the brains were dissected,

the Danio rerio brain samples were prepared
for sodium dodecyl sulfate- polyacrylamide
gel electrophoresis (SDS-PAGE). 250 L of
Laemmli buffer was added to the
microcentrifuge tubes containing each brain
sample. Each tube was then flicked several
times to agitate the brain tissue. Next, the
samples were allowed to incubate at room

micropipette tips. Due to a shortage of

protein micropipette tips, brain samples 3B
and 4B were loaded with regular
micropipette tips. In the ten-well gel, Lanes
1 and 10 were left blank. Lanes 2-5 held fish
1A-4A and Lanes 6-9 held fish 1B-4B. Gel
electrophoresis was started at 3:25 pm at
200 V and stopped at 3:40 pm.

EFFECTS OF VITAMIN D3 ON THE -AMYLOID CONCENTRATION OF BRAINS

The gel was taken out of the PAGE

11

When the Danio rerio were

apparatus and placed on a flat surface with

transferred to a beaker for euthanization, the

the short plate facing up. The gel plates were

fish were noticeably more red than while in

pried apart, and the gel was transferred to a

the tanks. During the dissection of the

stain-free gel-imaging tray. During gel

Danio rerio brains, the mass of the brain

electrophoresis, a standard and molecular

was miniscule compared to the mass of the

weight ruler were not available; therefore,

entire fish head. The brain was a white mass

these lanes were approximated using an

at the forefront of the head. The eyes of

online source (Gel Electrophoresis of

some of the fish during dissection were

Proteins, n.d.). The molecular weights of

punctured, which resulted in a black liquid

the bands were analyzed in relation to the

being released into the head, making it more

approximated standard. A method of

difficult to navigate to the brain during

analysis was comparing the bands with

dissection. As the brains were extracted and

similar treatment (similar sleep cycles and

placed in the microcentrifuge tubes, the

presence of Vitamin D3). Relative molecular

brain mass was estimated as being less than

weights were analyzed to show any

one mm3 in volume. In addition, when the

similarities and differences of the bands

samples were loaded into the gels, samples

among the lanes. Implications were then

3B and 4B were not loaded properly due to

drawn from these similarities and

the use of regular 10 L micropipette tips.

differences to conclude the effectiveness of

The well did not hold the entire injected

Vitamin D3 on Danio rerio brains,

sample. After the gel was run at 200 V, the

specifically -amyloid.

dye front seemed to move faster than from

Results:

past experience.

EFFECTS OF VITAMIN D3 ON THE -AMYLOID CONCENTRATION OF BRAINS

Bands with similar positions among

12

difference between 1A and 4A was 12.8

the 8 lanes were compared through

KDa (the second largest difference of A

molecular weights. Lanes, which contained

tanks) and the difference between 1B and

samples on the same sleep cycle, were

4B was 1.44 KDa (largest difference of B

compared as well as samples given the same

tanks). The tanks with no treatment (Tanks 2

treatment (presence of Vitamin D3). Table 1

and 3) also had relatively large differences

shows the molecular weights of all the bands

in molecular weightthe difference

analyzed by the gel imager. The bands are

between 2A and 3A was 20.54 KDa (largest

organized by similar positions along the

of A tanks) and the difference between 2B

gels. The gel imager did not detect bands in

and 3B was 0.6 KDa (second largest of B

some positions in Lanes 4, 7, and 8. Table 2

tanks).

uses the data found in Table 1 and calculates

the differences in the molecular weights of
the tanks with similar conditions and
treatment. In addition, Table 2 includes an
average distance of molecular weights
between similar bands. This table shows that
Tanks 1 and 2 had the most similar bands.
Tanks 3 and 4 also had similar molecular
weights, although less similar than that of
Tanks 1 and 2. The tanks with similar
treatment (Tanks 1 and 4) had a relatively
large difference in molecular weightsthe

The bands of tanks 1 and 4 had the

most similar bands in terms of level of
darkness. Tank 3 is somewhat similar to
Tanks 1 and 4; however, Tank 2 is very
different in level of darkness in its bands
than that of Tanks 1, 3, and 4. Tanks with
similar treatment (1 and 4) do not have a
large difference in number of bands and
level of darkness1A and 4A are similar
(second largest difference in level of
darkness in bands of A tanks) while 1B and
4B are still somewhat similar (largest

EFFECTS OF VITAMIN D3 ON THE -AMYLOID CONCENTRATION OF BRAINS

13

difference in level of darkness in bands of

sleep cycles of: fourteen-hour lightten-

A tanks). Tanks with no treatment (2 and 3)

hour dark, eighteen-hour lightsix-hour

have a relatively large difference in level of

dark, and a twenty-one hour lightthree

darkness2A is higher in level of darkness

hour dark cycle. Because Tank 1 was treated

than 3A (largest difference in level of

with Vitamin D3 and Tank 2 was not, the

darkness in bands of A tanks) while 2B and

similarity suggests that the supplement did

3B are somewhat similar in level of

not impact the molecular weights of the -

darkness (second largest difference in level

amyloid in the Danio rerio brains.

of darkness in bands of B tanks; however,

these lanes (7 and 8) were not detected well
by the gel imager.
Discussion:
Tanks 1 and 2 were the most similar
in both trials (A and B). The average
difference between 1A and 2A was 11.08
KDa, and the average difference between 1B
and 2B was 0.325 KDa. This similarity was
most likely due to these tanks having the
same environmental conditions, in which all
facets of the environment aside from
treatment were controlled. Tanks 1 and 2
were given the same sleep cycle and were
placed in a storage closet. Both experienced

The molecular weights of Tanks 3

and 4 were not as similar as that of Tanks 1
and 2 despite having the same sleep cycles.
The average difference of molecular weights
between 3A and 4A was 16.03 KDa, and that
between 3B and 4B was 0.58 KDa.
Relatively, these differences are larger than
expected. The deviation from what was
expected resulted from the environment in
which Tanks 3 and 4 were placed. These
tanks were stored in cabinets inside of a
classroom. Light entering the cabinets could
not be completely blocked as well as other
disturbances such as noise. This differs from
the environment in which Tanks 1 and 2

EFFECTS OF VITAMIN D3 ON THE -AMYLOID CONCENTRATION OF BRAINS

were kept because the storage closet, in

which these tanks were placed, was isolated
from interference; therefore, no factors
affected the environment of the tanks other
than what was controlled. Inside of a
classroom, complete isolation could not be
guaranteed. In addition, Tanks 3 and 4 were
kept in different cabinets. The outside
pressures may have independently affected
one tank or the other. Differing
environmental conditions could strain the
fish differently, leading to different brain
development between the two tanks.
Furthermore, the difference may have been
caused by the treatment of Vitamin D3 in
Tank 4. This explanation supports the
hypothesis that Vitamin D3 does affect
protein levels within the brain. Additional
analysis of the protein concentrations from
the fish in Tanks 3 and 4 would show greater
protein concentration in Tank 3. The proven
effects of Vitamin D3 on the brain would
allow the fish in Tank 4 to reduce -amyloid

14

agglomeration.
The two tanks with the treatment of
Vitamin D3, Tanks 1 and 4, did not seem to
be similar in a significant manner. The
average difference of molecular weights
between 1A and 4A was 12.8 KDa, and the
average difference between 1B and 4B was
1.44 KDa. These values are relatively large,
which means there is a difference in the
protein concentrations of fish between the
two tanks. This difference leads to the
conclusion that the sleep cycle does
influence the amount of proteins in the
brains of Danio rerio. Tank 1 had a
manipulated sleep cycle of changing lightdark ratios, whereas Tank 4 had a natural
sleep cycle common to Danio rerio. Further
analysis of the protein concentration
between these two tanks would most likely
yield a larger protein concentration in the
fish in Tank 1 than that in Tank 4 because
the fish in Tank 1 were deprived of sleep.
Sleep deprivation meant that the brain could

EFFECTS OF VITAMIN D3 ON THE -AMYLOID CONCENTRATION OF BRAINS

15

not naturally drain itself of -amyloid

were on a fourteen-hour lightten-hour

agglomeration.

dark sleep cycle but only Tank 4 was treated

The summary of the controls

described in Table 3 suggest that each tank
produced very similar fish in terms of
protein concentrations. All the tanks had a
variation of less than 5 KDa, which means
that the two fish from each tank were very
similar. This similarity isolates any
differences seen in protein molecular
weights between tanks. The differences in
protein molecular weights between the fish
from separate tanks can be attributed more
reliably to the presence of Vitamin D3 or
variation in sleep cycle.

with Vitamin D3. The difference therefore

suggests that the presence of the Vitamin D3
had a certain degree of influence on the
brains of Danio rerio. Conversely, the lack
of difference in Tanks 1 and 2 do not support
this notion. Furthermore, when a difference
was observed between the protein molecular
weights from fish in Tanks 1 and 4, it was
concluded that there was a difference in
protein concentrations between fish that
were sleep deprived and those that were not.
Because both tanks received Vitamin D3
treatment, a difference in protein
concentrations can be attributed to varying

With the similarities and differences

between the protein molecular weights of
the fish in the tanks, the previous notion that
-amyloid agglomeration was reduced with
the treatment of Vitamin D3 was supported.
This is seen with the difference present in
the molecular weight of the proteins of the
fish between Tanks 3 and 4. These tanks

sleep cycles. -Amyloid agglomeration

would be lower in Tank 4 because the Danio
rerio in that tank were on a normal sleep
cycle. Therefore, the Danio rerios natural
mechanism to remove the amyloid plaques
during sleep worked more effectively in
Tank 4 rather than in Tank 1. Moreover, the

EFFECTS OF VITAMIN D3 ON THE -AMYLOID CONCENTRATION OF BRAINS

16

large difference seen between Tanks 2 and 3

therefore does not affect data in relation to

(an average difference of 20.54 KDa in Trial

each other. During the five-day acclimation

A and 0.6 in Trial B), show that even

period, the fish were placed inside a dark

without Vitamin D3 treatment, protein

classroom with little sunlight access. The

concentration and molecular weight differ.

sleep cycles of the fish were not strictly

This difference can be accounted for through

regulated. After the acclimation period,

the difference in sleep cycles: Tank 2 had a

when the fish were subject to a strict

manipulated sleep cycle whereas Tank 3 had

sleeping cycle, the transition could have

a natural sleep cycle.

produced a type of strain on the fish

Discrepancies in the results can be

accounted for throughout the experiment.
At the beginning of the experiment, the
Danio rerio were placed in two tanks. These
tanks were filled with tap water, which was
slightly colder than the ideal temperature for
such tropical fish (27C). In addition, after
the fish were placed in these two tanks,
within the five days they were allowed to
acclimate, a buildup of excess food and
waste collected in the filter. However,
because these factors occurred at the
beginning of the experiment, the affect is the
same across all the Danio rerio, and

resulting production of more or less protein.

Another type of strain that was observed
occurred the day the fish were divided into
four tanks. The water heater in Tank 3
malfunctioned, resulting in a water
temperature of 32C. On the day of the first
treatment, the air pump on Tank 1
malfunctioned. Enough oxygen may not
have been supplied. However, a new air
pump replaced the defective pump the next
day (the day of the second treatment).
Another error that was encountered was the
leaking of the filter in Tank 1 into Tank 2.
As shown in Picture 4, this error caused the

EFFECTS OF VITAMIN D3 ON THE -AMYLOID CONCENTRATION OF BRAINS

17

water level in Tank 1 to greatly decrease,

Tissue surrounding the brain may have been

and the water level in Tank 2 to increase

included in the sample. In addition, due to

slightly.

the miniscule size of the brain, the extraction

of the complete brain was difficult, and
therefore unlikely that all the brain samples
included a complete brain. The lack of a
complete brain led to a lower protein
concentration in some brains.

Picture 4. Tank Water Level Source of Error. Picture

above displays the decreased water level; this was
noted as a source of error in Tank 1.

The water level was, nonetheless,

During the gel electrophoresis, the

dye front moved faster than observed in
previous experiences. Therefore, the process
was stopped to account for the quick

still adequate to sustain the fish for forty-

process. However, the dye front was not as

eight hours, at which time the levels were

close to the end of the gel as expected. The

balanced. These strains may have influenced

distances that the bands would have traveled

the protein concentrations in the brain of the

down the gel are therefore less than had the

Danio rerio. Strains could have incited an

gel electrophoresis been stopped at the

increase in production or a decrease in

correct time. In addition, due to the

production of certain proteins.

unavailability of the actin and myosin

Due to time constraints, the brains of

standard and the Precision Plus Protein

the Danio rerio could not be extracted as

Kaleidoscope prestained standards, the

cautiously. The mass of the sample that was

molecular weights of the sample proteins

taken did not consist entirely of brain matter.

were estimated using an online

EFFECTS OF VITAMIN D3 ON THE -AMYLOID CONCENTRATION OF BRAINS

18

approximation. Due to the error with

fish had oil-like properties. Injection under

stopping the gel electrophoresis before

the surface of the water resulted in the

completion, the estimate may have led to a

supplement floating on the surface in a

large degree of inaccuracy with the

single spot. This method of delivery may not

molecular weights of the proteins shown in

have been as effective to observe the short-

this experiment.

term effects of Vitamin D3 on protein

Lastly, the treatment used to reduce

the agglomeration of -amyloid, Vitamin
D3, is commonly found in the liver of fish.
Because Vitamin D3 is naturally produced
by Danio rerio, the treatment given to the
fish in this experiment simply increased the
dosage rather than introducing a supplement.
The protein concentration, and more
specifically the concentration of -amyloid,
may not have decreased if the fish were not
affected with the increase in dosage.
Therefore, the differences between protein
concentration from the tanks with and
without Vitamin D3 would not be able to be

concentration. A longer study with the same

delivery system may have more reliable
data. A more efficient method of supplying
the Danio rerio with Vitamin D3 would be
to completely submerge these fish in a
beaker containing the vitamin, rather than to
pipette the Vitamin D3 into the fish tanks. In
addition, having dissected more Danio rerio
brains from each tank would have created a
more effective experiment because more amyloid samples would have been analyzed
to further observe a trend or relationship
between Vitamin D3 and -amyloid.
Vitamin D3 can be further applicable

attributed to the ability of Vitamin D3 to

to the curing of the Alzheimers disease

reduce -amyloid agglomeration.

because -amyloid is an important factor in

Furthermore, the Vitamin D3 given to the

driving the pathogenesis of the Alzheimers

EFFECTS OF VITAMIN D3 ON THE -AMYLOID CONCENTRATION OF BRAINS

19

disease. Vitamin D3 could potentially

the sole protein. In addition, prolonging the

become an essential component in

treatment for more than three days could be

decreasing the neurodegenerative effects of

an extension of this experiment because an

Alzheimers. Vitamin D3 can also be

extended treatment would allow the Danio

pertinent to other aggregation-associated

rerio to more effectively uptake the Vitamin

neurodegenerative diseases such as,

D3, which in turn would produce more

Parkinsons and Huntingtons. These

conclusive results on the effect of Vitamin

diseases are aggregation-associated

D3 on -amyloid.

neurodegenerative diseases, which have

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been found to be comparable in that there is

a deposition of aggregated proteins;
therefore, Vitamin D3 may be pertinent to
the diseases Parkinsons and Huntingtons as
an essential part of treatment.

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Tangles. (2011, January 1). Retrieved
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_tangles.asp

A more precise method of analysis

rather than SDS-PAGE would have created
more effective results. A more effective
analysis method of the -amyloid
concentrations within the Danio rerio would
be using the Western Blot Method as this
method involves the use of antibodies that
specifically bind to the protein, which would
essentially allow for the precise detection of

Kim, H. (2003). Behavior analysis method

for fishes in a tankusing image
processsing technology.International
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Kulie, T. (2009). Vitamin d: An evidence-

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10.3122/jabfm.2009.06.090037

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Yokogawa, T. (2007, October 16).
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Sanders, G. (2009, February 17). Zebrafish

workshop: Water quality parameters
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Kang, J. (2009, September 24). Amyloid-

Dynamics are Regulated by Orexin
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Champeau, R. (2013, February 5). Vitamin

http://www.ncbi.nlm.nih.gov/pmc/art

D, omega-3 may help clear amyloid

icles/PMC2789838/

plaques found in Alzheimer's.

Gupta, T., & Mullins, M. (2010, March 4).

Dissection of Organs from the Adult
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Chiu, C., & Prober, D. (2013, April 9).

section-of-organs-from-the-adult-

Regulation of zebrafish sleep and

zebrafish

arousal states: Current and

Masters, C., & Selkoe, D. (2012, June 1).

Biochemistry of Amyloid -Protein
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prospective approaches. Retrieved

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10.3389/fncir.2013.00058/full
Singh, A. (2013, June 26). Zebrafish, 82%

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Similar To Humans, Reveal New

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Insights On Neurological Disorders

Mastin, L. (2013, January 1). Sleep - How

Sleep Works - Neurological
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Hamilton, J. (2013, October 17). Brains

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extended light and extended dark

Sweep Themselves Clean Of Toxins

conditions in adult zebrafish.

During Sleep. Retrieved December

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Konnikova, M. (2014, January 11).

Goodnight. Sleep Clean. Retrieved
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flush out toxins during sleep.

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Table 1
Molecular Weight of the Danio rerio brain samples

Vitamin D. (2014, November 14). Retrieved

December 14, 2014, from
http://ods.od.nih.gov/factsheets/Vita
minD-HealthProfessional/

EFFECTS OF VITAMIN D3 ON THE -AMYLOID CONCENTRATION OF BRAINS

23

Band

Lane 2

Lane 3

Lane 4

Lane 5

Lane 6

Lane 7

Lane 8

Lane 9

No.
1
2
3
4
5
6
7
8
9
10

(1A)
195.8
175.2
146.4
126
102.5
97.9
94.7
90.9
89.4
82.2

(2A)
212.5
188.3
172.0
147.8
124.2
102.0
97.7
94.5
90.6
82.2

(3A)
217.2
166.1
145.7
123.5
100.7
97.1
93.8
90.0
81.7

(4A)
207.2
180.2
144.4
136.2
122.1
101.4
97.3
94.0
88.8
81.9

(1B)
193.4
166.1
154.6
141.0
120.4
100.9
97.0
93.8
88.1
81.5

(2B)

(3B)

(4B)
195.6
174.5

101.5
97.1
93.9
81.0

120.2
100.6
97.9
93.5
90.1
81.3

140.0
120.5
101.2
97.1
93.6
88.7
81.6

EFFECTS OF VITAMIN D3 ON THE -AMYLOID CONCENTRATION OF BRAINS

24

Table 2
Change in Molecular Weight of the Danio rerio brain samples (KDa)

Band No.
1
2
3
4
5
6
7
8
9
10
Average

1A-4A
21.4
9.1
0.7
2.5
1.8
0.8
0.9
0.9
7.7
82.2

1A-2A
16.7
13.1
25.6
21.8
21.7
4.1
3.0
3.6
1.2
0.0

3A-4A
10.0
14.1
1.3
12.7
21.4
4.3
3.5
4.0
7.1
81.9

2A-3A
4.7
22.2
26.3
24.3
23.5
4.9
3.9
4.5
8.9
82.2

1B-4B
2.2
8.4

1B-2B

3B-4B

1.0
0.1
0.3
0.1
0.2
0.6
0.1

0.6
0.1
0.1
0.5

0.3
0.6
0.8
0.1
1.4
0.3

Difference

12.8

11.08

16.03

20.54

1.44

0.325

0.58

2B-3B

0.9
0.8
0.4
0.3
0.6

EFFECTS OF VITAMIN D3 ON THE -AMYLOID CONCENTRATION OF BRAINS

25

Table 3
Controls of the Danio rerio brain samples (KDa)

Band No.
1
2
3
4
5
6
7
8
9
10
Average
Difference

1A-1B
2.4
9.1
8.2
15

2A-2B

3.0
2.3
2.9
1.3
0.7

0.5
0.6
0.6

4.99

0.725

3A-3B

3.5
4.1
3.5
8.4

1.2
4.88

4A-4B
11.6
5.7
3.8
1.6
0.2
0.2
0.4
0.1
0.3
2.66