The inhibition of dopamine degradation, facilitated using MAO-inhibitor pirlindole mesylate, did

not significantly alter reward-seeking behavior in larval D. rerio.

Rithika Senthilkumar, Siobhan Curran

Massachusetts Academy of Mathematics and Science at WPI, Worcester, Massachusetts

Senthilkumar 2

Summary

The release of the neurotransmitter dopamine plays a major role in reward and motivation;

however, there is no consensus among scientists regarding the effects of dopamine degradation on

reward-seeking behavior (2). The actions of dopamine can be terminated by two processes:

reuptake and degradation. During reuptake, the excess dopamine is recalled by the neuron and

stored for later use. During degradation, an enzyme, monoamine oxidase (MAO), breaks down

dopamine (5). It was hypothesized that the inhibition of dopamine degradation would increase

reward-seeking behavior in larval Danio rerio. The larvae were conditioned to GBR-12909, a

dopamine reuptake inhibitor, and water in separate environments. They were then exposed to

different concentrations of pirlindole mesylate, a MAO inhibitor. Conditioned Place Preference

Testing was used to determine if preference for GBR-12909 increased with exposure to pirlindole

mesylate. The inhibition of dopamine degradation did not significantly alter the reward-seeking

behavior of larval D. rerio exposed to 10 µM pirlindole mesylate (p = 0.471) or 75 µM pirlindole

mesylate (p = 0.462).

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Introduction

Dopamine, a neurotransmitter that plays a key role in memory formation, attention,

movement, motivation, and rewarding behavior, is formed from the breakdown of the amino acid

tyrosine. In response to certain chemical signals, dopamine is released into the synapse, where it

reacts with other dopaminergic neurons to facilitate various biological processes (2). Dopamine’s

actions in the synapses can be terminated by two processes: reuptake and degradation. Reuptake

is the recalling of the dopamine in the synapse back into the synaptic vesicles of the original cell

through a structure called the dopamine transporter. When dopamine reuptakes, it is stored in the

synaptic vesicles for future use and does not interact with any other receptors, thereby terminating

its effects (5). During degradation, dopamine is broken down into various substances by enzymes,

one of which is monoamine oxidase (MAO) (3). Psychostimulant substances have the ability to

artificially increase dopamine levels in the synapse and block reuptake, reinforcing reward-seeking

behavior in the user (7). While some studies report that dopamine degradation does not have a

significant effect on reward-seeking behavior, other studies reported the opposite. How does the

inhibition of dopamine degradation affect reward-seeking behavior in larval D. rerio? It was

hypothesized that the inhibition of dopamine degradation would increase reward-seeking behavior

in larval Danio rerio. This study found that the inhibition of dopamine degradation, facilitated by

MAO-inhibitor pirlindole mesylate, did not significantly alter reward-seeking behavior, achieved

with the psychostimulant GBR12909, in larval Danio rerio.

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Results

Initial Preference Testing

When tested for initial preference, the average percentage of time (out of total 5 minutes)

spent in the dotted environment was 43.5% and in the white environment was 56.5%. The data had

a standard deviation of 27.2%. The measured difference in average percentage of time spent in

dotted environment and white environment was 13.0%, with a standard deviation of 54.3% (See

Table 1). The shape of the distribution of the histogram of differences was approximately unimodal

(See Figure 1).

Table 1: Initial Preference averages and standard deviation.

Dotted Time (%) White Time (%) Difference (%)

AVG 0.435 0.565 0.130

STDEV 0.272 0.272 0.543

Figure 1: Histogram of differences in percentages of time.

The results of the two-tailed t-test for means with unequal variances yielded a p-value of 0.069.

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Final Preference Testing

0 µM pirlindole mesylate Treatment

With 0 µM pirlindole mesylate treatment, the average percentage of time spent in the GBR-

12909 associated environment was 72.4%, with a standard deviation of 30.1% (See Table 2). The

distribution of the histogram of 0 µM pirlindole mesylate treatment was unimodal and symmetric

(See Figure 2).

Table 2: 0 µM pirlindole mesylate treatment average and standard deviation.

Dotted Time (%)

AVG 0.724

STDEV 0.301

Figure 2: Histogram of 0 µM treatment percentages of time.

10 µM pirlindole mesylate Treatment

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With 10 µM pirlindole mesylate treatment, the average percentage of time spent in the

GBR-12909 associated environment was 71.6%, with a standard deviation of 29.9% (See Table

3). The distribution of the histogram of 0 µM pirlindole mesylate treatment was approximately

unimodal and symmetric (See Figure 3).

Table 3: 10 µM pirlindole mesylate treatment average and standard deviation.

Dotted Time (%)

AVG 0.716

STDEV 0.299

Figure 3: Histogram of 10 µM treatment percentages of time.

75 µM pirlindole mesylate Treatment

With 75 µM pirlindole mesylate treatment, the average percentage of time spent in the

GBR-12909 associated environment was 71.4%, with a standard deviation of 25.8% (See Table

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4). The distribution of the histogram of 0 µM pirlindole mesylate treatment was approximately

unimodal and symmetric (See Figure 4).

Table 4: 75 µM pirlindole mesylate treatment average and standard deviation.

Dotted Time (%)

AVG 0.714

STDEV 0.258

Figure 4: Histogram of 75 µM treatment percentages of time.

Comparison of Samples

A two-tailed t-test was performed on the percentages of time between the 0 µM treatment

and the 10 µM treatment, which yielded a p-value of 0.471. A two-tailed t-test was performed on

the percentages of time between the 0 µM treatment and the 75 µM treatment, which yielded a p-

value of 0.462.

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Discussions

Initial Preference Testing

Since the histogram of the differences in percentages of time was shaped unimodally,

normalcy and statistical tests were assumed to be suitable. The resulting p-value, 0.069, from the

two-tailed t-test for means suggested that the difference between the preference for the dotted

environment and the white environment was not significant during initial preference testing. It was

concluded that the CPP apparatus was not biased in any way and that random assignment could be

used to assign environments to drug treatment.

Final Preference Testing

Since the histograms of the percentages of time of 0 µM, 10 µM, and 75 µM pirlindole

mesylate treatments were shaped unimodally and were symmetric, normalcy and statistical tests

were assumed to be suitable. The average percentage of time spent in the GBR-12909 associated

environment with the 0 µM treatment, 72.4%, was higher than the average percentage of time

spent in in the GBR-12909 associated environment with the 10 µM and 75 µM treatments, which

were 71.6% and 71.4% respectively. These summary statistics seemed to not support the

hypothesis; statistical tests were performed to test for any significant differences in the data.

Comparison of Samples

When a two-tailed t-test was performed on the percentages of time between the 0 µM

treatment and the 10 µM treatment, a p-value of 0.471 was obtained. Since the p-value was less

than 0.05, it was concluded that increasing the pirlindole mesylate concentration to which the

larvae were exposed to from 0 µM to 10 µM did not have a significant effect on the larvae’s

reward-seeking behavior. When a two-tailed t-test was performed on the percentages of time

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between the 0 µM treatment and the 75 µM treatment, a p-value of 0.462 was obtained. Since the

p-value was less than 0.05, it was concluded that increasing the pirlindole mesylate concentration

to which the larvae were exposed to from 0 µM to 75 µM did not have a significant effect on the

larvae’s reward-seeking behavior. The results from the experiments did not support the hypothesis

that dopamine degradation will increase reward-seeking behavior in larval Danio rerio.

Sources of Error

Possible sources of error include the handling of the D. rerio larvae, the measurement of time spent

in each chamber, and the administration of the chemical substances to the water.

Conclusions

The results indicate that the inhibition of dopamine degradation using the MAO inhibitor

pirlindole mesylate did not have a significant effect on reward-seeking behavior in larval Danio

rerio, induced by dopamine reuptake inhibitor GBR-12909. This project aimed to provide

information about the role of dopamine degradation in reward-seeking behavior. It has managed

to accomplish that goal and has provided substantial evidence that could be used to reach a

consensus about the role of dopamine degradation in reward-seeking behavior.

Future Extensions

The scope of this project can be extended to provide more information about dopamine

degradation. Future work on this topic includes using other organisms, such as mice, to test the

hypothesis. Other MAO inhibitors could be used to regulate dopamine degradation; MAO inhibitor

of type B could be used to inhibit degradation. Danio rerio that are more mature, such as adults or

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juveniles, can also be used to test for increased reward-seeking behavior. Danio rerio, genetically

modified so that dopamine is the only monoamine inhibited, could be used to test the hypothesis.

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Methodology

Materials

Table 5: The table lists the materials and the quantities that were used, and from where the materials were obtained from.

Product Quantity Source

Edvotek Digital Water Bath 1 Mass Academy

TopFin Twinsies 0.8-gallon tank 1 PetSmart

TopFin Stackable Studio 0.25-gallon tank 4 PetSmart

1000 mL beaker 5 Mass Academy

250 mL beaker 2 Mass Academy

AUCH Disposable Plastic Pipettes 1 mL 100 Amazon

Petri dish 5 Mass Academy

Zebrafish larvae (4-7 dpf) 160 Timothy Beane, UMASS Medical

School

Pirlindole mesylate CAT. sc-203664 10 mg Santa Cruz Biotechnology Inc.

GBR-12909 dihydrochloride CAT. sc-200398 5 mg Santa Cruz Biotechnology Inc.

Parafilm Mass Academy

Large Strip Thermometer 1 PetSmart

Zebrafish Care

Edvotek Digital Water Bath, 1000-mL beakers, and 250-mL beakers were obtained from

Mass Academy. The zebrafish were obtained from Mr. Timothy Beane, UMASS Medical School.

1-mL AUCH disposable plastic pipettes were purchased from Amazon. The fish were transferred

from the test tubes they arrived in into 1000-mL beakers using pipettes. Approximately 100 fish

were kept in a 1000-mL beaker filled with 400-mL system water. The beaker was placed in the

Edvotek Water Bath and the temperature was set to 28 ºC (8). The temperature was monitored

using Large Strip Thermometers, obtained from PetSmart. The thermometer was placed in the

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water inside the water bath. 1/3 of the water in the beaker was replaced with new water once every

day. The new water was given a good shake before replacement (8).

In order to euthanize the fish, the beaker with the fish was removed from the water bath

and ice cubes were added to the water. The temperature was dropped to 0 - 4 ºC and the beaker

was left alone for at least 30 minutes. The fish were then removed from the beaker with a pipette

and placed in a Petri dish, which was then covered with parafilm. The deceased fish were then

disposed of per national, state, and local laws (8).

In the case of dead larvae, they were removed from the rest of the sample with a pipette

and placed in a Petri dish. This process was repeated when necessary throughout the course of the

experiment.

Constructing Conditioning Tanks

Two TopFin Stackable Studio 0.25 gallon tanks were purchased from PetSmart. Plain

white paper was cut in the shape of the bottom side of the 0.25-gallon tank. The cut piece of paper

was pasted on the outside of the bottom side of one of the 0.25-gallon tanks. The sides of the tank

remained transparent (6). (Figure 5)

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Figure 5: Side view of white conditioning tank.

Plain white paper with black dots, 1.10 inches in diameter and in a 4-3-4-3 pattern was

printed. The dotted paper was cut in the shape of the bottom of the 0.25-gallon tank and included

3 1/3 dots. The cut piece of paper was pasted on the outside of the bottom side of the other 0.25-

gallon tank. The sides of the tank remained transparent (6). (Figure 6)

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Figure 6: Side view dotted conditioning tank.

Constructing Conditioned Place Preference Testing Tank

A TopFin Twinsies 0.8-gallon tank with divider was purchased from PetSmart. The divider

was removed from the tank, since it was not required. The tank was placed with the curved side

facing forward. Plain white paper was cut in the shape of the right half of the bottom side of the

tank. The cut piece of paper was pasted on the outside right bottom half of the tank. Plain white

paper with black dots, similar to the paper printed when constructing the conditioning chamber

was printed. The dotted paper was cut in the shape of the left half of the bottom side of the tank

and included 4 dots. The cut piece of paper was pasted on the outside left bottom half of the tank.

The sides of the tank remained transparent (6). (Figure 7)

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Figure 7: Conditioned Place Preference Testing Tank.

Testing Initial Preference

The Conditioned Place Preference (CPP) Testing Tank was filled with 600 mL of system

water. Fish were individually removed from the beaker with a pipette and placed into the CPP

Testing Tank. The fish was allowed to roam freely in the tank for 5 minutes. The amount of time

the fish physically spent in the dotted chamber was recorded using the stopwatch application on

the Apple IPhone 6S. The fish was returned to a 250-mL beaker with 100-mL system water, which

was placed inside the water bath. This process was repeated 29 more times. Data for 30 fish was

collected (4).

Conditioning Tank Assignment

The amount of time the fish spent in the dotted chamber was converted to seconds and the

amount of time spent in the white chamber was determined. Average times spent in the dotted

chamber and the white chamber (in seconds) were computed and converted to percentages. A two-

tailed t-test was performed on the data to determine if the difference was statistically significant.

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The chemical substance GBR 12909 dihydrochloride was paired with the pattern (dotted or white)

associated with the side where the fish spent significantly less time. Conversely, system water was

paired with the pattern associated with the side where the fish spent significantly more time. In the

case where the fish displayed no significant difference between the two patterns, the substances

were randomly assigned to the substance using a random number generator. The dotted chamber

was assigned the number 1 and the white chamber was assigned the number 2. GBR 12909

received the first assignment to the dotted environment and system water received the second

assignment to the white environment (4).

Conditioning

Two more TopFin Stackable Studio 0.25 gallon tanks were purchased from PetSmart. The

fish in the 250-mL beaker were transferred, using a pipette, to a 0.25 gallon fully transparent tank,

in which the substance pirlindole mesylate was dissolved with water. Three different

concentrations were used for three different settings. The concentrations of pirlindole mesylate

used were 0 µM, 10 µM, and 75 µM (1). The fish were exposed to the pirlindole mesylate solution

for 5 minutes (4).

The fish were then removed from the pirlindole mesylate solution using a pipette and

placed in the GBR 12909 conditioning tank. The fish were exposed to 10 µM GBR 12909 with

their sample group for 5 minutes (4).

The fish were then removed from the GBR 12909 tank using a pipette and placed in a 0.25

gallon fully transparent tank with 350 mL system water for 1 minute. The fish were then returned

to the 250-mL beaker where they were previously stored (4).

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At least 24 hours later, the fish were removed from the 250-mL tank using a pipette and

placed in the system water assigned conditioning tank. This conditioning tank contained 450 mL

system water. The fish were remained in the conditioning tank in their sample group for 10

minutes. The fish were then returned to the 250-mL beaker where they were previously stored.

Testing Final Preference

The Conditioned Place Preference (CPP) Testing Tank was filled with 600 mL of system

water. The fish were removed individually from the 250-mL beaker with a pipette and placed into

the CPP Testing Tank. The fish was allowed to roam freely in the tank for 3 minutes. The amount

of time the fish physically spent in the GBR 12909 associated chamber was recorded using the

stopwatch application on the Apple IPhone 6S. The fish was returned to a 250-mL beaker with

100 mL system water. This process was repeated for the rest of the fish in the sample (4).

Final Statistical Testing

Average times were calculated for each treatment group and the times were converted into

percentages. T-tests were performed on the percentages of average time the fish spent in the GBR

12909 associated chamber during Final Preference Testing. The average time the control group (0

µM) spent in the GBR 12909 associated chamber was compared to the average time the treatment

groups (10 µM, 75 µM) spent in the GBR associated chamber.

Senthilkumar 18

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Collier, Adam D., et al. “Zebrafish and Conditioned Place Preference: A Translational Model of

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Mandal, Ananya. "Dopamine Biochemistry." News-Medical.net. N.p., 14 November. 2012. Web.

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Senthilkumar 19

Acknowledgements

I sincerely thank the following people for their support and help with the research and the

manuscript. Mr. Timothy Beane, University of Massachusetts Medical School, provided zebrafish

embryos. Dr. Haley Melikian, University of Massachusetts, provided her professional opinion on

the procedure. Dr. William Norton, University of Leicester, also offered helpful suggestions on

the procedure. Ms. Siobhan Curran, Massachusetts Academy of Math and Science, read and

commented on the manuscript. Mr. Senthilkumar Manickam and Mrs. Anuradha Senthilkumar

provided materials and space to carry out the experiment.