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“Dopamine Influence on Behavior Change in Fruit flies”
An Honors Thesis
by
Lanieta Waqanivalu
California, Pennsylvania
2021
Dopamine Influence on Behavior
Change in Fruit Flies
Lanieta K. Waqanivalu
Advised by Dr. Louise Nicholson
University Honors Program: Biology Department, 250 University Ave., California
University of PA, California, PA 15419
KEYWORDS: Dopamine influence, kavalactones, fruit fly locomotion, fruit fly survival
rate
ABSTRACT: Dopamine levels in fruit flies have been linked to a number of specific
mutations that have influenced acquired and learned behavior. Increased dopamine
levels have altered normal behavior patterns in that they have been found to decrease in
offspring larval locomotion. The purpose of this research is to identify locomotion
activity in offspring of fruit flies with different dopamine levels through several
generations, as well as to identify the boundary at which dopamine influences different
locomotion activities between fruit fly (parents) and larvae (offspring). To conduct this
experiment, several mortality and locomotion assays will be utilized to examine
dopamine influence in kavalactones and determine specific behaviors it inhibits or
initiates.
Introduction
Drosophila melanogaster, commonly known as
fruit flies, are an Insecta class derived from the
phylum Arthropoda and are often found infesting
homes by feeding on ripe or decayed fruits and
produce. Fruit flies have been widely used as
model systems in many branches of biology,
including research pertaining to psychological and
social behaviors. These studies have examined a
wide range of behaviors, and determined patterns
of aggression, excitement, courtship, and mating
abilities in generations of fruit flies1. Studies have
also investigated the roles of specific neuronal
signaling pathways and have found that the
neurotransmitter known as dopamine influences
specific behaviors such as regulating arousal,
promoting risk taking behavior, influencing malemale courtship, and altering locomotor activity
between parents and offspring2.
Dopamine is known to be an important chemical
messenger in the brain, involved in reward
motivation, motor function, compulsion, perseveration, sensation and regulating body movement3. This neurotransmitter is found in several
areas of the brain including frontal cortex, nucleus
accumbens, striatum, and hippocampus. The
distinctive molecular structure of dopamine adds
to its unique nature as it is derived from a
catecholamine family, which makes up approximately 80% of the catecholamine content in
the brain4. Dopamine is featured by a single amine
group that is connected to a benzene ring with two
adjacent hydroxyl groups and a side chain of
ethylamine5. Because dopamine is synthesized in
the brain, it is unable to pass through the blood
brain barrier that inhibits the interaction of
neurotransmitters and foreign substances trying to
enter the central nervous system. A precursor of
dopamine known as L-DOPA is where this
neurotransmitter is originally derived from. To
produce dopamine, a phenylalanine hydroxylase
converts phenylalanine to tyrosine, which is then
converted to L-DOPA, then later altered to
dopamine through decarboxylation of the aromatic
aminoacid.
Currently, three major brain dopamine
pathways have been determined to be involved
in brain actions: the tuberoinfundibular hypothalamic system, the mesolimbic-cortical of the
ventral tegmentum, and the nigrostriatal of the
brain’s A9 region7. Dopamine has been found to
function heavily in altering behavioral patterns
which are both impulsive and inflexible, and
drive a key role in pleasure related behaviors
such as sex, social interaction, food, and even
drug addiction8. Recent studies in dopamine
signaling has showed that dopamine regulates
behavior through the mesolimbic dopaminergic pathway, specifically within the D1
and D2 receptors9. D1 receptors stimulate
Figure 1. Dopamine Synthesis Pathway6.
intracellular cyclic adenosine monophosphate (cAMP)
levels, which are ATP derivatives used for intracellular
signal transduction, while D2 receptors inhibit cAMP.
When dopamine binds to the D1 receptor, an enzyme
known as adenylate cyclase is activated, increasing cAMP
synthesis, but binding to D2 receptor inactivates adenylate
cyclase, decreasing production of cAMP10. Inhibition of
cAMP then activates protein kinase A, but its regulation is
based on the dissociation of the D2 receptor whichin itself
is inhibited by Ca2+ . Affinity of these receptors influence
D2 to have a 10-100 fold greater affinity than that of D1,
which has the lowest affinity across all dopamine
receptors. D1 receptors are coupled with G proteins
(guanine-binding proteins) to activate adenylyl cyclase
activity and increase cAMP production. Activation of
cAMP induces the activation of protein kinase A, allowing
phosphorylation to occur as well as adjustment of various
ion channels. D2 receptors are then coupled to the
previously mentioned G proteins, and due to its inhibition
characteristic, it decreases protein kinase A activity by
regulation of cAMP levels, thus activating potassium ion
channels and regulating others. These D2 receptors which
are coupled to G alpha I and G alpha o activate Calcineurin, a protein phosphatase in charge of dephosphorylating DARPP-32 at Thr3412.
Figure 2. D2 Dopamine Receptor Signaling Pathway11.
D1 receptor stimulation have therefore been linked to
1
DARPP-32
(cAMP
regulated
neuronal
phosphoprotein) phosphorylation during protein
kinase A activation, while D2 reduced phosphorylation, presenting a bidirectional pathway on
dopamine signaling molecules.
Because dopamine has been used in pharmaceuticals as an antipsychotic drug, its components
of the D1 and D2 like receptors play majors roles
in motor function, specifically within the striatal
out- flow pathways13. In this pathway, D1
receptors are attached to GA- BAergic medium
spiny neurons (direct pathway) which project out
to the substantia nigra and medial globus pallidus,
while D2 receptors are attached to the lateral
globus pallidus, which is the indirect pathway7.
Studies on antidopa-minergic drugs have found
D1 and D2 receptors to be located on separate
neurons as activation of D1 enhance motor
function, wile D2 regulates behavioral response14.
These receptor signals of the D1 and D2
communicate through the βγ signaling portion of
the Gα subunits, allowing for their agonist/
antagonistic relationship.
Figure 3. Schematic of the striatal outflow
pathway15.
Before the D1 and D2 receptor characteristics
were fully known, functional selectivity was
found through experimentation of antipsychotic
dug aripiprazole16. This finding suggested that
different signaling pathways could be mediated
by a single receptor, i.e. influencing different
outcomes within different pathways. D1- like
receptors have been shown to mediate voltagegated ion channels both directly and indirectly
within the striatal outflow pathway. These
receptors are dependent on factors such as G
proteins, G protein receptor kinases (GRK), βarrestins and more17. In addition to their
involvement in signaling and striatal outflow
pathways, they have also been found to evoke
functionality within mitogen-activated protein
(MAP) kinase and calcium signaling pathways. In
MAP kinase, D1 receptors function as inhibitors
to inactivate p38 MAP kinase as well as c-Jun
amino terminal kinase (JNK) through its agonist,
SKF383918. Similarly in the calcium signaling
pathway, D1 receptors directly interact with
calcium channels, inhibiting N-type channels
which consist of α1B primary subunit, as well as
α2δ and β auxiliary subunits, which are localized
to the nerve and dendritic terminals of neurons 19.
This functional selectivity characteristic of D1
receptors mediating different signaling pathways
provide varying inactivation and inhibition properties,
suggesting its function to be selective upon proteins the D1
receptor is coupled toand the signaling pathway it undergoes.
Unlike D1, D2- like receptors are adenylate cyclase
inhibitors in dopamine signaling pathways, inactivated by
pertussis toxin through catalyzation of ADP-ribosylation20. In addition to this signaling pathway, D2
receptors are also involved in regulation of sodium
channels and play a major role in G- protein independent
signaling. In sodium channels, depending on the type of
D2 receptor involved, sodium currents can be increased or
decreased to allow access of potassium channels in
neuron21. Within G proteins, independent signaling is
promoted by GRK through phosphorylation of D2
receptors, which leads to binding of β-arrestins7. Because
β- arrestin signaling is a time-consuming mechanism, in
some cases, G- proteins initiate response through
utilization of cAMP and protein kinase A in order to
accelerate the response propagated from signaling
pathways22.
While dopamine can be obtained from readily available
supplements, it can also be obtained from other natural
sources. To provide and obtain natural dopamine,
kavalactones that are extracted from kava root (Piper
methysticum) will be utilized as a dopamine source in this
study. Grown and harvested in the South Pacific Islands,
kavalactones in kava have been shown to increase
dopamine concentration in mice through blockage in
reuptake of norepinephrine, regulating serotonin and
increasing dopamine levels23. While kava is normally
consumed as an alcoholic drink, it’s calming effect
contrasts that of alcohol as it reduces stress-induced
anxiety and insomnia24. Kavalactone compounds are
currently under pharmaceutical research for psychotropic
effects, but its complex structure suggests enzymatic
activity. Kavalactones are derived from lipophilic
compounds, which are molecules able to be dissolved in
lipids, as well as non- polar solvents25. Currently, six
major kavalactones have been identified: kavain,
methysticin, yangonin, dihydrokavain, dihydromethysticin, and desmethoxyyangonin.
Figure 4. Chemical structures of the six major kavalactones26
In addition to these kavalactones, studies conducted on
their structure determined them to be molecular targets
of proteins such as histamine type 1 and 2 receptors,
dopamine receptors, opioid μ and δ receptors, and γaminobutyric acid type A receptors27-28. Through
kavalactone’s natural metabolic pathway in humans,
hydroxylation of the C-12 aromatic and lactone rings
occur, followed by demethylation of the 4-methoxyl
group, and reduction of the 7,8- double bonds26. These
hydroxylation and demethylation processes are conducted by CYP2D6, which are dopamine metabolizing
enzymes involved in the reversal of kava hepatoxicity29. Studies conducted have found kava-lactones
to be effective in treating psychotropic effects such as
anxiety and insomnia as they maintain similar changesin
brain wave activity as Valium30. In addition to this
finding, a study conducted in 2004 found that at the
specific concentration of300 mg, kavalactones improve
mood and cognitive performance, similar to the effects
of dopamine related behaviors30. It is inferred that
kavalactone will increase dopamine concentrations in
fruit flies and enhance locomotion activity in its
offspring and future generations. This study focuses on
the behaviors influenced by different kavalactone
concentrations, as well as validating the hypothesis
previously stated.
Methods
Fruit fly Strains
In this study, fruit fly strains used include wild type,
ebony, and mutants of the R1 and R2 types. Wild types
are those of the typical phenotypic fruit fly forms as it
occurs in nature, and ebony are mutants that encode
specific proteins, linking beta-alanine to amines like
dopamine31. Mutants of the R1 and R2 types
correspond to D1 and D2 receptors of the dopamine
signaling pathway.
Kavalactone Treatment
Flies were raised on standard cornmeal-agar fly food at
25C, with either 0 (control), 100mg/ml, 300mg/ml or
500 mg/ml kavalactone. For wild type flies and ebony
(dopamine synthesis mutants), three 100 mg, 300 mg,
and control vials were prepared with five females and
five males in each, totaling ten fruit flies in each vial.
Wild type flies were then left to feed and mate for a
period of 30 days with observations conducted on the
12th, 22nd, and 26th day. Ebony flies were left to grow
for a period of several days with observations made on
the 5th, 10th, and 15th day (data still being collected for
later time points).
For assays involving the R1 and R2 dopamine receptor
mutants, due to lowered viability and the larval
maturation process of R1 type, only two were placed in
each of these vials, one male and one female as there
were only six fully mature. These flies were then also
left to grow and mate then observed on the 4th day.
Light Assay for Anxiety-like Behaviors
For light assays32, fruit flies from the wild type vials
were extracted and placed into a clear box that was
taped on one half to provide adark setting and left clear
on the other half to provide for light set ting. Once the
fly was placed in and the box closed, five minutes was
given for the fly to adapt to its new environment then
observed for a period of ten minutes and timed on how
long it spent in the light. Altogether, three runs of each
concentrations were conductedon both male and female
flies then averaged for data analysis. As for the light
assay involving different fruit fly variants, one female
and one male fly of ebony, R1, and R2 were observed
from each concentration. Similar to the wild type
assay, three runs were conducted across each fly type
in each concentrations then averaged for data analysis.
Locomotion Assay
For locomotion assay33, a vial was measured to 5 cm
from the bottom and marked with a sharpie to
emphasize boundary line marker.After preparation, wild
type and fruit fly variants in different kavalactone
concentrations previously used on light assay were then
tested by placing each group in the prepared vial, then
observed for how many crossed the boundary marker.
To maintain consistency across all groups, the vials
were tapped on a surface to allow all flies to start from
the bottom then timed for ten seconds and observed.
Data was obtained from the number of flies of each
group that crossed the boundary marker then averaged
from three runs.
Results
Mortality/ Survival Rate
Figure 5. Fruit fly mortality in wild type flies.
Average survival rates of wild type flies on different
concentrations of kavalactones are shown over three
trials. To first determine whether kavalactones were
effective dopamine sources for this study, a mortality
assay was conducted to observe fruit fly survivalrates
in those placed in kavalactone infused food,
compared to the control group with no kavalactone
concentrations. Mortality observation in wild type
fruit flies displayed a constant depletion throughout
time. Flies in kavalactone concentrations appeared to
have a slightly higher survival rate compared to nonkavalactone concentration groups. Flies in 100 mg
kavalactone concentrations showed the highest
survival rate compared to the higher concentrations
and to controls. While concentrations in 300 mg
kavalactones produced a lower survival rate then its
lesser counterpart (100 mg), it ranked this
concentration higher than the control group with no
kavalactones.
Figure 6. Fruit fly mortality in ebony flies.
Ebony fruit flies carry a specific feature as they contain
a defect in their ebony gene, which encodes a β-alanyldopamine (DA) synthase (BAS), thus increasing
dopamine concentration within them34. In these mutants, the presence of kavalactones reduced theirsurvival,
compared to flies reared on control food. Observation
of these dopamine mutants yielded 300 mg kavalactone
concentrations to contain the highest mortality across
all other concentrations. 100 mg concentrations
followed shortly after in mortality rate with a difference
less than 10% compared to 300 mg. 500 mg
kavalactones produced the lowest rate of all three
kavalactone concentrations as it maintained a constant
rate throughout all observations. Although 500 mg
were lower in rate, flies placed in control group yielded
a result slightly lower than this, leaving a difference of
more than 10% mortality rate between its flies and
those in 300mg concentrations.
Figure 7. Survival rate in different fruit fly variants.
In preliminary observations of dopamine receptor
mutants and kavalactone concentrations, R1 flies
displayed the highest survival rate as there were only
two placed in each vial compared to the ten placed in
vials of other fruit fly types. R2 flies produced second
highest survival rate as all flies were alive during
observation and some had even begun to produce
larvae. As these are preliminary results, summary
figures of flies in 300 mg concentrations could not be
drawn due to difference in sample sizes. This portion
of the assay is currently ongoing, therefore yielding no
conclusions currently. For the current data however,
results suggest control and 100 mg concentration flies
had the highest survival rate when compared to other
fruit fly types in 500 mg concentration.
Light Assay
Table 1. Light assay of wild type flies.
In this assay, kavalactones were used to determine
anxiety-like behaviors in fruit flies by comparing male
and female wild type flies, with an average of three data
runs taken and placed in the table above. Differences
between male and female anatomy did not affect this
study, but concentrations did as data varied across this
category. According to the data obtained, flies in 300
mg concentrations spent more time in light with male
averaging 87s (47%) and females at 47 s (23%).
Comparatively, flies in control group with no kavalactone concentrations yielded the least amount with
male averaging 11.75s (0.06%) and females at 23s
(0.11%). Flies in 100 and 500 mg concentrations were
similar in that the difference in amount of time were
proportionate, b u t diverged in that males spent more
time in light in 100 mg concentration compared to females in 500 mg.
Table 2. Light assay of male variant flies.
In this assay comparing wild, R1, and R2 type flies of
different kavalactone concentrations, sex anatomy
played a major role in that female flies spent little to
no time in light environments (table omitted due to no
data) and rather preferred dark settings throughout
the observation time frame. Wild and R2 female flies
in the control group with no kavalactones produced
minimal data, which is substantiated in the table
above. As for males, flies in 100 mg concentrations
spent the most amount of time in light as wild type
averaged 262 s (65%) and R1 at 32 s (62%).
Following 100 mg concentration, flies placed in the
control group yielded the second highest number of
male flies in light with wild type at 14 s (0.03%) and
R2 at 168 s (79%). Flies in 300 and 500 mg
concentrations then followed after with the 500 mg
kavalactone concentration ranking above 300 mg in
the number of flies preferring light environment.
Figure 8. Locomotion assay of fruit fly parents.
Due to lack of time in conducting this study, not
enough R1 and R2 type flies arrived in time to be able
to fully complete this assay. My data for this portion
of the assay are inconclusive but the statements made
below only pertain to wild type flies and 100-300 mg
concentrations of the R2 type.
In this assay, R2 fruit flies displayed the highest
amount of locomotion when averaged with fruit fly
types. Although the 500 mg concentration was tested
against the wild type, its result is not included in this
analysis as it was not tested against R1 and R2 type
flies. Exclusion of this concentration therefore
provided the R2 type with the highest locomotion in
fruit fly parent category. Wild type fruit flies
produced the second highest locomotion with both
100 mg and control groups yielding more locomotion activity, although R2 type leads in 300 mg
concentrations when compared to wild types.
Figure 9. Locomotion assay of fruit fly offspring.
In this category of locomotion assay of only wild type
offspring fruit flies, the control group with no
kavalactones produced the highest amount of locomotion activity by more than five times than that of the
second highest yielding which is the 300 mg concentration. Offspring fruit flies from the 100 mg
concentration yielded the least amount of locomotion
activity, which is only about 1.5 times less than that of
the 500 mg kavalactone concentration.
Discussion
As seen in the results of fruit fly mortality or survival
rate, wild type fruit flies in 100 mg kavalactone
concentrations had a better survival rate when
compared to other concentrations. This suggests that
those less than 100 mg kavalactone concentrations
were not enough to influence any activity in fruit flies,
such as that of the control vials. This neuroprotective
effect may be due to the mediation by several factors
such as P38, nuclear factor-kappaB, and cyclooxygenase2 signaling pathways35. Similarly how-ever,
they could also be mediated by the upregulation of
antioxidant enzymes by ERK phosphorylation, translocation of the transcription factor Nrf2, binding of the
antioxidant responsive element (ARE), and activation
of the heme oxygenase I (HO1) enzyme to induce
nitric oxide synthase (iNOS) which allows for cell
survival35.
Figure 10. Neuroprotective pathways of kavalactones35.
For the ebony dopamine mutant flies, 300 mg yielded
the highest survival rate, thus suggesting the 100 mg
concentration to not be enough, and the 500 mg
concentration to be too much to the extent that it killed
some of the flies. Because these dopamine mutant flies
were already concentrated with dopamine, its effects at
500 mg concentrations are valid as it correlates to the
underlying biology that too much of anything could be
lethal. This suggests that kavalactones are dosedependent and that at lower concentrations are not as
effective, but at too much concentration could prove
lethal. In the assay involving several fruit fly variants
in different dopamine concentrations, the results were
inconclusive as there was not enough of the R1 and R2
type flies available to be able to accurately conduct the
assay without skewing the results. Therefore, for the
fruit fly parent locomotion assay, no conclusion can be
drawn due to lack of data.
As for the light assay of wild type flies, sex anatomy
did not make a difference as data was dependent on
the kavalactone concentrations. Flies in 300 mg
kavalactone concentrations were seen to have spent
the most amount of time in light, whereas the control
group spent little to no time in light. This data
suggests that at the specifickavalactone concentration
of 300 mg, normal behavior was impacted in that
flies that would normally prefer dark settings have
been influenced to change behavior and spend more
time in light. Due to these results, it is suggested that
kavalactones are anxiolytic at 300 mg concentrations,
as they were the most interactive in light
environment. This behavior change looks to have
been initiated however at the 100 and 500 mg
concentrations as their data correlated to each other
but differed in the sex that it accounted for.
In the light assay of fruit fly variants and concentrations, sex anatomy looks to not have played a role
based on the results obtained from data. Female fruit
flies of the wild, R1, and R2 type were notinfluenced
in the behavior change to conform to light
environments as they spent no time in light, except
for its control group which spent less than five
seconds in light, thus substantiating this theory.As for
the male flies however, behavior change was
dependent on the kavalactone concentration rather
than the type of fruit fly involved. As suggested in
previous studies, anxiety behaviors in fruit flies are
not sex-dependent36, suggesting that anxiety is not
one of the behavioral patterns influenced by the flies’
sex but rather by external factors, which in this case
are kavalactones. Flies in 100 mg concentrations
averaged the most amount of time in light com pared
to the 300 mg concentration which averaged the least.
Results from this assay suggested that 100 mg
kavalactone was the right amount of concentration
needed to influence behavior change and that 300 mg
could most probably have been too much. 500 mg
concentrations however yielded the third highest in
the amount of time these flies spent in light,
suggesting that another behavior change may have
been initiated at this concentration level within the
different type flies.
In locomotion assay of parent fruit flies, analysis was
made without consideration of the 500 mg
concentration as there was not enough of this fly type
to test across all concentrations. Results obtained
from the parent locomotion assay showed R2 type to
have produced the greatest amount of locomotion
when averaged with flies from other groups. R2 type
yielded the greatest locomotion activity in parent
flies, suggesting that R2 type without kavalactones
was the most efficient concentration to influence this
behavior.
In locomotion of offspring fruit flies, the control
group containing no kavalactones produced the
highest amount of locomotion activity in wild type
flies with 300 mg being the second highest. This
assay was only conducted on offspring of wild type
flies as there were not any offspring yet mature from
R1 and R2 type. Data obtained from this assay
suggests that dopamine concentration has no effect on
offspring larval locomotion, thus invalidating the
initial theory of correlation between increased
dopamine concentration leading to increased larval
locomotion. In fact, studies conducted suggest an
inverse correlation in that increased dopamine levels
led to reduction of larval locomotion as signaling
efficiencies diverged37.
While this study proved successful in validating and
rejecting the initial hypothesis, certain changes would
have to be made when reconducting this experiment.
Due to the time frame needed for fruit flies to mate and
produce larvae, a time frame of two to three semesters
should be allotted to allow for accurate development of
results. As there was only one semester allotted for this
experiment, some results obtained may not be as
accurate as fruit flies needed more time to produce
larvae. Another change that would also haveto be made
would be maintaining a constant rate of observations
across all assays compared to the varied rate in this
study. Although changes would have to be made, further
enhancements of this study could be conducted through
exploration of other behaviors influenced by
kavalactones, and testing for several other concentrations in addition to the ones used in this study.
Conclusion
From the results obtained in this experiment, it is
evident that kavalactones are effective dopamine
enhancers as it was able to influence certain behavior
changes in fruit flies. Flies in 100 mg kavalactone
concentrations produced the highest survival rate in
wild type flies while 300 mg concentration produced
the highest in ebony dopamine mutant flies. Fruit fly
variants placed in different concentrations suggested
R1 to be the highest yielding in mortality rate, which
inferred that survival rate is based first on the fruit fly
type (i.e. wild, R1, AND R2), then based secondary on
what kavalactone concentration it was placed in.
As for light assays, wild type flies in 300 mg
kavalactone concentrations spent the most amount of
time in light, and upon further analysis, male flies were
seen to make up most if not all of this group. This data
suggests that only at the 300 mg concentration was this
behavior fully adopted, and only within male flies.
Correlating to the study conducted in 2004, the specific
concentration of 300 mg kavalactones were effective in
treating anxiety by improving mood and cognitive
performance30. This effect is due to the modulation of
gamma-aminobutyric acid (GABA) receptors as well
as inhibition of inflammation and monoamine oxidase
B38. GABA r ceptor functions in kavalactones mimic
function in benzodiazepines (tranquilizer) as increase
in GABA activity inhibits calcium channels, therefore
inhibiting process of neuronal firing30.
The locomotion assay however fully rejected the initial
hypothesis of increased dopamine concentration
influencing larval locomotion within fruit fly offspring
as the control group containing no kavalactones
yielded the greatest amount of locomotion activity.
While the hypothesis may have been reasonable given
previous studies of dopamine increasing locomotion
activity in fruit flies, the results of this study suggests an
inverse relationship instead where increased dopamine
inhibits locomotion in larvae.
ACKNOWLEDGMENT
This study was supported by the California University
of Pennsylvania Biology department through use of its
labs and equipment. I thank my advisor, Dr. Nicholson
as well as my thesis committee for providing necessary
support and feedback throughout this study.
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article/pii/B978012374927700008X
An Honors Thesis
by
Lanieta Waqanivalu
California, Pennsylvania
2021
Dopamine Influence on Behavior
Change in Fruit Flies
Lanieta K. Waqanivalu
Advised by Dr. Louise Nicholson
University Honors Program: Biology Department, 250 University Ave., California
University of PA, California, PA 15419
KEYWORDS: Dopamine influence, kavalactones, fruit fly locomotion, fruit fly survival
rate
ABSTRACT: Dopamine levels in fruit flies have been linked to a number of specific
mutations that have influenced acquired and learned behavior. Increased dopamine
levels have altered normal behavior patterns in that they have been found to decrease in
offspring larval locomotion. The purpose of this research is to identify locomotion
activity in offspring of fruit flies with different dopamine levels through several
generations, as well as to identify the boundary at which dopamine influences different
locomotion activities between fruit fly (parents) and larvae (offspring). To conduct this
experiment, several mortality and locomotion assays will be utilized to examine
dopamine influence in kavalactones and determine specific behaviors it inhibits or
initiates.
Introduction
Drosophila melanogaster, commonly known as
fruit flies, are an Insecta class derived from the
phylum Arthropoda and are often found infesting
homes by feeding on ripe or decayed fruits and
produce. Fruit flies have been widely used as
model systems in many branches of biology,
including research pertaining to psychological and
social behaviors. These studies have examined a
wide range of behaviors, and determined patterns
of aggression, excitement, courtship, and mating
abilities in generations of fruit flies1. Studies have
also investigated the roles of specific neuronal
signaling pathways and have found that the
neurotransmitter known as dopamine influences
specific behaviors such as regulating arousal,
promoting risk taking behavior, influencing malemale courtship, and altering locomotor activity
between parents and offspring2.
Dopamine is known to be an important chemical
messenger in the brain, involved in reward
motivation, motor function, compulsion, perseveration, sensation and regulating body movement3. This neurotransmitter is found in several
areas of the brain including frontal cortex, nucleus
accumbens, striatum, and hippocampus. The
distinctive molecular structure of dopamine adds
to its unique nature as it is derived from a
catecholamine family, which makes up approximately 80% of the catecholamine content in
the brain4. Dopamine is featured by a single amine
group that is connected to a benzene ring with two
adjacent hydroxyl groups and a side chain of
ethylamine5. Because dopamine is synthesized in
the brain, it is unable to pass through the blood
brain barrier that inhibits the interaction of
neurotransmitters and foreign substances trying to
enter the central nervous system. A precursor of
dopamine known as L-DOPA is where this
neurotransmitter is originally derived from. To
produce dopamine, a phenylalanine hydroxylase
converts phenylalanine to tyrosine, which is then
converted to L-DOPA, then later altered to
dopamine through decarboxylation of the aromatic
aminoacid.
Currently, three major brain dopamine
pathways have been determined to be involved
in brain actions: the tuberoinfundibular hypothalamic system, the mesolimbic-cortical of the
ventral tegmentum, and the nigrostriatal of the
brain’s A9 region7. Dopamine has been found to
function heavily in altering behavioral patterns
which are both impulsive and inflexible, and
drive a key role in pleasure related behaviors
such as sex, social interaction, food, and even
drug addiction8. Recent studies in dopamine
signaling has showed that dopamine regulates
behavior through the mesolimbic dopaminergic pathway, specifically within the D1
and D2 receptors9. D1 receptors stimulate
Figure 1. Dopamine Synthesis Pathway6.
intracellular cyclic adenosine monophosphate (cAMP)
levels, which are ATP derivatives used for intracellular
signal transduction, while D2 receptors inhibit cAMP.
When dopamine binds to the D1 receptor, an enzyme
known as adenylate cyclase is activated, increasing cAMP
synthesis, but binding to D2 receptor inactivates adenylate
cyclase, decreasing production of cAMP10. Inhibition of
cAMP then activates protein kinase A, but its regulation is
based on the dissociation of the D2 receptor whichin itself
is inhibited by Ca2+ . Affinity of these receptors influence
D2 to have a 10-100 fold greater affinity than that of D1,
which has the lowest affinity across all dopamine
receptors. D1 receptors are coupled with G proteins
(guanine-binding proteins) to activate adenylyl cyclase
activity and increase cAMP production. Activation of
cAMP induces the activation of protein kinase A, allowing
phosphorylation to occur as well as adjustment of various
ion channels. D2 receptors are then coupled to the
previously mentioned G proteins, and due to its inhibition
characteristic, it decreases protein kinase A activity by
regulation of cAMP levels, thus activating potassium ion
channels and regulating others. These D2 receptors which
are coupled to G alpha I and G alpha o activate Calcineurin, a protein phosphatase in charge of dephosphorylating DARPP-32 at Thr3412.
Figure 2. D2 Dopamine Receptor Signaling Pathway11.
D1 receptor stimulation have therefore been linked to
1
DARPP-32
(cAMP
regulated
neuronal
phosphoprotein) phosphorylation during protein
kinase A activation, while D2 reduced phosphorylation, presenting a bidirectional pathway on
dopamine signaling molecules.
Because dopamine has been used in pharmaceuticals as an antipsychotic drug, its components
of the D1 and D2 like receptors play majors roles
in motor function, specifically within the striatal
out- flow pathways13. In this pathway, D1
receptors are attached to GA- BAergic medium
spiny neurons (direct pathway) which project out
to the substantia nigra and medial globus pallidus,
while D2 receptors are attached to the lateral
globus pallidus, which is the indirect pathway7.
Studies on antidopa-minergic drugs have found
D1 and D2 receptors to be located on separate
neurons as activation of D1 enhance motor
function, wile D2 regulates behavioral response14.
These receptor signals of the D1 and D2
communicate through the βγ signaling portion of
the Gα subunits, allowing for their agonist/
antagonistic relationship.
Figure 3. Schematic of the striatal outflow
pathway15.
Before the D1 and D2 receptor characteristics
were fully known, functional selectivity was
found through experimentation of antipsychotic
dug aripiprazole16. This finding suggested that
different signaling pathways could be mediated
by a single receptor, i.e. influencing different
outcomes within different pathways. D1- like
receptors have been shown to mediate voltagegated ion channels both directly and indirectly
within the striatal outflow pathway. These
receptors are dependent on factors such as G
proteins, G protein receptor kinases (GRK), βarrestins and more17. In addition to their
involvement in signaling and striatal outflow
pathways, they have also been found to evoke
functionality within mitogen-activated protein
(MAP) kinase and calcium signaling pathways. In
MAP kinase, D1 receptors function as inhibitors
to inactivate p38 MAP kinase as well as c-Jun
amino terminal kinase (JNK) through its agonist,
SKF383918. Similarly in the calcium signaling
pathway, D1 receptors directly interact with
calcium channels, inhibiting N-type channels
which consist of α1B primary subunit, as well as
α2δ and β auxiliary subunits, which are localized
to the nerve and dendritic terminals of neurons 19.
This functional selectivity characteristic of D1
receptors mediating different signaling pathways
provide varying inactivation and inhibition properties,
suggesting its function to be selective upon proteins the D1
receptor is coupled toand the signaling pathway it undergoes.
Unlike D1, D2- like receptors are adenylate cyclase
inhibitors in dopamine signaling pathways, inactivated by
pertussis toxin through catalyzation of ADP-ribosylation20. In addition to this signaling pathway, D2
receptors are also involved in regulation of sodium
channels and play a major role in G- protein independent
signaling. In sodium channels, depending on the type of
D2 receptor involved, sodium currents can be increased or
decreased to allow access of potassium channels in
neuron21. Within G proteins, independent signaling is
promoted by GRK through phosphorylation of D2
receptors, which leads to binding of β-arrestins7. Because
β- arrestin signaling is a time-consuming mechanism, in
some cases, G- proteins initiate response through
utilization of cAMP and protein kinase A in order to
accelerate the response propagated from signaling
pathways22.
While dopamine can be obtained from readily available
supplements, it can also be obtained from other natural
sources. To provide and obtain natural dopamine,
kavalactones that are extracted from kava root (Piper
methysticum) will be utilized as a dopamine source in this
study. Grown and harvested in the South Pacific Islands,
kavalactones in kava have been shown to increase
dopamine concentration in mice through blockage in
reuptake of norepinephrine, regulating serotonin and
increasing dopamine levels23. While kava is normally
consumed as an alcoholic drink, it’s calming effect
contrasts that of alcohol as it reduces stress-induced
anxiety and insomnia24. Kavalactone compounds are
currently under pharmaceutical research for psychotropic
effects, but its complex structure suggests enzymatic
activity. Kavalactones are derived from lipophilic
compounds, which are molecules able to be dissolved in
lipids, as well as non- polar solvents25. Currently, six
major kavalactones have been identified: kavain,
methysticin, yangonin, dihydrokavain, dihydromethysticin, and desmethoxyyangonin.
Figure 4. Chemical structures of the six major kavalactones26
In addition to these kavalactones, studies conducted on
their structure determined them to be molecular targets
of proteins such as histamine type 1 and 2 receptors,
dopamine receptors, opioid μ and δ receptors, and γaminobutyric acid type A receptors27-28. Through
kavalactone’s natural metabolic pathway in humans,
hydroxylation of the C-12 aromatic and lactone rings
occur, followed by demethylation of the 4-methoxyl
group, and reduction of the 7,8- double bonds26. These
hydroxylation and demethylation processes are conducted by CYP2D6, which are dopamine metabolizing
enzymes involved in the reversal of kava hepatoxicity29. Studies conducted have found kava-lactones
to be effective in treating psychotropic effects such as
anxiety and insomnia as they maintain similar changesin
brain wave activity as Valium30. In addition to this
finding, a study conducted in 2004 found that at the
specific concentration of300 mg, kavalactones improve
mood and cognitive performance, similar to the effects
of dopamine related behaviors30. It is inferred that
kavalactone will increase dopamine concentrations in
fruit flies and enhance locomotion activity in its
offspring and future generations. This study focuses on
the behaviors influenced by different kavalactone
concentrations, as well as validating the hypothesis
previously stated.
Methods
Fruit fly Strains
In this study, fruit fly strains used include wild type,
ebony, and mutants of the R1 and R2 types. Wild types
are those of the typical phenotypic fruit fly forms as it
occurs in nature, and ebony are mutants that encode
specific proteins, linking beta-alanine to amines like
dopamine31. Mutants of the R1 and R2 types
correspond to D1 and D2 receptors of the dopamine
signaling pathway.
Kavalactone Treatment
Flies were raised on standard cornmeal-agar fly food at
25C, with either 0 (control), 100mg/ml, 300mg/ml or
500 mg/ml kavalactone. For wild type flies and ebony
(dopamine synthesis mutants), three 100 mg, 300 mg,
and control vials were prepared with five females and
five males in each, totaling ten fruit flies in each vial.
Wild type flies were then left to feed and mate for a
period of 30 days with observations conducted on the
12th, 22nd, and 26th day. Ebony flies were left to grow
for a period of several days with observations made on
the 5th, 10th, and 15th day (data still being collected for
later time points).
For assays involving the R1 and R2 dopamine receptor
mutants, due to lowered viability and the larval
maturation process of R1 type, only two were placed in
each of these vials, one male and one female as there
were only six fully mature. These flies were then also
left to grow and mate then observed on the 4th day.
Light Assay for Anxiety-like Behaviors
For light assays32, fruit flies from the wild type vials
were extracted and placed into a clear box that was
taped on one half to provide adark setting and left clear
on the other half to provide for light set ting. Once the
fly was placed in and the box closed, five minutes was
given for the fly to adapt to its new environment then
observed for a period of ten minutes and timed on how
long it spent in the light. Altogether, three runs of each
concentrations were conductedon both male and female
flies then averaged for data analysis. As for the light
assay involving different fruit fly variants, one female
and one male fly of ebony, R1, and R2 were observed
from each concentration. Similar to the wild type
assay, three runs were conducted across each fly type
in each concentrations then averaged for data analysis.
Locomotion Assay
For locomotion assay33, a vial was measured to 5 cm
from the bottom and marked with a sharpie to
emphasize boundary line marker.After preparation, wild
type and fruit fly variants in different kavalactone
concentrations previously used on light assay were then
tested by placing each group in the prepared vial, then
observed for how many crossed the boundary marker.
To maintain consistency across all groups, the vials
were tapped on a surface to allow all flies to start from
the bottom then timed for ten seconds and observed.
Data was obtained from the number of flies of each
group that crossed the boundary marker then averaged
from three runs.
Results
Mortality/ Survival Rate
Figure 5. Fruit fly mortality in wild type flies.
Average survival rates of wild type flies on different
concentrations of kavalactones are shown over three
trials. To first determine whether kavalactones were
effective dopamine sources for this study, a mortality
assay was conducted to observe fruit fly survivalrates
in those placed in kavalactone infused food,
compared to the control group with no kavalactone
concentrations. Mortality observation in wild type
fruit flies displayed a constant depletion throughout
time. Flies in kavalactone concentrations appeared to
have a slightly higher survival rate compared to nonkavalactone concentration groups. Flies in 100 mg
kavalactone concentrations showed the highest
survival rate compared to the higher concentrations
and to controls. While concentrations in 300 mg
kavalactones produced a lower survival rate then its
lesser counterpart (100 mg), it ranked this
concentration higher than the control group with no
kavalactones.
Figure 6. Fruit fly mortality in ebony flies.
Ebony fruit flies carry a specific feature as they contain
a defect in their ebony gene, which encodes a β-alanyldopamine (DA) synthase (BAS), thus increasing
dopamine concentration within them34. In these mutants, the presence of kavalactones reduced theirsurvival,
compared to flies reared on control food. Observation
of these dopamine mutants yielded 300 mg kavalactone
concentrations to contain the highest mortality across
all other concentrations. 100 mg concentrations
followed shortly after in mortality rate with a difference
less than 10% compared to 300 mg. 500 mg
kavalactones produced the lowest rate of all three
kavalactone concentrations as it maintained a constant
rate throughout all observations. Although 500 mg
were lower in rate, flies placed in control group yielded
a result slightly lower than this, leaving a difference of
more than 10% mortality rate between its flies and
those in 300mg concentrations.
Figure 7. Survival rate in different fruit fly variants.
In preliminary observations of dopamine receptor
mutants and kavalactone concentrations, R1 flies
displayed the highest survival rate as there were only
two placed in each vial compared to the ten placed in
vials of other fruit fly types. R2 flies produced second
highest survival rate as all flies were alive during
observation and some had even begun to produce
larvae. As these are preliminary results, summary
figures of flies in 300 mg concentrations could not be
drawn due to difference in sample sizes. This portion
of the assay is currently ongoing, therefore yielding no
conclusions currently. For the current data however,
results suggest control and 100 mg concentration flies
had the highest survival rate when compared to other
fruit fly types in 500 mg concentration.
Light Assay
Table 1. Light assay of wild type flies.
In this assay, kavalactones were used to determine
anxiety-like behaviors in fruit flies by comparing male
and female wild type flies, with an average of three data
runs taken and placed in the table above. Differences
between male and female anatomy did not affect this
study, but concentrations did as data varied across this
category. According to the data obtained, flies in 300
mg concentrations spent more time in light with male
averaging 87s (47%) and females at 47 s (23%).
Comparatively, flies in control group with no kavalactone concentrations yielded the least amount with
male averaging 11.75s (0.06%) and females at 23s
(0.11%). Flies in 100 and 500 mg concentrations were
similar in that the difference in amount of time were
proportionate, b u t diverged in that males spent more
time in light in 100 mg concentration compared to females in 500 mg.
Table 2. Light assay of male variant flies.
In this assay comparing wild, R1, and R2 type flies of
different kavalactone concentrations, sex anatomy
played a major role in that female flies spent little to
no time in light environments (table omitted due to no
data) and rather preferred dark settings throughout
the observation time frame. Wild and R2 female flies
in the control group with no kavalactones produced
minimal data, which is substantiated in the table
above. As for males, flies in 100 mg concentrations
spent the most amount of time in light as wild type
averaged 262 s (65%) and R1 at 32 s (62%).
Following 100 mg concentration, flies placed in the
control group yielded the second highest number of
male flies in light with wild type at 14 s (0.03%) and
R2 at 168 s (79%). Flies in 300 and 500 mg
concentrations then followed after with the 500 mg
kavalactone concentration ranking above 300 mg in
the number of flies preferring light environment.
Figure 8. Locomotion assay of fruit fly parents.
Due to lack of time in conducting this study, not
enough R1 and R2 type flies arrived in time to be able
to fully complete this assay. My data for this portion
of the assay are inconclusive but the statements made
below only pertain to wild type flies and 100-300 mg
concentrations of the R2 type.
In this assay, R2 fruit flies displayed the highest
amount of locomotion when averaged with fruit fly
types. Although the 500 mg concentration was tested
against the wild type, its result is not included in this
analysis as it was not tested against R1 and R2 type
flies. Exclusion of this concentration therefore
provided the R2 type with the highest locomotion in
fruit fly parent category. Wild type fruit flies
produced the second highest locomotion with both
100 mg and control groups yielding more locomotion activity, although R2 type leads in 300 mg
concentrations when compared to wild types.
Figure 9. Locomotion assay of fruit fly offspring.
In this category of locomotion assay of only wild type
offspring fruit flies, the control group with no
kavalactones produced the highest amount of locomotion activity by more than five times than that of the
second highest yielding which is the 300 mg concentration. Offspring fruit flies from the 100 mg
concentration yielded the least amount of locomotion
activity, which is only about 1.5 times less than that of
the 500 mg kavalactone concentration.
Discussion
As seen in the results of fruit fly mortality or survival
rate, wild type fruit flies in 100 mg kavalactone
concentrations had a better survival rate when
compared to other concentrations. This suggests that
those less than 100 mg kavalactone concentrations
were not enough to influence any activity in fruit flies,
such as that of the control vials. This neuroprotective
effect may be due to the mediation by several factors
such as P38, nuclear factor-kappaB, and cyclooxygenase2 signaling pathways35. Similarly how-ever,
they could also be mediated by the upregulation of
antioxidant enzymes by ERK phosphorylation, translocation of the transcription factor Nrf2, binding of the
antioxidant responsive element (ARE), and activation
of the heme oxygenase I (HO1) enzyme to induce
nitric oxide synthase (iNOS) which allows for cell
survival35.
Figure 10. Neuroprotective pathways of kavalactones35.
For the ebony dopamine mutant flies, 300 mg yielded
the highest survival rate, thus suggesting the 100 mg
concentration to not be enough, and the 500 mg
concentration to be too much to the extent that it killed
some of the flies. Because these dopamine mutant flies
were already concentrated with dopamine, its effects at
500 mg concentrations are valid as it correlates to the
underlying biology that too much of anything could be
lethal. This suggests that kavalactones are dosedependent and that at lower concentrations are not as
effective, but at too much concentration could prove
lethal. In the assay involving several fruit fly variants
in different dopamine concentrations, the results were
inconclusive as there was not enough of the R1 and R2
type flies available to be able to accurately conduct the
assay without skewing the results. Therefore, for the
fruit fly parent locomotion assay, no conclusion can be
drawn due to lack of data.
As for the light assay of wild type flies, sex anatomy
did not make a difference as data was dependent on
the kavalactone concentrations. Flies in 300 mg
kavalactone concentrations were seen to have spent
the most amount of time in light, whereas the control
group spent little to no time in light. This data
suggests that at the specifickavalactone concentration
of 300 mg, normal behavior was impacted in that
flies that would normally prefer dark settings have
been influenced to change behavior and spend more
time in light. Due to these results, it is suggested that
kavalactones are anxiolytic at 300 mg concentrations,
as they were the most interactive in light
environment. This behavior change looks to have
been initiated however at the 100 and 500 mg
concentrations as their data correlated to each other
but differed in the sex that it accounted for.
In the light assay of fruit fly variants and concentrations, sex anatomy looks to not have played a role
based on the results obtained from data. Female fruit
flies of the wild, R1, and R2 type were notinfluenced
in the behavior change to conform to light
environments as they spent no time in light, except
for its control group which spent less than five
seconds in light, thus substantiating this theory.As for
the male flies however, behavior change was
dependent on the kavalactone concentration rather
than the type of fruit fly involved. As suggested in
previous studies, anxiety behaviors in fruit flies are
not sex-dependent36, suggesting that anxiety is not
one of the behavioral patterns influenced by the flies’
sex but rather by external factors, which in this case
are kavalactones. Flies in 100 mg concentrations
averaged the most amount of time in light com pared
to the 300 mg concentration which averaged the least.
Results from this assay suggested that 100 mg
kavalactone was the right amount of concentration
needed to influence behavior change and that 300 mg
could most probably have been too much. 500 mg
concentrations however yielded the third highest in
the amount of time these flies spent in light,
suggesting that another behavior change may have
been initiated at this concentration level within the
different type flies.
In locomotion assay of parent fruit flies, analysis was
made without consideration of the 500 mg
concentration as there was not enough of this fly type
to test across all concentrations. Results obtained
from the parent locomotion assay showed R2 type to
have produced the greatest amount of locomotion
when averaged with flies from other groups. R2 type
yielded the greatest locomotion activity in parent
flies, suggesting that R2 type without kavalactones
was the most efficient concentration to influence this
behavior.
In locomotion of offspring fruit flies, the control
group containing no kavalactones produced the
highest amount of locomotion activity in wild type
flies with 300 mg being the second highest. This
assay was only conducted on offspring of wild type
flies as there were not any offspring yet mature from
R1 and R2 type. Data obtained from this assay
suggests that dopamine concentration has no effect on
offspring larval locomotion, thus invalidating the
initial theory of correlation between increased
dopamine concentration leading to increased larval
locomotion. In fact, studies conducted suggest an
inverse correlation in that increased dopamine levels
led to reduction of larval locomotion as signaling
efficiencies diverged37.
While this study proved successful in validating and
rejecting the initial hypothesis, certain changes would
have to be made when reconducting this experiment.
Due to the time frame needed for fruit flies to mate and
produce larvae, a time frame of two to three semesters
should be allotted to allow for accurate development of
results. As there was only one semester allotted for this
experiment, some results obtained may not be as
accurate as fruit flies needed more time to produce
larvae. Another change that would also haveto be made
would be maintaining a constant rate of observations
across all assays compared to the varied rate in this
study. Although changes would have to be made, further
enhancements of this study could be conducted through
exploration of other behaviors influenced by
kavalactones, and testing for several other concentrations in addition to the ones used in this study.
Conclusion
From the results obtained in this experiment, it is
evident that kavalactones are effective dopamine
enhancers as it was able to influence certain behavior
changes in fruit flies. Flies in 100 mg kavalactone
concentrations produced the highest survival rate in
wild type flies while 300 mg concentration produced
the highest in ebony dopamine mutant flies. Fruit fly
variants placed in different concentrations suggested
R1 to be the highest yielding in mortality rate, which
inferred that survival rate is based first on the fruit fly
type (i.e. wild, R1, AND R2), then based secondary on
what kavalactone concentration it was placed in.
As for light assays, wild type flies in 300 mg
kavalactone concentrations spent the most amount of
time in light, and upon further analysis, male flies were
seen to make up most if not all of this group. This data
suggests that only at the 300 mg concentration was this
behavior fully adopted, and only within male flies.
Correlating to the study conducted in 2004, the specific
concentration of 300 mg kavalactones were effective in
treating anxiety by improving mood and cognitive
performance30. This effect is due to the modulation of
gamma-aminobutyric acid (GABA) receptors as well
as inhibition of inflammation and monoamine oxidase
B38. GABA r ceptor functions in kavalactones mimic
function in benzodiazepines (tranquilizer) as increase
in GABA activity inhibits calcium channels, therefore
inhibiting process of neuronal firing30.
The locomotion assay however fully rejected the initial
hypothesis of increased dopamine concentration
influencing larval locomotion within fruit fly offspring
as the control group containing no kavalactones
yielded the greatest amount of locomotion activity.
While the hypothesis may have been reasonable given
previous studies of dopamine increasing locomotion
activity in fruit flies, the results of this study suggests an
inverse relationship instead where increased dopamine
inhibits locomotion in larvae.
ACKNOWLEDGMENT
This study was supported by the California University
of Pennsylvania Biology department through use of its
labs and equipment. I thank my advisor, Dr. Nicholson
as well as my thesis committee for providing necessary
support and feedback throughout this study.
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