“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. REFERENCES 1. Dukas, R. (2020). Natural history of social and sexual behavior in fruit flies. Scientific Reports,10(1),21932. https://doi.org/10.1038/s41598-02079075-7 2. .Kume, K. (2005). 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