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BEHAVIORAL EFFECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster BY CRISTINA CAMAYD A Thesis Submitted to the Division of Natural Sciences New College of Florida In partial fulfillment of the requirements for the degree B achelor of Arts Under the sponsorship of Professor Sandra Gilchrist Sarasota, Florida April, 2010
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster ii ACKNOWLEDGEMENTS The following individuals deserve immense credit and gratitude for their contributions to this project: Professor Edward Kravitz not only for opening his lab up to me and finding summer funding for my work, but for fostering each of the lab's members in an environment where his guidance and support was never hard to find. Dr. Olga Alekseenko for suggesting this project to me, taking t he time to oversee its completion, creating the TRH driver, providing me with all true breeding fly lines, and patiently answering all of my questions. The laboratory of Dr. David Corey for use of their confocal microscope and Dr. Adelaine Leung for obta ining the confocal images. Dani Sanchez for being an exceptional lab bench mate and laughing with me when experiments went horribly wrong. Jacob Long for his encouragement and accuracy check. All Members of the Kravitz lab for their advice, discourse and companionship. Professors Amy Clore, and Alfred Beulig for their review and feedback.
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster iii ABSTRACT Serotonin is the molecule most frequently implicated in the modulation of aggression in mammals. Recent findings have brought into q uestion previous suspicions that aggression in Drosophila melanogaster is unaffected by the serotonergic system. Development of a novel transgene based on the promoter of tryptophan hydroxylase can now be used to spatially restrict genetic manipulations to serotonin positive cells in the central nervous system (CNS) of Drosophila melanogaster By pairing this promoter with the GAL4 UAS system endogenous overexpression of serotonin transporter (SERT) was driven in the CNS, and behavioral effects were charact erized using an aggression assay. SERT overexpressing flies showed a predisposition to male male courtship, exemplified by an increase in mutual singing during the beginning of the observation period. Overexpression of SERT resulting in altered fight dynam ics. SERT overexpressing flies were less aggressive during the middle of the observation period in regards to lunging behavior. Provisional examination of temperature sensitive GAL80 dependant overexpression of SERT found the period of heat shock necessary to cause temporally restricted overexpression. Future comparisons between the behavioral phenotype that has been characterized and behavioral phenotypes yet to be characterized may provide insights into the dynamics of the serotonergic system.
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster i v TABLE OF CONTENTS Acknowledgments ii Abstract iii Chapter 1 Introduction 1 Model Organisms 1 Behavior 3 Neurochemistry 8 Transgenic Flies 21 Overexpression of Serotonin Transporter 23 Chapter 2 Materials and Met hods 27 General 27 Aggression Assay 32 Antibody Labeling and Microscopy 37 Chapter 3 Results 38 Statistics 38 Fly Pair Activity and Mutual Behavior 40 Individual Fly Behaviors and Dominance H ie rarchies 42 Preliminary GAL80ts Temporal Control 46 Chapter 4 Discussion 48 Male Male Courtship 49 Altered Fight Dynamics 50 Directions of Future Studies 53 Conclusions 54 Cited References 55 Appendix 60
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster v LIST OF FIGURES 1.1 Ethogram of male Canton S wild type Drosophila melanogaster aggression 4 1.2 Diagram of synaptic trafficking of serotonin 10 1.3 Diagram of serotonin (5 HT) synthesis 11 1.4 A current model of serotonin transporter fu nction 17 2.1 Diagram of the genetic mosaic used to induce chronic SERT overexpression 30 2.2 Diagram of the genetic mosaic used to induce acute SERT overexpression 31 2.3 Photographs of the observation chamber used in aggression assays 33 3. 1 Overall frequency of mutual singing in all flies 40 3.2 Temporal dynamics of mutual singing in all flies 41 3.3 Overall frequency of lunging in flies that demonstrated the behavior 43 3.4 Temporal dynamics of lunging in flies that demonstrat ed the behavior 44 3.5 Overall frequency of lunges in all flies 45 3.6 Temporal dynamics of lunging in all flies 46 3.7 CNS confocal images of temporal SERT overexpression after varying lengths of heat shock 47
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster vi LIST OF TABLES 1.1 The study of aggression in Drosophila melanogaster 7 1.2 The study of serotonin manipulations and aggression 12 1.3 The study of serotonin receptor manipulations and aggression 15 1.4 The study of serotonin transporter manipulations and behavior 19 1.5 Downstream neurochemical observations in SERT deficient mice 21 2.1 The true breeding parental lines crossed to form each genotype 28 2.2 Results from yeast paste trials 60 2.3 Classifications and operational d efinitions of the behaviors noted 36 3.1 Mean ranks of statistically significant findings 60
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 1 CHAPTER 1 INTRODUCTION The following is an examination the behavioral effect of spatially restricting overexpression of serotonin transporter in Drosophila melanogaster to serotonin positive cells in the central nervous system (CNS). It is important to explore serotonin transporter (dSERT) in D. melanogaster because it is very similar to serotonin transporter (SERT) in huma ns. Serotonin transporter is the target of many drugs including serotonin reuptake inhibitors, yet little is known as to why altering the function of serotonin transporter is able to treat depression and alter behaviors. For this reason, the behavioral eff ects as seen in aggression assays are examined. Coupling the tryptophan hydroxylase enhancer sequence with the driver in a GAL4/UAS system spatially restricts serotonin transporter overexpression to serotonergic cells. By characterizing the behavioral phen otype of serotonin transporter overexpression in D. melanogaster one should be able to determine correlations with the behavioral phenotypes observed when novel elements of the serotonergic system are altered. In so doing behavioral characterization will be used to observe dynamic molecular processes. I. Model Organisms By genetically manipulating model organisms it becomes possible to identify, characterize, and manipulate circuit elements to establish the causal relationship between the biochemistry of in dividual neurons, the function of multiple neurons within a circuit, and the animal's overall behavior (Dickson, 2008). Drosophila melanogaster is a natural choice for this purpose owing to its success as a genetic model organism. The resulting
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 2 availabilit y of genetic constructs renders targeted manipulations possible. More generally, the overall knowledge of the organism allows results to be placed within the context of a rich body of knowledge dedicated to this organism. Model organisms serve by represent ing a larger group of organisms or a specific organism for which direct observations of the kind being undertaken are not practical. The model is often selected based on how well it can be expected to represent that group or specific organism, but care mus t always be taken when attempting to extrapolate a finding to signify something outside of the parameters within which that observation was made. For instance, the behavioral phenotypes observed in this study are not suspected to represent what would resul t if the genetic manipulations performed here could be faithfully recreated in a human subject. Rather, these behavioral phenotypes have been characterized to observe manipulations of the serotonergic system in the CNS at an organismal level in the form of action selections. It is the dynamics of the neuronal serotonergic system that are of primary interest, and not the nature of aggression within Drosophila melanogaster The latter of which must be concerned with the extent to which laboratory observations reflect those that take place in nature, while the former is more concerned with the extent to which elements of the serotonergic system are conserved.
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 3 II. Behavior Aggression and courtship are species specific innate behaviors composed of discernable act ions available for quantitative and qualitative analysis (Kitamoto, 2002; Chen et al., 2002). Due to the strong genetic determination of these innate behaviors in socially nave flies, behavioral phenotypes can be used to characterize a genetic manipulatio n. While molecular analysis is required to confirm the immediate effects of a genetic construct, analysis at an organismal level avoids in vitro artifacts and is sensitive to the possible secondary or downstream effects of a genetic construct or expressed molecules. Additionally, it is important to understand what constitutes a behavior and how an observed behavior can be consistently defined. While the classification of an action as a behavior may seem intuitive to an observer, there must be some non arbi trary external criteria to assess the temporal span of the behavior and its definition if qualitative and quantitative analysis is to be done. For instance, the original classification of aggression behaviors for this purpose was done mathematically with t ransition matrices and Markov chain analyses (Nilsen et al., 2004).
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 4 Figure 1.1 : Ethogram of male Canton S wild type Drosophila melanogaster aggression collected by Nilsen and colleagues (2002). Gender specific aggressive behaviors are shown i n blue. (Image from Nilsen et al., 2004) The size of a box indicates the average amount of time spent performing the behavior, and the size of the arrow indicates the frequency with which one behavior led into the other. Larger boxes indicate more comm on behaviors and thicker arrows indicate more common transitions. Behaviors encased in blue boxes where found to be specific to males. The large circular arrows indicate common sequences of behaviors, where blue circular arrows are male specific. The dist inct modules that were found were named and defined as aggressive behaviors (see boxes in Figure 1.1). Nonetheless, even the most standardized behavioral analysis will have slight differences between observers. To ameliorate this concern, analysis begins b y delineating the operational definitions of the behaviors of interest (see Chapter 2, Table 2.1 for more detail), and an observer strives to remain consistent throughout the analysis. The addition of a second observer to evaluate a portion of the
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 5 data may serve as an accuracy check. The operational definitions of each behavior provide reproducibility of findings, and observer consistency ensures that differences between experimental and control groups reflect the underlying circuitry behind actions selecti ons. Since the overall purpose of this study is to use behavior to observe molecular differences, the exact definition of a behavior is far less important to findings than the differences between behavioral phenotypes when a given definition is used. Befor e discussing sexually dimorphic behaviors such as courtship and, as will later be discussed, aggression, it is worth reviewing how sex is expressed in Drosophila melanogaster Sex specific splicing of the fruitless and double sex genes result in the putati ve transcription factors fru and dsx respectively. Fru is expressed exclusively in the nervous system, while dsx is expressed throughout the organism. As the basis for sexual differentiation in the developing fly, study of these transcription factors has linked them both to the sex specific patterns of aggression, and to the pheromonal cues that mediate the decision to court (Vrontou et al., 2006; Dickson, 2008). Sex is also encoded by a sex chromosome. While he male bares a single sex chromosome, the fema le possesses two. This means than the female has a higher gene dosage of sex linked traits. While this effects the breeding of flies, the gene dosage is consistent among the male flies examined by this experiment. An addition consideration is the effect of inbreeding on the variability of genes within the fly populations that are studied. While standard fly husbandry attempts to limit inbreeding, there is no doubt that it persists within the laboratory strains of D. melanogaster and other model organisms. I nbreeding will result in a decrease of genetic variance within the population and an increase in the observation of recessive
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 6 phenotypes. This may signify that a greater variation of a trait being examined may exist than is seen in the laboratory. However, the use of the laboratory strain of wildtype Canton S flies is useful because the aggression and courtship patterns have been so well established, and studies are able to compare findings within a single strain. The use of the laboratory strain of D. mela nogaster is common but the resulting limit to genetic variability should not be overlooked. a. Courtship The courtship ritual consists of a series of actions that can be grouped into behaviors, with the ultimate goal of successful copulation. The courtshi p ritual can be characterized by a fly orienting to the posterior of its courtship target, singing (unilateral wing extension and vibration), tapping (the abdomen of the target with an anterior leg), licking (extending the proboscis to the abdomen of the c ourtship target), and mounting. A male fruit fly may or may not court an encountered fly based on the male's previous courtship experience, the other fly's pheromonal cues, and the male's interpretation of those cues. A female chooses whether to accept cou rtship based on the male's pheromones and acoustic signals, as well as the female's readiness to mate. Pheromonal cues consist of volatile pheromones detected by the olfactory system and nonvolatile pheromones detected by the gustatory system (Dickson, 200 8). b. Aggression Aggression and territorial behavior, as part of the pursuit and defense of resources, has been well characterized in fruit flies (Table 1.1). An aggressive encounter is composed of a series of actions, postures, and signals rather than i njury inflicting blows
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 7 (Price, 1988). While its natural occurrence has long since been noted, adaptive aggression in Drosophila melanogaster has only recently been experimentally harnessed by Chen (2002) as a tool for observing the neurological and genetic basis of aggression, as well as the molecular processes leading to an observed aggression phenotype. Table 1.1 : The study of aggression in Drosophila melanogaster. Author Historical Milestones Sturtevant, 1915 Found aggressive behavior in Drosophila J acobs, 1960 Described aggressive behavior in Drosophila Spieth H, 1968 Species specific patterns of mating may have given rise to behavioral differentiation. Dow, 1975 Characterization of aggression in Drosophila and its evolutionary importance. Hoffman 1987 Aggression differs between fly strains, and appears under certain ecological conditions. Price and Boake, 1995 Analysis of courtship and aggression between two different species of Drosophila and their hybrids. Found that behavioral isolation was l ikely due to early interactions, rather than sexual selection against hybrids. Boake, Price and Andreadis, 1998 Analyzed the courtship and aggression behavioral differences between two species of Drosophila and their hybrids. The use of behavioral pheno types to deduce molecular events was framed by the discovery by Nilsen and colleagues (2004) that wild type Drosophila melanogaster follow a temporal and sequential pattern of aggression. The results show that females do not form clear hierarchical relatio nships, that certain modules are shared between the sexes while others are highly sex specific, and that transitions between modules common to both sexes differ in accordance with gender. In short it was found that aggression is sexually dimorphic. Further more, the discovery that manipulating the fruitless and
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 8 transformer genes can exchange these sex specific aggressive phenotypes highlights the genetic basis of aggression in socially nave flies (Vrontou et al., 2006). In addition to genetic determinants o f aggression, previous experiences have been shown to determine patterns of male aggression in cases where hierarchical relationships are established. The behavior of the fly after dominance is established, within that fight and in any fight that takes pla ce within the following two hours is altered in a learning dependant manner. This "loser effect" typically results in the perpetuation of the fly's hierarchical status into future aggressive encounters (Yurkovic et al., 2006). The aggression of a male frui t fly serves as an ideal candidate for observing the effects of molecular events on an organismal level, because it is able to establish complex dominance hierarchies, the fly's aggressive encounters exclude the physical effect and ethical dilemma of injur y to the subjects, and aggression assays do not require manipulation of otherwise placid animals. III. Neurochemistry While the impact of serotonin (5 HT) on aggression in Drosophila melanogaster has yet to be fully characterized, it is the molecule most frequ ently implicated in the neuronal basis of aggression (reviewed by Miczek et al., 2007). Previous studies have found opposing and at times no effect of manipulating molecular elements of the serotinergic system ( see T ables 1.2 1.5)
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 9 a. Serotinergic Syste m After the precursor to neuronal serotonin (5 HTP) is synthesized in the cell body, it is pumped into secretory vesicles by a vesicular monoamine transporter, and converted into neuronal serotonin (5 HT). The two mammalian vesicular monoamine transporters VMAT1 and VMAT2, are replaced by a single ortholog, dVMAT, in Drosophila dVMAT does this for 5 HT, dopamine, and octopamine (the invertebrate homolog of noradrenaline). Vesicles transport the neurotransmitter to the nerve terminal. Here the vesicles fus e with the cell membrane releasing 5 HT into the synapse. Dynamin allows reformation of the vesicle after exocytosis. Once in the synapse, serotonin can bind to one of four types of serotonin receptors in flies (over fourteen in humans) on the membrane of post synaptic cells. The action of 5 HT binding to one of its receptors prompts a signal within the postsynaptic cell. 5 HT in the synapse may also bind to the serotonin membrane transporter (SERT), which terminates its synaptic transmission by 5 HT reupt ake into the presynaptic cell in an ion dependant manner. The transport of 5 HT between pre and post synaptic cells is depicted in figure 1.2.
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 10 Figure 1.2 : Diagram of synaptic trafficking of serotonin (Image by Camayd C) A schematic representation of how molecular elements of the serotinergic system facilitate synaptic transmission. b. Serotonin Synthesis Within the cytoplasm of serotinergic neurons, the enzyme tryptophan hydroxylase hydrolyzes L tryptophan to produce 5 hydroxy L tryptophan (5 HTP). Thi s is the rate limiting step of neuronal serotonin (5 HT) synthesis. 5 HTP is then transported to axon terminals and is decarboxylated by 5 hydroxytryptophan decarboxylase also referred to
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 11 as dopa decarboxylase or Ddc (Bender et al., 1987 as reviewed by Ba o et al., 2010). The activated form of neuronal serotonin is more formally called 5 hydroxytryptamine, indolealkylamine serotonin, or 5 HT. The process of 5 HT synthesis is outlined below in figure 1.3. Figure 1.3 : Diagram of serotonin (5 HT) synthesis. (Image by Camayd C) The steps completed, enzymes required, and chemical structures formed in the synthesis of serotonin (5 HT). The synthesis of 5 HT has been a popular target for observing the effect of 5 HT on aggression (Table 1.2). Altering the av ailability of synthetic precursors, or affecting the activity of enzymes involved in 5 HT biosynthesis manipulates endogenous levels of
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 12 5 HT. Other approaches have introduced exogenous 5 HT causing a rapid increase in its availability. Table 1.2 : The stu dy of serotonin manipulations and aggression Model Organism Author Method Effect on Aggression Livingston et al, 1980 Assume socially dominant posture. Lobster Huber et al., 1997 Exogenous 5 HT by infusion into the hemolymph. Subordinate animals en gage dominant ones. Crayfish Yeh, Fricke & Edwards 2007 Exogenous 5 HT by bath of isolated, dominant and submissive crayfish. The response of the lateral giant tail flip command neuron to sensory stimuli was differentially affected based on social ex perience. Endogenous 5 HT by feeding 5 HTP None. Baier et al., 2002 Endogenous 5 HT by feeding pCPA None. 5 HT pharmacologically # Of pairs that lunge 5 HT genetically in cells e xpressing Ddc # Of pairs that lunge Drosophila melanogaster Dierick and Greenspan, 2007 Silencing serotinergic circuit Show aggression but pharmacological effects on the behavior are lost The initial finding by Baier and colleagues (2002) supported the belief that in D. melanogaster serotonin had no modulatory effect on aggression. By feeding flies the synthetic precursor to 5 HT (5 HTP) or an inhibitor of 5 HT biosynthesis (pCPA), Baier raised or lowered endogenous levels of 5 HT. In contrast, earlier crayfish studies introduced exogenous 5 HT and ca used a rapid shift in available 5 HT. With this difference in mind, it is possible that Baier found no serotonergic effect on aggression in
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 13 D. melanogaster because the study caused prolonged alterations in total 5 HT levels, rather then rapid changes in 5 HT availability. The more recent finding by Dierick and Greenspan (2007) of a link between 5 HT and aggression has renewed interest in examining this within Drosophila melanogaster The increase of 5 HT was done in cells expressing dopa decarboxylase (Ddc) of which both dopaminergic and serotinergic cells are members, and fighting frequency was taken as the proportion of fly pairs that exhibited a lunging behavior. The limited behavioral analysis and ectopic expression of 5 HT in dopaminergic cells begs fo r additional investigation into the link between 5 HT and aggression. As was discussed earlier, aggression in male fruit flies is a complex behavior that includes many different postures, actions, and communicative signals of varying aggressive intensity. Lunging is a single one of these behaviors that can be seen to occur once or over a hundred times within an observation period. That is not to say that noting fighting frequency has no value. In fact this approach allowed Dierick to expand his sample size to one hundred fights. The study by Dierick rejected previous statements that 5 HT was not involved in the modulation of aggression in flies and renewed interest in analyzing the extent to which aggression is effected. The findings shown in table 1.2 that altering the rate of change in 5 HT levels leads to aggression effects in lobster and crayfish studies suggests that regulators of 5 HT levels may be important candidates for these studies. Examples of molecular elements that control the rate of change of 5 HT include 5 HT receptors, SERT, and VMAT. Since VMAT is not specific to 5 HT, the following two sections will focus on investigations of 5 HT receptors and membrane serotonin transporter molecules.
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 14 c. Serotinergic Receptors Drosophila 5 HT receptors ar e functional orthologues of human 5 HT receptors. Of the 5 HT receptors in Drosophila 5 HT 2 Dro is homologous to 5 HT 2 5 HT 1A Dro and 5 HT 1B Dro (thought to be duplicated genes) are homologous to 5 HT 1A and 5 HT 7 Dro is homologous to 5 HT 7 The possibility of opposing effects between different types of 5 HT receptors, an observation that has been made in both mammalian and fly systems (Table 1.3), complicates analysis. Even within a single type of 5 HT receptor, effects may vary in accordance with positionin g on the post or pre synaptic membrane. That is, 5 HT binding to the receptor may prompt a signal cascade within the postsynaptic cell, or 5 HT binding to the receptor may prompt a regulatory signal within the presynaptic cell. Finally, all of theses diffe rences in 5 HT receptor function may also differ depending on the brain region or subset of cells being observed.
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 15 Table 1.3 : The study of serotonin receptor manipulation and aggression Model Organism Author Receptor Serotonin Receptor Manipulation Aggression Effect Rats Reviewed by Miczek et al, 2007 5 HT 1A & 5 HT 1B Agonist on somatodendric autoreceptors & post synaptic receptors Aggression 5 HT 2 agonist Frequency of aggressive behaviors by 50% (lunging and boxing specifically) 5 HT 2 Dro 5 HT 2 antagonist None 5 HT 1A agonist Frequency of aggressive behaviors by 50% (wing threats and fencing specifically) 5 H T 1A antagonist Frequency of aggressive behaviors (wing threats, lunging, and boxing specifically) Drosophila melanogaster Johnson, Becnel and Nichols, 2009 5 HT 1A Dro & 5 HT 1B Dro Both 5 HT 1A agonist and antagonist Frequency of aggressive behaviors; near no lunging and boxing The finding of aggression effects resulting from 5 HT receptor manipulations in D. melanogaster further confirms a role for the serotinergic system in aggression. Based on the findings depicted in Table 1.3, increasing and decreasing the activity of 5 HT receptors in Drosophila differentially affects the level of total aggression, and primarily effects distinct behaviors. Increasing the activity of 5 HT 2 Dro and decreasing the activity
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 16 of 5 HT 1A like receptors seems to decrease aggression. However the agonist used to increase the activity of 5 HT 1A like re ceptors is also a 5 HT 7 agonist. Further decreased aggression levels upon agonist and antagonist treatment suggest that it is the 5 HT 7 agonist function that led to increased levels of aggression. The analysis of exactly how each receptor effects aggressio n may be made more clear as methods develop to specifically target each receptor, and the conditions under which each receptor serves a specific function become known. d. Serotonin Membrane Transporter Serotonin Membrane Transporter (SERT) is an integral membrane protein that recycles 5 HT from the synapse back into the presynaptic neuron through the active pump, Na + ATPase (Chen et al., 2004). In this it is considered to be a member of the transmembrane sodium symporter family, and of the two subclasses i t belongs to the SLC6 transporters. Other members of this subclass include the biogenic monoamine transporters of dopamine and noradrenaline/octopamine, as well as the amino acid transporters of GABA and glycine. The energetically unfavorable transport of 5 HT into the cell is driven by the influx of sodium ions across their concentration gradient. Transport is dependant on extracellular sodium ions, and intracellular chloride ions. A current model of SERT suggests that the binding of sodium ion to the carr ier protein, 5 HT to the transporter, and then chloride ion to the transporter, causes a conformation shift of SERT to its inward facing position (Figure 1.4). The Na + ATPase creates a concentration gradient, which is further established by the resulting l ose of intracellular chloride ions.
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 17 The SLC6 transporters are all characterized by 12 transmembrane helices, glycosylation sites in the loop between the third and fourth of these helices, potential for oligomerisation, phosphorylation sites, and both N an d C terminals reside intracellularly. All of these transporters are suspected to have a similar binding pocket that functions differently between amino acid and monoamine substrates (Wang and Lewis, 2010). Based on what is known of SERT, the crystallized structure of bacterial homologues within this subclass, and the expected differences based on non conserved sequences, a provisional model has been constructed of SERT function (Figure 1.4). Figure 1.4 : A current model of serotonin transporter function. (Image from Wang and Lewis, 2010) This figure demonstrates the binding of two sodium ions in red to the extracellular face of SERT in its proposed substrate binding conformation (A). Upon 5 HT binding salt bridges function as a gating structure to inhibi t extracellular 5 HT release (B). The binding of one extracellular (C) and one intracellular (D) chloride ions are thought to facilitate the conformational change between the substrate binding conformation and the open to in conformation. This hypothesized model is based on the crystallization of bacterial orthologues, chemical studies of SERT, and conserved or non conserved regions.
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 18 The Drosophila homolog (dSERT) to human serotonin transporter (SERT) demonstrates structural and functional conservation, wh ich give reason to expect similar dynamics of 5 HT reuptake in the D. melanogaster model. The initial characterization of dSERT, done by Demchyshyn and colleagues (1994), found only slight differences from human SERT. The 69kDa integral membrane protein sp ans 622 residues and shares its highest conservation, 51%, with human SERT. The structural and functional similarity between dSERT and SERT may be best exemplified by the ability of dSERT to be expressed in mammalian cell cultures, where it demonstrates ac tivity that is inhibited by cocaine (Park et al., 2006). However, dSERT is not as dependent on extracellular chloride ions, though they do facilitate 5 HT transport. Additionally anti depressants have been found to be between three and three hundred fold l ess potent in the case of dSERT (Demchyshyn et al., 1994). The decreased dependence of dSERT on extracellular chloride ions, and decreased sensitivity to certain antidepressants suggest that, while homologous, there are slight functional differences betwee n the two transporters. Additionally, the overall kinetics of the serotonergic system in the larval ventral chord of D. melanog aster were found to be analogous to that of mammals by real time measurement of extracellular 5 HT levels. This finding by Bourne and colleagues (2009) is especially significant to the investigation at hand because of the similarity with which serotonin positive cells where targeted via a promoter targeting tryptophan hydroxylase. The results demonstrated the similarities in 5 HT k inetics between species, suggesting that Drosophila may be an effective model for studying serotonergic plasticity. SERT has previously been examined with the intention of informing treatment and understanding of human disorders, and certain drugs of abuse that involve altered
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 19 SERT function. There have even been mutant mice models of inhibited SERT expression created to examine the downstream effects of restricted SERT function (the mode of SSRI and SRI antidepressant function). Unfortunately, Drosophila ag gression as a means of analyzing the serotonergic system has only recently been revisited. A limited availability of studies that link aggression to SERT in Drosophila is probably due to previous evidence by Baier (2002) suggesting that the serotonergic sy stem in Drosophila had no effect on aggression. However, there are behavioral effects of SERT manipulation documented in mice models (Figure 1.4). Table 1.4 : The study of serotonin transporter manipulations and behavior Model Organism Author Method Effe ct SERT knockout mice (Slc6a4 / ) in an isolated resident intruder test Aggressive Holmes et al., 2002 SERT deficient mutants (Slc6a4 / & Slc6a4 +/ ) in subsequent resident intruder test Latency to attack did not shorten as it did in the wildt ype. Mice Jennings et al., 2006 2 3x overexpression of SERT using a yeast artificial chromosome. Extracellular fluid 5 HT levels SERT binding sites Anxiety The SERT knockout mice described in Table 1.4 demonstrated decreased aggression. While it may be t empting to expect SERT overexpression to result in the inverse of an observation made in the case of inhibited SERT expression, such expectations would overlook the functional character of SERT. As one of several regulatory molecules of the serotonergic sy stem there is no static correlation between the
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 20 number of SERT binding sites and the level of 5 HT signal transduction. Additionally, an observed behavioral phenotype could be due to any number of effects to molecular elements the serotonergic system. The absence of an aggression effect in mice overexpressing SERT demonstrates a case where there does not appear to be a direct correlation between the level of SERT expression and aggression behavior (Table 1.4). To predict the effects of SERT overexpression, or any other manipulation that might lead to this, the molecular and organismal effects of SERT overexpression must be characterized. Table 1.5 provides an example of extensive characterization of the effects of the aforementioned mice mutants with restri cted SERT expression. By identifying the elements of the serotonergic system expected to effect aggression in Drosophila and characterizing their behavioral phenotypes the results may suggest 5 HT dynamics that have yet to be observed molecularly.
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 21 Table 1.5 : Downstream neurochemical observations in SERT deficient mice Effect Heterozygote Slc6a4 +/ Null Slc6a4 / Extracellular Fluid [5 HT] Basal [ ] in striatum and cortex Brain Tissue [5 HT] Unchanged 40 60% 5 HT Release Altered dynamics u nder KCl induced depolarization 5 HT Clearance Prolonged Indistinguishable from diffusion in the CA3 region of hippocampus 5 HT Synthesis and Turnover Unchanged Across brain Expression of Organic Cation Transporters Unchanged OCT1 & OCT3 5 HT 2A Unc hanged Binding sites in striatum, claustrum & cortex Binding sites in septum & hypothalamus 5 HT 2C Unchanged Amygdala & choroids plexus 5 HT 1A At presynapse of somatodendrites This table serves as an example of the detail with which downstream effec ts of manipulating SERT can be characterized. The studies herein were described in a review by Murphy and colleagues (2008). While the findings shown in figure 1.5 do not provide a predictive framework for the overexpression of SERT in Drosophila they do suggest possible downstream effects for future investigation. These studies also show how gene dosage, or the degree to which the expression of SERT is restricted can cause variable effects. IV. Transgenic Flies Genetically modified flies can be used to in troduce a genetic change to a subset of cells, and limit the time period over which the genetic change is experienced. Though a variety of transgenic fly lines exist and are commonly used, this study takes advantage of spatial restriction by the GAL4 UAS s ystem.
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 22 a. Spatial Restriction The general benefit to spatially restricting a genetic change is that observations account for the cell autonomy of a mutant phenotype. These observations are significant if mutant cells are effected by nearby wildtype cells if mutant cells effect the development of wildtype cells through domineering non autonomy, or if wildtype cells inhibit observation of the mutant phenotype (Blair, 2003). The limitation of dSERT overexpression to serotonin positive cells in the CNS signi fies an ability to attribute the phenotype to endogenous dSERT overexpression. The spatial restriction of overexpression to serotonin positive cells is accomplished using the GAL4 UAS system (see Chapter 2, Figure 2.1 for more detail). This is a genetic te chnique in which the yeast derived GAL4 encodes for a transcription factor, and the Upstream Activation Sequence (UAS) enhancer element forms a regulatory GAL4 binding site. GAL4 drives expression in a subset of cells as defined by its adjacent promoter. T he UAS enhancer element is inserted in the 5' region flanking the target (effector) sequence. In addition to this, linking the effector sequence to a benign marker on the same chromosome can be used to identify mutant cells and confirm that the genetic con struct is functioning as expected. b. Temporal Restriction Temporally restricting a genetic change allows observation of the resulting physiological changes in the neurons, without observing associated defects in the development of the nervous system (McG uire et al, 2003). This is especially important when observing alterations to the serotonergic system because of the manifold functions of 5 HT during development. Within the CNS, these include the modulation of
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 23 serotinergic varicosity development, and neu ronal branch spacing (Skykes and Condron, 2005). GAL80 normally functions as a repressor of GAL4 in yeast. GAL80ts represses GAL4 at low temperatures (+19 C), but becomes inactive at high temperatures (+30 C) allowing the expression of the UAS effector gen e upon the temperature shift (see Chapter 2, Figure 2.2 for more detail). In 2003, McGuire and colleagues demonstrated the use of Gal80ts construct to allow simultaneous observation of where and when a gene product leads to a mutant phenotype. They used RT PCR to measure effector mRNA levels after varying periods of heat shock in flies carrying GAL80ts as compared to flies that expressed the effector independently of GAL80ts. This demonstrated that high temperature induced inactivation of GAL80ts can lead t o effector gene expression levels equivalent to those of GAL80ts independent effector expression. Furthermore, the expression levels can be lowered by a period of heat shock recovery to those of GAL80ts flies that never experienced heat shock. While the ex act levels of expression are in part dependent on the strength of the promoter coupled to GAL80ts and GAL4, these findings demonstrate that GAL80ts can be used to genetically restrict the transgene expression in time. V. Overexpression of Serotonin Membrane Transporter a. Pan neuronal Overexpression When dSERT is overexpressed pan neuronally the concern is that the ectopic expression of dSERT will lead to phenotypic changes that obscure what would otherwise be seen. However, in 2006 Park and colleagues demon strated that pan neuronal overexpression of dSERT using the actin driver in the larval central nervous system only
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 24 leads to accumulation of 5 HT in serotonergic, dopaminergic, and a small number of other cells which are not substantially broadened by addit ion of exogenous 5 HT. This cannot be taken to mean that dSERT is only reuptaking 5 HT in these cells; for 5 HT may be metabolized at such a rapid rate that accumulation is rendered impossible. The finding does signify that ectopic overexpression of dSERT in these cell types leads to a functional protein pump. The consistency of these findings even after increasing the availability of 5 HT suggests that the observation of limited 5 HT storage was not due to a limited availability of synaptic 5 HT, but may r eflect the chemical behavior of cells with ectopic expression of dSERT. The synthesis and storage of monoamines requires a number of conserved proteins, such as the enzymes required for the synthesis of 5 hydroxytryptophan (5 HTP), dopa decarboxylase (Ddc) required for conversion of 5 HTP to 5 HT (Figure 1.2), and the VMATs required for pumping 5 HT into vesicles for exocytosis (Liu & Edwards, 1997). Due to the high conservation between dopaminergic and serotonergic proteins, the similarities in their biosy nthesis, and their shared monoamine character, it stands to reason that dSERT would have increased function in these cells. However, dopaminergic neurons remain unable to synthesize 5 HT. While the limited ectopic storage of 5 HT may be taken as support fo r the reliability of pan neuronal overexpression, it is also evidence of secondary effects of dSERT in the subset of cells where ectopic expression leads to 5 HT storage. In the absence of evidence to suggest that the effects of targeting dopaminergic and serotonergic cells are additive, and in the presence of evidence that dSERT is functioning in dopaminergic cells robust results require more targeted approaches to manipulating dSERT expression. The
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 25 limitation of dSERT overexpression to serotonin positive cells in the CNS signifies an ability to attribute the phenotype to endogenous dSERT overexpression. b. Tryptophan Hydroxylase Promoter As is the case in mammals, there are two types of tryptophan hydroxylase in Drosophila with varying expression and func tion (Walther et al., 2003; Coleman and Neckameyer, 2005). Drosophila tryptophan hydroxylase (DTRHn, or TRH) is homologues to mammalian TPH2, and is exclusively neuronal in its expression and function. Drosophila tryptophan phenylalanine hydroxylase (DTPHu ) functions as a tryptophan hydroxylase in the brain periphery, but can be found functioning as a phenylalanine hydroxylase in both the brain and its periphery (Neckameyer, 2007). The promoter used to define the subset of cells targeted in this investiga tion was TRH. TRH, called TPH in mammals, stands for tryptophan hydroxylase, which is the enzyme involved in the rate limiting step of 5 HT synthesis (Figure 1.3). This driver was created from long regulatory sequences of the TRH gene (Alekseenko, unpublis hed data). Unlike the enzymes required for dopamine synthesis, which are expressed both in the nervous system and cuticle, TRH is expressed exclusively in the nervous system. As such it is the ideal target for promoting endogenous overexpression of seroton ergic molecules within the CNS. By targeting the CNS manipulations exclude effects to the peripheral functions of 5 HTP, such as ovarian follicle formation (Willard et al., 2006). This construct was attempted previously in the creation of a TPH driver. Unf ortunately, the TPH driver was never properly characterized, and is not selective to as many 5 HT positive cells (Alekseenko, unpublished data). While incomplete targeting of 5 HT positive cells is not a concern for chemical studies, such as the one by Bou rne and
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 26 colleagues in 2009, a behavioral phenotype may be the result of cells not targeted by the TPH driver. While both constructs target the gene for tryptophan hydroxylase in flies, the somewhat misnamed TPH driver was made without any publication of wh ich region of the promoter was used in its creation, and is not as reliable for behavioral studies. c. Fluorescent Tag The green fluorescent protein (GFP) dSERT hybrid gene was made by fusing the fluorescent tag to the N terminus of dSERT and the addition was not seen to interfere with dSERT function (Park et al., 2006). This is to be expected, given that the binding kinetics, transport activity, and localization of monoamine transporters tagged with GFP at the N terminus were unaffected in cultured cells (Daniels et al., 1999; Chang et al., 2001).
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 27 CHAPTER 2 MATERIALS AND METHODS The goal of this research was to ascertain whether endogenous overexpression of SERT in the CNS of Drosophila melanogaster would lead to altered action selections when males compete for food, territory, and a female. Based on previous evidence of a serotonergic effect on aggression in Drosophila melanogaster and other model organisms (Tables 1.2, 1.3, 1.4,1.5), the expectation was that there would be an a ltered behavioral phenotype in the case of SERT overexpression. A pilot study suggested that this behavioral phenotype might be centered on altered levels of aggression and male male courtship. However, there was not a reliable basis upon which to predict how aggression would differ in the case of SERT overexpression, because this study u s ed a novel combination of transgenes applied in an organism that until recently was thought to show aggression independently of 5 HT. The following was an exploratory stud y into the behavioral effects of SERT overexpression with the expectation that a difference would be found. I. General The fly strains (Table 2.1) were kept in vials with a standard cornmeal medium i These vials were then incubated with a 12hr: 12hr ligh t/dark cycle at 50% humidity and 25 degrees Celsius. Flies with the temperature sensitive allele (GAL80ts) were incubated at 19 degrees Celsius (Figure 2.2 A), followed by a shift to +30 C. (Figure 2.2 B). Prior to eclossion, progeny pupae were individuall y isolated into vials of 16 mm diameter containing approximately 2 mm of food. The b eh avior of the flies was analyzed at 4 8
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 28 days of age. All experiments were recorded using Sony Video8 digital tapes in a Sony digital8 Han d ycam so that tapes could be exami ned using iMovie on Macintosh computers. Table 2.1 : The True Breeding Parental Lines Crossed to Form Each Genotype. GENETIC CROSSES ¡ C w ;+;UAS SERT:GFP w ;+;+ w ;+;TRH GAL4 Spatially Restricted Overexpression TRH Control +25 w ;+; + SERT:GFP Control Wildtype Background +19 +30 w ;GAL80ts;TRH GAL4 Temporally Restricted Overexpression Control While a true control would be the wildtype background, the isolated transgenes serve as controls for all but the possible position al effects of the transgene. The wildtype background is of mini white (w ) Canton S flies. Homologous mini white flies lack pigments in the compound eye and suffer from a decreased sense of sight. While the white color of their compound eyes is beneficial in confirming the accuracy of genetic crosses, the decreased sense of sight introduces a physical impediment absent in heterozygous mini white flies that would interfere with the comparability of behavioral phenotypes. The mini white gene was necessary bec ause it was used in the creation of the
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 29 TRH construct. Essentially the wildtype is not a suitable control in this case, so the isolated transgenes have been used instead. If there is a positional effect resulting from the mere insertion of these genetic co nstructs the following analysis will be unable to characterize it, but analysis will be able to characterize the effects of SERT overexpression that are seen in cross comparisons with each isolated transgene. The following figures depict the function of th e transgenes in flies that overexpress SERT throughout the life cycle (Figure 2.1) and flies that overexpress SERT after a period of heat shock (Figure 2.2).
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 30 Figure 2.1 : Diagram of the genetic mosaic used to induce chronic SERT overexpressi on (Image by Camayd C) This figure demonstrates the genetic mosaic used to induce endogenous overexpression of SERT throughout the life cycle. When the fly possesses a copy of both the top and bottom sequences the event depicted by the red arrow may occ ur, in contrast to control strains which posses either the top or the bottom sequence. The GAL4 driver (yellow) (Figure 2.1) sits downstream of the Tryptophan Hydroxylase promoter (violet) conferring expression that is specific to serotonin positive cell s. The upstream activation sequence (light blue) enhances downstream expression when bound to the GAL4 transcription factor (yellow circle). The serotonin transporter sequence (light green) is overexpressed (light green circles) along with the Green fluore scent protein insert (dark green).
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 31 Figure 2.2 : Diagram of the genetic manipulation used to induce acute SERT overexpression (A) (B) (Images by Camayd C) Temperature sensitive GAL80 (pink) encodes a repressor of GAL4 function at 19 degrees C elsius (A), but is inactive at 30 degrees Celsius (B).
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 32 II. Aggression Assay The genotypes examined in the aggression assay are those with SERT overexpression throughout the life cycle, and those with isolated insertion of the transgenes (Table 2.1, +25 ¡ C and Figure 2.1). Approximately two days prior to experimentation, flies were anesthetized with CO 2 and a dot of acrylic paint was applied to the cuticle of each dorsal thorax. The dots differentiated the flies so that dominance relationships could be obse rved. Socially na ve males of the same age and similar size were randomly paired and simultaneously introduced into the chamber. Filming began immediately after flies were introduced into the chamber and continued for one hour. Aggression assay observatio n chambers consisted of four clear glass microscope slides adhered at the edges (Figure 2.3 A) and propped up in a petri dish of 5mm deep 2% agarose (Figure 2.3 B). The chamber was covered by an inverted petri dish with small holes for ventilation and a la rger hole for the gentle aspiration of flies into the chamber. Within the chamber lay a 1 cm high food cup with a diameter of 1.5 cm (Figure 2.3 C). The cup was filled with fresh food before every experiment, and an attractant was applied to the center of the cup.
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 33 Figure 2.3 : Photographs of the observation chamber used in aggression assays (A) (B) (C) (Images from Mundiyanapurath Certel & Kravitz, 2007) In aggression assays, the attractant was a decapitated female or dot o f yeast paste. These are equally attractive to Canton S wildtype flies (Nilsen et al., 2004). The decapitated female is oriented with the anterior thorax and posterior abdomen both inundated in the food to inhibit successful copulation. These attractants s erve as desirable objects, which draw the flies onto the food cup and provide an opportunity for aggression. The choice of which attractant to use was made based on trials conducted under both conditions. An initial pilot study, not included in these resu lts, used decapitated
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 34 females. It was the behavioral phenotype observed in the pilot study that spurred an interest in conducting a full experiment. However, since this behavioral phenotype was heavily characterized by male male courtship, the experimenter s initially began using yeast paste. This was done to eliminate any uncertainties about the intended object of a courtship behavior. The results of these yeast paste trials can be seen in the appendix (Table 2.2) ii Yeast paste was determined to be an insuf ficient attractant for both control and experimental flies. The remainder of aggression assays were performed using decapitated females. a. Scoring It was decided that the movement of the experimental flies was too rapid to identify lunges with confidenc e in real time. To account for this all fights were clipped into encounters using iMovie. This is a standard practice of the Kravitz laboratory that allows video to be viewed at tenths of a second. A clip was started whenever the flies interacted on the fo od cup and ended after three seconds of no interaction. This was done for a thirty minute observation period, which began when both flies landed on the food cup and remained there for at least twenty seconds. If the flies failed to do this within the first twenty minutes of the tape, it was discarded and the failure of the flies to land was noted. The twenty minute time frame within which the flies must land for the tape to be scored serves to eliminate fly pairs that have had substantial interactions off t he food cup by the start of the observation period. A record is kept of when the flies first landed on the food cup and the clips, which span each ten minute interval of the observation period. Separating the encounters out into time intervals allowed for an idea of the fight dynamics that would otherwise be lost when taking clips with no time stamp.
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 35 Scoring the behaviors of interest was done by observing the clips and recording the clip number, time past in the clip, and color of the dot on the fly that pe rformed the behavior. This provided a chronology and frequency of the behaviors of interest. Should dominance be established, it was also recorded. Any additional interesting behaviors or trends were noted, as where any behaviors whose categorization was n ot clear to the observer. The scoring of any unclear behaviors was a joint assessment made by multiple researchers. b. Behaviors While the exact definitions of a behavior are likely to vary between observers the descriptions in table 2.3 were used to ens ure that this variability was not reflected in results. A blind observer was trained to score behaviors and dominance by the original observer. The blind observer scored one randomly selected tape from each genotype, and the results were used as a consiste ncy check.
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 36 Table 2.3 : The classifications and operational definitions of the behaviors noted Type Behavior Operational Definition Aggression Lunge Fly rears up with anterior legs raised. The raised anterior end of the body is thrust d own on the target and raised up in a single motion. Aggression Hold Lasts for at least a second and is preceded by a lunge behavior. Fly may curve abdomen for balance, but this should not be interpreted as an attempt to copulate. Courtship Mount Fly atte mpts to copulate by curving abdomen and is preceded by a courtship behavior. Since the abdomen begins to bend during licking and tapping the head must be above the other fly!s abdomen for attempted mounting to occur. There must be a behavior in between mou nts to distinguish them as separate attempts. Courtship (specifically male male) Mutual Singing Male sings to male and visa versa while side stepping in a semi circle. If a decapitated female is near she is outside of the circle of motion. The flies must be facing one another and it must last for a second at the least. While the criteria for establishing a dominance hierarchy differs, for the purposes of these experiments dominance was established after a fly lunges three or more times in rapid successi on such that the other fly leaves the food cup. The lunging fly is considered the winner, unless retaliation occurs. A retaliation is when the loser returns to the food cup and lunges back at the winner. When this occurs the pair is not considered to have established a clear dominance hierarchy. The purpose of noting the interaction and occurrence of dominance hierarchies is to assess whether the behavior of the winner and loser changes afterwards, as is the case with wildtype flies.
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 37 III. Antibody La beling and Microscopy CNS immunofluorescent staining of flies with GAL80ts regulated overexpression of SERT was done using standard methods after varying periods of heat shock to determine the necessary duration for overexpression to occur. Once establish ed, it was possible to characterize the behavioral phenotype resulting from endogenous overexpression of SERT in the CNS of adult flies. The CNS of flies were dissected in a phosphate buffer, and then fixed in 4% paraformaldehyde (PFA) for twenty minutes. Samples were kept overnight at +4 degrees C in a thousand fold dilution of primary antibodies (a mix of rabbit anti 5 HT and mouse anti GFP antibodies). This was followed by two hours in a five hundred fold dilution of secondary antibodies (a mix of anti m ouse AlexaFlour 488 and anti rabbit AlexaFluor 594). Dissections were performed by this author and Dr. Olga Alekseenko. The resulting fluorescence was observed using a Nikon Eclipse 90i fluorescent microscope, and Confocal Z stacks were acquired using an O lympus Fluoview FV1000 confocal microscope with a UAPO 20x water immersion objective, and processed with ImageJ software
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 38 CHAPTER 3 RESULTS The following sections detail aspects of the novel behavioral phenotype characterized in SERT overex pressing flies, and present preliminary findings to inform future efforts at temporal restriction of SERT overexpression. In assessing the consistency with which observations were made a blind observer showed a 6% deviation from the observations made by th e original observer when scoring ~10% of the data. Observations were split into three ten minute intervals, so differences between groups are evaluated over specific time intervals as well as over the whole of the observation period. Although inbreeding in laboratory stocks of model organisms inhibits the extent to which transgenic strains from the same wildtype line can be called independent, for the purpose of statistical analysis each genotype was treated as an independent sample. I. Statistics Analysis s et out to determine whether the experimental observations were due to the nature of the inserted genetic constructs or the resulting overexpression of SERT. The absence of information that might justify assuming a normal probability distribution necessitat ed the use of nonparametric statistics. Since two transgenes were used, experimental observations could be the result of insertion of either transgene or the overexpression of SERT. For this reason, it was necessary to use a statistical test for three or m ore independent samples. The Kruskal Wallis test is a nonparametric method for testing equality of population medians I t is virtually identical to a parametric one way analysis of variance with the data replaced by their ranks. When the data lacked an
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 39 ord inal scale, differences were not found. This ordinal analysis is preferable to a Chi Squared test because of the small cell sizes in the data. In all the Kruskal Wallis test was chosen based on the unknown probability distribution, the number of groups, an d small cell sizes of the data. Post hoc analysis was done with the Mann Whitney U rank sum test, which compares differences across two groups. Post hoc analysis is done in cases of three or more groups in order to verify between which groups a difference is observed. While the Kruskal Wallis post test (Dunn's) is used more commonly, it is not applicable to cases where groups have unequal sizes. The Mann Whitney U is a nonparametric test similar to a parametric Student's t test after ranking the samples; no netheless it is used here solely for post hoc analysis The p value resulting from the Mann Whitney U test can be adjusted using the Bonferroni method. However this is a very conservative approach to Mann Whitney U post hoc analysis that may overlook a sig nificant result. Therefore, it was decided that the Mann Whitney U rank sum test was best suited for post hoc comparisons because of the data's unequal group size, and the test's sensitivity to significant results. In the convention set forth by the litera ture previously cited the figures that follow display mean values with an error bar that denotes standard error of the mean. However, it should be noted that nonparametric statistics are based on variance in medians rather than means. A table listing the m ean ranks from which significance was assessed can be found in the appendix (Table 3.1). iii
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 40 II. Fly Pair Activity and Mutual Behavior The latency to land, time spent interacting, number of encounters, and frequency of mutual singing were noted for each intera cting fly pair. No significant differences were found in the activity level of the groups. However, the total occurrence of mutual singing and the frequency of mutual singing within the first ten minutes were significantly greater in the SERT overexpressin g flies (Figures 3.1& 3.2). *** = 99.9% confidence (p < .001) The findings depicted in Figure 3.2 demonstrate that the significant increase of mutual singing within SERT overexpressing flies is the result of early interactions and is not perpetuated i nto the observation period.
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 41 *** = 99.9% confidence (p < .001) The total occurrence of mutual singing and frequency of mutual singing during the first ten minutes are significantly greater in SERT overexpressing flies at a 99.9% confidence interval. Diffe rences in activity level would have signified a chance in the opportunity of each group to perform a given behavior. The time spent interacting is based on the total lengths of all clips taken, while the number of encounters is taken as the number of clips made. The absence of significant findings in this area signifies that differences in action selections are not paired with differences in activity level.
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 42 III. Individual Fly Behaviors and Dominance Hierarchies Lunges, holds, and mounts were noted for all individual flies, and then adjustments had to be made in analysis to correct for the effect of dominance hierarchy on fight dynamics and the ties between interacting flies. No significant differences were found in holding and mounting behaviors. The follow ing sections describe the differences in aggression as seen through lunging behavior. a. Dominance Hierarchies Dominance was established in 17% of SERT: GFP control pairs, 38% of TRH control pairs, and 8% of the experimental pairs. Due to the low percenta ge of flies that established dominance relative to wild type Canton S flies, the common practice of drawing comparisons across genotypes between groups of the same dominance status was not possible in this case. This is typically done in order to exclude t he effect of ties between the two interacting flies, and the expected effect of dominance status on behavior. With this limitation in mind, comparisons of individual fly behaviors were drawn between the flies that demonstrated lunging behavior (Figures 3.5 and 3.6). Since holds are by definition preceded by a lunge, there was no instance in which these might be excluded from the data based on the parameters used. Of the 24 SERT: GFP control flies, 38% demonstrated lunging behavior; of the 26 TRH control fli es, 58% demonstrated lunging behavior; and of the 26 SERT overexpressing flies, only 35% demonstrated lunging behavior. Since exclusion of flies that never lunged eliminated a substantial portion of the sample size, individual behaviors were also assessed by including all flies in order to see what differences, if any, may emerge (Figures 3.7 and 3.8).
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 43 b. Individual Fly Behaviors In Those That Lunged Comparison of all genotypes found a significant difference in the total number of lunges, and in the numbe r of lunges during the second ten minute interval (Figures 3.5 and 3.6). That is, the TRH control flies lunged more than other flies. Mann Whitney U analysis found that TRH control flies lunged more frequently than the SERT overexpressing flies, but not mo re frequently than the SERT: GFP control flies. There were no significant differences or trends in individual behaviors found between control genotypes when samples excluded flies that never lunged. = 95% (p < .05)
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 44 SERT overexpressing flies lunged l ess than controls during the middle of the observation period (Figure 3.6). Post hoc analysis demonstrated that this difference exists between SERT overexpressing flies and both control groups. = 95% (p < .05) c. Individual Fly Behaviors In All Flie s Comparison of all genotypes resulted in the same significant differences: TRH control flies lunged more in total and during the middle of the observation period (Figure 3.7 and 3.8). However analysis also uncovered a new tendency (p=0.088) of TRH contro l flies to lunge more frequency towards the end of the observation period (Figure 3.8). Once again post hoc analysis determined that none of these findings are reflected in comparisons between control genotypes.
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 45 = 95% confidence (p < .05) Temporal ana lysis of lunging behavior demonstrates that TRH control flies are more likely to lunge during the second ten minutes relative to SERT overexpressing flies, and trend (p=0.088) towards the same during the last ten minutes relative to SERT overexpressing fli es (Figure 3.6).
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 46 ** = 95% (p < .05) No significant differences were found in holding behavior. IV. Preliminary GAL80ts Temporal Control At +30 C GAL80ts is inactive, so GAL4 proceeds to encode the transcription factor driving dSERT overexpression. The dura tion of the heat shock necessary for overexpression was determined by confocal images of antibody labeled 5 HT and GFP (Figure 3.9). The regions of overlap between 5 HT and GFP indicated those where the action of dSERT had resulted in an accumulation of 5 HT. Based on figure 3.9 three days was determined to be a sufficient period of heat shock for SERT overexpression to occur.
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 47 Figure 3.7 : CNS confocal images of temporal SERT overexpression after varying length of heat shock The primary antibody for 5 HT was labeled with a fluorescent secondary antibody with an emission peak at 594 nm. In contrast, the primary antibody for the GFP tag on SERT was labeled with a secondary fluorescent antibody with an emission at 488 nm.
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 48 CHAPTER 5 DISCUSSION Exploration of this novel combination of transgenes in a model organism that until recently was thought to show aggression unaffected by the serotonergic system has provided important insights. These insights will prove even more valuable as additional molecular elements of the serotonergic system are targeted in this manner and behavioral effects are quantified. While many previous studies have restricted the expression of SERT in order to model the action of popular fo rms of antidepressants, a full picture of this dynamic system begs for a predictive framework of the affects of SERT function. In order to pursue this purpose the overexpression of SERT ought to be examined. None of the significant differences or trends we re found to between control flies as was expected of isolated transgenes. Differences found between control groups would suggest the presence of an additional variable, such as dominance relationships or a positional effect of inserting the transgene. Exc lusion of flies that did not lunge at least in part corrected for the expected differences in aggression between flies that established dominance hierarchies and those that did not. However, it should be noted that analysis did not fully correct for associ ations between interacting fly pairs. This limitation must be kept in mind, since the behavior of one fly most surely influenced the behavior of the other fly involved in the encounter.
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 49 I. Male Male Courtship SERT overexpressing flies are more p rone to male male courtship, as seen by their increased frequency of mutual singing (p < .001) (Figure 3.1). This is interesting not only because it is male male courtship, but also the mutual nature of this behavior incorporates auditory and visual confir mation that the target of the courtship behavior is male. The persistence of this behavior in dSERT overexpressing flies in spite of this suggests that there are underlying differences in the sexually dimorphic circuits controlling the decision to court. SERT overexpressing flies do not perpetuate this behavior past the beginning of the observation period (p < .001) (Figure 3.2). This suggests that SERT overexpressing flies display male male courtship in an experience dependant manner, but are predisposed to higher initial levels of male male courtship. Furthermore, this temporal trend is not surprising given that even naive wildtype flies will display male male singing among initial encounters. Male male courtship does not naturally occur in adult wildtype flies, but it is seen to occur in juvenile flies. In the absence of social contact SERT overexpressing flies display a higher initial predisposition to mutual singing, but these experiences likely inform later choices of a courtship targets lowering male male courtship to control levels. The primary error inherent in this observation was the determination of the intended target of singing. Had yeast paste been used as the attractant, this would not have been a concern. But the inactivity of both experimen tal and control genotypes suggested that yeast paste was not a sufficient attractant for the wildtype background used ( ii Table 2.2). In order to confidently identify mutual singing the behavior was only
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 50 noted when it was performed distal from the decapita ted female. These criteria likely led to underscoring of mutual singing. Nonetheless, if methods of observation were consistent between genotypes, as was suggested by the mere 6% difference in noted behaviors in comparison to a blind observer, consistency in the observation of all flies will render reliable differences between genotypes even in the presence of less reliable absolute frequency of mutual singing. The assessment of the consistency with which behaviors were scored could be better determined if inter observer reliability had been established by statistical analysis using Person Product Moment correlations. Had this been established the second observer could have served as an accuracy check of all observations. The degree of inter observer reliab ility may have been compromised due to the blind observer's lack of experience identifying behaviors in Drosophila At the same time the blind observer could not have been biased by knowledge of the transgenes expressed by the subjects. In addition to this blind observer, Dr. Olga Alekseenko reviewed all trials to confirm that dominance hierarchies were established. II. Altered Fight Dynamics It follows that flies that have a greater number of encounters may have increased opportunity to perform a give n behavior. Conversely, flies that are likely to perform a behavior more frequently will display a greater number of encounters, or a greater length of time spent interacting if the behaviors are displayed consecutively. However, the absence of significant findings regarding activity level signifies that the differences in lunging behavior are due to a preference towards that action selection.
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 51 Analysis of flies that demonstrated lunging showed that SERT overexpressing flies are less aggressive during the m iddle of the observation period. This assessment is based on their decreased lunging during the middle of the observation period. These results suggest that SERT overexpressing flies are less aggressive in the middle of the observation period. While this m ight suggest that aggression is decreased in response to SERT overexpression, such a conclusion cannot be definitively drawn from this sample due to the temporal restriction of these differences. Analysis of all flies reconfirmed the differences found whe n analysis excluded flies that did not lunge. The exclusion of flies that did not lunge substantially decreased the sample size, so analysis of all flies was able to find differences over a higher confidence interval. For instance, the decreased lunging of SERT overexpressing flies during the middle of the observation period had a higher degree of significance (p<0.01). Unfortunately, dominance would bias the findings towards decreased aggression in the genotype where it is established least, and SERT overe xpressing flies seem to show decreased aggression during the middle of the observation period. Since the differences that are being analyzed are in the same direction as the expected bias resulting for differences in dominance, analysis that best corrects for the effect of dominance hierarchies will be the most reliable. Had a greater number of pairs been observed analysis could have randomly considered a single fly from each pair in addition to excluding non lunging flies. Unfortunately, the most efficien t method of correcting for both ties and dominance hierarchies would have required a much greater percentage of dominance.
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 52 A decrease in total aggression when manipulating SERT has been found previously in SERT knockout mice mutants (Table 1.4). A similar behavioral phenotype in response to SERT manipulation, whether it be restricting or increasing SERT expression, suggests that deviations from normal levels of SERT binding sites may modulate aggression. This stands to reason given the findings in lobster a nd crayfish studies that rapid increases in 5 HT availability cause increases in certain measures of aggression (Table 1.2). Increased SERT binging sites may inhibit rapid increases in 5 HT levels in the synapse, since SERT terminates the activity of 5 HT in the synaptic cleft. The absence of SERT in mice mutants has been seen to cause higher in extracellular 5 HT concentrations in certain brain regions, as well as increased 5 HT synthesis and turnover (Table 1.5). If the concentration of 5 HT in the synapt ic cleft is consistently above normal levels this may similarly inhibit rapid shifts in extracellular 5 HT availability and possible associated increases in aggression. The observation in Drosophila melanogaster of altered aggression levels in response to 5 HT receptor treatment with agonists or antagonists of mammalian orthologs suggests that the effect of SERT on aggression may be due to its role as a regulator of 5 HT signal transmission. By altering the availability of 5 HT in the synapse the manipulat ions discussed above also affect the binding frequency of 5 HT to presynaptic and postsynaptic receptors. Therefore, the decease in aggression upon increased SERT binding sites may be due to the effects it has on 5 HT signal transduction.
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 53 III. Directi ons of Future Studies Future investigations should examine suspicions of an altered circuit effecting the decision to court in SERT overexpressing flies. Mutual singing has been observed previously in fruitless mutants (Vrontou et al., 2006). Future inves tigations might determine whether this transcription factor is affected by SERT overexpression. It may also be interesting to determine whether SERT overexpressing flies preferentially court males, or have a greater drive to court overall. A choice courtsh ip assay of the SERT overexpressing flies would provide a ratio of male male to male female courtship using a decapitated courtship target of each gender. Choice courtship data is already being collected to examine these questions. Finally, traditional cou rtship assays using females with altered pheromonal profiles may be able to determine whether this male male courtship is due to an altered pheromonal profile in male SERT overexpressing flies, or due to an inhibited ability of SERT overexpressing flies to interpret pheromonal cues. The observation of GAL80ts dependant overexpression of SERT (Figure 3.9) is the start of future investigations into endogenous overexpression of SERT in the CNS of adult flies. These findings will be independent of possible eff ects the current manipulation might have on the function of 5 HT during development. Observations made in flies that overexpress SERT during the adult life stage can exclude developmental effects, such as the modulation of neuronal branch spacing and serot inergic varicosity formation. Also, comparison of the behavioral phenotypes resulting from chronic and acute overexpression of SERT may serve to identify possible mechanisms of functional synaptic plasticity. For instance the difference in the two behavior al phenotypes may be due to remodeling of the postsynaptic cell membrane to
BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 54 more efficiently receive 5 HT dependant signals in the face of rapid 5 HT turnover. Another possibility might be that the presynaptic cell regulates the levels of 5 HT available in the synapse by developing a faster rate of 5 HT release. These are only a few of the dynamic changes that might be occurring. Other molecular elements of the serotinergic system that may serve as promising targets for future manipulation include the 5 HT7 Dro receptor. Characterization of the behavioral phenotype resulting for 5 HT7Dro manipulation appears to be a promising target based on the findings by Johnson (2009) that suggested it played a role in the modulation of aggression in Drosophila By compar ing the behavioral phenotype resulting from manipulations of serotinergic receptors to the overexpression of SERT, one may be able to predict whether postsynaptic 5 HT receptors are overexpressed in response to the increase in SERT. IV. Conclusions En dogenous overexpression of SERT in the CNS of Drosophila melanogaster led to a greater predisposition towards male male courtship (Figure 3.1 and 3.2), and decreased aggression during the middle of the observation period (Figure 3.3, 3.4, 3.5 and 3.6). Thi s evidence confirms expectations that overexpressing SERT leads to a behavioral effect in socially nave flies competing for territory, food, and a female. This conclusion is consistent with previous findings by Dierick and Greenspan (2007) regarding 5 HT levels, and those of Johnson (2009) concerning 5 HT receptors.
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BEHAVIORAL EF FECTS OF SEROTONIN TRANSPORTER OVEREXPRESSION IN Drosophila melanogaster 60 APPENDIX i 0.9% agar medium containing 10.5% dextrose, 5% cornmeal, 2.6% baker's yeast and 0.23% tegocept ii Table 2.2 : Results from yeast paste trials Genotype Observations SERT Overexpression All of five pairs demonstrated no courtship and no escalation beyond fencing. SERT:GFP Control All six pairs demonstrated no courtship and no escalation beyond fencing. TRH Control Two out of four pairs demonstrated lunges. iii Table 3.1 : Mean ranks of statistically significant findings Genotype N Mean Rank Asymptotic Significance 1 13 27.88 2 12 15.83 3 13 14.50 Part 1 Mutual Singing Total 38 0.000 1 13 27.62 2 12 15.33 3 13 15.23 Total Mutual Singing Total 38 0.000 1 9 9.11 2 9 20.39 3 15 19.70 Part 2 Lunging (flies that lunged) Total 33 0.011 1 9 11.33 2 9 15.83 3 15 21.10 Total Lunging (flies that lunged) Total 33 0.050