Large analysis of genetic manipulations reveals an inverse correlation between initial alcohol resistance and rapid tolerance phenotypes.

Tolerance occurs when, following an initial experience with a substance, more of the substance is required subsequently to induce identical behavioral effects. Tolerance is not well-understood, and numerous researchers have turned to model organisms, particularly Drosophila melanogaster, to unravel its mechanisms. Flies have high translational relevance for human alcohol responses, and there is substantial overlap in disease-causing genes between flies and humans, including those associated with Alcohol Use Disorder. Numerous Drosophila tolerance mutants have been described; however, approaches used to identify and characterize these mutants have varied across time and labs and have mostly disregarded any impact of initial resistance/sensitivity to ethanol on subsequent tolerance development. Here, we analyzed our own, as well as data published by other labs to uncover an inverse correlation between initial ethanol resistance and tolerance phenotypes. This inverse correlation suggests that initial resistance phenotypes can explain many 'perceived' tolerance phenotypes, thus classifying such mutants as 'secondary' tolerance mutants. Additionally, we show that tolerance should be measured as a relative increase in time to sedation between an initial and second exposure rather than an absolute change in time to sedation. Finally, based on our analysis, we provide a method for using a linear regression equation to assess the residuals of potential tolerance mutants. These residuals provide predictive insight into the likelihood of a mutant being a 'primary' tolerance mutant, where a tolerance phenotype is not solely a consequence of initial resistance, and we offer a framework for understanding the relationship between initial resistance and tolerance.

Synaptic Mechanisms of Ethanol Tolerance and Neuroplasticity: Insights from Invertebrate Models.

Alcohol tolerance is a neuroadaptive response that leads to a reduction in the effects of alcohol caused by previous exposure. Tolerance plays a critical role in the development of alcohol use disorder (AUD) because it leads to the escalation of drinking and dependence. Understanding the molecular mechanisms underlying alcohol tolerance is therefore important for the development of effective therapeutics and for understanding addiction in general. This review explores the molecular basis of alcohol tolerance in invertebrate models, Drosophila and C. elegans, focusing on synaptic transmission. Both organisms exhibit biphasic responses to ethanol and develop tolerance similar to that of mammals. Furthermore, the availability of several genetic tools makes them a great candidate to study the molecular basis of ethanol response. Studies in invertebrate models show that tolerance involves conserved changes in the neurotransmitter systems, ion channels, and synaptic proteins. These neuroadaptive changes lead to a change in neuronal excitability, most likely to compensate for the enhanced inhibition by ethanol.

Neuronal E93 is required for adaptation to adult metabolism and behavior.

OBJECTIVE: Metamorphosis is a transition from growth to reproduction, through which an animal adopts adult behavior and metabolism. Yet the neural mechanisms underlying the switch are unclear. Here we report that neuronal E93, a transcription factor essential for metamorphosis, regulates the adult metabolism, physiology, and behavior in Drosophila melanogaster. METHODS: To find new neuronal regulators of metabolism, we performed a targeted RNAi-based screen of 70 Drosophila orthologs of the mammalian genes enriched in ventromedial hypothalamus (VMH). Once E93 was identified from the screen, we characterized changes in physiology and behavior when neuronal expression of E93 is knocked down. To identify the neurons where E93 acts, we performed an additional screen targeting subsets of neurons or endocrine cells. RESULTS: E93 is required to control appetite, metabolism, exercise endurance, and circadian rhythms. The diverse phenotypes caused by pan-neuronal knockdown of E93, including obesity, exercise intolerance and circadian disruption, can all be phenocopied by knockdown of E93 specifically in either GABA or MIP neurons, suggesting these neurons are key sites of E93 action. Knockdown of the Ecdysone Receptor specifically in MIP neurons partially phenocopies the MIP neuron-specific knockdown of E93, suggesting the steroid signal coordinates adult metabolism via E93 and a neuropeptidergic signal. Finally, E93 expression in GABA and MIP neurons also serves as a key switch for the adaptation to adult behavior, as animals with reduced expression of E93 in the two subsets of neurons exhibit reduced reproductive activity. CONCLUSIONS: Our study reveals that E93 is a new monogenic factor essential for metabolic, physiological, and behavioral adaptation from larval behavior to adult behavior.

Iterative assay for transposase-accessible chromatin by sequencing to isolate functionally relevant neuronal subtypes.

The Drosophila brain contains tens of thousands of distinct cell types. Thousands of different transgenic lines reproducibly target specific neuron subsets, yet most still express in several cell types. Furthermore, most lines were developed without a priori knowledge of where the transgenes would be expressed. To aid in the development of cell type-specific tools for neuronal identification and manipulation, we developed an iterative assay for transposase-accessible chromatin (ATAC) approach. Open chromatin regions (OCRs) enriched in neurons, compared to whole bodies, drove transgene expression preferentially in subsets of neurons. A second round of ATAC-seq from these specific neuron subsets revealed additional enriched OCR2s that further restricted transgene expression within the chosen neuron subset. This approach allows for continued refinement of transgene expression, and we used it to identify neurons relevant for sleep behavior. Furthermore, this approach is widely applicable to other cell types and to other organisms.

Large genetic analysis of alcohol resistance and tolerance reveals an inverse correlation and suggests 'true' tolerance mutants.

Tolerance occurs when, following an initial experience with a substance, more of the substance is required subsequently to induce the same behavioral effects. Tolerance is historically not well-understood, and numerous researchers have turned to model organisms, particularly Drosophila melanogaster, to unravel its mechanisms. Flies have high translational relevance for human alcohol responses, and there is substantial overlap in disease-causing genes between flies and humans, including those associated with Alcohol Use Disorder. Numerous Drosophila tolerance mutants have been described; however, approaches used to identify and characterize these mutants have varied across time and between labs and have mostly disregarded any impact of initial resistance/sensitivity to ethanol on subsequent tolerance development. Here, we have analyzed a large amount of data - our own published and unpublished data and data published by other labs - to uncover an inverse correlation between initial ethanol resistance and tolerance phenotypes. This inverse correlation suggests that initial resistance phenotypes can explain many 'perceived' tolerance phenotypes. Additionally, we show that tolerance should be measured as a relative increase in time to sedation between an initial and second exposure rather than an absolute change in time to sedation. Finally, based on our analysis, we provide a method for using a linear regression equation to assess the residuals of potential tolerance mutants. We show that these residuals provide predictive insight into the likelihood of a mutant being a 'true' tolerance mutant, and we offer a framework for understanding the relationship between initial resistance and tolerance.

The Stage-Based Model of Addiction-Using Drosophila to Investigate Alcohol and Psychostimulant Responses.

Addiction is a progressive and complex disease that encompasses a wide range of disorders and symptoms, including substance use disorder (SUD), for which there are few therapeutic treatments. SUD is the uncontrolled and chronic use of substances despite the negative consequences resulting from this use. The progressive nature of addiction is organized into a testable framework, the neurobiological stage-based model, that includes three behavioral stages: (1) binge/intoxication, (2) withdrawal/negative affect, and (3) preoccupation/anticipation. Human studies offer limited opportunities for mechanistic insights into these; therefore, model organisms, like Drosophila melanogaster, are necessary for understanding SUD. Drosophila is a powerful model organism that displays a variety of SUD-like behaviors consistent with human and mammalian substance use, making flies a great candidate to study mechanisms of behavior. Additionally, there are an abundance of genetic tools like the GAL4/UAS and CRISPR/Cas9 systems that can be used to gain insight into the molecular mechanisms underlying the endophenotypes of the three-stage model. This review uses the three-stage framework and discusses how easily testable endophenotypes have been examined with experiments using Drosophila, and it outlines their potential for investigating other endophenotypes.

Harnessing changes in open chromatin determined by ATAC-seq to generate insulin-responsive reporter constructs.

BACKGROUND: Gene regulation is critical for proper cellular function. Next-generation sequencing technology has revealed the presence of regulatory networks that regulate gene expression and essential cellular functions. Studies investigating the epigenome have begun to uncover the complex mechanisms regulating transcription. Assay for transposase-accessible chromatin by sequencing (ATAC-seq) is quickly becoming the assay of choice for many epigenomic investigations. However, whether intervention-mediated changes in accessible chromatin determined by ATAC-seq can be harnessed to generate intervention-inducible reporter constructs has not been systematically assayed. RESULTS: We used the insulin signaling pathway as a model to investigate chromatin regions and gene expression changes using ATAC- and RNA-seq in insulin-treated Drosophila S2 cells. We found correlations between ATAC- and RNA-seq data, especially when stratifying differentially-accessible chromatin regions by annotated feature type. In particular, our data demonstrated a weak but significant correlation between chromatin regions annotated to enhancers (1-2 kb from the transcription start site) and downstream gene expression. We cloned candidate enhancer regions upstream of luciferase and demonstrate insulin-inducibility of several of these reporters. CONCLUSIONS: Insulin-induced chromatin accessibility determined by ATAC-seq reveals enhancer regions that drive insulin-inducible reporter gene expression.

Optimized assay for transposase-accessible chromatin by sequencing (ATAC-seq) library preparation from adult Drosophila melanogaster neurons.

Assay for transposase-accessible chromatin by sequencing (ATAC-seq) is rapidly becoming the assay of choice to investigate chromatin-mediated gene regulation, largely because of low input requirements, a fast workflow, and the ability to interrogate the entire genome in an untargeted manner. Many studies using ATAC-seq use mammalian or human-derived tissues, and established protocols work well in these systems. However, ATAC-seq is not yet widely used in Drosophila. Vinegar flies present several advantages over mammalian systems that make them an excellent model for ATAC-seq studies, including abundant genetic tools that allow straightforward targeting, transgene expression, and genetic manipulation that are not available in mammalian models. Because current ATAC-seq protocols are not optimized to use flies, we developed an optimized workflow that accounts for several complicating factors present in Drosophila. We examined parameters affecting nuclei isolation, including input size, freezing time, washing, and possible confounds from retinal pigments. Then, we optimized the enzymatic steps of library construction to account for the smaller Drosophila genome size. Finally, we used our optimized protocol to generate ATAC-seq libraries that meet ENCODE quality metrics. Our optimized protocol enables extensive ATAC-seq experiments in Drosophila, thereby leveraging the advantages of this powerful model system to understand chromatin-mediated gene regulation.

Chloride oscillation in pacemaker neurons regulates circadian rhythms through a chloride-sensing WNK kinase signaling cascade.

Central pacemaker neurons regulate circadian rhythms and undergo diurnal variation in electrical activity in mammals and flies.1,2 Circadian variation in the intracellular chloride concentration of mammalian pacemaker neurons has been proposed to influence the response to GABAergic neurotransmission through GABAA receptor chloride channels.3 However, results have been contradictory,4-9 and a recent study demonstrated circadian variation in pacemaker neuron chloride without an effect on GABA response.10 Therefore, whether and how intracellular chloride regulates circadian rhythms remains controversial. Here, we demonstrate a signaling role for intracellular chloride in the Drosophila small ventral lateral (sLNv) pacemaker neurons. In control flies, intracellular chloride increases in sLNvs over the course of the morning. Chloride transport through sodium-potassium-2-chloride (NKCC) and potassium-chloride (KCC) cotransporters is a major determinant of intracellular chloride concentrations.11Drosophila melanogaster with loss-of-function mutations in the NKCC encoded by Ncc69 have abnormally low intracellular chloride 6 h after lights on, loss of morning anticipation, and a prolonged circadian period. Loss of kcc, which is expected to increase intracellular chloride, suppresses the long-period phenotype of Ncc69 mutant flies. Activation of a chloride-inhibited kinase cascade, consisting of WNK (with no lysine [K]) kinase and its downstream substrate, Fray, is necessary and sufficient to prolong period length. Fray activation of an inwardly rectifying potassium channel, Irk1, is also required for the long-period phenotype. These results indicate that the NKCC-dependent rise in intracellular chloride in Drosophila sLNv pacemakers restrains WNK-Fray signaling and overactivation of an inwardly rectifying potassium channel to maintain normal circadian period length.

The Use of Drosophila to Understand Psychostimulant Responses.

The addictive properties of psychostimulants such as cocaine, amphetamine, methamphetamine, and methylphenidate are based on their ability to increase dopaminergic neurotransmission in the reward system. While cocaine and methamphetamine are predominately used recreationally, amphetamine and methylphenidate also work as effective therapeutics to treat symptoms of disorders including attention deficit and hyperactivity disorder (ADHD) and autism spectrum disorder (ASD). Although both the addictive properties of psychostimulant drugs and their therapeutic efficacy are influenced by genetic variation, very few genes that regulate these processes in humans have been identified. This is largely due to population heterogeneity which entails a requirement for large samples. Drosophila melanogaster exhibits similar psychostimulant responses to humans, a high degree of gene conservation, and allow performance of behavioral assays in a large population. Additionally, amphetamine and methylphenidate reduce impairments in fly models of ADHD-like behavior. Therefore, Drosophila represents an ideal translational model organism to tackle the genetic components underlying the effects of psychostimulants. Here, we break down the many assays that reliably quantify the effects of cocaine, amphetamine, methamphetamine, and methylphenidate in Drosophila. We also discuss how Drosophila is an efficient and cost-effective model organism for identifying novel candidate genes and molecular mechanisms involved in the behavioral responses to psychostimulant drugs.

The fly liquid-food electroshock assay (FLEA) suggests opposite roles for neuropeptide F in avoidance of bitterness and shock.

BACKGROUND: Proper regulation of feeding is important for an organism's well-being and survival and involves a motivational component directing the search for food. Dissecting the molecular and neural mechanisms of motivated feeding behavior requires assays that allow quantification of both motivation and food intake. Measurements of motivated behavior usually involve assessing physical effort or overcoming an aversive stimulus. Food intake in Drosophila can be determined in a number of ways, including by measuring the time a fly's proboscis interacts with a food source associated with an electrical current in the fly liquid-food interaction counter (FLIC). Here, we show that electrical current flowing through flies during this interaction is aversive, and we describe a modified assay to measure motivation in Drosophila. RESULTS: Food intake is reduced during the interaction with FLIC when the electrical current is turned on, which provides a confounding variable in studies of motivated behavior. Based on the FLIC, we engineer a novel assay, the fly liquid-food electroshock assay (FLEA), which allows for current adjustments for each feeding well. Using the FLEA, we show that both external incentives and internal motivational state can serve as drivers for flies to overcome higher current (electric shock) to obtain superior food. Unlike similar assays in which bitterness is the aversive stimulus for the fly to overcome, we show that current perception is not discounted as flies become more food-deprived. Finally, we use genetically manipulated flies to show that neuropeptide F, an orthologue of mammalian NPY previously implicated in regulation of feeding motivation, is required for sensory processing of electrical current. CONCLUSION: The FLEA is therefore a novel assay to accurately measure incentive motivation in Drosophila. Using the FLEA, we also show that neuropeptide F is required for proper perception or processing of an electroshock, a novel function for this neuropeptide involved in the processing of external and internal stimuli.

Flying Together: Drosophila as a Tool to Understand the Genetics of Human Alcoholism.

Alcohol use disorder (AUD) exacts an immense toll on individuals, families, and society. Genetic factors determine up to 60% of an individual's risk of developing problematic alcohol habits. Effective AUD prevention and treatment requires knowledge of the genes that predispose people to alcoholism, play a role in alcohol responses, and/or contribute to the development of addiction. As a highly tractable and translatable genetic and behavioral model organism, Drosophila melanogaster has proven valuable to uncover important genes and mechanistic pathways that have obvious orthologs in humans and that help explain the complexities of addiction. Vinegar flies exhibit remarkably strong face and mechanistic validity as a model for AUDs, permitting many advancements in the quest to understand human genetic involvement in this disease. These advancements occur via approaches that essentially fall into one of two categories: (1) discovering candidate genes via human genome-wide association studies (GWAS), transcriptomics on post-mortem tissue from AUD patients, or relevant physiological connections, then using reverse genetics in flies to validate candidate genes' roles and investigate their molecular function in the context of alcohol. (2) Utilizing flies to discover candidate genes through unbiased screens, GWAS, quantitative trait locus analyses, transcriptomics, or single-gene studies, then validating their translational role in human genetic surveys. In this review, we highlight the utility of Drosophila as a model for alcoholism by surveying recent advances in our understanding of human AUDs that resulted from these various approaches. We summarize the genes that are conserved in alcohol-related function between humans and flies. We also provide insight into some advantages and limitations of these approaches. Overall, this review demonstrates how Drosophila have and can be used to answer important genetic questions about alcohol addiction.

The Neurotransmitters Involved in Drosophila Alcohol-Induced Behaviors.

Alcohol is a widely used and abused substance with numerous negative consequences for human health and safety. Historically, alcohol's widespread, non-specific neurobiological effects have made it a challenge to study in humans. Therefore, model organisms are a critical tool for unraveling the mechanisms of alcohol action and subsequent effects on behavior. Drosophila melanogaster is genetically tractable and displays a vast behavioral repertoire, making it a particularly good candidate for examining the neurobiology of alcohol responses. In addition to being experimentally amenable, Drosophila have high face and mechanistic validity: their alcohol-related behaviors are remarkably consistent with humans and other mammalian species, and they share numerous conserved neurotransmitters and signaling pathways. Flies have a long history in alcohol research, which has been enhanced in recent years by the development of tools that allow for manipulating individual Drosophila neurotransmitters. Through advancements such as the GAL4/UAS system and CRISPR/Cas9 mutagenesis, investigation of specific neurotransmitters in small subsets of neurons has become ever more achievable. In this review, we describe recent progress in understanding the contribution of seven neurotransmitters to fly behavior, focusing on their roles in alcohol response: dopamine, octopamine, tyramine, serotonin, glutamate, GABA, and acetylcholine. We chose these small-molecule neurotransmitters due to their conservation in mammals and their importance for behavior. While neurotransmitters like dopamine and octopamine have received significant research emphasis regarding their contributions to behavior, others, like glutamate, GABA, and acetylcholine, remain relatively unexplored. Here, we summarize recent genetic and behavioral findings concerning these seven neurotransmitters and their roles in the behavioral response to alcohol, highlighting the fitness of the fly as a model for human alcohol use.

Altered Actin Filament Dynamics in the Drosophila Mushroom Bodies Lead to Fast Acquisition of Alcohol Consumption Preference.

Alcohol use is highly prevalent in the United States and across the world, and every year millions of people suffer from alcohol use disorders (AUDs). Although the genetic contribution to developing AUDs is estimated to be 50-60%, many of the underlying molecular mechanisms remain unclear. Previous studies from our laboratory revealed that Drosophila melanogaster lacking RhoGAP18B and Ras Suppressor 1 (Rsu1) display reduced sensitivity to ethanol-induced sedation. Both Rsu1 and RhoGAP18B are negative regulators of the small Rho-family GTPase, Rac1, a modulator of actin dynamics. Here we investigate the role of Rac1 and its downstream target, the actin-severing protein cofilin, in alcohol consumption preference. We show that these two regulators of actin dynamics can alter male experience-dependent alcohol preference in a bidirectional manner: expressing either activated Rac1 or dominant-negative cofilin in the mushroom bodies (MBs) abolishes experience-dependent alcohol preference. Conversely, dominant-negative Rac1 or activated cofilin MB expression lead to faster acquisition of alcohol preference. Our data show that Rac1 and cofilin activity are key to determining the rate of acquisition of alcohol preference, revealing a critical role of actin dynamics regulation in the development of voluntary self-administration in DrosophilaSIGNIFICANCE STATEMENT The risks for developing an alcohol use disorder (AUD) are strongly determined by genetic factors. Understanding the genes and molecular mechanisms that contribute to that risk is therefore a necessary first step for the development of targeted therapeutic intervention. Here we show that regulators of actin cytoskeleton dynamics can bidirectionally determine the acquisition rate of alcohol self-administration, highlighting this process as a key mechanism contributing to the risk of AUD development.

JmjC domain proteins modulate circadian behaviors and sleep in Drosophila.

Jumonji (JmjC) domain proteins are known regulators of gene expression and chromatin organization by way of histone demethylation. Chromatin modification and remodeling provides a means to modulate the activity of large numbers of genes, but the importance of this class of predicted histone-modifying enzymes for different aspects of post-developmental processes remains poorly understood. Here we test the function of all 11 non-lethal members in the regulation of circadian rhythms and sleep. We find loss of every Drosophila JmjC gene affects different aspects of circadian behavior and sleep in a specific manner. Together these findings suggest that the majority of JmjC proteins function as regulators of behavior, rather than controlling essential developmental programs.

Alcohol-Induced Behaviors Require a Subset of Drosophila JmjC-Domain Histone Demethylases in the Nervous System.

BACKGROUND: Long-lasting transcriptional changes underlie a number of adaptations that contribute to alcohol use disorders (AUD). Chromatin remodeling, including histone methylation, can confer distinct, long-lasting transcriptional changes, and histone methylases are known to play a role in the development of addiction. Conversely, little is known about the relevance of Jumonji (JmjC) domain-containing demethylases in AUDs. We systematically surveyed the alcohol-induced phenotypes of null mutations in all 13 Drosophila JmjC genes. METHODS: We used a collection of JmjC mutants, the majority of which we generated by homologous recombination, and assayed them in the Booze-o-mat to determine their naïve sensitivity to sedation and their tolerance (change in sensitivity upon repeat exposure). Mutants with reproducible phenotypes had their phenotypes rescued with tagged genomic transgenes, and/or phenocopied by nervous system-specific knockdown using RNA interference (RNAi). RESULTS: Four of the 13 JmjC genes (KDM3, lid, NO66, and HSPBAP1) showed reproducible ethanol (EtOH) sensitivity phenotypes. Some of the phenotypes were observed across doses, for example, the enhanced EtOH sensitivity of KDM3KO and NO66KO , but others were dose dependent, such as the reduced EtOH sensitivity of HSPBAP1KO , or the enhanced EtOH tolerance of NO66KO . These phenotypes were rescued by their respective genomic transgenes in KDM3KO and NO66KO mutants. While we were unable to rescue lidk mutants, knockdown of lid in the nervous system recapitulated the lidk phenotype, as was observed for KDM3KO and NO66KO RNAi-mediated knockdown. CONCLUSIONS: Our study reveals that the Drosophila JmjC-domain histone demethylases Lid, KDM3, NO66, and HSPBAP1 are required for normal EtOH-induced sedation and tolerance. Three of 3 tested of those 4 JmjC genes are required in the nervous system for normal alcohol-induced behavioral responses, suggesting that this gene family is an intriguing avenue for future research.

Systematic discovery of genetic modulation by Jumonji histone demethylases in Drosophila.

Jumonji (JmjC) domain proteins influence gene expression and chromatin organization by way of histone demethylation, which provides a means to regulate the activity of genes across the genome. JmjC proteins have been associated with many human diseases including various cancers, developmental and neurological disorders, however, the shared biology and possible common contribution to organismal development and tissue homeostasis of all JmjC proteins remains unclear. Here, we systematically tested the function of all 13 Drosophila JmjC genes. Generation of molecularly defined null mutants revealed that loss of 8 out of 13 JmjC genes modify position effect variegation (PEV) phenotypes, consistent with their ascribed role in regulating chromatin organization. However, most JmjC genes do not critically regulate development, as 10 members are viable and fertile with no obvious developmental defects. Rather, we find that different JmjC mutants specifically alter the phenotypic outcomes in various sensitized genetic backgrounds. Our data demonstrate that, rather than controlling essential gene expression programs, Drosophila JmjC proteins generally act to "fine-tune" different biological processes.

Dopaminergic rules of engagement for memory in Drosophila.

Dopamine is associated with a variety of conserved responses across species including locomotion, sleep, food consumption, aggression, courtship, addiction and several forms of appetitive and aversive memory. Historically, dopamine has been most prominently associated with dynamics underlying reward, punishment, or salience. Recent emerging evidence from Drosophila supports a role in all of these functions, as well as additional roles in the interplay between external sensation and internal states and forgetting of the very memories dopamine helped encode. We discuss how cell-specific resolution and manipulation are elucidating the rules of dopamine's involvement in encoding valence and memory.

Neural basis of reward anticipation and its genetic determinants.

Dysfunctional reward processing is implicated in various mental disorders, including attention deficit hyperactivity disorder (ADHD) and addictions. Such impairments might involve different components of the reward process, including brain activity during reward anticipation. We examined brain nodes engaged by reward anticipation in 1,544 adolescents and identified a network containing a core striatal node and cortical nodes facilitating outcome prediction and response preparation. Distinct nodes and functional connections were preferentially associated with either adolescent hyperactivity or alcohol consumption, thus conveying specificity of reward processing to clinically relevant behavior. We observed associations between the striatal node, hyperactivity, and the vacuolar protein sorting-associated protein 4A (VPS4A) gene in humans, and the causal role of Vps4 for hyperactivity was validated in Drosophila Our data provide a neurobehavioral model explaining the heterogeneity of reward-related behaviors and generate a hypothesis accounting for their enduring nature.