Biology subjects

Rothenfluh, A.

Publications and source records attributed to Rothenfluh, A..

13 recordsLinked to original sources

bioRxiv2026

Motivation to work for amino acids requires acute dopaminergic activity.

Aberrant motivation is a hallmark of many psychiatric disorders, and studying motivation in model systems can be useful to understand the neural mechanisms of these disorders. Having versatile, ethologically relevant assays with which to measure motivation is therefore important. However, motivational assays in Drosophila have not been able to measure willingness to exert effort to consume food. Here, we took advantage of the fact that flies prefer to feed on liquid over solid foods to develop a new assay using agarose as a barrier to feeding, which we termed the Fly Liquid vs Agarose Motivation Evaluation (FLAME). Unlike motivational assays based on overcoming bitterness, the FLAME measures willingness to work and is less confounded by deprivation acting on peripheral sensing of the modality to overcome. By depriving flies of specific nutrients and measuring their choice to overcome semi-solid agarose food to get those nutrients, we show that the FLAME is a versatile assay for measuring motivation. We show that flies choose to overcome agarose to consume sugar, salt, or amino acids, depending on the specific of prior deprivation. We then show that show that flies rely on acute activity in a subset of their dopamine neurons during the choice period in order to work for amino acids when deprived. The FLAME thus allows the investigation of specific neural mechanisms of motivation.

neuroscience↗

bioRxiv2026

A Microglial Regulatory Program Linked to Neuropsychiatric Disorders

Hoxb8 is a transcription factor required for the normal function of a specialized microglial population. Loss of Hoxb8 causes compulsive overgrooming and anxiety-like behaviors in mice, with greater severity in females after sexual maturity. However, the Hoxb8-dependent transcriptional program in microglia remains poorly understood. Here, we integrated Hoxb8 chromatin occupancy, transcriptional responses, and chromatin contacts to classify genes by their spatial relationship to Hoxb8 binding. Hoxb8 occupied thousands of genomic regions, but only a subset of associated genes responded transcriptionally. Locally associated, Hoxb8-activated genes were linked to immune signaling and hormone responsiveness, whereas locally associated, Hoxb8-suppressed genes were linked to cell-cycle and genome-maintenance processes. Distally associated genes contributed to neuronal and intercellular communication and were enriched for genes associated with obsessive-compulsive disorder and anxiety. These findings reveal a functionally organized Hoxb8-dependent transcriptional program and identify potential connections between Hoxb8 activity in microglia, hormone responsiveness, intercellular communication, and neuropsychiatric disease risk.

neuroscience↗

bioRxiv2023

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.

neuroscience↗

bioRxiv2023

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.

neuroscience↗

bioRxiv2022

One-carbon Pathways and Methylation Potential in Glutamatergic Neurons Regulate Behavioral Alcohol Responses

Despite the enormous harms of alcohol use disorders (AUDs), many mechanisms, as well as effective prevention or treatment strategies remain elusive. Genetic factors dictate a majority of AUD risk. These risk factors can manifest as reduced naive sensitivity to alcohols intoxicating effects and increased functional tolerance, i.e., brain-mediated decreases in sensitivity upon repeat exposure. The underlying neurobiology of how AUD-associated genes alter these endophenotypes remains poorly understood. Genes implicated in AUDs include epigenetic modifiers, such as histone demethylases, including Kdm3. We previously showed that whole-body and neuronal Kdm3 strongly affect ethanol sensitivity and tolerance in Drosophila. Here, we investigate the mechanisms of these effects, and, by extension, mechanisms of sensitivity and tolerance. RNA-seq and pathway analysis on Kdm3KO flies revealed disproportionate upregulation of genes involved in amino acid metabolism, including 1-carbon pathways. We show that acute amino acid feeding modulates sensitivity and tolerance in a Kdm3-dependent manner. Global manipulation of 1-carbon genes, especially glycine N-methyltransferase (Gnmt), glycine decarboxylase (Gldc), and sarcosine dehydrogenase (Sardh), alters alcohol sensitivity and tolerance. These changes in alcohol responses are likely mediated by global glycine levels (a substrate of these enzymes) rather than by 1-carbon input. Conversely, neuronal manipulations of 1-carbon pathways change alcohol sensitivity and tolerance in a pattern that suggests a mechanism through S-adenosyl methionine (SAM), a 1-carbon metabolite that is the universal methyl donor required for epigenetic methylation. Increasing SAM production specifically in glutamatergic neurons increases sensitivity and tolerance. Together, these findings reveal distinct mechanisms affecting alcohol sensitivity and tolerance globally (via glycine) and neuronally (via SAM), thus revealing an important and complex role of 1-carbon metabolism in mediating AUD phenotypes.

neuroscience↗

bioRxiv2022

Neuronal E93 regulates metabolic homeostasis

Metamorphosis is a transition from growth to reproduction, through which an animal adopts adult behavior and metabolism. Yet the mechanisms underlying the switch is unclear. Here we report that neuronal E93, a transcription factor essential for metamorphosis, regulates the adult metabolism and circadian rhythm in Drosophila melanogaster. When E93 is specifically knocked down in neurons, the flies become hyperphagic and obese with increased energy stores and disrupted circadian rhythms. A screen of Gal4 lines targeting subsets of neurons and endocrine cells identified neurons producing GABA and myoinhibitory peptide (MIP) as the main sites of E93 action. Knockdown of the ecdysone receptor specifically in MIP neurons partly phenocopies the MIP neuron-specific knockdown of E93 suggesting the steroid signal coordinates adult metabolism via E93. The circadian disruption caused by neuronal knockdown of E93 is also observed when E93 is knockdown in GABA and MIP neurons. Based on these results we suggest that neuronal E93 is a key switch for metabolic transition representing an intersectional node between metabolism and circadian biology.

physiology↗

bioRxiv2022

Drosophila Cocaine Avoidance is Mediated by Peripheral Bitter Gustatory Neurons

Genetic variation contributes to heterogeneity in the prevalence of complex disorders such as addiction. The genetic risk for developing a substance use disorder can vary between drugs. The estimated heritability rate of cocaine addiction is 72%, higher than any other drug. Despite recognition of this significant genetic component, little is known about the specific genes and mechanisms that lead to the development of cocaine addiction. Drosophila is an effective model organism for identifying the genes that underlie complex behaviors, including addiction. While Drosophila exposed to cocaine display features of intoxication similar to those observed in mammals, there is currently no model of cocaine self-administration in flies. Because cocaine is a natural insecticide, we wondered if Drosophila might naively avoid it through bitter chemosensory detection. To answer this question, we performed cocaine consumption and preference assays comparing wild-type flies and bitter-taste mutants. Our results demonstrate that Drosophila detect and avoid cocaine through bitter sensing gustatory neurons, and that this process requires gustatory receptor 66a (Gr66a). Additionally, we identify a peripheral mechanism of avoidance through cocaine detection with Drosophila legs. Our findings reveal that preingestive mechanisms of toxin detection play a significant role in Drosophila cocaine avoidance and provide evidence that disrupting gustatory perception of cocaine is essential for self-administration and, therefore, developing a model of self-administration in Drosophila.

neuroscience↗

bioRxiv2021

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. In mammals, circadian variation in the intracellular chloride concentration of pacemaker neurons has been proposed to influence the response to GABAergic neurotransmission through GABAA receptor chloride channels. However, results have been contradictory, and a recent study demonstrated circadian variation in pacemaker neuron chloride without an effect on GABA response. Therefore, whether and how intracellular chloride regulates circadian rhythms remains controversial. Here, we demonstrate a signaling role for intracellular chloride in the Drosophila ventral lateral (LNv) pacemaker neurons. In control flies, intracellular chloride increases in LNv neurons over the course of the morning. Chloride transport through the sodium-potassium-2-chloride (NKCC) and potassium-chloride (KCC) cotransporters is a major determinant of intracellular chloride concentrations. Drosophila melanogaster with loss-of-function mutations in the NKCC encoded by Ncc69 have abnormally low intracellular chloride six hours after lights on, and a lengthened 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 the 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 LNv pacemaker neurons restrains WNK-Fray signaling and overactivation of an inwardly rectifying potassium channel to maintain normal circadian period length.

neuroscience↗

bioRxiv2021

Integrating RNA-seq and assay for transposase-accessible chromatin by sequencing (ATAC-seq) predicts functionally-relevant chromatin regions

BackgroundGene 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. ResultsWe 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 strong correlation between chromatin regions annotated to distal promoters (1-2 kb from the transcription start site) and downstream gene expression. We cloned candidate distal promoter regions upstream of luciferase and demonstrate insulin-inducibility of several of these reporters. ConclusionsInsulin-induced chromatin accessibility determined by ATAC-seq reveals enhancer regions that drive insulin-inducible reporter gene expression.

cell biology↗

bioRxiv2021

An optimized protocol for neuronal assay for transposase-accessible chromatin by sequencing (ATAC-seq) library preparation using Drosophila melanogaster

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 unbiased 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, and 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.

neuroscience↗

bioRxiv2020

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

Proper regulation of feeding is important for an organisms well-being and survival. Food intake in Drosophila can be determined in a number of ways, including by measuring the time a flys proboscis interacts with a food source in the fly liquid-food interaction counter (FLIC). Here, we show that electrical current flowing through flies during this interaction is aversive and leads to a reduction in food intake. 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 as well as 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. 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 processing of external and internal stimuli. Significance StatementMany neuropsychiatric disorders, such as depression or addiction, are associated with alterations in motivated behavior. Assays measuring incentive motivation determine how driven an organism is to attain a goal, like food, or how attractive an incentive is. These tests require the animal to put effort into obtaining the reward, which can include physical work or overcoming an aversive stimulus. Such assays for Drosophila feeding have relied on flies overcoming bitterness to obtain their food. However, the perception of bitterness is discounted as flies become food deprived, confounding the interpretation. Here, we developed a novel assay that does not suffer from the same shortcomings and thus allows for more accurate assessments of incentive motivation in this widely used model organism.

neuroscience↗

bioRxiv2019

Dopaminergic learning and arousal circuits mediate opposing effects on alcohol consumption in Drosophila

The response to drugs of abuse is a combination of aversive and reinforcing reactions. While much is known about the role of dopamine in mammalian drug reinforcement, we know little about the brain circuits mediating drug aversion. Here we show that two distinct dopaminergic circuits mediate reinforcing and acute aversive responses to alcohol consumption in Drosophila. Protocerebral anterior medial dopamine neurons projecting to the mushroom bodies are required for flies to acquire alcohol preference. Conversely, a bilateral pair of dopamine neurons projecting to the dorsal fan-shaped body (dFSB) mediates acute alcohol avoidance. Alcohol consumption can be reduced by decreasing the activity of the appetitive reinforcement-circuit to the mushroom bodies, or by increasing activity in the dopamine neurons projecting to the dFSB. Thus, distinct dopaminergic pathways can be targeted to reduce the intake of harmful drugs.

neuroscience↗

bioRxiv2019

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). While the genetic contribution to developing AUDs is estimated to be 50-60%, many of the underlying molecular mechanisms remain unclear. Previous studies from our lab revealed that Drosophila 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 experience-dependent alcohol preference in a bidirectional manner: expressing either activated Rac1 or dominant-negative cofilin in the mushroom bodies (MB) 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 Drosophila.\n\nSignificance StatementThe 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.

neuroscience↗