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Official websites use. Share sensitive information only on official, secure websites. Corresponding authors: ranholt clemson. Whereas the neurological effects of cocaine have been well documented, effects of acute cocaine consumption on genome-wide gene expression across the brain remain largely unexplored. This question cannot be readily addressed in humans but can be approached using the Drosophila melanogaster model, where gene expression in the entire brain can be surveyed at once. Flies exposed to cocaine show impaired locomotor activity, including climbing behavior and startle response a measure of sensorimotor integration , and increased incidence of seizures and compulsive grooming. To identify specific cell populations that respond to acute cocaine exposure, we analyzed single-cell transcriptional responses in duplicate samples of flies that consumed fixed amounts of sucrose or sucrose supplemented with cocaine, in both sexes. Unsupervised clustering of the transcriptional profiles of a total of 86, cells yielded 36 distinct clusters. Annotation of clusters based on gene markers revealed that all major cell types neuronal and glial as well as neurotransmitter types from most brain regions were represented. The brain transcriptional responses to cocaine showed profound sexual dimorphism and were considerably more pronounced in males than females. Differential expression analysis within individual clusters indicated cluster-specific responses to cocaine. Clusters corresponding to Kenyon cells of the mushroom bodies and glia showed especially large transcriptional responses following cocaine exposure. Cluster specific coexpression networks and global interaction networks revealed a diverse array of cellular processes affected by acute cocaine exposure. These results provide an atlas of sexually dimorphic cocaine-modulated gene expression in a model brain. Cocaine use presents a significant socioeconomic health problem Kariisa et al. Although cocaine use results in arousal and euphoria, side effects include accelerated heart rate, mood swings, difficulty sleeping, loss of appetite, and cognitive distortions. Escalated consumption of cocaine can result in psychosis, cardiovascular disease, and stroke. The propensity for cocaine use depends on genetic and environmental factors. Whereas much is known about the neurological effects of cocaine, information about genetic variants that are associated with variation in individual susceptibility to psychostimulant use remains incomplete. Furthermore, little is known about acute effects of cocaine consumption on genome-wide gene expression across the brain. Drosophila melanogaster presents an advantageous model system for systems genetic analyses of cocaine consumption Kaun et al. The crystal structure of the Drosophila dopamine transporter has been obtained and its binding site can accommodate cocaine Wang et al. Dopamine Bainton et al. Consistent with the actions of these neurotransmitters, overexpression of the vesicular monoamine transporter in both dopaminergic and serotonergic neurons decreases the response to cocaine Chang et al. Thus, fundamental neural mechanisms affected by exposure to psychostimulants are conserved across phyla, from flies to humans. Studies on inbred wild-derived, fully sequenced lines of the Drosophila melanogaster Genetic Reference Panel Mackay et al. Targeted RNA interference RNAi of gene expression implicated dopaminergic neurons and the mushroom bodies, central brain structures associated with experience-dependent modification of behavior, with consumption and development of preference for these psychostimulants Highfill et al. However, RNAi-mediated reduction in expression of candidate genes in glia also affected cocaine consumption, suggesting that widespread brain regions contribute to cocaine-associated behavioral phenotypes. The present study aims to delineate the effects of acute cocaine consumption on genome-wide gene expression across the Drosophila brain. To assess the effects of acute cocaine exposure on fly behavior, we allowed males and females to ingest a fixed amount of sucrose or sucrose supplemented with cocaine within a maximal 2-h time period. We measured negative geotaxis, an innate locomotor behavior, to assess locomotion impairments, and startle behavior as a measure of sensorimotor integration Fig. Male flies exposed to cocaine took longer to climb in the negative geotaxis assay than control flies, whereas females appeared unaffected Fig. Both male and female flies exposed to cocaine spent less time moving after being subjected to a mechanical disturbance Fig. The average reduced locomotor activity in both assays might result from excessive grooming behavior in a fraction of male flies exposed to cocaine Fig. In addition, we observed seizures in a small percentage of flies after cocaine intake during the negative geotaxis assay Supplemental Video S3. Seizures rarely occurred in controls Fig. Collectively, these experiments provide evidence that acute exposure to cocaine results in neurological impairments. Behavioral characterization of Canton S B flies after cocaine exposure. A Negative geotaxis. The 7. Horizontal lines represent means with standard error. Male flies exposed to cocaine took longer on average to climb compared to controls. B Startle response. The percent time out of 45 sec that each fly spent moving following a cm drop was measured. Flies exposed to cocaine spent less time moving on average than controls. C Seizures and grooming activity during negative geotaxis. The percent of flies that exhibited seizures or grooming activity during the negative geotaxis assay after exposure to cocaine was measured. Both females and males exposed to cocaine exhibited seizure activity more than controls. D Seizures and grooming activity during the startle response. The percent of flies that exhibited seizures or grooming activity during the startle assay after exposure to cocaine was measured. To assess effects of cocaine consumption on brain gene expression, we analyzed single-cell transcriptional responses in duplicate samples of flies that consumed fixed amounts of sucrose or sucrose supplemented with cocaine in both males and females Supplemental Table S2. Unsupervised clustering of the integrated data set based on the expression profiles of individual cells resulted in 36 distinct, stable clusters Fig. We assessed the stability of clustering by examining the relationship between the number of new clusters identified and the granularity resolution parameter Butler et al. At a resolution of 0. UMAP visualization and clustering of single-cell expression data. Cells were clustered based on their expression pattern using the unsupervised shared nearest neighbor SNN clustering algorithm. Individual dots represent each cell and the colors of the dots represent the cluster to which the cells belong. Identification of cell types from clusters was performed by cross-referencing cluster-defining genes across FlyBase Thurmond et al. Annotation of clusters based on their gene markers revealed that all major cell types neuronal and glial as well as neurotransmitter types from most brain regions, including the mushroom bodies, were represented Fig. Differential expression analysis within individual clusters indicated cluster-specific transcriptional responses to cocaine. Especially, clusters corresponding to glia and Kenyon cells of the mushroom bodies showed transcriptional responses following cocaine exposure Fig. Thus, acute exposure to cocaine elicits rapid widespread changes in gene expression throughout the brain. Distribution of differentially expressed genes across clusters in males A and females B exposed to cocaine, and Venn diagrams showing overlap between differentially expressed genes in males and females C. Differentially expressed genes are listed on the top columns and cell clusters are represented by the rows. Magenta boxes show up-regulation and turquoise boxes show down-regulation of gene expression as a result of exposure to cocaine. Panel C shows Venn diagrams of clusters with sexually dimorphic responses to cocaine exposure. We first analyzed differential expression by combining the male and female samples. There was a significantly greater number of genes up-regulated than down-regulated across all clusters in response to cocaine. In addition, a core set of genes, selected based on their ranks from the TopKLists consistency analysis Schimek et al. S2 ; Supplemental Tables S5, S6. Examination of males and females separately revealed extensive sexual dimorphism in the response to cocaine. Consistent with effects on organismal phenotypes Fig. Clusters C11 Kenyon cells , C16 not annotated , C17 astrocytes , and C22 surface glia had unique responses compared to the rest of the clusters in both males and females; C22 shows the most extensive cocaine-induced changes in transcript abundances in males Fig. In addition to differences in the magnitude of cocaine-modulated gene expression between the sexes, we also observe differences in direction, in which up-regulation in one sex corresponds to down-regulation in the other. Thus, although cocaine-modulated changes in gene expression are widespread throughout the brain in both sexes, specific changes in transcript abundances are distinct between males and females. Reactome analysis of pathway enrichment Fabregat et al. We were unable to assign a specific identity to C16, which might be comprised of a mixture of neurons from the antennal lobe and optic lobe Supplemental Table S4. In contrast to the signal transduction elements associated with the neuronal C11 and C16 clusters, different cellular mechanisms are associated with cocaine exposure in C17 and C22, which represent astrocytes and surface glia, which comprise the blood-brain barrier in the fly, respectively Supplemental Table S Functional enrichment analysis yielded few differentially expressed genes for females, but for C22 in the male data set, it revealed enrichment of Notch activation and signaling, degradation of GABA, immune pathways related to NF-kB activation, cytokine production and Toll-like receptor signaling, and nonsense-mediated decay and translation initiation Supplemental Table S These observations are in line with expected functions of glia Kremer et al. Across all cell clusters, we find genes of unknown function and genes encoding long noncoding RNAs, which are likely to play a regulatory role Everett et al. We present examples of coexpression networks for C16 males Fig. S3 and C22 males Fig. Coexpressions have been filtered using Random Matrix Theory. The inset in A corresponds to a subset of genes within the subnetwork that have very strong correlation coefficient values with each other compared to the rest of the data set. Colors of edges represent the positive red and negative green correlations. Central genes include transcriptional regulators associated with development, including dendrite morphogenesis Lim1 , jim as well as signal transduction 5-HT2A, CNMaR Fig. Random Matrix Theory identified two major subnetworks within the interaction network that represents C22 Fig. Of the three genes, CG , which belongs to the SLC22 family of organic ion transporters, had the largest number of negative correlations linking the two large subnetworks. This gene is expressed in the blood-brain barrier of flies and postulated to play a role in chemoprotection of the brain Hindle and Bainton Finally, we assessed interaction networks among differentially expressed genes across all cell clusters separately for males and females Figs. The global transcriptional response to cocaine in males is captured by a complex network of interconnected modules Fig. Functional analyses reveal modules associated with Toll-like receptor signaling, ABC xenobiotic transporters and ATPase ion pumps, translation initiation, and hexose transport, G protein-coupled receptor signaling and clathrin-mediated endocytosis. The female network has fewer genes and contains modules associated with phototransduction, lipid receptors and transport, and glutathione metabolism and neurotransmission Fig. In each network, multiple cell clusters contribute to the organization of each network module, indicating that the transcriptional response to cocaine is coordinated not only within but also across different cells throughout the brain. Interaction network analysis of DEGs from all clusters in the male data set. Gray edges represent interactions. Genes that were differentially expressed in multiple clusters are depicted as pie charts with each color representing the respective cluster. Annotations of these circular groups represent the pathways that are enriched for the genes within these groups. Interaction network analysis of DEGs from all clusters in the female data set. Unlike humans and rodent models of substance abuse, Drosophila enables comprehensive single-cell transcriptomics analyses of living cells across the entire brain in a single analysis Davie et al. We generated an atlas of cocaine-modulated gene expression changes in the fly brain and found that transcriptional changes in response to acute consumption of cocaine are rapid, widespread in both neurons and glia, and sexually dimorphic. We performed the experiments in duplicate to establish cross-validation. Transcript abundance levels are influenced by circadian time Claridge-Chang et al. Therefore, we performed all experiments within a defined window of circadian time. Also, we cannot draw inferences as to which changes in gene expression might lay a foundation for development of cocaine preference Highfill et al. However, sexual dimorphism in the transcriptional response following acute exposure to cocaine is extreme compared to any previous studies and is mirrored and consistent with the behavioral phenotypes Fig. It is possible that differences in cocaine metabolism between males and females may contribute to this sexual dimorphism. The sexual dimorphism we observe is in line with previous studies that show reduced locomotion and increased grooming in flies given low doses of cocaine, with males showing greater impairments in behavior McClung and Hirsch We note, however, that changes in gene expression are not, a priori, necessary for cocaine to elicit behavioral effects. Transcriptional effects of cocaine exposure are evident in all cell clusters, but among neuronal populations, the Kenyon cells of the mushroom bodies represented by C11 and C20 have especially large responses to cocaine. The mushroom bodies are integrative centers associated with experience-dependent modulation of behavior and have previously been implicated in development of preference for cocaine intake Highfill et al. Acute cocaine consumption leads to changes in gene products associated with signal transduction, including phosphatidyl inositol-mediated signaling in C11 in males Supplemental Table S10 , as well as cyclic AMP-mediated signaling, which is evident from increased expression of rutabaga rut , in C20 in females Supplemental Tables S8, S Kenyon cells receive dopaminergic input, and acute exposure to cocaine results in altered expression of Ddc , which encodes Dopa decarboxylase, and down-regulation of Dop2R , which encodes a dopamine receptor in C11 in females Supplemental Table S8. Down-regulation of jdp can lead to dopamine blockade through its activity as a cochaperone in synaptic vesicle release Ye et al. Based on the collective data, cocaine-induced modulation of gene expression appears to result in altered synaptic regulation in the mushroom bodies. In addition to cocaine-modulated changes in gene expression in neurons, acute exposure to cocaine results in altered transcript abundances in different populations of glia C13, C17, C19, C22 , including surface glia C22 and astrocytes C Mutants of moody , which encodes two G protein-coupled receptor isoforms localized to surface glia, have increased sensitivity to cocaine Bainton et al. The surface glia, represented by perineurial and subperineurial glia, act as the blood-brain barrier DeSalvo et al. Genes associated with the blood-brain barrier in Drosophila are also up-regulated in response to cocaine. This includes ogre, which regulates gap junction channel activity Holcroft et al. Acute exposure to cocaine causes changes in expression of genes involved with Toll-like receptor TLR signaling, nuclear factor kappa B NF-kB activation, cytokine production, and glutathione metabolism Figs. TLR signaling has been associated with response to cocaine Zhu et al. Astrocytes provide metabolic support for neurons Tsacopoulos and Magistretti and regulate neuronal NMDA receptors and synaptic plasticity Haydon et al. Glutamatergic neurons feature prominently in C1, C3, C21, and C Studies on rats have shown that cocaine is toxic to astrocytes and that loss of astrocytes leads to dysfunctional neuron-glia communication Badisa et al. Eaat1 , which is highly expressed in astrocytes Supplemental Table S4 , encodes a transmembrane glutamate transporter involved in glia-neuron communication MacNamee et al. Eaat1 is down-regulated in response to cocaine in males and has been associated with lifespan Mazaud et al. Episodic ataxia due to a mutation in this gene Parinejad et al. Like flies, people show sexually dimorphic effects of cocaine use. Although substance use disorders are more prevalent in males, females are more likely to escalate their drug usage to the compulsive stage faster than males Haas and Peters ; Westermeyer and Boedicker , report more negative effects during withdrawal, and have greater relapse than males Becker and Koob Females metabolize cocaine faster than males, as is evident from lower levels of cocaine metabolites in the bloodstream of females compared to males who have consumed equal amounts of cocaine Lukas et al. In this same study, males experienced the effects of cocaine faster and with greater intensity than females. In rats, differences in sex hormones and the estrous cycle contribute to differences in sensitivity to cocaine Becker and Koob ; Cao et al. Thus, sexual dimorphism is a universal feature of the physiological response to cocaine. Although the Drosophila brain is anatomically distinct from the vertebrate brain, fundamental neural processes are evolutionarily conserved. Functions of the dopamine reward pathway in humans are analogous to experience-dependent modulation of behaviors by the mushroom bodies. Dop2R encodes a dopamine receptor, and its human ortholog, DRD2 , is a well-characterized component of the dopamine reward pathway, which mediates development of cocaine dependence Noble et al. The glutamate receptor genes GluRIA and GluRIB are associated with glutamatergic neurotransmission, which is altered following exposure to cocaine and has been linked to cocaine sensitization and cocaine-induced behavioral effects Ghasemzadeh et al. Vmat encodes the vesicular monoamine transporter responsible for packaging the neurotransmitters dopamine, serotonin, and octopamine in synaptic vesicles Greer et al. These functional parallels between the fly model and human studies provide proof-of-principle that results from cocaine exposure obtained from the fly model can be translated to human populations. Thus, the comprehensive documentation of cocaine-mediated modulation of gene expression which we have derived can serve as a contextual framework for future human studies. Canton S B flies Norga et al. Briefly, five males and five females were placed into a vial and allowed to mate for 2 d before being cleared. Progeny from these vials were collected after eclosion and aged for 3—5 d before experimentation. To expose flies to cocaine, we performed a modified version of the capillary feeder CAFE assay Ja et al. We collected the first 40 flies that consumed 0. All experiments were carried out between 8 a. Flies were allowed to feed for no more than 2 h. We measured negative geotaxis and startle response of individual flies within a min time frame immediately following acute exposure to cocaine in the CAFE assay. We quantified grooming and seizures in addition to measuring the behavioral response in each assay. Excessive grooming was defined as more than 10 sec of constant grooming Supplemental Video S2. Seizure activity was defined as severe muscle tremors that prevented the fly from moving normally Supplemental Video S3. Brains were dissected from each fly immediately after it consumed the designated amount of sucrose or cocaine solution, and we used a dissociation protocol modified from Croset et al. We collected eight samples of 20 brains from males and females exposed to cocaine or sucrose, with two biological replicates per treatment and sex. We made libraries after GEM generation in accordance with 10x Genomics v3. The mkfastq pipeline within Cell Ranger v3. The sequencing and alignment summary is given in Supplemental Table S2. Raw expression counts output for each sample from the Cell Ranger pipeline was imported and analyzed using the Seurat v3 package in R R core team ; Butler et al. Genes expressed in less than five cells and cells with less than or greater than RNA features were filtered out. The upper and lower thresholds for the RNA features per cell were chosen based on the recommendations from the developers of the Seurat v3 pipeline. The recommendation is based on the multitude of observations indicating that cells with less than RNA features tend to have very sparse and unreliable signal and those with greater than RNA features tend to be miscalled multiplet cells. Normalization and subsequent integration were performed using the sctransform pipeline Hafemeister and Satija To identify the cell type clusters within the data set, unsupervised clustering using the FindClusters function and a resolution of 0. Cluster marker genes were identified using the FindAllMarker function min. The top three genes with positive expression for each cluster were extracted and used for cell type characterization. Differential expression was performed for each cluster in two ways: 1 after combining male and female samples together to test for effects of cocaine that are common to both sexes; and 2 testing for effects of cocaine in males and females separately to identify sexually dimorphic responses. The Pearson residuals output from the sctransform pipeline was used as input for differential expression DE calculation Hafemeister and Satija The results from DE calculation from the combined data set were used to determine which genes were consistently up-regulated and down-regulated, respectively, across all clusters as a result of exposure to cocaine. The top 50 ranked differentially up-regulated genes for each cluster and the top 20 ranked differentially down-regulated genes for each cluster were input into the TopKLists R package Schimek et al. The scaled data from the sctransform pipeline for differentially expressed genes from clusters 16 and 22 were extracted for the male samples. These scaled data were used as input for filtering through the Random Matrix Theory Gibson et al. The correlations that passed the filtering process were visualized using Cytoscape version 3. To identify specific pathways that are enriched in genes within each of the circular groups, we performed statistical overrepresentation tests on the gene IDs from each group using PANTHER Thomas et al. We thank Marion Campbell for technical assistance. Article published online before print. This section collects any data citations, data availability statements, or supplementary materials included in this article. As a library, NLM provides access to scientific literature. Genome Res. Find articles by Brandon M Baker. Find articles by Sneha S Mokashi. Find articles by Vijay Shankar. Find articles by Jeffrey S Hatfield. Find articles by Rachel C Hannah. Find articles by Trudy FC Mackay. Find articles by Robert RH Anholt. Received Jun 29; Accepted Feb 2. Open in a new tab. Freely available online through the Genome Research Open Access option. Download video file Similar articles. Add to Collections. Create a new collection. Add to an existing collection. Choose a collection Unable to load your collection due to an error Please try again. Add Cancel.
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