The Drosophila brain on cocaine at single-cell resolution

Abstract

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 86,224 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.

Developmental Alcohol Exposure in Drosophila: Effects on Adult Phenotypes and Gene Expression in the Brain

“…Single cell RNA sequencing on adult fly brains following developmental alcohol exposure shows widespread sexually dimorphic changes in gene expression. These changes in gene expression resemble changes observed previously following cocaine exposure (19), indicating common neuronal and glial elements that respond to alcohol and cocaine consumption.…”

“…Increasing the stringency to |log e FC| > 1.0 (Bonferroni adjusted p value <0.05) retained 36 upregulated and 10 downregulated genes in males and 32 upregulated and 20 downregulated genes in females (Supplementary Figure S1). Differential expression patterns are sexually dimorphic, as observed previously for cocaine-induced modulation of gene expression (19), with only 32 differentially expressed genes in common between the sexes. Changes in gene expression in the mushroom bodies, represented by cluster C12, are primarily observed in females.…”

“…No reuse allowed without permission. observed changes in transcript abundances following consumption of cocaine (19). Sexual dimorphism is also a hallmark of FASD, with different effects of fetal alcohol exposure on neural development and cognitive abilities between males and females (32)(33)(34)(35).…”

“…We noticed that many genes that are differentially expressed following developmental exposure to ethanol correspond to genes that undergo altered expression when flies are exposed to cocaine (19). However, the transcriptional response to acute exposure to cocaine is larger than the transcriptional response to developmental alcohol exposure.…”

“…The number of flies that emerged from vials into which 50 eggs had been placed were counted and the data were analyzed using the "PROC GLM" command (Type III) in SAS v3.8 (Cary, NC) For single cell RNA sequencing, we collected duplicate samples of 20 brains for each sex from flies reared on control or ethanol medium. We dissociated the brains as previously described after incubation with 450µl of collagenase solution (50 ul of fresh 25mg/ml collagenase (Gibco) in sterile water + 400µl of Schneider's medium) for 30 minutes followed by stepwise trituration -P200 pipette 5 times, 23G needle pre-wetted with PBS + BSA 5 times, and 27G pre-wetted needle 5 times (19). The resulting suspension was passed through a pre-wetted 10µm strainer (Celltrics, Görlitz, Germany) with gentle tapping.…”