Scientists at the Clemson University Center for Human Genetics used single cell transcriptomics technology to identify how acute cocaine exposure affects specific cell clusters in the brain of the common fruit fly, Drosophila melanogaster. The studies, headed by geneticists Trudy Mackay, PhD, and Robert Anholt, PhD, found that cocaine use by the fruit flies elicited rapid, widespread changes in gene expression throughout the brain, and that the differences were more pronounced in males than in females. The investigators hope that the resulting “atlas of sexually dimorphic cocaine-modulated gene expression” could potentially lay the groundwork for developing drugs that would treat or prevent cocaine addiction in humans.

“This research identifies the regions of the brain which are important,” said Mackay, the Self Family Endowed Chair in Human Genetics. “Now, we can see what genes are expressed when exposed to cocaine and whether there are Federal Drug Administration-approved drugs that could be tested, perhaps first in the fly model. We’ve already spotted several of these genes. This is a baseline. We can now leverage this work to understand potential therapy.”

Mackay, Anholt and colleagues report on their findings in Genome Research, in a paper titled, “The Drosophila brain on cocaine at single cell resolution.”

The propensity for cocaine use depends on both genetic and environmental factors, making it hard to study. And while the neurological effects of the drug are well known, scientists know much less about how gene variation may impact on sensitivity to the drug’s effects. “Furthermore, little is known about acute effects of cocaine consumption on genome-wide gene expression across the brain,” they continued.

The fruit fly Drosophila melanogaster is a useful model for systems genetic analysis of cocaine consumption. The majority of the fruit fly genes have human counterparts, providing researchers with a comparable model when studying complex genetic traits. “Flies can be reared rapidly in large numbers at low cost in defined genetic backgrounds and under controlled environmental conditions, and about 75% of disease-causing genes in humans have fly orthologs,” the team pointed out.

Fruit flies exposed to cocaine showed impaired locomotor activity and increased seizures and, as the authors explained, exposure to cocaine also elicits motor responses in the fruit fly that resemble behaviors observed in rodents. “… flies in addition develop sensitization to repeated intermittent exposure to cocaine.”

For their reported studies, the investigators allowed male and female flies to ingest a fixed amount of sucrose or sucrose supplemented with cocaine, over no more than two hours. Observation of the flies’ behavior after cocaine ingestion showed evidence that the drug exposure resulted in physiological and behavioral effects, including seizures and compulsive grooming.

To assess the effects of cocaine consumption on gene expression in the brain, the researchers dissected the fly brains into single cells. Using next-generation RNA sequencing technology they were able to make libraries of the expressed genes for individual cells.

“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,” the scientists explained. The single-cell technique is ultra-powerful and offers advantages over standard gene expression profile studies. “If an entire brain is used and there’s heterogeneity of gene expression, such that it’s up in one cell and down in another, you don’t see any signal,” Mackay commented. “But with the single cell analysis, we’re able to capture those very, very fine details that reflect heterogeneity in gene expression among different cell types. It is very exciting to apply this advanced technology here at the CHG.”

The investigators looked at 88,991 cells, with each cell having thousands of transcripts. Through sophisticated statistical analysis, the researchers were able to categorize the cells into 36 distinct cell clusters. 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 mushroom bodies, were represented. The study results showed that all types of fly brain cells were affected, especially Kenyon cells in the fly brain’s mushroom bodies, and some glia cells. Mushroom bodies, which get their name because they look like a pair of mushrooms, are integrative brain centers that are associated with experience-dependent behavioral modifications.

Interestingly, the study highlighted extensive sexual dimorphism in the response to cocaine. “We found the effects of cocaine in the brain are very widespread, and there are distinct differences between males and females,” added Anholt, Provost’s Distinguished Professor of Genetics and Biochemistry. The investigators further stated, “… 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 …“We identified 691 differentially expressed genes in males and 322 in females following acute exposure to cocaine, of which ~69% have human orthologs.” The scientists say the observed sexual dimorphism is in line with previous studies that showed reduced locomotion and increased grooming in flies given low doses of cocaine, with males showing more profound effects than femails.

The collective results of the team’s analyses were used to generate what they say is “… an atlas of sexually dimorphic cocaine-modulated gene expression in a model brain.” Ahnolt said it’s hoped that the atlas will serve as a resource for the research community.

“ … 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,” the investigators stated. “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.”

Mackay is one of the world’s leading authorities on the genetics of complex traits. She has a longstanding interest in behavioral genetics and developing the fruit fly as a model for understanding the genetic basis of complex behaviors. Her laboratory developed the Drosophila melanogaster Genetic Reference Panel (DGRP), which now consists of 1,000 inbred fly lines with fully sequenced genomes derived from a natural population. The DGRP allows researchers to use naturally occurring variation to examine genetic variants that contribute to susceptibility to various stressors.

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