Study of Individual Neurons in Flies Reveals Memory Related Changes in Gene Activity

Summary: Study identifies new patterns of gene expression that drives long-term memory formation.

Source: Princeton University.

Researchers at Princeton University have developed a highly sensitive and precise method to explore genes important for memory formation within single neurons of the Drosophila fly brain. With this method, the researchers found an unexpected result: certain genes involved in creating long-term memories in the brain are the same ones that the eye uses for sensing light.

The study, published in the May 17 issue of the journal Cell Reports, demonstrated the utility of the new method and also identified new patterns of gene expression that drive long-term memory formation.

“Ultimately, to understand the brain, we want to know what individual neurons are doing,” said Mala Murthy, assistant professor in the Princeton Neuroscience Institute and the Department of Molecular Biology. “We found that single neurons can be defined by their pattern of their gene expression, even if they’re all in the same brain network.”

To their surprise, the researchers found that many of the active genes in these neurons produce proteins that are best known for their roles in detecting light in the fly’s eye or sensing odor in the fly’s nose. “It is possible that these sensory proteins have been repurposed by the brain for a different function,” Murthy said.

“Even though the paper is focused on the methodology, which I think will be impactful for the field, there is this new science here—a whole new class of molecules we found that is in the central brain and seems to be involved in memory formation,” Murthy said.

Researchers have known that genes “turn on,” or start making proteins, during the formation of long-term memories in Drosophila, a widely used organism in studies of neurobiology, but they didn’t know exactly which genes in which neurons were involved.

To investigate this question, the researchers first trained flies to form long-term memories. Then they extracted single neurons from the fly brains and evaluated all of the gene readouts, or transcripts, which encode proteins. By comparing the transcripts of the memory-trained flies to those of non-trained flies, researchers were able to identify genes involved in long-term memory formation.

The task was complicated by the tiny size of the fly’s head, which is just one millimeter across, and contains fewer than 100,000 neurons. Murthy’s team focused on neuron types in one part of the brain, the mushroom body, named for its distinctive shape.

First author Amanda Crocker, a former postdoctoral fellow in Murthy’s lab and now an assistant professor of neuroscience at Middlebury College, conducted the experiments in collaboration with co-authors Xiao-Juan Guan, a senior research specialist in the Princeton Neuroscience Institute; Coleen Murphy, professor of molecular biology and the Lewis-Sigler Institute for Integrative Genomics; and Murthy.

“Our work opens up the ability to use Drosophila as a way to study how gene expression in single neurons relates to brain function,” Crocker said. “This has been a challenge because the fly brain is very small and contains fewer neurons than other organisms that neuroscientists study. The advantage of using flies is that they have significantly less redundancy in the neurons that they do have. We can look at specific neurons and gene expression, and ask what the genes are doing in that cell to cause the behavior.”

The researchers trained the flies to form long-term memories by exposing them to an odor – either an earthy, mushroom-like smell (3-octanol) or a menthol-like smell (4-methylcyclohexanol) – while simultaneously delivering a negative stimulus in the form of an electric shock.

The training took place in a tube containing the two odors, one at each end of the tube. Researchers paired one of the odors with the electric shock, and as a result the fly avoided that end of the tube. The assay was conducted in the dark, so that the flies could use only their sense of smell, not their vision, to navigate the tube.

A second group of flies received the electric shock and the odor, but not at the same time, so they did not form the memory that linked odor to shock.

The researchers then isolated single neurons from the fly brains using tiny glass tubes to suction out the cells. Harvesting neurons using this technique is not common, Murthy said, and it had not been combined with a complete analysis of gene activity in fly neurons before. With this novel method, they were able to use only 10 to 90 femtograms – a quintillionth of a kilogram – of genetic material.

They evaluated gene activity by looking at the production of messenger ribonucleic acid (mRNA), an intermediary between DNA and proteins. The result is a “transcriptome,” or readout of all of the genetic messages that the cell uses to produce proteins. The researchers then read the transcriptome to see which genes produced proteins in the memory-trained flies versus the non-trained flies, and found that some of the active genes in memory-trained flies were the same as ones used in the sensory organs to detect light, odors and taste.

Image shows a drosophila brain.
Image of the Drosophila brain (magenta) with a subset of mushroom body neurons expressing green fluorescent protein (GFP) via a genetic marker. NeuroscienceNews.com image is credited to Murthy lab, Princeton University.

To follow-up, the researchers bred mutant flies that lacked genes for some of the light-sensing proteins and thus could not see. The same memory experiments as before were carried out, and the researchers confirmed that the flies lacking light-sensing proteins were both unable to see and unable to form long-term memories.

The discovery of the expression of genes for classical ‘light-sensing’ proteins, such as rhodopsin, as well as other sensory-related proteins for odor and taste detection, was unexpected because these proteins were not known to be utilized in mushroom bodies, Murthy said. Although studies in other organisms, including humans, have detected sensory genes in areas of the brain unrelated to the sensory organ itself, this may be the first study to link these genes to memory formation.

About this genetics and memory research article

Funding: The study was funded by a Ruth L. Kirschstein Institutional National Research Service Award, the Alfred P. Sloan Foundation, the Human Frontier Science Program, a National Science Foundation (NSF) CAREER award, the McKnight Endowment Fund for Neuroscience, the Klingenstein Foundation, a National Institutes of Health New Innovator award, and an NSF BRAIN Initiative EAGER award. The study was also funded in part through the Glenn Center for Quantitative Aging Research at Princeton University, whose director is Coleen Murphy.

Source: Kristin Qian – Princeton University
Image Source: This NeuroscienceNews.com image is credited to Murthy lab, Princeton University.
Original Research:Full open access research “Cell-Type-Specific Transcriptome Analysis in the Drosophila Mushroom Body Reveals Memory-Related Changes in Gene Expression” by Amanda Crocker, Xiao-Juan Guan, Coleen T. Murphy, and Mala Murthy in Cell Reports. Published online June 9 2016 doi:10.1016/j.celrep.2016.04.046

Cite This NeuroscienceNews.com Article

[cbtabs][cbtab title=”MLA”]Princeton University. “Study of Individual Neurons in Flies Reveals Memory Related Changes in Gene Activity.” NeuroscienceNews. NeuroscienceNews, 13 June 2016.
<https://neurosciencenews.com/memory-genes-vision-4447/>.[/cbtab][cbtab title=”APA”]Princeton University. (2016, June 13). Study of Individual Neurons in Flies Reveals Memory Related Changes in Gene Activity. NeuroscienceNews. Retrieved June 13, 2016 from https://neurosciencenews.com/memory-genes-vision-4447/[/cbtab][cbtab title=”Chicago”]Princeton University. “Study of Individual Neurons in Flies Reveals Memory Related Changes in Gene Activity.” https://neurosciencenews.com/memory-genes-vision-4447/ (accessed June 13, 2016).[/cbtab][/cbtabs]


Abstract

Cell-Type-Specific Transcriptome Analysis in the Drosophila Mushroom Body Reveals Memory-Related Changes in Gene Expression

Highlights
•Low cell number RNA sequencing identifies neuron types in the MB based on gene expression
•The genes encoding cell surface receptors are sufficient for identifying cell types
•Differential gene expression following LTM induction identifies memory-related genes
•Several phototransduction genes are expressed in MB neurons and required for LTM

Summary
Learning and memory formation in Drosophila rely on a network of neurons in the mushroom bodies (MBs). Whereas numerous studies have delineated roles for individual cell types within this network in aspects of learning or memory, whether or not these cells can also be distinguished by the genes they express remains unresolved. In addition, the changes in gene expression that accompany long-term memory formation within the MBs have not yet been studied by neuron type. Here, we address both issues by performing RNA sequencing on single cell types (harvested via patch pipets) within the MB. We discover that the expression of genes that encode cell surface receptors is sufficient to identify cell types and that a subset of these genes, required for sensory transduction in peripheral sensory neurons, is not only expressed within individual neurons of the MB in the central brain, but is also critical for memory formation.

“Cell-Type-Specific Transcriptome Analysis in the Drosophila Mushroom Body Reveals Memory-Related Changes in Gene Expression” by Amanda Crocker, Xiao-Juan Guan, Coleen T. Murphy, and Mala Murthy in Cell Reports. Published online June 9 2016 doi:10.1016/j.celrep.2016.04.046

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