2023 is shaping up to be a busy year for the fly connectome.

Already this year, researchers have released a wiring diagram of the larval fruit fly brain. Connectomes of the complete adult female fly brain and the optic lobe are expected in 2023, with the complete male fly nervous system connectome following soon. 

On June 6, Janelia scientists and collaborators in the US and UK added another piece to the connectome puzzle with the unveiling of the wiring diagram of the male adult nerve cord, dubbed the MANC. The connectome, a joint effort by Janelia’s FlyEM Project Team and collaborators, is detailed in preprints on bioRxiv and is freely available to researchers worldwide through Janelia websites.

With about 23,000 neurons, 10 million pre-synaptic sites, and 74 million post-synaptic densities, the MANC is the most in-depth and complete connectome of an adult fruit fly nerve cord – a structure analogous to the human spinal cord that controls most of the fly’s motor functions. The unprecedented detail in this map of neurons and their connections will help scientists figure out how a fly moves its legs or flaps its wings.

Preprints released alongside the connectome data describe the different cell types, their origins and connections, and the biological insights starting to emerge from the data. The fruit fly is a key organism that neuroscientists use to probe how the nervous system works, so having connectomes is critical for uncovering how cells work together to enable behavior.

“Once you can see a whole network, you can start to ask big organizational questions,” says Gwyneth Card, an HHMI Investigator at Columbia University’s Zuckerman Institute and former Janelia group leader, who helped lead the project.

The MANC and the other connectomes being released follow in the footsteps of the hemibrain connectome released by Janelia scientists in 2020. At the time, the hemibrain – a portion of the adult fly brain – was the largest and most comprehensive wiring diagram ever completed, showing that a feat many thought impossible could be done.

The release of the hemibrain led to additional support for and interest in connectome efforts. Researchers are now filling in pieces missing from the hemibrain, and the goal of mapping the entire central nervous system of both a male and female adult fruit fly is within reach.

“This train is going to keep rolling,” Card says. “You are just seeing the beginning.”

Constructing the MANC

The MANC connectome was constructed using methods akin to those used to map the hemibrain, with the Janelia team preparing the nerve cord sample and imaging layer after layer of nanometer-thick slices on focused ion beam scanning electron microscopes. Google’s algorithms and computers stitched the images together and did a first pass at identifying neurons. 

Then, a team of Janelians and collaborators set about proofreading the data – a manual effort to ensure that the shape and connectivity of neurons are correct, and one of the most time-consuming parts of the process. Because of the COVID-19 pandemic, the team developed software to work on home computers. That, along with additional funding from the Wellcome Trust, meant international collaborators could more easily help with the effort.

“Since it has been completely proofread and we can find all the same neurons on the fly’s left and right, we can tell colleagues, ‘You can trust this,’” says Greg Jefferis, a neuroscientist at the MRC Laboratory of Molecular Biology and University of Cambridge and another project leader who is part of the FlyEM Project Team Steering Committee.

Researchers at Cambridge also identified the different cell types, where they are found along the fly’s body, and from which stem cells they originated, helping to tease out some of the organizational principles.

“The ventral nerve cord has basically been seen as a black box,” says Lisa Marin, a research associate at the University of Cambridge who led the cell-typing effort. “A large majority of the neurons have never been identified. So a big part of our process was to divide these up into smaller populations and then dig into the connectivity.”

Examination of the connectome data has already started to uncover some surprises. Card and her team found that some behaviors involving the same muscles use distinct pre-motor microcircuits, not the same circuits, as previously thought. Jefferis and his team described the complex repeated circuits that control the legs and found, surprisingly, that the interconnections coordinating the legs differ from existing models.

Many more insights from the MANC will happen as other researchers start to probe the data, which can be accessed through neuPrint and Clio, online tools developed at Janelia.

“It’s clear that these connectomes are so rich and that they’re really only the starting point for trying to understand how this system works,” Card says. “It will take the whole community to dig in to get the breadth of different behaviors that people study in different contexts, to probe this network. That’s how we are going to tease out the higher principles.”

Along with the scientific insights to be gained, the project also serves as one model for other groups undertaking connectome efforts.

“This kind of cooperation is going to be absolutely necessary when people start moving to the mouse connectome and things like that,” says Lou Scheffer, a principal scientist at Janelia and a member of the FlyEM team. “There’s no conceivable way any single organization could do it, and so this is a prototype for that sort of cooperation.”

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Citation:

Shin-ya Takemura et al. "A Connectome of the Male Drosophila Ventral Nerve Cord." Posted on bioRxiv.org on June 6, 2023. DOI: 10.1101/2023.06.05.543757

Elizabeth C. Marin et al. "Systematic annotation of a complete adult male Drosophila nerve cord connectome reveals principles of functional organisation." Posted on bioRxiv.org on June 6, 2023. DOI: 10.1101/2023.06.05.543407

H.S.J. Cheong et al. “Transforming descending input into behavior: The organization of premotor circuits in the Drosophila Male Adult Nerve Cord connectome.” Posted on bioRxiv.org on June 7, 2023. DOI: 10.1101/2023.06.07.543976

Datasets: https://www.janelia.org/project-team/flyem/manc-connectome