Scientists Complete Second Full Map Of Fruit Fly Brain, Charting 300 Million Connections In New Study

ASHBURN, Va. — Researchers have completed a full map of every neuron and connection in the brain of a male fruit fly, marking the second complete “connectome” of the species and giving scientists an unprecedented tool for understanding how brains process information at the most fundamental level.

The project, announced Friday, was a collaboration between biologists at the Howard Hughes Medical Institute’s Janelia Research Campus and computer scientists at Google. Both teams have said neither could have completed the effort without the other. The new male fly connectome follows the completion of a female Drosophila connectome earlier this year by university-based researchers, giving scientists their first opportunity to compare complete neural wiring diagrams between the sexes of the same species.

A connectome is a comprehensive catalog of every neuron in a brain, including its precise location in three-dimensional space and every connection, or synapse, it forms with other neurons. While the fruit fly’s entire nervous system contains roughly 150,000 neurons, with only a fraction of those located in the brain itself, the new research identified more than 300 million individual synaptic connections within that relatively small structure, illustrating just how densely interconnected even a comparatively simple nervous system can be.

Gerry Rubin, a senior group leader at the Janelia Research Campus and one of the senior authors on the new study, described how dramatically the tools available for this kind of work have changed since he began his career studying an even simpler organism.

“I was a graduate student at the UK’s Laboratory of Molecular Biology … and when I got there in ’71, they already bought this giant computer, and they had the idea that they were going to use machine vision and computers to assemble the C. elegans connectome,” Rubin said. C. elegans is a small, transparent worm with just over 300 neurons.

“It took them about two years to realize that the computers were nowhere near powerful enough,” Rubin said, “and so they went with printing everything out on photographic prints and colored magic markers and circling neurons and tracing it by hand.”

That painstaking manual approach would have been entirely impractical for the fruit fly, a species that is both far more complex than C. elegans and, unlike the transparent worm, opaque, making its neurons far more difficult to image directly. To build the new connectome, researchers dissected a fruit fly brain along with part of its ventral nerve cord and sliced it into an enormous series of evenly spaced sections, which were then imaged using electron microscopy at a resolution fine enough to identify small cellular structures. From there, the reconstruction of the brain’s three-dimensional architecture depended almost entirely on artificial intelligence.

Michal Januszewski, a staff scientist at Google Research, told Ars Technica that the first computational step involves using a form of generative AI to seamlessly reconnect the tissue across the boundaries created by each physical slice.

“When you take those blocks and you stitch them back together computationally, there’s a little bit of a gap in between them so the tissue doesn’t completely smoothen,” Januszewski said. “We use a generative model to make the tissue look as if the seams were not there, and that then makes all the downstream processing easier because you can basically ignore the problem to a large degree.”

A separate AI model then works to fill in the spaces defined by cellular membranes, allowing the system to track individual neurons continuously across three-dimensional space. Januszewski described that model as functioning differently than many people typically imagine when they think about artificial intelligence.

“It is different in a number of ways from what people commonly think when they talk about AI,” Januszewski said. “One is that it is actually a recurrent process, so it literally moves through space as it makes the outline of the neurons, and it is a visual model, so it converts voxels out of the images from the microscope, converting them into a 3D presentation of the neurons.”

Additional models were used to identify synapses and classify their specific type once detected, with their sensitivity levels adjustable based on ongoing feedback from human proofreaders reviewing the AI’s output. Rubin described the process as a continuous back-and-forth between researchers and engineers.

“This has to be tuned by going back and forth between the proofreaders and Mikhail to say, ‘Oh, give us a version where you were less greedy because it’s harder to disassemble than it is to assemble,’” Rubin said. “So it’s an iterative process between the humans giving feedback and the algorithms getting tuned.”

The full project took roughly four years to complete, from the initial dissection of an intact fly brain to the finished connectome. Rubin said the techniques and software developed along the way are intended to scale toward far more complex nervous systems in the future, potentially including those of vertebrates such as mice.

“Our view is we did Drosophila with a team of 50 people,” Rubin said. “The hope is, by the time someone does a mouse, they’ll also need a team of 50 people, even though there are a thousand times more neurons in there. The people will never go away, but the people will not need to scale with the number of neurons, which would be economically not feasible.”

Januszewski said the scale and difficulty of the challenge is precisely what drew Google into the collaboration in the first place.

“The reason I think Google and we were interested in this is because this is this type of grand challenge that just cannot be done in any other way,” Januszewski said. “We knew we need AI for this. This cannot be solved by having more humans or by any other technology. And it’s important.”

Beyond serving as a foundational tool for future neuroscience research, the completed male connectome, when compared against the earlier female connectome, has already yielded new insight into how sex differences are wired into the fly brain. Building on decades of prior genetic research identifying two genes, doublesex and fruitless, as key regulators converting chromosomal sex into behavioral differences, researchers identified 289 neurons unique to males, 71 unique to females, and 138 present in both sexes but differing in shape and connectivity. Notably, roughly 90% of the male-specific neurons expressed the doublesex or fruitless genes, but 10% did not, suggesting some neurons may acquire sex-specific characteristics indirectly, through interactions with neighboring cells that do express those genes.

Researchers also found that sex-specific neurons tended to cluster together and operate at higher levels of neural processing, rather than directly within basic sensory detection or motor control circuits, with a few notable exceptions such as neurons that detect sex-specific pheromones. As the research team wrote in their published paper, the pattern suggests “a hierarchy in which sex differences primarily modify integrative and decision-making areas while sensory detection and the highly tuned motor interface remain more constant.”

Rubin said the completed connectome has already begun reshaping theoretical neuroscience by giving researchers a concrete wiring diagram to test their models against, rather than working from open-ended speculation about how a brain might function.

“Before this, most neuro theorists were very much like, ‘How could a brain work?’” Rubin said. “And they didn’t have a constraint. Once they had the connectome, they could say, ‘The brain does this and here’s the wiring diagram. How can this wiring diagram allow this function?’ So this has been a major — I’d say this is the biggest change in having the connectome.”

Looking ahead, researchers say a key open question is whether the cost and time required to build additional connectomes will fall sharply as techniques continue to improve, similar to the trajectory seen in genome sequencing over the past two decades. With only two complete fly connectomes currently available, including one notable case in which a specific neuron found in the female fly was entirely missing from one hemisphere of the male fly’s brain, researchers say dozens of additional connectomes will likely be needed before scientists can draw statistically meaningful conclusions about how much natural variability exists between individual brains, even within the same species.