A perfect day for zebrafish

On a warm sunny morning last September, eight 4- and 5-year-olds in Phyllis Bodel Childcare Center’s kindergarten class on the campus of Yale School of Medicine (YSM) sat outside at picnic tables with their eyes glued skyward. In the middle of the courtyard in front of them a large crane was slowly lifting enormous crates from a giant delivery pallet to a doorway in the Sterling Hall of Medicine. For over an hour, these youngsters sat nearly still with razor-like focus on the large machinery, discussing what was happening—and what was inside those crates.

The enormous crane was unloading parts of a microscope into YSM, one that would be built by Joel Greenwood, PhD, associate research scientist in neuroscience and director of the Neurotechnology Core in YSM’s Kavli Institute for Neuroscience. Greenwood established the core in 2017, a design and fabrication facility that is free for other researchers to use. At the same time, Greenwood had just started to construct another microscope, one that also required special delivery of multiple component parts, a two-photon laser, and rigging for a large optical table. This microscope was being built from scratch, and is the only one of its kind in New England.

The gargantuan microscope that’s being built? One dedicated to the study of tiny organisms—zebrafish. Ellen Hoffman, MD, PhD ’14, assistant professor in the Child Study Center and of neuroscience at YSM, will use this microscope to perform whole-brain functional imaging in zebrafish to better understand the function of genes that increase the risk of autism. This microscope will enable her to look into the fishes’ brains throughout development—their embryos are transparent—to examine brain structure in zebrafish lacking functional autism risk genes. Using this specialized microscope, Hoffman will be able to collect live images of the fish while watching neural signaling before, during, and after drug treatment. In this way, she will be able to glean information that wasn’t possible before she acquired this microscope.

“This microscope will enable us to visualize how loss of function of autism risk genes leads to differences in neural signaling in the developing vertebrate brain in real time at the single-cell level,” says Hoffman.

As with other colleagues at YSM, Greenwood is teaching members of Hoffman’s lab to build and use components of the microscope while providing crucial oversight in the process. In this way, scientists will have an intricate understanding of this specialized piece of technology. This understanding also helps scientists think critically about their work and the specifics of how experiments can and should be conducted, Greenwood says.

This microscope had to overcome a primary challenge of working with zebrafish—how to record the animal quickly and without its knowing. Hoffman’s microscope is a two-photon light sheet microscope, meaning that sheets of light rapidly “scan” through the entire fish brain to record whole-brain activity in less than one second. Traditional two-photon microscopes are slow, scanning the brain point by point. With a small zebrafish, the animal must be held still in a natural position and scanned quickly enough that it can’t detect the light. Otherwise, its natural behaviors would be compromised.

To solve this problem, Hoffman and Greenwood relied on existing research publications to build the novel instrument that will precisely answer Hoffman’s questions. They modified attributes of existing models to make her microscope a “double whammy”—one that increases the speed of the light sheet and is also invisible to the fish. The two-photon light sheets are outside the visual spectrum of the fish, making the light undetectable to the animal, and the light can penetrate deeper, giving better resolution. The microscope will enable Hoffman and her team to see more neurons simultaneously in a live, functioning animal. This clarity will give her studies both ethological and ecological relevance.

Hoffman already knows the first experiments she will conduct with this microscope, which was funded by the National Genetics Foundation, the SPECTOR Fund, the Kavli Foundation, and the National Institute of Mental Health. She plans to study baseline brain activity in fish that have mutant forms of the CNTNAP2 gene, which is strongly associated with autism and epilepsy in humans. Using transgenic fish expressing a form of green fluorescent protein that turns on when brain cells become active, Hoffman will learn what is happening at the cellular level. She then hopes to determine how compounds identified in related drug screening experiments affect brain signaling. For these studies, she will be able to use her new microscope to understand how changes occurring at the circuit level in the zebrafishes’ brains might relate to behavioral differences resulting from loss of autism risk gene function.

With this new technology, Hoffman aims to answer fundamental questions about the mechanisms underlying brain and behavioral development so she can lay the groundwork for developing improved treatments for children with developmental disabilities. In so doing, she may also lay the groundwork for a future scientist—perhaps even one of the children admiring how carefully the crane’s long arm moved microscope parts into YSM—to develop the next generation of technologies to make life-altering discoveries.