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Using a technique dubbed "brainbow," scientists tagged synapse terminals with proteins that
fluoresce different colors. Researchers expected one color to dominate, representing a single
source for the many terminals. Instead, several colors appeared together, intertwined yet distinct.
Image Credit: Virginia Tech

'Brainbow' reveals a surprising visual connection

Neuroscientists know some connections in the brain are pruned during infant growth and neural development. According to the textbooks, function drives new neural growth. But scientists are discovering the textbooks might be wrong.

"Retinal neurons associated with vision generate connections in the brain, and as the brain develops it strengthens and maintains some of those connections more than others, while unused connections are eliminated," says Michael Fox, Associate Professor at the Virginia Tech Carilion Research Institute and leader of the study. However, "We found that activity-dependent pruning might not be as simple as we believed."

Their results were published in Cell Reports.

Fox and his team of researchers used two different techniques to examine how retinal ganglion cells — neurons in the retina transmitting information to the visual centers in the brain — develop in a mouse. Mice are used to model the human brain.

"It's widely accepted that synaptic connections from about 20 retinal ganglion cells converge onto cells in the lateral geniculate nucleus during development. That number reduces to just one or two by the third week in a mouse's life," Fox said. "It was also thought that mature retinal ganglion develop several synaptic terminals clustered around information exchange points."

But as the theory of several terminals blossoming from one retinal ganglion cell had not yet been proved, Fox and his researchers decided to follow these terminals back to their roots.

Using a technique dubbed "brainbow," the scientists tagged the terminals with fluorescent proteins of different colors, expecting one color to dominate. Instead, several colors appeared, intertwined and distinct from each other.

"A true 'brainbow. I could see right in front of me something very different than the concept I learned from my textbooks."

Aboozar Monavarfeshani, graduate student who tagged the terminals in Fox's laboratory .

The results showed individual terminals come from more than one retinal ganglion in a mature mouse brain. This is in direct contradiction to previous research indicating neural development weeds out most connections between retinal ganglion and target cells in the brain.

Fox: "Is this a discrepancy - a technical issue with the different types of approaches applied in all of these disparate studies? Possibly, but more likely it represents that retinal ganglion cells are more complex than previously thought."

Along with the brainbow technique, Fox's team also imaged the connections with electron microscopy. Sarah Hammer, currently a sophomore at Virginia Tech, traced individual retinal terminals through hundreds of serial images. The data confirmed the "brainbow" analysis results - retinal axons from numerous retinal ganglion cells remained present on adult brain cells.

"These results are not what we expected, and they will force us to reevaluate our understanding of the architecture and flow of visual information through neural pathways," Fox added. "The dichotomy of these results also sheds important light on the benefits of combining approaches to understand complicated problems in science."

"The research provides strong evidence for a multiple innervation [supply of nerves] and calls for a reevaluation of our current understanding of information flow and neural circuit maturation in our visual system."

Albert Pan, Assistant Professor, Medical College of Georgia at Georgia Regents University, an expert in neural circuitry development who was not involved in the research.

Fox's team is continuing research to understand exactly how many retinal terminals converge and if the information each conveys is unique. Once they understand the intricacy of the brain's visual circuitry, the scientists might be able to develop therapies for when errors occur.

Fox adds: "The lesson in this particular study is that no single technique gives us all the right answers. Science is never as simple as we'd like it to be."

Abstract Highlights
•Brainbow-based technology can be used to differentially label retinal terminals
•Clusters of retinal terminals originate from numerous uniquely labeled RGCs
•Complex retinogeniculate synapses contain terminals from >10 distinct retinal axons
•A higher level of convergence exists on relay cells than previously appreciated

Summary
Activity-dependent refinement of neural circuits is a fundamental principle of neural development. This process has been well studied at retinogeniculate synapses—synapses that form between retinal ganglion cells (RGCs) and relay cells within the dorsal lateral geniculate nucleus. Physiological studies suggest that shortly after birth, inputs from ∼20 RGCs converge onto relay cells. Subsequently, all but just one to two of these inputs are eliminated. Despite widespread acceptance, this notion is at odds with ultrastructural studies showing numerous retinal terminals clustering onto relay cell dendrites in the adult. Here, we explored this discrepancy using brainbow AAVs and serial block face scanning electron microscopy (SBFSEM). Results with both approaches demonstrate that terminals from numerous RGCs cluster onto relay cell dendrites, challenging the notion that only one to two RGCs innervate each relay cell. These findings force us to re-evaluate our understanding of subcortical visual circuitry.

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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