By Timothy Balmer, Ph.D.
Expressing genes in neurons has become an essential technique that is used to explore the neurophysiological mechanisms of brain function and disease. For example, neuroscientists can control the expression of specific genes to determine how they affect a neuron’s development or physiology.
This visual abstract in eNeuro summarizes the results of testing six AAV serotypes for efficacy in delivering a protein. Credit: Witteveen and Balmer/eNeuro
Neuroscientists often express fluorescent proteins in populations of neurons to identify them and to determine where they send their signals. Adeno-associated viruses (AAVs) have become a common tool to deliver genes to neurons in laboratories.
AAVs are also becoming more common in clinical treatments. For example, AAVs have been used to recover hearing in congenitally deaf children by expressing a gene that is rendered dysfunctional by mutation.
AAVs are available in several serotypes that have varying abilities to infect neurons and cause them to produce proteins of interest. The efficacy of AAV transduction in specific cell types depends on many factors and remains difficult to predict, so an empirical approach is often required to determine the best performing serotype in each population of cells.
Typically, individual labs may test a few serotypes but the results are not usually shared. In this project we tested the six most commonly available serotypes in the inferior colliculus and cerebellum of the mouse, and we reported our results in eNeuro, an open-access journal, in November 2024, in order to limit redundant experiments and save resources for the numerous labs studying these brain regions.
The AAVs that we used delivered the gene for green fluorescent protein (GFP), a modified version of a gene that is naturally expressed in the jellyfish Aequorea victoria. In the inferior colliculus, which is a midbrain auditory region that is essential for hearing, we found that AAV1 produced the brightest labeling, indicating the highest expression of GFP. AAV1 also labeled more neurons than the five other serotypes.
In the cerebellum we found that AAV1 also produced the best labeling. The cerebellar cortex has several cell types that can be easily identified by their shape and location. This allowed us to test which serotypes labeled each cell type most effectively.
Cerebellar granule cells make up about half of the total neurons in the brain, and they appear more resistant to AAVs than other cell types. While AAV1 labeled most of the cell types better than the other serotypes (more Purkinje cells, unipolar brush cells, and molecular layer interneurons than the others), we found AAV2 labeled more granule cells.
We expect that these results will help guide the use of AAVs as gene delivery tools in these regions of the brain. By understanding which AAV serotype works best for delivering genetic instructions to specific brain cells and sharing this information in an open-access journal, researchers can design better experiments and potentially develop treatments for brain-related conditions.
Timothy Balmer, Ph.D., is an assistant professor in the School of Life Sciences at Arizona State University. He is a 2025 Emerging Research Grants (ERG) scientist generously funded by the Salice Family Foundation. This paper is funded in part by his 2022–2023 ERG grant generously underwritten by an anonymous donor. Balmer is also a 2017 ERG scientist generously funded by the Les Paul Foundation.
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By understanding which AAV serotype works best for delivering genetic instructions to specific brain cells and sharing this information in an open-access journal, researchers can design better experiments and potentially develop treatments for brain-related conditions.