Methodology tracks neuron paths, gene expression concurrently | Spectrum
Mapping of neurons: BARseq2 can track neuronal pathways and at the same time measure gene expression within cells. In this mouse brain slice, each point represents the location of a unique mRNA molecule and each color represents a different gene.
Courtesy of Xiaoyin Chen
A new method can map the pathways of thousands of neurons in mice while quantifying the expression of dozens of genes in these cells.
The technique, called BARseq2 and described in Nature Neuroscience in May, could help researchers create a large-scale map of the neuronal circuits relevant to autism in mice, including at different stages of development, according to study researcher Xiaoyin Chen, a postdoctoral fellow in Anthony Zador’s lab at Cold Spring Harbor Laboratory in New York. Chen will join the Allen Institute in Seattle, Washington, in August.
BARseq2 extends the team’s original BARseq method that they reported on in 2019. This version maps neural projections – or where the axon of each neuron crosses the brain – and could measure the level of expression of up to three genes in those cells.
For BARseq2, the researchers first have to create a set of short strands of DNA with random sequences and convert them into RNA. They then package each RNA sequence in a Sindbis virus, an alphavirus that was first isolated in Egypt in 1952. Each modified virus infects a neuron in the animal and delivers its RNA load, giving each cell a unique molecular barcode.
“As soon as the barcode infects a neuron, it begins to replicate and makes millions of copies,” says Chen. “The barcodes are transported to the axons, so you essentially fill the entire neuron and all axons and dendrites with that particular barcode.”
The next day, researchers use chemicals to fix the RNA molecules in brain slices. Then they artificially duplicate the barcode in each neuron to create more easily recognizable clusters. The sequencing of the RNA in the clusters shows the barcode present in each neuron. To demonstrate the technique, Chen and his colleagues created tens of millions of RNA barcodes and injected them into individual mice.
Using a similar approach, researchers can measure the expression levels of dozen of different genes in neurons. They wash the brain slices of the mice with a short DNA sequence – a so-called primer – that binds to RNA copies of the gene of interest. They then convert the target RNAs into DNA and sequence them within the brain slices. Each RNA molecule forms a fluorescent spot; the number of spots reflects the level of expression of the gene in the cell.
Researchers can reassemble the brain slices on the computer to create a 3D map of the brain in which the barcode sequences intertwine and show where neurons are projecting.
With BARseq2, Chen and his colleagues mapped the projections of more than 3,000 cells in the mouse’s motor and auditory cortex, a region of the brain that can slowly mature in autistic children. They also measured the expression levels of cadherin genes – which help cells connect and communicate – in nearly 30,000 cells.
Chen, who has been working on BARseq and BARseq2 for almost seven years, says that he and his colleagues developed the methods out of frustration. All of the neural circuitry for simpler organisms, such as the roundworm Caenorhabditis elegans, has been carefully mapped, but only a small subset has been mapped for the mouse brain because it is more complicated.
“It’s very intimidating to work on the mouse nervous system because it’s pretty messy,” says Chen. “My initial motivation was to find a way to understand the mouse nervous system, similar to how we understand the simple C. elegans system. I think BARseq is one way of doing this. “