Researchers at the Salk Institute for Biological Studies have jumped what many believe to be a major hurdle to preparing a detailed wiring diagram of the brain: identifying all of the connections to a single neuron. The researchers describe how they modified the deadly rabies virus, turning it into a tool that can cross the synaptic space of a targeted nerve cell just once to identify all the neurons to which it is directly connected.
Viruses that naturally spread between neurons have previously been used to outline the flow of nerve cell communication, but they have two drawbacks. First, once inside the brain, they keep spreading from cell to cell without stopping. Second, they cross different synapses – the specialized junctions between nerve cells – at different rates, crossing bigger, stronger synapses faster than smaller, weaker ones. Together these attributes make these viruses unable to determine exactly which cells are connected to which. The team of Salk researchers sought to create a modified virus whose spread could be limited to a single synaptic connection.
“The core idea is to use a virus that is missing a gene required for spreading across synapses but to provide the missing gene by some other means within the initially infected cells,” says Ian Wickersham, Ph.D., postdoctoral researcher and lead author on the project.
With the critical gene deleted from its genome, the virus is marooned inside a cell, unable to spread beyond it. However, supplying the missing gene in that same cell allows the virus to spread to cells that are directly connected to it. Since these neighboring cells lack the gene supplied in the first cell, the virus is stuck. Only the cells connected directly to the original cell are labeled.
You need two genes expressed in the cell or cell type of interest: TVA, to get the rabies virus in, and the missing viral gene so the virus can spread to connected cells,” says Wickersham.
They experimented on a neonatal rat brain: The result was spectacular: as expected, these red cells were selectively infected by the virus, which spread to hundreds of surrounding cells, turning them brilliantly fluorescent green. Once scientists can identify a neural circuit, they can then deactivate it, and test for changes in brain function.