Neuroscientists published in Neuron details of a new way of mapping the brain at the resolution of individual neurons, which they have successfully demonstrated in the mouse brain.
The new method, called MAPseq (Multiplexed Analysis of Projections by Sequencing), makes it possible in a single experiment to trace the long-range projections of large numbers of individual neurons from a specific region or regions to wherever they lead in the brain in experiments that are many times less expensive, labor-intensive and time-consuming than current mapping technologies allow.
Although a number of important brain-mapping projects are now under way, all of these efforts to obtain “connectomes,” or wiring maps, rely upon microscopes and related optical equipment to trace the myriad thread-like projections that link neurons to other neurons, near and far. For the first time ever, MAPseq “converts the task of brain mapping into one of RNA sequencing,” said its inventor, Anthony Zador, M.D., Ph.D., professor at Cold Spring Harbor Laboratory.
“The RNA sequences, or ‘barcodes,’ that we deliver to individual neurons are unmistakably unique,” Zador said, “and this enables us to determine if individual neurons, as opposed to entire regions, are tailored to specific targets.”
MAPseq differs from so-called “bulk tracing” methods now in common use, in which a marker, typically a fluorescent protein, is expressed by neurons and carried along their axons. Such markers are good at determining all of the regions where neurons in the source region project to, but they cannot tell scientists that any two neurons in the source region project to the same region, to different regions, or to some of the same regions, and some different ones. That inability to resolve a neuron’s axonal destinations, cell by cell in a given region, is what motivated Zador to come up with a new technique.
Zador and his team, including Justus Kebschull, a graduate student in his lab, have spent several years working out a technology that enables them to assign unique barcode-like identifiers to large numbers of individual neurons via a single injection in any brain region of interest. Each injection consists of a deactivated virus that has been engineered to contain massive pools of individually unique RNA molecules, each of whose sequence – consisting of 30 “letters,” or nucleotides – is taken up by single neurons. Thirty letters yield many, many times more barcode sequences (1018) than there are neurons in either the mouse or human brain, so this method is especially well suited to the massive complexity problem that brain mapping presents.
An injection into a “source” region of the brain contains a viral library encoding a diverse collection of barcode sequences, which are hitched to an engineered protein that is designed to carry the barcode along axonal pathways. The barcode RNA is expressed at high levels and transported into the terminals of axons in the source region where the injection is made. In each neuron, it travels to the point where the axon forms a synapse with a projection from another neuron. Tests show that the technology works – the barcodes travel reliably and evenly throughout the brain, along the “trunklines” that are the axons, and out to the “branch points” where synapses form.
About two days after one or more injections are made in a region of interest, the brain is dissected and RNA is collected and sequenced. RNA barcodes in the “source” area are now matched with the same barcodes collected in distant parts of the brain.
In their demonstration experiment, only RNA that ended up in the cortex or olfactory bulb was sequenced, along with that of the source region in the LC where the barcodes were originally injected. The team divided the cortex into 22 slices, each about 300 microns thick, and dissected the slices. The results were exciting to the team.
The team showed that results could be obtained in experiments based on one injection in the LC, and also two injections, on opposite sides. Already in progress are experiments in which the entire cortex is being “tiled” with injections. It is hoped this will yield the first connectome of the entire cortex at single-neuron resolution.
Zador’s next goal with MAPseq is to map the brains of animals that model various neurodevelopmental and neuropsychiatric illnesses, to see how gene mutations strongly associated with causality alter the structure of brain circuits, and thus, presumably, brain function.
