黑料吃瓜资源

MAPseq uses RNA sequencing to rapidly and inexpensively find the diverse destinations of thousands of neurons in a single experiment in a single animal

Cold Spring Harbor, NY — Neuroscientists today publish in Neuron details of a revolutionary 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鈥攊n 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 鈥渃onnectomes,鈥� 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 鈥渃onverts the task of brain mapping into one of RNA sequencing,鈥� says its inventor, Anthony Zador, M.D., Ph.D., professor at 黑料吃瓜资源.

鈥淭he RNA sequences, or 鈥榖arcodes,鈥� that we deliver to individual neurons are unmistakably unique,鈥� Zador explains, 鈥渁nd this enables us to determine if individual neurons, as opposed to entire regions, are tailored to specific targets.鈥�

MAPseq differs from so-called 鈥渂ulk tracing鈥� methods now in common use, in which a marker—typically a fluorescent protein鈥攊s 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鈥檚 axonal destinations, cell by cell in a given region, is what motivated Zador to come up with a new technique.

One way of explaining the advantage of MAPseq over bulk tracing methods is to imagine being at an international airport, with the intention of getting on a flight to, say, Germany. 鈥淚f you go to the international terminal, you see a long line of ticket counters,鈥� Zador explains. 鈥淚f you want to go to Germany, it鈥檚 not enough to take any airline at the international terminal. If you stand in line at the counter for Air Chile, you鈥檙e probably not going to be able to buy a ticket for Germany.鈥�

鈥淭hose many airlines whose counters are adjacent serve many destinations, some of which overlap, some of which are unique. You can print out a map showing all of the foreign countries that all of the airlines serve from your airport, but that doesn鈥檛 tell you anything at all about individual airlines and where they go. This is the difference between current labeling methods and MAPseq. The 鈥榠ndividual airlines鈥� in my example are adjacent neurons in a part of the brain whose 鈥榬outes鈥� we want to trace.鈥�

Zador and his team, including Justus Kebschull, a graduate student in his lab who is first author on the Neuron paper introducing the new method, 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鈥攃onsisting of 30 鈥渓etters,鈥� or nucleotides鈥攊s taken up by single neurons. Thirty letters yields 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 鈥渟ource鈥� 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鈥攖he barcodes travel reliably and evenly throughout the brain, along the 鈥渢runklines鈥� that are the axons, and out to the 鈥渂ranch points鈥� 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 鈥渟ource鈥� area are now matched with the same barcodes collected in distant parts of the brain.

鈥淪equencing the RNA is a highly efficient, automated process, which makes MAPseq such a potentially radical tool,鈥� Kebschull says. 鈥淚n addition to the speed and economy of RNA sequencing, it has the great advantage of making it possible for researchers to distinguish between individual neurons within the same region that project to different parts of the brain.鈥�

To demonstrate MAPseq鈥檚 capabilities, Zador鈥檚 team injected a part of the mouse brain called the locus coeruleus (LC), located in the brain stem. It is the cortex鈥檚 sole source of noradrenaline, a hormone that signals surprise. Zador鈥檚 team used MAPseq to address an old question: does the 鈥渟urprise鈥� signal get broadcast everywhere in the cortex, or only to particular places, where, perhaps, it is most needed or relevant?

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.

鈥淲e found that neurons in the LC have a variety of idiosyncratic projection patterns,鈥� Zador says. 鈥淪ome neurons project almost exclusively to a single preferred target in the cortex or olfactory bulb. Other neurons project more broadly, although weakly.鈥�

These results, he adds, 鈥渁re consistent with, and reconcile, previous seemingly contradictory results about LC projections.鈥� The surprise signal can reach most parts of the brain, but there are very specific parts of the brain where the signal is especially focused.

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 鈥渢iled鈥� with injections. It is hoped this will yield the first connectome of the entire cortex at single-neuron resolution.

鈥淥nce we automate the process of using many injections, we think this kind of experiment can be completed by a single person in just a week or two, and at a cost of only a few thousand dollars,鈥� Zador says. 鈥淲e are very keen on being able to do these kind of studies in a single animal, which will eliminate the past problem of injecting multiple animals to trace multiple neurons, a method that requires one to make a single map based on many brains, each of which is somewhat different.鈥�

Zador鈥檚 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.

Written by: Peter Tarr, Senior Science Writer | [email protected] | 516-367-8455

Funding

The research discussed here was supported by: National Institutes of Health [5RO1NS073129, 5RO1DA036913]; Brain Research Foundation [BRF-SIA-2014-03]; Simons Foundation [382793/SIMONS]; Boehringer Ingelheim Fonds; Genentech Foundation; Funda莽茫o para a Ci锚ncia e Tecnologia, Portugal; Pew Scholarship and CSHL startup funds; CSHL Cancer Center Support Grant 5P30CA045508.

Citation

鈥淗igh-throughput mapping of single neuron projections by sequencing of barcoded RNA鈥� appears online in advance in Neuron August 18, 2016 The authors are: Justus M. Kebschull, Pedro Garcia da Silva, Ashlan P Reid, Ian D Peikon, Dinu F. Albeanu and Anthony M. Zador. The paper can be accessed at: