Wiring the Brain: How Viruses Can Be Engineered to Trace Neural Connections

The fact that the rabies virus can spread from an infected neuron to other neurons connected to it makes it an almost perfect vector for tracing connections in the brain.

Dr. Kevin Mitchell is a geneticist who studies neural development. In this guest post, he explains how viruses can be used to map connections in the brain.

What's the Big Idea?

It is hard to overstate the complexity of the brain. Not only are there tens of billions of individual nerve cells, or neurons, which make literally trillions ofconnections between each other. The complexity really lies in the fact that there are hundreds or maybe even thousands of different types of neurons, which are arranged in highly-organised patterns, and which connect to each other in very specific ways. Now, a new technique is emerging that enables neuroscientists to trace these kinds of connections by enlisting help from an unlikely source – viruses.

To understand why this approach is so exciting, we need to consider the scope of the problem it can help solve. This begins with individual neurons themselves. Neurons are polarised – they have an end for inputs and an end for outputs. Each of these may be branched to give thousands of independent sites of input and output. For any given neuron, there are other neurons that connect to it (information flows from all those neurons into our subject neuron) and other neurons that it connects to (information flows from our subject neuron out to all these neurons).

But neurons are not all the same. The most obvious and perhaps most important difference between neurons is that some are excitatory and some inhibitory. When an excitatory neuron is activated, it releases neurotransmitter at the connections it makes with its output neurons – this neurotransmitter tends to make those other cells electrically active. The exact opposite happens when an inhibitory cell is activated – it releases a different neurotransmitter onto its target neurons, which makes them less electrically active.

Neurons That Fire Together Wire Together

At any given moment, each single neuron is integrating the incoming information from typically hundreds of excitatory and inhibitory neurons – the balance between these and their precise timing will determine whether it becomes active enough to “fire” a signal to its own output neurons. (Without the inhibitory neurons, any electrical activity would rapidly spread through the entire brain in an epileptic-like firestorm).

There are hundreds of subtypes of excitatory and inhibitory neurons, all with different jobs to do. The way in which these different cell types are interconnected determines the functional properties of each little microcircuit in the brain – the type of information that comes into the system, how it filters and transforms that information, how long a neuron will be active before it's shut off, whether it will fire with a rhythm and at what frequency, etc.

These parameters determine the computations that the circuit can perform. While some of the details of such microcircuits have been worked out, for the most part we remain unblissfully ignorant of this crucial information. But we cannot hope to understand how the brain functions without knowing how the basic units of computation are put together.

How to solve this problem? In looking for ways to map the connections of neurons, scientists are turning to viruses – in particular, those that have evolved to infect neurons and to spread from one to another through the synaptic connections between them. One of these is the deadly rabies virus – a specialist in infecting neurons.

Neural connections in the brain

Rabies is typically transmitted from one infected animal to another through saliva, often via a bite, which releases viral particles that infect peripheral neurons. From there, it spreads backwards into the spinal cord and brain, passing from the initially infected neurons into every neuron that connects to them. This continues in the next neurons, resulting in the rapid spread of infection throughout the enture nervous system. This has a striking effect onthe host – among other symptoms, increasing aggression and attack behaviors, along with salivation, resulting in spread to a new host.

The fact that the virus can spread from an infected neuron to other neuronsconnected to it makes it an almost perfect vector for tracing these connections in the brains of experimental animals. To make it perfect required some modifications.

Modifying the Virus

First, using the tools of molecular biology, researchers have modified the genomeof the rabies virus, so that, as well as its own genes, the virus now carries so-called marker proteins, like the well-known green fluorescent protein from jellyfish. When ultraviolet light is shone on this protein, it fluoresces, giving off avivid green light. Neurons infected with the virus (either directly or via synaptic connections) can thus be beautifully visualised.

A major problem, however, is the rabies virus is too efficient – it continues to spread to all the neurons connected to each of the neurons connected to the first neuron, obscuring the pattern we are interested in. To get around this, the virus had to be crippled by removing one of the genes it needs to spread.

The sequence of the rabies virus genome - which is made from RNA, not DNA - encodes five proteins that are essential for the virus to replicate and be transmitted from one cell to another. When a rabies virus infects a neuron, it starts to express these proteins and replicate its genome. These replicates are packaged into a viral particle, which includes an envelope of host cell membrane studded with viral proteins.

One of these is the rabies glycoprotein, encoded by the G gene. This protein is essential for the viral particle to infect another neuron – it accomplishes this by binding to receptor proteins on the surface ofthese neurons, which leads to it being sucked into the cell (tricksy!).

A Solution: Delete the Genes

Even more tricksy is how scientists have taken advantage of this system to control which cells can be infected and to limit the spread of virus to only one synapse. This involves modifying both the virus and the cells of the host animal (usually a transgenic mouse). First, the G gene can be deleted from the viral genome – these viral particles can infect neurons when the virus is grown ina dish with the G protein added. But after they infect one neuron they cannotspread to others because they can’t make more G protein. The trick is to makethe infected host cell supply that protein – this can be achieved by adding DNA encoding this protein directly into a single cell.

When that single cell is infected, it leads to replication of the virus and transfer to only those cells with directinputs on the primary neuron. Scientists can thus visualise all the inputs ontoa single neuron, and painstakingly reconstruct the connectivity of an entiremicrocircuit.

What's the Significance?

Since the development of these techniques there have been a whole range of further modifications, allowing infection of specific cell types, the tracing of connections in the other direction (to all the output neurons) using different viruses, and the transfer of other cargo proteins to allow all kinds of additional analyses of the functions of these microcircuits.

In combination with tremendous advances in microscopy, these approaches are leading to a resurgence of “descriptive” neuroanatomy – just the kinds of studies that fueled the insights of pioneering neuroscientists like Ramon y Cajal 100 years ago. 

Those insights derived from the application of new technologies forvisualising single neurons. These new techniques take us the next step to see themicrocircuits that make up the computational units of the brain.

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New fossils suggest human ancestors evolved in Europe, not Africa

Experts argue the jaws of an ancient European ape reveal a key human ancestor.

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  • The jaw bones of an 8-million-year-old ape were discovered at Nikiti, Greece, in the '90s.
  • Researchers speculate it could be a previously unknown species and one of humanity's earliest evolutionary ancestors.
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Homo sapiens have been on earth for 200,000 years — give or take a few ten-thousand-year stretches. Much of that time is shrouded in the fog of prehistory. What we do know has been pieced together by deciphering the fossil record through the principles of evolutionary theory. Yet new discoveries contain the potential to refashion that knowledge and lead scientists to new, previously unconsidered conclusions.

A set of 8-million-year-old teeth may have done just that. Researchers recently inspected the upper and lower jaw of an ancient European ape. Their conclusions suggest that humanity's forebearers may have arisen in Europe before migrating to Africa, potentially upending a scientific consensus that has stood since Darwin's day.

Rethinking humanity's origin story

The frontispiece of Thomas Huxley's Evidence as to Man's Place in Nature (1863) sketched by natural history artist Benjamin Waterhouse Hawkins. (Photo: Wikimedia Commons)

As reported in New Scientist, the 8- to 9-million-year-old hominin jaw bones were found at Nikiti, northern Greece, in the '90s. Scientists originally pegged the chompers as belonging to a member of Ouranopithecus, an genus of extinct Eurasian ape.

David Begun, an anthropologist at the University of Toronto, and his team recently reexamined the jaw bones. They argue that the original identification was incorrect. Based on the fossil's hominin-like canines and premolar roots, they identify that the ape belongs to a previously unknown proto-hominin.

The researchers hypothesize that these proto-hominins were the evolutionary ancestors of another European great ape Graecopithecus, which the same team tentatively identified as an early hominin in 2017. Graecopithecus lived in south-east Europe 7.2 million years ago. If the premise is correct, these hominins would have migrated to Africa 7 million years ago, after undergoing much of their evolutionary development in Europe.

Begun points out that south-east Europe was once occupied by the ancestors of animals like the giraffe and rhino, too. "It's widely agreed that this was the found fauna of most of what we see in Africa today," he told New Scientists. "If the antelopes and giraffes could get into Africa 7 million years ago, why not the apes?"

He recently outlined this idea at a conference of the American Association of Physical Anthropologists.

It's worth noting that Begun has made similar hypotheses before. Writing for the Journal of Human Evolution in 2002, Begun and Elmar Heizmann of the Natural history Museum of Stuttgart discussed a great ape fossil found in Germany that they argued could be the ancestor (broadly speaking) of all living great apes and humans.

"Found in Germany 20 years ago, this specimen is about 16.5 million years old, some 1.5 million years older than similar species from East Africa," Begun said in a statement then. "It suggests that the great ape and human lineage first appeared in Eurasia and not Africa."

Migrating out of Africa

In the Descent of Man, Charles Darwin proposed that hominins descended out of Africa. Considering the relatively few fossils available at the time, it is a testament to Darwin's astuteness that his hypothesis remains the leading theory.

Since Darwin's time, we have unearthed many more fossils and discovered new evidence in genetics. As such, our African-origin story has undergone many updates and revisions since 1871. Today, it has splintered into two theories: the "out of Africa" theory and the "multi-regional" theory.

The out of Africa theory suggests that the cradle of all humanity was Africa. Homo sapiens evolved exclusively and recently on that continent. At some point in prehistory, our ancestors migrated from Africa to Eurasia and replaced other subspecies of the genus Homo, such as Neanderthals. This is the dominant theory among scientists, and current evidence seems to support it best — though, say that in some circles and be prepared for a late-night debate that goes well past last call.

The multi-regional theory suggests that humans evolved in parallel across various regions. According to this model, the hominins Homo erectus left Africa to settle across Eurasia and (maybe) Australia. These disparate populations eventually evolved into modern humans thanks to a helping dollop of gene flow.

Of course, there are the broad strokes of very nuanced models, and we're leaving a lot of discussion out. There is, for example, a debate as to whether African Homo erectus fossils should be considered alongside Asian ones or should be labeled as a different subspecies, Homo ergaster.

Proponents of the out-of-Africa model aren't sure whether non-African humans descended from a single migration out of Africa or at least two major waves of migration followed by a lot of interbreeding.

Did we head east or south of Eden?

Not all anthropologists agree with Begun and his team's conclusions. As noted by New Scientist, it is possible that the Nikiti ape is not related to hominins at all. It may have evolved similar features independently, developing teeth to eat similar foods or chew in a similar manner as early hominins.

Ultimately, Nikiti ape alone doesn't offer enough evidence to upend the out of Africa model, which is supported by a more robust fossil record and DNA evidence. But additional evidence may be uncovered to lend further credence to Begun's hypothesis or lead us to yet unconsidered ideas about humanity's evolution.