Tuesday, November 23, 2010

Behind the NeuroArt #1 @ TheBeautifulBrain

Brain slice preparations, which make up the bulk of neural microcircuitry research to date, have greatly added to our understanding of how distinct neuronal cell types are connected in different parts of the brain. However this method is limited because it gives only a snapshot of a cold lifeless brain. That is, it can not show the relationship between connectivity and function in the intact functioning brain.

However a recently developed technique by researches at the Salk Institute which is being displayed at The Beautiful Brain promises to unleash a wealth of new understanding by opening up the functioning brain to traditional brain connectivity study. For the first time an analysis will be able to be made concerning the fine-scale connectivity of the brain in living organisms.

And it has all been made possible using a dash of fluorescence and smigin of Rabies virus.

Monosynaptic tracing rabies virus with EGFP gene | The Beautiful Brain
As a neurotropic virus, rabies prefers to live inside the nervous system. Once a neuron is infected, the Wild-type strain used by the Salk Institute, travels in the retrograde direction - from host to it's inputs - where it attempts to cross the synapse and infect the input neurons.

In this case the rabies virus is a little too leggy. In order to understand how a single neuron collates information from its inputs in order to fire an output, it will be necessary to isolate a target neuron and its input neurons from the rest of the brain.

To accomplish this it will be necessary to genetically modify the rabies virus so that it does not continue to infect neurons after it has infected the targets presynaptic neurons. Further more, the infected and non infected neurons will need to be identified, it is essential to have a suitable visual clue.

By swapping the glycoprotein gene responsible for rabies ability to infect neurons with the EGFP gene researches can disable the virus ability to infect whilst simultaneously giving it a fluorescence green glow. EGFP use is a common practice in science and has been included in many instances of BioArt, of which the most famous example is GFP Bunny.

However, before the EGFP gene is introduced, the glycoprotein gene responsible for rabies transsynaptic spread must be removed.

Monosynaptic tracing rabies virus with EGFP gene | The Beautiful Brain
The ability of rabies to bind to and infect neurons is provided by the glycoprotein covering on it's viral-envelope for which mammalian neurons contain a corresponding glycoprotein receptor. Rabies can produce this glycoprotein coating and furthermore pass this function on to daughter cells (clones) during the process of mitosis as the glycoprotein gene exists within its RNA/genome. This allows the virus to infect the host unabated.

To stop this runaway infection, it is necessary to remove the glycoprotein gene from the rabies genome. However, doing so will remove any chance of infecting the target neuron. Thus researches have had to manufacture an ability that allows the virus to infect only the target and its input neurons.

To allow the virus to infect the target neuron, both the neuron and the rabies virus in question must be modified in a complementary way. Known as "targeting for infection", by coating the virus with the envelope protein EnvA through a process known as pseudotyping and biolistically transfecting the target neuron with the gene for the TVA receptor a method is provided for the virus to bind to the target neuron. The EnvA envelope protein is capable of binding to the TVA receptor and thus when the virus, coated in EnvA binds to the TVA receptors on the target neuron, it infects it.

However, once the virus invades the target neuron and replicates, the 'viral particles' or 'daughter cells' will have no way of infecting further neurons because their mother, devoid of the glycoprotein gene could not produce glycoprotein for their viral envelopes and secondly any remnants of the EnvA protein from their mother existing on their viral envelopes is useless as only the target neurons were transfected with the TVA receptor gene.

Monosynaptic tracing rabies virus with EGFP gene | The Beautiful Brain
This is where the rabies virus glycoprotein gene comes back into the picture. Even though the virus does not contain the gene in it's genome, it can still produce the glycoprotein if it is in the neuron during infection.

Thus in order for the daughter cells to transsynaptically infect the input neurons, the target neurons must also be transfected with the gene for the rabies glycoprotein. This was done through a process known as Single-Cell Electroporation. Then as the replicated virus particles break out of the neuron, through a process known as viral shedding, they will acquire the produced glycoprotien as it was deposited within the membrain of the host cell.

This allows the daughter cells to transsynaptically infect the input neurons because as I detailed in the earlier part of this article, all the neurons contain the glycoprotein receptor that allows the rabies virus to bind to it. Further more, because they do not contain the glycoprotien gene and only the covering on their envelopes, they can not produce the glycoprotien when they divide/replicate in the input neurons. Thus the virus is trapped, glowing green through the expression of the EGFP gene.

The researches last step was to coinfect the post synaptic neurons with a gene for red fluorescence in the biolistics transfection step mentioned earlier. This gives a strong contrast between the infected and non infected neurons.

By tracing the monosynaptic inputs of the target neuron in this manner, the pre-synaptic and target neurons have been visually isolated from the rest of the millions of neurons throughout the brain.

This research, which is presented in the abstract Targeting Single Neuronal Networks for Gene Expression and Cell Labeling In Vivo concludes:

"Combining the power of anatomical tracing with genetic manipulation and existing assays of neuronal function, it should be possible to gain greater insights into the mechanisms supporting information processing by single neuronal networks and determine the link between connectivity and function of single neurons throughout the brain."

Using the technique researches will for the first time be able to understand how distinct cell types, particular genes and patterns of synaptic input influence a single neurons function of information processing and perhaps allow those researchers to eventually do away with the limited usefulness of brain slice preparations.

Today, I have featured images from the Art of Neuroscience volume 1 from The Beautiful Brain. In the coming weeks, if your not still reading this post, I want to bring you more science behind the Art of Neuroscience volumes two and three. Feel free to have a look at these before i get stuck into them.

I'd like to thank the Beautiful Brain who has given me access to their fantastic images including the information for last weeks post of The Amygdaloids.

Stay tuned for more Art of Neuroscience in the coming weeks.

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