MOLECULES OF THE MIND Such descriptions hardly depict the neurological reality of the brain, of course. A better metaphor for 2 neu- rons might be 2 trees that have been uprooted and turned 90 degrees so their root systems face each other. Then, some Paul Bunyan–type char- acter jams both ends together. Thou- sands of connections from 1 tree now face thousands of connections from the other. Multiply those 2 neurons by thousands while all their root systems are still jammed together with the same Bunyanesque enthusiasm and you can visualize an approximation of the real world of brain wiring. Not as elegant as 2 limbs—and not as simple either. How can we understand the way all those myriad connections work to- gether to produce the various neuro- logical abilities of the brain? Given its complexity, the task is enormous. Since we are only in the initial stages, it will take a long time before we will be able to map structure to function. Like any exploration in its initial stages, we first need a good map—a schematic that shows how each slen- der dendritic branch interacts with a specific nerve cell. From Nissl and Weigert stains to the canonical Golgi stain, we have traditionally used dye technology to help us visualize these interactions. However, there are se- vere limitations to most of these stain- ing technologies that center around their inability to make visible all the connections that actual neurons pos- sess. We need something with far greater resolution and, perhaps, with a bit more elegance. The topic of this column and the next is a technology that promises to deliver just such circuit diagrams at a very high level of resolution. The technology involves the exploration of viruses, which in their native form cause some pretty tough diseases (eg, rabies, cancer). Thoughtful genetic engineering has transformed these viruses’ job description from fearful disease inducer to doughty cartogra- pher. We are going to follow how this transformation has occurred. I give you fair warning that it is a complex manipulation. I encourage you to closely follow the Figure that accompanies this column as well as the figure in Part 2. Both diagrams describe the story of hypothetical “Neuron A” and our attempts to un- derstand all its many intricacies and connections. To explain Neuron A’s interactions, I need to review not on- ly 2 pathogenic viruses but also sev- eral manipulated proteins, genetical- ly engineered neural cells, and protein-based fluorescent dyes. In Part 1, I will discuss some back- ground, which I have divided into 3 parts. In Part 2, I will explain the data. The actual experiment that took place asks and answers the simple question: Are the neighbors green yet? Why rabies virus? The rabies virus has 2 characteristics that make it an ideal “truck” for the delivery of molecular products to mammalian neural interiors. The first has to do with its legendary life cycle. Once inside a nerve cell, the virus sets up a manufacturing site to create more viruses, like any typical virus. At maturity, these progeny can jump to other neurons, spreading the virus along specific neural routes. If you could find a way to follow the virus, you could identify the routes. The second characteristic has to do with access to the CNS, which is a topic of great concern to researchers who are interested in drug delivery, W e often describe neural connections in the brain as if they were a cellular version of Michelan- gelo’s famous painting “Hand of God Giving Life to Adam,” on the ceiling of the Sistine Chapel. Like the painting, the neural explanations usually in- voke 2 outstretched limbs nearly touching each other—one presynaptic, one postsynaptic—that are separated by their 20- nanometer synaptic cleft. Soon, Neuron A will be flooded with the glycopro- tein. Why do we go through these steps? This double manipulation al- lows the virus to set up a productive infection in just 1 cell. If the hobbled ra- bies virus is allowed to infect the modified Neuron A, the cell will allow the virus to make new progeny, which can then escape Neuron A and infect all its neighbors. But that is where everything stops. The neighbors will be able to create a new virus just like Neuron A; how- ever, these progeny are still reproduc- tively incompetent (remember, the vi- ral genetic information is gutted from the glycoprotein). Neuron A will serve as the birth mother, and the virus will spread to neighboring nerve cells. If the neighbors do not have the viral glycoprotein already working in the background, the virus will not be able to spread outward from them. The buck, as it were, stops there. This finding could be of great val- ue to a neurobiologist, especially if Neuron A is embedded in a thicket of normal neurons that are not geneti- cally engineered. It is the neurobiolo- gist’s job to find out how they are connected. This concept will become very important as I further discuss this technology. ASLV-A The second virus in our discussion goes by the tongue-twisting name avian sarcoma and leukosis virus subgroup A (ASLV-A). The virus at- tacks via a surface grappling hook— a protein called EnvA. If EnvA finds a receptor protein called TVA,ASLV- A will set up an infection. This will occur even if the TVA protein is found on a cell other than an avian cell. Why am I bringing this up? It is possible to mix and match viral pro- teins and to alter their host ranges. For example, a researcher could remove an EnvA gene from ASLV-A and stick it to the genetic background of a rabies virus. The rabies virus now has a grappling hook for avian cells. This means that the rabies virus is now ful- ly capable of binding to an avian cell or any cell that carries the TVA re- for example. The rabies virus is not a respecter of neural borders. It can in- fect neurons in the peripheral nervous system and then jump the neurologi- cal border to enter the CNS. That is why a bite anywhere on the body can result in a catastrophic brain infec- tion. By investigating how this trans- fer occurs at the molecular level, bi- ologists can exploit the viral life cycle to understand neural interactions. I will now discuss how a modified rabies virus could confront our Neuron A described previously. We will “genetically engineer” a ra- bies virus and, at the same time, ge- netically engineer its neural target to make this happen. The gene we are going to engineer is one of the proteins that the rabies virus needs to set up a productive in- fection. It is a glycoprotein, which is carried in gene form by the virus that infects Neuron A. If the virus can manufacture this glycoprotein after it enters Neuron A, it can make proge- ny and spread the infection to an ad- jacent nerve cell. If the virus cannot make the glycoprotein, it will not be able to jump to an adjacent nerve cell, even if Neuron A is otherwise infect- ed. The virus will simply stay inside its host. Today, researchers can be quite clever about forcing the hand of the rabies virus. For example, they can gut the gene that encodes the glyco- protein from the virus, rendering it impotent to spread infection. The virus can get into Neuron A but it can- not get out. While we are working on the ra- bies virus, we are also going to ge- netically modify Neuron A. We will engineer the nerve cell to make the glycoprotein by extracting its gene from the virus and inserting it into the nucleus of Neuron A. This process is called transfection, and there are many ways to perform the procedure. by John J. Medina, PhD Painting Neural Circuitry With a Viral Brush 16 OCTOBER 2008 www.psychiatrictimes.com PSYCHIATRIC TIMES Part 1 Like any exploration in its initial stages, we first need a good map— a schematic that shows how each slender dendritic branch interacts with a specific nerve cell.
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MOLECULES OF THE MIND
Such descriptions hardly depict theneurological reality of the brain, ofcourse. A better metaphor for 2 neu-rons might be 2 trees that have beenuprooted and turned 90 degrees sotheir root systems face each other.Then, some Paul Bunyan–type char-acter jams both ends together. Thou-sands of connections from 1 tree nowface thousands of connections fromthe other. Multiply those 2 neurons bythousands while all their root systemsare still jammed together with thesame Bunyanesque enthusiasm andyou can visualize an approximationof the real world of brain wiring. Notas elegant as 2 limbs—and not assimple either.
How can we understand the wayall those myriad connections work to-gether to produce the various neuro-logical abilities of the brain? Given its complexity, the task is enormous.Since we are only in the initial stages,it will take a long time before we willbe able to map structure to function.
Like any exploration in its initialstages, we first need a good map—aschematic that shows how each slen-der dendritic branch interacts with aspecific nerve cell. From Nissl andWeigert stains to the canonical Golgistain, we have traditionally used dyetechnology to help us visualize theseinteractions. However, there are se-vere limitations to most of these stain-ing technologies that center aroundtheir inability to make visible all theconnections that actual neurons pos-sess. We need something with fargreater resolution and, perhaps, witha bit more elegance.
The topic of this column and thenext is a technology that promises todeliver just such circuit diagrams at a very high level of resolution. Thetechnology involves the explorationof viruses, which in their native formcause some pretty tough diseases (eg,rabies, cancer). Thoughtful geneticengineering has transformed theseviruses’ job description from fearfuldisease inducer to doughty cartogra-
pher. We are going to follow how thistransformation has occurred.
I give you fair warning that it is acomplex manipulation. I encourageyou to closely follow the Figure thataccompanies this column as well asthe figure in Part 2. Both diagramsdescribe the story of hypothetical“Neuron A” and our attempts to un-
derstand all its many intricacies andconnections. To explain Neuron A’sinteractions, I need to review not on-ly 2 pathogenic viruses but also sev-eral manipulated proteins, genetical-ly engineered neural cells, andprotein-based fluorescent dyes. InPart 1, I will discuss some back-ground, which I have divided into 3parts. In Part 2, I will explain the data.
The actual experiment that tookplace asks and answers the simplequestion:Are the neighbors green yet?
Why rabies virus?The rabies virus has 2 characteristicsthat make it an ideal “truck” for thedelivery of molecular products tomammalian neural interiors. The firsthas to do with its legendary life cycle.Once inside a nerve cell, the virus setsup a manufacturing site to createmore viruses, like any typical virus.At maturity, these progeny can jumpto other neurons, spreading the virusalong specific neural routes. If youcould find a way to follow the virus,you could identify the routes.
The second characteristic has to dowith access to the CNS, which is atopic of great concern to researcherswho are interested in drug delivery,
We often describe neural connections in the brainas if they were a cellular version of Michelan-gelo’s famous painting “Hand of God GivingLife to Adam,” on the ceiling of the Sistine
Chapel. Like the painting, the neural explanations usually in-voke 2 outstretched limbs nearly touching each other—onepresynaptic, one postsynaptic—that are separated by their 20-nanometer synaptic cleft.
Soon, Neuron Awill be floodedwith the glycopro-tein.
Why do we go through thesesteps? This doublemanipulation al-lows the virus toset up a productive infection in just 1
cell. If the hobbled ra-bies virus is allowed toinfect the modifiedNeuron A, the cell willallow the virus to make
new progeny, which can then escapeNeuron A and infect all its neighbors.But that is where everything stops.The neighbors will be able to createa new virus just like Neuron A; how-ever, these progeny are still reproduc-tively incompetent (remember, the vi-ral genetic information is gutted fromthe glycoprotein). Neuron A willserve as the birth mother, and thevirus will spread to neighboring nervecells. If the neighbors do not have theviral glycoprotein already working inthe background, the virus will not beable to spread outward from them.The buck, as it were, stops there.
This finding could be of great val-ue to a neurobiologist, especially ifNeuron A is embedded in a thicket ofnormal neurons that are not geneti-cally engineered. It is the neurobiolo-gist’s job to find out how they areconnected. This concept will becomevery important as I further discussthis technology.
ASLV-AThe second virus in our discussiongoes by the tongue-twisting nameavian sarcoma and leukosis virussubgroup A (ASLV-A). The virus at-tacks via a surface grappling hook—a protein called EnvA. If EnvA findsa receptor protein called TVA,ASLV-A will set up an infection. This willoccur even if the TVA protein isfound on a cell other than an aviancell.
Why am I bringing this up? It ispossible to mix and match viral pro-teins and to alter their host ranges. Forexample, a researcher could removean EnvA gene from ASLV-A andstick it to the genetic background of arabies virus. The rabies virus now hasa grappling hook for avian cells. Thismeans that the rabies virus is now ful-ly capable of binding to an avian cellor any cell that carries the TVA re-
for example. The rabies virus is not arespecter of neural borders. It can in-fect neurons in the peripheral nervoussystem and then jump the neurologi-cal border to enter the CNS. That iswhy a bite anywhere on the body canresult in a catastrophic brain infec-tion. By investigating how this trans-fer occurs at the molecular level, bi-ologists can exploit the viral life cycleto understand neuralinteractions.
I will now discusshow a modified rabiesvirus could confrontour Neuron A described previously.We will “genetically engineer” a ra-bies virus and, at the same time, ge-netically engineer its neural target tomake this happen.
The gene we are going to engineeris one of the proteins that the rabiesvirus needs to set up a productive in-fection. It is a glycoprotein, which iscarried in gene form by the virus thatinfects Neuron A. If the virus canmanufacture this glycoprotein after itenters Neuron A, it can make proge-ny and spread the infection to an ad-jacent nerve cell. If the virus cannotmake the glycoprotein, it will not beable to jump to an adjacent nerve cell,even if Neuron A is otherwise infect-ed. The virus will simply stay insideits host.
Today, researchers can be quiteclever about forcing the hand of therabies virus. For example, they cangut the gene that encodes the glyco-protein from the virus, rendering itimpotent to spread infection. Thevirus can get into Neuron A but it can-not get out.
While we are working on the ra-bies virus, we are also going to ge-netically modify Neuron A. We willengineer the nerve cell to make theglycoprotein by extracting its genefrom the virus and inserting it into thenucleus of Neuron A. This process iscalled transfection, and there aremany ways to perform the procedure.
by John J. Medina, PhD
Painting Neural Circuitry With a Viral Brush
16 OCTOBER 2008
www.psychiatr ict imes.com
PSYCHIATRIC TIMES
Part 1
Like any exploration in its initial
stages, we first need a good map—
a schematic that shows how each
slender dendritic branch interacts
with a specific nerve cell.
0810PT203967MEDlay 9/24/08 12:14 PM Page 16
MOLECULES OF THE MIND
dark if given the proper substrates atdifferent wavelengths when insertedinto cells. There are genes whose pro-teins glow red and genes whose pro-teins glow green (green fluorescentprotein, GFP). These genes can bestitched into the genetic machinery ofviruses, which are then allowed to in-fect cells. Researchers can trace notonly the success of the infection butalso the routes of infection just bylooking for the presence of the fluo-
ceptor protein. The virus could be di-rected to go anywhere the researcherwanted it to, as long as the EnvA-TVA matching conditions were met.This, too, turns out to be a critical partof this story.
Two fluorescent proteinsThe fluorescent proteins are probablythe least complex piece of molecularbiology in our discussion. There are anumber of proteins that glow in the
rescent color in the cell. For example,if the GFP gene is inserted into therabies genetic information, every cellin which the rabies virus sets up an in-fection will turn green. As you mightsuspect, this phenomenon provid-ed researchers with everything theyneeded to trace neural interactions.
With this information in mind, weare prepped to discuss the data, whichwill occur in Part 2. We will see thatthe clever use of these viruses, genes,
and manipulated cellular back-grounds has allowed researchers tocreate their own Lilliputian Sistinemasterpiece.
They did it all by answering thatsimple question, “Are the neighborsgreen yet?”
Dr Medina is a developmental molecular
biologist and private consultant, with research
interests in the genetics of psychiatric
disorders. �
17PSYCHIATRIC TIMES OCTOBER 2008
www.psychiatr ict imes.com
Rabies viruses and wiring diagrams
1
2
3
The goal
NEURON “A”
NEURON “A”
TVAgene
added
Rabiesglycoproteingene added Red
“glow”geneadded
T G R
G
Rabiesglycoproteingene deleted
EnvAgene
addedGFPgene
added
E G
ERabies viral particle with
surface EnvA protein
??
?
?
??
?
?
????
Engineering the virus
Engineering the neuron
The use of rabies viruses to map synaptic interactions between neurons involves the complex engineering of both virus and target neurons. Divided into 3 parts, here’s a graphic overview of the manipulations described in the text.
Figure
Neuron A is surrounded by hundreds of cells and thousands of synaptic connections. Which connections interact directly with Neuron A? The goal of the technology described below is to find out.
Three genetic manipulations were performed on a rabies virus. (1) The rabies’ glycoprotein (which allows progeny virus to setup and propagate their own infections) was deleted; (2) a gene encoding the EnvA protein of the avian sarcoma and leukosis virus subgroup A was inserted; (3) the green fluorescent protein gene (GFP) was added. The cell carrying this gene will fluoresce green when the GFP gene is expressed.
Three genetic manipulations were performed on Neuron A. (1) The TVA receptor gene was inserted into the cell. This allows a virus carrying EnvA to bind to the cell; (2) the rabies’ glycoprotein was inserted into the neural genome; (3) a red fluorescing gene was added. The cell carrying this gene will fluoresce red when the gene is expressed.
Virus can now bind to any cell carrying EnvA receptor (receptor called TVA)
0810PT203967MEDlay 9/24/08 12:14 PM Page 17
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