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Scientists make major discovery to advance regenerative medicine

Scientists at Forsyth may have moved one step closer to regenerating

human spinal cord tissue by artificially inducing a frog tadpole to

re-grow its tail at a stage in its development when it is normally

impossible. Using a variety of methods including a kind of gene therapy,

the scientists altered the electrical properties of cells thus inducing

regeneration. This discovery may provide clues about how bioelectricity

can be used to help humans regenerate.

This study, for the first time, gave scientists a direct glimpse of the

source of natural electric fields that are crucial for regeneration, as

well as revealing how these are produced. In addition, the findings

provide the first detailed mechanistic synthesis of bioelectrical,

molecular-genetic, and cell-biological events underlying the

regeneration of a complex vertebrate structure that includes skin,

muscle, vasculature and critically spinal cord.

Although the Xenopus (frog) tadpole sometimes has the ability to re-grow

its tail, there are specific times during its development that

regeneration does not take place (much as human children lose the

ability to regenerate finger-tips after 7 years of age). During the

Forsyth study, the activity of a yeast proton pump (which produces H+

ion flow and thus sets up regions of higher and lower pH) triggered the

regeneration of the frog's tail during the normally quiescent time.

This research will be published in the April issue of Development and

will appear online on February 28, 2007.

According to the publication's first author, Dany , Ph.D.,

Assistant Research Investigator at the Forsyth Institute, applied

electric fields have long been known to enhance regeneration in

amphibia, and in fact have led to clinical trials in human patients.

" However, the molecular sources of relevant currents and the mechanisms

underlying their control have remained poorly understood, " said .

" To truly make strides in regenerative medicine, we need to understand

the innate components that underlie bioelectrical events during normal

development and regeneration. Our ability to stop regeneration by

blocking a particular H+ pump and to induce regeneration when it is

normally absent, means we have found at least one critical component. "

The research team, led by Levin, Ph.D., Director of the Forsyth

Center for Regenerative and Developmental Biology has been using the

Xenopus tadpole to study regeneration because it provides an opportunity

to see how much can be done with non-embryonic (somatic) cells during

regeneration, and it is a perfect model system in which to understand

how movement of electric charges leads to the ability to re-grow a fully

functioning tail. Furthermore, said Dr. Levin, tail regeneration in

Xenopus is more likely to be similar to tissue renewal in human beings

than some other regenerative model systems. The Forsyth scientists

previously studied the role that apoptosis, a process of programmed cell

death in multi-cellular organisms, plays in regeneration.

Levin, PhD. is an Associate Member of the Staff in The Forsyth

Institute Department of Cytokine Biology and the Director of the Forsyth

Center for Regenerative and Developmental Biology. Through experimental

approaches and mathematical modeling, Dr. Levin and his team examine the

processes governing large-scale pattern formation and biological

information storage during animal embryogenesis. The lab investigates

mechanisms of signaling between cells and tissues that allows a living

system to reliably generate and maintain a complex morphology. The Levin

team studies these processes in the context of embryonic development and

regeneration, with a particular focus on the biophysics of cell behavior.

Source: Forsyth Institute

This news is brought to you by PhysOrg.com

http://www.physorg.com/news91878580.html

Deconstructing Brain Wiring, One Neuron at a Time

Researchers have long said they won't be able to understand the brain

until they can put together a " wiring diagram " - a map of how billions

of neurons are interconnected. Now, researchers at the Salk Institute

for Biological Studies have jumped what many believe to be a major

hurdle to preparing that chart: identifying all of the connections to a

single neuron.

In the March 1 issue of the journal Neuron, the researchers describe how

they modified the deadly rabies virus, turning it into a tool that can

cross the synaptic space of a targeted nerve cell just once to identify

all the neurons to which it is directly connected.

" We've wanted to do this for a very long time and finally found a way to

make it possible, " says the study's senior author, M. Callaway,

Ph.D., a professor in the Systems Neurobiology Laboratories. " It will

offer us an unprecedented view of the brain. "

The problem neuroscientists are confronted with " is akin to a computer

user who tries to figure out how the machine's electronic chip works by

looking down at it; there is no way to figure out how things are

connected, " Callaway says. " If you were given a wiring diagram, you

could begin to understand how the chip moves electricity and how that

operates the computer. "

Neuroscientists also want to deconstruct the flow of electrical signals

in the extraordinarily complex architecture of the brain and then

correlate these neural circuits with such brain functions as perception

and behavior. But these circuits are difficult to unravel because dozens

of different neuronal types are entangled within a precisely connected

network, and even neighboring neurons of the same type differ in

connectivity and function.

So, researchers have been trying to figure out the pattern of

connections typical of a type of neuron, to see which other cell types

they connect with and how those connections are configured. To do this,

they need a tracer that can tease apart the chain of directly connecting

neurons, identifying them one by one.

Viruses that naturally spread between neurons have previously been used

to outline the flow of nerve cell communication, but they have two

drawbacks. First, once inside the brain, they keep spreading from cell

to cell without stopping. Second, they cross different synapses - the

specialized junctions between nerve cells - at different rates, crossing

bigger, stronger synapses faster than smaller, weaker ones. Together

these attributes make these viruses unable to determine exactly which

cells are connected to which. The team of Salk researchers sought to

create a modified virus whose spread could be limited to a single

synaptic connection.

" The core idea is to use a virus that is missing a gene required for

spreading across synapses but to provide the missing gene by some other

means within the initially infected cells, " says Ian Wickersham, Ph.D.,

postdoctoral researcher and lead author on the project.

With the critical gene deleted from its genome, the virus is marooned

inside a cell, unable to spread beyond it. However, supplying the

missing gene in that same cell allows the virus to spread to cells that

are directly connected to it. Since these neighboring cells lack the

gene supplied in the first cell, the virus is stuck. Only the cells

connected directly to the original cell are labeled.

The team's second challenge was to find a way of targeting the viral

infection specifically to particular cells, so that the virus could be

used to map the connections of cell types of interest or even of single

cells. The solution came from a conversation between Callaway and

Young, Ph.D., a Professor in the Infectious Disease Laboratory, and

co-author of the study. When Callaway described the problem, Young

immediately suggested the answer, which is based on an avian viral

receptor that Young discovered as a postdoctoral fellow at UCSF. The

protein that ordinarily coats rabies virus particles was replaced with

its equivalent from a bird virus. This prevented the modified rabies

virus from infecting mammalian neurons at all - unless they had been

engineered to express the bird virus's receptor, a bird cell surface

protein known as TVA. Such neurons will then be " disguised " as bird

cells and the rabies virus - now acting like a bird virus - will infect

them.

" The bottom line is that you need two genes expressed in the cell or

cell type of interest: TVA, to get the rabies virus in, and the missing

viral gene so the virus can spread to connected cells, " says Wickersham.

Experimenting on slices of neonatal rat brain, the Salk researchers

inserted these two genes into selected neurons - as well as a gene that

fluoresces red when expressed. Then they applied the modified rabies

virus, which had furthermore been given the ability to make infected

cells fluoresce green. The result was spectacular: as expected, these

red cells were selectively infected by the virus, which spread to

hundreds of surrounding cells, turning them brilliantly fluorescent green.

While these experiments were conducted using slices of brain, it is

possible to produce transgenic mice that will express specific genes in

a targeted class of neurons, Callaway says. " All neurons of the type we

select will then express the avian viral receptor and the rabies virus

protein, allowing the modified rabies virus to infect targeted cells and

spread only once to connecting cells, " he adds. The wiring map can be

constructed step by step as subsequent populations of cells are imaged.

The recombinant rabies virus could contain genes for any proteins of

interest, Callaway says, and he adds that once scientists can identify a

neural circuit, they can then deactivate it, and test for changes in

brain function.

Source: Salk Institute for Biological Studies

http://www.physorg.com/news91958783.html