Nerve-muscle Junction - Fast-freeze Snapshot Yields New Picture

[Guest guest]

Fast-freeze Snapshot Yields New Picture Of Nerve-muscle Junction

http://www.medicalnewstoday.com/medicalnews.php?newsid=51443

When nerve cells excite muscle fibers to flex, getting synaptic

proteins and components into the right place can mean the difference

between feats of strength or lapses of drowsy lethargy.

Several proteins that have been shown to be major players in synaptic

transmission have now been studied using a flash-freeze physical-

fixation technique that reveals new details of their location and

function in neuromuscular synapses. The technique was used with tiny,

one-millimeter-long nematode worms, a lab animal widely studied by

neuroscientists.

Investigators report the finding in the Aug. 2 issue of the Journal

of Neuroscience. Janet Richmond, associate professor of biological

sciences at the University of Illinois at Chicago, is the

corresponding author.

Previously, Richmond developed a technique that allows a more precise

understanding of how synaptic proteins affect release of

neurotransmitter chemicals at the junctions -- the signal that

enables nerve cells to issue commands.

The technique described in the new study, high-pressure freeze

electron microscopy and immuno-gold staining, now provides an

accurate picture of where these synaptic proteins cluster --

information previously unknown to scientists.

Co-author Robby Weimer, a post-doctoral fellow at the Cold Stream

Harbor Laboratory in New York, developed the high-pressure freeze

technique to view synapses while working in the laboratory of

coauthor Jean-Louis Bessereau at INSERM Ecole Normale Superieure in

Paris. Richmond's graduate student Elena Gracheva introduced the

technique at UIC.

" It's a new technique that allows us to take a snapshot of what's

going on at the neuromuscular junction and actually physically view

the consequences of losing these proteins, " said Richmond.

The conventional technique is to use gluteraldhyde fixation, which

takes seconds or minutes to complete -- unlike the fraction of a

second when using the high-pressure freeze method. What's more,

during gluteraldhyde fixation the nematodes writhe around, releasing

neurotransmitters while cells become dehydrated, causing synaptic

components to get misplaced and synapses to take on a wrinkly

appearance.

While slow freezing can create ice crystals that tear cell structures

apart, the high-pressure technique, using liquid nitrogen to flash-

freeze at minus-180 degrees Celsius, makes ice appear like liquid

glass and devoid of destructive crystals.

Cross-sections taken of synapses reveal that membrane packets, or

vesicles, of neurotransmitter localize in places scientists have

never before seen.

" It tells us that specific proteins are required to transition

vesicles in close apposition to pre-synaptic membranes, " said

Richmond. " That prediction had been made, but hasn't before been

demonstrated. "

Richmond said the conventional gluteraldhyde fixation technique was

the problem with the earlier view of vesicle positioning in nerve

synapses, and she predicted that future use of the new technique will

open up new discoveries of the roles various proteins play in nerve

synapses.

" It's going to revolutionize the way we do this kind of analysis, "

she said.

###

Other contributors to the paper include Olivier Meyrignac, a medical

student at INSERM, and Ken , assistant member in the molecular

and cell biology research program at the Oklahoma Medical Research

Foundation in Oklahoma City.

Funding was provided by the National Institutes of Health.