X-Message-Number: 28418
From: "Basie" <>
Subject: Fast-freeze Snapshot Yields New Picture Of Nerve-muscle Junction
Date: Sun, 10 Sep 2006 13:42:25 -0400

Fast-freeze Snapshot Yields New Picture Of Nerve-muscle Junction
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 Miller, 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.

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