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

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.