New methods for revealing nerve-muscle activity in live animals

Picking up a pencil, running a marathon, even the simple act of breathing - every time we move a muscle we depend on a series of finely-tuned interactions at the junction of nerve fiber and muscle cell.

When something goes wrong in that pathway, as happens in diseases such as myasthenia gravis or with exposure to nerve gas, the result can be paralysis or death by asphyxiation.

In a recent series of papers, the latest published in the Journal of Biological Chemistry, University of Michigan researcher Mohammed Akaaboune and colleagues describe how they've used innovative imaging techniques to study nerve-muscle interactions in living animals, yielding some surprising findings….

When an impulse comes zipping down a nerve fiber, hundreds of microscopic sacs at the end of the fiber spew molecules of the chemical messenger acetylcholine (ACh) into the gap between the nerve cell and the nearest muscle cell. The messenger molecules cross the gap and attach to receptor proteins on the muscle cell membrane, triggering an electrical impulse that makes the muscle contract. An essential enzyme called acetylcholinesterase (AChE) then breaks down the messenger molecules in the gap, clearing the way for the next impulse.

"However, in some diseases, such as myasthenia gravis, this enzyme is somehow caused to malfunction," said Akaaboune, assistant professor of molecular, cellular and developmental biology. "The same enzyme is also the target of nerve gases, such as Sarin." When the enzyme is inactivated, ACh messenger molecules build up to such high levels that receptors become desensitized and stop responding. "If they don't respond, muscles don't contract, and because muscles control breathing, breathing stops."

Akaaboune and colleagues have developed techniques for spying on nerve-muscle junction activity in living animals, using fluorescently-tagged molecules that bind tightly to AChE and receptors. They focus on an easily accessed muscle in the necks of mice where all the nerve-muscle connections, called synapses, are bundled together. This makes it easier to single out a particular synapse and study it over the course of several days, Akaaboune said.

In a study published last fall in the Journal of Neuroscience, Akaaboune's group tagged receptors to get a closer look at their activity. They found that, instead of being degraded after they do their job and travel into the interior of the cell, some 50 percent of receptors are recycled back to the membrane to start the process all over again. "Although recycling of receptors occurs in the central nervous system, we never thought it occurred here, but we found that it does," Akaaboune said. "In this way, the cell maintains the number and density of receptors necessary for efficient synaptic transmission."

When the researchers blocked activity in the synapse with curare, a paralyzing poison that Native South Americans use on arrow tips when hunting, receptor recycling was prevented. Such studies may aid in understanding diseases such as myasthenia gravis, which affect receptor number, density and turnover, Akaaboune said.

In the latest work, the researchers tagged AChE with a fluorescently-labeled toxin from the green mamba snake. The toxin blocks AChE's activity, making it a powerful tool for exploring what happens when the enzyme is inactivated by disease or toxic gas. One interesting finding was that blocking the enzyme's activity increased the rate at which receptors were lost from the membrane.

Studies such as these are important, Akaaboune said, because they can lead to insights that will help researchers target treatments more precisely. "Understanding the biology of acetylcholinesterase and acetylcholine receptors will help us design new drugs for either curing disease or changing the course of response to nerve gas exposure."

Akaaboune collaborated on the latest research with Eric Krejci of the French health research institute INSERM, postdoctoral fellow Isabel Martinez-Pena y Valenzuela and research associate Rafiqa Ameziane. The researchers received funding from the University of Michigan, the National Institutes of Health, Centre Nationale de Recherche Scientific, Association Francaise contre les Myopathies and Fondation pour la Recherche Medicale.