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Finding the Message Essential to Neuron-Muscle Signal Transmission

Clark Lindgren, professor of neuroscience, studied physics as an undergraduate at Wheaton College. One day he picked up his girlfriend’s physiology textbook. “I was just captivated by it. It was the coolest thing,” he remembers. He realized that physiology is fundamentally applying physical theories to biological problems. With his understanding of physical theories, he says, “I could remember really distinctly reading something and then knowing what was going to be on the next page.”

Many pages and some years later, Lindgren’s curiosity led him to doctoral studies in physiology at the University of Wisconsin–Madison. There he became interested in neural synapses, the spaces where neurons transmit signals to each other and to other types of cells.

Humans have trillions of synapses, and Lindgren could not possibly study them all. For its relative simplicity, he chose to study neuromuscular junctions, the synapses between neurons and muscles. Research on neuromuscular junctions can also have important implications for disease prevention and treatment. However, he says, “What I’ve learned over 30 years is that this simple synapse is not that simple. It is incredibly complex.”

Normally, at the neuromuscular junction and at other synapses, a neuron releases a chemical signal called a neurotransmitter. The neurotransmitter moves from the neuron to receptors on another cell, and the signal is transmitted. The neuron sending the signal is the presynaptic cell, and the cell receiving the signal is the postsynaptic cell.

Occasionally, some of the receptors on the postsynaptic cell are blocked. In response, the presynaptic cell releases more neurotransmitter to increase the likelihood that the signal is transmitted. This process is called presynaptic homeostatic potentiation (PHP).

Research to Understand Our World and Ourselves

We know PHP happens, but many questions about how it happens remain. In “Extracellular Protons Mediate Presynaptic Homeostatic Potentiation at the Mouse Neuromuscular Junction,” the 2021 paper Lindgren co-published in Neuroscience, he focused on the question of “How does the presynaptic cell know that the postsynaptic cell is not getting its message?” There must be a retrograde signal, a message informing the presynaptic cell that some of the receptors on the postsynaptic cell are blocked. What that retrograde signal is, we do not know. “That’s the mystery,” he says.

Knowing what the retrograde signal is would have important implications for preventing and treating diseases related to PHP, such as myasthenia gravis and amyotrophic lateral sclerosis. “The more basic knowledge we have about how we work when we’re working normally, the better we’re going to be able to deal with those limitations we encounter because of disease,” says Lindgren.

This research is also important simply because it improves our understanding of the world. Lindgren explains, “We have this world we live in. Don’t we want to know more about it?”

Students on a Quest

Yiyang Zhu ’21 and Claire Warrenfelt ’21, two students at the College who carried out Mentored Advanced Projects with Lindgren, began the search for the retrograde signal by isolating mice muscles and neurons. They blocked some receptors on the muscle and observed the muscle being activated despite that, which demonstrated that PHP was occurring normally.

Then, Zhu and Warrenfelt tried to block the retrograde signal. If they blocked it successfully, PHP would not occur normally: the neuron would not know that some of the receptors on the postsynaptic cell were blocked, it would not release more neurotransmitter, and the muscle would not be activated.

Zhu and Warrenfelt tried blocking nitric oxide production. They tried blocking endocannabinoids. But PHP still happened; neither was the retrograde signal.

Then, Warrenfelt tried blocking protons.

Breaking PHP, Finding the Retrograde Signal

Lindgren compares the scientific method to finding a contraption in the desert and discovering how it works by trying to break it. Only after breaking the contraption do you know how a part affects the function of the whole. After Warrenfelt tried blocking protons, she ran into Lindgren’s office and exclaimed, “I broke it!”

With less protons, PHP broke. The neuron did not know some of the receptors on the postsynaptic cell were blocked, it did not release more neurotransmitter, and the muscle was not activated.

Lindgren did not expect protons to be the retrograde signal. “There is no simpler chemical,” so “the idea [a proton] could be a signaling molecule is cool.”

Now that they had some evidence that protons are the retrograde signal, Lindgren’s team had to test that hypothesis in different ways. They artificially raised the concentration of protons at the neuromuscular junction and observed whether neurons released more neurotransmitter in response. They did.

Lindgren’s team then blocked the receptors that receive protons on the neurons. Unable to receive the retrograde signal, the neurons did not release more neurotransmitter.

Lastly, using a method called immunofluorescence, Jill Flannery ’21, another student at the College who carried out a Mentored Advanced Project with Lindgren, found a protein at the neuromuscular junction that transmits the retrograde signal to the neuron — more evidence in support of their hypothesis.

Dedication to the Ever-Elusive Truth

Lindgren and, he hopes, other researchers will continue to test his hypothesis. He is now working to measure the proton concentration in the synapse before and after some of the receptors on the postsynaptic cell are blocked. If the proton concentration decreases after the receptors are blocked, that would be another important piece of evidence in support of his hypothesis.

“All you can do is keep trying to disprove [your hypothesis]. … Every time you don’t disprove it, you gain more confidence in your hypothesis,” Lindgren explains. As he does more experiments and generates more confidence in his hypotheses, he gets closer and closer to the truth. However, with the possibility that one experiment will disprove a hypothesis, the truth is not a place he can ever arrive, he says. His work is a journey without an end in sight, but it’s an irresistible, necessary journey, nevertheless.

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Vishva Nalamalapu ’20 is the content specialist fellow in the Office of Communications and Marketing at 99��Ʒ��Ƶ College. She loves writing about scientific research in a way that is accessible and interesting to readers with or without science backgrounds. Her series on scientific research projects focuses on doing just that. If you are a 99��Ʒ��Ƶ College professor or student interested in having your scientific research project featured or think someone else’s project would be a good fit, please contact her.