We don’t have to think about breathing, but that doesn’t mean respiration is a no-brainer.
Automatic respiration is controlled by a surprisingly small area in the brain stem known as the pre-Bötzinger complex. An interdisciplinary team of researchers based at William & Mary has been drilling down on the workings of the pre-Bötzinger complex for more than a decade.
Christopher Del Negro explains that the team’s goal was to get a fuller understanding of the neurophysiology that generates respiratory rhythm. But in pursuit of rhythm, the team documented an understanding of the neural workings of motor pattern, another aspect of breathing.
They’ve published their findings in the open-access, online journal PLoS Biology in the paper “Trpm4 ion channels in pre-Bötzinger complex interneurons are essential for breathing motor pattern but not rhythm.”
The work is an important milepost in neuroscience, and Del Negro points out that it may have clinical implications for a number of respiratory ailments, notably obstructive sleep apnea.
“I rely on Margaret to help me understand some things in molecular biology and genetics,” Del Negro explained. “I just don’t have the same training. Likewise, when it comes to physiology and breathing, she would rely on me.”
The work built on a 2007 discovery of a pathway of electrically charged particles that is important to the rhythm of respiration. The next step was to map the specific pathway — called an ion channel.
“Margaret and I, working with a very talented undergraduate student, came to the conclusion that it could be a kind of a ‘Trp’ — transient receptor potential — ion channel,” he explained.
It was a big step, one that required years of wet-lab work by an interdisciplinary team whose membership included every level of William & Mary’s scientific community.
Del Negro put together a team of researchers centered around Tina Picardo, a research scientist who managed the entire project. An early member of the team was the “very talented undergraduate student” Del Negro mentioned, Erin Crowder ’07, who walked as a brand new freshman into Del Negro’s brand new lab and announced that she wanted to do research.
Access to better lab instrumentation allowed Saha and Picardo to start a series of RNA-sequencing experiments to characterize the transcriptome — essentially mapping out the structure — of all the expressed genes that make up the particular neurons that are essential to the generation of breathing.
Picardo explained that an ion channel is essentially a molecular gatekeeper.
“It’s a protein that allows passage of electrically charged ions,” she said. The passage of ions — sodium and potassium ions in this case — is vital to cellular communications.
Del Negro explained that the team had identified two likely ion channels for further study. Once the transcriptome was in hand, in vivo experiments could begin to test the role of the identified ion channels in breathing behavior.
Picardo led a group that used a reverse-genetic technique known as short-hairpin RNA to “knock down” the expression of the genes that govern the two ion channels. Her group comprised a platoon of graduate and undergraduate students performing a series of anatomy and physiology experiments.
They found something interesting from the studies of one class of ion channels, labeled Trpm4.
“Knocking down this one class of ion channels did not have the predicted effect,” Del Negro said. “It didn’t affect the breathing rhythm, but it had a dramatic impact on the amplitude or the volume of breath.”
The Trpm4 class of ion channels is the first stage in the process, pushing out the neurophysiological rhythm impulses to the circuits that move the muscles that perform breathing movements.
The discovery has relevance to obstructive sleep apnea. Del Negro went on to explain that the motor program driving amplitude of breaths also includes movements that keep the airways open during inspiration. Action of the Trpm4 class of ion channels is the first stage in the process.
“The same information that is sent to the diaphragm to inhale is also sent to the pharynx, larynx and tongue, to maintain airway diameter openness and to move the tongue out of the way, so that the airways stay clear for the incoming breath,” he said.
Picardo, Del Negro and Saha were fixtures in a team that saw a number of comings and goings of members of the 13 years of work that led to the paper.
A pyramid chart of the team would show Del Negro and Saha, tenured faculty, at the apex, working with senior scientists, post-docs, and visiting scientists. Then would come graduate students, and the base of the pyramid would be occupied by undergraduate researchers.
But Saha says such a traditional top-down view mischaracterizes the entire nature of the collaboration. For starters, Picardo — a senior scientist — is the lead author on the paper, not the tenured faculty. Del Negro and Saha both lauded Picardo for her dedication and the mentorship of her squad of student researchers.
“It’s a really layered, interesting team,” Del Negro said. Saha agreed, and went on to describe the “horizontality” of the actual working relationship.
“Christopher and I don’t interact with just the post-docs,” Saha said. “It’s a network. Faculty are involved in mentoring all levels. And the students often develop new ways of doing things in the lab. So, the students end up mentoring us sometimes.”
The team included a large number of William & Mary undergraduates, cycling in and out of the lab during their four years at William & Mary. Two undergraduates are co-authors on the paper. Kaitlyn E. Dorst ’17 is now in the Ph.D. program in Boston University. Xingru Ma is a current biology major and expects to graduate in 2020
The network is international as well as horizontal. Most of the authors on the paper have some William & Mary affiliation, past or present. Yae K. Sugimura of Jikei School of Medicine in Tokyo spent some time in the Del Negro lab as a visiting postdoctoral scholar. Likewise, Romain Guinamard of Normandie Université in Caen, France, was a visiting scholar in Del Negro’s lab in 2014-15.
“He invented the drug 9-phenanthrol that was key to this study,” Del Negro said. “That’s his drug.”
Not all co-authors had William & Mary affiliations. Other contributors were Ryoichi Teruyama from Louisiana State University and Kaiwen Kam of the Chicago Medical School at Rosalind Franklin University.
And not all contributors became co-authors on the paper, not even the student who walked into the lab and asked to be involved.“Erin Crowder is not an author, because she completed her portion in 2007, when she graduated,” Del Negro said. “But she definitely planted a seed.”