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‘That was terrifying:’ Ph.D. student begins research career by unsettling ‘settled science’

  • sigh475.jpg
    Collaborators:  Authors on the neurophysiology paper are (from left) Greg Conradi Smith and Christopher Del Negro, professors in W&M’s applied science department; and Daniel Borrus and Cameron Grover, graduate students in the department.  Submitted photo
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Daniel Borrus began his Ph.D. research at William & Mary with what he thought was going to be an easy experiment, just to get his feet wet in neurophysiology research.

That “easy experiment” turned into a two-year collaborative project and a first-authorship on a paper that has challenged what had, for 20 years, been considered settled science concerning the neural control of respiration.

Borrus’s co-authors are Christopher Del Negro and Greg Conradi Smith, professors in William & Mary’s Department of Applied Science, and Cameron Grover, another graduate student in the department. Their paper, “Role of synaptic inhibition in the coupling of the respiratory rhythms that underlie eupnea and sigh behaviors,” was recently published in the Society for Neuroscience’s open-access journal, eNeuro.

“Everyone knows we all breathe,” Borrus explained — but he added that not everyone realizes that we take two different kinds of breaths. “Regular” breaths are known to scientists as eupnea. It’s the bread-and-butter automatic respiratory behavior.

“We have this other regular respiratory behavior called sighing,” he said. “It happens every few minutes, about an order of magnitude or so slower than normal breathing. It's just a large volume breath.”

Del Negro points out that this sigh should not be confused with the more familiar, emotional, woe-is-me sigh. Borrus went on to explain that the purpose of the sigh in respiration is to fully inflate the lungs, down to the alveoli, the tiny sacs in the lungs where the exchange of oxygen and carbon dioxide takes place.

“So it's a pretty important behavior physiologically,” Borrus said. “And interestingly, it actually works kind of in concert with normal breathing. So you're kind of going along, taking normal breaths. And the sigh — this large, sigh breath — usually happens relatively close after a normal breath.”

He went on to explain that the sigh is followed by a pause — known as the post-sigh apnea — before eupnea resumes. The two forms of respiration run on separate rhythms, but the rhythms are coupled.

“If you trace back the neural origins for these two breathing behaviors, you actually find that they come from the same area of the brain. It’s called the pre-Bötzinger complex, in the brainstem area — the medulla,” Borrus said. “And so not only do the rhythms originate there, but the coupling originates there as well.”

The William & Mary paper points out an important aspect of the eupnea-sigh rhythm coupling. It challenges — and is likely to completely upset — conventional understanding of how eupnea and sighing are coupled.

“We basically told them everything they believed for the past 20 years was wrong,” Del Negro said.

What previous researchers got wrong was the role of synaptic inhibition in the coupling of the eupnea and sigh rhythms. Think of inhibition as a kind of neural braking system. Inhibition is the opposite of excitation, and like a car, the brain needs braking as well as acceleration to do its job.

“So this previous group, about 20 years ago, said that if you blocked inhibition in the pre-Bötzinger complex, you could uncouple the sigh and eupnea rhythms,” Borrus said. “You could get a free-running sigh rhythm.”

Early in his work, Borrus set out to duplicate the 20-year-old inhibition-blocking experiments. He wasn’t getting the same results, and it made him nervous.

“That was terrifying for sure. I mean, it was my first time doing electrophysiology experiments. The date on the paper is 2000, and it’s from big, big names in the field,” he said. “I had no reason to doubt those findings. I thought for sure that something I was doing was wrong: Honestly, I didn't believe it. But I had Christopher to keep me going.”

Borrus kept working, encouraged by both Del Negro and Conradi Smith. He said his confidence grew as the work progressed, especially after he began using a different drug to block inhibition.

“The results only got more clear,” he said. “That’s when I became really convinced that we were on the right path. But for a while there it was a little scary. I was sure that my first six months of Ph.D. research were going to be refuted because we had screwed something up.”

Their paper naturally met some resistance in the review process, as would be expected of any challenge to the accepted understanding of a physiological process. But the William & Mary collaborators answered all queries and made a convincing case that synaptic inhibition does not couple the rhythms driving eupnea and sigh behavior.

As with scientific findings, their results are provisional, and subject to duplication, revision and testing. And Borrus et al do not offer an alternative explanation for the neural origins of the sigh rhythm. Yet.

“That question is definitely still up in the air,” Borrus said. “And it’s something we’re tackling.”

The paper made a number of other contributions to the understanding of the eupnea-sigh connection. Borrus noted that his work challenged another long-standing notion: that the coupling is such that the sigh always happens quickly after a eupnea breath.

“We found that there is actually a bit of variability in the timing between the sigh and the preceding eupnea event,” he noted.

Conradi Smith pointed out that the findings regarding the variability of timing raise an interesting question.

“It sort of suggests that the two rhythms are less coupled than people originally thought,” Smith said. “But it's still the same neural network. The same collection of cells is producing the two different rhythms. The pre-Bötzinger complex is producing one regular rhythm and then this, this sigh overtone rhythm. And so how does one neural network produce those two different types of oscillations? I think that's an important theoretical question.”

As with scientific findings, their results are provisional, and subject to duplication, revision and testing. And Borrus et al do not offer an alternative explanation for the neural origins of the sigh rhythm. Yet.

“That question is definitely still up in the air,” Borrus said. “And it’s something we’re tackling.”

The pre-Bötzinger complex, seat of neural control of respiration, is surprisingly small. Del Negro said there are fewer than 1,000 cells involved. “For brain systems, less than 1,000 cells is minuscule,” he added.

The paper is a continuation of a larger ongoing exploration of neural respiration being conducted by Conradi Smith, a computational biologist, and Del Negro, an experimental neuroscientist. Their project, Collaborative Research in Computational Neuroscience, is jointly funded by both the National Institutes of Health and the National Science Foundation.

“The whole nature of these grants is that an experimentalist teams up with a mathematical modeler or a computationalist and then they wed their skills together with the aim of solving a problem that neither branch of technical expertise could solve on its own,” Del Negro explained.

Those problems have real life — even life-and-death — relevance. Del Negro described the group’s paper as an “obligatory first step” in a research progression that will lead to computational modeling by Smith and Borrus and eventually to clinical applications.

“The biophysical basis of the sigh rhythm is important to understand,” Del Negro said. “For instance, physicians have to program in sighing when they program a ventilator.”

Borrus entered the field through the computational modeling side. He is a 2017 graduate of William & Mary’s neuroscience program, which has a required course called Cellular Biophysics and Modeling. The course design arose from Smith’s CAREER grant, supporting for education and research from the National Science Foundation.

“It’s essentially an introduction to electrophysiology,” Smith explained. “But it takes the perspective that you should understand how to mathematically model these things, to best explain how everything works. And that’s where Dan, I believe, first learned how to do this type of modeling when he was an undergraduate research student.”

Borrus said he took the course as a sophomore, only because another course was full.

“It was a great accidental move on my part,” he said. “It was the first time I had seen biology wedded with math. And so, yeah, I was hooked. I loved the course, consumed all the content.

“And then at the end of the semester, I went up to Greg and said, ‘Do you actually get to do this? Like, for your job? Can I do research with you?’”