Most of us think of respiration as a two-part process: We breathe in, then breathe out.
Christopher Del Negro says that’s not how it works.
“Under resting conditions, for most terrestrial mammals including humans, inspiration is the active process and expiration is passive. It’s mostly accomplished through passive recoil of the rib cage and the diaphragm,” he explained. “The only active part of breathing is the force to inspire, or breathe air in. Under standard conditions, the rhythm of breathing is the rhythm of inspiration.”
By the same token, most scientists had thought for years that the rhythm of breathing is generated by a set of specialized cells, cells with intrinsic rhythmic characteristics. The rhythm of the heart is regulated by such “pacemaker” cells and scientists believed the rhythm of breathing had a similar genesis.
Heart rhythm not a good model
This time, the scientists—or at least most of them—were wrong.
“The heart was the model for how we thought about breathing,” Del Negro said. “We disproved that theory in 2002 in a sequence of papers.”
Now, Del Negro and his colleagues have identified a promising candidate mechanism for the rhythm underlying respiration. In a paper published recently in the Proceedings of the National Academies of the Sciences of the USA, they describe how a comparatively small network of neurons in the brain stem work as a team to generate the rhythm of breathing.
Del Negro, an associate professor in the William & Mary Department of Applied Science, is co-first author on the paper, a culmination of five years of work, both experimental and theoretical. The experimental portions of the project were done in Del Negro’s lab and involved several people, including John Hayes. Hayes did his Ph.D. work in the Del Negro lab and is now a post-doc in biology. The theoretical work consisted of mathematical modeling done by Hayes and Jonathan Rubin, a William & Mary math major who graduated in 1991 and is now a faculty member in mathematics at the University of Pittsburgh.
The neural spark plug behind the rhythm that is transferred to the diaphragm and other respiratory infrastructure is an example of a CPG—a central pattern generator. CPGs are also involved in locomotion and chewing behaviors in humans and other mammals. Del Negro said the respiratory CPG is a rhythmically active network of neurons located in an area of the brainstem known as the preBötzinger Complex, abbreviated as preBötC.
The preBötC network has two interesting properties. First, there’s its size; Del Negro says the network may be as small as 200 neurons.
“We think that that’s the lowest possible estimate; it must be at least 200,” he said. “Other estimates have been around 1,000. So at this point, our estimate is somewhere between 200 and 1,000 neurons. This is not a large network. I would describe this as the worst possible estimation, except for all the others. We are one of the few groups who have actually taken the risk of saying, in print, how big we think it is.”
The second aspect is how Del Negro’s group believes the network does its job. Neurons, the nervous system’s cellular messengers, can produce “bursts” of activity through specialized proteins known as ion channels. The neurons in the preBötC act in the same way, except they need to cooperate with each other to do their job of “bursting.” He said that each neuron can’t activate its own ion channels, but needs its neighbor to fire the necessary electrochemical bursts.
“It’s as if you had a hundred dollars stapled to your back, so you can’t reach it. But you could reach mine and I could reach yours,” he explained. “These cells have sets of ion channels that they themselves can’t access without synaptic input from a neighbor. So it’s only once you connect the network that all the cells can have access to these burst-generating properties.”
Labwork combined with modeling
The findings were a result of years of experimental observation coupled with mathematical modeling. In the lab, Del Negro recorded neural activity from the preBötzinger Complex from tissue slices. After enough experimental data are collected, a mathematical model can be constructed.
“Because breathing is so important, the preBötC retains its rhythmicity, even in the very thin specimen slices, which we preserve and look at under the microscope,” he explained. “Because it retains its rhythmicity, we can go in with microelectrodes and make recordings from neurons that comprise the preBötC. If we can identify cells that are rhythmically active, we can record them, test them and find out what their properties are like. So on that basis, we can build mathematical models of how they operate.”
Reconciling the model with the observed
Jonathan Rubin, at Pitt, took the lead on building the mathematical model, to formulate a theory that would explain and predict the behavior of the data collected in Del Negro’s lab. For a time, Del Negro says, the model was “sort of working, but it really didn’t look right.” There was a calcium-activated burst of neural activity, but the model fit awkwardly with experimental observations.
It was Hayes who refined the model—and the group’s understanding of respiration—rethinking how calcium-activated bursts could start and stop. “Calcium elevates within the cell when the cells communicate with one another,” Hayes explained. “But, what happens is the cells get too excited and then consequently they stop communicating very well. So the calcium drops and the bursts end.”
The interaction of the calcium ebb and flow with the cells in the CPG not only instigate the electrochemical burst that triggers inspiration, but also sows the biochemical seeds to turn the burst off.
“That was the innovative breakthrough,” Del Negro said. “The reason we got motivated on this project is that the respiratory system can’t be attributed to canonical, classical mechanisms of rhythm generation. This system is different from all the others that we know of.”
Del Negro said this concept of collective network properties that drive rhythmic breathing may serve as a model for understanding the cellular bases of other rhythmic behaviors and functions.
“We believe that the essence of breathing comes from this network in the preBötzinger Complex, which generates the rhythm to inspire. That rhythm then is distributed to the muscle groups, like the diaphragm, that do the dominant work to breathe in. But, it’s not just about breathing,” he said. “There are so many different mammalian and human behaviors that are rhythmic: locomotion, chewing, swimming, breathing. The preBötzinger Complex provides us a model for us to study them.”