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         This summer I held a research fellowship position in the Cell Biology and Anatomy department of Rosalind Franklin University of Medicine and Science (RFUMS). The research was conducted under the auspices of Dr. Kaiwen Kam, a researcher in the field of circuit neuroscience that has spent years unravelling the mysteries of the pre-Bötzinger Complex (preBötC), a medullary neuronal circuit. The preBötC is the hypothesized center for respiratory rhythmogenesis in rodents, converting tonic excitatory drive into rhythmic inspiratory bursting. Essentially, the preBötC generates rhythms that control breathing. Now this information alone is enough (at least for me) to explain why researchers would study the preBötC. Such a vital neural circuit deserves the attention of scientists who are eager to understand the neural mechanisms underlying breathing, as well as clinical researchers who are looking to improve the quality of human pulmonary health. However, to me the preBötC’s importance extends well beyond its function. What I want to do here is highlight how scientists discovered the preBötC’s function, discuss my own work with the circuit, and show what research regarding the preBötC can (and has) taught us about neuroscience as a whole.

            Circuit neuroscience is a cutting-edge and relatively new division of the neuroscience field. It involves studying both the structure and function of neuronal circuitry, and uses anatomical, physiological, and computational methods to elucidate this structure and function (Yuste, 2008). Neural circuits themselves can be defined as the set of neurons involved in a particular behavior, a necessarily broad concept to understand the sheer complexity of the mammalian nervous system. Since mammalian neuronal circuits have large numbers of neurons and synaptic connections, it is difficult to understand the general rules of neural circuits when that circuitry involves complex behavior. To circumvent this complication, a coterie of neuroscientists turn to the relatively simple neurocircuitry of mammalian breathing.

            Breathing is one of the most essential and simple mammalian behaviors. It is necessary for the maintenance of O₂ and CO₂ homeostasis in the bloodstream, which, in turn, allows for the survival of the organism. Breathing at rest occurs rhythmically, with cycles of inhalation and exhalation following a fairly constant frequency. Inhalation is caused by the contraction of the diaphragm (and the resulting expansion of the lungs into the thoracic cavity), whereas exhalation (at rest) is caused by the elastic recoil of the lung tissue and diaphragmic relaxation. Although exhalation can occur passively, inhalation requires active motoneuron input into the diaphragm. For many years, the source of this inspiratory input was unknown, understandably frustrating quite a few scientists. Here was one of the most simple, elegant, and essential mammalian behaviors, yet the source of this behavior was unknown.

One of the earliest and most prominent neuroscientists, Marie-Jean-Pierre Flourens (1794-1867) set out to investigate the localization of particular behaviors to specific brain regions (Pearce, 2009). Flourens’ work was centered upon another famous scientist, Franz Joseph Gall (1758-1828). Gall asserted that specific regions of the brain were responsible for particular functions, thereby developing the discipline of phrenology based on this idea. Flourens was skeptical of Gall’s work and investigated the function of the brain by ablating (removing) portions of the brain and then studying the impact on behavior. For example, ablation of the cerebellum resulted in a loss of coordinated motor activity, and ablation of the posterior medulla impacted respiration. Despite these observations, Flourens rejected Gall’s ideas of cerebral localization, stating that “a large section of the cerebral lobes can be removed without loss of function. As more is removed, all functions weaken and gradually disappear. Thus, the cerebral lobes operate in unison for the full exercise of their functions…” (Changeux, 1985). Essentially, Flourens is saying that the brain is more than just a sum of its parts (the cerebral lobes), and that behavior is an emergent property of the interaction among various brain regions.

           But I digress. Flourens pointed out that the medulla was important for breathing; in fact, he used the term noeud vital to refer to the mysterious medullary source of respiration (Smith et al., 1991). It wasn’t until the late 20th century that neuroscientists identified the exact brain region responsible for respiration. In the laboratory of Dr. Jack L. Feldman, Smith et al. conducted research on neonatal (newborn) rats. They performed in vitro electrophysiological recordings on the XIIth (hypoglossal) cranial nerve while taking serial microsections of the medulla. Essentially, they were recording the output of the noeud vital while simultaneously removing nearby brain regions. When the researchers observed a loss of respiratory rhythm in the XIIth nerve, they knew they had removed part of the brain essential for breathing. Next, the researchers took a slice of the medulla that could generate respiratory rhythms (and thus would contain the noeud vital). Electrophysiological recordings from the slice revealed that the preBötC generates these respiratory rhythms, a function upregulated by extracellular K+. These results jumpstarted scientific inquiry into the inner workings of the preBötC that continues to this day.

           In 1999, Feldman’s lab published another article detailing several molecules that, when administrated to the preBötC, modulate respiratory rhythmogenesis (Gray et al., 1999). Of particular interest was [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin acetate (DAMGO), a μ-opioid receptor (μOR) agonist. When applied to in vitro medullary slices, DAMGO decreased the frequency of preBötC inspiratory bursts. This is important because opioids (the activity of which DAMGO mimics in the preBötC) are known to decrease the frequency of breathing in humans, a side-effect with high clinical significance. This work by Feldman’s lab may have pinned down the mechanism by which opioids slow breathing in humans (although it is unclear wheather a parallel to the preBötC actually exists in humans), and (at least for me) highlights just how important understanding the preBötC is to medicine. In the same paper, the results of immunohistochemical staining on and near the preBötC were published. As it turns out, the preBötC is anatomically distinct from surrounding brain regions, and has differing levels of several molecules (including μOR) compared to surrounding regions. 

           My work with the preBötC involved conducting electrophysiological recordings on in vitro preBötC sandwich slices in the hopes of testing the effects of phenytoin, an anti-epileptic drug, on rhythmogenesis. Phenytoin selectively binds to and inhibits the voltage-gated Nav1.1 and Nav1.5 sodium ion channels, resulting in decreased neuronal firing rates (Yaari et al., 1986). I hypothesized that the addition of phenytoin to preBötC sandwich slices would decrease the frequency of preBötC bursts. This is because phenytoin’s reduction of neuronal firing rates would likely reduce the cumulative firing rate of this region. Although my experiments (and those previously conducted) did show a decreased rate (and occasional ablation) of preBötC bursting upon application of phenytoin, statistical analysis revealed that the impact of phenytoin on steady-state burst frequency was not significant. In the future, more research will have to be conducted to understand exactly how phenytoin impacts the preBötC neural network, and why it ablates preBötC bursting.

           Overall, Feldman’s research (and the work of other labs, including Dr. Kam’s) has taught us much about the inner workings of the preBötC. Even this relatively simple neural circuit is quite complex, and there are still many avenues of research being conducted to understand the preBötC. For example, Dr. Kam’s lab uses the anatomical, physiological, and computational facets of circuit neuroscience to investigate the preBötC from many angles. Anatomically, it is important to understand what types of neurons reside in and around the preBötC and how these neurons connect to and impact the preBötC. Physiologically, the inner workings of the preBötC can be investigated and better understood through pharmacological manipulation. And computationally, the preBötC can be modeled in silico to highlight what we know (and don’t know) about the circuit.

           In conclusion, this research isn’t just oriented towards breathing, rather, it is used to understand how the entire brain works. My own research highlights how we still have much to learn about pharmacological actions at the circuit level, and how these interactions inform us about the circuit itself. It is my opinion that to understand how the brain processes information (which is, arguably, the goal of the field of neuroscience) we must first understand the simplest systems of the brain (such as the preBötC). Then, we can work our way up to more complex systems (such as those governing emotion) building off of what we know about the simpler ones. This will require scientists to think of the brain and behavior as an emergent property of many neural circuits (instead of a set of distinct regions), an idea which Marie-Jean-Pierre Flourens hinted at centuries ago.

 References cited:

Changeux Jean-Pierre: Neuronal Man. The Biology of Mind, transl by Garey L. Princeton,

Princeton University Press, 1985.

Gray, P. A., Rekling, J. C., Bocchiaro, C. M., & Feldman, J. L. (1999). Modulation of respiratory

frequency by peptidergic input to rhythmogenic neurons in the preBötzinger

complex. Science, 286(5444), 1566-1568.

Pattinson, K. T. S. (2008). Opioids and the control of respiration. British journal of

anaesthesia, 100(6), 747-758.

Pearce, J. M. S. (2009). Marie-Jean-Pierre Flourens (1794–1867) and cortical

localization. European neurology, 61(5), 311-314.

Smith, J. C., Ellenberger, H. H., Ballanyi, K., Richter, D. W., & Feldman, J. L. (1991). Pre-Botzinger

complex: a brainstem region that may generate respiratory rhythm in

mammals. Science, 254(5032), 726-729.

Yaari, Y., Selzer, M. E., & Pincus, J. H. (1986). Phenytoin: mechanisms of its anticonvulsant

action. Annals of Neurology: Official Journal of the American Neurological Association and

the Child Neurology Society, 20(2), 171-184.

Yuste, R. (2008). Circuit neuroscience: the road ahead. Frontiers in neuroscience, 2, 17.