Figuring out how our brains keep from ‘getting the wires crossed’

From the Moens Lab, Basic Sciences Division

It’s a familiar feeling—you get to work in the morning, sit down at your desk (or bench), and before you know it, the clock reads 5 P.M. and it’s dark outside. You wonder where the time went and marvel at the feeling of what can only be described as ‘autopilot.’ As it turns out, the autopilot metaphor is not terribly inaccurate—in order to function effectively, our bodies constantly survey our internal and external states and accordingly alter our physiology to accommodate any changes; often, this happens without our conscious awareness. On an anatomical level, this feat is accomplished by neural circuits consisting of sensory neurons that—as their name suggests—carry information obtained from the world to the brain, which processes these signals and conveys a response via motor neurons to muscles that move to respond appropriately to the stimulus.

The Moens Lab at Fred Hutch Cancer Center studies neural development—how neural circuits like the ones described above are formed, refined, and maintained. Dr. Takuya Kaneko, a postdoctoral fellow in the lab, is particularly interested in a specific neural circuit featuring the vagus nerve (also called the tenth cranial nerve). You may be unfamiliar with the vagus nerve, but your body knows it well: if you’ve ever felt your heart rate slow after a long, deep breath, or accidentally gagged on your toothbrush, you have your vagus nerve to thank. Among neural circuits, the vagal circuit is somewhat of an overachiever—it receives sensory input from throughout your body and subsequently dispatches signals along vagal motor neurons to diverse body systems (your heart, throat muscles, digestive enzyme-secreting cells, and even the muscles which keep food moving through your gut, among others). It juggles these diverse signals all while maintaining an exquisite coherency among them, and it’s this coherency that Dr. Kaneko is fascinated by. “We know in several model organisms that the different vagal motor neurons are spatially organized in the brainstem according to their function,” Kaneko begins, “but this spatial organization is imperfect, and different groups of motor neurons projecting to different organs frequently overlap and intermingle. How the brain is able to maintain coherency between these different neural circuits despite their close proximity is a fundamental question in neuroscience.”

To study this process, Kaneko needed a model system. For this, he turned to the humble zebrafish, the organism of choice for the Moens Lab and a powerhouse in neuroscience research. As it turns out, zebrafish have similar vagal circuit architecture to humans, and their larval optical transparency and ease of genetic manipulation mean that Kaneko can ask and answer detailed mechanistic questions about this circuit. To narrow his approach further, Kaneko mostly focused on the pharyngeal arches, a system of sequential organs that fish use for swallowing and gill manipulation, the last four of which are innervated by four distinct groups of vagal motor neurons. The Moens lab and others had previously shown that these four motor neuron groups show a loose spatial organization in the fish brainstem based on the arch to which they project—that is, motor neurons projecting to the most anterior (closest to the front) arch live in the more anterior part of the motor nucleus, while those project to more posterior arches live in the posterior part. To start, Kaneko used sophisticated optical imaging tools in larval zebrafish to establish that—while this loose spatial organization was present, the different groups of motor neurons in fact showed significant overlap, especially in the case of the first three pharyngeal arches.

a microscopy image depicting a cluster of neurons stained different shades of the rainbow
Vagal motor neuron bodies in a larval zebrafish brain stained according to which pharyngeal arch they project to. Motor neurons projecting to the first three arches are found to the anterior (leftmost) side of the image. Image courtesy of Dr. Takuya Kaneko

So, vagus motor neuron groups projecting to discrete pharyngeal arches are spatially overlapping—but what does this mean for the functional connectivity of this neural circuit? To test this, Kaneko needed a way to precisely stimulate one pharyngeal arch and measure the responses of the various vagus motor neuron populations. Zebrafish to the rescue! Kaneko and colleagues placed larval zebrafish into media containing a photo-activatable ligand for TrpA1 channels, which normally sense noxious stimuli. By delivering a precise pulse of laser light, the team was able to mimic a noxious stimulus in a specific pharyngeal arch—combining this technique with neuronal activity imaging using a fluorescent calcium reporter allowed the team to stress-test this neural circuit in living larval zebrafish. As Kaneko puts it, “we found that, despite their close spatial proximity, specific populations of vagal motor neurons in the brainstem responded to stimulation of specific pharyngeal arches.” The team referred to this feature of vagal motor neurons as a functional topography.

The fact that groups of intermingled vagal motor neurons maintained stereotyped, coherent responses to distinct stimuli suggested that this functional topography was independent of neuron location; however, the ultimate test of this hypothesis would necessitate altering the spatial orientation of these neurons and seeing whether this would break their functional associations. In what was the key experiment of the study, Kaneko leveraged the awesome power of the zebrafish model system to do just that: he transplanted vagal motor neurons which usually respond to stimulus at the third pharyngeal arch from a donor fish into a recipient fish, with one twist—he misplaced the neurons from their normal anterior position to the middle of the motor nucleus. Allowing the transplanted neurons to regrow their connections and performing stimulus-response measurements revealed that—even though these vagus motor neurons inhabited different cellular locales—they retained their functional association with the third pharyngeal arch!

Follow-up experiments revealed clues as to how these misplaced neurons maintained their functional identities—a process which likely involves the neurons remodeling their dendrite architectures and requires motor neuron output. Overall, this study establishes a new system in which to study the development of motor neuron wiring circuits and uses the system to highlight an astonishing position-independent plasticity in the wiring of vagus motor neurons. “Part of what made these results really striking,” Kaneko notes, “was that they were in direct opposition for that of motor neurons in our spinal cord: our spinal motor neurons form distinct functional groups like vagus motor neurons, but these groups are positionally defined; switching their location switches their functional identities and causes motor defects. Thus, we define a new paradigm for motor neuron wiring, and we’re excited to keep studying this system to better understand the mechanisms by which these circuits establish, and whether this paradigm applies to other neural circuits as well.”

The spotlighted research was funded by the National Institutes of Health, an American Heart Association postdoctoral fellowship, and a Japan Society for the Promotion of Science Overseas Research Fellowship.

Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium member Dr. Cecilia Moens contributed to this study.

Kaneko, T., Boulanger-Weill, J., Isabella, A. J., & Moens, C. B. (2023). Position-independent refinement of vagus motor neuron wiring [Preprint]. Neuroscience.