“One of the ways I often motivate our work,” begins Dr. Aakanksha Singhvi, an Assistant Professor in the Basic Sciences Division at Fred Hutch, “is by drawing attention to the fact that everything we know about neurons—how they develop, function, interact with each other, modulate behavior, and change over time—altogether only accounts for the biology of roughly half of our brains.” If this fact is surprising to you, then welcome to the wonderful world of glia. Indeed, when it comes to neuroscience, neurons often get most of the attention even though they wouldn’t be able to perform their crucial cognitive functions without the support of glia—specialized neuronal support cells which collectively mediate the structure and function of their neuron companions. Put another way, if your brain is a movie and neurons are the cast, then glia encompass everything else—the supporting staff, set designers, scene directors, and score composers—without which we would be stuck watching an improv skit at the local community center instead of Dune 2. Laboratories around the world, including the Singhvi Lab, are now focusing full attention on glia in order to better understand the mechanisms by which they support neural function as well as how they contribute to neural dysfunction in disease.
“One feature of neuron-glia circuits that has long been known is that, in many cases, one glia makes functionally meaningful contacts with more than one neuron—sometimes as many as one thousand!” explains Dr. Sneha Ray, a previous graduate student and current post-doctoral fellow in the Singhvi Lab. “In this project, we studied a particular neuron-glia circuit in order to ask a pretty basic question that arises out of this observation: if one glia contacts multiple neurons, does it regulate them all in a similar manner? And if it doesn’t, how does it manage multiple distinct functional interactions at once?” To address this question, the team leveraged the awesome power of the nematode, Caenorhabditis elegans (C. elegans), which is not only easy to raise in large quantities, genetically tractable, and optically transparent, but has the notable advantage of having a fully mapped nervous system. This meant that Dr. Ray and colleagues could zoom in (literally and figuratively) on a single glial cell—named the amphid sheath (AMsh) glia—and study its interactions with its twelve invariant neuron partners.
To address the specificity of the interactions between this glia and its neuron partners, the team took advantage of a previous discovery made by Dr. Singhvi during her postdoc: a membrane-bound potassium chloride transporter called KCC-3 on AMsh, whose loss impaired the function of a sensory neuron called AFD—one of the twelve with which AMsh interfaces. The fact that KCC-3 loss impaired AFD but not the other eleven AMsh partners suggested that KCC-3 somehow specifically enabled this AMsh-AFD interaction—so, what could explain this? By labeling AMsh KCC-3 and performing high-resolution fluorescence microscopy, the team got their answer: KCC-3 wasn’t expressed uniformly on the membrane of AMsh, but instead was restricted to specific membrane microdomains whose locations exactly corresponded to the contact sites between AMsh and AFD. What’s more, Ray and team found that VAP-1 (a different glial protein implicated in AMsh’s communication with its partner neurons) formed a distinct microdomain on AMsh different from KCC-3, surrounding different combinations of AMsh partner neurons. Thus, it appears that AMsh forms architecturally distinct molecular interactions with its different partner neurons; by analogy, imagine that you have one friend who you call up on the phone, another friend who you call and email, and a third friend who you only see in person.
Focusing back on the AMsh-AFD interaction, the team asked a follow-up question: KCC-3 appeared to mediate this glia-neuron interaction by localizing specifically to their interface, but how was this specific KCC-3 localization established in the first place? “One tempting hypothesis that we had for a while,” noted Dr. Ray, “was that the AFD sensory neuron was responsible for somehow recruiting KCC-3 to the AMsh-AFD interface.” To test this, the team used genetic methods to completely ablate the AFD neuron and examined the resulting KCC-3 localization on AMsh. Strikingly, even when AFD was completely gone, KCC-3 microdomains on AMsh were still enriched at the (would-have-been) AMsh-AFD interface, indicating that it likely wasn’t AFD dictating KCC-3’s location.
If AFD wasn’t recruiting KCC-3 to talk to AMsh, then what was? A different experiment that the team performed offered an important clue. This experiment used mutant animals with defective neuronal primary cilia—specialized cellular structures which are essentially tiny signaling antennae that neurons use to communicate with each other in certain cases. Interestingly, messing with these cilia (on AFD but simultaneously on other AMsh neuron partners) did prevent specific KCC-3 enrichment at the AMsh-AFD interface, indicating that cilia are likely involved in KCC-3 localization but also posing a puzzle to the team. As Dr. Ray put it, “Basically, we knew that removing cilia from all of AMsh’s neuron partners abolished KCC-3 localization to the AFD neuron interface, but we also showed that entirely deleting the AFD neuron didn’t keep KCC-3 from localizing to where it would have contacted AMsh. So, we were left with the conclusion that cilia on AMsh partner neurons other than AFD were the crucial factor in localizing KCC-3.” Indeed, after extensive follow-up experiments where the team systematically impaired cilia in different combinations of the eleven other AMsh neuron partners, they found a surprising result: proper KCC-3 localization to the AFD neuron depended on functional cilia in not one, but two other AMsh partner neurons.
So, KCC-3 localizes specifically to the AMsh-AFD interface, and establishing this localization appears to be a team effort between AMsh partner neurons other than AFD—but what does this mean for the function of these neurons? To get at this question, the team used a combination of behavioral and single-neuron recording strategies to examine the function of the different AMsh partner neurons in conditions where KCC-3 was mislocalized. This led the team to perhaps the most surprising result of the entire study—while impairing KCC-3 localization predictably inhibited the function of the AFD sensory neuron, it also impaired the function of two other AMsh partners (called AWA and AWC), which don’t rely on KCC-3! Keeping with the analogy above, this would represent a situation where cutting off communication with one friend affects your relationship with a different friend (who doesn’t talk to the first friend)!
Overall, by taking a deep dive into a single neuron-glia interaction network in living, behaving animals, the team’s findings provide unprecedented insight into the ways that neurons and glia might interact in more diverse contexts. “These unexpected results generated two exciting theories,” noted Dr. Singhvi, “one, that a single glia can differentially modulate its interactions with multiple partner neurons in a manner which depends—not only on one specific glia-neuron pair—but on the large-scale properties of the neural-glial circuit. And two, that neurons which don’t directly interact can nonetheless modulate each other’s function through a shared glial partner.” Certainly, neuron-glia circuits are a good deal more complicated in our brains compared to those of C. elegans; nevertheless, many properties of these circuits are conserved in higher animals, and the Singhvi Lab is excited keep leveraging this system to reveal the subtle (and complex) regulatory logic that ultimately keeps the two halves of our brains on speaking terms.
The spotlighted work was funded by the National Institutes of Health, the Simons Foundation, the Esther A. & Joseph Klingenstein Fund, the Brain Research Foundation, philanthropic support from the Stephanus, George Brown, and Van Sloun Foundations, and the Cellular Imaging and Genomics Shared Resources of the Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium.
Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium member Dr. Aakanksha Singhvi contributed to this study.
Ray, S., Gurung, P., Manning, R. S., Kravchuk, A. A., & Singhvi, A. (2024). Neuron cilia restrain glial KCC-3 to a microdomain to regulate multisensory processing. Cell Reports, 43(3), 113844.