Our nervous system is constantly at work, so that we can meaningfully engage in real-time with the world around us, life-long. It achieves this remarkable feat through the combined effort of its two major cell-types, glia and neurons, which exist in about equal numbers in our brain.

It is now clear that glia modify neuron development, connectivity, and functions life-long (Figure 1). Glia dysfunctions are implicated in a growing list of neurological disorders, of development (e.g. Autism), function (sensory deficits, cognitive impairment), and aging (e.g. Alzheimer’s dementia, Parkinson’s disease, age-related decline). So, understanding how glia impact neuron and circuit functions is critical if we are to decipher how our brains make sense of the world, store memories, and execute meaningful behaviors; or how this is altered in disease-states. If not, our knowledge of the brain will remain akin to reading only a random half of a mystery novel or seeing only half of a master’s painting!

Our philosophy is to adopt or develop any tools and techniques needed to tackle the exciting question at hand. Glia are technically challenging to study in intact animals at single-cell resolution. So, to do so rapidly and with reproducible single-cell and gene precision, we previously helped establish C. elegans as a powerful model (Singhvi et al 2016; Singhvi and Shaham 2019; Singhvi et al 2024; Purice et al 2024) (Figure 2). Leveraging the unique properties of this experimental platform for unprecedented speed and molecular precision, we seamlessly probe glia at multiple levels of inquiry:

• Molecules: gene function and networks by tools like advanced genetics, transgenics/CRISPR, -omics (single-cell/RNAseq), protein biochemistry
• Cells: organelle biology, cell polarity, shape and contacts by conventional, super-resolution and/or electron microscopy 
• Circuits and Behavior: functional imaging of curated circuits by microfluidics or whole-brain imaging of freely behaving animals; quantitative study of behaviors like sensory processing, memory, sleep, and organismal aging
• Disease Models: C. elegans and mammalian cell-culture studies

We overlay these multi-disciplinary wet-lab studies also with computational/machine-learning modeling to comprehensively study glia. Most projects deploy varying combinations of approaches. 

1. The complete molecular atlas of glia (global, -omics)

How different are glia across the nervous system, or by sex? Most glia-implicated neurological diseases (neurodevelopmental or neurodegenerative) have brain region and sex bias. However, the molecular reasons for this remain unclear.

To address this, we have built the molecular atlas of glia across the entire nervous system of C. elegans, by sex, using single-nuclear RNA sequencing (snRNAseq) technology  (Purice et al, bioRxiv, 2023) (wormglia.org) (Singhvi Lab GitHub). This is the first such validated atlas for any metazoan animal. Collaborating with the Setty Lab, we also built computational and ML analytics to define different glia properties and identities (Figure 3), and validated these in silico studies in vivo by transgenic approaches. This global “gliome” map unlocks for us, and the field, single-cell precision to ask how glia differ across the nervous system and sex (Purice et al, bioRxiv, 2023). Molecular insights from this global atlas we built now allows us unprecedented insight into how glial heterogeneity and sex-dimorphism drive nervous system function, aging, and disease.

2. Specificity in glia-neuron interactions (circuits, functional imaging)

Each glia in our brain contacts thousands of neurons, and vice versa. This raises a fundamental logic question: how does one glia talk to different neurons? This core organization of our neural circuits remains less-defined; we do not yet know if each glia talks to all associated neurons similarly or differently.

We have uncovered a mechanism by which glia regulate multisensory processing. This has implications to diseases like autism and epilepsy, which have defects in circuit dynamics. Interestingly, molecules we found are linked to these diseases, but glial roles in these diseases are poorly defined.

Briefly, we focused on a single curated C. elegans glia, the AMsh. Each AMsh glial cell interacts with twelve neurons, offering us an experimental paradigm to compare its interactions between neurons (Singhvi et al 2016; Ray et al 2021) (Figure 4). We found that the glia can differentiate neurons with sub-micrometer resolution. It does so by constraining specific regulatory cues to individual neuron-contact sites, that we termed “molecular microdomains”. We also found that this glia-neuron specificity is critical for cross-modal processing of information flow across the different sensory neurons (Ray et al 2024). Finally, these studies shed light on novel aspects of glial cell biology and glia-neuronal cilia signaling.

3. Glial pruning (single glial functions, disease models)

Multiple glial cell-types across species (astrocytes, microglia, RPE, OPC) prune, or engulf, neuron-fragments. This disease-relevant glial function is implicated in circuit refinement, learning & memory, and in diseases (autism, Alzheimer’s, Parkinson’s macular generation). However, molecular mechanisms regulating this process are not well-detailed.

Our lab discovered that C. elegans glia execute this conserved function to engulf/prune neuron-endings (Raiders et al eLife 2021; Raiders et al J Neuro 2021). Focusing on a single glia-neuron pair, we found that the glia actively and dynamically prune a neuron-ending to control its function and associated animal sensory behavior. Current work is focused on understanding how this process is regulated in neurodegenerative diseases like Parkinson’s and macular degeneration in C. elegans and mammals.

4. Epithelia-glia interactions and mechanical stress (inter-organ communication, neural aging)

How do nervous systems maintain themselves, or not, with age? In vivo, glia don’t just contact neurons but also other non-neural tissues like blood-vessel endothelia, brain meninges, sensory epithelia, and skin. We found that external tissue (epithelia) or insults (mechanical stress) affect neuronal properties with age, with glia acting as the transmitting intermediary (Martin et al 2024).  

Briefly, we found that glia coordinate their cell shape and polarity with contacting epithelia. This is important for the entire sense organ in the adult to subsequently withstand mechanical stress through animal life. We then uncovered the molecular mechanism for this tri-cellular communication, requiring the proteostatic regulator UNC-23/BAG2 Hsp-co-chaperone in epithelia and EGL-15/FGFR and SMA-1/β_H-spectrin in glia. In animals with unc-23 lesions, defective epithelia-glia coupling dramatically impacts neuron shape, function, and aging. More curiously, we found that this epithelia-glia cross talk occurs with remarkable specificity – only in some parts of the nervous system, and only in some stages of animal life (specifically juvenile-adult transition).

Our current studies are focused on further studies of neural aging, and also extending this study to pain perception and diseases like brain cancer (glioblastoma/glioma).