Subthreshold Voltage Analysis Demonstrates Neuronal Cell-Surface Sialic Acids Modulate Excitability and Network Integration

Abstract

All neurons are covered in a thick layer of carbohydrates called glycans. Glycans are modified during neurological processes and are thought to play a role in neuronal communication. We develop a voltage imaging platform for analyzing functional connectivity changes using simultaneous voltage recordings in small populations of neurons. We validate this platform using a culture model of development as well as with several pharmacological interventions. Using this platform, we show that ablation of SNA-binding glycans results in loss of functional connectivity in mouse hippocampal neurons, while ablation of MAL II binding glycans minimally perturbs functional connectivity. Altogether, our data reveal that subpopulations of glycans play different roles in maintenance of electrophysiology and provide a platform for modeling changes in functional connectivity with simultaneous voltage recordings. Scheme 1:The neuronal cell membrane is densely coated with the glycans. Sialic acid (Sia) is a negatively charged residue typically found at the non-reducing end of glycans and is a significant contributor to the surface charge density of the neuronal membrane.

Microglia Mediate Contact-Independent Neuronal Network Remodeling via Secreted Neuraminidase-3 Associated with Extracellular Vesicles

“…Our lab has recently demonstrated that desialylation of primary neurons in culture by the highly promiscuous Arthrobacter ureafaciens (Au) sialidase results in decreased cell surface sialic acids and neuronal network integration. 21 We hypothesized that Neu3 would have a similar effect. To assay this, we performed voltage imaging of primary neurons in culture using BeRST1, 38 a membrane-localized voltage-sensitive fluorophore ( Figure 3 a,b).…”

“…This technique enables simultaneous high-quality measurements of membrane potential in larger groups of neurons compared to traditional electrophysiology, enabling studies of network connectivity by comparing when multiple neurons in a given field of view fire . We have previously used this method in combination with factor analysis to quantify neuronal network connectivity as the “shared variance” of the network . In brief, the covariance in the firing activity of measured neurons may reflect variation in synaptic input (factors), while unexplained variance reflects the fraction of the neuron’s activity that arises spontaneously.…”

“…Our lab has recently demonstrated that desialylation of primary neurons in culture by the highly promiscuous Arthrobacter ureafaciens (Au) sialidase results in decreased cell surface sialic acids and neuronal network integration . We hypothesized that Neu3 would have a similar effect.…”

“…Neuroinflammation has been correlated with changes in the glycocalyxthe coating of sugars on cell surfacesof both neurons and microglia. − Sialic acids are a particular subset of bioactive sugars in the glycocalyx. They are known to modulate neuronal excitability and plasticity, , and changes in the sialylation state are associated with neuroinflammation and microglial activation. − Upon exposure to inflammatory stimuli, microglia have been observed to release sialidase activity into the surrounding media, which effects desialylation, ,, deposition of opsonizing factors, , microglial activation, , and phagocytosis of neurons. , Additionally, our lab has recently identified the sialylation state as a critical factor in maintaining neuronal excitability and network integration . Collectively, these observations point to the glycocalyx as a regulator of neuronal activity.…”

“… 17 , 23 Additionally, our lab has recently identified the sialylation state as a critical factor in maintaining neuronal excitability and network integration. 21 Collectively, these observations point to the glycocalyx as a regulator of neuronal activity.…”