Super‐resolution shadow imaging reveals local remodeling of astrocytic microstructures and brain extracellular space after osmotic challenge

Abstract

The extracellular space (ECS) plays a central role in brain physiology, shaping the time course and spread of neurochemicals, ions, and nutrients that ensure proper brain homeostasis and neuronal communication. Astrocytes are the most abundant type of glia cell in the brain, whose processes densely infiltrate the brain's parenchyma. As astrocytes are highly sensitive to changes in osmotic pressure, they are capable of exerting a potent physiological influence on the ECS. However, little is known about the spatial distribution and temporal dynamics of the ECS that surrounds astrocytes, owing mostly to a lack of appropriate techniques to visualize the ECS in live brain tissue. Mitigating this technical limitation, we applied the recent SUper‐resolution SHadow Imaging technique (SUSHI) to astrocyte‐labeled organotypic hippocampal brain slices, which allowed us to concurrently image the complex morphology of astrocytes and the ECS with unprecedented spatial resolution in a live experimental setting. Focusing on ring‐like astrocytic microstructures in the spongiform domain, we found them to enclose sizable pools of interstitial fluid and cellular structures like dendritic spines. Upon experimental osmotic challenge, these microstructures remodeled and swelled up at the expense of the pools, effectively increasing the physical interface between astrocytic and cellular structures. Our study reveals novel facets of the dynamic microanatomical relationships between astrocytes, neuropil, and the ECS in living brain tissue, which could be of functional relevance for neuron–glia communication in a variety of (patho)physiological settings, for example, LTP induction, epileptic seizures or acute ischemic stroke, where osmotic disturbances are known to occur.

Heterogeneity and Development of Fine Astrocyte Morphology Captured by Diffraction-Limited Microscopy

“…This approach should also be useful in other preparations, in fixed tissue and for microscopy methods such as confocal microscopy, if the aforementioned conditions are met. Despite these advantages, our approach cannot replace the methods that accurately quantify the geometry and branching patterns of large astrocyte processes, see for instance Tavares et al (2017), or that resolve the fine geometric details of astrocyte structure such as electron (Ventura and Harris, 1999), STED (Arizono et al, 2020(Arizono et al, , 2021 or expansion microscopy (Herde et al, 2020), among other techniques as recently reviewed (Heller and Rusakov, 2017). However, the approach can be used to efficiently screen for biologically relevant scenarios by comparing large populations of astrocytes in different conditions or by online monitoring of astrocyte morphology changes, their triggers and functional consequences.…”

“…Second, it is incompatible with real-time monitoring of astrocytic morphology changes. These obstacles can be overcome by stimulated-emission depletion (STED) imaging, which has been successfully used to visualize astrocytes at the necessary resolution in living brain tissue (Tønnesen et al, 2018;Arizono et al, 2020Arizono et al, , 2021Henneberger et al, 2020). However, threedimensional STED imaging typically requires high illumination powers, which can limit the number of image acquisitions, and is at least currently not widely available.…”

Super-resolution imaging to reveal the nanostructure of tripartite synapses

“…For example, Verkman and colleagues investigated the clustering behaviour of aquaporin 4 and of the inwardly rectifying potassium channel Kir4.1 using SMLM techniques in cultured cells and in brain sections [98][99][100]. Moreover, thin astroglial processes have been illuminated by expressing genetically-encoded fluorescent proteins and by labelling glutamine synthetase and S100β in cultured cells and brain sections [34,[101][102][103][104][105].…”

“…The researchers saw that the aforementioned ring-like structures that are formed by the reticular network of astrocytic processes, encircle spines and areas of interstitial fluid (Figure 3, D-G) [104]. After osmotic STED image of astrocytes (gold) and inverted signal of extracellular space (ECS); highlighting astrocytic processes penetrating the neuropil; modified from [104] with permission. (E) STED images of positively labelled astrocyte (gold) and stained ECS (grey); modified from [104] with permission.…”

“…Downloaded from http://portlandpress.com/neuronalsignal/article-pdf/doi/10.1042/NS20210003/921250/ns-2021-0003c.pdf by guest on 11 October 2021 12 challenge, the astrocyte structures swell, increasing the interface between astrocytic and other cellular structures but decreasing the size of interstitial pools[104].…”