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

Daniel Minge,Polina Gulakova,Andrea Delekate,Gabor C Petzold,Christian Henneberger,Petr Unichenko,Alberto Pauletti,Stefanie Anders,Charlotte Behringer,Cátia Domingos,Susanne Schoch,Michel K Herde

doi:10.3389/fncel.2021.669280

Abstract

The fine processes of single astrocytes can contact many thousands of synapses whose function they can modulate through bi-directional signaling. The spatial arrangement of astrocytic processes and neuronal structures is relevant for such interactions and for the support of neuronal signaling by astrocytes. At the same time, the geometry of perisynaptic astrocyte processes is variable and dynamically regulated. Studying these fine astrocyte processes represents a technical challenge, because many of them cannot be fully resolved by diffraction-limited microscopy. Therefore, we have established two indirect parameters of astrocyte morphology, which, while not fully resolving local geometry by design, provide statistical measures of astrocyte morphology: the fraction of tissue volume that astrocytes occupy and the density of resolvable astrocytic processes. Both are straightforward to obtain using widely available microscopy techniques. We here present the approach and demonstrate its robustness across various experimental conditions using mainly two-photon excitation fluorescence microscopy in acute slices and in vivo as well as modeling. Using these indirect measures allowed us to analyze the morphology of relatively large populations of astrocytes. Doing so we captured the heterogeneity of astrocytes within and between the layers of the hippocampal CA1 region and the developmental profile of astrocyte morphology. This demonstrates that volume fraction (VF) and segment density are useful parameters for describing the structure of astrocytes. They are also suitable for online monitoring of astrocyte morphology with widely available microscopy techniques.

Highlights

Astrocytes have several distinct morphological features that set them apart from other brain cells
When diffraction-limited microscopy of the same section is simulated by convolving the image with a typical point spread function (PSF), a blurry image reminiscent of the periphery of the astrocyte in Figure 1A is obtained and astrocyte segments cannot be clearly identified any longer (Figure 1B, right panel)
If all astrocyte processes in a tissue section are filled with a dye/label, the fraction of volume they occupy is given by the cumulative intensity of that label divided by the value corresponding to the tissue section being entirely filled by astrocytic structures

Summary

Introduction

Astrocytes have several distinct morphological features that set them apart from other brain cells. Individual astrocytes and their processes occupy territories that largely do not overlap between neighbors (Bushong et al, 2002). Within those territories, their processes branch out extensively forming the so-called gliapil, into which many thousand synapses can be embedded. The processes that approach synapses are called perisynaptic astrocyte processes, which can be very thin (∼100–200 nm; Ventura and Harris, 1999; Witcher et al, 2007). Because of their proximity to synapses, they are believed to be important sites for neuron-astrocyte interactions

Methods

Results

Conclusion