Characterization of Astrocyte Morphology and Function Using a Fast and Reliable Tissue Clearing Technique

Abstract

Astrocytic processes interact with synapses throughout the brain modulating neurotransmitter signaling and synaptic communication. During conditions such as exposure to drugs of abuse and neurological diseases, astrocytes respond by altering their morphological and functional properties. Reactive astrocyte phenotypes exhibit a bushy morphology with altered soma volume and an increased number of processes compared to resting astrocytes. The reactive astrocytic phenotype also overexpresses proteins one of which can be glial fibrillary acidic protein (GFAP). Fluorescence microscopy on thin tissue sections (<20 μm) requires reconstruction, often through multiple sections, to delineate the full astrocytic morphology. In contrast, tissue clearing methods have been developed that enable imaging of larger sections including the whole brain, providing an opportunity to see in-depth changes in single cell structure. In this article, a detailed protocol for studying astrocyte morphology using tissue clearing and subsequent imaging of whole brains as well as region-specific slices is provided. This method is ideal for understanding the effect of different physiological conditions on astrocyte morphology. A standard biochemistry laboratory has the resources to accomplish tissue clearing using this protocol and most universities have the required imaging facilities. Protocols to study brains from both genetically modified mice that contain an astrocyte-specific marker and from wild-type mice using antibody labeling steps after tissue clearing are provided. We also describe general protocols to conduct fluorescence imaging of astrocytes in cleared tissue to characterize their morphology. This protocol could be useful for researchers working in the rapidly growing field of astrocyte biology.

Astrocytic modulation of the synaptic and the extrasynaptic space

“…Dye-filling (intracellular iontophoresis of astrocytes using for instance Lucifer yellow), tissue clearing, and genetic approaches (viruses and transgenic mouse lines) combined with confocal microscopy have successfully been used to visualize and characterize astrocyte territory (~0.1 µm-50 µm) (18,19,140,373,374). In chapter 3 we aimed to examine the effects of Ezrin deletion on hippocampal astrocytes territory and how this manipulation affects astrocyte synaptic coverage.…”