Analysis of Astrocyte Territory Volume and Tiling in Thick Free-Floating Tissue Sections

This protocol describes methods for sectioning, staining, and imaging free-floating tissue sections of the mouse brain, followed by a detailed description of the analysis of astrocyte territory volume and astrocyte territory overlap or tiling.

Astrocytes possess an astounding degree of morphological complexity that enables them to interact with nearly every type of cell and structure within the brain. Through these interactions, astrocytes actively regulate many critical brain functions, including synapse formation, neurotransmission, and ion homeostasis. In the rodent brain, astrocytes grow in size and complexity during the first three postnatal weeks and establish distinct, non-overlapping territories to tile the brain. This protocol provides an established method for analyzing astrocyte territory volume and astrocyte tiling using free-floating tissue sections from the mouse brain. First, this protocol describes the steps for tissue collection, cryosectioning, and immunostaining of free-floating tissue sections. Second, this protocol describes image acquisition and analysis of astrocyte territory volume and territory overlap volume, using commercially available image analysis software. Lastly, this manuscript discusses the advantages, important considerations, common pitfalls, and limitations of these methods. This protocol requires brain tissue with sparse or mosaic fluorescent labeling of astrocytes, and is designed to be used with common lab equipment, confocal microscopy, and commercially available image analysis software.

Astrocytes are elaborately branched cells that perform many important functions in the brain¹. In the mouse cortex, radial glial stem cells give rise to astrocytes during the late embryonic and early postnatal stages². During the first three postnatal weeks, astrocytes grow in size and complexity, developing thousands of fine branches that directly interact with synapses¹. Concurrently, astrocytes interact with neighboring astrocytes to establish discrete, non-overlapping territories to tile the brain³, while maintaining communication via gap junction channels

All mice were used in accordance with the Institutional Animal Care and Use Committee (IACUC) at the University of North Carolina at Chapel Hill and the Division of Comparative Medicine (IACUC protocol number 21-116.0). Mice of both sexes at postnatal day 21 (P21) were used for these experiments. CD1 mice were obtained commercially (Table of Materials), and MADM9 WT:WT and MADM9 WT:KO mice were described previously⁹.

NOTE: This protocol requires brains .......

Figure 1 presents a schematic outline of the major steps and workflow for this protocol. Figure 2 shows screenshots of key steps using the image analysis software to generate a surface, generate spots close to the surface, and generate a convex hull. Figure 3 demonstrates the application of this technique to determine astrocyte territory overlap/tiling. In Figure 4, representative results from a pr.......

This protocol describes an established method for analyzing astrocyte territory volume and astrocyte tiling in the mouse cortex, detailing all of the major steps beginning with perfusion and ending with image analysis. This protocol requires brains from mice that express fluorescent proteins in a sparse or mosaic population of astrocytes. Outside of this requirement, mice of any age may be used for this protocol, with only minor adjustments to perfusion settings and the volume of freezing media added to the embedding mol.......

Microscopy was performed at the UNC Neuroscience Microscopy Core (RRID:SCR_019060), supported in part by funding from the NIH-NINDS Neuroscience Center Support Grant P30 NS045892 and the NIH-NICHD Intellectual and Developmental Disabilities Research Center Support Grant U54 HD079124. Figure 1 was created with BioRender.com. The images and data in Figure 4 are reprinted from a previous publication⁹ with permission from the publisher.