Visualizing the Brain’s Astrocytes with Diverse Chemical Scaffolds

Abstract

Astrocytes are the most abundant cells in the brain. They support neurons, adjust synaptic strength, and modulate neuronal signaling, yet the full extent of their functions is obscured by the dearth of methods for their visualization and analysis. Here, we report a chemical reporter that targets small molecules specifically to astrocytes both in vitro and in vivo. Fluorescent versions of this tag are imported through an organic cation transporter to label glia across species. The structural modularity of this approach will enable wide-ranging applications for understanding astrocyte biology.

Design Principles for Cationic, Astrocyte‐Targeted Probes

“…Primary mouse cortical astrocytes were bathed for 20 min at 37 °C in 1 μ m of the free acid compound, then the labeling was analyzed by confocal microscopy. In these experiments, the lack of a targeting moiety led to minimal labeling of the astrocytes due to its net neutral molecular charge (see Figures A and S1 for brightfield and individual channel images, quantified in Figure B); this is consistent with results we observed with pyridinium‐containing fluorophores bearing a net negative charge, like sulfo‐cy3 and fluorescein …”

“…We recently described a class of cationic dyes that take advantage of a pendant pyridinium moiety to promote astrocyte‐specific targeting via astrocyte‐resident organic cation transporters (Scheme A). This strategy distinguished itself from sulforhodamine 101 and β‐Ala‐Lys‐Nϵ‐coumarin by its broad accommodation of diverse fluorophore structures.…”

“…Rhodamines, cyanines, and BODIPYs, tagged with a pyridinium moiety, produced strong labeling of primary mouse and rat astrocytes and were excluded from neurons as well as other brain‐resident cells like microglia or oligodendrocyte precursor cells. This strategy extended into other vertebrate glia as well, marking GFAP‐positive cells in the brains of zebrafish larvae but not neurons, other glial cell types, or other organ systems in zebrafish …”

“…Using the most successful fluorescent dye scaffold, rhodamine B, we systematically altered and tested the targeting group, exploring astrocyte labeling's dependence on overall molecular charge and structure. Importantly, we did not explore anionic species because a model anionic compound did not label astrocytes in a previous report; this was expected given that the uptake mechanism relies on an organic cation transporter. The scaffolds tested can be broken down into four structural classes of targeting moiety (Table ).…”

High‐Resolution Three‐Dimensional Imaging of Individual Astrocytes Using Confocal Microscopy

“…Information regarding astrocyte volume is also lost (or highly simplified) when using Golgi stain. The same caveat applies for GFAP, the most widely used structural astrocyte marker, and for the newest smallmolecule astrocyte markers, like the cationicpyridinium family of markers (Preston et al, 2018;Preston, Cervasio, & Laughlin, 2019). The majority of structural astrocyte analyses are performed using GFAP exclusively (SheikhBahaei et al, 2018;Viola et al, 2009) or injectable intracellular fluorescent dyes (Butt & Ransom, 1989;Moye, Diaz-Castro, Gangwani, & Khakh, 2019).…”