Abstract
Purpose :
Quantifying astrocyte morphology is challenging due to their heterogeneity and a lack of cell-specific membrane markers. Because of their role in neurovascular coupling and the fact that their morphology changes with health and injury, we sought to describe single cell astrocyte morphology using a novel transgenic mouse that reveals complex morphological features.
Methods :
MORF3 and GFAP-Cre 77.6 mice on a C57BL6 background were crossed together to form a novel mouse line (G-MORF). Retinas from 8 wk. naïve mice were dissected and whole-mounted for confocal imaging. Over one thousand single cells were imaged for the V5 reporter, glial fibrillary acidic protein (GFAP), and isolectin-B4. Cell images were processed for morphological characterization using both custom and open-source software.
Results :
G-MORF mice exhibit sparse labeling of retinal astrocytes (80-150 well separated cells per retina, ~2% of total population) with full membranous morphology at a quality comparable to whole-cell patch. All astrocytes observed with the V5 label are GFAP+, but GFAP represents only 19.0 ± 0.6 % of the total cell volume. These cells display recurring structural motifs and each one imaged contacts at least one vascular element. In addition to the expected protoplasmic-appearing astrocytes, V5 labeling reveals a novel retinal cell with a more fibrous appearance observed from the peripapillary region to mid-retina as well as the extreme periphery. These GFAP+ fibrous cells have soma located in the inner plexiform layer (IPL) and send thin projections throughout the IPL where they contact one another, blood vessels, Muller Glia, and smi-31-positive dendrites. Additionally, they send thick processes into the nerve fiber layer which contact canonical astrocytes.
Conclusions :
Immunolabeling retinas from the G-MORF reporter mouse demonstrates that GFAP significantly underestimates the degree of structural diversity in the astrocyte population. Furthermore, G-MORF mice allow for a detailed morphological characterization of retinal astrocytes and reveal a novel GFAP-positive cell of the IPL.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.