Abstract
Purpose :
Astrocytes modulate neurovascular coupling in health and disease. In the retina, astrocytes form a dense plexus that intercalates within the nerve fiber layer (NFL), where retinal ganglion cell (RGC) axons emanate. Recently we discovered that retinal astrocytes in the mouse retina, visualized through a stochastic membrane marker, demonstrate diverse morphological features which are predictive of local neuronal and vascular structures in the inner retina. Using this technique, our goal is to characterize retinal astrocytes using single-cell morphology and physiology to determine if discrete subtypes exist.
Methods :
MORF3 and GFAP-Cre mice were crossed to stochastically label individual retinal astrocytes for full membranous morphology, followed by confocal imaging. GFAP-Cre and floxed tdTomato mice were crossed to label all astrocytes for targeting with patch clamp electrophysiology. We classified cells on the basis of retinal layer localization, radial distribution, immunohistochemistry, and electrophysiology.
Results :
Immunolabeling GFAP-Cre/MORF3 retina revealed an unexpected group of fibrous cells in the inner plexiform layer (IPL) with a distribution distinct from those in the NFL. These concentrate within 1.5mm of the optic nerve head, appearing in all sublamina of the IPL, while also distributing to the mid-retina and far periphery at a lower concentration. Despite their appearance and localization, these cells express canonical astrocyte markers (GFAP, S100β, Sox2, Sox9 and connexin-43) and lack expression of amacrine markers (syntaxin-1a, Glyt1, GAD, ChAT and TH). Both groups depolarize to both light onset and offset and have similar resting membrane potentials (NFL: -74.5 ± 0.8 mV, IPL: -75.7 ± 1.4 mV, p=0.79). Interestingly, they are functionally connected to amacrine cells and Müller glia, making contact with their processes and permitting dye spread.
Conclusions :
Our data distinguish two morphological classes of retinal astrocytes, the usual in the NFL, but the other in the IPL. Protein expression, electrophysiology and intercellular interactions suggest a similar function for both groups, each with a specialized morphology to suit their retinal layer localization.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.