Abstract
Purpose :
Primary cell culture of dissociated adult mouse retinas provides specific advantages over stem cell- or neonatal-derived systems in that they enable incorporation of factors such as age-dependent pathology or genetic manipulation. We previously demonstrated that in primary retinal co-cultures, some retinal ganglion cells (RGCs) spontaneously generate various types of neurite extensions, however, whether these neurites represent axonal or dendritic processes was not determined.
Methods :
We dissociated adult Vglut2-cre; Ai9 retinas and collected all cells using our published protocol (Park, et al 2020 ). To enrich for RGCs, we performed negative immunopanning against rods using an antibody to CD73. We cultured the remaining heterogeneous population of cells for seven days under standard culture conditions in 384 well plates containing Neurobasal media augmented with various growth factors and supplements. The media was changed every other day. After seven days, immunostaining for neurite components and nuclei was performed.
Results :
Rod-depletion removed about 50% of total cells with no effect on RGC numbers. At one week, about 60% of the RGCs at plating survived and many grew neurites. Of these, long neurite extensions (presumed axons) expressed growth-associated protein 43 (GAP43), a neuronal marker for axonal growth and synaptic plasticity. Short neurite extensions (presumed dendrites) expressed microtubule-associated protein 2 (MAP2), a neuronal marker for dendrites. We will continue to test additional neuronal markers to define the cellular features of RGCs in culture.
Conclusions :
Our work demonstrates that adult mouse RGCs survive in mixed, rod-depleted retinal cultures and regenerate neurites robustly. Neurites from cultured RGCs express classic axonal and dendritic markers. This system can potentially be used to determine pro-axonal and pro-dendritic factors and better understand the impact of in vivo manipulations prior to dissociation.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.