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C. Guo, S. L. Stella, Jr., N. C. Brecha; Evidence for Vesicle Endo-/Exocytosis at the Horizontal Cell Processes Using a Monolayer Retinal Sandwich Culture Preparation. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3284.
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© ARVO (1962-2015); The Authors (2016-present)
The mechanism of transmitter release from horizontal cells has remained unclear. Some evidence favor a vesicular release pathway in the mammalian horizontal cells, however, no evidence have shown vesicle endo-/exocytosis activity at the terminals of the horizontal cell processes. The aim of our study is to develop a retinal monolayer culture system in which differentiated horizontal cells can be identified and used for functional analysis.
Retinas from guinea pig pups from postnatal day 5 (P5) to P9 were digested by typsin and triturated into single cell suspension. Cells were plated into 12-well plates pre-placed with Poly-L-Lysine coated coverslips and incubated in DMEM medium containing 10% FBS for 1-3 hours. Coverslips were then transferred upside down into a new culture plate so that the cells are "sandwiched" in between the coverslip and the plate. Cells were incubated in defined medium and allowed for differentiation for 5-7 days. Horizontal cells were identified in the culture by morphology and characteristic markers including calbindin, GABA, GAD65, VGAT, GAT-1, SV2A and syntaxin-4. Fixable fluorescent styryl dyes with different absorbance/emission spectrums (AM4-65 and AM1-44) were used for labeling endo-/exocytosed vesicles at the horizontal cell processes by 55mM high K+ stimulation.
Different types of retinal neurons were observed in the retinal culture. A-type horizontal cells were identified by morphological and molecular characteristics. These cells retain their in-vivo like morphology and molecular features including the expression of calbindin, GABA, GAD65, VGAT, SV2A and syntaxin-4, and the lack of GAT-1. The cultured cells can survive for up to two months. Activity-dependent dye loading indicated vesicle endo-/exocytosis at processes of the cultured horizontal cell. Subsequential uptake of AM4-65 and AM1-44 into the same set of vesicles suggested vesicle recycling activity at the horizontal cell processes. In addition, some large cellular acidic organelles (mostly late endosomes and lysosomes) also internalized the dyes as confirmed by co-labeling of LysoTracker Green.
This study developed a monolayer retinal sandwich culture system in which horizontal cells can be identified, and provided direct evidence for vesicle endo-/exocytosis at the horizontal cell processes, supporting the vesicular mechanism for transmitter release.
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