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Paul G. Layer, Gesine Bachmann, Florian Frohns, Gopenath Thangaraj; DL-Alpha Aminoadipate Disrupts Cholinergic Stratification and Increases Inner Retinal Cells in 3D Chick Retinospheroids. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3962.
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© ARVO (1962-2015); The Authors (2016-present)
To elucidate in vitro developmental roles of Müller glial cells (MCs) in the histotypical organization of retinal cells using reaggregated retinospheroids as a suitable 3-dimensional model system.
Enzymatically dissociated retinal cells from embryonic day 6 chicks were cultured on a rotating shaker to produce histotypic retinal spheroids (1). To depict the role of MCs in the organization of laminar areas, we ablated or damaged them partially by applying 1 and 0.4 mM of the specific gliotoxin DL-alpha aminoadipate (AAA). Cryostat sections of fixed spheres were stained by DAPI, or were subjected to immunohistochemistry for choline acetyltransferase (ChAT), Pax6, axonin1, glutamine synthetase (GS), CERN901 and CERN906.
Our reaggregated retinal spheroids present laminar areas corresponding to an ONL (rosettes) and an INL, and neuropil-rich areas corresponding to an IPL (called IPLcircles), which are comprised mainly of processes from amacrine cells and from MCs. Pairs of starburst amacrine cells (SACs) differentiate first, their processes forming 1-2 cholinergic subbands in IPLcircles. AAA at 1 mM proved to be highly toxic to the spheres, while at 0.4 mM AAA, the shape of the spheres was highly irregular, compared with controls. MCs appeared swollen, presenting shortened processes, as revealed by GS immunostaining. IPLcircles were still formed, type II SACs migrated into the IPLcircles, but neuropil there was non-organized and cholinergic subbands were absent. Immunostaining for Pax6 and axonin1, both specific for inner retinal cells, showed that AAA treatment increased their numbers. Concomitantly, formation of photoreceptor rosettes was negligible, as revealed by CERN901 and CERN906 antibodies.
MCs and their processes - possibly in a premature state - play a major role in IPL substratification, since a subtoxic concentration of AAA disrupts it. Further, MCs affect retinal cell lineages and photoreceptor differentiation.
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