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IPL Sublamination in Chicken Retinal Spheroids Is Initiated via Müller Cells and Cholinergic Differentiation, and Is Disrupted by NMDA Signaling.
Invest Ophthalmol Vis Sci 2019; 60(14):4759-4773IO

Abstract

Purpose

Reaggregates from E6 embryonic chicken retina exhibit areas corresponding to an inner plexiform layer (IPL), which presents an ideal in vitro model to test conditions and constraints of cholinergic and glutamatergic network formation, providing a basis for retinal tissue engineering. Here, we show that ipl formation is regulated by cholinergic starburst amacrine cells (SACs), a glial scaffold and by L-glutamate.

Methods

Rosetted spheroids were cultured in absence or presence of 0.2 to 0.4 mM L-glutamate and analyzed by immuno- and enzyme histochemistry, proliferation, and apoptosis assays.

Results

After 2 days in vitro (div), ipl formation was announced by acetylcholinesterase+ (AChE) and choline acetyltransferase+ (ChAT) cells. Individual vimentin+ or transitin+ Müller glial cell precursors (MCPs) in ipl centers coexpressed ChAT. Comparable to in vivo, pairwise arranged ChAT+ SACs formed two laminar subbands. Projections of calretinin+ amacrine cells (ACs) into ipl associated with MCP processes. In L-glutamate-, or NMDA-treated spheroids ipls were disrupted, including loss of SACs and MCs; coincubation with NMDA receptor inhibitor MK-801 prevented these effects. Also, many Pax6+ cells, comprising most ACs, were lost, while rho4D2+ rod photoreceptors were increased. Cell proliferation was slightly increased, while apoptosis remained unaffected.

Conclusions

This demonstrated: (1) a far-advanced differentiation of an IPL in retinal spheroids, as never described before; (2) ipl sublamination was initiated by cholinergic precursor cells, which-functioning as "ipl founder cells"-(3) gave rise to neurons and glial cells; (4) these SACs and MCPs together organized ipl formation; and (5) this process was counteracted by NMDA-dependent glutamate actions.

Authors+Show Affiliations

Developmental Biology and Neurogenetics, Technische Universität Darmstadt, Darmstadt, Germany.Developmental Biology and Neurogenetics, Technische Universität Darmstadt, Darmstadt, Germany. Radiation Biology, Technische Universität Darmstadt, Darmstadt, Germany.Developmental Biology and Neurogenetics, Technische Universität Darmstadt, Darmstadt, Germany. Division of Biotechnology, Faculty of Life Sciences, JSS Academy of Higher Education & Research, Mysuru, India.Developmental Biology and Neurogenetics, Technische Universität Darmstadt, Darmstadt, Germany.Developmental Biology and Neurogenetics, Technische Universität Darmstadt, Darmstadt, Germany.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31738824

Citation

Bachmann, Gesine, et al. "IPL Sublamination in Chicken Retinal Spheroids Is Initiated Via Müller Cells and Cholinergic Differentiation, and Is Disrupted By NMDA Signaling." Investigative Ophthalmology & Visual Science, vol. 60, no. 14, 2019, pp. 4759-4773.
Bachmann G, Frohns F, Thangaraj G, et al. IPL Sublamination in Chicken Retinal Spheroids Is Initiated via Müller Cells and Cholinergic Differentiation, and Is Disrupted by NMDA Signaling. Invest Ophthalmol Vis Sci. 2019;60(14):4759-4773.
Bachmann, G., Frohns, F., Thangaraj, G., Bausch, A., & Layer, P. G. (2019). IPL Sublamination in Chicken Retinal Spheroids Is Initiated via Müller Cells and Cholinergic Differentiation, and Is Disrupted by NMDA Signaling. Investigative Ophthalmology & Visual Science, 60(14), pp. 4759-4773. doi:10.1167/iovs.18-24952.
Bachmann G, et al. IPL Sublamination in Chicken Retinal Spheroids Is Initiated Via Müller Cells and Cholinergic Differentiation, and Is Disrupted By NMDA Signaling. Invest Ophthalmol Vis Sci. 2019 Nov 1;60(14):4759-4773. PubMed PMID: 31738824.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - IPL Sublamination in Chicken Retinal Spheroids Is Initiated via Müller Cells and Cholinergic Differentiation, and Is Disrupted by NMDA Signaling. AU - Bachmann,Gesine, AU - Frohns,Florian, AU - Thangaraj,Gopenath, AU - Bausch,Alexander, AU - Layer,Paul G, PY - 2019/11/19/entrez PY - 2019/11/19/pubmed PY - 2019/11/19/medline SP - 4759 EP - 4773 JF - Investigative ophthalmology & visual science JO - Invest. Ophthalmol. Vis. Sci. VL - 60 IS - 14 N2 - Purpose: Reaggregates from E6 embryonic chicken retina exhibit areas corresponding to an inner plexiform layer (IPL), which presents an ideal in vitro model to test conditions and constraints of cholinergic and glutamatergic network formation, providing a basis for retinal tissue engineering. Here, we show that ipl formation is regulated by cholinergic starburst amacrine cells (SACs), a glial scaffold and by L-glutamate. Methods: Rosetted spheroids were cultured in absence or presence of 0.2 to 0.4 mM L-glutamate and analyzed by immuno- and enzyme histochemistry, proliferation, and apoptosis assays. Results: After 2 days in vitro (div), ipl formation was announced by acetylcholinesterase+ (AChE) and choline acetyltransferase+ (ChAT) cells. Individual vimentin+ or transitin+ Müller glial cell precursors (MCPs) in ipl centers coexpressed ChAT. Comparable to in vivo, pairwise arranged ChAT+ SACs formed two laminar subbands. Projections of calretinin+ amacrine cells (ACs) into ipl associated with MCP processes. In L-glutamate-, or NMDA-treated spheroids ipls were disrupted, including loss of SACs and MCs; coincubation with NMDA receptor inhibitor MK-801 prevented these effects. Also, many Pax6+ cells, comprising most ACs, were lost, while rho4D2+ rod photoreceptors were increased. Cell proliferation was slightly increased, while apoptosis remained unaffected. Conclusions: This demonstrated: (1) a far-advanced differentiation of an IPL in retinal spheroids, as never described before; (2) ipl sublamination was initiated by cholinergic precursor cells, which-functioning as "ipl founder cells"-(3) gave rise to neurons and glial cells; (4) these SACs and MCPs together organized ipl formation; and (5) this process was counteracted by NMDA-dependent glutamate actions. SN - 1552-5783 UR - https://www.unboundmedicine.com/medline/citation/31738824/IPL_Sublamination_in_Chicken_Retinal_Spheroids_Is_Initiated_via_Müller_Cells_and_Cholinergic_Differentiation,_and_Is_Disrupted_by_NMDA_Signaling L2 - http://iovs.arvojournals.org/article.aspx?doi=10.1167/iovs.18-24952 DB - PRIME DP - Unbound Medicine ER -