IPL sublamination in chicken retinal spheroids is initiated via Müller cells and cholinergic differentiation, and is disrupted by NMDA signaling

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 acetylcholinesteraseþ (AChE) and choline acetyltransferaseþ (ChAT) cells. Individual vimentinþ or transitinþ Müller glial cell precursors (MCPs) in ipl centers coexpressed ChAT. Comparable to in vivo, pairwise arranged ChATþ SACs formed two laminar subbands. Projections of calretininþ 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 Pax6þ cells, comprising most ACs, were lost, while rho4D2þ 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.