Rapid increase in cytosolic calcium ion concentration mediated by acetylcholine receptors in cultured retinal neurons and Muller cells

Purpose: This study was conducted to detect the presence of muscarinic or n icotinic receptors in cultured retinal neurons and Muller cells. Methods: P ure Muller cell cultures and cocultures of retinal neurons and Muller cells were used; the former, obtained from adult rabbit retinas, and the latter, retinal neurons from neonatal rats, were cocultured with Muller cells. Int racellular calcium ion concentration ([Ca2+](i)) following the administrati on of acetylcholine, a cholinesterase inhibitor (trichlorfon), nicotine or muscarinic agonist with or without a receptor antagonist was monitored usin g the calcium ion indicator, fura-2. a Results: Acetylcholine and trichlorf on induced rapid increase in [Ca2+](i) in half of either cell type. Trichlo rfon induced positive response in coculture but not in the pure Muller cell cultures. This positive response was blocked only partially in the presenc e of atropine. Approximately 30-40% of neurons responded to nicotine at 5 m u M, which was significantly blocked by alpha-bungarotoxin at 50 nM. No res ponse to nicotine could be detected in Muller cells. Approximately 50% of n eurons responded to muscarine at 50 mu M, but 500 mu M was required for the formation of calcium transients in 50% of Muller cells. The muscarine indu cement of rapid increase in [Ca2+](i) was blocked by atropine. The agonist of M1 (a muscarinic receptor subtype), McN-A-343, at 0.5 mu M induced the m ost significant and rapid increase in [Ca2+](i) both in neurons and Muller cells. McN-A-343 administration at 0.05 mu M induced positive response in h alf the neurons, but only in approximately 10% of Muller cells. Such positi ve response was not observed following preincubation with the M1 antagonist , pirenzepine, at 50 mu M. Conclusions: Cocultured retinal neurons enhance the release of acetylcholine following anticholinesterase administration, a nd approximately half the neurons were found to possess muscarinic and nico tinic receptors. However. Muller cells appeared to possess only the less se nsitive muscarinic receptor. Muscarinic receptor subtypes on either type of cell contained at least MI.