title: New insight into the functional role of acetylcholine in developing embryonic rat retinal neurons.
abstract: OBJECTIVE
  To examine the effects of acetylcholine (ACh) on glutamate-induced neurotoxicity in embryonic rat retinal neurons.
  
  
  METHODS
  Primary cultures were obtained from rat retinas at embryonic days 17 to 19. Cultured cells were exposed to glutamate for 10 minutes, followed by incubation in glutamate-free medium for 1 hour. Drugs were added to the incubation medium for 1 to 24 hours until immediately before glutamate exposure and were removed from culture medium during glutamate exposure and the postincubation period. The neurotoxic effects on retinal cultures were quantitatively assessed by the trypan blue exclusion method.
  
  
  RESULTS
  Cell viability was markedly reduced by 10-minute exposure to 500 microM glutamate followed by a 1-hour incubation in glutamate-free medium. Incubating the cultures with 1 microM ACh for 12 hours before glutamate exposure reduced glutamate neurotoxicity. A similar effect was induced by application of carbachol (1 microM). The protective effect of ACh against glutamate neurotoxicity was inhibited by a nicotinic acetylcholine receptor (nAChR) antagonist, mecamylamine (0.5 microM), whereas a muscarinic acetylcholine receptor (mAChR) antagonist, atropine (0.5 microM) did not affect ACh-induced protection. In addition, a similar protection was induced by application of nicotine (1 microM), but not by muscarine (1 microM). Pretreatment with nicotine induced a protective effect in a time-dependent manner, ranging from 1 to 12 hours. Pretreatment with nicotine at concentrations ranging from 0.001 to 1 microM induced dose-dependent protection against glutamate neurotoxicity. Furthermore, the protective action of nicotine was inhibited by simultaneous application of dopamine D1 receptor antagonist, SCH23390 (1 microM), with nicotine, whereas a dopamine D2 receptor antagonist, domperidone (1 microM), did not affect nicotine-induced protection.
  
  
  CONCLUSIONS
  These results suggest that pretreatment of cultured rat retinal neurons with ACh or the nAChR agonists, nicotine and carbachol, has a protective action against glutamate neurotoxicity through nAChRs and that the dopamine release induced by nicotinic stimulation subsequently protects the retinal neurons by way of dopamine D1 receptors.
authors.affiliation: Department of Ophthalmology and Visual Sciences, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Yasuyoshi
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kashii
authors.forename: Masashi
authors.initials: M
authors.lastname: Kikuchi
authors.forename: Shen
authors.initials: S
authors.lastname: Zhang
authors.forename: Yoshihito
authors.initials: Y
authors.lastname: Honda
authors.forename: Akinori
authors.initials: A
authors.lastname: Akaike
mesh_terms: D000109:Acetylcholine / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D000818:Animals; D002470:Cell Survival / Q000187:drug effects*; D002478:Cells, Cultured; D018679:Cholinergic Agonists / Q000494:pharmacology; D019610:Cytoprotection / Q000187:drug effects; D018492:Dopamine Antagonists / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D018698:Glutamic Acid / Q000633:toxicity*; D008464:Mecamylamine / Q000494:pharmacology; D018727:Muscarinic Antagonists / Q000494:pharmacology; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D018696:Neuroprotective Agents / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D018733:Nicotinic Antagonists / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism; D012160:Retina / Q000187:drug effects / Q000196:embryology* / Q000378:metabolism; D013997:Time Factors
chemical_list: D018679:Cholinergic Agonists; D018492:Dopamine Antagonists; D018727:Muscarinic Antagonists; D018696:Neuroprotective Agents; D018733:Nicotinic Antagonists; D016194:Receptors, N-Methyl-D-Aspartate; D018698:Glutamic Acid; D008464:Mecamylamine; D000109:Acetylcholine