title: NMDA potentiation by visible light in the presence of a fluorescent neurosteroid analogue.
abstract: N-Methyl-D-aspartate (NMDA) receptors are widely studied because of their importance in synaptic plasticity and excitotoxic cell death. Here we report a novel method of potentiating NMDA receptors with fluorescence excited by blue (480 nm) light. In the presence of 300 nM of a (7-nitro-2,1,3-benzoxadiazol-4-yl) amino (NBD)-tagged neuroactive steroid carrier C2-NBD-(3alpha,5alpha)-3-hydroxypregnan-20-one (C2-NBD 3alpha5alphaP), responses of cultured hippocampal neurons to 10 microM NMDA were potentiated to 219.2 +/- 9.2% of the baseline response (100%) by a 30 s exposure to 480 nm light. The potentiation decayed back to baseline with a time constant of 80.6 s. Responses to 1 microM and 100 microM NMDA were potentiated to 147.9 +/- 9.6% and 174.1 +/- 15.6% of baseline, respectively, suggesting that visible-light potentiation is relatively insensitive to NMDA concentration. Peak autaptic NMDA responses were potentiated to 178.9 +/- 22.4% of baseline. Similar potentiation was seen with 10 microM NBD-lysine, suggesting that visible-light potentiation is not a steroid effect. Potentiation was also seen with a steroid analogue in which the NBD was replaced with fluorescein, suggesting that NBD is not the only fluorophore capable of supporting visible-light potentiation. UV light and redox potentiation of NMDA receptors largely occluded subsequent blue light potentiation (127.7 +/- 7.4% and 120.2 +/- 6.2% of baseline, respectively). The NR1a(C744A,C798A) mutant that is insensitive to redox and UV potentiation was also largely unaffected by visible-light potentiation (135.0 +/- 10.0% of baseline). Finally, we found that the singlet oxygen scavenger furfuryl alcohol decreased visible-light potentiation. Collectively, these data suggest that visible-light potentiation of NMDA receptors by fluorescence excitation shares mechanisms with UV and redox potentiation and may involve singlet oxygen production.
authors.affiliation: Department of Neurology, Washington University School of Medicine, 660 S. Euclid Ave, Box 8111, St Louis, MO 63110, USA. leisenman@wustl.edu
authors.forename: Lawrence N
authors.initials: LN
authors.lastname: Eisenman
authors.forename: Hong-Jin
authors.initials: HJ
authors.lastname: Shu
authors.forename: Cunde
authors.initials: C
authors.lastname: Wang
authors.forename: Elias
authors.initials: E
authors.lastname: Aizenman
authors.forename: Douglas F
authors.initials: DF
authors.lastname: Covey
authors.forename: Charles F
authors.initials: CF
authors.lastname: Zorumski
authors.forename: Steven
authors.initials: S
authors.lastname: Mennerick
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004594:Electrophysiology; D005456:Fluorescent Dyes; D016166:Free Radical Scavengers / Q000494:pharmacology; D005663:Furans / Q000494:pharmacology; D006624:Hippocampus / Q000737:chemistry / Q000502:physiology; D008027:Light; D010069:Oxadiazoles; D010084:Oxidation-Reduction; D018408:Patch-Clamp Techniques; D011280:Pregnanolone / Q000031:analogs & derivatives*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D011963:Receptors, GABA-A / Q000187:drug effects; D016194:Receptors, N-Methyl-D-Aspartate / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects*; D014466:Ultraviolet Rays
chemical_list: D005456:Fluorescent Dyes; D016166:Free Radical Scavengers; D005663:Furans; D010069:Oxadiazoles; D017382:Reactive Oxygen Species; D011963:Receptors, GABA-A; D016194:Receptors, N-Methyl-D-Aspartate; D011280:Pregnanolone; C012986:furfuryl alcohol