NMDA potentiation by visible light in the presence of a fluorescent neurosteroid analogue

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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-(3α,5α)-3-hydroxypregnan-20-one (C2-NBD 3α5αP), responses of cultured hippocampal neurons to 10 μM 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 μM and 100 μM 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 μM 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.

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