Blue light stopped potassium-induced epileptiform firing in hippocampal neurons treated with NBD-allopregnanolone.
Researchers intended to design a fluorescent compound to shine a light on neurosteroid function, but instead they lit the way for photoactivated therapies. Like barbiturates, neurosteroids bind to sites in the transmembrane domain of GABAA receptors, potentiating GABAA response. Eisenman et al. fluorescently tagged the neurosteroid allopregnanolone (also known as 3α5αP) to localize its sites of action, but mistakenly disabled its activity at GABAA receptors. However, they report that the same wavelength of visible light that causes the tag to fluoresce specifically activates the neurosteroid at GABAA receptors in a recent article in Nature Neuroscience.
'Caged' compounds are photoactivatable. Spatially restricted application of ultraviolet light 'uncages' and therefore focally activates the compound. However, ultraviolet light is toxic and does not penetrate well through tissue. Therefore, caged compounds cannot be used therapeutically.
The authors tagged allopregnanolone with the fluorescent marker NBD. NBD-allopregnanolone absorbed blue light (400-480 nm). In cultured hippocampal cells exposed to GABA and NBD-allopregnanolone, 30 seconds of blue light illumination increased the current through GABAA receptors by over 400% relative to control. Within 30 seconds of full illumination, photopotentiation reached a plateau. By the time photopotentiation reached a plateau, fluorescence declined, suggesting that photodepletion of the active compound limits potentiation.
NBD-allopregnanolone behaved similarly to classical neurosteroids. Like other neurosteroids, it increased the amount of time individual GABAA receptors were open and decreased the amount of time they were closed, as shown by whole-cell patch-clamp recordings. Before photodepletion, re-exposure to light repeatedly potentiated the same cells, suggesting that NBD-allopregnanolone is difficult to wash out, similar to other neurosteroids. The beta epimer of allopregnanolone (3
5αP) decreased GABAA receptor potentiation relative to the photoactive alpha epimer (3α5αP), suggesting that photopotentiation is stereoselective. However, 35αP slightly potentiated the GABAA receptor response.
Although not as potent as NBD-allopregnanolone, molecules unrelated to neurosteroids, including lysine and fatty acids, potentiated GABAA receptor responses when tagged with NBD and stimulated with light. NBD-allopregnanolone and a membrane-impermeable form of NBD-allopregnanolone photopotentiated GABAA receptor responses similarly. Therefore, the neurosteroid-binding site is not necessary for photopotentiation. However, photopotentiation is limited to GABAA receptors. In hippocampal neurons, NBD-allopregnanolone potentiated GABA-mediated inhibitory postsynaptic currents, but did not affect glutamate-mediated excitatory postsynaptic currents.
What are the potential in vivo applications for photopotentiation? Blue light sedated tadpoles in water containing NBD-allopregnanolone. Potassium induces epilepsy-like trains of activity in hippocampal neurons. In cells preincubated with NBD-allopregnanalone, light stopped potassium-induced epileptiform firing, suggesting that photopotentiation can silence selected neurons. Some people with severe epilepsy are treated with electrical stimulation from implanted electrodes. The authors propose that NBD-allopregnanolone and implanted light sources might silence epileptic loci in the human brain.