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Anticonvulsant and anesthetic effects of a fluorescent neurosteroid analog activated by visible light

Nature Neuroscience volume 10, pages 523–530 (2007)Cite this article

Abstract

Most photoactivatable compounds suffer from the limitations of the ultraviolet wavelengths that are required for activation. We synthesized a neuroactive steroid analog with a fluorescent (7-nitro-2,1,3-benzoxadiazol-4-yl) amino (NBD) group in the β configuration at the C2 position of (3α,5α)-3-hydroxypregnan-20-one (allopregnanolone, 3α5αP). Light wavelengths (480 nm) that excite compound fluorescence strongly potentiate GABAA receptor function. Potentiation is limited by photodepletion of the receptor-active species. Photopotentiation is long-lived and stereoselective and shows single-channel hallmarks similar to steroid potentiation. Other NBD-conjugated compounds also generate photopotentiation, albeit with lower potency. Thus, photopotentiation does not require a known ligand for neurosteroid potentiating sites on the GABAA receptor. Photoactivation of a membrane-impermeant, fluorescent steroid analog demonstrates that membrane localization is critical for activity. The photoactivatable steroid silences pathological spiking in cultured rat hippocampal neurons and anesthetizes tadpoles. Fluorescent steroids photoactivated by visible light may be useful for modulating GABAA receptor function in a spatiotemporally defined manner.

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Figure 1: C2-NBD 3α5αP photoactivation.
Figure 2: C2-NBD 3α5αP potentiation characteristics.
Figure 3: Single-channel current photopotentiation.
Figure 4: C2-NBD 3α5αP potentiation is stereospecific.
Figure 5: Effects of NBD-tagged lysine and a membrane-impermeant NBD-tagged steroid.
Figure 6: Physiological uses of C2-NBD 3α5αP.

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Acknowledgements

We thank A. Taylor, A. Benz and B.-W. Ma for technical help and laboratory members for advice and discussion. This work was supported by a gift from the Bantly Foundation (C.F.Z.) and by US National Institutes of Health grants NS44041 (L.N.E), GM 47969 (D.F.C., C.F.Z., A.S.E, J.H.S), AA12951 (C.F.Z.), NS54174, AA12952 and MH78823 (S.M.) and AA14707 (G.A.).

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Authors and Affiliations

  1. Department of Neurology, Washington University School of Medicine, 660 S. Euclid Ave, St. Louis, 63110, Missouri, USA

    Lawrence N Eisenman

  2. Department of Psychiatry, Washington University School of Medicine, 660 S. Euclid Ave, St. Louis, 63110, Missouri, USA

    Hong-Jin Shu, Geraldine J Kress, Charles F Zorumski & Steven Mennerick

  3. Department of Anesthesiology, Washington University School of Medicine, 660 S. Euclid Ave, St. Louis, 63110, Missouri, USA

    Gustav Akk, Brad D Manion, Alex S Evers & Joe Henry Steinbach

  4. Molecular Biology and Pharmacology, Washington University School of Medicine, 660 S. Euclid Ave, St. Louis, 63110, Missouri, USA

    Cunde Wang, Alex S Evers & Douglas F Covey

  5. Anatomy and Neurobiology, Washington University School of Medicine, 660 S. Euclid Ave, St. Louis, 63110, Missouri, USA

    Joe Henry Steinbach, Charles F Zorumski & Steven Mennerick

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Contributions

Neurosteroid analogs were synthesized by C.W. and D.F.C. Experiments were performed by L.N.E., H.-J.S., G.A., B.D.M, G.J.K. and S.M., and the resulting data were analyzed by L.N.E., G.A., A.S.E., J.H.S., C.F.Z. and S.M. The manuscript was written by L.N.E., J.H.S., D.F.C., C.F.Z. and S.M.

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Correspondence to Steven Mennerick.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Photoactivation of two additional 3α-OH NBD steroid conjugates. (PDF 703 kb)

Supplementary Table 1

The results of the analysis of open and closed duration. (PDF 36 kb)

Supplementary Video 1

Effects of C2-NBD 3α5αP and C2-NBD 3β5αP on tadpole swimming behavior after dim light and after 470 nm light exposure. (WMV 5107 kb)

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Eisenman, L., Shu, HJ., Akk, G. et al. Anticonvulsant and anesthetic effects of a fluorescent neurosteroid analog activated by visible light. Nat Neurosci 10, 523–530 (2007). https://doi.org/10.1038/nn1862

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