Protocol | Published:

A practical guide to the synthesis of dinitroindolinyl-caged neurotransmitters

Nature Protocols volume 6, pages 314326 (2011) | Download Citation

Abstract

This protocol describes a method for efficient chemical synthesis of dinitroindolinyl derivatives of glutamate and γ-aminobutyric acid. These caged neurotransmitters are currently the most chemically and photochemically efficient probes for two-photon photolysis in living brain slices. The protocol only requires basic organic synthesis equipment, and no silica gel column chromatography or NMR spectroscopy is needed at any stage. HPLC is used to purify the caged transmitters at the end of the syntheses. Thus, the synthesis of dinitroindolinyl-caged neurotransmitters is within the scope of a modestly equipped chemistry laboratory.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    , & Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).

  2. 2.

    & Principles of two-photon excitation microscopy and its applications to neuroscience. Neuron 50, 823–839 (2006).

  3. 3.

    Constructing and exploiting the fluorescent protein paintbox (Nobel Lecture). Angew. Chem. Int. Ed. Engl. 48, 5612–5626 (2009).

  4. 4.

    Caged compounds: photorelease technology for control of cellular chemistry and physiology. Nat. Methods 4, 619–628 (2007).

  5. 5.

    et al. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28, 41–51 (2000).

  6. 6.

    et al. Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex. Nature 420, 788–794 (2002).

  7. 7.

    , & Long-term dendritic spine stability in the adult cortex. Nature 420, 812–816 (2002).

  8. 8.

    , & Dendritic spine dynamics. Annu. Rev. Physiol. 71, 261–282 (2009).

  9. 9.

    & Experience-dependent structural synaptic plasticity in the mammalian brain. Nat. Rev. Neurosci. 10, 647–658 (2009).

  10. 10.

    & Genetically encoded calcium indicators. Chem. Rev. 108, 1550–1564 (2008).

  11. 11.

    , , & Calcium dynamics of cortical astrocytic networks in vivo. PLoS Biol. 2, E96 (2004).

  12. 12.

    et al. An astrocytic basis of epilepsy. Nat. Med. 11, 973–981 (2005).

  13. 13.

    , , & Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo. Nat. Methods 1, 31–37 (2004).

  14. 14.

    , , & In vivo two-photon calcium imaging of neuronal networks. Proc. Natl. Acad. Sci. USA 100, 7319–7324 (2003).

  15. 15.

    et al. A genetically encoded calcium indicator for chronic in vivo two-photon imaging. Nat. Methods 5, 805–811 (2008).

  16. 16.

    et al. Single-spike detection in vitro and in vivo with a genetic Ca2+ sensor. Nat. Methods 5, 797–804 (2008).

  17. 17.

    , , & Minimally invasive high-speed imaging of sarcomere contractile dynamics in mice and humans. Nature 454, 784–788 (2008).

  18. 18.

    et al. Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons. Nat. Neurosci. 4, 1086–1092 (2001).

  19. 19.

    & Optical sectioning microscopy. Nat. Methods 2, 920–931 (2005).

  20. 20.

    Two-photon scanning photochemical microscopy: mapping ligand-gated ion channel distributions. Proc. Natl. Acad. Sci. USA 91, 6629–6633 (1994).

  21. 21.

    , & Mechanism of the distance-dependent scaling of Schaffer collateral synapses in rat CA1 pyramidal neurons. J. Physiol. 548, 245–258 (2003).

  22. 22.

    & State-dependent calcium signaling in dendritic spines of striatal medium spiny neurons. Neuron 44, 483–493 (2004).

  23. 23.

    , & NMDA receptor subunit-dependent [Ca2+] signaling in individual hippocampal dendritic spines. J. Neurosci. 25, 6037–6046 (2005).

  24. 24.

    et al. Synapse-specific regulation of AMPA receptor function by PSD-95. Proc. Natl. Acad. Sci. USA 103, 19535–19540 (2006).

  25. 25.

    , & Synaptic strength of individual spines correlates with bound Ca2+-calmodulin-dependent kinase II. J. Neurosci. 27, 14007–14011 (2007).

  26. 26.

    et al. Surface mobility of postsynaptic AMPARs tunes synaptic transmission. Science 320, 201–205 (2008).

  27. 27.

    , & Optical induction of plasticity at single synapses reveals input-specific accumulation of alphaCaMKII. Proc. Natl. Acad. Sci. USA 105, 12039–12044 (2008).

  28. 28.

    , , & Connectivity patterns revealed by mapping of active inputs on dendrites of thalamorecipient neurons in the auditory cortex. J. Neurosci. 29, 6406–6417 (2009).

  29. 29.

    , , , & 4-Carboxymethoxy-5,7-dinitroindolinyl-Glu: an improved caged glutamate for expeditious ultraviolet and two-photon photolysis in brain slices. J. Neurosci. 27, 6601–664 (2007).

  30. 30.

    , , & Two-photon uncaging of gamma-aminobutyric acid in intact brain tissue. Nat. Chem. Biol. 6, 255–257 (2010).

  31. 31.

    , , & A practical guide to the synthesis and use of membrane-permeant acetoxymethyl esters of caged inositol-polyphosphates. Nat. Protoc. 6, 327–337 (2011).

  32. 32.

    , & Targeted bulk-loading of fluorescent indicators for two-photon brain imaging in vivo. Nat. Protoc. 1, 380–386 (2006).

  33. 33.

    , & Three-dimensional mapping of unitary synaptic connections by two-photon macro photolysis of caged glutamate. J. Neurophysiol. 99, 1535–1544 (2008).

  34. 34.

    , , , & Two-color, two-photon uncaging of glutamate and GABA. Nat. Meth. 7, 123–125 (2010).

  35. 35.

    , , & An antenna triplet sensitiser for 1-acyl-7-nitroindolines improves the efficiency of carboxylic acid photorelease. Photochem. Photobiol. Sci. 3, 366–373 (2004).

  36. 36.

    , & An antenna-sensitized nitroindoline precursor to enable photorelease of L-glutamate in high concentrations. J. Org. Chem. 69, 7228–7233 (2004).

  37. 37.

    , , , & Questioning the role of rebound firing in the cerebellum. Nat. Neurosci. 11, 1256–1258 (2008).

  38. 38.

    , , , & Synthesis and characterization of 4-methoxy-7-nitroindolinyl-D-aspartate, a caged compound for selective activation of glutamate transporters and N-methyl-D-aspartate receptors in brain tissue. Biochemistry 44, 3316–3326 (2005).

Download references

Acknowledgements

This work was supported by a grant from the US National Institutes of Health (GM53395) to G.C.R.E.-D.

Author information

Affiliations

  1. Department of Neuroscience, Mount Sinai School of Medicine, New York, New York, USA.

    • Graham C R Ellis-Davies

Authors

  1. Search for Graham C R Ellis-Davies in:

Contributions

G.C.R.E.-D. performed the synthesis and wrote the paper.

Competing interests

G.C.R.E.-D. has filed a patent in the United States of America on the synthesis of dinitroindolinyl-caged neurotransmitters.

Corresponding author

Correspondence to Graham C R Ellis-Davies.

About this article

Publication history

Published

DOI

https://doi.org/10.1038/nprot.2010.193

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.