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An in vivo biosensor for neurotransmitter release and in situ receptor activity

Abstract

Tools from molecular biology, combined with in vivo optical imaging techniques, provide new mechanisms for noninvasively observing brain processes. Current approaches primarily probe cell-based variables, such as cytosolic calcium or membrane potential, but not cell-to-cell signaling. We devised cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) to address this challenge and monitor in situ neurotransmitter receptor activation. CNiFERs are cultured cells that are engineered to express a chosen metabotropic receptor, use the Gq protein–coupled receptor cascade to transform receptor activity into a rise in cytosolic [Ca2+] and report [Ca2+] with a genetically encoded fluorescent Ca2+ sensor. The initial realization of CNiFERs detected acetylcholine release via activation of M1 muscarinic receptors. We used chronic implantation of M1-CNiFERs in frontal cortex of the adult rat to elucidate the muscarinic action of the atypical neuroleptics clozapine and olanzapine. We found that these drugs potently inhibited in situ muscarinic receptor activity.

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Figure 1: Design and in vitro characterization of CNiFERs.
Figure 2: In vivo characterization of acutely implanted M1-CNiFERs.
Figure 3: Chronic implantation of CNiFERs.
Figure 4: In vivo pharmacology of chronically implanted M1-CNiFERs.

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Acknowledgements

We are grateful to T. Bartfai, D.K. Berg, J.-P. Changeux, J.M. Edeline, A.L. Fairhall, B. Hille, H.J. Karten, R. Metherate, P.A. Slesinger, T. Talley, R.Y. Tsien and M. Tuszynski for valuable discussions. We thank R. Figueroa for maintaining the cell culture facility, J. Groisman for preparing the artwork in Figure 2a and A. Miyanohara (Vector Development Laboratory, Human Gene Therapy Program, University of California San Diego) for producing the lentiviruses. This work was supported by the US National Institutes of Health Medical Scientist Training Program (L.F.S.), the Max Planck Society (O.G.) and grants from the US National Institutes of Health (DA024206, EB003832 and MH085499 to D.K., GM18360 and DA19372 to P.T., and MH070655 to Q.-T.N.).

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Authors

Contributions

D.K., Q.-T.N. and L.F.S. designed and made the CNiFERs, O.G. and M.M. synthesized the calcium indicator and consulted on molecular biology, D.K., A.M., Q.-T.N., L.F.S. and P.T. characterized and applied the CNiFERS, and D.K., Q.-T.N. and L.F.S. analyzed the data and wrote the manuscript.

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Correspondence to David Kleinfeld.

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David Kleinfeld, Lee F. Schroeder and Quoc-Thang Nguyen are the authors of a patent application related to the paper and are entitled to receive royalties if a patent is granted. FemtoScience, of which Quoc-Thang Nguyen is CEO and founder, has licensed the intellectual property from the University of California San Diego.

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Nguyen, QT., Schroeder, L., Mank, M. et al. An in vivo biosensor for neurotransmitter release and in situ receptor activity. Nat Neurosci 13, 127–132 (2010). https://doi.org/10.1038/nn.2469

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