Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Technical Report
  • Published:

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

Nature Neuroscience volume 13, pages 127–132 (2010)Cite this article

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

References

  1. Agnati, L.F. et al. Volume transmission and wiring transmission from cellular to molecular networks: history and perspectives. Acta Physiol. (Oxf.) 187, 329–344 (2006).

    Article  CAS  Google Scholar 

  2. Beaudet, A. & Descarries, L. The monoamine innervation of rat cerebral cortex: synaptic and nonsynaptic axon terminals. Neuroscience 3, 851–860 (1978).

    Article  CAS  Google Scholar 

  3. Tsien, R.Y. Building and breeding molecules to spy on cells and tumors. FEBS Lett. 579, 927–932 (2005).

    Article  CAS  Google Scholar 

  4. Hires, S.A., Zhu, Y. & Tsien, R.Y. Optical measurement of synaptic glutamate spillover and reuptake by linker optimized glutamate-sensitive fluorescent reporters. Proc. Natl. Acad. Sci. USA 105, 4411–4416 (2008).

    Article  CAS  Google Scholar 

  5. Okumoto, S. et al. Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors. Proc. Natl. Acad. Sci. USA 102, 8740–8745 (2005).

    Article  CAS  Google Scholar 

  6. Vilardaga, J.-P., Bünemann, M., Krasel, C., Castro, M. & Lohse, M.J. Measurement of the millisecond activation switch of G protein−coupled receptors in living cells. Nat. Biotechnol. 21, 807–812 (2003).

    Article  CAS  Google Scholar 

  7. Young, S.H. & Poo, M.M. Spontaneous release of transmitter from growth cones of embryonic neurones. Nature 305, 634–637 (1983).

    Article  CAS  Google Scholar 

  8. Haas, B. et al. Activity-dependent ATP-waves in the mouse neocortex are independent from astrocytic calcium waves. Cereb. Cortex 16, 237–246 (2006).

    Article  Google Scholar 

  9. Schroeder, K.S. & Neagle, B. FLIPR: A new instrument for accurate, high throughput optical screening. J. Biomol. Screen. 1, 75–80 (1995).

    Article  Google Scholar 

  10. Duff Davis, M. & Schmidt, J.J. In vivo spectrometric calcium flux recordings of intrinsic Caudate-Putamen cells and transplanted IMR-32 neuroblastoma cells using miniature fiber optrodes in anesthetized and awake rats and monkeys. J. Neurosci. Methods 99, 9–23 (2000).

    Article  CAS  Google Scholar 

  11. Umbriaco, D., Watkins, K.C., Descarries, L., Cozzari, C. & Hartman, B.K. Ultrastructural and morphometric features of the acetylcholine innervation in adult rat parietal cortex: an electron microscopic study in serial sections. J. Comp. Neurol. 348, 351–373 (1994).

    Article  CAS  Google Scholar 

  12. Everitt, B.J. & Robbins, T.W. Central cholinergic systems and cognition. Annu. Rev. Psychol. 48, 649–684 (1997).

    Article  CAS  Google Scholar 

  13. Raedler, T.J. et al. Towards a muscarinic hypothesis of schizophrenia. Mol. Psychiatry 12, 232–246 (2007).

    Article  CAS  Google Scholar 

  14. Levey, A.I., Kitt, C.A., Simonds, W.F., Price, D.L. & Brann, M.R. Identification and localization of muscarinic acetylcholine receptor proteins in brain with subtype-specific antibodies. J. Neurosci. 11, 3218–3226 (1991).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  16. Denk, W., Strickler, J.H. & Webb, W.W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).

    Article  CAS  Google Scholar 

  17. Rasmusson, D.D., Clow, K. & Szerb, J.C. Frequency-dependent increase in cortical acetylcholine release evoked by stimulation of the nucleus basalis magnocellularis in the rat. Brain Res. 594, 150–154 (1992).

    Article  CAS  Google Scholar 

  18. Day, J.C., Kornecook, T.J. & Quirion, R. Application of in vivo microdialysis to the study of cholinergic systems. Methods 23, 21–39 (2001).

    Article  CAS  Google Scholar 

  19. Rasmusson, D.D., Clow, K. & Szerb, J.C. Modification of neocortical acetylcholine release and electroencephalogram desynchronization due to brainstem stimulation by drugs applied to the basal forebrain. Neuroscience 60, 665–677 (1994).

    Article  CAS  Google Scholar 

  20. Berg, R.W., Friedman, B., Schroeder, L.F. & Kleinfeld, D. Activation of nucleus basalis facilitates cortical control of a brainstem motor program. J. Neurophysiol. 94, 699–711 (2005).

    Article  Google Scholar 

  21. Meltzer, H.Y. What's atypical about atypical antipsychotic drugs? Curr. Opin. Pharmacol. 4, 53–57 (2004).

    Article  CAS  Google Scholar 

  22. Snyder, E.M. & Murphy, M.R. Schizophrenia therapy: beyond atypical antipsychotics. Nat. Rev. Drug Discov. 7, 471–472 (2008).

    Article  CAS  Google Scholar 

  23. Bymaster, F.P. et al. Muscarinic mechanisms of antipsychotic atypicality. Prog. Neuropsychopharmacol. Biol. Psychiatry 27, 1125–1143 (2003).

    Article  CAS  Google Scholar 

  24. Nguyen, Q.T., Yang, J. & Miledi, R. Effects of atypical antipsychotics on vertebrate neuromuscular transmission. Neuropharmacology 42, 670–676 (2002).

    Article  CAS  Google Scholar 

  25. Parada, M.A. & Hernandez, L. Selective action of acute systemic clozapine on acetylcholine release in the rat prefrontal cortex by reference to the nucleus accumbens and striatum. J. Pharmacol. Exp. Ther. 281, 582–588 (1997).

    CAS  PubMed  Google Scholar 

  26. Ichikawa, J., Dai, J., O'Laughlin, I.A., Fowler, W.L. & Meltzer, H.Y. Atypical, but not typical, antipsychotic drugs increase cortical acetylcholine release without an effect in the nucleus accumbens or striatum. Neuropsychopharmacology 26, 325–339 (2002).

    Article  CAS  Google Scholar 

  27. Bolden, C., Cusack, B. & Richelson, E. Antagonism by antimuscarinic and neuroleptic compounds at the five cloned human muscarinic cholinergic receptors expressed in Chinese hamster ovary cells. J. Pharmacol. Exp. Ther. 260, 576–580 (1992).

    CAS  PubMed  Google Scholar 

  28. Chew, M.L. et al. A model of anticholinergic activity of atypical antipsychotic medications. Schizophr. Res. 88, 63–72 (2006).

    Article  Google Scholar 

  29. Davies, M.A., Compton-Toth, B.A., Hufeisen, S.J., Meltzer, H.Y. & Roth, B.L. The highly efficacious actions of N-desmethylclozapine at muscarinic receptors are unique and not a common property of either typical or atypical antipsychotic drugs: Is M1 agonism a pre-requisite for mimicking clozapine's actions. Psychopharmacology (Berl.) 178, 451–460 (2005).

    Article  CAS  Google Scholar 

  30. Johnson, D.E. et al. The role of muscarinic receptor antagonism in antipsychotic-induced hippocampal acetylcholine release. Eur. J. Pharmacol. 506, 209–219 (2005).

    Article  CAS  Google Scholar 

  31. Gray, J.A. & Roth, B.L. Molecular targets for treating cognitive dysfunction in schizophrenia. Schizophr. Bull. 33, 1100–1119 (2007).

    Article  Google Scholar 

  32. Tani, Y., Saito, K., Imoto, M. & Ohno, T. Pharmacological characterization of nicotinic receptor-mediated acetylcholine release in rat brain: an in vivo microdialysis study. Eur. J. Pharmacol. 351, 181–188 (1998).

    Article  CAS  Google Scholar 

  33. Snyder, S., Greenberg, D. & Yamamura, H.I. Antischizophrenic drugs and brain cholinergic receptors. Affinity for muscarinic sites predicts extrapyramidal effects. Arch. Gen. Psychiatry 31, 58–61 (1974).

    Article  CAS  Google Scholar 

  34. Krasel, C., Vilardaga, J.P., Bunemann, M. & Lohse, M.J. Kinetics of G protein–coupled receptor signaling and desensitization. Biochem. Soc. Trans. 32, 1029–1031 (2004).

    Article  CAS  Google Scholar 

  35. Vögler, O. et al. Receptor subtype-specific regulation of muscarinic acetylcholine receptor sequestration by dynamin. Distinct sequestration of m2 receptors. J. Biol. Chem. 273, 12155–12160 (1998).

    Article  Google Scholar 

  36. Willars, G.B. & Nahorski, S.R. Quantitative comparisons of muscarinic and bradykinin receptor–mediated Ins (1,4,5)P3 accumulation and Ca2+ signaling in human neuroblastoma cells. Br. J. Pharmacol. 114, 1133–1142 (1995).

    Article  CAS  Google Scholar 

  37. Horowitz, L.F. et al. Phospholipase C in living cells: Activation, inhibition, Ca2+ requirement, and regulation of M current. J. Gen. Physiol. 126, 243–262 (2005).

    Article  CAS  Google Scholar 

  38. Zhang, J., Hupfeld, C.J., Taylor, S.S., Olefsky, J.M. & Tsien, R.Y. Insulin disrupts beta-adrenergic signaling to protein kinase A in adipocytes. Nature 437, 569–573 (2005).

    Article  CAS  Google Scholar 

  39. Kostenis, E., Waelbroeck, M. & Milligan, G. Techniques: promiscuous Gα proteins in basic research and drug discovery. Trends Pharmacol. Sci. 26, 595–602 (2005).

    Article  CAS  Google Scholar 

  40. Coward, P., Chan, S.D., Wada, H.G., Humphries, G.M. & Conklin, B.R. Chimeric G proteins allow a high-throughput signaling assay of Gi-coupled receptors. Anal. Biochem. 270, 242–248 (1999).

    Article  CAS  Google Scholar 

  41. Shaner, N.C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–1572 (2004).

    Article  CAS  Google Scholar 

  42. Tsai, P.S. & Kleinfeld, D. In vivo two-photon laser-scanning microscopy with concurrent plasma-mediated ablation: principles and hardware realization. in Methods for In vivo Optical Imaging, 2nd edn (ed. R.D. Frostig) 59–115 (CRC Press, Boca Raton, 2009).

  43. Nguyen, Q.-T., Dolnick, E.M., Driscoll, J. & Kleinfeld, D. MPScope 2.0: A computer system for two-photon laser scanning microscopy with concurrent plasma-mediated ablation and electrophysiology. in Methods for In vivo Optical Imaging 2nd edn (ed. R.D. Frostig) 117–142 (CRC Press, Boca Raton, 2009).

  44. Drobizhev, M., Tillo, S., Makarov, N.S., Hughes, T.E. & Rebane, A. Absolute two-photon absorption spectra and two-photon brightness of orange and red fluorescent proteins. J. Phys. Chem. B 113, 855–859 (2009).

    Article  CAS  Google Scholar 

  45. Schaffer, C.B. et al. Two-photon imaging of cortical surface microvessels reveals a robust redistribution in blood flow after vascular occlusion. PLoS Biol. 4, e22 (2006).

    Article  Google Scholar 

  46. Metherate, R. & Ashe, J.H. Ionic flux contributions to neocortical slow waves and nucleus basalis-mediated activation: whole-cell recordings in vivo. J. Neurosci. 12, 5312–5323 (1993).

    Article  Google Scholar 

  47. Kawaja, M.D. & Gage, F.H. Morphological and neurochemical features of cultured primary skin fibroblasts of Fischer 344 rats following striatal implantation. J. Comp. Neurol. 317, 102–116 (1992).

    Article  CAS  Google Scholar 

  48. Nishimura, N. et al. Targeted insult to individual subsurface cortical blood vessels using ultrashort laser pulses: three models of stroke. Nat. Methods 3, 99–108 (2006).

    Article  CAS  Google Scholar 

  49. Shirazi-Southall, S., Rodriguez, D.E. & Nomikos, G.G. Effects of typical and atypical antipsychotics and receptor selective compounds on acetylcholine efflux in the hippocampus of the rat. Neuropsychopharmacology 26, 583–594 (2002).

    Article  CAS  Google Scholar 

  50. DiMatteo, I., Genovese, C.R. & Kass, R.E. Bayesian curve-fitting with free-knot splines. Biometrika 88, 1055–1071 (2001).

    Article  Google Scholar 

Download references

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.).

Author information

Author notes

  1. Quoc-Thang Nguyen and Lee F Schroeder: These authors contributed equally to this work.

Authors and Affiliations

  1. Physics Department, University of California San Diego, La Jolla, California, USA

    Quoc-Thang Nguyen, Arnaud Muller & David Kleinfeld

  2. Medical Scientist Training Program, University of California San Diego, La Jolla, California, USA

    Lee F Schroeder

  3. Graduate Program in Neurosciences, University of California San Diego, La Jolla, California, USA

    Lee F Schroeder & David Kleinfeld

  4. Max-Planck Institut für Neurobiologie, Martinsried, Germany

    Marco Mank & Oliver Griesbeck

  5. Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, USA

    Palmer Taylor

  6. Center for Neural Circuits and Behavior, University of California San Diego, La Jolla, California, USA

    David Kleinfeld

Authors
  1. Quoc-Thang Nguyen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lee F Schroeder
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Marco Mank
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Arnaud Muller
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Palmer Taylor
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Oliver Griesbeck
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. David Kleinfeld
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Corresponding author

Correspondence to David Kleinfeld.

Ethics declarations

Competing interests

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.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9 (PDF 3225 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nn.2469

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing