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Cell-based reporters reveal in vivo dynamics of dopamine and norepinephrine release in murine cortex

Nature Methods volume 11, pages 1245–1252 (2014)Cite this article

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Abstract

The neuronal coding of stimulus-to-action sequences is believed to involve the release of dopamine (DA) and norepinephrine (NE). The electrochemical similarity of these monoamines, however, confounds real-time measurements of their release. Here we report cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) that use the specificity of G protein–coupled receptors (GPCRs) to discriminate nanomolar concentrations of DA and NE. CNiFERs were implanted into the frontal cortex of mice to measure the timing of neurotransmitter release during classical conditioning with the use of two-photon microscopy. The onset of DA release correlated with that of licking and shifted from the time of the reward toward that of the cue upon conditioning. In contrast, concurrent release of NE did not correlate with licking or the cue. This generation of CNiFERs provides unique tools to assess the release of monoamines. The molecular design of these CNiFERs may be generalized to realize CNiFERs for any molecule that activates a GPCR.

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Figure 1: Design of D2 and α1A CNiFERs and their in vitro characterization using a high-throughput plate reader.
Figure 2: In vitro characterization of CNiFER response to agonist pulses.
Figure 3: In vivo characterization of D2 and α1A CNiFERs.
Figure 4: Simultaneous detection of DA, NE and ACh release during behavioral conditioning.
Figure 5: Shift in DA but not NE release with behavioral conditioning.

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  • 10 November 2014

    In the version of this article initially published online, the trace for "Day 3" in Figure 4f was incorrect. The error has been corrected for the print, PDF and HTML versions of this article.

References

  1. Adamantidis, A.R. et al. Optogenetic interrogation of dopaminergic modulation of the multiple phases of reward-seeking behavior. J. Neurosci. 31, 10829–10835 (2011).

    Article  CAS  Google Scholar 

  2. Tsai, H.C. et al. Phasic firing in dopaminergic neurons is sufficient for behavioral conditioning. Science 324, 1080–1084 (2009).

    Article  CAS  Google Scholar 

  3. Aston-Jones, G. & Cohen, J.D. Adaptive gain and the role of the locus coeruleus-norepinephrine system in optimal performance. J. Comp. Neurol. 493, 99–110 (2005).

    Article  CAS  Google Scholar 

  4. Bouret, S. & Sara, S.J. Network reset: a simplified overarching theory of locus coeruleus noradrenaline function. Trends Neurosci. 28, 574–582 (2005).

    Article  CAS  Google Scholar 

  5. Floresco, S.B. Prefrontal dopamine and behavioral flexibility: shifting from an “inverted-U” toward a family of functions. Front. Neurosci. 7, 62 (2013).

    Article  Google Scholar 

  6. Edeline, J.M., Manunta, Y. & Hennevin, E. Induction of selective plasticity in the frequency tuning of auditory cortex and auditory thalamus neurons by locus coeruleus stimulation. Hear. Res. 274, 75–84 (2011).

    Article  Google Scholar 

  7. Bao, S., Chan, V.T. & Merzenich, M.M. Cortical remodelling induced by activity of ventral tegmental dopamine neurons. Nature 412, 79–83 (2001).

    Article  CAS  Google Scholar 

  8. 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 

  9. Greco, S., Danysz, W., Zivkovic, A., Gross, R. & Stark, H. Microdialysate analysis of monoamine neurotransmitters—a versatile and sensitive LC-MS/MS method. Anal. Chim. Acta 771, 65–72 (2013).

    Article  CAS  Google Scholar 

  10. Ji, C. et al. Diethylation labeling combined with UPLC/MS/MS for simultaneous determination of a panel of monoamine neurotransmitters in rat prefrontal cortex microdialysates. Anal. Chem. 80, 9195–9203 (2008).

    Article  CAS  Google Scholar 

  11. Mingote, S., de Bruin, J.P.C. & Feenstra, M.G. Noradrenaline and dopamine efflux in the prefrontal cortex in relation to appetitive classical conditioning. J. Neurosci. 24, 2475–2480 (2004).

    Article  CAS  Google Scholar 

  12. Wang, Y. & Michael, A.C. Microdialysis probes alter presynaptic regulation of dopamine terminals in rat striatum. J. Neurosci. Methods 208, 34–39 (2012).

    Article  CAS  Google Scholar 

  13. Robinson, D.L., Venton, B.J., Heien, M.L. & Wightman, R.M. Detecting subsecond dopamine release with fast-scan cyclic voltammetry in vivo. Clin. Chem. 49, 1763–1773 (2003).

    Article  CAS  Google Scholar 

  14. Park, J., Takmakov, P. & Wightman, R.M. In vivo comparison of norepinephrine and dopamine release in rat brain by simultaneous measurements with fast-scan cyclic voltammetry. J. Neurochem. 119, 932–944 (2011).

    Article  CAS  Google Scholar 

  15. Nguyen, Q.-T. et al. An in vivo biosensor for neurotransmitter release and in situ receptor activity. Nat. Neurosci. 13, 127–132 (2010).

    Article  CAS  Google Scholar 

  16. Svoboda, K., Denk, W., Kleinfeld, D. & Tank, D.W. In vivo dendritic calcium dynamics in neocortical pyramidal neurons. Nature 385, 161–165 (1997).

    Article  CAS  Google Scholar 

  17. Drew, P.J. et al. Chronic optical access through a polished and reinforced thinned skull. Nat. Methods 7, 981–984 (2010).

    Article  CAS  Google Scholar 

  18. Schultz, W. Updating dopamine reward signals. Curr. Opin. Neurobiol. 23, 229–238 (2013).

    Article  CAS  Google Scholar 

  19. Feenstra, M.G. Dopamine and noradrenaline release in the prefrontal cortex in relation to unconditioned and conditioned stress and reward. Prog. Brain Res. 126, 133–163 (2000).

    Article  CAS  Google Scholar 

  20. Schultz, W., Dayan, P. & Montague, P.R. A neural substrate of prediction and reward. Science 275, 1593–1599 (1997).

    Article  CAS  Google Scholar 

  21. Bouret, S. & Richmond, B.J. Relation of locus coeruleus neurons in monkeys to Pavlovian and operant behaviors. J. Neurophysiol. 101, 898–911 (2009).

    Article  Google Scholar 

  22. Yamauchi, J.G. et al. Characterizing ligand-gated ion channel receptors with genetically encoded Ca2+ sensors. PLoS ONE 6, e16519 (2011).

    Article  CAS  Google Scholar 

  23. Conklin, B.R., Farfel, Z., Lustig, K.D., Julius, D. & Bourne, H.R. Substitution of three amino acids switches receptor specificity of Gqα (to that of Giα. Nature 363, 274–276 (1993).

    Article  CAS  Google Scholar 

  24. Engleman, E.A., Ingraham, C.M., McBride, W.J., Lumeng, L. & Murphy, J.M. Extracellular dopamine levels are lower in the medial prefrontal cortex of alcohol-preferring rats compared to Wistar rats. Alcohol 38, 5–12 (2006).

    Article  CAS  Google Scholar 

  25. Ihalainen, J.A., Riekkinen, P. Jr. & Feenstra, G.P. Comparison of dopamine and noradrenaline release in mouse prefrontal cortex, striatum and hippocampus using microdialysis. Neurosci. Lett. 277, 71–74 (1999).

    Article  CAS  Google Scholar 

  26. Clark, J.J. et al. Chronic microsensors for longitudinal, subsecond dopamine detection in behaving animals. Nat. Methods 7, 126–129 (2010).

    Article  CAS  Google Scholar 

  27. Day, J.J., Roitman, M.F., Wightman, R.M. & Carelli, R.M. Associative learning mediates dynamic shifts in dopamine signaling in the nucleus accumbens. Nat. Neurosci. 10, 1020–1028 (2007).

    Article  CAS  Google Scholar 

  28. Hoffmann, C. et al. A FlAsH-based FRET approach to determine G protein–coupled receptor activation in living cells. Nat. Methods 2, 171–176 (2005).

    Article  CAS  Google Scholar 

  29. Falkenburger, B.H., Jensen, J.B. & Hille, B. Kinetics of M1 muscarinic receptor and G protein signaling to phospholipase C in living cells. J. Gen. Physiol. 135, 81–97 (2010).

    Article  CAS  Google Scholar 

  30. Loughlin, S.E. & Fallon, J.H. Substantia nigra and ventral tegmental area projections to cortex: topography and collateralization. Neuroscience 11, 425–435 (1984).

    Article  CAS  Google Scholar 

  31. Hoover, J.E. & Strick, P.L. The organization of cerebellar and basal ganglia outputs to primary motor cortex as revealed by retrograde transneuronal transport of herpes simplex virus type 1. J. Neurosci. 19, 1446–1463 (1999).

    Article  CAS  Google Scholar 

  32. Middleton, F.A. & Strick, P.L. Basal-ganglia 'projections' to the prefrontal cortex of the primate. Cereb. Cortex 12, 926–935 (2002).

    Article  Google Scholar 

  33. Hosp, J.A., Pekanovic, A., Rioult-Pedotti, M.S. & Luft, A.R. Dopaminergic projections from midbrain to primary motor cortex mediate motor skill learning. J. Neurosci. 31, 2481–2487 (2011).

    Article  CAS  Google Scholar 

  34. Gatter, K.C. & Powell, T.P. The projection of the locus coeruleus upon the neocortex in the macaque monkey. Neuroscience 2, 441–445 (1977).

    Article  CAS  Google Scholar 

  35. Loughlin, S.E., Foote, S.L. & Bloom, F.E. Efferent projections of nucleus locus coeruleus: topographic organization of cells of origin demonstrated by three-dimensional reconstruction. Neuroscience 18, 291–306 (1986).

    Article  CAS  Google Scholar 

  36. Zheng, J.Q., Felder, M., Connor, J.A. & Poo, M.-m. Turning of growth cones induced by neurotransmitters. Nature 368, 140–144 (1994).

    Article  CAS  Google Scholar 

  37. 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 

  38. Schulz, K. et al. Simultaneous BOLD fMRI and fiber-optic calcium recording in rat neocortex. Nat. Methods 9, 597–602 (2012).

    Article  CAS  Google Scholar 

  39. Stroh, A. et al. Making waves: Initiation and propagation of corticothalamic Ca2+ waves in vivo. Neuron 77, 1136–1150 (2013).

    Article  CAS  Google Scholar 

  40. Levene, M.J., Dombeck, D.A., Kasischke, K.A., Molloy, R.P. & Webb, W.W. In vivo multiphoton microscopy of deep brain tissue. J. Neurophysiol. 91, 1908–1912 (2004).

    Article  Google Scholar 

  41. Jung, J.C., Mehta, A.D., Aksay, E., Stepnoski, R. & Schnitzer, M.J. In vivo mammalian brain imaging using one- and two-photon fluorescence microendoscopy. J. Neurophysiol. 92, 3121–3133 (2004).

    Article  Google Scholar 

  42. Robinson, D.L. & Wightman, R.M. in Electrochemical Methods for Neuroscience Ch. 2, 17–34 (CRC Press, 2007).

  43. Okubo, Y. et al. Imaging extrasynaptic glutamate dynamics in the brain. Proc. Natl. Acad. Sci. USA 107, 6526–6531 (2010).

    Article  CAS  Google Scholar 

  44. Marvin, J.S. et al. An optimized fluorescent probe for visualizing glutamate neurotransmission. Nat. Methods 10, 162–170 (2013).

    Article  CAS  Google Scholar 

  45. Romo, R. & Schultz, W. Dopamine neurons of the monkey midbrain: contingencies of responses to active touch during self-initiated arm movements. J. Neurophysiol. 63, 592–606 (1990).

    Article  CAS  Google Scholar 

  46. Schultz, W., Apicella, P. & Ljungberg, T. Responses of monkey dopamine neurons to reward and conditioned stimuli during successive steps of learning a delayed response task. J. Neurosci. 13, 900–913 (1993).

    Article  CAS  Google Scholar 

  47. Kosobud, A.E., Harris, G.C. & Chapin, J.K. Behavioral associations of neuronal activity in the ventral tegmental area of the rat. J. Neurosci. 14, 7117–7129 (1994).

    Article  CAS  Google Scholar 

  48. Miller, J.D., Sanghera, M.K. & German, D.C. Mesencephalic dopaminergic unit activity in the behaviorally conditioned rat. Life Sci. 29, 1255–1263 (1981).

    Article  CAS  Google Scholar 

  49. Montague, P.R., Dayan, P. & Sejnowski, T.J. A framework for mesencephalic dopamine systems based on predictive Hebbian learning. J. Neurosci. 16, 1936–1947 (1996).

    Article  CAS  Google Scholar 

  50. Pan, W.X., Schmidt, R., Wickens, J.R. & Hyland, B.I. Dopamine cells respond to predicted events during classical conditioning: evidence for eligibility traces in the reward-learning network. J. Neurosci. 25, 6235–6242 (2005).

    Article  CAS  Google Scholar 

  51. 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 

  52. Nguyen, Q.-T., Driscoll, J., Dolnick, E.M. & Kleinfeld, D. in In Vivo. Optical Imaging of Brain Function 2nd edn. (ed. Frostig, R.D.) Ch. 4, 117–142 (CRC Press, 2009).

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Acknowledgements

We thank B. Conklin (University of California, San Francisco) for providing the Gqi5 cDNA, A. Schweitzer for assistance with the electronics, N. Taylor for assistance with screening of clones, and T. Komiyama and W. Schultz for discussions. This work was supported by research grants through the US National Institute on Drug Abuse (NIDA) (DA029706), the National Institute of Biomedical Imaging and Bioengineering (NIBIB) (EB003832), Hoffman-La Roche (88610A) and the “Neuroscience Related to Drugs of Abuse” training grant through NIDA (DA007315).

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Author notes

  1. Arnaud Muller and Victory Joseph: These authors contributed equally to this work.

Authors and Affiliations

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

    Arnaud Muller & David Kleinfeld

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

    Victory Joseph, Paul A Slesinger & David Kleinfeld

  3. Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, New York, USA

    Paul A Slesinger

  4. Section of Neurobiology, University of California at San Diego, La Jolla, California, USA

    David Kleinfeld

  5. Department of Electrical and Computer Engineering, University of California at San Diego, La Jolla, California, USA

    David Kleinfeld

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Contributions

All authors contributed to the experimental design and realization, analysis of the data and writing of the paper. A.M. and V.J. performed the in vitro testing and in vivo imaging and behavioral experiments. D.K. and P.A.S. dealt with the myriad of university organizations that govern animal health and welfare, surgical procedures, and laboratory health and safety issues that include specific oversight of chemicals, controlled substances, human cell lines, lasers and viruses.

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Correspondence to Paul A Slesinger or David Kleinfeld.

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Integrated supplementary information

Supplementary Figure 1 Selectivity of D2 and α1A CNiFERs characterized in vitro.

(a) D2‑CNiFER FRET response to 20 nM DA alone or in the presence of D1-receptor antagonist SCH 23390 (100nM, blue, n = 5; p = 0.89, t-test) or D2-receptor antagonist eticlopride (50nM, red, n = 5, p = 0.0003, t-test). (b) α1A-CNiFER response to 50 nM NE alone or in the presence of β-adrenergic receptor antagonist sotatol (5 µM, blue, n = 4; p = 0.17, t-test), or α1A‑receptor antagonist WB4101 (50 nM, red, n = 4, p = 0.0001, t-test). CNiFER response to agonist alone normalized to one, ** p < 0.001.

Supplementary Figure 2 In vitro characterization of CNiFERs to repeated pulses of agonist.

(a) Single-trial response of seven individual D2 CNiFERs and (b) seven individual α1A CNiFERs to a single 2.5 s pulse of 100 nM DA (left) or 100 nM NE (right).

Supplementary Figure 3 Identification of dopaminergic and noradrenergic projections to frontal cortex.

(a) Immunostaining for tyrosine hydroxylase (green), Fluorogold™ tracer (magenta) injected ~ 200 µm deep into frontal cortex (+1.5 mm A/P, +1.5 mm M/L), and NeuroTrace®, a Nissl stain that labels neurons (blue). Coronal sections including substantia nigra (SN) (left, A/P -3.5 mm) or locus coeruleus (LC) (right, A/P -5.6 mm). (b) Co-labeling of tyrosine hydroxylase (green) and Fluorogold™ (magenta) in SN (left) or LC (right), magnified from cyan boxes in (a). (c) Position of co-labeled cell bodies in SN (left) or LC (right) indicated by magenta dots imposed on three-dimensional reconstructions as outlined by grey in (a).

Supplementary Figure 4 Individual mouse FRET onset times plotted as a function of conditioning day.

Error bars represent standard error (n = 13). (a) Licking onset times during conditioning trials (CS, grey bar; US, dashed red line) across five days of conditioning. (b) D2‑CNiFER FRET response onset times during conditioning. FRET onset times are measured relative to CS onset (n = 13). (c) α1A‑CNIFER onset times during conditioning (n = 7). (d) M1‑CNiFER onset times during conditioning (n = 4). (e) Example of M1-CNiFER FRET response in a trial where the animal engaged in high frequency licking but there was no CS or US presentation.

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Muller, A., Joseph, V., Slesinger, P. et al. Cell-based reporters reveal in vivo dynamics of dopamine and norepinephrine release in murine cortex. Nat Methods 11, 1245–1252 (2014). https://doi.org/10.1038/nmeth.3151

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