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

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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.

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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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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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The authors declare no competing financial interests.

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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