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      Next-generation GRAB sensors for monitoring dopaminergic activity in vivo


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          Dopamine (DA) plays a critical role in the brain, and the ability to directly measure dopaminergic activity is essential for understanding its physiological functions. We therefore developed red fluorescent GPCR-activation–based DA (GRAB DA) sensors and optimized versions of green fluorescent GRAB DA sensors. In response to extracellular DA, both the red and green GRAB DA sensors exhibit a large increase in fluorescence, with subcellular resolution, subsecond kinetics, and nanomolar to submicromolar affinity. Moreover, the GRAB DA sensors resolve evoked DA release in mouse brain slices, detect evoked compartmental DA release from a single neuron in live flies, and report optogenetically elicited nigrostriatal DA release as well as mesoaccumbens dopaminergic activity during sexual behavior in freely behaving mice. Co-expressing red GRAB DA with either green GRAB DA or the calcium indicator GCaMP6s allows simultaneously tracking neuronal activity and dopaminergic signaling in distinct circuits in vivo.

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          Most cited references56

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          Enzymatic assembly of DNA molecules up to several hundred kilobases.

          We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular engineering tool.
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            Neocortical excitation/inhibition balance in information processing and social dysfunction.

            Severe behavioural deficits in psychiatric diseases such as autism and schizophrenia have been hypothesized to arise from elevations in the cellular balance of excitation and inhibition (E/I balance) within neural microcircuitry. This hypothesis could unify diverse streams of pathophysiological and genetic evidence, but has not been susceptible to direct testing. Here we design and use several novel optogenetic tools to causally investigate the cellular E/I balance hypothesis in freely moving mammals, and explore the associated circuit physiology. Elevation, but not reduction, of cellular E/I balance within the mouse medial prefrontal cortex was found to elicit a profound impairment in cellular information processing, associated with specific behavioural impairments and increased high-frequency power in the 30-80 Hz range, which have both been observed in clinical conditions in humans. Consistent with the E/I balance hypothesis, compensatory elevation of inhibitory cell excitability partially rescued social deficits caused by E/I balance elevation. These results provide support for the elevated cellular E/I balance hypothesis of severe neuropsychiatric disease-related symptoms.
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              Optimization of a GCaMP calcium indicator for neural activity imaging.

              Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of "GCaMP5" sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.

                Author and article information

                Nat Methods
                Nat Methods
                Nature methods
                20 September 2020
                21 October 2020
                November 2020
                21 April 2021
                : 17
                : 11
                : 1156-1166
                [1 ]State Key Laboratory of Membrane Biology, Peking University School of Life Sciences, 100871 Beijing, China
                [2 ]PKU-IDG/McGovern Institute for Brain Research, 100871 Beijing, China
                [3 ]Peking-Tsinghua Center for Life Sciences, 100871 Beijing, China
                [4 ]School of Life Sciences, Tsinghua University, Beijing 100084, China
                [5 ]Neurobiology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA
                [6 ]Neuroscience Institute, Department of Psychiatry, New York University School of Medicine, New York, NY 10016, USA
                [7 ]These authors contributed equally.
                Author notes

                Author contributions

                Y.L. supervised the study. F.S., Y.L. designed the study. F.S., Y. Zhuo, Y. Zhang and C.Q. performed the experiments related to developing, optimizing, and characterizing the sensors in cultured HEK293T cells and neurons with help from J.F. and H.D.. F.S. and T.Q. performed the surgery and 2-photon imaging experiments related to the validation of the sensors in acute brain slices. J. Zeng, X.L., Y.W., and K.T. performed the 2-photon imaging experiments in transgenic flies. J. Zhou performed the fiber photometry recordings during optogenetics in freely moving mice under the supervision of G.C.. B.D. performed the fiber photometry recordings in the mouse NAc during sexual behavior under the supervision of D.L.. All authors contributed to the data interpretation and analysis. F.S. and Y.L. wrote the manuscript with input from all authors.


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