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      ReaChR: A red-shifted variant of channelrhodopsin enables deep transcranial optogenetic excitation


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          Channelrhodopsins are used to optogenetically depolarize neurons. We engineered a variant of channelrhodopsin, denoted Re d- a ctivatable Ch annel r hodopsin (ReaChR), that is optimally excited with orange to red light (λ ~ 590 to 630 nm) and offers improved membrane trafficking, higher photocurrents, and faster kinetics compared with existing red-shifted channelrhodopsins. Red light is more weakly scattered by tissue and absorbed less by blood than the blue to green wavelengths required by other channelrhodopsin variants. ReaChR expressed in vibrissa motor cortex was used to drive spiking and vibrissa motion in awake mice when excited with red light through intact skull. Precise vibrissa movements were evoked by expressing ReaChR in the facial motor nucleus in the brainstem and illuminating with red light through the external auditory canal. Thus, ReaChR enables transcranial optical activation of neurons in deep brain structures without the need to surgically thin the skull, form a transcranial window, or implant optical fibers.

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          Most cited references 33

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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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            Neural substrates of awakening probed with optogenetic control of hypocretin neurons.

            The neural underpinnings of sleep involve interactions between sleep-promoting areas such as the anterior hypothalamus, and arousal systems located in the posterior hypothalamus, the basal forebrain and the brainstem. Hypocretin (Hcrt, also known as orexin)-producing neurons in the lateral hypothalamus are important for arousal stability, and loss of Hcrt function has been linked to narcolepsy. However, it is unknown whether electrical activity arising from Hcrt neurons is sufficient to drive awakening from sleep states or is simply correlated with it. Here we directly probed the impact of Hcrt neuron activity on sleep state transitions with in vivo neural photostimulation, genetically targeting channelrhodopsin-2 to Hcrt cells and using an optical fibre to deliver light deep in the brain, directly into the lateral hypothalamus, of freely moving mice. We found that direct, selective, optogenetic photostimulation of Hcrt neurons increased the probability of transition to wakefulness from either slow wave sleep or rapid eye movement sleep. Notably, photostimulation using 5-30 Hz light pulse trains reduced latency to wakefulness, whereas 1 Hz trains did not. This study establishes a causal relationship between frequency-dependent activity of a genetically defined neural cell type and a specific mammalian behaviour central to clinical conditions and neurobehavioural physiology.
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              Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications.

              Yellow mutants of the green fluorescent protein (YFP) are crucial constituents of genetically encoded indicators of signal transduction and fusions to monitor protein-protein interactions. However, previous YFPs show excessive pH sensitivity, chloride interference, poor photostability, or poor expression at 37 degrees C. Protein evolution in Escherichia coli has produced a new YFP named Citrine, in which the mutation Q69M confers a much lower pK(a) (5.7) than for previous YFPs, indifference to chloride, twice the photostability of previous YFPs, and much better expression at 37 degrees C and in organelles. The halide resistance is explained by a 2.2-A x-ray crystal structure of Citrine, showing that the methionine side chain fills what was once a large halide-binding cavity adjacent to the chromophore. Insertion of calmodulin within Citrine or fusion of cyan fluorescent protein, calmodulin, a calmodulin-binding peptide and Citrine has generated improved calcium indicators. These chimeras can be targeted to multiple cellular locations and have permitted the first single-cell imaging of free [Ca(2+)] in the Golgi. Citrine is superior to all previous YFPs except when pH or halide sensitivity is desired and is particularly advantageous within genetically encoded fluorescent indicators of physiological signals.

                Author and article information

                Nat Neurosci
                Nat. Neurosci.
                Nature neuroscience
                25 September 2013
                01 September 2013
                October 2013
                01 April 2014
                : 16
                : 10
                : 1499-1508
                [1 ]Department of Pharmacology, University of California, San Diego
                [2 ]Department of Physics, University of California, San Diego
                [3 ]Section of Neurobiology, University of California, San Diego
                [4 ]Howard Hughes Medical Institute
                Author notes
                Correspondence: John Y. Lin, Department of Pharmacology, University of California, 9500 Gilman Drive, j8lin@ 123456ucsd.edu

                The authors contributed equally to this work

                Funded by: National Institute of Neurological Disorders and Stroke : NINDS
                Award ID: R01 NS058668 || NS
                Funded by: National Institute of Neurological Disorders and Stroke : NINDS
                Award ID: R01 NS027177 || NS
                Funded by: National Institute of Mental Health : NIMH
                Award ID: R01 MH059867 || MH
                Funded by: National Institute of Biomedical Imaging and Bioengineering : NIBIB
                Award ID: R01 EB003832 || EB
                Funded by: National Institute on Drug Abuse : NIDA
                Award ID: R01 DA029706 || DA
                Funded by: Office of the Director : NIH
                Award ID: DP1 OD006831 || OD
                Funded by: National Institute of Neurological Disorders and Stroke : NINDS
                Award ID: DP1 NS082097 || NS
                Funded by: Howard Hughes Medical Institute :
                Award ID: || HHMI_


                brainstem, cell culture, light-activated, motor cortex, vibrissae


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