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      All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins.

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          Abstract

          All-optical electrophysiology-spatially resolved simultaneous optical perturbation and measurement of membrane voltage-would open new vistas in neuroscience research. We evolved two archaerhodopsin-based voltage indicators, QuasAr1 and QuasAr2, which show improved brightness and voltage sensitivity, have microsecond response times and produce no photocurrent. We engineered a channelrhodopsin actuator, CheRiff, which shows high light sensitivity and rapid kinetics and is spectrally orthogonal to the QuasArs. A coexpression vector, Optopatch, enabled cross-talk-free genetically targeted all-optical electrophysiology. In cultured rat neurons, we combined Optopatch with patterned optical excitation to probe back-propagating action potentials (APs) in dendritic spines, synaptic transmission, subcellular microsecond-timescale details of AP propagation, and simultaneous firing of many neurons in a network. Optopatch measurements revealed homeostatic tuning of intrinsic excitability in human stem cell-derived neurons. In rat brain slices, Optopatch induced and reported APs and subthreshold events with high signal-to-noise ratios. The Optopatch platform enables high-throughput, spatially resolved electrophysiology without the use of conventional electrodes.

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          A simple method for organotypic cultures of nervous tissue.

          L Stoppini, P A Buchs, D. Müller (1991)
          Hippocampal slices prepared from 2-23-day-old neonates were maintained in culture at the interface between air and a culture medium. They were placed on a sterile, transparent and porous membrane and kept in petri dishes in an incubator. No plasma clot or roller drum were used. This method yields thin slices which remain 1-4 cell layers thick and are characterized by a well preserved organotypic organization. Pyramidal neurons labelled by extra- and intracellular application of horse radish peroxidase resemble by the organization and complexity of their dendritic processes those observed in situ at a comparable developmental stage. Excitatory and inhibitory synaptic potentials can easily be analysed using extra- or intracellular recording techniques. After a few days in culture, long-term potentiation of synaptic responses can reproducibly be induced. Evidence for a sprouting response during the first days in culture or following sections is illustrated. This technique may represent an interesting alternative to roller tube cultures for studies of the developmental changes occurring during the first days or weeks in culture.
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            A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells.

            Maria C. N. Marchetto, Cassiano Carromeu, Allan Acab … (2010)
            Autism spectrum disorders (ASD) are complex neurodevelopmental diseases in which different combinations of genetic mutations may contribute to the phenotype. Using Rett syndrome (RTT) as an ASD genetic model, we developed a culture system using induced pluripotent stem cells (iPSCs) from RTT patients' fibroblasts. RTT patients' iPSCs are able to undergo X-inactivation and generate functional neurons. Neurons derived from RTT-iPSCs had fewer synapses, reduced spine density, smaller soma size, altered calcium signaling and electrophysiological defects when compared to controls. Our data uncovered early alterations in developing human RTT neurons. Finally, we used RTT neurons to test the effects of drugs in rescuing synaptic defects. Our data provide evidence of an unexplored developmental window, before disease onset, in RTT syndrome where potential therapies could be successfully employed. Our model recapitulates early stages of a human neurodevelopmental disease and represents a promising cellular tool for drug screening, diagnosis and personalized treatment. Copyright © 2010 Elsevier Inc. All rights reserved.
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              Automated analysis of cellular signals from large-scale calcium imaging data.

              Eran Mukamel, Axel Nimmerjahn, Mark J. Schnitzer (2009)
              Recent advances in fluorescence imaging permit studies of Ca(2+) dynamics in large numbers of cells, in anesthetized and awake behaving animals. However, unlike for electrophysiological signals, standardized algorithms for assigning optically recorded signals to individual cells have not yet emerged. Here, we describe an automated sorting procedure that combines independent component analysis and image segmentation for extracting cells' locations and their dynamics with minimal human supervision. In validation studies using simulated data, automated sorting significantly improved estimation of cellular signals compared to conventional analysis based on image regions of interest. We used automated procedures to analyze data recorded by two-photon Ca(2+) imaging in the cerebellar vermis of awake behaving mice. Our analysis yielded simultaneous Ca(2+) activity traces for up to >100 Purkinje cells and Bergmann glia from single recordings. Using this approach, we found microzones of Purkinje cells that were stable across behavioral states and in which synchronous Ca(2+) spiking rose significantly during locomotion.
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                Author and article information

                Journal
                Journal ID (iso-abbrev): Nat. Methods
                Title: Nature methods
                Publisher: Springer Nature
                ISSN (Electronic): 1548-7105
                ISSN (Print): 1548-7091
                Publication date (Electronic): Aug 2014
                Volume: 11
                Issue: 8
                Affiliations
                [1 ] 1] Applied Physics Program, School of Engineering and Applied Science, Harvard University, Cambridge, Massachusetts, USA. [2].
                [2 ] 1] Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada. [2].
                [3 ] Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.
                [4 ] 1] The MIT Media Laboratory, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA. [2] Department of Biological Engineering, MIT, Cambridge, Massachusetts, USA. [3] Department of Brain and Cognitive Sciences, MIT, Cambridge, Massachusetts, USA. [4] McGovern Institute for Brain Research, MIT, Cambridge, Massachusetts, USA.
                [5 ] Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA.
                [6 ] Department of Physics, Harvard University, Cambridge, Massachusetts, USA.
                [7 ] Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, USA.
                [8 ] Institute of Botany, Cologne Biocenter, University of Cologne, Cologne, Germany.
                [9 ] 1] Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada. [2] Department of Medicine, University of Alberta, Edmonton, Alberta, Canada. [3] Beijing Genomics Institute-Shenzhen, Shenzhen, China.
                [10 ] Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
                [11 ] 1] Department of Neurobiology, Harvard Medical School, Boston, Massachusetts, USA. [2] Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.
                [12 ] 1] The MIT Media Laboratory, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA. [2] Department of Biological Engineering, MIT, Cambridge, Massachusetts, USA. [3] Department of Brain and Cognitive Sciences, MIT, Cambridge, Massachusetts, USA. [4] McGovern Institute for Brain Research, MIT, Cambridge, Massachusetts, USA. [5].
                [13 ] 1] Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA. [2] Department of Physics, Harvard University, Cambridge, Massachusetts, USA. [3] Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.
                Article
                Publisher Item ID: nmeth.3000 Mid ID: NIHMS600566
                DOI: 10.1038/nmeth.3000
                PMC ID: 4117813
                PubMed ID: 24952910
                SO-VID: 73d769c2-3a19-4f91-8103-c6fc8541b824
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