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      Gap Junctions in the Nervous System: Probing Functional Connections Using New Imaging Approaches

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          Gap junctions are channels that physically connect adjacent cells, mediating the rapid exchange of small molecules, and playing an essential role in a wide range of physiological processes in nearly every system in the body, including the nervous system. Thus, altered function of gap junctions has been linked with a plethora of diseases and pathological conditions. Being able to measure and characterize the distribution, function, and regulation of gap junctions in intact tissue is therefore essential for understanding the physiological and pathophysiological roles that gap junctions play. In recent decades, several robust in vitro and in vivo methods have been developed for detecting and characterizing gap junctions. Here, we review the currently available methods with respect to invasiveness, signal-to-noise ratio, temporal resolution and others, highlighting the recently developed chemical tracers and hybrid imaging systems that use novel chemical compounds and/or genetically encoded enzymes, transporters, channels, and fluorescent proteins in order to map gap junctions. Finally, we discuss possible avenues for further improving existing techniques in order to achieve highly sensitive, cell type-specific, non-invasive measures of in vivo gap junction function with high throughput and high spatiotemporal resolution.

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          Most cited references 89

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          Ultra-sensitive fluorescent proteins for imaging neuronal activity

          Summary Fluorescent calcium sensors are widely used to image neural activity. Using structure-based mutagenesis and neuron-based screening, we developed a family of ultra-sensitive protein calcium sensors (GCaMP6) that outperformed other sensors in cultured neurons and in zebrafish, flies, and mice in vivo. In layer 2/3 pyramidal neurons of the mouse visual cortex, GCaMP6 reliably detected single action potentials in neuronal somata and orientation-tuned synaptic calcium transients in individual dendritic spines. The orientation tuning of structurally persistent spines was largely stable over timescales of weeks. Orientation tuning averaged across spine populations predicted the tuning of their parent cell. Although the somata of GABAergic neurons showed little orientation tuning, their dendrites included highly tuned dendritic segments (5 - 40 micrometers long). GCaMP6 sensors thus provide new windows into the organization and dynamics of neural circuits over multiple spatial and temporal scales.
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            Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.

            Microbial-type rhodopsins are found in archaea, prokaryotes, and eukaryotes. Some of them represent membrane ion transport proteins such as bacteriorhodopsin, a light-driven proton pump, or channelrhodopsin-1 (ChR1), a recently identified light-gated proton channel from the green alga Chlamydomonas reinhardtii. ChR1 and ChR2, a related microbial-type rhodopsin from C. reinhardtii, were shown to be involved in generation of photocurrents of this green alga. We demonstrate by functional expression, both in oocytes of Xenopus laevis and mammalian cells, that ChR2 is a directly light-switched cation-selective ion channel. This channel opens rapidly after absorption of a photon to generate a large permeability for monovalent and divalent cations. ChR2 desensitizes in continuous light to a smaller steady-state conductance. Recovery from desensitization is accelerated by extracellular H+ and negative membrane potential, whereas closing of the ChR2 ion channel is decelerated by intracellular H+. ChR2 is expressed mainly in C. reinhardtii under low-light conditions, suggesting involvement in photoreception in dark-adapted cells. The predicted seven-transmembrane alpha helices of ChR2 are characteristic for G protein-coupled receptors but reflect a different motif for a cation-selective ion channel. Finally, we demonstrate that ChR2 may be used to depolarize small or large cells, simply by illumination.
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              Bioorthogonal chemistry: fishing for selectivity in a sea of functionality.

              The study of biomolecules in their native environments is a challenging task because of the vast complexity of cellular systems. Technologies developed in the last few years for the selective modification of biological species in living systems have yielded new insights into cellular processes. Key to these new techniques are bioorthogonal chemical reactions, whose components must react rapidly and selectively with each other under physiological conditions in the presence of the plethora of functionality necessary to sustain life. Herein we describe the bioorthogonal chemical reactions developed to date and how they can be used to study biomolecules.

                Author and article information

                Front Cell Neurosci
                Front Cell Neurosci
                Front. Cell. Neurosci.
                Frontiers in Cellular Neuroscience
                Frontiers Media S.A.
                19 September 2018
                : 12
                1State Key Laboratory of Membrane Biology, Peking University School of Life Sciences , Beijing, China
                2PKU-IDG/McGovern Institute for Brain Research , Beijing, China
                3Peking-Tsinghua Center for Life Sciences , Beijing, China
                Author notes

                Edited by: Bradley James Baker, Korea Institute of Science and Technology (KIST), South Korea

                Reviewed by: Bela Volgyi, University of Pécs, Hungary; Pablo Jose Saez, Institut Curie, France

                Copyright © 2018 Dong, Liu and Li.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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                Figures: 2, Tables: 1, Equations: 0, References: 89, Pages: 9, Words: 0
                Cellular Neuroscience
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