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      Differential Calcium Signaling Mediated by Voltage-Gated Calcium Channels in Rat Retinal Ganglion Cells and Their Unmyelinated Axons

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          Abstract

          Aberrant calcium regulation has been implicated as a causative factor in the degeneration of retinal ganglion cells (RGCs) in numerous injury models of optic neuropathy. Since calcium has dual roles in maintaining homeostasis and triggering apoptotic pathways in healthy and injured cells, respectively, investigation of voltage-gated Ca channel (VGCC) regulation as a potential strategy to reduce the loss of RGCs is warranted. The accessibility and structure of the retina provide advantages for the investigation of the mechanisms of calcium signalling in both the somata of ganglion cells as well as their unmyelinated axons. The goal of the present study was to determine the distribution of VGCC subtypes in the cell bodies and axons of ganglion cells in the normal retina and to define their contribution to calcium signals in these cellular compartments. We report L-type Ca channel α1C and α1D subunit immunoreactivity in rat RGC somata and axons. The N-type Ca channel α1B subunit was in RGC somata and axons, while the P/Q-type Ca channel α1A subunit was only in the RGC somata. We patch clamped isolated ganglion cells and biophysically identified T-type Ca channels. Calcium imaging studies of RGCs in wholemounted retinas showed that selective Ca channel antagonists reduced depolarization-evoked calcium signals mediated by L-, N-, P/Q- and T-type Ca channels in the cell bodies but only by L-type Ca channels in the axons. This differential contribution of VGCC subtypes to calcium signals in RGC somata and their axons may provide insight into the development of target-specific strategies to spare the loss of RGCs and their axons following injury.

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          Most cited references48

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          Identification and differential subcellular localization of the neuronal class C and class D L-type calcium channel alpha 1 subunits

          To identify and localize the protein products of genes encoding distinct L-type calcium channels in central neurons, anti-peptide antibodies specific for the class C and class D alpha 1 subunits were produced. Anti-CNC1 directed against class C immunoprecipitated 75% of the L-type channels solubilized from rat cerebral cortex and hippocampus. Anti-CND1 directed against class D immunoprecipitated only 20% of the L-type calcium channels. Immunoblotting revealed two size forms of the class C L-type alpha 1 subunit, LC1 and LC2, and two size forms of the class D L-type alpha 1 subunit, LD1 and LD2. The larger isoforms had apparent molecular masses of approximately 200-210 kD while the smaller isoforms were 180-190 kD, as estimated from electrophoresis in gels polymerized from 5% acrylamide. Immunocytochemical studies using CNC1 and CND1 antibodies revealed that the alpha 1 subunits of both L-type calcium channel subtypes are localized mainly in neuronal cell bodies and proximal dendrites. Relatively dense labeling was observed at the base of major dendrites in many neurons. Staining in more distal dendritic regions was faint or undetectable with CND1, while a more significant level of staining of distal dendrites was observed with CNC1, particularly in the dentate gyrus and the CA2 and CA3 areas of the hippocampus. Class C calcium channels were concentrated in clusters, while class D calcium channels were generally distributed in the cell surface membrane of cell bodies and proximal dendrites. Our results demonstrate multiple size forms and differential localization of two subtypes of L-type calcium channels in the cell bodies and proximal dendrites of central neurons. The differential localization and multiple size forms may allow these two channel subtypes to participate in distinct aspects of electrical signal integration and intracellular calcium signaling in neuronal cell bodies. The preferential localization of these calcium channels in cell bodies and proximal dendrites implies their involvement in regulation of calcium-dependent functions occurring in those cellular compartments such as protein phosphorylation, enzyme activity, and gene expression.
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            Axonal self-destruction and neurodegeneration.

            Neurons seem to have at least two self-destruct programs. Like other cell types, they have an intracellular death program for undergoing apoptosis when they are injured, infected, or not needed. In addition, they apparently have a second, molecularly distinct self-destruct program in their axon. This program is activated when the axon is severed and leads to the rapid degeneration of the isolated part of the cut axon. Do neurons also use this second program to prune their axonal tree during development and to conserve resources in response to chronic insults?
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              Calcium microdomains: organization and function.

              Microdomains of Ca(2+), which are formed at sites where Ca(2+) enters the cytoplasm either at the cell surface or at the internal stores, are a key element of Ca(2+) signalling. The term microdomain includes the elementary events that are the basic building blocks of Ca(2+) signals. As Ca(2+) enters the cytoplasm, it produces a local plume of Ca(2+) that has been given different names (sparks, puffs, sparklets and syntillas). These elementary events can combine to produce larger microdomains. The significance of these localized domains of Ca(2+) is that they can regulate specific cellular processes in different regions of the cell. Such microdomains are particularly evident in neurons where both pre- and postsynaptic events are controlled by highly localized pulses of Ca(2+). The ability of single neurons to process enormous amounts of information depends upon such miniaturization of the Ca(2+) signalling system. Control of cardiac cell contraction and gene transcription provides another example of how the parallel processing of Ca(2+) signalling can occur through microdomains of intracellular Ca(2+).
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2014
                8 January 2014
                : 9
                : 1
                : e84507
                Affiliations
                [1 ]Department of Neurobiology and Jules Stein Eye Institute, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America
                [2 ]Veterans Administration Greater Los Angeles Healthcare System, Los Angeles, California, United States of America
                [3 ]Departments of Physiology & Biophysics and Ophthalmology & Visual Sciences, Dalhousie University, Halifax, Nova Scotia, Canada
                Vanderbilt University Medical Center, United States of America
                Author notes

                Competing Interests: Steven Barnes, is a PLOS ONE Editorial Board member. The authors confirm that this fact does not alter their adherence to all the PLOS ONE policies on sharing data and materials.

                Conceived and designed the experiments: AS SB NB. Performed the experiments: AS XS. Analyzed the data: AS XS SB. Wrote the paper: AS SB NB. Funding: NB.

                Article
                PONE-D-13-29719
                10.1371/journal.pone.0084507
                3885580
                24416240
                09a6d459-b945-4401-bb32-f6d9d1606601
                Copyright @ 2014

                This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

                History
                : 18 July 2013
                : 20 November 2013
                Page count
                Pages: 10
                Funding
                Support for these studies came from NIH EY04067 (NCB), a VA Merit Review (NCB), a CIHR and NSHRF Regional Partnership Program grant (SB), and NSERC (SB). Part of this work was made possible by a contract agreement awarded to NCB and administered by the U.S. Army Medical Research & Materiel Command (USAMRMC) and the Telemedicine & Advanced Technology Research Center (TATRC) at Fort Detrick, MD under Contract Number: W81XWH-10-2-0077. NCB is a VA Senior Career Research Scientist. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Anatomy and Physiology
                Neurological System
                Sensory Physiology
                Ocular System
                Ocular Anatomy
                Cell Physiology
                Electrophysiology
                Model Organisms
                Animal Models
                Rat
                Neuroscience
                Cellular Neuroscience
                Ion Channels
                Neuronal Morphology
                Sensory Systems
                Visual System
                Medicine
                Ophthalmology
                Glaucoma
                Retinal Disorders

                Uncategorized
                Uncategorized

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