A genetically targeted optical sensor to monitor calcium signals in astrocyte processes

Calcium signaling is studied as a potential form of astrocyte excitability that may control astrocyte involvement in synaptic and cerebrovascular regulation. Fundamental questions remain unanswered about astrocyte calcium signaling, as current methods can not resolve calcium in small volume compartments, such as near the cell membrane and in distal cell processes. We modified the genetically encoded calcium sensor GCaMP2 with a membrane-tethering domain, Lck, increasing the level of Lck-GCaMP2 near the plasma membrane tenfold as compared with conventional GCaMP2. Using Lck-GCaMP2 in rat hippocampal astrocyte-neuron cocultures, we measured near-membrane calcium signals that were evoked pharmacologically or by single action potential–mediated neurotransmitter release. Moreover, we identified highly localized and frequent spontaneous calcium signals in astrocyte somata and processes that conventional GCaMP2 failed to detect. Lck-GCaMP2 acts as a genetically targeted calcium sensor for monitoring calcium signals in previously inaccessible parts of astrocytes, including fine processes.

[1]  M. Sofroniew.  Molecular dissection of reactive astrogliosis and glial scar formation , 2009, Trends in Neurosciences.

[2]  B. Barres.  The Mystery and Magic of Glia: A Perspective on Their Roles in Health and Disease , 2008, Neuron.

[3]  Sreekanth H. Chalasani,et al.  Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators , 2009, Nature Methods.

[4]  Michael M. Halassa,et al.  Synaptic Islands Defined by the Territory of a Single Astrocyte , 2007, The Journal of Neuroscience.

[5]  D. Tank,et al.  Imaging Large-Scale Neural Activity with Cellular Resolution in Awake, Mobile Mice , 2007, Neuron.

[6]  H. Berg,et al.  Supporting Online Material Materials and Methods Som Text Figs. S1 to S7 Tables S1 to S3 References Movies S1 to S6 Tuned Responses of Astrocytes and Their Influence on Hemodynamic Signals in the Visual Cortex , 2022 .

[7]  E. Shigetomi,et al.  Tracking transmitter-gated P2X cation channel activation in vitro and in vivo , 2008, Nature Methods.

[8]  S. Oloff,et al.  Hippocampal astrocytes in situ exhibit calcium oscillations that occur independent of neuronal activity. , 2002, Journal of neurophysiology.

[9]  T. Pozzan,et al.  Intracellular Calcium Oscillations in Astrocytes: A Highly Plastic, Bidirectional Form of Communication between Neurons and Astrocytes In Situ , 1997, The Journal of Neuroscience.

[10]  L. Mucke,et al.  Fulminant Jejuno-Ileitis following Ablation of Enteric Glia in Adult Transgenic Mice , 1998, Cell.

[11]  Wei He,et al.  Locus coeruleus alpha-adrenergic-mediated activation of cortical astrocytes in vivo. , 2008, Cerebral cortex.

[12]  E. Shigetomi,et al.  Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes , 2009, Journal of visualized experiments : JoVE.

[13]  J. Ojemann,et al.  Uniquely Hominid Features of Adult Human Astrocytes , 2009, The Journal of Neuroscience.

[14]  T. Takano,et al.  Astrocytic Ca2+ signaling evoked by sensory stimulation in vivo , 2006, Nature Neuroscience.

[15]  J. Goldman,et al.  Cyclic AMP-induced shape changes of astrocytes are accompanied by rapid depolymerization of actin , 1990, Brain Research.

[16]  Neuronal Synchrony Mediated by Astrocytic Glutamate through Activation of Extrasynaptic NMDA Receptors , 2005, Neuron.

[17]  J. Jaiswal,et al.  Imaging single events at the cell membrane. , 2007, Nature chemical biology.

[18]  E. Newman,et al.  Potassium buffering in the central nervous system , 2004, Neuroscience.

[19]  G. Buzsáki,et al.  Calcium Dynamics of Cortical Astrocytic Networks In Vivo , 2004, PLoS biology.

[20]  G. Perea,et al.  Tripartite synapses: astrocytes process and control synaptic information , 2009, Trends in Neurosciences.

[21]  S. Finkbeiner,et al.  Glutamate induces calcium waves in cultured astrocytes: long-range glial signaling. , 1990, Science.

[22]  B. MacVicar,et al.  Astrocyte control of the cerebrovasculature , 2007, Glia.

[23]  Todd A Fiacco,et al.  Loss of IP3 Receptor-Dependent Ca2+ Increases in Hippocampal Astrocytes Does Not Affect Baseline CA1 Pyramidal Neuron Synaptic Activity , 2008, The Journal of Neuroscience.

[24]  S. Green,et al.  Ballistic labeling and dynamic imaging of astrocytes in organotypic hippocampal slice cultures , 2005, Journal of Neuroscience Methods.

[25]  Cathryn L. Kubera,et al.  Astrocytic Purinergic Signaling Coordinates Synaptic Networks , 2005, Science.

[26]  A. Araque,et al.  Tripartite synapses: glia, the unacknowledged partner , 1999, Trends in Neurosciences.

[27]  C. Iadecola,et al.  Glial regulation of the cerebral microvasculature , 2007, Nature Neuroscience.

[28]  Takahiro Takano,et al.  Loss of Astrocytic Domain Organization in the Epileptic Brain , 2008, The Journal of Neuroscience.

[29]  Todd A Fiacco,et al.  Selective Stimulation of Astrocyte Calcium In Situ Does Not Affect Neuronal Excitatory Synaptic Activity , 2007, Neuron.

[30]  A. Kocharyan,et al.  Transgenic mice expressing a cameleon fluorescent Ca2+ indicator in astrocytes and Schwann cells allow study of glial cell Ca2+ signals in situ and in vivo , 2009, Journal of Neuroscience Methods.

[31]  M. Ohkura,et al.  A high signal-to-noise Ca2+ probe composed of a single green fluorescent protein , 2001, Nature Biotechnology.

[32]  Dwight E. Bergles,et al.  Defining the Role of Astrocytes in Neuromodulation , 2007, Neuron.

[33]  E. Shigetomi,et al.  Two Forms of Astrocyte Calcium Excitability Have Distinct Effects on NMDA Receptor-Mediated Slow Inward Currents in Pyramidal Neurons , 2008, The Journal of Neuroscience.

[34]  A. Magee,et al.  Retargeting of cytosolic proteins to the plasma membrane by the Lck protein tyrosine kinase dual acylation motif. , 1997, Journal of cell science.

[35]  Vladimir Parpura,et al.  The trinity of Ca2+ sources for the exocytotic glutamate release from astrocytes , 2009, Neurochemistry International.

[36]  Y. Xing,et al.  A Transcriptome Database for Astrocytes, Neurons, and Oligodendrocytes: A New Resource for Understanding Brain Development and Function , 2008, The Journal of Neuroscience.

[37]  P. Haydon,et al.  Astrocytic glutamate targets NMDA receptors , 2007, The Journal of physiology.

[38]  Mark Ellisman,et al.  Protoplasmic Astrocytes in CA1 Stratum Radiatum Occupy Separate Anatomical Domains , 2002, The Journal of Neuroscience.

[39]  K. McCarthy,et al.  Hippocampal Astrocytes In Situ Respond to Glutamate Released from Synaptic Terminals , 1996, The Journal of Neuroscience.

[40]  P. Haydon,et al.  Physiological astrocytic calcium levels stimulate glutamate release to modulate adjacent neurons. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[41]  A. Reichenbach,et al.  Microdomains for neuron–glia interaction: parallel fiber signaling to Bergmann glial cells , 1999, Nature Neuroscience.

[42]  Grant R. Gordon,et al.  Astrocyte-Mediated Distributed Plasticity at Hypothalamic Glutamate Synapses , 2009, Neuron.

[43]  Todd A Fiacco,et al.  What Is the Role of Astrocyte Calcium in Neurophysiology? , 2008, Neuron.

[44]  C. J. Lee,et al.  Astrocytic control of synaptic NMDA receptors , 2007, The Journal of physiology.

[45]  Fang Liu,et al.  Glutamate-mediated astrocyte–neuron signalling , 1994, Nature.

[46]  F. Helmchen,et al.  Imaging cellular network dynamics in three dimensions using fast 3D laser scanning , 2007, Nature Methods.

[47]  Junichi Nakai,et al.  Characterization and Subcellular Targeting of GCaMP-Type Genetically-Encoded Calcium Indicators , 2008, PloS one.

[48]  K. McCarthy,et al.  Sorting out astrocyte physiology from pharmacology. , 2009, Annual review of pharmacology and toxicology.

[49]  Guy Salama,et al.  Imaging cellular signals in the heart in vivo: Cardiac expression of the high-signal Ca2+ indicator GCaMP2. , 2006, Proceedings of the National Academy of Sciences of the United States of America.