Calcium dynamics are altered in cortical neurons lacking the calmodulin-binding protein RC3

Authors

  • Jacqueline J. W. Van Dalen,

    1. Division of Pharmacology and Anatomy, Rudolf Magnus Institute for Neurosciences, University Medical Center, Utrecht, The Netherlands
    2. Department of Molecular Biology and
    3. Department of Neuropharmacology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
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  • Dan D. Gerendasy,

    1. Department of Molecular Biology and
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  • Pierre N. E. De Graan,

    1. Division of Pharmacology and Anatomy, Rudolf Magnus Institute for Neurosciences, University Medical Center, Utrecht, The Netherlands
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  • Loes H. Schrama,

    1. Division of Pharmacology and Anatomy, Rudolf Magnus Institute for Neurosciences, University Medical Center, Utrecht, The Netherlands
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  • Donna L. Gruol

    1. Department of Neuropharmacology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
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: Dr Donna L. Gruol, Department of Neuropharmacology, as above.
E-mail: gruol@scripps.edu

Abstract

RC3 is a neuronal calmodulin-binding protein and protein kinase C substrate that is thought to play an important regulatory role in synaptic transmission and neuronal plasticity. Two molecules known to regulate synaptic transmission and neuronal plasticity are Ca2+ and calmodulin, and proposed mechanisms of RC3 action involve both molecules. However, physiological evidence for a role of RC3 in neuronal Ca2+ dynamics is limited. In the current study we utilized cultured cortical neurons obtained from RC3 knockout (RC3−/−) and wildtype mice (RC3+/+) and fura-2-based microscopic Ca2+ imaging to investigate a role for RC3 in neuronal Ca2+ dynamics. Immunocytochemical characterization showed that the RC3−/− cultures lack RC3 immunoreactivity, whereas cultures prepared from wildtype mice showed RC3 immunoreactivity at all ages studied. RC3+/+ and RC3−/− cultures were indistinguishable with respect to neuron density, neuronal morphology, the formation of extensive neuritic networks and the presence of glial fibrillary acidic protein (GFAP)-positive astrocytes and γ-aminobutyric acid (GABA)ergic neurons. However, the absence of RC3 in the RC3−/− neurons was found to alter neuronal Ca2+ dynamics including baseline Ca2+ levels measured under normal physiological conditions or after blockade of synaptic transmission, spontaneous intracellular Ca2+ oscillations generated by network synaptic activity, and Ca2+ responses elicited by exogenous application of N-methyl-d-aspartate (NMDA) or class I metabotropic glutamate receptor agonists. Thus, significant changes in Ca2+ dynamics occur in cortical neurons when RC3 is absent and these changes do not involve changes in gross neuronal morphology or neuronal maturation. These data provide direct physiological evidence for a regulatory role of RC3 in neuronal Ca2+ dynamics.

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