title: Calcium dynamics are altered in cortical neurons lacking the calmodulin-binding protein RC3.
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 Ca(2+) and calmodulin, and proposed mechanisms of RC3 action involve both molecules. However, physiological evidence for a role of RC3 in neuronal Ca(2+) 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 Ca(2+) imaging to investigate a role for RC3 in neuronal Ca(2+) 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 gamma-aminobutyric acid (GABA)ergic neurons. However, the absence of RC3 in the RC3-/- neurons was found to alter neuronal Ca(2+) dynamics including baseline Ca(2+) levels measured under normal physiological conditions or after blockade of synaptic transmission, spontaneous intracellular Ca(2+) oscillations generated by network synaptic activity, and Ca(2+) responses elicited by exogenous application of N-methyl-D-aspartate (NMDA) or class I metabotropic glutamate receptor agonists. Thus, significant changes in Ca(2+) 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 Ca(2+) dynamics.
authors.affiliation: Division of Pharmacology and Anatomy, Rudolf Magnus Institute for Neurosciences, University Medical Center, Utrecht, The Netherlands.
authors.forename: Jacqueline J W
authors.initials: JJ
authors.lastname: van Dalen
authors.forename: Dan D
authors.initials: DD
authors.lastname: Gerendasy
authors.forename: Pierre N E
authors.initials: PN
authors.lastname: de Graan
authors.forename: Loes H
authors.initials: LH
authors.lastname: Schrama
authors.forename: Donna L
authors.initials: DL
authors.lastname: Gruol
mesh_terms: D000818:Animals; D002118:Calcium / Q000502:physiology*; D020013:Calcium Signaling / Q000502:physiology; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000502:physiology*; D005456:Fluorescent Dyes; D016257:Fura-2; D007150:Immunohistochemistry; D008274:Magnesium / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D051579:Neurogranin; D009473:Neuronal Plasticity / Q000502:physiology; D009474:Neurons / Q000378:metabolism*; D010641:Phenotype; D018094:Receptors, Metabotropic Glutamate / Q000819:agonists; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism / Q000502:physiology; D009435:Synaptic Transmission / Q000187:drug effects; D013779:Tetrodotoxin / Q000494:pharmacology
chemical_list: D002148:Calmodulin-Binding Proteins; D005456:Fluorescent Dyes; D009419:Nerve Tissue Proteins; C496650:Nrgn protein, mouse; D018094:Receptors, Metabotropic Glutamate; D016194:Receptors, N-Methyl-D-Aspartate; C104077:metabotropic glutamate receptor type 1; D051579:Neurogranin; D013779:Tetrodotoxin; D008274:Magnesium; D002118:Calcium; D016257:Fura-2