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 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.