Contribution of Cav1.2 channels to neuronal excitability and mitochondrial dynamics in primary culture of hippocampal neurons

The calcium ion is the main second messenger that helps to transmit depolarization status and synaptic activity to the biochemical machinery of neurons. Therefore Ca²⁺ regulation is a critical process in neurons, which have developed extensive and intricate Ca²⁺ signaling pathways. The Ca²⁺ ions enter the neuronal cytoplasm via voltage-gated calcium channels which are rather specifically distributed within brain sub-regions as well as within individual neurons. L-type calcium channels are known to play specific and important role in regulation of many signaling cascades in neurons. Therefore we analysed the contribution of Ca_V1.2 channel to the regulation of neuron excitability, calcium homeostasis, mitochondrial dynamics and neuron morphology in primary culture of hippocampal neurons from new born Wistar rats. We used siRNA technique to downregulate gene transcription. Neurons transfected with random siRNA  mixture were compared with neurons transfected with a mixture of three siRNAs specific for Ca_V1.2 channel. Gene expression was evaluated by RT-PCR. Patch clamp method in current-clamp mode was used to observe individual and series of action potentials (APs). Mitochondrial dynamics was estimated by confocal microscope using cotransfection with mitochondrially targeted KikumeGR1 protein. Intracellular calcium concentration was monitored using the ratiometric Ca²⁺ indicator dye Fluoro-4, AM. Experiments were performed with a confocal laser scanning microscope. Our results showed, that downregulation of Ca_V1.2 with siRNA significantly affected parameters of single AP. Number of APs in a series significantly decreased in siRNA trasfected cells compare to negative control. Transfection with siRNAs for Ca_V1.2 channel significantly decreased the average mitochondria lenght compare to mock-transfected neurons. The neurite lenght in all group was not affected neither was changed the number of mitochondrias in each neurite. Altered calcium signaling significantly decreased the lenght of the mitochondria and the frequency of mitochondrial fusion in Ca_V1.2  siRNA transfected neurons. Ca²⁺ respond was significantly lower in siRNA-Cav1.2 transfected neurons, compare to negative control. Our results showed, that Ca_V1.2 channel participate in observed processes.