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

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

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ISBN: 978-80-970712-6-4

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

Lucia Lichvárová1 , Michal Cagalinec2 , Zuzana Hodúrová , Allen Kaasik , Ľubica Lacinová
1 Institute of Molecular Physiology and Genetics, Slovak academy of Sciences, Vlárska 5, 833 34 Bratislava, Slovak Republic
2 Department of Pharmacology, Faculty of Medicine, University of Tartu, Ravila 19, 504 11 Tartu, Estonia
lucia.lichvarova@savba.sk

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 CaV1.2 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 CaV1.2 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 CaV1.2 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 CaV1.2 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 CaV1.2  siRNA transfected neurons. Ca2+ respond was significantly lower in siRNA-Cav1.2 transfected neurons, compare to negative control. Our results showed, that CaV1.2 channel participate in observed processes.

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Acknowledgements: Supported by APVV-0212-10 and by DoRa 5 scholarship.

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Diskusia

LINK PATCH CLAMP TECHNIGQUE
http://www.science-display.com/new/flashfile/pc.swf

LINK PATCH CLAMP TECHNIGQUE
http://www.science-display.com/new/flashfile/pc.swf

https://www.youtube.com/watch?v=bjPaZ6Gz5ck&feature=youtu.be

Hippocampal neurons were loaded with ratiometric Ca2+ indicator dye - Fluoro-4,AM.
Calcium uptake was induced by infusion of KCl (in 3rd second of the movie)
that causes plasma membrane depolarization and increase of fluorescence intensity ratio.

https://www.youtube.com/watch?v=A0FFDxaXQgk&feature=youtu.be

Example of the mitochondrial fusion and fission process.
For visualisation of fusion and fission events, we used mitochondrially targeted Kikume-Green protein. Mitochondria expressing Kikume-Green were irradiated using a 405-nm laser line and mito-Kikume-Green was converted into mito-Kikume-Red. The fusion events between red (photoactivated) and green (non-activated) mitochondria were easily recognizable because the contacting mitochondria turn yellow after exchanging their matrices.

I want to ask what´s the name of a device with which you analyse the electric potential in cell ? Could you analyse the electric potential in some intervals, for example every 5 seconds ?

Thank you very much for your question.
For studying the electrophysiological properties of biological membranes the patch clamp technique was used. The patch clamp technique permits high-resolution recording of the ionic currents flowing through plasma membrane of the cell and for recording electric potential. The principle of the method is to isolate a patch of membrane electrically from the external solution and to record current flowing into the patch. This is achieved by pressing a fire-polished glass pipette, which has been filled with a suitable electrolyte solution, against the surface of a cell and applying light suction. In different configurations, this technique has
allowed experimenters to record and manipulate the currents that flow either through single ion channels or those that flow across the whole plasma membrane. It is also possible to measure action potential or membrane potential of excitable cells, when different protocols with interval of recording are used.

Thank you very much for your answer :)