The Role Of Calmodulin In The Release Of Synaptic Vesicles From Mouse Primary Hippocampal Neurons

Background and Objective: Synapse is a key part of information transmission between neurons,which is composed of presynaptic membrane,synaptic cleft and postsynaptic membrane.The unidirectional flow of information between neurons is mainly mediated by the release of neurotransmitters from synaptic vesicles located in presynapses.When the presynaptic membrane is stimulated to depolarize,the calcium channels of the presynaptic membrane are opened,and calcium ions influx into the presynapses,causing the synaptic vesicles to fuse with the presynaptic membrane to release neurotransmitters through exocytosis.Then the vesicle components are recovered through endocytosis to maintain the sustainable release of synaptic vesicles.Therefore,the release of neurotransmitters by synaptic vesicles through exocytosis is essential for the transmission of information between neurons.Calmodulin(CaM)is a calcium-binding protein expressed in almost all eukaryotic cells.In mammals,CaM is mainly encoded by 3 different genes.Studies have found that CaM plays an important role in the process of synaptic information transmission,and its role in the recovery of synaptic vesicles has been reported.However,whether CaM regulates the release of synaptic vesicles is still controversial.Therefore,this project intends to design si RNA to specifically interfere with the CaM encoded by the three genes,and further analyze the effect of RNA interference with CaM on the release of neuronal synaptic vesicles and possible regulatory mechanisms.Methods: 1.Primary culture mouse hippocampal neurons,and use immunofluorescence experiment to analyze the proportion of mature neurons,which is convenient for followup experimental research;2.Use cell transfection,western blot and immunofluorescence experiments to analyze si RNA targeting 3 type CaM genes interference efficiency.3.Transfect the plasmid Synapto-p Hluorin that can label vesicle release into primary mouse hippocampal neurons,and use live cell real-time microscopic imaging technology to detect the release of synaptic vesicles in hippocampal neurons after electrical stimulation,and analyze knockdown The effect of CaM on the release of synaptic vesicles.4.Using transmission electron microscopy to detect the changes in the number of presynaptic membrane vesicles of hippocampal neurons after inhibiting CaM.5.Using whole-cell patch clamp technology,record the resting potential and action potential changes of primary hippocampal neurons,and analyze the effect of knocking down CaM on neuronal excitability.6.Using the methods of nascent RNA determination and nascent protein determination,detect the new ribosomal RNA and nascent protein synthesis of hippocampal neurons after knocking down CaM.Results: 1.Among the primary neurons extracted from the hippocampus,mature neurons can reach 95%,and glutamatergic neurons account for 100% of mature neurons.2.Construct siRNA interference sequence and successfully knock down the expression of CaM in mouse hippocampal neurons.3.After RNA interference to CaM,the exocytosis of synaptic vesicles was inhibited.4.After down-regulating the expression of CaM,the generation of synaptic vesicles decreases.5.Inhibiting the function of CaM causes the excitability of neurons to decrease.6.Inhibition of CaM reduces neuronal protein synthesis.Conclusion: 1.Using si RNA can effectively knock down the expression of CaM in primary hippocampal neurons.2.Knockdown of CaM can inhibit the release of synaptic vesicles in hippocampal neurons.3.CaM may regulate synaptic transmission in two ways,one is to regulate the excitability of neurons and affect the release of synaptic vesicles,and the other is to regulate protein regeneration and affect the generation of synaptic vesicles.