The Establishment Of An Optogenetic Model In Order To Research On The Function Of Hippocampal Neural Circuit

Neural circuit, being constituted by a series of linked nerve cells, has been identified asthe construction of functional nerve fiber connections. The synaptic connections could bedefined as a circuit typically containing a trisynaptic loop with a dense tangle of dendrites,axons terminals, and glial cell processes. The synaptic connections constituted neural circuitscontain extreme complexity in information transferring and processing to bring out thefunction of the brain. The hippocampus is a brain region of the central nervous system playingan important role in learning and memory. The neural circuit of hippocampal dentategyrus-CA3-CA1region is one of the most important neuronal circuits involved in epilepsyand drug addiction.Optogenetic manipulation is a new kind of experimental technique being applied in thestudy of neural circuits function. The neurons expressing the light-sensitive protein can beactivated by illumination, which could be employed in the study of the establishment,functions, and associated proteins of a specific neural circuit in a certain physiological orpathological processes in vivo. The Thy1-ChR2-EYFP is a transgenic mouse strain, in whichthe Channelrhodopsin-2is integrated into the mouse genome. Channelrhodopsin-2is a kind oflight sensitive protein, which can be achieved to be stably expressed in the offspring of thetransgenic mouse. In this study, we identified the genotype of the Thy1-ChR2-EYFPtransgenic mouse strain and detected the expression of ChR2protein in the hippocampal CA3and CA1pyramidal neurons. This transgenic mouse strain laid the foundation for thefunctional study in the neural circuits of hippocampal dentate gyrus-CA3-CA1region.Myosin X (Myo X) is a newly discovered unconventional myosin being only expressedin vertebrates. Our former works have tested the expression of Myo X in axons and dendritesof hippocampal neurons cultured in vitro and silent the expression of Myo X affected thedevelopment of the axon; in vivo experiments showed that the silence of Myo X impacthippocampal function. In order to study the functions of Myo X in the neural circuits of thehippocampal dentate gyrus-CA3-CA1, we made the rabbit polyclonal antibodies againstMyosin X. In conclusion, the Myosin X polyclonal antibody was successfully prepared,which provided an efficient material for further studies in the expression and distribution ofMyosin X both in tissues and cells.Two kinds of Myo X have been found expressed in the process of brain development,including the full-length Myo X, which is approximately240kDa, and the headless Myo X,which is about160kDa. Compared to the full-length Myo X, the headless Myo X lacks a myosin head domain. This kind of headless Myo X is thus unable to function as a molecularmotor.In order to analyze the function of two different forms of Myo X in the neural circuit ofhippocampal dentate gyrus-CA3-CA1region in the brain development, we designed twokinds of lentivirus silencing plasmids that targeted the5’UTR of the full-length and headlessMyo X respectively. These core viral plasmids were employed to transfect293FT cellstogether with another two kinds of packaging plasmids. After then, the virus was collectedfrom the culture media., To test the virus titer, HeLa cells were transfected with the virus, andthe virus were finally purified and concentrated.We plan to use the positioning injection, fiber embedding technology and multi-channelrecording system to map the neural circuits of the hippocampus dentate gyrus-CA3-CA1region of the mouse brain in vivo. We will further study the function of myosin X in thehippocampus dentate gyrus-CA3-CA1region.