Analysis of local neural circuit in the central nervous system

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To reveal the local circuit in the central nervous system, we examine the circuit under the strategy of 'From one to group'.1) Single GABAergic neurons were labeled by intracellular staining after whole cell clamp in the cortical slices of adult VGAT/GFP transgenic rats. We analyzed the contacts of these GABAergic axons onto the dendrites of corticospinal neurons, which were retrogradely labeled before the slice preparation.2) To visualize the whole dendrites of a specific group of neurons, we developed a dendritic membrane-targeted reporter protein (myrGFP-LDLRCT) using a test system of lentiviral vectors. We further produced three kinds of transgenic mice with Thy1 promoter, GAD67 short promoter, and parvalbumin BAC promoter. In all the mice, we found that the dendrites and cell bodies of a specific neuron group were completely labeled with GFP fluorescence or immunofluorescence. Thus, we can now use these mice in the 'From one to group' study combining with an intracellular staining technique.3) To visualize the whole dendrites of a specific group of neurons, we also developed a viral vectors for retrograde labeling. We have tried pseudorabies virus, rabies virus glycoprotein (RVG)-pseudotyped Sindbis virus, and RVG-pseudotyped lentivirus, and got a partial success in Sindbis virus and lentivirus. In addition, when myrGFP-LDLRCT-expressing adenovirus was injected into the thalamus with 0.6 M NaCl, the dendrites of corticothalamic neurons were efficiently labeled. Thus, we have started the analysis on the local connection of a pyramidal cell to corticothalamic neurons.4) In the striatum, single medium-sized spiny neurons were labeled with Sindbis virus exressing membrane-targeted GFP. Cortical and thalamic inputs to the single neurons were examined with VGluT1 and VGluT2 immunoreactivities. This may be a 'From group to one' analysis of neural circuit.