Abstract:

Objective To observe the development process of the neuronal synapse in cerebral cortex and basal ganglionic eminence （GE） regions of the mice， and to clarify the differences in the development of excitatory and inhibitory synapses in different brain regions in vivo and in vitro. Methods The female C57BL/6 mice were euthanized by cervical dislocation from the 13.5th day to the 15.5th day during the pregnancy， and the embryos were collected under the sterile conditions. The cortex and GE regions of brain tissue of the embryonic mice were gradually isolated under microscope. The primary neurons from the embryonic mice were cultured in vitro， and the cell samples were collected on the 3rd， 7th， 14th， and 21th days， respectively， and regarded as culture 3 d， 7 d， 14 d， and 21 d groups. The expression levels of postsynaptic density 95 （PSD95） and Gephyrin mRNA in the primary neurons from the cortex and GE regions of the mice in various groups were detected by real-time fluorescence quantitative PCR （RT-qPCR）method. Immunofluorescence method was used to detect the expression levels of vesicular glutamate transporter 1 （vGLUT1）， PSD95， vesicular GABA transporter （vGAT）， and Gephyrin proteins in the neurons from the cortex and GE regions of the mice in various groups. Immunofluorescence method was also used to detect the expression levels of vGLUT1 and vGAT proteins in the neurons from the cortical and GE regions in brain tissue of the embryonic mice. Results Compared with culture 3 d group， the expression levels of PSD95 and Gephyrin mRNA in cortex and GE regions of the mice in culture 14 d and 21 d groups were significantly increased （P<0.01）. Compared with cortex area， the expression level of Gephyrin mRNA in the neurons from GE region of the mice in culture 14 d group was significantly decreased （P<0.01）. The microscope observation results showed that the excitatory and inhibitory synapses in the neurons from cortex and GE regions of the mice in culture 14 d group showed preliminary development， with positive expression of relevant proteins； among them， the excitatory synaptic proteins showed more distinct positive expression in the cortex neurons， and the presynaptic vGLUT1 and postsynaptic PSD95 molecules exhibited co-localization in the cell bodies and protrusions of the cortical neurons； the inhibitory presynaptic vGAT protein and postsynaptic Gephyrin protein in the neurons from GE region also exhibited co-localization in the cell bodies and protrusions， and there were more distinct expressions of the presynaptic molecule proteins than postsynaptic molecule proteins. Compared with cortex region， the levels of vGLUT1 and PSD95 proteins in the neurons from GE region of the mice in culture 14 d group were significantly decreased （P<0.01）， while the levels of vGAT and gephyrin proteins were significantly increased （P<0.01）. In culture 21 d group， the positive expressions of synaptic protein in the neurons from cortex and GE regions were increased， and the excitatory and inhibitory synapses further matured and enhanced. In the neurons from cortex and GE regions， rich patterns of corresponding pre- and postsynaptic expression were formed in the cell bodies and protrusions， and synapse structures showed gradual， positive development， with more apparent expression of presynaptic molecules compared wih postsynaptic proteins. Compared with cortex region， the levels of vGLUT1 and PSD95 proteins in the neurons from GE region of the mice in culture 21 d group were significantly decreased （P<0.01）， and the levels of vGAT and Gephyrin proteins were significantly increased （P<0.01）. Compared with cortex region， the expression level of vGLUT1 protein in the neurons from GE region in brain tissue of the embryonic mice was significantly decreased （P<0.01）， while the expression level of vGAT protein was significantly increased （P<0.05）. Conclusion There are distinct differences in synaptic development between the neurons from cortex and GE regions, the excitatory synapses develope earlier in the cortical region and the inhibitory synapses develope earlier in the GE region. The region-specific development of synapses suggests that different types of neural diseases with different cell types might originate from different developmental processes.

Key words: Neuronal development, Excitatory synapses, Inhibitory synapses, Cortex, Ganglionic eminence