| BackgroundAutism is a kind of severe neurodevelopmental diseases caused by neurodevelopmnental disfunction which is characterized by impaired social interaction,language communication skills,and repeated behavior and narrow interests.In recent years,due to its high incidence and great damage,autism was caught great attention.At present,it is accepted that autism is a kind of neurodevelopmental disorder,but the neural development mechanism of autism has still not yet been elucidated.Thus,exploration of autism behaviors related neural circuits,especially research from developmental perspective has become an important direction to explore the pathogenesis and treatment of autism.At present,progress has been made on studies of autism genetics which found many autism-associated risk genes,such as MeCP2,FMR1,PTEN and NF1,UBE3A,Shank3,etc.Based on researches on genetics of autism patients,Shank3 gene mutation is recognized as one of the important factors that lead to the onset of autism.Our research selected Shank3 gene as the breakthrough point.Shank3 protein is a kind of excitatory postsynaptic protein which can interact with a variety of postsynaptic proteins,postsynaptic glutamate receptors,signal molecules and skeleton proteins.Research shows that Shank3 plays an important role in excitatory synapse formation and glutamate.receptors function maintenance Shank3 gene knockout mice can perform obvious autistic behaviors.However,how does Shank3 participate in the development of brain,especially cerebral cortex?And in autism model,how does the involvement of Shank3 in the process of cortex neurons development induce the autistic behavior?Answers to these questions are very important to clarify the developmental mechanism of autism.This topic will be discussed.Objectives1.Observe the distribution and cellular chemical characteristics of Shank3 in adult mice cerebral cortex.2.Study the expression changes and distribution characteristics of Shank3 gene in mice cerebral cortex of different developmental stages.3.Explore the effect of Shank3 on the development of pyramidal neurons dendrites of cerebral cortex in mice.4.Explore the effect of Shank3 on pyramidal neuronal function in mice cerebral cortex of different developmental stages.Methods1.Applying immunofluorescence staining techniques to observe the distribution and chemical characteristics of Shank3 positive immune cells in the cerebral cortex.2.Using fluorescence quantitative PCR technique to detect Shank3 mRNA expression in the cerebral cortex of different developmental periods.3.Using Western Blot to detect Shank3 protein expression in cerebral cortex of different developmental periods.4.Construction of Shank3 knockdown plasmids and through in utero electroporation technology to observe the effects of Shank3 knockdown on the neuronal development.5.Introduction of Shank3B KO mice,application of Golgi staining techniques and through the sholl analysis to observe the effects of Shank3 knockout on pyramidal neurons dendritic branches in different developmental stages.6.Application of traditional Golgi staining technology-confocal imaging technology-Imaris restoring technology to observe the influence of Shank3 gene knockout on pyramidal neurons dendritic spines in different developmental stages.7.Based on the Shank3 KO genetically modified animal models and using in vitro brain slice electrophysiological patch clamp recording method to discusses the effect of Shank3 knockout on excitatory synaptic function in cerebral cortex of different developmental stages.Results1.In adult mice cerebral cortex,Shank3 immune positive cells were widely distributed in the anterior cingulate cortex(ACC)and sensory area(S)with high expression;while in other parts of the cerebral cortex,the expression of shank3 is very low.2.In adult mice cerebral cortex,shank3 did not coexist with Ibal and GFAP in cells.All cells expressing the shank3 expressed NeuN,but not all cells expressing NeuN expressed the shank3.Shank3 coexisted with the excitatory neurons marker CaMK Ⅱin neurons,and didn’t coexist with inhibitory GABA neurons marker GAD67.3.In the ACC area and S area,Shank3 molecules were located in pyramidal neurons’cell body,apical dendrites and basal dendrites;while in other areas,Shank3 molecules were mainly located in the neurites.4.Shank3 mRNA expression was very low in cerebral cortex of brephic mice;its expression began to increase in 1 week after birth,increased significantly at postnatal 2 weeks and leveled off aferwards.From embryonic stage to postnatal 2 weeks,the amount of shank3 protein expression were stable and low in mice cerebral cortex.Since postnatal 3 weeks,its expression increased significantly and then leveled off.5.In the mice cerebral cortex,Shank3 immune positive cells were mainly concentrated in M2 area and S area at postnatal 1 week;its positive immune cells begin to mainly focus on the ACC area,M and S areas from postnatal 2 weeks and to postnatal4 weeks,its distribution was uniformed with that in adult mice.6.Using in utero electroporation technology to knock down the Shank3 gene.expression in the pyramidal neurons of cerebral cortex.Results showed that the morphology of the cerebral cortex pyramidal neurons did not change at postnatal 1 week..From postnatal 2 weeks,the morphology of pyramidal neurons began to change characterized by shortened neurites and abnomal neurites.The phenomenon was more obvious at postnatal 3 weeks.7.Observation of the morphology of the cerebral cortex pyramidal neurons in Shank3B KO mice of the different developmental stages showed the dendritic length and branch number and density of dendr:itic spines of pyramidal neurons were not changed at postnatal 1 week.Shortened length of apical and base dendrites,reduced dendritic branch number and dendritic spines of basal dendrites of pyramidal neurons in Shank3B KO mice at postnatal 2 weeks were observed.At postnatal 3 weeks,the length of the apical dendrites was significantly shortened,the dendrites branch numbers were also significantly reduced,and the dendritic spines density of apical and basal dendrites of pyramidal neurons were significantly reduced in Shank3B KO mice.8.The amplitude and frequency of sEPSC of the pyramidal neurons had no significant change,but AMPA receptor mediated I-O curve apparently shifted to the left in Shank3B KO mice at postnatal 1 week.The amplitude and frequency of sEPSC of the pyramidal neuron in ACC area were reduced but the AMPA receptor mediated I-O curve had no obvious change in Shank3B KO mice at postnatal 2 weeks.The amplitude and frequency of sEPSC of the pyramidal neuron in ACC area were reduced and AMPA receptor mediated I-O curve apparently shifted to the left in Shank3B KO mice at postnatal 3 weeks.Conclusions1.The distribution of Shank3 molecules are different in different areas of the cerebral cortex with high expression in.particular areas.2.The distribution pattern and expression of shank3 molecules are different in different developmental stages of cerebral cortex.3.Shank3 has little impact on dendritic growth and functions of the cerebral cortex pyramidal neurons in mice at postnatal 1 week.4.The effects of shank3 gene on dendritic development and neuronal functions of cerebral cortex pyramidal neurons are remarkable from postnatal 2 weeks and afterwards. |
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