Differential Gene Regulatory Plasticity Between Upper And Lower Layer Cortical Excitatory Neurons

Neocortical projection neurons are consisted of intracortical connected upper layer(UL,layer II-IV)neurons and subcortical connected lower layer(LL,layer V-VI)neurons.Thalamus afferent activity is critical in the neocortical cytoarchitecture formation through regulating layer specific gene expression during postnatal development.The neocortex can express many layer specific genes,those genes can regulated by neuron activity.Activity dependent gene regulation are essential for neuronal control of adaptive behavior and experience dependent plasticity.Objective: our study here aim to1? the expression of layer specific genes in the absence of intrinsic thalamic afferent activity2?The expression of layer specific genes in the absence of external perception sensory activity3?The intrinsic molecular mechanism of activity dependent gene regulation methods:1 ? We had generated thalamus specific knock out of munc18 mice line,a mouse model which sensory cortices lack thalamic axon innervations,and examined expressions of the layer genes in absence of thalamic afferent input.2?We deprived peripheral afferent activity to visual cortex during development by enucleating both eyes immediately after birth and examined the expressions of laminar specific transcription factors at P19.3?We cultured primary cortical neurons from pups at P0 in neurobasal medium with increased KCl concentration(10m M)to mimic hyperactivity condition.High potassium medium was changed to cultured cortical neurons at the 7 th day in vitro and neurons were continued to culture in presence of high K+ for 5 days before fixation,then examined the expressions of laminar specific transcription factors4?We detect whether EGR1 and c-FOS can bind to the promoter of neocortex specific gene by luciferase and CHIP-Qpcr assay5 ? To investigate whether epigenetic modifications are differentially expressed in upper and lower layer cortical neurons,we examined the epigenetic modification of H3 histones and H4 histones using Epi Quik Histone H3 Modification Multiplex Assay Kit and Epi Quik Histone H4 Modification Multiplex Assay Kit from UL and LL tissue lysatesResults:After analyzing the results,we find that the regulation is confined to UL cortical neurons and the regulatory plasticity occurs in vivo but not in vitro.However,immediate early genes(IEGs): EGR1 and C-FOS are down regulated in all cortical laminar layers in absence of afferent activity in vivo.Transcriptional assay demonstrated that EGR1 and c-FOS are able to bind the promoters of UL and LL specific genes and induce transcription.Finally,we discovered that LL neurons have higher expressions of heterochromatin markers such as H3K9m3 and H4K20m3 than UL neurons.Our results suggests that epigenetic modifications of chromatin may be an intrinsic mechanism underlying the differential sensitivity of cortical neurons to activity dependent gene regulation.