Effects of normal aging on the transcriptomic and physiological profiles of layer 3 pyramidal neurons in diverse neocortical areas of the monkey

正常衰老对猴子不同新皮质区域第 3 层锥体神经元转录组和生理特征的影响

• 批准号: 10194940
• 负责人: JENNIFER I LUEBKE
• 金额: $ 24.75万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-30 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10194940
• 关键词: 12 year old; Action Potentials; Acute; Address; Aging; Anterior; Area; Behavioral; Biological Assay; Boston; Brain; CASP3 gene; Caliber; Cell physiology; Cells; Cognitive; Data; Dendritic Spines; Dependence; Electrophysiology (science); Exhibits; Functional disorder; Future; Gene Expression; Genes; Genetic; Harvest; Immunohistochemistry; Impaired cognition; In Situ Hybridization; Individual; Inflammation; Inflammatory; Ion Channel; Laser Scanning Confocal Microscopy; Link; Macaca mulatta; Mediating; Monkeys; Morphology; Mutation; NR1 gene; Nerve Degeneration; Neuronal Dysfunction; Neurons; Neuropil; Oxidative Stress; Oxidative Stress Pathway; Phenotype; Physiological; Prefrontal Cortex; Primates; Property; Proteins; Publishing; Pyramidal Cells; Resolution; Slice; Specificity; Structure; Synapses; Synaptic Receptors; TNF gene; Therapeutic; Tissue-Specific Gene Expression; Universities; V1 neuron; Vertebral column; Work; age related; aged; area striata; base; biocytin; cell type; cingulate cortex; cognitive function; cognitive performance; density; differential expression; executive function; hippocampal pyramidal neuron; human old age (65+); interest; neocortical; neural network; normal aging; patch clamp; patch sequencing; protein expression; receptor; reconstruction; response; sensory cortex; transcriptome; transcriptome sequencing; transcriptomics

The higher order dorsolateral prefrontal cortex (LPFC) and the limbic anterior cingulate cortex (ACC) are key areas in the frontal neural network that mediates executive cognitive functions, which often decline during normal aging. There is strong evidence that layer 3 (L3) pyramidal cells in these higher-order areas are selectively vulnerable during normal aging in the primate, especially compared to those in sensory cortices such as the primary visual cortex (V1). Indeed, extensive morphologic, electrophysiological and structural age-related changes are present in LPFC but not V1 L3 pyramidal cells in the rhesus monkey. However, the mechanisms underlying these area-specific vulnerabilities, and whether LPFC and ACC exhibit similar vulnerability to aging, is not known. The overall hypothesis of this proposal is that LPFC and ACC L3 pyramidal cells share transcriptomic and phenotypic profiles that are highly distinct from V1 neurons, and that underlie selective vulnerability of these frontal areas to age-related synaptic dysfunction and hyperexcitability. The cognitive status of young and aged rhesus monkeys will be assessed on a battery of behavioral as part of other projects. Single-cell Patch-Seq transcriptomic assessment of physiologically characterized L3 pyramidal cells in acute slices of LPFC, ACC and V1 prepared from these monkeys will then be performed. Transcriptomic findings will be validated with RNAscope in situ hybridization and immunohistochemical assessment of proteins on/in biocytin filled, morphologically characterized neurons. This project has two aims: 1) assessment of the transcriptomic profiles of physiologically-characterized pyramidal cells in young vs. aged LPFC, ACC and V1. We will use whole-cell patch-clamp recordings to quantify over 30 physiological variables in L3 pyramidal cells and then harvest these cells for Patch-Seq to determine their transcriptomic profiles. 2) assessment of the morphology and protein expression of pyramidal cells in young vs. aged LPFC, ACC and V1. We will characterize protein expression on a separate subset of non-harvested but biocytin-filled morphologically characterized cells and thus validate Aim 1 gene expression findings. Data on specific age-related genetic changes in expression of ion channels and synaptic markers in individual L3 neurons will be related to age-related changes in genes for oxidative stress, inflammation, and neurodegeneration such as caspase 3 and TNFα. The project will reveal mechanisms underlying differential age-related neuronal dysfunction and mechanisms that can compensate for changes to restore cellular function, and thus has broad implications for therapeutic strategies to reduce cognitive decline during normal aging. This study will form the basis of future studies to investigate relationships and co-dependence of age-related cellular changes in a variety of cell types, laminae and cortical areas during aging that can be correlated with cognitive performance in rhesus monkeys.

高级背外侧前额叶皮层（LPFC）和边缘前扣带皮层（ACC）是关键 额叶神经网络中调节执行认知功能的区域，在正常情况下， 衰老有强有力的证据表明，这些高级区域中的第3层（L3）锥体细胞选择性地被激活。 在灵长类动物的正常衰老过程中，特别是与感觉皮层中的那些相比， 初级视皮层（V1）。事实上，广泛的形态，电生理和结构的年龄相关的 恒河猴的LPFC中存在变化，但V1 L3锥体细胞中不存在变化。然而，机制 潜在的这些特定地区的脆弱性，以及是否LPFC和ACC表现出类似的脆弱性老化， 是未知的。该提议的总体假设是LPFC和ACC L3锥体细胞共享 与V1神经元高度不同的转录组学和表型谱， 这些额叶区域对年龄相关的突触功能障碍和过度兴奋的脆弱性。认知状态 作为其他项目的一部分，将对年轻和老年恒河猴的行为进行评估。单细胞Patch-Seq转录组学评估急性脑切片中生理学表征的L3锥体细胞 然后将对这些猴制备的LPFC、ACC和V1进行检测。转录组学研究结果将是 通过RNAscope原位杂交和生物胞素上/内蛋白质的免疫组织化学评估进行验证 充满的，形态特征的神经元。该项目有两个目的：1）评估转录组学 年轻与老年LPFC、ACC和V1中生理特征的锥体细胞概况。我们将使用全细胞膜片钳记录来量化L3锥体细胞中的30多个生理变量，然后收集 将这些细胞用于Patch-Seq以确定它们的转录组谱。2)形态学和蛋白质评估 年轻与老年LPFC、ACC和V1中锥体细胞的表达。我们将描述蛋白质的表达， 未收获但生物胞素填充的形态学表征细胞的单独子集，从而验证Aim 1基因表达结果。关于离子通道表达的特定年龄相关遗传变化的数据， 单个L3神经元中的突触标记物将与氧化应激基因的年龄相关变化相关， 炎症和神经变性，如caspase 3和TNFα。该项目将揭示 潜在的差异年龄相关的神经元功能障碍和机制，可以补偿的变化， 恢复细胞功能，因此对减少认知能力下降的治疗策略具有广泛的意义。 在正常的老化过程中。本研究将为进一步研究衰老过程中各种细胞类型、层和皮层区域中与年龄相关的细胞变化的关系和相互依赖性奠定基础 这与恒河猴的认知能力有关。