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: 10491682
• 负责人: JENNIFER I LUEBKE
• 金额: $ 20.63万
• 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 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10491682
• 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; 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; gene network; 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.

高阶背外侧前额叶皮层 (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，在正常情况下这些功能通常会下降 有强有力的证据表明，这些高级区域的第 3 层 (L3) 锥体细胞是有选择性的。 灵长类动物在正常衰老过程中很脆弱，特别是与感觉皮层（例如 事实上，初级视觉皮层（V1）具有广泛的形态学、电生理学和结构年龄相关性。 恒河猴的 LPFC 中存在变化，但 V1 L3 锥体细胞中不存在变化。 这些特定领域的脆弱性背后，以及 LPFC 和 ACC 是否表现出类似的老化脆弱性， 该提案的总体假设是 LPFC 和 ACC L3 锥体细胞共享。 转录组和表型特征与 V1 神经元高度不同，并且是选择性的基础 这些额叶区域容易受到与年龄相关的突触功能障碍和过度兴奋的影响。 作为其他项目的一部分，将对年轻和老年恒河猴的一系列行为进行评估，对急性切片中生理特征的 L3 锥体细胞进行单细胞 Patch-Seq 转录组评估。 然后将对这些猴子制备的 LPFC、ACC 和 V1 进行转录组学研究。 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 该项目有两个目标：1）评估转录组。 年轻与老年 LPFC、ACC 和 V1 中的生理特征锥体细胞的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，然后收获。 对这些细胞进行 Patch-Seq 以确定其转录组图谱 2) 评估形态和蛋白质。 年轻与老年 LPFC、ACC 和 V1 中锥体细胞的表达 我们将表征其蛋白质表达。 未收获但充满生物胞素的形态特征细胞的单独子集，从而验证 Aim 1 基因表达研究结果。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将与氧化应激基因的年龄相关变化相关， 炎症和神经退行性疾病，如 caspase 3 和 TNFα，该项目将揭示机制。 潜在的差异性年龄相关神经功能障碍和可以补偿变化的机制 恢复细胞功能，因此对减少衰退的治疗策略具有广泛的影响 这项研究将为未来研究衰老过程中各种细胞类型、细胞层和皮质区域与年龄相关的细胞变化的关系和相互依赖性奠定基础。 这可能与恒河猴的认知表现相关。