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.

高阶背侧前额叶皮层（LPFC）和边缘前扣带回皮层（ACC）是关键 额神经元网络中介导执行认知功能的领域，通常在正常情况下下降 老化。有充分的证据表明，这些高阶区域中的第3层（L3）锥体细胞有选择性 在灵长类动物的正常衰老期间易受伤害，尤其是与感觉皮质中的那些 一级视觉皮层（V1）。确实，广泛的形态学，电生理和结构年龄相关 LPFC中存在变化，但恒河猴中的V1 L3锥体细胞不存在。但是，机制 这些特定区域的漏洞以及LPFC和ACC是否暴露了与衰老相似的脆弱性， 不知道。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和表型轮廓，这是选择性的基础 这些额叶区域与年龄相关的突触功能障碍和过度兴奋性的脆弱性。认知状况 作为其他项目的一部分，将在一系列行为上评估年轻和老化的恒河猴的猴子。急性切片中物理表征的L3锥体细胞的单细胞斑块ze转录组评估 然后将执行由这些猴子制备的LPFC，ACC和V1。转录组发现将是 用rnascope验证蛋白质/蛋白质的原位杂交和免疫组织化学评估 填充，形态学特征的神经元。该项目有两个目的：1）转录组的评估 年轻人与老年LPFC，ACC和V1中物理表征的锥体细胞的谱。我们将使用全细胞贴片钳记录来量化L3锥体细胞中的30多个生理变量，然后收获 这些用于斑块序列的细胞以确定其转录组曲线。 2）评估形态和蛋白质 锥体细胞在年轻的LPFC，ACC和V1中的表达。我们将表征蛋白质的表达 单独的非收获但生物细胞充满形态学的细胞的子集，从而验证目标 1个基因表达发现。有关特定年龄相关基因的数据变化的数据，而离子通道表达的变化和 单个L3神经元中的突触标记将与氧化应激的基因相关的变化有关， 炎症和神经变性，例如caspase 3和TNFα。该项目将揭示机制 与年龄相关的基本神经元功能障碍和可以补偿变化的机制 恢复细胞功能，因此对减少认知能力下降的治疗策略具有广泛的影响 在正常衰老期间。这项研究将构成未来研究的基础，以研究各种细胞类型，层层和皮质区域中与年龄相关的细胞变化的关系和共依赖性。 这可以与恒河猴的认知表现相关。