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

高级背外侧前额叶皮质(LPFC)和边缘前扣带回(ACC)是关键 额叶神经网络中调节执行认知功能的区域，这些功能在正常情况下通常会下降 衰老。有强有力的证据表明，这些高阶区域中的第3层(L3)锥体细胞选择性地 灵长类动物在正常衰老过程中很脆弱，特别是与感觉皮质中的那些相比，如 初级视皮层(V1)。事实上，广泛的形态、电生理和结构与年龄有关 恒河猴LPFC有变化，但V1、L3锥体细胞无变化。然而，这些机制 这些特定区域脆弱性的潜在原因，以及LPFC和ACC是否表现出类似的老化脆弱性， 是未知的。这一提议的总体假设是LPFC和ACC L3锥体细胞共享 与V1神经元截然不同的转录和表型特征，是选择性的基础 这些额叶区域对年龄相关的突触功能障碍和过度兴奋性的脆弱性。认知状态 作为其他项目的一部分，将根据一系列行为指标对幼年和老年恒河猴进行评估。急性脑片L3锥体细胞生理特征的单细胞Patch-Seq转录分析 然后对这些猴子进行LPFC、ACC和V1检查。转录本发现将是 RNAScope原位杂交和免疫组织化学检测生物细胞蛋白 充满形态特征的神经元。这个项目有两个目标：1)评估转录 青年与老年LPFC、ACC和V1锥体细胞的生理特征。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 这些细胞用于Patch-Seq以确定它们的转录图谱。2)形态和蛋白质的评价 青年与老年LPFC、ACC和V1锥体细胞的表达我们将对蛋白质表达进行表征 一组单独的未收获但充满生物细胞素的细胞的形态特征，从而验证AIM 1基因表达发现。离子通道和离子通道基因表达的特定年龄相关基因变化的数据 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有关， 炎症和神经退行性变，如半胱氨酸天冬氨酸蛋白酶3和肿瘤坏死因子α。该项目将揭示机制 潜在的不同年龄相关的神经元功能障碍和可以补偿变化的机制 恢复细胞功能，因此对减少认知衰退的治疗策略具有广泛的意义 在正常老化过程中。这项研究将构成未来研究的基础，以探讨衰老过程中各种细胞类型、板层和皮质区中与年龄相关的细胞变化的关系和相互依赖 这可能与恒河猴的认知表现有关。

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.