Slow Outward Currents and Learning In Aging Hippocampus

海马体关键地参与了陈述性学习的早期阶段，它的功能和 在导致与年龄相关的学习障碍的正常衰老过程中，能力会退化。一直以来 反复证明，这种与年龄相关的学习缺陷的一个细胞生物标志物是扩大的 钙依赖的阵发性后超极化(AHP)降低CA1的内在兴奋性 老年人的锥体神经元。因此，我们假设恢复老年人的内在兴奋性 通过遗传操作降低AHP，使蔡氏神经元达到年轻状态，将拯救年龄- 相关的学习缺陷。因此，我们设计了一个研究计划来确定候选蛋白质 使用重组腺相关病毒(AAV)载体进行基因操作。在最初的3.5中 多年来，我们已经确定：1)由火车引起的胞浆中钙离子的积累 在衰老的CA1神经元中，动作电位的频率显著升高，这可能是这些神经元AHP扩大的基础 2)老龄CA1神经元的钙缓冲能力增强，这可能是一种细胞机制 3)CREB的激活(一个重要的细胞机制 学习和降低AHP所必需的蛋白质合成)在老年大鼠的海马区受损； 4)老龄大鼠大脑皮质CA1区神经元表面L型钙通道表达增加， 这为报道的老年性脑梗塞患者LTCC钙离子内流增加提供了分子机制 神经元。基于这些发现，我们确定了钙结合蛋白、CREB和LTCC为 通过使用AAV载体操纵他们的功能来挽救与年龄相关的缺陷的候选人。我们有 创建了针对CREB和LTCC的AAV载体，并将继续系统地表征其 作为修复与年龄相关的缺陷的治疗方法的潜力。与钙结合蛋白相关的候选基因 操作将从蛋白质微阵列实验中确定(一种新的强大的筛选方法 数百种蛋白质的表达水平变化)，并通过文献回顾和进一步的 分子(例如，蛋白质印迹)分析。此外，我们还将确定升高的钙离子的来源(S) 用双光子激光扫描显微镜钙离子成像技术研究老化的CA1区神经元的积聚； 因此，揭示了更多潜在的干预治疗靶点。我们的目标不变：确认 AHP是内在兴奋性的关键调节因子，靶向分子方法可以减少 老年受试者CA1区神经元中的AHP将导致学习成功。持续的成功将表明 正在被操纵的蛋白质是一个可行的候选对象，可以作为年龄治疗干预点- 相关的学习障碍。这项研究计划与理解和治疗有明显的相关性 神经退行性疾病，如阿尔茨海默病，其中衰老是主要的风险因素。

The hippocampus is critically involved in the early stages of declarative learning, and its function and capacity are degraded during normal aging that causes age-associated learning impairments. It has been repeatedly demonstrated that a cellular biomarker of this age-associated learning deficit is the enlarged Ca2+-dependent postburst afterhyperpolarization (AHP) that reduces the intrinsic excitability of CA1 pyramidal neurons in aged subjects. Thus, we have hypothesized that restoring intrinsic excitability of aged CAI neurons to a young-like state by reducing the AHP using genetic manipulations would rescue the age- related learning deficits. Hence we have designed a research program to identify the candidate proteins for genetic manipulation with the use of recombinant adeno-associated viral (AAV) vectors. In the initial 3.5 years of this MERIT award, we have determined that 1) Ca2+ accumulation in the cytosol evoked with trains of action potentials is greatly elevated in aged CA1 neurons and may underlie the enlarged AHP in these neurons; 2) Ca2+ buffer capacity is increased in aged CAI neurons, potentially as a cellular mechanism to counteract the increased Ca2+ accumulation; 3) CREB activation (an important cellular mechanism for protein synthesis necessary for learning and for AHP reduction) is impaired in hippocampus of aged rats; and 4) L-type Ca2+ channel (LTCC) expression on the surface of CAI neurons is elevated in aged rats, which provides a molecular mechanism for the reported increased Ca2+ influx through LTCC in aged CAI neurons. Based on these findings, we have identified Ca2+ binding proteins, CREB, and LTCC as candidates to rescue the age-related deficits by manipulating their function with AAV vectors. We have created AAV vectors targeting CREB and LTCC, and will continue the systematic characterization of their potential as therapeutics for restoring the age-related deficits. The candidate Ca2+ binding protein genes to manipulate will be determined from protein microarray experiments (a new powerful method to screen expression level changes in hundreds of proteins), and confirmed through literature review and further molecular (e.g., western blot) assays. In addition, we will identify the source(s) ofthe elevated Ca2+ accumulation in aged CAI neurons using Ca2+ imaging with two-photon laser scanning microscopy; and thus, reveal additional potential therapeutic targets for intervention. Our goals remain unchanged: to confirm that the AHP is the key regulator of intrinsic excitability and that targeted molecular methods to reduce the AHP in CAI neurons in aged subjects will lead to successful learning. Continued success will indicate that the protein being manipulated is a viable candidate to target as a therapeutic intervention point for age- associated learning impairments. This research program has clear relevance to understanding and treating neurodegenerative diseases such as Alzheimer's Disease, in which aging is the principal risk factor.