Slow Outward Currents and Learning In Aging Hippocampus

海马体在陈述性学习的早期阶段至关重要，它的功能和 能力在正常老化过程中退化，导致与年龄相关的学习障碍。已经 反复证明，这种与年龄相关的学习缺陷的细胞生物标志物是扩大的 Ca 2+依赖性爆发后后超极化（AHP）降低CA 1的内在兴奋性 老年人锥体神经元。因此，我们假设，恢复老年人的内在兴奋性， 通过使用遗传操作减少AHP，CAI神经元达到类似于神经元的状态，将挽救年龄- 相关学习缺陷。因此，我们设计了一个研究计划，以确定候选蛋白质， 使用重组腺相关病毒（AAV）载体进行遗传操作。在最初的3.5 多年的MERIT奖，我们已经确定1）Ca 2+积累在细胞质中诱发列车 在老年CA 1神经元中，动作电位的水平大大升高，可能是这些神经元中扩大的AHP的基础。 2）Ca 2+缓冲能力在老化的CAI神经元中增加，可能是一种细胞机制， 抵消增加的Ca 2+积累; 3）CREB激活（CREB激活的重要细胞机制， 学习和AHP还原所必需的蛋白质合成）在老年大鼠的海马中受损; （4）老年大鼠CAI神经元表面L型钙通道（LTCC）表达增加， 这为报道的老年CAI中通过LTCC增加的Ca 2+内流提供了分子机制 神经元基于这些发现，我们鉴定了Ca 2+结合蛋白CREB和LTCC， 候选人通过用AAV载体操纵其功能来挽救年龄相关的缺陷。我们有 创建了靶向CREB和LTCC的AAV载体，并将继续对其进行系统表征。 作为治疗剂用于恢复与年龄相关的缺陷的潜力。候选的Ca 2+结合蛋白基因， 操纵将从蛋白质微阵列实验（一种新的强大的方法来筛选 在数百种蛋白质中的表达水平变化），并通过文献综述和进一步的 分子（例如，蛋白质印迹）测定。此外，我们将确定升高的Ca 2+的来源 使用双光子激光扫描显微镜的Ca 2+成像在老化CAI神经元中的积累;以及 从而揭示了其他潜在的治疗靶点。我们的目标保持不变：确认 AHP是内在兴奋性的关键调节剂，并且有针对性的分子方法可以减少 在老年人的CAI神经元层次分析法将导致成功的学习。持续的成功将表明， 被操纵的蛋白质是一个可行的候选目标，作为年龄的治疗干预点， 相关的学习障碍。这项研究计划与理解和治疗 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.