Slow Outward Currents and Learning In Aging Hippocampus

描述（由申请人提供）：海马体在陈述性学习的早期阶段起着至关重要的作用，这种能力随着年龄的增长而退化，导致与年龄相关的学习障碍。衰老过程中Ca2+依赖性爆发后超极化（AHP）的增加降低了CA1锥体神经元的固有兴奋性以及海马CA1区域的信息处理能力，并有助于年龄相关的学习障碍。我们的初步数据强烈表明，与学习和年龄相关的AHP变化可能部分直接归因于Ca2+本身的改变。静息Ca2+和内源性Ca2+缓冲能力深刻影响神经元功能。但是，它们是否会随着学习和衰老而改变，从而成为AHP改变的机制呢？我们将使用Ca2+成像技术来了解Ca2+处理变化对AHP随着学习和衰老的整体改变的潜在贡献。我们将确定AHP的Ca2+来源，确定树突中的Ca2+来源是否与体细胞附近的Ca2+来源对AHP有相同的影响，以及这些Ca2+来源是否通过学习痕迹blink调节和衰老而改变。学习依赖海马体的任务需要蛋白质合成。我们最近的研究表明，在年轻成年大鼠中，学习相关的AHP减少部分是由蛋白激酶A （PKA）活性介导的，已知PKA可以激活CREB和随后的基因转录/翻译，并减少爆发后AHP。系统的学习和年龄相关的分子分析涉及亚细胞级联的蛋白质，导致CREB激活和AHP的改变，将继续使用western blot和免疫组织化学实验。如果与年龄相关的学习障碍确实是由于爆发后AHP的扩大，那么通过基因沉默一种蛋白质的表达来导致AHP的减少（从而增加神经元的兴奋性）应该可以逆转与年龄相关的学习障碍。我们将使用重组腺相关病毒载体在调节过程中沉默海马中的特定蛋白表达。我们还将比较转染（用荧光指示剂标记）和未转染的大鼠CA1神经元的生物物理和Ca2+特性，以验证转染神经元中Ca2+瞬态和爆发后AHP的减少。候选沉默基因将从文献和本研究项目早期完成的分子分析中确定。目的是确认AHP是内在兴奋性的关键调节因子，并通过靶向分子方法降低老年受试者CA1神经元中的AHP将导致学习成功。成功将表明被沉默的蛋白质是一个可行的候选目标，作为年龄相关学习障碍的治疗干预点。

DESCRIPTION (provided by applicant): The hippocampus is critically involved in the early stages of declarative learning, a capacity degraded during aging, contributing to age-associated learning impairments. Enlarged Ca2+-dependent postburst afterhyperpolarization (AHP) during aging reduces the intrinsic excitability of CA1 pyramidal neurons as well as the information handling capacity of the CA1 region of the hippocampus and contributes to the age-associated learning impairment. Our preliminary data strongly suggest that the learning- and age-related AHP changes may, in part, be directly due to alterations in Ca2+ itself. Resting Ca2+ and endogenous Ca2+ buffering capacity profoundly influence neuronal function. But are they altered by learning and aging, serving as the mechanism by which the AHP is changed? We will use Ca2+ imaging techniques to understand the potential contribution of change in Ca2+ handling to the overall alterations in the AHP with learning and aging. We will determine the Ca2+ sources for the AHP, determine if sources in dendrites have the same impact on the AHP as sources near the soma, and if these Ca2+ sources are altered by learning trace eyeblink conditioning and aging. Learning hippocampus-dependent tasks require protein synthesis. We have recently shown that the learning-related AHP reduction in young adult rats is mediated in part by protein kinase A (PKA) activity, known to activate CREB and subsequent gene transcription/translation, and reduce the postburst AHP. Systematic learning- & age-related molecular assays for proteins involved in the subcellular cascades that lead to CREB activation and alterations in the AHP with western blot and immunohistochemistry experiments will be continued. If age related learning impairment is truly due to the enlarged postburst AHP, then genetically silencing the expression of a protein to cause AHP reduction (and thus, increase neuronal excitability) should reverse the age-related learning impairment. We will use recombinant adeno-associated viral vectors to silence specific protein expression in the hippocampus during conditioning. We will also compare the biophysical and Ca2+ properties of transfected (tagged with fluorescent indicators) and untransfected CA1 neurons from treated rats to verify that the Ca2+ transient and the postburst AHP are reduced in the transfected neurons. Candidate genes to silence will be determined from the literature and molecular assays done earlier in this research program. The goals are to confirm that the AHP is the key regulator of intrinsic excitability and that targeted molecular methods to reduce the AHP in CA1 neurons in aged subjects will lead to successful learning. Success will indicate that the protein being silenced is a viable candidate to target as a therapeutic intervention point for age-associated learning impairments. PUBLIC HEALTH RELEVANCE: Behavioral, calcium imaging, molecular and biophysical experimental approaches will be used to investigate the role of neuronal calcium processing in control of learning in young and aging rats. The goal is to determine if molecular genetic interventions developed from these approaches reverse age-associated learning impairments in rats. Successful experiments will have direct translatability to humans, as molecular genetic approaches are being developed to treat neurodegeneration in aging humans and the hippocampus dependent eyeblink conditioning task has direct parallels between experimental animals and humans.