Hippocampal dendritic spines tracked in learning and memory processing using in v

描述(申请人提供)：长期以来，人们一直认为记忆会在大脑上留下一个物理签名，但目前还不清楚这个签名可能是什么。在海马区，CA1区是情景记忆所必需的，与其他形式的记忆一样，CA1神经元的突触连接是记忆获得和保持的关键底物。大多数兴奋性突触出现在树突棘上，树突棘可以在几个小时内出现和消失，并经历突触强度的长期增强或抑制。学习和损害记忆功能的疾病都会改变海马棘的密度。为了检验脊柱作为记忆存储基本元素的想法，我们工作的一个目标是开发一种延时成像技术，能够在活着的小鼠身上跟踪单个海马棘数周的时间。然后，我们将使用这项技术来观察CA1中脊椎的动态变化，包括对海马体依赖记忆的获取、保持、回忆和消退。之前的研究已经通过成像固定组织来比较幼稚动物和训练有素动物的海马棘。体外研究也将神经元活动与切片培养中的短期脊柱动力学联系起来。然而，这些方法不允许对脊椎动力学进行长达数周的研究，也不能将脊椎动力学与动物正在进行的生活经验联系起来。为了克服这些限制，我们将利用一种新的光学技术-双光子显微内窥镜，这将为我们提供第一次机会来纵向跟踪活体动物的海马棘，并了解脊柱动力学如何与情景记忆相关。这种方法将使我们能够解决尚未得到回答的基本问题。在学习过程中，海马棘是稳定还是不稳定？学习过程中的脊柱动力学表现出任何可观察到的空间组织吗？例如，我们将检查新的脊椎是否倾向于沿着相同的树突聚集在一起。因此，我们的目标是：目标1：建立一种慢性小鼠准备，用于在活体内对CA1海马棘动态进行延时显微内窥镜成像，持续数周和数月。目的2：在活体小鼠中检测CA1脊椎在海马区依赖的情境恐惧条件反射过程中的动态变化。我们将在恐惧条件作用后和记忆消失后，跟踪单个脊椎及其在基线条件下的动态。由于我们将在学习前后观察相同的神经元，我们将能够测试细胞变化与受试者行为之间的关系，作为时间的函数。由于海马和新皮质记忆形式的不同，我们预计CA1脊椎动力学将显示出与以前用活体显微镜研究的新皮质脊椎不同的时间和空间特征。在未来，我们的成像方法将适用于其他大脑深层区域和其他亚细胞元素的研究，如细胞器或突触超分子结构，这些结构也与学习以及记忆和认知疾病有关。 与公共卫生相关：在神经科学中，目前的一个限制是无法可视化隐藏在活着的哺乳动物大脑深处的神经元。我们的研究旨在创造一种脑成像技术，使研究人员能够观察活着的小鼠体内如此深埋的神经元，并在动物生命的几周和几个月内跟踪它们的亚细胞特性，无论是在正常动物还是脑疾病动物模型中。然后，我们将使用这种成像技术来检查海马体中的树突棘是否表现出与它们是长期记忆存储的重要细胞底物和困扰记忆的疾病的关键部位一致的动力学特性。

DESCRIPTION (provided by applicant): It has long been thought memories leave a physical signature on the brain, but it remains unclear precisely what this signature might be. In the hippocampus, area CA1 is necessary for episodic memory, and as with other forms of memory the synaptic connections of CA1 neurons are attractive candidates for being key substrates of memory acquisition and retention. Most excitatory synapses arise on dendritic spines, which can appear and disappear within hours and undergo long-term potentiation or depression of their synaptic strength. Both learning and diseases that impair memory function can alter hippocampal spine density. To examine the idea spines serve as basic elements of memory storage, a goal of our work is to develop a time-lapse imaging technique capable of tracking individual hippocampal spines over weeks in live mice. We will then use this technique to watch the dynamics of spines in CA1 across the acquisition, retention, recall, and extinction of a hippocampal-dependent memory. Prior studies have compared hippocampal spines of naive versus trained animals by imaging fixed tissue. In vitro studies have also correlated neuronal activity to short-term spine dynamics in slice culture. However, these approaches did not permit studies of spine dynamics over many weeks, nor did they correlate spine dynamics to an animal's ongoing life experiences. To overcome these limitations, we will capitalize on a novel optical technology, two-photon microendoscopy that will provide us the first opportunity to track hippocampal spines longitudinally in live animals and to see how spine dynamics relate to episodic memory. This approach will allow us to address basic questions that have gone unanswered. Are hippocampal spines stabilized or destabilized during learning? Do spine dynamics during learning exhibit any observable spatial organization? For example, we will examine if new spines tend to arise clustered together along the same dendrites. Thus, our aims are: Aim 1: Develop a chronic mouse preparation for time-lapse in vivo microendoscopy imaging of CA1 hippocampal spine dynamics over weeks and months. Aim 2: Examine CA1 spine dynamics over the course of hippocampal-dependent contextual fear conditioning in live mice. We will track individual spines and their dynamics across baseline conditions, following fear conditioning, and after memory extinction. Since we will observe the same neurons before and after learning, we will be able to test for relationships between cellular changes and subjects' behavior as a function of time. Due to the differences between hippocampal and neocortical forms of memory, we expect CA1 spine dynamics will display temporal and spatial characteristics distinct from those of neocortical spines, which have been studied previously by intravital microscopy. In the future, our imaging methodology will be applicable to other deep lying brain areas and to the study of other sub-cellular elements, such as organelles or synaptic supramolecular structures that have also been implicated in learning and diseases of memory and cognition. PUBLIC HEALTH RELEVANCE: In neuroscience, a current limitation is the inability to visualize neurons that lie deep within the brains of living mammals. Our research seeks to create a brain-imaging technique that will allow researchers to observe such deep lying neurons in live mice and to track their sub-cellular properties over weeks and months in an animal's life, in both normal animals and animal models of brain disease. We will then use this imaging technique to examine whether dendritic spines in the hippocampus show dynamical properties consistent with their being an important cellular substrate for long-term memory storage and a key locus for diseases afflicting memory.