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

描述（由申请人提供）：长期以来，人们一直认为记忆会在大脑中留下一个物理印记，但目前还不清楚这个印记到底是什么。在海马体中，CA1区域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基础。大多数兴奋性突触出现在树突棘上，可在数小时内出现和消失，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可以改变海马棘密度。为了验证棘是记忆储存的基本要素，我们的工作目标是开发一种延时成像技术，能够在几周内跟踪活老鼠的海马棘。然后，我们将使用这种技术来观察CA1中的棘在海马依赖记忆的获取、保留、回忆和消失过程中的动态。先前的研究通过固定组织成像比较了幼稚动物和训练动物的海马棘。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的持续生活经历联系起来。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，这将使我们第一次有机会纵向跟踪活体动物的海马棘，并了解脊柱动力学与情景记忆的关系。这种方法将使我们能够解决尚未得到解答的基本问题。学习过程中海马棘是稳定的还是不稳定的？学习过程中脊柱的动态表现出任何可观察到的空间组织吗？例如，我们将检查新刺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：开发一种慢性小鼠制剂，用于在数周和数月内对CA1海马脊柱动态进行延时显微内镜成像。目的2：在活体小鼠海马依赖情境恐惧条件反射过程中检测CA1脊柱动态。我们将追踪个体脊柱及其在基线条件下的动态，在恐惧条件下，在记忆消失后。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。由于海马和新皮层记忆形式的差异，我们预计CA1脊柱动力学将显示不同于新皮层脊柱的时间和空间特征，这些特征已经被活体显微镜研究过。在未来，我们的成像方法将适用于其他深层大脑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，也涉及学习和记忆和认知疾病。

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.