Neurophysiological mechanisms of memory formation investigated with two-photon calcium imaging in the behaving mouse

了解记忆是如何编码的对于增加我们对大脑功能的了解很重要，但也将为记忆障碍的新治疗方法的发展提供信息。记忆被认为是由在记忆提取过程中重新参与的稀疏神经元集合(“engram”)来表示的。在乔斯林博士的实验室里，我们试图了解决定选择哪些神经元对记忆进行编码的规则。在这条静脉中，最近研究表明，杏仁核外侧(LA)神经元具有高水平的内在兴奋性，比邻近神经元竞争更多地重新进入相应的纹状体。然而，(1)调节记忆分配的电生理活动的特定时空模式仍然未知。此外，恐惧记忆的强度与LA中记忆的大小无关，这引发了以下问题：(2)LA中弱记忆与强记忆的神经生理学差异，以及(3)LA记忆如何随时间变化或演变。解决这些问题在过去一直受到阻碍，因为在几天和几周内重复记录深部神经区的相同神经元存在技术困难。然而，如果我们要捕捉跨越时间的记忆编码、存储和检索背后的动态细胞过程，这种空间和时间精度是必不可少的。我的项目将通过使用我在博士期间开发的最先进的成像技术来解决这个问题。之前，我建造了一个双光子显微镜来研究活体小鼠大脑皮质感觉处理过程中涉及的细胞和亚细胞过程。用荧光钙指示剂对细胞进行染色，并通过在清醒小鼠中记录的荧光变化来报告神经元活动。在Josselyn实验室的博士后工作期间，我将以这一经验为基础，长期成像小鼠LA中的单细胞活动--这是一个关键参与编码和保留恐惧联系的大脑区域。这些录音将在恐惧学习和提取恐惧记忆的过程中进行。我将在记忆编码事件之前记录自发活动作为细胞兴奋性的读数，以调查印记兴奋性对记忆分配的影响。为了更好地了解杏仁核局部回路在印记形成中的作用，我将靶向和光遗传学操作特定的细胞亚型，并将其与功能性双光子成像相结合。总之，这些实验将进一步加深我们对跨时间调节记忆编码、存储和提取的细胞机制的理解，并将为研究阿尔茨海默病(恐惧记忆较弱)和创伤后应激障碍(恐惧记忆较强)小鼠模型中这些过程如何出错奠定基础。

Understanding how memories are encoded is important for increasing our knowledge of brain function but will also inform the development of novel treatment for memory disorders. Memory is thought to be represented by sparse neuronal ensembles ('engrams') that are reengaged during memory retrieval. In Dr. Josselyn’s laboratory, we try to understand the rules that determine which neurons are chosen to encode a memory. In this vein, it was recently demonstrated that lateral amygdala (LA) neurons with high levels of intrinsic excitability out-compete neighboring neurons for recruitment into the corresponding engram. However, (1) the specific spatial-temporal patterns of electrophysiological activity that mediate memory allocation remain unknown. Moreover, fear memory strength is independent from the size of the engram in the LA, raising the questions as to (2) the neurophysiological difference in how a weak vs strong memory is represented in the LA, and (3) how LA engrams transform or evolve across time. Addressing these questions has been hindered in the past due to the technical difficulty of repeatedly recording the same neurons in deep neural regions across multiple days and weeks. However, this level of spatial and temporal precision is essential if we are to capture the dynamic cellular processes that underlie memory encoding, storage, and retrieval across time. My project will address this problem by using state-of-the-art imaging techniques I developed in my PhD. Previously, I built a two-photon microscope to study cellular and subcellular processes involved in sensory processing of mouse cortex in vivo. The cells were stained with a fluorescent calcium indicator and neuronal activity was reported by changes in fluorescence that were recorded in awake mice. During my post-doctoral work in the Josselyn lab, I will build upon this experience and chronically image single-cell activity in the mouse LA – a brain region crucially involved in the encoding and retention of fear associations. These recordings will be performed both during fear learning and fear memory retrieval. I will record spontaneous activity as a read-out for cellular excitability just before the memory encoding event, to investigate the influence of engram excitability on memory allocation. To better understand the role of local amygdala circuitry in engram formation, I will target and optogenetically manipulate specific cellular subtypes and combine this with functional two-photon imaging. Together, these experiments will further our understanding of the cellular mechanisms that mediate memory encoding, storage, and retrieval across time, and will lay the groundwork for investigating how these processes go awry in mouse models of Alzheimer’s disease (in which fear memories are weaker) and posttraumatic stress disorder (in which fear memories are stronger).