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非常重要，但也将为记忆障碍的新疗法的开发提供信息。记忆被认为是由稀疏的神经元集合（“痕迹”）表示的，这些神经元集合在记忆提取过程中重新参与。在Josselyn博士的实验室里，我们试图了解决定选择哪些神经元来编码记忆的规则。在这方面，最近的研究表明，具有高水平的内在兴奋性的外侧杏仁核（LA）神经元在招募到相应的记忆痕迹中的竞争中胜过邻近的神经元。然而，（1）介导记忆分配的电生理活动的特定时空模式仍然未知。此外，恐惧记忆强度与左心房内记忆痕迹的大小无关，这就提出了以下问题：（2）左心房内弱记忆与强记忆的神经生理学差异，以及（3）左心房内记忆痕迹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，因为在多天和多周内重复记录深层神经区域中的相同神经元存在技术困难。然而，如果我们要捕捉记忆编码、存储和提取的动态细胞过程，这种水平的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阶段开发的最先进的成像技术来解决这个问题。在此之前，我建立了一个双光子显微镜，研究细胞和亚细胞过程中参与感觉处理的小鼠大脑皮层在体内。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性。在Josselyn实验室的博士后工作期间，我将建立在这一经验的基础上，并长期成像小鼠LA中的单细胞活动-这是一个与恐惧关联的编码和保留至关重要的大脑区域。这些记录将在恐惧学习和恐惧记忆提取期间进行。我将记录自发活动作为读出的细胞兴奋性之前的记忆编码事件，调查的影响，记忆印迹兴奋性对记忆分配。为了更好地理解局部杏仁核回路在记忆印迹形成中的作用，我将瞄准并光遗传学操纵特定的细胞亚型，并将其与功能性双光子成像联合收割机相结合。总之，这些实验将进一步加深我们对介导记忆编码，存储和检索的细胞机制的理解，并将为研究这些过程如何在阿尔茨海默病（恐惧记忆较弱）和创伤后应激障碍（恐惧记忆较强）的小鼠模型中出错奠定基础。

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).