Multiplex imaging of neuronal activity and signaling dynamics underlying learning in discrete amygdala circuits of behaving mice.

项目概要 杏仁核在多种学习行为中发挥着核心作用。通过将感官信息与 压力、惩罚和奖励信号，杏仁核内的电路被认为在 学习调解特定的行为结果。然而，控制变化的电路原理 不同类型的学习如何产生性质不同的行为仍然很大程度上未知。它有 人们认识到，这是剖析杏仁核基础电路机制的重要一步 依赖性学习是在之前确定离散杏仁核回路内单个神经元的活动， 在学习任务期间和之后。然而，由于技术原因，这一目标很难实现。 首先，杏仁核深埋在大脑内部，因此很难通过成像方法进入，例如 钙成像，已成为检查神经元动作电位活动的首选技术 具有对大量神经元群体的细胞分辨率。其次，压力和奖励信号部分是 编码为神经调节活动，通常不会导致神经元电的直接变化 活动，不能通过钙成像或电压测量来测量。测量神经调节 在体内，特别是在行为过程中，仍然具有挑战性。增加难度的是个人的身份 杏仁核电路以及每个电路接收输入和发送输出的位置仅部分 明白了。 我们计划通过整合最新的、互补的技术进步来应对这些挑战 三位共同PI。在定义的行为范式中，我们将把钙想象为神经元放电的代表。 通过微小的 GRIN 透镜（Φ~0.5 mm）进行双光子成像，对行为小鼠的杏仁核进行成像， 光学进入大脑深层结构，损伤相对较小。同时通过同一个GRIN 镜头，我们将对 cAMP/蛋白激酶 A (PKA) 信号通路的活动动态进行成像，这是一个 许多神经调节剂的共同下游信号传导途径，包括去甲肾上腺素和多巴胺， 通过使用双光子荧光寿命读出压力/奖励诱导的神经调节信号 成像显微镜。同时，我们将执行基于计算的解剖电路分析 剖析杏仁核的新功能细分，并识别每个细分的输入输出 细胞类型特异性。基于这些技术，我们将系统地绘制电路图，包括以前的 杏仁核内的未知回路，并确定每个回路的神经元如何被招募 有助于特定行为的产生。

PROJECT SUMMARY The amygdala plays a central role in diverse learned behaviors. By integrating the sensory information with stress, punishment, and reward signals, the circuitry within the amygdala is thought to be modified during learning to mediate specific behavioral outcomes. However, the circuit principles governing what is changed and how different types of learning give rise to qualitatively distinct behaviors remains largely unknown. It has been recognized that an important step towards dissecting the circuitry mechanism underlying amygdala- dependent learning is to determine the activities of individual neurons within discrete amygdala circuits before, during, and after a learning task. However, this goal has been challenging to achieve for technical reasons. First, the amygdala is buried deep within the brain, making it difficult to access by imaging methods, such as calcium imaging, which has become a technique of choice for interrogating neuronal action potential activities with cellular resolution over large neuronal populations. Second, the stress and reward signals are in part encoded as neuromodulatory activities, which do not usually result in direct changes in neuronal electrical activities and cannot be measured by calcium imaging or voltage measurements. Measuring neuromodulation in vivo, especially during behavior, remains challenging. Adding to the difficulty, the identity of individual amygdala circuits, as well as where each circuit receives input and where it sends output, are only partially understood. We plan to meet these challenges by integrating the most recent, complementary technological advances from the three co-PIs. In defined behavioral paradigms we will image calcium as a proxy for neuronal firing in the amygdalae of behaving mice by performing two-photon imaging via a tiny GRIN lens (Φ~0.5 mm), which offers optical access to deep brain structures with relatively little damage. Simultaneously through the same GRIN lens, we will image the activity dynamics of the cAMP/protein kinase A (PKA) signaling pathway, which is a common downstream signaling pathway for many neuromodulators, including norepinephrine and dopamine, as readout for stress/reward-induced neuromodulatory signals by using two-photon fluorescence lifetime imaging microscopy. In conjunction, we will perform computation-based anatomical circuitry analyses to dissect novel functional subdivisions of the amygdala, and identify the input-output of each subdivision with cell-type specificity. Based on these techniques, we will systematically map circuits, including previously unknown circuits, within the amygdala and determine how neurons from each circuit are recruited by and contribute to the generation of specific behaviors.