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

项目总结 杏仁核在不同的习得行为中起着核心作用。通过将感官信息与 应激、惩罚和奖励信号，杏仁核内的电路被认为在 学会调节特定的行为结果。然而，控制更改内容的电路原理 而不同类型的学习如何导致不同性质的行为在很大程度上仍是个未知数。它有 人们已经认识到，解剖杏仁核背后的回路机制的重要一步- 依赖学习是确定之前杏仁核离散回路中单个神经元的活动， 在学习任务期间和之后。然而，由于技术原因，这一目标的实现一直具有挑战性。 首先，杏仁核深埋在大脑中，很难通过成像方法进入，例如 钙成像已成为询问神经元动作电位活动的一种首选技术 以细胞分辨率覆盖大的神经元群体。其次，压力和奖励信号在一定程度上 编码为神经调节活动，通常不会导致神经元电的直接变化 钙离子是一种活性物质，无法通过钙成像或电压测量进行测量。测量神经调节 在体内，尤其是在行为过程中，仍然具有挑战性。增加了难度，个人的身份认同 杏仁核回路以及每个回路接收输入和发送输出的位置只是部分的 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 三个共同的私家侦探。在已定义的行为范式中，我们将把钙想象为神经元放电的代用品 通过一个微小的GRIN透镜(Φ~0.5mm)进行双光子成像，对行为正常的小鼠的杏仁核进行成像，这提供了 光学接触到大脑深层结构，损伤相对较小。同时带着同样的笑容 镜头，我们将成像cAMP/蛋白激酶A(PKA)信号通路的活性动态，这是一种 许多神经调节剂的共同下游信号通路，包括去甲肾上腺素和多巴胺， 利用双光子荧光寿命读出应激/奖赏诱导的神经调制信号 成像显微镜。同时，我们将进行基于计算的解剖电路分析，以 解剖杏仁核新的功能分区，并用以下方法识别每个分区的输入输出 细胞类型特异性。基于这些技术，我们将系统地映射电路，包括前面 杏仁核内的未知回路，并决定每个回路的神经元是如何被和招募的 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.