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

项目摘要 杏仁核在各种学习行为中起着核心作用。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是修改过程中 学习调节特定的行为结果。然而，控制改变的电路原理 不同类型的学习如何产生不同的行为，这在很大程度上仍是未知的。它有 已经认识到，解剖杏仁核下的电路机制的重要一步- 依赖性学习是确定离散杏仁核回路内的单个神经元的活动， 在学习任务期间和之后。然而，由于技术原因，这一目标一直难以实现。 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，这已成为一种技术的选择询问神经元动作电位活动 在大的神经元群体上具有细胞分辨率。第二，压力和奖励信号在一定程度上 编码为神经调节活动，这通常不会导致神经元电活动的直接变化。 活动，并且不能通过钙成像或电压测量来测量。测量神经调节 在体内，特别是在行为期间，仍然具有挑战性。更困难的是，个人的身份 杏仁核回路，以及每个回路接收输入和发送输出的地方，只是部分地 明白 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， 三位联合私家侦探在定义的行为范例中，我们将钙想象为神经元放电的代理， 通过微小GRIN透镜（Φ~0.5 mm）进行双光子成像， 以相对较小的损伤进入脑深部结构的光学通路。同时通过同一个GRIN 透镜，我们将图像的cAMP/蛋白激酶A（PKA）信号通路的活性动力学，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， 作为通过使用双光子荧光寿命的应激/奖励诱导的神经调节信号的读出 成像显微术。同时，我们将进行基于计算的解剖电路分析， 解剖杏仁核的新功能分区，并确定每个分区的输入输出， 细胞类型特异性。基于这些技术，我们将系统地绘制电路，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 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.