A novel approach to examine slow synaptic transmission in vivo

描述(申请人提供)：大脑中神经元之间存在两种主要的化学交流模式：快速突触传递，如谷氨酸和GABA介导的，直接控制神经元电活动的突触传递；慢突触传递，如去甲肾上腺素和多巴胺介导的，调节无法直接从神经细胞电活动测量的亚细胞信号事件。慢突触传递又称神经调节，在调节兴奋性、突触可塑性等神经功能方面起着重要的调节作用，最终对快速突触传递功能产生强大的调控作用。然而，与快速突触传递不同的是，快速突触传递可以通过越来越多的现代方法直接监测，如多电极记录、电压成像和钙成像方法，而对活体动物中发生的准确的神经调制事件知之甚少，因为还没有一种确定的方法来可靠地记录体内单个神经元中神经调制引发的相关活动。为了克服这一问题，我们提出了一种新的方法，通过成像环磷酸腺苷(CAMP)和蛋白激酶A(PKA)的活性来检测体内单个神经元的神经调节活性。CAMP/PKA通路是多巴胺和去甲肾上腺素共同的下游信号转导通路。尽管基于Förster共振能量转移(FRET)的基因编码的cAMP/PKA传感器已被用于体外实验，但由于在更具挑战性的体内成像条件下信噪比较低，它们在体内的应用一直很困难。我们提出了一种多管齐下的方法来消除当前FRET成像方法遇到的几个瓶颈，以最大限度地提高信噪比。我们的方法包括：1)开发和改进cAMP/PKA传感器，2)在光散射脑组织中实现比传统FRET更有效的FRET成像方式，3)校正与活体成像条件相关的光像差，以及4)开发用于高对比度、可重复性的FRET成像的新型小鼠试剂。我们将通过使用光遗传学方法在麻醉小鼠和使用不同应激刺激的行为小鼠中确定去甲肾上腺素作用的时空模式，来验证该方法在监测神经调节活动方面的有效性。如果成功，我们的努力将提供一种以前无法实现的能力，在细胞和电路水平上对大脑中的神经调节活动进行大规模监测。这种量化神经调节的能力将补充快速突触传递的测量，以增强我们对动物行为背后的大脑功能的理解。

DESCRIPTION (provided by applicant): Two primary modes of chemical communication occur between neurons in the brain: fast synaptic transmission, such as that mediated by glutamate and GABA, which directly control the electrical activities of neurons, and slow synaptic transmission, such as that mediated by norepinephrine and dopamine, which regulate subcellular signaling events that cannot be measured directly from neuronal electrical activities. Slow synaptic transmission, which is also called neuromodulation, plays important modulatory roles in regulating excitability, synaptic plasticity and other aspects of neuronal function, and eventually imposes powerful control over the function of fast synaptic transmission. However, unlike fast synaptic transmission, which can be monitored directly via an increasing number of modern approaches such as multi-electrode recording, voltage imaging and calcium imaging methods, much less is known about the precise neuromodulatory events that occur in living animals because there has not been an established method to reliably record the relevant activities triggered by neuromodulation in individual neurons in vivo. To overcome this problem, we propose a novel approach for examining neuromodulatory activities with single-neuron resolution in vivo by imaging the activity of cyclic AMP (cAMP) and protein kinase A (PKA). The cAMP/PKA pathway is a common downstream signal transduction pathway for both dopamine and norepinephrine. Although genetically encoded cAMP/PKA sensors based on Förster resonance energy transfer (FRET) have been used for experiments in vitro, their application in vivo has been difficult due to lower signal-to-noise ratios under the more challenging in vivo imaging conditions. We propose a multipronged approach to eliminate several bottlenecks encountered with current FRET imaging approaches to maximize the signal-to-noise ratio. Our approach includes: 1) developing and improving cAMP/PKA sensors, 2) implementing a FRET imaging modality that is more effective than conventional FRET measures in light-scattering brain tissue, 3) correcting light aberrations associated with in vivo imaging conditions, and 4) developing novel mouse reagents for high-contrast, reproducible FRET imaging. We will validate the utility of this method for monitoring neuromodulatory activities by determining the spatiotemporal patterns of norepinephrine action in anesthetized mice using optogenetic approaches and in behaving mice using different stress stimulations. If successful, our efforts will provide a previously unattainable ability to conduct large- scale monitoring of neuromodulatory activities in the brain at the cellular and circuitry levels. This ability to quantitate neuromodulation will complement the measurements of fast synaptic transmission to enhance our understanding of brain function underlying animal behavior.