Multiphoton Imaging of Synaptic Processes in Neuron-Glia Networks with Novel Transgenic Fluorescent Protein Probes

蛋白质在活细胞中有许多功能。最近，自然发光（荧光）水母蛋白被设计成可视化细胞生化过程。这大大提高了活细胞成像的重要性，并获得了2008年诺贝尔化学奖。在阿尔伯塔大学，由罗伯特·E.坎贝尔在开发这种基于荧光蛋白的遗传编码生物传感器方面是高产的。我的团队将使用他们的最新工具来成像脑细胞相互交谈的电活动。具体来说，我们将使用钙和电压传感器蛋白来成像大脑电活动如何瞬时增加细胞钙。这对研究很重要，因为与活动相关的钙增加会触发化学神经递质的释放，这对神经细胞（称为神经元）的通信至关重要。发生这种情况的结构是突触。在这里，一个活跃的突触前神经元释放神经递质，然后结合到突触后神经元上的受体，引起该细胞的电活动。突触释放的神经递质也可以提高另一种称为星形胶质细胞的脑细胞类型中的钙。因此，星形胶质细胞可能会释放自己的递质，然后影响两个神经元之间的相互作用。研究突触中的这些相互作用对于理解2013年诺贝尔医学奖所反映的脑细胞功能的基本过程至关重要。 我的小组将在新生啮齿动物的神经元-星形胶质细胞网络中对荧光蛋白传感器基因插入靶细胞后的突触过程进行成像。这种成像是用我们最先进的多光子显微镜进行的。我们的研究的短期目标是实现新的荧光蛋白传感器成像脑细胞通信的生化过程。作为长期目标，我们将同时将几个遗传传感器插入脑细胞中，以进行多参数分析，了解各种细胞生化过程如何相互作用，从而产生复杂的神经功能。这项研究可能会发表在领先的科学期刊上，从而将加拿大神经科学进一步推向国际神经科学舞台。虽然这项研究的重点是一个基本的神经生物学现象，我们的创新方法的成功实施将适用于其他器官细胞的实时成像。这也为研究器官功能的病理扰动机制铺平了道路。对于大脑来说，这可能会对开发治疗神经疾病的新治疗方法产生重大影响。这方面的例子是影响许多加拿大人的神经性疼痛、阿尔茨海默氏症或早产儿的自发性呼吸抑制。这些脑部疾病是我们与合作者研究的研究课题之一。

Proteins serve many functions in living cells. Recently, naturally glowing (fluorescent) jellyfish proteins were engineered to visualize cellular biochemical processes. This enhanced greatly the importance of live cell imaging as recognized with the 2008 Nobel Prize in Chemistry. At the University of Alberta, the NSERC-supported group of Dr. Robert E. Campbell is highly productive in developing such fluorescent protein based genetically encoded biosensors. Their latest tools will be used by my group to image electrical activity by which brain cells talk to each other. Specifically, we will use calcium and voltage sensor proteins to image how electrical brain activity transiently increases cellular calcium. This is important to study because activity-related calcium increases trigger the release of chemical neurotransmitter that is pivotal for communication of the nerve cells, called neurons. The structure at which that happens is the synapse. Here, an active presynaptic neuron releases neurotransmitter which then binds to receptors on the postsynaptic neuron to evoke electrical activity in that cell. The synaptically released neurotransmitter can also raise calcium in another brain cell type called astrocyte. Consequently, the astrocyte may release its own transmitter which then influences the interaction between both neurons. Studying these interactions at synapses is of utmost importance for understanding the fundamental processes of how brain cells function as reflected by the 2013 Nobel Prize in Medicine. My group will image synaptic processes in neuron-astrocyte networks from newborn rodents after the fluorescent protein sensors are genetically inserted into the target cells. Such imaging is performed with our state-of-the-art multiphoton microscopes. The short-term objective of our research is to implement the novel fluorescent protein sensors for imaging biochemical processes in brain cell communication. As the long-term objective, we will insert several of the genetic sensors at the same time into brain cells for a multi-parameter analysis of how various cellular biochemical processes interact to give rise to complex nervous functions. This research will likely be published in leading scientific journals and will thus put Canadian neuroscience further on the international neuroscience stage. Although this research focuses on a fundamental neurobiological phenomenon, successful implementation of our innovative approaches will be applicable to live imaging in cells of other organs. This paves way to study also mechanisms of pathological perturbation of organ function. For the brain, this will likely have a major impact on developing novel therapeutic approaches for treatment of nervous diseases. Examples for this are neuropathic pain, Alzheimer’s or spontaneous depression of breathing in preterm infants that affect many Canadians. These brain diseases are among the research topics that we study with our collaborators.