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）水母蛋白被用于可视化细胞生化过程。这大大增强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nserc支持的Robert E. Campbell博士组成的小组在开发这种基于荧光蛋白的基因编码生物传感器方面成效显著。他们最新的工具将被我的小组用来成像脑细胞相互交流的电活动。具体来说，我们将使用钙和电压传感器蛋白来成像脑电活动如何瞬间增加细胞钙。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）的交流至关重要。发生这种情况的结构是突触。在这里，活跃的突触前神经元释放神经递质，然后与突触后神经元上的受体结合，引起该细胞的电活动。突触释放的神经递质也能提高另一种叫做星形胶质细胞的脑细胞中的钙含量。因此，星形胶质细胞可能释放自己的递质，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脑细胞功能的基本过程至关重要，正如2013年诺贝尔医学奖所反映的那样。我的小组将在荧光蛋白传感器基因插入靶细胞后，对新生啮齿动物的神经元-星形胶质细胞网络中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。我们研究的短期目标是实现新型荧光蛋白传感器，用于成像脑细胞通信中的生化过程。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，对各种细胞生化过程如何相互作用以产生复杂的神经功能进行多参数分析。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推向国际神经科学的舞台。虽然这项研究关注的是一种基本的神经生物学现象，但我们的创新方法的成功实施将适用于其他器官细胞的活体成像。这也为研究器官功能病理性扰动的机制铺平了道路。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。这方面的例子包括神经性疼痛、阿尔茨海默氏症或影响许多加拿大人的早产儿自发性呼吸抑制。这些脑部疾病是我们与合作者共同研究的课题之一。

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.