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

蛋白质在活细胞中具有多种功能。最近，天然发光(荧光)的水母蛋白被设计成可视化细胞生化过程。这极大地增强了活细胞成像的重要性，正如2008年诺贝尔化学奖所承认的那样。在艾伯塔大学，NSERC支持的Robert E.Campbell博士团队在开发这种基于荧光蛋白的基因编码生物传感器方面非常有成效。他们的最新工具将被我的团队用来成像脑细胞相互对话的电活动。具体地说，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。这一点值得研究，因为与活动相关的钙离子增加会触发化学神经递质的释放，这种化学递质对神经细胞(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，一个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，在该细胞中激发电活动。突触释放的神经递质也可以提高另一种类型的脑细胞--星形胶质细胞的钙含量。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。 我的团队将在将荧光蛋白传感器基因插入目标细胞后，从新生啮齿动物身上拍摄神经元-星形胶质细胞网络中的突触过程。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的荧光蛋白传感器，用于脑细胞通讯中的生化过程成像。作为长期目标，我们将同时将几个遗传传感器植入脑细胞，以多参数分析各种细胞生化过程如何相互作用，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，从而使加拿大神经科学在国际神经科学舞台上更进一步。虽然这项研究侧重于一种基本的神经生物学现象，但我们创新方法的成功实施将适用于其他器官细胞的活体成像。这也为进一步研究器官功能病理性紊乱的机制奠定了基础。对于大脑来说，这可能会对开发治疗神经疾病的新疗法产生重大影响。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.