Training in hIPSC differentiation protocols to generate motor neuron-muscle cultures to replace rat models in study of mitochondria on axon physiology

神经元是大脑中的主要计算单位。它们形成了一个由相互连接的细胞组成的网络，这些细胞在称为突触的专门位点上相互传递信息。被称为动作电位的电信号是神经元中信息的主要流通方式，它沿着被称为轴突的索状薄结构传播到突触。在这里，它们引起化学物质的释放，然后在接收神经元中产生电信号。在过去的100年里，许多研究人员都对电信号沿轴突传播的研究感兴趣，霍奇金和赫胥黎在1963年获得了诺贝尔奖，因为他们发现了这些电信号传播的机制。虽然他们的模型一直是电信号传播的基石，但这些方程依赖于在相对较大的宏观尺度上观察轴突。在这里，我们提供了初步的证据，当在更微观的尺度上观察时，以前未被识别的因素对电信号如何沿着轴突传播具有重要影响。线粒体是存在于细胞内的结构，可以产生能量并吸收多余的钙。线粒体出现在沿着轴突的规则点处，并且由于轴突的相对细的直径，当观察横截面时，线粒体几乎充满整个线缆。根据Hodgkin-Huxely模型，这会增加电信号传播的阻力，就像一个堵塞的管道会减少水的流量一样。然而，通过使用最新的显微镜技术，使我们能够跟踪电压的传播作为光强度的变化，我们观察到线粒体增加了沿着轴突的电信号传输。这一惊人的发现似乎是由线粒体产生的电压和轴突的电压之间的相互作用引起的，在我们目前的模型中，扩展这些结果的能力相对有限。从大脑海马区解剖的大鼠神经元的轴突在培养时形成复杂的模式，以随机方向编织并相互重叠。在这项提议中，Rigby博士希望接受培养人类诱导多能干细胞（hIPSC）衍生的运动神经元的培训，这些神经元具有非常直的轴突，很少分支，并且允许更简单地获取和分析电信号和钙信号的传播。hIPSC是一些非凡发现的结果，即人类皮肤细胞可以变回干细胞样状态，然后它们就可以转变成任何类型的细胞。运动神经元是位于脊柱中的细胞，引起肌肉的收缩和舒张。在过去的10年里，将hIPSCs转化为运动神经元和肌肉的方案已经得到了改进，现在包括Rigby博士的合作者Ivo Lieberam博士在内的研究人员可以可靠地生产人类神经肌肉培养物，而无需使用胚胎或动物。Rigby博士希望学习的正是这些实验方案，hIPSCs的使用带来了许多优势。首先，里格比博士有机会在他的工作中完全停止使用动物，从而解决3R的“替代”问题。此外，通过在微流控设备上生长神经元和肌肉，他将能够制作一个可定制和可转移的人类神经肌肉系统模型，这对其他研究人员来说是有益的，从而进一步减少神经科学研究中动物的使用。hIPSC也可以来源于患有特定神经退行性疾病的人类患者。在我们的提案中，我们还计划通过比较来自健康患者的hIPSC衍生的运动神经元中的电压沿沿着轴突的传播与来自肌萎缩侧索硬化症（ALS）患者的神经元（ALS是运动神经元疾病的最常见形式）来受益于hIPSC模型的这种多功能性

Neurons are the main computational units in the brain. They form a network of interconnected cells that pass information to each other at specialised sites called synapses. Electrical signals known as action potentials, the main currency of information in the neuron, travel down cable-like thin structure called the axon to reach the synapse. Here, they cause the release of chemicals that then result in electrical signals in the receiving neuron. The study of the spreading of electrical signals down axons has been of interest to many researchers over the past 100 years, and Hodgkin and Huxley won the Nobel Prize in 1963 for their identification of the mechanisms that underlie the propagation of these electrical signals. Though their model has been the cornerstone of electrical signal propagation ever since, the equations rely viewing the axon on a relatively large, macroscopic scale. Here we provide preliminary evidence that when viewed on a more microscopic scale, that previously unidentified factors have important influences on how electrical signals are spread down the axon.Mitochondria are structures that reside within cells to produce energy and soak up excess calcium. Mitochondria occur at regular points along an axon, and due to the relative thin diameter of axons mitochondria almost fill the entire cable when looked at a cross section. According to the Hodgkin-Huxely model this would increase the resistance in the spread of electrical signals, much like a pipe with a blockage reduces the flow of water. However, by using the very latest techniques in microscopy that allow us to track the spread of voltage as a change in intensity of light, we observed that mitochondria increase the transmission of electrical signals along the axon. This surprising discovery seems to result from the interaction between the electrical voltage generated by mitochondria and the electrical voltage of the axon.The ability to expand on these results is relatively limited in our current model. The axons of rat neurons that are dissected from the hippocampus region of the brain form complex patterns when they are cultured, weaving in random directions and overlapping with each other. In this proposal Dr Rigby wishes to be trained in the culturing of human induced pluripotent stem cell (hIPSC) derived motor neurons, which have very straight axons that infrequently branch and allow for much simpler acquisition and analysis of the spread of electrical and calcium signals.hIPSCs are the result of some extraordinary discoveries that skin cells from humans can be turned back into a stem cell-like state, from which they can then be turned into any cell type. Motor neurons are the cells which reside in the spine and cause the contraction and relaxation of muscle. Over the past 10 years the protocols to transform hIPSCs into motor neurons and muscle have been refined so that now researchers, including Dr Rigby's collaborator Dr Ivo Lieberam, can reliably produce human neuromuscular cultures without the need of using embryos or animals. It is these protocols that Dr Rigby wishes to learn.The use of hIPSCs confer many advantages. Firstly, Dr Rigby has the opportunity to completely cease animal usage in his work and thus address the 'Replacement' aspect of the 3Rs. In addition, by growing the neurons and muscle on microfluidic devices he will be able to make a customizable and transferable model of the human neuromuscular system, that can be of benefit to other researchers, and thereby further reduce the use of animals in neuroscience research. hIPSCs can also be sourced from human patients with particular neurodegenerative disorders. In our proposal we also plan to benefit from this versatility of the hIPSC model by comparing the spread of voltage along axons in hIPSC-derived motor neurons from healthy patients with neurons derived from patients with Amyotrophic Lateral Sclerosis (ALS), the most common form of motor neuron disease