Neurotransmitter imaging to understand seizure mechanisms

癫痫影响了多达1%的人口，即使使用最佳药物，30%的患者仍会癫痫发作。耐药癫痫患者遭受药物的副作用，有很大的抑郁和其他合并症的风险，并且由于癫痫事故、自杀和突然意外死亡，死亡率比一般人群高出几倍。治疗癫痫的唯一可行方法是通过手术切除癫痫发作的区域，但这只适用于一小部分患者，因为癫痫发作区通常与大脑中控制语言、记忆、运动、感觉或其他认知功能的区域混杂在一起。目前令人沮丧的状况在很大程度上是由于对癫痫发作的机制了解不足。不同类型的癫痫发作通常用大规模的神经网络来描述，而哪些神经元放电，如何放电以及在哪里放电的细节，人们只能以非常肤浅的方式了解，因为直到最近，人们还只能通过电方法间接地研究这些问题。这意味着，当癫痫发作时，大脑不同区域的兴奋性和抑制性神经元的哪些亚型变得活跃或沉默，以及它们的顺序仍然很不清楚。一个主要的进步是光学方法的发展，通过使用响应钙离子水平变化的荧光分子，可以可视化神经元群体的活动。这种方法可以在实验性啮齿动物癫痫模型中实施，但非常缓慢，并且不能通过释放主要的兴奋性和抑制性神经递质（分别为谷氨酸和GABA）来检测神经元之间的信号。我们最近通过使用另一种荧光显微镜来检测神经元释放的谷氨酸和GABA的水平，克服了这一限制。这可以更快地读出兴奋性和抑制性神经元群体是如何被招募的，并对神经元群体之间的信号传导有了直接的了解。我们还可以同时对两种神经递质进行成像，或者对一种神经递质和钙进行成像，方法是记录不同波长的荧光灯，同时记录通常用来定义癫痫发作的放电。我们的建议建立在方法论上的突破和初步数据的基础上，这些数据表明，围绕病理性放电起始部位的抑制性“光环”逐渐失效，导致全面发作，并扩散到整个大脑。接下来，我们将询问哪些抑制性神经元亚群失效，并验证这样的假设：它们之所以失效，是因为它们变得过度兴奋，无法放电。这将通过使用模拟激发或抑制的光激活蛋白来操纵它们的电学特性来实现（“光遗传学”）。此外，我们将从癫痫模型扩展到癫痫发作是由化学物质应用于大脑引起的啮齿动物模型，癫痫发作是自发发生的。这再现了一种与脑发育局灶性畸形相关的常见耐药性人类癫痫，可对受影响的儿童和成人造成灾难性后果。最终，我们的研究计划将阐明不同的神经元群体在癫痫发作的过渡过程中是如何放电的，以及它们的活动与兴奋性和抑制性信号的关系。改进的认识对于改进策略，使用先进的工具来改变神经元群的兴奋性是至关重要的。我们的实验室在癫痫基因治疗方面处于领先地位，其中一个项目将于2021年进入临床试验，因此我们有能力将当前研究提案的发现转化为患者的利益。

Epilepsy affects up to 1% of the population, and even with optimal medication 30% of affected people continue to have seizures. People with drug-resistant epilepsy suffer the side effects of drugs, have a substantial risk of depression and other comorbidities, and have a several-fold increased mortality over the general population through accidents, suicide and sudden unexpected death in epilepsy. The only realistic prospect of seizure freedom is surgery to remove the region where seizures arise, but this is only possible in a small subset of affected people because the seizure-onset zone is often intermingled with brain areas that are necessary for language, memory, movement, sensation or other cognitive functions. The dismal current state of affairs is, to a great extent, due to a poor understanding of the mechanisms by which seizures occur. Different types of seizures are generally described in terms of large-scale networks, and the fine details of which neurons fire, how and where, are only understood in a very superficial way, because until very recently they could only be studied very indirectly using electrical methods. This means that it is still quite unclear which subtypes of excitatory and inhibitory neurons in different regions of the brain become active, or become silent, as seizures initiate, and in which order. A major advance has been the development of optical methods whereby the activity of populations of neurons can be visualised by using molecules that fluoresce in response to changes in the levels of calcium ions. This method can be implemented in experimental rodent models of epilepsy but is quite sluggish and does not detect how neurons signal among themselves by releasing the main excitatory and inhibitory neurotransmitters (glutamate and GABA respectively). We have recently overcome this limitation by using another type of fluorescence microscopy that detects the levels of glutamate and GABA as they are released by neurons. This gives a faster read-out of how populations of excitatory and inhibitory neurons are recruited and gives a direct insight into signalling among populations of neurons. We can also combine imaging of two neurotransmitters simultaneously, or of a neurotransmitter and calcium, by recording fluorescent light of different wavelengths, and in parallel record the electrical discharges that conventionally define seizures. Our proposal builds on our methodological breakthroughs and preliminary data that indicate that an inhibitory 'halo' surrounding the site of initiation of pathological discharges gradually fails in the lead-up to a full-blown seizure that escapes to spread across the brain. We will follow this up by asking which sub-populations of inhibitory neurons fail, and test the hypothesis that they do so because they become over-excited and unable to fire. This will be achieved by manipulating their electrical properties using light-activated proteins that mimic excitation or inhibition ('optogenetics'). We will, moreover, extend from models of epilepsy where seizures are evoked by the application of chemicals to the brain to a rodent model where seizures arise spontaneously. This reproduces a frequently drug-resistant form of human epilepsy associated with focal malformations of brain development that can have catastrophic outcomes for affected children and adults. Ultimately our research proposal will shed light on how different populations of neurons fire at the transition to seizures, and how their activity relates to excitatory and inhibitory signalling. An improved understanding is essential to refine strategies to treat drug-resistant epilepsy with advanced tools to alter the excitability of populations of neurons. Our laboratory is at the vanguard of gene therapy for epilepsy, with one programme entering clinical trials in 2021, and so we are well positioned to translate the findings of the present research proposal for patient benefit.