Platform for in vivo analysis of early GABA synapse function in neural circuitry formation

在我们资助的NSERC发现资助项目“大脑发育期间抑制性和兴奋性神经递质系统之间的串扰”中，我们正在研究不同类型的突触连接之间的相互作用如何有助于控制神经回路的发育和组织。在发育过程中，神经元建立了兴奋性和抑制性突触连接的复杂平衡，这种平衡对于大脑中正确编码信息至关重要。过度的抑制对大脑的可塑性有害，而过度的兴奋则对神经元有害。究竟如何实现这种平衡仍然知之甚少。我们已经发现，通过神经递质GABA的早期信号控制发育中海马神经元上兴奋性突触的数量。使用体外方法和高分辨率延时共聚焦显微镜的组合，我们已经取得了重大进展，在确定GABA传输如何帮助设置兴奋性突触的数量在神经元的早期发育。我们现在正处于该项目的关键阶段，我们需要了解这些机制如何在活体大脑中工作。在本研究中，我们提出建立一个先进的平台，通过结合子宫内电穿孔和膜片钳电生理学在小鼠体内进行实验分析。子宫内电穿孔是需要在体内提供新的分子生物学技术，并精确地控制突变型GABA受体的基因表达的时间，而膜片钳电生理学是需要了解修改早期GABA传递对突触组织的功能影响。我们已经获得了子宫内电穿孔设备，膜片钳放大器和操纵器，部分是通过我们的发现补助基金。然而，为了完成平台，我们想购买一台与电生理兼容的显微镜。这种显微镜与其他组件相结合，将为我们的实验提供前所未有的分析水平，同时为学生提供一个独特的机会，使用尖端的方法来回答大脑研究中的一个重要问题。

In our funded NSERC Discovery Grant titled, "Crosstalk between inhibitory and excitatory neurotransmitter systems during brain development" we are investigating how interactions between different types of synaptic connections may help control the development and organization of neural circuitry. During development, neurons establish an intricate balance of excitatory and inhibitory synaptic connections and this balance is essential for properly encoding information in the brain. Too much inhibition is detrimental for brain plasticity, whereas over-excitation is toxic for neurons. Exactly how this equilibrium is achieved remains poorly understood. We have discovered that early signaling through the neurotransmitter GABA controls the number of excitatory synapses on developing hippocampal neurons. Using a combination of in vitro methods and high-resolution time-lapse confocal microscopy, we have made significant progress in determining how GABA transmission helps set the number of excitatory synapses during early development of neurons. We are now at a critical stage of the project where we need to understand how these mechanisms work in the living brain. In this grant, we propose to establish an advanced platform for in vivo experimental analysis in mice through combined in utero electroporation and patch clamp electrophysiology. In utero electroporation is needed to deliver novel molecular biology technologies in vivo and to precisely control the timing of gene expression of mutant GABA receptors, while patch clamp electrophysiology is needed to understand the functional impact of modifying early GABA transmission on synaptic organization. We have already secured in utero electroporation equipment, a patch-clamp amplifier, and manipulators, in part, through our Discovery Grant funds. However, to complete the platform, we would like to purchase a microscope that is compatible with electrophysiology. This microscope, when combined with the other components, will provide an unprecedented level of analysis for our experiments while, at the same time, offer a unique opportunity for students to use a cutting-edge approach to answer an important question in brain research.