Deciphering the genomic mechanisms underlying the physiology of human brain stimulation

人类大脑刺激的潜在机制尚不清楚，尤其是在基因表达水平上。为了解决这一知识缺口，我们提出了一系列三个实验，利用从神经外科患者那里获得高质量的人类神经组织的机会，以衡量脑刺激对基因表达的影响。我们的实验将产生数据，以解释刺激引发的大脑回路变化背后的基因表达水平的变化。我们的研究团队拥有七年的经验，使用已建立的研究神经外科患者大脑皮质组织的管道来分析基因表达，包括应用尖端方法测量基因表达。这些方法包括单核RNA测序(SnRNA-seq)和激动型单核添加ATAC测序(nATAC-seq)，以了解与刺激相关的转录因子变化和染色质重塑。我们关于特定基因类别的假设是从我们发表的数据中发展出来的，这些数据将基因表达的变化与与成功记忆形成有关的神经生理特征(大脑振荡)联系在一起。在这个建议中，我们的实验解决了复杂的问题，即刺激如何使用三种互补的方法改变神经回路。首先，我们将在颞叶切除患者的脑组织切除前立即在体内使用直接皮质刺激，随后进行基因表达分析。我们的计划得到了初步数据的支持，这些数据显示了皮质刺激后即刻早期基因(IEGs)表达的差异，这与从动物模型中得出的预测一致。其次，我们将建立在我们已经实施的人类神经组织培养技术(来自神经外科患者)的基础上，来测量慢性体外刺激引起的基因表达变化。本实验将利用我们对基因信息的时间序列建模的经验，阐明刺激环境中基因表达的时间动力学，包括转录因子的变化。最后，我们将使用多电极阵列(MEA)来测量体外刺激对共放电神经元网络的影响，直接测试刺激诱导局部电路变化的模型。我们将把这些电生理措施与刺激引起的基因表达变化联系起来。这个实验建立在我们发表的研究人类单个单位记录中的网络活动的工作基础上，以及我们的初步数据证明了在培养中记录来自人类神经组织的电生理信号的能力。为了便于比较，体内和体外实验中的刺激参数被设计成对齐的。综上所述，我们的实验将提供开创性的数据，阐明大脑刺激如何引发神经调节的遗传基础。研究团队的经验和在使用神经外科组织标本发布数据方面的可靠记录支持了我们对成功的期望。

The underlying mechanisms of brain stimulation in humans are poorly understood, especially at the level of gene expression. To address this gap in knowledge, we propose a series of three experiments that take advantage of the opportunity to obtain high-quality human neural tissue from neurosurgical patients in order to measure the impact of brain stimulation on gene expression. Our experiments will generate data to explicate changes at the level of gene expression that underlie brain circuit changes elicited by stimulation. Our study team has seven years of experience analyzing gene expression using an established pipeline for studying human cortical tissue from neurosurgical patients, including application of cutting-edge methods for measuring gene expression. These methods include single nuclei RNA-sequencing (snRNA-seq) and the exciting addition of single nuclei ATAC-sequencing (snATAC-seq) to understand stimulation-related changes in transcription factors and chromatin remodeling. Our hypotheses regarding specific gene classes were developed from our published data correlating gene expression changes with neurophysiological signatures (brain oscillations) linked with successful memory formation. In this proposal, our experiments address the complex problem of how stimulation alters neural circuits using three complementary approaches. First, we will use direct cortical stimulation in vivo immediately prior to resection of brain tissue in temporal lobectomy patients, followed by gene expression analysis. Our plans are supported by preliminary data showing differences in expression of immediate early genes (IEGs) following cortical stimulation, in line with predictions drawn from animal models. Second, we will build on techniques we have implemented for culture of human neural tissue (from neurosurgical patients) to measure gene expression changes elicited by chronic ex vivo stimulation. This experiment will elucidate the temporal dynamics of gene expression in the setting of stimulation, including transcription factor changes, using our experience with time series modeling of gene information. Finally, we will use multi-electrode arrays (MEAs) to measure the impact of ex vivo stimulation on networks of co-firing neurons, directly testing models of stimulation-induced changes in local circuits. We will connect these electrophysiological measures with gene expression changes elicited by stimulation. This experiment builds on our published work studying network activity in single unit recordings in humans, as well as our preliminary data demonstrating the ability to record electrophysiological signals from human neural tissue in culture. The stimulation parameters were developed to be aligned across in vivo and in vitro experiments to facilitate comparison. Taken together, our experiments will provide ground-breaking data elucidating the genetic underpinnings of how brain stimulation elicits neuromodulation. The experience of research team and proven record in publishing data using neurosurgical tissue specimens supports our expectations of success.