Department of Biomedical Engineering, Rutgers University, 599 Taylor Road, Piscataway, NJ 08854, USA.
Child Health Institute of New Jersey, Robert Wood Johnson Medical School, 89 French Street, New Brunswick, NJ 08901, USA and Department of Neuroscience and Cell Biology, 675 Hoes Lane West, Research Tower, Third Floor, Piscataway, NJ 08854, USA and Pediatrics, Robert Wood Johnson Medical School, Rutgers University, One Robert Wood Johnson Place, MEB Third, PO Box 19, New Brunswick, NJ 08903, USA.
Lab Chip. 2020 Mar 17;20(6):1140-1152. doi: 10.1039/c9lc01179j.
Proper brain function relies on the precise arrangement and flow of information between diverse neural subtypes. Developing improved human cell-based models which faithfully mimic biologically relevant connectivity patterns may improve drug screening efforts given the limited success of animal models to predict safety and efficacy of therapeutics in human clinical trials. To address this need, we have developed experimental models of defined neural circuitries through the compartmentalization of neuronal cell subtypes in a 96 well plate-based platform where each microwell is divided into two compartments connected by microchannels allowing high-throughput screening (HTS) of small molecules. We demonstrate that we can generate subtype-specific excitatory and inhibitory induced neuronal cells (iNs) from human stem cell lines and that these neurons form robust functional circuits with defined connectivity. Through the use of the genetically encoded calcium indicator GCaMP6f, we monitor calcium ion transients generated during neuronal firing between and within compartments. We further demonstrate functionality of the circuit by perturbing network activity through the addition of glutamate receptor blockers using automated liquid handling. Lastly, we show that we can stimulate network activity in defined neuronal subtypes through the expression of the designer receptor exclusively activated by designer drugs (DREADD) hM3Dq and application of the ligand clozapine-N-oxide (CNO). Our results demonstrate the formation of functional neural circuits in a high-throughput platform that is compatible with compound screening, representing an important step towards developing new screening platforms for studying and ultimately treating psychiatric brain disorders that arise from disordered neural circuit function.
正常的大脑功能依赖于不同神经亚型之间信息的精确排列和流动。鉴于动物模型在预测治疗药物在人类临床试验中的安全性和疗效方面的有限成功，开发能够忠实地模拟生物学相关连接模式的改进的人类基于细胞的模型可能会改善药物筛选工作。为了解决这一需求，我们通过在基于 96 孔板的平台上将神经元细胞亚型分隔在微腔内，开发了定义明确的神经回路实验模型，其中每个微孔被分成两个通过微通道连接的腔室，从而实现高通量筛选（HTS）小分子。我们证明，我们可以从人类干细胞系中生成具有特定亚型的兴奋性和抑制性诱导神经元（iNs），并且这些神经元形成具有定义连接性的稳健功能回路。通过使用基因编码钙指示剂 GCaMP6f，我们监测在腔室之间和内部神经元放电过程中产生的钙离子瞬变。我们通过使用自动液体处理添加谷氨酸受体阻滞剂来干扰网络活动，进一步证明了电路的功能。最后，我们通过表达专门被设计药物激活的设计受体（DREADD）hM3Dq 并应用配体氯氮平-N-氧化物（CNO）来刺激特定神经元亚型的网络活动，展示了我们可以在高吞吐量平台上形成功能神经回路，该平台与化合物筛选兼容，这是朝着开发用于研究和最终治疗源自神经回路功能障碍的精神疾病的新型筛选平台迈出的重要一步。
