Monreal-Trigo Javier, Terres-Haro Jose Manuel, Martinez-Rojas Beatriz, Sanchez-Martin Maria Del Mar, Giraldo Esther, Manzano Victoria Moreno, Fillol Miguel Alcaniz
IEEE Trans Biomed Circuits Syst. 2022 Dec;16(6):1397-1405. doi: 10.1109/TBCAS.2022.3226558. Epub 2023 Feb 14.
Optogenetics is an emerging discipline with multiple applications in neuroscience, allowing to study neuronal pathways or serving for therapeutic applications such as in the treatment of anxiety disorder, autism spectrum disorders (ASDs), or Parkinson's disease. More recently optogenetics is opening its way also to stem cell-based therapeutic applications for neuronal regeneration after stroke or spinal cord injury. The results of optogenetic stimulation are usually evaluated by immunofluorescence or flow cytometry, and the observation of transient responses after stimulation, as in cardiac electrophysiology studies, by optical microscopy. However, certain phenomena, such as the ultra-fast calcium waves acquisition upon simultaneous optogenetics, are beyond the scope of current instrumentation, since they require higher image resolution in real-time, employing for instance time-lapse confocal microscopy. Therefore, in this work, an optogenetic stimulation matrix controllable from a graphical user interface has been developed for its use with a standard 24-well plate for an inverted confocal microscope use and validated by using a photoactivable adenyl cyclase (bPAC) overexpressed in rat fetal cortical neurons and the consequent calcium waves propagation upon 100 ms pulsed blue light stimulation.
光遗传学是一门在神经科学中有多种应用的新兴学科，可用于研究神经元通路或用于治疗应用，如治疗焦虑症、自闭症谱系障碍（ASD）或帕金森病。最近，光遗传学也正在为中风或脊髓损伤后基于干细胞的神经元再生治疗应用开辟道路。光遗传学刺激的结果通常通过免疫荧光或流式细胞术进行评估，而对于刺激后的瞬态反应的观察，如同在心脏电生理学研究中那样，通过光学显微镜进行。然而，某些现象，如在同时进行光遗传学操作时超快速钙波的获取，超出了当前仪器的范围，因为它们需要更高的实时图像分辨率，例如采用延时共聚焦显微镜。因此，在这项工作中，已经开发了一种可通过图形用户界面控制的光遗传学刺激矩阵，用于与倒置共聚焦显微镜使用的标准24孔板配合使用，并通过在大鼠胎儿皮质神经元中过表达的光激活腺苷酸环化酶（bPAC）以及在100毫秒脉冲蓝光刺激后随之而来的钙波传播进行了验证。
