Automated cell-specific laser detection and ablation of neural circuits in neonatal brain tissue.
Automated cell-specific laser detection and ablation of neural circuits in neonatal brain tissue.
复制标题
自动细胞特异性激光检测和新生儿脑组织神经回路消融。
DOI：
10.1113/jphysiol.2012.247338
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
DelNegro,ChristopherA
中科院分区：
文献类型：
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作者：
Wang,Xueying;Hayes,JohnA;Picardo,MariaCristinaD;DelNegro,ChristopherA
•我们开发了一种自动化系统，可在体外原位检测属于特定群体的神经元，在三维组织标本中绘制其物理位置，然后依次激光消融细胞“目标”，同时从电生理学上监测神经群体的活动。•基于节律性Ca2+荧光活动模式的双光子Ca2+成像和图像处理程序检测和验证目标神经元。•可见波长共聚焦成像和图像处理程序检测和验证表达遗传编码荧光蛋白的目标神经元。•高强度双光子点扫描具有特异性地蒸发目标神经元，同时最大限度地减少对邻近组织的损伤。•在细胞特异性激光消融之前、期间和之后进行网络功能的生理监测，以实时测量对网络功能的影响。神经退行性疾病的一个关键特征是参与产生行为的神经元的病理丧失。为了研究神经回路的网络特性，并为体外原位研究神经退行性变提供补充工具，我们开发了一种自动细胞特异性激光检测和消融系统。该仪器由双光子和可见波长共聚焦成像装置组成，由执行软件控制，根据遗传编码的荧光蛋白或Ca2+成像识别制剂中的神经元，然后在监测网络功能的同时依次切除细胞靶点。网络功能的病理变化可直接归因于消融的细胞，并实时记录。在这里，我们研究了脑干呼吸回路，以证明生理网络活动期间消融的单细胞精度，但该技术可以应用于询问神经系统中的网络特性，这些神经系统在体外蛋白原位还原制备中保留了网络功能。
Key points•We developed an automated system that detects neurons belonging to specific populationsin vitroorin situ, maps their physical locations in three‐dimensional tissue specimens and then laser ablates the cell ‘targets’ one at a time, in sequence, while monitoring neural population activity electrophysiologically.•Two‐photon Ca2+imaging and image processing routines detect and validate target neurons based on rhythmic Ca2+fluorescence activity patterns.•Visible‐wavelength confocal imaging and image processing routines detect and validate target neurons that express genetically encoded fluorescent proteins.•High‐intensity two‐photon spot scanning vaporizes target neurons with specificity while minimizing damage to neighbouring tissue.•Physiological monitoring of network function is performed before, during and after the cell‐specific laser ablations to measure the effects on network functionality in real time.AbstractA key feature of neurodegenerative disease is the pathological loss of neurons that participate in generating behaviour. To investigate network properties of neural circuits and provide a complementary tool to study neurodegenerationin vitroorin situ, we developed an automated cell‐specific laser detection and ablation system. The instrument consists of a two‐photon and visible‐wavelength confocal imaging setup, controlled by executive software, that identifies neurons in preparations based on genetically encoded fluorescent proteins or Ca2+imaging, and then sequentially ablates cell targets while monitoring network function concurrently. Pathological changes in network function can be directly attributed to ablated cells, which are logged in real time. Here, we investigated brainstem respiratory circuits to demonstrate single‐cell precision in ablation during physiological network activity, but the technique could be applied to interrogate network properties in neural systems that retain network functionality in reduced preparationsin vitroorin situ.