Department of Chemical and Biomolecular Engineering, University of California, Berkeley, Berkeley, CA, USA.
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA.
Nat Protoc. 2021 Jun;16(6):3026-3048. doi: 10.1038/s41596-021-00530-4. Epub 2021 May 21.
Dopamine neuromodulation of neural synapses is a process implicated in a number of critical brain functions and diseases. Development of protocols to visualize this dynamic neurochemical process is essential to understanding how dopamine modulates brain function. We have developed a non-genetically encoded, near-IR (nIR) catecholamine nanosensor (nIRCat) capable of identifying ~2-µm dopamine release hotspots in dorsal striatal brain slices. nIRCat is readily synthesized through sonication of single walled carbon nanotubes with DNA oligos, can be readily introduced into both genetically tractable and intractable organisms and is compatible with a number of dopamine receptor agonists and antagonists. Here we describe the synthesis, characterization and implementation of nIRCat in acute mouse brain slices. We demonstrate how nIRCat can be used to image electrically or optogenetically stimulated dopamine release, and how these procedures can be leveraged to study the effects of dopamine receptor pharmacology. In addition, we provide suggestions for building or adapting wide-field microscopy to be compatible with nIRCat nIR fluorescence imaging. We discuss strategies for analyzing nIR video data to identify dopamine release hotspots and quantify their kinetics. This protocol can be adapted and implemented for imaging other neuromodulators by using probes of this class and can be used in a broad range of species without genetic manipulation. The synthesis and characterization protocols for nIRCat take ~5 h, and the preparation and fluorescence imaging of live brain slices by using nIRCats require ~6 h.
多巴胺能神经元突触的神经调制是许多关键大脑功能和疾病所涉及的过程。开发可视化这种动态神经化学过程的方案对于理解多巴胺如何调节大脑功能至关重要。我们已经开发出一种非基因编码的近红外（nIR）儿茶酚胺纳米传感器（nIRCat），能够识别背侧纹状体脑片中约 2 µm 的多巴胺释放热点。nIRCat 通过超声处理单壁碳纳米管与 DNA 寡核苷酸很容易合成，可以很容易地引入遗传上可追踪和不可追踪的生物体，并且与许多多巴胺受体激动剂和拮抗剂兼容。在这里，我们描述了 nIRCat 在急性小鼠脑片中的合成、表征和应用。我们展示了如何使用 nIRCat 来成像电或光遗传学刺激的多巴胺释放，以及这些程序如何用于研究多巴胺受体药理学的影响。此外，我们还提供了有关构建或适应宽场显微镜以与 nIRCat 的 nIR 荧光成像兼容的建议。我们讨论了分析 nIR 视频数据以识别多巴胺释放热点并量化其动力学的策略。通过使用此类探针，可以对其他神经调节剂进行调整和实施此方案，并且可以在无需遗传操作的情况下在广泛的物种中使用。nIRCat 的合成和表征方案需要约 5 小时，而使用 nIRCat 制备和荧光成像活脑片需要约 6 小时。
