Cruz-Martin Alberto, Portera-Cailliau Carlos
Cold Spring Harb Protoc. 2014 Jan 1;2014(1):57-64. doi: 10.1101/pdb.prot080150.
Time-lapse in vivo imaging of neuronal structures is critical for understanding the assembly of neural circuits during development. Imaging developing neurons in vivo can be performed with relative ease in lower vertebrates, but ideally, one would also like to image the developing mammalian brain. In vivo chronic imaging of mice is particularly desirable because of the availability of transgenic lines that model human neuropsychiatric disease or those that allow cell- or region-specific expression of fluorescent proteins (e.g., green fluorescent protein [GFP], channelrhodopsins, and genetically encoded calcium indicators). Unfortunately, although chronic imaging of neural structures in adult mice that express GFP is now commonplace, similar approaches in neonatal mice face several additional challenges. First, the small size of the animal complicates the cranial window surgery. Second, there is a tendency for dams to cannibalize pups with head caps. Third, the head cap can impede the normal growth of the skull in neonates, which can limit the duration of imaging. Here, we describe a method for implanting chronic glass-covered cranial windows in the skulls of early postnatal mice through which axonal and dendritic structures can be imaged in vivo over a period of hours or even days.
对神经元结构进行延时活体成像对于理解发育过程中神经回路的组装至关重要。在低等脊椎动物中，对发育中的神经元进行活体成像相对容易，但理想情况下，人们也希望对发育中的哺乳动物大脑进行成像。由于存在模拟人类神经精神疾病的转基因品系，或允许荧光蛋白（如绿色荧光蛋白[GFP]、通道视紫红质和基因编码钙指示剂）进行细胞或区域特异性表达的转基因品系，对小鼠进行活体长期成像尤为可取。不幸的是，尽管目前对表达GFP的成年小鼠的神经结构进行长期成像已很常见，但在新生小鼠中采用类似方法面临一些额外挑战。首先，动物体型小使颅骨开窗手术变得复杂。其次，母鼠有吃掉带帽幼崽的倾向。第三，头帽会阻碍新生小鼠颅骨的正常生长，这可能会限制成像持续时间。在此，我们描述一种在出生后早期小鼠颅骨中植入慢性玻璃覆盖颅骨窗的方法，通过该窗口可在数小时甚至数天内对轴突和树突结构进行活体成像。
