In vivo imaging of axonal and dendritic structures in neonatal mouse cortex

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.