Imaging neocortical neurons through a chronic cranial window

The rich structural dynamics of axonal arbors and neuronal circuitry can only be revealed through direct and repeated observations of the same neuron(s) over time, preferably in vivo. This protocol describes a long-term, high-resolution method for imaging neocortical neurons in vivo, using a combination of two-photon laser scanning microscopy (2PLSM) and a surgically implanted chronic cranial window. The window is used because the skull of most mammals is too opaque to allow high-resolution imaging of cortical neurons. Using this method, it is feasible to image the smallest neuronal structures in the superficial layers of the neocortex, such as dendritic spines and axonal boutons. Because the surface area of the craniotomy is relatively large, this technique is even suitable for use when labeled neurons are relatively uncommon. The surgery and imaging procedures are illustrated with examples from our studies of structural plasticity in the developing or adult mouse brain. The protocol is optimized for adult mice; we have used mice up to postnatal day 511 (P511). With minor modifications, it is possible to image neurons in rats and mice from P2. Most of our studies have used the Thy1 promoter to drive expression of fluorophores in subsets of cortical neurons.