To increase the intensity of fluorescent labeling, we designed an AAV viral vector containing three copies of the YFP coding sequence connected by 2A sequences. In vivo imaging 4 weeks find more after P0 injection demonstrated that all major anatomical features of cortical pyramidal neurons could be readily resolved in AAV8-triple-YFP-infected cells (Fig. 11). Cell bodies, apical and basal dendrites, axons, and even individual spines were visible in our preparations (Figs 11A–C). In many cases, apical dendrites

could be traced all the way to their origin in cortical layer 5 (500–600 μm depth). An important advantage of this labeling technique compared with the Thy1-GFP mice is the relatively large number of labeled pyramidal cells in L2/3. Labeled L2/3 pyramids could be imaged in their entirety (Fig. 11D), allowing in vivo comparisons of apical (the primary recipients of feedback inputs) and basal (the primary recipients of feedforward inputs) dendritic

arbors, which has not yet been possible in the Thy1-GFP lines (Holtmaat et al., 2009). These data, along with the finding that fluorescence endures for more than 12 months in injected mice, indicate that P0 injection with AAV-triple-YFP provides an efficient method for labeling the processes of cortical pyramidal neurons for chronic in vivo two-photon imaging. In addition to transducing cortical IWR1 layers that are not labeled in the Thy1-XFP transgenic lines, neonatal viral injection also reaches areas of the brain that are not visible in the Thy1 mice. Specifically, as shown in Figs 2-5, viral transgenesis strongly labels cerebellar Purkinje neurons in both the juvenile and adult. Moreover, viral expression begins within days after injection, at a time when Purkinje neurons are just beginning to form their

mature dendritic arbors. Compared with Endonuclease cortical neurons, few tools exist to sparsely label or genetically manipulate Purkinje neurons. The natural tropism of several AAV serotypes for these cells might offer an easy way to overcome this limitation. We injected AAV8-triple-YFP (109 particles/hemisphere) or AAV1-YFP (1010 particles/hemisphere) at P0 and harvested pups 2, 4, 7, and 14 days later (Fig. 12). Although arborisation is still immature, individual cells can be easily identified at these dilutions. The selection, extension, and elaboration of dendritic processes can be followed from shortly after birth when multiple small neurites are present until a single dendrite develops into its final shape weeks later. With further dilution of the virus, even mature Purkinje cells could be fully identified. Sagittal sections from mice injected with low-titer AAV8-triple-YFP (between 1.0 × 108 and 4.