AAV-based bright and sparse labeling of versatile neurons adaptable in Cre-dependent genetic backgrounds

Sparse labeling techniques are essential for morphological analysis of the central nervous system. Various sophisticated strategies have been developed, but conventional methods, such as Golgi-Cox staining and biocytin injection, remain widely used. Recent advances in adeno-associated virus (AAV) technology have enabled sparse neuronal labeling in rodents, either through postnatal AAV injection or by co-injecting a dilute Cre recombinase-expressing driver with a Cre-dependent amplifier. However, postnatal AAV injection requires prior preparation, and Cre-dependent systems are incompatible with Cre-expressing or floxed genetic backgrounds. Here, we present a Cre-orthogonal AAV-based sparse labeling method that utilizes Supernova technology in male and female mice. We employed Flpe recombinase to achieve Cre-independent labeling. An intra-retro-orbital injection of PHP.eB AAVs failed to label neurons, whereas local injection enabled the bright and sparse labeling of multiple neuronal types, including cerebellar neurons. The labeled neurons were sufficiently bright to visualize fine structures, such as dendritic spines, without the need for immunostaining. This method is compatible with both Cre-expressing and floxed genetic backgrounds. Furthermore, we demonstrated that the labeled neurons can be manipulated by the co-injection of a Flpe-dependent third virus. This straightforward and flexible approach is applicable across various genetic backgrounds and can be readily integrated into existing experimental systems. Significance Statement This method enables the bright and sparse labeling of diverse neuronal populations in rodents. A simple injection of two AAV vectors in the target region labels a small subset of neurons in 2-3 weeks. The labeled neurons are sufficiently bright to visualize fine subcellular structures, such as dendritic spines, without the need for immunostaining. This approach does not require complex transgenic strategies and is readily applicable to existing experimental systems. The labeling is independent of Cre recombinase, and it is broadly compatible with a wide range of Cre-expressing and floxed animals. Moreover, the labeled cells express tTA and Flpe recombinase, thereby enabling further genetic manipulation through co-infection with additional viruses.