Intraventricular injection of AAV8 at 1010 particles/ventricle transduced 88 ± 3% of NeuN-positive pyramidal neurons in the cerebral cortex, 93.3 ± 0.7% of NeuN-positive pyramidal neurons in the CA1 region of the hippocampus and 87 ± 2% of calbindin D-28k-positive Purkinje cells in the cerebellum (mean ± SEM, n = 20–28, three to five sections/brain from five to six animals). Labeling was also detected in the superior and inferior colliculus, pons, and medulla, with more transduction along superficial structures as

expected from viral diffusion in the cerebrospinal fluid through the fourth ventricle and subarachnoid space (Figs 2G and H). Fluorescence was occasionally detected in the thalamus, but the density of labeled cells was

lower than in other regions of the same brain. To compare the efficiency of neonatal injection with adult injection, adult Epigenetics inhibitor mice were stereotaxically injected with the same titer and volume of virus. This resulted in a much more limited pattern of viral transduction immediately adjacent to the injection site (n = 3, Figs 2I and J vs. K and L). To examine whether viral transgene expression could be maintained long-term, mice were injected bilaterally at birth and harvested 3 weeks or 12 months later to examine the extent and intensity of the fluorescence label (n = 5 for each group). Even 12 months after injection, fluorescence remained at qualitatively Dabrafenib manufacturer similar levels and was located in the same structures as at 3 weeks postinjection. In contrast to the localised injury often seen following adult intracranial injections, we observed no sign of overt malformations or injury to the brain in animals that had undergone neonatal intraventricular injection. The gross neuroanatomy was normal in the mice that we studied (> 100 P0 injections to date), and virally-labeled neurons displayed normal morphology in all brain regions examined. Immunostaining

for astrocytic and microglial markers looked similar to uninjected wild-type animals (data not shown). The injected pups appeared to mature normally and were indistinguishable from uninjected litters at weaning. Although we have not conducted rigorous behavioral assessments, the neonatally injected mice did before not display obvious behavioral abnormalities. One potential disadvantage of transduction with AAV is its delayed onset of transgene expression, which may limit studies on postnatal development in juvenile mice after P0 injection (Sarra et al., 2002; McCarty et al., 2003; Natkunarajah et al., 2008). We wanted to determine in our own hands when transgene expression began and when it peaked, harvesting brains at P2, P4, P7 and P14 after intraventricular P0 injection with AAV8. We selected a virus encoding both tdTomato and Cre-recombinase under the control of the elongation factor 1α (EF1α) promoter, and injected it into Cre-reporter Ai3 mice in which YFP expression is restricted by a loxP-flanked stop cassette (n = 2–5) (Madisen et al., 2010).