Cellular analyses

Brains were post-fixed overnight in 4% PFA at 4 °C and cut in coronal 40 μm thick vibratome sections that were serially collected along the rostro-caudal axis of the hippocampus and stored at −20 °C in cryoprotectant solution (25% ethylene glycol and 25% glycerol in PBS). Immunohistochemistry was performed stereologically 1 every 6 sections (i.e. 10–12 sections analysed per brain) after blocking and permeabilization with 10% donkey serum in 0.3% Triton X-100 in PBS for 1.5 h at room temperature as previously described²⁵. Primary and secondary antibodies (Table ^S1) were incubated in 3% donkey serum in 0.3% Triton X-100 in PBS overnight at 4 °C. For BrdU detection sections were exposed to HCl 2M for 25 min at 37 °C and EdU detected following the manufacturer’s guidelines (Click-iT EdU, Life technologies). DAPI was used to counterstain nuclei. Pictures were acquired using an automated Zeiss ApoTome or confocal (LSM 780) microscopes (Carl Zeiss) and maximal intensity projections of three optical sections (10 μm thick in total) taken and quantified using Photoshop CS5 (Adobe). In all cases, GFP immunohistochemistry was performed to assess infectivity, which was confirmed in the vast majority (>90%) of the cases. Neurogenesis quantifications were performed on one complete DG series per animal considering the subgranular zone (NSC) or the whole thickness of the granular cell layer (immature and mature neurons) of both the dorsal and ventral hippocampus, whereas neuronal activity (c-Fos) quantifications were restricted to representative sub-areas of the dorsal hippocampus (−1.3 to −2.3 from Bregma) selected in the DAPI channel and identified anatomically (Allen Brain Atlas). Quantifications in the striatum were performed in the lateral and medial portions of the dorsal caudate/putamen (+1.1 to 0.0 from Bregma). Areas were measured using Fiji 1.45b (ImageJ) and morphometric analyses performed on confocal reconstructions through the entire section to include most processes that were later traced and visualised using Fiji (ImageJ).