Figure 5.

Neonatal intraventricular injection of adeno-associated virus induces efficient recombination events in hippocampal Cajal–Retzius cells in the ΔNp73-Cre mice. A, Schematic shows the experimental procedure and brain region of interest. The lateral ventricles of P0 ΔNp73-Cre mouse pups were freehand injected with different titers of AAV serotype 8 carrying a Cre-dependent expression construct for the ChR2-mCherry fusion protein under the EF1α promoter (AAV8/EF1α-DIO-ChR2-mCherry). Injected mice were analyzed at P14. Three representative confocal images show the hippocampal fissure (HF) area after intraventricular injection of three titers of the AAV8 for comparison of transduction efficiency. Viral-mediated, Cre-dependent transgene expression is visualized by immunostaining for mCherry. Note the lack of transduction using the 1.0 × 10¹¹ viral titer. Cre-independent spontaneous recombination events were observed in some cortical and hippocampal neurons in mice injected with the 1.0 × 10¹³ titer (white arrows). Scale bars, 200 μm. B, Quantification of viral transduction efficiency in CR cells and recombination events in granule neurons. Data are presented as scatter plots with all data points shown and error bars representing ±SD. Each data point is an individual animal, whereby three sections were measured for each animal. The total number of CR cells per animal analyzed along the hippocampal fissure of different animals were as follows: 1.0 × 10¹¹ titer, 90–174 cells; 1.0 × 10¹² titer, 82–152 cells; 1.0 × 10¹³ titer, 108–188 cells. The total number of CR cells per animal analyzed in the molecular layer of the infrapyramidal blade of different animals were as follows: 1.0 × 10¹¹ titer, 47–83 cells; 1.0 × 10¹² titer, 39–82 cells; 1.0 × 10¹³ titer, 42–77 cells. Statistical analyses were performed using nested one-way ANOVA with Tukey’s post hoc test. **p < 0.01; ***p < 0.001; ****p < 0.0001. C, Immunostaining for TRP73, mCherry, and RELN in P14 1.0 × 10¹² GC/ml virus-injected tissue confirmed that mCherry-expressing cells (yellow arrows) are CR cells. Some CR cells are not transduced as they do not express mCherry (white arrowheads). Scale bar, 20 μm. GL, Granular layer; ML, molecular layer; SLM, stratum lacunosum-moleculare; CA1, Cornu Ammonis 1; CA3, Cornu Ammonis 3; loML, lower molecular layer (i.e., molecular layer of the infrapyramidal blade of the dentate gyrus). The images that illustrate the result using AAV/CAG-YFP are shown in Extended Data Figure 5-1. The images that show the specificity of our neonatal AAV injection approach in the hippocampus are provided in Extended Data Figure 5-2. The data that demonstrate the absence of Cre-mediated recombination in the wild-type mice are shown in Extended Data Figure 5-3.