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. 2023 Oct 16;10(10):ENEURO.0054-23.2023. doi: 10.1523/ENEURO.0054-23.2023

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Copyright © 2023 van Bruggen et al.

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Figure 7. — Neonatal intraventricular injection of AAV8/hSyn-DIO-hM3D-mCherry results in robust and highly specific transgene expression in Cajal–Retzius cells. A, Neonatal ΔNp73-Cre pups were injected with adeno-associated virus serotype 8 carrying a Cre-dependent expression construct for the hM3D-mCherry fusion protein under the hSyn promoter (AAV8/hSyn-DIO-hM3D-mCherry) and brain tissue analyzed at P14. mCherry-expressing cells also express TRP73 and RELN, confirming their identity as CR cells (yellow arrows). Some CR cells do not express mCherry as they were not transduced by the injected AAV8 (white arrowhead). Scale bars: top, 200 μm; bottom, 20 μm. B, Comparison of viral transduction efficiency in CR cells at P14 of the two different viral constructs (AAV8/EF1α-DIO-ChR2-mCherry and AAV8/hSyn-DIO-hM3D-mCherry). C, Reporter expression persists for 7 weeks after neonatal injection. A representative image of a ΔNp73-Cre mouse hippocampus 7 weeks post injection with AAV8/hSyn-DIO-hM3D-mCherry is shown. Scale bar, 200 μm. D, Comparison of viral transduction efficiency in CR cells at 7 weeks of age of the two different viral constructs (AAV8/EF1α-DIO-ChR2-mCherry and AAV8/hSyn-DIO-hM3D-mCherry). E, Comparison of the number of mCherry⁺ granule neurons in P14 and 7-week-old ΔNp73-Cre mice neonatally injected with AAV8/hSyn-DIO-hM3D-mCherry. F, Quantification of the densities of TRP73⁺ CR cells in P14 and 7-week-old wild-type or ΔNp73-Cre mice neonatally injected with AAV8/hSyn-DIO-hM3D-mCherry. SLM, Stratum lacunosum-moleculare; HF, hippocampal fissure; ML, molecular layer; GL, granular layer; CA1, Cornu Ammonis 1; loML, lower molecular layer (i.e., molecular layer of the infrapyramidal blade of the dentate gyrus). Data are presented in B, D, and E as scatter plots with all data points shown and error bars representing ±SD; statistical analyses were performed using nested t tests. Each data point is an individual animal, whereby three sections were measured for each animal. Data are presented as summary data in F with mean ± SD, and statistical analyses were performed using two-way ANOVA with Sidak test to correct for multiple comparisons. **p < 0.01; ***p < 0.001; ****p < 0.0001. The data that demonstrate the absence of Cre-mediated recombination in the wild-type mice are shown in Extended Data Figure 7-1. Analyses of the effect of sex on AAV transduction efficiency are provided in Extended Data Figure 7-2.