shRNA#14 strongly reduces Na⁺-currents and abolishes action potential firing in GCs.

(A) Representative traces of whole-cell voltage-clamp recordings of control cells (black), rAAV-mmshRNA#14-infected (gray), and rAAV-shRNA#14-infected (red) GCs. All recordings were done at 34 ± 1 °C. Voltage-clamp recordings were made in bath solution containing 10 mM TEA. The voltage traces depict voltage steps from −30 mV to +10 mV. (B) Quantification of the Na+ currents (color code as in A). No differences were observed between control cells (n = 6) and rAAV-mmshRNA#14-infected cells (n = 6; ANOVA, F = 64.59, p + currents in GCs infected with rAAV-shRNA#14 (n = 6) were strongly reduced (ANOVA, F = 64.59, p V1.2 expression in the OB. Maximum-intensity projections of confocal stacks extending over 20 consecutive image frames of stainings against NaV1.2 in control (upper panel), shRNAmm (middle panel), and shRNA#14 (lower panel). Grayscale levels adjusted for display. (F) Quantification of the intensity ratio relative to control levels of the NaV1.2 subtype in control, shRNAmm, and shRNA#14. MIPs of 37.53 μm × 37.53 μm (100 × 100 pixels), as in (E), were used (n = 12 per condition), obtained from two independent experiments. The intensity ratio in the shRNA#14 condition was reduced in comparison to the control and shRNAmm conditions (ANOVA, F = 265.2, p n = 4 mice), rAAV-mmshRNA#14 (“mm,” n = 6 mice), or rAAV-shRNA#14 (“kd,” n = 6 mice). DAPI-stained nuclei (blue), anti-NeuN antibody labels neuronal nuclei (magenta), and GFP reveal transduced GCs (green). (H) Efficiency of neuronal transduction by rAAV: control: 47.57% ± 0.93%, mm: 54.92% ± 1.73%, kd: 55.45% ± 2.35%; ANOVA, F = 2.69, p = 0.12. Data used in the generation of this figure can be found in S1 Data. DAPI, 4,6-diamidino-2-phenylindole; GC, granule cell; GCL, granule cell layer; GFP, green fluorescent protein; mGFP, membrane-bound GFP; MIP, maximum intensity projection; shRNAmm, rAAV, recombinant adeno-associated virus; shRNA, short hairpin RNA; TEA, tetraethylammonium