Figure 1: Cell- and layer-specific transfection of neonatal prelimbic cortex by site-directed in utero electroporation.

(a) Structure of the ChR2(ET/TC)-containing and opsin-free constructs. (b) Schematic drawing illustrating the orientation of electrode paddles for specific targeting of pyramidal neurons in layer II/III and V/VI of PL. (c) tDimer2-expressing cells (red) in a 50-μm-thick coronal section of a P10 mouse at the level of the prefrontal cortex after IUE at E15.5. (d) Photographs displaying NeuN (blue), Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) (green) and GABA (green) immunohistochemistry in relationship to tDimer2 expression (red) at P10 after IUE at E15.5. Note that the transfection is restricted to CaMKII-positive and GABA-negative neurons in layer II/III. (e) Bar diagrams displaying the mean density of NeuN-, CaMKII-, GABA-, tDimer2-, tDimer2+NeuN-, tDimer2+CaMKII- as well as tDimer2+GABA-positive cells in layer I (top), II/III (middle) and V/VI (bottom) of PL from P8 to P10 mice after IUE with constructs containing CAG-ChR2(ET/TC)-2A-tDimer2 (dark blue bars, n=28 slices from 7 pups for tDimer2, NeuN, GABA; n=20 slices from 5 pups for CaMKII) or opsin-free constructs (light blue bars, n=28 slices from 7 pups for tDimer2, NeuN, GABA; n=20 slices from 5 pups for CaMKII). (f–h) Same as (c–e) for IUE at E12.5 with constructs containing CAG-ChR2(ET/TC)-2A-tDimer2 (dark red bars, n=20 slices from 5 pups for tDimer2, NeuN, GABA; n=20 slices from 5 pups for CaMKII) or opsin-free constructs (light red bars, n=20 slices from 5 pups for tDimer2, NeuN, GABA; n=16 slices from 4 pups for CaMKII). Note that the transfection is restricted to CaMKII-positive and GABA-negative cells mainly located in layer V/VI. Data are presented as mean±s.e.m. ITR, inverted terminal repeat. WRE, woodchuck hepatitis virus posttranscriptional regulatory element.