Characterization of rAAV-mediated transgene expression. A) Schematic illustration of the rAAV-dnCaMKIV expression cassette, for nomenclature see Methods section. B) Immunoblot analysis of neuronal activity-dependent induction of c-Fos expression in uninfected cultured mouse hippocampal neurons or in cultured mouse hippocampal neurons infected with rAAV-dnCaMKIV or rAAV-hrGFP. The neurons were treated for 4 hr with the GABAA receptor bicuculline (50 μM) to induce action potential bursting [2, 19], or were left untreated. Expression of c-Fos, hrGFP, Flag-tagged dnCaMKIV, and calmodulin (loading control) was analyzed. C) Immunohistochemical analysis of dnCaMKIV expression (using antibodies to the Flag-tag) and expression of the neuronal marker NeuN in the NAc of animals infected in the NAc with rAAV-dnCaMKIV. The overlay of representative photomicrographs (10× objective) of Flag-immunohistochemistry (green) and NeuN (red) is shown. ac, anterior commissure. D) Photomicrograph (100× objective) showing overlay of Flag-immunoreactivity (green) and Hoechst stain (blue). Arrows indicate the cytosolic localization of Flag-tagged dnCaMKIV in two representative neurons; the nuclei in those neurons are indicated with asterisks. E) Schematic diagrams showing the approximate extension of transgene expression (gray shading) in rAAV-dnCaMKIV injected mice. Numbers, distance from bregma .