Promoter directs tTA expression in forebrain principal neurons (Mayford et al., ). The luciferase (luc) and H1aV genes are transcribed from a bidirectional unit activated upon binding of tTA to heptamerized tet operator sequences (tetO) flanked by CMV minimal promoters. Dox prevents tTA binding to tetO, thus switching off expression of luc and H1aV transgenes. () Screening of transgenic founders for dox-dependent gene regulation. Relative light units corresponding to ratios of firefly to renilla luciferase (rlu-FL/rlu-RL) activity, measured in mouse ear fibroblast cell cultures in absence (black) and presence (white) of dox (Hasan et al., ), are plotted logarithmically on the Y-axis. Circles (solid and open) indicate transgenic lines selected for crossing with Tgα-CaMKII-tTA mice. The open circle indicates the line #14 used in this study. () Para-saggital brain section of a P42 mouse positive for both transgenes (TgH1aV.Fb), in absence of dox. Robust Venus epi-fluorescence was detected in cortical and subcortical regions, especially in the striatum (STR) and the hippocampal formation (HP). Boxed regions (CA1, CA3) represent areas for quantitative H1aV expression analysis. Inset upper left: high-power confocal image of the boxed regions in CA1 and CA3. Epi-fluorescence is robust in CA1 pyramidal cell bodies and dendritic trees but sparse in CA3. Note the intense fluorescence of mossy fiber axons. Inset lower right: proximal dendritic shaft segment from a CA1 neuron after deconvolution, with the fusion-protein seen in spines. () Overexpressed H1aV fusion protein has similar binding properties as endogenous Homer1a. Representative IP-Western blot from forebrain lysates of TgH1aV.Fb (N = 2) and wild-type (N = 2) P42 mice. (1), Immunoprecipitation with anti-GFP antibody pulled mGluR1 from lysates of TgH1aV.Fb mice, indicating binding of H1aV to mGluR1. (2), mGluR1 was also detected with Homer1a-specific antibody. (3, 4), No association with mGluR1 was detected with antibodies against Homer2 or Homer1b/c, indicating efficient competition by overexpressed H1aV. (5), In brains of wild-type mice the GFP antibody did not pull mGluR1. (6, 7), Moderate binding was seen with antibodies against Homer1a and Homer2 in lysates of wild-type brains. (8), Antibody against Homer1b/c robustly pulled mGluR1 from lysates of wild-type brains, indicating strong association. P38 was used to show equal overall levels of protein in the lysates (OB, olfactory bulb; CTX, cerebral cor...