PMC:2518050 / 22339-28312 JSONTXT

{"project":"0_colil","denotations":[{"id":"18982121-12429690-359875","span":{"begin":192,"end":196},"obj":"12429690"},{"id":"18982121-11431570-359876","span":{"begin":211,"end":215},"obj":"11431570"},{"id":"18982121-11753368-359877","span":{"begin":312,"end":316},"obj":"11753368"},{"id":"18982121-7567477-359878","span":{"begin":455,"end":459},"obj":"7567477"},{"id":"18982121-12429690-359879","span":{"begin":480,"end":484},"obj":"12429690"},{"id":"18982121-15208716-359880","span":{"begin":662,"end":666},"obj":"15208716"},{"id":"18982121-8939850-359881","span":{"begin":1080,"end":1084},"obj":"8939850"},{"id":"18982121-15208716-359882","span":{"begin":1654,"end":1658},"obj":"15208716"},{"id":"18982121-8939850-359883","span":{"begin":3756,"end":3760},"obj":"8939850"},{"id":"18982121-15548671-359884","span":{"begin":3966,"end":3970},"obj":"15548671"},{"id":"18982121-12357963-359885","span":{"begin":3992,"end":3996},"obj":"12357963"},{"id":"18982121-11431570-359886","span":{"begin":5029,"end":5033},"obj":"11431570"},{"id":"18982121-10559416-359887","span":{"begin":5831,"end":5835},"obj":"10559416"}],"text":"Conditional Homer1a expression in forebrain\nTo regulate Homer1a expression in adult mouse forebrain, we utilized the tet-off version of the tet-dependent expression system (Gossen and Bujard, 2002; Mack et al., 2001). The rat Homer1a cDNA was fused at its 3′ end to Venus fluorescent protein cDNA (Nagai et al., 2002) and expressed from a tet-responsive bidirectional transgene harboring in addition the firefly luciferase gene (Figure 1A) (Baron et al., 1995; Gossen and Bujard, 2002). Expression of the luciferase gene permitted the use of ear fibroblasts in screening founders for the presence of a functional tet-responsive transcription unit (Hasan et al., 2004). The highest expresser of 18 candidate founders (Figure 1B and Materials and Methods) was chosen for our studies, and the corresponding transgenic line is here referred to as TgH1aV.\nFigure 1 Tetracycline-dependent forebrain expression of H1aV. (A) Transgenes for tet-dependent H1aV expression in the mouse forebrain. The α-CaMKII promoter directs tTA expression in forebrain principal neurons (Mayford et al., 1996). The luciferase (luc) and H1aV genes are transcribed from a bidirectional unit activated upon binding of tTA to heptamerized tet operator sequences (tetO7) flanked by CMV minimal promoters. Dox prevents tTA binding to tetO7, thus switching off expression of luc and H1aV transgenes. (B) 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., 2004), 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. (C) 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. (D) 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 cortex; STR, striatum; HP, hippocampus; MW., molecular weight). Overexpression in forebrain was in response to α-calcium/calmodulin-dependent kinase II (α-CaMKII) promoter-driven expression of the tet-dependent tTA (Tgα-CaMKII-tTA) (Mayford et al., 1996) (Figure 1A). Double transgenic Tgα-CaMKII-tTA/TgH1aV (abbreviated here as TgH1aV.Fb) mice (studied at P42) displayed robust Venus epi-fluorescence in all forebrain structures (Figure 1C) (Krestel et al., 2004; Schonig and Bujard, 2003). Strong fluorescence was seen in cortical and subcortical brain regions, with homogeneous distribution in the CA1 region of the hippocampus. Antibody stainings with a Homer1a-specific antibody detected the H1aV fusion protein in fluorescent but not in non-fluorescent cells, indicating sustained expression of the IEG fusion (data not shown). TgH1aV.Fb transgenic mice, when supplied with the tet derivative doxycycline in the drinking water (dox, 0.1 g/l) for 3 weeks, showed efficient down-regulation of H1aV. Thus, TgH1aV.Fb mice allowed us to investigate the effect of sustained H1aV expression on hippocampal synaptic plasticity and spatial learning.\nInspection of sagittal hippocampal sections of TgH1aV.Fb mice revealed intense fluorescence in the somata and dendrites of pyramidal neurons in the subiculum, the CA1 region and the dentate gyrus (Figure 1C and inset, upper left side). The cell bodies of CA3 neurons displayed only sparse fluorescence, as reported for other α-CaMKII promoter-driven constructs (Mack et al., 2001) (Figure 1C and inset, upper left side). However, robust staining was detected in CA1 pyramidal cell dendrites of stratum radiatum and oriens, mossy fiber projections from the dentate gyrus (Figure 1C and inset, upper left side), and dendritic spines after enlargement of a dendritic segment following deconvolution (Figure 1C and inset, lower right side). To quantify the proportion of H1aV expressing neurons, we counted the number of H1aV-expressing cells by direct epi-fluorescence or after staining with fluorescent or HRP conjugated antibody against Venus in randomly chosen sections of CA1 and CA3 by confocal microscopy (Figure 1C, white boxes). Of ∼800 CA1 cells, 80 ± 9% (mean ± SD) expressed H1aV. The remaining, non-expressing 15–20% of CA1 neurons may be interneurons (Gulyas et al., 1999), indicating that the vast majority of CA1 principal cells expressed H1aV. In contrast, only ∼15% of the CA3 neurons were Venus positive."}

{"project":"TEST0","denotations":[{"id":"18982121-148-156-359875","span":{"begin":192,"end":196},"obj":"[\"12429690\"]"},{"id":"18982121-167-175-359876","span":{"begin":211,"end":215},"obj":"[\"11431570\"]"},{"id":"18982121-94-102-359877","span":{"begin":312,"end":316},"obj":"[\"11753368\"]"},{"id":"18982121-233-241-359878","span":{"begin":455,"end":459},"obj":"[\"7567477\"]"},{"id":"18982121-231-239-359879","span":{"begin":480,"end":484},"obj":"[\"12429690\"]"},{"id":"18982121-175-183-359880","span":{"begin":662,"end":666},"obj":"[\"15208716\"]"},{"id":"18982121-93-101-359881","span":{"begin":1080,"end":1084},"obj":"[\"8939850\"]"},{"id":"18982121-213-221-359882","span":{"begin":1654,"end":1658},"obj":"[\"15208716\"]"},{"id":"18982121-185-193-359883","span":{"begin":3756,"end":3760},"obj":"[\"8939850\"]"},{"id":"18982121-123-131-359884","span":{"begin":3966,"end":3970},"obj":"[\"15548671\"]"},{"id":"18982121-149-157-359885","span":{"begin":3992,"end":3996},"obj":"[\"12357963\"]"},{"id":"18982121-139-147-359886","span":{"begin":5029,"end":5033},"obj":"[\"11431570\"]"},{"id":"18982121-88-96-359887","span":{"begin":5831,"end":5835},"obj":"[\"10559416\"]"}],"text":"Conditional Homer1a expression in forebrain\nTo regulate Homer1a expression in adult mouse forebrain, we utilized the tet-off version of the tet-dependent expression system (Gossen and Bujard, 2002; Mack et al., 2001). The rat Homer1a cDNA was fused at its 3′ end to Venus fluorescent protein cDNA (Nagai et al., 2002) and expressed from a tet-responsive bidirectional transgene harboring in addition the firefly luciferase gene (Figure 1A) (Baron et al., 1995; Gossen and Bujard, 2002). Expression of the luciferase gene permitted the use of ear fibroblasts in screening founders for the presence of a functional tet-responsive transcription unit (Hasan et al., 2004). The highest expresser of 18 candidate founders (Figure 1B and Materials and Methods) was chosen for our studies, and the corresponding transgenic line is here referred to as TgH1aV.\nFigure 1 Tetracycline-dependent forebrain expression of H1aV. (A) Transgenes for tet-dependent H1aV expression in the mouse forebrain. The α-CaMKII promoter directs tTA expression in forebrain principal neurons (Mayford et al., 1996). The luciferase (luc) and H1aV genes are transcribed from a bidirectional unit activated upon binding of tTA to heptamerized tet operator sequences (tetO7) flanked by CMV minimal promoters. Dox prevents tTA binding to tetO7, thus switching off expression of luc and H1aV transgenes. (B) 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., 2004), 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. (C) 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. (D) 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 cortex; STR, striatum; HP, hippocampus; MW., molecular weight). Overexpression in forebrain was in response to α-calcium/calmodulin-dependent kinase II (α-CaMKII) promoter-driven expression of the tet-dependent tTA (Tgα-CaMKII-tTA) (Mayford et al., 1996) (Figure 1A). Double transgenic Tgα-CaMKII-tTA/TgH1aV (abbreviated here as TgH1aV.Fb) mice (studied at P42) displayed robust Venus epi-fluorescence in all forebrain structures (Figure 1C) (Krestel et al., 2004; Schonig and Bujard, 2003). Strong fluorescence was seen in cortical and subcortical brain regions, with homogeneous distribution in the CA1 region of the hippocampus. Antibody stainings with a Homer1a-specific antibody detected the H1aV fusion protein in fluorescent but not in non-fluorescent cells, indicating sustained expression of the IEG fusion (data not shown). TgH1aV.Fb transgenic mice, when supplied with the tet derivative doxycycline in the drinking water (dox, 0.1 g/l) for 3 weeks, showed efficient down-regulation of H1aV. Thus, TgH1aV.Fb mice allowed us to investigate the effect of sustained H1aV expression on hippocampal synaptic plasticity and spatial learning.\nInspection of sagittal hippocampal sections of TgH1aV.Fb mice revealed intense fluorescence in the somata and dendrites of pyramidal neurons in the subiculum, the CA1 region and the dentate gyrus (Figure 1C and inset, upper left side). The cell bodies of CA3 neurons displayed only sparse fluorescence, as reported for other α-CaMKII promoter-driven constructs (Mack et al., 2001) (Figure 1C and inset, upper left side). However, robust staining was detected in CA1 pyramidal cell dendrites of stratum radiatum and oriens, mossy fiber projections from the dentate gyrus (Figure 1C and inset, upper left side), and dendritic spines after enlargement of a dendritic segment following deconvolution (Figure 1C and inset, lower right side). To quantify the proportion of H1aV expressing neurons, we counted the number of H1aV-expressing cells by direct epi-fluorescence or after staining with fluorescent or HRP conjugated antibody against Venus in randomly chosen sections of CA1 and CA3 by confocal microscopy (Figure 1C, white boxes). Of ∼800 CA1 cells, 80 ± 9% (mean ± SD) expressed H1aV. The remaining, non-expressing 15–20% of CA1 neurons may be interneurons (Gulyas et al., 1999), indicating that the vast majority of CA1 principal cells expressed H1aV. In contrast, only ∼15% of the CA3 neurons were Venus positive."}

{"project":"2_test","denotations":[{"id":"18982121-12429690-38286620","span":{"begin":192,"end":196},"obj":"12429690"},{"id":"18982121-11431570-38286621","span":{"begin":211,"end":215},"obj":"11431570"},{"id":"18982121-11753368-38286622","span":{"begin":312,"end":316},"obj":"11753368"},{"id":"18982121-7567477-38286623","span":{"begin":455,"end":459},"obj":"7567477"},{"id":"18982121-12429690-38286624","span":{"begin":480,"end":484},"obj":"12429690"},{"id":"18982121-15208716-38286625","span":{"begin":662,"end":666},"obj":"15208716"},{"id":"18982121-8939850-38286626","span":{"begin":1080,"end":1084},"obj":"8939850"},{"id":"18982121-15208716-38286627","span":{"begin":1654,"end":1658},"obj":"15208716"},{"id":"18982121-8939850-38286628","span":{"begin":3756,"end":3760},"obj":"8939850"},{"id":"18982121-15548671-38286629","span":{"begin":3966,"end":3970},"obj":"15548671"},{"id":"18982121-12357963-38286630","span":{"begin":3992,"end":3996},"obj":"12357963"},{"id":"18982121-11431570-38286631","span":{"begin":5029,"end":5033},"obj":"11431570"},{"id":"18982121-10559416-38286632","span":{"begin":5831,"end":5835},"obj":"10559416"}],"text":"Conditional Homer1a expression in forebrain\nTo regulate Homer1a expression in adult mouse forebrain, we utilized the tet-off version of the tet-dependent expression system (Gossen and Bujard, 2002; Mack et al., 2001). The rat Homer1a cDNA was fused at its 3′ end to Venus fluorescent protein cDNA (Nagai et al., 2002) and expressed from a tet-responsive bidirectional transgene harboring in addition the firefly luciferase gene (Figure 1A) (Baron et al., 1995; Gossen and Bujard, 2002). Expression of the luciferase gene permitted the use of ear fibroblasts in screening founders for the presence of a functional tet-responsive transcription unit (Hasan et al., 2004). The highest expresser of 18 candidate founders (Figure 1B and Materials and Methods) was chosen for our studies, and the corresponding transgenic line is here referred to as TgH1aV.\nFigure 1 Tetracycline-dependent forebrain expression of H1aV. (A) Transgenes for tet-dependent H1aV expression in the mouse forebrain. The α-CaMKII promoter directs tTA expression in forebrain principal neurons (Mayford et al., 1996). The luciferase (luc) and H1aV genes are transcribed from a bidirectional unit activated upon binding of tTA to heptamerized tet operator sequences (tetO7) flanked by CMV minimal promoters. Dox prevents tTA binding to tetO7, thus switching off expression of luc and H1aV transgenes. (B) 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., 2004), 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. (C) 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. (D) 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 cortex; STR, striatum; HP, hippocampus; MW., molecular weight). Overexpression in forebrain was in response to α-calcium/calmodulin-dependent kinase II (α-CaMKII) promoter-driven expression of the tet-dependent tTA (Tgα-CaMKII-tTA) (Mayford et al., 1996) (Figure 1A). Double transgenic Tgα-CaMKII-tTA/TgH1aV (abbreviated here as TgH1aV.Fb) mice (studied at P42) displayed robust Venus epi-fluorescence in all forebrain structures (Figure 1C) (Krestel et al., 2004; Schonig and Bujard, 2003). Strong fluorescence was seen in cortical and subcortical brain regions, with homogeneous distribution in the CA1 region of the hippocampus. Antibody stainings with a Homer1a-specific antibody detected the H1aV fusion protein in fluorescent but not in non-fluorescent cells, indicating sustained expression of the IEG fusion (data not shown). TgH1aV.Fb transgenic mice, when supplied with the tet derivative doxycycline in the drinking water (dox, 0.1 g/l) for 3 weeks, showed efficient down-regulation of H1aV. Thus, TgH1aV.Fb mice allowed us to investigate the effect of sustained H1aV expression on hippocampal synaptic plasticity and spatial learning.\nInspection of sagittal hippocampal sections of TgH1aV.Fb mice revealed intense fluorescence in the somata and dendrites of pyramidal neurons in the subiculum, the CA1 region and the dentate gyrus (Figure 1C and inset, upper left side). The cell bodies of CA3 neurons displayed only sparse fluorescence, as reported for other α-CaMKII promoter-driven constructs (Mack et al., 2001) (Figure 1C and inset, upper left side). However, robust staining was detected in CA1 pyramidal cell dendrites of stratum radiatum and oriens, mossy fiber projections from the dentate gyrus (Figure 1C and inset, upper left side), and dendritic spines after enlargement of a dendritic segment following deconvolution (Figure 1C and inset, lower right side). To quantify the proportion of H1aV expressing neurons, we counted the number of H1aV-expressing cells by direct epi-fluorescence or after staining with fluorescent or HRP conjugated antibody against Venus in randomly chosen sections of CA1 and CA3 by confocal microscopy (Figure 1C, white boxes). Of ∼800 CA1 cells, 80 ± 9% (mean ± SD) expressed H1aV. The remaining, non-expressing 15–20% of CA1 neurons may be interneurons (Gulyas et al., 1999), indicating that the vast majority of CA1 principal cells expressed H1aV. In contrast, only ∼15% of the CA3 neurons were Venus positive."}