Journal: Frontiers in Behavioral Neuroscience

Article Title: Increased conditioned place preference for cocaine in high anxiety related behavior (HAB) mice is associated with an increased activation in the accumbens corridor

doi: 10.3389/fnbeh.2014.00441

Figure Lengend Snippet: Colocalization of neuronal markers with EGR1 expression 1 h after cocaine CPP is restricted to D1- and D2-medium spiny neurons . Brains were harvested for double fluorescence immunohistochemistry 1 h after the start of a 15-min cocaine CPP test session. EGR1 immunoreactivity (shown in green) remained restricted to nuclei. All other neuronal markers are shown in red. Colocalization of immunoreactivity is found only in cells positive for the neuronal nuclear protein NeuN (A) In contrast, no colocalization with EGR1 was observed in glial cells. The employed glial markers were glial fibrillary acidic protein for astrocytes (GFAP, B ), myelin basic protein for oligodendrocytes (MBP, C ), and tomato lectin for microglia (TL, D ). Colocalization of immunoreactivity was found only in neurons immunoreactive against an anti-dynorphin antibody (DYN, E ), i.e., most likely dopamine D1 receptor expressing medium spiny neurons (D1-MSNs) or in neurons immunoreactive against an anti-dopamine D2 receptor antibody (DRD2, D2-MSNs, F ). No colocalization with EGR1 was observed in cholinergic interneurons (marker: choline acetyltransferase ChAT, G ) or GABAergic interneurons positive for parvalbumin (PV, H ), neuropeptide Y (NPY, I ) or calretinin (CR, J ) Images were taken with a laser scanning confocal microscope with a magnification of 100× (bar size, 10 μm).

Article Snippet: Additionally we processed three slices each for double staining of EGR1 with dynorphin (goat polyclonal anti-DYN; 1:50, Santa Cruz Biotechnology, sc-46313) to presumably label dopamine D1 receptor expressing medium spiny neurons (D1-MSNs) or the dopamine D2 receptor expressing neurons (mouse polyclonal anti-DRD2, 1:50, Santa Cruz Biotechnology, sc-5303) to label D2-MSNs, choline acetyltransferase (goat polyclonal anti-ChAT, 1:166, Millipore, AB144p) to label cholinergic interneurons; or parvalbumin (goat polyclonal anti-PV, 1:1000, Swant, PVG-214), calretinin (goat polyclonal anti-CR, 1:1000, Swant, CG1) and neuropeptide Y (goat polyclonal anti-NPY, 1:500, Novus, NBP1-46535) to label GABAergic interneurons.

Techniques: Expressing, Fluorescence, Immunohistochemistry, Marker, Microscopy

Journal: Frontiers in Behavioral Neuroscience

Article Title: Increased conditioned place preference for cocaine in high anxiety related behavior (HAB) mice is associated with an increased activation in the accumbens corridor

doi: 10.3389/fnbeh.2014.00441

Figure Lengend Snippet: Cocaine CPP-induced EGR1- and c-Fos expression are increased in the accumbens corridor . Shown for each ( A–D ) are representative images of the individual 190 μm counting bins of the different accumbens corridor regions comprising, from medial to the lateral, VDB + MS, ICjM + LSI, AcbShm, and AcbCm. For abbreviations see Materials and Methods. In (A) Cocaine CPP-induced EGR1 expression is shown for NAB (A) vs. HAB (B) mice. Cocaine CPP-induced c-Fos expression is shown for NAB (C) vs. HAB (D) mice. Images were taken with a laser scanning confocal microscope with a magnification of 20× (bar size, 50 μm).

Article Snippet: Additionally we processed three slices each for double staining of EGR1 with dynorphin (goat polyclonal anti-DYN; 1:50, Santa Cruz Biotechnology, sc-46313) to presumably label dopamine D1 receptor expressing medium spiny neurons (D1-MSNs) or the dopamine D2 receptor expressing neurons (mouse polyclonal anti-DRD2, 1:50, Santa Cruz Biotechnology, sc-5303) to label D2-MSNs, choline acetyltransferase (goat polyclonal anti-ChAT, 1:166, Millipore, AB144p) to label cholinergic interneurons; or parvalbumin (goat polyclonal anti-PV, 1:1000, Swant, PVG-214), calretinin (goat polyclonal anti-CR, 1:1000, Swant, CG1) and neuropeptide Y (goat polyclonal anti-NPY, 1:500, Novus, NBP1-46535) to label GABAergic interneurons.

Techniques: Expressing, Microscopy

Journal: Frontiers in Behavioral Neuroscience

Article Title: Increased conditioned place preference for cocaine in high anxiety related behavior (HAB) mice is associated with an increased activation in the accumbens corridor

doi: 10.3389/fnbeh.2014.00441

Figure Lengend Snippet: Cocaine CPP-induced EGR1 and c-Fos expression is increased in the accumbens corridor of HAB mice. (A) shows group means ± SEMs of EGR1-positive nuclei per mm 2 , whereas (B) shows group means ± SEM of c-Fos-positive nuclei per mm 2 . On the x-axis the individual regions of the accumbens corridor, the AcbCl and the CPu are displayed. The accumbens corridor comprises the VDB + MS, ICjM + LSI, AcbShm, and AcbCm (for abbreviations see Materials and Methods). The graphs in the left panel show NAB animals trained for cocaine CPP (COCAINE NAB n = 7, green filled squares), treated with non-contingent cocaine (NONCONT NAB n = 8, green unfilled squares) and NAIVE NAB animals ( n = 8, black small dots). The graphs in the middle panel display HAB animals trained for cocaine CPP (COCAINE HAB n = 8, red filled squares), treated with non-contingent cocaine (NONCONT HAB n = 8, red unfiled squares), and NAIVE HAB animals ( n = 8, black small dots). For reasons of clarity, significant differences (indicated by an asterisks) between treatment groups are only displayed for the comparison of animals conditioned for cocaine CPP and animals that received cocaine non-contingently ( * p < 0.05; ** p < 0.01; *** p < 0.001, and **** p < 0.0001). Comparison between the NAB COCAINE and HAB COCAINE group is shown in the right panel of the figure. Statistical differences assessed by a one-tailed t -test between these two groups for each region of the accumbens corridor is indicated by the number sign (# p < 0.05; ## p < 0.01). The complete statistical analysis and the individual p- values are shown in Table .

Article Snippet: Additionally we processed three slices each for double staining of EGR1 with dynorphin (goat polyclonal anti-DYN; 1:50, Santa Cruz Biotechnology, sc-46313) to presumably label dopamine D1 receptor expressing medium spiny neurons (D1-MSNs) or the dopamine D2 receptor expressing neurons (mouse polyclonal anti-DRD2, 1:50, Santa Cruz Biotechnology, sc-5303) to label D2-MSNs, choline acetyltransferase (goat polyclonal anti-ChAT, 1:166, Millipore, AB144p) to label cholinergic interneurons; or parvalbumin (goat polyclonal anti-PV, 1:1000, Swant, PVG-214), calretinin (goat polyclonal anti-CR, 1:1000, Swant, CG1) and neuropeptide Y (goat polyclonal anti-NPY, 1:500, Novus, NBP1-46535) to label GABAergic interneurons.

Techniques: Expressing, Comparison, One-tailed Test

Journal: Frontiers in Behavioral Neuroscience

Article Title: Increased conditioned place preference for cocaine in high anxiety related behavior (HAB) mice is associated with an increased activation in the accumbens corridor

doi: 10.3389/fnbeh.2014.00441

Figure Lengend Snippet: Cocaine CPP-induced increase in EGR1 and c-Fos expression .

Article Snippet: Additionally we processed three slices each for double staining of EGR1 with dynorphin (goat polyclonal anti-DYN; 1:50, Santa Cruz Biotechnology, sc-46313) to presumably label dopamine D1 receptor expressing medium spiny neurons (D1-MSNs) or the dopamine D2 receptor expressing neurons (mouse polyclonal anti-DRD2, 1:50, Santa Cruz Biotechnology, sc-5303) to label D2-MSNs, choline acetyltransferase (goat polyclonal anti-ChAT, 1:166, Millipore, AB144p) to label cholinergic interneurons; or parvalbumin (goat polyclonal anti-PV, 1:1000, Swant, PVG-214), calretinin (goat polyclonal anti-CR, 1:1000, Swant, CG1) and neuropeptide Y (goat polyclonal anti-NPY, 1:500, Novus, NBP1-46535) to label GABAergic interneurons.

Techniques: Expressing, Comparison

Journal: Frontiers in Behavioral Neuroscience

Article Title: Increased conditioned place preference for cocaine in high anxiety related behavior (HAB) mice is associated with an increased activation in the accumbens corridor

doi: 10.3389/fnbeh.2014.00441

Figure Lengend Snippet: Correlation of EGR1 and c-Fos expression in the accumbens corridor regions 1 h after the cocaine CPP test with the time spent in the cocaine associated compartment . The correlation of EGR1 expression (A) or c-Fos expression (B) per mm 2 vs. the time spent in the cocaine compartment during the cocaine CPP test is shown by pooling data from NAB and HAB animals. The correlation is given for each region of the accumbens corridor, the AcbCl and the CPu for animals that had undergone cocaine CPP (COCAINE NAB n = 7, filled green squares, filled; COCAINE HAB n = 8, filled red squares) as a continuous line and animals treated with non-contingent cocaine shown as dashed line (NONCONT NAB n = 8, unfilled green squares; NONCONT HAB n = 8, unfilled red squares).

Article Snippet: Additionally we processed three slices each for double staining of EGR1 with dynorphin (goat polyclonal anti-DYN; 1:50, Santa Cruz Biotechnology, sc-46313) to presumably label dopamine D1 receptor expressing medium spiny neurons (D1-MSNs) or the dopamine D2 receptor expressing neurons (mouse polyclonal anti-DRD2, 1:50, Santa Cruz Biotechnology, sc-5303) to label D2-MSNs, choline acetyltransferase (goat polyclonal anti-ChAT, 1:166, Millipore, AB144p) to label cholinergic interneurons; or parvalbumin (goat polyclonal anti-PV, 1:1000, Swant, PVG-214), calretinin (goat polyclonal anti-CR, 1:1000, Swant, CG1) and neuropeptide Y (goat polyclonal anti-NPY, 1:500, Novus, NBP1-46535) to label GABAergic interneurons.

Techniques: Expressing

Journal: Frontiers in Behavioral Neuroscience

Article Title: Increased conditioned place preference for cocaine in high anxiety related behavior (HAB) mice is associated with an increased activation in the accumbens corridor

doi: 10.3389/fnbeh.2014.00441

Figure Lengend Snippet: Correlation of cocaine CPP-induced EGR1 and c-Fos expression with time spent in the cocaine compartment .

Article Snippet: Additionally we processed three slices each for double staining of EGR1 with dynorphin (goat polyclonal anti-DYN; 1:50, Santa Cruz Biotechnology, sc-46313) to presumably label dopamine D1 receptor expressing medium spiny neurons (D1-MSNs) or the dopamine D2 receptor expressing neurons (mouse polyclonal anti-DRD2, 1:50, Santa Cruz Biotechnology, sc-5303) to label D2-MSNs, choline acetyltransferase (goat polyclonal anti-ChAT, 1:166, Millipore, AB144p) to label cholinergic interneurons; or parvalbumin (goat polyclonal anti-PV, 1:1000, Swant, PVG-214), calretinin (goat polyclonal anti-CR, 1:1000, Swant, CG1) and neuropeptide Y (goat polyclonal anti-NPY, 1:500, Novus, NBP1-46535) to label GABAergic interneurons.

Techniques: Expressing

Journal: The Journal of Clinical Investigation

Article Title: Podocyte histone deacetylase activity regulates murine and human glomerular diseases

doi: 10.1172/JCI124030

Figure Lengend Snippet: (A) Heatmap representing color-coded differential glomerular genes analyzed by Z ratio of Tln1fl/fl Pod-rtTA TetO-Cre mice with vehicle to those with VPA, 2 weeks after completion of Dox induction. (B) Reverse transcriptase PCR of the candidate genes in control, and Tln1fl/fl Pod-rtTA TetO-Cre mice treated or not treated with VPA 2 weeks after completion of Dox induction. *P < 0.05 vs. control mice, #P < 0.05 vs. Tln1fl/fl Pod-rtTA TetO-Cre mice; n = 3. (C) Representative immunoblots of EGR1 and GAPDH in glomeruli of control mice, Tln1fl/fl Pod-rtTA TetO-Cre mice treated or not treated with VPA or SAHA, and Hdac1fl/fl Hdac2fl/fl Tln1fl/fl Pod-rtTA TetO-Cre mice. (D) Quantification of EGR1 immunoblots in C. *P < 0.05 vs. control mice, #P < 0.05 vs. Tln1fl/fl Pod-rtTA TetO-Cre mice; n = 3. (E) Representative immunofluorescence images of EGR1 (green) and nephrin (red) in control and focal segmental glomerulosclerosis (FSGS) patient glomeruli. Arrows display podocyte EGR1 staining. Scale bar: 20 μm. (F) Quantification of podocyte EGR1 immunofluorescence intensity in E. *P < 0.05 vs. control; n = 3. (B and D) Statistically analyzed by 1-way ANOVA with Dunnett’s correction. (F) Statistically analyzed by 2-tailed Student’s t test.

Article Snippet: Mouse anti–Wilms tumor 1 (anti-WT1) (EMD Millipore Corp., catalog 05-753); rabbit anti-WT1 (Santa Cruz Biotechnology, catalog sc-192); mouse anti-HDAC1 (catalog 5356), mouse anti-HDAC2 (catalog 5113), rabbit anti–serum response factor (anti-SRF) (catalog 5147), rabbit anti–CRE-binding protein (anti-CREB) (catalog 9197), rabbit anti–phosphorylated CREB (Ser133) (catalog 9198), rabbit anti-EGR1 (catalog 4154), and rabbit anti-GAPDH (catalog 5174) (Cell Signaling Technology); mouse anti–talin-1 (Bio-Rad, catalog MCA4770); guinea pig anti-nephrin (Progen, catalog GP-N2); rabbit anti-EGR1 (Protein Tech, catalog 55117-1-AP); Alexa Fluor 594 phalloidin (catalog A12381), Alexa Fluor 488 goat anti-mouse IgG antibody (catalog A-11001), Alexa Fluor 594 goat anti–guinea pig IgG antibody (catalog A-11076), and Alexa Fluor 594 goat anti-rabbit IgG antibody (catalog A-11037) (Invitrogen) were purchased commercially. eGFP-EGR1 plasmid was purchased from Addgene.

Techniques: Western Blot, Immunofluorescence, Staining

Journal: The Journal of Clinical Investigation

Article Title: Podocyte histone deacetylase activity regulates murine and human glomerular diseases

doi: 10.1172/JCI124030

Figure Lengend Snippet: (A) Representative immunoblots of EGR1 and WT1 in primary podocytes with or without LPS or PS, treated or not treated with VPA or SAHA. (B and C) Quantification of EGR1 immunoblots in primary podocytes with LPS (B) or PS (C) treated or not treated with VPA or SAHA. *P < 0.05 vs. control, #P < 0.05 vs. LPS- or PS-treated primary podocytes; n = 3. (D and E) ChIP assay using CREB antibody and primer sets for Egr1 promoter in primary podocytes with LPS (D) or PS (E) treated or not treated with VPA or SAHA. DNA binding was determined by PCR. *P < 0.05 vs. control, #P < 0.05 vs. LPS- or PS-treated primary podocytes; n = 3. (F) Representative immunoblots of phosphorylated CREB (p-CREB), CREB, and WT1 in LPS- or PS-treated primary podocytes with or without VPA or SAHA. (G and H) Quantification of p-CREB immunoblots in primary podocytes with LPS (G) or PS (H) treated or not treated with VPA or SAHA. *P < 0.05 vs. control, #P < 0.05 vs. LPS- or PS-treated primary podocytes; n = 3. (B–E, G, and H) Statistically analyzed by 1-way ANOVA with Dunnett’s correction.

Article Snippet: Mouse anti–Wilms tumor 1 (anti-WT1) (EMD Millipore Corp., catalog 05-753); rabbit anti-WT1 (Santa Cruz Biotechnology, catalog sc-192); mouse anti-HDAC1 (catalog 5356), mouse anti-HDAC2 (catalog 5113), rabbit anti–serum response factor (anti-SRF) (catalog 5147), rabbit anti–CRE-binding protein (anti-CREB) (catalog 9197), rabbit anti–phosphorylated CREB (Ser133) (catalog 9198), rabbit anti-EGR1 (catalog 4154), and rabbit anti-GAPDH (catalog 5174) (Cell Signaling Technology); mouse anti–talin-1 (Bio-Rad, catalog MCA4770); guinea pig anti-nephrin (Progen, catalog GP-N2); rabbit anti-EGR1 (Protein Tech, catalog 55117-1-AP); Alexa Fluor 594 phalloidin (catalog A12381), Alexa Fluor 488 goat anti-mouse IgG antibody (catalog A-11001), Alexa Fluor 594 goat anti–guinea pig IgG antibody (catalog A-11076), and Alexa Fluor 594 goat anti-rabbit IgG antibody (catalog A-11037) (Invitrogen) were purchased commercially. eGFP-EGR1 plasmid was purchased from Addgene.

Techniques: Western Blot, Binding Assay

Journal: The Journal of Clinical Investigation

Article Title: Podocyte histone deacetylase activity regulates murine and human glomerular diseases

doi: 10.1172/JCI124030

Figure Lengend Snippet: (A) Quantification of urine albumin/creatinine ratio in control (black), Tln1fl/fl Pod-rtTA TetO-Cre (red), and Egr1–/– Tln1fl/fl Pod-rtTA TetO-Cre (blue) mice at 0, 2, and 4 weeks after completion of Dox induction. *P < 0.05 vs. control mice, #P < 0.05 vs. Tln1fl/fl Pod-rtTA TetO-Cre mice; n = 5. (B) Plasma creatinine in control, Tln1fl/fl Pod-rtTA TetO-Cre, and Egr1–/– Tln1fl/fl Pod-rtTA TetO-Cre mice 0 and 4 weeks after completion of Dox induction. *P < 0.05 vs. control mice, #P < 0.05 vs. Tln1fl/fl Pod-rtTA TetO-Cre mice; n = 5. (C) Representative light microscope images (H&E, PAS, and trichrome) of glomeruli from control, Tln1fl/fl Pod-rtTA TetO-Cre, and Egr1–/– Tln1fl/fl Pod-rtTA TetO-Cre mice 4 weeks after completion of Dox induction. Arrowheads show mesangial matrix deposition and mesangial cell proliferation. Scale bar: 25 μm. (D) Representative trichrome staining in control, Tln1fl/fl Pod-rtTA TetO-Cre, and Egr1–/– Tln1fl/fl Pod-rtTA TetO-Cre mouse kidneys 4 weeks after completion of Dox induction. Arrowheads show dilated tubules and proteinaceous casts; arrows display interstitial fibrosis. Scale bar: 50 μm. (E) Quantification of glomerulosclerosis in C. *P < 0.05 vs. control mice, #P < 0.05 vs. Tln1fl/fl Pod-rtTA TetO-Cre mice. (F) Quantification of interstitial fibrosis in D. *P < 0.05 vs. control mice, #P < 0.05 vs. Tln1fl/fl Pod-rtTA TetO-Cre mice. (A, B, E, and F) Statistically analyzed by 1-way ANOVA with Dunnett’s correction.

Article Snippet: Mouse anti–Wilms tumor 1 (anti-WT1) (EMD Millipore Corp., catalog 05-753); rabbit anti-WT1 (Santa Cruz Biotechnology, catalog sc-192); mouse anti-HDAC1 (catalog 5356), mouse anti-HDAC2 (catalog 5113), rabbit anti–serum response factor (anti-SRF) (catalog 5147), rabbit anti–CRE-binding protein (anti-CREB) (catalog 9197), rabbit anti–phosphorylated CREB (Ser133) (catalog 9198), rabbit anti-EGR1 (catalog 4154), and rabbit anti-GAPDH (catalog 5174) (Cell Signaling Technology); mouse anti–talin-1 (Bio-Rad, catalog MCA4770); guinea pig anti-nephrin (Progen, catalog GP-N2); rabbit anti-EGR1 (Protein Tech, catalog 55117-1-AP); Alexa Fluor 594 phalloidin (catalog A12381), Alexa Fluor 488 goat anti-mouse IgG antibody (catalog A-11001), Alexa Fluor 594 goat anti–guinea pig IgG antibody (catalog A-11076), and Alexa Fluor 594 goat anti-rabbit IgG antibody (catalog A-11037) (Invitrogen) were purchased commercially. eGFP-EGR1 plasmid was purchased from Addgene.

Techniques: Light Microscopy, Staining

Journal: The Journal of Clinical Investigation

Article Title: Podocyte histone deacetylase activity regulates murine and human glomerular diseases

doi: 10.1172/JCI124030

Figure Lengend Snippet: (A) Representative immunostaining for phalloidin (red) and WT1 (green) in control Pod-Cre Rosa-DTRflox or Egr1–/– Pod-Cre Rosa-DTRflox mouse primary podocytes treated with LPS or PS. Scale bar: 10 μm. (B–D) Quantification of phalloidin staining in control (black) or Egr1–/– Pod-Cre Rosa-DTRflox (blue) mouse primary podocytes with vehicle (B), LPS (C), and PS (D). *P < 0.05 vs. control mouse primary podocytes; n = 3. (E) Cell surface area in primary podocytes overexpressing GFP or GFP-EGR1 following LPS with or without VPA treatment. *P < 0.05 vs. control primary podocytes, #P < 0.05 vs. LPS-treated primary podocytes; n = 3. Panels C and D statistically analyzed by 2-tailed Student’s t test. Panel E statistically analyzed by 1-way ANOVA with Dunnett’s correction.

Article Snippet: Mouse anti–Wilms tumor 1 (anti-WT1) (EMD Millipore Corp., catalog 05-753); rabbit anti-WT1 (Santa Cruz Biotechnology, catalog sc-192); mouse anti-HDAC1 (catalog 5356), mouse anti-HDAC2 (catalog 5113), rabbit anti–serum response factor (anti-SRF) (catalog 5147), rabbit anti–CRE-binding protein (anti-CREB) (catalog 9197), rabbit anti–phosphorylated CREB (Ser133) (catalog 9198), rabbit anti-EGR1 (catalog 4154), and rabbit anti-GAPDH (catalog 5174) (Cell Signaling Technology); mouse anti–talin-1 (Bio-Rad, catalog MCA4770); guinea pig anti-nephrin (Progen, catalog GP-N2); rabbit anti-EGR1 (Protein Tech, catalog 55117-1-AP); Alexa Fluor 594 phalloidin (catalog A12381), Alexa Fluor 488 goat anti-mouse IgG antibody (catalog A-11001), Alexa Fluor 594 goat anti–guinea pig IgG antibody (catalog A-11076), and Alexa Fluor 594 goat anti-rabbit IgG antibody (catalog A-11037) (Invitrogen) were purchased commercially. eGFP-EGR1 plasmid was purchased from Addgene.

Techniques: Immunostaining, Staining