Journal: Nature Communications

Article Title: Prefrontal cortex molecular clock modulates development of depression-like phenotype and rapid antidepressant response in mice

doi: 10.1038/s41467-024-51716-9

Figure Lengend Snippet: a , b Representative western blots and quantitative data of synaptic AMPA receptors subunits GluA1 and GluA2 levels normalized to Homer1b/c and PSD95 in mPFC of WT mice 24 h after SR10067 application ( a ), and 6 h and 24 h post SR1078 injection ( b ). ( n = 5, a : two-tailed Student’s t test: * P = 0.0422 for GluA1, ** P = 0.0069 for GluA2, b : one-way ANOVA with Bonferroni post hoc test: * P < 0.05, *** P < 0.001). c Experimental design: WT mice were subjected to the CDM protocol before undergoing implantation of ECoG, LFP and EMG recording electrodes. After recovery, mice were recorded in their home cage over 32 h from ZT0, with treatment (vehicle/ketamine/SR1078) administered at ZT06. d , e Delta power (0.5–4 Hz) of LFP signal recorded from the mPFC ( d ) and ECoG signal ( e ) recorded during slow wave sleep (SWS) across 12 h:12 h LD conditions ( n = 14 mice CDM/vehicle; n = 6 SR1078; n = 4 KET; repeated measures mixed-effects model two-way ANOVA with Bonferroni post hoc test * P < 0.05, ** P < 0.01 vs CDM/vehicle). Black arrow indicates time of treatment administration. f Schema summarizing the effects of RORα/γ activation on BMAL1, Homer1a and synaptic AMPAR expression in the mPFC and the relation to depression-like behavior. Data are presented as mean ± SEM and the individual data points are depicted. See also Supplementary Fig. and Supplementary Data . Source data are provided as a Source Data file.

Article Snippet: The membranes were blocked with 5% non-fat dry milk in TBS-T (1% Tween20 in Tris-buffered saline (TBS)) and afterward incubated with the respective primary antibodies diluted in TBS (mouse anti-GluR1-NT, Merck (MAB2263), 1:1000; rabbit anti-GluR2, Merck (AB1768-I), 1:1000; rabbit anti-Homer1, Merck (ABN37), 1:1000; mouse anti-PSD95, Merck (MABN68), 1:1000; rabbit anti-mouse Per2, Alpha Diagnostics (PER21-A), 1:1000; rabbit anti-BMAL1, Thermo Fisher Scientific PA1-523, 1:1000; mouse anti-Histone H2b, Euromedex (IG-H2-2A8), 1:500 overnight at 4 °C.

Techniques: Western Blot, Injection, Two Tailed Test, Activation Assay, Expressing

Journal: The Journal of Neuroscience

Article Title: β-Secretase BACE1 Is Required for Normal Cochlear Function

doi: 10.1523/JNEUROSCI.0028-19.2019

Figure Lengend Snippet: The architecture of ribbon synapses appears normal in BACE1−/− mice. A, B, Representative maximum intensity projections of confocal z-stacks from the IHC region in the organ of Corti of the apical turn of (A) wild-type and (B) and BACE1−/− mice stained with antibodies directed against presynaptic CtBP2 (red) and postsynaptic GluR2/3 (green). Dotted lines visualize individual IHCs in the region. C, CtBP2 and GluR2/3 puncta were quantified by blinded counting of immunopositive clusters from images as shown in (A+B) in BACE1+/+ (n = 41 cells of 3 mice) and BACE1−/− mice (n = 57 cells of 3 mice). There was no significant difference between genotypes. D, There was no obvious difference in the number of CtBP2-positive clusters in OHCs of BACE1+/+ (left) and BACE1−/− (right) mice. D, Representative confocal projections through the OHC region in the apical turn of BACE1+/+ (left) and BACE1−/− (right) mice stained with anti-CtBP2 antibodies (red) and DAPI (blue).

Article Snippet: Immunostaining was performed overnight at 4°C with the following primary antibodies (diluted in blocking solution): rabbit-anti-BACE1 (Abcam, ab108394; 1:50), guinea pig-anti-synapsin1,2 (Synaptic Systems, 106004; 1:500), rabbit-anti-KCNQ1 (K v 7.1) (Abcam, ab135737; 1:200), goat-anti-prestin (N-20; Santa Cruz Biotechnology, sc-22692; 1:400), rabbit-anti-KCNQ4 (K v 7.4) (H-130; Santa Cruz Biotechnology, sc-50417; 1:400), rabbit-anti-NF-200 (neurofilament heavy chain polypeptide, Sigma-Aldrich, N4142; 1:600) and chicken-anti-NF-H (neurofilament heavy chain polypeptide, Abcam, ab4680; 1:400), mouse-anti-CtBP2 (C-terminal binding protein; BD Bioscience, 612044; 1:200), rabbit-anti-GluR2/3 (Glutamate receptor subunits 2/3, Merck, ab1506; 1:200), rabbit-anti-PSD95 (postsynaptic density 95, Abcam, ab18258; 1:200), mouse-anti-MBP (myelin basic protein; F-6; Santa Cruz Biotechnology, sc-271524; 1:400), and rabbit-anti-B-FABP (brain-type fatty acid binding protein, Kurtz et al., 1994 , 1:1000).

Techniques: Staining

Journal: The Journal of Neuroscience

Article Title: β-Secretase BACE1 Is Required for Normal Cochlear Function

doi: 10.1523/JNEUROSCI.0028-19.2019

Figure Lengend Snippet: Disorganization of nerve fibers and ectopic overexpression of PSD95 in BACE1−/− mice. Auditory nerve fibers and the synaptic arrangement were analyzed in whole-mount organ of Corti preparations of the apical cochlear turn stained with primary antibodies targeted against neurofilament (NF-200 or NF-H, both are directed against neurofilament heavy chain polypeptide) and CtBP2 or PSD95, respectively. A, B, Auditory nerve fibers in the osseus spiral lamina (OSL) of BACE1−/− mice (in B; P48) showed more intense anti-NF-200 immunofluorescence, and appeared disorganized and swollen. In the OSL of BACE1−/− mice, we detected NF-200-positive patches with very high immunofluorescence (arrowheads in B) that were completely absent in wild-types. Dashed lines in A and B indicate the habenula perforata. Smaller images in A and B display magnifications of the OSL (top) and the IHC (bottom) region. C, D, Triple staining with antibodies directed against neurofilament (NF-H; blue), CtBP2 (presynaptic ribbon; red) and anti-PSD95 (postsynaptic density; green) showed that (C) in BACE1+/+ mice the presynaptic and postsynaptic terminals were well aligned in close proximity. D, In BACE1−/− mice a substantial number of PSD95-positive clusters was located far outside the synaptic region and not aligned at all with presynaptic anti-CtBP2 signals. Maximum intensity projections of confocal z-stacks shown in the acquisition plane (x–y, left) and in the orthogonal plane (z–y, right). E, Blinded counting of CtBP2- and PSD95-positive signals revealed significantly more PSD95 clusters in BACE1−/− than in BACE1+/+ mice (p < 0.001). In BACE1−/− mice, postsynaptic PSD95 clusters significantly outnumbered presynaptic CtBP2 clusters (p < 0.001). F, Schematic drawing summarizing our findings (neurofilament-positive fibers, blue; PSD95, orange; CtBP2, red; GluR2/3, green). (A–D) All images are representative confocal projections of the apical turn of the organ of Corti. Corresponding images (A,B and C,D) were taken in parallel under identical experimental conditions (See Materials and Methods). ***p<0.001.

Article Snippet: Immunostaining was performed overnight at 4°C with the following primary antibodies (diluted in blocking solution): rabbit-anti-BACE1 (Abcam, ab108394; 1:50), guinea pig-anti-synapsin1,2 (Synaptic Systems, 106004; 1:500), rabbit-anti-KCNQ1 (K v 7.1) (Abcam, ab135737; 1:200), goat-anti-prestin (N-20; Santa Cruz Biotechnology, sc-22692; 1:400), rabbit-anti-KCNQ4 (K v 7.4) (H-130; Santa Cruz Biotechnology, sc-50417; 1:400), rabbit-anti-NF-200 (neurofilament heavy chain polypeptide, Sigma-Aldrich, N4142; 1:600) and chicken-anti-NF-H (neurofilament heavy chain polypeptide, Abcam, ab4680; 1:400), mouse-anti-CtBP2 (C-terminal binding protein; BD Bioscience, 612044; 1:200), rabbit-anti-GluR2/3 (Glutamate receptor subunits 2/3, Merck, ab1506; 1:200), rabbit-anti-PSD95 (postsynaptic density 95, Abcam, ab18258; 1:200), mouse-anti-MBP (myelin basic protein; F-6; Santa Cruz Biotechnology, sc-271524; 1:400), and rabbit-anti-B-FABP (brain-type fatty acid binding protein, Kurtz et al., 1994 , 1:1000).

Techniques: Over Expression, Staining, Immunofluorescence

Journal: Aging (Albany NY)

Article Title: FGF22 promotes generation of ribbon synapses through downregulating MEF2D

doi: 10.18632/aging.103042

Figure Lengend Snippet: FGF22 depletion reduces ribbon synapse number in vivo. AAV-shFGF22 and AAV-FGF22 were administrated to mouse cochlea to deplete or overexpress FGF22 in hair cells, respectively. Co-administration of AAV-shMEF2D with AAV-shFGF22 was also performed to assess the regulatory relationship between FGF22 and MEF2D in hair cells and ribbon synapses. Similarly, co-administration of AAV-MEF2D with AAV-FGF22 was performed, also to assess the regulatory relationship between FGF22 and MEF2D in hair cells and ribbon synapses. ( A , B ) TUNEL staining was performed on cochlea, showing by representative images ( A ), and by quantification ( B ). ( C , D ) The ribbon synapses were determined by co-staining for CtBP2 (in green) and GluR2&3 (in red). Cochlear ribbon synapse number was assessed, shown by representative images ( C ), and by quantification ( D ). *p<0.05. NS: non-significant. N=5. Scale bar is 20μm.

Article Snippet: Primary antibodies are rabbit anti-CtBP2 polyclonal antibody (ab128871, 1:100, Abcam, Cambridge, MA, USA), rabbit anti-GluR2&3 polyclonal antibody (2mg/mL, Fisher Scientific, CA, USA) and rabbit anti-Myosin7a (1:200, ab3481, Abcam).

Techniques: In Vivo, TUNEL Assay, Staining

Journal: Toxicology Reports

Article Title: Development of a simple measurement method for GluR2 protein expression as an index of neuronal vulnerability

doi: 10.1016/j.toxrep.2014.12.014

Figure Lengend Snippet: AlphaLISA assay of GluR2 protein expression. (A) Principles of the AlphaLISA assay; biotinylated anti-GluR2 mouse monoclonal antibody binds streptavidin-coated alpha donor beads and anti-GluR2 rabbit polyclonal antibody binds anti-rabbit IgG-coated alpha acceptor beads. Both antibodies recognize GluR2, allowing the beads to come into close proximity. The excitation of the donor beads at 680 nm provokes the release of singlet oxygen molecules that trigger an energy transfer cascade in acceptor beads, resulting in a sharp emission peak at 615 nm. (B) AlphaLISA assay protocol.

Article Snippet: Rabbit anti-GluR2 pAb (BS3658) was purchased from Bioworld Technology, Inc. (St. Louis, MN, USA).

Techniques: Expressing

Journal: Toxicology Reports

Article Title: Development of a simple measurement method for GluR2 protein expression as an index of neuronal vulnerability

doi: 10.1016/j.toxrep.2014.12.014

Figure Lengend Snippet: GluR2 complex formation for AlphaLISA assays. (A) Cell lysates, anti-GluR2 antibodies, and AlphaLISA beads were incubated in microtubes without anti-rabbit IgG-coated alpha acceptor beads or without streptavidin-coated alpha donor beads. After incubation, bead fractions were separated by centrifugation at 15,000 rpm for 15 min. (B) Western blotting of fraction buffer with extra antibodies and extra GluR2; fraction beads bound with antibodies and GluR2. TNE buffer with cell lysis buffer was used as a control. (B) Western blotting of fraction , buffer with extra antibodies and extra GluR2 fraction , beads bound with antibodies and GluR2. TNE buffer with cell lysis buffer was used as a control.

Article Snippet: Rabbit anti-GluR2 pAb (BS3658) was purchased from Bioworld Technology, Inc. (St. Louis, MN, USA).

Techniques: Incubation, Centrifugation, Western Blot, Lysis

Journal: Toxicology Reports

Article Title: Development of a simple measurement method for GluR2 protein expression as an index of neuronal vulnerability

doi: 10.1016/j.toxrep.2014.12.014

Figure Lengend Snippet: Optimized assay conditions for detecting GluR2 expression. (A) Determination of optimum concentrations of biotinylated anti-GluR2 mouse monoclonal antibody, and (B) anti-GluR2 rabbit polyclonal antibody; (C) determination of optimum protein concentrations of cell lysates; rat primary cerebral cortical neurons were lysed in TNE buffer, and total protein abundance was quantified using bicinchoninic acid (BCA) protein assays; (D) enlarged view of (C); (E) time course of incubation: 1st incubation, binding of GluR2 protein and anti-GluR2 antibodies; 3rd incubation, binding of acceptor beads and anti-GluR2 rabbit polyclonal antibody. Data are expressed as the mean ± standard error of the mean ( n = 3).

Article Snippet: Rabbit anti-GluR2 pAb (BS3658) was purchased from Bioworld Technology, Inc. (St. Louis, MN, USA).

Techniques: Expressing, Incubation, Binding Assay

Journal: Toxicology Reports

Article Title: Development of a simple measurement method for GluR2 protein expression as an index of neuronal vulnerability

doi: 10.1016/j.toxrep.2014.12.014

Figure Lengend Snippet: Schematic representation of GluR2 protein and AlphaLISA assay. (A) GluR2 is a predicted six-transmembrane protein; (B) Schematics of the GluR2Δ1–169, 539–834 construct; numbers above the amino acid residues refer to residues of the full-length receptor; (C) measurement of purified GluR2 standard using AlphaLISA. Data are expressed as the mean ± the standard error of the mean ( n = 3). (D) Correlation between western blotting analyses and AlphaLISA assays. Quantitative western blotting analyses of GluR2 protein expression were performed using image J software. (E) Interference of the other proteins except GluR2 protein by means of the lysate of C6 cells. AlphaLISA was conducted using identical samples which contain the GluR2 standard and the lysates of C6 cells.

Article Snippet: Rabbit anti-GluR2 pAb (BS3658) was purchased from Bioworld Technology, Inc. (St. Louis, MN, USA).

Techniques: Construct, Purification, Western Blot, Expressing, Software

Journal: Toxicology Reports

Article Title: Development of a simple measurement method for GluR2 protein expression as an index of neuronal vulnerability

doi: 10.1016/j.toxrep.2014.12.014

Figure Lengend Snippet: Test compounds for the screening of GluR2 decrease.

Article Snippet: Rabbit anti-GluR2 pAb (BS3658) was purchased from Bioworld Technology, Inc. (St. Louis, MN, USA).

Techniques: Inhibition

Journal: Toxicology Reports

Article Title: Development of a simple measurement method for GluR2 protein expression as an index of neuronal vulnerability

doi: 10.1016/j.toxrep.2014.12.014

Figure Lengend Snippet: Changes in protein expression of GluR2 following long-term exposure to nitenpyram or TBT. (A) Cortical neurons were exposed to 0.1–100 μM NIT or 50 nM TBT for 9 days from 2 days in vitro (DIV) to 10 DIV, and GluR2 was then detected using western blotting. Quantitative analysis of GluR2 western blots were performed using ImageJ software, and GluR2 protein levels were normalized to those of β-actin. Data are expressed as the mean + standard error of the mean ( n = 4) * p < 0.05, ** p < 0.01 vs. DMSO. (B) Correlation between western blotting analyses and AlphaLISA assays. Quantitative western blotting analyses of GluR2 protein expression were performed using image J software.

Article Snippet: Rabbit anti-GluR2 pAb (BS3658) was purchased from Bioworld Technology, Inc. (St. Louis, MN, USA).

Techniques: Expressing, In Vitro, Western Blot, Software