Journal: EBioMedicine

Article Title: Inhibition of Nwd1 activity attenuates neuronal hyperexcitability and GluN2B phosphorylation in the hippocampus

doi: 10.1016/j.ebiom.2019.08.050

Figure Lengend Snippet: Nwd1 location in excitatory synapses acute seizures brain tissues. (a–b) Immunofluorescence staining for Nwd1 in the cerebral cortex of individuals with TLE patients. Nwd1 co-localizes with excitatory synapses marker postsynaptic density protein PSD95 but presynaptic vesicular marker Vglut1 (a) and less in inhibitory synapses marker GAD67 (b). (c–d) Immunofluorescence of Nwd1 in the hippocampi from KA-induced acute seizures mice show that Nwd1 colocalizes with PSD95 and lacks of colocalization of Nwd1with Vglut1 (c) and less in GAD67 (d) in the neuronal cells.

Article Snippet: The following primary antibodies were used in this study: a rabbit anti-NWD1 pAb (1:500, Proteintech Group, Inc., Cat# 25025–1-AP); a rabbit anti-GAPDH mAb (1:1000, Proteintech Group, Inc., Cat# 10494–1-AP); a guinea pig anti-MAP2 mAb (1:300, Synaptic Systems, Cat# 188004); a mouse anti-GFAP mAb (1:200, Proteintech Group, Inc., Cat# 60190–1-Ig); a chicken anti-GAD67 mAb (1:300; Synaptic Systems, Cat# 198006); a goat anti-PSD95 mAb (1:300, Abcam, Cat# ab12093); a mouse anti-PSD95 mAb (1:300, Abcam, Cat# ab13552); a guinea pig anti-Vglut1 mAb (1:300; Synaptic Systems, Cat# 135304); DAPI (1:50, Sigma-Aldrich, Cat# D9542); a rabbit anti-GluN2A pAb (1:500, Proteintech Group, Inc., Cat# 19953-1-AP); a mouse anti-GluN2B mAb (1:500, Abcam, Cat# ab28373); a rabbit anti-GluN2B pAb (1:500, Abcam, Cat# ab65783); a rabbit anti-GluN2B-Tyr1472 pAb (1:500, Abcam, Cat# ab3856,); and a rabbit anti-GluN2B-Ser1480 pAb (1:200, Bioss, Cat# bs-5382R); a mouse anti-Beta Actin Antibody mAb (1:1000, Proteintech Group, Inc., Cat# 60008-1-Ig).

Techniques: Immunofluorescence, Staining, Marker

Journal: Journal of neurochemistry

Article Title: Reduction in NPY-positive neurons and dysregulation of excitability in young senescence-accelerated mouse prone 8 (SAMP8) hippocampus precede the onset of cognitive impairment.

doi: 10.1111/jnc.13274

Figure Lengend Snippet: Fig. 3 Immunohistochemical staining with anti-glutamic acid decar- boxylase (GAD)67, anti-parvalbumin (PV), and anti-neuropeptide Y (NPY) antibodies in the hippocampus of SAMP8 and SAMR1 at 2w. Hippocampal sections from SAMP8 and SAMR1 at 2w were reacted with anti-GAD67, anti-PV, or anti-NPY antibody, then with appropriate secondary antibody and visualized with diaminobenzidine. Represen- tative photomicrographs of the hippocampal subregions [a: CA3, b: dentate gyrus (DG)] of SAMR1 (R1) and SAMP8 (P8) are shown. o: stratum oriens, py: stratum pyramidale, r: stratum radiatum, luc: stratum lucidum, m: stratum moleculare, g: stratum granulare, h: hilus.

Article Snippet: For double immunofluorescence staining, rabbit monoclonal antiNPY antibody (1 : 100; Cell Signaling) or rabbit polyclonal anti-PV antibody (1 : 1000; Abcam), was first applied to each section followed by mouse monoclonal anti-GAD67 antibody (1 : 200; Abcam), and the sections were incubated overnight at 4°C.

Techniques: Immunohistochemical staining, Staining

Journal: Journal of neurochemistry

Article Title: Reduction in NPY-positive neurons and dysregulation of excitability in young senescence-accelerated mouse prone 8 (SAMP8) hippocampus precede the onset of cognitive impairment.

doi: 10.1111/jnc.13274

Figure Lengend Snippet: Fig. 4 Immunohistochemical staining with anti-glutamic acid decar- boxylase (GAD)67, anti-parvalbumin (PV), and anti-neuropeptide Y (NPY) antibodies in the hippocampus of SAMP8 and SAMR1 at 4w. Hippocampal sections from SAMP8 and SAMR1 at 4w were reacted with anti-GAD67, anti-PV, or anti-NPY antibody, then with appropriate secondary antibody and visualized with diaminobenzidine. Represen- tative photomicrographs of the hippocampal subregions [(a) CA3, (b) dentate gyrus (DG)] of SAMR1 (R1) and SAMP8 (P8) are shown. o: stratum oriens, py: stratum pyramidale, r: stratum radiatum, luc: stratum lucidum, m: stratum moleculare, g: stratum granulare, h: hilus.

Article Snippet: For double immunofluorescence staining, rabbit monoclonal antiNPY antibody (1 : 100; Cell Signaling) or rabbit polyclonal anti-PV antibody (1 : 1000; Abcam), was first applied to each section followed by mouse monoclonal anti-GAD67 antibody (1 : 200; Abcam), and the sections were incubated overnight at 4°C.

Techniques: Immunohistochemical staining, Staining

Journal: Journal of neurochemistry

Article Title: Reduction in NPY-positive neurons and dysregulation of excitability in young senescence-accelerated mouse prone 8 (SAMP8) hippocampus precede the onset of cognitive impairment.

doi: 10.1111/jnc.13274

Figure Lengend Snippet: Fig. 5 Numbers of glutamic acid decarboxylase (GAD)67-positive, PV-positive, and neuropeptide Y (NPY)-positive cells in the hippocam- pal subregions of SAMP8 and SAMR1 during development. Numbers of (a) GAD67-positive, (b) PV-positive, and (c) NPY-positive cells in the hippocampal subregions [CA1, CA3, and dentate gyrus (DG)] of SAMR1 (open bars) and SAMP8 (filled bars) were counted in each section obtained at 2, 3, and 4w and expressed as means + SEM. n = 6 (SAMP8 and SAMR1). *p < 0.05 between age-matched SAMP8 versus SAMR1.

Article Snippet: For double immunofluorescence staining, rabbit monoclonal antiNPY antibody (1 : 100; Cell Signaling) or rabbit polyclonal anti-PV antibody (1 : 1000; Abcam), was first applied to each section followed by mouse monoclonal anti-GAD67 antibody (1 : 200; Abcam), and the sections were incubated overnight at 4°C.

Techniques:

Journal: Journal of neurochemistry

Article Title: Reduction in NPY-positive neurons and dysregulation of excitability in young senescence-accelerated mouse prone 8 (SAMP8) hippocampus precede the onset of cognitive impairment.

doi: 10.1111/jnc.13274

Figure Lengend Snippet: Fig. 6 Double immunofluorescence staining of neuropeptide Y (NPY)/glutamic acid decarboxylase (GAD)67 and PV/ GAD67 in the hippocampus of SAMP8 and SAMR1 at 2w. Hippocampal sections obtained from SAMR1 (R1) and SAMP8 (P8) were first reacted with rabbit monoclonal anti-NPY antibody (a) or rabbit polyclonal anti-PV antibody (b) followed by mouse monoclonal anti-GAD67 antibody, and visualized by staining with fluorescently labeled secondary antibodies. Representative micrographs of CA3 and dentate gyrus (DG) subfields are shown in (a) and (b). Numbers of cells positively stained with anti-NPY antibody (NPY+ cells; c), doubly stained with anti-NPY and anti- GAD67 antibodies (NPY+/GAD67+ cells; d), positively stained with anti-PV antibody (PV+ cells; e), and doubly stained with anti-PV and anti-GAD67 antibodies (PV+/ GAD67+ cells; f) in CA3 and DG regions of SAMR1 (open bars) and SAMP8 (filled bars) were counted in each section and expressed as means + SEM. n = 6 (SAMP8 and SAMR1). *p < 0.05 between age- matched SAMP8 versus SAMR1.

Article Snippet: For double immunofluorescence staining, rabbit monoclonal antiNPY antibody (1 : 100; Cell Signaling) or rabbit polyclonal anti-PV antibody (1 : 1000; Abcam), was first applied to each section followed by mouse monoclonal anti-GAD67 antibody (1 : 200; Abcam), and the sections were incubated overnight at 4°C.

Techniques: Staining, Labeling

Journal: bioRxiv

Article Title: A selective top-down pathway from anterior cingulate cortex embeds a representation of saliency in hippocampus

doi: 10.1101/2025.06.26.661852

Figure Lengend Snippet: (A) Dual retrograde labeling of ACC neurons. Left: The injection sites of retrograde AAVs in RSCd, RSCg and dmSTR. Right: The distribution of fluorescently tagged neurons in the ACC. (B) The proportion of dmSTR-projecting, RSCd-projecting, RSCg-projecting ACC neurons located in ACC layer 2/3 or layer 5. (C) The fraction of ACC projection neurons which project to each area. (D) Example images of sections of ACC labeled with retrograde AAV injected in RSCd/g and immunostained with anti-CaMKIIα or anti-GAD67 antibodies. (E) Similar to (D) but injections of retrograde AAV were targeted to dmSTR. (F) The proportion of RSCd/g-projecting or dmSTR-projecting ACC neurons which were labeled with anti-CaMKIIα or anti-GAD67.

Article Snippet: Prior to staining, slices were blocked in blocking buffer (0.1 M Tris-HCl, 0.15 M NaCl, 0.5% (w/v) blocking reagent (41116100, Roche)) at room temperature (RT) for 1 h. Slices were then washed with TNB-T (0.1 M Tris-HCl (pH 7.5), 0.15 M NaCl, 0.5% (w/v) blocking reagent, 1% Triton (R) X-100) twice for 2 min each and incubated with a monoclonal mouse anti-CaMKIIα antibody (1:250; 6G9, Abcam), or a polyclonal rabbit anti-GAD67 antibody (1:300; GTX 101881), or a monoclonal rabbit anti-c-Fos antibody (1:1,000; 9F6 #2250, Cell Signaling) in TNB-T overnight at 4 °C.

Techniques: Labeling, Injection

Journal: Molecular Psychiatry

Article Title: A novel approach to PTSD modeling in rats reveals alternating patterns of limbic activity in different types of stress reaction

doi: 10.1038/mp.2015.169

Figure Lengend Snippet: Differential activation of medial prefrontal cortex (mPFC) and amygdala in the different phenotypes of underwater trauma (UWT) and control rats. ( a ) Diagram of analyzed regions. Labeled cells were quantified bilaterally and averaged from 3 × 30 μm sections per region. ( b ) An example of dual-colored immunohistochemical labeling for c-Fos expression (magenta) as a biochemical marker of cellular activation and GAD67 (green) as a biochemical marker of inhibitory GABAergic cells in the BLA. White arrows point to dual-labeled (DL) cells, co-expressing GAD67 and c-Fos, which are considered as activation of inhibition. ( c ) Differences in activation (total c-Fos-expressing cells) and activation of inhibition (DL cell count) in the sub-divisions of the mPFC between the different phenotypes of UWT and control rats. ( d ) Differences in activation and activation of inhibition in the BLA and CeA nuclei of the amygdala between the different phenotypes of UWT and control rats. Bars represent the groups mean±s.e.m. Significant Bonferroni post-hoc results with P <0.05 are flagged as: *different from control; #different from unaffected; $different from affected anxious; and &different from affected anhedonic. BLA, basolateral amygdala; CeA, central amygdala; IL, infralimbic cortex; PL, perilimbic cortex.

Article Snippet: Sections were then incubated with the primary antibodies for c-Fos and GAD67 (rabbit anti c-Fos: 1:1000 dilution, Cell Signaling Technology, Danvers, MA, USA, catalog no. 2250S; mouse anti GAD67: 1:400, EMD Millipore, Billerica, MA, USA, catalog no. MAB5406) in 3% bovine serum albumin in PBS with 0.3% Triton X-100 (PBST) for 1.5 h on a shaker at room temperature followed by an overnight incubation at 4 °C.

Techniques: Activation Assay, Labeling, Immunohistochemical staining, Expressing, Marker, Inhibition, Cell Counting