Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Na V 1.1 channels are critical for intercellular communication in the suprachiasmatic nucleus and for normal circadian rhythms

doi: 10.1073/pnas.1115729109

Figure Lengend Snippet: NaV1.1 voltage-gated Na+ channels are expressed in SCN GABAergic cells. (A) Fluorescent in situ hybridization with an antisense probe against the NaV1.1 mRNA labels cells in the SCN of WT mice (Left) but not mice homozygote for a KO mutation in the Scn1a gene (Right), which encodes the pore-forming α-subunit of the channel. Upper shows red immunofluorescence after antibody detection of a digoxigenin-labeled antisense riboprobe; Lower represents the same sections stained with DAPI. OC, optic chiasm. (B) Immunoblotting of SCN proteins with antibodies against the NaV1.1 reveals a 250-kDa band in WT mice, a lighter band in Scn1a+/− mice (with one copy of the WT Scn1a gene), and no immunoreactivity in KO mice. The lower band shows immunoblotting of actin as a loading control. (C) In situ hybridization against the NaV1.1 mRNA combined with GAD67 immunohistochemistry reveals expression of the channel only in GABAergic cells. Top shows fluorescent in situ hybridization for the Scn1a mRNA. Middle shows fluorescent immunolabeling of GAD. Bottom shows the merge of both fluorescence wavelengths. Eighty percent of GAD+ cells express the channel; 100% of NaV1.1+ cells express GAD. (Scale bar: A, 70 μm; B, 35 μm.)

Article Snippet: For double-labeling experiments, monoclonal anti-GAD67 (diluted 1:200; Chemicon) was added at the same time as antidigoxigenin antibody.

Techniques: In Situ Hybridization, Mutagenesis, Immunofluorescence, Labeling, Staining, Western Blot, Immunohistochemistry, Expressing, Immunolabeling, Fluorescence

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: Frontiers in Molecular Neuroscience

Article Title: Altered exocytosis of inhibitory synaptic vesicles at single presynaptic terminals of cultured striatal neurons in a knock-in mouse model of Huntington’s disease

doi: 10.3389/fnmol.2023.1175522

Figure Lengend Snippet: Cultured striatal neurons were predominantly medium spiny neurons (MSNs). (A) Representative confocal images of WT and HD cultured striatal neurons immunostained for MAP2 (green), GAD67 (red), and DARPP32 (yellow). Nuclei were counterstained with DAPI (blue). WT and HD cultured striatal neurons were isolated from WT and heterozygous zQ175 mice and were grown on coverslips, respectively. The scale bars represent 20 μm. (B) Percentage of GAD67-positive cells in MAP2-positive cells, which represents the ratio of the inhibitory neurons in cultured striatal neurons. The percentage was not significantly different between WT and HD cultured striatal neurons ( p = 0.62, independent two-tailed Student’s t -test). (C) Percentage of DARPP32-positive cells in GAD67-positive cells, which represents the ratio of the MSNs. A high percentage of DARPP32-positive cells implies that cultured striatal neurons were predominantly MSNs. The percentage showed no significant difference between WT and HD striatal neurons ( p = 0.21, independent two-tailed Student’s t -test).

Article Snippet: Fixation of cultured striatal neurons was performed using ice-cold 100% methanol for 10 min. After washing with PBS three times and blocking with 5% goat serum in staining buffer (0.2% BSA, 0.8 M NaCl, 0.5% Triton X-100, 30 mM phosphate buffer, pH7.4), 50 μL of primary antibody mixtures containing polyclonal anti-MAP2 (1:1000, Ab5392 (Abcam)), monoclonal anti-DARPP32 (1:500, Ab40801 (Abcam)) and monoclonal anti-GAD67 (1:500, MAB5406 (Merck)) or monoclonal anti-VGLUT1 (1:500, MAB5502 (Merck)) were added to coverslips, and then striatal neurons were incubated at 4°C overnight.

Techniques: Cell Culture, Isolation, Two Tailed Test

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