Journal: bioRxiv

Article Title: Unravelling the molecular players at the cholinergic efferent synapse of the zebrafish lateral line

doi: 10.1101/2020.07.09.196246

Figure Lengend Snippet: Zebrafish α9 and α9α10 nAChRs are reversibly blocked by ɑ-Btx. Responses to 10 μM (α9) or 300 μM (α9α10) ACh either alone, in the presence of 100 nM α-Btx or after washing with control bath solution for 5 min, in oocytes expressing zebrafish α9 or α9α10 nAChRs are shown. Oocytes were pre-incubated with 100 nM α-Btx for 1 min prior to the addition of the agonist. α-Btx inhibited ACh responses by 93.70 ± 2.17% (n = 2) and 85.16 ± 6.86% (n= 2), respectively.

Article Snippet: All drugs were obtained from Sigma-Aldrich, except α-Btx and apamin that were purchased from Alomone.

Techniques: Expressing, Incubation

Journal: bioRxiv

Article Title: Unravelling the molecular players at the cholinergic efferent synapse of the zebrafish lateral line

doi: 10.1101/2020.07.09.196246

Figure Lengend Snippet: ACh-mediated inhibition of evoked Ca 2+ signals is blocked by α-Btx and apamin. A , Top , Representative temporal curves of mechanosensitive Ca 2+ responses of 4 HC, before (red) and after the application of 10 μM α-Btx (green). Bottom , Mechanosensitive Ca 2+ signals show no significant difference before and after 10 μM α-Btx treatment (Extra: Mdn ΔF/F0= 0.509 IQR: 0.252-1.134 Vs. α-Btx: Mdn ΔF/F0= 0.534 IQR: 0.331-1.325, n = 25, W= −45, p= 0.5449, MPRBC = 0.138). B , Top , Representative temporal curves of mechanosensitive Ca 2+ responses of 4 HC, after the application of 10 μM α-Btx (green) and after the co-application of 1 mM ACh and 10 μM α-Btx (blue). Bottom , When co-applied with 10 μM α-Btx, ACh-mediated inhibition is blocked (n = 25, W= −87, p= 0.2521, MPRBC = 0.268). C , Top , Representative temporal curves of mechanosensitive Ca 2+ responses of 4 hair cells, before (red) and after the application of 10 μM apamin (orange). Bottom , Mechanosensitive Ca 2+ signals show no significant difference before and after 10 μM apamin treatment (Extra: Mdn ΔF/F0= 0.599 IQR: 0.243-1.216 Vs. Apa: Mdn ΔF/F0= 0.567 IQR: 0.191-1.040, n = 41, W= −91, p= 0.5554, MPRBC = 0.106). D , Top , Representative temporal curves of mechanosensitive Ca 2+ responses of 4 HC, after the application of 10 μM apamin (orange) and after the co-application of 1 mM ACh and 10 μM apamin (blue). Bottom , ACh-mediated inhibition is blocked by 10 μM apamin (n = 60, W= −322, p= 0.2359, MPRBC = 0.1759). A Wilcoxon matched-pairs signed rank test was used in all cases. Extra: Extracellular imaging solution, ACh: Acetylcholine, α-Btx: α-bungarotoxin, Apa: Apamin. Horizontal scale bar in A , B , C and D : 1.5 sec, vertical scale bar in A , B , C and D : 25% ΔF/F0. Duration of the stimulus is indicated by gray lines below each trace.

Article Snippet: All drugs were obtained from Sigma-Aldrich, except α-Btx and apamin that were purchased from Alomone.

Techniques: Inhibition, Imaging

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: MRI Characteristics of Autoimmune Encephalitis With Autoantibodies to GABAA Receptor

doi: 10.1212/NXI.0000000000001158

Figure Lengend Snippet: Note that multiple cortical and subcortical lesions show hyperintense signal on FLAIR imaging (A.a), hypointense signal on T1-weighted imaging without evident mass effect and gadolinium enhancement (A.b, A.c), and no significant restricted diffusion on diffusion-weighted imaging and apparent diffusion coefficient maps (A.d, A.e). Long-term follow-up FLAIR scans in 1 representative patient (patient 1) show confluent lesions predominantly involved bilateral frontal and temporal lobes at the acute stage (B.a, B.b). Some lesions disappeared, but new multifocal lesions emerged 1.5 months later (C.a, C.b). Seven months after onset, most intracranial lesions at acute stage resolved, but signs of cortical atrophy and enlarged lateral ventricles appeared (D.a, D.b). Three years after onset, this patient had stable clinical symptoms without any lesion but dilated lateral ventricles (red circle) and brain atrophy predominantly involved in temporal and frontal lobes (red arrow) (E.a, E.b). Of note, the FLAIR scans at the follow-ups were registered in the native FLAIR space at baseline to ensure that the scans from 4 time points were comparable anatomically with a side-by-side inspection. FLAIR = fluid-attenuated inversion recovery; GABA A R = γ-aminobutyric acid type A receptor.

Article Snippet: In experiments of subunit-binding specificity, cells were incubated with corresponding commercial antibodies to α1 subunit (Proteintech Cat# 12410-1-AP, RRID: AB_2108692), β3 subunit (Millipore Cat# MAB341, RRID: AB_11214320), or γ2 subunit (Proteintech Cat# 14104-1-AP, RRID: AB_10693527) for 2 hours at RT.

Techniques: Imaging, Diffusion-based Assay

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: MRI Characteristics of Autoimmune Encephalitis With Autoantibodies to GABAA Receptor

doi: 10.1212/NXI.0000000000001158

Figure Lengend Snippet: Type 1: confluent type (A); type 2: spotted type (B). Note that patients with both types had bilateral but asymmetric lesion involvement in cingulate gyri (red arrow). GABA A R = γ-aminobutyric acid type A receptor.

Article Snippet: In experiments of subunit-binding specificity, cells were incubated with corresponding commercial antibodies to α1 subunit (Proteintech Cat# 12410-1-AP, RRID: AB_2108692), β3 subunit (Millipore Cat# MAB341, RRID: AB_11214320), or γ2 subunit (Proteintech Cat# 14104-1-AP, RRID: AB_10693527) for 2 hours at RT.

Techniques:

Journal: Basic and Clinical Neuroscience

Article Title: Evaluation of the GABAA Receptor Expression and the Effects of Muscimol on the Activity of Wide Dynamic Range Neurons Following Chronic Constriction Injury of Sciatic Nerve in Rats

doi: 10.32598/bcn.2021.1726.1

Figure Lengend Snippet: Comparison of the GABAA receptor α1 subunit expression between the sham and CCI groups (n=8) on POD 14 The expression of the GABAA receptor α1 subunit was assessed by immunoblot analysis. An equal amount (50 μg/lane) of each sample was loaded on an SDS-PAGE gel. The results were normalized to β-actin, and statistical significance was assessed by Student’s t-test. Note that there is no significant difference in the GABAA receptor content between the sham and CCI rats. Representative immunoblots are shown above the graph. GABAA: γ-Aminobutyric Acid Type A receptor; CCI: Chronic Constriction Injury; POD: Postoperative Day.

Article Snippet: After that, the membranes were incubated overnight at 4°C with mouse monoclonal primary antibody of the GABAA receptor α1 subunit (diluted 1:5000, Abcam, Cambridge, UK).

Techniques: Expressing, Western Blot, SDS Page

Journal: Brain research

Article Title: Surface expression of GABAA receptors in the rat nucleus accumbens is increased in early but not late withdrawal from extended-access cocaine self-administration.

doi: 10.1016/j.brainres.2016.04.014

Figure Lengend Snippet: Fig. 1. Effect of extended-access cocaine self-administration and different periods of withdrawal on the expression of GABAA α2 receptor subunits in the NAc. (a, b) On WD2 after discontinuing cocaine self-administration, total expression of GABAA α2 subunits was unchanged (t-test, p40.05), while there was a significant increase in surface expression of this subunit (t-test, *po0.05). (c, d) On WD25, total expression of GABAA α2 subunits remained unchanged while surface expression of the subunit returned to a level comparable to saline controls (t-tests, p40.05). (e, f) On WD48, total and cell surface levels of GABAA α2 subunits did not differ between cocaine and saline groups (t- tests, p40.05). Two-way ANOVA revealed no significant difference between cocaine and saline groups over different withdrawal times (p40.05). Representative blots show data from 4 different animals: 2 cocaine animals and 2 saline animals. Numbers within the bars indicate the number of rats in each experimental group.

Article Snippet: Using the SNAP-ID 2.0 protein detection system (EMD Millipore, Billerica, MA), PVDF membranes were incubated with blocking solution (0.5% non-fat milk and 1% goat serum in TBS-Tween20 (TBS-T); 10 min), which was followed by incubation with primary antibodies for GABAA receptor subunits α1 (1:600; 75-136; Neuromab, Davis, CA), α2 (1:333; AB72445; ABCAM, Cambridge, MA), or α4 (1:10; 73-383; Neuromab, Davis, CA) for 30 min. Membranes were washed 4 times with TBS-T, followed by incubation with secondary antibodies (HRPconjugated anti-rabbit or anti-mouse; 1:3000; Invitrogen, Carlsbad, CA).

Techniques: Expressing, Saline

Journal: Brain research

Article Title: Surface expression of GABAA receptors in the rat nucleus accumbens is increased in early but not late withdrawal from extended-access cocaine self-administration.

doi: 10.1016/j.brainres.2016.04.014

Figure Lengend Snippet: Fig. 2. Effect of extended-access cocaine self-administration and different periods of withdrawal on the expression of GABAA α4 subunits in the NAc. Saline and cocaine rats did not differ in either surface or total expression of GABAA α4 subunits on WD2 (a, b), WD25 (c, d), or WD48 (e, f) (t-tests, p40.05). Two-way ANOVA revealed no significant difference between cocaine and saline groups over different withdrawal times (p40.05). Representative blots show data from 4 different animals: 2 cocaine animals and 2 saline animals. Numbers within the bars indicate the number of rats in each experimental group.

Article Snippet: Using the SNAP-ID 2.0 protein detection system (EMD Millipore, Billerica, MA), PVDF membranes were incubated with blocking solution (0.5% non-fat milk and 1% goat serum in TBS-Tween20 (TBS-T); 10 min), which was followed by incubation with primary antibodies for GABAA receptor subunits α1 (1:600; 75-136; Neuromab, Davis, CA), α2 (1:333; AB72445; ABCAM, Cambridge, MA), or α4 (1:10; 73-383; Neuromab, Davis, CA) for 30 min. Membranes were washed 4 times with TBS-T, followed by incubation with secondary antibodies (HRPconjugated anti-rabbit or anti-mouse; 1:3000; Invitrogen, Carlsbad, CA).

Techniques: Expressing, Saline

Journal: Brain research

Article Title: Surface expression of GABAA receptors in the rat nucleus accumbens is increased in early but not late withdrawal from extended-access cocaine self-administration.

doi: 10.1016/j.brainres.2016.04.014

Figure Lengend Snippet: Fig. 3. Effect of extended-access cocaine self-administration and different periods of withdrawal on the expression of GABAA α1 subunits in the NAc. Saline and cocaine rats did not differ in either surface or total expression of GABAA α1 subunits on WD2 (a, b), WD25 (c, d), or WD48 (e, f) (t-tests, p40.05). Two-way ANOVA revealed no significant difference between cocaine and saline groups over different withdrawal times (p40.05). Representative blots show data from 4 different animals: 2 cocaine animals and 2 saline animals. Vertical line in the surface blot in the middle panel indicates that lanes were not adjacent in the original immunoblot. Numbers within the bars indicate the number of rats in each experimental group.

Article Snippet: Using the SNAP-ID 2.0 protein detection system (EMD Millipore, Billerica, MA), PVDF membranes were incubated with blocking solution (0.5% non-fat milk and 1% goat serum in TBS-Tween20 (TBS-T); 10 min), which was followed by incubation with primary antibodies for GABAA receptor subunits α1 (1:600; 75-136; Neuromab, Davis, CA), α2 (1:333; AB72445; ABCAM, Cambridge, MA), or α4 (1:10; 73-383; Neuromab, Davis, CA) for 30 min. Membranes were washed 4 times with TBS-T, followed by incubation with secondary antibodies (HRPconjugated anti-rabbit or anti-mouse; 1:3000; Invitrogen, Carlsbad, CA).

Techniques: Expressing, Saline, Western Blot

Journal: Cell research

Article Title: Gephyrin-mediated formation of inhibitory postsynaptic density sheet via phase separation.

doi: 10.1038/s41422-020-00433-1

Figure Lengend Snippet: Fig. 1 Phase separation of the GPHN-E/GlyR-βLD complex. a Schematic diagrams showing the pentameric subunit assembly of GlyR or GABAAR. The gephyrin binding core sequences in the cytoplasmic TM3–4 loops of the receptors are highlighted by an orange line and the amino acid sequences are shown. The figure also shows the domain organization of gephyrin. The positions of mutations introduced into the recombinant receptor loops and gephyrin proteins, as well as three potential phosphorylation sites in the gephyrin C-domain are indicated. b Representative SDS-PAGE of sedimentation experiments (upper panel) and quantification of relative (line graph, left y-axis) and absolute (bar graph, right y-axis) amount of proteins recovered in the supernatant (S, gray columns) and pellet (P, blue columns) (lower panel) in the assay. Proteins were mixed at the indicated concentrations. Data from three different batches of experiments were presented as means ± SD. c DIC and fluorescence images showing that mixtures of Cy3-GPHN-E and 488-GlyR-βLD at the 1:1 molar ratio and indicated concentrations formed phase-separated droplets. The boxes show a 5× zoom-in analysis of a droplet at the 5 μM of the protein concentration. Scale bars, 10 μm. d Time-lapse imaging showing the fusion of two small droplets into a larger one. The concentration of the protein mixture was 20 μM. Scale bars, 5 μm. e Time-lapse imaging showing that membrane-tethered 488-GlyR-βLD gradually formed clusters on lipid bilayers upon the addition of 100 nM Cy3-GPHN-E. Scale bars, 2 μm. f Fluorescence images showing that the clustering patterns of membrane-tethered 488- GlyR-βLD on lipid bilayers depended on the concentration of Cy3-GPHN-E. Cy3-GPHN-E was added at the indicated concentrations. Scale bars, 2 μm. g Top panel: representative fluorescence images showing that co-expression of mCherry-GPHN-E with GFP-GlyR-βLD in HeLa cells led to the formation of many puncta with the two proteins colocalized together in each punctum. These puncta are not enriched with membranes (Fig. S4a). The dashed box is magnified and shown at right. Scale bars, 20 μm. Scale bars for the zoomed-in images are 2 μm. Bottom panel: fluorescence intensity line-scanning plots showing that both proteins were concentrated and colocalized together in the two bright puncta shown in the zoomed-in images.

Article Snippet: The genes encoding GABAAR α1-3 subunits (from Addgene plasmid # 49168, # 49169, and custom-synthesized based on the sequence of NM_000808, respectively) were each subcloned into pET.32M3C with an N-terminal His6-tag and a dimeric (or trimeric) GCN4 coiled-coil domain69 (referred to as His6-GlyR-βLD or His6-GABAAR-αLD).

Techniques: Binding Assay, Recombinant, Phospho-proteomics, SDS Page, Sedimentation, Protein Concentration, Imaging, Concentration Assay, Membrane, Fluorescence, Expressing

Journal: Cell research

Article Title: Gephyrin-mediated formation of inhibitory postsynaptic density sheet via phase separation.

doi: 10.1038/s41422-020-00433-1

Figure Lengend Snippet: Fig. 7 A schematic model showing GABAAR/GlyR binding-induced iPSD formation by gephyrin. a Clustering of GABAAR/GlyR on the synaptic plasma membrane and formation of the gephyrin sheet beneath the synaptic membrane via receptor binding-induced phase separation. Note that the width of the gephyrin E-domain dimer is ~11 nm, and the distance between the two GABAAR receptors in iPSD is also ~11 nm.28 b A top-down view from the extracellular side of the synaptic membranes showing the phase separation-mediated GABAAR/ GlyR clustering and organization by the gephyrin condensates. c A schematic diagram showing progressive steps of zoomed-in views of an inhibitory synapse to show the formation of the iPSD sheet via phase separation.

Article Snippet: The genes encoding GABAAR α1-3 subunits (from Addgene plasmid # 49168, # 49169, and custom-synthesized based on the sequence of NM_000808, respectively) were each subcloned into pET.32M3C with an N-terminal His6-tag and a dimeric (or trimeric) GCN4 coiled-coil domain69 (referred to as His6-GlyR-βLD or His6-GABAAR-αLD).

Techniques: Binding Assay, Clinical Proteomics, Membrane