Journal:

Article Title:

doi:

Figure Lengend Snippet: iGluR activity, but not LRP6 overexpression, control cell polarization to Wnt3a beads, and pESC–TSC approximation. (A and B) Representative images of pESCs stained with antibodies against LRP6 (A) and β-catenin (B), and with phalloidin (F-actin). Inserts are magnification of boxes, contrast enhanced for clarity. Scale bars, 20 µm for larger images, 5 µm for inserts. (C) The percentage of cytonemes positive for LRP6 or β-catenin in pESCs. n = 62 cells. (D) Lrp6 RNA expression levels in control (CNTRL) pESCs or pESCs transiently overexpressing LRP6-eGFP (LRP6 OE), presented as fold-change to CNTRL pESCs. Bars are mean of n = 3 experiments. Error bars are SEM. Statistical significance calculated by unpaired two-sided t test: ***, P < 0.001. (E) Representative images of CNTRL or LRP6-eGFP overexpressing pESCs, stained with antibodies against LRP6. BF is brightfield. Yellow arrowhead indicates high levels of LRP6 in the cytoneme. Images are maximum-intensity projections presented at equal intensity range to allow comparison between panels. Scale bars, 20 µm. (F) The percentage of reactive interactions (defined in ) between ESCs, CNTRL pESCs, pESCs overexpressing LRP6 (LRP6 OE), or pESCs treated with 100 µM kainate and TSCs. n ≥ 58 cells pooled from ≥3 independent experiments. Statistical significance calculated by multiple Fisher’s exact two-sided tests: ****, P < 0.0001. (G) Representative images of ESCs contacting a Wnt3a bead at the base of the cytonemes, stained with antibodies against LRP6 or β-catenin (cyan) and GluA3, GluA4, GluK1, and GluK3. Bead is black sphere in brightfield (BF) panel and is highlighted with a dashed yellow circle. Scale bars, 10 µm. (H and I) The percentage of control ESCs (CTRL, blue), 10 µM CNQX-treated ESCs (green), pESCs (CTRL, orange), or pESCs treated with 100 µM kainate (pink) with polarized presentation of LRP6 (H) or β-catenin (I) upon Wnt3a bead contact. n ≥ 41 cells. Further quantification is shown in . Statistical significance calculated by multiple Fisher’s exact two-sided tests: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (J) The percentage of ESCs (blue) or pESCs (orange) with polarized distribution of both Wnt/β-catenin pathway components and GluA3, GluA4, GluK1, or GluK3. n ≥ 26 cells. Statistical significance calculated by multiple Fisher’s exact two-sided tests: **, P < 0.01. (K and L) Representative frames of a time course live-cell imaging experiment showing a pESC (magenta) treated with 100 µM kainate contacting a TSC (green) through a cytoneme, approaching it, and then separating (K). Time is minutes, and yellow dashed line indicates distance between cells. Scare bar, 20 µm. Plot on L indicates pESC-TSC distance over time (cell in K only). Arrows point to distance at initial contact (X c ) and distance at 50 min after initial cytoneme-mediated contact (X c+50 ). (M) The difference in distance between CNTRL pESCs (orange) or 100 µM kainate–treated pESCs (KA, pink) and TSCs at the initial cytoneme-mediated contact (X c ) or 50 min after contact (X c+50 , Δ Distance = X c+50 – X c ). Bars are mean of n ≥ 58 cells pooled from ≥3 experiments. Error bars are SEM. Asterisks indicate statistical significance calculated by unpaired two-sided t test: **, P < 0.01.

Article Snippet: The antibodies used were anti-α-tubulin (YL1/2; rat; ab6160; Abcam), anti–β-catenin (mouse; #610154; BD Transduction), anti-LRP6 (EPR2423(2); rabbit; ab134146; Abcam), anti-NANOG (rabbit; RCAB002P-F; Reprocell), anti-OCT3/4 (mouse; #611202; BD Transduction), anti-EOMES (rabbit; ab183991; Abcam), anti-GriA3 (mouse; MAB5416; Sigma-Aldrich), anti-GriA4 (rabbit; AB1508; Sigma-Aldrich), anti-GriK1 (rabbit; AGC-008; Alomone Laboratories), anti-GriK3 (rabbit; AGC-040; Alomone Laboratories), anti–N-cadherin (mouse; #33-3900; Thermo Fisher Scientific), anti–E-cadherin (DECMA-1; rat; ab11512; Abcam), anti-GFP (chicken; GFP-1020; Aves), anti-mCherry (goat; #200-101-379; Rockland), and AF488, AF555, or AF647-conjugated secondary antibodies (Thermo Fisher Scientific).

Techniques: Activity Assay, Over Expression, Staining, RNA Expression, Live Cell Imaging

Journal:

Article Title:

doi:

Figure Lengend Snippet: Primers used for RT-qPCR

Article Snippet: The antibodies used were anti-α-tubulin (YL1/2; rat; ab6160; Abcam), anti–β-catenin (mouse; #610154; BD Transduction), anti-LRP6 (EPR2423(2); rabbit; ab134146; Abcam), anti-NANOG (rabbit; RCAB002P-F; Reprocell), anti-OCT3/4 (mouse; #611202; BD Transduction), anti-EOMES (rabbit; ab183991; Abcam), anti-GriA3 (mouse; MAB5416; Sigma-Aldrich), anti-GriA4 (rabbit; AB1508; Sigma-Aldrich), anti-GriK1 (rabbit; AGC-008; Alomone Laboratories), anti-GriK3 (rabbit; AGC-040; Alomone Laboratories), anti–N-cadherin (mouse; #33-3900; Thermo Fisher Scientific), anti–E-cadherin (DECMA-1; rat; ab11512; Abcam), anti-GFP (chicken; GFP-1020; Aves), anti-mCherry (goat; #200-101-379; Rockland), and AF488, AF555, or AF647-conjugated secondary antibodies (Thermo Fisher Scientific).

Techniques: Sequencing

Journal:

Article Title:

doi:

Figure Lengend Snippet: ( A ) Representative images of hippocampal synaptoneurosomes (prepared from adult Sprague-Dawley rats 6-8 weeks old) incubated with BDNF (50 ng/mL). Synaptoneurosomes were immunoassayed for GluN2B using an antibody against an extracellular epitope in the GluN2B N-terminus and immunoassayed for vGlut1, PSD-95. Merge scale bar, 10 μm. Insert scale bar, 0.5 µm. Images illustrated in ( A ) were analyzed for the GluN2B integrated density ( B ) and GluN2B mean gray value ( C ). Data are the means ± SEM of 779 - 840 synaptoneurosomes per condition, in at least three independent experiments performed in different preparations. ****p < 0.0001, by Kruskal-Wallis’s test and Dunn’s multiple comparisons test.

Article Snippet: To label surface GluN2B-containing NMDAR, live neurons (low-density hippocampal cultures) were incubated for 10 min at room temperature (RT) with an antibody against an extracellular epitope of the GluN2B N-terminus (1:100; AGC-003, Alomone Labs) diluted in a saline buffer (145 mM NaCl, 5 mM glucose, 10 mM HEPES, 5 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2, [pH 7.3]) (Sham) solution, as previously described ( ).

Techniques: Incubation

Journal:

Article Title:

doi:

Figure Lengend Snippet: ( A ) Representative images of hippocampal neurons (DIV 14 - 15) that were stimulated with BDNF (50 ng/ml for 10 or 30 min), as indicated. Neurons were then live-immunoassayed for GluN2B using an antibody against an extracellular epitope in the GluN2B N-terminus, fixed, and then further immunoassayed for PSD-95, vGlut1, and MAP2. Scale bar, 5 μm. Images illustrated in ( A) , were analyzed for the total number ( B ), intensity ( C ), and area ( D ) of surface GluN2B puncta per µm. Synaptic (PSD-95- and vGlut1-colocalized) surface GluN2B number ( E ), area ( F ), and intensity ( G ) of puncta per density of excitatory synapses (number of puncta PSD-95–vGlut1 colocalized per µm), were also analyzed. Data are normalized to the means of the control and are the means ± SEM of 43-45 cells per condition, from at least three independent experiments performed in different preparations. *p < 0.05, **p < 0.01 by one-way analysis of variance (ANOVA) followed by Bonferroni post-test.

Article Snippet: To label surface GluN2B-containing NMDAR, live neurons (low-density hippocampal cultures) were incubated for 10 min at room temperature (RT) with an antibody against an extracellular epitope of the GluN2B N-terminus (1:100; AGC-003, Alomone Labs) diluted in a saline buffer (145 mM NaCl, 5 mM glucose, 10 mM HEPES, 5 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2, [pH 7.3]) (Sham) solution, as previously described ( ).

Techniques: Control

Journal:

Article Title:

doi:

Figure Lengend Snippet: ( A ) Representative images of hippocampal neurons (DIV 14 - 15) pre-incubated with GÖ 6983 (100 nM) or vehicle (DMSO; 1:1000 dilution for 40 min) and then either maintained under the same conditions or stimulated with BDNF (50 ng/ml for 30 min), as indicated. Neurons were live-immunoassayed for GluN2B using an antibody against an extracellular epitope in the GluN2B N-terminus, fixed, and then further immunoassayed for PSD-95, vGlut1, and MAP2. Scale bar, 5 μm. Images illustrated in ( A ) were analyzed for the total number ( B ), area ( C ), and intensity ( D ) of surface GluN2B puncta per µm. Synaptic (PSD-95- and vGlut1-colocalized) surface GluN2B number ( E ), area ( F ), and intensity ( G ) of puncta per µm of excitatory synapses (number of puncta PSD-95–vGlut1 colocalized per µm), were also analyzed. Data are normalized to the mean of the DMSO control and are the means ± SEM of 28 - 30 cells per condition, in at least three independent experiments performed in different preparations. ***p < 0.001, ****p < 0.0001 by one-way analysis of variance (ANOVA) followed by Bonferroni post-test.

Article Snippet: To label surface GluN2B-containing NMDAR, live neurons (low-density hippocampal cultures) were incubated for 10 min at room temperature (RT) with an antibody against an extracellular epitope of the GluN2B N-terminus (1:100; AGC-003, Alomone Labs) diluted in a saline buffer (145 mM NaCl, 5 mM glucose, 10 mM HEPES, 5 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2, [pH 7.3]) (Sham) solution, as previously described ( ).

Techniques: Incubation, Control

Journal:

Article Title:

doi:

Figure Lengend Snippet: ( A ) Experimental design for the lithium-pilocarpine model of Status Epilepticus. ( B ) Representative images of hippocampal synaptoneurosomes (prepared from adult Sprague-Dawley rats 6-8 weeks old, treated with saline, saline and ANA-12, Pilocarpine or Pilocarpine and ANA-12). Synaptoneurosomes were live-immunoassayed for GluN2B using an antibody against an extracellular epitope in the GluN2B N-terminus and immunoassayed for vGlut1 and PSD-95. Merge scale bar, 10 μm. Insert scale bar, 0.5 µm. Images illustrated in ( B ) were analyzed for the GluN2B integrated density ( C ) and GluN2B mean gray value ( D ). Data are the means ± SEM of 779 - 840 synaptoneurosomes per condition, from at least four animals for each experimental condition. ****p < 0.0001, **p<0.01 as determined by Kruskal Wallis’s test and Dunn’s multiple comparisons test. Representative western blot and analysis ( E-G ). Proteins were extracted from synaptoneurosomes prepared from the same animals used in the immunocytochemistry experiments. For the immunoblot, antibodies against pTrkB, total TrKB and β-Tubulin were used. In this analysis, pTrkB levels were normalized to total TrkB levels and total TrkB levels were normalized to β-tubulin. Data are means ± SEM of at least four animals for each experimental condition. *p < 0.05, by one-way analysis of variance (ANOVA) followed by Dunnett’s post-test.

Article Snippet: To label surface GluN2B-containing NMDAR, live neurons (low-density hippocampal cultures) were incubated for 10 min at room temperature (RT) with an antibody against an extracellular epitope of the GluN2B N-terminus (1:100; AGC-003, Alomone Labs) diluted in a saline buffer (145 mM NaCl, 5 mM glucose, 10 mM HEPES, 5 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2, [pH 7.3]) (Sham) solution, as previously described ( ).

Techniques: Saline, Western Blot, Immunocytochemistry

Journal: Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology

Article Title: Glutamatergic Dysfunction of Astrocytes in Paraventricular Nucleus of Thalamus Contributes to Adult Anxiety Susceptibility in Adolescent Ethanol Exposed Mice

doi: 10.1038/s41386-025-02264-3

Figure Lengend Snippet: (a) The quantification of glutamate levels in the dorsal thalamic area including PVT. (b-d) Representative expression of GLT1 (as known as EAAT2), GLAST (as known as EAAT1), and GAPDH (b) and pooled data (c-d) of western blots showing that the GLT1 expression in the PVT of AIE mice is selectively reduced compared to that of the CON mice. (e-f) Representative figures (e) and pooled data (f) confirming the reduction of GLT1 in the conditional GLT1 knockdown mice (GLT1 cHET ). (g-h) Representative figures (g) and pooled data (h) showing the selective reduction of GLT1 in astrocytes, not in neurons of PVT. (i-k) Non-invasive magnetic resonance spectroscopy (MRS) measurement showing the increase in glutamate levels in the dorsal thalamic area including PVT of GLT1 cHET . (l-q) Representative traces (l, o) and pooled data (m-n, p-q) showing the anxiogenic profiles of GLT1 cHET in the open field test (l-n) and elevated plus maze test (o-q). Female (circles) and male (triangles). Data represented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figure 3i , 3l , and 3o were created using BioRender.com .

Article Snippet: These membranes were incubated with anti-GLT1 antibody (1:2000, Guinea pig, Millipore Sigma Cat# AB1783, RRID:AB_90949), anti-GLAST antibody (1:2000, Rabbit, Alomone Labs Cat# AGC-021, RRID:AB_2039885), anti-GAPDH antibody (1:2000, Mouse, Sigma Aldrich Cat# MAB374, RRID:AB_2107445), and anti-xCT antibody (1:1000, Rabbit, ABclonal Cat# A2413, RRID:AB_2863004) overnight at 4 °C.

Techniques: Expressing, Knockdown, Western Blot, Spectroscopy

Journal: Annals of neurology

Article Title: Absence of Neuronal Autoantibodies in Neuropsychiatric Systemic Lupus Erythematosus.

doi: 10.1002/ana.25908

Figure Lengend Snippet: Figure 1. Clinical features and immunoglobulin G (IgG) binding to conformational neuronal surface epitopes from plasma and cerebrospinal fluid (CSF) of patients with neuropsychiatric lupus (NPSLE). A. Clinical features of 35 patients with SLE, including 15 with active NPSLE at the time of sampling. 32/34 (94%) were on immunotherapies, including prednisolone (71%), hydroxychloroquine (41%), belimumab (6%) and rituximab (3%). ANA = anti-nuclear antibody. B. Live cell-based assays employing HEK293T cells with surface expressed N-methyl D-aspartate receptors (NMDARs) as NR1-NR2A or NR1-NR2B heteromers (nuclei highlighted with DAPI, 4′,6-diamidino-2-phenylindole). First panel shows that a commercial antibody directed against the extracellular domain of the NR2A-subunit (red) binds to HEK293T cells which express NR1-NR2A heteromers. NMDAR-antibody encephalitis (NMDAR-Ab-E) patient serum IgGs (n=11) bind to the NR1-NR2A heteromers (second panel), and to NR1-NR2B heteromers / NR1 homomers (data not shown). NMDAR-antibody patient CSFs show similar binding (as Irani et al, 2010).10 Serum from healthy controls (n=36), plasma and CSFs from SLE patients (n=35 of each) show no binding. No binding was observed with the G11 antibody (data not shown, lower panel representative). Throughout, IgG binding was visualized with an anti-human 568-Alexaflour antibody (1:750 dilution, A-21090) C. Commercial antibodies against the extracellular domains of NR1 (Alomone, AGC-001), NR2A (Alomone,

Article Snippet: Throughout, IgG binding was visualized with an anti-human 568-Alexaflour antibody (1:750 dilution, A-21090) C. Commercial antibodies against the extracellular domains of NR1 (Alomone, AGC-001), NR2A (Alomone, AGC-002) and NR2B (kind gift from Prof Stephenson, London) subunits bound to the surface of live HEK293T cells co-transfected with EGFP plus NR1 and NR2B subunits.

Techniques: Binding Assay, Clinical Proteomics, Sampling

Journal: Neuroscience

Article Title: Presynaptic PICK1 facilitates trafficking of AMPA-receptors between active zone and synaptic vesicle pool.

doi: 10.1016/j.neuroscience.2016.12.042

Figure Lengend Snippet: Fig. 3. Electron micrographs of asymmetric synapses in the stratum radiatum in CA1 of the hippocampus. Immunogold labeling with anti-GluA2 (A) and anti-PICK1 (B). (C, D) Double immunogold labeling for PICK1 (20 nm) and GluA2 (10 nm). Note labeling of pre- and postsynaptic vesicles. Abbreviations: AT, axon terminal; PS, postsynaptic; AZ, active zone; PSD, postsynaptic density. Scale bars: A, B, C: 125 nm. D: 50 nm.

Article Snippet: Anti-GluA2 (Alomone labs, Jerusalem, Israel, Cat# AGC-005) was used at 1:75 for LM and at 1:40 for EM.

Techniques: Labeling

Journal: Neuroscience

Article Title: Presynaptic PICK1 facilitates trafficking of AMPA-receptors between active zone and synaptic vesicle pool.

doi: 10.1016/j.neuroscience.2016.12.042

Figure Lengend Snippet: Fig. 2. Light micrographs of rat brain vibratome sections, stained with immunoperoxidase method, with anti-GluA2 (A) and anti-PICK1 (B). The upper left image in each of the panels is coronal sections showing cortex, hippocampus, and thalamus. Hc: hippocampus. CA1, CA3 and DG (dentate gyrus) are subregions of the hippocampus. Cx: cerebral cortex. Th: thalamus. Cb: cerebellum. Note neurons with dense staining throughout the cytoplasm and large dendrites. Scale bars: Brain 2 mm. Hc 500 lm. CA1, CA3, DG, Th 20 lm. Cx 50 lm. Cb 25 lm.

Article Snippet: Anti-GluA2 (Alomone labs, Jerusalem, Israel, Cat# AGC-005) was used at 1:75 for LM and at 1:40 for EM.

Techniques: Staining, Immunohistochemistry

Journal: Channels

Article Title: Differential expression of GluN2 NMDA receptor subunits in the dorsal horn of male and female rats

doi: 10.1080/19336950.2020.1871205

Figure Lengend Snippet: List of antibodies used for immunohistochemistry

Article Snippet: Rabbit anti-GluN2D , 1:1000 , Alomone Labs , ACG-020.

Techniques:

Journal: Channels

Article Title: Differential expression of GluN2 NMDA receptor subunits in the dorsal horn of male and female rats

doi: 10.1080/19336950.2020.1871205

Figure Lengend Snippet: Immunolabelling of GluN2A, GluN2B and GluN2D subunits in the SDH and DDH of male and female rats. The CGRP+ marginal region of the SDH as well as the remaining DDH was outlined as described in . For immunoreactive quantification in subsequent figures, SDH and DDH staining (per area) were normalized to background (BG) signal in a boxed region of the immuno-negative dorsal column. A, bottom) Representative confocal images (20x objective) illustrating the fluorescence staining patterns for GluN2A (left), GluN2B (middle) and GluN2D (right) in male juvenile rats. Scale bar: 200 μm. A, top) Higher magnification insets illustrating the distribution of immunoreactivity in the marginal SDH for all three GluN2 subtypes. GluN2A immunoreactivity is uniformly distributed across the neuropil of the SDH and DDH laminae, while GluN2B and GluN2D signal is also widespread but with differential expression in SDH versus DDH laminae, with staining of both neuropil and cell bodies. Scale bar: 50 µm. B, bottom) Representative confocal images (20x objective) illustrating the fluorescence staining patterns for GluN2A (left), GluN2B (middle) and GluN2D (right) in female juvenile rats. Scale bar: 200 μm. A, top) Higher magnification insets illustrating the distribution of immunoreactivity in the marginal SDH for all three GluN2 subtypes. GluN2A immunoreactivity is distributed mainly in the neuropil of the SDH and DDH laminae, while GluN2B and GluN2D signal demonstrates staining of both neuropil and cell bodies in the SDH and DDH. Scale bar: 50 µm

Article Snippet: Rabbit anti-GluN2D , 1:1000 , Alomone Labs , ACG-020.

Techniques: Staining, Fluorescence, Expressing

Journal: Channels

Article Title: Differential expression of GluN2 NMDA receptor subunits in the dorsal horn of male and female rats

doi: 10.1080/19336950.2020.1871205

Figure Lengend Snippet: Relative expression of GluN2A, GluN2B and GluN2D in the SDH versus the DDH of males and female rats. Optical density of a given GluN2 signal per area for the SDH as well as for the DDH was normalized to background optical density per area (see box in ). Average SDH and DDH measures were derived from multiple spinal sections for each animal (shown as individual data points), followed by a paired comparison between the normalized SDH and DDH for each GluN2 subtype. (a) In males, there is significantly higher expression in the SDH (dark blue bars) versus the DDH (light blue bars) for all the three GluN2 subunits, but with more pronounced differential expression for GluN2B (middle) and GluN2D (right). (b) In females, the GluN2B isoform (middle) is the only GluN2 subunit that exhibits significantly greater expression in the SDH (dark purple) versus the DDH (light purple). *p < 0.05

Article Snippet: Rabbit anti-GluN2D , 1:1000 , Alomone Labs , ACG-020.

Techniques: Expressing, Derivative Assay

Journal: Channels

Article Title: Differential expression of GluN2 NMDA receptor subunits in the dorsal horn of male and female rats

doi: 10.1080/19336950.2020.1871205

Figure Lengend Snippet: The GluN2B, but not GluN2A or GluN2D, isoform is preferentially expressed in the medial versus the lateral region of the SDH in male rats. (a) Representative confocal images (20x objective) illustrating the immunofluorescence for GluN2A (left), GluN2B (middle), and GluN2D (left). For each isoform, the OD per area was quantified in selected oval regions positioned within the medial, central and lateral regions of the CGRP+ SDH, as shown. There was a visible increase in intensity and distribution of fluorescent puncta in the medial SDH compared to the lateral SDH for GluN2B but not GluN2A or GluN2D. Scale bar: 200 µm. (b) Statistical analysis showing the mean medial vs lateral OD ratio for GluN2A, GluN2B and GluN2D. The medial/lateral OD ratio was significantly higher for GluN2B compared to both GluN2A and GluN2D. *p < 0.05

Article Snippet: Rabbit anti-GluN2D , 1:1000 , Alomone Labs , ACG-020.

Techniques: Immunofluorescence

Journal: Channels

Article Title: Differential expression of GluN2 NMDA receptor subunits in the dorsal horn of male and female rats

doi: 10.1080/19336950.2020.1871205

Figure Lengend Snippet: Comparison of the medial vs lateral OD ratio of GluN2 subunit isoforms between males and female rats. A, B) Representative confocal images showing GluN2B immunofluorescence in a male (a) and female rat (b), Scale bar: 200 μm, as well as higher magnification insets of the SDH for both male (top left) and female (top right), Scale bar: 50 μm. GluN2B signal is preferentially localized to the medial portion of the SDH in males (a) but not females (b). (c–e) Quantitative statistical analysis of the mean medial vs lateral OD ratio for GluN2A (c), GluN2B (d) and GluN2D (e) in males versus females. The medial/lateral ratio for GluN2B was significantly greater in males compared to females (d), while the medial/lateral ratio was not significantly different between males and females and near values of 1 for both GluN2A (c) and GluN2D (e). *p < 0.05

Article Snippet: Rabbit anti-GluN2D , 1:1000 , Alomone Labs , ACG-020.

Techniques: Immunofluorescence