Journal: bioRxiv

Article Title: Metaplastic priming enables non-ionotropic NMDA receptor-mediated synaptic depotentiation in the hippocampus

doi: 10.1101/2025.02.28.640846

Figure Lengend Snippet: ( A ) Representative confocal microscopy images of hippocampal slices following electrophysiology stained for NeuN (grey, AF-647), GluA1 (blue, AF-488), GluA2 (red, AF-594), and DAPI (green) and corresponding merged images for naïve, cLTP, sLTP, cDEP, and sDEP slices (maximum intensity projection). The dotted white line shown in the merged image represents the region of interest (stratum radiatum, SR) for each slice. Scalebar = 200 µm. ( B ) After normalization to naïve slices, the GluA1/GluA2 ratio in the SR was higher following cLTP compared to sLTP (unpaired Student’s t test, mean difference = 0.277 ± 0.084 times the naïve GluA1/GluA2 ratio, p = 0.022). Data are means ± SEM from 4 (sLTP) and 3 (cLTP) biological replicates. ( C ) No differences in the GluA1/GluA2 ratio were observed following cDEP compared to sDEP (unpaired Student’s t test, mean difference = −0.002 ± 0.186 times the naïve GluA1/GluA2 ratio, p = 0.993). Data are means ± SEM from 5 (sDEP) and 3 (cDEP) biological replicates. ( D ) Fluorescence intensity profiles normalized to the maximum fluorescence intensity as a function of distance along the SR (normalized to 1) for GluA1 (left) and GluA2 (right) in naïve slices. Data are means ± SEM from 10 biological replicates. ( E ) Normalized fluorescence intensity profiles for GluA1 (left) and GluA2 (right) in slices fixed after cLTP or sLTP. Data are means ± SEM from 4 (sLTP) and 3 (cLTP) biological replicates. Differences in GluA1 fluorescence intensity along the SR relative to the maxima were observed between cLTP and sLTP (mixed effects model, distance x LTP F(99, 494) = 1.11, p = 0.239; main effect of LTP type F(1,5) = 9.43, p = 0.028). No statistically significant differences in GluA2 distribution were observed between cLTP and sLTP (mixed effects model, distance x LTP F(99, 494) = 1.23, p = 0.081, LTP type F(1, 5) = 3.11, p = 0.138). ( F ) Normalized fluorescence intensity profiles for GluA1 (left) and GluA2 (right) in slices fixed after cDEP or sDEP. Data are means ± SEM from 5 (sDEP) and 3 (cDEP) biological replicates. No statistically significant differences were observed between cDEP and sDEP for GluA1 (distance x DEP F(99, 591) = 0.459, p > 0.999, DEP type F(1, 6) = 2.07, p = 0.200) or GluA2 (distance x DEP F(99, 591) = 0.680, p = 0.991, DEP type F(1, 6) = 1.69, p = 0.242) using mixed effects analysis. Data from male (closed circles) and female (open circles) mice combined, 1-2 technical replicates per biological replicate.

Article Snippet: Primary antibodies used include anti-GluA1 (1:400, guinea pig host, Alomone labs, Jerusalem, Israel, cat nr #AGC-004-GP, RRID: AB_2340961), anti-GluA2 (1:1000, rabbit host, Abcam, cat nr #ab206293, RRID: AB_2800401), and NeuN (1:1000, mouse host, Millipore-Sigma, Burlington, MA, United States, cat nr #MAB377, RRID: AB_2298772).

Techniques: Confocal Microscopy, Staining, Fluorescence

Journal: bioRxiv

Article Title: Metaplastic priming enables non-ionotropic NMDA receptor-mediated synaptic depotentiation in the hippocampus

doi: 10.1101/2025.02.28.640846

Figure Lengend Snippet: ( A ) GluA1 normalized to naïve hippocampal slices following cDEP and sDEP in control conditions (black), in the presence of 100 µM 7-CK (purple) and in the presence of 50 µM APV (blue). DEP x treatment F(2, 15) = 0.240, p = 0.790. ( B ) GluA2 normalized to naïve slices following cDEP and sDEP in control conditions and in the presence of 7-CK or APV. DEP x treatment F(2, 15) = 0.220, p = 0.805. ( C ) pGluA1 S831/GluA1 ratio normalized to naïve hippocampal slices following cDEP and sDEP in control conditions and in the presence of 7-CK or APV. DEP x treatment F(2, 15) = 0.583, p = 0.571, main effect of DEP type F(1, 15) = 12.27, p = 0.003. Post-hoc cDEP+7-CK vs. sDEP+7-CK least squares (LS) mean difference = 1.3 ± 0.6 times naïve pGluA1 S831/GluA1 expression, p = 0.034; cDEP vs. sDEP LS mean difference = 1.4 ± 0.6 times naïve pGluA1 S831/GluA1 expression, p = 0.021. ( D ) pGluA1 S845/GluA1 ratio normalized to naïve hippocampal slices following cDEP and sDEP in control conditions and in the presence of 7-CK or APV. DEP x treatment F(2, 15) = 1.62, p = 0.231, main effect of DEP type (F(1, 15) = 5.88, p = 0.028) and drug treatment (F(2, 15) = 6.57, p = 0.0089). Post-hoc cDEP+7CK vs. cDEP+APV LS mean difference = 3.7 ± 1.4 times naïve pGluA1 S845/GluA1 expression, p = 0.054; sDEP vs. sDEP+7-CK LS mean difference = −4.4 ± 1.6 times naïve pGluA1 S845/GluA1 expression, p = 0.043; cDEP vs. sDEP LS mean difference = 4.3 ± 1.5 times naïve pGluA1 S845/GluA1 expression, p = 0.012. Data in (A-D) are means ± SEM from 4 (cDEP) and 3 (sDEP) biological replicates. ( E-H ) AMPAR expression and phosphorylation states are unaltered following cDEP in the presence of C1.1 compared to C1.1Scr. ( E ) GluA1 normalized to naïve hippocampal slices following cDEP in the presence of C1.1Scr (black) versus C1.1 (grey) (p = 0.555). ( F ) GluA2 normalized to naïve hippocampal slices following cDEP in the presence of C1.1Scr versus C1.1 (p = 0.876). ( G ) pGluA1 S831/GluA1 ratio normalized to naïve hippocampal slices following cDEP with C1.1Scr versus C1.1 (p = 0.238) ( H ) pGluA1 S845/GluA1 ratio normalized to naïve hippocampal slices following cDEP with C1.1Scr versus C1.1 (p = 0.067). Data in (E-G) are means ± SEM from 4 biological replicates per group. 1-2 technical replicates per biological replicate. Blots were cut to probe for each protein (see Fig. S5A-C). Each drug treatment condition was normalized to a naïve slice run in the same blot. Statistical comparisons were made using ordinary two-way ANOVA followed by Holm-Šídák post-hoc comparisons within and across drug treatment conditions (A-D) or unpaired Student’s t test (E-H) as appropriate.

Article Snippet: Primary antibodies used include anti-GluA1 (1:400, guinea pig host, Alomone labs, Jerusalem, Israel, cat nr #AGC-004-GP, RRID: AB_2340961), anti-GluA2 (1:1000, rabbit host, Abcam, cat nr #ab206293, RRID: AB_2800401), and NeuN (1:1000, mouse host, Millipore-Sigma, Burlington, MA, United States, cat nr #MAB377, RRID: AB_2298772).

Techniques: Control, Expressing

Journal: bioRxiv

Article Title: Metaplastic priming enables non-ionotropic NMDA receptor-mediated synaptic depotentiation in the hippocampus

doi: 10.1101/2025.02.28.640846

Figure Lengend Snippet: ( A ) Full blots stained for GluA1 (top left-hand side of blots), pGluA1 S831 (top right-hand side of blots) and GAPDH (bottom section of blots) after membranes were cut as indicated along dotted lines. Blots containing samples frozen after cDEP (left) and sDEP (right) are shown. Samples were treated during electrophysiology experiments with 7-CK or APV as indicated. ( B ) Full blots stained for GluA2 (top left-hand side of blots), pGluA1 S845 (top right-hand side of blots) and GAPDH (bottom of blots) after blots were cut along dotted lines. Blots containing samples frozen after cDEP (left) and sDEP (right) are shown with corresponding treatments indicated. ( C ) Full blots stained for GluA1 (top left-hand side of left blot), pGluA1 S831 (top right-hand side of left blot), GluA2 (top left-hand side of right blot) and pGluA1 S845 (top right-hand side of right blot). Cropped blots from main are indicated with the corresponding colour-coded boxes. Images of blots stained for pGluA1 S831 and S845 were mirrored in for clarity.

Article Snippet: Primary antibodies used include anti-GluA1 (1:400, guinea pig host, Alomone labs, Jerusalem, Israel, cat nr #AGC-004-GP, RRID: AB_2340961), anti-GluA2 (1:1000, rabbit host, Abcam, cat nr #ab206293, RRID: AB_2800401), and NeuN (1:1000, mouse host, Millipore-Sigma, Burlington, MA, United States, cat nr #MAB377, RRID: AB_2298772).

Techniques: Staining

Journal: Cell Reports

Article Title: A High-Resolution Method for Quantitative Molecular Analysis of Functionally Characterized Individual Synapses

doi: 10.1016/j.celrep.2020.107968

Figure Lengend Snippet:

Article Snippet: Guinea pig polyclonal anti-Kv4.2 , Alomone , Cat#AGP-038; RRID: AB_2340969.

Techniques: Virus, Plasmid Preparation, Recombinant, Software, Microscopy

Journal: iScience

Article Title: Placode and neural crest origins of congenital deafness in mouse models of Waardenburg-Shah syndrome

doi: 10.1016/j.isci.2024.111680

Figure Lengend Snippet: Ednrb expression in P7 cochlea (A–G) Sagittal sections of cochlea isolated from P7 Ednrb-EGFP BAC transgenic mice stained for GFP only (green; A) or co-stained for neuronal Tuj1 (magenta; B and C), glial BLBP (magenta; D and E), inward rectifier potassium channel Kir4.1 (magenta; F), and vascular endothelial PECAM (magenta; G), and counterstained with DAPI (blue). High magnification views demonstrate Ednrb expression in SGN cell bodies (B) and peripheral nerve terminals (C), in satellite glia in the spiral ganglia (D) and peripheral axon bundles (E), in Kir4.1 + intermediate and Kir4.1 - basal stria cells (F), and in vascular endothelial cells of the Reissner’s membrane (G, flat mount view). “N” and “g” in (B) and (D) denote spiral ganglion neurons and glia, respectively. Yellow asterisks in (B) denote GFP + ; Tuj1 + spiral ganglion neurons. Yellow arrowheads in (C) point to GFP + ; Tuj1 + axons of spiral ganglion neurons that innervate the inner and outer hair cells corresponding to the boxed area in (A). GFP signal is much stronger in glia than in neurons. Yellow asterisks in (D) denote GFP + ; BLBP + satellite glia in the spiral ganglion. Among all Schwann cells that are associated with auditory nerve axonal bundles in (E), GFP + ; BLBP + cells and GFP + ; BLBP - cells are denoted by yellow and white asterisks, respectively. White and gray arrows in (F) point to GFP + ; Kir4.1 - basal stria cells and GFP - ; Kir4.1 - marginal stria cells, respectively. Arrows in (G) denote GFP + ; PECAM + vascular endothelial cells of the Reissner’s membrane. Abbreviations: ANF, auditory nerve fibers; bc, basal cell; ic, intermediate cell; IHC, inner hair cell; isb, inner spiral bundle; mc, marginal cell; OHC, outer hair cell; osb, outer spiral bundle; OSL, osseous spiral lamina; RM, Reissner’s membrane; SG, spiral ganglion; SV, stria vascularis. Scale bars: 200μm (A), 10μm (B–D), 25μm (E), 100μm (F), 50μm (G).

Article Snippet: Guinea pig Anti-Kir4.1 (KCNJ10) Antibody , Alomone Labs , Cat# APC-035-GP; RRID: AB_2340962.

Techniques: Expressing, Isolation, Transgenic Assay, Staining, Membrane, Immunohistochemistry, Immunofluorescence

Journal: iScience

Article Title: Placode and neural crest origins of congenital deafness in mouse models of Waardenburg-Shah syndrome

doi: 10.1016/j.isci.2024.111680

Figure Lengend Snippet: Wnt1Cre and Pax2Cre delineate distinct cell types in the cochlea (A–G) Sagittal sections of cochlea isolated from P7 Wnt1Cre/R26 nT-nG (A–D) and Pax2Cre/R26 nT-nG (E–G) mice stained for GFP (A), costained for neuronal HuD (B, C, and F), glial BLBP (B, C, and F), Kir4.1 (D), type II neuron-specific Peripherin (G), and counterstained with DAPI (blue). (A) Reporter recombination (nuclear GFP) driven by Wnt1Cre occurs in the stria vascularis (arrowheads), and in the spiral ganglion and along their peripheral nerve trajectories (arrows). (B) High magnification view of spiral ganglia in (A) shows Wnt1Cre is active in BLBP + satellite cells (arrowheads) but not in HuD + SGNs. (C) A magnified view of the boxed area in (A). Wnt1Cre labels BLBP + (arrowheads) and BLBP - (black asterisks) Schwann cells (HuD - ) along the peripheral nerve bundles. (D) A magnified view of the stria vascularis in (A) shows the activity of Wnt1Cre in Kir4.1 + intermediate stria cells, but not in Kir4.1 - basal (white arrows) and medial (gray arrows) stria cells. (E) Reporter recombination by Pax2Cre occurs in vast majority of cochlear epithelium (arrowheads) and in the spiral ganglion. (F and G) High magnification views of spiral ganglia in (E) demonstrate that Pax2Cre is exclusively active in HuD + SGNs (F; white arrows) including Peripherin + type II neurons (G; yellow arrows) but not in BLBP + satellite cells (F; asterisks). Abbreviations: SL, spiral lamina. Scale bars; 200μm (A, E), 10μm (B and C, F and G), 100μm (D).

Article Snippet: Guinea pig Anti-Kir4.1 (KCNJ10) Antibody , Alomone Labs , Cat# APC-035-GP; RRID: AB_2340962.

Techniques: Isolation, Staining, Activity Assay, Immunofluorescence

Journal: iScience

Article Title: Placode and neural crest origins of congenital deafness in mouse models of Waardenburg-Shah syndrome

doi: 10.1016/j.isci.2024.111680

Figure Lengend Snippet: Contribution of Wnt1Cre -labeled melanocytes to the Kir4.1 + intermediate stria cells in hearing and hearing impaired Ednrb -deficient mice (A–G) Confocal (z stack) images of the stria vascularis of P19 hearing (ABR threshold≤70dB) Wnt1Cre/Ednrb/R26 td-Tomato mutant (B and C), hearing impaired (ABR threshold = NR) Wnt1Cre/Ednrb/R26 td-Tomato mutants (D and E), a littermate control (A), and hearing impaired (ABR threshold = NR) global Ednrb mutant crossed into the Wnt1Cre/R26 td-Tomato background (F) immunostained for Kir4.1 (green) and counterstained with DAPI (blue). (C and E) Asterisks denote the portion of the stria vascularis that does not contain Wnt1Cre lineage derived Kir4.1 + intermediate cells. Arrows point out Wnt1Cre lineage + cells that are lacking Kir4.1 expression. Arrowheads point out misexpression or mislocalization of Kir4.1 at the apical surface of the marginal stria cells. Scale bar: 100μm. (G) Table summarizing the number of animals tested and the phenotype (intermediate cell number and maturation) frequency for Wnt1Cre/Ednrb and global Ednrb mutant mice.

Article Snippet: Guinea pig Anti-Kir4.1 (KCNJ10) Antibody , Alomone Labs , Cat# APC-035-GP; RRID: AB_2340962.

Techniques: Labeling, Confocal, Mutagenesis, Control, Derivative Assay, Expressing, Immunofluorescence

Journal: iScience

Article Title: Placode and neural crest origins of congenital deafness in mouse models of Waardenburg-Shah syndrome

doi: 10.1016/j.isci.2024.111680

Figure Lengend Snippet:

Article Snippet: Guinea pig Anti-Kir4.1 (KCNJ10) Antibody , Alomone Labs , Cat# APC-035-GP; RRID: AB_2340962.

Techniques: Recombinant, Purification, Software