Journal: BMC Medicine

Article Title: Involvement of sphingosine-1-phosphate receptor 1 in pain insensitivity in a BTBR mouse model of autism spectrum disorder

doi: 10.1186/s12916-024-03722-3

Figure Lengend Snippet: Overall workflow diagram for discovering and verifying the roles and mechanisms of PAI in ASD

Article Snippet: The monoclonal antibodies used for western blotting were as follows: anti-S1PR1 (Rabbit, 1:1000, 55,133–1-Ap, Proteintech, USA); anti-S1PR3 (Rabbit, 1:1000; NBP2-24,762, Novus, USA); anti-KCNQ2 (Guinea pig, 1:200; AGP-065, Alomone, Israel); anti-KCNQ3 (Rabbit, 1:200; APC-051, Alomone, Israel); anti-KCNQ5 (Rabbit, 1:1000; YT2460, Proteintech, USA); anti-phospho-p44/42 MAPK (Rabbit, 1:2000; 7370S, CST, USA); anti-p44/42 MAPK (Erk1/2) (Rabbit, 1:2000; 4695, CST, USA); anti-phospho-p38 MAPK (Rabbit, 1:1000; 4511S, CST, USA); anti-p38 MAPK (Rabbit, 1:1000; 8690S, CST, USA); anti-PKA (Rabbit, 1:1000; 4782S, CST, USA); anti-phospho-PKA C (Rabbit, 1:1000; 5561S, CST, USA); anti-PKC (Rabbit, 1:1000; ab181558, Abcam, UK); anti-phospho-PKC (Rabbit, 1:1000; 38938S, CST, USA); anti-GADPH (Rabbit, 1:1000; 5174S, CST, USA); anti-β-tubulin (Rabbit, 1:2000; 10,094–1-AP, Proteintech, USA); and anti-β-actin (Rabbit, 1:1000, 4970 s, CST, USA).

Techniques: RNA Sequencing, Quantitative RT-PCR, Western Blot, Real-time Polymerase Chain Reaction, Co-Immunoprecipitation Assay

Journal: BMC Medicine

Article Title: Involvement of sphingosine-1-phosphate receptor 1 in pain insensitivity in a BTBR mouse model of autism spectrum disorder

doi: 10.1186/s12916-024-03722-3

Figure Lengend Snippet: Increased expression of KCNQ2 and KCNQ5 neurons in DRG of BTBR mice. a – c Quantification of KCNQ2 , KCNQ5 , and KCNQ3 mRNA; n = 8–11 mice per group. d – f Representative western blotting results and quantification of the KCNQ2, KCNQ3, and KCNQ5 proteins. n = 8 mice per group. g and h Comparison of the percentage of KCNQ2 + and KCNQ5 + neurons in the BTBR and Con group. n = 8–14 mice per group. i and j Representative images of KCNQ2 and KCNQ5 immunofluorescence in mice DRGs. Scale bars = 100 μm. k and l Distribution of KCNQ2 + and KCNQ5 + neurons of different diameters in the Con and BTBR group. n = 5 mice per group. m XE-991 induced nociceptive behavior in BTBR mice. n = 6 mice per group. All data are shown in bar diagrams, which reflect the arithmetic mean ± standard error of the mean. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n.s. not significantly different

Article Snippet: The monoclonal antibodies used for western blotting were as follows: anti-S1PR1 (Rabbit, 1:1000, 55,133–1-Ap, Proteintech, USA); anti-S1PR3 (Rabbit, 1:1000; NBP2-24,762, Novus, USA); anti-KCNQ2 (Guinea pig, 1:200; AGP-065, Alomone, Israel); anti-KCNQ3 (Rabbit, 1:200; APC-051, Alomone, Israel); anti-KCNQ5 (Rabbit, 1:1000; YT2460, Proteintech, USA); anti-phospho-p44/42 MAPK (Rabbit, 1:2000; 7370S, CST, USA); anti-p44/42 MAPK (Erk1/2) (Rabbit, 1:2000; 4695, CST, USA); anti-phospho-p38 MAPK (Rabbit, 1:1000; 4511S, CST, USA); anti-p38 MAPK (Rabbit, 1:1000; 8690S, CST, USA); anti-PKA (Rabbit, 1:1000; 4782S, CST, USA); anti-phospho-PKA C (Rabbit, 1:1000; 5561S, CST, USA); anti-PKC (Rabbit, 1:1000; ab181558, Abcam, UK); anti-phospho-PKC (Rabbit, 1:1000; 38938S, CST, USA); anti-GADPH (Rabbit, 1:1000; 5174S, CST, USA); anti-β-tubulin (Rabbit, 1:2000; 10,094–1-AP, Proteintech, USA); and anti-β-actin (Rabbit, 1:1000, 4970 s, CST, USA).

Techniques: Expressing, Western Blot, Comparison, Immunofluorescence, Real-time Polymerase Chain Reaction

Journal: BMC Medicine

Article Title: Involvement of sphingosine-1-phosphate receptor 1 in pain insensitivity in a BTBR mouse model of autism spectrum disorder

doi: 10.1186/s12916-024-03722-3

Figure Lengend Snippet: M channel and abnormal MAPK cAMP/PKA signaling pathways rescued by inhibition of S1PR1 in mice DRGs. a – c Quantification of KCNQ2 , KCNQ3 , and KCNQ5 mRNA levels after the W146 intervention. n = 6–12 mice per group. d – f Representative western blotting results and quantification of the KCNQ2, KCNQ3, and KCNQ5 proteins after W146 intervention. n = 8 mice per group. g and h Representative images of KCNQ2 and KCNQ5 immunofluorescence in mice DRGs. Scale bars = 100 μm. Comparison of the percentage of KCNQ2 + neurons in the BTBR + W146 and BTBR group; n = 6–9 mice per group. i – l Representative western blotting results and quantification of P-ERK/ERK, P-P38/P38, P-PKA/PKA, and P-PKC/PKC proteins. n = 8 mice per group. All data are shown in bar diagrams, which reflect the arithmetic mean ± standard error of the mean. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. not significantly different

Article Snippet: The monoclonal antibodies used for western blotting were as follows: anti-S1PR1 (Rabbit, 1:1000, 55,133–1-Ap, Proteintech, USA); anti-S1PR3 (Rabbit, 1:1000; NBP2-24,762, Novus, USA); anti-KCNQ2 (Guinea pig, 1:200; AGP-065, Alomone, Israel); anti-KCNQ3 (Rabbit, 1:200; APC-051, Alomone, Israel); anti-KCNQ5 (Rabbit, 1:1000; YT2460, Proteintech, USA); anti-phospho-p44/42 MAPK (Rabbit, 1:2000; 7370S, CST, USA); anti-p44/42 MAPK (Erk1/2) (Rabbit, 1:2000; 4695, CST, USA); anti-phospho-p38 MAPK (Rabbit, 1:1000; 4511S, CST, USA); anti-p38 MAPK (Rabbit, 1:1000; 8690S, CST, USA); anti-PKA (Rabbit, 1:1000; 4782S, CST, USA); anti-phospho-PKA C (Rabbit, 1:1000; 5561S, CST, USA); anti-PKC (Rabbit, 1:1000; ab181558, Abcam, UK); anti-phospho-PKC (Rabbit, 1:1000; 38938S, CST, USA); anti-GADPH (Rabbit, 1:1000; 5174S, CST, USA); anti-β-tubulin (Rabbit, 1:2000; 10,094–1-AP, Proteintech, USA); and anti-β-actin (Rabbit, 1:1000, 4970 s, CST, USA).

Techniques: Protein-Protein interactions, Inhibition, Western Blot, Immunofluorescence, Comparison, Real-time Polymerase Chain Reaction

Journal: The Journal of Neuroscience

Article Title: Merkel Cell-Driven BDNF Signaling Specifies SAI Neuron Molecular and Electrophysiological Phenotypes

doi: 10.1523/JNEUROSCI.3781-15.2016

Figure Lengend Snippet: Model for how Merkel cell-derived BDNF specifies SAI neuron molecular and electrophysiological phenotypes. In wild-type mice (left side), BDNF released from embryonic Merkel cells binds TrkB expressed on SAI neuron peripheral terminals, initiating signaling cascades that induce expression of the transcription factors Egr1, Fos, Nr4a1, Tbx3, Cux2, MafA, and Sall1. Fos maintains TrkC expression (Hughes and Dragunow, 1995), Egr1 initiates and MafA maintains Ret expression (Andrew et al., 2002; Bourane et al., 2009), and transcription factors like Cux2 coordinate ion channel expression (Bachy et al., 2011), all of which contribute to SAI neuron excitability and mature electrophysiological properties. In contrast, BDNF deletion during the embryonic period (right side) prevents the activation of these signaling cascades, which in turn leads to failed expression of TrkC and Ret along with misexpression of ion channels.

Article Snippet: The primary antibodies used were mouse anti-Islet1/2 (1:50; catalog #39.4D5, Developmental Studies Hybridoma Bank), rabbit anti-NF200 (1:1000; N4142, Sigma-Aldrich), goat anti-TrkB (1:200; AF1494, R&D Systems), goat anti-TrkC [AF1404 (1:100) and BAF1404 (1:20), R&D Systems], rabbit anti-Ret (1:50; catalog #18121, Immuno-Biological Laboratories), rabbit anti-parvalbumin (PV; 1:1000; PV25, Swant), rabbit anti-CGRP (1:1000; T-4032, Peninsula Laboratories), rabbit anti-phospho-SMAD1/5/8 (1:250; catalog 9511, Cell Signaling Technology), goat anti-TBX3 (1:100; sc-31656, Santa Cruz Biotechnology), rat anti-Keratin8 (1:20, TROMA-1, Developmental Studies Hybridoma Bank), guinea pig anti-Asic1 (1:250; Alomone Labs), rabbit anti-δ opioid receptor (DOR; 1:500; Alomone Labs), and rabbit anti-γENaC (1:500; StressMarq Biosciences).

Techniques: Derivative Assay, Expressing, Activation Assay

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: Pharmaceuticals

Article Title: The Inhibition of the Small-Conductance Ca 2+ -Activated Potassium Channels Decreases the Sinus Node Pacemaking during Beta-Adrenergic Activation

doi: 10.3390/ph15030313

Figure Lengend Snippet: ( A ) shows representative immunofluorescent images of rabbit sinus node cells with SK2 and HCN4 immunolabelling. ( B ) shows the relative expression of SK2 and HCN4. There were no significant changes between the SK2 and HCN4 fluorescence intensity. ( C ) shows fluorescence colocalization analysis. One data point represents one animal. Four cells were evaluated and averaged from each animal. Data are presented as mean ± SEM, applied statistical probe was unpaired Student’s t -test ( p ≤ 0.05).

Article Snippet: SK2 and HCN4 were labelled with anti-KCa2.2 (SK2) (Alomone, Jerusalem, Israel; 1:50) and anti-HCN4 (Alomone; 1:50) primary antibody overnight at 4 °C.

Techniques: Expressing, Fluorescence