tetrodotoxin  (Alomone Labs)

Journal: Scientific Reports

Article Title: Hyaluronic acid based extracellular matrix regulates surface expression of GluN2B containing NMDA receptors

doi: 10.1038/s41598-017-07003-3

Figure Lengend Snippet: ECM digestion increases p1472-GluN2B level and decreases the endocytosis of GluN2B. ( A )Dissociated hippocampal cultures at DIV21-24 were treated with Hya over night and endocytosed GluN2B (green) was quantified using Map2 staining as mask (red). ( B ) There is less endocytosis of GluN2B after ECM removal within 30 minutes (Ctl 1.00 ± 0.02, n = 79; Hya 0.9 ± 0.02, n = 80; average ± SEM, Unpaired t-test, **P = 0.0015. Scale bar: 5 µm). ( C ) Quantitative WB from lysates of acute hippocampal slices treated with Ctl or Hya probed with an antibody against pGluN2B pTyr1472 (AP2 binding site) and GluN2B. ( D ) Quantification of WB of acute hippocampal slices and cortical cultures (DIV 21–24) revealed that the amount of phosphorylated GluN2B, normalized to the total amount of GluN2B, is increased after Hya treatment (overnight for cultures, 3 h for slices; slices: Ctl 1.00 ± 0.06, n = 4; Hya 1.23 ± 0.09, n = 4; cultures: Ctl 1.00 ± 0.05, n = 9; Hya 1.26 ± 0.1, n = 9; Unpaired t-test, cultures: P = 0.0332, slices P = 0.0837, ***P

Article Snippet: Antibodies and drugs The following commercial antibodies were used for Immunocytochemistry (ICC) and Western blot (WB) in the concentrations indicated: rabbit (rb) antibodies against GluN2B (alomone labs; ICC live staining: 1:200, fixed staining.

Techniques: Staining, CTL Assay, Western Blot, Binding Assay

Journal: Scientific Reports

Article Title: Hyaluronic acid based extracellular matrix regulates surface expression of GluN2B containing NMDA receptors

doi: 10.1038/s41598-017-07003-3

Figure Lengend Snippet: ECM removal enhances GluN2B-NMDAR mediated synaptic currents. ( A ) Example traces of NMDAR - mediated sEPCSs before and after Hya treatment in dissociated hippocampal cultures DIV21-24. ( B ) Amplitudes of single peaks show no significant differences between Hya treated or Hya plus Ifenprodil treated cultures (Ctl, −905.5 ± 179.4, n = 10; Hya, −776.2 ± 174.8, n = 10; Hya + Ifen, −758.2 ± 161.7, n = 11; average ± SEM; One-way ANOVA, P = 0.7991). ( C ) Average of single peaks before and after Hya treatment and after Ifenprodil application. Normalization of the amplitude illustrates the increased decay-time after Hya treatment (red line) in comparison to Ctl (black line). This can be restored after Ifenprodil application (green line). Ctl traces are identical. ( D ) Quantification of the area under the curve (AUC) of averaged and normalized events (left), which represent the total charge transfer revealed bigger charge transfer after ECM removal, which was reduced to control levels after blocking GluN2B-NMDAR with Ifen (Ctl, 1 ± 0.02, n = 10; Hya, 1.38 ± 0.09, n = 10; Hya + Ifenprodil, 0.98 ± 0.05, n = 11; average ± SEM; One-way ANOVA, P

Article Snippet: Antibodies and drugs The following commercial antibodies were used for Immunocytochemistry (ICC) and Western blot (WB) in the concentrations indicated: rabbit (rb) antibodies against GluN2B (alomone labs; ICC live staining: 1:200, fixed staining.

Techniques: CTL Assay, Blocking Assay

Journal: Scientific Reports

Article Title: Hyaluronic acid based extracellular matrix regulates surface expression of GluN2B containing NMDA receptors

doi: 10.1038/s41598-017-07003-3

Figure Lengend Snippet: ECM removal leads to increased surface expression of GluN2B in a β1 - integrin dependent manner. ( A ) Dissociated hippocampal cultures were treated with Hya over night and stained against the total amount of GluN2B and the dendritic marker Map2 (scale bar: 10 μm. ( B ) Total GluN2B expression is not affected by ECM removal (Dendrites: Ctl 1 ± 0.10, n = 30; Hya 0.89 ± 0.03, n = 30, P = 0.31; Synapses: Ctl: 1 ± 0.03, n = 30; Hya: 1.05 ± 0.03, n = 30, P = 0.27; average ± SEM; unpaired t-test). ( C ) Quantitative WB of lysed cortical cultures (DIV21) pretreated with Hya over night show no significant change in GluN2B immunoreactivity. ( D ) Dissociated hippocampal cultures at DIV21-24 were treated with Hya over night and stained against surface GluN2B (green) and the synaptic marker PSD-95 (scale bar: 10 μm). ( E ) Synaptic GluN2B surface expression at various time points after Hya treatment (Ctl: 1 ± 0.04, n = 24; Hya 1,5 h: 1.08 ± 0.04, n = 22, P = 0.76; Hya 3 h: 1.40 ± 0.09, n = 30, P = 0.0001; Hya 6 h: 1.41 ± 0.13, n = 9, P = 0.002; Hya 12 h: 1.35 ± 0.08, n = 8, P = 0.01; Hya 48 h: 1.18 ± 0.05, n = 8, P = 0.04 average ± SEM; One way-ANOVA, Dunnett’s Multiple Comparison Test). ( F,G ) GluN2B surface expression at synapses and dendrites increases after ECM degradation and can be restored by simultaneous application of the β1-integrin function blocking antibody CD29. ( F ) Synapses: Ctl: 1.0 ± 0.05, n = 68; Hya: 1.3 ± 0.05, n = 70; Hya + CD29: 0.93 ± 0.03, n = 51. ( G ) Dendrites: Ctl 1.00 ± 0.04, n = 36; Hya 1.78 ± 0.11, n = 35; Hya + CD29 0.96 ± 0.03, n = 34; average ± SEM; One-way ANOVA, P

Article Snippet: Antibodies and drugs The following commercial antibodies were used for Immunocytochemistry (ICC) and Western blot (WB) in the concentrations indicated: rabbit (rb) antibodies against GluN2B (alomone labs; ICC live staining: 1:200, fixed staining.

Techniques: Expressing, Staining, Marker, CTL Assay, Western Blot, Blocking Assay

Journal: Journal of Neurophysiology

Article Title: Parvalbumin-Positive Basket Interneurons in Monkey and Rat Prefrontal Cortex

doi: 10.1152/jn.90396.2008

Figure Lengend Snippet: Properties of the first evoked spike in monkey and rat basket cells. A : first suprathreshold response to the depolarizing current was significantly delayed in rat, but not in monkey basket cells. B : spike threshold was considerably lower in monkey basket cells than that in rat (APs are truncated). C : spikes evoked by rectangular current pulses with the latency of 8 ms still had more negative threshold in monkey ( n = 15) basket cells than that in rat ( n = 14). D : threshold of the 1st spikes and 8-ms-latency spikes is lower in monkeys than that in rats. Error bars represent SE. E : α-dendrotoxin (α-DTX) decreased spike threshold for the 1st spikes evoked in monkey ( n = 4) and rat basket cells ( n = 5).

Article Snippet: During some of the experiments, gabazine (4.5–6 μM; Sigma, St. Louis, MO) was added to the bath to block GABAA receptors. d -2-Amino-5-phospho-pentanoic acid (APV, 50 μM; Sigma) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20 μM; Sigma) were included in the bath to block N -methyl- d -aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, respectively. α-Dendrotoxin (α-DTX, 150–200 nM; Alomone Labs, Jerusalem, Israel) was used to block Kv1 channels and 4-( N -ethyl- N -phenylamino-1,2-dimethyl-6-methylamino)pyrimidinium chloride (ZD7288, 20 μM; Alomone Labs) was used to block I h channels.

Techniques: Mass Spectrometry

Journal: PeerJ

Article Title: Hypoxia and metabolic inhibitors alter the intracellular ATP:ADP ratio and membrane potential in human coronary artery smooth muscle cells

doi: 10.7717/peerj.10344

Figure Lengend Snippet: The identification of K + channels in setting HCASMC resting membrane potential. (A–F) The change in the fractional fluorescence of DiBAC 4 (3) treated with 10 µM glibenclamide (A), 200 nM penitrem A (B), 1 µM tram34 (C), 200 nM apamin (D), 25 µM BaCl 2 (E), and 10 µM XE991 (F). (G) A summary of percentchange in fractional fluorescence of DiBAC 4 (3) after application of K + channel inhibitors: glibenclamide ( p

Article Snippet: These included glibenclamide (10 µM), penitrem A (200 nM), tram34 (1 µM), apamin (200 nM), BaCl2 (25 µM) and XE991 (10 µM) which are inhibitors of KATP channels, calcium-activated large, intermediate and small conductance potassium (BKCa , IKCa , SKCa ) channels, inward rectifying potassium (Kir ) channels and voltage-gated potassium (Kv 7) channels respectively ( ; ; ; ; ; ; ).

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Long-term dynamic changes of NMDA receptors following an excitotoxic challenge

doi: 10.1101/2022.01.03.474836

Figure Lengend Snippet: nNOS (+) neurons show a decreased expression of the NMDA receptor subunit GluN1. (A) The pictogram illustrates the “patch-like” procedure employed to isolate mRNA obtained from single nNOS (+) neurons. The same approach was employed to isolate nNOS (-) neurons employed as control. (B) Bar graphs illustrate changes in mRNA levels of the indicated genes measured by real-time PCR in nNOS (+) neurons when compared to the general population of nNOS (-) cells (relative Nos1 expression in nNOS (-): 1.07 ± 0.22 vs. 2.92± 0.25 in nNOS (+) neurons, p

Article Snippet: Cells were incubated for 1 h at RT with anti-GluN1 antibody (1:200, Alomone) and anti-NOS1 antibody (1:50, Santa Cruz Biotechnology) in the blocking solution.

Techniques: Expressing, Real-time Polymerase Chain Reaction