Journal:

Article Title: Modulation of Astrocyte P2Y 1 Receptors by the Carboxyl Terminal Domain of the Gap Junction Protein Cx43

doi: 10.1002/glia.20598

Figure Lengend Snippet: (A) Western blot showing expression of Cx43 in WT (lane 1) and in Cx43-null astrocytes transfected with full length Cx43 (lane 3), with Cx43 carboxyl terminus (lane 4) and with Cx43M257 (lane 5). Lane 2 corresponds to untransfected Cx43-null cells. Lanes 1–4 correspond to immunoblots performed with Cx43-18A antibody and lane 5 with Cx43-16A. (B) Time courses of calcein FRAP obtained from untreated (black squares) and 48 h carbenoxolone-treated (open triangles) WT astrocytes, and from untransfected (open circles) and transfected Cx43-null astrocytes with full length Cx43 (open squares) and Cx43M257 (black circles). Data were obtained from astrocytes cultured from a minimum of 3 litters of mice.

Article Snippet: The following primary antibodies were employed: polyclonal antiP2Y1 (1:200; Alomone Labs), and two different polyclonal antiCx43 antibodies (gifts from Dr. E.L. Hertzberg, Albert Einstein College of Medicine), one recognizing Cx43 at position 241–261 (16A) and the other at position 345–360 (18A).

Techniques: Western Blot, Expressing, Transfection, Cell Culture

Journal:

Article Title: Modulation of Astrocyte P2Y 1 Receptors by the Carboxyl Terminal Domain of the Gap Junction Protein Cx43

doi: 10.1002/glia.20598

Figure Lengend Snippet: (A) Dose-response curves obtained for untreated (black squares) and 48 h carbenoxolone-treated (open triangles) WT and untransfected Cx43-null spinal cord astrocytes exposed to the P2Y1R agonist 2-MeS-ATP. Note that blockade of gap junctional communication in WT astrocytes does not alter the half-maximal response (EC50 value) induced by the P2Y1R agonist. Mean values were obtained from four to seven independent experiments. (B) Dose-response curves obtained for WT (black squares), untransfected (open circles) and transfected Cx43-null astrocytes with full length Cx43 (open squares), Cx43M257 (black circles) and Cx43CT (black triangles). Note that both full length Cx43 and Cx43CT but not Cx43M257 shifted the EC50 values of 2-MeS-ATP obtained for Cx43-null astrocytes to those obtained in WT cells. Data were obtained from 5 to 8 litters.

Article Snippet: The following primary antibodies were employed: polyclonal antiP2Y1 (1:200; Alomone Labs), and two different polyclonal antiCx43 antibodies (gifts from Dr. E.L. Hertzberg, Albert Einstein College of Medicine), one recognizing Cx43 at position 241–261 (16A) and the other at position 345–360 (18A).

Techniques: Transfection

Journal:

Article Title: Modulation of Astrocyte P2Y 1 Receptors by the Carboxyl Terminal Domain of the Gap Junction Protein Cx43

doi: 10.1002/glia.20598

Figure Lengend Snippet: Bar histograms showing the mean values of P2Y1R expression levels in WT, untransfected Cx43-null (KO), and in Cx43-null transfected with Cx43 CT, Cx43 truncated at position 257 (M257) and with full length Cx43. An example of western blots for Cx43 and β-actin is shown above.

Article Snippet: The following primary antibodies were employed: polyclonal antiP2Y1 (1:200; Alomone Labs), and two different polyclonal antiCx43 antibodies (gifts from Dr. E.L. Hertzberg, Albert Einstein College of Medicine), one recognizing Cx43 at position 241–261 (16A) and the other at position 345–360 (18A).

Techniques: Expressing, Transfection, Western Blot

Journal:

Article Title: Modulation of Astrocyte P2Y 1 Receptors by the Carboxyl Terminal Domain of the Gap Junction Protein Cx43

doi: 10.1002/glia.20598

Figure Lengend Snippet: (A) Dose-response curves obtained for WT (black squares) and untransfected (open circles) and Cx43Δ260–280 transfected (black triangles) Cx43-null astrocytes exposed to 2-MeS-ATP, showing that deletion of the Cx43 SH3 domain does not rescue P2Y1 receptor function. Mean±SE values are from 100 to 200 cells obtained from three litters. (B) Dose-response curves obtained from WT (black squares), and Cx43-null astrocytes untreated (open circles) and treated (black triangles) with a membrane permeant peptide corresponding to amino acids 260–280 of Cx43CT. (C) Bar histograms of the mean±SE values of intracellular calcium mobilization induced by 100 nM 2-MeS-ATP recorded from WT, Cx43-null and Cx43-null transfected with Cx43CT and Cx43M257 in the absence and presence of 5 µM PP2. Note that only untransfected and Cx43M257 transfected Cx43-null astrocytes that were not exposed to PP2 did not respond to agonist with intracellular calcium levels similar to WT astrocytes. Mean ± SE values are from 4 litters (**P < 0.001; ANOVA followed by Newman-Keuls’ Multiple Comparison Test).

Article Snippet: The following primary antibodies were employed: polyclonal antiP2Y1 (1:200; Alomone Labs), and two different polyclonal antiCx43 antibodies (gifts from Dr. E.L. Hertzberg, Albert Einstein College of Medicine), one recognizing Cx43 at position 241–261 (16A) and the other at position 345–360 (18A).

Techniques: Transfection

Journal:

Article Title: Modulation of Astrocyte P2Y 1 Receptors by the Carboxyl Terminal Domain of the Gap Junction Protein Cx43

doi: 10.1002/glia.20598

Figure Lengend Snippet: (A) Western blot showing decreased expression levels of Cx43 following exposure of spinal cord astrocytes to IL-1β (B) Dose-response curves obtained for 2-MeS-ATP performed on Fura-2 loaded WT and Cx43 KO astrocytes treated for 24 h with IL-1β (20 ng/mL). Note that exposure to the cytokine altered the agonist EC50 values in WT astrocytes. About 180 cells from three independent experiments were used in each condition.

Article Snippet: The following primary antibodies were employed: polyclonal antiP2Y1 (1:200; Alomone Labs), and two different polyclonal antiCx43 antibodies (gifts from Dr. E.L. Hertzberg, Albert Einstein College of Medicine), one recognizing Cx43 at position 241–261 (16A) and the other at position 345–360 (18A).

Techniques: Western Blot, Expressing

Journal:

Article Title: Protein Kinase C? Mediates Salutary Effects on Electrical Coupling Induced by Ischemic Preconditioning

doi: 10.1016/j.hrthm.2007.05.030

Figure Lengend Snippet: (A) Top: Representative confocal microscopy images showing Cx43 signal at cell-cell junctions in control wildtype (WT) and PKCε-KO hearts. Bottom: Quantitative confocal microscopy measurements showing amount of Cx43 signal as a percent of total tissue area in WT and PKCε-KO hearts; n for each group indicated in bar. *p<0.001 vs WT. (B) Densitometric measurements from immunoblots of total tissue Cx43 in WT and PKCε-KO hearts under basal conditions. Representative immunoblots are shown below each measurement; n for each group indicated in bar.

Article Snippet: Antibodies used in this study included a rabbit polyclonal antibody (Zymed) directed against epitopes in the C-terminus of rat Cx43 (immunoblotting, 1:5000 dilution); a mouse monoclonal anti-Cx43 antibody (Chemicon MAB3068) (immunohistochemistry and confocal microscopy, 1:400 dilution); a rabbit polyclonal anti-phospho-Cx43 antibody directed against Ser368 (Cell Signaling) (immunoblotting, 1:500 dilution; immunostaining,1:100 dilution); rabbit polyclonal anti-phospho-Cx43 antibodies directed against Ser262, Ser279/282, Ser255 or Tyr265 (Santa Cruz) (immunoblotting); a monoclonal anti-PKCε antibody (BD Biosciences) (immunoblotting, 1:250 dilution); a polyclonal anti-PKCδ antibody (Santa Cruz) (immunoblotting, 1:750 dilution); a monoclonal anti-GAPDH antibody (RDI) (immunoblotting, 1:5000 dilution), and a polyclonal anti-actin antibody (Santa Cruz) (immunoblotting, 1:1000 dilution).

Techniques: Confocal Microscopy, Control, Western Blot

Journal:

Article Title: Protein Kinase C? Mediates Salutary Effects on Electrical Coupling Induced by Ischemic Preconditioning

doi: 10.1016/j.hrthm.2007.05.030

Figure Lengend Snippet: Top: Representative confocal microscopy images showing Cx43 signal at cell-cell junctions in wildtype (WT) and PKCε-KO hearts subjected to 30 min ischemia without (Non-PC) or with preconditioning (PC). Bottom: Quantitative confocal microscopy measurements showing amount of Cx43 signal as a percent of total tissue area. Values are expressed as percent of basal values to account for differences in basal Cx43 signal between WT and PKCε-KO animals; n for each group indicated in bar. *p<0.001 vs. WT non-PC; #p<0.001 vs. PKCε-KO PC.

Article Snippet: Antibodies used in this study included a rabbit polyclonal antibody (Zymed) directed against epitopes in the C-terminus of rat Cx43 (immunoblotting, 1:5000 dilution); a mouse monoclonal anti-Cx43 antibody (Chemicon MAB3068) (immunohistochemistry and confocal microscopy, 1:400 dilution); a rabbit polyclonal anti-phospho-Cx43 antibody directed against Ser368 (Cell Signaling) (immunoblotting, 1:500 dilution; immunostaining,1:100 dilution); rabbit polyclonal anti-phospho-Cx43 antibodies directed against Ser262, Ser279/282, Ser255 or Tyr265 (Santa Cruz) (immunoblotting); a monoclonal anti-PKCε antibody (BD Biosciences) (immunoblotting, 1:250 dilution); a polyclonal anti-PKCδ antibody (Santa Cruz) (immunoblotting, 1:750 dilution); a monoclonal anti-GAPDH antibody (RDI) (immunoblotting, 1:5000 dilution), and a polyclonal anti-actin antibody (Santa Cruz) (immunoblotting, 1:1000 dilution).

Techniques: Confocal Microscopy

Journal:

Article Title: Protein Kinase C? Mediates Salutary Effects on Electrical Coupling Induced by Ischemic Preconditioning

doi: 10.1016/j.hrthm.2007.05.030

Figure Lengend Snippet: (A) Densitometric measurements from immunoblots of total tissue Cx43 in control, non-PC, and PC hearts from WT animals. (B) Densitometric measurements from immunoblots of total tissue Cx43 in control, non-PC, and PC hearts from PKCε-KO animals. Representative immunoblots are shown below each measurement; n for each group indicated in bar.

Article Snippet: Antibodies used in this study included a rabbit polyclonal antibody (Zymed) directed against epitopes in the C-terminus of rat Cx43 (immunoblotting, 1:5000 dilution); a mouse monoclonal anti-Cx43 antibody (Chemicon MAB3068) (immunohistochemistry and confocal microscopy, 1:400 dilution); a rabbit polyclonal anti-phospho-Cx43 antibody directed against Ser368 (Cell Signaling) (immunoblotting, 1:500 dilution; immunostaining,1:100 dilution); rabbit polyclonal anti-phospho-Cx43 antibodies directed against Ser262, Ser279/282, Ser255 or Tyr265 (Santa Cruz) (immunoblotting); a monoclonal anti-PKCε antibody (BD Biosciences) (immunoblotting, 1:250 dilution); a polyclonal anti-PKCδ antibody (Santa Cruz) (immunoblotting, 1:750 dilution); a monoclonal anti-GAPDH antibody (RDI) (immunoblotting, 1:5000 dilution), and a polyclonal anti-actin antibody (Santa Cruz) (immunoblotting, 1:1000 dilution).

Techniques: Western Blot, Control

Journal:

Article Title: Protein Kinase C? Mediates Salutary Effects on Electrical Coupling Induced by Ischemic Preconditioning

doi: 10.1016/j.hrthm.2007.05.030

Figure Lengend Snippet: Top: Representative confocal microscopy images showing Cx43 signal at cell-cell junctions in WT hearts perfused for 10 min with a PKCε activator, PKCδ activator, or control peptide before undergoing 30 min of global ischemia. Bottom: Quantitative confocal microscopy measurements showing amount of Cx43 signal as a percent of total tissue area in hearts treated with control peptide (CC), PKCε activator or PKCδ activator; n=3 for each group. *p=0.013 vs. control; #p=0.006 vs. PKCδ activator.

Article Snippet: Antibodies used in this study included a rabbit polyclonal antibody (Zymed) directed against epitopes in the C-terminus of rat Cx43 (immunoblotting, 1:5000 dilution); a mouse monoclonal anti-Cx43 antibody (Chemicon MAB3068) (immunohistochemistry and confocal microscopy, 1:400 dilution); a rabbit polyclonal anti-phospho-Cx43 antibody directed against Ser368 (Cell Signaling) (immunoblotting, 1:500 dilution; immunostaining,1:100 dilution); rabbit polyclonal anti-phospho-Cx43 antibodies directed against Ser262, Ser279/282, Ser255 or Tyr265 (Santa Cruz) (immunoblotting); a monoclonal anti-PKCε antibody (BD Biosciences) (immunoblotting, 1:250 dilution); a polyclonal anti-PKCδ antibody (Santa Cruz) (immunoblotting, 1:750 dilution); a monoclonal anti-GAPDH antibody (RDI) (immunoblotting, 1:5000 dilution), and a polyclonal anti-actin antibody (Santa Cruz) (immunoblotting, 1:1000 dilution).

Techniques: Confocal Microscopy, Control

Journal:

Article Title: Protein Kinase C? Mediates Salutary Effects on Electrical Coupling Induced by Ischemic Preconditioning

doi: 10.1016/j.hrthm.2007.05.030

Figure Lengend Snippet: Densitometric measurements from immunoblots of Cx43 phosphorylated at (A) Ser368, (B) Ser 262, (C) Ser255 and Ser279/282, and (D) Tyr265 in control, non-PC and PC hearts from WT and PKCε-KO animals; n for each group indicated in bar. #p<0.03 vs. WT control; *p<0.001 vs. PKCε-KO control; †p<0.02 vs. WT non-PC; §p=0.003 vs. WT PC. Representative immunoblots are shown below each measurement.

Article Snippet: Antibodies used in this study included a rabbit polyclonal antibody (Zymed) directed against epitopes in the C-terminus of rat Cx43 (immunoblotting, 1:5000 dilution); a mouse monoclonal anti-Cx43 antibody (Chemicon MAB3068) (immunohistochemistry and confocal microscopy, 1:400 dilution); a rabbit polyclonal anti-phospho-Cx43 antibody directed against Ser368 (Cell Signaling) (immunoblotting, 1:500 dilution; immunostaining,1:100 dilution); rabbit polyclonal anti-phospho-Cx43 antibodies directed against Ser262, Ser279/282, Ser255 or Tyr265 (Santa Cruz) (immunoblotting); a monoclonal anti-PKCε antibody (BD Biosciences) (immunoblotting, 1:250 dilution); a polyclonal anti-PKCδ antibody (Santa Cruz) (immunoblotting, 1:750 dilution); a monoclonal anti-GAPDH antibody (RDI) (immunoblotting, 1:5000 dilution), and a polyclonal anti-actin antibody (Santa Cruz) (immunoblotting, 1:1000 dilution).

Techniques: Western Blot, Control

Journal:

Article Title: Protein Kinase C? Mediates Salutary Effects on Electrical Coupling Induced by Ischemic Preconditioning

doi: 10.1016/j.hrthm.2007.05.030

Figure Lengend Snippet: (A) Representative confocal microscopy images showing amount of Cx43 phosphorylated at Ser368 in cell-cell junctions. (B) Quantitative confocal microscopy measurements showing amount of phosphorylated at Ser368 in cell-cell junctions expressed as a proportion of total Cx43 in junctions; n for each group indicated in bar. *p=0.005 vs. WT control; #p=0.015 vs. WT PC; §p=0.049 vs. PKCε-KO control.

Article Snippet: Antibodies used in this study included a rabbit polyclonal antibody (Zymed) directed against epitopes in the C-terminus of rat Cx43 (immunoblotting, 1:5000 dilution); a mouse monoclonal anti-Cx43 antibody (Chemicon MAB3068) (immunohistochemistry and confocal microscopy, 1:400 dilution); a rabbit polyclonal anti-phospho-Cx43 antibody directed against Ser368 (Cell Signaling) (immunoblotting, 1:500 dilution; immunostaining,1:100 dilution); rabbit polyclonal anti-phospho-Cx43 antibodies directed against Ser262, Ser279/282, Ser255 or Tyr265 (Santa Cruz) (immunoblotting); a monoclonal anti-PKCε antibody (BD Biosciences) (immunoblotting, 1:250 dilution); a polyclonal anti-PKCδ antibody (Santa Cruz) (immunoblotting, 1:750 dilution); a monoclonal anti-GAPDH antibody (RDI) (immunoblotting, 1:5000 dilution), and a polyclonal anti-actin antibody (Santa Cruz) (immunoblotting, 1:1000 dilution).

Techniques: Confocal Microscopy, Control

Journal: Korean Circulation Journal

Article Title: Preventive Effects of the Angiotensin-II Receptor Blocker on Atrial Remodeling in an Ischemic Heart Failure Model of Rats

doi: 10.4070/kcj.2013.43.10.686

Figure Lengend Snippet: Even distribution of connexin 43 protein on the intercalated disc was observed in the sham group (A: magnification: ×400). Connexin 43 protein stain was not observed on the intercalated disc in the heart failure group (B: magnification: ×400). A slight connexin 43 protein stain was observed on the intercalated disc in the heart failure-ARB group (C: magnification: ×400). Western blot of connexin 43 protein showed a lower expression in the heart failure-ARB group (D). ARB: angiotensin-II receptor blocker.

Article Snippet: The immunohistochemical stain for connexin 43 (rabbit polyclonal anti-Cx43 antibody, 1/100, Zymed-laboratories, CliniSciences, France) showed a significant amount of plasmalemmal distribution of connexin 43 in the heart failure group.

Techniques: Staining, Western Blot, Expressing

Journal: Nature Communications

Article Title: Physiological synaptic activity and recognition memory require astroglial glutamine

doi: 10.1038/s41467-022-28331-7

Figure Lengend Snippet: a Representative confocal images showing close proximity of Cx43 (cyan) in GFAP-eGFP positive astrocytes (yellow) to presynaptic structures immunolabeled for VGlut1 (magenta). Higher magnification images of a region containing astroglial processes (blue square) are shown in the middle row. Arrowheads denote points of close contact. Masks showing co-localized area of GFAP (yellow) and Cx43 (cyan) as total Cx43 (binary inverse image) and co-localized area of total Cx43 (cyan) and VGlut1(magenta) as presynaptic Cx43 (binary inverse image). b Bar graph (mean ± SEM) showing % Perisynaptic Cx43 normalized to total Cx43 area ( n = 13 fields, 3 independent experiments). c Schematic illustration of co-purification of perisynaptic astroglial processes in crude synaptosomes. d Representative western blots showing an enrichment of Cx43 protein in synaptosomal preparations (Syn) compared to total hippocampal lysates (Hip) in wild type (+/+), but not in glial conditional Cx43 knockout (−/−) mice. GAPDH was used as a loading control. e Representative high magnification electron micrographs showing the presence of Cx43 protein labeled by immunogold particles in astroglial processes near synaptic complexes. f Distribution histogram of distance between Cx43 gold grains and the nearest active zone. Scale bars: a 5 µm, e 0.5 µm (left), 0.3 µm (right). Representative images ( a ) are from replicates described in ( b ); d are from n = 3 replicates; and e are from n = 8 replicates. Source data are provided as a Source data file.

Article Snippet: The following primary antibodies were used: polyclonal chicken anti-GFP (1:500, AB13970, Abcam), monoclonal mouse anti-VGlut1 (1:200, 135511, Synaptic Systems), monoclonal mouse anti-Cx43 (1:500, 610-062, BD Biosciences), polyclonal rabbit anti-Cx43 (1:500, 71-2200, Zymed Laboratories), monoclonal mouse anti-GFAP (1:500, G3893, Sigma), and monoclonal mouse anti-GAPDH-peroxidase (1:1000, G9295, Sigma).

Techniques: Immunolabeling, Copurification, Western Blot, Knock-Out, Labeling

Journal: Nature Communications

Article Title: Physiological synaptic activity and recognition memory require astroglial glutamine

doi: 10.1038/s41467-022-28331-7

Figure Lengend Snippet: Acute hippocampal slices were loaded with ethidium bromide (EtBr) under different experimental conditions. a Sample images of EtBr uptake (magenta) in hippocampal GFAP-immunolabeled astrocytes (green) are shown for Control, Stim (10 Hz, 30 s every 3 min for 20 min) in the absence or presence of the Cx43 HC blocker Gap26 or a Gap26 scramble version (Src). Higher magnifications of the CA1 stratum radiatum subregion are shown in bottom two rows. b Schematic illustrating stimulation of hippocampal Schaffer collaterals and EtBr uptake in neighboring astrocytes. c Quantification of EtBr uptake normalized to 100% control (dotted line) is shown. Stimulation-enhanced EtBr uptake by nearly 2-fold (mean ± SEM; Control, n = 6; Stim, n = 7, p = 0.0002 between Control and Stim, one-sampled t -test). This enhanced uptake was not observed in the presence of Gap26 (Stim + Gap26, n = 4, p < 0.0001 between Stim and Stim+Gap26, one-way ANOVA with Bonferroni’s post hoc test; p = 0.0168 with control, one-sampled t -test) but persisted with Src (Stim + Src, n = 4, p = 0.6857 between Stim and Stim+Src, one-way ANOVA with Bonferroni’s post hoc test; p = 0.0125 with control, one-sampled t -test), while Gap26 alone decreases EtBr uptake from control level ( n = 5, p = 0.014, one-sampled t -test) but not Src ( n = 5, p = 0.4443, one-sampled t -test). Scale bars: a 450 µm (top), 50 µm (middle and bottom). Asterisks indicate statistical significance (*** p < 0.001; * p < 0.05). Source data are provided as a Source data file.

Article Snippet: The following primary antibodies were used: polyclonal chicken anti-GFP (1:500, AB13970, Abcam), monoclonal mouse anti-VGlut1 (1:200, 135511, Synaptic Systems), monoclonal mouse anti-Cx43 (1:500, 610-062, BD Biosciences), polyclonal rabbit anti-Cx43 (1:500, 71-2200, Zymed Laboratories), monoclonal mouse anti-GFAP (1:500, G3893, Sigma), and monoclonal mouse anti-GAPDH-peroxidase (1:1000, G9295, Sigma).

Techniques: Immunolabeling

Journal: Nature Communications

Article Title: Physiological synaptic activity and recognition memory require astroglial glutamine

doi: 10.1038/s41467-022-28331-7

Figure Lengend Snippet: Acute hippocampal slices were loaded with ethidium bromide (EtBr) under stimulated conditions in the absence or presence of various blockers. a , b Sample images of EtBr uptake (magenta) in hippocampal GFAP-immunolabeled astrocytes (green) are shown in ( a ) and quantified in ( b ). Stimulation-enhanced uptake was blocked in the presence of NBQX + CPP (20 μM, n = 6, p = 0.0112 with wild-type control, +/+, n = 11, one-way ANOVA with Bonferroni’s post hoc test), but not LY341495 (20 μM; n = 5, p = 0.2433 with +/+, one-way ANOVA with Bonferroni’s post hoc test), indicating the specific involvement of ionotropic glutamate receptor activity. This stimulation-dependent uptake was also blocked in the presence of BaCl 2 (200 μM; n = 7, p = 0.0004 with +/+, one-way ANOVA with Bonferroni’s post hoc test) and in acute slices prepared from Kir4.1−/− mice ( n = 5, p = 0.0086 with +/+, one-way ANOVA with Bonferroni’s post hoc test), indicating the specific involvement of Kir4.1 activity. c , d Incubation of acute slices with either 2 mM K + or 1 μM glutamate (Glut) alone enhanced basal EtBr uptake (K + + Src, n = 9, 3 experiments, p = 0.0061; Glut+Src, n = 9, 3 experiments, p = 0.0056 with Src, n = 14, 5 experiments; Kruskal–Wallis test with Dunn’s post hoc test) in a Cx43 HC-dependent manner (K + + Gap26, n = 20, 7 experiments, p < 0.0001 with K + + Src; Glut + Gap26, n = 11, 5 experiments, p < 0.0001 with Glut+Src; Kruskal–Wallis test with Dunn’s post hoc test). In Kir4.1−/− hippocampal slices, neither K + nor Glut were able to enhance EtBr uptake (K + Kir4.1−/−, n = 49, 17 experiments, p < 0.0001 with K + +/+, n = 22, 8 experiments; Glut Kir4.1−/−, n = 27, 9 experiments, p = 0.0001 with Glut +/+, n = 23, 8 ex p eriments, Mann–Whitney test). Mean ± SEM in ( b ) and ( d ). Scale bars: a and c , 50 µm. Asterisks indicate statistical significance (*** p < 0.001; ** p < 0.01; * p < 0.05). NBQX = 2,3-Dihydroxy-6-nitro-7-sulfamoyl-benzo[f]chinoxalin-2,3-dion; CPP = (3-((R)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid. Source data are provided as a Source data file.

Article Snippet: The following primary antibodies were used: polyclonal chicken anti-GFP (1:500, AB13970, Abcam), monoclonal mouse anti-VGlut1 (1:200, 135511, Synaptic Systems), monoclonal mouse anti-Cx43 (1:500, 610-062, BD Biosciences), polyclonal rabbit anti-Cx43 (1:500, 71-2200, Zymed Laboratories), monoclonal mouse anti-GFAP (1:500, G3893, Sigma), and monoclonal mouse anti-GAPDH-peroxidase (1:1000, G9295, Sigma).

Techniques: Immunolabeling, Activity Assay, Incubation, MANN-WHITNEY

Journal: Nature Communications

Article Title: Physiological synaptic activity and recognition memory require astroglial glutamine

doi: 10.1038/s41467-022-28331-7

Figure Lengend Snippet: a Representative confocal (dark background) and thresholded binary (white background) images of hippocampal CA1 astrocytes dialyzed with RhGln (0.8 mM, 20 min) via the patch pipette under control or stimulated (10 Hz, 30 s every 3 min for 20 min) conditions in acute slices obtained from: wild-type mice (+/+), glial conditional Cx43 knockout mice (−/−) or wild-type mice exposed to Gap26 (+/+ Gap26) or Gap26 scramble (+/+Src) peptides. The binary images were quantified by Sholl analysis ( b ) and total punctate area ( c ). In +/+ mice, repetitive synaptic stimulation strongly increased the punctate RhGln-labeling compared to control as shown by both Sholl analysis (+/+: Ct, n = 12; Stim, n = 9, p < 0.0001, two-way ANOVA in b ) and total punctate area ( p < 0.0001 between Ct and Stim in +/+, one-way ANOVA with Bonferroni’s post hoc test in c ). This was abolished in −/− mice (−/−: Ct, n = 7; Stim, n = 5, p = 0.0826, two-way ANOVA in b ; p > 0.999 between Ct and Stim in −/−, and p = 0.0002 between +/+ Stim and −/− Stim, one-way ANOVA with Bonferroni’s post hoc test in c ) or in the presence of Gap26 (+/+ Gap26: Ct, n = 8; Stim, n = 5, p = 0.0651, two-way ANOVA in b ; p > 0.999 between Ct and Stim in +/+ Gap26, and p < 0.0001 between Stim of +/+ and +/+ Gap26, one-way ANOVA with Bonferroni’s post hoc test in c ), but unchanged in the presence of the scramble Gap26 peptide (+/+Src: Ct, n = 5; Stim, n = 4, p < 0.0001, two-way ANOVA in b ; p = 0.025 between Ct and Stim with +/+ Src, and p < 0.0001 between Stim of +/+ Gap26 and +/+ Src, one-way ANOVA with Bonferroni’s post hoc test in c ). Gap26 alone also inhibited the spread of glutamine, suggesting a basal transfer of glutamine into synaptic structures which is dependent on Cx43 HC activity ( p < 0.0001 between +/+ Gap26 Ct and +/+ Gap26 Stim, two-way ANOVA in b ). Mean ± SEM in ( b ), ( c ). Scale bar: a 20 µm. Asterisks indicate statistical significance (*** p < 0.001; * p < 0.05). Source data are provided as a Source data file.

Article Snippet: The following primary antibodies were used: polyclonal chicken anti-GFP (1:500, AB13970, Abcam), monoclonal mouse anti-VGlut1 (1:200, 135511, Synaptic Systems), monoclonal mouse anti-Cx43 (1:500, 610-062, BD Biosciences), polyclonal rabbit anti-Cx43 (1:500, 71-2200, Zymed Laboratories), monoclonal mouse anti-GFAP (1:500, G3893, Sigma), and monoclonal mouse anti-GAPDH-peroxidase (1:1000, G9295, Sigma).

Techniques: Transferring, Knock-Out, Labeling, Activity Assay

Journal: Nature Communications

Article Title: Physiological synaptic activity and recognition memory require astroglial glutamine

doi: 10.1038/s41467-022-28331-7

Figure Lengend Snippet: a Sample image of the hippocampus of Cx43−/− mice injected intra-hippocampally with rAAV2/9-GFAP-Cx43-GFP virus, showing numerous cells expressing Cx43-GFP in the CA1 area. The blue box is magnified on the right. b RhGln (magenta) was loaded into a Cx43-GFP expressing astrocyte (yellow) via a patch pipette as shown. Arrow head indicates the patched cell. c Sample images after immunostaining showing specific expression of Cx43 (cyan) in Cx43-GFP-positive (yellow) astrocytes (GFAP, magenta). The yellow box is magnified in the bottom row. Solid and dotted white lines outline GFP-positive and -negative astrocytes, respectively. d – g Cx43−/− mice first received either rAAV2/9-GFAP-Cx43-GFP (−/− Cx43 Rescue, d left, e and g ) or rAAV2/9-GFAP-GFP (−/− GFP Control, d right, f and g ) virus. Hippocampal astrocytes were then dialyzed with RhGln under either control or synaptic stimulation (10 Hz, 30 s) conditions for 20 min. Both representative confocal (dark background) and thresholded binary (white background) images are shown in ( d ) for each condition. The binary images were quantified by Sholl analysis ( e , f ) and total punctate area ( g ). The stimulation-induced transfer of RhGln was rescued in Cx43−/− mice ( n = 5) by restoring Cx43 expression selectively in astrocytes via viral infection shown by both Sholl analysis (−/− Cx43 rescue, n = 4, p < 0.0001, two-way ANOVA for e ) and total punctate area ( p = 0.0031 between Ct and Stim with −/− Cx43 Rescue, one-way ANOVA with Bonferroni’s post hoc test for g ) as compared to the GFP control infection (−/− GFP Ct, n = 3; Stim, n = 3, p = 0.9856, two-way ANOVA for f , p > 0.999 between Ct and Stim with −/−GFP Control and p < 0.0001 between Stim of −/− Cx43 Rescue and −/− GFP Control, one-way ANOVA with Bonferroni’s post hoc test for g ). Mean ± SEM in ( e )–( g ). Scale bars: a 200 µm (left) and 50 µm (right); b 50 µm (top) and 20 µm (bottom); c , d 20 µm. Asterisks indicate statistical significance (*** p < 0.001; ** p < 0.01). Representative images a , b from replicates described in ( g ); c are from n = 3 replicates. Source data are provided as a Source data file.

Article Snippet: The following primary antibodies were used: polyclonal chicken anti-GFP (1:500, AB13970, Abcam), monoclonal mouse anti-VGlut1 (1:200, 135511, Synaptic Systems), monoclonal mouse anti-Cx43 (1:500, 610-062, BD Biosciences), polyclonal rabbit anti-Cx43 (1:500, 71-2200, Zymed Laboratories), monoclonal mouse anti-GFAP (1:500, G3893, Sigma), and monoclonal mouse anti-GAPDH-peroxidase (1:1000, G9295, Sigma).

Techniques: Injection, Expressing, Transferring, Immunostaining, Infection