glt1  (Alomone Labs)


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    Name:
    Anti EAAT2 GLT 1 extracellular ATTO Fluor 633 Antibody
    Description:
    Anti EAAT2 GLT 1 extracellular Antibody AGC 022 is a highly specific antibody directed against an epitope of the rat Excitatory amino acid transporter 2 The antibody can be used in western blot live cell imaging and immunohistochemistry applications It has been designed to recognize EAAT2 from human rat and mouse samples nAnti EAAT2 GLT 1 extracellular ATTO Fluor 633 Antibody AGC 022 FR is directly labeled with an ATTO 633 fluorescent dye ATTO dyes are characterized by strong absorption high extinction coefficient high fluorescence quantum yield and high photo stability ATTO 633 has a maximum absorption at 629 nm and a maximum fluorescence at 657 nm The fluorescence is excited most efficiently in the range 610 to 645 nm This label is analogous to the well known dyes Alexa 647 Alexa 633 and Cy5 Anti EAAT2 GLT 1 extracellular ATTO Fluor 633 Antibody has been tested in immunohistochemical staining and is specially suited for experiments requiring simultaneous labeling of different markers
    Catalog Number:
    AGC-022-FR
    Price:
    686.0
    Category:
    Primary Antibody
    Applications:
    Immunofluorescence, Immunohistochemistry
    Purity:
    Affinity purified on immobilized antigen.
    Immunogen:
    Synthetic peptide
    Size:
    50 mcl
    Antibody Type:
    Polyclonal ATTO 633 (Red) Conjugated Primary Antibody
    Format:
    Lyophilized Powder
    Host:
    Rabbit
    Isotype:
    Rabbit IgG
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    Structured Review

    Alomone Labs glt1
    Anti EAAT2 GLT 1 extracellular ATTO Fluor 633 Antibody
    Anti EAAT2 GLT 1 extracellular Antibody AGC 022 is a highly specific antibody directed against an epitope of the rat Excitatory amino acid transporter 2 The antibody can be used in western blot live cell imaging and immunohistochemistry applications It has been designed to recognize EAAT2 from human rat and mouse samples nAnti EAAT2 GLT 1 extracellular ATTO Fluor 633 Antibody AGC 022 FR is directly labeled with an ATTO 633 fluorescent dye ATTO dyes are characterized by strong absorption high extinction coefficient high fluorescence quantum yield and high photo stability ATTO 633 has a maximum absorption at 629 nm and a maximum fluorescence at 657 nm The fluorescence is excited most efficiently in the range 610 to 645 nm This label is analogous to the well known dyes Alexa 647 Alexa 633 and Cy5 Anti EAAT2 GLT 1 extracellular ATTO Fluor 633 Antibody has been tested in immunohistochemical staining and is specially suited for experiments requiring simultaneous labeling of different markers
    https://www.bioz.com/result/glt1/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    glt1 - by Bioz Stars, 2021-09
    93/100 stars

    Images

    1) Product Images from "Deletion of the Clock Gene Period2 (Per2) in Glial Cells Alters Mood-Related Behavior in Mice"

    Article Title: Deletion of the Clock Gene Period2 (Per2) in Glial Cells Alters Mood-Related Behavior in Mice

    Journal: bioRxiv

    doi: 10.1101/2020.12.09.417162

    Adeno-associated virus (AAV)-mediated deletion of Per2 in glial cells of adult mice leads to a depression resistant behavior. (A) Fluorescent imaging of whole brains 3 weeks after no injection (left), intravenous (i.v.) injection of the engineered AAV-PHP.eB, which can pass the blood-brain barrier (BBB), containing the general CAG driver (second from left) or the glial Gfap driver (middle). The second to last brain is from an animal with i.v. injected AAV-9, which does not pass the BBB, containing the general CAG driver. The last brain (right) is from an animal injected intraperitoneally (i.p.) with the AAV-PHP.eB Gfap -driven construct. Note that only the brains of animals that received the AAV-PHP.eb i.v. display significant fluorescent signal after 3 weeks (orange and yellow color). (B) Fluorescent imaging of whole brains 2 months after injection of the AAV-PHP.eB. Note that the fluorescence is still maintained after 2 months post injection and that even the i.p. injected AAV-PHP.eB Gfap is showing signal in the brain now. (C) Sorting of neurons and astrocytes by flow cytometry from brain tissue including the nucleus accumbens (NAc). The left panel shows the removal of debris from a single cell suspension, showing the distribution of debris in the forward as well as in the side scatter (FSC and SSC, respectively). The middle panel shows the removal of CD11b + cells (microglia) from the cell suspension (lower left corner from left panel). The CD11b - cells (bottom half from middle panel) were then sorted into two distinct cell populations corresponding to astrocytes (GLT1 + /CD90.2 - ) and neurons (CD90.2 + /GLT1 - ) (right panel). (D) PCR analysis of astrocytes and neurons from the cell sorting. Microglia (CD11 + ) as well as astrocytes (GLT1 + /CD90.2 - ), but not neurons (CD90.2 + /GLT1 - ) from PHP.eB Gfap-iCre infected animals show the presence of iCre , indicating that only glia and not neurons could express iCre in order to delete Per2 in the Per2 fl/fl mice. (E) Immunohistochemistry of vG Per2 brain tissue from nucleus accumbens (NAc) isolated at ZT6. The signal for PER2 (green) mainly overlaps with neuronal NeuN signal (red) giving rise to the yellow color. Scale bar: 100 µm. (F) Immobility time in the forced swim test (FST) of vG Per2 (PHP.eB Gfap-iCre , green) and control (PHP.eB control, blue) animals are shown (n = 5, two-tailed t-test, *p
    Figure Legend Snippet: Adeno-associated virus (AAV)-mediated deletion of Per2 in glial cells of adult mice leads to a depression resistant behavior. (A) Fluorescent imaging of whole brains 3 weeks after no injection (left), intravenous (i.v.) injection of the engineered AAV-PHP.eB, which can pass the blood-brain barrier (BBB), containing the general CAG driver (second from left) or the glial Gfap driver (middle). The second to last brain is from an animal with i.v. injected AAV-9, which does not pass the BBB, containing the general CAG driver. The last brain (right) is from an animal injected intraperitoneally (i.p.) with the AAV-PHP.eB Gfap -driven construct. Note that only the brains of animals that received the AAV-PHP.eb i.v. display significant fluorescent signal after 3 weeks (orange and yellow color). (B) Fluorescent imaging of whole brains 2 months after injection of the AAV-PHP.eB. Note that the fluorescence is still maintained after 2 months post injection and that even the i.p. injected AAV-PHP.eB Gfap is showing signal in the brain now. (C) Sorting of neurons and astrocytes by flow cytometry from brain tissue including the nucleus accumbens (NAc). The left panel shows the removal of debris from a single cell suspension, showing the distribution of debris in the forward as well as in the side scatter (FSC and SSC, respectively). The middle panel shows the removal of CD11b + cells (microglia) from the cell suspension (lower left corner from left panel). The CD11b - cells (bottom half from middle panel) were then sorted into two distinct cell populations corresponding to astrocytes (GLT1 + /CD90.2 - ) and neurons (CD90.2 + /GLT1 - ) (right panel). (D) PCR analysis of astrocytes and neurons from the cell sorting. Microglia (CD11 + ) as well as astrocytes (GLT1 + /CD90.2 - ), but not neurons (CD90.2 + /GLT1 - ) from PHP.eB Gfap-iCre infected animals show the presence of iCre , indicating that only glia and not neurons could express iCre in order to delete Per2 in the Per2 fl/fl mice. (E) Immunohistochemistry of vG Per2 brain tissue from nucleus accumbens (NAc) isolated at ZT6. The signal for PER2 (green) mainly overlaps with neuronal NeuN signal (red) giving rise to the yellow color. Scale bar: 100 µm. (F) Immobility time in the forced swim test (FST) of vG Per2 (PHP.eB Gfap-iCre , green) and control (PHP.eB control, blue) animals are shown (n = 5, two-tailed t-test, *p

    Techniques Used: Mouse Assay, Imaging, Injection, Construct, Fluorescence, Flow Cytometry, Polymerase Chain Reaction, FACS, Infection, Immunohistochemistry, Isolation, Two Tailed Test

    Related Articles

    Staining:

    Article Title: High-Throughput Liquid Chromatography–Tandem Mass Spectrometry Quantification of Glycosaminoglycans as Biomarkers of Mucopolysaccharidosis II
    Article Snippet: .. Cells were Fc blocked (Biolegend #101320, 1:100) and stained for flow cytometric analysis with Fixable Viability Stain BV510 (BD Biosciences #564406, 1:100) to exclude dead cells, CD11b-BV421 (BD Biosciences 562605, 1:100), CD31-PerCP Cy5.5 (BD Biosciences #562861, 1:100), O1-488 (Thermo/eBio #14-6506-82, 1:37.5), Thy1-PE (R & D #FAB7335P, 1:100), and EAAT2-633 (Alomone #AGC-022-FR, 1:50). ..

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    Alomone Labs glt1
    Adeno-associated virus (AAV)-mediated deletion of Per2 in glial cells of adult mice leads to a depression resistant behavior. (A) Fluorescent imaging of whole brains 3 weeks after no injection (left), intravenous (i.v.) injection of the engineered AAV-PHP.eB, which can pass the blood-brain barrier (BBB), containing the general CAG driver (second from left) or the glial Gfap driver (middle). The second to last brain is from an animal with i.v. injected AAV-9, which does not pass the BBB, containing the general CAG driver. The last brain (right) is from an animal injected intraperitoneally (i.p.) with the AAV-PHP.eB Gfap -driven construct. Note that only the brains of animals that received the AAV-PHP.eb i.v. display significant fluorescent signal after 3 weeks (orange and yellow color). (B) Fluorescent imaging of whole brains 2 months after injection of the AAV-PHP.eB. Note that the fluorescence is still maintained after 2 months post injection and that even the i.p. injected AAV-PHP.eB Gfap is showing signal in the brain now. (C) Sorting of neurons and astrocytes by flow cytometry from brain tissue including the nucleus accumbens (NAc). The left panel shows the removal of debris from a single cell suspension, showing the distribution of debris in the forward as well as in the side scatter (FSC and SSC, respectively). The middle panel shows the removal of CD11b + cells (microglia) from the cell suspension (lower left corner from left panel). The CD11b - cells (bottom half from middle panel) were then sorted into two distinct cell populations corresponding to astrocytes <t>(GLT1</t> + /CD90.2 - ) and neurons (CD90.2 + /GLT1 - ) (right panel). (D) PCR analysis of astrocytes and neurons from the cell sorting. Microglia (CD11 + ) as well as astrocytes (GLT1 + /CD90.2 - ), but not neurons (CD90.2 + /GLT1 - ) from PHP.eB Gfap-iCre infected animals show the presence of iCre , indicating that only glia and not neurons could express iCre in order to delete Per2 in the Per2 fl/fl mice. (E) Immunohistochemistry of vG Per2 brain tissue from nucleus accumbens (NAc) isolated at ZT6. The signal for PER2 (green) mainly overlaps with neuronal NeuN signal (red) giving rise to the yellow color. Scale bar: 100 µm. (F) Immobility time in the forced swim test (FST) of vG Per2 (PHP.eB Gfap-iCre , green) and control (PHP.eB control, blue) animals are shown (n = 5, two-tailed t-test, *p
    Glt1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/glt1/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    glt1 - by Bioz Stars, 2021-09
    93/100 stars
      Buy from Supplier

    93
    Alomone Labs rabbit α glt1
    <t>GLT1</t> HET mice have higher ipsilateral eye responses, lower contralateral eye bias and disrupted experience-dependent binocular matching of orientation-selective responses. A) Schematic of experimental design. Top: Visual gratings were separately presented to the contra (green) and ipsi (blue) eyes in P28 mice and neuronal responses recorded. Bottom: schematic of measures. Ocular dominance index (ODI) was calculated as (max Contra − max Ipsi ) / max Contra +max Ipsi . Orientation Selectivity Index (OSI) was calculated as described previously ( Banerjee et al., 2016 ). Difference in preferred orientation (ΔPO) was calculated as the difference between preferred orientations of the max contralateral and ipsilateral responses. B) Example cells in GLT1 WT (top) and GLT1 HET (bottom) animals. Left: in vivo images of neuronal somas measured in binocular visual cortex using the calcium indicator, GCaMP6f. Right: Tuning curves of three cells (white circles in left) to contra (green) and ipsi (blue) stimulation. Note the matched tuning and contralateral bias in WT animals and the mismatched tuning curves and lack of contralateral bias in GLT1 HETs. C) Quantification of the average response to PO in GLT1 WT and HET mice. WT mice have a significantly higher contralateral response than ipsilateral response while HET mice have approximately equal contralateral and ipsilateral responses (n=4-6 animals, 23-52 cells per animal, two-way ANOVA, genotype F(1,674)=7.72, p=0.0056, interaction F(1,674)=4.243, p=0.040). D) Quantification of ocular dominance index showing that GLT1 HET mice have significantly decreased ODI (n=4-6 animals, 23-52 cells per animal, t-test, p=0.0018). E) Quantification of OSI showing that GLT1 HET mice have a significantly decreased OSI of ipsilateral responses compared to both contra and ipsi responses in GLT1 WT animals (n=4-6 animals, 23-52 cells per animal, two-way ANOVA, genotype F(1,674)=12.46, p=4.5×10 −4 ). F) Quantification of ΔPO showing an increased difference in the preferred orientations between contralateral and ipsilateral inputs to neurons in GLT1 HET animals (n=4-6 animals, 23-52 cells per animal, t-test, p=1.0×10 −4 ). *p
    Rabbit α Glt1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit α glt1/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit α glt1 - by Bioz Stars, 2021-09
    93/100 stars
      Buy from Supplier

    Image Search Results


    Adeno-associated virus (AAV)-mediated deletion of Per2 in glial cells of adult mice leads to a depression resistant behavior. (A) Fluorescent imaging of whole brains 3 weeks after no injection (left), intravenous (i.v.) injection of the engineered AAV-PHP.eB, which can pass the blood-brain barrier (BBB), containing the general CAG driver (second from left) or the glial Gfap driver (middle). The second to last brain is from an animal with i.v. injected AAV-9, which does not pass the BBB, containing the general CAG driver. The last brain (right) is from an animal injected intraperitoneally (i.p.) with the AAV-PHP.eB Gfap -driven construct. Note that only the brains of animals that received the AAV-PHP.eb i.v. display significant fluorescent signal after 3 weeks (orange and yellow color). (B) Fluorescent imaging of whole brains 2 months after injection of the AAV-PHP.eB. Note that the fluorescence is still maintained after 2 months post injection and that even the i.p. injected AAV-PHP.eB Gfap is showing signal in the brain now. (C) Sorting of neurons and astrocytes by flow cytometry from brain tissue including the nucleus accumbens (NAc). The left panel shows the removal of debris from a single cell suspension, showing the distribution of debris in the forward as well as in the side scatter (FSC and SSC, respectively). The middle panel shows the removal of CD11b + cells (microglia) from the cell suspension (lower left corner from left panel). The CD11b - cells (bottom half from middle panel) were then sorted into two distinct cell populations corresponding to astrocytes (GLT1 + /CD90.2 - ) and neurons (CD90.2 + /GLT1 - ) (right panel). (D) PCR analysis of astrocytes and neurons from the cell sorting. Microglia (CD11 + ) as well as astrocytes (GLT1 + /CD90.2 - ), but not neurons (CD90.2 + /GLT1 - ) from PHP.eB Gfap-iCre infected animals show the presence of iCre , indicating that only glia and not neurons could express iCre in order to delete Per2 in the Per2 fl/fl mice. (E) Immunohistochemistry of vG Per2 brain tissue from nucleus accumbens (NAc) isolated at ZT6. The signal for PER2 (green) mainly overlaps with neuronal NeuN signal (red) giving rise to the yellow color. Scale bar: 100 µm. (F) Immobility time in the forced swim test (FST) of vG Per2 (PHP.eB Gfap-iCre , green) and control (PHP.eB control, blue) animals are shown (n = 5, two-tailed t-test, *p

    Journal: bioRxiv

    Article Title: Deletion of the Clock Gene Period2 (Per2) in Glial Cells Alters Mood-Related Behavior in Mice

    doi: 10.1101/2020.12.09.417162

    Figure Lengend Snippet: Adeno-associated virus (AAV)-mediated deletion of Per2 in glial cells of adult mice leads to a depression resistant behavior. (A) Fluorescent imaging of whole brains 3 weeks after no injection (left), intravenous (i.v.) injection of the engineered AAV-PHP.eB, which can pass the blood-brain barrier (BBB), containing the general CAG driver (second from left) or the glial Gfap driver (middle). The second to last brain is from an animal with i.v. injected AAV-9, which does not pass the BBB, containing the general CAG driver. The last brain (right) is from an animal injected intraperitoneally (i.p.) with the AAV-PHP.eB Gfap -driven construct. Note that only the brains of animals that received the AAV-PHP.eb i.v. display significant fluorescent signal after 3 weeks (orange and yellow color). (B) Fluorescent imaging of whole brains 2 months after injection of the AAV-PHP.eB. Note that the fluorescence is still maintained after 2 months post injection and that even the i.p. injected AAV-PHP.eB Gfap is showing signal in the brain now. (C) Sorting of neurons and astrocytes by flow cytometry from brain tissue including the nucleus accumbens (NAc). The left panel shows the removal of debris from a single cell suspension, showing the distribution of debris in the forward as well as in the side scatter (FSC and SSC, respectively). The middle panel shows the removal of CD11b + cells (microglia) from the cell suspension (lower left corner from left panel). The CD11b - cells (bottom half from middle panel) were then sorted into two distinct cell populations corresponding to astrocytes (GLT1 + /CD90.2 - ) and neurons (CD90.2 + /GLT1 - ) (right panel). (D) PCR analysis of astrocytes and neurons from the cell sorting. Microglia (CD11 + ) as well as astrocytes (GLT1 + /CD90.2 - ), but not neurons (CD90.2 + /GLT1 - ) from PHP.eB Gfap-iCre infected animals show the presence of iCre , indicating that only glia and not neurons could express iCre in order to delete Per2 in the Per2 fl/fl mice. (E) Immunohistochemistry of vG Per2 brain tissue from nucleus accumbens (NAc) isolated at ZT6. The signal for PER2 (green) mainly overlaps with neuronal NeuN signal (red) giving rise to the yellow color. Scale bar: 100 µm. (F) Immobility time in the forced swim test (FST) of vG Per2 (PHP.eB Gfap-iCre , green) and control (PHP.eB control, blue) animals are shown (n = 5, two-tailed t-test, *p

    Article Snippet: Antibodies targeted CD11b (fluorophore PECy7, ref. 552850, BD Pharmingen), CD90.2 (fluorophore PE, ref., 130-102-489, Miltenyi Biotech) and GLT1 (fluorophore ATTO 633, ref., AGC-022-FR, Alomone lab), and were used in a 1:100 dilution.

    Techniques: Mouse Assay, Imaging, Injection, Construct, Fluorescence, Flow Cytometry, Polymerase Chain Reaction, FACS, Infection, Immunohistochemistry, Isolation, Two Tailed Test

    GLT1 HET mice have higher ipsilateral eye responses, lower contralateral eye bias and disrupted experience-dependent binocular matching of orientation-selective responses. A) Schematic of experimental design. Top: Visual gratings were separately presented to the contra (green) and ipsi (blue) eyes in P28 mice and neuronal responses recorded. Bottom: schematic of measures. Ocular dominance index (ODI) was calculated as (max Contra − max Ipsi ) / max Contra +max Ipsi . Orientation Selectivity Index (OSI) was calculated as described previously ( Banerjee et al., 2016 ). Difference in preferred orientation (ΔPO) was calculated as the difference between preferred orientations of the max contralateral and ipsilateral responses. B) Example cells in GLT1 WT (top) and GLT1 HET (bottom) animals. Left: in vivo images of neuronal somas measured in binocular visual cortex using the calcium indicator, GCaMP6f. Right: Tuning curves of three cells (white circles in left) to contra (green) and ipsi (blue) stimulation. Note the matched tuning and contralateral bias in WT animals and the mismatched tuning curves and lack of contralateral bias in GLT1 HETs. C) Quantification of the average response to PO in GLT1 WT and HET mice. WT mice have a significantly higher contralateral response than ipsilateral response while HET mice have approximately equal contralateral and ipsilateral responses (n=4-6 animals, 23-52 cells per animal, two-way ANOVA, genotype F(1,674)=7.72, p=0.0056, interaction F(1,674)=4.243, p=0.040). D) Quantification of ocular dominance index showing that GLT1 HET mice have significantly decreased ODI (n=4-6 animals, 23-52 cells per animal, t-test, p=0.0018). E) Quantification of OSI showing that GLT1 HET mice have a significantly decreased OSI of ipsilateral responses compared to both contra and ipsi responses in GLT1 WT animals (n=4-6 animals, 23-52 cells per animal, two-way ANOVA, genotype F(1,674)=12.46, p=4.5×10 −4 ). F) Quantification of ΔPO showing an increased difference in the preferred orientations between contralateral and ipsilateral inputs to neurons in GLT1 HET animals (n=4-6 animals, 23-52 cells per animal, t-test, p=1.0×10 −4 ). *p

    Journal: bioRxiv

    Article Title: Astrocytic glutamate uptake coordinates experience-dependent, eye-specific refinement in developing visual cortex

    doi: 10.1101/2020.05.25.113613

    Figure Lengend Snippet: GLT1 HET mice have higher ipsilateral eye responses, lower contralateral eye bias and disrupted experience-dependent binocular matching of orientation-selective responses. A) Schematic of experimental design. Top: Visual gratings were separately presented to the contra (green) and ipsi (blue) eyes in P28 mice and neuronal responses recorded. Bottom: schematic of measures. Ocular dominance index (ODI) was calculated as (max Contra − max Ipsi ) / max Contra +max Ipsi . Orientation Selectivity Index (OSI) was calculated as described previously ( Banerjee et al., 2016 ). Difference in preferred orientation (ΔPO) was calculated as the difference between preferred orientations of the max contralateral and ipsilateral responses. B) Example cells in GLT1 WT (top) and GLT1 HET (bottom) animals. Left: in vivo images of neuronal somas measured in binocular visual cortex using the calcium indicator, GCaMP6f. Right: Tuning curves of three cells (white circles in left) to contra (green) and ipsi (blue) stimulation. Note the matched tuning and contralateral bias in WT animals and the mismatched tuning curves and lack of contralateral bias in GLT1 HETs. C) Quantification of the average response to PO in GLT1 WT and HET mice. WT mice have a significantly higher contralateral response than ipsilateral response while HET mice have approximately equal contralateral and ipsilateral responses (n=4-6 animals, 23-52 cells per animal, two-way ANOVA, genotype F(1,674)=7.72, p=0.0056, interaction F(1,674)=4.243, p=0.040). D) Quantification of ocular dominance index showing that GLT1 HET mice have significantly decreased ODI (n=4-6 animals, 23-52 cells per animal, t-test, p=0.0018). E) Quantification of OSI showing that GLT1 HET mice have a significantly decreased OSI of ipsilateral responses compared to both contra and ipsi responses in GLT1 WT animals (n=4-6 animals, 23-52 cells per animal, two-way ANOVA, genotype F(1,674)=12.46, p=4.5×10 −4 ). F) Quantification of ΔPO showing an increased difference in the preferred orientations between contralateral and ipsilateral inputs to neurons in GLT1 HET animals (n=4-6 animals, 23-52 cells per animal, t-test, p=1.0×10 −4 ). *p

    Article Snippet: The following primary antibodies and dilutions were used: rabbit α-GLT1 (1:500, AGC-022, Alamone Labs), guinea pig α-GFAP (1:2000, 173 004, Synaptic Systems), chicken α-GFP (1:1000, GFP-1020, Aves Labs), α-rabbit α-SST-14 (1:500, T-4103, Peninsula Labs), mouse α-PV (1:500, MAB1572, EMD Millipore).

    Techniques: Mouse Assay, In Vivo

    GLT1 is upregulated in the developing visual cortex concurrent with visual experience. A) Immunohistochemical stain of GLT1 (red), astrocytes (green), and DAPI (blue) at ~P28 in mouse visual cortex. GLT1 is expressed by astrocytes throughout cortical layers. B) High-magnification image of a single GFAP-labelled astrocyte with surrounding GLT1 expression. C) Quantification of GLT1-mRNA across developmental time points showing a significant increase from birth and peaking at P21. Levels are normalized to P28 (P0 (n=4), P7 (n=4), P14 (n=3), P21 (n=3), P28 (n=4), P42 (n=3), P60 (n=4); one-way ANOVA, F(6,18)=86.7, p=2.7×10 −12 ). D) Western blot quantification showing that transgenic mice with heterozygous expression of GLT1 (HET) have significantly less GLT1 expression compared to wildtype (WT) littermates (n=WT(11), HET(11); unpaired t-test, t=4.64, p=2.7×10 −4 ), but comparable expression of GLAST (WT (n=8), HET (n=4); unpaired t-test, t=0.62, p=0.55). E) Example images of GLT1 WT and HET astrocytes labeled using a custom GFAP-tdTomato transgenic mouse line (red). Astrocyte volume is reconstruction from imaged z-stacks (green). F) Quantification of astrocyte volume from 3D reconstructions show no difference between GLT1 WT and HET animals (WT (n=3), HET (n=13); unpaired t-test, t=0.53, p=0.60). G) Images of the lateral geniculate nucleus (LGN) after CTB-594 (red) and CTB-488 (green) injection into the contralateral and ipsilateral eyes respectively. Merged overlays from GLT1 WT and HET mice show normal retinothalamic axon segregation. H) Quantification of ipsilateral area in GLT1 WT and HETs across several binary thresholds (0, 5, 30%) showing no difference in absolute ipsilateral area (WT (n=3), HET (n=6); two-way ANOVA, genotype effect F(1,7)=1.93, p=0.21). I) Quantification of contra/ipsi projection overlap showing no difference in contra/ipsi segregation (WT (n=3), HET (n=6); two-way ANOVA, genotype effect, F(1,7)=0.43, p=0.53). *p

    Journal: bioRxiv

    Article Title: Astrocytic glutamate uptake coordinates experience-dependent, eye-specific refinement in developing visual cortex

    doi: 10.1101/2020.05.25.113613

    Figure Lengend Snippet: GLT1 is upregulated in the developing visual cortex concurrent with visual experience. A) Immunohistochemical stain of GLT1 (red), astrocytes (green), and DAPI (blue) at ~P28 in mouse visual cortex. GLT1 is expressed by astrocytes throughout cortical layers. B) High-magnification image of a single GFAP-labelled astrocyte with surrounding GLT1 expression. C) Quantification of GLT1-mRNA across developmental time points showing a significant increase from birth and peaking at P21. Levels are normalized to P28 (P0 (n=4), P7 (n=4), P14 (n=3), P21 (n=3), P28 (n=4), P42 (n=3), P60 (n=4); one-way ANOVA, F(6,18)=86.7, p=2.7×10 −12 ). D) Western blot quantification showing that transgenic mice with heterozygous expression of GLT1 (HET) have significantly less GLT1 expression compared to wildtype (WT) littermates (n=WT(11), HET(11); unpaired t-test, t=4.64, p=2.7×10 −4 ), but comparable expression of GLAST (WT (n=8), HET (n=4); unpaired t-test, t=0.62, p=0.55). E) Example images of GLT1 WT and HET astrocytes labeled using a custom GFAP-tdTomato transgenic mouse line (red). Astrocyte volume is reconstruction from imaged z-stacks (green). F) Quantification of astrocyte volume from 3D reconstructions show no difference between GLT1 WT and HET animals (WT (n=3), HET (n=13); unpaired t-test, t=0.53, p=0.60). G) Images of the lateral geniculate nucleus (LGN) after CTB-594 (red) and CTB-488 (green) injection into the contralateral and ipsilateral eyes respectively. Merged overlays from GLT1 WT and HET mice show normal retinothalamic axon segregation. H) Quantification of ipsilateral area in GLT1 WT and HETs across several binary thresholds (0, 5, 30%) showing no difference in absolute ipsilateral area (WT (n=3), HET (n=6); two-way ANOVA, genotype effect F(1,7)=1.93, p=0.21). I) Quantification of contra/ipsi projection overlap showing no difference in contra/ipsi segregation (WT (n=3), HET (n=6); two-way ANOVA, genotype effect, F(1,7)=0.43, p=0.53). *p

    Article Snippet: The following primary antibodies and dilutions were used: rabbit α-GLT1 (1:500, AGC-022, Alamone Labs), guinea pig α-GFAP (1:2000, 173 004, Synaptic Systems), chicken α-GFP (1:1000, GFP-1020, Aves Labs), α-rabbit α-SST-14 (1:500, T-4103, Peninsula Labs), mouse α-PV (1:500, MAB1572, EMD Millipore).

    Techniques: Immunohistochemistry, Staining, Expressing, Western Blot, Transgenic Assay, Mouse Assay, Labeling, CtB Assay, Injection

    GLT1 HET mice have upregulation of GLT1 expression during monocular deprivation. A) Quantification of GLT1 mRNA in WT (grays) and HET (reds) mice in ND, 4dMD, and 7dMD conditions. GLT1 HET mice have significantly less GLT1 mRNA in ND conditions, but no difference at 4dMD and 7dMD compared to WT mice (n=2-3 animals per group, two-way ANOVA, Holm-Sidak post-hoc comparisons). B) Example western blots for GLT1 protein in WT (top) and HET (bottom) mice in ND, 4dMD, and 7dMD conditions. C) Quantification of western blots showing significantly less GLT1 protein in HET mice in ND, but no difference in 4dMD and 7dMD compared to WT littermates (n=4-9 animals per group, two-way ANOVA, Holm-Sidak post-hoc comparisons). *p

    Journal: bioRxiv

    Article Title: Astrocytic glutamate uptake coordinates experience-dependent, eye-specific refinement in developing visual cortex

    doi: 10.1101/2020.05.25.113613

    Figure Lengend Snippet: GLT1 HET mice have upregulation of GLT1 expression during monocular deprivation. A) Quantification of GLT1 mRNA in WT (grays) and HET (reds) mice in ND, 4dMD, and 7dMD conditions. GLT1 HET mice have significantly less GLT1 mRNA in ND conditions, but no difference at 4dMD and 7dMD compared to WT mice (n=2-3 animals per group, two-way ANOVA, Holm-Sidak post-hoc comparisons). B) Example western blots for GLT1 protein in WT (top) and HET (bottom) mice in ND, 4dMD, and 7dMD conditions. C) Quantification of western blots showing significantly less GLT1 protein in HET mice in ND, but no difference in 4dMD and 7dMD compared to WT littermates (n=4-9 animals per group, two-way ANOVA, Holm-Sidak post-hoc comparisons). *p

    Article Snippet: The following primary antibodies and dilutions were used: rabbit α-GLT1 (1:500, AGC-022, Alamone Labs), guinea pig α-GFAP (1:2000, 173 004, Synaptic Systems), chicken α-GFP (1:1000, GFP-1020, Aves Labs), α-rabbit α-SST-14 (1:500, T-4103, Peninsula Labs), mouse α-PV (1:500, MAB1572, EMD Millipore).

    Techniques: Mouse Assay, Expressing, Western Blot

    GLT1 HET mice have abnormal maturation of excitatory and inhibitory circuits. A) Left: low-magnification image of neurons in visual cortex of GFP-M transgenic mice. Right: higher-magnification image of layer 2/3 neurons (dotted box in left). B) Images of basal dendrites of layer 2/3 neurons in GLT1 WT (top) and HET (bottom) mice. WT example is from dotted box in right panel of A. C) GLT1 HET mice have increased spine density on basal dendrites of layer 2/3 neurons in visual cortex (n=4 animals, 5 slices, 10 dendrites per animal, t-test, p=0.041). D) Example traces of miniature excitatory post-synaptic currents (mEPSCs) from voltage-clamped layer 2/3 neurons in the visual cortex of GLT1 WT and HET mice. E) Quantification of mEPSC amplitude showing no difference in magnitude of mEPSCs (n=8-13 cell, t-test, p=0.49). F) Neurons from GLT1 HET mice have a trend towards increased mEPSC frequency (n=8-13 cells, t-test, p=0.052). G) Example images of parvalbumin positive (PV+, green) and somatostatin positive (SST+, magenta) interneurons in visual cortex of GLT1 WT and HET mice). H) GLT1 HET mice have a trend towards decreased PV+ neuron density (n=4 animals, 5 slices per animal, t-test, p=0.12). I) GLT1 HET mice have a significant increase in SST+ cell density (n=4 animals, 5 slices per animal, t-test, p=0.0023). J) Example images of perineuronal nets (PNNs) visualized using wisteria floribunda agglutin (WFA) staining. K) GLT1 HET mice have significantly decreased PNN density compared to WT littermates (n=9 animals, 5 slices per animal, t-test, p=0.0068). L) Model of net decrease in cortical inhibition through increased SST+ cell density inhibiting PV+ interneurons yielding increase in excitatory pyramidal neuron responses (Pyr). *p

    Journal: bioRxiv

    Article Title: Astrocytic glutamate uptake coordinates experience-dependent, eye-specific refinement in developing visual cortex

    doi: 10.1101/2020.05.25.113613

    Figure Lengend Snippet: GLT1 HET mice have abnormal maturation of excitatory and inhibitory circuits. A) Left: low-magnification image of neurons in visual cortex of GFP-M transgenic mice. Right: higher-magnification image of layer 2/3 neurons (dotted box in left). B) Images of basal dendrites of layer 2/3 neurons in GLT1 WT (top) and HET (bottom) mice. WT example is from dotted box in right panel of A. C) GLT1 HET mice have increased spine density on basal dendrites of layer 2/3 neurons in visual cortex (n=4 animals, 5 slices, 10 dendrites per animal, t-test, p=0.041). D) Example traces of miniature excitatory post-synaptic currents (mEPSCs) from voltage-clamped layer 2/3 neurons in the visual cortex of GLT1 WT and HET mice. E) Quantification of mEPSC amplitude showing no difference in magnitude of mEPSCs (n=8-13 cell, t-test, p=0.49). F) Neurons from GLT1 HET mice have a trend towards increased mEPSC frequency (n=8-13 cells, t-test, p=0.052). G) Example images of parvalbumin positive (PV+, green) and somatostatin positive (SST+, magenta) interneurons in visual cortex of GLT1 WT and HET mice). H) GLT1 HET mice have a trend towards decreased PV+ neuron density (n=4 animals, 5 slices per animal, t-test, p=0.12). I) GLT1 HET mice have a significant increase in SST+ cell density (n=4 animals, 5 slices per animal, t-test, p=0.0023). J) Example images of perineuronal nets (PNNs) visualized using wisteria floribunda agglutin (WFA) staining. K) GLT1 HET mice have significantly decreased PNN density compared to WT littermates (n=9 animals, 5 slices per animal, t-test, p=0.0068). L) Model of net decrease in cortical inhibition through increased SST+ cell density inhibiting PV+ interneurons yielding increase in excitatory pyramidal neuron responses (Pyr). *p

    Article Snippet: The following primary antibodies and dilutions were used: rabbit α-GLT1 (1:500, AGC-022, Alamone Labs), guinea pig α-GFAP (1:2000, 173 004, Synaptic Systems), chicken α-GFP (1:1000, GFP-1020, Aves Labs), α-rabbit α-SST-14 (1:500, T-4103, Peninsula Labs), mouse α-PV (1:500, MAB1572, EMD Millipore).

    Techniques: Mouse Assay, Transgenic Assay, Staining, Inhibition

    GLT1 HET mice have disrupted ocular dominance plasticity. A) Schematic of experimental setup for intrinsic signal optical imaging. Drifting bars are presented to each eye individually and phase maps are generated by the retinotopic activity in visual cortex. Averaged responses of multiple sweeps yield an amplitude map. Ocular Dominance Index (ODI) is calculated as the contralateral response − ipsilateral response / contralateral + ipsilateral responses. B) ODI for GLT1 WT (grays) and GLT1 HET (reds) mice that were either non-deprived (ND), or had the contralateral eye monocularly deprived for 4 days (4dMD) or 7 days (7dMD). GLT1 WT mice display a typical contralateral bias in ND conditions. After 4dMD and 7dMD the ODI significantly decreases demonstrating intact ocular dominance plasticity. GLT1 HET mice display an abnormal lack of contralateral ODI bias under ND conditions, a significant decrease in ODI at 4dMD, and a return to no bias at 7dMD (n=4 animals per group, two-way ANOVA, Holm-Sidak post-hoc comparisons). C) Comparison of eye-specific amplitudes for GLT1 WT and HET mice. ND GLT1 HET mice have approximately equal responses to contralateral and ipsilateral inputs. After 4dMD, GLT1 HET mice have a significant decrease in contralateral responses and at 7dMD, significant decrease in both contralateral and ipsilateral responses (n=4 animals per group, two-way ANOVA, Holm-Sidak post-hoc comparisons). *p

    Journal: bioRxiv

    Article Title: Astrocytic glutamate uptake coordinates experience-dependent, eye-specific refinement in developing visual cortex

    doi: 10.1101/2020.05.25.113613

    Figure Lengend Snippet: GLT1 HET mice have disrupted ocular dominance plasticity. A) Schematic of experimental setup for intrinsic signal optical imaging. Drifting bars are presented to each eye individually and phase maps are generated by the retinotopic activity in visual cortex. Averaged responses of multiple sweeps yield an amplitude map. Ocular Dominance Index (ODI) is calculated as the contralateral response − ipsilateral response / contralateral + ipsilateral responses. B) ODI for GLT1 WT (grays) and GLT1 HET (reds) mice that were either non-deprived (ND), or had the contralateral eye monocularly deprived for 4 days (4dMD) or 7 days (7dMD). GLT1 WT mice display a typical contralateral bias in ND conditions. After 4dMD and 7dMD the ODI significantly decreases demonstrating intact ocular dominance plasticity. GLT1 HET mice display an abnormal lack of contralateral ODI bias under ND conditions, a significant decrease in ODI at 4dMD, and a return to no bias at 7dMD (n=4 animals per group, two-way ANOVA, Holm-Sidak post-hoc comparisons). C) Comparison of eye-specific amplitudes for GLT1 WT and HET mice. ND GLT1 HET mice have approximately equal responses to contralateral and ipsilateral inputs. After 4dMD, GLT1 HET mice have a significant decrease in contralateral responses and at 7dMD, significant decrease in both contralateral and ipsilateral responses (n=4 animals per group, two-way ANOVA, Holm-Sidak post-hoc comparisons). *p

    Article Snippet: The following primary antibodies and dilutions were used: rabbit α-GLT1 (1:500, AGC-022, Alamone Labs), guinea pig α-GFAP (1:2000, 173 004, Synaptic Systems), chicken α-GFP (1:1000, GFP-1020, Aves Labs), α-rabbit α-SST-14 (1:500, T-4103, Peninsula Labs), mouse α-PV (1:500, MAB1572, EMD Millipore).

    Techniques: Mouse Assay, Optical Imaging, Generated, Activity Assay

    Summary and model of experimental results. A) Schematic showing the contralateral (contra, green) and ipsilateral (ipsi, blue) inputs to binocular visual cortex synapsing onto layer 2/3 pyramidal cells (L2/3, Pyr, gray). Astrocytes (Ast, orange) have fine processes that surround excitatory synapses. Dashed box is magnified below, with details. B) At eye-opening, GLT1 WT and HET animals have similar astrocyte volume and LGN refinement. With visual experience, GLT1-WT animals undergo activity-dependent plasticity resulting in decreased spine density, binocular matching of preferred orientation, and a contralaterally biased ocular dominance. GLT1-HET animals have comparatively decreased GLT1 protein resulting in increased dendritic spines, increased ipsi responses, reduced contra bias and orientation tuning, and decreased binocular matching of orientation preference. Following 4 days of MD in GLT1-WT mice, contralateral responses decrease and after 7 days of MD, ipsilateral responses increase. In GLT1-HET animals, after 4 days of MD contralateral responses decrease resulting in a negative ODI. However, after 7 days of MD, increased GLT1 expression also decreases ipsilateral inputs resulting in no ocular dominance bias. These results are reasonably explained by a selective influence of GLT1 on ipsilateral inputs and responses during development.

    Journal: bioRxiv

    Article Title: Astrocytic glutamate uptake coordinates experience-dependent, eye-specific refinement in developing visual cortex

    doi: 10.1101/2020.05.25.113613

    Figure Lengend Snippet: Summary and model of experimental results. A) Schematic showing the contralateral (contra, green) and ipsilateral (ipsi, blue) inputs to binocular visual cortex synapsing onto layer 2/3 pyramidal cells (L2/3, Pyr, gray). Astrocytes (Ast, orange) have fine processes that surround excitatory synapses. Dashed box is magnified below, with details. B) At eye-opening, GLT1 WT and HET animals have similar astrocyte volume and LGN refinement. With visual experience, GLT1-WT animals undergo activity-dependent plasticity resulting in decreased spine density, binocular matching of preferred orientation, and a contralaterally biased ocular dominance. GLT1-HET animals have comparatively decreased GLT1 protein resulting in increased dendritic spines, increased ipsi responses, reduced contra bias and orientation tuning, and decreased binocular matching of orientation preference. Following 4 days of MD in GLT1-WT mice, contralateral responses decrease and after 7 days of MD, ipsilateral responses increase. In GLT1-HET animals, after 4 days of MD contralateral responses decrease resulting in a negative ODI. However, after 7 days of MD, increased GLT1 expression also decreases ipsilateral inputs resulting in no ocular dominance bias. These results are reasonably explained by a selective influence of GLT1 on ipsilateral inputs and responses during development.

    Article Snippet: The following primary antibodies and dilutions were used: rabbit α-GLT1 (1:500, AGC-022, Alamone Labs), guinea pig α-GFAP (1:2000, 173 004, Synaptic Systems), chicken α-GFP (1:1000, GFP-1020, Aves Labs), α-rabbit α-SST-14 (1:500, T-4103, Peninsula Labs), mouse α-PV (1:500, MAB1572, EMD Millipore).

    Techniques: AST Assay, Activity Assay, Mouse Assay, Expressing

    Neuroprotective properties of GT949 in mixed neuron-glia cultures after prolonged exposure to glutamate. A. Representative images of mixed neuron-glia cultures after treatments with 10 μM L-glutamate, and a compound of interest for 24 hours. Cells were fixed and immunostained for the neuronal marker MAP-2 (green) and the glial marker GFAP (red), and counterstaining with the nuclear marker DAPI (blue). Vehicle and GT949 treated cultures in the absence of L-glutamate do not display obvious degeneration of cell death. Cell death was evident in L-glutamate treated cultures, as assessed by the increased number of DAPI positive, MAP-2 and GFAP negative cells. Co-treatment with 100 nM GT949 reversed a portion of the L-glutamate toxicity by increasing MAP-2 and GFAP expression and reducing the number of DAPI-only cells. All images are shown at 40x magnification. Scale bar: 50 μm. B: Quantification of neuronal survival in mixed cultures (normalized to control, vehicle-treated cultures). Treatment with 100 nM GT949, GT996, or 100 μM TBOA (glutamate transporter inhibitor) alone did not affect neuronal survival levels. L-glutamate significantly reduced neuronal survival, which was mitigated by co-treatment with GT949 and the NMDA antagonist AP-V. The 100 nM concentration of GT949 produced a more significant neuroprotective effect than the 10 nM concentration. Co-treatment with GT949 and the glutamate transporter inhibitor TBOA and the selective EAAT2 inhibitor WAY 23 213613 did not rescue neuronal damage in L-glutamate exposed cultures, suggesting that GT949 neuroprotection requires active glutamate transporters, specifically EAAT2. Co-treatment with inactive analog GT996 did not alter glutamate toxicity. 3–4 coverslips were assessed per treatment condition, and 100–150 cells were manually counted per treatment. Neuronal survival data is representative of 3 independent experiments and control levels were not statistically different for normalization purposes. ***p

    Journal: ACS chemical neuroscience

    Article Title: Novel positive allosteric modulators of glutamate transport have neuroprotective properties in an in vitro excitotoxic model

    doi: 10.1021/acschemneuro.9b00061

    Figure Lengend Snippet: Neuroprotective properties of GT949 in mixed neuron-glia cultures after prolonged exposure to glutamate. A. Representative images of mixed neuron-glia cultures after treatments with 10 μM L-glutamate, and a compound of interest for 24 hours. Cells were fixed and immunostained for the neuronal marker MAP-2 (green) and the glial marker GFAP (red), and counterstaining with the nuclear marker DAPI (blue). Vehicle and GT949 treated cultures in the absence of L-glutamate do not display obvious degeneration of cell death. Cell death was evident in L-glutamate treated cultures, as assessed by the increased number of DAPI positive, MAP-2 and GFAP negative cells. Co-treatment with 100 nM GT949 reversed a portion of the L-glutamate toxicity by increasing MAP-2 and GFAP expression and reducing the number of DAPI-only cells. All images are shown at 40x magnification. Scale bar: 50 μm. B: Quantification of neuronal survival in mixed cultures (normalized to control, vehicle-treated cultures). Treatment with 100 nM GT949, GT996, or 100 μM TBOA (glutamate transporter inhibitor) alone did not affect neuronal survival levels. L-glutamate significantly reduced neuronal survival, which was mitigated by co-treatment with GT949 and the NMDA antagonist AP-V. The 100 nM concentration of GT949 produced a more significant neuroprotective effect than the 10 nM concentration. Co-treatment with GT949 and the glutamate transporter inhibitor TBOA and the selective EAAT2 inhibitor WAY 23 213613 did not rescue neuronal damage in L-glutamate exposed cultures, suggesting that GT949 neuroprotection requires active glutamate transporters, specifically EAAT2. Co-treatment with inactive analog GT996 did not alter glutamate toxicity. 3–4 coverslips were assessed per treatment condition, and 100–150 cells were manually counted per treatment. Neuronal survival data is representative of 3 independent experiments and control levels were not statistically different for normalization purposes. ***p

    Article Snippet: EAAT2 rabbit polyclonal (1: 1,000, cat: AGC-022) was purchased from Alomone, Jerusalem, Israel.

    Techniques: Marker, Expressing, Concentration Assay, Produced

    Aldolase C tagged with green fluorescent protein (C-GFP) expressed in forebrain astrocytes is detected in extracellular vesicles (EVs) isolated from the blood. (a) Scheme of the in utero electroporation. Forebrain astrocytes were transduced by in utero electroporation with aldolase C-GFP or GFP. Plasmids were injected into the left lateral ventricle on embryonic day 18.5. The orientation of the electrodes used to apply the voltage pulse is shown. (b) Immunohistofluorescent detection of glial fibrillary acid protein (GFAP) and GFP in coronal brain slices indicated that cells positive for both proteins were detected in the borders of the lateral ventricles (LV, indicated by arrows), and (c) in the hilus of the dentate gyrus (DG). Gr, granule cells. (d) Aldolase C (left) or GFP (right) was detected in astrocyte homogenates (AH) electroporated with aldolase C-GFP or in sEVs isolated from the serum of these animals. Note that in the sEVs, the modified form of aldolase C was detected (~ 55 kDa), as well as the recombinant protein (~ 70 kDa), which was also visible with the GFP antibody. (e) Aldolase C (left) or GFP (right) was detected in the astrocyte homogenates (AH) that were electroporated with GFP or in extracellular vesicles (sEVs) isolated from the serum of these animals. Note that in sEVs, the modified form of aldolase C was detected (~ 55 kDa), while in these animals, no GFP could be detected in the EVs. Observations were conducted in n = 5 independent animal groups (n = 4 rats per group for blood collection). (f) EVs bearing the glial glutamate transporter EAAT2 in their membrane are enriched in aldolase C. EVs were immunoisolated in nondenaturating conditions with an antibody directed against an extracellular epitope of EAAT2. The input lane was loaded with 8% of the EVs used for the precipitation procedure (i.e., 20 µg). Note that EAAT2 can only be detected in the input after overexposure of the same blot (right lanes), revealing a huge enrichment of EAAT2-bearing vesicles in the isolated material that in turn also contains higher aldolase C levels compared with the input EVs.

    Journal: International Journal of Neuropsychopharmacology

    Article Title: Small Extracellular Vesicles in Rat Serum Contain Astrocyte-Derived Protein Biomarkers of Repetitive Stress

    doi: 10.1093/ijnp/pyy098

    Figure Lengend Snippet: Aldolase C tagged with green fluorescent protein (C-GFP) expressed in forebrain astrocytes is detected in extracellular vesicles (EVs) isolated from the blood. (a) Scheme of the in utero electroporation. Forebrain astrocytes were transduced by in utero electroporation with aldolase C-GFP or GFP. Plasmids were injected into the left lateral ventricle on embryonic day 18.5. The orientation of the electrodes used to apply the voltage pulse is shown. (b) Immunohistofluorescent detection of glial fibrillary acid protein (GFAP) and GFP in coronal brain slices indicated that cells positive for both proteins were detected in the borders of the lateral ventricles (LV, indicated by arrows), and (c) in the hilus of the dentate gyrus (DG). Gr, granule cells. (d) Aldolase C (left) or GFP (right) was detected in astrocyte homogenates (AH) electroporated with aldolase C-GFP or in sEVs isolated from the serum of these animals. Note that in the sEVs, the modified form of aldolase C was detected (~ 55 kDa), as well as the recombinant protein (~ 70 kDa), which was also visible with the GFP antibody. (e) Aldolase C (left) or GFP (right) was detected in the astrocyte homogenates (AH) that were electroporated with GFP or in extracellular vesicles (sEVs) isolated from the serum of these animals. Note that in sEVs, the modified form of aldolase C was detected (~ 55 kDa), while in these animals, no GFP could be detected in the EVs. Observations were conducted in n = 5 independent animal groups (n = 4 rats per group for blood collection). (f) EVs bearing the glial glutamate transporter EAAT2 in their membrane are enriched in aldolase C. EVs were immunoisolated in nondenaturating conditions with an antibody directed against an extracellular epitope of EAAT2. The input lane was loaded with 8% of the EVs used for the precipitation procedure (i.e., 20 µg). Note that EAAT2 can only be detected in the input after overexposure of the same blot (right lanes), revealing a huge enrichment of EAAT2-bearing vesicles in the isolated material that in turn also contains higher aldolase C levels compared with the input EVs.

    Article Snippet: Primary antibodies were used at dilutions of 1:1000 against MAP2 (MAB378, Merck Millipore); TSG101 (Ab83, Abcam); Flotillin-1 (610821, BD Transduction Laboratories); CD63 (sc-15363, Santa Cruz Biotechnology); GM130 (Ab1299, Abcam); GFAP (Mab C 2032-28B, US Biological); GFP (Ab6673, Abcam); GFP (MAB3580, Merck Millipore); EAAT2 (AGC022, Alomone, Israel); SUMO 1 (Ab32058, Abcam); SUMO 2/3 (Ab109005, Abcam), and aldolase C (sc12065, Santa Cruz Biotechnology).

    Techniques: Isolation, In Utero, Electroporation, Injection, Modification, Recombinant