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rabbit polyclonal anti vesicular glutamate transporter 1 vglut1 synaptic systems 135 303 ab 887875  (Synaptic Systems)


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    Synaptic Systems rabbit polyclonal anti vesicular glutamate transporter 1 vglut1 synaptic systems 135 303 ab 887875
    Rabbit Polyclonal Anti Vesicular Glutamate Transporter 1 Vglut1 Synaptic Systems 135 303 Ab 887875, supplied by Synaptic Systems, used in various techniques. Bioz Stars score: 96/100, based on 211 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/rabbit+polyclonal+anti-vesicular+glutamate+transporter+1+(vglut1/135+303/pmc09683396__fcac286_supplementary_data-20-124-131
    Average 96 stars, based on 211 article reviews
    rabbit polyclonal anti vesicular glutamate transporter 1 vglut1 synaptic systems 135 303 ab 887875 - by Bioz Stars, 2026-10
    96/100 stars

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    Related Articles

    Staining:

    Article Title: SLC4A10 mutation causes a neurological disorder associated with impaired GABAergic transmission
    Article Snippet: For co-staining, the following primary antibodies were used: polyclonal guinea pig anti-vesicular GABA transporter (VGAT, 1:250, Synaptic Systems), polyclonal guinea pig anti-vesicular glutamate transporter 1 (VGLUT1, 1:500, Synaptic Systems).

    Article Title: Long-Term Culture of Astrocytes Attenuates the Readily Releasable Pool of Synaptic Vesicles
    Article Snippet: Autaptic hippocampal neurons were fixed in PBS containing 4% PFA for 20 min at room temperature, and then blocked and permeabilized with PBS containing 5% normal goat serum and 0.1% Triton X-100, for 30 min. After blocking, the samples were incubated overnight at 4°C with the following primary antibodies: anti-microtubule-associated protein 2 (MAP 2, guinea-pig polyclonal, antiserum, Synaptic Systems, 1∶1000 dilution), anti-vesicular glutamate transporter 1 (VGLUT 1, rabbit polyclonal, affinity purified, Synaptic Systems, 1∶2000 dilution), anti-bassoon (guinea-pig polyclonal, Synaptic Systems, 1∶2000 dilution) or anti-glial fibrillary acidic protein (GFAP, rabbit polyclonal, Synaptic Systems, 1∶1000 dilution) in a humidity chamber.

    Article Title: SLC4A10 mutation causes a neurological disorder associated with impaired GABAergic transmission.
    Article Snippet: For immunofluorescence, brains of 2 to 3-month-old wild-type mice were prepared and fixed as described previously.8 Free-floating cryosections (50 μm) were stained with a polyclonal rabbit anti-NeuN antibody (1:1000, Abcam, ab104225) or polyclonal rabbit anti-SLC4A10 antibody.22 For co-staining, the following primary antibodies were used: polyclonal guinea pig anti-vesicular GABA transporter (VGAT, 1:250, Synaptic Systems), polyclonal guinea pig anti-vesicular glutamate transporter 1 (VGLUT1, 1:500, Synaptic Systems).

    Article Title: Dendritic degeneration and altered synaptic innervation of a central auditory neuron during age-related hearing loss
    Article Snippet: Slices were then immune-stained as previously described ( ; ), using primary antibodies against vesicular glutamate transporter 1 (VGluT1) (polyclonal Guinea pig anti-vGluT1; Cat#: 135304, Synaptic Systems, 1:500) and calretinin (rabbit ant-CR; Cat# 214102, Synaptic Systems, 1:500).

    Article Title: Vesicular Glutamate Transporter 3 Is Involved in Glutamatergic Signalling in Podocytes.
    Article Snippet: Rabbit polyclonal anti-synaptobrevin 2 (anti-VAMP2) antibodies (cat# 104202), rabbit polyclonal anti-synaptophysin 1 antibodies (cat# 101002), and rabbit polyclonal anti-vesicular glutamate transporter 1 (anti-VGLUT1) antibodies (cat#135303) were purchased from Synaptic Systems GmbH (Göttingen, Germany).

    Article Title: The deficiency of KIF2C leads to abnormal distribution of cortical neurons and behavioral deficits in mice
    Article Snippet: Rabbit polyclonal Anti-VGLUT 1 , Synaptic Systems , Cat# 135 303C5; RRID: AB_2744598.

    Marker:

    Article Title: SLC4A10 mutation causes a neurological disorder associated with impaired GABAergic transmission
    Article Snippet: For co-staining, the following primary antibodies were used: polyclonal guinea pig anti-vesicular GABA transporter (VGAT, 1:250, Synaptic Systems), polyclonal guinea pig anti-vesicular glutamate transporter 1 (VGLUT1, 1:500, Synaptic Systems).

    Article Title: Long-Term Culture of Astrocytes Attenuates the Readily Releasable Pool of Synaptic Vesicles
    Article Snippet: Autaptic hippocampal neurons were fixed in PBS containing 4% PFA for 20 min at room temperature, and then blocked and permeabilized with PBS containing 5% normal goat serum and 0.1% Triton X-100, for 30 min. After blocking, the samples were incubated overnight at 4°C with the following primary antibodies: anti-microtubule-associated protein 2 (MAP 2, guinea-pig polyclonal, antiserum, Synaptic Systems, 1∶1000 dilution), anti-vesicular glutamate transporter 1 (VGLUT 1, rabbit polyclonal, affinity purified, Synaptic Systems, 1∶2000 dilution), anti-bassoon (guinea-pig polyclonal, Synaptic Systems, 1∶2000 dilution) or anti-glial fibrillary acidic protein (GFAP, rabbit polyclonal, Synaptic Systems, 1∶1000 dilution) in a humidity chamber.

    Article Title: SLC4A10 mutation causes a neurological disorder associated with impaired GABAergic transmission.
    Article Snippet: For immunofluorescence, brains of 2 to 3-month-old wild-type mice were prepared and fixed as described previously.8 Free-floating cryosections (50 μm) were stained with a polyclonal rabbit anti-NeuN antibody (1:1000, Abcam, ab104225) or polyclonal rabbit anti-SLC4A10 antibody.22 For co-staining, the following primary antibodies were used: polyclonal guinea pig anti-vesicular GABA transporter (VGAT, 1:250, Synaptic Systems), polyclonal guinea pig anti-vesicular glutamate transporter 1 (VGLUT1, 1:500, Synaptic Systems).

    Article Title: Dendritic degeneration and altered synaptic innervation of a central auditory neuron during age-related hearing loss
    Article Snippet: Slices were then immune-stained as previously described ( ; ), using primary antibodies against vesicular glutamate transporter 1 (VGluT1) (polyclonal Guinea pig anti-vGluT1; Cat#: 135304, Synaptic Systems, 1:500) and calretinin (rabbit ant-CR; Cat# 214102, Synaptic Systems, 1:500).

    Article Title: Vesicular Glutamate Transporter 3 Is Involved in Glutamatergic Signalling in Podocytes.
    Article Snippet: Rabbit polyclonal anti-synaptobrevin 2 (anti-VAMP2) antibodies (cat# 104202), rabbit polyclonal anti-synaptophysin 1 antibodies (cat# 101002), and rabbit polyclonal anti-vesicular glutamate transporter 1 (anti-VGLUT1) antibodies (cat#135303) were purchased from Synaptic Systems GmbH (Göttingen, Germany).

    Article Title: The deficiency of KIF2C leads to abnormal distribution of cortical neurons and behavioral deficits in mice
    Article Snippet: Rabbit polyclonal Anti-VGLUT 1 , Synaptic Systems , Cat# 135 303C5; RRID: AB_2744598.

    Labeling:

    Article Title: SLC4A10 mutation causes a neurological disorder associated with impaired GABAergic transmission
    Article Snippet: For co-staining, the following primary antibodies were used: polyclonal guinea pig anti-vesicular GABA transporter (VGAT, 1:250, Synaptic Systems), polyclonal guinea pig anti-vesicular glutamate transporter 1 (VGLUT1, 1:500, Synaptic Systems).

    Article Title: Long-Term Culture of Astrocytes Attenuates the Readily Releasable Pool of Synaptic Vesicles
    Article Snippet: Autaptic hippocampal neurons were fixed in PBS containing 4% PFA for 20 min at room temperature, and then blocked and permeabilized with PBS containing 5% normal goat serum and 0.1% Triton X-100, for 30 min. After blocking, the samples were incubated overnight at 4°C with the following primary antibodies: anti-microtubule-associated protein 2 (MAP 2, guinea-pig polyclonal, antiserum, Synaptic Systems, 1∶1000 dilution), anti-vesicular glutamate transporter 1 (VGLUT 1, rabbit polyclonal, affinity purified, Synaptic Systems, 1∶2000 dilution), anti-bassoon (guinea-pig polyclonal, Synaptic Systems, 1∶2000 dilution) or anti-glial fibrillary acidic protein (GFAP, rabbit polyclonal, Synaptic Systems, 1∶1000 dilution) in a humidity chamber.

    Article Title: SLC4A10 mutation causes a neurological disorder associated with impaired GABAergic transmission.
    Article Snippet: For immunofluorescence, brains of 2 to 3-month-old wild-type mice were prepared and fixed as described previously.8 Free-floating cryosections (50 μm) were stained with a polyclonal rabbit anti-NeuN antibody (1:1000, Abcam, ab104225) or polyclonal rabbit anti-SLC4A10 antibody.22 For co-staining, the following primary antibodies were used: polyclonal guinea pig anti-vesicular GABA transporter (VGAT, 1:250, Synaptic Systems), polyclonal guinea pig anti-vesicular glutamate transporter 1 (VGLUT1, 1:500, Synaptic Systems).

    Article Title: Dendritic degeneration and altered synaptic innervation of a central auditory neuron during age-related hearing loss
    Article Snippet: Slices were then immune-stained as previously described ( ; ), using primary antibodies against vesicular glutamate transporter 1 (VGluT1) (polyclonal Guinea pig anti-vGluT1; Cat#: 135304, Synaptic Systems, 1:500) and calretinin (rabbit ant-CR; Cat# 214102, Synaptic Systems, 1:500).

    Article Title: Vesicular Glutamate Transporter 3 Is Involved in Glutamatergic Signalling in Podocytes.
    Article Snippet: Rabbit polyclonal anti-synaptobrevin 2 (anti-VAMP2) antibodies (cat# 104202), rabbit polyclonal anti-synaptophysin 1 antibodies (cat# 101002), and rabbit polyclonal anti-vesicular glutamate transporter 1 (anti-VGLUT1) antibodies (cat#135303) were purchased from Synaptic Systems GmbH (Göttingen, Germany).

    Article Title: The deficiency of KIF2C leads to abnormal distribution of cortical neurons and behavioral deficits in mice
    Article Snippet: Rabbit polyclonal Anti-VGLUT 1 , Synaptic Systems , Cat# 135 303C5; RRID: AB_2744598.

    Expressing:

    Article Title: SLC4A10 mutation causes a neurological disorder associated with impaired GABAergic transmission
    Article Snippet: For co-staining, the following primary antibodies were used: polyclonal guinea pig anti-vesicular GABA transporter (VGAT, 1:250, Synaptic Systems), polyclonal guinea pig anti-vesicular glutamate transporter 1 (VGLUT1, 1:500, Synaptic Systems).

    Article Title: Long-Term Culture of Astrocytes Attenuates the Readily Releasable Pool of Synaptic Vesicles
    Article Snippet: Autaptic hippocampal neurons were fixed in PBS containing 4% PFA for 20 min at room temperature, and then blocked and permeabilized with PBS containing 5% normal goat serum and 0.1% Triton X-100, for 30 min. After blocking, the samples were incubated overnight at 4°C with the following primary antibodies: anti-microtubule-associated protein 2 (MAP 2, guinea-pig polyclonal, antiserum, Synaptic Systems, 1∶1000 dilution), anti-vesicular glutamate transporter 1 (VGLUT 1, rabbit polyclonal, affinity purified, Synaptic Systems, 1∶2000 dilution), anti-bassoon (guinea-pig polyclonal, Synaptic Systems, 1∶2000 dilution) or anti-glial fibrillary acidic protein (GFAP, rabbit polyclonal, Synaptic Systems, 1∶1000 dilution) in a humidity chamber.

    Article Title: SLC4A10 mutation causes a neurological disorder associated with impaired GABAergic transmission.
    Article Snippet: For immunofluorescence, brains of 2 to 3-month-old wild-type mice were prepared and fixed as described previously.8 Free-floating cryosections (50 μm) were stained with a polyclonal rabbit anti-NeuN antibody (1:1000, Abcam, ab104225) or polyclonal rabbit anti-SLC4A10 antibody.22 For co-staining, the following primary antibodies were used: polyclonal guinea pig anti-vesicular GABA transporter (VGAT, 1:250, Synaptic Systems), polyclonal guinea pig anti-vesicular glutamate transporter 1 (VGLUT1, 1:500, Synaptic Systems).

    Article Title: Dendritic degeneration and altered synaptic innervation of a central auditory neuron during age-related hearing loss
    Article Snippet: Slices were then immune-stained as previously described ( ; ), using primary antibodies against vesicular glutamate transporter 1 (VGluT1) (polyclonal Guinea pig anti-vGluT1; Cat#: 135304, Synaptic Systems, 1:500) and calretinin (rabbit ant-CR; Cat# 214102, Synaptic Systems, 1:500).

    Article Title: Vesicular Glutamate Transporter 3 Is Involved in Glutamatergic Signalling in Podocytes.
    Article Snippet: Rabbit polyclonal anti-synaptobrevin 2 (anti-VAMP2) antibodies (cat# 104202), rabbit polyclonal anti-synaptophysin 1 antibodies (cat# 101002), and rabbit polyclonal anti-vesicular glutamate transporter 1 (anti-VGLUT1) antibodies (cat#135303) were purchased from Synaptic Systems GmbH (Göttingen, Germany).

    Article Title: The deficiency of KIF2C leads to abnormal distribution of cortical neurons and behavioral deficits in mice
    Article Snippet: Rabbit polyclonal Anti-VGLUT 1 , Synaptic Systems , Cat# 135 303C5; RRID: AB_2744598.

    Immunofluorescence:

    Article Title: SLC4A10 mutation causes a neurological disorder associated with impaired GABAergic transmission
    Article Snippet: For co-staining, the following primary antibodies were used: polyclonal guinea pig anti-vesicular GABA transporter (VGAT, 1:250, Synaptic Systems), polyclonal guinea pig anti-vesicular glutamate transporter 1 (VGLUT1, 1:500, Synaptic Systems).

    Article Title: Long-Term Culture of Astrocytes Attenuates the Readily Releasable Pool of Synaptic Vesicles
    Article Snippet: Autaptic hippocampal neurons were fixed in PBS containing 4% PFA for 20 min at room temperature, and then blocked and permeabilized with PBS containing 5% normal goat serum and 0.1% Triton X-100, for 30 min. After blocking, the samples were incubated overnight at 4°C with the following primary antibodies: anti-microtubule-associated protein 2 (MAP 2, guinea-pig polyclonal, antiserum, Synaptic Systems, 1∶1000 dilution), anti-vesicular glutamate transporter 1 (VGLUT 1, rabbit polyclonal, affinity purified, Synaptic Systems, 1∶2000 dilution), anti-bassoon (guinea-pig polyclonal, Synaptic Systems, 1∶2000 dilution) or anti-glial fibrillary acidic protein (GFAP, rabbit polyclonal, Synaptic Systems, 1∶1000 dilution) in a humidity chamber.

    Article Title: SLC4A10 mutation causes a neurological disorder associated with impaired GABAergic transmission.
    Article Snippet: For immunofluorescence, brains of 2 to 3-month-old wild-type mice were prepared and fixed as described previously.8 Free-floating cryosections (50 μm) were stained with a polyclonal rabbit anti-NeuN antibody (1:1000, Abcam, ab104225) or polyclonal rabbit anti-SLC4A10 antibody.22 For co-staining, the following primary antibodies were used: polyclonal guinea pig anti-vesicular GABA transporter (VGAT, 1:250, Synaptic Systems), polyclonal guinea pig anti-vesicular glutamate transporter 1 (VGLUT1, 1:500, Synaptic Systems).

    Article Title: Dendritic degeneration and altered synaptic innervation of a central auditory neuron during age-related hearing loss
    Article Snippet: Slices were then immune-stained as previously described ( ; ), using primary antibodies against vesicular glutamate transporter 1 (VGluT1) (polyclonal Guinea pig anti-vGluT1; Cat#: 135304, Synaptic Systems, 1:500) and calretinin (rabbit ant-CR; Cat# 214102, Synaptic Systems, 1:500).

    Article Title: Vesicular Glutamate Transporter 3 Is Involved in Glutamatergic Signalling in Podocytes.
    Article Snippet: Rabbit polyclonal anti-synaptobrevin 2 (anti-VAMP2) antibodies (cat# 104202), rabbit polyclonal anti-synaptophysin 1 antibodies (cat# 101002), and rabbit polyclonal anti-vesicular glutamate transporter 1 (anti-VGLUT1) antibodies (cat#135303) were purchased from Synaptic Systems GmbH (Göttingen, Germany).

    Article Title: The deficiency of KIF2C leads to abnormal distribution of cortical neurons and behavioral deficits in mice
    Article Snippet: Rabbit polyclonal Anti-VGLUT 1 , Synaptic Systems , Cat# 135 303C5; RRID: AB_2744598.

    Blocking Assay:

    Article Title: SLC4A10 mutation causes a neurological disorder associated with impaired GABAergic transmission
    Article Snippet: For co-staining, the following primary antibodies were used: polyclonal guinea pig anti-vesicular GABA transporter (VGAT, 1:250, Synaptic Systems), polyclonal guinea pig anti-vesicular glutamate transporter 1 (VGLUT1, 1:500, Synaptic Systems).

    Article Title: Long-Term Culture of Astrocytes Attenuates the Readily Releasable Pool of Synaptic Vesicles
    Article Snippet: Autaptic hippocampal neurons were fixed in PBS containing 4% PFA for 20 min at room temperature, and then blocked and permeabilized with PBS containing 5% normal goat serum and 0.1% Triton X-100, for 30 min. After blocking, the samples were incubated overnight at 4°C with the following primary antibodies: anti-microtubule-associated protein 2 (MAP 2, guinea-pig polyclonal, antiserum, Synaptic Systems, 1∶1000 dilution), anti-vesicular glutamate transporter 1 (VGLUT 1, rabbit polyclonal, affinity purified, Synaptic Systems, 1∶2000 dilution), anti-bassoon (guinea-pig polyclonal, Synaptic Systems, 1∶2000 dilution) or anti-glial fibrillary acidic protein (GFAP, rabbit polyclonal, Synaptic Systems, 1∶1000 dilution) in a humidity chamber.

    Article Title: SLC4A10 mutation causes a neurological disorder associated with impaired GABAergic transmission.
    Article Snippet: For immunofluorescence, brains of 2 to 3-month-old wild-type mice were prepared and fixed as described previously.8 Free-floating cryosections (50 μm) were stained with a polyclonal rabbit anti-NeuN antibody (1:1000, Abcam, ab104225) or polyclonal rabbit anti-SLC4A10 antibody.22 For co-staining, the following primary antibodies were used: polyclonal guinea pig anti-vesicular GABA transporter (VGAT, 1:250, Synaptic Systems), polyclonal guinea pig anti-vesicular glutamate transporter 1 (VGLUT1, 1:500, Synaptic Systems).

    Article Title: Dendritic degeneration and altered synaptic innervation of a central auditory neuron during age-related hearing loss
    Article Snippet: Slices were then immune-stained as previously described ( ; ), using primary antibodies against vesicular glutamate transporter 1 (VGluT1) (polyclonal Guinea pig anti-vGluT1; Cat#: 135304, Synaptic Systems, 1:500) and calretinin (rabbit ant-CR; Cat# 214102, Synaptic Systems, 1:500).

    Article Title: Vesicular Glutamate Transporter 3 Is Involved in Glutamatergic Signalling in Podocytes.
    Article Snippet: Rabbit polyclonal anti-synaptobrevin 2 (anti-VAMP2) antibodies (cat# 104202), rabbit polyclonal anti-synaptophysin 1 antibodies (cat# 101002), and rabbit polyclonal anti-vesicular glutamate transporter 1 (anti-VGLUT1) antibodies (cat#135303) were purchased from Synaptic Systems GmbH (Göttingen, Germany).

    Article Title: The deficiency of KIF2C leads to abnormal distribution of cortical neurons and behavioral deficits in mice
    Article Snippet: Rabbit polyclonal Anti-VGLUT 1 , Synaptic Systems , Cat# 135 303C5; RRID: AB_2744598.

    Incubation:

    Article Title: SLC4A10 mutation causes a neurological disorder associated with impaired GABAergic transmission
    Article Snippet: For co-staining, the following primary antibodies were used: polyclonal guinea pig anti-vesicular GABA transporter (VGAT, 1:250, Synaptic Systems), polyclonal guinea pig anti-vesicular glutamate transporter 1 (VGLUT1, 1:500, Synaptic Systems).

    Article Title: Long-Term Culture of Astrocytes Attenuates the Readily Releasable Pool of Synaptic Vesicles
    Article Snippet: Autaptic hippocampal neurons were fixed in PBS containing 4% PFA for 20 min at room temperature, and then blocked and permeabilized with PBS containing 5% normal goat serum and 0.1% Triton X-100, for 30 min. After blocking, the samples were incubated overnight at 4°C with the following primary antibodies: anti-microtubule-associated protein 2 (MAP 2, guinea-pig polyclonal, antiserum, Synaptic Systems, 1∶1000 dilution), anti-vesicular glutamate transporter 1 (VGLUT 1, rabbit polyclonal, affinity purified, Synaptic Systems, 1∶2000 dilution), anti-bassoon (guinea-pig polyclonal, Synaptic Systems, 1∶2000 dilution) or anti-glial fibrillary acidic protein (GFAP, rabbit polyclonal, Synaptic Systems, 1∶1000 dilution) in a humidity chamber.

    Article Title: SLC4A10 mutation causes a neurological disorder associated with impaired GABAergic transmission.
    Article Snippet: For immunofluorescence, brains of 2 to 3-month-old wild-type mice were prepared and fixed as described previously.8 Free-floating cryosections (50 μm) were stained with a polyclonal rabbit anti-NeuN antibody (1:1000, Abcam, ab104225) or polyclonal rabbit anti-SLC4A10 antibody.22 For co-staining, the following primary antibodies were used: polyclonal guinea pig anti-vesicular GABA transporter (VGAT, 1:250, Synaptic Systems), polyclonal guinea pig anti-vesicular glutamate transporter 1 (VGLUT1, 1:500, Synaptic Systems).

    Article Title: Dendritic degeneration and altered synaptic innervation of a central auditory neuron during age-related hearing loss
    Article Snippet: Slices were then immune-stained as previously described ( ; ), using primary antibodies against vesicular glutamate transporter 1 (VGluT1) (polyclonal Guinea pig anti-vGluT1; Cat#: 135304, Synaptic Systems, 1:500) and calretinin (rabbit ant-CR; Cat# 214102, Synaptic Systems, 1:500).

    Article Title: Vesicular Glutamate Transporter 3 Is Involved in Glutamatergic Signalling in Podocytes.
    Article Snippet: Rabbit polyclonal anti-synaptobrevin 2 (anti-VAMP2) antibodies (cat# 104202), rabbit polyclonal anti-synaptophysin 1 antibodies (cat# 101002), and rabbit polyclonal anti-vesicular glutamate transporter 1 (anti-VGLUT1) antibodies (cat#135303) were purchased from Synaptic Systems GmbH (Göttingen, Germany).

    Article Title: The deficiency of KIF2C leads to abnormal distribution of cortical neurons and behavioral deficits in mice
    Article Snippet: Rabbit polyclonal Anti-VGLUT 1 , Synaptic Systems , Cat# 135 303C5; RRID: AB_2744598.

    Affinity Purification:

    Article Title: SLC4A10 mutation causes a neurological disorder associated with impaired GABAergic transmission
    Article Snippet: For co-staining, the following primary antibodies were used: polyclonal guinea pig anti-vesicular GABA transporter (VGAT, 1:250, Synaptic Systems), polyclonal guinea pig anti-vesicular glutamate transporter 1 (VGLUT1, 1:500, Synaptic Systems).

    Article Title: Long-Term Culture of Astrocytes Attenuates the Readily Releasable Pool of Synaptic Vesicles
    Article Snippet: Autaptic hippocampal neurons were fixed in PBS containing 4% PFA for 20 min at room temperature, and then blocked and permeabilized with PBS containing 5% normal goat serum and 0.1% Triton X-100, for 30 min. After blocking, the samples were incubated overnight at 4°C with the following primary antibodies: anti-microtubule-associated protein 2 (MAP 2, guinea-pig polyclonal, antiserum, Synaptic Systems, 1∶1000 dilution), anti-vesicular glutamate transporter 1 (VGLUT 1, rabbit polyclonal, affinity purified, Synaptic Systems, 1∶2000 dilution), anti-bassoon (guinea-pig polyclonal, Synaptic Systems, 1∶2000 dilution) or anti-glial fibrillary acidic protein (GFAP, rabbit polyclonal, Synaptic Systems, 1∶1000 dilution) in a humidity chamber.

    Article Title: SLC4A10 mutation causes a neurological disorder associated with impaired GABAergic transmission.
    Article Snippet: For immunofluorescence, brains of 2 to 3-month-old wild-type mice were prepared and fixed as described previously.8 Free-floating cryosections (50 μm) were stained with a polyclonal rabbit anti-NeuN antibody (1:1000, Abcam, ab104225) or polyclonal rabbit anti-SLC4A10 antibody.22 For co-staining, the following primary antibodies were used: polyclonal guinea pig anti-vesicular GABA transporter (VGAT, 1:250, Synaptic Systems), polyclonal guinea pig anti-vesicular glutamate transporter 1 (VGLUT1, 1:500, Synaptic Systems).

    Article Title: Dendritic degeneration and altered synaptic innervation of a central auditory neuron during age-related hearing loss
    Article Snippet: Slices were then immune-stained as previously described ( ; ), using primary antibodies against vesicular glutamate transporter 1 (VGluT1) (polyclonal Guinea pig anti-vGluT1; Cat#: 135304, Synaptic Systems, 1:500) and calretinin (rabbit ant-CR; Cat# 214102, Synaptic Systems, 1:500).

    Article Title: Vesicular Glutamate Transporter 3 Is Involved in Glutamatergic Signalling in Podocytes.
    Article Snippet: Rabbit polyclonal anti-synaptobrevin 2 (anti-VAMP2) antibodies (cat# 104202), rabbit polyclonal anti-synaptophysin 1 antibodies (cat# 101002), and rabbit polyclonal anti-vesicular glutamate transporter 1 (anti-VGLUT1) antibodies (cat#135303) were purchased from Synaptic Systems GmbH (Göttingen, Germany).

    Article Title: The deficiency of KIF2C leads to abnormal distribution of cortical neurons and behavioral deficits in mice
    Article Snippet: Rabbit polyclonal Anti-VGLUT 1 , Synaptic Systems , Cat# 135 303C5; RRID: AB_2744598.



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    Rabbit Polyclonal Anti Vesicular Glutamate Transporter 1 Vglut1 Synaptic Systems 135 303 Ab 887875, supplied by Synaptic Systems, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BDNF is enriched in glutamatergic corticostriatal presynaptic terminals. A , Confocal (top) and SIM (bottom) microscopic images showing BDNF-IR in the same section in glutamatergic (left) versus dopaminergic terminals (right) in the dorsal striatum. B , BDNF-IR is present in <t>VGluT1-positive</t> terminals (magenta arrows). Single BDNF-IR signals overlap with TH (white arrows). VGluT1- and TH-positive terminals reside in direct regional proximity but do not overlap. C , Quantification of BDNF signals in VGluT1-positive terminals and TH-positive terminals. True colocalization between BDNF/VGluT1 was confirmed by Costes p value (Costes p > 0.95) but not between BDNF/TH (Costes p ≪ 0.95). D , Representative Western blots showing recombinant BDNF (lanes 1, 2) versus endogenous BDNF derived from anterior cortex or striatum of P21 NFL-Cre BDNF fl/ko mice (lane 3), P21 sedentary mice (lanes 4, 5), and P21 runners after 72 h voluntary running-wheel exercise (lanes 6, 7); 30 µg of protein lysate was loaded for each sample. BDNF levels were normalized to cytochrome C. Band intensities were determined from extracts of 9 independent mice and presented in % of P21 sedentary mice. Statistical analysis reveals significant increase in BDNF protein levels in both brain areas after running-wheel exercise. Statistical analysis: unpaired t test (anterior CTX: t = 5,312, p < 0.0001; striatum: t = 2,784, p = 0.0133). E , SIM images showing BDNF-IR in VGluT1-positive terminals in the dorsal striatum in sedentary mice (top row) and after 72 h of voluntary running-wheel exercise (bottom row). Data are presented as box and whiskers (Tukey). +, Mean. Vertical line indicates median. Black dots indicate outliers. n , number indicated below. Raw data are provided in Extended Data and . Scale bars: A , 2.5 µm; B , 1.5 µm; E , Overview, 2 µm; Detail, 1 µm. * p < 0.05; **** p < 0.0001.
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    Synaptic Systems rabbit polyclonal anti-vesicular glutamate transporter 1 (vglut1
    PTPσ and PTPδ are selectively required for the heterologous synapse-formation activities of distinct postsynaptogenic adhesion molecules. A, Representative images of the heterologous synapse-formation activities of various LAR-RPTP ligands. Neurons were infected at DIV4 with lentiviruses expressing sh-Control, sh-PTPσ, sh-PTPδ, sh-LAR, sh-LAR/sh-PTPσ, or sh-PTPσ/PTPδ/LAR as indicated, and then cocultured from DIV9 to DIV11 with HEK293T cells expressing various postsynaptic ligands. Neurons were stained with antibodies against HA or EGFP (blue) and <t>VGLUT1</t> (red). Scale bar (all images), 10 μm. B, The synapse-forming activity in A was quantified by measuring the ratio of VGLUT1 staining intensity (red) to HA or EGFP immunoreactivity intensity (blue). Data are mean ± SEM. Mann–Whitney U test: ****p < 0.0001. n = number of neurons as follows: sh-Control/Control, n = 13; sh-PTPσ/Control, n = 15; sh-PTPδ/Control, n = 17; sh-LAR/Control, n = 15; sh-LAR/sh-PTPσ/Control, n = 11; sh-LAR/PTPσ/PTPδ/Control, n = 12; sh-Control/NGL-3, n = 18; sh-PTPσ/NGL-3, n = 17; sh-PTPδ/NGL-3, n = 16; sh-LAR/NGL-3, n = 16; sh-LAR/sh-PTPσ/NGL-3, n = 16; sh-LAR/PTPσ/PTPδ/NGL-3, n = 16; sh-Control/Slitrk1, n = 13; sh-PTPσ/Slitrk1, n = 15; sh-PTPδ/Slitrk1, n = 13; sh-LAR/Slitrk1, n = 11; sh-Control/TrkC, n = 15; sh-PTPσ/TrkC, n = 11; sh-PTPδ/TrkC, n = 12; sh-LAR/ TrkC, n = 11; sh-Control/IL1RAPL1, n = 11; sh-PTPσ/IL1RAPL1, n = 13; sh-PTPδ/IL1RAPL1, n = 14; sh-LAR/IL1RAPL1, n = 12; sh-Control/NL-1, n = 11; sh-PTPσ/NL-1, n = 12; sh-PTPδ/NL-1, n = 10; and sh-LAR/NL-1, n = 11. p values for Control condition: sh-Control vs sh-PTPσ, p = 0.439; sh-Control vs sh-PTPδ, p = 0.5604; sh-Control vs sh-LAR, p = 0.1835; sh-Control vs sh-LAR/sh-PTPσ, p = 0.9534; and sh-Control vs sh-LAR/PTPσ/PTPδ, p = 0.5641. p values for NGL-3 condition: sh-Control vs sh-PTPσ, p = 0.4129; sh-Control vs sh-PTPδ, p = 0.7706; sh-Control vs sh-LAR, p = 0.5832; sh-Control vs sh-LAR/sh-PTPσ, p = 0.8166; and sh-Control vs sh-LAR/PTPσ/PTPδ, p = 0.9921. p values for Slitrk1 condition: sh-Control vs sh-PTPσ, p < 0.0001; sh-Control vs sh-PTPδ, p = 0.3394; and sh-Control vs sh-LAR, p = 0.684. p values for TrkC condition: sh-Control vs sh-PTPσ, p < 0.0001; sh-Control vs sh-PTPδ, p = 0.1433; and sh-Control vs sh-LAR, p = 0.2958. p values for IL1RAPL1 condition: sh-Control vs sh-PTPσ, p = 0.7832; sh-Control vs sh-PTPδ, p < 0.01; and sh-Control vs sh-LAR, p = 0.5212. p values for NL1 condition: sh-Control vs sh-PTPσ, p = 0.9537; sh-Control vs sh-PTPδ, p = 0.8919; and sh-Control vs sh-LAR, p = 0.6262. C, Levels of PTPσ, PTPδ, and LAR mRNAs were measured at DIV12-DV13 by qRT-PCR in cultured cortical neurons infected at DIV4 with lentiviruses expressing the indicated shRNAs. Dashed line indicates 70% knockdown cutoff level for tests of biological effects.
    Rabbit Polyclonal Anti Vesicular Glutamate Transporter 1 (Vglut1, supplied by Synaptic Systems, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Synaptic Systems rabbit polyclonal anti-vesicular glutamate transporter 1 (vglut1)
    PTPσ and PTPδ are selectively required for the heterologous synapse-formation activities of distinct postsynaptogenic adhesion molecules. A, Representative images of the heterologous synapse-formation activities of various LAR-RPTP ligands. Neurons were infected at DIV4 with lentiviruses expressing sh-Control, sh-PTPσ, sh-PTPδ, sh-LAR, sh-LAR/sh-PTPσ, or sh-PTPσ/PTPδ/LAR as indicated, and then cocultured from DIV9 to DIV11 with HEK293T cells expressing various postsynaptic ligands. Neurons were stained with antibodies against HA or EGFP (blue) and <t>VGLUT1</t> (red). Scale bar (all images), 10 μm. B, The synapse-forming activity in A was quantified by measuring the ratio of VGLUT1 staining intensity (red) to HA or EGFP immunoreactivity intensity (blue). Data are mean ± SEM. Mann–Whitney U test: ****p < 0.0001. n = number of neurons as follows: sh-Control/Control, n = 13; sh-PTPσ/Control, n = 15; sh-PTPδ/Control, n = 17; sh-LAR/Control, n = 15; sh-LAR/sh-PTPσ/Control, n = 11; sh-LAR/PTPσ/PTPδ/Control, n = 12; sh-Control/NGL-3, n = 18; sh-PTPσ/NGL-3, n = 17; sh-PTPδ/NGL-3, n = 16; sh-LAR/NGL-3, n = 16; sh-LAR/sh-PTPσ/NGL-3, n = 16; sh-LAR/PTPσ/PTPδ/NGL-3, n = 16; sh-Control/Slitrk1, n = 13; sh-PTPσ/Slitrk1, n = 15; sh-PTPδ/Slitrk1, n = 13; sh-LAR/Slitrk1, n = 11; sh-Control/TrkC, n = 15; sh-PTPσ/TrkC, n = 11; sh-PTPδ/TrkC, n = 12; sh-LAR/ TrkC, n = 11; sh-Control/IL1RAPL1, n = 11; sh-PTPσ/IL1RAPL1, n = 13; sh-PTPδ/IL1RAPL1, n = 14; sh-LAR/IL1RAPL1, n = 12; sh-Control/NL-1, n = 11; sh-PTPσ/NL-1, n = 12; sh-PTPδ/NL-1, n = 10; and sh-LAR/NL-1, n = 11. p values for Control condition: sh-Control vs sh-PTPσ, p = 0.439; sh-Control vs sh-PTPδ, p = 0.5604; sh-Control vs sh-LAR, p = 0.1835; sh-Control vs sh-LAR/sh-PTPσ, p = 0.9534; and sh-Control vs sh-LAR/PTPσ/PTPδ, p = 0.5641. p values for NGL-3 condition: sh-Control vs sh-PTPσ, p = 0.4129; sh-Control vs sh-PTPδ, p = 0.7706; sh-Control vs sh-LAR, p = 0.5832; sh-Control vs sh-LAR/sh-PTPσ, p = 0.8166; and sh-Control vs sh-LAR/PTPσ/PTPδ, p = 0.9921. p values for Slitrk1 condition: sh-Control vs sh-PTPσ, p < 0.0001; sh-Control vs sh-PTPδ, p = 0.3394; and sh-Control vs sh-LAR, p = 0.684. p values for TrkC condition: sh-Control vs sh-PTPσ, p < 0.0001; sh-Control vs sh-PTPδ, p = 0.1433; and sh-Control vs sh-LAR, p = 0.2958. p values for IL1RAPL1 condition: sh-Control vs sh-PTPσ, p = 0.7832; sh-Control vs sh-PTPδ, p < 0.01; and sh-Control vs sh-LAR, p = 0.5212. p values for NL1 condition: sh-Control vs sh-PTPσ, p = 0.9537; sh-Control vs sh-PTPδ, p = 0.8919; and sh-Control vs sh-LAR, p = 0.6262. C, Levels of PTPσ, PTPδ, and LAR mRNAs were measured at DIV12-DV13 by qRT-PCR in cultured cortical neurons infected at DIV4 with lentiviruses expressing the indicated shRNAs. Dashed line indicates 70% knockdown cutoff level for tests of biological effects.
    Rabbit Polyclonal Anti Vesicular Glutamate Transporter 1 (Vglut1), supplied by Synaptic Systems, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    (A) Confocal images of muscle spindles in the tibialis anterior muscle from P13 5-ht1d+/+ (left) and 5-ht1d−/− (right) mice. Sensory axons <t>(VGLUT1+TUJ1+)</t> and gamma motor axons (VGLUT1−TUJ1+) were present and distributed similarly in the muscle spindles of control and 5-ht1d−/− mice . Bungarotoxin (BTX) binds to postsynaptic receptors of motor synapses. (B) Confocal images of motor neuron synapses in muscle spindles visualized with Calbindin (muscle spindle fiber), VAChT (presynaptic motor terminal) and BTX (postsynaptic motor terminal). Quantification of VAChT+ BTX+ neuromuscular junctions on muscle spindles showed a similar motor innervation between 5-ht1d−/− mice and control mice (mean ± SEM, two-tailed student's t-test, p = 0.86).
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    (A) Confocal images of muscle spindles in the tibialis anterior muscle from P13 5-ht1d+/+ (left) and 5-ht1d−/− (right) mice. Sensory axons <t>(VGLUT1+TUJ1+)</t> and gamma motor axons (VGLUT1−TUJ1+) were present and distributed similarly in the muscle spindles of control and 5-ht1d−/− mice . Bungarotoxin (BTX) binds to postsynaptic receptors of motor synapses. (B) Confocal images of motor neuron synapses in muscle spindles visualized with Calbindin (muscle spindle fiber), VAChT (presynaptic motor terminal) and BTX (postsynaptic motor terminal). Quantification of VAChT+ BTX+ neuromuscular junctions on muscle spindles showed a similar motor innervation between 5-ht1d−/− mice and control mice (mean ± SEM, two-tailed student's t-test, p = 0.86).
    Polyclonal Rabbit Anti Vesicular Glutamate Transporter 1 (Vglut1) 135302, supplied by Synaptic Systems, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    (A) Confocal images of muscle spindles in the tibialis anterior muscle from P13 5-ht1d+/+ (left) and 5-ht1d−/− (right) mice. Sensory axons <t>(VGLUT1+TUJ1+)</t> and gamma motor axons (VGLUT1−TUJ1+) were present and distributed similarly in the muscle spindles of control and 5-ht1d−/− mice . Bungarotoxin (BTX) binds to postsynaptic receptors of motor synapses. (B) Confocal images of motor neuron synapses in muscle spindles visualized with Calbindin (muscle spindle fiber), VAChT (presynaptic motor terminal) and BTX (postsynaptic motor terminal). Quantification of VAChT+ BTX+ neuromuscular junctions on muscle spindles showed a similar motor innervation between 5-ht1d−/− mice and control mice (mean ± SEM, two-tailed student's t-test, p = 0.86).
    Polyclonal Rabbit Anti Vesicular Glutamate Transporter 1 (Vglut1; 135302), supplied by Synaptic Systems, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    BDNF is enriched in glutamatergic corticostriatal presynaptic terminals. A , Confocal (top) and SIM (bottom) microscopic images showing BDNF-IR in the same section in glutamatergic (left) versus dopaminergic terminals (right) in the dorsal striatum. B , BDNF-IR is present in VGluT1-positive terminals (magenta arrows). Single BDNF-IR signals overlap with TH (white arrows). VGluT1- and TH-positive terminals reside in direct regional proximity but do not overlap. C , Quantification of BDNF signals in VGluT1-positive terminals and TH-positive terminals. True colocalization between BDNF/VGluT1 was confirmed by Costes p value (Costes p > 0.95) but not between BDNF/TH (Costes p ≪ 0.95). D , Representative Western blots showing recombinant BDNF (lanes 1, 2) versus endogenous BDNF derived from anterior cortex or striatum of P21 NFL-Cre BDNF fl/ko mice (lane 3), P21 sedentary mice (lanes 4, 5), and P21 runners after 72 h voluntary running-wheel exercise (lanes 6, 7); 30 µg of protein lysate was loaded for each sample. BDNF levels were normalized to cytochrome C. Band intensities were determined from extracts of 9 independent mice and presented in % of P21 sedentary mice. Statistical analysis reveals significant increase in BDNF protein levels in both brain areas after running-wheel exercise. Statistical analysis: unpaired t test (anterior CTX: t = 5,312, p < 0.0001; striatum: t = 2,784, p = 0.0133). E , SIM images showing BDNF-IR in VGluT1-positive terminals in the dorsal striatum in sedentary mice (top row) and after 72 h of voluntary running-wheel exercise (bottom row). Data are presented as box and whiskers (Tukey). +, Mean. Vertical line indicates median. Black dots indicate outliers. n , number indicated below. Raw data are provided in Extended Data and . Scale bars: A , 2.5 µm; B , 1.5 µm; E , Overview, 2 µm; Detail, 1 µm. * p < 0.05; **** p < 0.0001.

    Journal: The Journal of Neuroscience

    Article Title: Induction of BDNF Expression in Layer II/III and Layer V Neurons of the Motor Cortex Is Essential for Motor Learning

    doi: 10.1523/JNEUROSCI.0288-20.2020

    Figure Lengend Snippet: BDNF is enriched in glutamatergic corticostriatal presynaptic terminals. A , Confocal (top) and SIM (bottom) microscopic images showing BDNF-IR in the same section in glutamatergic (left) versus dopaminergic terminals (right) in the dorsal striatum. B , BDNF-IR is present in VGluT1-positive terminals (magenta arrows). Single BDNF-IR signals overlap with TH (white arrows). VGluT1- and TH-positive terminals reside in direct regional proximity but do not overlap. C , Quantification of BDNF signals in VGluT1-positive terminals and TH-positive terminals. True colocalization between BDNF/VGluT1 was confirmed by Costes p value (Costes p > 0.95) but not between BDNF/TH (Costes p ≪ 0.95). D , Representative Western blots showing recombinant BDNF (lanes 1, 2) versus endogenous BDNF derived from anterior cortex or striatum of P21 NFL-Cre BDNF fl/ko mice (lane 3), P21 sedentary mice (lanes 4, 5), and P21 runners after 72 h voluntary running-wheel exercise (lanes 6, 7); 30 µg of protein lysate was loaded for each sample. BDNF levels were normalized to cytochrome C. Band intensities were determined from extracts of 9 independent mice and presented in % of P21 sedentary mice. Statistical analysis reveals significant increase in BDNF protein levels in both brain areas after running-wheel exercise. Statistical analysis: unpaired t test (anterior CTX: t = 5,312, p < 0.0001; striatum: t = 2,784, p = 0.0133). E , SIM images showing BDNF-IR in VGluT1-positive terminals in the dorsal striatum in sedentary mice (top row) and after 72 h of voluntary running-wheel exercise (bottom row). Data are presented as box and whiskers (Tukey). +, Mean. Vertical line indicates median. Black dots indicate outliers. n , number indicated below. Raw data are provided in Extended Data and . Scale bars: A , 2.5 µm; B , 1.5 µm; E , Overview, 2 µm; Detail, 1 µm. * p < 0.05; **** p < 0.0001.

    Article Snippet: Presynaptic corticostriatal terminals were labeled with rabbit polyclonal antibodies against vesicular glutamate transporter 1 (VGluT1) (Synaptic Systems, #135302, RRID: AB_887877 ).

    Techniques: Western Blot, Recombinant, Derivative Assay

    Image preparation

    Journal: The Journal of Neuroscience

    Article Title: Induction of BDNF Expression in Layer II/III and Layer V Neurons of the Motor Cortex Is Essential for Motor Learning

    doi: 10.1523/JNEUROSCI.0288-20.2020

    Figure Lengend Snippet: Image preparation

    Article Snippet: Presynaptic corticostriatal terminals were labeled with rabbit polyclonal antibodies against vesicular glutamate transporter 1 (VGluT1) (Synaptic Systems, #135302, RRID: AB_887877 ).

    Techniques:

    PTPσ and PTPδ are selectively required for the heterologous synapse-formation activities of distinct postsynaptogenic adhesion molecules. A, Representative images of the heterologous synapse-formation activities of various LAR-RPTP ligands. Neurons were infected at DIV4 with lentiviruses expressing sh-Control, sh-PTPσ, sh-PTPδ, sh-LAR, sh-LAR/sh-PTPσ, or sh-PTPσ/PTPδ/LAR as indicated, and then cocultured from DIV9 to DIV11 with HEK293T cells expressing various postsynaptic ligands. Neurons were stained with antibodies against HA or EGFP (blue) and VGLUT1 (red). Scale bar (all images), 10 μm. B, The synapse-forming activity in A was quantified by measuring the ratio of VGLUT1 staining intensity (red) to HA or EGFP immunoreactivity intensity (blue). Data are mean ± SEM. Mann–Whitney U test: ****p < 0.0001. n = number of neurons as follows: sh-Control/Control, n = 13; sh-PTPσ/Control, n = 15; sh-PTPδ/Control, n = 17; sh-LAR/Control, n = 15; sh-LAR/sh-PTPσ/Control, n = 11; sh-LAR/PTPσ/PTPδ/Control, n = 12; sh-Control/NGL-3, n = 18; sh-PTPσ/NGL-3, n = 17; sh-PTPδ/NGL-3, n = 16; sh-LAR/NGL-3, n = 16; sh-LAR/sh-PTPσ/NGL-3, n = 16; sh-LAR/PTPσ/PTPδ/NGL-3, n = 16; sh-Control/Slitrk1, n = 13; sh-PTPσ/Slitrk1, n = 15; sh-PTPδ/Slitrk1, n = 13; sh-LAR/Slitrk1, n = 11; sh-Control/TrkC, n = 15; sh-PTPσ/TrkC, n = 11; sh-PTPδ/TrkC, n = 12; sh-LAR/ TrkC, n = 11; sh-Control/IL1RAPL1, n = 11; sh-PTPσ/IL1RAPL1, n = 13; sh-PTPδ/IL1RAPL1, n = 14; sh-LAR/IL1RAPL1, n = 12; sh-Control/NL-1, n = 11; sh-PTPσ/NL-1, n = 12; sh-PTPδ/NL-1, n = 10; and sh-LAR/NL-1, n = 11. p values for Control condition: sh-Control vs sh-PTPσ, p = 0.439; sh-Control vs sh-PTPδ, p = 0.5604; sh-Control vs sh-LAR, p = 0.1835; sh-Control vs sh-LAR/sh-PTPσ, p = 0.9534; and sh-Control vs sh-LAR/PTPσ/PTPδ, p = 0.5641. p values for NGL-3 condition: sh-Control vs sh-PTPσ, p = 0.4129; sh-Control vs sh-PTPδ, p = 0.7706; sh-Control vs sh-LAR, p = 0.5832; sh-Control vs sh-LAR/sh-PTPσ, p = 0.8166; and sh-Control vs sh-LAR/PTPσ/PTPδ, p = 0.9921. p values for Slitrk1 condition: sh-Control vs sh-PTPσ, p < 0.0001; sh-Control vs sh-PTPδ, p = 0.3394; and sh-Control vs sh-LAR, p = 0.684. p values for TrkC condition: sh-Control vs sh-PTPσ, p < 0.0001; sh-Control vs sh-PTPδ, p = 0.1433; and sh-Control vs sh-LAR, p = 0.2958. p values for IL1RAPL1 condition: sh-Control vs sh-PTPσ, p = 0.7832; sh-Control vs sh-PTPδ, p < 0.01; and sh-Control vs sh-LAR, p = 0.5212. p values for NL1 condition: sh-Control vs sh-PTPσ, p = 0.9537; sh-Control vs sh-PTPδ, p = 0.8919; and sh-Control vs sh-LAR, p = 0.6262. C, Levels of PTPσ, PTPδ, and LAR mRNAs were measured at DIV12-DV13 by qRT-PCR in cultured cortical neurons infected at DIV4 with lentiviruses expressing the indicated shRNAs. Dashed line indicates 70% knockdown cutoff level for tests of biological effects.

    Journal: The Journal of Neuroscience

    Article Title: PTPσ Drives Excitatory Presynaptic Assembly via Various Extracellular and Intracellular Mechanisms

    doi: 10.1523/JNEUROSCI.0672-18.2018

    Figure Lengend Snippet: PTPσ and PTPδ are selectively required for the heterologous synapse-formation activities of distinct postsynaptogenic adhesion molecules. A, Representative images of the heterologous synapse-formation activities of various LAR-RPTP ligands. Neurons were infected at DIV4 with lentiviruses expressing sh-Control, sh-PTPσ, sh-PTPδ, sh-LAR, sh-LAR/sh-PTPσ, or sh-PTPσ/PTPδ/LAR as indicated, and then cocultured from DIV9 to DIV11 with HEK293T cells expressing various postsynaptic ligands. Neurons were stained with antibodies against HA or EGFP (blue) and VGLUT1 (red). Scale bar (all images), 10 μm. B, The synapse-forming activity in A was quantified by measuring the ratio of VGLUT1 staining intensity (red) to HA or EGFP immunoreactivity intensity (blue). Data are mean ± SEM. Mann–Whitney U test: ****p < 0.0001. n = number of neurons as follows: sh-Control/Control, n = 13; sh-PTPσ/Control, n = 15; sh-PTPδ/Control, n = 17; sh-LAR/Control, n = 15; sh-LAR/sh-PTPσ/Control, n = 11; sh-LAR/PTPσ/PTPδ/Control, n = 12; sh-Control/NGL-3, n = 18; sh-PTPσ/NGL-3, n = 17; sh-PTPδ/NGL-3, n = 16; sh-LAR/NGL-3, n = 16; sh-LAR/sh-PTPσ/NGL-3, n = 16; sh-LAR/PTPσ/PTPδ/NGL-3, n = 16; sh-Control/Slitrk1, n = 13; sh-PTPσ/Slitrk1, n = 15; sh-PTPδ/Slitrk1, n = 13; sh-LAR/Slitrk1, n = 11; sh-Control/TrkC, n = 15; sh-PTPσ/TrkC, n = 11; sh-PTPδ/TrkC, n = 12; sh-LAR/ TrkC, n = 11; sh-Control/IL1RAPL1, n = 11; sh-PTPσ/IL1RAPL1, n = 13; sh-PTPδ/IL1RAPL1, n = 14; sh-LAR/IL1RAPL1, n = 12; sh-Control/NL-1, n = 11; sh-PTPσ/NL-1, n = 12; sh-PTPδ/NL-1, n = 10; and sh-LAR/NL-1, n = 11. p values for Control condition: sh-Control vs sh-PTPσ, p = 0.439; sh-Control vs sh-PTPδ, p = 0.5604; sh-Control vs sh-LAR, p = 0.1835; sh-Control vs sh-LAR/sh-PTPσ, p = 0.9534; and sh-Control vs sh-LAR/PTPσ/PTPδ, p = 0.5641. p values for NGL-3 condition: sh-Control vs sh-PTPσ, p = 0.4129; sh-Control vs sh-PTPδ, p = 0.7706; sh-Control vs sh-LAR, p = 0.5832; sh-Control vs sh-LAR/sh-PTPσ, p = 0.8166; and sh-Control vs sh-LAR/PTPσ/PTPδ, p = 0.9921. p values for Slitrk1 condition: sh-Control vs sh-PTPσ, p < 0.0001; sh-Control vs sh-PTPδ, p = 0.3394; and sh-Control vs sh-LAR, p = 0.684. p values for TrkC condition: sh-Control vs sh-PTPσ, p < 0.0001; sh-Control vs sh-PTPδ, p = 0.1433; and sh-Control vs sh-LAR, p = 0.2958. p values for IL1RAPL1 condition: sh-Control vs sh-PTPσ, p = 0.7832; sh-Control vs sh-PTPδ, p < 0.01; and sh-Control vs sh-LAR, p = 0.5212. p values for NL1 condition: sh-Control vs sh-PTPσ, p = 0.9537; sh-Control vs sh-PTPδ, p = 0.8919; and sh-Control vs sh-LAR, p = 0.6262. C, Levels of PTPσ, PTPδ, and LAR mRNAs were measured at DIV12-DV13 by qRT-PCR in cultured cortical neurons infected at DIV4 with lentiviruses expressing the indicated shRNAs. Dashed line indicates 70% knockdown cutoff level for tests of biological effects.

    Article Snippet: The following antibodies were obtained commercially: mouse monoclonal anti-GAD67 (clone 1G10.2; Millipore, RRID: AB_2278725 ); rabbit polyclonal anti-vesicular glutamate transporter 1 (VGLUT1) (Synaptic Systems, RRID: AB_887880 ); mouse monoclonal anti-ELKS1 (clone ELKS-30; Sigma-Aldrich, RRID: AB_2100013 ), and rabbit polyclonal anti-hemagglutinin (HA) (Sigma-Aldrich, RRID: AB_260070 ); mouse monoclonal anti-CASK (clone K56A/50; NeuroMab, RRID: AB_2068730 ), mouse monoclonal anti-PSD-95 (clone K28/43; NeuroMab, RRID: AB_2307331 ), and mouse monoclonal anti-GluN2B (clone BWJHL; Millipore, RRID: AB_417391 ); mouse monoclonal anti-PTPσ (MediMabs, RRID: AB_1808357 ); goat polyclonal anti-EGFP (Rockland, RRID: AB_218182 ); mouse monoclonal anti-HA (clone 16B12; Covance, RRID: AB_2314672 ); mouse monoclonal anti-Caskin-2 (Santa Cruz Biotechnology, RRID: AB_2713992 ); and mouse monoclonal anti-β-actin (clone C4; Santa Cruz Biotechnology, RRID: AB_626632 ).

    Techniques: Infection, Expressing, Staining, Activity Assay, MANN-WHITNEY, Quantitative RT-PCR, Cell Culture

    Specific localization of PTPσ and PTPδ at excitatory and inhibitory synaptic sites in cultured hippocampal neurons. A, Immunoblot analyses of PTPδ and PTPσ using lysates from HEK293T cells transfected with HA-tagged PTPδ (HA- PTPδ) or PTPσ (HA-PTPσ). The expression of HA-tagged PTP isoforms was confirmed by immunoblotting with anti-HA antibodies. Unt., Untransfected HEK293T cell lysates; Trans. (HA-PTPδ), lysates from HEK293T cells transfected with HA-PTPδ; Trans. (HA-PTPσ), lysates from HEK293T cells transfected with HA-PTPσ. B, Authenticity testing of anti-PTPδ and anti-PTPσ antibodies. Mature cultured hippocampal neurons (DIV14) infected with lentiviruses expressing sh-Control, sh-PTPδ (PTPδ KD), or sh-PTPσ (PTPσ KD) were labeled by single immunofluorescence staining for the indicated PTP antibodies (gray). Scale bar (all images), 10 μm. C, Summary graphs of B. Data are mean ± SEM. **p < 0.01 (Mann–Whitney U test). n = number of analyzed neurons as follows: sh-Control/PTPσ, n = 10; sh-PTPσ/PTPσ, n = 10; sh-Control/PTPδ, n = 10; and sh-PTPδ/PTPδ, n = 11. p value for PTPσ antibody: sh-Control vs sh-PTPσ, p = 0.0052. p value for PTPδ antibody: sh-Control vs sh-PTPδ, p = 0.0048. D, F, Immunolocalization of PTPδ (D) and PTPσ (F) in cultured hippocampal neurons, along with colocalization of GAD67, VGAT, or VGLUT1 as indicated. Scale bar (all images), 10 μm. E, G, Quantification of colocalization of endogenous PTPδ (E) and PTPσ (G) with synaptic markers.

    Journal: The Journal of Neuroscience

    Article Title: PTPσ Drives Excitatory Presynaptic Assembly via Various Extracellular and Intracellular Mechanisms

    doi: 10.1523/JNEUROSCI.0672-18.2018

    Figure Lengend Snippet: Specific localization of PTPσ and PTPδ at excitatory and inhibitory synaptic sites in cultured hippocampal neurons. A, Immunoblot analyses of PTPδ and PTPσ using lysates from HEK293T cells transfected with HA-tagged PTPδ (HA- PTPδ) or PTPσ (HA-PTPσ). The expression of HA-tagged PTP isoforms was confirmed by immunoblotting with anti-HA antibodies. Unt., Untransfected HEK293T cell lysates; Trans. (HA-PTPδ), lysates from HEK293T cells transfected with HA-PTPδ; Trans. (HA-PTPσ), lysates from HEK293T cells transfected with HA-PTPσ. B, Authenticity testing of anti-PTPδ and anti-PTPσ antibodies. Mature cultured hippocampal neurons (DIV14) infected with lentiviruses expressing sh-Control, sh-PTPδ (PTPδ KD), or sh-PTPσ (PTPσ KD) were labeled by single immunofluorescence staining for the indicated PTP antibodies (gray). Scale bar (all images), 10 μm. C, Summary graphs of B. Data are mean ± SEM. **p < 0.01 (Mann–Whitney U test). n = number of analyzed neurons as follows: sh-Control/PTPσ, n = 10; sh-PTPσ/PTPσ, n = 10; sh-Control/PTPδ, n = 10; and sh-PTPδ/PTPδ, n = 11. p value for PTPσ antibody: sh-Control vs sh-PTPσ, p = 0.0052. p value for PTPδ antibody: sh-Control vs sh-PTPδ, p = 0.0048. D, F, Immunolocalization of PTPδ (D) and PTPσ (F) in cultured hippocampal neurons, along with colocalization of GAD67, VGAT, or VGLUT1 as indicated. Scale bar (all images), 10 μm. E, G, Quantification of colocalization of endogenous PTPδ (E) and PTPσ (G) with synaptic markers.

    Article Snippet: The following antibodies were obtained commercially: mouse monoclonal anti-GAD67 (clone 1G10.2; Millipore, RRID: AB_2278725 ); rabbit polyclonal anti-vesicular glutamate transporter 1 (VGLUT1) (Synaptic Systems, RRID: AB_887880 ); mouse monoclonal anti-ELKS1 (clone ELKS-30; Sigma-Aldrich, RRID: AB_2100013 ), and rabbit polyclonal anti-hemagglutinin (HA) (Sigma-Aldrich, RRID: AB_260070 ); mouse monoclonal anti-CASK (clone K56A/50; NeuroMab, RRID: AB_2068730 ), mouse monoclonal anti-PSD-95 (clone K28/43; NeuroMab, RRID: AB_2307331 ), and mouse monoclonal anti-GluN2B (clone BWJHL; Millipore, RRID: AB_417391 ); mouse monoclonal anti-PTPσ (MediMabs, RRID: AB_1808357 ); goat polyclonal anti-EGFP (Rockland, RRID: AB_218182 ); mouse monoclonal anti-HA (clone 16B12; Covance, RRID: AB_2314672 ); mouse monoclonal anti-Caskin-2 (Santa Cruz Biotechnology, RRID: AB_2713992 ); and mouse monoclonal anti-β-actin (clone C4; Santa Cruz Biotechnology, RRID: AB_626632 ).

    Techniques: Cell Culture, Western Blot, Transfection, Expressing, Infection, Labeling, Immunofluorescence, Staining, MANN-WHITNEY

    Effects of PTPσ and PTPδ KD on synapse density and synaptic transmission in cultured neurons. A, B, Cultured hippocampal neurons were infected with lentiviruses expressing sh-Control, sh-LAR, sh-PTPσ, or sh-PTPδ, or were coinfected with lentiviruses expressing sh-PTPσ or sh-PTPδ together with shRNA-resistant full-length PTPσ (+ PTPσ WT) or sh-PTPδ (+ PTPδ WT) at DIV4, and analyzed at DIV14 by double-immunofluorescence detection of MAP2 and VGLUT1 (A) or VGAT (B). Scale bar (all images), 10 μm. C–E, Summary graphs of A, B. Synaptic puncta density (C), synaptic puncta size (D), and synaptic puncta intensity (E) were measured using VGLUT1 and VGAT as excitatory and inhibitory synaptic markers, respectively. Two or three dendrites per infected neurons were analyzed and group-averaged. Data are mean ± SEM. Mann–Whitney U test: **p < 0.01; ***p < 0.001. n = number of neurons as follows: (E) sh-Control, n = 17; sh-PTPσ, n = 16; sh-PTPδ, n = 15; sh-LAR, n = 15; and sh-PTPσ+PTPσ WT, n = 13. F, sh-Control, n = 13; sh-PTPσ, n = 14; sh-PTPδ, n = 14; sh-LAR, n = 14; and sh-PTPδ+PTPδ WT, n = 14. p values for VGLUT1 puncta density: sh-Control vs sh-PTPσ, p < 0.001; sh-Control vs sh-PTPδ, p = 0.3322; sh-Control vs sh-LAR, p = 0.3765; and sh-Control vs sh-PTPσ (+PTPσ), p = 0.8210. p values for VGAT puncta density: sh-Control vs sh-PTPσ, p = 0.9781; sh-Control vs sh-PTPδ, p < 0.001; sh-Control vs sh-LAR, p = 0.1958; and sh-Control vs sh-PTPδ (+PTPδ), p = 0.8124. p values for VGLUT1 puncta size: sh-Control vs sh-PTPσ, p = 0.0820; sh-Control vs sh-PTPδ, p > 0.9999; sh-Control vs sh-LAR, p = 0.9999; and sh-Control vs sh-PTPσ (+PTPσ), p = 0.9212. p values for VGAT puncta size: sh-Control vs sh-PTPσ, p = 0.1248; sh-Control vs sh-PTPδ, p < 0.01; sh-Control vs sh-LAR, p > 0.9999; and sh-Control vs sh-PTPδ (+PTPδ), p = 0.8790. p values for VGLUT1 puncta intensity: sh-Control vs sh-PTPσ, p = 0.7176; sh-Control vs sh-PTPδ, p = 0.8392; sh-Control vs sh-LAR, p = 0.9115; and sh-Control vs sh-PTPσ (+PTPσ), p = 0.7938. p values for VGAT puncta intensity: sh-Control vs sh-PTPσ, p = 0.2190; sh-Control vs sh-PTPδ, p = 0.4771; sh-Control vs sh-LAR, p = 0.3198; and sh-Control vs sh-PTPδ (+PTPδ), p = 0.6787. F, I, Representative traces of mEPSCs (F) and mIPSCs (I) in neurons infected with control, shPTPσ, sh-PTPδ, or sh-LAR lentiviruses. Neurons were infected with lentiviruses at DIV4, and electrophysiological recordings were obtained at DIV14–16. G, H, Summary graphs of the frequencies (G) and amplitudes (H) of mEPSCs in neurons infected with control, shPTPσ, sh-PTPδ, or sh-LAR lentiviruses. Data are mean ± SEM. *p < 0.05 (Student's t test). n = number of neurons as follows: sh-Control 1, n = 16; sh-PTPσ, n = 21; sh-Control 2, n = 13; sh-PTPδ, n = 30; sh-Control 3, n = 11; and sh-LAR, n = 16. p values for mEPSC frequency: sh-Control 1 vs sh-PTPσ, p < 0.05; sh-Control 2 vs sh-PTPδ, p = 0.4237; and sh-Control 3 vs sh-LAR, p = 0.5211. p values for mEPSC amplitude: sh-Control 1 vs sh-PTPσ, p < 0.05; sh-Control 2 vs sh-PTPδ, p = 0.8921; and sh-Control 3 vs sh-LAR, p = 0.9783. J, K, Same as G, H, except that mIPSCs were measured. Data are mean ± SEM. *p < 0.05 (Student's t test). n = number of neurons as follows: sh-Control 1, n = 16; sh-PTPσ, n = 23; sh-Control 2, n = 11; sh-PTPδ, n = 20; sh-Control 3, n = 16; and sh-LAR, n = 23. p values for mIPSC frequency: sh-Control 1 vs sh-PTPσ, p = 0.3576; sh-Control 2 vs sh-PTPδ, p < 0.05; and sh-Control 3 vs sh-LAR, p = 0.6183. p values for mIPSC amplitude: sh-Control 1 vs sh-PTPσ, p = 0.7120; sh-Control 2 vs sh-PTPδ, p < 0.05; and sh-Control 3 vs sh-LAR, p = 0.8217.

    Journal: The Journal of Neuroscience

    Article Title: PTPσ Drives Excitatory Presynaptic Assembly via Various Extracellular and Intracellular Mechanisms

    doi: 10.1523/JNEUROSCI.0672-18.2018

    Figure Lengend Snippet: Effects of PTPσ and PTPδ KD on synapse density and synaptic transmission in cultured neurons. A, B, Cultured hippocampal neurons were infected with lentiviruses expressing sh-Control, sh-LAR, sh-PTPσ, or sh-PTPδ, or were coinfected with lentiviruses expressing sh-PTPσ or sh-PTPδ together with shRNA-resistant full-length PTPσ (+ PTPσ WT) or sh-PTPδ (+ PTPδ WT) at DIV4, and analyzed at DIV14 by double-immunofluorescence detection of MAP2 and VGLUT1 (A) or VGAT (B). Scale bar (all images), 10 μm. C–E, Summary graphs of A, B. Synaptic puncta density (C), synaptic puncta size (D), and synaptic puncta intensity (E) were measured using VGLUT1 and VGAT as excitatory and inhibitory synaptic markers, respectively. Two or three dendrites per infected neurons were analyzed and group-averaged. Data are mean ± SEM. Mann–Whitney U test: **p < 0.01; ***p < 0.001. n = number of neurons as follows: (E) sh-Control, n = 17; sh-PTPσ, n = 16; sh-PTPδ, n = 15; sh-LAR, n = 15; and sh-PTPσ+PTPσ WT, n = 13. F, sh-Control, n = 13; sh-PTPσ, n = 14; sh-PTPδ, n = 14; sh-LAR, n = 14; and sh-PTPδ+PTPδ WT, n = 14. p values for VGLUT1 puncta density: sh-Control vs sh-PTPσ, p < 0.001; sh-Control vs sh-PTPδ, p = 0.3322; sh-Control vs sh-LAR, p = 0.3765; and sh-Control vs sh-PTPσ (+PTPσ), p = 0.8210. p values for VGAT puncta density: sh-Control vs sh-PTPσ, p = 0.9781; sh-Control vs sh-PTPδ, p < 0.001; sh-Control vs sh-LAR, p = 0.1958; and sh-Control vs sh-PTPδ (+PTPδ), p = 0.8124. p values for VGLUT1 puncta size: sh-Control vs sh-PTPσ, p = 0.0820; sh-Control vs sh-PTPδ, p > 0.9999; sh-Control vs sh-LAR, p = 0.9999; and sh-Control vs sh-PTPσ (+PTPσ), p = 0.9212. p values for VGAT puncta size: sh-Control vs sh-PTPσ, p = 0.1248; sh-Control vs sh-PTPδ, p < 0.01; sh-Control vs sh-LAR, p > 0.9999; and sh-Control vs sh-PTPδ (+PTPδ), p = 0.8790. p values for VGLUT1 puncta intensity: sh-Control vs sh-PTPσ, p = 0.7176; sh-Control vs sh-PTPδ, p = 0.8392; sh-Control vs sh-LAR, p = 0.9115; and sh-Control vs sh-PTPσ (+PTPσ), p = 0.7938. p values for VGAT puncta intensity: sh-Control vs sh-PTPσ, p = 0.2190; sh-Control vs sh-PTPδ, p = 0.4771; sh-Control vs sh-LAR, p = 0.3198; and sh-Control vs sh-PTPδ (+PTPδ), p = 0.6787. F, I, Representative traces of mEPSCs (F) and mIPSCs (I) in neurons infected with control, shPTPσ, sh-PTPδ, or sh-LAR lentiviruses. Neurons were infected with lentiviruses at DIV4, and electrophysiological recordings were obtained at DIV14–16. G, H, Summary graphs of the frequencies (G) and amplitudes (H) of mEPSCs in neurons infected with control, shPTPσ, sh-PTPδ, or sh-LAR lentiviruses. Data are mean ± SEM. *p < 0.05 (Student's t test). n = number of neurons as follows: sh-Control 1, n = 16; sh-PTPσ, n = 21; sh-Control 2, n = 13; sh-PTPδ, n = 30; sh-Control 3, n = 11; and sh-LAR, n = 16. p values for mEPSC frequency: sh-Control 1 vs sh-PTPσ, p < 0.05; sh-Control 2 vs sh-PTPδ, p = 0.4237; and sh-Control 3 vs sh-LAR, p = 0.5211. p values for mEPSC amplitude: sh-Control 1 vs sh-PTPσ, p < 0.05; sh-Control 2 vs sh-PTPδ, p = 0.8921; and sh-Control 3 vs sh-LAR, p = 0.9783. J, K, Same as G, H, except that mIPSCs were measured. Data are mean ± SEM. *p < 0.05 (Student's t test). n = number of neurons as follows: sh-Control 1, n = 16; sh-PTPσ, n = 23; sh-Control 2, n = 11; sh-PTPδ, n = 20; sh-Control 3, n = 16; and sh-LAR, n = 23. p values for mIPSC frequency: sh-Control 1 vs sh-PTPσ, p = 0.3576; sh-Control 2 vs sh-PTPδ, p < 0.05; and sh-Control 3 vs sh-LAR, p = 0.6183. p values for mIPSC amplitude: sh-Control 1 vs sh-PTPσ, p = 0.7120; sh-Control 2 vs sh-PTPδ, p < 0.05; and sh-Control 3 vs sh-LAR, p = 0.8217.

    Article Snippet: The following antibodies were obtained commercially: mouse monoclonal anti-GAD67 (clone 1G10.2; Millipore, RRID: AB_2278725 ); rabbit polyclonal anti-vesicular glutamate transporter 1 (VGLUT1) (Synaptic Systems, RRID: AB_887880 ); mouse monoclonal anti-ELKS1 (clone ELKS-30; Sigma-Aldrich, RRID: AB_2100013 ), and rabbit polyclonal anti-hemagglutinin (HA) (Sigma-Aldrich, RRID: AB_260070 ); mouse monoclonal anti-CASK (clone K56A/50; NeuroMab, RRID: AB_2068730 ), mouse monoclonal anti-PSD-95 (clone K28/43; NeuroMab, RRID: AB_2307331 ), and mouse monoclonal anti-GluN2B (clone BWJHL; Millipore, RRID: AB_417391 ); mouse monoclonal anti-PTPσ (MediMabs, RRID: AB_1808357 ); goat polyclonal anti-EGFP (Rockland, RRID: AB_218182 ); mouse monoclonal anti-HA (clone 16B12; Covance, RRID: AB_2314672 ); mouse monoclonal anti-Caskin-2 (Santa Cruz Biotechnology, RRID: AB_2713992 ); and mouse monoclonal anti-β-actin (clone C4; Santa Cruz Biotechnology, RRID: AB_626632 ).

    Techniques: Transmission Assay, Cell Culture, Infection, Expressing, shRNA, Immunofluorescence, MANN-WHITNEY

    Effect of PTPσ alternative splicing on excitatory synapse development in cultured neurons. A, Schematic diagrams of PTPσ alternative splice variants used in the current study. D1, first catalytic domain of LAR-RPTPs; D2, second catalytic domain of LAR-RPTPs; F, fibronectin Type III repeat; Ig, Ig domain; MeA, mini-exon A; MeB; mini-exon B; SP, IgκB signal peptide contained in the pDisplay vector (Invitrogen); TM, transmembrane region. B, Representative images from cultured hippocampal neurons infected at DIV4 with lentiviruses expressing sh-Control, sh-PTPσ, or coinfected with lentiviruses expressing sh-PTPσ and the human PTPσ alternatively spliced variant rescue viruses, PTPσMeA+MeB+, PTPσMeA−MeB−, PTPσMeA−MeB+, or PTPσMeA+MeB−, and analyzed at DIV14 by double-immunofluorescence detection of MAP2 (blue) and the excitatory synaptic marker VGLUT1 (red). Scale bar (all images), 10 μm. C, Summary graphs of the effects of PTPσ splice variants in neurons on puncta density (left) and puncta size (right), measured using VGLUT1 as an excitatory synaptic marker. Two or three dendrites per infected neuron were analyzed and group-averaged. Data are mean ± SEM. ANOVA with a nonparametric Kruskal–Wallis test: **p < 0.01; #p < 0.05; ##p < 0.01; ###p < 0.001. n = number of neurons as follows: sh-Control/ VGLUT1, n = 14; sh-PTPσ/ VGLUT1, n = 15; PTPσMeA+MeB+ rescue/VGLUT1, n = 15; PTPσMeA−MeB− rescue/VGLUT1, n = 13; PTPσMeA−MeB+ rescue/VGLUT1, n = 15; and PTPσMeA+MeB− rescue/VGLUT1, n = 15. p values for puncta density: sh-Control vs sh-PTPσ, p = 0.0065; sh-Control vs PTPσMeA+MeB+ rescue, p > 0.9999; sh-Control vs PTPσMeA−MeB− rescue, p > 0.9999; sh-Control vs PTPσMeA−MeB+ rescue, p > 0.9999; sh-Control vs PTPσMeA+MeB rescue, p > 0.9999; sh-PTPσ vs PTPσMeA+MeB+ rescue, p = 0.0304; sh-PTPσ vs PTPσMeA−MeB− rescue, p = 0.0002; sh-PTPσ vs PTPσMeA−MeB+ rescue, p = 0.0015; and sh-PTPσ vs PTPσMeA+MeB rescue, p = 0.002. p values for puncta size: sh-Control vs sh-PTPσ, p > 0.9999; sh-Control vs PTPσMeA+MeB+ rescue, p > 0.9999; sh-Control vs PTPσMeA−MeB− rescue, p > 0.9999; sh-Control vs PTPσMeA−MeB+ rescue, p > 0.9999; sh-Control vs PTPσMeA+MeB rescue, p > 0.9999; sh-PTPσ vs PTPσMeA+MeB+ rescue, p > 0.9999; sh-PTPσ vs PTPσMeA−MeB− rescue, p > 0.9999; sh-PTPσ vs PTPσMeA−MeB+ rescue, p > 0.9999; and sh-PTPσ vs PTPσMeA+MeB rescue, p > 0.9999.

    Journal: The Journal of Neuroscience

    Article Title: PTPσ Drives Excitatory Presynaptic Assembly via Various Extracellular and Intracellular Mechanisms

    doi: 10.1523/JNEUROSCI.0672-18.2018

    Figure Lengend Snippet: Effect of PTPσ alternative splicing on excitatory synapse development in cultured neurons. A, Schematic diagrams of PTPσ alternative splice variants used in the current study. D1, first catalytic domain of LAR-RPTPs; D2, second catalytic domain of LAR-RPTPs; F, fibronectin Type III repeat; Ig, Ig domain; MeA, mini-exon A; MeB; mini-exon B; SP, IgκB signal peptide contained in the pDisplay vector (Invitrogen); TM, transmembrane region. B, Representative images from cultured hippocampal neurons infected at DIV4 with lentiviruses expressing sh-Control, sh-PTPσ, or coinfected with lentiviruses expressing sh-PTPσ and the human PTPσ alternatively spliced variant rescue viruses, PTPσMeA+MeB+, PTPσMeA−MeB−, PTPσMeA−MeB+, or PTPσMeA+MeB−, and analyzed at DIV14 by double-immunofluorescence detection of MAP2 (blue) and the excitatory synaptic marker VGLUT1 (red). Scale bar (all images), 10 μm. C, Summary graphs of the effects of PTPσ splice variants in neurons on puncta density (left) and puncta size (right), measured using VGLUT1 as an excitatory synaptic marker. Two or three dendrites per infected neuron were analyzed and group-averaged. Data are mean ± SEM. ANOVA with a nonparametric Kruskal–Wallis test: **p < 0.01; #p < 0.05; ##p < 0.01; ###p < 0.001. n = number of neurons as follows: sh-Control/ VGLUT1, n = 14; sh-PTPσ/ VGLUT1, n = 15; PTPσMeA+MeB+ rescue/VGLUT1, n = 15; PTPσMeA−MeB− rescue/VGLUT1, n = 13; PTPσMeA−MeB+ rescue/VGLUT1, n = 15; and PTPσMeA+MeB− rescue/VGLUT1, n = 15. p values for puncta density: sh-Control vs sh-PTPσ, p = 0.0065; sh-Control vs PTPσMeA+MeB+ rescue, p > 0.9999; sh-Control vs PTPσMeA−MeB− rescue, p > 0.9999; sh-Control vs PTPσMeA−MeB+ rescue, p > 0.9999; sh-Control vs PTPσMeA+MeB rescue, p > 0.9999; sh-PTPσ vs PTPσMeA+MeB+ rescue, p = 0.0304; sh-PTPσ vs PTPσMeA−MeB− rescue, p = 0.0002; sh-PTPσ vs PTPσMeA−MeB+ rescue, p = 0.0015; and sh-PTPσ vs PTPσMeA+MeB rescue, p = 0.002. p values for puncta size: sh-Control vs sh-PTPσ, p > 0.9999; sh-Control vs PTPσMeA+MeB+ rescue, p > 0.9999; sh-Control vs PTPσMeA−MeB− rescue, p > 0.9999; sh-Control vs PTPσMeA−MeB+ rescue, p > 0.9999; sh-Control vs PTPσMeA+MeB rescue, p > 0.9999; sh-PTPσ vs PTPσMeA+MeB+ rescue, p > 0.9999; sh-PTPσ vs PTPσMeA−MeB− rescue, p > 0.9999; sh-PTPσ vs PTPσMeA−MeB+ rescue, p > 0.9999; and sh-PTPσ vs PTPσMeA+MeB rescue, p > 0.9999.

    Article Snippet: The following antibodies were obtained commercially: mouse monoclonal anti-GAD67 (clone 1G10.2; Millipore, RRID: AB_2278725 ); rabbit polyclonal anti-vesicular glutamate transporter 1 (VGLUT1) (Synaptic Systems, RRID: AB_887880 ); mouse monoclonal anti-ELKS1 (clone ELKS-30; Sigma-Aldrich, RRID: AB_2100013 ), and rabbit polyclonal anti-hemagglutinin (HA) (Sigma-Aldrich, RRID: AB_260070 ); mouse monoclonal anti-CASK (clone K56A/50; NeuroMab, RRID: AB_2068730 ), mouse monoclonal anti-PSD-95 (clone K28/43; NeuroMab, RRID: AB_2307331 ), and mouse monoclonal anti-GluN2B (clone BWJHL; Millipore, RRID: AB_417391 ); mouse monoclonal anti-PTPσ (MediMabs, RRID: AB_1808357 ); goat polyclonal anti-EGFP (Rockland, RRID: AB_218182 ); mouse monoclonal anti-HA (clone 16B12; Covance, RRID: AB_2314672 ); mouse monoclonal anti-Caskin-2 (Santa Cruz Biotechnology, RRID: AB_2713992 ); and mouse monoclonal anti-β-actin (clone C4; Santa Cruz Biotechnology, RRID: AB_626632 ).

    Techniques: Cell Culture, Plasmid Preparation, Infection, Expressing, Variant Assay, Immunofluorescence, Marker

    Analysis of PTPσ extracellular mechanisms involved in heterologous synapse formation and excitatory synapse development in cultured neurons. A, Schematic diagrams of a series of PTPσ constructs for deletion variants of extracellular domains or point mutants at extracellular residues. AAAA, Quadruple alanine mutant; D1, first catalytic domain of LAR-RPTPs; D2, second catalytic domain of LAR-RPTPs; F, fibronectin Type III repeat; Ig, Ig domain; MeA, mini-exon A; MeB; mini-exon B; SP, IgκB signal peptide; TM, transmembrane region. B, Representative images of heterologous synapse-formation activities of PTPσ WT and the indicated extracellular domain variants and point mutants. Neurons were infected at DIV4 with lentiviruses expressing sh-Control or sh-PTPσ, or coexpressing sh-PTPσ and the various PTPσ mutant constructs (WT, deletion, or point mutants presented in A), and then cocultured from DIV9 to DIV11 with HEK293T cells expressing EGFP alone (Control) or HA-Slitrk1 (Slitrk1). Neurons were stained with antibodies against EGFP or HA (blue) and synapsin (red). Scale bar (all images), 10 μm. C, Synapse-formation activity in B was quantified by measuring the ratio of synapsin staining intensity (red) to HA/EGFP intensity (blue). Data are mean ± SEM. ANOVA with a nonparametric Kruskal–Wallis test: **p < 0.01; ***p < 0.001; #p < 0.05; ###p < 0.001. n = number of neurons as follows: sh-Control/Control, n = 9; sh-PTPσ/Control, n = 9; +WT/Control, n = 9; +ΔIg/Control, n = 9; +ΔFN1–2/Control, n = 15; +ΔEcto/Control, n = 10; +R781A/Control, n = 11; +AAAA/Control, n = 10; sh-Control/Slitrk1, n = 16; sh-PTPσ/Slitrk1, n = 18; +WT/Slitrk1, n = 16; +ΔIg/Slitrk1, n = 12; +ΔFN1–2/Slitrk1, n = 15; +ΔEcto/Slitrk1, n = 12; +R781A/Slitrk1, n = 15; and +AAAA/Slitrk1, n = 14. p values for Control condition: sh-Control vs sh-PTPσ, p > 0.9999; sh-Control vs +WT, p > 0.9999; sh-Control vs +ΔIg, p > 0.9999; sh-Control vs +ΔFN1–2, p > 0.9999; sh-Control vs +ΔEcto, p > 0.9999; sh-Control vs +R781A, p = 0.3835; sh-Control vs +AAAA, p = 0.7746; sh-PTPσ vs +WT, p > 0.9999; sh-PTPσ vs +ΔIg, p > 0.9999; sh-PTPσ vs +ΔFN1–2, p > 0.9999; sh-PTPσ vs +ΔEcto, p > 0.9999; sh-PTPσ vs +R781A, p = 0.9262; and sh-PTPσ vs +AAAA, p > 0.9999. p values for Slitrk1 condition: sh-Control vs sh-PTPσ, p < 0.0001; sh-Control vs +WT, p > 0.9999; sh-Control vs +ΔIg, p = 0.008; sh-Control vs +ΔFN1–2, p > 0.9999; sh-Control vs +ΔEcto, p = 0.0002; sh-Control vs +R781A, p > 0.9999; sh-Control vs +AAAA, p > 0.9999; sh-PTPσ vs +WT, p < 0.0001; sh-PTPσ vs +ΔIg, p > 0.9999; sh-PTPσ vs +ΔFN1–2, p = 0.0348; sh-PTPσ vs +ΔEcto, p > 0.9999; sh-PTPσ vs +R781A, p = 0.0341; and sh-PTPσ vs +AAAA, p = 0.0132. D, Representative images from cultured hippocampal neurons infected at DIV4 with lentiviruses expressing sh-Control or sh-PTPσ, or coinfected with lentiviruses expressing sh-PTPσ and the indicated rescue viruses for PTPσ WT and extracellular domain mutants and analyzed at DIV14 by double-immunofluorescence detection of MAP2 (blue) and the excitatory synaptic marker VGLUT1 (red). Scale bar (all images), 10 μm. E, F, Summary graphs of the effects of PTPσ molecular replacement in neurons on puncta density (E) and puncta size (F), measured using VGLUT1 as an excitatory synaptic marker. Two or three dendrites per transfected neuron were analyzed and group-averaged. Data are mean ± SEM. ANOVA with a nonparametric Kruskal–Wallis test: *p < 0.05; **p < 0.01; #p < 0.05; ##p < 0.01. n = number of neurons as follows: sh-Control, n = 28; sh-PTPσ, n = 20; +WT, n = 20; +ΔIg, n = 20; +ΔFN1–2, n = 21; +ΔEcto, n = 20; +R781A, n = 14; and +AAAA, n = 20. p values for puncta density: sh-Control vs sh-PTPσ, p = 0.0063; sh-Control vs +WT, p > 0.9999; sh-Control vs +ΔIg, p = 0.0178; sh-Control vs +ΔFN1–2, p > 0.9999; sh-Control vs +ΔEcto, p = 0.0493; sh-Control vs +R781A, p > 0.9999; sh-Control vs +AAAA, p = 0.0132; sh-PTPσ vs +WT, p = 0.0281; sh-PTPσ vs +ΔIg, p > 0.9999; sh-PTPσ vs +ΔFN1–2, p = 0.0073; sh-PTPσ vs +ΔEcto, p > 0.9999; sh-PTPσ vs +R781A, p = 0.0086; and sh-PTPσ vs +AAAA, p > 0.9999. p values for puncta sizes: sh-Control vs sh-PTPσ, p > 0.9999; sh-Control vs +WT, p > 0.9999; sh-Control vs +ΔIg, p > 0.9999; sh-Control vs +ΔFN1–2, p > 0.9999; sh-Control vs +ΔEcto, p > 0.9999; sh-Control vs +R781A, p > 0.9999; sh-Control vs +AAAA, p > 0.9999; sh-PTPσ vs +WT, p > 0.9999; sh-PTPσ vs +ΔIg, p > 0.9999; sh-PTPσ vs +ΔFN1–2, p > 0.9999; sh-PTPσ vs +ΔEcto, p > 0.9999; sh-PTPσ vs +R781A, p > 0.9999; and sh-PTPσ vs +AAAA, p > 0.9999.

    Journal: The Journal of Neuroscience

    Article Title: PTPσ Drives Excitatory Presynaptic Assembly via Various Extracellular and Intracellular Mechanisms

    doi: 10.1523/JNEUROSCI.0672-18.2018

    Figure Lengend Snippet: Analysis of PTPσ extracellular mechanisms involved in heterologous synapse formation and excitatory synapse development in cultured neurons. A, Schematic diagrams of a series of PTPσ constructs for deletion variants of extracellular domains or point mutants at extracellular residues. AAAA, Quadruple alanine mutant; D1, first catalytic domain of LAR-RPTPs; D2, second catalytic domain of LAR-RPTPs; F, fibronectin Type III repeat; Ig, Ig domain; MeA, mini-exon A; MeB; mini-exon B; SP, IgκB signal peptide; TM, transmembrane region. B, Representative images of heterologous synapse-formation activities of PTPσ WT and the indicated extracellular domain variants and point mutants. Neurons were infected at DIV4 with lentiviruses expressing sh-Control or sh-PTPσ, or coexpressing sh-PTPσ and the various PTPσ mutant constructs (WT, deletion, or point mutants presented in A), and then cocultured from DIV9 to DIV11 with HEK293T cells expressing EGFP alone (Control) or HA-Slitrk1 (Slitrk1). Neurons were stained with antibodies against EGFP or HA (blue) and synapsin (red). Scale bar (all images), 10 μm. C, Synapse-formation activity in B was quantified by measuring the ratio of synapsin staining intensity (red) to HA/EGFP intensity (blue). Data are mean ± SEM. ANOVA with a nonparametric Kruskal–Wallis test: **p < 0.01; ***p < 0.001; #p < 0.05; ###p < 0.001. n = number of neurons as follows: sh-Control/Control, n = 9; sh-PTPσ/Control, n = 9; +WT/Control, n = 9; +ΔIg/Control, n = 9; +ΔFN1–2/Control, n = 15; +ΔEcto/Control, n = 10; +R781A/Control, n = 11; +AAAA/Control, n = 10; sh-Control/Slitrk1, n = 16; sh-PTPσ/Slitrk1, n = 18; +WT/Slitrk1, n = 16; +ΔIg/Slitrk1, n = 12; +ΔFN1–2/Slitrk1, n = 15; +ΔEcto/Slitrk1, n = 12; +R781A/Slitrk1, n = 15; and +AAAA/Slitrk1, n = 14. p values for Control condition: sh-Control vs sh-PTPσ, p > 0.9999; sh-Control vs +WT, p > 0.9999; sh-Control vs +ΔIg, p > 0.9999; sh-Control vs +ΔFN1–2, p > 0.9999; sh-Control vs +ΔEcto, p > 0.9999; sh-Control vs +R781A, p = 0.3835; sh-Control vs +AAAA, p = 0.7746; sh-PTPσ vs +WT, p > 0.9999; sh-PTPσ vs +ΔIg, p > 0.9999; sh-PTPσ vs +ΔFN1–2, p > 0.9999; sh-PTPσ vs +ΔEcto, p > 0.9999; sh-PTPσ vs +R781A, p = 0.9262; and sh-PTPσ vs +AAAA, p > 0.9999. p values for Slitrk1 condition: sh-Control vs sh-PTPσ, p < 0.0001; sh-Control vs +WT, p > 0.9999; sh-Control vs +ΔIg, p = 0.008; sh-Control vs +ΔFN1–2, p > 0.9999; sh-Control vs +ΔEcto, p = 0.0002; sh-Control vs +R781A, p > 0.9999; sh-Control vs +AAAA, p > 0.9999; sh-PTPσ vs +WT, p < 0.0001; sh-PTPσ vs +ΔIg, p > 0.9999; sh-PTPσ vs +ΔFN1–2, p = 0.0348; sh-PTPσ vs +ΔEcto, p > 0.9999; sh-PTPσ vs +R781A, p = 0.0341; and sh-PTPσ vs +AAAA, p = 0.0132. D, Representative images from cultured hippocampal neurons infected at DIV4 with lentiviruses expressing sh-Control or sh-PTPσ, or coinfected with lentiviruses expressing sh-PTPσ and the indicated rescue viruses for PTPσ WT and extracellular domain mutants and analyzed at DIV14 by double-immunofluorescence detection of MAP2 (blue) and the excitatory synaptic marker VGLUT1 (red). Scale bar (all images), 10 μm. E, F, Summary graphs of the effects of PTPσ molecular replacement in neurons on puncta density (E) and puncta size (F), measured using VGLUT1 as an excitatory synaptic marker. Two or three dendrites per transfected neuron were analyzed and group-averaged. Data are mean ± SEM. ANOVA with a nonparametric Kruskal–Wallis test: *p < 0.05; **p < 0.01; #p < 0.05; ##p < 0.01. n = number of neurons as follows: sh-Control, n = 28; sh-PTPσ, n = 20; +WT, n = 20; +ΔIg, n = 20; +ΔFN1–2, n = 21; +ΔEcto, n = 20; +R781A, n = 14; and +AAAA, n = 20. p values for puncta density: sh-Control vs sh-PTPσ, p = 0.0063; sh-Control vs +WT, p > 0.9999; sh-Control vs +ΔIg, p = 0.0178; sh-Control vs +ΔFN1–2, p > 0.9999; sh-Control vs +ΔEcto, p = 0.0493; sh-Control vs +R781A, p > 0.9999; sh-Control vs +AAAA, p = 0.0132; sh-PTPσ vs +WT, p = 0.0281; sh-PTPσ vs +ΔIg, p > 0.9999; sh-PTPσ vs +ΔFN1–2, p = 0.0073; sh-PTPσ vs +ΔEcto, p > 0.9999; sh-PTPσ vs +R781A, p = 0.0086; and sh-PTPσ vs +AAAA, p > 0.9999. p values for puncta sizes: sh-Control vs sh-PTPσ, p > 0.9999; sh-Control vs +WT, p > 0.9999; sh-Control vs +ΔIg, p > 0.9999; sh-Control vs +ΔFN1–2, p > 0.9999; sh-Control vs +ΔEcto, p > 0.9999; sh-Control vs +R781A, p > 0.9999; sh-Control vs +AAAA, p > 0.9999; sh-PTPσ vs +WT, p > 0.9999; sh-PTPσ vs +ΔIg, p > 0.9999; sh-PTPσ vs +ΔFN1–2, p > 0.9999; sh-PTPσ vs +ΔEcto, p > 0.9999; sh-PTPσ vs +R781A, p > 0.9999; and sh-PTPσ vs +AAAA, p > 0.9999.

    Article Snippet: The following antibodies were obtained commercially: mouse monoclonal anti-GAD67 (clone 1G10.2; Millipore, RRID: AB_2278725 ); rabbit polyclonal anti-vesicular glutamate transporter 1 (VGLUT1) (Synaptic Systems, RRID: AB_887880 ); mouse monoclonal anti-ELKS1 (clone ELKS-30; Sigma-Aldrich, RRID: AB_2100013 ), and rabbit polyclonal anti-hemagglutinin (HA) (Sigma-Aldrich, RRID: AB_260070 ); mouse monoclonal anti-CASK (clone K56A/50; NeuroMab, RRID: AB_2068730 ), mouse monoclonal anti-PSD-95 (clone K28/43; NeuroMab, RRID: AB_2307331 ), and mouse monoclonal anti-GluN2B (clone BWJHL; Millipore, RRID: AB_417391 ); mouse monoclonal anti-PTPσ (MediMabs, RRID: AB_1808357 ); goat polyclonal anti-EGFP (Rockland, RRID: AB_218182 ); mouse monoclonal anti-HA (clone 16B12; Covance, RRID: AB_2314672 ); mouse monoclonal anti-Caskin-2 (Santa Cruz Biotechnology, RRID: AB_2713992 ); and mouse monoclonal anti-β-actin (clone C4; Santa Cruz Biotechnology, RRID: AB_626632 ).

    Techniques: Cell Culture, Construct, Mutagenesis, Infection, Expressing, Staining, Activity Assay, Immunofluorescence, Marker, Transfection

    Analysis of PTPσ intracellular mechanisms involved in heterologous synapse formation and excitatory synapse development in cultured neurons. A, Schematic diagrams of a series of PTPσ constructs for deletion variants of intracellular domains or point mutants at intracellular residues. Cyto, Cytoplasmic; D1, first catalytic domain of LAR-RPTPs; D2, second catalytic domain of LAR-RPTPs; F, fibronectin Type III repeat; Ig, Ig domain; MeA, mini-exon A; MeB; mini-exon B; SP, IgκB signal peptide; TM, transmembrane region. B, Representative images of the heterologous synapse-formation activities of PTPσ WT and intracellular domain mutants. Neurons were infected at DIV4 with lentiviruses expressing sh-Control or sh-PTPσ, or coexpressing sh-PTPσ and the various PTPσ variants (WT, deletion variants, and point mutants presented in A), and then cocultured from DIV9 to DIV11 with HEK293T cells expressing EGFP alone (Control) or HA-Slitrk1 (Slitrk1). Neurons were stained with antibodies against EGFP or HA (blue) and synapsin (red). Scale bar (all images), 10 μm. C, The synapse-formation activity in B was quantified by measuring the ratio of synapsin staining intensity (red) to HA/EGFP intensity (blue). Data are mean ± SEM. ANOVA with a nonparametric Kruskal–Wallis test: ***p < 0.001; ****p < 0.0001; ##p < 0.01; ####p < 0.0001. n = number of neurons as follows: sh-Control/Control, n = 19; sh-PTPσ/Control, n = 15; +WT/Control, n = 16; +ΔCyto/Control, n = 9; +ΔD2/Control, n = 10; +Swap/Control, n = 11; +C1157S/Control, n = 10; and +D1125A/Control, n = 10; sh-Control/Slitrk1, n = 15; sh-PTPσ/Slitrk1, n = 15; +WT/Slitrk1, n = 15; +ΔCyto/Slitrk1, n = 16; +ΔD2/Slitrk1, n = 14; +Swap/Slitrk1, n = 21; +C1157S/Slitrk1, n = 21; and +D1125A/Slitrk1, n = 22. p values for Control condition: sh-Control vs sh-PTPσ, p > 0.9999; sh-Control vs +WT, p > 0.9999; sh-Control vs +ΔCyto, p = 0.2621; sh-Control vs +ΔD2, p = 0.0915; sh-Control vs +Swap, p > 0.9999; sh-Control vs +C1157S, p > 0.9999; sh-Control vs +D1125A, p > 0.9999; sh-PTPσ vs +WT, p > 0.9999; sh-PTPσ vs +ΔCyto, p > 0.9999; sh-PTPσ vs +ΔD2, p > 0.9999; sh-PTPσ vs +Swap, p > 0.9999; sh-PTPσ vs +C1157S, p > 0.9999; and sh-PTPσ vs +D1125A, p > 0.9999. p values for Slitrk1 condition: sh-Control vs sh-PTPσ, p < 0.0001; sh-Control vs +WT, p > 0.9999; sh-Control vs +ΔCyto, p < 0.0001; sh-Control vs +ΔD2, p = 0.002; sh-Control vs +Swap, p > 0.9999; sh-Control vs +C1157S, p = 0.0445; sh-Control vs +D1125A, p = 0.0045; sh-PTPσ vs +WT, p < 0.0001; sh-PTPσ vs +ΔCyto, p > 0.9999; sh-PTPσ vs +ΔD2, p > 0.9999; sh-PTPσ vs +Swap, p = 0.0023; sh-PTPσ vs +C1157S, p = 0.2585; and sh-PTPσ vs +D1125A, p = 0.6586. D, Representative images of cultured hippocampal neurons infected at DIV4 with lentiviruses expressing sh-Control or sh-PTPσ, or coinfected with lentiviruses expressing sh-PTPσ and the indicated rescue viruses for PTPσ WT and intracellular domain mutants. Images were obtained on DIV14 following double-immunofluorescence labeling of MAP2 (blue) and the excitatory synaptic marker VGLUT1 (red). Scale bar (all images), 10 μm. E, F, Summary graphs of the effects of PTPσ molecular replacement in neurons on puncta density (E) and puncta size (F), measured using VGLUT1 as an excitatory synaptic marker. Two or three dendrites per transfected neuron were analyzed and group-averaged. Data are mean ± SEM. ANOVA with a nonparametric Kruskal–Wallis test: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; #p < 0.05. n = number of neurons as follows: sh-Control, n = 17; sh-PTPσ, n = 21; +WT, n = 15; +ΔCyto, n = 15; +ΔD2, n = 14; +SWAP, n = 20; +C1157S, n = 25; and +D1125A, n = 26. p values for puncta density: sh-Control vs sh-PTPσ, p < 0.0001; sh-Control vs +WT, p > 0.9999; sh-Control vs +ΔCyto, p < 0.0001; sh-Control vs +ΔD2, p = 0.0184; sh-Control vs +Swap, p < 0.0001; sh-Control vs +C1157S, p = 0.0083; sh-Control vs +D1125A, p = 0.0147; sh-PTPσ vs +WT, p = 0.0105; sh-PTPσ vs +ΔCyto, p > 0.9999; sh-PTPσ vs +ΔD2, p > 0.9999; sh-PTPσ vs +Swap, p > 0.9999; sh-PTPσ vs +C1157S, p > 0.9999; and sh-PTPσ vs +D1125A, p > 0.9999. p values for puncta size: sh-Control vs sh-PTPσ, p > 0.9999; sh-Control vs +WT, p > 0.9999; sh-Control vs +ΔCyto, p < 0.0161; sh-Control vs +ΔD2, p > 0.9999; sh-Control vs +Swap, p = 0.0003; sh-Control vs +C1157S, p > 0.9999; sh-Control vs +D1125A, p > 0.9999; sh-PTPσ vs +WT, p > 0.9999; sh-PTPσ vs +ΔCyto, p > 0.9999; sh-PTPσ vs +ΔD2, p > 0.9999; sh-PTPσ vs +Swap, p = 0.266; sh-PTPσ vs +C1157S, p > 0.9999; and sh-PTPσ vs +D1125A, p > 0.9999.

    Journal: The Journal of Neuroscience

    Article Title: PTPσ Drives Excitatory Presynaptic Assembly via Various Extracellular and Intracellular Mechanisms

    doi: 10.1523/JNEUROSCI.0672-18.2018

    Figure Lengend Snippet: Analysis of PTPσ intracellular mechanisms involved in heterologous synapse formation and excitatory synapse development in cultured neurons. A, Schematic diagrams of a series of PTPσ constructs for deletion variants of intracellular domains or point mutants at intracellular residues. Cyto, Cytoplasmic; D1, first catalytic domain of LAR-RPTPs; D2, second catalytic domain of LAR-RPTPs; F, fibronectin Type III repeat; Ig, Ig domain; MeA, mini-exon A; MeB; mini-exon B; SP, IgκB signal peptide; TM, transmembrane region. B, Representative images of the heterologous synapse-formation activities of PTPσ WT and intracellular domain mutants. Neurons were infected at DIV4 with lentiviruses expressing sh-Control or sh-PTPσ, or coexpressing sh-PTPσ and the various PTPσ variants (WT, deletion variants, and point mutants presented in A), and then cocultured from DIV9 to DIV11 with HEK293T cells expressing EGFP alone (Control) or HA-Slitrk1 (Slitrk1). Neurons were stained with antibodies against EGFP or HA (blue) and synapsin (red). Scale bar (all images), 10 μm. C, The synapse-formation activity in B was quantified by measuring the ratio of synapsin staining intensity (red) to HA/EGFP intensity (blue). Data are mean ± SEM. ANOVA with a nonparametric Kruskal–Wallis test: ***p < 0.001; ****p < 0.0001; ##p < 0.01; ####p < 0.0001. n = number of neurons as follows: sh-Control/Control, n = 19; sh-PTPσ/Control, n = 15; +WT/Control, n = 16; +ΔCyto/Control, n = 9; +ΔD2/Control, n = 10; +Swap/Control, n = 11; +C1157S/Control, n = 10; and +D1125A/Control, n = 10; sh-Control/Slitrk1, n = 15; sh-PTPσ/Slitrk1, n = 15; +WT/Slitrk1, n = 15; +ΔCyto/Slitrk1, n = 16; +ΔD2/Slitrk1, n = 14; +Swap/Slitrk1, n = 21; +C1157S/Slitrk1, n = 21; and +D1125A/Slitrk1, n = 22. p values for Control condition: sh-Control vs sh-PTPσ, p > 0.9999; sh-Control vs +WT, p > 0.9999; sh-Control vs +ΔCyto, p = 0.2621; sh-Control vs +ΔD2, p = 0.0915; sh-Control vs +Swap, p > 0.9999; sh-Control vs +C1157S, p > 0.9999; sh-Control vs +D1125A, p > 0.9999; sh-PTPσ vs +WT, p > 0.9999; sh-PTPσ vs +ΔCyto, p > 0.9999; sh-PTPσ vs +ΔD2, p > 0.9999; sh-PTPσ vs +Swap, p > 0.9999; sh-PTPσ vs +C1157S, p > 0.9999; and sh-PTPσ vs +D1125A, p > 0.9999. p values for Slitrk1 condition: sh-Control vs sh-PTPσ, p < 0.0001; sh-Control vs +WT, p > 0.9999; sh-Control vs +ΔCyto, p < 0.0001; sh-Control vs +ΔD2, p = 0.002; sh-Control vs +Swap, p > 0.9999; sh-Control vs +C1157S, p = 0.0445; sh-Control vs +D1125A, p = 0.0045; sh-PTPσ vs +WT, p < 0.0001; sh-PTPσ vs +ΔCyto, p > 0.9999; sh-PTPσ vs +ΔD2, p > 0.9999; sh-PTPσ vs +Swap, p = 0.0023; sh-PTPσ vs +C1157S, p = 0.2585; and sh-PTPσ vs +D1125A, p = 0.6586. D, Representative images of cultured hippocampal neurons infected at DIV4 with lentiviruses expressing sh-Control or sh-PTPσ, or coinfected with lentiviruses expressing sh-PTPσ and the indicated rescue viruses for PTPσ WT and intracellular domain mutants. Images were obtained on DIV14 following double-immunofluorescence labeling of MAP2 (blue) and the excitatory synaptic marker VGLUT1 (red). Scale bar (all images), 10 μm. E, F, Summary graphs of the effects of PTPσ molecular replacement in neurons on puncta density (E) and puncta size (F), measured using VGLUT1 as an excitatory synaptic marker. Two or three dendrites per transfected neuron were analyzed and group-averaged. Data are mean ± SEM. ANOVA with a nonparametric Kruskal–Wallis test: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; #p < 0.05. n = number of neurons as follows: sh-Control, n = 17; sh-PTPσ, n = 21; +WT, n = 15; +ΔCyto, n = 15; +ΔD2, n = 14; +SWAP, n = 20; +C1157S, n = 25; and +D1125A, n = 26. p values for puncta density: sh-Control vs sh-PTPσ, p < 0.0001; sh-Control vs +WT, p > 0.9999; sh-Control vs +ΔCyto, p < 0.0001; sh-Control vs +ΔD2, p = 0.0184; sh-Control vs +Swap, p < 0.0001; sh-Control vs +C1157S, p = 0.0083; sh-Control vs +D1125A, p = 0.0147; sh-PTPσ vs +WT, p = 0.0105; sh-PTPσ vs +ΔCyto, p > 0.9999; sh-PTPσ vs +ΔD2, p > 0.9999; sh-PTPσ vs +Swap, p > 0.9999; sh-PTPσ vs +C1157S, p > 0.9999; and sh-PTPσ vs +D1125A, p > 0.9999. p values for puncta size: sh-Control vs sh-PTPσ, p > 0.9999; sh-Control vs +WT, p > 0.9999; sh-Control vs +ΔCyto, p < 0.0161; sh-Control vs +ΔD2, p > 0.9999; sh-Control vs +Swap, p = 0.0003; sh-Control vs +C1157S, p > 0.9999; sh-Control vs +D1125A, p > 0.9999; sh-PTPσ vs +WT, p > 0.9999; sh-PTPσ vs +ΔCyto, p > 0.9999; sh-PTPσ vs +ΔD2, p > 0.9999; sh-PTPσ vs +Swap, p = 0.266; sh-PTPσ vs +C1157S, p > 0.9999; and sh-PTPσ vs +D1125A, p > 0.9999.

    Article Snippet: The following antibodies were obtained commercially: mouse monoclonal anti-GAD67 (clone 1G10.2; Millipore, RRID: AB_2278725 ); rabbit polyclonal anti-vesicular glutamate transporter 1 (VGLUT1) (Synaptic Systems, RRID: AB_887880 ); mouse monoclonal anti-ELKS1 (clone ELKS-30; Sigma-Aldrich, RRID: AB_2100013 ), and rabbit polyclonal anti-hemagglutinin (HA) (Sigma-Aldrich, RRID: AB_260070 ); mouse monoclonal anti-CASK (clone K56A/50; NeuroMab, RRID: AB_2068730 ), mouse monoclonal anti-PSD-95 (clone K28/43; NeuroMab, RRID: AB_2307331 ), and mouse monoclonal anti-GluN2B (clone BWJHL; Millipore, RRID: AB_417391 ); mouse monoclonal anti-PTPσ (MediMabs, RRID: AB_1808357 ); goat polyclonal anti-EGFP (Rockland, RRID: AB_218182 ); mouse monoclonal anti-HA (clone 16B12; Covance, RRID: AB_2314672 ); mouse monoclonal anti-Caskin-2 (Santa Cruz Biotechnology, RRID: AB_2713992 ); and mouse monoclonal anti-β-actin (clone C4; Santa Cruz Biotechnology, RRID: AB_626632 ).

    Techniques: Cell Culture, Construct, Infection, Expressing, Staining, Activity Assay, Immunofluorescence, Labeling, Marker, Transfection

    Effects of PTPσ extracellular domain and PTPδ intracellular domain on PTPσ-mediated heterologous synapse formation. A, Schematic illustration of PTPσ WT and mutants used in the experiments presented in B–D. B, Representative images of the heterologous excitatory or inhibitory synapse-formation activities of PTPσ WT and PTPσ/PTPδ Swap mutants. Neurons were infected at DIV4 with lentiviruses expressing sh-Control, sh-PTPσ, or sh-PTPδ, or coexpressing sh-PTPσ or sh-PTPδ with the indicated PTPσ or PTPδ expression vectors, and then cocultured from DIV9 to DIV11 with HEK293T cells expressing HA-Slitrk6 (Slitrk6) or neuroligin-2 fused to mVenus (NL-2). Neurons were stained with antibodies against HA (blue) and GAD67 or VGLUT1 (red). Scale bar (all images), 10 μm. C, D, The synapse-formation activity in B was quantified by measuring the ratio of GAD67 (C) or VGLUT1 (D) staining intensity (red) to HA/EGFP intensity (blue). Data are mean ± SEM. ANOVA with a nonparametric Kruskal–Wallis test: ***p < 0.001; ****p < 0.0001; ##p < 0.01; ###p < 0.001. n = number of neurons as follows: sh-Control/GAD67/Slitrk6, n = 16; sh-PTPδ/GAD67/Slitrk6, n = 15; + PTPδ/GAD67/Slitrk6, n = 16; + PTPσ Swap/GAD67/Slitrk6, n = 17; sh-Control/VGLUT1/Slitrk6, n = 15; sh-PTPσ/VGLUT1/Slitrk6, n = 15; + PTPσ WT/VGLUT1/Slitrk6, n = 12; + PTPσ Swap/VGLUT1/Slitrk6, n = 13; sh-Control/GAD67/NL-2, n = 21; sh-PTPδ/GAD67/NL-2, n = 23; sh-Control/VGLUT1/NL-2, n = 12; and sh-PTPσ/VGLUT1/NL-2, n = 11. p values for Slitrk6/GAD67 condition: sh-Control vs sh-PTPδ, p < 0.0001; sh-Control vs + PTPδ, p = 0.4456; sh-Control vs + PTPσ Swap, p = 0.4583; sh-PTPδ vs + PTPδ, p = 0.0064; and sh-PTPδ vs + PTPσ Swap, p = 0.0047. p values for Slitrk6/VGLUT1 condition: sh-Control vs sh-PTPσ, p = 0.0004; sh-Control vs + PTPσ, p > 0.9999; sh-control vs + PTPσ Swap, p > 0.9999; sh-PTPσ vs + PTPσ, p = 0.0019; and sh-PTPσ vs + PTPσ Swap, p = 0.0002. p values for NL-2/GAD67 condition: sh-Control vs sh-PTPδ, p > 0.9999. p values for NL-2/VGLUT1 condition: sh-Control vs sh-PTPσ, p > 0.9999.

    Journal: The Journal of Neuroscience

    Article Title: PTPσ Drives Excitatory Presynaptic Assembly via Various Extracellular and Intracellular Mechanisms

    doi: 10.1523/JNEUROSCI.0672-18.2018

    Figure Lengend Snippet: Effects of PTPσ extracellular domain and PTPδ intracellular domain on PTPσ-mediated heterologous synapse formation. A, Schematic illustration of PTPσ WT and mutants used in the experiments presented in B–D. B, Representative images of the heterologous excitatory or inhibitory synapse-formation activities of PTPσ WT and PTPσ/PTPδ Swap mutants. Neurons were infected at DIV4 with lentiviruses expressing sh-Control, sh-PTPσ, or sh-PTPδ, or coexpressing sh-PTPσ or sh-PTPδ with the indicated PTPσ or PTPδ expression vectors, and then cocultured from DIV9 to DIV11 with HEK293T cells expressing HA-Slitrk6 (Slitrk6) or neuroligin-2 fused to mVenus (NL-2). Neurons were stained with antibodies against HA (blue) and GAD67 or VGLUT1 (red). Scale bar (all images), 10 μm. C, D, The synapse-formation activity in B was quantified by measuring the ratio of GAD67 (C) or VGLUT1 (D) staining intensity (red) to HA/EGFP intensity (blue). Data are mean ± SEM. ANOVA with a nonparametric Kruskal–Wallis test: ***p < 0.001; ****p < 0.0001; ##p < 0.01; ###p < 0.001. n = number of neurons as follows: sh-Control/GAD67/Slitrk6, n = 16; sh-PTPδ/GAD67/Slitrk6, n = 15; + PTPδ/GAD67/Slitrk6, n = 16; + PTPσ Swap/GAD67/Slitrk6, n = 17; sh-Control/VGLUT1/Slitrk6, n = 15; sh-PTPσ/VGLUT1/Slitrk6, n = 15; + PTPσ WT/VGLUT1/Slitrk6, n = 12; + PTPσ Swap/VGLUT1/Slitrk6, n = 13; sh-Control/GAD67/NL-2, n = 21; sh-PTPδ/GAD67/NL-2, n = 23; sh-Control/VGLUT1/NL-2, n = 12; and sh-PTPσ/VGLUT1/NL-2, n = 11. p values for Slitrk6/GAD67 condition: sh-Control vs sh-PTPδ, p < 0.0001; sh-Control vs + PTPδ, p = 0.4456; sh-Control vs + PTPσ Swap, p = 0.4583; sh-PTPδ vs + PTPδ, p = 0.0064; and sh-PTPδ vs + PTPσ Swap, p = 0.0047. p values for Slitrk6/VGLUT1 condition: sh-Control vs sh-PTPσ, p = 0.0004; sh-Control vs + PTPσ, p > 0.9999; sh-control vs + PTPσ Swap, p > 0.9999; sh-PTPσ vs + PTPσ, p = 0.0019; and sh-PTPσ vs + PTPσ Swap, p = 0.0002. p values for NL-2/GAD67 condition: sh-Control vs sh-PTPδ, p > 0.9999. p values for NL-2/VGLUT1 condition: sh-Control vs sh-PTPσ, p > 0.9999.

    Article Snippet: The following antibodies were obtained commercially: mouse monoclonal anti-GAD67 (clone 1G10.2; Millipore, RRID: AB_2278725 ); rabbit polyclonal anti-vesicular glutamate transporter 1 (VGLUT1) (Synaptic Systems, RRID: AB_887880 ); mouse monoclonal anti-ELKS1 (clone ELKS-30; Sigma-Aldrich, RRID: AB_2100013 ), and rabbit polyclonal anti-hemagglutinin (HA) (Sigma-Aldrich, RRID: AB_260070 ); mouse monoclonal anti-CASK (clone K56A/50; NeuroMab, RRID: AB_2068730 ), mouse monoclonal anti-PSD-95 (clone K28/43; NeuroMab, RRID: AB_2307331 ), and mouse monoclonal anti-GluN2B (clone BWJHL; Millipore, RRID: AB_417391 ); mouse monoclonal anti-PTPσ (MediMabs, RRID: AB_1808357 ); goat polyclonal anti-EGFP (Rockland, RRID: AB_218182 ); mouse monoclonal anti-HA (clone 16B12; Covance, RRID: AB_2314672 ); mouse monoclonal anti-Caskin-2 (Santa Cruz Biotechnology, RRID: AB_2713992 ); and mouse monoclonal anti-β-actin (clone C4; Santa Cruz Biotechnology, RRID: AB_626632 ).

    Techniques: Infection, Expressing, Staining, Activity Assay

    Analysis of the effects of PTPσ extracellular point mutants that selectively abolish interactions with TrkC or Slitrks on heterologous synapse formation. A, Schematic illustration of PTPσ WT and point mutants that exhibit defective binding to the specific postsynaptic ligands, TrkC or Slitrks. D1, First catalytic domain of LAR-RPTPs; D2, second catalytic domain of LAR-RPTPs; F, fibronectin Type III repeat; Ig, Ig domain; MeA, mini-exon A; MeB; mini-exon B; SP, IgκB signal peptide; TM, transmembrane region. B, Representative images of cell surface-binding assays. HEK293T cells expressing HA-tagged PTPσ WT or its various point mutants were incubated with 10 μg/ml control IgC (Control), Ig-GPC-4, or Ig-NGL-3, and then analyzed by immunofluorescence imaging of Ig-fusion proteins (red) and HA antibodies (green). Scale bar (all images), 10 μm. C, Representative images of the heterologous synapse-formation activities of PTPσ WT and specific ligand-binding-defective mutants. Neurons were infected at DIV4 with lentiviruses expressing sh-Control or sh-PTPσ, or coexpressing various PTPσ mutant constructs (WT and ligand-binding-defective mutants presented in A), and then cocultured from DIV9 to DIV11 with HEK293T cells expressing EGFP alone (Control) or HA-Slitrk1 (Slitrk1). Neurons were stained with antibodies against EGFP or HA (blue) and synapsin (red). Scale bar (all images), 10 μm. D, The synapse-formation activity in C was quantified by measuring the ratio of synapsin staining intensity (red) to HA/EGFP intensity (blue). Data are mean ± SEM. ANOVA with a nonparametric Kruskal–Wallis test: **p < 0.01; ***p < 0.001; ####p < 0.0001. n = number of neurons as follows: sh-Control/Slitrk1, n = 19; sh-PTPσ/Control, n = 15; +WT/Control, n = 16; +R97/100A/Control, n = 10; +Y233S/Control, n = 9; and +R235D/Control, n = 10; sh-Control/Slitrk1, n = 20; sh-PTPσ/Slitrk1, n = 25; +WT/Slitrk1, n = 25; +R97/100A/Slitrk1, n = 22; +Y233S/Slitrk1, n = 16; and +R235D/Slitrk1, n = 24. p values for Control condition: sh-Control vs sh-PTPσ, p > 0.9999; sh-Control vs +WT, p > 0.9999; sh-Control vs +R97/100A, p > 0.9999; sh-Control vs +Y233S, p = 0.3522; sh-Control vs +R235D, p = 0.1213; sh-PTPσ vs +WT, p > 0.9999; sh-PTPσ vs +R97/100A, p > 0.9999; sh-PTPσ vs +Y233S, p = 0.1596; and sh-PTPσ vs +R235D, p = 0.0504. p values for Slitrk1 condition: sh-Control vs sh-PTPσ, p < 0.0001; sh-Control vs +WT, p > 0.9999; sh-Control vs +R97/100A, p > 0.9999; sh-Control vs +Y233S, p = 0.0015; sh-Control vs +R235D, p = 0.0004; sh-PTPσ vs +WT, p < 0.0001; sh-PTPσ vs +R97/100A, p < 0.0001; sh-PTPσ vs +Y233S, p > 0.9999; and sh-PTPσ vs +R235D, p > 0.9999. E, Representative images of cultured hippocampal neurons infected at DIV4 with lentiviruses expressing sh-Control or sh-PTPσ, or coinfected with lentiviruses expressing sh-PTPσ and the indicated rescue viruses for human PTPσ alternative splicing variants. These images were taken on DIV14 following double-immunofluorescence labeling of MAP2 (blue) and the excitatory synaptic marker VGLUT1 (red). Scale bar (all images), 10 μm. F, Summary graphs of the effects of PTPσ molecular replacement in neurons on puncta density (left) and puncta size (right), measured using VGLUT1 as an excitatory synaptic marker. Two or three dendrites per transfected neuron were analyzed and group-averaged. Data are mean ± SEM. ANOVA with a nonparametric Kruskal–Wallis test: **p < 0.01; ***p < 0.001; #p < 0.05; ##p < 0.01. n = number of neurons as follows: sh-Control, n = 21; sh-PTPσ, n = 20; +WT, n = 16; +R97/100A, n = 21; +Y233S, n = 15; and +R235D, n = 15. p values for puncta density: sh-Control vs sh-PTPσ, p < 0.0001; sh-Control vs +WT, p > 0.9999; sh-control vs +R97/100A, p > 0.9999; sh-Control vs +Y233S, p = 0.0036; sh-Control vs +R235D, p = 0.0059; sh-PTPσ vs +WT, p = 0.0074; sh-PTPσ vs +R97/100A, p = 0.0179; sh-PTPσ vs +Y233S, p > 0.9999; and sh-PTPσ vs +R235D, p > 0.9999. p values for puncta size: sh-Control vs sh-PTPσ, p > 0.9999; sh-Control vs +WT, p > 0.9999; sh-Control vs +R97/100A, p > 0.9999; sh-Control vs +Y233S, p > 0.9999; sh-Control vs +R235D, p = 0.0177; sh-PTPσ vs +WT, p > 0.9999; sh-PTPσ vs +R97/100A, p > 0.9999; sh-PTPσ vs +Y233S, p > 0.9999; and sh-PTPσ vs +R235D, p = 0.2712.

    Journal: The Journal of Neuroscience

    Article Title: PTPσ Drives Excitatory Presynaptic Assembly via Various Extracellular and Intracellular Mechanisms

    doi: 10.1523/JNEUROSCI.0672-18.2018

    Figure Lengend Snippet: Analysis of the effects of PTPσ extracellular point mutants that selectively abolish interactions with TrkC or Slitrks on heterologous synapse formation. A, Schematic illustration of PTPσ WT and point mutants that exhibit defective binding to the specific postsynaptic ligands, TrkC or Slitrks. D1, First catalytic domain of LAR-RPTPs; D2, second catalytic domain of LAR-RPTPs; F, fibronectin Type III repeat; Ig, Ig domain; MeA, mini-exon A; MeB; mini-exon B; SP, IgκB signal peptide; TM, transmembrane region. B, Representative images of cell surface-binding assays. HEK293T cells expressing HA-tagged PTPσ WT or its various point mutants were incubated with 10 μg/ml control IgC (Control), Ig-GPC-4, or Ig-NGL-3, and then analyzed by immunofluorescence imaging of Ig-fusion proteins (red) and HA antibodies (green). Scale bar (all images), 10 μm. C, Representative images of the heterologous synapse-formation activities of PTPσ WT and specific ligand-binding-defective mutants. Neurons were infected at DIV4 with lentiviruses expressing sh-Control or sh-PTPσ, or coexpressing various PTPσ mutant constructs (WT and ligand-binding-defective mutants presented in A), and then cocultured from DIV9 to DIV11 with HEK293T cells expressing EGFP alone (Control) or HA-Slitrk1 (Slitrk1). Neurons were stained with antibodies against EGFP or HA (blue) and synapsin (red). Scale bar (all images), 10 μm. D, The synapse-formation activity in C was quantified by measuring the ratio of synapsin staining intensity (red) to HA/EGFP intensity (blue). Data are mean ± SEM. ANOVA with a nonparametric Kruskal–Wallis test: **p < 0.01; ***p < 0.001; ####p < 0.0001. n = number of neurons as follows: sh-Control/Slitrk1, n = 19; sh-PTPσ/Control, n = 15; +WT/Control, n = 16; +R97/100A/Control, n = 10; +Y233S/Control, n = 9; and +R235D/Control, n = 10; sh-Control/Slitrk1, n = 20; sh-PTPσ/Slitrk1, n = 25; +WT/Slitrk1, n = 25; +R97/100A/Slitrk1, n = 22; +Y233S/Slitrk1, n = 16; and +R235D/Slitrk1, n = 24. p values for Control condition: sh-Control vs sh-PTPσ, p > 0.9999; sh-Control vs +WT, p > 0.9999; sh-Control vs +R97/100A, p > 0.9999; sh-Control vs +Y233S, p = 0.3522; sh-Control vs +R235D, p = 0.1213; sh-PTPσ vs +WT, p > 0.9999; sh-PTPσ vs +R97/100A, p > 0.9999; sh-PTPσ vs +Y233S, p = 0.1596; and sh-PTPσ vs +R235D, p = 0.0504. p values for Slitrk1 condition: sh-Control vs sh-PTPσ, p < 0.0001; sh-Control vs +WT, p > 0.9999; sh-Control vs +R97/100A, p > 0.9999; sh-Control vs +Y233S, p = 0.0015; sh-Control vs +R235D, p = 0.0004; sh-PTPσ vs +WT, p < 0.0001; sh-PTPσ vs +R97/100A, p < 0.0001; sh-PTPσ vs +Y233S, p > 0.9999; and sh-PTPσ vs +R235D, p > 0.9999. E, Representative images of cultured hippocampal neurons infected at DIV4 with lentiviruses expressing sh-Control or sh-PTPσ, or coinfected with lentiviruses expressing sh-PTPσ and the indicated rescue viruses for human PTPσ alternative splicing variants. These images were taken on DIV14 following double-immunofluorescence labeling of MAP2 (blue) and the excitatory synaptic marker VGLUT1 (red). Scale bar (all images), 10 μm. F, Summary graphs of the effects of PTPσ molecular replacement in neurons on puncta density (left) and puncta size (right), measured using VGLUT1 as an excitatory synaptic marker. Two or three dendrites per transfected neuron were analyzed and group-averaged. Data are mean ± SEM. ANOVA with a nonparametric Kruskal–Wallis test: **p < 0.01; ***p < 0.001; #p < 0.05; ##p < 0.01. n = number of neurons as follows: sh-Control, n = 21; sh-PTPσ, n = 20; +WT, n = 16; +R97/100A, n = 21; +Y233S, n = 15; and +R235D, n = 15. p values for puncta density: sh-Control vs sh-PTPσ, p < 0.0001; sh-Control vs +WT, p > 0.9999; sh-control vs +R97/100A, p > 0.9999; sh-Control vs +Y233S, p = 0.0036; sh-Control vs +R235D, p = 0.0059; sh-PTPσ vs +WT, p = 0.0074; sh-PTPσ vs +R97/100A, p = 0.0179; sh-PTPσ vs +Y233S, p > 0.9999; and sh-PTPσ vs +R235D, p > 0.9999. p values for puncta size: sh-Control vs sh-PTPσ, p > 0.9999; sh-Control vs +WT, p > 0.9999; sh-Control vs +R97/100A, p > 0.9999; sh-Control vs +Y233S, p > 0.9999; sh-Control vs +R235D, p = 0.0177; sh-PTPσ vs +WT, p > 0.9999; sh-PTPσ vs +R97/100A, p > 0.9999; sh-PTPσ vs +Y233S, p > 0.9999; and sh-PTPσ vs +R235D, p = 0.2712.

    Article Snippet: The following antibodies were obtained commercially: mouse monoclonal anti-GAD67 (clone 1G10.2; Millipore, RRID: AB_2278725 ); rabbit polyclonal anti-vesicular glutamate transporter 1 (VGLUT1) (Synaptic Systems, RRID: AB_887880 ); mouse monoclonal anti-ELKS1 (clone ELKS-30; Sigma-Aldrich, RRID: AB_2100013 ), and rabbit polyclonal anti-hemagglutinin (HA) (Sigma-Aldrich, RRID: AB_260070 ); mouse monoclonal anti-CASK (clone K56A/50; NeuroMab, RRID: AB_2068730 ), mouse monoclonal anti-PSD-95 (clone K28/43; NeuroMab, RRID: AB_2307331 ), and mouse monoclonal anti-GluN2B (clone BWJHL; Millipore, RRID: AB_417391 ); mouse monoclonal anti-PTPσ (MediMabs, RRID: AB_1808357 ); goat polyclonal anti-EGFP (Rockland, RRID: AB_218182 ); mouse monoclonal anti-HA (clone 16B12; Covance, RRID: AB_2314672 ); mouse monoclonal anti-Caskin-2 (Santa Cruz Biotechnology, RRID: AB_2713992 ); and mouse monoclonal anti-β-actin (clone C4; Santa Cruz Biotechnology, RRID: AB_626632 ).

    Techniques: Binding Assay, Expressing, Incubation, Immunofluorescence, Imaging, Ligand Binding Assay, Infection, Mutagenesis, Construct, Staining, Activity Assay, Cell Culture, Labeling, Marker, Transfection

    (A) Confocal images of muscle spindles in the tibialis anterior muscle from P13 5-ht1d+/+ (left) and 5-ht1d−/− (right) mice. Sensory axons (VGLUT1+TUJ1+) and gamma motor axons (VGLUT1−TUJ1+) were present and distributed similarly in the muscle spindles of control and 5-ht1d−/− mice . Bungarotoxin (BTX) binds to postsynaptic receptors of motor synapses. (B) Confocal images of motor neuron synapses in muscle spindles visualized with Calbindin (muscle spindle fiber), VAChT (presynaptic motor terminal) and BTX (postsynaptic motor terminal). Quantification of VAChT+ BTX+ neuromuscular junctions on muscle spindles showed a similar motor innervation between 5-ht1d−/− mice and control mice (mean ± SEM, two-tailed student's t-test, p = 0.86).

    Journal: Molecular and Cellular Neurosciences

    Article Title: Sensorimotor function is modulated by the serotonin receptor 1d, a novel marker for gamma motor neurons

    doi: 10.1016/j.mcn.2012.01.003

    Figure Lengend Snippet: (A) Confocal images of muscle spindles in the tibialis anterior muscle from P13 5-ht1d+/+ (left) and 5-ht1d−/− (right) mice. Sensory axons (VGLUT1+TUJ1+) and gamma motor axons (VGLUT1−TUJ1+) were present and distributed similarly in the muscle spindles of control and 5-ht1d−/− mice . Bungarotoxin (BTX) binds to postsynaptic receptors of motor synapses. (B) Confocal images of motor neuron synapses in muscle spindles visualized with Calbindin (muscle spindle fiber), VAChT (presynaptic motor terminal) and BTX (postsynaptic motor terminal). Quantification of VAChT+ BTX+ neuromuscular junctions on muscle spindles showed a similar motor innervation between 5-ht1d−/− mice and control mice (mean ± SEM, two-tailed student's t-test, p = 0.86).

    Article Snippet: The following antibodies were used; polyclonal goat anti-Choline Acetyltransferase (ChAT) (1:100; AB144P, Milipore), polyclonal goat anti- VAChT (1:500, AB1578, Milipore), polyclonal rabbit anti-Vesicular glutamate transporter 1 (VGLUT1) (1:500, 135002, Synaptic Systems), polyclonal rabbit- and guinea pig anti-VGLUT1 (1:300 both, ( Fujiyama et al., 2001 )), polyclonal rabbit anti-Calbindin (1:5000, CB38, Swant), monoclonal mouse anti-Parvalbumin (1:500, MAB1572, Chemicon), monoclonal rabbit anti-β-tubulin III (TUJ1) (1:1000, TUJ1-1-15-79, Covance) and Alexa 488-conjugated bungarotoxin (1:1000, B13422, Invitrogen).

    Techniques: Two Tailed Test

    (A) VGLUT1+ contacts on ChAT+ motor neurons in adult (P30) thoracic spinal cord of 5-ht1d+/+ and 5-ht1d−/− mice (left). (B) Quantification of VGLUT1+ contacts on motor neurons (left; 5-ht1d+/+, n = 57, and 5-ht1d−/−, n = 51, from 3 animals per genotype; Mann-Whitney U-test, p = 0.33). Bar graph illustrating the ratio of motor neurons with (black) and without (white) detectable VGLUT1 contacts (right, 5-ht1d+/+, n = 57, and 5-ht1d−/−, n = 51, from 3 animals per genotype; Chi-squared test, p = 0.17). (C) Schematic of experimental setup for recording responses in ventral root after dorsal root stimulations. (D) Example of averaged responses in L3 ventral roots from a P5 5-ht1d+/+ (left) and a P3 5-ht1d−/− (right) mouse. Horizontal arrows indicate monosynaptic and polysynaptic peaks, vertical arrows indicate onset of monosynaptic and polysynaptic episodes. Shaded area indicates part of plot used to estimate polysynaptic area. (E) Graph of amplitude (left) and latency (right) of monosynaptic peak in 5-ht1d+/+ mice and 5-ht1d−/− mice (Mean ± SEM, students t-test, p = 0.034 (amplitude), p = 0.17 (latency), n=10 5-ht1d+/+ mice and n=7 5-ht1d−/− mice). (F) Graph of amplitude (left), latency (middle) and area (right) of polysynaptic peak in neonatal 5-ht1d+/+ mice and 5-ht1d−/− mice (Mean ± SEM, students t-test, p = 0.35 (amplitude), p = 0.88 (latency), p = 0.16 (area) n=10 5-ht1d+/+mice and n=7 5-ht1d−/− mice).

    Journal: Molecular and Cellular Neurosciences

    Article Title: Sensorimotor function is modulated by the serotonin receptor 1d, a novel marker for gamma motor neurons

    doi: 10.1016/j.mcn.2012.01.003

    Figure Lengend Snippet: (A) VGLUT1+ contacts on ChAT+ motor neurons in adult (P30) thoracic spinal cord of 5-ht1d+/+ and 5-ht1d−/− mice (left). (B) Quantification of VGLUT1+ contacts on motor neurons (left; 5-ht1d+/+, n = 57, and 5-ht1d−/−, n = 51, from 3 animals per genotype; Mann-Whitney U-test, p = 0.33). Bar graph illustrating the ratio of motor neurons with (black) and without (white) detectable VGLUT1 contacts (right, 5-ht1d+/+, n = 57, and 5-ht1d−/−, n = 51, from 3 animals per genotype; Chi-squared test, p = 0.17). (C) Schematic of experimental setup for recording responses in ventral root after dorsal root stimulations. (D) Example of averaged responses in L3 ventral roots from a P5 5-ht1d+/+ (left) and a P3 5-ht1d−/− (right) mouse. Horizontal arrows indicate monosynaptic and polysynaptic peaks, vertical arrows indicate onset of monosynaptic and polysynaptic episodes. Shaded area indicates part of plot used to estimate polysynaptic area. (E) Graph of amplitude (left) and latency (right) of monosynaptic peak in 5-ht1d+/+ mice and 5-ht1d−/− mice (Mean ± SEM, students t-test, p = 0.034 (amplitude), p = 0.17 (latency), n=10 5-ht1d+/+ mice and n=7 5-ht1d−/− mice). (F) Graph of amplitude (left), latency (middle) and area (right) of polysynaptic peak in neonatal 5-ht1d+/+ mice and 5-ht1d−/− mice (Mean ± SEM, students t-test, p = 0.35 (amplitude), p = 0.88 (latency), p = 0.16 (area) n=10 5-ht1d+/+mice and n=7 5-ht1d−/− mice).

    Article Snippet: The following antibodies were used; polyclonal goat anti-Choline Acetyltransferase (ChAT) (1:100; AB144P, Milipore), polyclonal goat anti- VAChT (1:500, AB1578, Milipore), polyclonal rabbit anti-Vesicular glutamate transporter 1 (VGLUT1) (1:500, 135002, Synaptic Systems), polyclonal rabbit- and guinea pig anti-VGLUT1 (1:300 both, ( Fujiyama et al., 2001 )), polyclonal rabbit anti-Calbindin (1:5000, CB38, Swant), monoclonal mouse anti-Parvalbumin (1:500, MAB1572, Chemicon), monoclonal rabbit anti-β-tubulin III (TUJ1) (1:1000, TUJ1-1-15-79, Covance) and Alexa 488-conjugated bungarotoxin (1:1000, B13422, Invitrogen).

    Techniques: MANN-WHITNEY