Journal: PLoS ONE

Article Title: Diffusion Barriers Constrain Receptors at Synapses

doi: 10.1371/journal.pone.0043032

Figure Lengend Snippet: Diffusion properties of GABAAR γ2 and α5 subunits, and of AMPAR GluA2 subunit in relation with excitatory and inhibitory synapses.

Article Snippet: Fritschy), rabbit anti- GABA A R γ2 subunit (1∶100; Alomone Labs, Jerusalem, Israel), mouse anti-gephyrin (mAb7a, 1.25 µg/ml; Synaptic Systems, Gottingen, Germany) and mouse anti-GluA2 (1∶200; BD Pharmingen, Franklin Lakes, USA).

Techniques: Diffusion-based Assay

Journal: PLoS ONE

Article Title: Diffusion Barriers Constrain Receptors at Synapses

doi: 10.1371/journal.pone.0043032

Figure Lengend Snippet: (A) Examples of GABA A R γ2 trajectories at inhibitory synapses for passing (top) and trapped (bottom) receptors outside (black) or inside (grey) synapses. Scale, 0.5 µm. (B) Proportion (mean ± SEM) of γ2 (black) and α5 (white) trajectories classified as trapped at inhibitory synapses (t-test, **p<10 −2 ). (C) Trapped (trap) γ2 (black) and α5 (white) have lower mobility (median D ±25%–75% IQR) in inhibitory synapses than passing ones (KS test, ***p<10 −3 ). (D) Passing (pass) γ2 (black) and α5 (white) explored a similar surface area (median EA ±25%–75% IQR) while trapped γ2 explored a smaller surface area than α5 (KS test, *p<5×10 −2 ; ***p<10 −3 ).

Article Snippet: Fritschy), rabbit anti- GABA A R γ2 subunit (1∶100; Alomone Labs, Jerusalem, Israel), mouse anti-gephyrin (mAb7a, 1.25 µg/ml; Synaptic Systems, Gottingen, Germany) and mouse anti-GluA2 (1∶200; BD Pharmingen, Franklin Lakes, USA).

Techniques:

Journal: PLoS ONE

Article Title: Diffusion Barriers Constrain Receptors at Synapses

doi: 10.1371/journal.pone.0043032

Figure Lengend Snippet: (A) Dendritic portion of a neuron co-transfected with SEP-GABA A Rγ2 (green) and DsRed-homer1c (red) constructs. Scale, 1 µm. Note that SEP-GABA A R γ2 (green) form clusters that are not colocalized with DsRed-homer1c (red) fluorescent clusters. (B) Normalized SEP-GABA A Rγ2 FRAP fluorescence recovery curves at inhibitory synapses (green, inh), excitatory synapses (red, exc), and in extrasynaptic compartment (black, ex). (C) Percentage (mean ± SEM) of SEP-γ2 fluorescence recovery at inhibitory synapses (inh, green), excitatory synapses (exc, red) and extrasynaptically (ex, black). Note the increase in the size of the pool of slowly mobile γ2 at excitatory synapses as compared with the extrasynaptic compartment (t-test, ***p<10 −3 ). (D–E) Time constants (mean ± SEM) for the fast (D) and slow (E) pool, obtained from the double exponential fit (see Materials and Methods) applied to the data in B for inhibitory (green, inh), excitatory (red, exc) synapses or extrasynaptic area (black, ex) (t test, ns: not significant, *: p<0.05, **: p<0.01; ***: p<0.001).

Article Snippet: Fritschy), rabbit anti- GABA A R γ2 subunit (1∶100; Alomone Labs, Jerusalem, Israel), mouse anti-gephyrin (mAb7a, 1.25 µg/ml; Synaptic Systems, Gottingen, Germany) and mouse anti-GluA2 (1∶200; BD Pharmingen, Franklin Lakes, USA).

Techniques: Transfection, Construct, Fluorescence

Journal: PLoS ONE

Article Title: Diffusion Barriers Constrain Receptors at Synapses

doi: 10.1371/journal.pone.0043032

Figure Lengend Snippet: (A) Hippocampal cultured neurons from mRFP-gephyrin knock-in mice transfected with GFP, venus-G(2), or venus-E constructs, and stained for γ2. Scale, 2 µm. Note that mRFP-gephyrin and GABA A R γ2 formed numerous clusters along dendrites of GFP transfected neurons. Many mRFP-gephyrin clusters were colocalized with GABA A R γ2 clusters (arrows). Over-expression of the venus-G(2) and venus-E chimera resulted in a marked reduction of the number and intensity of mRFP-gephyrin and GABA A R γ2 clusters. The remaining mRFP-gephyrin clusters were rarely colocalized with γ2 clusters (arrows). (B-E) Quantifications showing venus-G(2) and venus-E constructs reduced both the number of gephyrin (B) and γ2 (D) clusters per 10 µm dendritic length and the fluorescence intensity of the corresponding clusters (C, E). Values are mean ± SEM. t-test, ns: not significant, **: p<0.01; ***: p<10 −3 .

Article Snippet: Fritschy), rabbit anti- GABA A R γ2 subunit (1∶100; Alomone Labs, Jerusalem, Israel), mouse anti-gephyrin (mAb7a, 1.25 µg/ml; Synaptic Systems, Gottingen, Germany) and mouse anti-GluA2 (1∶200; BD Pharmingen, Franklin Lakes, USA).

Techniques: Cell Culture, Knock-In, Transfection, Construct, Staining, Over Expression, Fluorescence

Journal: The Journal of Cell Biology

Article Title: The adhesion protein IgSF9b is coupled to neuroligin 2 via S-SCAM to promote inhibitory synapse development

doi: 10.1083/jcb.201209132

Figure Lengend Snippet: IgSF9b is abundant in GABAergic interneurons and mainly present at inhibitory synapses. (A) Domain structure of IgSF9b. SP, signal peptide; Ig, immunoglobulin domain; FNIII, fibronectin type III domain; TM, transmembrane domain; PB, PDZ domain-binding motif. (B) IgSF9b signals are more abundant in interneurons than in pyramidal neurons. Cultured hippocampal neurons (DIV 28, a stage after IgSF9b reaches a plateau of its expression) were labeled by triple immunofluorescence staining for IgSF9b, GAD67, and MAP2. Yellow and red asterisks indicate cell bodies of GAD67-positive interneurons and GAD67-negative pyramidal neurons, respectively. (C and D) IgSF9b colocalizes with VGAT (an inhibitory presynaptic protein) both in pyramidal neurons (C) and in interneurons (D). The bottom images are enlarged views of the boxed regions. (E–G) IgSF9b colocalizes with gephyrin (E) and the α1 subunit of GABA A receptors (F) at VGAT- or GAD65 (an inhibitory presynaptic protein)-positive inhibitory synapses, but not with vGlut1 (an excitatory presynaptic protein)- or Shank (an excitatory postsynaptic protein)-positive excitatory synapses (G) in interneurons. (H–J) In cultured cortical neurons at DIV 28, IgSF9b is more abundantly expressed in GAD65-positive interneurons than in GAD65-negative pyramidal neurons, and colocalizes with GAD65-positive inhibitory synapses (I and J). (K and L) IgSF9b colocalizes with gephyrin and GAD65 but not with PSD-95 in dendrites of cortical interneurons. Bars: (B) 20 µm; (C and D, top rows) 20 µm; (C and D, bottom rows) 5 µm; (E–G) 10 µm; (H) 20 µm; (I–L) 5 µm.

Article Snippet: The following antibodies are commercially available: GABA A Rγ2, vGlut1, VGAT, gephyrin, neuroligin 2 (Synaptic Systems); neuroligin1, Myc, and HA (Santa Cruz Biotechnology, Inc.); IgSF9b (HPA010802; rabbit), MAP2, α-tubulin, Flag, and synaptophysin (Sigma-Aldrich); PSD-95 (Thermo Fisher Scientific); GABA A Rα1 (Alomone Labs); GAD67, synapsin I (EMD Millipore), and GAD65 (Developmental Studies Hybridoma Bank); GAD65/67 (Enzo Life Sciences); and Cy3-conjugated anti-Fc (Jackson ImmunoResearch Laboratories, Inc.).

Techniques: Binding Assay, Cell Culture, Expressing, Labeling, Immunofluorescence, Staining

Journal: The Journal of Cell Biology

Article Title: The adhesion protein IgSF9b is coupled to neuroligin 2 via S-SCAM to promote inhibitory synapse development

doi: 10.1083/jcb.201209132

Figure Lengend Snippet: Gephyrin colocalizes with neuroligin 2 and the γ2 subunit of GABA A receptors, whereas IgSF9b displays close but minimally overlapping localization with gephyrin or the γ2 subunit of GABA A receptors. (A) In normal-resolution immunofluorescence imaging, IgSF9b and gephyrin show closely associated but distinct distribution patterns at GAD65-positive inhibitory synapses. Cultured hippocampal neurons (DIV 21) were triply stained for IgSF9b, gephyrin, and GAD65. (B and C) Cultured hippocampal neurons (DIV 21) were visualized by the combinations of double staining indicated and normal (non-STORM) light microscopy. Bars: (A, left) 20 µm; (A, right) 2 µm; (B and C) 1 µm.

Article Snippet: The following antibodies are commercially available: GABA A Rγ2, vGlut1, VGAT, gephyrin, neuroligin 2 (Synaptic Systems); neuroligin1, Myc, and HA (Santa Cruz Biotechnology, Inc.); IgSF9b (HPA010802; rabbit), MAP2, α-tubulin, Flag, and synaptophysin (Sigma-Aldrich); PSD-95 (Thermo Fisher Scientific); GABA A Rα1 (Alomone Labs); GAD67, synapsin I (EMD Millipore), and GAD65 (Developmental Studies Hybridoma Bank); GAD65/67 (Enzo Life Sciences); and Cy3-conjugated anti-Fc (Jackson ImmunoResearch Laboratories, Inc.).

Techniques: Immunofluorescence, Imaging, Cell Culture, Staining, Double Staining, Light Microscopy