Journal: Neuroscience

Article Title: Connexin and AMPA Receptor Expression Changes Over Time in the Rat Olfactory Bulb

doi: 10.1016/j.neuroscience.2012.06.070

Figure Lengend Snippet: Statistical results for measured mRNA

Article Snippet: Primary antisera used in this study were purchased from Cell Signaling Technology (CST; Danvers, MA, USA), Invitrogen, Millipore (Billerica, MA, USA), and Sigma–Aldrich and are as follows, with the epitope, dilution/buffer, and catalog number in parentheses: Invitrogen rabbit anti-Cx36 (cytoplasmic loop between second and third transmembrane domains; 1:250 in 4% milk/TBST; 51-6200), Invitrogen rabbit anti-Cx36 (C-terminus; 1:250 in 1% milk/TBST; 51-6300), Invitrogen mouse anti-Cx36 (C-terminus; 1:500 in 4% milk/TBST; 37-4600), Invitrogen rabbit anti-Cx43 (C-terminus; 1:500 in OBBT; 71-0700), Invitrogen rabbit anti-Cx45 (C-terminus; 1:250/OBBT; 40-7000), Millipore mouse anti-GluR1 (N-terminus; 1:500/OBBT; MAB2263), CST rabbit anti-GluR2 (N-terminus; 1:500/OBBT; 5306S), CST rabbit anti-GluR3 (residues surrounding Pro590 of human GluR3; 1:1500/OBBT; 4676S), CST rabbit anti-GluR4 (residues surrounding Gln890 of human GluR4; 1:1000 in 5% bovine serum albumin/TBST*; 8070S), CST mouse anti-β-III-tubulin (C-terminus; 1:2000/OBBT; 4466S), and Sigma–Aldrich rabbit anti-β-III-tubulin (residues 442–446; 1:5000 in 4% milk/TBST; SAB4300623).

Techniques:

Journal: Neuroscience

Article Title: Connexin and AMPA Receptor Expression Changes Over Time in the Rat Olfactory Bulb

doi: 10.1016/j.neuroscience.2012.06.070

Figure Lengend Snippet: Changes in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunit mRNA across dark and light phases in rat OB. Line plots of (A) GluR1, (B) GluR2, (C) GluR3, and (D) GluR4 mRNA expression over 48-h. Sample size and notation as in Fig. 1. GluR1, GluR2, and GluR3 mRNA changes were statistically significant across the 48-h (Kruskal–Wallis H test, P < 0.05) but only GluR1 was rhythmic for both 24-h periods (harmonic regression analysis, P < 0.05). GluR1 and GluR4 were rhythmic according to JTK_CYCLE analysis (P < 0.05).

Article Snippet: Primary antisera used in this study were purchased from Cell Signaling Technology (CST; Danvers, MA, USA), Invitrogen, Millipore (Billerica, MA, USA), and Sigma–Aldrich and are as follows, with the epitope, dilution/buffer, and catalog number in parentheses: Invitrogen rabbit anti-Cx36 (cytoplasmic loop between second and third transmembrane domains; 1:250 in 4% milk/TBST; 51-6200), Invitrogen rabbit anti-Cx36 (C-terminus; 1:250 in 1% milk/TBST; 51-6300), Invitrogen mouse anti-Cx36 (C-terminus; 1:500 in 4% milk/TBST; 37-4600), Invitrogen rabbit anti-Cx43 (C-terminus; 1:500 in OBBT; 71-0700), Invitrogen rabbit anti-Cx45 (C-terminus; 1:250/OBBT; 40-7000), Millipore mouse anti-GluR1 (N-terminus; 1:500/OBBT; MAB2263), CST rabbit anti-GluR2 (N-terminus; 1:500/OBBT; 5306S), CST rabbit anti-GluR3 (residues surrounding Pro590 of human GluR3; 1:1500/OBBT; 4676S), CST rabbit anti-GluR4 (residues surrounding Gln890 of human GluR4; 1:1000 in 5% bovine serum albumin/TBST*; 8070S), CST mouse anti-β-III-tubulin (C-terminus; 1:2000/OBBT; 4466S), and Sigma–Aldrich rabbit anti-β-III-tubulin (residues 442–446; 1:5000 in 4% milk/TBST; SAB4300623).

Techniques: Expressing

Journal: Neuroscience

Article Title: Connexin and AMPA Receptor Expression Changes Over Time in the Rat Olfactory Bulb

doi: 10.1016/j.neuroscience.2012.06.070

Figure Lengend Snippet: Changes in AMPAR subunit protein expressed in the rat OB membrane across light and dark phases in the rat OB. (A) Anti-GluR3 (1:1500) and anti-beta-III-tubulin (1:2000) and (B) anti-GluR4 (1:1000) using experimental design, analysis, notation, sample size, and statistical metric as in Fig. 4. Nitrocellulose blots probed with anti-GluR4 were stripped and reprobed with anti-β-III-tubulin (1:2000). Arrows indicate expected size in kilodalton (Mr = 100 kDa for GluR3 and GluR4, and 50 kDa for tubulin). GluR3 and GluR4 protein changes were statistically significant across the 48-h (Kruskal–Wallis H test, P < 0.05) but not rhythmic across either 24-h tested (harmonic regression analysis, P > 0.05). GluR3 protein expression was statistically significant for JTK_CYCLE analysis (P < 0.05).

Article Snippet: Primary antisera used in this study were purchased from Cell Signaling Technology (CST; Danvers, MA, USA), Invitrogen, Millipore (Billerica, MA, USA), and Sigma–Aldrich and are as follows, with the epitope, dilution/buffer, and catalog number in parentheses: Invitrogen rabbit anti-Cx36 (cytoplasmic loop between second and third transmembrane domains; 1:250 in 4% milk/TBST; 51-6200), Invitrogen rabbit anti-Cx36 (C-terminus; 1:250 in 1% milk/TBST; 51-6300), Invitrogen mouse anti-Cx36 (C-terminus; 1:500 in 4% milk/TBST; 37-4600), Invitrogen rabbit anti-Cx43 (C-terminus; 1:500 in OBBT; 71-0700), Invitrogen rabbit anti-Cx45 (C-terminus; 1:250/OBBT; 40-7000), Millipore mouse anti-GluR1 (N-terminus; 1:500/OBBT; MAB2263), CST rabbit anti-GluR2 (N-terminus; 1:500/OBBT; 5306S), CST rabbit anti-GluR3 (residues surrounding Pro590 of human GluR3; 1:1500/OBBT; 4676S), CST rabbit anti-GluR4 (residues surrounding Gln890 of human GluR4; 1:1000 in 5% bovine serum albumin/TBST*; 8070S), CST mouse anti-β-III-tubulin (C-terminus; 1:2000/OBBT; 4466S), and Sigma–Aldrich rabbit anti-β-III-tubulin (residues 442–446; 1:5000 in 4% milk/TBST; SAB4300623).

Techniques: Expressing

Journal: Neuroscience

Article Title: Connexin and AMPA Receptor Expression Changes Over Time in the Rat Olfactory Bulb

doi: 10.1016/j.neuroscience.2012.06.070

Figure Lengend Snippet: Statistical results for measured membrane/organelle protein

Article Snippet: Primary antisera used in this study were purchased from Cell Signaling Technology (CST; Danvers, MA, USA), Invitrogen, Millipore (Billerica, MA, USA), and Sigma–Aldrich and are as follows, with the epitope, dilution/buffer, and catalog number in parentheses: Invitrogen rabbit anti-Cx36 (cytoplasmic loop between second and third transmembrane domains; 1:250 in 4% milk/TBST; 51-6200), Invitrogen rabbit anti-Cx36 (C-terminus; 1:250 in 1% milk/TBST; 51-6300), Invitrogen mouse anti-Cx36 (C-terminus; 1:500 in 4% milk/TBST; 37-4600), Invitrogen rabbit anti-Cx43 (C-terminus; 1:500 in OBBT; 71-0700), Invitrogen rabbit anti-Cx45 (C-terminus; 1:250/OBBT; 40-7000), Millipore mouse anti-GluR1 (N-terminus; 1:500/OBBT; MAB2263), CST rabbit anti-GluR2 (N-terminus; 1:500/OBBT; 5306S), CST rabbit anti-GluR3 (residues surrounding Pro590 of human GluR3; 1:1500/OBBT; 4676S), CST rabbit anti-GluR4 (residues surrounding Gln890 of human GluR4; 1:1000 in 5% bovine serum albumin/TBST*; 8070S), CST mouse anti-β-III-tubulin (C-terminus; 1:2000/OBBT; 4466S), and Sigma–Aldrich rabbit anti-β-III-tubulin (residues 442–446; 1:5000 in 4% milk/TBST; SAB4300623).

Techniques:

Journal: bioRxiv

Article Title: A synthetic synaptic organizer protein restores glutamatergic neuronal circuits

doi: 10.1101/2020.02.27.967836

Figure Lengend Snippet: (A) The CPTX concept. While Cbln1 induces synapse formation by binding to postsynaptic GluD2 and presynaptic Nrx(+4), NP1 induces clustering of postsynaptic AMPARs without inducing presynaptic elements. CPTX was designed as a chimeric ESP of Cbln1 and NP1. (B) Crystal structure of the pentraxin domain of human NP1. The N- and C-termini are annotated. Calcium (Ca) atoms and water molecules are shown as green and red spheres, respectively. 2mFo-DFc electron density is contoured at 1.0 σ. The Ca 2+ coordination shell, which includes a cacodylate buffer molecule from the crystallization solution, is indicated by black lines. (C) Diagram of CPTX and related constructs. CC, coiled-coil domain; PTX, pentraxin domain, CRR, cysteine-rich region; gC1q, global C1q domain; 3Cl, triple coil. The right graph shows molecular masses of monomeric NP1 PTX , trimeric NP1 PTX-3Cl and hexameric CPTX. (D) Binding isotherms for the interactions between GluA1–4 ATD or GluD2 ATD and immobilized CPTX, NP1 PTX and Cbln1. The ATD of GluA4 interacted strongest with CPTX ( K D ∼ 4.5 ± 0.5 μM) and NP1 PTX ( K D ∼ 21.5 ± 1.0 μM), but not with Cbln1. In contrast, the ATD of GluD2 only interacted with Cbln1 ( K D >100 μM). (E) Single-cycle kinetic sensorgrams for the interaction between the ectodomain of Nrx1β(±4) and immobilized CPTX. CPTX directly bound to Nrx1β(+4) ( K D ∼ 4.90 ± 0.90 nM), but not to Nrx1β(–4).

Article Snippet: The origin, dilution, company and catalog number are described as follows: Anti-calbindin (Goat, 1:500, Frontier Institute, Af1040), anti-HIS (Mouse, 1:1000, MBL, D291-3 or Rabbit, 1:1000, CST, #2365), anti-Myc (Rabbit, 1:1000, MBL, 562), anti-FLAG (Rabbit, 1:1000, Sigma-Aldrich, F7425), anti-HA (Mouse, 1:1000, BAbCO, MMS-101P), anti-synaptophysin (Mouse, 1:500, Sigma-Aldrich, S5768 or G, 1:500, Frontier Institute, Af300), anti-neurexin (Chicken, 1:500, a gift from Peter Scheiffele , anti-GFP (Rabbit 1:1000, Frontier Institute, Af-2020Frontier Institute), anti-GluA1 (Rabbit, 1:100, Calbiochem, PC246 or Guinea Pig, 1:500, Frontier Institute, Af380), anti-GluA2/3 (Rabbit, 1:1000, Chemicon, AB1506), anti-GluA4 (Rabbit, 1:1000, Pharmingen, 60666N or Guinea Pig, 1:500, Frontier Institute, Af640), anti-VGluT1 (Rabbit, 1:500, Frontier Institute, Af570 or Goat, 1:500, Frontier Institute, Af310), anti-VGluT2 (Guinea Pig, 1:500, Frontier Institute, Af720 or Goat, 1:500, Frontier Institute, Af310), anti-VGAT (Goat, 1:500, Frontier Institute, Af620)), anti-Parvalbumin (Goat, 1:500, Frontier Institute, Af460), anti-Homer 1 (Guinea pig, 1:1000, Synaptic Systems, 160-004) and anti-Bassoon (Rabbit, 1:500, Synaptic Systems, 141-003).

Techniques: Binding Assay, Crystallization Assay, Construct

Journal: bioRxiv

Article Title: A synthetic synaptic organizer protein restores glutamatergic neuronal circuits

doi: 10.1101/2020.02.27.967836

Figure Lengend Snippet: ( A, B ) CPTX binds AMPARs and Nrx(+4) GluD2. Vehicle (mock treatment), HIS-tagged Cbln1 or CPTX-HIS (23.6 nM) was incubated with HEK cells expressing GFP or myc-tagged ATDs of GluA1–GluA4 AMPARs or the myc-tagged ATD of GluD2 ( A ). Cbln1-HIS or CPTX-HIS was incubated with HEK cells expressing Flag-tagged various Nrx isoforms with or without S4 or GFP ( B ). The intensities of HIS immunoreactivity (magenta) on green-positive cells were quantified in the right graphs. The bars represent mean ± SEM values. *** P < 0.001, n.s. means not significant, n = 5–15 fields in more than 3 independent experiments, one-way ANOVA followed by Bonferroni test (compared with the other ligand treatment in each receptor). ( C ) CPTX mediates a tripartite complex consisting of Nrx(+4), CPTX and GluAs. CPTX-HIS (23.6 nM) was incubated with HEK cells expressing GFP or Myc-tagged GluA4-ATD, followed by the conditioned medium of HEK cells transfected with the human Fc (hFc)–tagged extracellular domain (ECD) of Nrx1β(+4) or empty vector (mock treatment). The intensities of hFc (red) and HIS (blue) immunoreactivity on Myc-positive cells (green) were quantified in the right graph. The bars represent mean ± SEM values. * P < 0.05, *** P < 0.001, n = 10–15 fields in 3 independent experiments, one-way ANOVA followed by Tukey’s test (compared with GFP + CPTX-HIS + Nrx-hFc).

Article Snippet: The origin, dilution, company and catalog number are described as follows: Anti-calbindin (Goat, 1:500, Frontier Institute, Af1040), anti-HIS (Mouse, 1:1000, MBL, D291-3 or Rabbit, 1:1000, CST, #2365), anti-Myc (Rabbit, 1:1000, MBL, 562), anti-FLAG (Rabbit, 1:1000, Sigma-Aldrich, F7425), anti-HA (Mouse, 1:1000, BAbCO, MMS-101P), anti-synaptophysin (Mouse, 1:500, Sigma-Aldrich, S5768 or G, 1:500, Frontier Institute, Af300), anti-neurexin (Chicken, 1:500, a gift from Peter Scheiffele , anti-GFP (Rabbit 1:1000, Frontier Institute, Af-2020Frontier Institute), anti-GluA1 (Rabbit, 1:100, Calbiochem, PC246 or Guinea Pig, 1:500, Frontier Institute, Af380), anti-GluA2/3 (Rabbit, 1:1000, Chemicon, AB1506), anti-GluA4 (Rabbit, 1:1000, Pharmingen, 60666N or Guinea Pig, 1:500, Frontier Institute, Af640), anti-VGluT1 (Rabbit, 1:500, Frontier Institute, Af570 or Goat, 1:500, Frontier Institute, Af310), anti-VGluT2 (Guinea Pig, 1:500, Frontier Institute, Af720 or Goat, 1:500, Frontier Institute, Af310), anti-VGAT (Goat, 1:500, Frontier Institute, Af620)), anti-Parvalbumin (Goat, 1:500, Frontier Institute, Af460), anti-Homer 1 (Guinea pig, 1:1000, Synaptic Systems, 160-004) and anti-Bassoon (Rabbit, 1:500, Synaptic Systems, 141-003).

Techniques: Incubation, Expressing, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: A synthetic synaptic organizer protein restores glutamatergic neuronal circuits

doi: 10.1101/2020.02.27.967836

Figure Lengend Snippet: ( A ) Application of recombinant CPTX, but not Cbln1 or vehicle (mock treatment), induces accumulation of synaptophysin (Syp). Representative immunocytochemical images show accumulation of endogenous presynaptic sites (magenta; Syp, upper panels; Nrx, lower panels) of co-cultured hippocampal neurons on HEK cells displaying the ATDs of AMPA receptors, but not GluD2 (green). Scale bars, 30 μm. The right graph shows averaged intensities of Syp immunoreactivity on HEK cells expressing GluA or GluD2 ATDs. The bars represent mean ± SEM values. *** P < 0.001, n = 10–15 fields in 4 independent experiments, Bonferroni test (compared with the other ligand treatment in each receptor). ( B ) Application of recombinant NP1 does not induce accumulation of Syp. HEK cells overexpressing GluA4-ATD were treated with the conditioned medium from HEK cell overexpressing empty vector (mock treatment), HA-tagged full-length NP1 or HIS-tagged CPTX (blue). Representative immunocytochemical images show that while both NP1 and CPTX (blue) bind to HEK cells expressing GluA4-ATD, only CPTX accumulates endogenous Syp (red). The right graph shows averaged intensities of Syp and HIS/HA immunoreactivity on HEK cells expressing GluA4-ATD. The bars represent mean ± SEM values. n = 15 fields in 3 independent experiments, one-way ANOVA followed by Tukey’s test (* P < 0.001 compared with the mock ligand treatment). ( C ) Beads coated with Cbln1 or CPTX, but not mock (beads treated with vehicle), accumulate endogenous Nrx (blue) and VGluT1 (red) immunoreactivities in axons of co-cultured wild-type hippocampal neurons. The right graph shows averaged intensities of VGluT1 and Nrx immunoreactivities on beads. * P < 0.05, ** P < 0.001, n = 20 fields in 3 independent experiments, one-way ANOVA followed by Tukey’s test (compared with mock).

Article Snippet: The origin, dilution, company and catalog number are described as follows: Anti-calbindin (Goat, 1:500, Frontier Institute, Af1040), anti-HIS (Mouse, 1:1000, MBL, D291-3 or Rabbit, 1:1000, CST, #2365), anti-Myc (Rabbit, 1:1000, MBL, 562), anti-FLAG (Rabbit, 1:1000, Sigma-Aldrich, F7425), anti-HA (Mouse, 1:1000, BAbCO, MMS-101P), anti-synaptophysin (Mouse, 1:500, Sigma-Aldrich, S5768 or G, 1:500, Frontier Institute, Af300), anti-neurexin (Chicken, 1:500, a gift from Peter Scheiffele , anti-GFP (Rabbit 1:1000, Frontier Institute, Af-2020Frontier Institute), anti-GluA1 (Rabbit, 1:100, Calbiochem, PC246 or Guinea Pig, 1:500, Frontier Institute, Af380), anti-GluA2/3 (Rabbit, 1:1000, Chemicon, AB1506), anti-GluA4 (Rabbit, 1:1000, Pharmingen, 60666N or Guinea Pig, 1:500, Frontier Institute, Af640), anti-VGluT1 (Rabbit, 1:500, Frontier Institute, Af570 or Goat, 1:500, Frontier Institute, Af310), anti-VGluT2 (Guinea Pig, 1:500, Frontier Institute, Af720 or Goat, 1:500, Frontier Institute, Af310), anti-VGAT (Goat, 1:500, Frontier Institute, Af620)), anti-Parvalbumin (Goat, 1:500, Frontier Institute, Af460), anti-Homer 1 (Guinea pig, 1:1000, Synaptic Systems, 160-004) and anti-Bassoon (Rabbit, 1:500, Synaptic Systems, 141-003).

Techniques: Recombinant, Cell Culture, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: A synthetic synaptic organizer protein restores glutamatergic neuronal circuits

doi: 10.1101/2020.02.27.967836

Figure Lengend Snippet: ( A ) Co-expression of CPTX and Nrx1β(+4) facilitates the accumulation of endogenous AMPARs in co-cultured hippocampal neurons. Hippocampal neurons were co-cultured with HEK cells expressing a combination of Nrx1β(+4) and HIS-tagged CPTX or Cbln1-HIS or empty vector (mock). Representative immunocytochemical staining shows that endogenous GluA1–3 (red) in hippocampal neurons accumulated on HEK cells co-expressing Nrx (blue) and CPTX (green). The right graphs show quantification of GFP/HIS, Nrx (middle) and GluA1–3 (bottom) on HEK cells. The bars represent mean ± SEM. *** P < 0.001, n.s., not significant, n = 15 fields from 3 independent experiments, one-way ANOVA followed by Tukey’s test. ( B ) Beads coated with recombinant CPTX recruit GluA4 in dendrites of parvalbumin-positive (PV + ) interneurons. Representative immunocytochemical staining shows that endogenous GluA4 (magenta) in PV + interneurons (green) highly accumulated on contacting beads (arrows) coated with CPTX, but not Cbln1 or vehicle (mock). The right graph shows the quantification of GluA4 immunoreactivities on beads contacting dendrites of PV + interneurons (arrow) subtracted by that on beads non-contacting. The bars represent mean ± SEM values. *** P < 0.001, n = 25 fields from 4 independent experiments, one-way ANOVA followed by Tukey’s test. ( C ) CPTX, but not Cbln1, colocalize with AMPARs. Representative immunocytochemical staining shows endogenous GluA1–3 (red) and pan-Nrx (green) in hippocampal neurons incubated with vehicle (mock), Cbln1-HIS or CPTX-HIS (blue). Scale bar, 2 μm.

Article Snippet: The origin, dilution, company and catalog number are described as follows: Anti-calbindin (Goat, 1:500, Frontier Institute, Af1040), anti-HIS (Mouse, 1:1000, MBL, D291-3 or Rabbit, 1:1000, CST, #2365), anti-Myc (Rabbit, 1:1000, MBL, 562), anti-FLAG (Rabbit, 1:1000, Sigma-Aldrich, F7425), anti-HA (Mouse, 1:1000, BAbCO, MMS-101P), anti-synaptophysin (Mouse, 1:500, Sigma-Aldrich, S5768 or G, 1:500, Frontier Institute, Af300), anti-neurexin (Chicken, 1:500, a gift from Peter Scheiffele , anti-GFP (Rabbit 1:1000, Frontier Institute, Af-2020Frontier Institute), anti-GluA1 (Rabbit, 1:100, Calbiochem, PC246 or Guinea Pig, 1:500, Frontier Institute, Af380), anti-GluA2/3 (Rabbit, 1:1000, Chemicon, AB1506), anti-GluA4 (Rabbit, 1:1000, Pharmingen, 60666N or Guinea Pig, 1:500, Frontier Institute, Af640), anti-VGluT1 (Rabbit, 1:500, Frontier Institute, Af570 or Goat, 1:500, Frontier Institute, Af310), anti-VGluT2 (Guinea Pig, 1:500, Frontier Institute, Af720 or Goat, 1:500, Frontier Institute, Af310), anti-VGAT (Goat, 1:500, Frontier Institute, Af620)), anti-Parvalbumin (Goat, 1:500, Frontier Institute, Af460), anti-Homer 1 (Guinea pig, 1:1000, Synaptic Systems, 160-004) and anti-Bassoon (Rabbit, 1:500, Synaptic Systems, 141-003).

Techniques: Expressing, Cell Culture, Plasmid Preparation, Staining, Recombinant, Incubation

Journal: bioRxiv

Article Title: A synthetic synaptic organizer protein restores glutamatergic neuronal circuits

doi: 10.1101/2020.02.27.967836

Figure Lengend Snippet: ( A ) Co-immunostaining of HIS (CPTX) and each GluA subunit in the CA1 region of wild-type hippocampus ( stratum radiatum ) 1 d after CPTX injection. White arrowhead indicates the colocalization while yellow arrowhead indicates the CPTX puncta without colocalization with AMPA receptor. Scale bar, 3 μm. ( B ) Quantification of the mean intensity and variance of each GluA subunit. The bar represents the mean ± SEM. * P < 0.05, by Student’s t -test with Holm correction, n = 8–11 images from 3–5 mice. ( C ) Representative GluA1, GluA2/3 and GluA4 immunoreactivities. Contralateral uninjected regions are shown for comparison. Scale bars, 5 μm. ( D ) Normalized intensity histograms of GluA1, GluA2/3 and GluA4 in injected (CPTX) and contralateral (Contra) CA1 regions. While CPTX shifted the histogram of GluA1 and GluA4 to the higher value, it increased the fraction of darker GluA2/3 signals (bins 14–18). Inset shows the magnified area of the histogram corresponding to the bins > mean + 3 SD intensity. * P < 0.05, by multi-way analysis of variance (in the bin of mean + 3 SD intensity for GluA1 and GluA4, and in the whole bin for GluA2/3) with post-hoc paired t-test between contra and +CPTX at bins 23–35 in GluA1, bins 13–15 and 27–40 in GluA2/3. Each point represents mean ± SEM. n = 8–11 images from 3–5 mice. ( E ) Representative high-intensity immunopositive puncta of each GluA subunit. Pixels that had > mean intensity + 3 SD of the contralateral area are shown in red. ( F ) Quantification of the size, number, total area occupied by particles and intensity of high-intensity (mean + 3 x SD) puncta of each GluA subunit. CPTX increased the size of high-intensity particles of GluA1 and GluA2/3, but not GluA4. Particle number, area and intensity were normalized with those in the contralateral images. * P < 0.05, ** P < 0.01, by Student’s t -test with Holm correction. The bar represents the mean ± SEM. n = 8–11 images from 3–5 mice.

Article Snippet: The origin, dilution, company and catalog number are described as follows: Anti-calbindin (Goat, 1:500, Frontier Institute, Af1040), anti-HIS (Mouse, 1:1000, MBL, D291-3 or Rabbit, 1:1000, CST, #2365), anti-Myc (Rabbit, 1:1000, MBL, 562), anti-FLAG (Rabbit, 1:1000, Sigma-Aldrich, F7425), anti-HA (Mouse, 1:1000, BAbCO, MMS-101P), anti-synaptophysin (Mouse, 1:500, Sigma-Aldrich, S5768 or G, 1:500, Frontier Institute, Af300), anti-neurexin (Chicken, 1:500, a gift from Peter Scheiffele , anti-GFP (Rabbit 1:1000, Frontier Institute, Af-2020Frontier Institute), anti-GluA1 (Rabbit, 1:100, Calbiochem, PC246 or Guinea Pig, 1:500, Frontier Institute, Af380), anti-GluA2/3 (Rabbit, 1:1000, Chemicon, AB1506), anti-GluA4 (Rabbit, 1:1000, Pharmingen, 60666N or Guinea Pig, 1:500, Frontier Institute, Af640), anti-VGluT1 (Rabbit, 1:500, Frontier Institute, Af570 or Goat, 1:500, Frontier Institute, Af310), anti-VGluT2 (Guinea Pig, 1:500, Frontier Institute, Af720 or Goat, 1:500, Frontier Institute, Af310), anti-VGAT (Goat, 1:500, Frontier Institute, Af620)), anti-Parvalbumin (Goat, 1:500, Frontier Institute, Af460), anti-Homer 1 (Guinea pig, 1:1000, Synaptic Systems, 160-004) and anti-Bassoon (Rabbit, 1:500, Synaptic Systems, 141-003).

Techniques: Immunostaining, Injection

Journal: bioRxiv

Article Title: A synthetic synaptic organizer protein restores glutamatergic neuronal circuits

doi: 10.1101/2020.02.27.967836

Figure Lengend Snippet: ( A, B ) Endogenous NPs highly colocalize with GluA4 in PV + interneurons. Representative immunohistochemical images show endogenous NPs (green; NP1 [A], NPR [B]) colocalize with VGluT1 (blue) and GluA4 (red) in PV + interneurons in the hippocampus. Scale bar, 1 μm ( C–F ). Validation of antibodies against NP1 and NPR. ( C ) Conditioned medium (CM) containing NP1, NP2 or NPR lacking transmembrane domain (ΔTM) expressed in HEK293 cells and hippocampal tissue lysates were analyzed by western blotting with the antibodies against HA, NP1 or NPR. Anti-NP1 and NPR showed the selective band for the overexpressed NP1 or NPR, respectively, and similar size of band in the hippocampal lysate. ( D ) Immunocytochemical analysis of HA-tagged NP1, NP2 and NPR (full-length) expressed in HEK293 cells. Anti-NP1 and NPR antibody showed selective signals for NP1 or NPR, respectively. ( E, F ) Specific immunostaining of endogenous NP1 and NPR in the cortex. cDNAs encoding GFP and control miRNA or miRNAs against NP1 ( E ) or NPR ( F ) were expressed in layer 2/3 cortical neurons by in utero electroporation. Representative immunohistochemical images after 4 weeks show a reduction of NPs immunoreactivities (magenta) in neurons (green) expressing miRNA against NPs compared to control miRNAs. Scale bars, 10 μm. The graphs show the quantification of the immunoreactivity of NP1 ( E ) or NPR ( F ). The bars represent mean ± SEM. *** P < 0.001, n = 15–47 cells, Student’s t -test. ( G ) Representative NP1 and NPR immunoreactivities in the hippocampal CA1 region. The intensity histogram is shown for each layer. Scale bar, 50 μm.

Article Snippet: The origin, dilution, company and catalog number are described as follows: Anti-calbindin (Goat, 1:500, Frontier Institute, Af1040), anti-HIS (Mouse, 1:1000, MBL, D291-3 or Rabbit, 1:1000, CST, #2365), anti-Myc (Rabbit, 1:1000, MBL, 562), anti-FLAG (Rabbit, 1:1000, Sigma-Aldrich, F7425), anti-HA (Mouse, 1:1000, BAbCO, MMS-101P), anti-synaptophysin (Mouse, 1:500, Sigma-Aldrich, S5768 or G, 1:500, Frontier Institute, Af300), anti-neurexin (Chicken, 1:500, a gift from Peter Scheiffele , anti-GFP (Rabbit 1:1000, Frontier Institute, Af-2020Frontier Institute), anti-GluA1 (Rabbit, 1:100, Calbiochem, PC246 or Guinea Pig, 1:500, Frontier Institute, Af380), anti-GluA2/3 (Rabbit, 1:1000, Chemicon, AB1506), anti-GluA4 (Rabbit, 1:1000, Pharmingen, 60666N or Guinea Pig, 1:500, Frontier Institute, Af640), anti-VGluT1 (Rabbit, 1:500, Frontier Institute, Af570 or Goat, 1:500, Frontier Institute, Af310), anti-VGluT2 (Guinea Pig, 1:500, Frontier Institute, Af720 or Goat, 1:500, Frontier Institute, Af310), anti-VGAT (Goat, 1:500, Frontier Institute, Af620)), anti-Parvalbumin (Goat, 1:500, Frontier Institute, Af460), anti-Homer 1 (Guinea pig, 1:1000, Synaptic Systems, 160-004) and anti-Bassoon (Rabbit, 1:500, Synaptic Systems, 141-003).

Techniques: Immunohistochemical staining, Western Blot, Immunostaining, In Utero, Electroporation, Expressing

Journal: bioRxiv

Article Title: A synthetic synaptic organizer protein restores glutamatergic neuronal circuits

doi: 10.1101/2020.02.27.967836

Figure Lengend Snippet: ( A ) Co-immunostaining of HIS (CPTX) and each GluA subunit in the CA1 region of wild-type hippocampus ( stratum lacunosum moleculare ) 1 d after CPTX injection. White arrowhead indicates the colocalization of CPTX and GluA1 or GluA2/3. GluA4 signals are weak in this layer. Scale bar, 3 μm. ( B ) Quantification of the mean intensity and variance of each GluA subunit. The bar represents the mean ± SEM. * P < 0.05, by Student’s t -test with Holm correction, n = 8–11 images from 3–5 mice. ( C ) Representative GluA1, GluA2/3 and GluA4 immunoreactivities. Contralateral uninjected regions are shown for comparison. Scale bars, 5 μm. ( D ) Normalized intensity histograms of GluA1, GluA2/3 and GluA4 in injected (CPTX) and contralateral (Contra) CA1 regions. While CPTX shifted the histogram of GluA1 to the higher value, it increased the fraction of darker GluA2/3 signals (bins 14–18). Inset shows the magnified area of the histogram corresponding to the bins > mean + 3 SD intensity. * P < 0.05, by multi-way analysis of variance (in the bin of mean + 3 SD intensity for GluA1 and GluA4, and in the whole bin for GluA2/3) with post-hoc paired t-test between contra and +CPTX at bins 22–35 in GluA1, bins 11-13, 18-20 and 25–33 in GluA2/3. Each point represents the mean ± SEM. n = 8–11 images from 3–5 mice. The bar represents the mean ± SEM. ( E ) Representative high-intensity immunopositive puncta of each GluA subunit. Pixels that had > mean intensity + 3 SD of the contralateral area are shown in red. ( F ) Quantification of the size, number, total area occupied by particles and intensity of high-intensity (mean + 3 x SD) puncta of each GluA subunit. CPTX increased the size of high-intensity particles of GluA1 and GluA2/3, but not GluA4. Particle number, area and intensity were normalized with those in the contralateral images. * P < 0.05, ** P < 0.01, by Student’s t -test with Holm correction. The bar represents the mean ± SEM. n = 8–11 images from 3–5 mice.

Article Snippet: The origin, dilution, company and catalog number are described as follows: Anti-calbindin (Goat, 1:500, Frontier Institute, Af1040), anti-HIS (Mouse, 1:1000, MBL, D291-3 or Rabbit, 1:1000, CST, #2365), anti-Myc (Rabbit, 1:1000, MBL, 562), anti-FLAG (Rabbit, 1:1000, Sigma-Aldrich, F7425), anti-HA (Mouse, 1:1000, BAbCO, MMS-101P), anti-synaptophysin (Mouse, 1:500, Sigma-Aldrich, S5768 or G, 1:500, Frontier Institute, Af300), anti-neurexin (Chicken, 1:500, a gift from Peter Scheiffele , anti-GFP (Rabbit 1:1000, Frontier Institute, Af-2020Frontier Institute), anti-GluA1 (Rabbit, 1:100, Calbiochem, PC246 or Guinea Pig, 1:500, Frontier Institute, Af380), anti-GluA2/3 (Rabbit, 1:1000, Chemicon, AB1506), anti-GluA4 (Rabbit, 1:1000, Pharmingen, 60666N or Guinea Pig, 1:500, Frontier Institute, Af640), anti-VGluT1 (Rabbit, 1:500, Frontier Institute, Af570 or Goat, 1:500, Frontier Institute, Af310), anti-VGluT2 (Guinea Pig, 1:500, Frontier Institute, Af720 or Goat, 1:500, Frontier Institute, Af310), anti-VGAT (Goat, 1:500, Frontier Institute, Af620)), anti-Parvalbumin (Goat, 1:500, Frontier Institute, Af460), anti-Homer 1 (Guinea pig, 1:1000, Synaptic Systems, 160-004) and anti-Bassoon (Rabbit, 1:500, Synaptic Systems, 141-003).

Techniques: Immunostaining, Injection

Journal: bioRxiv

Article Title: A synthetic synaptic organizer protein restores glutamatergic neuronal circuits

doi: 10.1101/2020.02.27.967836

Figure Lengend Snippet: ( A ) Schematic depiction of SCI caused by hemisection at T10. Lower panels indicate representative horizontal sections of spinal cord immunostained for VGluT2, HIS (CPTX) and GluA4 upon mock (HBS) or CPTX injection. ( B ) Representative coronal sections at approximately 1.5 mm from the injury epicenter immunostained for VGluT2, HIS and GluA4 upon mock (HBS) or CPTX injection. Right panels show high-magnification views in the dorsal region. Scale bars, 100 μm. ( C ) A representative orthogonal image obtained by Airyscan super-resolution microscopy indicating the localization of CPTX in the proximity of VGluT2- and GluA4-immunopositive puncta. Scale bar, 0.5 μm. ( D–F ) Quantification of excitatory synapses induced by CPTX. Representative immunohistochemical images of coronal sections stained for VGluT2 (blue), GluA4 (green) and HIS (red; CPTX) in the HBS buffer (Mock) or CPTX injection ( D ). Scale bar, 5 μm. The size and the signal intensity of GluA4, VGluT2 and HIS were quantified in each region of interest (ROI; GluA4+ (A4+), VGluT2+ (VT2+) or GluA4+/VGluT2+ (A4/VT2+)) ( E ). The percentage of VGluT2+ puncta that were accompanied with GluA4+ puncta is also shown ( F ). * P < 0.05, ** P < 0.01, *** P < 0.001 compared with mock by Student’s t -test, # P < 0.05 compared with GluA4 or VGluT2 by Student’s paired t -test, n = 16 slices each for mock and CPTX from 8 mice. ( G–I ) Time-course analyses of locomotion (BMS score) in SCI mice after the injection. Mock (HBS buffer vehicle control), ChABC, Cbln1 or CTPX was injected into the spinal cord immediately ( G ) or 1 week ( H ) after hemisection, or immediately after contusion by impactors ( I , 90 kdyn). For sham controls, the spinal cord was surgically exposed without imposing injury or injection. Mice that showed BMS score = 1.5 at 1 week after hemisection were selected for H. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with mock and # P < 0.05, ** P < 0.01 compared with ChABC by repeated two-way ANOVA with post-hoc Bonferroni-Dunn test, n = 9 mice for each treatment.

Article Snippet: The origin, dilution, company and catalog number are described as follows: Anti-calbindin (Goat, 1:500, Frontier Institute, Af1040), anti-HIS (Mouse, 1:1000, MBL, D291-3 or Rabbit, 1:1000, CST, #2365), anti-Myc (Rabbit, 1:1000, MBL, 562), anti-FLAG (Rabbit, 1:1000, Sigma-Aldrich, F7425), anti-HA (Mouse, 1:1000, BAbCO, MMS-101P), anti-synaptophysin (Mouse, 1:500, Sigma-Aldrich, S5768 or G, 1:500, Frontier Institute, Af300), anti-neurexin (Chicken, 1:500, a gift from Peter Scheiffele , anti-GFP (Rabbit 1:1000, Frontier Institute, Af-2020Frontier Institute), anti-GluA1 (Rabbit, 1:100, Calbiochem, PC246 or Guinea Pig, 1:500, Frontier Institute, Af380), anti-GluA2/3 (Rabbit, 1:1000, Chemicon, AB1506), anti-GluA4 (Rabbit, 1:1000, Pharmingen, 60666N or Guinea Pig, 1:500, Frontier Institute, Af640), anti-VGluT1 (Rabbit, 1:500, Frontier Institute, Af570 or Goat, 1:500, Frontier Institute, Af310), anti-VGluT2 (Guinea Pig, 1:500, Frontier Institute, Af720 or Goat, 1:500, Frontier Institute, Af310), anti-VGAT (Goat, 1:500, Frontier Institute, Af620)), anti-Parvalbumin (Goat, 1:500, Frontier Institute, Af460), anti-Homer 1 (Guinea pig, 1:1000, Synaptic Systems, 160-004) and anti-Bassoon (Rabbit, 1:500, Synaptic Systems, 141-003).

Techniques: Injection, Microscopy, Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: A synthetic synaptic organizer protein restores glutamatergic neuronal circuits

doi: 10.1101/2020.02.27.967836

Figure Lengend Snippet: ( A ) Determination of spared region following SCI. One week after dorsal hemisections, CPTX was injected into the rostral region. Representative immunostaining images of coronal serial sections (20 μm thickness, 200 μm intervals) revealed that sections > 1.1 mm rostral to the epicenter of the injured region showed intact VGluT2 immunoreactivity. Scale bar, 0.5 mm. ( B ) Representative confocal immunohistochemical images of CPTX and synaptic markers. CPTX was mostly colocalized with a postsynaptic marker GluA4 (top) and a presynaptic marker VGluT2 (bottom). Scale bar, 2 μm. ( C ) Rapid recovery of motor performance by CPTX. Mock (HBS buffer, vehicle of CPTX), CPTX or ChABC were delivered 1 week after hemisections and the initial recovery of the motor function in the subsequent week was calculated as ΔBMS/week. The graph shows the mean ± SEM. ** P < 0.01, *** P < 0.001, one-way ANOVA followed by Student t -test, n = 21 mice for each treatment. ( D and E ) Time-course of footfall test after hemisection ( C ) and contusion ( D ). Cbln1, CPTX, ChABC or mock (vehicle control) was injected immediately after hemisections. For sham controls, the spinal cord was surgically exposed without imposing injury or injection. The graphs show the mean ± SEM. * P < 0.05, Repeated-measures ANOVA followed by Bonferroni-Dunn test, n = 6 mice for each treatment.

Article Snippet: The origin, dilution, company and catalog number are described as follows: Anti-calbindin (Goat, 1:500, Frontier Institute, Af1040), anti-HIS (Mouse, 1:1000, MBL, D291-3 or Rabbit, 1:1000, CST, #2365), anti-Myc (Rabbit, 1:1000, MBL, 562), anti-FLAG (Rabbit, 1:1000, Sigma-Aldrich, F7425), anti-HA (Mouse, 1:1000, BAbCO, MMS-101P), anti-synaptophysin (Mouse, 1:500, Sigma-Aldrich, S5768 or G, 1:500, Frontier Institute, Af300), anti-neurexin (Chicken, 1:500, a gift from Peter Scheiffele , anti-GFP (Rabbit 1:1000, Frontier Institute, Af-2020Frontier Institute), anti-GluA1 (Rabbit, 1:100, Calbiochem, PC246 or Guinea Pig, 1:500, Frontier Institute, Af380), anti-GluA2/3 (Rabbit, 1:1000, Chemicon, AB1506), anti-GluA4 (Rabbit, 1:1000, Pharmingen, 60666N or Guinea Pig, 1:500, Frontier Institute, Af640), anti-VGluT1 (Rabbit, 1:500, Frontier Institute, Af570 or Goat, 1:500, Frontier Institute, Af310), anti-VGluT2 (Guinea Pig, 1:500, Frontier Institute, Af720 or Goat, 1:500, Frontier Institute, Af310), anti-VGAT (Goat, 1:500, Frontier Institute, Af620)), anti-Parvalbumin (Goat, 1:500, Frontier Institute, Af460), anti-Homer 1 (Guinea pig, 1:1000, Synaptic Systems, 160-004) and anti-Bassoon (Rabbit, 1:500, Synaptic Systems, 141-003).

Techniques: Injection, Immunostaining, Immunohistochemical staining, Marker

Journal: eLife

Article Title: GluA4 facilitates cerebellar expansion coding and enables associative memory formation

doi: 10.7554/eLife.65152

Figure Lengend Snippet: High-speed (400 fps) video of a mouse crossing the LocoMouse corridor, displayed at 30 fps. Side and bottom (via mirror reflection) views of the mouse are captured in a single camera. Top: Raw video of a WT mouse. Bottom: Raw video of a GluA4-knockout (GluA4-KO) mouse.

Article Snippet: Antibody , Anti-GluA4 F-9 (mouse monoclonal) , Santa Cruz Biotechnology , Cat. # sc-271894 RRID: AB_10715096 , WB (1:100–1:200).

Techniques:

Journal: Frontiers in Behavioral Neuroscience

Article Title: Effect of Aggressive Experience in Female Syrian Hamsters on Glutamate Receptor Expression in the Nucleus Accumbens

doi: 10.3389/fnbeh.2020.583395

Figure Lengend Snippet: The effect of aggression on AMPAR subunit expression in the NAc. (A) Aggression resulted in an increase in GluA1 expression in the NAc 24 h following aggressive experience compared to 1 week following aggressive experience ( p < 0.05). (B) There was no effect of aggressive experience on GluA2 expression. (C) There was no effect of aggressive experience on GluA4 expression. (Control n = 7–9, 24 h n = 10–11, 1 Week n = 9). (*Indicates significant difference between groups, p < 0.05).

Article Snippet: After transfer, membranes were then blocked in Odyssey Blocking Buffer (Li-Cor Biosiences, Lincoln, NE, United States) for 1 h. Membranes were then incubated overnight in primary antibodies against mouse anti-PSD-95 (1:1000; Invitrogen, Rockford, IL, United States, REF MA 1-045), rabbit anti-Caskin I (1:2000; Synaptic Systems, Göttingen, Germany, Cat. # 185 002), rabbit anti-GluA1 (1:2000; Abcam, Cambridge, MA, United States, ab31232), rabbit anti-GluA2 (1:1000; Cell Signaling, Danvers, MA, United States, Ref: 04/2019 5306S), rabbit anti-GluA4 (1:1000; Abcam, Cambridge, MA, United States, ab109431), rabbit anti-NR2A (1:500; Abcam, Cambridge, MA, United States, ab124913), rabbit anti-NR2B (1:1000; Abcam, Cambridge, MA, United States, ab65783), rabbit anti-mGluR1a (1:1000; Millipore, Billerica, MA, United States, 07-617), rabbit anti-mGluR5 (1:2000; Millipore, Billerica, MA, United States, AB5675) and mouse anti-GAPDH (1:10,000; Abcam, Cambridge, MA, United States, ab8245).

Techniques: Expressing