Journal: Neuropsychopharmacology

Article Title: The Membrane Proximal Region of AMPA Receptors in Lateral Amygdala is Essential for Fear Memory Formation

doi: 10.1038/npp.2015.121

Figure Lengend Snippet: TAT-MPR(AA) in LA impairs long-term but not short-term fear memory formation. (a) The MPR(AA) peptide is derived from the MPR region of GluA4. The GluA4 MPR is different from GluA1 MPR region at positions 816 and 818 where the GluA4 contains alanines (highlighted by squares). (b) Microinjection of biotin-labeled TAT-conjugated MPR(AA) into the LA (50 μg/μl, 0.5 μl per LA) led to its insertion into cells when tested 30 min later as detected by the Alexa Fluor 568-streptavidin (red) labeling (blue—DAPI staining). (c) Rat were microinjected with fluorescent (FITC)-labeled TAT-MPR(AA) into LA 30 min before fear conditioning. Two hours after training, surface expression of the GluA4 (upper panels) or GluA2 (lower panels) receptors was monitored. We used antibodies against the extracellular portion of the GluA4 or GluA2 to detect surface-expressing receptors. As seen, the surface expression of GluA4 (open arrows) 2 h after fear conditioning training is reduced in neurons that contain the TAT-MPR(AA) peptide (closed arrows) compared with neurons that do not contain the peptide. The GluA2 (open arrow) is expressed at the surface of neurons that contain the TAT-MPR(AA) peptide (closed arrow), indicating that the peptide is not effective in these neurons. (d) Freezing throughout the tone presentations during training of the same animals as in panel (e) was analyzed. There is no significant difference between animals injected with TAT-MPR(AA) peptide and rats injected with saline (F(4)=0.649, p>0.6). (e) Microinjection of the TAT-MPR(AA) peptide (50 μg/μl, 0.5 μl per LA n=10) into LA 30 min before fear conditioning significantly impaired long-term fear memory formation tested 24 h after training when compared with saline-injected rats (0.5 μl saline per LA n=9) (F(1)=9.128, p<0.009). (f) Freezing throughout the tone presentations during training of the same animals as in panel (g) was analyzed. There is no significant difference between animals injected with TAT-MPR(AA) peptide and rats injected with saline (F(2.535)=1.228, p>0.3). (g) Microinjection of the TAT-MPR(AA) peptide (50 μg/μl, 0.5 μl per LA n=11) into LA 30 min before fear conditioning had no significant effect on short-term fear memory formation tested 2 h after training when compared with saline-injected rats (0.5 μl saline per LA n=8) (F(1)=0.679. p>0.4). □, Saline; ▴, TAT-MPR(AA).

Article Snippet: Sections were then incubated overnight at 4 °C with the anti-GluA1, anti-GluA2, or anti-GluA4 primary antibodies that recognize the extracellular portion of the receptors (1:200; Alomone Labs, Israel) in 0.01 M PBS containing 1% BSA.

Techniques: Derivative Assay, Labeling, Staining, Expressing, Injection

Journal: EMBO Reports

Article Title: Hyperfunction of post-synaptic density protein 95 promotes seizure response in early-stage aβ pathology

doi: 10.1038/s44319-024-00090-0

Figure Lengend Snippet: ( A , B ) Immunocytochemistry and quantification showing dendritic marker MAP2, total (t) and surface (s) GluA1 ( A ) and GluA2 ( B ) from WT primary cortical neuron cultures treated with Aβ 1-42 (Aβ42, 1 µM) or scrambled peptide (Scr, 1 µM) for 2 h at DIV 12–14. For GluA1, * P = 0.0192 for surface level, P = 0.42 for total level, and n = 17 and 18 cells from two independent cultures treated with Scr and Aβ42, respectively. For GluA2, ** P = 0.0025 for surface level, P = 0.1584 for total level, and n = 16 and 20 cells from two independent cultures treated with Scr and Aβ42, respectively. ( C , D ) Immunocytochemistry and quantification showing dendritic marker MAP2, total (t) and surface (s) GluA1 ( C ) and GluA2 ( D ) from PSD-95 +/− primary cortical neuron cultures treated with Aβ 1-42 (Aβ42, 1 µM) or scrambled peptide (Scr, 1 µM) for 2 h at DIV 12–14. For GluA1, P = 0.4539 for surface level, P = 0.1311 for total level, and n = 14 and 13 cells from two independent cultures treated with Scr and Aβ42, respectively. For GluA2, P = 0.6643 for surface level, P = 0.659 for total level, and n = 13 and 14 cells from two independent cultures treated with Scr and Aβ42, respectively. Data Information: significance was determined by Student’s t test ( A , C ) or Mann–Whitney U test ( B , D ). Data are represented as mean ± SEM with * P < 0.05, ** P < 0.01, ns non-significant. Scale bar: 5 μm. .

Article Snippet: Rabbit anti-GluA2-C-terminus antibody (#SAB4501295) is from Sigma.

Techniques: Immunocytochemistry, Marker, MANN-WHITNEY

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 rat OB. (A) Anti-GluR1 (1:500) and (B) anti-GluR2 (1:500) using experimental design, analysis, notation, sample size, and statistical metric as in Fig. 4. Nitrocellulose blots were stripped and reprobed with anti-β-III-tubulin (1:2000). Arrows indicate expected size in kilodaltons (Mr = 110 kDa for GluR1, 100 kDa for GluR2, and 50 kDa for tubulin). GluR1 and GluR2 protein changes were statistically significant across the 48 h (Kruskal–Wallis H test, P < 0.05) but only GluR2 protein expression was rhythmic in the 24-h tested (harmonic regression analysis, P < 0.05). GluR1 and GluR2 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: