Journal: Cell reports

Article Title: CPEB3-dependent increase in GluA2 subunits impairs excitatory transmission onto inhibitory interneurons in a mouse model of fragile X

doi: 10.1016/j.celrep.2022.110853

Figure Lengend Snippet: (A and B) Upper: representative images of individual endogenous GluA1 (red) (A) or GluA2 (red) (B) mRNA molecules within a single hippocampal neuron detected by single-molecule FISH, followed by immunofluorescence with antibodies directed to the presynaptic marker bassoon (green), the dendritic marker MAP2 (blue), and merge. Lower: higher-magnification images of boxed region in the upper panel. (C) Upper: representative images of individual GluA1 (red) and GluA2 (green) mRNA molecules in the same neuron. Lower: higher-magnification images. (D) Summary data of images like those in (A and B) showing the number of AMPAR GluA1 (black) and GluA2 (red) mRNAs that juxtapose to synaptic sites marked by basoon (first two bars) or that colocalize with each other (third bar). GluA1 and GluA2, 30dendrites, 30 neurons, n = 4. (E) Summary data for images like those in (C) showing the number of GluA1 and GluA2 molecules per dendrite. (F) GluA1 and GluA2 mRNA molecules as a function of the distance from the soma (GluA1, 101 dendrites, 35 neurons, n = 4; GluA2, 190 dendrites, 82 neurons, n = 4). The number of individual GluA1 and GluA2 mRNA molecules in all dendrites of all hippocampal neurons that met the criteria for identification as mRNAs were analyzed by an individual blinded to the treatment. Scale bar, 10 μm. For (D–F): Data are mean ± SEM. **p < 0.01. NS, not significant. Here and in , , (with the exception of ), , and , n is defined as number of independent experiments each involving a different batch of neurons. In , n is defined as number of animals.

Article Snippet: Rabbit polyclonal anti-AMPA Receptor 1 (GluR1)/GluA1 , Alomone Labs , Cat# AGC-004; RRID:AB_2039878.

Techniques: Immunofluorescence, Marker

Journal: Cell reports

Article Title: CPEB3-dependent increase in GluA2 subunits impairs excitatory transmission onto inhibitory interneurons in a mouse model of fragile X

doi: 10.1016/j.celrep.2022.110853

Figure Lengend Snippet: (A–D) Representative images of endogenous single mRNAs molecules encoding GluA1 were detected by single-molecule FISH in primary cultures of hippocampal neurons from WT and Fmr1 KO mice (A) quantified in (B). (C) Higher-magnification images of boxed region in (A) quantified in (D). (E–H) Representative images of endogenous single mRNAs molecules encoding GluA2 were detected by single-molecule FISH in primary cultures of hippocampal neurons from WT and Fmr1 KO mice (E) quantified in (F). (G) Higher-magnification images of boxed region in (E) quantified in (H) (GluA1, WT: 101 dendrites, 35 neurons; KO: 134 dendrites, 63 neurons, n = 4 per group; GluA2, WT: 190 dendrites, 82 neurons; KO: 99 dendrites, 82 neurons, n = 4 per group). (I) Representative images of a dual FISH experiment with probes targeting either the GluA2 coding (upper left) or intronic (upper right) sequences. (J) Representative images showing the number of GluA1 and GluA2 mRNA molecules at transcription hotspots in the nucleus of neurons from WT and Fmr1 KO mice. (K) Summary data of images like those illustrated in (J) (GluA1, WT: 35 neurons; KO: 63 neurons; GluA2, WT: 82 neurons, KO: 86 neurons, n = 4). (L) Summary qRT-PCR data from whole hippocampus showing GluA1 (upper) and GluA2 (lower) mRNA expression (n = 4 per group). For all the quantitative graphs: Data are mean ± SEM. *p < 0.05; **p < 0.01.

Article Snippet: Rabbit polyclonal anti-AMPA Receptor 1 (GluR1)/GluA1 , Alomone Labs , Cat# AGC-004; RRID:AB_2039878.

Techniques: Quantitative RT-PCR, Expressing

Journal: Cell reports

Article Title: CPEB3-dependent increase in GluA2 subunits impairs excitatory transmission onto inhibitory interneurons in a mouse model of fragile X

doi: 10.1016/j.celrep.2022.110853

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-AMPA Receptor 1 (GluR1)/GluA1 , Alomone Labs , Cat# AGC-004; RRID:AB_2039878.

Techniques: shRNA, Recombinant, Sequencing, Binding Assay, Plasmid Preparation, Software

Journal: Journal of Neuroinflammation

Article Title: Oxidative stress and inflammation cause auditory system damage via glial cell activation and dysregulated expression of gap junction proteins in an experimental model of styrene-induced oto/neurotoxicity

doi: 10.1186/s12974-023-02996-3

Figure Lengend Snippet: List of reagents and antibodies used

Article Snippet: Antibody , Anti-AMPA Receptor 1 (GluA1) (rabbit polyclonal) , Cell signaling , Cat. No. #13185 , WB (1:1000) , Total GluA1 levels.

Techniques: Marker, Immunofluorescence, Activation Assay, Nitration, Diagnostic Assay, Control

Journal: Journal of Neuroinflammation

Article Title: Oxidative stress and inflammation cause auditory system damage via glial cell activation and dysregulated expression of gap junction proteins in an experimental model of styrene-induced oto/neurotoxicity

doi: 10.1186/s12974-023-02996-3

Figure Lengend Snippet: Morphological and functional damage induced by the oto/neurotoxic insult in the auditory cortex (ACx). A Graph shows results of field excitatory post-synaptic potential (fEPSP) amplitude measured following stimulation of afferent fibers in ACx layer II/III at increasing intensities. Statistical analysis by two-way ANOVA followed by Tukey’s post-hoc revealed significant differences between groups ( p < 0.001; n = 17 slices from 4 Styrene and n = 16 slices from 4 Ctrl rats). B – E Representative images of Golgi-stained segments from apical ( B , D ) and basal dendrites ( C , E ) of pyramidal neurons of layers II/III in Ctrl and Styrene groups. Scale bar: 10 μm. F Bar graphs showing values of spine density in apical and basal dendrites of neurons of layer II/III of the ACx in the experimental groups ( n = at least 30 segments from 30 different neurons were analyzed from three animals/groups; two-way ANOVA, apical dendrites p < 0.0001, basal dendrites p < 0.0001). G , H Images of western immunoblot indicating lower pGluA1 Ser845 ( G ) and higher cleaved caspase-3 ( H ) levels in the ACx of styrene-treated rats compared to controls. Histograms show densitometric analyses in all samples normalized to total protein amount (GluA1 and GAPDH/Caspase-3) ( n = 3 animals for each group; Student’s t test, pGluA1 p = 0.022; Caspase-3 p = 0.0004). Data are expressed as mean ± SEM. Asterisks indicate statistical significance (* p < 0.05; *** p < 0.001)

Article Snippet: Antibody , Anti-AMPA Receptor 1 (GluA1) (rabbit polyclonal) , Cell signaling , Cat. No. #13185 , WB (1:1000) , Total GluA1 levels.

Techniques: Functional Assay, Staining, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: A vital role for PICK1 in the differential regulation of metabotropic glutamate receptor internalization and synaptic AMPA receptor endocytosis

doi: 10.1016/j.jbc.2023.104837

Figure Lengend Snippet: Knockdown of endogenous PICK1 does not have any effect on mGluR-mediated signaling and AMPAR endocytosis when both mGluR1 and mGluR5 are activated. A and B , Western blot ( A ) and quantitation of the western blots ( B ) showing that in control cells, application of 100 μM R,S-DHPG for 5 min increased the phosphorylation of MAP kinases. The receptors that recycled to the cell surface in 2.5 h also induced phosphorylation of MAP kinases upon application of the agonist (N = 3). C and D , Western blot ( C ) and quantitation of the western blots ( D ) suggested that similar to control cells, in PICK1 knockdown cells, initial application of 100 μM R,S-DHPG for 5 min increased the phosphorylation of MAP kinases and application of the agonist after 2.5 h also resulted in the increase in the phosphorylation of MAP kinases (N = 3). E and F , representative images ( E ) and quantitation of the mGluR-mediated AMPAR endocytosis ( F ) suggested that in control cells, initial application of 100 μM R,S-DHPG for 5 min led to the endocytosis of AMPARs. The mGluRs that recycled back to the cell surface in 2.5 h were able to induce the endocytosis of GluA1-containing receptors when they were stimulated with 100 μM R,S-DHPG for 5 min. On the other hand, application of 100 μM R,S-DHPG did not cause endocytosis of GluA1-containing receptors when the mGluR recycling was inhibited with okadaic acid and FK-506 (N: control:: untreated = 37; DHPG = 36; 2.5 h untreated = 37; 2.5 h DHPG = 41; 2.5 h (OA + FK-506) untreated = 39; 2.5 h (OA + FK-506) DHPG = 36). G and H , representative images ( G ) and quantitation of the mGluR-mediated AMPAR endocytosis ( H ) showed that in shPICK1 transfected cells, initial application of 100 μM R,S-DHPG for 5 min led to the endocytosis of GluA1-containing receptors, and the receptors that recycled back to the cell surface in 2.5 h post agonist application also induced the internalization of GluA1-containing receptors. When the mGluR recycling was inhibited with okadaic acid and FK-506, application of 100 μM R,S-DHPG could not induce endocytosis of GluA1-containing receptors (N: shPICK1:: untreated = 31; DHPG = 31; 2.5 h untreated = 39; 2.5 h DHPG = 37; 2.5 h (OA + FK-506) untreated = 33; 2.5 h (OA + FK-506) DHPG = 35). Results are presented as means ± SD. Scale bar represents 10 μm. ∗∗∗ p < 0.001; ∗∗ p < 0.01; ∗ p < 0.05; n.s, p > 0.05. DHPG, 3, 5-dihydroxyphenylglycine; mGluR, metabotropic glutamate receptor; PICK1, protein interacting with C kinase 1.

Article Snippet: Anti-GluA1 rabbit polyclonal antibody was purchased from Millipore.

Techniques: Knockdown, Western Blot, Quantitation Assay, Control, Phospho-proteomics, Transfection

Journal: The Journal of Biological Chemistry

Article Title: A vital role for PICK1 in the differential regulation of metabotropic glutamate receptor internalization and synaptic AMPA receptor endocytosis

doi: 10.1016/j.jbc.2023.104837

Figure Lengend Snippet: PICK1 is specifically involved in the mGluR1-mediated signaling and mGluR1-mediated AMPAR endocytosis. A and B , Western blot ( A ) and quantitation of the western blots ( B ) showing that in control cells that were treated with the mGluR5 antagonist MTEP, application of 100 μM R,S-DHPG for 5 min led to the phosphorylation of MAP kinases. Furthermore, the receptors recycled to the cell surface in 2.5 h and also showed the ability to induce phosphorylation of MAP kinases on application of 100 μM R,S-DHPG for 5 min (N = 3). C and D , Western blot ( C ) and quantitation of the western blots ( D ) suggested that although in PICK1 knockdown cells, in presence of MTEP, initial application of 100 μM R,S-DHPG for 5 min led to the phosphorylation of MAP kinases, but application of 100 μM R,S-DHPG after 2.5 h did not increase the phosphorylation of MAP kinases (N = 3). E and F , representative images ( E ) and quantitation of the endocytosis index ( F ) suggested that in control cells (treated with MTEP), the receptors that recycled back to the cell surface in 2.5 h were able to induce the internalization of GluA1-containing receptors when they were stimulated with 100 μM R,S-DHPG for 5 min. Application of 100 μM R,S-DHPG did not cause endocytosis of GluA1-containing receptors when the mGluR1 recycling was inhibited with okadaic acid and FK-506 (N: control:: untreated = 30; DHPG = 32; 2.5 h untreated = 30; 2.5 h DHPG = 31; 2.5 h (OA + FK-506) untreated = 29; 2.5 h (OA + FK-506) DHPG = 29). G and H , representative images ( G ) and quantitation of the mGluR-mediated AMPAR endocytosis ( H ) showed that in shPICK1 transfected cells that were treated with mGluR5 antagonist MTEP, although initial application of 100 μM R,S-DHPG for 5 min led to the endocytosis of GluA1-containing receptors, application of 100 μM R,S-DHPG after 2.5 h did not induce the internalization of GluA1-containing receptors. Expectedly, in okadaic acid and FK-506–treated cells, 100 μM R,S-DHPG did not cause endocytosis of GluA1-containing receptors (N: shPICK1:: untreated = 31; DHPG = 32; 2.5 h untreated = 34; 2.5 h DHPG = 33; 2.5 h (OA + FK-506) untreated = 33; 2.5 h (OA + FK-506) DHPG = 32). Results are presented as means ± SD. Scale bar represents 10 μm. ∗∗∗ p < 0.001; ∗∗ p < 0.01; ∗ p < 0.05; n.s, p > 0.05. DHPG, 3, 5-dihydroxyphenylglycine; mGluR, metabotropic glutamate receptor; PICK1, protein interacting with C kinase 1; MTEP, (3-((2-methyl-1,3-thiazol-4-yl) ethynyl) pyridine).

Article Snippet: Anti-GluA1 rabbit polyclonal antibody was purchased from Millipore.

Techniques: Western Blot, Quantitation Assay, Control, Phospho-proteomics, Knockdown, Transfection