Journal: Annals of neurology

Article Title: Direct Current Stimulation Induces mGluR5-Dependent Neocortical Plasticity

doi: 10.1002/ana.24708

Figure Lengend Snippet: Direct current stimulation-induced long-term depression (DCS-LTD) depends on metabotropic glutamate receptor 5 (mGluR5) activation in mouse M1 slices. (A, B) DCS-LTD effect can be elicited by cathodal DCS with treatment by either 50μM D-(–)-2-amino-5-phosphonopentanoic acid (D-AP5; slopes were decreased to 84.3 ± 0.7% of baseline; n = 5 mice, 7 slices; p < 0.001) or 25μM bicuculline (BIC; 79.6 ± 0.7% of baseline; n = 5 mice, 6 slices; p < 0.001), antagonists of N-methyl-D-aspartate receptor and γ-aminobutyric acid type A receptor, respectively. (C) Bath application of mGluR5 negative allosteric modulator (2-chloro-4-[(2,5-dimethyl-1-[4-(trifluoromethoxy)phenyl]-1H-imidazol-4-yl)ethynyl]pyridine [CTEP], 10μM) completely abolished DCS-LTD in M1 slices (98.3 ± 0.7% of baseline; n = 7 mice, 10 slices; p > 0.05). Black bars under the traces in A–C indicate timing of drug exposure. (D) Statistical analysis of DCS-LTD changes on specific drug conditions (F7,56 = 423.2, p < 0.001); 1-way analysis of variance (ANOVA) post-test between each treatment and its baseline, ### = p < 0.001 and ns = p > 0.05; 1-way ANOVA post-test between 2 means as indicated in the graph, ***p < 0.001. fEPSP = field excitatory postsynaptic potential.

Article Snippet: NMDAR antagonist D-(–)-2-amino-5-phosphonopentanoic acid (D-AP5, Cat#-0106), GABA type A receptor (GABA A R) antagonist bicuculline (BIC; Cat#-0130), and positive mGluR5 allosteric modulator 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB, Cat#-3235) were purchased from Tocris Bioscience (Minneapolis, MN).

Techniques: Activation Assay

Journal: Annals of neurology

Article Title: Direct Current Stimulation Induces mGluR5-Dependent Neocortical Plasticity

doi: 10.1002/ana.24708

Figure Lengend Snippet: Metabotropic glutamate receptor 5 (mGluR5) positive allosteric modulator (PAM) promotes direct current stimulation-induced long-term depression (DCS-LTD) in mouse M1 slices. (A) Short-term depression induced by 10-minute 400μA cathodal DCS (99.6 ± 0.7% of baseline 1 hour after DCS; n = 5 mice, 5 slices; p > 0.05) was enhanced to DCS-LTD (83.3 ± 0.4% of baseline 1 hour after DCS; n = 4 mice, 9 slices; p < 0.001) by exposure of M1 slices to an mGluR5 PAM (3-cyano-N-[1,3-diphenyl-1H-pyrazol-5-yl]benzamide [CDPPB], 10μM). (B) Statistic analysis of the aftereffects induced by cathodal DCS (400μA, 10 minutes) with and without CDPPB compared to normal DCS-LTD (F5,42 = 1317, p < 0.001); 1-way analysis of variance (ANOVA) post-test between each treatment and its respective baseline, ### = p < 0.001 and ns = p > 0.05; 1-way ANOVA post-test between 2 means as indicated in the graph, ***p < 0.001. fEPSP = field excitatory postsynaptic potential.

Article Snippet: NMDAR antagonist D-(–)-2-amino-5-phosphonopentanoic acid (D-AP5, Cat#-0106), GABA type A receptor (GABA A R) antagonist bicuculline (BIC; Cat#-0130), and positive mGluR5 allosteric modulator 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB, Cat#-3235) were purchased from Tocris Bioscience (Minneapolis, MN).

Techniques:

Journal: Annals of neurology

Article Title: Direct Current Stimulation Induces mGluR5-Dependent Neocortical Plasticity

doi: 10.1002/ana.24708

Figure Lengend Snippet: Cathodal direct current stimulation (DCS) increases phosphorylated (p) ribosomal protein S6 via metabotropic glutamate receptor 5 and mechanistic target of rapamycin activation in mouse neocortex. (A) p-S6 in mouse M1 slices was measured 0, 15, 30, and 60 minutes after cathodal DCS (400μA, 25 minutes) by immunoblot, and a time-dependent increase of p-S6 was found. Actin was used as loading reference. Control slices (CT) were collected without DCS treatment. Total S6, normalized to actin, was unchanged (F4,15 = 0.85, p = 0.52 by 1-way analysis of variance [ANOVA]). (B) Statistical analysis of normalized p-S6:S6 ratio from M1 slices collected at different time points (F4,15 = 3.35, p < 0.05 by 1-way ANOVA). The normalized p-S6:S6 ratio is significantly increased 60 minutes after DCS (213.0 ± 26.9% of control; n = 4 mice, 4 slices; **p < 0.01 by 1-way ANOVA post-test). (C, D) p-S6 and S6 levels in M1 collected 1 hour after in vivo cathodal transcranial DCS (tDCS; 1mA, 25 minutes) with or without pretreatment with rapamycin (Rapa; C) or 2-chloro-4-([2,5-dimethyl-1-(4-[trifluoromethoxy]phenyl)-1H-imidazol-4-yl]ethynyl)pyridine (CTEP; D). p-S6 increases in the vehicle (Veh) + tDCS condition, and is otherwise stable. Total S6, normalized to actin, was unchanged (F5,24 = 1.29, p = 0.30 by 1-way ANOVA). (E) Statistical analysis of normalized p-S6:S6 ratio from M1 tissues of in vivo tDCS work (F5,40 = 4.76, p < 0.01). Normalized p-S6:S6 is increased (227.8 ± 32.0% of control) after cathodal tDCS (control [±vehicle]: n = 9 mice, tDCS [±vehicle]: n = 9 mice; ***p < 0.001). This increase is abolished by either rapamycin (73.7 ± 21.2% of control; n = 6 mice; p > 0.05 as compared to the control; ***p < 0.001 as compared to tDCS group) or CTEP pretreatment (135.8 ± 21.7% of control; n = 6 mice; p > 0.05 as compared to the control; *p < 0.05 as compared to tDCS group). Rapamycin (48.7 ± 5.4% of control; n = 3 mice; p > 0.05) or CTEP (122.0 ± 58.6% of control; n = 3 mice; p > 0.05) exposure without tDCS did not significantly affect the p-S6:S6 relative to the control group. ***p < 0.001 and *p < 0.05 by 1-way ANOVA post-test.

Article Snippet: NMDAR antagonist D-(–)-2-amino-5-phosphonopentanoic acid (D-AP5, Cat#-0106), GABA type A receptor (GABA A R) antagonist bicuculline (BIC; Cat#-0130), and positive mGluR5 allosteric modulator 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB, Cat#-3235) were purchased from Tocris Bioscience (Minneapolis, MN).

Techniques: Activation Assay, Western Blot, Control, In Vivo

Journal: The Journal of Neuroscience

Article Title: Mitogen-Activated Protein Kinase Signaling in the Hippocampus and Its Modulation by Corticotropin-Releasing Factor Receptor 2: A Possible Link between Stress and Fear Memory

doi: 10.1523/JNEUROSCI.23-36-11436.2003

Figure Lengend Snippet: PKA-dependent upregulation of pErk-1/2 in response to fear conditioning. A, Experimental design. Injection of the PKA inhibitor Rp-cAMPS (18 μg per mouse) into the dorsal hippocampus was performed 15 min before the context-dependent fear conditioning of mice of the FC and stress/FC groups. B, Rp-cAMPS significantly reduced freezing behavior when compared with vehicle-injected animals. C, Mice injected intrahippocampally with Rp-cAMPS exhibited significantly lower pErk-1/2 levels than vehicle-injected animals. D, Representative immunoblots. Statistically significant differences: *p < 0.01 versus corresponding FC- or stress/FC-vehicle group.

Article Snippet: The PKA inhibitor Rp-cAMPS was purchased from Calbiochem (La Jolla, CA).

Techniques: Injection, Western Blot

Journal: Genes and immunity

Article Title: Genetic variations in GPSM3 associated with protection from rheumatoid arthritis affect its transcript abundance

doi: 10.1038/gene.2016.3

Figure Lengend Snippet: GPSM3 transcript abundance in whole blood from individuals homozygous for the minor allele of the rs204989/rs204991 haploblock (the “m/m” genotype) is significantly lower than in whole blood from individuals homozygous for the major allele of the haploblock (the “M/M” genotype). ( A ) Schematic representation of identified polymorphisms with respect to the predicted transcription start site of the human GPSM3 gene. ( B ) Scatter plot representation of GPSM3 mRNA abundance, measured in whole blood by qRT-PCR, as normalized to the average level measured in M/M individuals. Individual results for homozygous major allele samples (M/M; n = 53) are depicted by solid circles, and homozygous minor allele samples (m/m; n=11) are depicted by open circles. An average decrease (Δμ) in GPSM3 transcript abundance of 24.1% (t = 3.80, df = 62, p = 0.0003) was observed in homozygous minor allele samples. Error bars represent S.E.M. ( C ) Descriptive statistics of the three cohorts of volunteers genotyped in this study: Minor allele frequencies were 23% for rs204989 and rs204991 in the rheumatoid arthritis samples versus 18% in the disease-free controls (Fisher's exact test; p = 0.4839). Risk/minor allele frequency for rs2812378 was 30.0% in the rheumatoid arthris samples versus 25.0% in the disease-free controls (p = 0.5267). Risk/minor allele frequency for the HLA gene region biallelic SNP rs6457620 was 35.0% in the rheumatoid arthritis samples and 45.0% in the disease-free matched controls (p = 0.1938).

Article Snippet: SNP genotypes were determined using the Type-it Fast Probe PCR Kit (Qiagen), TaqMan SNP genotyping probes, and primers for rs204989 ( GPSM3 ), rs204991 ( GPSM3 ), rs2812378 ( CCL21 ), and rs6457620 ( HLA gene region) (Life Technologies; cat. #C_3293828_20, C_2412452_20, C_16113556_10, and C_29315329_10, respectively) using the manufacturer's suggested protocols.

Techniques: Quantitative RT-PCR

Journal: Genes and immunity

Article Title: Genetic variations in GPSM3 associated with protection from rheumatoid arthritis affect its transcript abundance

doi: 10.1038/gene.2016.3

Figure Lengend Snippet: Linkage of the GPSM3 SNP haploblock and the known RA risk allele in the HLA gene region, rs6457620 [C>G], is more pronounced in RA samples than controls, but this linkage does not result in a rs6457620 genotype-dependent effect on GPSM3 transcript abundance in whole blood. ( A ) Descriptive statistics of all 200 volunteers, including the RA (n = 50), disease-free matched control (n = 50), and young healthy control volunteers recruited for the current study. These data are stratified by GPSM3 SNP haplotype, showing that the rs6457620 [G] risk allele frequency amongst homozygous ancestral GPSM3 haploblock individuals (M/M) is 41.3%, heterozygous individuals (M/m) is 56.3%, and homozygous minor GPSM3 haploblock (m/m) is 60.7%. These data are significantly different from chance distribution (Fisher-Freeman-Halton exact test; p = 0.0066). ( B ) Analyses of GPSM3 SNP haploblock linkage within RA samples stratified by rs6457620 genotype, again exhibiting statistically significant difference from chance distribution (Fisher-Freeman-Halton exact test; p = 0.0123). ( C ) Within the disease-free matched control samples, GPSM3 SNP minor allele (m) frequency, as stratified by rs6457620 genotype, is not significantly different from chance distribution as determined by Fisher-Freeman-Halton exact test (p = 0.2739). ( D ) Graphical representation of minor allele frequencies (MAFs; of European [‘eu’] population), physical distance (in basepairs), and linkage disequilibrium (LD) value (as quantified by heatmap) between the three linked SNPs of the GPSM3 SNP haploblock (rs204989, rs204990, rs204991) and the HLA gene region SNP rs6547620, as obtained using the NIEHS SNPinfo webserver ( http://snpinfo.niehs.nih.gov/ ) from NCBI dbSNP data (ref. ). ( E ) Despite modest linkage between GPSM3 SNP minor alleles (m) and the rs6457620 risk allele (G), there is no effect of the genotype at HLA gene region SNP rs6457620 on GPSM3 transcript abundance. Significance determined by one-way ANOVA with Bonferroni post-hoc .

Article Snippet: SNP genotypes were determined using the Type-it Fast Probe PCR Kit (Qiagen), TaqMan SNP genotyping probes, and primers for rs204989 ( GPSM3 ), rs204991 ( GPSM3 ), rs2812378 ( CCL21 ), and rs6457620 ( HLA gene region) (Life Technologies; cat. #C_3293828_20, C_2412452_20, C_16113556_10, and C_29315329_10, respectively) using the manufacturer's suggested protocols.

Techniques: Control

Journal: Genes and immunity

Article Title: Genetic variations in GPSM3 associated with protection from rheumatoid arthritis affect its transcript abundance

doi: 10.1038/gene.2016.3

Figure Lengend Snippet: Functional consequences of SNPs rs204989, rs204990, rs204991, and rs3096688 on GPSM3 promoter activity. ( A ) Functional results upon subcloning a 3.5 kb-region immediately 5′ to the GPSM3 transcription start site from M/M and m/m genotype volunteers, leading to wild type (pGL3-M) and “minor”/polymorphic (pGL3-m) promoter-driven expression of firefly luciferase in HEK293T cells. Luciferase activity is reported as a ratio of GPSM3 promoter-dependent firefly luciferase activity (FLuc) to control Renilla luciferase activity (pRL-TK control vector) and normalized to the average level measured from the wild type pGL3-M vector (set to 1.00; dotted line). Introduction of independent minor alleles (denoted “+###”) into the wild type pGL3-M vector is seen to differentially affect resultant firefly luciferase activity. ( B ) Restoration of the major allele at rs204989 in the pGL3-m vector ( i.e. , “pGL-m-989”) restores wild type GPSM3 promoter activity. All data are compiled from three independent experiments. Error measure is S.E.M. Significance was determined using one-way ANOVA after controlling for multiple comparisons with Dunnett's test using pGL3-M as the defined control.

Article Snippet: SNP genotypes were determined using the Type-it Fast Probe PCR Kit (Qiagen), TaqMan SNP genotyping probes, and primers for rs204989 ( GPSM3 ), rs204991 ( GPSM3 ), rs2812378 ( CCL21 ), and rs6457620 ( HLA gene region) (Life Technologies; cat. #C_3293828_20, C_2412452_20, C_16113556_10, and C_29315329_10, respectively) using the manufacturer's suggested protocols.

Techniques: Functional Assay, Activity Assay, Subcloning, Expressing, Luciferase, Control, Plasmid Preparation

Journal: Genes and immunity

Article Title: Genetic variations in GPSM3 associated with protection from rheumatoid arthritis affect its transcript abundance

doi: 10.1038/gene.2016.3

Figure Lengend Snippet: Putative transcription factor binding sites within the human GPSM3 promoter potentially disrupted by the minor allele of SNP rs204989 (arrows), as predicted by the JASPAR database: ( A ) Fos/Jun (AP1) heterodimer; (B) C/EBP-β.

Article Snippet: SNP genotypes were determined using the Type-it Fast Probe PCR Kit (Qiagen), TaqMan SNP genotyping probes, and primers for rs204989 ( GPSM3 ), rs204991 ( GPSM3 ), rs2812378 ( CCL21 ), and rs6457620 ( HLA gene region) (Life Technologies; cat. #C_3293828_20, C_2412452_20, C_16113556_10, and C_29315329_10, respectively) using the manufacturer's suggested protocols.

Techniques: Binding Assay

Journal: Genes and immunity

Article Title: Genetic variations in GPSM3 associated with protection from rheumatoid arthritis affect its transcript abundance

doi: 10.1038/gene.2016.3

Figure Lengend Snippet: Lentiviral-mediated shRNA knockdown of endogenous GPSM3 expression in the human monocytic THP-1 cell line disrupts migration toward MCP-1 ( A ) Western blot showing whole lysate (40 μg) immunoreactivity with mouse monoclonal anti-GPSM3 antibody 35.5.1 (ref. ) and with anti-β-actin as a loading control. ( B ) Real-time Transwell migration analysis of indicated calcein-AM stained THP-1 cell lines as shown by change in the ratio of MCP-1-induced to vehicle-induced relative fluorescence units (RFU) over a 30 minute timeframe. ( C ) qRT-PCR data from indicated stable knockdown or scrambled control THP-1 cell line total RNA preparations. There was no significant difference in CCR2 mRNA abundance in the THP-1 cell lines as determined by one-way ANOVA (F = 2.40; p = 0.119). ( D ) Regression analysis-derived rate of THP-1 cell line transmigration over initial migratory period; differences between each cell line was calculated by one-way ANOVA. ( E ) Regression analysis-derived maximum THP-1 cell transmigration over 30 minute timecourese; differences between each cell line were calculated by one-way ANOVA. All data are reported as means with error bars representing S.E.M.

Article Snippet: SNP genotypes were determined using the Type-it Fast Probe PCR Kit (Qiagen), TaqMan SNP genotyping probes, and primers for rs204989 ( GPSM3 ), rs204991 ( GPSM3 ), rs2812378 ( CCL21 ), and rs6457620 ( HLA gene region) (Life Technologies; cat. #C_3293828_20, C_2412452_20, C_16113556_10, and C_29315329_10, respectively) using the manufacturer's suggested protocols.

Techniques: shRNA, Knockdown, Expressing, Migration, Western Blot, Control, Staining, Fluorescence, Quantitative RT-PCR, Derivative Assay, Transmigration Assay

Journal: The European journal of neuroscience

Article Title: Transient viral-mediated overexpression of α-calcium/calmodulin-dependent protein kinase II in the nucleus accumbens shell leads to long-lasting functional upregulation of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors: dopamine type-1 receptor and protein kinase A dependence

doi: 10.1111/j.1460-9568.2010.07155.x

Figure Lengend Snippet: Location of microinjection cannula tips in the NAcc shell for all rats included in the data analyses. Line drawings of coronal sections (adapted from Paxinos and Watson, 1997) show the location of the microinjection cannula tips in the NAcc shell for rats tested with no inhibitor (A), with SCH23390 (B) or with Rp-cAMPS (C). Numbers to the right indicate mm from bregma. Symbols denote group affiliation: filled circles, αCaMKII-AMPA; open circles, control-AMPA; filled squares, αCaMKII-saline; open ssquares, control-saline.

Article Snippet: Rats in separate groups were administered saline (0.5 µL/side) or the AMPA receptor agonist (±) AMPA hydrobromide (0.8 nmol/0.5 µL/side; Sigma-Aldrich Inc.) infused either alone or with the D1 receptor antagonist SCH23390 HCl (0.8 nmol/0.5 µL/side; Tocris Bioscience, Ellisville, MO, USA) or the PKA inhibitor Rp-cAMPS (80 nmol/1 µL/side; Axxora LLC, San Diego, CA, USA).

Techniques:

Journal: The European journal of neuroscience

Article Title: Transient viral-mediated overexpression of α-calcium/calmodulin-dependent protein kinase II in the nucleus accumbens shell leads to long-lasting functional upregulation of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors: dopamine type-1 receptor and protein kinase A dependence

doi: 10.1111/j.1460-9568.2010.07155.x

Figure Lengend Snippet: Enhanced locomotor responding to NAcc shell AMPA at 20 days post-infection requires D1 receptor and PKA activation. Data in (A–F) are shown as group mean (±SEM) locomotor counts obtained before and after the challenge injection (arrows). (A and B) The locomotor response to NAcc shell AMPA was significantly enhanced in αCaMKII compared with control group rats. Little locomotor activity was observed in either group following NAcc shell saline. Coadministration of either SCH23390 (C and D) or Rp-cAMPS (E and F) with AMPA into the NAcc shell blocked the enhanced locomotor response in αCaMKII compared with control group rats but spared the locomotor effects of AMPA in both groups compared with NAcc shell saline. Neither SCH23390 nor Rp-cAMPS produced effects that differed significantly from saline when administered alone. (G) Summary of post-challenge injection results illustrated as group mean (+SEM) 2 h total locomotor counts. *P < 0.001, AMPA vs. saline; †P < 0.01, αCaMKII vs. control; revealed by post-hoc Scheffé comparisons following anova.

Article Snippet: Rats in separate groups were administered saline (0.5 µL/side) or the AMPA receptor agonist (±) AMPA hydrobromide (0.8 nmol/0.5 µL/side; Sigma-Aldrich Inc.) infused either alone or with the D1 receptor antagonist SCH23390 HCl (0.8 nmol/0.5 µL/side; Tocris Bioscience, Ellisville, MO, USA) or the PKA inhibitor Rp-cAMPS (80 nmol/1 µL/side; Axxora LLC, San Diego, CA, USA).

Techniques: Infection, Activation Assay, Injection, Activity Assay, Produced