Journal: Nature Communications

Article Title: Neuropeptide SP protects against colitis and linked anxiety-like behavior through the putative roles of gut microbiota and metabolite inositol

doi: 10.1038/s41467-025-67904-0

Figure Lengend Snippet: a , b Heat maps revealing differential gene expression in FMT-DSS vs FMT-CON, FMT-DSS + SP vs FMT-DSS groups. n = 4 mice/group. The pathway-related genes were selected (log2 fold change at least > 1, p < 0.05). c , d KEGG analysis of total-, up- and down-regulated genes in the FMT-DSS group compared with the FMT-CON group (Top 20). n = 4 mice/group. e , f KEGG analysis of total-, up- and down-regulated genes in the FMT-DSS + SP group vs the FMT-DSS group (Top 20). n = 4 mice/group. g Quantitative real-time PCR analysis of mRNA expressions of NF-κB signaling pathway genes ( p65 and IKBα ) in the hippocampus. n = 4 mice/group. The whiskers indicate the minimum and maximum values observed within the range of Q1 − 1.5 × IQR to Q3 + 1.5 × IQR. The box reveals the interquartile range (IQR) between the 25th (Q1) and 75th (Q3) percentiles, and the line inside the box represents the median (50th percentile). h The protein expression of p-p65 and p-IKBα in hippocampus tissue, as determined by western blotting. n = 3 independent experiments. i Quantitative real-t i me PCR analysis of mRNA expressions of GABA receptor and Ca 2+ signaling genes ( Gabra1 , Gabra3 , Gabrg2 , Gabrb2 and Camk2d ). n = 4 mice/group. The whiskers indicate the minimum and maximum values observed within the range of Q1 − 1.5 × IQR to Q3 + 1.5 × IQR. The box reveals the interquartile range (IQR) between the 25th (Q1) and 75th (Q3) percentiles, and the line inside the box represents the median (50th percentile). j Protein expressions of GABA A R, GABA B R, GAD65 and CaMKII were measured in hippocampus tissue by western blot. n = 3 independent experiments. k Representative immunofluorescence images of double-labeling for p-IKBα (red)/Iba1 (green) in hippocampus tissues. Scale bar = 50 μm. n = 3 independent experiments. I Representative immunofluorescence images of double-labeling for GABA A R (green)/GFAP (red) in hippocampus tissues. Scale bar = 50 μm. n = 3 independent experiments. m Double immunofluorescence staining for CaMKII (green)/GFAP (red) in hippocampus tissues and statistical analysis. Scale bar = 50 μm. n = 3 independent experiments. n Differential gene expressions were presented by the heatmap in microglia of three groups, and GSEA analysis of microglia from FMT-DSS + SP group vs the FMT-DSS group. n = 4 mice/group. o Heat maps of differential gene expression in astrocytes from three groups and GSEA analysis of astrocytes from FMT-DSS + SP group vs the FMT-DSS group. n = 4 mice/group. Data were presented as means ± SD. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Source data are provided as a file.

Article Snippet: Next, primary antibody against GFAP (1:1000, A8335, 1:200, NB100-53809, USA), GABA A Rα1 (1:1000, 12708-1-AP, Proteintech, China), GABA B R2 (1:1000, 27567-1-AP, Proteintech, China), CaMKII (1:1000, 12716, Cell Signaling Technology, USA), GAD65 (1:1000, ab239372, Abcam, USA), CD206 (1:1000, ab64693, Abcam, USA), iNOS (1:1000, 18985-1-AP, Proteintech, China), CD80 (1:1000, 66406-1, Proteintech, China), p-p65 (1:1000, A00284-1, Boster, China), p-IKBα (1:1000, WLH3930, Wanleibio, China), and β-actin (50201, 1:1000, Kemei Borui Science and Technology, China) were incubated with the membranes overnight at 4 °C.

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Immunofluorescence, Labeling, Double Immunofluorescence Staining

Journal: Nature Communications

Article Title: Neuropeptide SP protects against colitis and linked anxiety-like behavior through the putative roles of gut microbiota and metabolite inositol

doi: 10.1038/s41467-025-67904-0

Figure Lengend Snippet: a Diagram illustrating the mouse model of colitis employed in this study, and inositol treatments are indicated (Some schematic elements were created by Figdraw.com). b Daily body weight changes. n = 8 mice/group. c Diseases activity index. n = 6 mice/group. d Macroscopic pictures of colons. e H&E-stained colon sections. Scale bar = 100 μm. n = 5 independent experiments. f Immunofluorescence staining for E-cadherin (red)/DAPI (blue) in colon tissues. Scale bar = 100 μm. n = 4 independent experiments. g The mRNA level of inflammatory cytokines ( IL-1β , IL-6 and TNF-α ) in the colon. n = 4 mice/group. h , i Representative movement tracks in the open field test and related bar graphs (Distance traveled, distance of center region and speed). n = 6 mice/group. j , k Track plot of the elevated plus maze, and statistical analysis including percentage of time spent in the open arms and percentage of times entering the open arms. n = 6 mice/group. l Representative H&E-stained hippocampus sections of three groups. Scale bar = 100 μm. n = 6 independent experiments. m Photomicrographs of Nissl staining in the hippocampus. Scale bar = 100 μm. n = 6 independent experiments. n The mRNA expressions of downstream cytokines ( IL-6 , IL-1β , TNF-α and IL-10 ) in the hippocampus tissue. n = 5 mice/group. o Representative immunohistochemistry images of Iba-1 in hippocampus, Scale bar = 100 μm. n = 3 independent experiments. p The concentration of GABA in hippocampus tissue. n = 6 mice/group. q The mRNA expression of GABA A Rα1 in hippocampus. n = 5 mice/group. r The protein level of GABA B R detected by western blot. n = 3 independent experiments. s Double immunofluorescence staining for GFAP (red)/GAT1 (green) in the DG and CA1 regions of hippocampus tissue. Scale bar = 50 μm. n = 3 independent experiments. Data were presented as means ± SD. For body weight change, two-way repeated-measures ANOVA was performed and the rest of the statistics was analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Source data are provided as a file.

Article Snippet: Next, primary antibody against GFAP (1:1000, A8335, 1:200, NB100-53809, USA), GABA A Rα1 (1:1000, 12708-1-AP, Proteintech, China), GABA B R2 (1:1000, 27567-1-AP, Proteintech, China), CaMKII (1:1000, 12716, Cell Signaling Technology, USA), GAD65 (1:1000, ab239372, Abcam, USA), CD206 (1:1000, ab64693, Abcam, USA), iNOS (1:1000, 18985-1-AP, Proteintech, China), CD80 (1:1000, 66406-1, Proteintech, China), p-p65 (1:1000, A00284-1, Boster, China), p-IKBα (1:1000, WLH3930, Wanleibio, China), and β-actin (50201, 1:1000, Kemei Borui Science and Technology, China) were incubated with the membranes overnight at 4 °C.

Techniques: Activity Assay, Staining, Immunofluorescence, Immunohistochemistry, Concentration Assay, Expressing, Western Blot, Double Immunofluorescence Staining

Journal: Nature Communications

Article Title: Neuropeptide SP protects against colitis and linked anxiety-like behavior through the putative roles of gut microbiota and metabolite inositol

doi: 10.1038/s41467-025-67904-0

Figure Lengend Snippet: a Schematic illustration of experimental design (Some schematic elements were created by Figdraw.com). b , c Track plot of the elevated plus maze, and statistical analysis including percentage of time spent in the open arms, percentage of distance traveled in the open arms and percentage of times entering the open arms. n = 6 mice/group. d , e Track plot of the elevated zero maze, and statistical analysis including percentage of time spent in the open zones and percentage of times entering the open zones. n = 6 mice/group. f Statistical analysis, including percentage of distance traveled in the light area, percentage of time spent in the light area, and times of transitions in the light-dark box test. n = 6 mice/group. g Representative images of H&E-staining for the hippocampus sections. Scale bar = 100 μm. n = 6 independent experiments. h Representative images of Nissl staining for the hippocampus sections. Scale bar = 100 μm. n = 4 independent experiments. i The content of inositol in hippocampus tissue tested by ELISA. n = 8 mice/group. j The content of inositol in serum tested by ELISA. n = 8 mice/group. k–n The mRNA expression of TNF-α , IL-1β , IL-6 and iNOS in BV-2 cells. n = 3 mice/group. o, p The mRNA expression of Gabra3 and Camk2d in C8D1A cells. n = 3 mice/group. q The protein level of GABA A R, CaMKII, GABA B R detected by western blot. n = 3 independent experiments. Data were presented as means ± SD. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Source data are provided as a file.

Article Snippet: Next, primary antibody against GFAP (1:1000, A8335, 1:200, NB100-53809, USA), GABA A Rα1 (1:1000, 12708-1-AP, Proteintech, China), GABA B R2 (1:1000, 27567-1-AP, Proteintech, China), CaMKII (1:1000, 12716, Cell Signaling Technology, USA), GAD65 (1:1000, ab239372, Abcam, USA), CD206 (1:1000, ab64693, Abcam, USA), iNOS (1:1000, 18985-1-AP, Proteintech, China), CD80 (1:1000, 66406-1, Proteintech, China), p-p65 (1:1000, A00284-1, Boster, China), p-IKBα (1:1000, WLH3930, Wanleibio, China), and β-actin (50201, 1:1000, Kemei Borui Science and Technology, China) were incubated with the membranes overnight at 4 °C.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot

Journal: bioRxiv

Article Title: Analgesic α-conotoxins modulate GIRK1/2 channels via GABA B receptor activation and reduce neuroexcitability

doi: 10.1101/2020.12.02.407627

Figure Lengend Snippet: A, C Representative K + currents recorded from GIRK1/2 channels co-expressed either with GABA B R ( A ) or alone ( C ) in response to a 50 ms voltage ramp protocol (−100 to +40 mV; see inset) applied at 0.1 Hz from a holding potential of −40 mV in the absence (control) and presence of 1 μM Vc1.1 (blue), 100 μM baclofen (red) or 1 mM Ba 2+ (grey). B, D Corresponding diary plots of K + current amplitude at −100 mV as a function of time in cells expressing GIRK1/2 channels and GABA B R (B) and GIRK1/2 channels alone D Responses to sequential bath application of Vc1.1 (blue), baclofen (red), and Ba 2+ (grey) are indicated by the bars above. E Vc1.1 potentiates also homomeric GIRK2 channel when co-expressed with GABA B R. Representative human GIRK2-mediated K + currents obtained in the absence (Control, black), and presence of 1 μM Vc1.1 (blue), 100 μM baclofen (red) or 1 mM Ba 2+ (grey). F Corresponding diary plot to (E) showing peak K + current amplitude at −100 mV as a function of time in response to bath application of Vc1.1 (blue), baclofen (red) or Ba 2+ (grey). G α-Conotoxin Vc1.1 concentration-response relationship obtained for potentiation of GIRK1/2 co-expressed with GABA B R in HEK293T cells. ΔI Kir represents total I Kir (basal + potentiation) minus basal I Kir . H Bar graph of ΔI Kir density (pA/pF) at −100 mV in response to 100 μM baclofen (red), 100 μM GABA (orange), 1 μM Vc1.1 (blue), 1 μM RgIA (purple) and 1 μM PeIA (yellow) recorded from cells expressing either heteromeric GIRK1/2 (solid) or homomeric GIRK2 (hashed) co-expressed with GABA B R. Neither Vc1.1 nor baclofen potentiates K + currents in cells expressing GIRK1/2 alone. The vertical dotted line (40 pA/pF) is for reference. Data represent mean ± SEM and the number of experiments is given in parentheses.

Article Snippet: HEK293T cells were transiently co-transfected with plasmid cDNAs encoding human GIRK 1 (KCNJ3 or Kir3.1) and/or GIRK 2 (KCNJ6 or Kir3.2) (both pcDNA3 based and were kindly provided by Dr Paul Slesinger, Mt Sinai, New York, USA) and human GABA B1 and GABA B2 subunits (OriGene Technologies, Inc, Rockville, MD USA) using Lipofectamine 2000 (ThermoFisher Scientific, Australia).

Techniques: Control, Expressing, Concentration Assay

Journal: bioRxiv

Article Title: Analgesic α-conotoxins modulate GIRK1/2 channels via GABA B receptor activation and reduce neuroexcitability

doi: 10.1101/2020.12.02.407627

Figure Lengend Snippet: A Representative current-voltage relationship elicited from HEK293 cells co-expressing GABA B R and GIRK1/2 channels in the absence (Control, black) and presence of 1 μM Vc1.1 reduced by 1 mM DTT/reduced Vc1.1 (Vc1.1 RA ) (pink), 100 μM baclofen (red), 1 μM wild-type Vc1.1 (blue) or 1 mM Ba 2+ (grey). B GIRK1/2-mediated K + current amplitude measured at −100 mV plotted as a function of time in the presence of sequential application of reduced Vc1.1 (DTT treatment/Vc1.1 RA ), baclofen, globular Vc1.1 and Ba 2+ . C Representative current-voltage relationships obtained from HEK293T cells co-expressing GABA B R and GIRK1/2 channels in the absence (Control, black) and presence of 1 μM ImI (light blue), 1 μM Vc1.1 (blue), 100 μM baclofen (red) or 1 mM Ba 2+ (grey). D Peak K + current amplitude plotted as a function of time during sequential application of ImI, Vc1.1, baclofen, and Ba 2+ . E Quantification of the effect of reduced (linear) Vc1.1 and ImI compared to globular Vc1.1 and baclofen. Data represent mean ± SEM. Statistical significance, **** p < 0.0001; one-way ANOVA. Number of experiments is given in parentheses.

Article Snippet: HEK293T cells were transiently co-transfected with plasmid cDNAs encoding human GIRK 1 (KCNJ3 or Kir3.1) and/or GIRK 2 (KCNJ6 or Kir3.2) (both pcDNA3 based and were kindly provided by Dr Paul Slesinger, Mt Sinai, New York, USA) and human GABA B1 and GABA B2 subunits (OriGene Technologies, Inc, Rockville, MD USA) using Lipofectamine 2000 (ThermoFisher Scientific, Australia).

Techniques: Expressing, Control

Journal: bioRxiv

Article Title: Analgesic α-conotoxins modulate GIRK1/2 channels via GABA B receptor activation and reduce neuroexcitability

doi: 10.1101/2020.12.02.407627

Figure Lengend Snippet: A Bath application of the selective GABA B R antagonist CGP 55845 (1 μM) antagonizes Vc1.1 potentiation of GIRK1/2 channels. Representative K + currents recorded from HEK293T cells co-expressing GABA B R and GIRK 1/2 channels in the absence (control; black) or presence of 1 μM CGP 55845 alone (green), and co-application of CGP 55845 plus 1 μM Vc1.1 (light green), Vc1.1 alone (blue) or 1 mM Ba 2+ (grey). B Corresponding dairy plot of K + current amplitude at −100 mV (A) as a function of time. C Representative K + currents elicited from cells co-expressing GABA B R and GIRK1/2 channels with 24 hr pre-treatment with 1 μg/ml Pertussis toxin (PTX). D Corresponding diary plots of peak K + currents at −100 mV (C) as a function of time. E Inclusion of 500 μM GDP-β-S in the intracellular patch pipette solution abolished Vc1.1 potentiation of GIRK1/2 current. Representative K + current traces in the absence (control; black) or presence of Vc1.1 (blue), baclofen (red) or Ba 2+ (grey). F Corresponding diary plot of (E) as a function of time. G Inclusion of Gβγ scavenger, GRK2i (10 μM) inhibited potentiation by both Vc1.1 and baclofen in cells co-expressing GABA B R and GIRK1/2. H Corresponding time plot of (G) showing peak I Kir potentiation by Vc1.1 and baclofen at −100 mV. I Bar graph of ΔI Kir density recorded at −100 mV in response to 1 μM Vc1.1 (−29.5 ± 1.52 pA/pF, blue) or 100 μM baclofen (−68.4 ± 5 pA/pF, red) in HEK293T cells co-expressing GIRK1/2 channels and GABA B R (control). Potentiation of ΔI Kir density by Vc1.1 and baclofen was significantly reduced by 1 μM CGP 55845. Following pre-treatment with PTX (1 μg/ml) or inclusion of either GDP-β-S (500 μM) or Gβγ scavenger, GRK2i (10 μM), in the intracellular pipette solution, ΔI Kir density was similarly attenuated compared to peak current density measured for Vc1.1 or baclofen in control condition. Data are expressed as mean ± SEM and statistical significance, **** p < 0.0001 vs control; one-way ANOVA followed by Tukey’s post hoc test; number of experiments is given in parentheses.

Article Snippet: HEK293T cells were transiently co-transfected with plasmid cDNAs encoding human GIRK 1 (KCNJ3 or Kir3.1) and/or GIRK 2 (KCNJ6 or Kir3.2) (both pcDNA3 based and were kindly provided by Dr Paul Slesinger, Mt Sinai, New York, USA) and human GABA B1 and GABA B2 subunits (OriGene Technologies, Inc, Rockville, MD USA) using Lipofectamine 2000 (ThermoFisher Scientific, Australia).

Techniques: Expressing, Control, Transferring

Journal: bioRxiv

Article Title: Analgesic α-conotoxins modulate GIRK1/2 channels via GABA B receptor activation and reduce neuroexcitability

doi: 10.1101/2020.12.02.407627

Figure Lengend Snippet: A Immunolabeling of mouse DRG neurons and visual inspection by confocal microscopy revealed both GIRK1 and GIRK2 channels express and show co-localization with each other using antibodies directed against GIRK1 and GIRK2 channels. B DRG neurons showing expression of GABA B2 subunit and GIRK2 channel and both GIRK2 and GABA B2 co-localize in mouse DRG neurons. Co-expression of GIRK 1 and GIRK 2 or GIRK2 and GABA B R immunoreactivity in mouse DRG neurons was independent of cell diameter. Scale bars: 100 μm. DAPI (blue) staining marks nucleus. C Diary plot of peak inward K + currents recorded at −100 mV in small to medium diameter (<30 μm) mouse DRG neurons as a function of time. Whole-cell K + currents obtained in response to voltage ramps (−100 mV to +40 mV) applied at 0.1 Hz in the absence (basal control taken after baclofen washout, black) and presence of 1 μM Vc1.1 (blue), 100 μM baclofen (red) or 500 nM Tertiapin-Q (brown). D Bar graph of inward K + current density measured at −100 mV in response to Vc1.1 (1 μM, blue) and baclofen (100 μM, red). Data represent mean ± SEM. Number of experiments are shown in parentheses. p = 0.011, paired t-test. E Summary of the effects of Tertiapin-Q (500 nM, brown) and Ba 2+ (1 mM, grey) on whole-cell inward I K . Data represent mean ± SEM. p = 0.001, paired t-test. Number of experiments is given in parentheses.

Article Snippet: HEK293T cells were transiently co-transfected with plasmid cDNAs encoding human GIRK 1 (KCNJ3 or Kir3.1) and/or GIRK 2 (KCNJ6 or Kir3.2) (both pcDNA3 based and were kindly provided by Dr Paul Slesinger, Mt Sinai, New York, USA) and human GABA B1 and GABA B2 subunits (OriGene Technologies, Inc, Rockville, MD USA) using Lipofectamine 2000 (ThermoFisher Scientific, Australia).

Techniques: Immunolabeling, Confocal Microscopy, Expressing, Staining, Control

Journal: bioRxiv

Article Title: Analgesic α-conotoxins modulate GIRK1/2 channels via GABA B receptor activation and reduce neuroexcitability

doi: 10.1101/2020.12.02.407627

Figure Lengend Snippet: A-C GIRK1 (red), GIRK2 (green) and GABA B R2 (red) exhibited immunoreactivity in HEK293T cells and DAPI (blue) staining denotes the nucleus of the cells. D-E DAPI (blue), GIRK1 (red), GIRK2 (green) and GABA B R2 (red) immunoreactivity in HEK293T cells. The merged/overlay images depict co-localization of GIRK1 and GIRK2 or GIRK2 and GABA B R2 upon transfection. Scale bars: 100 μm.

Article Snippet: HEK293T cells were transiently co-transfected with plasmid cDNAs encoding human GIRK 1 (KCNJ3 or Kir3.1) and/or GIRK 2 (KCNJ6 or Kir3.2) (both pcDNA3 based and were kindly provided by Dr Paul Slesinger, Mt Sinai, New York, USA) and human GABA B1 and GABA B2 subunits (OriGene Technologies, Inc, Rockville, MD USA) using Lipofectamine 2000 (ThermoFisher Scientific, Australia).

Techniques: Staining, Transfection

Journal: bioRxiv

Article Title: Analgesic α-conotoxins modulate GIRK1/2 channels via GABA B receptor activation and reduce neuroexcitability

doi: 10.1101/2020.12.02.407627

Figure Lengend Snippet: Allosteric binding of Vc1.1 to GABA B R activates heterotrimeric G protein. Dissociation of Gα i/o and heterodimer Gβγ (red line) of G protein activates heteromeric GIRK1/2 channel. Similarly, baclofen binding to orthosteric binding site, Venus Fly Trap (VFT) of GABA B1 subunit of GABA B R can also potentiate GIRK channel via dissociation of Gα i/o and Gβγ heterodimer of G protein.

Article Snippet: HEK293T cells were transiently co-transfected with plasmid cDNAs encoding human GIRK 1 (KCNJ3 or Kir3.1) and/or GIRK 2 (KCNJ6 or Kir3.2) (both pcDNA3 based and were kindly provided by Dr Paul Slesinger, Mt Sinai, New York, USA) and human GABA B1 and GABA B2 subunits (OriGene Technologies, Inc, Rockville, MD USA) using Lipofectamine 2000 (ThermoFisher Scientific, Australia).

Techniques: Binding Assay

Journal: Scientific Reports

Article Title: Extracellular Signal-Regulated Kinases Mediate an Autoregulation of GABA B -Receptor-Activated Whole-Cell Current in Locus Coeruleus Neurons

doi: 10.1038/s41598-020-64292-x

Figure Lengend Snippet: The activation of GABA B Rs increases pERK 1 levels in LC tissue. ( A ) The images show two sagittal brainstem slices from an animal. The LC in the left slice was punched (A1) for western blot analysis, and the right slice was used for comparison (A2). IHC with anti-TH antibody was performed for the two slices, as shown in the insets showing merged fluorescence images of anti-TH (red) and DAPI (blue) staining of the dashed rectangular areas at high magnification. A comparison of the two slices shows that the punched area contained mostly TH-ir tissue. ( B ) Images show representative western blot analysis results for pERK 1/2 in LC tissue punched from slices bathed in vehicle or baclofen (B1) and from slices bathed in baclofen or baclofen plus CGP54626 (B2). The plot in the right panels summarizes the results. Each paired circle and line indicates the result of a single experiment; bars and capped lines denote the mean and SEM, respectively. The asterisks denote significant differences compared to the control at p < 0.05 (*) and p < 0.01 (**); ns denotes no significance compared to the control.

Article Snippet: In Fig. , the primary antibodies were rabbit polyclonal antibodies against p44/42 MAPK (ERK 1/2 ) (1/1000, Cell Signaling Technology) or rabbit monoclonal antibodies against phospho-p44/42 MAPK (ERK 1/2 ) (1/750, Cell Signaling Technology); in Fig. , the primary antibodies were rabbit monoclonal antibodies against the GABA B2 receptor (1/1000, Cell Signaling Technology) and rabbit polyclonal antibodies against phospho-S783 of the GABA B2 receptor (1/1000, Rockland Immunochemicals).

Techniques: Activation Assay, Western Blot, Comparison, Fluorescence, Staining, Control

Journal: Scientific Reports

Article Title: Extracellular Signal-Regulated Kinases Mediate an Autoregulation of GABA B -Receptor-Activated Whole-Cell Current in Locus Coeruleus Neurons

doi: 10.1038/s41598-020-64292-x

Figure Lengend Snippet: The inhibition of ERK 1/2 attenuates GABA B R-mediated tonic inhibition of LC neurons. ( A ) The top bar shows the experimental protocol, and the bottom traces show representative episodes of recordings with baclofen (left panel) and CGP54626 (right panel) from an LC neuron in control slices (upper raw), U0126-treated (middle), and FR180204-treated slices (bottom raw). The recordings were performed using the cell-attached configuration to record spontaneous APs. (B & C) Summarized results show the change in the spontaneous firing rate upon CGP54626 application (B) and GABA B R-mediated tonic inhibition (C). Each dashed line (B) or cross (C) shows the results of an individual experiment; the circles (B) or bars (C) denote the mean, and capped lines denote the SEM. The asterisks indicate a significant difference compared to the control at p < 0.05 (*).

Article Snippet: In Fig. , the primary antibodies were rabbit polyclonal antibodies against p44/42 MAPK (ERK 1/2 ) (1/1000, Cell Signaling Technology) or rabbit monoclonal antibodies against phospho-p44/42 MAPK (ERK 1/2 ) (1/750, Cell Signaling Technology); in Fig. , the primary antibodies were rabbit monoclonal antibodies against the GABA B2 receptor (1/1000, Cell Signaling Technology) and rabbit polyclonal antibodies against phospho-S783 of the GABA B2 receptor (1/1000, Rockland Immunochemicals).

Techniques: Inhibition, Control

Journal: Scientific Reports

Article Title: Extracellular Signal-Regulated Kinases Mediate an Autoregulation of GABA B -Receptor-Activated Whole-Cell Current in Locus Coeruleus Neurons

doi: 10.1038/s41598-020-64292-x

Figure Lengend Snippet: ERK 1/2 activated by GABA B R does not have an effect on pGABA B2 R. Left images show representative western blot analysis results for pGABA B2 R in LC tissue punched from slices bathed in baclofen or in baclofen plus FR180204. The plot in the right panel summarizes the results. Each paired circle and line indicates the result of a single experiment; bars and vertical lines denote the mean and SEM, respectively. ns denotes no significance compared to the control.

Article Snippet: In Fig. , the primary antibodies were rabbit polyclonal antibodies against p44/42 MAPK (ERK 1/2 ) (1/1000, Cell Signaling Technology) or rabbit monoclonal antibodies against phospho-p44/42 MAPK (ERK 1/2 ) (1/750, Cell Signaling Technology); in Fig. , the primary antibodies were rabbit monoclonal antibodies against the GABA B2 receptor (1/1000, Cell Signaling Technology) and rabbit polyclonal antibodies against phospho-S783 of the GABA B2 receptor (1/1000, Rockland Immunochemicals).

Techniques: Western Blot, Control

Journal: Journal of Neurochemistry

Article Title: GABA B Receptor Modulation of Membrane Excitability in Human Pluripotent Stem Cell‐Derived Sensory Neurons by Baclofen and α‐Conotoxin Vc1.1

doi: 10.1111/jnc.70004

Figure Lengend Snippet: Expression of ion channels and membrane proteins in H9 NGN2 ‐induced sensory neurons (iSNs) that modulate neuronal excitability. (A–E) Immunocytochemistry staining in H9 NGN2 iSNs: (A) GABA B receptor subunits GABBR2 (green) and GABBR1 (red). (B) G protein–coupled inwardly rectifying potassium channel subunit 1 (GIRK1/KCNJ3, green) and GABBR1 (red). (C) Voltage‐gated calcium channel (Ca V 2.2/CACNA1B, green) and GABBR1 (red). (D) Voltage‐gated sodium channel (Na V 1.7/SCN9A, green) and neuronal marker TUJ1 (red). (E) Voltage‐gated sodium channel (Na V 1.8/SCN10A, green) and neuronal marker TUJ1 (red). (F–I) Scaled transcriptomic expression (TPM) of curated genes in discrete classes of ion channels and membrane receptors. Expression levels in iSNs (dark blue, TPM values denoted) are compared to primary human sensory neurons (light blue), with genes pertinent to this study highlighted in red. (F) G protein–coupled inwardly rectifying K + channel (GIRK) subunit expression. (G) GABA B receptor subunit expression. (H) Calcium channel subunit expression. (I) Hyperpolarisation‐activated, cyclic nucleotide‐gated (HCN) channel subunit expression.

Article Snippet: Antibodies used for immunocytochemistry were those against SOX2 (AF2018; R&D Systems), OCT4 (sc‐5279 Santa Cruz Biotechnology, Dallas, TX, USA), CD271 (M‐1818‐100; Biosensis, Thebarton, SA, Australia), SOX10 (AF2684; R&D Systems, Minneapolis, MN, USA), BRN3A (MAB1585; Millipore, St. Louis, MO, USA), ISLET1 (ab20670; Abcam, Cambridge, UK), TUJ1 (MAB1637, Millipore), TUJ1 (ab18207, Abcam), Peripherin (ab4666, Abcam), NGN1 (MA5‐24900, Invitrogen), NGN2 (PA5‐78556, Invitrogen), GABBR1 (AP23115PU‐N; OriGene, Rockville, MD, USA), GABBR2 (ab75838, Abcam), Ca V 2.2 (KP10001, CALBIOCHEM), GIRK1 (APC‐005; Alomone Labs, Jerusalem, Israel), Na V 1.7 (ab65167, Abcam), Na V 1.8 (ab66743, Abcam), S100β (ab52642, Abcam), MAP2 (M4403, Sigma‐Aldrich), and PSD95 (#51–6900, Invitrogen).

Techniques: Expressing, Membrane, Immunocytochemistry, Staining, Marker

Journal: Journal of Biological Chemistry

Article Title: γ-Aminobutyric Acid Type B (GABAB) Receptor Expression Is Needed for Inhibition of N-type (Cav2.2) Calcium Channels by Analgesic α-Conotoxins

doi: 10.1074/jbc.m112.342998

Figure Lengend Snippet: FIGURE 1. GABAB receptor mRNA knockdown by siRNA treatment in rat DRG neurons. Transfection of siRNA (100 nM) targeting GABAB receptor sub- units GABAB R1 (R1 KD) and GABAB R2 (R2 KD) resulted in a knockdown by 67 3% (n 4) of R1 (A) and 46 8% (n 4) for R2 mRNA levels (B), respectively. This is compared with a negative control in which cells were transfected with an equal concentration of a scrambled, nontargeting siRNA. Co-transfection of R1- and R2-targeting siRNAs (R1R2 KD) resulted in knock- down levels similar to single subunit knockdowns (66 6 and 47 13%, respectively; n 5). mRNA levels were determined by qRT-PCR, calculated by comparative quantitation analysis, and normalized to the expression levels of water-transfected cells (*, p 0.05; ***, p 0.0001 versus control).

Article Snippet: HEK293 cells stably expressing humanCav2.2 channels ( 1B, 2 1 and 3 subunits) (17) were transiently co-transfected with cDNAs encoding human GABAB R1 (10 g), human GABAB R2 (10 g) (OriGene Technologies, Inc., Rockville, MD), and enhanced green fluorescent protein (1 g) (a reporter gene construct), using calcium phosphate precipitation (18).

Techniques: Knockdown, Transfection, Negative Control, Concentration Assay, Cotransfection, Quantitative RT-PCR, Quantitation Assay, Expressing, Control

Journal: Journal of Biological Chemistry

Article Title: γ-Aminobutyric Acid Type B (GABAB) Receptor Expression Is Needed for Inhibition of N-type (Cav2.2) Calcium Channels by Analgesic α-Conotoxins

doi: 10.1074/jbc.m112.342998

Figure Lengend Snippet: FIGURE 2. GABAB receptor immunofluorescence is reduced in GABAB knockdownDRGneuronsasobservedusingconfocalmicroscopy.Immu- nolabeling of DRG neurons and visual inspection by confocal microscopy revealed an apparent decrease in fluorescence intensities for GABAB R1 and R2inneuronstransfectedwithsiRNAstodisruptGABABR1andR2expression. This is compared with scrambled, nontargeting siRNA-transfected DRG neu- rons. Cells were immunostained 3 days after transfection, using antibodies directed against R1 (green) (A) or R2 (green) (B). Immunostaining of III-tubu- lin is shown in red. Representative images are shown. Scale bars, 50 m.

Article Snippet: HEK293 cells stably expressing humanCav2.2 channels ( 1B, 2 1 and 3 subunits) (17) were transiently co-transfected with cDNAs encoding human GABAB R1 (10 g), human GABAB R2 (10 g) (OriGene Technologies, Inc., Rockville, MD), and enhanced green fluorescent protein (1 g) (a reporter gene construct), using calcium phosphate precipitation (18).

Techniques: Immunofluorescence, Confocal Microscopy, Fluorescence, Transfection, Immunostaining

Journal: Journal of Biological Chemistry

Article Title: γ-Aminobutyric Acid Type B (GABAB) Receptor Expression Is Needed for Inhibition of N-type (Cav2.2) Calcium Channels by Analgesic α-Conotoxins

doi: 10.1074/jbc.m112.342998

Figure Lengend Snippet: FIGURE 3. Quantitative evaluation of protein levels in GABAB R1 knock- down DRG neurons by high content imaging analysis revealed signifi- cantly reduced GABAB R1 expression. We compared DRG cells transfected with siRNAs targeting both GABAB receptor subunits (R1R2 KD) with control cells transfected with scrambled, nontargeting siRNA (control). Quantitative analysis of immunofluorescence-labeled R1 proteins in DRG neurons 2–3 days after transfection revealed significantly reduced R1 protein expression in knockdown cells (R1R2 KD). A, representative sites of the high content scan of a total of 756 sites per treatment group. DRG neurons showing weak green fluorescence, indicating low expression of R1 (white arrowheads), were countedasneuronswithR1expressionbelowthresholdinthestatisticalanal- ysis. Scale bars, 50 m. B, percentage of neurons exhibiting R1 immunofluo- rescence above threshold, which correlates to high R1 expression levels. C, R1 expression levels of neurons with R1 expression above threshold calculated from fluorescence intensities. Number of experiments indicated in parenthe- ses (*, p 0.05; ***, p 0.0001 versus control).

Article Snippet: HEK293 cells stably expressing humanCav2.2 channels ( 1B, 2 1 and 3 subunits) (17) were transiently co-transfected with cDNAs encoding human GABAB R1 (10 g), human GABAB R2 (10 g) (OriGene Technologies, Inc., Rockville, MD), and enhanced green fluorescent protein (1 g) (a reporter gene construct), using calcium phosphate precipitation (18).

Techniques: Knockdown, Imaging, Expressing, Transfection, Control, Immunofluorescence, Labeling, Fluorescence

Journal: Journal of Biological Chemistry

Article Title: γ-Aminobutyric Acid Type B (GABAB) Receptor Expression Is Needed for Inhibition of N-type (Cav2.2) Calcium Channels by Analgesic α-Conotoxins

doi: 10.1074/jbc.m112.342998

Figure Lengend Snippet: FIGURE 4. Inhibition of N-type calcium channels in response to baclofen and Vc1. 1 is lower in DRG neurons transfected with siRNAs that target the GABAB receptor. A and B show superimposed traces of depolarization-activated VGCC currents in scrambled, nontargeting siRNA-transfected (A) and GABABR knockdown siRNA-transfected (B) rat DRG neurons, in the absence (control) and presence of Vc1.1 (100 nM) and baclofen (30 M). C and D, bar graph summary ofdataonpeakVGCCcurrentamplitudereductioninthepresenceofbaclofen(C)andVc1.1(D).Vc1.1inhibitionofVGCCinR1R2KDneuronswassignificantly lower than in control neurons. Number of experiments is indicated in parentheses (**, p 0.01 versus control).

Article Snippet: HEK293 cells stably expressing humanCav2.2 channels ( 1B, 2 1 and 3 subunits) (17) were transiently co-transfected with cDNAs encoding human GABAB R1 (10 g), human GABAB R2 (10 g) (OriGene Technologies, Inc., Rockville, MD), and enhanced green fluorescent protein (1 g) (a reporter gene construct), using calcium phosphate precipitation (18).

Techniques: Inhibition, Transfection, Knockdown, Control

Journal: Journal of Biological Chemistry

Article Title: γ-Aminobutyric Acid Type B (GABAB) Receptor Expression Is Needed for Inhibition of N-type (Cav2.2) Calcium Channels by Analgesic α-Conotoxins

doi: 10.1074/jbc.m112.342998

Figure Lengend Snippet: FIGURE 5. Inhibition of stably expressed Cav2. 2 channels in HEK 293 cells transiently co-transfected with human GABAB receptor subunits R1 R2 by -conotoxin Vc1.1 and baclofen. A–C, peak Ba2 current amplitude elicited by step depolarization (0.1 Hz) to 10 mV from a holding potential of 80 mV plotted as a function of time. Bars indicate bath application of various compounds. Superimposed inward Ba2 currents (top insets) are shown at the times marked by lowercase letters (see below). Dotted line indicates zero current level. A, in HEK 293 cells expressing Cav2.2 channels alone, depolarization-activated Ba2 currents are not affected by -conotoxin Vc1.1 (200 nM) but are almost completely inhibited by -conotoxin MVIIA (200 nM). B, in Cav2.2R1R2 cells, bath application of Vc1.1 (200 nM) inhibits inward Ba2 current amplitude. C, in Cav2.2R1R2 cells, the effect of Vc1.1 was antagonized by bath application of the selective GABAB receptor antagonist CGP55845 (1 M), whereas baclofen (50 M), applied after the washout of CGP55845, inhibited the inward Ba2 current. D, bar graph summary of the inhibition of peak Ba2 current by Vc1.1 (200 nM) and baclofen (50 M) in the absence of GABAB receptor subunits (Cav2.2 alone; control) or presence of Cav2.2 channels and R1, R2, or R1R2. Number of experiments indicated in parentheses (*, p 0.0001 versus control).

Article Snippet: HEK293 cells stably expressing humanCav2.2 channels ( 1B, 2 1 and 3 subunits) (17) were transiently co-transfected with cDNAs encoding human GABAB R1 (10 g), human GABAB R2 (10 g) (OriGene Technologies, Inc., Rockville, MD), and enhanced green fluorescent protein (1 g) (a reporter gene construct), using calcium phosphate precipitation (18).

Techniques: Inhibition, Stable Transfection, Transfection, Expressing, Control