r d systems af2086  (R&D Systems)

Journal: ACS Omega

Article Title: Electroacupuncture Attenuates Cyclophosphamide-Induced Bladder Pain by Restoring Autophagy in Spinal Inhibitory Interneurons

doi: 10.1021/acsomega.6c01060

Figure Lengend Snippet: Time-dependent increase in oxidative DNA damage in the spinal dorsal horn and its cellular distribution. (A) Representative images of 8-OHdG immunofluorescence in the spinal dorsal horn of control rats and CYP-treated rats at 4, 7, and 15 days. (B) Quantification of 8-OHdG integrated density. ( n = 5 rats/group; per-rat averages of 4–5 sections; Kruskal–Wallis test followed by Dunn’s multiple comparisons test.) (C) Representative double immunofluorescence images of 8-OHdG (red) with GFAP, Iba1, or NeuN (green) in CYP-treated rats at 4, 7, and 15 days (left). Quantification of the proportion of double-labeled cells among GFAP + , Iba1 + , or NeuN + cells (middle). Colocalization was also quantified by Pearson’s correlation coefficient ( r ) (right). ( n = 5 rats/time point; per-rat averages of 4–5 sections; Kruskal–Wallis test followed by Dunn’s multiple comparisons test.). (D) Representative images of 8-OHdG (red) and GAD67-GFP (green) in the spinal dorsal horn of control mice and CYP-treated GAD67-GFP mice at 15 days after CYP injection. Quantification of the proportion of double-labeled cells among GAD67-GFP-positive neurons (middle) and Pearson’s correlation coefficient ( r ) for 8-OHdG and GAD67-GFP colocalization (right). ( n = 5 mice/group; per-mouse averages of 4–5 sections; Mann–Whitney U test.) Scale bar: 100 μm. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: GAD67-GFP mice were acquired from The Jackson Laboratory and were used for immunofluorescence identification of inhibitory neurons.

Techniques: Immunofluorescence, Control, Labeling, Injection, MANN-WHITNEY

Journal: ACS Omega

Article Title: Electroacupuncture Attenuates Cyclophosphamide-Induced Bladder Pain by Restoring Autophagy in Spinal Inhibitory Interneurons

doi: 10.1021/acsomega.6c01060

Figure Lengend Snippet: Autophagy impairment is mainly observed in neurons and involves inhibitory neurons. (A) Representative double immunofluorescence images of p62 (red) with Iba1, GFAP, or NeuN (green) in the spinal dorsal horn of rats at 4, 7, and 15 days after CYP injection (left). Quantification of the proportion of double-labeled cells among GFAP-positive, Iba1-positive, or NeuN-positive cells (middle) and Pearson’s correlation coefficient ( r ) for colocalization between p62 and each marker (right). ( n = 5 rats/time point; per-rat averages of 4–5 sections; Kruskal–Wallis test followed by Dunn’s multiple comparisons test.) (B) Representative transmission electron microscopy (TEM) images of neurons in the spinal dorsal horn from control and CYP-treated rats (left), with quantification of the number of autophagosomes (right). Low-magnification images show overview fields, and high-magnification images show the corresponding zoom-ins of the boxed regions. Green arrows indicate ribosomes, and white arrows indicate autophagosomes/phagophores. Scale bars: 2 μm (low magnification) and 1 μm (high magnification). ( n = 6 rats/group; per-rat averages of 4–5 sections; Mann–Whitney U test.) (C) Representative immunofluorescence images showing p62 (red) and GAD67-GFP (green) in the spinal dorsal horn of control and CYP-treated mice at 15 days after CYP injection (left). Quantification of the proportion of double-labeled cells among GAD67-GFP-positive neurons (middle) and Pearson’s correlation coefficient ( r ) for colocalization between p62 and GAD67-GFP (right). ( n = 5 mice/group; per-mouse averages of 4–5 sections; Mann–Whitney U test.) Scale bar: 100 μm for immunofluorescence images. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: GAD67-GFP mice were acquired from The Jackson Laboratory and were used for immunofluorescence identification of inhibitory neurons.

Techniques: Immunofluorescence, Injection, Labeling, Marker, Transmission Assay, Electron Microscopy, Control, MANN-WHITNEY

Journal: Science Advances

Article Title: A causal coding variant regulating alternative splicing of DOC2A at 16p.11.2 GWAS locus influences susceptibility to schizophrenia

doi: 10.1126/sciadv.adw7667

Figure Lengend Snippet: ( A ) Representative traces of mEPSC in primary hippocampal neuron infected with control (empty vector), DOC2A Full-Length , or DOC2A ∆Val217-Pro218 AAV. ( B and C ) Quantification of mEPSC amplitude and frequency. N ≥ 29 neurons. ( D ) Representative traces of mIPSC in in primary hippocampal neuron infected with control, DOC2A Full-Length , or DOC2A ∆Val217-Pro218 AAV. ( E and F ) Quantification of mIPSC amplitude and frequency. N ≥ 20 neurons. ( G ) Representative gels of Western blot. ( H to K ) Quantification of synaptophysin, VAMP-2, GAD65, and GAD67 expression in hippocampus tissue overexpressing control, DOC2A Full-Length , or DOC2A ∆Val217-Pro218 . N = 3 in each group. Full Western blotting images are shown in fig. S16. Data are presented as the means ± SEM; * P < 0.05, ** P < 0.01, and *** P < 0.001. Error bars indicate the SEM.

Article Snippet: The primary antibodies were as follows: FLAG (Cell Signaling Technology, no. 8146S; 1:1000) and DOC2A (Invitrogen, no. PA5-31345; 1:1000), GAD67 (Proteintech, no. 10408-1-AP; 1:5000), GAD65 (Cell Signaling Technology, no. 5843T; 1:1000), synaptophysin (Sigma-Aldrich, no. S5768; 1:500), VAMP-2 (Synaptic Systems, no. 104211; 1:2000), α-tubulin (Proteintech, no. 66031-1-Ig; 1:20,000), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Proteintech, no. 10494-1-AP; 1:20,000).

Techniques: Infection, Control, Plasmid Preparation, Western Blot, Expressing

Journal: Brain Communications

Article Title: Gonadotropin-releasing hormone alleviates chronic pain-related depression in male mice by rebalancing the anterior cingulate cortex excitatory-inhibitory processes via the protein kinase C/Erb-B2 receptor tyrosine kinase 4 pathway

doi: 10.1093/braincomms/fcag138

Figure Lengend Snippet: Effect of GnRH on the E/I balance of ACC neurons induced by CFA injection . ( A ) The localization of GnRHR (red) with CamKII (green) and GAD67 (green) in the ACC of mice, bar = 200 μm. ( B ) Effects of Trip treatment on the protein levels of GAD67, VGAT, vGluT1 and vGluT2 are demonstrated by western blot and the semiquantitative analysis of these proteins relative to β-actin protein, and the original uncropped blots are shown in . ( C ) Effects of GnRH overexpression on the protein levels of GAD67, VGAT, vGluT1 and vGluT2 are demonstrated by western blot and the semiquantitative analysis of these proteins relative to β-actin, and the original uncropped blots are shown in . n = 6 per group for western blot. Two-way ANOVAs followed by Bonferroni’s post hoc testing for multiple comparisons among the groups. Each data point represents the data of one mouse. * P < 0.05, ** P < 0.01 and *** P < 0.001. Abbreviations: ACC, anterior cingulate cortex; NS, normal saline; CFA, complete Freund’s adjuvant; Lv, lentivirus; GFP, green fluorescent protein; GnRH, gonadotropin-releasing hormone; GnRHR, gonadotropin-releasing hormone receptor; OE, over expression; Trip, triptorelin; CaMKII, calcium/calmodulin-dependent protein kinase II; GAD67, glutamate decarboxylase 67; VGAT, vesicular γ-aminobutyric acid transporter; vGluT1, vesicular glutamate transporter 1; vGluT2, vesicular glutamate transporter 2.

Article Snippet: GAD67 , Boster , Rabbit , 67 , 1:500 , BA0603-2.

Techniques: Injection, Western Blot, Over Expression, Saline, Adjuvant

Journal: Brain Communications

Article Title: Gonadotropin-releasing hormone alleviates chronic pain-related depression in male mice by rebalancing the anterior cingulate cortex excitatory-inhibitory processes via the protein kinase C/Erb-B2 receptor tyrosine kinase 4 pathway

doi: 10.1093/braincomms/fcag138

Figure Lengend Snippet: ErbB4 is the downstream target of GnRH signalling to regulate the E/I balance and pain-related depression-like behaviour induced by CFA injection . ( A ) The localization of GnRHR (green) with ErbB4 (red) in the ACC of mice, bar = 200 μm. ( B ) Effects of Trip treatment on the protein level of p-ErbB4 are demonstrated by western blot and the semiquantitative analysis of p-ErbB4 relative to ErbB4, and the original uncropped blots are shown in . ( C ) Effects of GnRH overexpression on the protein levels of p-ErbB4 are demonstrated by western blot and the semiquantitative analysis of p-ErbB4 relative to ErbB4, and the original uncropped blots are shown in . ( D ) The experimental timeline for Trip and dacomitinib treatment after CFA injection. ( E ) Effects of Trip and dacomitinib treatment on the total movement distance of mice in the OFT. ( F ) Effects of Trip and dacomitinib treatment on the sucrose preference rate of 1% sucrose solution in the SCPT. ( G ) Effects of Trip and dacomitinib treatment on the immobility time within 4 min in the TST. ( H ) Effects of Trip and dacomitinib treatment on the immobility time within 4 min in the FST. ( I ) Effects of Trip and dacomitinib treatment on the protein levels of p-ErbB4, GAD67, VGAT, vGluT1 and vGluT2 are demonstrated by western blot and the semiquantitative analysis of these proteins relative to ErbB4 or β-actin, and the original uncropped blots are shown in . n = 6 mice per group for western blot test and n = 4–5 per group for behaviour tests and any value >2 SD from the group mean was considered an outlier and excluded from the analysis. One-way ANOVAs followed by Bonferroni’s post hoc testing for multiple comparisons among Trip or dacomitinib treatment groups. Two-way ANOVAs followed by Bonferroni’s post hoc testing for multiple comparisons among Lv-GnRH overexpression groups. Each data point represents the data of one mouse. * P < 0.05, ** P < 0.01 and *** P < 0.001. Abbreviations: ACC, anterior cingulate cortex; NS, normal saline; CFA, complete Freund’s adjuvant; ErbB4, Erb-B2 receptor tyrosine kinase 4; p-ErbB4, phosphorylated-Erb-B2 receptor tyrosine kinase 4; Lv, lentivirus; GFP, green fluorescent protein; GnRH, gonadotropin-releasing hormone; GnRHR, gonadotropin-releasing hormone receptor; OE, over expression; Trip, triptorelin; Dac, dacomitinib; GAD67, glutamate decarboxylase 67; VGAT, vesicular γ-aminobutyric acid transporter; vGluT1, vesicular glutamate transporter 1; vGluT2, vesicular glutamate transporter 2; OFT, open field test; SCPT, sucrose preference test; TST, tail suspension test; FST, forced swimming test.

Article Snippet: GAD67 , Boster , Rabbit , 67 , 1:500 , BA0603-2.

Techniques: Injection, Western Blot, Over Expression, Saline, Adjuvant, Suspension

Journal: Brain Communications

Article Title: Gonadotropin-releasing hormone alleviates chronic pain-related depression in male mice by rebalancing the anterior cingulate cortex excitatory-inhibitory processes via the protein kinase C/Erb-B2 receptor tyrosine kinase 4 pathway

doi: 10.1093/braincomms/fcag138

Figure Lengend Snippet: GnRH activates ErbB4 via the PKC pathway in the ACC of mice after CFA injection . ( A ) Effects of Trip treatment on the protein levels of PKCα and p-MARCKS are demonstrated by western blot and the semiquantitative analysis of PKCα and p-MARCKS relative to β-actin and MARCKS, and the original uncropped blots are shown in . ( B ) The experimental timeline for Trip and bisindolylmaleimide I treatment after CFA injection. ( C ) Effects of Trip and bisindolylmaleimide I treatment on the total movement distance by mice in the OFT. ( D ) Effects of Trip and bisindolylmaleimide I treatment on the sucrose preference rate of 1% sucrose solution in the SCPT. ( E ) Effects of Trip and bisindolylmaleimide I treatment on the immobility time within 4 min in the TST. ( F ) Effects of Trip and bisindolylmaleimide I treatment on the immobility time within 4 min in the FST. ( G ) Effects of Trip and bisindolylmaleimide I treatment on the protein levels of PKCα, p-MARCKS and p-ErbB4 were demonstrated by western blot and the semiquantitative analysis of these proteins relative to β-actin, MARCKS and ErbB4, and the original uncropped blots are shown in . ( H ) Effects of Trip and bisindolylmaleimide I treatment on the protein levels of GAD67, VGAT, vGluT1 and vGluT2 demonstrated by western blot and the semiquantitative analysis of these proteins relative to β-actin, and the original uncropped blots are shown in . n = 6 mice for western blot test and n = 6–9 mice per group for behaviour tests and any value >2 SD from the group mean was considered an outlier and excluded from the analysis. One-way ANOVAs followed by Bonferroni’s post hoc testing for multiple comparisons among the groups. Each data point represents the data of one mouse. * P < 0.05, ** P < 0.01 and *** P < 0.001. Abbreviations: ACC, anterior cingulate cortex; NS, normal saline; CFA, complete Freund’s adjuvant; ErbB4, Erb-B2 receptor tyrosine kinase 4; p-ErbB4, phosphorylated-Erb-B2 receptor tyrosine kinase 4; GnRH, gonadotropin-releasing hormone; GnRHR, gonadotropin-releasing hormone receptor; PKCα, protein kinase C α; MARCKS, myristoylated alanine-rich C kinase substrate; p-MARCKS, phosphorylated-myristoylated alanine-rich C kinase substrate; Trip, triptorelin; Bis I, bisindolylmaleimide I; GAD67, glutamate decarboxylase 67; VGAT, vesicular γ-aminobutyric acid transporter; vGluT1, vesicular glutamate transporter 1; vGluT2, vesicular glutamate transporter 2; OFT, open field test; SCPT, sucrose preference test; TST, tail suspension test; FST, forced swimming test.

Article Snippet: GAD67 , Boster , Rabbit , 67 , 1:500 , BA0603-2.

Techniques: Injection, Western Blot, Saline, Adjuvant, Suspension