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