Journal: Cells

Article Title: The Novel Monoacylglycerol Lipase Inhibitor MJN110 Suppresses Neuroinflammation, Normalizes Synaptic Composition and Improves Behavioral Performance in the Repetitive Traumatic Brain Injury Mouse Model

doi: 10.3390/cells10123454

Figure Lengend Snippet: MJN110 treatment restored the altered expression of the ionotropic glutamate and GABA receptor components and the survival signaling molecules in the TBI mouse hippocampus. At 8 days post-TBI, the expression of GABA A -α1, GABA A -β2,3, GABA A -γ2, GluR1, GluR2, NR2A, NR2B, pERK and pAKT in the ipsilateral hippocampal tissues was significantly reduced in the TBI vehicle group, and reversed by the MJN110 (2.5 mg/kg) treatment ( A , B , D , F ). The expression of GAD67 and NMDAR1 was upregulated in the TBI/vehicle group and reduced by the MJN110 treatment ( A , C , D ). There were no changes in the expression of VGLUT2 in all the experimental groups ( E ). The expression of COX-1, but not COX-2 was increased in the TBI/vehicle group ( G ). (** p < 0.01, *** p < 0.001, and **** p < 0.0001 comparison between sham and TBI/vehicle; # p < 0.05, ## p < 0.01 and ### p < 0.001 comparing TBI/vehicle vs. TBI + MJN110 groups. n = 5 animals in each group). Bars represent means ± SEM).

Article Snippet: These antibodies included anti-rabbit phosphorylated (p-) p44/42 MAPK (T202/Y204) (1:3000; cat# 9101S, Cell Signaling, Danvers, MA, USA), anti-rabbit p-AKT (S473) (1:2500; cat# 9271S, Cell Signaling), anti-mouse COX1 (1:500; cat#160109, Cayman Chemical), anti-mouse COX2 (1:500; cat#160109, Cayman Chemical), anti-Rabbit NR2A (1:1000; cat# 07-632, Millipore, Burlington, MA, USA), anti-Rabbit NR2B (1:1000; cat# AB1557P, Millipore), anti-Rabbit GluR1 clone C3T (1:1000; cat# 04-855, Millipore), anti-rabbit ionotropic glutamate receptor 2 (1:4000; cat# ab20673, Abcam, Cambridge, MA, USA), anti-mouse VGLUT2 (1:2500; cat# MAB5504, Millipore), anti-NMDAR1 (1:2000; cat# ab109182, Abcam), anti-mouse GAD67 clone 1G102 (1:4000; cat# MAB5406, Millipore), anti-mouse GABA A receptor α1 protein clone N95/35 (1:2500; cat# MABN489, Millipore), anti-mouse GABA A receptor β2,3 chain clone BD17 (1:2500; cat# MAB341, Millipore) and anti-mouse GABA A γ2 subunit clone KC4-8A7 (1:2500; cat# MABN875, Millipore).

Techniques: Expressing, Comparison

Journal: Cells

Article Title: The Novel Monoacylglycerol Lipase Inhibitor MJN110 Suppresses Neuroinflammation, Normalizes Synaptic Composition and Improves Behavioral Performance in the Repetitive Traumatic Brain Injury Mouse Model

doi: 10.3390/cells10123454

Figure Lengend Snippet: The reversed expression of glutamate and GABA A receptor subunits by MJN110 was independent of cannabinoid receptor signaling. ( A – F ) Western blot analysis of hippocampal GABA A -α1, GABA A -β 2,3, GABA A -γ2, GluR1, GluR2, NMDAR1, NR2A, NR2B, GAD67, VGLUT2, COX1 and COX2 at 30 days post-TBI. (* p < 0.05, ** p < 0.01 and *** p < 0.001 comparing sham vs. TBI/vehicle groups; # p < 0.05, ## p < 0.01 and ### p < 0.001 comparing TBI vs. TBI + MJN110 (2.5 mg/kg) groups). n = 5 animals in each group; Bars represent means ± SEM.

Article Snippet: These antibodies included anti-rabbit phosphorylated (p-) p44/42 MAPK (T202/Y204) (1:3000; cat# 9101S, Cell Signaling, Danvers, MA, USA), anti-rabbit p-AKT (S473) (1:2500; cat# 9271S, Cell Signaling), anti-mouse COX1 (1:500; cat#160109, Cayman Chemical), anti-mouse COX2 (1:500; cat#160109, Cayman Chemical), anti-Rabbit NR2A (1:1000; cat# 07-632, Millipore, Burlington, MA, USA), anti-Rabbit NR2B (1:1000; cat# AB1557P, Millipore), anti-Rabbit GluR1 clone C3T (1:1000; cat# 04-855, Millipore), anti-rabbit ionotropic glutamate receptor 2 (1:4000; cat# ab20673, Abcam, Cambridge, MA, USA), anti-mouse VGLUT2 (1:2500; cat# MAB5504, Millipore), anti-NMDAR1 (1:2000; cat# ab109182, Abcam), anti-mouse GAD67 clone 1G102 (1:4000; cat# MAB5406, Millipore), anti-mouse GABA A receptor α1 protein clone N95/35 (1:2500; cat# MABN489, Millipore), anti-mouse GABA A receptor β2,3 chain clone BD17 (1:2500; cat# MAB341, Millipore) and anti-mouse GABA A γ2 subunit clone KC4-8A7 (1:2500; cat# MABN875, Millipore).

Techniques: Expressing, Western Blot

Journal: Cells

Article Title: The Novel Monoacylglycerol Lipase Inhibitor MJN110 Suppresses Neuroinflammation, Normalizes Synaptic Composition and Improves Behavioral Performance in the Repetitive Traumatic Brain Injury Mouse Model

doi: 10.3390/cells10123454

Figure Lengend Snippet: Improvement on locomotor function, working memory and the expression of synaptic components by MJN110 was reversed by blockade of 2-AG synthesis. ( A ) Beam walk test was conducted at 4, 6 and 10 days post-TBI (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.001). ( B ) On day 13 post injury, spontaneous alternation Y maze test was performed (** p < 0.01). ( C – E ) Ipsilateral cortical tissues on day 4 post-TBI were subjected to LC-MS/MS analysis to measure 2-AG, AEA and AA. (## p < 0.01 and #### p < 0.001 comparing TBI vs. MJN110 (1 mg/kg); $ p < 0.05, $$ p < 0.01, $$$ p < 0.001 and $$$$ p < 0.001 comparing MJN110 (1 mg/kg) vs. DO34 (30 mg/kg) or DO34 + MJN110). ( F ) The ipsilateral cortical tissues at day 4 post-TBI were assessed for PGE 2 production by ELISA. (* p < 0.05 comparing TBI vs. DO34 or DO34 + MJN110). ( G ) Western blot analysis of the hippocampal GABA A -β2,3, GABA A -γ2, GluR1, GluR2, NMDAR1, NR2A and NR2B expression at 14 days post-TBI (* p < 0.05 and ** p < 0.01 comparing sham vs. TBI; # p < 0.05 and ## p < 0.01 comparing TBI vs. TBI + MJN110; $ p < 0.05 and $$ p < 0.01 comparing MJN110 vs. DO34 and DO34 + MJN110). n = 5–10 animals in each group; Bars represent means ± SEM.

Article Snippet: These antibodies included anti-rabbit phosphorylated (p-) p44/42 MAPK (T202/Y204) (1:3000; cat# 9101S, Cell Signaling, Danvers, MA, USA), anti-rabbit p-AKT (S473) (1:2500; cat# 9271S, Cell Signaling), anti-mouse COX1 (1:500; cat#160109, Cayman Chemical), anti-mouse COX2 (1:500; cat#160109, Cayman Chemical), anti-Rabbit NR2A (1:1000; cat# 07-632, Millipore, Burlington, MA, USA), anti-Rabbit NR2B (1:1000; cat# AB1557P, Millipore), anti-Rabbit GluR1 clone C3T (1:1000; cat# 04-855, Millipore), anti-rabbit ionotropic glutamate receptor 2 (1:4000; cat# ab20673, Abcam, Cambridge, MA, USA), anti-mouse VGLUT2 (1:2500; cat# MAB5504, Millipore), anti-NMDAR1 (1:2000; cat# ab109182, Abcam), anti-mouse GAD67 clone 1G102 (1:4000; cat# MAB5406, Millipore), anti-mouse GABA A receptor α1 protein clone N95/35 (1:2500; cat# MABN489, Millipore), anti-mouse GABA A receptor β2,3 chain clone BD17 (1:2500; cat# MAB341, Millipore) and anti-mouse GABA A γ2 subunit clone KC4-8A7 (1:2500; cat# MABN875, Millipore).

Techniques: Expressing, Liquid Chromatography with Mass Spectroscopy, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: The Journal of Neuroscience

Article Title: Increased Expression of Fibronectin Leucine-Rich Transmembrane Protein 3 in the Dorsal Root Ganglion Induces Neuropathic Pain in Rats

doi: 10.1523/JNEUROSCI.0295-19.2019

Figure Lengend Snippet: FLRT3 induces mechanical allodynia in vivo. A, Schematic and timeline of recombinant FLRT3 administration and behavioral testing. B, Relative hindpaw-withdrawal threshold after intrathecal administration of recombinant FLRT3 protein. The mean threshold in the FLRT3 group was significantly decreased compared with that in the saline (control) group, indicating that FLRT3 induced mechanical allodynia (n = 12–14 rats per group). *p < 0.05 (two-way repeated-measures ANOVA followed by Tukey–Kramer test). C, Representative immunoblots of NMDA-type glutamate receptor subunit GluN2B and pGluN2B in spinal cord lysates prepared at 12 h after FLRT3 administration. The pGluN2B signal intensity was normalized to that of total GluN2B (n = 3 rats per group), and signal intensity differences were analyzed by Student's t test. D, Double-immunofluorescence staining for the neuronal activation marker c-Fos (green) and neuronal marker NeuN (red) in the dorsal horn. Intrathecal FLRT3 administration increased c-Fos expression in dorsal horn neurons (arrows). Scale bar, 200 μm. E, Magnification images of Figure 4D. Scale bar, 10 μm. F, Total numbers of c-Fos-positive cells in dorsal horn laminae I-III at 12 h after administration in each group (n = 5 rats per group). *p < 0.05 (Student's t test). G, The c-fos-labeled cell profiles in lamina I-II and lamina III-V (n = 5 rats per group). *p < 0.05 (Student's t test). H, The frequency distribution of c-fos-positive cells is classified by nucleus diameter. n.s., not significant.

Article Snippet: The membranes were then incubated overnight at 4°C with antibodies against FLRT3 (1:500; AF2795, R&D Systems), NR2B (GluN2B) (1:500; TA309191, OriGene), phosphorylated (Tyr1472) NR2B (GluN2B; 1:500; TA309196, OriGene), β-actin (1:1000; 4970L, Cell Signaling Technology), or α-tubulin (1:1000; sc-5286, Santa Cruz Biotechnology).

Techniques: In Vivo, Recombinant, Western Blot, Double Immunofluorescence Staining, Activation Assay, Marker, Expressing, Labeling

Journal: Frontiers in Neuroscience

Article Title: Additive Effects of Environmental Enrichment and Ketamine on Neuropathic Pain Relief by Reducing Glutamatergic Activation in Spinal Cord Injury in Rats

doi: 10.3389/fnins.2021.635187

Figure Lengend Snippet: Primary antibodies for western blotting.

Article Snippet: Rabbit anti-phospho-NMDAR2B Tyr 1472 (p-NR2B) , 1:1,000 , Merck Millipore, Darmstadt, Germany.

Techniques: Western Blot

Journal: eNeuro

Article Title: Localized Calcium Signaling and the Control of Coupling at Cx36 Gap Junctions

doi: 10.1523/ENEURO.0445-19.2020

Figure Lengend Snippet: Primers used for cloning

Article Snippet: Cells were then immunolabeled using polyclonal antibodies for rabbit anti-NR2A (PhosphoSolutions; 1:500 dilution), rabbit anti-NR2B (Biosensis; 1:500 dilution), rabbit anti-NR2C (PhosphoSolutions; 1:500 dilution), and rat anti-NMDA NR1 (UC Davis NeuroMab; 1:500 dilution).

Techniques: Sequencing, Clone Assay, Modification, Plasmid Preparation

Journal: eNeuro

Article Title: Localized Calcium Signaling and the Control of Coupling at Cx36 Gap Junctions

doi: 10.1523/ENEURO.0445-19.2020

Figure Lengend Snippet: Immunofluorescence labeling of Cx36-GCaMP and NMDA receptors transfected into HEK293 cells. For each transfection combination, Cx36-GCaMP is shown in green, NR1 in red, and NR2x in blue. A , Cx36-GCaMP + NR1. B , Cx36-GCaMP + NR1 + NR2A. C , Cx36-GCaMP + NR1 + NR2B. D , Cx36-GCaMP + NR1 + NR2C. Well-formed gap junctions at cell-cell boundaries were used for live imaging experiments, while overexpressing cells with diffusely distributed Cx36-GCaMP were avoided.

Article Snippet: Cells were then immunolabeled using polyclonal antibodies for rabbit anti-NR2A (PhosphoSolutions; 1:500 dilution), rabbit anti-NR2B (Biosensis; 1:500 dilution), rabbit anti-NR2C (PhosphoSolutions; 1:500 dilution), and rat anti-NMDA NR1 (UC Davis NeuroMab; 1:500 dilution).

Techniques: Immunofluorescence, Labeling, Transfection, Imaging