Journal: Molecular Neurobiology

Article Title: Electroacupuncture Exerts Analgesic Effects by Restoring Hyperactivity via Cannabinoid Type 1 Receptors in the Anterior Cingulate Cortex in Chronic Inflammatory Pain

doi: 10.1007/s12035-023-03760-7

Figure Lengend Snippet: Endocannabinoids may be involved in electroacupuncture analgesia. ( A ) The mRNA level of CB1R in ACC was quantitatively analyzed. EA significantly increased CB1R mRNA downregulation in the ACC of model mice. Sham group: 1.09 ± 0.14, Model group: 0.5 ± 0.91, Model + EA group: 0.94 ± 0.90. (One-way ANOVA with Bonferroni test, F (2,15) = 7.5, P < 0.01. N = 6 mice for each group. ** vs. Sham group, * vs. Model + EA group). ( B ) Representative gel image of CB1R protein levels in ACC. ( C ) Statistical map of relative expression levels of CB1R protein. EA significantly increased CB1R protein downregulation in the ACC of model mice. Sham group: 1.0 ± 0.41, Model group: 0.8 ± 0.52, Model + EA group: 1.02 ± 0.44. EA significantly increased the fluorescence intensity of CB1R in the ACC of model mice. (One-way ANOVA with Bonferroni test, F (2,15) = 7.06, * P < 0.05. N = 6 mice for each group. * Sham group and Model + EA group, respectively). ( D ) The distribution of CB1R in ACC was displayed by immunofluorescence staining and 3D reconstruction using Imairs 9.0 software, Scale bars: 50 μm in immunofluorescence image and 30 μm in 3D reconstruction image. ( E ) Statistical graph of mean fluorescence intensity of CB1R in ACC. EA significantly increased the fluorescence intensity of CB1R in the ACC of model mice. Sham group: 67.82 ± 4.3, Model group: 37.91 ± 1.69, Model + EA group: 78.18 ± 10.92. (Dunnett T3 test, N = 5 mice for each group. * Sham group and Model + EA group, respectively). ( F ) The statistical plot represents the area of the CB1R in ACC. EA significantly increased the area of CB1R in the ACC of model mice. Sham group: 266621.32 ± 10803.03, Model group: 210534.81 ± 9188.47, Model + EA group: 283744 ± 19673.11. (One-way ANOVA with Bonferroni test, F (2,12) = 7.51, P < 0.01. N = 5 mice for each group. * vs. Sham group, ** vs. Model + EA group). ( G ) The statistical graph represents the volume of the CB1R in ACC. EA significantly increased the volume of CB1R in the ACC of model mice. Sham group: 145177.97 ± 6740.74, Model group: 102766.02 ± 4407.62, Model + EA group: 181473.25 ± 16117.87. (Tamhane T2 test, N = 5 mice for each group. ** vs. Sham group, * vs. Model + EA group). ( H ) A timeline of in vivo electrophysiological LFP recording. The electrodes were implanted in the ACC of mice by stereotaxic, and CFA was injected 21 days after recovery and EA was performed for 7 days. In vivo recording was performed at the end of EA treatment. ( I ) Alpha band in local field location recording. EA modulated the abnormal elevations in the alpha, bands. Sham group: (0.93 ± 0.10)*10 –6 , Model group: (7.32 ± 0.88)*10 –6 , Model + EA group: (4.98 ± 0.59)*10 –6 . (Kruskal–wallis test, ** vs. the Model + EA group, *** Sham group vs. Model + EA group, **** vs. Sham group). ( J ) Delta band in local field location recording. EA modulated the abnormal elevations in the delta, bands. Sham group: (0.30 ± 0.03)*10 –4 , Model group: (4.78 ± 1.01)*10 –4 , Model + EA group: (0.40 ± 0.06)*10 –4 . (Kruskal–wallis test, **** vs. Sham group and Model + EA group, respectively). ( K ) Theta band in local field location recording. EA modulated the abnormal elevations in the theta bands. Sham group: (0.29 ± 0.03)*10 –5 , Model group: (2.52 ± 0.33)*10 –5 , Model + EA group: (1.45 ± 0.17)*10 –5 . (Kruskal–wallis test, ** Sham group vs. Model + EA group, **** vs. Sham group and Model + EA group, respectively). (I-K) N = 3 for Sham, Model, Model + EA group. Data are presented as mean ± SEM

Article Snippet: Ten minutes before EA, 300 nl of CB1R antagonist AM251 was diluted to a concentration of 2.5 mg/mL (4.50 mM) using 100 μl Dimethyl sulfoxide (DMSO) + 400 μl PEG300 + 50 μl Tween-80 (MedChemExpress, United States) injected into the ACC region at a rate of 30 nl/min through a microinjection pump (RWD, China) connecting the cannula.

Techniques: Expressing, Fluorescence, Immunofluorescence, Staining, Software, In Vivo, Injection

Journal: Molecular Neurobiology

Article Title: Electroacupuncture Exerts Analgesic Effects by Restoring Hyperactivity via Cannabinoid Type 1 Receptors in the Anterior Cingulate Cortex in Chronic Inflammatory Pain

doi: 10.1007/s12035-023-03760-7

Figure Lengend Snippet: CB1R in ACC was necessary in the analgesic effect of electroacupuncture. (A) Schematic injection of AM251 or ACSF (300 μl) into ACC through cannula. ( B ) The mechanical threshold after EA was determined after injection of AM251 into ACC. Injection of AM251 in ACC reverses EA-mediated analgesia. (Two-way ANOVA with Bonferroni test, F (10, 120) = 6.04, P < 0.0001. N = 8, 10, 9 for Model + EA + ACSF, Model + EA + AM251 and Model + ACSF group. *, **, ***, **** vs. Model + EA + AM251 group. #, ##, ### vs. Model + ACSF group). ( C ) Statistical graph showing mechanical threshold determination on day 7 of EA after AM251 injection into ACC. Injection of AM251 in ACC reversed the effects of EA-mediated analgesia at day 7. Model + EA + ACSF: 0.85 ± 0.07, Model + EA + AM251: 0.26 ± 0.04 and Model + ACSF group: 0.32 ± 0.04. (One-way ANOVA with Bonferroni test, F (2, 24) = 39.2, P < 0.0001. N = 8, 10, 9 for Model + EA + ACSF, Model + EA + AM251 and Model + ACSF group. **, *** vs. Model + ACSF and Model + EA + AM251 group, respectively). ( D ) Immunofluorescence images showed the CB1R expression after bilateral ACC injection with rAAV-CMV-CRE knockout CB1R. ( E ) Representative gel image of CB1R protein levels in ACC after knockout. ( F ) Statistical diagram of relative expression level of CB1R protein in ACC of knockout mice. ACC Flox−CB1+/+ group: 1.0 ± 0.06, ACC Flox−CB1−/− group: 0.48 ± 0.03. (Independent-samples T test, t (10) = 7.68. N = 6 mice for each group. **** vs. ACC Flox−CB1+/+ group). ( G ) Mechanical threshold was used to determine the change of EA for 7 days after knocking down CB1R in ACC. Knockout of CB1R in ACC reverses the effects of EA-mediated analgesia. (Two-way ANOVA with Bonferroni test, F (20, 175) = 9.08, P < 0.0001. N = 8 for each group. ¥, ¥¥ ACC Flox−CB1+/+ + Model vs. ACC Flox−CB1+/+ + Model + EA group. #, ## ACC Flox−CB1−/− + Model + EA vs. ACC Flox−CB1+/+ + Model + EA group. *, **, *** ACC Flox−CB1−/− vs. ACC Flox−CB1+/+ + Model and ACC Flox−CB1−/− + Model + EA group, respectively. &&, &&& ACC Flox−CB1+/+ group vs. ACC Flox−CB1+/+ + Model and ACC Flox−CB1−/− + Model + EA group, respectively). ( H ) Mechanical threshold was determined in ACC-CB1R knockout mice on day 7 of EA. Knockout of CB1R in ACC reverses the effects of EA-mediated analgesia at day 7. ACC Flox−CB1−/− group: 0.9 ± 0.1, ACC Flox−CB1+/+ group: 0.9 ± 0.1, ACC Flox−CB1+/+ + Model + EA group: 0.85 ± 0.73, ACC Flox−CB1−/− + Model + EA group: 0.31 ± 0.44, ACC Flox−CB1+/+ + Model group: 0.25 ± 0.44. (One-way ANOVA with Bonferroni test, F (4, 35) = 18.84, P < 0.0001. N = 8 for each group. *, ** ACC Flox−CB1−/− + Model + EA group vs. ACC Flox−CB1+/+ , ACC Flox−CB1−/− and ACC Flox−CB1+/+ + Model + EA group, respectively. ACC Flox−CB1+/+ + Model group vs. ACC Flox−CB1+/+ , ACC Flox−CB1−/− , and ACC Flox−CB1+/+ + Model + EA group, respectively). Data are presented as mean ± SEM

Article Snippet: Ten minutes before EA, 300 nl of CB1R antagonist AM251 was diluted to a concentration of 2.5 mg/mL (4.50 mM) using 100 μl Dimethyl sulfoxide (DMSO) + 400 μl PEG300 + 50 μl Tween-80 (MedChemExpress, United States) injected into the ACC region at a rate of 30 nl/min through a microinjection pump (RWD, China) connecting the cannula.

Techniques: Injection, Immunofluorescence, Expressing, Knock-Out

Journal: Molecular Neurobiology

Article Title: Electroacupuncture Exerts Analgesic Effects by Restoring Hyperactivity via Cannabinoid Type 1 Receptors in the Anterior Cingulate Cortex in Chronic Inflammatory Pain

doi: 10.1007/s12035-023-03760-7

Figure Lengend Snippet: Electroacupuncture inhibits NMDAR through the interaction between CB1R and NR1 in ACC. ( A ) Representative gel image of NR1, NR2A, and NR2B protein levels in ACC. ( B ) Statistical diagram of relative expression level of NR1 protein in ACC of mice. EA reduces elevated NR1 protein expression in the ACC of model mice. Sham group: 1.0 ± 0.03, Model group: 1.28 ± 0.08, Model + EA group: 1.05 ± 0.06. (One-way ANOVA with LSD test, F (2,12) = 5.95, * P < 0.05 . N = 5 for each group. * vs. Model + EA group, ** vs. Sham group). ( C ) Statistical diagram of relative expression level of NR2A protein in ACC of mice. EA did not produce differences in NR2A expression in the ACC of the three groups of mice. Sham group: 1.03 ± 0.08, Model group: 0.86 ± 0.11, Model + EA group: 1.11 ± 0.08. (One-way ANOVA with Bonferroni test, F (2,15) = 1.99, P > 0.05. N = 6 for each group. ( D ) Statistical diagram of relative expression level of NR2B protein in ACC of mice. EA reduces elevated NR2B protein expression in the ACC of model mice. Sham group: 1.0 ± 0.03, Model group: 1.49 ± 0.10, Model + EA group: 0.91 ± 0.16. (Tamhane T2 test, N = 6 for each group. * vs. Sham group and Model + EA group). ( E ) Representative gel image of NR1, and NR2B protein levels in mice with knockout of CB1R in ACC. ( F ) Statistical diagram of relative expression level of NR1 protein in mice with knockout of CB1R in ACC. Disappearance of EA effects on NR1 protein in three groups of mice after knockout of CB1R in ACC. ACC Flox−CB1−/− group: 1.0 ± 0.02, ACC Flox−CB1−/− + Model group: 1.04 ± 0.06, ACC Flox−CB1−/− + Model + EA group: 1.01 ± 0.07. (One-way ANOVA with Bonferroni test, F (2,15) = 0.202, P > 0.05. N = 6 for each group.). ( G ) Statistical diagram of relative expression level of NR2B protein in mice with knockout of CB1R in ACC. EA still reduces elevated NR2B in model mice after knockout of CB1R in ACC. ACC Flox−CB1−/− group: 1.0 ± 0.05, ACC Flox−CB1−/− + Model group: 1.32 ± 0.06, ACC Flox−CB1−/− + Model + EA group: 0.86 ± 0.11. (One-way ANOVA with Bonferroni test, F (2,15) = 8.77, P < 0.01. N = 6 for each group. * vs. ACC Flox−CB1−/− group, ** vs. ACC Flox−CB1−/− + Model + EA group). ( H ) Representative gel images of HINT1 protein levels in ACC in WT mice and mice that have knocked out the CB1R in ACC. ( I ) Statistical diagram of relative expression level of HINT1 protein in ACC of mice. EA was able to increase the decrease of HINT1 in the ACC of model mice. Sham group: 1.0 ± 0.03, Model group: 0.77 ± 0.07, Model + EA group: 1.02 ± 0.04. (One-way ANOVA with Bonferroni test, F (2,15) = 8.169, P < 0.01. N = 6 for each group. * vs. Sham group, ** vs. Model + EA group). ( J ) Statistical diagram of relative expression level of HINT1 protein in mice with knockout of CB1R in ACC. Disappearance of EA effects on HINT1 protein in three groups of mice after knockout of CB1R in ACC. ACC Flox−CB1−/− group: 1.0 ± 0.02, ACC Flox−CB1−/− + Model group: 1.03 ± 0.16, ACC Flox−CB1−/− + Model + EA group: 1.15 ± 0.16. (One-way ANOVA with Bonferroni test, F (2,15) = 0.374, P > 0.05. N = 6 for each group). Data are presented as mean ± SEM

Article Snippet: Ten minutes before EA, 300 nl of CB1R antagonist AM251 was diluted to a concentration of 2.5 mg/mL (4.50 mM) using 100 μl Dimethyl sulfoxide (DMSO) + 400 μl PEG300 + 50 μl Tween-80 (MedChemExpress, United States) injected into the ACC region at a rate of 30 nl/min through a microinjection pump (RWD, China) connecting the cannula.

Techniques: Expressing, Knock-Out

Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: CB1 and CB2 cannabinoid receptor antagonists prevent minocycline-induced neuroprotection following traumatic brain injury in mice.

doi: 10.1093/cercor/bht202

Figure Lengend Snippet: Figure 2. Effects of TBI, minocycline, and cannabinoid receptor antagonists on brain edema and neurological score, 24 h after lesion. Histograms represent the mean + SEM (n = 12) of the percentage of water (A) and of the neurological score (B). Naïve, noninjured animals; TBI, traumatic brain injury; AM251, CB1R antagonist; AM630, CB2R antagonist; Mino, minocycline. Significant differences, P < 0.05: * versus naïve; # versus TBI-Mino.

Article Snippet: The CB1R antagonist AM251 (1 mg/kg; Tocris Bioscience, Bristol, UK), the CB2R receptor antagonist AM630 (1 mg/kg, Tocris), or their vehicle [ethanol:cremophor:saline (1:1:18), Cremophor® EL Fluka, Sigma-Aldrich, Madrid, Spain] were intraperitoneally (i.p.) administered 1 h before the lesion.

Techniques:

Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: CB1 and CB2 cannabinoid receptor antagonists prevent minocycline-induced neuroprotection following traumatic brain injury in mice.

doi: 10.1093/cercor/bht202

Figure Lengend Snippet: Figure 3. Effects of TBI, minocycline, and cannabinoid receptor antagonists on the number of microglial cells in the border of the lesion, 24 h after lesion. Representative examples of Iba-1-immunoreactive microglia cells in the injured brain area. (A) Naïve; (B) TBI; (C) TBI-Mino, (D) TBI-AM251-Mino; (E) TBI-AM630-Mino; and (F) histogram representing the mean + SEM (n = 8) of the percentage of microglial cells with the reactive phenotype. Naïve, noninjured animals; TBI, traumatic brain injury; AM251, CB1R antagonist; AM630, CB2R antagonist. Significant differences, P < 0.05: * versus naïve; # versus TBI-Mino.

Article Snippet: The CB1R antagonist AM251 (1 mg/kg; Tocris Bioscience, Bristol, UK), the CB2R receptor antagonist AM630 (1 mg/kg, Tocris), or their vehicle [ethanol:cremophor:saline (1:1:18), Cremophor® EL Fluka, Sigma-Aldrich, Madrid, Spain] were intraperitoneally (i.p.) administered 1 h before the lesion.

Techniques:

Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: CB1 and CB2 cannabinoid receptor antagonists prevent minocycline-induced neuroprotection following traumatic brain injury in mice.

doi: 10.1093/cercor/bht202

Figure Lengend Snippet: Figure 4. Effects of TBI, minocycline, and cannabinoid receptor antagonists on microglial reactivity in the border of the lesion, 24 h after lesion. Representative examples of Iba-1-immunoreactive microglia cells. (A) Naïve; (B) TBI; (C) TBI-Mino, (D) TBI-AM251-Mino; (E) TBI-AM630-Mino; and (F) histogram representing the mean + SEM (n = 8) of the percentage of microglial cells with the reactive phenotype. Naïve, noninjured animals; TBI, traumatic brain injury; AM251, CB1R antagonist; AM630, CB2R antagonist; Mino, minocycline. Significant differences, P < 0.05: * versus naïve; # versus TBI-Mino.

Article Snippet: The CB1R antagonist AM251 (1 mg/kg; Tocris Bioscience, Bristol, UK), the CB2R receptor antagonist AM630 (1 mg/kg, Tocris), or their vehicle [ethanol:cremophor:saline (1:1:18), Cremophor® EL Fluka, Sigma-Aldrich, Madrid, Spain] were intraperitoneally (i.p.) administered 1 h before the lesion.

Techniques:

Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: CB1 and CB2 cannabinoid receptor antagonists prevent minocycline-induced neuroprotection following traumatic brain injury in mice.

doi: 10.1093/cercor/bht202

Figure Lengend Snippet: Figure 5. Effects of TBI, minocycline, and cannabinoid receptor antagonists on microglial reactivity in the corpus callosum, 24 h after lesion. Representative examples of Iba-1-immunoreactive microglia cells. (A) Naïve; (B) TBI; (C) TBI-Mino; (D) TBI-AM251-Mino; (E) TBI-AM630-Mino; and (F) histogram representing the mean + SEM (n = 4) of the percentage of microglial cells with the reactive phenotype. Naïve, noninjured animals; TBI, traumatic brain injury; AM251, CB1R antagonist; AM630, CB2R antagonist; Mino, minocycline; IS, interhemispheric space. Significant differences, P < 0.05: * versus naïve; # versus TBI-Mino.

Article Snippet: The CB1R antagonist AM251 (1 mg/kg; Tocris Bioscience, Bristol, UK), the CB2R receptor antagonist AM630 (1 mg/kg, Tocris), or their vehicle [ethanol:cremophor:saline (1:1:18), Cremophor® EL Fluka, Sigma-Aldrich, Madrid, Spain] were intraperitoneally (i.p.) administered 1 h before the lesion.

Techniques:

Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: CB1 and CB2 cannabinoid receptor antagonists prevent minocycline-induced neuroprotection following traumatic brain injury in mice.

doi: 10.1093/cercor/bht202

Figure Lengend Snippet: Figure 6. Effects of TBI, minocycline, and cannabinoid receptor antagonists on DAI, 24 h after lesion. Representative images of β-APP immunoreactivity in the corpus callosum. (A) Naïve; (B) TBI; (C) TBI-AM630; (D) TBI-Mino; (E) TBI-AM630-Mino; and (F) histograms represent the mean + SEM (n = 8) of the surface density of β-APP-immunolabeled axons. Naïve, noninjured animals; TBI, traumatic brain injury; AM251, CB1 receptor antagonist; AM630, CB2 receptor antagonist; Mino, minocycline. Significant differences, P < 0.05: * versus naïve; # versus TBI-Mino; + versus TBI.

Article Snippet: The CB1R antagonist AM251 (1 mg/kg; Tocris Bioscience, Bristol, UK), the CB2R receptor antagonist AM630 (1 mg/kg, Tocris), or their vehicle [ethanol:cremophor:saline (1:1:18), Cremophor® EL Fluka, Sigma-Aldrich, Madrid, Spain] were intraperitoneally (i.p.) administered 1 h before the lesion.

Techniques: Immunolabeling

Journal: Physiological Reports

Article Title: The endocannabinoid systemʼs involvement in motor development relies on cannabinoid receptors, TRP channels, and Sonic Hedgehog signaling

doi: 10.14814/phy2.15565

Figure Lengend Snippet: Escape swimming deficits caused by singular MAGL or dual FAAH/MAGL inhibition is rescued by blocking CB1R. Escape swimming in response to an acute touch stimulus was assessed in zebrafish embryos at 2 days post‐fertilization (dpf). The effect of ~24‐h drug treatment on embryonic escape swimming distance (cm) and mean velocity (cm/s) was measured, and mean ranks of treatment groups were compared against each other. In each respective treatment group, the horizontal line within its boxplot represents the median value which is surrounded by the interquartile range, while the error bars represent the maximum and minimum values. (A, B) The effects of 5 μM URB 597 ( n = 38) on escape swimming are compared relative to the vehicle control ( n = 34), where ns = not statistically significant (Mann–Whitney test—where statistical significance was determined as p < 0.05). (C, D) The effects of the vehicle control ( n = 34), 5 μM JZL 184 ( n = 30), 10 nM AM 251 ( n = 33), and 5 μM JZL 184 in the presence of 10 nM AM 251 ( n = 43) on escape swimming was compared. (E, F) The effects the vehicle control ( n = 34), 2 μM JZL 195 ( n = 39), 10 nM AM 251 ( n = 33), and 2 μM JZL 195 in the presence of 10 nM AM 251 ( n = 34) on escape swimming was compared. N = 4 experiments with 8–11 animals per experiment for each treatment. (G) Representative tracings of the swimming paths are shown for vehicle, 10 nM AM 251, 5 μM JZL 184, 5 μM JZL 184 + 10 nM AM 251, 2 μM JZL 195, and 2 μM JZL 195 + 10 nM AM 251. For (C–F), columns which share the same letter(s) of the alphabet are not statistically different from one another (Kruskal–Wallis, followed by Dunn's multiple comparisons test—where statistical significance was determined as p < 0.05).

Article Snippet: Along with these compounds, animals were co‐treated with the CB1R antagonist AM 251 (Selleck Chemicals), the CB2R antagonist AM630 (Adooq Bioscience), the TRPV1/TRPA1/TRPM8 inhibitor AMG 9090 (Alomone Labs), or the SMO agonist Purmorphamine (Sigma‐Aldrich).

Techniques: Inhibition, Blocking Assay, Control, MANN-WHITNEY

Journal: The Journal of comparative neurology

Article Title: Cannabinoid receptor Type 1 densities reflect social organization in Microtus.

doi: 10.1002/cne.24996

Figure Lengend Snippet: FIGURE 1 Validation of [3H] CP-55,940 for autoradiography in prairie vole brains. Three adjacent sections of brain tissue were incubated with [3H] CP-55,940 with or without an unlabeled receptor competitor. Coincubation of the radioligand with the CB2 blocker (HU308) had no discernible impact on binding in the hippocampus (HPC) but increased binding in the prefrontal cortex (PFC) relative to control. Coincubation of the radioligand with the CB1 blocker (AM251) significantly decreased signal in both regions. Representative autoradiographs depict each condition at the level of the HPC. *p < .05 [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: These radioligands were coincubated with or without one of three unlabeled ligands to produce competitor conditions in order to assess nonspecific binding: the CB1 ligand AM251 (Tocris, Minneapolis, MN) and the CB2 ligands HU308 (Tocris) or SR144528 (Tocris) (Table 1).

Techniques: Biomarker Discovery, Autoradiography, Incubation, Binding Assay, Control

Journal: The Journal of comparative neurology

Article Title: Cannabinoid receptor Type 1 densities reflect social organization in Microtus.

doi: 10.1002/cne.24996

Figure Lengend Snippet: FIGURE 2 Validation of [3H] SR141716A and [3H] A-836,339 for autoradiography in prairie vole brains. Four adjacent sections of brain tissue were incubated with radioligand that was specific for CB1 ([3H] SR141716A) or CB2 ([3H] A-836,339) with complementary blockers for the same receptor. Coincubation of [3H] SR141716A with AM251 decreased signal in the hippocampus (HPC) but not in the prefrontal cortex (PFC). Neither the [3H] A-836,339 condition with or without the competitor (HU308) produced quantifiable levels of signal. Representative autoradiographs depict each condition at the level of the HPC. *p < .05 [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: These radioligands were coincubated with or without one of three unlabeled ligands to produce competitor conditions in order to assess nonspecific binding: the CB1 ligand AM251 (Tocris, Minneapolis, MN) and the CB2 ligands HU308 (Tocris) or SR144528 (Tocris) (Table 1).

Techniques: Biomarker Discovery, Autoradiography, Incubation, Produced

Journal: The Journal of comparative neurology

Article Title: Cannabinoid receptor Type 1 densities reflect social organization in Microtus.

doi: 10.1002/cne.24996

Figure Lengend Snippet: FIGURE 3 Validation of [3H] CP-55,940, [3H] SR141716A and [3H] A-836,339 for autoradiography in prairie vole spleens. Seven adjacent sections of spleen tissue were incubated with radioligand that was coincubated with or without unlabeled blockers. [3H] CP- 55,940 signal was decreased when coincubated with HU308 (CB2 blocker) but not with AM251 (CB1 blocker). [3H] SR141716A signal was not significantly impacted by AM251 competition. We found no evidence of [3H] A-836,339 signal in spleen assay with or without a CB2 competitor (SR144528). *p < .05 [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: These radioligands were coincubated with or without one of three unlabeled ligands to produce competitor conditions in order to assess nonspecific binding: the CB1 ligand AM251 (Tocris, Minneapolis, MN) and the CB2 ligands HU308 (Tocris) or SR144528 (Tocris) (Table 1).

Techniques: Biomarker Discovery, Autoradiography, Incubation

Journal: The Journal of comparative neurology

Article Title: Cannabinoid receptor Type 1 densities reflect social organization in Microtus.

doi: 10.1002/cne.24996

Figure Lengend Snippet: FIGURE 4 Representative autoradiograms of CB1 receptors, mapped with [3H] CP-55,940, throughout prairie vole brain. A1, primary auditory cortex; ACC, anterior cingulate cortex; AH, anterior hypothalamus; AOB, accessory olfactory bulb; AON, anterior olfactory nucleus; BLA, basolateral amygdala; BMA, basomedial amygdala; BST, bed nucleus of the stria terminalis; CBX, cerebellar cortex; CEA, central amygdala; CP, caudate-putamen; DG, dentate gyrus; ENT, entorhinal cortex; GPe, globus pallidus external; GPi, globus pallidum internal; IC, inferior colliculus; IPN, interpeduncular nucleus; LDTh, laterodorsal thalamus; LH, lateral hypothalamus; LS, lateral septum; MBO, mammillary bodies; MDTh, mediodorsal thalamus; MEA, medial amygdala; M1, primary motor area; MOB, main olfactory bulb; MPA, medial preoptic area; MRF, reticular formation; MRN, median raphe nucleus; NAc, nucleus accumbens; ORB, orbitofrontal cortex; PAG, periaqueductal gray; PCC, posterior cingulate cortex; PFC, prefrontal cortex; PG, pontine gray; PIR, piriform cortex; RSP, retrosplenial cortex; S1, primary somatosensory cortex; SC, superior colliculus; SN, substantia nigra; SUB, subiculum; V1, primary visual cortex; VP, ventral pallidum; VPTh, ventroposterior thalamus; VTA, ventral tegmental area

Article Snippet: These radioligands were coincubated with or without one of three unlabeled ligands to produce competitor conditions in order to assess nonspecific binding: the CB1 ligand AM251 (Tocris, Minneapolis, MN) and the CB2 ligands HU308 (Tocris) or SR144528 (Tocris) (Table 1).

Techniques:

Journal: The Journal of comparative neurology

Article Title: Cannabinoid receptor Type 1 densities reflect social organization in Microtus.

doi: 10.1002/cne.24996

Figure Lengend Snippet: FIGURE 5 Comparisons of CB1 density in prairie and meadow voles. Prairie voles had higher CB1 density in CA2 and CA3 (a); DG and PAG (b); LDTh, MDTh, and VPTh (c); and in MBO (d). *p < .05. DG, dentate gyrus; LDTh, laterodorsal thalamus; MBO, mammillary bodies; MDTh, mediodorsal thalamus; PAG, periaqueductal gray; VPTh, ventroposterior thalamus [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: These radioligands were coincubated with or without one of three unlabeled ligands to produce competitor conditions in order to assess nonspecific binding: the CB1 ligand AM251 (Tocris, Minneapolis, MN) and the CB2 ligands HU308 (Tocris) or SR144528 (Tocris) (Table 1).

Techniques:

Journal: The Journal of Neuroscience

Article Title: CB1-Dependent Long-Term Depression in Ventral Tegmental Area GABA Neurons: A Novel Target for Marijuana

doi: 10.1523/JNEUROSCI.0190-17.2017

Figure Lengend Snippet: CB1 mediates HFS-LTD. Two candidate receptors that could act as a presynaptic target for eCBs include TRPV1 and CB1. A, The TRPV1 antagonist capsazepine failed to block LTD, suggesting that TRPV1 is not involved in this HFS-LTD (n = 9; p > 0.05 compared with control LTD). B, The CB1 antagonist AM-251 significantly blocked LTD (n = 9, p < 0.05 compared with control LTD).

Article Snippet: However, application of the CB1 antagonist AM-251 effectively blocked LTD ( B ), suggesting that CB1 is required for HFS-LTD. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 3. caption a7 CB1 mediates HFS-LTD. Two candidate receptors that could act as a presynaptic target for eCBs include TRPV1 and CB1.

Techniques: Blocking Assay, Control

Journal: The Journal of Neuroscience

Article Title: CB1-Dependent Long-Term Depression in Ventral Tegmental Area GABA Neurons: A Novel Target for Marijuana

doi: 10.1523/JNEUROSCI.0190-17.2017

Figure Lengend Snippet: Postsynaptically produced 2-AG mediates LTD of VTA GABA cells. A, Application of the CB1 agonist Win55,212-2 depresses glutamate transmission at this synapse (n = 8). There is a significant depression between baseline and drug application (p < 0.05), but further HFS-induced depression was occluded (arrow; p > 0.5; WIN55,212-2 depression compared with post-HFS, ANOVA). B, When the CB1 antagonist AM-251 was present, Win55,212-2 caused no significant depression, demonstrating the specificity of Win 55,212-2 for CB1 (n = 5, p < 0.05 compared with Win 55,212-2 alone). C, Application of endogenous CB1 ligand 2-AG also induced significant depression (n = 9; p < 0.05 at both 15–20 and 20–25 min). D, When the DAGLα inhibitor RHC 80267 was applied to block postsynaptic production of 2-AG, HFS failed to produce a significant depression, suggesting that 2-AG is produced postsynaptically in the GABA neuron (n = 6, p > 0.5). E, To control for drug specificity, we applied a second DAGL antagonist, THL (10–20 μm) to the bath for at least 30–60 min before HFS. THL significantly blocked HFS-induced LTD (p < 0.05 compared with control LTD, n = 7). F, To further tie mGluR activation to CB1-induced LTD, we applied DHPG in the presence of CB1 antagonist AM-251 (2 μm). AM-251 significantly blocked DHPG-induced depression (n = 8; p < 0.05, DHPG-induced depression compared with DHPG-induced depression in the presence of AM-251 at 15–20 and 20–25 min after DHPG application; compare with Fig. 2B). G, H, DSE was used to further test for postsynaptic eCB production. VTA GABA neurons were depolarized to 0 mV for 10 s (arrowhead) to induce DSE. We observed a persistent depression following the DSE protocol (n = 7, p < 0.05), which was CB1-dependent, as it was blocked by AM-251 (n = 7; p < 0.05, DSE compared with DSE with AM-251).

Article Snippet: However, application of the CB1 antagonist AM-251 effectively blocked LTD ( B ), suggesting that CB1 is required for HFS-LTD. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 3. caption a7 CB1 mediates HFS-LTD. Two candidate receptors that could act as a presynaptic target for eCBs include TRPV1 and CB1.

Techniques: Produced, Transmission Assay, Blocking Assay, Control, Activation Assay

Journal: The Journal of Neuroscience

Article Title: CB1-Dependent Long-Term Depression in Ventral Tegmental Area GABA Neurons: A Novel Target for Marijuana

doi: 10.1523/JNEUROSCI.0190-17.2017

Figure Lengend Snippet: HFS-LTD is blocked in CB1 knock-out mice. A, HFS induced a significant LTD in heterozygous mice compared with baseline (n = 7, p < 0.05). B, In CB1−/−, HFS failed to produce LTD, further demonstrating the importance of CB1 in this plasticity (n = 9, p < 0.05 compared with CB1+/−).

Article Snippet: However, application of the CB1 antagonist AM-251 effectively blocked LTD ( B ), suggesting that CB1 is required for HFS-LTD. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 3. caption a7 CB1 mediates HFS-LTD. Two candidate receptors that could act as a presynaptic target for eCBs include TRPV1 and CB1.

Techniques: Knock-Out

Journal: The Journal of Neuroscience

Article Title: CB1-Dependent Long-Term Depression in Ventral Tegmental Area GABA Neurons: A Novel Target for Marijuana

doi: 10.1523/JNEUROSCI.0190-17.2017

Figure Lengend Snippet: THC induces CB1-dependent depression. A, THC is the psychoactive component in marijuana, and a known CB1 agonist. When THC is applied to the extracellular bath, it produced a lasting depression (n = 6, p < 0.05). B, The CB1 antagonist AM-251 significantly blocked THC-induced depression (n = 6, p < 0.05, compared with THC without AM251). C, CB1 knock-out and heterozygous mice were also used to confirm CB1 involvement, where THC induced depression in heterozygous mice (n = 7, p < 0.05 compared with baseline). D, In CB1 KO mice, THC failed to produce significant depression (n = 9, p < 0.05; compared with THC in heterozygote mice). E, THC occluded HFS-LTD, suggesting that THC and HFS-LTD use the same pathway (p < 0.05 compared with control LTD).

Article Snippet: However, application of the CB1 antagonist AM-251 effectively blocked LTD ( B ), suggesting that CB1 is required for HFS-LTD. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 3. caption a7 CB1 mediates HFS-LTD. Two candidate receptors that could act as a presynaptic target for eCBs include TRPV1 and CB1.

Techniques: Produced, Knock-Out, Control

Journal: The Journal of Neuroscience

Article Title: CB1-Dependent Long-Term Depression in Ventral Tegmental Area GABA Neurons: A Novel Target for Marijuana

doi: 10.1523/JNEUROSCI.0190-17.2017

Figure Lengend Snippet: Current working model. HFS-LTD is induced by CB1 activation that is mediated by postsynaptically produced 2-AG, which is formed by DAGL and mGluR5 activation to reduce presynaptic neurotransmitter release.

Article Snippet: However, application of the CB1 antagonist AM-251 effectively blocked LTD ( B ), suggesting that CB1 is required for HFS-LTD. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 3. caption a7 CB1 mediates HFS-LTD. Two candidate receptors that could act as a presynaptic target for eCBs include TRPV1 and CB1.

Techniques: Activation Assay, Produced

Journal: PLoS ONE

Article Title: Suppression of CB1 Cannabinoid Receptor by Lentivirus Mediated Small Interfering RNA Ameliorates Hepatic Fibrosis in Rats

doi: 10.1371/journal.pone.0050850

Figure Lengend Snippet: PCR primer sequences for RT-PCR detection.

Article Snippet: 60 hours later, freshmedium with CB1 antagonists AM251 (1 μM, Santa Cruz, Santa Cruz, USA) was added to the cultured cells.

Techniques:

Journal: PLoS ONE

Article Title: Suppression of CB1 Cannabinoid Receptor by Lentivirus Mediated Small Interfering RNA Ameliorates Hepatic Fibrosis in Rats

doi: 10.1371/journal.pone.0050850

Figure Lengend Snippet: ( A ) The statistical results of CB1 mRNA expression in primary hepatic stellate cells (HSCs) transfected with 4 pairs of shRNA expressing lentivirus named KD1, KD2, KD3 and KD4 by RT-PCR detection. The pGCSIL/U6 mock vector named Con was used as a negative control. KD4 displayed the highest gene-silencing efficacy among all the 4 pairs of lentivirus. Relative expression levels of mRNA were normalized against those of GAPDH mRNA. ( B ) The CB1 protein expression in HSCs transfected with 4 pairs of shRNA expressing lentivirus and the negative control lentivirus, respectively. The results demonstrated that KD4 deduced CB1 mRNA expression by about 75%, and protein expression by about 80%, which had the highest gene-silencing efficacy among all the four pairs of lentivirus vectors.

Article Snippet: 60 hours later, freshmedium with CB1 antagonists AM251 (1 μM, Santa Cruz, Santa Cruz, USA) was added to the cultured cells.

Techniques: Expressing, Transfection, shRNA, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Negative Control

Journal: PLoS ONE

Article Title: Suppression of CB1 Cannabinoid Receptor by Lentivirus Mediated Small Interfering RNA Ameliorates Hepatic Fibrosis in Rats

doi: 10.1371/journal.pone.0050850

Figure Lengend Snippet: ( A ) The statistical results of α-SMA, TGF-β1, TGF-β RII, fibronectin and procollagen type I mRNA expression in HSCs (named Con), HSCs with 2-AG (named Con+2AG), HSCs with NC-LV and 2-AG (named NC+2AG), HSCs with CB1-RNAi-LV and 2-AG (named siRNA+2AG), HSCs with 2-AG and AM251 (named Con+2AG+AM251) by RT-PCR analysis. Relative expression levels of mRNA were normalized against those of GAPDH mRNA. ( B ) shows representative graphs of α-SMA, TGF-β1, TGF-β RII, fibronectin and procollagen type I protein expression by Western blot analysis and ( C ) shows the statistical results. Results demonstrated that the mRNA and protein expressions of α-SMA, TGF-β1, TGF-β RII, fibronectin and procollagen type I in HSCs were all increased significantly after 2-AG stimulation, which were all decreased significantly when stimulated with AM251 or transfected with CB1-RNAi-LV. Furthermore, the mRNA expression of α-SMA, TGF-β1, TGF-β RII and fibronectin in HSCs stimulated by 2-AG and AM251 was higher significantly than that in HSCs stimulated by 2-AG and transfected with CB1-RNAi-LV. “#” p <0.05, “# #” p <0.01.

Article Snippet: 60 hours later, freshmedium with CB1 antagonists AM251 (1 μM, Santa Cruz, Santa Cruz, USA) was added to the cultured cells.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection

Journal: PLoS ONE

Article Title: Suppression of CB1 Cannabinoid Receptor by Lentivirus Mediated Small Interfering RNA Ameliorates Hepatic Fibrosis in Rats

doi: 10.1371/journal.pone.0050850

Figure Lengend Snippet: ( A ) The statistical results of the optical density (OD) value of BrdU incorporative cells in HSCs (named Con), HSCs with PDGF (named Con+PDGF), HSCs with NC-LV and PDGF (named NC+PDGF), HSCs with CB1-RNAi-LV and PDGF (named siRNA+PDGF) by ELISA method. “# #” p <0.01. Results showed that the proliferation of HSCs stimulated by PDGF-BB elevated significantly than HSCs alone. NC-LV transfection did not affect the proliferation of HSCs stimulated by PDGF-BB. Furthermore, the proliferation of HSCs stimulated with PDGF-BB and transfected with CB1-RNAi-LV decreased markedly than that of HSCs stimulated with PDGF-BB and transfected with NC-LV. ( B ) The statistical results of PDGFR-β mRNA expression in HSCs (named Con), HSCs with PDGF (named Con+PDGF), HSCs with NC-LV and PDGF (named NC+PDGF), HSCs with CB1-RNAi-LV and PDGF (named siRNA+PDGF) by RT-PCR analysis. Relative expression levels of mRNA were normalized against those of GAPDH mRNA. “# #” p <0.01. Results showed that PDGFR-β mRNA expression increased markedly by PDGF-BB pretreatment or by PDGF-BB pretreatment plus NC-LV transfection, while PDGFR-β mRNA expression in HSCs pretreated with PDGF-BB and transfected with CB1-RNAi-LV decreased markedly than that in HSCs pretreated with PDGF-BB and transfected with NC-LV.

Article Snippet: 60 hours later, freshmedium with CB1 antagonists AM251 (1 μM, Santa Cruz, Santa Cruz, USA) was added to the cultured cells.

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Suppression of CB1 Cannabinoid Receptor by Lentivirus Mediated Small Interfering RNA Ameliorates Hepatic Fibrosis in Rats

doi: 10.1371/journal.pone.0050850

Figure Lengend Snippet: ( A ) The statistical results of E-cadherin, Snail and Vimentin mRNA expression in HSCs (named Con), HSCs with 2-AG (named Con+2AG), HSCs with NC-LV and 2-AG (named NC+2AG), HSCs with CB1-RNAi-LV and 2-AG (named siRNA+2AG), HSCs with 2-AG and AM251 (named Con+2AG+AM251) by RT-PCR analysis. Relative expression levels of mRNA were normalized against those of GAPDH mRNA. “# #” p <0.01. ( B ) shows representative graphs of E-cadherin, Snail and Vimentin protein expression by Western blot analysis and ( C ) shows the statistical results. These data indicated that the mRNA and protein expressions of E-cadherin in HSCs were down-regulated significantly by 2-AG stimulation, while the levels of Snail and Vimentin were up-regulated significantly by 2-AG stimulation. On the other hand, the expression level of E-cadherin in HSCs with CB1-RNAi-LV transfection and 2-AG stimulation were increased markedly than that in HSCs with NC-LV transfection and 2-AG stimulation, while the expression levels of Vimentin and Snail in HSCs with CB1-RNAi-LV transfection and 2-AG stimulation were decreased markedly than that in HSCs with NC-LV transfection and 2-AG stimulation. The results also revealed that the expression levels of E-cadherin, Snail and Vimentin in HSCs stimulated by 2-AG were not affected by AM251 treatment.

Article Snippet: 60 hours later, freshmedium with CB1 antagonists AM251 (1 μM, Santa Cruz, Santa Cruz, USA) was added to the cultured cells.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection

Journal: PLoS ONE

Article Title: Suppression of CB1 Cannabinoid Receptor by Lentivirus Mediated Small Interfering RNA Ameliorates Hepatic Fibrosis in Rats

doi: 10.1371/journal.pone.0050850

Figure Lengend Snippet: The statistical results of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in normal rats, rats with DMN treatment and Ringer's solution (RS) injected, rats with DMN treatment and NC-LV injected, rats with DMN treatment and CB1-RNAi-LV injected. “#” p <0.05 and “# #” p <0.01. The data demonstrated that serum ALT and AST elevated significantly in DMN rats as compared with the normal rats. However, CB1-RNAi-LV treatment markedly reduced the serum ALT and AST as compared with NC-LV treatment rats.

Article Snippet: 60 hours later, freshmedium with CB1 antagonists AM251 (1 μM, Santa Cruz, Santa Cruz, USA) was added to the cultured cells.

Techniques: Injection

Journal: PLoS ONE

Article Title: Suppression of CB1 Cannabinoid Receptor by Lentivirus Mediated Small Interfering RNA Ameliorates Hepatic Fibrosis in Rats

doi: 10.1371/journal.pone.0050850

Figure Lengend Snippet: ( A ) The first line showed the representative graph of Masson's trichrome staining (magnification of 20×) in normal rats (Normal), rats with DMN treatment and Ringer's solution injected (DMN+RS), rats with DMN treatment and NC-LV injected (DMN+NC-LV), rats with DMN treatment and CB1-RNAi-LV injected (DMN+RNAi-LV). The second, the third and the fourth line showed the representative graph of CB1, α-SMA and E-cadherin immunohistochemical examination (magnification of 20×) in normal rats (Normal), rats with DMN treatment and Ringer's solution injected (DMN+RS), rats with DMN treatment and NC-LV injected (DMN+NC-LV), rats with DMN treatment and CB1-RNAi-LV injected (DMN+RNAi-LV). ( B ) Shows statistical results of CB1, α-SMA and E-cadherin immunohistochemical staining. Masson's trichrome staining of liver sections in the DMN rats and the NC-LV treatment rats showed periportal fibrosis with short septa extending into lobules or porto-portal septa, and severe cholestasis and bile duct hyperplasia were also observed, whereas hepatic fibrosis was significantly ameliorated in the CB1-RNAi-LV treatment rats as compared with the NC-LV treatment rats. Immunohistochemical examination revealed that CB1 and α-SMA protein expressions increased significantly in the DMN rats, which were suppressed by CB1-RNAi-LV treatment. Immunochemical examination also showed that DMN treatment declined E-cadherin expression significantly compared with normal rats, while E-cadherin expression elevated significantly in DMN rats transfected with CB1-RNAi-LV, compared with that in DMN rats transfected with NC-LV. “# #” p <0.01.

Article Snippet: 60 hours later, freshmedium with CB1 antagonists AM251 (1 μM, Santa Cruz, Santa Cruz, USA) was added to the cultured cells.

Techniques: Staining, Injection, Immunohistochemical staining, Expressing, Transfection

Journal: PLoS ONE

Article Title: Suppression of CB1 Cannabinoid Receptor by Lentivirus Mediated Small Interfering RNA Ameliorates Hepatic Fibrosis in Rats

doi: 10.1371/journal.pone.0050850

Figure Lengend Snippet: Effect of CB1 shRNA on hepatic fibrosis of DMN rats.

Article Snippet: 60 hours later, freshmedium with CB1 antagonists AM251 (1 μM, Santa Cruz, Santa Cruz, USA) was added to the cultured cells.

Techniques: shRNA