Journal: International Journal of Molecular Sciences

Article Title: Brexpiprazole Reduces 5-HT7 Receptor Function on Astroglial Transmission Systems

doi: 10.3390/ijms23126571

Figure Lengend Snippet: Receptor binding profiles of mood-stabilising atypical antipsychotics. Brexpiprazole (Brex), aripiprazole (APZ), clozapine (CLZ), lurasidone (LUR), quetiapine (QTP), ziprasidone (ZIP), zotepine (ZTP) against serotonin (5-HT) type 1A (5-HT1AR), type 2A (5-HT2AR), type 7 (5-HT7R) receptor, histamine type 1 (H1R) receptor and dopamine receptors type 1 (D1R) and 2 (D2R). Data are equilibrium constant (Ki) values (nM).

Article Snippet: Antibodies against GAPDH (NB300-327, 1:300, Novus Biologicals, Littleton, CO, USA), connexin43 (C6219, 1:100, Sigma-Aldrich, St. Louis, MO, USA), 5-HT1AR (NBP2-21590, 1:00, Novus Biologicals, Littleton, CO, USA), 5-HT7R (NB100-56352, 1:00, Novus Biologicals, Littleton, CO, USA), Erk (AF1576, 10 μg/mL, R&D systems, Minneapolis, MN, USA), phosphorylated Erk (AF1018, 5 μg/mL, R&D Systems), Akt (AF1775, 1 μg/mL, R&D Systems), phosphorylated Akt (AF877, 5 μg/mL, R&D Systems), AMPKα (2603, 1:50, Cell Signalling Technology, Danvers, MA, USA), and phosphorylated-AMPKα (2535, 1:50, Cell Signalling Technology, Danvers, MA, USA) were diluted in an antibody diluent (Immuno Shot Platinum, CosmoBio, Tokyo, Japan) [ ].

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Brexpiprazole Reduces 5-HT7 Receptor Function on Astroglial Transmission Systems

doi: 10.3390/ijms23126571

Figure Lengend Snippet: Effects of subchronic administration (7 and 14 days) of therapeutic relevant concentration of Brex (Brex: 300 nM) and interaction between Brex and 5-HT1A receptor (5-HT1AR) antagonist WAY100635 (WAY: 10 μM) on protein expression of 5-HT1A (panel A ) and 5-HT7 (panel B ) receptor in the plasma membrane fraction of cortical primary cultured astrocytes. In left side histograms, ordinate: mean ± SD ( n = 6) of the relative protein level of 5-HT1AR and 5-HT7R per GAPDH. * p < 0.05, ** p < 0.01: relative to control (Brex-free) by one-way analysis of variance (ANOVA) with Tukey’s post-hoc test, and @ p < 0.05: relative to Brex for 14 days by Student’s T -test. Right side panels indicate their pseudo-gel images using capillary immunoblotting.

Article Snippet: Antibodies against GAPDH (NB300-327, 1:300, Novus Biologicals, Littleton, CO, USA), connexin43 (C6219, 1:100, Sigma-Aldrich, St. Louis, MO, USA), 5-HT1AR (NBP2-21590, 1:00, Novus Biologicals, Littleton, CO, USA), 5-HT7R (NB100-56352, 1:00, Novus Biologicals, Littleton, CO, USA), Erk (AF1576, 10 μg/mL, R&D systems, Minneapolis, MN, USA), phosphorylated Erk (AF1018, 5 μg/mL, R&D Systems), Akt (AF1775, 1 μg/mL, R&D Systems), phosphorylated Akt (AF877, 5 μg/mL, R&D Systems), AMPKα (2603, 1:50, Cell Signalling Technology, Danvers, MA, USA), and phosphorylated-AMPKα (2535, 1:50, Cell Signalling Technology, Danvers, MA, USA) were diluted in an antibody diluent (Immuno Shot Platinum, CosmoBio, Tokyo, Japan) [ ].

Techniques: Concentration Assay, Expressing, Clinical Proteomics, Membrane, Cell Culture, Control, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Brexpiprazole Reduces 5-HT7 Receptor Function on Astroglial Transmission Systems

doi: 10.3390/ijms23126571

Figure Lengend Snippet: Concentration-dependent acute effects of Brex (100 and 300 nM) on intracellular cAMP level in astrocytes (panel A ). Interaction among therapeutic relevant concentration of Brex (100 and 300 nM), 5 μM AS19 (5-HT7R agonist) (panel B ) and 10 μM WAY100635 (5-HT1AR antagonist) (panel C ) on intracellular cAMP level in astrocytes. Ordinates indicate mean ± SD ( n = 6) of intracellular cAMP level in cultured astrocytes (fmol/mg). * p < 0.05, ** p < 0.01: relative to control (Brex-free) by one-way ANOVA with Tukey’s post-hoc test.

Article Snippet: Antibodies against GAPDH (NB300-327, 1:300, Novus Biologicals, Littleton, CO, USA), connexin43 (C6219, 1:100, Sigma-Aldrich, St. Louis, MO, USA), 5-HT1AR (NBP2-21590, 1:00, Novus Biologicals, Littleton, CO, USA), 5-HT7R (NB100-56352, 1:00, Novus Biologicals, Littleton, CO, USA), Erk (AF1576, 10 μg/mL, R&D systems, Minneapolis, MN, USA), phosphorylated Erk (AF1018, 5 μg/mL, R&D Systems), Akt (AF1775, 1 μg/mL, R&D Systems), phosphorylated Akt (AF877, 5 μg/mL, R&D Systems), AMPKα (2603, 1:50, Cell Signalling Technology, Danvers, MA, USA), and phosphorylated-AMPKα (2535, 1:50, Cell Signalling Technology, Danvers, MA, USA) were diluted in an antibody diluent (Immuno Shot Platinum, CosmoBio, Tokyo, Japan) [ ].

Techniques: Concentration Assay, Cell Culture, Control

Journal: International Journal of Molecular Sciences

Article Title: Brexpiprazole Reduces 5-HT7 Receptor Function on Astroglial Transmission Systems

doi: 10.3390/ijms23126571

Figure Lengend Snippet: Proposed hypothesis of the mechanisms of Brex on intracellular signalling in astrocytes. Chronic administration of a therapeutically relevant concentration of Brex downregulates both 5-HT1AR and 5-HT7R via its 5-HT1AR partial agonistic and possible 5-HT7R inverse agonistic actions, respectively. The downregulation of 5-HT1AR and 5-HT7R by Brex attenuates AMPK, Erk, and Akt signalling in astrocytes.

Article Snippet: Antibodies against GAPDH (NB300-327, 1:300, Novus Biologicals, Littleton, CO, USA), connexin43 (C6219, 1:100, Sigma-Aldrich, St. Louis, MO, USA), 5-HT1AR (NBP2-21590, 1:00, Novus Biologicals, Littleton, CO, USA), 5-HT7R (NB100-56352, 1:00, Novus Biologicals, Littleton, CO, USA), Erk (AF1576, 10 μg/mL, R&D systems, Minneapolis, MN, USA), phosphorylated Erk (AF1018, 5 μg/mL, R&D Systems), Akt (AF1775, 1 μg/mL, R&D Systems), phosphorylated Akt (AF877, 5 μg/mL, R&D Systems), AMPKα (2603, 1:50, Cell Signalling Technology, Danvers, MA, USA), and phosphorylated-AMPKα (2535, 1:50, Cell Signalling Technology, Danvers, MA, USA) were diluted in an antibody diluent (Immuno Shot Platinum, CosmoBio, Tokyo, Japan) [ ].

Techniques: Concentration Assay

Journal: Research

Article Title: Platelets Orchestrate a Neuroimmune Axis Driving Cutaneous Inflammation and Itch

doi: 10.34133/research.1294

Figure Lengend Snippet: Platelet-derived serotonin activated TRPV1 + pruriceptors via HTR2B to promote MC903-induced itch. (A) Bubble plot showed the transcript expression of Htr2b in various itch-related neurons based on DRG scRNA-seq data. n = 5 mice. (B) Representative images of HTR2B and TRPV1 fluorescence intensity in DRG. n = 3 to 5 sections from 3 mice. Scale bar, 50 μm. (C) Schematic diagram of the generation of TRPV1 Cre ; HTR2B f/f mice ( HTR2B cko ). (D) Quantification of spontaneous scratching bouts in HTR2B cko mice and control littermates. n = 5 HTR2B cko mice, n = 6 control littermates. F 6,63 = 9.668, P < 0.0001. Day 7: P = 0.0012, day 9: P < 0.0001, day 11: P < 0.0001 (2-way ANOVA followed by Šídák’s multiple comparisons test). (E and F) Representative traces of spontaneous C-fiber firings recorded ex vivo from the control littermates (left) and HTR2B cko mice (right). (G and H) Representative traces of mechanically evoked C-fiber firings in response to 50-mN force. (I to K) Summarized data showing spontaneous C-fiber firing frequency (I), mechanically evoked C-fiber firing frequency (J), and mechanical firing threshold (K). n = 6 units from 4 mice. For (I), t 10 = 6.604, P < 0.0001 (2-tailed unpaired Student’s t test). For (J), F 8,90 = 23.39, P < 0.0001. 50 mN: P = 0.0007, 75 mN: P < 0.0001, 100 mN: P < 0.0001 (2-way ANOVA followed by Šídák’s multiple comparisons test). For (K), P = 0.0325 (2-tailed Mann–Whitney test). * P < 0.05, ** P < 0.01, *** P < 0.001. Individual data points in (I) to (K) represent single animals, and bars show mean ± SD from 2 independent experiments. Result in (D), individual data points represent single animals and are shown as mean ± SD from one representative of 2 independent experiments with consistent results.

Article Snippet: To evaluate the therapeutic effect of HTR2B and/or HTR7 inhibition, mice were randomly assigned to receive vehicle [1% dimethyl sulfoxide (DMSO) in PBS], HTR2B inhibitor ( LY266097 , 0.5 mg/kg, MCE, NJ, USA), HTR7 inhibitor (SB269970, 5 mg/kg, MCE, NJ, USA), or a combination of HTR2B and HTR7 inhibitors.

Techniques: Derivative Assay, Expressing, Fluorescence, Control, Ex Vivo, MANN-WHITNEY

Journal: Research

Article Title: Platelets Orchestrate a Neuroimmune Axis Driving Cutaneous Inflammation and Itch

doi: 10.34133/research.1294

Figure Lengend Snippet: HTR antagonist treatment attenuates pruritus and skin inflammation in MC903-induced AD model. (A) Schematic illustration of HTR2B inhibitor ( LY266097 ) and/or HTR7 inhibitor (SB269970) applications in MC903-induced AD model. (B) Representative images of ears from MC903-induced AD model after 11 d of HTR2B inhibitor and/or HTR7 inhibitor applications. Scale bar, 1 mm. (C and D) Ear thickness (C) and inflammation score (D) measured at the indicated time points. n = 6 mice per group. For (C), F 18,140 = 3.900, P < 0.0001. Vehicle versus LY266097 : day 7: P = 0.0174, day 9: P = 0.0050, day 11: P = 0.0110. Vehicle versus SB269970: day 9: P = 0.0050, day 11: P = 0.0174. Vehicle versus LY266097 + SB269970: day 5: P < 0.0001, day 7: P < 0.0001, day 9: P < 0.0001, day 11: P < 0.0001. For (D), F 18,140 = 8.957, P < 0.0001. Vehicle versus LY266097 : day 7: P = 0.0256, day 9: P < 0.0001, day 11: P < 0.0001. Vehicle versus SB269970: day 7: P < 0.0001, day 9: P < 0.0001, day 11: P < 0.0001. Vehicle versus LY266097 + SB269970: day 5: P = 0.0064, day 7: P < 0.0001, day 9: P < 0.0001, day 11: P < 0.0001 (2-way ANOVA followed by Šídák’s multiple comparisons test). (E) Quantification of spontaneous scratching bouts. n = 6 mice per group. F 18,140 = 21.76, P < 0.0001. Vehicle versus LY266097 : day 7: P < 0.0001, day 9: P < 0.0001, day 11: P < 0.0001. Vehicle versus SB269970: day 7: P < 0.0001, day 9: P < 0.0001, day 11: P < 0.0001. Vehicle versus LY266097 + SB269970: day 5: P < 0.0001, day 7: P < 0.0001, day 9: P < 0.0001, day 11: P < 0.0001 (2-way ANOVA followed by Šídák’s multiple comparisons test). (F) Representative H&E histopathology images of ear sections. n = 3 to 5 sections from 3 mice. Scale bar, 100 μm. (G and H) Quantification of dermal thickness (G) and dermal immune cell infiltration (H). n = 6 mice per group. For (G), F 3,20 = 28.03, P < 0.0001. Vehicle versus LY266097 : P = 0.0032. Vehicle versus SB269970: P = 0.0002. Vehicle versus LY266097 + SB269970: P = 0.0002. For (H), F 3,20 = 45.05, P < 0.0001. Vehicle versus LY266097 : P = 0.0124. Vehicle versus SB269970: P = 0.0018. Vehicle versus LY266097 + SB269970: P < 0.0001 (one-way ANOVA followed by Holm–Šídák’s multiple comparisons test). (I and J) Representative images of F4/80 + macrophages in the skin, which were quantified in (J). Scale bar, 20 μm. n = 6 mice per group. For (J), F 3,20 = 55.62, P < 0.0001. Vehicle versus LY266097 : P < 0.0001. Vehicle versus SB269970: P < 0.0001. Vehicle versus LY266097 + SB269970: P < 0.0001 (one-way ANOVA followed by Holm–Šídák’s multiple comparisons test). * P < 0.05, ** P < 0.01, *** P < 0.001. Individual data points in the remaining panels represent single animals and are shown as mean ± SD from 2 independent experiments.

Article Snippet: To evaluate the therapeutic effect of HTR2B and/or HTR7 inhibition, mice were randomly assigned to receive vehicle [1% dimethyl sulfoxide (DMSO) in PBS], HTR2B inhibitor ( LY266097 , 0.5 mg/kg, MCE, NJ, USA), HTR7 inhibitor (SB269970, 5 mg/kg, MCE, NJ, USA), or a combination of HTR2B and HTR7 inhibitors.

Techniques: Histopathology