Journal: bioRxiv

Article Title: Spinal reconsolidation engages non-ionotropic NMDA receptor signaling to reverse pain hypersensitivity

doi: 10.1101/2021.09.08.458120

Figure Lengend Snippet: a) Mechanical withdrawal thresholds were assessed in mice at baseline (Base) and after injection of capsaicin (‘Cap’, red arrows). NI NMDA activation (i.t. 7-CK + NMDA) partially reversed sensitization caused by capsaicin, while i.t. 7-CK or NMDA alone did not. n = 12 per group, ** P < 0.01. b) NI-NMDA can also be induced by potent glycine site antagonist, L-689,560 (i.t.; L689) and a 2 nd intraplantar capsaicin injection to cause the reversal of hyperalgesia. n=8 per group, ***P < 0.001 c) Similar to 7-CK and L689, AV-101 caused reversal of hyperalgesia when combined with a second capsaicin injection, but not when given without capsaicin injection. n = 5, ** P <0.01 d) NI-NMDA induced by i.t. 7-CK and a 2nd intraplantar capsaicin injection was prevented by co-administration of 7-CK and APV (i.t). n = 6, ** P <0.01 e) Antihyperalgesic effect mediated by AV-101 at different concentrations of 2 nd intraplantar capsaicin injection (0; 0.1; 0.3; 0.5 w/v%) expressed as percentage of maximum possible effect (MPE) n = 4 per group, ** P <0.01. Two days after the induction of hyperalgesia by plantar CFA, i.t. injection of (f) 7-CK n = 6 per group, ** P <0.01 or (g) MK-801 n=6, ***P <0.001, 30 min prior to a plantar injection of capsaicin caused a significant reduction of hyperalgesia when mechanical sensitivity was tested the following day. h) LTP of fPSPs induced by 2-Hz stimulation at time = 0 min, followed by 7-CK administration from 90 min to 150 min (shaded area) with (orange circles) or without (blue circles) electrical stimulation of dorsal roots. i) Representative traces of fPSPs recorded during baseline (0 min; top), at 90 min (middle) and 210 min (bottom) post 2-Hz stimulation. j-k) The magnitude of fPSP potentiation was compared before (LTP) and after (Post Treatment) 7-CK administration. Stim: n = 14, t (13) = 3.514, ** p = 0.0038; No Stim: n = 6, t (5) = 0.4343, p = 0.6822.

Article Snippet: L689-560 was obtained from Alomone Labs (Jerusalem, Israel).

Techniques: Injection, Activation Assay

Journal: bioRxiv

Article Title: Spinal reconsolidation engages non-ionotropic NMDA receptor signaling to reverse pain hypersensitivity

doi: 10.1101/2021.09.08.458120

Figure Lengend Snippet: a) Bath application of L689 during stimulation induces activity-dependent depotentiation of dorsal horn LTP in spinal cord explants from female C57BL/6 mice. b) Antagonism at the NMDA receptor glutamate-binding site via APV does not reverse dorsal horn LTP, but instead further potentiates fPSPs after drug administration. 7-CK with stimulation: n = 14, t (13) = 3.514, ** p = 0.0038; L689 with stimulation: n = 7, t (6) = 2.845, * p = 0.0294; APV with stimulation: n = 7, t (6) = 4.033, ** p = 0.0069.

Article Snippet: L689-560 was obtained from Alomone Labs (Jerusalem, Israel).

Techniques: Activity Assay, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Bipartite Activation of Sensory Neurons by a TRPA1 Agonist Allyl Isothiocyanate Is Reflected by Complex Ca 2+ Influx and CGRP Release Patterns: Enhancement by NGF and Inhibition with VAMP and SNAP-25 Cleaving Botulinum Neurotoxins

doi: 10.3390/ijms24021338

Figure Lengend Snippet: Allyl isothiocyanate (AITC) induces Ca 2+ -regulated release of calcitonin gene-related peptide (CGRP) from trigeminal ganglion neurons (TGNs) apparently via high- and low-affinity mechanisms, which are susceptible to TRPA1 antagonists HC-030031 or A967079. ( A ) Immuno-cytochemistry with confocal microscope imaging was performed on the TGNs as described in the to detect the expression of CGRP (red) as well as TRPA1 (green) and assess their co-expression (merged). Scale bars represent 20 μm. ( B , C ) Cultured TGNs were exposed for 30 min to each [AITC]. Secreted and residual intracellular CGRP were quantified by ELISA as detailed in the . ( B ) The dose-response relationship between [AITC] and CGRP release (expressed as a % of the total CGRP content) was fitted with two separate four-parameter logistic functions (green, 0.0001–0.05 mM AITC and black, 0.01–0.5 mM; see ). The response peaked at 0.35 mM AITC and declined for concentrations higher than 0.5 mM; data points between 0.5 and 1 mM AITC, connected with a broken line, were not included in the fitting. The quantities of CGRP released into Ca 2+ -free HEPES buffered saline (HBS) containing 2 mM EGTA by the presence of 0.01, 0.1, 0.5, or 1 mM AITC are plotted with squares; N ≥ 3, n ≥ 6. ( C ) TGNs were exposed to 100 µM HC-030031, 100 µM A967079, or vehicle only for 30 min before and during stimulation with various [AITC]. After subtraction of the spontaneous efflux, the amounts of CGRP released were calculated as detailed in the and plotted (blue bars, HC-030031; grey bars, A967079) as a % of the requisite control (vehicle only) level elicited by each [AITC]; N = 2, n ≥ 6. Data are presented as mean ± standard error of the mean (s.e.m.); error bars are not shown where they are smaller than the associated symbol.

Article Snippet: NGF 2.5S (N-100), anti-NGF (AN-240) and anti-TRPA1 (ACC-037) antibodies, TRPA1 antagonists A967079 (A-225) and HC-030031 (H-105) were supplied by Alomone Labs (Jerusalem, Israel).

Techniques: Immunocytochemistry, Microscopy, Imaging, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Bipartite Activation of Sensory Neurons by a TRPA1 Agonist Allyl Isothiocyanate Is Reflected by Complex Ca 2+ Influx and CGRP Release Patterns: Enhancement by NGF and Inhibition with VAMP and SNAP-25 Cleaving Botulinum Neurotoxins

doi: 10.3390/ijms24021338

Figure Lengend Snippet: AITC elicits bi-phasic increases in [Ca 2+ ] i with a complex relationship to agonist concentration. Cultured TGNs were loaded with Fluo-4 AM and fluorescence intensity was recorded by time-lapse confocal microscopy. ( A , B ) Solid lines indicate the mean increases of fluorescence intensity (F − F 0 ) evoked by various [AITC] relative to initial fluorescence (F 0 ), plotted against time. Dotted lines above and below the solid traces indicate ± s.e.m. The black lines above the traces indicate the period AITC was present. ( C ) Area under the curve (AUC) of mean fluorescence change recorded over 30 min for each [AITC] in the absence (N = 3, n ≥ 100) or presence of 100 µM A967079 [(A96), N = 2, n ≥ 30]; data were analysed using unpaired t -test with Welch’s correction; **** p ≤ 0.0001, 0.05 mM AITC vs. 0.05 mM AITC + A967079; #### p ≤ 0.0001 1 mM AITC vs. 1 mM AITC + A967079. Error bars represent standard error. ( D ) The % of excitable cells (±s.e.m.; N = 3, n ≥ 80) that responded to different concentrations of AITC.

Article Snippet: NGF 2.5S (N-100), anti-NGF (AN-240) and anti-TRPA1 (ACC-037) antibodies, TRPA1 antagonists A967079 (A-225) and HC-030031 (H-105) were supplied by Alomone Labs (Jerusalem, Israel).

Techniques: Concentration Assay, Cell Culture, Fluorescence, Confocal Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Bipartite Activation of Sensory Neurons by a TRPA1 Agonist Allyl Isothiocyanate Is Reflected by Complex Ca 2+ Influx and CGRP Release Patterns: Enhancement by NGF and Inhibition with VAMP and SNAP-25 Cleaving Botulinum Neurotoxins

doi: 10.3390/ijms24021338

Figure Lengend Snippet: The hypothetical model for [AITC]-dependence of CGRP release from neonatal rat TGNs in vitro, the enhancement of low [AITC]-evoked exocytosis by acute NGF and the blockade of these pain-related processes by BoNTs that inhibit membrane trafficking. ( A ) At low concentrations of AITC (0.01 mM; yellow background), in accordance with those reported to activate TRPA1 by the modification of strongly nucleophilic cysteine residues ( ● ) in the channel , a relatively small increase in [Ca 2+ ] i evokes a modest amount of CGRP exocytosis. Moderate increases to 0.05 mM AITC (grey background) cause [Ca 2+ ] i to rise more rapidly to higher levels initially, but do not elevate the AUC of Ca 2+ signal over the full 30 min time course; this may be reconciled with the known delayed desensitisation of TRPA1 mediated by an intracellular Ca 2+ -binding domain proximal to the channel pore [ , ]. High [AITC] (>0.1 mM; green background) induces large increases in [Ca 2+ ] i , which seems consistent with the modification of a weaker nucleophilic K708 ( ◆ ) that causes a non-desensitising TRPA1 activation , thereby, provoking the exocytosis of a larger amount of CGRP. At >0.35 mM AITC, large increases of [Ca 2+ ] i are elicited without corresponding increments in CGRP exocytosis, likely due to the cation concentration exceeding the optimum level for catalysis of membrane fusion. ( B ) NGF induces a small amount of CGRP release and it is presumed that TRPA1 on the peptidergic granules is trafficked to the plasma membrane. This enhances subsequent responses to 0.01 mM AITC, but not higher concentrations due to various possible reasons; for example, increased Ca 2+ entry might be attenuated by faster Ca 2+ -dependent desensitisation, small NGF-induced enhancements might be obscured by large AITC-evoked signals at high agonist concentrations or increases in plasma membrane TRPA1 might be non-productive at high [AITC] because the optimum [Ca 2+ ] i for triggering CGRP release has been exceeded. ( C ) CGRP exocytosis and the trafficking of TRPA1 to the plasma membrane are inhibited by BoNT/A and BoNT/DA that cleave (scissors) SNAP-25 and VAMP1/2/3, respectively. Some clinical evidence supports the notion that BoNT/A can provide migraine relief to patients by reducing excess CGRP exocytosis . No VAMP-cleaving toxin is licensed for migraine treatment but a few clinical trials have been performed, with some evidence of symptom improvements . Nevertheless, a definitive mechanism of action for BoNT treatment of migraine remains elusive.

Article Snippet: NGF 2.5S (N-100), anti-NGF (AN-240) and anti-TRPA1 (ACC-037) antibodies, TRPA1 antagonists A967079 (A-225) and HC-030031 (H-105) were supplied by Alomone Labs (Jerusalem, Israel).

Techniques: In Vitro, Modification, Binding Assay, Activation Assay, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Reactive Oxygen Species Cause Exercise-Induced Angina in a Myocardial Ischaemia-Reperfusion Injury Model

doi: 10.3390/ijms23052820

Figure Lengend Snippet: Pharmacological and genetic inhibitions of TRPA1 attenuate exercise-induced angina in I/R model animals. ( A , B ) Double immunofluorescence histochemistry of p-ERK (magenta) and IB4 (green) in the T4–T5 dorsal horn of the I/R group ( n = 4), I/R + FTE + vehicle group ( n = 4) and I/R + FTE + A96 group (A-967079, 20 mg/kg, n = 7), and summary of p-ERK-immunoreactive spinal neurons in rats ( n = 4 each). ( C , D ) Immunofluorescence histochemistry of p-ERK (red) in the T4–T5 dorsal horn of WT I/R group ( n = 6), TRPA1 −/− I/R group ( n = 5), WT I/R + FTE group ( n = 6), and TRPA1 −/− I/R + FTE group ( n = 5), and summary of p-ERK-immunoreactive spinal neurons. ( E , F ) Time ( E ) and number ( F ) of immobility during FTE in WT I/R + FTE group ( n = 6) and TRPA1 −/− I/R + FTE group ( n = 5). All animals were used for the experiments 2 days after the surgery. Data are presented as mean ± SE, unpaired Student’s t -test and one-way analysis on variance with Bonferroni analysis. * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bar = 50 μm ( A , C ). I/R, ischaemia–reperfusion; ns, not significant; FTE, forced treadmill exercise; p-ERK, phosphorylated extracellular signal-regulated kinase; #, number.

Article Snippet: We used 1 mL of 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPOL) (250 mg/kg, H0865; Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) and its vehicle (0.9% saline), or A967079 (A96, 20 mg/kg, A-225, Alomone Labs) and its vehicle (10% DMSO, 5% Tween 80, 0.5% methylcellulose).

Techniques: Immunofluorescence