Computational protocol: High concentrations of morphine sensitize and activate mouse dorsal root ganglia via TRPV1 and TRPA1 receptors

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Protocol publication

[…] HEK293t cells were transfected with plasmids of hTRPV1 (John Davis) or mTRPA1 (Ardem Patapoutian) along with GFP as reporter plasmid using Polyfect (Qiagen, Hilden, Germany) according to the manufacturer's protocol. After incubation for one day, the cells were replated in 35 mm culture dishes and used for experiments within 1–2 days. GFP-expressing cells were identified by fluorescence.Recording electrodes were pulled from borosilicate glass tubes (GB150T-8P, Science Products, Hofheim, Germany) to give a resistance of 3.5 – 5.0 MΩ (P97, Sutter, Novato, CA). The external solution contained (in mM) 140 NaCl, 4 KCl, 1.8 CaCl2, 1 MgCl2, 10 HEPES, 4 glucose, adjusted to pH 7.4. The internal solution contained (in mM) 140 KCl, 1.6 MgCl2, 2 EGTA and 10 HEPES and was adjusted to pH 7.4. Recordings were performed at room temperature and cells were held at -60 mV. Membrane currents were acquired with an Axopatch 200B amplifier and pClamp 10 software (Molecular Devices, Sunnyvale, CA). Solutions were applied with a RSC-100 gravity-driven solution changer (Bio-Logic, Claix, France). All cells were first exposed to morphine, then to capsaicin or mustard oil. TRPA1 was sensitized by PLC activator m-3M3FBS (1 μM, Sigma). [...] The calcium influx elicited by a stimulus was quantified by evaluating the area under the curve (AUC) of the application period. Absolute increases in calcium concentrations were calculated based on Rmin and Rmax []. Compared to the foregoing reference period a mean increase of the intracellular calcium concentration of at least 15 nM throughout the application period was considered as activation. At the end of all protocols a 10 s stimulus of KCl 60 mM was applied as control and normalization reference. Cell diameter was calculated as the mean of two orthogonal axes. Repeated measurements and independent groups in calcium imaging experiments were compared by ANOVA and HSD post-hoc test. CGRP release experiments were performed using both hindpaw skin flaps of the same animal and compared using the Wilcoxon matched pairs test. Statistical analysis was performed using Statistica 7 (Tulsa, OK, USA), sigmoidal dose-response curves were fitted by Origin 7.5 (Northhampton, MA, USA). Data are presented as mean ± SEM; p < 0.05 was considered significant. […]

Pipeline specifications

Software tools pCLAMP, Statistica
Applications Miscellaneous, Patch-clamp
Organisms Mus musculus
Chemicals Acrolein, Bradykinin, Calcium, Morphine, Naloxone