Menthol

In 24-well plates, 5 dpf larvae were individually exposed to four natural compounds—Eugenol 99% (CAS number: 97-53-0; Alfa Aesar, Oeiras, Portugal), Thymol 100% (CAS number: 89-83-8; Merck Millipore, Algés, Portugal), Menthol 98% (CAS number: 2216-51-5; Alfa Aesar, Oeiras, Portugal), and Carvacrol 98% (CAS number: 499-75-2; London, UK)—at three different concentrations (2, 5, and 10 mg/L), for a duration of 1 h (Figure 5) [26 (link)]. These concentrations were selected based on the substances 10-times higher anaesthetic doses [68 (link)] and prepared by diluting a previously made stock solution of 89 g/L for each substance (Menthol and Eugenol, pH 7.2 ± 0.1 and 7.1 ± 0.1, respectively) in 90% alcohol [69 (link)]. After exposure, the larvae were removed and subjected to a 1 min exposure to 0.05% AA (CAS number: 64-19-7; Fisher Scientific, Oeiras, Portugal) diluted with E3 buffer, to induce pain [26 (link)]. To ensure this method works, a positive control using paracetamol (CAS number: 103-90-2; extracted from tablets using propanone and dissolved in E3 buffer) at a concentration of 0.05 mg/L was also evaluated [27 (link),70 ].

Solutions for determining acid sensitivity (pH 7, pH 6, pH 5) and TRP-agonist sensitivity (capsaicin, cinnamaldehyde and menthol) were applied in a random order through a gravity-driven 12 barrel perfusion system (Dittert et al., 2006 (link)) to DRG neurons in 5 s pulses with at least 30 s wash period (with pH 7.4) between stimuli. Solutions of 10 μM capsaicin (1 mM stock in 100% ethanol; Sigma-Aldrich), 100 μM cinnamaldehyde (10 mM stock in 100% ethanol; Merck) and 100 μM menthol (20 mM stock in 100% ethanol; Alfa Aesar) were made up in pH 7.4 extracellular solution from their respective stock solutions. Neurons that produced an inward current, time-locked to drug application, were counted as responders; in control experiments using the vehicle control (1% EtOH, n = 3) no such inward currents were observed. Current amplitude was measured in Fitmaster (HEKA) by subtracting the maximum peak response from the baseline (average of the first 3 s before stimulation), which was then normalized by dividing by neuron capacitance to give current density.

The TRPA1 antagonist A967079 ((1E,3E)–1-(4-Fluorophenyl)–2-methyl-1-pentene-3-one oxime); (Alomone Labs, # A-225) was dissolved in 10 % DMSO (Dimethyl sulfoxide) with 10 % Tween-80 in saline. The TRPM8 antagonist AMTB (N-(3-aminopropyl)–2-[(3-methylphenyl) methyl] oxy-N-(2-thienylmethyl) benzamide hydrochloride salt; Alomone Labs, #A-305) was dissolved in 10 % DMSO in saline. Both antagonists were administered i.p. 30 min before the cold treatment. Cinnamaldehyde (Sigma-Aldrich, #W228613, > 95% purity), menthol (Alfa Aesar, #A18098, 98 % purity), capsaicin (Sigma-Aldrich, #M2028, >95% purity) were prepared with 10 % DMSO in ethanol solution. 1.25 ng/μl NA (Sigma-Aldrich) and 1.25 ng/μl medetomidine (Orion Pharma) were administered via intraplantar injection in 20 μl saline.

A week before the test, gently handling the mice for several times to reduce animals’ stress response. On the testing day, mice were placed into the transparent Perspex box for habituation until its exploratory behavior has ceased. Then took the mice from the box for injection. Insert the 0.3 ml disposable insulin syringe needle into the center of the hindpaw at a shallow angle and subcutaneously inject the indicated dose of different chemicals (capsaicin (500 µg/ml, Sigma-Aldrich), 5% mustard oil (Sigma-Aldrich), menthol (0.8 mg/ml, Fisher Scientific), or 2% formalin (Fisher Scientific) in a total volume of 50 μl dissolved in saline). The mice should not bleed during or after injection. Finally, the mice were placed back into the Perspex box and use the stopwatch to record the time spent conducting nocifensive behaviors (licking or biting) for the desired amount of time (menthol, mustard oil, capsaicin for 5 min and formalin for 60 min, respectively).