Planarians dugesia dorotocephala

planarians (Dugesia dorotocephala) were purchased from Carolina Biological Supply (Burlington, NC, USA). Upon arrival in the laboratory, planarians were maintained in the aqueous solution provided by Carolina Biological Supply, acclimated to room temperature (21 °C), and tested within 3 days of receipt. DAMGO and the kappa agonist U50,488 hydrochloride was purchased from Tocris Biosciences (St. Louis, MO, USA). Naltrexone hydrochloride (NTX) and the kappa opioid receptor antagonist, nor-Binaltorphimine dihydrochloride (nBNI), were purchased from Sigma-Aldrich (St. Louis, MO, USA). Morphine sulfate was provided by NIDA Drug Supply (Bethesda, MD, USA). All drugs were dissolved in spring water, with stock and working solutions prepared daily, and concentrations were based on prior planarian work and Kd values of DAMGO (Tallarida et al., 2014 (link); Zhao et al., 2003 (link)). Behavioral experiments were conducted between 10 AM and 4 PM. Behavioral responses were quantified by a trained observer with a stopwatch who was blinded to drug treatment. planarians are large enough (average length about 3–15 mm) so that behavior can be observed and quantified with the naked eye, so behavioral responses were quantified without the aid of a microscope. Experiments and behavioral observations were conducted in ‘real time’, and no videotaping was used.

planarians (Dugesia dorotocephala) were purchased from Carolina Biological Supply (Burlington, NC, USA). Upon arrival in the laboratory, planarians were maintained in the aqueous solution provided by Carolina Biological Supply, acclimated to room temperature (21 °C), and tested within 3 days of receipt. Nicotine hydrogen tartrate salt and naloxone hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO). DAMGO, HC 030031, pancuronium dibromide, and meloxicam sodium salt hydrate were purchased from Tocris Biosciences (St. Louis, MO, USA). All drugs except HC 030031 were dissolved in spring water, with stock and working solutions prepared daily, and concentrations were based on prior planarian work and K_d values (Tallarida et al., 2014 (link); Zhao et al., 2003 (link)). Because HC 030031 is insoluble in water, stock solutions were prepared in DMSO and diluted with water to the testing concentrations. Behavioral experiments were conducted between 10 AM and 4 PM. Behavioral responses were quantified by a trained observer who was blinded to drug treatment. Each planarian was used only once.