We estimated the pharmacologically relevant dose of menthol by analogy with nicotine doses used in mouse studies. Typical menthol cigarettes contain 1–5 mg of menthol (Ai et al, 2015 (link)) and ~1 mg of nicotine (Rodgman and Perfetti, 2009 ). Therefore, menthol is 1–5 times that of nicotine. Steady state and peak concentrations of nicotine in human smokers are replicated in mice using 0.4 and 2.0 mg/kg/h doses of nicotine, respectively (Matta et al, 2007 ). CPP assays use 0.5 mg/kg nicotine (Tapper et al, 2004 (link)). Assays for in vivo upregulation use 2.0 mg/kg/h nicotine (Henderson et al, 2014 (link)). We selected 1 mg/kg and 2 mg/kg/h menthol for CPP and in vivo upregulation assays, respectively. Both dose selections fall within the 1–5 menthol-to-nicotine ratio of menthol cigarettes.
We previously discussed our menthol dose selection for cultured cells and neurons (Henderson et al, 2016 (link)). In preliminary assays to determine the concentration of menthol in a mouse brain, our chronic dosing methods (2 mg/kg/h, osmotic pump) produced concentrations of menthol at 0.5–2.5 μM. Thus, 500 nM menthol is appropriate in studying cultured neurons and cells and is consistent with previous investigations (Henderson et al, 2016 (link)).
All material and methods are described in detail in theSupplementary Material .
We previously discussed our menthol dose selection for cultured cells and neurons (Henderson et al, 2016 (link)). In preliminary assays to determine the concentration of menthol in a mouse brain, our chronic dosing methods (2 mg/kg/h, osmotic pump) produced concentrations of menthol at 0.5–2.5 μM. Thus, 500 nM menthol is appropriate in studying cultured neurons and cells and is consistent with previous investigations (Henderson et al, 2016 (link)).
All material and methods are described in detail in the
