Menthol enhances nicotine-induced changes midbrain neuron firing frequency. (a1, b1) Neurons were identified as TH+/DA or TH-/non-DA using TH-eGFP fluorescence. Bars, 20 μm. (a2–3, b2–3, c1–3) Neurons were treated 10 days with control, 200 nM nicotine, or 500 nM menthol plus 200 nM nicotine and firing frequencies were recorded in current clamp mode. (a3, b3) Mean firing frequency of neurons (n=20–22 for TH+/DA neurons and 9–13 for TH−/non-DA neurons). (c1) Current-clamp recordings of TH+/DA neurons before and after ACh puff (100 ms, 300 μM at arrows). (c2) Mean firing frequency over time before and after ACh application. (c3) Mean ‘peak’ firing frequency during 2 s post-ACh puff. For (c1–3), number of neurons recorded is indicated in parenthesis of (c3). The data are mean±SEM: *, p<0.05, one-way ANOVA, post hoc Tukey.