Figure 3.

GFP labels TRPM8 neurons in vivo and in vitro. a, One-day-old cultured TG neurons show robust GFP fluorescence (green) that colocalizes with immunoreactivity for the pan-neuronal marker PGP 9.5 (red). b, Overlap in GFP fluorescence and TRPM8-immunoreactivity in sensory ganglia. Paraformaldehyde-fixed cryosections of trigeminal ganglia from adult Trpm8^GFP mice were immunostained with anti-TRPM8 antibodies and GFP fluorescence (green) was correlated with TRPM8- IR (red). c, Menthol sensitivity of Trpm8^GFP neurons. GFP⁺ trigeminal neurons (left) from Trpm8^GFP mice respond with robust increases in intracellular Ca²⁺ when challenged with 200 μm menthol. Pseudo-colored images (middle and right panels) of ratio values for the Ca²⁺ indicator fura-2 were used to identify menthol-sensitive neurons (right). d, Fura-2 ratio values for the images shown in c indicate the time course of Ca²⁺ responses in GFP⁺ neurons (arrowheads). Numbering corresponds to the cells shown in c. *Note that cell 6 (arrow) was negative for GFP and did not respond to menthol. e, A representative whole-cell voltage-clamp recording from a GFP⁺ TG neuron in culture. Menthol (200 μm) was applied during the time indicated by the green bars and the membrane potential was held at −60 mV. f, Current-voltage relationship of basal (black) and menthol-evoked (200 μm, green) currents in a GFP⁺ TG neuron in culture show the expected voltage dependence and outward rectification of TRPM8-mediated currents.