Figure 3.

The pharyngeal D1 GRN pair responds to caffeine stimulation. (A) Schematic drawing of the preparation used for calcium imaging. The location of the DPS organ is highlighted in green. MH, mouth hooks; CNS, central nervous system. The red line indicates the place where the preparation was cut (B) Calcium increases of pharyngeal D1 neurons of Gr93a-Gal4;UAS-GCaMP6m larvae upon caffeine stimulation were recorded as fluorescence increases. The panel depicts the raw fluorescence images before and during caffeine application (25 mM) in a single larva preparation as morphological images (upper row), and the corresponding ΔF/F false color coded images (lower row). (C) Caffeine (25 mM) induced strong calcium responses of pharyngeal D1 neuron (red time trace), even when presented a second time to the preparation after extensive washing (yellow time trace). Denatonium (10 mM, light green trace), quinine (5 mM, dark green trace), and theobromine (25 mM, light blue trace) also induced calcium responses of the D1 neuron. In contrast, canavinine (12.5 mM, dark blue), salicin (12.5 mM, purple trace), and fructose (25 mM, magenta trace) did not elicit any responses. Responses are plotted as the relative response strength ΔF/F (n = 39, 12, 7, 11, 6, 7, 8, and 38, respectively; different animals were used in each group, individual animals were used for several stimuli). The dark gray bars below each trace indicate stimulus solution flow into the application chamber. The light gray bar indicates when the stimulus solution was present in the application chamber without flow. During all other time points a saline flow through the chamber washed out gustatory stimuli. Saline buffer did not trigger any neuronal responses.