Figure 1.

A, The Ca²⁺ indicator YC2.1 is expressed in the orexin neurons in the orexin/YC2.1 mouse brain. YFP and CFP fluorescence were observed in the same neurons. The merged picture (orexin-ir and YFP) shows that the expression of YC2.1 is restricted to orexin-immunoreactive neurons in the LHA. Approximately 50% of orexin neurons expressed YC2.1 (n = 5). B, C, The function of YC2.1 was confirmed by glutamate application. B, A pseudocolor ratio image of orexin neuron during application of glutamate. The images were captured at the times indicated in the graph. Glutamate (300 μm) was applied by bath application during the period represented by the bar in the graph. Color represents a ratio of 0.85 (blue) to 1.05 (red). C, Glutamate application increased the YFP/CFP ratio in a concentration-dependent manner. The Δ ratio is normalized to a high concentration of glutamate application (1000 μm). D-F, Simultaneous recording of calcium imaging and slice patch clamp. The neuron subjected to calcium imaging was whole-cell patch clamped, and depolarizing pulses were applied through patch pipette for 10 s (60-90 pA; duration of 5 ms; 20, 50, and 100 Hz). D, Bottom trace shows electrical stimulation through pipette, top trace shows evoked action potentials in the recording neuron. E, Intracellular calcium concentration increased in a firing frequency-dependent manner. Blue and green lines at the top of the graph show CFP and YFP intensity, respectively. F, Bar graph summarizes the data in E (n = 6). Values are represented by mean ± SEM.