Fig. 1

Simultaneous real-time measurement of orexin neuronal activity, heart rate, and body temperature in freely moving mice using a fiber photometry and telemetry system. A Schematic drawing showing specific expression of G-CaMP6 and mCherry in orexin neurons by injecting AAV vectors into the hypothalamus of Orexin-tTA transgenic mice. B Immunohistochemical confirmation showing G-CaMP6 (B-i) and mCherry (B-ii) exclusively expressed in almost all orexin neurons (B-iii) that were visualized by anti-Orexin A-antibody in Orexin-tTA mice. G-CaMP6 and mCherry were identified by their own fluorescence. A composite image is shown in (B-iv). Images are a close-up of the white rectangle in D. B-v Quantification of G-CaMP-positive cells, mCherry-positive cells, and G-CaMP6 and mCherry double-positive cells in hypothalamic orexin neurons. C Schematic diagram of the dual-channel fiber photometry system. Blue excitation light from the 470 nm LED illuminates the G-CaMP6-expressing neurons in the hypothalamus via optical fiber. Green fluorescence from G-CaMP6 is then gathered by the same optical fiber and detected by the photomultiplier tube (PMT1). Body temperature and heart rate were measured by a telemetry system. The telemetry transmitter was intraperitoneally implanted into the mouse. D Confirmation of the fiber tract after fluorescent recordings. Coronal section of the brain from the orexin-tTA mouse injected with AAV-tetO(3G) G-CaMP6 and AAV-tetO(3G) mCherry mixture 2 weeks before recording. The dashed line indicates the location of the inserted guide cannula and optic fiber. The white square indicates the hypothalamus region. E Self-assembled silica connecting cable called the “patch cord” (i) and in vivo optical fiber cannula (ii). F Overall fiber photometry system. G Representative traces, in response to ultrasonic sound stress, of locomotor activity (activity count), body temperature, electrocardiography (ECG, expanded traces in the rectangle), heart rate (calculated as beats per minute), and the fluorescence intensity of G-CaMP6 and mCherry in the orexin neurons