Vitalrecorder

We used an LED (PlexonBright OPT/LED Blue_TT_FC; Plexon, Dallas, TX) for the light source. The intensity of blue light at the tip of the fiber was 0.07 mW (465 nm). The excitation light was applied through a dichroic mirror and GFP excitation bandpass filter (path, 472 ± 35 nm) and coupled to the silica fiber (NA of 0.39, 400 μm diameter). The G-CaMP6 signals from CRF neurons were collected by the same silica fiber passed through a bandpass emission filter (path, 525 ± 25 nm) and guided to a photomultiplier tube (1P28; Hamamatsu Photonics K.K., Hamamatsu, Japan). G-CaMP6 signals were recorded by VitalRecorder (Kissei Comtec), together with the EEG/EMG signals at a sampling frequency of 128 Hz.
The guide cannula was stereotaxically implanted with a dummy fiber above the PVN more than 2 weeks after AAV injection, and they were fixed to the skull with dental cement. More than 7 days after the surgery, an optical fiber was implanted (AP, +0.4 to 0.5 mm; ML, +0.2 mm; DV, −4.7 mm). Recorded G-CaMP6 signals were smoothed by a 33-point moving average and converted to ΔF/F (%) as follows: dF/F = 100*(F(t) − Fmin) / Fmin, where F(t) is the G-CaMP6 signal and Fmin is the minimum value of the signal. Calcium signals of 1 min before and after stage change were selected and calculated mean value for each animal in Fig. 3. Statistics was performed using mean data obtained from all animals.

Surface electromyography (EMG) (Vital Recorder; Kissei Comtec Co., Matsumoto, Japan) with a 1,000-Hz sampling frequency was used to measure muscle activities. The following muscles were measured: the bilateral rectus abdominis (RA: about 2–3 cm lateral to the umbilical region), the bilateral EO (15 cm lateral to the umbilicus), the bilateral IO (2 cm medial and inferior to the ASIS), rectus femoris (RF: at 50% on the line from the superior part of the patella to the ASIS) on the leg raising side, the biceps femoris (BF: at 50% on the line between the lateral epicondyle of the tibia and the ischial tuberosity) on the non-leg raising side. The reference electrode was placed on the patella of the leg raising side. Bipolar surface electrodes (P-00-S; Ambu Inc., Ballerup, Denmark) were applied after successful skin preparation using sanding and alcohol. Each pair of electrodes was attached 2.5 cm apart and aligned parallel to muscle fibers. All EMG data were processed through a bandpass filter (15–500 Hz) and full-wave rectified. The average EMG amplitudes of the medial 3 sec of the total 5-sec duration of ASLR were determined. Mean amplitudes of the maximal voluntary isometric contraction (MVIC) were used for normalizing the average EMG amplitudes recorded during ASLR (%MVIC). The MVIC values of each muscle were performed in the manual muscle testing positions.