Data acquisition of resting-state fMRI and optogenetic fMRI experiments

Data acquisition protocol for resting-state fMRI and optogenetic fMRI experiments in mice

Setup before fMRI data acquisition

1. The mice were anesthetized with 3.5% isoflurane for 5 minutes.
2. Then, the mice were injected with a bolus of dexmedetomidine (0.02 mg/kg) intraperitoneally.
3. One drop of 2.5% lidocaine was then applied to the chords of mice to provide local anesthesia before endotracheal intubation.
4. The mice were positioned on an MRI-compatible cradle, fixed by ear bars and a bite bar, and connected to a ventilator (TOPO, Kent Scientific, USA) using a homemade 3D-printed mouse breathing mask (Fig. 1, A and B), which was compatible with mechanical ventilation under the Bruker mouse head crycoil (MRI CryoProbe, Bruker BioSpin, Germany). The mice were mechanically ventilated at a rate of 80 breaths per minute, with a respiration cycle of 25% inhalation and 75% exhalation (the gas source is medical oxygen).
5. The mice were connected to a variety of physiological detectors, including ones that measured respiration, rectal temperature, and oxygen saturation. Mouse rectal temperature was maintained at 37 ± 0.1°C with an MR-compatible heater system (Heater system, Small Animal Instruments, USA). Continuous physiological monitoring was performed throughout the duration of the experiments.
6. After the above setup, isoflurane was reduced and maintained at 0.4%, and a continuous subcutaneous infusion of dexmedetomidine (0.04 mg/kg per hour) was started to maintain the sedation level.
7. The above setup of mice took approximately 15 minutes. During this period, the physiological states of mice gradually stabilized to normal ranges (rectal temperature: 37 ± 0.1°C, breathing: 80 breaths per minute, heart rate: 350 to 420 beats per minute, oxygen saturation: >95%) before the start of the fMRI data acquisition.

fMRI data acquisition

1. All fMRI experiments were performed on a Bruker 7T MRI scanner (Bruker BioSpin, Germany) equipped with a mouse head cryocoil.
2. Data acquisition includes localizer scanning, high-resolution anatomical image acquisition, B0 map acquisition, and fMRI image acquisition.

• Turbo-RARE: The anatomical images were acquired using a spin echo (Turbo-RARE) sequence [field of view (FOV) = 16 × 16 mm², matrix = 256 × 256, RARE factor = 8, repetition time (TR)/echo time (TE) = 2500/35 ms, slice thickness = 0.4 mm]. 
• GE-EPI: Local field homogeneity was optimized in the brain using the B0 field maps scanned in advance. fMRI images were then obtained using a single-shot gradient echo planar imaging (GE-EPI) sequence with FOV = 16 × 16 mm², matrix = 64 × 64, flip angle = 54.7°, slice thickness = 0.4 mm, dummy scans = 10, TE = 15 ms, TR = 750 ms (resting-state fMRI) or 1000 ms (optogenetic fMRI), averages = 1, Segments = 1, Slice gap = 0, repetitions = 450 (resting-state fMRI) or 360 (optogenetic fMRI). fMRI image acquisition has the same slice geometry imported from the previously acquired T2-weighted anatomical image.
• An Arduino programming board was used to synchronize the scanner trigger and the optogenetic stimulation laser for optogenetic experiments. A 473 nm blue light was delivered using a laser transmitter via an optical patch cable. The optogenetic stimulation (10 Hz; light power: 3.5 mW at the fiber tip; 15% duty cycle) was delivered with a duration of 20 s.

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