A single female C57BL6/J mouse of mass 21.6 g was used. Anaesthesia was induced with isoflurane (2%) and maintained with medetomidine (0.4 mg/kg bolus, 0.8 mg/kg/h infusion) through a subcutaneous injection to the flank. A gas mixture of 0.1 L/min O2 and 0.4 L/min medical air (BOC Healthcare (Linde AG)) was continuously supplied during imaging. Respiratory rate was measured using a pressure-sensitive pad, and core body temperature was measured using a rectal thermometer (SA Instruments). Core body temperature was maintained using a warm water pipe system. For the duration of imaging, respiration rate was in the range of 130–160 breaths per minute and temperature in the range 36.6–37.0°C.
MRI was performed using a 9.4 T VNMRS horizontal bore MRI scanner (Agilent Inc., Palo Alto, CA) with an Agilent 205/120HD gradient set, in conjunction with a 72 mm inner diameter volume coil for RF transmission (Rapid Biomedical) and a room temperature 2 channel array surface coil (Rapid Biomedical) for RF reception. Shimming was performed using a GE 3D protocol with first-and second-order shims optimised for the mouse brain with a user-defined shim voxel. The measured linewidth (FWHM) within this voxel was 62 Hz. To localise the line of interest, a single-slice Gradient Echo (GEMS) sequence was used (TR/TE = 200/18 ms, 1 average, matrix size 128×128, FOV = 35 mm2, 1 slice (1 mm thick), flip angle = 25°). Saturation bands and line-scanning FOV was orientated as shown in figure 1A. Once the line-scanning acquisition space was determined, phase encoding was turned off. The same sequence parameters were used as in the GEMS localiser, except the FOV in the read direction was set to 10 mm and a matrix size of 128×1. The effective FOV perpendicular to the read direction was 2 mm, with a final voxel dimension of 0.078×2×1 mm3. The line was acquired for a total of 605 s per fMRI run, and the initial 5 s of data per run were discarded to allow for net magnetisation to reach equilibrium.
Stimulation timings were triggered from the beginning of the pulse sequence using a POWER1401 control system (CED Ltd., UK) with Spike2 software. The stimulus was generated using a cold white LED (Thor Labs), transmitted into the scanner bore using a fibre optic cable. The cable was placed above the mouse head, secured to the top of the surface coil and aimed into the bore in order that light reflected off the surface of the coil interior for binocular stimulation. The stimulus design was as follows: a dim but non-zero baseline intensity (20 mA) with bright flashes (1000 mA) and dark intervals (0 mA). Pulse duration was 10 ms, and a 2 Hz pulse flashing frequency was used during periods of activation. Each run began with 10 s of baseline (of which the first 5 s was discarded to allow net magnetisation to reach equilibrium), and then 20 s of activation followed by 40 s of baseline were repeated 10 times. Eight line-scanning fMRI scans were conducted, for a total line-scanning imaging time of approximately 80 min.
fMRI data were reconstructed using the average magnitude of the Fourier-transformed signal from each channel of the RF coil. The resultant line profile for a single time point is shown in figure 1A. Using the Allen Mouse Brain Atlas, the thickness of the superficial layers of the superior colliculus was estimated to be approximately 0.4 mm. For a pixel width of 0.078125 mm, this corresponded to a ROI of approximately five pixels. Based on the positioning of the FOV and with reference to the 2D GEMS localiser scans, it was inferred that pixels 24–28 corresponded to the superior colliculus. A control region-of-interest (Ctrl) of the same dimensions was placed in the grey matter (pixels 64–68). These ROIs are shown in figure 1A (lower panel). In the temporal domain, each voxel time course was high-pass-filtered with a cut-off period of 128 s to account for signal drift and then normalised to the baseline signal before averaging. In figure 1B, the mean BOLD responses for each run are plotted in colour, with the grand mean BOLD response across all stimulus block trials plotted in black.