Base of Skull

PET scans were acquired using either Signa PET/MRI 3 Tesla system, GE Healthcare, Waukesha, WI, USA (N = 46) or PET/CT, Discovery-690, GE Healthcare (N = 39).
Fasting condition was requested on the day of ⁶⁸Ga-PSMA PET/MRI and PET/CT scan.
PET scans were acquired from the skull base to mid-thigh (5–6 FOVs, 4 min/FOV), and started approximately 60 min (mean ± SD, 63 ± 6 min) after injection of 111–273 MBq (Mean ± SD, 168 ± 33 MBq) of ⁶⁸Ga-PSMA. PET images, acquired with either PET/MRI or PET/CT scanner, were reconstructed using fully 3D ordered subset expectation-maximization (OSEM) algorithm, time-of-flight (TOF) and point-spread-function (PSF).
⁶⁸Ga PSMA PET image read-out was performed by two Nuclear Medicine physicians on an Advantage Workstation (AW, General Electric Healthcare, Waukesha, WI, USA) and the presence of ⁶⁸GA-PSMA intraprostatic increased uptake was considered positive for malignancy.

The mice with or without cardiac-targeted ChRmine expression were implanted with custom-made headplates, reference electrodes and cyanoacrylate-adhesive-based ‘clear-skull caps’ as previously described^{66 (link)}. After recovery, mice were water-restricted and habituated to head fixation, but they were allowed to drink water to satiate thirst before recording sessions. Craniotomies were made with a dental drill at least several hours before recording sessions and were sealed with Kwik-Cast (World Precision Instruments). Exposed craniotomies before, during and after recordings were kept moist with frequent application of saline until sealed with Kwik-Cast.
Before recordings, the mice were placed into the pacemaker vests and reliable pacing was confirmed by ECG under brief anaesthesia with isoflurane. Then the mice were head-fixed and allowed to recover. Next, one or two (for simultaneous bilateral recordings) four-shank Neuropixels 2.0 probes mounted on a multi-probe manipulator system (New Scale Technologies) and controlled by SpikeGLX software (Janelia Research Campus) were inserted through the craniotomies at variable angles (0–20°) depending on the recording geometry. Typically the probes were aimed to touch the skull around the insula, which could be inferred from probe bending or changes in local field potential, and then were retracted around 100 µm and allowed to sit in place for at least 15 min before recordings. Recordings were performed along each of the four shanks sequentially while mice received 5 s of optical stimulation (900 bpm (15 Hz)) with inter-trial intervals of at least 15–25 s. Probes were cleaned with trypsin between recording sessions. Spike sorting was performed by Kilosort 2.5 and auxiliary software as previously described^{66 (link)}.
After recordings, the brains were perfused, cleared, imaged and registered to the Allen Brain Atlas as previously described^{66 (link)}. Using the traces of lipophilic dye CM-DiI or DiD (which coated the probes before each insertion) and electrophysiological features, the atlas coordinates of the recorded single units were determined.
The spikes from single units were aligned to pacing onset, and the visualized peri-stimulus time histograms were calculated by subtracting 5 s baseline firing rate, 10 ms binning and 500 ms half-Gaussian filtering. The population-averaged firing rate of each region was calculated by combining z-scores (before filtering) over time for all single units in the region of interest. Specifically, we used hierarchical bootstrap to combine data from multiple levels as previously described^{66 (link)}. For each condition, 100 bootstrap datasets were generated, and their mean and s.d. represented the mean and s.e.m. of the initial dataset. For statistical tests comparing ChRmine and control groups, the one-sided P value for the null hypothesis (the ChRmine firing rate subtracted by the control firing rate is zero) was calculated as the fraction of these subtracted values from the pairs of the resampled means (averaged over the time window of interest) that were smaller than zero.

The delineated CSF space was separated manually from the final CSF mask in ITK-SNAP into seven compartments (Appendix 1—figure 3B– ‘Filtration and labeling’), for further statistical comparison: lateral ventricles; third ventricle; fourth ventricle; basilar artery; basal perivascular space at the skull base surrounding the Circle of Willis; parietal perivascular spaces and cisterns (ventrally from the position of posterior cerebral artery, via space neighboring the transverse sinuses and dorsally to the junction of the superior sagittal sinus and transverse sinuses); remaining perivascular space within the olfactory area, surrounding anterior cerebral and frontopolaris arteries, middle cerebral arteries branches, and posterior cisterns including pontine and cisterna magna. For supplementary comparison, the segmented lateral, third and fourth ventricular spaces were considered jointly as the ventricular space, and the basilar, basal and the remaining anterior/posterior CSF spaces were considered jointly as the whole perivascular space. Number of voxels was counted, and the volume of each segment was calculated by multiplying the voxels count by the voxel dimension from the original 3D-CISS image, for subsequent statistical comparison.
To compensate for the brain capsule volume differences and provide a reliable measure of the brain’s CSF space volume between animals, a ratio of the CSF to the brain volume (intracranial volume) was calculated for each delineated CSF segment as: $Rati{o}_{CSFspace}=\frac{CS{F}_{compartmentvolume}}{Brai{n}_{volume}-CS{F}_{wholesegmentedvolume}}$
The ratios obtained for each of the CSF compartments, as well as the segmented brain volumes were compared between KO and WT animals using nonparametric Mann-Whitney U-test.