Dental Anesthesia

Mice were randomized to receive either bilateral injections of AAV-YFP or AAV-TrkB.FL to the hypothalamus. Mice were anaesthetized with a single dose of ketamine/xylazine (100 and 20 mg kg⁻¹; i.p.) and secured via ear bars and incisor bar on a Kopf stereotaxic frame. A mid-line incision was made through the scalp to reveal the skull and two small holes were drilled into the skull with a dental drill above the injection sites (-1.2 AP, ±0.5 ML, -6.2 DV, mm from bregma). rAAV vectors (2.5 × 10⁹ genomic particles per site) were injected bilaterally into the hypothalamus at a rate of 0.1 μl minute⁻¹ using a 10 μl Hamilton syringe attached to Micro4 Micro Syringe Pump Controller (World Precision Instruments, Sarasota, FL). At the end of infusion, the syringe was slowly raised from the brain and the scalp was sutured. Animals were placed back into a clean cage and carefully monitored until recovery from anesthesia.

Under the premise of double-blind, the success of MCAO in rats was monitored by laser speckle flow imaging (Heeman et al., 2019 (link)). Briefly, after anesthesia and sterilization, and the skull is exposed. The skull is slowly polished by a high-speed electric skull drill until the epidural forms a 10x6mm skull window. Record the blood flow before and after MCAO using laser speckle flow imaging (SIM BFI-HR PRO, Wuhan, China).

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.