The New Zealand rabbits were purchased from Guangdong Medical Experimental Animal Center and housed in the Animal Center of Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences and Shenzhen Research Institute of The Hong Kong Polytechnic University. Seven adult female rabbits (13–20 weeks of age and weighing 3.0–6.0 kg) were used in the study. The macaque (Macaca fascicularis) monkey was purchased and housed in the Institute of Guangdong Laboratory Animals. The adult male monkey used in the study was 7 years of age and weighed 6.0 kg. All experiments were approved by both Peking University’s and Shenzhen Bay Laboratory’s Institutional Animal Care and Use Committee (IACUC). All the experiments were conducted under the approved animal protocol in accordance with the institutional guidelines. All the experiments involving animals in the study are reported in accordance with the ARRIVE guidelines (PLoS Bio 8(6), e1000412,2010).
Surgical procedures for capillary implantation and adeno-associated virus (AAV) injection
The following surgical procedures were performed for implanting a thin-walled capillary into the brains of rabbits and monkeys (Fig. S1) and for injecting AAV viruses to label neurons with fluorescent indicators around the capillary:
First, animals (both rabbit and monkey) were anesthetized with 2.0%~4.5% isoflurane, and the monkey was further anesthetized with additional Ketamine (10 mg/kg). The animal’s head was fixed with a large animal stereotaxic apparatus (RWD Life Science Co., Ltd). The skin of the animal’s head was cut to expose the skull region above the motor cortex in rabbits (identified according to the stereotaxic map of rabbits 27-32, 3 mm anterior to bregma, 3 mm lateral from midline). The skull area over the motor cortex was then drilled with a miniature dental drill. A piece of skull ~3 mm in diameter was carefully removed with a pair of forceps, and the meninges overlying the cortex were further removed with a pair of fine forceps and scissors.
To implant the quartz capillary inside the brain, a computer-controlled electric controller (Z825B, Thorlabs) was used to slowly insert a thin-walled quartz capillary (Hilgenberg GmbH, 8 mm in length, 0.70/1.09 mm of outer diameter, 15 μm in thickness) into the brain at the speed of 10 μm/sec. The location with few blood vessels was chosen for capillary insertion to minimize the bleeding. After the capillary reached the desired depth, the brain tissue inside the capillary was removed with a thin needle connected to a vacuum pump. ~10 min after the tissue inside the capillary was removed, the capillary was moved out of the brain at the speed of 10 μm/sec, leaving a hole of ~0.70/1.09 mm in diameter in the brain. Subsequently, a new quartz capillary (Hyde entrepreneurship Biotechnology Co., Ltd. or Hilgenberg GmbH, 8 mm in length, 0.70/1.09 mm of outer diameter, 15 μm in thickness) with one end sealed with silicone (KN-300N, KANGLIBANG) was pushed down into the hole at the speed of 10 μm/sec to the desired depth. Artificial cortical spinal fluid (ACSF) was used to flush the cortex if bleeding occurred.
Next, Adeno-associated viral (AAV) vectors encoding GFP (green fluorescent protein), td-Tomato, or genetically encoded calcium indicator GCaMP6s were injected near the capillary in the animal’s brain. AAV9-hSyn-GFP (Titer: 4.63×1013 v.g./ml) purchased from WZ Biosciences was used to label neuronal structure in the rabbit brains. A mixture of three different viruses (AAV2/9-CAG-flex-GCaMP6s (Titer: 7.31×1012 v.g./ml): AAV2/9-hSyn-Cre (Titer: 2.28×1013 v.g./ml): AAV2/9-CAG-flex- td-Tomato (Titer: 7.44×1012 v.g./ml) = 10:1:1, OBiO) were used for co-labeling of neurons with td-Tomato and GCaMP6s in the rabbit and monkey brains. These viruses were injected through a sharp glass electrode attached to a pressure injection device (AONIEN; 30 p.s.i., 20 ms, 0.33 Hz). The precise locations and depths of the injection sites were controlled by an electrically controlled micromanipulator (Z825B, Thorlabs). We injected 0.1 µL of the virus at each location, ~10 locations per animal. The electrode remained for 5 min at the injection site after each injection to minimize the spread of the viruses to other areas.
After virus injection, silicone adhesive (Kwik-SIL, WPI) was used to seal the meninges, and dental cement (NISSIN) was used to fix the capillary to the skull. To prevent the capillary from dust and damage, we placed a metal protective cap around the capillary. Finally, we sutured the skin and applied Lincomycin Hydrochloride and Lidocaine Hydrochloride Gel (New Asia Pharmaceutical, C18H34N2O6S: C14H22N2O-HCl = 5:4) to the surgical location to avoid pain and infection.
During the three-week recovery period after the surgery, the rabbits were intramuscularly injected with ceftriaxone sodium (Youcare pharmaceutical group, 50 mg/kg) every two days, and the animals were able to engage in routine activities without affecting the implanted capillary.
Surgery for imaging and EEG/EMG recording
Three weeks after AAV injection, the second surgery was performed to attach the head-posts for stable in vivo imaging and to implant four electrodes for EEG/EMG recording. The four-leg and three-leg head-posts were attached to the animal head by stainless steel cranial screws (Fig. S2) as well as by the dental cement.
For EEG recording in rabbits, two holes (1.5 mm in diameter) were drilled with an electric mill above the visual cortex and cerebellum33-36. Two silver-plated screws (2 mm in diameter) connected to two epoxy-coated silver wire (0.008 inch (0.203 mm) in diameter, A-M Systems) were inserted into the two holes separately and were used as electrodes to record cortical EEG. One end of the screws touched the meninges, and the other end of the silver wire was soldered to a connector pin. Two electrodes for EMG recording made of insulated copper wire (0.3 mm in diameter) were placed on the nuchal muscle. Finally, all electrodes were stabilized with dental cement for stable recording of EEG and EMG signals.
EEG/EMG recording and analysis
EEG/EMG was recorded using the BL-420F Biological Data Acquisition & Analysis System (Chengdu TME Technology Co., Ltd, China) with a bandpass setting of 0.5–100 Hz. Quiet awake state was identified by lower amplitude and higher frequency (>10 Hz) EEG activity and medium-to-high muscle activity. Slow wave sleep was identified by higher amplitude and lower frequency (<10 Hz) EEG activity and low muscle activity.
The imaging probe was made of a high refractive index (N-LASF31, Joptics) right-angle micro-prism and a 0.5/1-mm-diameter GRIN lens (GoFoton Nanjing Co., Ltd). To attach the GRIN lens with the microprism, one end of the GRIN lens was inserted into a sponge ~5 mm in thickness. A small amount of UV-curing optical adhesive (NOA81) was applied as the connecting glue between the GRIN lens and prism under a stereoscope. The right-angle microprism was placed in the center of the lens with a pair of forceps, and the optical adhesive was cured with UV light for 8 s. Before the use of the probe for brain imaging, the probe was first used to image 1-μm fluorescent beads in 1% agar to check the quality of the probe as described previously21.
Structural and calcium imaging
Neuronal structure and somatic calcium imaging in rabbits was performed using a two-photon microscope (Bruker Ultima Investigator equipped with TI: Sapphire Laser) with the laser tuned to 920 nm and with a 20X air objective (N.A. 0.5). A 2 X digital zoom was used to yield images (295 × 295 μm, 512 × 512 pixels) for the quantification of somatic calcium transients.
Neuronal structure and somatic calcium imaging in monkeys were performed using a Thorlabs Multiphoton Imaging Microscope with the laser tuned to 920 nm and with a 10X air objective (N.A. 0.5). A 3X digital zoom was used to yield images (443 × 443 μm, 256 × 256 pixels) for quantification of somatic calcium activity.
Calcium imaging data analysis
Changes in somatic calcium activity during quiet wakefulness and slow wave sleep were measured by changes in GCaMP6s fluorescence that were analyzed post hoc with ImageJ software (NIH) according to previous studies37,38. Regions of interests (ROIs) corresponding to visually identifiable somatic structure and calcium transients were selected for quantification. Changes of fluorescence ΔF/F0 was calculated as ΔF/F0 = (F−F0)/F0, in which F was measured by averaging pixels within each visually identifiable soma, and F0 was the average of 10% minimum F values during the imaging period. The threshold for detecting somatic calcium transients was >3 S.D. of baseline fluorescence F0 for GCaMP6s. The peak amplitude was the maximum ΔF/F0 value of each calcium transient during the imaging period. Frequency of calcium transients was defined as the number of calcium transients per minute. Duration referred to the full width of each calcium transient above the threshold. The total integrated calcium activity was the sum of calcium activity above the threshold over 1 min.
All statistical analyses were performed using Prism 8.3.0 (GraphPad Software). All data are presented as mean ± SEM. Paired t-tests were used for comparison between different groups. P < 0.05 indicated the level of significant difference (*P < 0.05, **P < 0.01, ***P < 0.001).