Animals
Adult (aged 6–8 weeks) C57BL/6 male mice were purchased from the Experimental Animal Center of Xi’an Jiaotong University. Experimental animals were housed in plastic cages with ad libitum access to enough water and mouse chow, and the holding room was kept under standard laboratory conditions (12 h/12 h day/night cycle, temperature of 22–25°C, air humidity of 55%-60%). Mice were raised under standard laboratory conditions at least one week before all animal experiments were carried out. All experimental procedures were performed in accordance with the guidelines approved by the Ethics Committee of Xi’an Jiaotong University.
Drug application
All the chemicals and drugs were obtained from Tocris Cookson (Bristol, UK). Selective competitive NMDA receptor antagonist D-AP5 (Cat. No.0106) was prepared in distilled water. Non-competitive AMPA receptor antagonist GYKI53655 hydrochloride (Cat. No.2555) and selective non-NMDA ionotropic glutamate receptors antagonist CNQX (Cat. No.0190) were dissolved in dimethyl sulfoxide (DMSO). GABAA receptor antagonist Picrotoxin (Cat. No.1128) was dissolved in ethanol as stock solution. All these stock solutions were diluted to the final desired concentration in the artificial cerebrospinal fluid (ACSF) before immediate use. The DMSO and ethanol diluted in ACSF had no effect on basal synaptic transmission and plasticity.
In vitro whole-cell patch-clamp recordings
Briefly, mice were anesthetized with 2% isoflurane and decapitated quickly. The whole brain was rapidly separated and transferred into ice-cold oxygenated (95% O2 and 5% CO2) cutting solution (in mM: 252 sucrose, 2.5 KCl, 6 MgSO4, 0.5 CaCl2, 25 NaHCO3, 1.2 NaH2PO4, and 10 glucose, pH 7.3 to 7.4) within a short time. The whole brain was then trimmed and glued onto the ice-cold platform of a vibrating tissue slicer (Leica VT1200S). Then 200 µm-thickness sagittal brain slices containing both the RSC and ACC regions were cut (about 4–5 slices) according to the Mouse Brain in Stereotaxic Coordinates, 4th edition and then transferred to a room temperature-submerged incubation chamber containing oxygenated ACSF (in mM: 124 NaCl, 2.5 KCl, 1 NaH2PO4, 1 MgSO4, 2 CaCl2, 25 NaHCO3 and 10 glucose, pH 7.3 to 7.4) for at least 1-h incubation before conducting experiments.
The whole-cell patch recordings were performed as previously described29. The recordings were performed in voltage- or current-clamp mode using a HEKA amplifier. PatchMaster and Clampfit 10.2 software were used to acquire and analyze the data. In the present study, the eEPSCs were recorded in the ACC with a HEKA amplifier, and the electrical stimulations were delivered by a bipolar tungsten stimulating electrode placed in the RSC regions. For AMPA receptor-EPSCs and action potential recordings, the recording pipettes (3–5 MΩ for pyramidal neurons) were filled with an internal solution containing 124 mM K-gluconate, 5 mM NaCl, 1 mM MgCl2, 0.2 mM EGTA, 2 mM MgATP, 0.1 mM Na3GTP and 10 mM HEPES (adjusted to pH 7.2 with KOH, 290 mOsmol). Picrotoxin (100 µM) was added to block GABAA receptor-mediated inhibitory synaptic currents for EPSCs recordings in all experiments. The neurons were voltage clamped at − 60 mV in the presence of D-AP5 (50 µM) for AMPA receptor-EPSCs recordings and both D-AP5 and GYKI53655 (100 µM) for KA receptor-EPSCs recordings. CNQX was added in ACSF to block selective non-NMDA ionotropic glutamate receptors. To examine synaptic responses, the I-O curves in the ACC pyramidal neurons were recorded at different stimulus intensities. To examine presynaptic functions, the paired-pulse ratios were tested at different time intervals (25, 50, 75, 100, and 150 ms intervals). Action potentials were recorded in current-clamp mode by delivering depolarizing currents of -200-300 pA (400 ms duration) in increments of 20 pA.
Multi-channel field potential recordings
For extracellular field potential recordings, we performed a 64-channel recording system (MED64, Alpha-Med Sciences, Japan) throughout the experiments as previously described42. The MED64 P5001A probe contained 64 planar microelectrodes (50 × 50 µm/each) with a 150-µm interpolar distance. Before experiments, the surface of the MED64 P5001A probe was pre-treated overnight with 0.1% polyethyleneimine (Sigma Aldrich, St. Louis, MO; P-3143) in 25 mM borate buffer (pH 8.4) at room temperature to enhance surface hydrophilicity. The sagittal brain slice was prepared as mentioned above and transferred into the recording chamber after 1-h incubation. The ACC and RSC regions were covered separately onto the microelectrodes of P5001A probe and a fine mesh anchor was used to ensure slice stability during entire recordings. The slice was continuously perfused with oxygenated, fresh ACSF at 28–30°C and maintained at the flow rate of 2–3 mL/min throughout the entire experimental period. After a minimum 1-h recovery period, one channel located in the RSC was chosen as the optimum stimulus site, which can induce the best synaptic response in the ACC after a biphasic constant current pulse test stimulus (0.2 ms) was delivered. The channel with fEPSP induced by electrical stimulus was regarded as an activated channel. And the fEPSP response was sampled once every minute and averaged every 2 traces.
Virus injection and surgery
For local optogenetic stimulation, 100 nL of AAV2/9-hSyn-hChR2(H134R)-EYFP-WPRE-hGHpA (1.2x1012 genomics copies per mL, brainvta, Wuhan, China) was unilaterally injected into the RSC. For two-photon calcium imaging, 150 nL of AAV2/9-hSyn-GCamp6s-WPRE-hGHpA (1.6x1012 genomics copies per mL, brainvta) was injected into the bilateral ACC. For trans-monosynaptic retrograde tracing experiments, 200 nL of AAV2/9-hSyn-EGFP-2a-TVA-2a-RVG-WPREs-pA (2.0x1012 genomics copies per mL, brainvta) and RV-EnvA-ΔG-DsRed (2.0x108 genomic copies per mL, brainvta) were injected into the right ACC separately.
Viral injection procedures were performed as previously described 9. The experimental mice were anaesthetized with 2% isoflurane and fixed on a stereotaxic frame to keep parallel to the reference panel. A midline incision was made in the skull and the skull was drilled on the RSC (1.70 mm posterior to the bregma, 0.20 mm lateral to the midline, 1.00 mm ventral to the skull surface) or the ACC (0.90 mm anterior to the bregma, 0.30 mm lateral to the midline, 1.40 mm ventral to the skull surface). The viruses were stereotactically pressure-injected into the target site with equal speed (23 nL/min, once every 10 seconds) using a microsyringe pump (Nanoject Ⅲ #3-000-207, DRUMMOND). Next, an additional 10 min was allowed for diffusion of viral particles before the microsyringe was slowly withdrawn. The experimental mice were allowed to recover for at least 2–3 weeks before all the experiments were performed, except that the virus of RV-EnvA-ΔG-DsRed was expressed for 7 days.
Optogenetic manipulations
As described above, 100 nL of anterograde tracer virus AAV2/9-hSyn-hChR2(H134R)-EYFP-WPRE-hGHpA was injected into the right RSC. For the in-vitro electrophysiological experiment, the optic fiber was placed into the surface of brain slice for photostimulation. For the behavioral test, optic fiber cannula (length, 2 mm; 200-µm core; NA = 0.37; THINKERTECH, Nanjing) were chronically implanted into the ipsilateral ACC (0.90 mm anterior to the bregma, 0.30 mm lateral to the midline, 1.40 mm ventral to the skull surface) to activate RSC terminals. Behavioral tests related with optogenetics were performed after 2-week recovery. The optic fiber was connected to a fiber patch cable with a rotary joint, which was in turn connected to a fiber-coupled laser (200 mW, 465 nm, Inper Studio, Hangzhou). Mice received 465-nm blue-light illumination (5–10 mW, 20 Hz, 5 ms pulse) for the light-on group throughout the entire experiments in the EPM, OF and marble-burying tests. For von-Frey, tail-flick and hot-plate tests, mice received blue-light illumination for 30–60 s before testing. Finally, all mice were sacrificed and the whole brain was sectioned to verify optic fiber implantation and viral expression. The data was excluded if the viral expression or optic fiber implantation had a deviation from the targeted regions.
Two-photon Ca2+ imaging
Two-photon Ca2+ imaging was performed by using a Scientifica Hyperscope with a 16 × 0.8 NA water-immersion lens (CFI75 LWD, Nikon) and Coherent laser (Chameleon Ultra II, tuning range from 680 to 1080 nm, average power > 3.5 W) tuned at 900 nm for two-photon excitation for GCaMP6s. During two-photon imaging, the fluorescent baseline was first recorded at least for 20 s with the scanning parameters (1 frame/s and 512 × 512 pixels). After the baseline recording, different stimuli were applied in the RSC, at least 1.2 mm far away from the imaging region in the ACC. The electrical stimulations (5 Hz, 5 ms pulse and 195 ms interval, 9 V, 10 s) were delivered by a bipolar tungsten stimulating electrode in the RSC. In the puff experiments, 1 mM glutamate was released for 10 seconds just above the adjacent RSC through the whole-cell recording pipettes by using MPPI-3 pressure injector (5 ~ 10 psi). For optogenetic experiments, the optic fiber was placed onto the sagittal slice close to the RSC for blue-light illumination (465-nm blue light, 5–10 mW, 20 Hz, 5 ms pulse). The brain slices were perfused until the fluorescence was restored to the basal level after stimulus treatments. Obtained image data was analyzed with Image J. The fluorescent signals were quantified by measuring the mean pixel intensities of the cell body of each neuron. The fluorescent change was defined as ΔF/F0 = (Ft − F0)/F0. Ft was the fluorescent intensity at time t, and F0 was the mean of the baseline intensity before the beginning of stimulus application.
Anatomy and imaging
The mice with the virus infection were deeply anaesthetized and perfused with 0.01 M PBS, followed by 100 mL of 4% PFA in PBS (pH 7.4). The whole brain was immediately separated and stored into 4% PFA solution for 4-h post fixation. And then, the whole brain was placed into 0.1 M PB containing 30% (w/v) sucrose solution for 3-d dehydration at 4 ℃ and cut into 30 µm-thickness coronal brain slices using a freezing microtome (Leica CM1900). Sections were collected in sequence and every third section was mounted onto the slides. These sections were counterstained with DAPI (ABS9235, absin, Shanghai) and observed using a laser scanning confocal microscope (FV3000, Olympus, Japan) or a slide scanner (Slideview VS200, Olympus).
For trans-monosynaptic retrograde tracing experiments, we used a fast and high-resolution VISoR imaging method as previously described23, 24. The separated whole brain was placed into 4% acrylamide hydrogel monomer solution (w/v, HMS) in PBS for 2 days at 4°C. Next, the whole brain was embedded with equal volume mixed solution containing 4% HMS and 20% bovine serum albumin (BSA) at 37°C for 4 h and cut into 300 µm-thickness coronal sections. These sections were transferred into 5% PBS-Triton clearing solution for 24 h at 37°C with gentle shaking to increase membrane permeability. After clearing, these sections were washed three times with PBS and mounted onto the quartz slides in sequence. The quartz slide with fixed sections was immersed into refractive-index-matching solution and these sections were visualized with synchronized beam-scan illumination and camera-frame readout (10x objective). The resultant voxel size is 0.5x0.5x3.5 µm3.
Mechanical withdrawal measurement
The mechanical hypersensitivity was determined using up-down method with von Frey filaments (Stoelting; Wood Dale, Illinois) applied perpendicularly to the plantar surface as previously reported29. Mice were individually placed into a plastic cage with wire mesh floors and allowed to acclimate for 30 min before testing. A series of filaments (0.008, 0.02, 0.04, 0.16, 0.4, 0.6, 1, 1.4, 2.0 g) with various bending forces were applied to the plantar surface of the hindpaw until it was bent slightly and held for 3 s. Licking, biting, or sudden withdrawal of the hindpaw was defined as positive responses. An initial filament force of 0.4g was applied to test if the mouse was sensitive to this force. If the positive response occurred, the filament force was incrementally decreased until a negative result was obtained with the interval of 3–5 min between two tests. If the mouse was insensitive to 0.4g filament force, a stronger filament force was applied until a positive response was obtained. The paw withdrawal thresholds were finally determined using up-down method until the positive/negative responses crossed five times.
Open field test
The open field test was performed as previously described29. The open field consisted of an opaque cube (40 × 40 × 30.5 cm), and was divided into a center zone (20 × 20 cm) and an outer zone as the periphery. A single mouse was placed into the arena center and allowed to explore freely for 15 min with dim illumination. The movement traces were tracked using tracking master v3.0 system and all measurements (total distance, time in center, entries) were quantified relative to the mouse body.
Elevated plus maze
The EPM apparatus consisted of two open arms (30 × 5 cm) and two closed arms (30 × 5 × 30 cm) which were perpendicular to each other and intersected by a central platform (5 × 5 cm). The maze was 70 cm high from the floor. For each test, the mouse was individually placed into the center of the apparatus, and allowed to explore freely for 5 min with dim illumination. A tracking master v3.0 system was used to track the mouse movement. The number of entries, time spent in open arm and total distance were quantified relative to the mouse body.
Hot plate test
The mouse was placed on a hot plate set at 50 ± 1°C or 55 ± 1°C. And the latency time was recorded when the reaction of the hind paw (licking, shaking or lifting) first appeared. The cut-off times (40 s for 50 ℃ and 20 s for 55 ℃) were used to avoid tissue damage. Mice were tested a total of three times with an inter-trial interval of 30 min. The average of three repeated measurements was calculated as the final latency time.
Tail-flick test
The tail-flick reflex was measured using a 50 W projector lamp which produced noxious radiant heat. The TF latencies to reflexive removal of the tail from the heat were recorded for three repeated measurements with an inter-trial interval of 30 min. The cut-off time of 10 s was used to avoid heat damage to the tail.
Marble-burying test
The marble-burying test was used to assess the anxiety behaviors of mice. In detail, a single mouse was positioned in a plastic cage (33 × 14 × 14 cm) which was filled with 5 cm-thickness sawdust bedding for 30 min. A total of 18 glass marbles (3 × 6 array) with 12-mm diameter were evenly placed in six rows on the sawdust. The number of buried marbles counted was more than two-thirds enclosed with sawdust.
Statistical analysis
All data was reported as means ± S.E.M. OriginPro 2021 and SPSS 22.0 softwares were separately used for plotting figures and data analysis. Statistical significance was assessed using two-tail paired or unpaired t-test. In all cases, p < 0.05 was considered to be the threshold for statistical significance.