Supraspinal facilitation of painful stimuli by glutamatergic innervation from the retrosplenial to the anterior cingulate cortex

Animals.

Adult (aged 6 to 8 weeks) C57BL/6 male mice were purchased from the Experimental Animal Center of Fujian Medical 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 to 25°C, air humidity of 55% to 60%). Mice were raised under standard laboratory conditions at least 1 week before all animal experiments were carried out. All experimental procedures were performed following the guidelines approved by the Ethics Committee of Fujian Medical University and Forevercheer Medicine Pharmac Inc. (Qingdao). The license number of the ethical approval for the animal experiments: IACUC FJMU 2024-Y^-0586 and ECAU FMPI 2023020627.

Drug application.

All the chemicals and drugs were obtained from Tocris Cookson (Bristol, United Kingdom) and MedChemExpress (New Jersey, United States of America). Selective competitive NMDA receptor antagonist D-AP5 was prepared in distilled water. Non-competitive AMPA receptor antagonist GYKI53655 hydrochloride and selective non-NMDA ionotropic glutamate receptors antagonist CNQX were dissolved in dimethyl sulfoxide (DMSO). GABA_A receptor antagonist Picrotoxin was dissolved in ethanol as a stock solution. 4-AP was dissolved in distilled water with ultrasonic assistance. 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 did not affect basal synaptic transmission and plasticity.

Anatomy and imaging.

In the trans-monosynaptic retrograde tracing experiments, the viruses AAV2/9-hSyn-EGFP-2a-TVA-2a-RVG-WPRE-pA (200 nL, 2.0 × 10¹² genomics copies/ml, brainvta) and RV-EnvA-ΔG-DsRed (2.0 × 10⁸ genomic copies/ml, brainvta) were separately injected into the right ACC. After a 3-week expression, the mice with the full viral infection were deeply anesthetized 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 in the 4% PFA solution for 4-h post-fixation. Then, the whole brain was placed into 0.1 M PB containing 30% (w/v) sucrose solution for 3-d dehydration at 4°C 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).

Multichannel 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 to 30°C and maintained at the flow rate of 2 to 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. The fEPSP response was sampled once every minute and averaged every 2 traces.

Two-photon Ca²⁺ imaging.

For two-photon calcium imaging, the virus AAV2/9-hSyn-GCamp6s-WPRE-hGHpA (150 nL, 1.6 × 10¹² genomics copies/ml, brainvta) was injected into the bilateral ACC and expressed for at least 7 days. 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 1,080 nm, averaged 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 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 s just above the adjacent RSC through the whole-cell recording pipettes by using the MPPI-3 pressure injector (5~10 psi). The brain slices were perfused until the fluorescence was restored to the basal level after stimulus treatments. The 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 = (F_t−F₀)/F₀. F_t was the fluorescent intensity at time t, and F₀ was the mean of the baseline intensity before the beginning of stimulus application.

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 to 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 to 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 GABA_A 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 PPRs were tested at different time intervals (25, 50, 75, 100, and 150 ms intervals). Action potentials were recorded in current-clamp mode by delivering stepped currents of −200 to 300 pA (400 ms duration) in increments of 20 pA. To verify the monosynaptic connections, TTX and 4-AP were added to unselectively block Na⁺ and K⁺ channels respectively for presynaptic depolarization which specifically promotes the monosynaptic glutamate release in the optogenetical experiment in vitro.

Virus injection and surgery.

Virus injection procedures were performed as previously described⁹. The experimental mice were anesthetized 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 (40 nL/min) using a microsyringe pump (Nanoject III #3-000-207, DRUMMOND). Next, a 10-min extension was allowed for diffusion of viral particles before the microsyringe was slowly withdrawn. The experimental mice were allowed to recover for at least 2 to 3 weeks before all the experiments were performed, except that the virus RV-EnvA-ΔG-DsRed was expressed for 7 days.

Optogenetic manipulations.

For the in vitro electrophysiological experiment, the virus AAV2/9-hSyn-hChR2(H134R)-EYFP-WPRE-hGHpA (150 nL, 1.2 × 10¹² genomics copies/ml, brainvta, Wuhan) was injected into the bilateral RSC and expressed for at least 2 weeks. The blue-light pulses (420~520 nm, 10~20 mW, 0.5 ms) were given by the pE-300 LED illumination system (CoolLED) for photostimulation. For the behavioral test, the virus AAV2/9-hSyn-hChR2(H134R)-EYFP-WPRE-hGHpA and AAV2/9-hSyn-EYFP-WPRE-hGHpA (150 nL, 1.2 × 10¹² genomics copies/ml) was injected into the right RSC. The optic fiber cannula (length, 2 mm; 200-μm core; NA = 0.37; THINKERTECH, Nanjing) was chronically implanted into the ipsilateral ACC (0.90 mm anterior to the bregma, 0.40 mm lateral to the midline, 1.40 mm ventral to the skull surface) to activate neuronal terminals. Behavioral tests related to optogenetics were performed after a 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 to 20 mW, 20 Hz, 5 ms pulse) for the light-on group throughout the entire experiments in the EPM, OF tests. For von-Frey, tail-flick, and hot-plate tests, mice received blue-light illumination for 30 to 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.

Chemogenetic manipulations.

For chemogenetic experiments, virus injection surgery was performed 2 weeks before the behavioral tests. The viruses AAV2/9-hEF1a-DIO-hM3Dq-mCherry-ER2-WPRE-pA / AAV2/9-hEF1a-DIO-mCherry-ER2-WPRE-pA and AAV2/2-Retro Plus-mCaMKIIa-Cre-WPRE-pA (120 nL, 2.0 × 10¹² genomic copies/ml, Taitool, Shanghai) were injected into the right RSC and ACC respectively to label the excitatory neurons in the RSC-ACC pathway. CNO (MedChemExpress, 2 mg/kg, 0.1 ml/20 g body weight) or vehicle (saline) was injected intraperitoneally at least 30 min before behavioral testing. All the behavioral experiments were finished in 2 h after the CNO or vehicle injection. CNO was dissolved in the saline with ultrasonic assistance.

Mechanical withdrawal measurement.

The mechanical hypersensitivity was determined using an 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 were defined as positive responses. An initial filament force of 0.4 g 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 an interval of 3 to 5 min between 2 tests. If the mouse was insensitive to 0.4 g filament force, a stronger filament force was applied until a positive response was obtained. The hindpaw withdrawal thresholds were finally determined using the up-down method until the positive/negative responses crossed 5 times.

Hot plate test.

The mouse was placed on a hot plate set at 50 ± 1°C or 55 ± 1°C. 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°C and 20 s for 55°C) were used to avoid tissue damage. Mice were tested a total of 3 times with an inter-trial interval of 10 min. The average of 3 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 3 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.

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 the center, entries) were quantified relative to the mouse body.

Elevated plus maze.

The EPM apparatus consisted of 2 open arms (30 × 5 cm) and 2 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 the open arm, and total distance were quantified relative to the mouse body.

Conditioned place aversion test.

In the conditioned place aversion test (CPA), the mice were preconditioned on the first days, and they were allowed to explore the chamber for 15 min freely. The time they spent in each chamber was recorded and analyzed. On the second day, the mice with the CNO injection were paired with a randomly chosen chamber for 15 min in the morning; 4 to 6 h later, the mice with the saline treatment were paired with the other chamber for 15 min in the afternoon. After 3 days of pairing, the mice were allowed to explore all the chambers for 15 min on the fifth day. The time spent in every chamber was analyzed to understand chamber preference. The preference index was calculated as the time spent in the CNO-paired chamber minus the time spent in the saline-paired chamber.

Statistical analysis.

All data was reported as means ± SEM. OriginPro 2021, GraphPad Prism 9, and SPSS 22.0 software were used for figure plotting and data analysis. Two-tail paired or unpaired t test was used to examine statistical differences between the 2 groups. One-way ANOVA followed by Dunnett T3 post hoc test was used for comparison among multiple groups. Two-way ANOVA followed by Sidak multiple comparisons test was used to identify significant differences among multiple groups with 2 impact factors. The significance levels of the statistical tests were presented as *p p p p > 0.05.