The experimental timeline of the study is shown in Fig. 1A. Male C57BL/6J mice (aged 6-8 weeks, weighing 22-26 g) were acquired from the Experimental Animal Center of Fourth Military Medical University. The animals were housed in the laboratory under controlled conditions (temperature: 22~26℃, humidity: 40%, light/dark cycle: lights on 9 a.m.-9 p.m.) with food and water available ad libitum. After one-week acclimatization, mice were randomly divided into two groups with six mice each. All operations and handling follow the guidelines of the Fourth Military Medical University Ethics Committee.
Neuropathic pain model
The spared nerve injury (SNI) model for neuropathic pain was established as described in our previous study (24). After mice were anesthetized by intraperitoneal injection of 2% pentobarbital sodium (5 mL/kg), three terminal branches of the left sciatic nerve were exposed by direct incision of the skin and a section of the biceps femoris muscle in the left thigh. The tibial nerve and the common peroneal nerve were ligated with 6-0 silk sutures and sectioned distal to the ligation as shown in Fig. 1B. After the ligating and cutting, the nerve was put back to its original position and the muscle and skin were sutured in two layers. In the sham operation group, three branches of sciatic nerve were exposed successively and then disinfected and sutured again, with the nerves not being lesioned.
Pain Behavior Tests
Measurements were based on previous reports (22). Mice were habituated to the testing environment for 3 days before baseline testing and then placed under inverted plastic boxes (7 × 7 × 10 cm) on an elevated mesh floor and allowed to habituate for 30 min before threshold testing. A logarithmic series of 8 calibrated Semmes-Weinstein monofilaments (von Frey hairs; Stoelting, Kiel, WI, USA) (0.008, 0.02, 0.04, 0.16, 0.4, 0.6, 1, 1.4, and 2 g) with various bending forces (0.078, 0.196, 0.392, 1.568, 3.92, 5.88, 9.8, 13.72, and 19.6 mN) was applied to the plantar surface of the hind paw until the mice withdrew from the stimulus. Positive responses included licking, biting, and sudden withdrawal of the hind paws. A von Frey filament was applied 5 times (3 seconds for each stimulus) to each tested area. The minimum bending force of the von Frey filament able to evoke 3 occurrences of the paw withdrawal reflex was considered the paw withdrawal threshold. All tests were performed in a blinded manner.
Paw withdrawal thermal latency (PWTL) was measured using the UGO BASILE plantar tenderness instrument (Ugo basile, Comerio, Italy) as described in previous study (25). The mice were put in plastic boxes and adapted to the surrounding environment for 20 minutes. After that, the radiation light spot of the stimulator was irradiated to the plantar of the mice on the surgical side, and appropriate light stimulation was initiated (the intensity of the beam was adjusted to result in a latency of 8-15 s in Sham mice). Then the latency was measured and recorded every 10 minutes and repeated for five times. The average value of the results was considered as the threshold of heat pain. In order to avoid tissue damage, the time limit for single irradiation of plantar detection should not exceed 30s.
The Dynamic mechanical allodynia was tested as described in previous study (26). The mice were placed on an elevated wire grid and covered with a transparent plastic frame. After 30 minutes of adapting to the surrounding environment in the frame, the plantar hindpaw was stimulated with a paintbrush from heel to toe. During the test, walking away or occasionally raising the foot score 0, raising the foot for more than 2 seconds or a single gentle retreat score 1, strongly raising the foot above the body level score 2, and continuously shrinking or licking the foot score 3. Each mouse was measured five times with intervals of 3 minutes, and the average score of the results was calculated.
Immunofluorescent histochemical staining
Immunofluorescent histochemical procedure was applied to evaluate the double-labeling of CaMKII/FOS and GAD67/FOS in the ACC of sham and SNI mice. Briefly, mice were perfused with 0.1 mol/L PBS and 4% paraformaldehyde for fixation, and then serially cut into transverse slices with 30 µm thickness. All serial sections were then incubated with primary antisera (1:400, ab11959, Abcam, MA, United Kingdom) for 18–24 h at 4°C in 0.01 M PBS containing 1% (v/v) normal donkey serum, 0.3% (v/v) Triton X-100, 0.02% (w/v) sodium azide, and 0.12% (w/v) carrageenan (pH 7.4). Then, the sections were incubated with Alexa 488 donkey anti-rabbit (1:500, A21206, Invitrogen) /Alexa 594 donkey anti-mouse (1:500, A21203, Invitrogen, CA), and Alexa 488 donkey anti-mouse (1:500, A21202, Invitrogen) /Alexa 594 donkey anti-rabbit (1:500, A21207, Invitrogen)) for 6–8 h at 4°C. If necessary, the sections were incubated with tertiary antisera for 2–4 h in 0.01 m PBS with 0.3% (v/v) Triton X-100 at 4°C. After the immunofluorescence histochemical staining, the sections were observed and images were captured using VS200 microscope (VS200, Olympus, Japan). Digital images were captured using VS200 software (Olympus). Image J (NIH Image) software was utilized to count the number of double labeled neurons using.
In vivo single-unit recording
In vivo single-unit recordings were performed as previous studies with minor modifications (14, 27). The four guide tubes contained 16-channel electrodes using 25.4-µm formvar-insulated nichrome wire (Cat No. 761500, A-M System, USA) and a 62.5-µm diameter optical multimode fiber in the center. As shown in Fig. 3A, rAAV-CaMKIIα-hChR2 (E123T/T159C)-mCherry was injected into right MD (ipsilateral to recorded ACC) (1.7 mm posterior to the bregma, 0.3 mm lateral to the midline and 3.43 mm vertical to the skull surface). After two weeks, the electrodes/optrode implantation was performed to the right ACC at the following stereotaxic coordinates: 1.1 mm anterior to the bregma, 0.3 mm lateral to the midline and 1.8 mm vertical to the skull surface. Head-fixation was utilized immediately after the implantation of electrode in the ACC, dental adhesive resin cement (Super-bond C&B, Japan) was used to stick the metal head bar to the exposed skull, to ensure the security of the head-fixed position when the head bar was held firmly by the behavioral apparatus during recording (28). The mice were given at least one week to recover after the implantation of the electrodes. Before recording, mice were habituated to the head-fixed setup for at least 4 sessions (15 min per session, twice/day). To activate MD-ACC projecting fibers, A series of high frequency opto-stimulation (100 Hz for 1 s, repeated 5 times with 20 s intervals) was applied by using a blue laser (470 nm, Inper Ltd, China) after 20 min baseline recording. Recording was then continued for 30 min after HFS induction. Single-unit recording were performed with an Neurostudio System (Jiangsu Brain Medical Technology Co. ltd, China).
Spike sorting and cell type identification
Single-unit spike sorting was performed by MClust-v4.4 toolbox with MATLAB software (MathWorks, USA). To be precise, based on amplitude and waveform energy features, spikes were manually sorted into clusters. A cluster of waveforms was considered as a single neuron if the ratio of its inter-spike intervals (ISI) under 2 ms was <1%, and the unit quality was quantified by isolation distance (>20) and L-ratio (<0.1) (27). Besides, the two units were considered as a single neuron when the spike time of all the units measured coincided via the cross-correlation comparison. Pyramidal neurons and gamma-aminobutyric acid (GABA) neurons are two main cell types in ACC region. The single neuron was classified as putative pyramidal neuron mainly based on the criteria as described in a previous study (29): trough-to-peak duration above 430 µs which exhibited long duration action potentials. GABA neurons were identified mainly based on criteria that trough-to-peak duration under 430 µs.
Statistical analysis was done using the GraphPad Prism (GraphPad Software, San Diego, CA, USA). The p values were calculated by Wilcox rank-sum test, paired and unpaired t-tests and one-way ANOVA with Dunnett post hoc analysis. Results are expressed as mean ± SEM. Statistical significance was set at p < 0.05.