Animals. C57BL/6J male wild-type mice (20–30 g, at least 8 weeks, NTU College
of Medicine Laboratory Animal Center or National applied research laboratories) were housed in groups of 5 per cage with food and water ad libitum on a 12 h light/12 h dark cycle.
Stereotaxic surgery. 8-week-old C57BL/6J mice were deeply anaesthetized by
isoflurane and fixed the position on the stereotaxic. An incision was made on the scalp and the skull was exposed. According to the coordinates of the targeted regions, corresponding holes were made with 0.4mm diameter drill bit for injections of optogenetic virus and fiber implantation. The coordinates used relative to Bregma were as follows: aACC (0.500 mm anterior, ±0.350 mm lateral, -1.750 mm depth), ZI (1.450 mm posterior, ±0.950 mm lateral, -4,850 mm depth), dlPAG (4.500 mm posterior, ± 0.500 mm lateral, -2.330 mm depth). For behavioral experiments, AAV virus (AAV1-Syn-eNpHR3.0-eYFP, AAV-Syn-ChR2-eYFP or AAV-Syn-eYFP from Penn Vector Core or Addgene) was injected bilaterally in the aACC. For pathway-specific behavioral experiments, Cre-dependent AAV virus (AAV-EF1-DIO-eNpHR3.0-eYFP, AAV-EF1-DIO-eYFP, AAV-EF1-DIO-Arch-eYFP from Penn Vector Core or Addgene) was injected bilaterally in the aACC and retrograde AAV virus encoding Cre recombinase (rgAAV2-Ef1a-eBFP-IRES-Cre, Addgene) was injected bilaterally in the ZI or dlPAG. 10 μl Nanofil syringe (Hamilton, Reno, NV or World Precision Instruments, Sarasota, FL) with 33 or 34gauge needle were used to inject virus. The injections were controlled by Legato syringe pump (0.25 - 0.35 μ-l, 100 nl/min) (KD scientific, Holliston, MA). For the
mice aim for optogenetic manipulations, fiber optics was implanted in the midline above the aACC (coordinates from Bregma: 0.500 mm anterior, 0.000 mm medial/lateral, -1.450 mm depth) after the injections. Dental cement and screw were used to chronic secured the fiber optics to the exposed skull. Following surgery, the mice were single housed and kept warm on a heat pad for recovery.
The sites of injection and viral expression were confirmed at the end of all experiments; animals displaying incorrect expression sites were excluded from all analysis.
Spared nerve injury (SNI) model. Mice were deeply anaesthetized with isoflurane,
and the fur of right thigh was shaved. A 1 cm incision was made on the lateral skin surface of the thigh through the longitudinal direction distal to the knee. The muscle layer was separated by micro forcep to reveal the sciatic nerve close to the thigh bone (femur). By the aid of stereo microscope, the operator identified the area where the sural nerve branches from the sciatic nerve. The sural nerve is the smallest of the three branches. A tight surgical knot was applied around the other two branches (the tibial and common peroneal nerves). Micro scissors were used to completely severe two branches distal to the knot leaving an intact sural nerve.
In sham operated animals, the sciatic nerve is exposed but not manipulated. The muscle layer was gently closed and wounds were sutured using a non-absorbable surgical suture. After the surgery, animals were left to recover in their heated home cage for several hours with easily accessible water and chow. Behavioral testing was carried out 7 d after the operation.
Von Frey measurement. Tactile thresholds were measured in all SNI and Sham animals at different time points post-injury.In this test, mice were placed in red colored Plexiglas boxes (9 cm length × 9 cm width × 9.5 cm height) on an elevated mesh platform. The red color of the boxes makes them unable to see each other and the operator. For each session, there were 15 min habituation before the first measurement. Mechanical threshold of hind paw withdrawal was measured by repeated manual applications of electronic von Frey filaments (Bioseb) with ascending forces to the lateral plantar surface of hind paws. The machine could gauge the force change and automatically record the value as soon as the mice withdrew their hind paws.
All tests are performed on the right (injured) and left (noninjured) hind paws (5 applications per paw with 1 m inter-trial interval) separately. The average PWT was defined by excluding the maximum and minimum values out of 5 collected trials for each paw.
Plantar test (Hargreaves’ method). To assess thermal hyperalgesia induced by SNI surgery, thermal withdrawal latency was measured using the thermal plantar test apparatus (Ugo Basile, Italy), according to the Hargreaves method. The mouse was placed individually into a red transparent acrylic box (10 cm length × 10 cm width × 14 cm height) on the elevated glass floor 12.5 cm from tabletop for 30-minute habituation. The operator located the mobile infrared heat source beneath the plantar surface of the hind paw, and started the heat-up program with automatic timer. The latency of the withdrawal response to thermal nociception was recorded by the machine. A 20 s cut-off was employed to prevent potential tissue damage in non-responsive subjects. 5 measurements were performed per hind paw (right: injured paw; left: non-injured paw) with 5 m inter-trial interval in
order to avoid accommodation or sensitization effects which might bias the results.
The average thermal withdrawal latencies were calculated after excluding the maximum and minimum values out of 5 collected data points.
Instrumental intracranial self-stimulation. To examine the motivation of earning
photoinhibition in the aACC for pain-relief as negative reward. The behavioral experiments were conducted using SNI or Sham mice that received Halo or control vectors. After fiber implantation and gene expression, the mice underwent a habituation session of two days, exploring the standard mouse operant chambers (Panlab, Barcelona, Spain) for 40 minutes individually with optical connection. The following training session was performed for a minimum of 5-7 days. On every training day, the mice were place into the operant chambers at the same daypart for 30 minutes (14:00-14:30). In operant chambers, mice were allowed freely access to two levers, and the active lever pressing triggered continuous 5s green laser light (532 nm, 5 mW) delivery via the implanted fiber at a fixed ratio 1 (FR1).
Simultaneously, the cue light illuminated for 1 s with the house light off. On the contrary, inactive lever pressing did not result in any event in the chamber but were counted and recorded. During the 5s stimulation, additional active lever pressings were recorded but did not further trigger or prolong the laser stimulation. The behavioral experiments were performed during the mice’s dark cycle.
Conditioned place preference (CPP). To test the motivational drives resulting
from the aversive state induced by spontaneous pain of chronic neuropathic pain, a single-trial CPP paradigm was used (King et al., 2009). This test examines whether animals develop a preference to a paired chamber due to pain relief in the
designate environment. The apparatus was positioned underneath a video camera and consisted of 3 Plexiglas chambers separated by 2 manually detachable doors.
Two chambers (25.5 cm length × 13 cm width × 17 cm height) distinguished by the texture of the floor and by the wall colors (white and black), were connected by a central chamber (12 cm length × 13 cm width × 17 cm height). The CPP procedures contained three phases: a single habituation/preconditioning phase, a conditioning phase composed by two conditioning sessions of 30 minutes, and a preference test of 15 minutes. Animals went through 1 d preconditioning period during which they had access to all chambers for 15 min and acclimated to the environment. Time spent in each chamber was recorded and analyzed by Any-maze (Stoelting Co., Illinois, U.S.A.) tracking software. On the conditioning day (day 2), before entering optical-stimulation-unpaired compartment, mice first received sham light stimulation (mice were connected to laser generator’s optic cable without turning on the laser power) for an hour. Four hours later, in the paring session, mice received continuous one-hour green light stimulation (532 nm, 5 mW) in the aACC and then were placed in the opposite optical-stimulation-paired compartment. Conditioning lasted for 30 min per chamber, without allowing the animal to access the other chambers. The third day was preference test phase, during which mice were placed in the center chamber, with free access to both conditioning chambers. The time spent in each chamber was recorded for 15 min by Any-maze (Stoelting Co., Illinois, U.S.A.) tracking software and analyzed offline.
c-Fos staining and quantification. Operated animals (SNI or Sham) were perfused 1 h after von Frey measurement. Coronal sections (30 um) were collected
containing the entire regions of interest and prepared for c-Fos immunohistochemistry staining. The free-floating slices were repeatedly rinsed in PBS, permeabilized with 0.2% Triton X-100/PBS for 15 min and blocked with 3%
normal goat serum (NGS) for 1 h. An overnight (16-24 h) free-floating in the mixture of rabbit anti-c-Fos antibody (1:1000 for fluorescent staining or 1:5000 for Nickel-DAB staining, ABE457, Merck Millipore) with 1% NGS, 0.2% Triton X-100 and 0.1% azide in 0.1 M PBS were subsequently performed at 4℃.
On the second day, for fluorescent staining preparation, the slices were first rinses extensively in PBS, and then incubated in PBS containing Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (1: 500, Jackson Immunoresearch) for 1 hour at room temperature. After PB rinses, the immunostaining accomplished.
The immunolabeled slices were mounted on gelatin-coated microscope slides and then cover slipped with Mowiol (Calbiochem) for visualization with a laser-scanning confocal microscope (Zeiss LSM880, Oberkochen, Germany). To obtain high resolution low-magnification images, mosaic stitching was performed with Zen software (Zeiss, Oberkochen, Germany).
If the slices were aimed for 3,3’-diaminobenzidine (DAB) staining with amplified signals by Nickel (Vector Nickel-DAB, Vector laboratories, Burlingame, California), following the overnight incubation in the primary antibody, the slices were rinsed in PBS several times on the second day. Next, operator incubated the slices with anti-rabbit IgG biotinylated secondary antibody (1:500 in PBS, Vector Laboratories) for 1 h at room temperature followed by extensive PBS washes. The slices were then incubated in PBS containing avidin-biotinylated horseradish peroxidase
complex (1:200, Elite ABC, Vector Laboratories) for 1h at room temperature. After extensive washes in PBS, c-Fos protein was visualized with Vector DAB Substrate Kit. The slices were then dehydrated and degreased for embedding in mounting medium Histokitt (Assistent, Sondheim vor der Rhön, Germany).
Nickel-DAB signals were examined and documented with a bright field light microscope (Motic RED233, Xiamen, China) equipped with CCD and integrated image processing software (SG-HD3600, SAGE Vision, Taiwan). The areas of region of interest were obtain when the images were acquired via image processing software. 3-5 sections were serially sampled from each region of interest. Image J software (version 1.52e, National Institutes of Health, USA) was used to count all c-Fos positive cells within the boundary of the defined area. A cell was considered positive only if it displayed an intensity value above the intensity threshold of the background.
Histology. For those optogentically-manipulated mice, the viral expression and
fiber placements were verified after all assigned experiments finished. Animals were deeply anaesthetized with isoflurane when transcardially perfused with ice-cold 0.9% saline followed by 4% paraformaldehyde (PFA) dissolved in PB. Brains were removed and stored in 4% PFA for 6h at 4 C. After cryoprotection by 20%
glycerol (wt/vol) for 16 h at 4 C, 30-100 μm coronal slices including the aACC, ZI and PAG were sectioned on a microtome (Leica SM 2010R, Wetzlar, Germany).
Collected slices were mounted on gelatin-coated microscope slides and then cover slipped with Mowiol (Calbiochem). The placements of intracranial virus injections and optical fiber implantations were validated under fluorescence microscope.
Either an insufficient bilateral viral expression in the targeted regions or a missed fiber track terminal location made the subject excluded from statistical analyses.
Statistics. All line charts and histograms are using mean ± s.e.m. to represent
summarized statistics results. The student’s t-test and one-way ANOVA test followed by post hoc Bonferroni test was employed to compare data obtained from c-Fos immunolabelling experiments, conditioned place preference paradigm and behavioral thresholds as indicated in the figure legends. Two-way ANOVA for repeated measures was used in the analysis of behavioral data with time courses;
post hoc analysis was determined using Bonferroni multiple comparisons. In all
tests, the significance was determined by a value of P < 0.05. All statistical analyses were performed on Prism (GraphPad Software). The F(df1, df2) or t statistical test values are reported in the legends.
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