Animals
Adult male C57BL/6 and DBH-cre mice (8-12 week old) were used. Male Sprague-Dawley rats (SD rats) were used for in vivo extracellular recording. Adenylyl cyclase type 1 (AC1) knockout (KO) and AC8 KO mice were a gift from Dr Daniel R. Storm (University of Washington, Seattle, WA) [32-34] and were maintained on a C57BL/6 background. All mice and rats were maintained on a 12-hlight/dark cycle (temperature 22-26 °C, air humidity 55-60%) with food and water provided ad libitum. The Animal Care and Use Committee at the Air Force Medical University and Xian Jiaotong University in China, National Institutes of Natural Sciences in Japan and Hyogo College of Medicine approved the experimental protocols.
In vitro whole-cell patch-clamp recordings in theACC slices
Experimental procedures were based on those described previously [35-37]. Briefly, mice were anesthetized with 1-2% isoflurane and coronal brain slices including the ACC (300 mm) were prepared using standard methods. Slices were transferred to a room temperature-submerged recovery chamber with an oxygenated (95% O2-5% CO2) artificial cerebrospinal fluid (ACSF) containing (in mM) 124 NaCl, 25 NaHCO3, 2.5 KCl, 1 KH2PO4, 2 CaCl2, 2 MgSO4 and 10 glucose. After a 1h recovery period, slices were transferred into a recording chamber on the stage of an Axioskop 2FS microscope (Zeiss) equipped with infrared DIC optics for visualized recordings. All experiments were recorded with an Axon 200B amplifier (Axon Instruments). In the voltage-clamp configuration, recording electrodes (2-5 MΩ) contained the pipette solution composed of (in mM) 120 K-gluconate, 5 NaCl, 1 MgCl2 0.5 EGTA, 2 Mg-ATP, 0.1 Na3GTP, and 10 HEPES; pH 7.2, 280-300 mOsm. The membrane potential was held at -60 mV for recording spontaneous excitatory post-synaptic currents (sEPSCs) and held at 0 mV for recording spontaneous inhibitory post-synaptic currents (sIPSCs). APV (50 mM) was always added in the ACSF. Picrotoxin (100 mM) was added into the ACSF for recording sEPSCs and CNQX (25 mM) was added into the ACSF for recording sIPSCs. The pipette solution was containing (in mM): Cs-MeSO3, 120; NaCl, 5; MgCl2 1; EGTA, 0.5; Mg-ATP, 2; Na3GTP, 0.1; HEPES, 10; pH 7.2; 280-300 mOsmol. Pyramidal neurons and interneurons were distinguished based on their morphology, membrane properties and firing pattern (Fig. 6A). The initial access resistance was 15-30 MΩ, and it was monitored throughout the experiment. Data were discarded if the access resistance changed >15% during experiment. Data were filtered at 1 kHz, and digitized at 10 kHz. Data were collected and analyzed with Clampex and Clampfit 10.2 software (Axon Instruments). NA and NA receptors agonist or antagonists were bath applied in ACSF.
In some cases, biocytin (0.5%) were added into the pipette solution for labeling the morphology of the recorded pyramidal cells or interneurons. After recording, the slices were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) for 1 hr at room temperature. Slices were then rinsed with 3% hydrogen peroxide in PBS for 30 min and throughly washed with 0.01 M PBS (pH 7.4). The tissue was then incubated with Alex594 conjugated Streptavidin (1:200, Jackson) for 4 hrs at room temperature. The immunofluorescence-labeled sections were then rinsed in PBS, mounted onto glass slides and visualized under confocal microscope under appropriate filter.
In vivo electrophysiology for ACC recording
In vivo preparations were made as described previously [38]. Under urethane anesthesia (1.2-1.5 g/kg, i.p.), rats were mechanically ventilated after tracheostomy and bilateral thoracotomy was performed. After the head was fixed in a stereotaxic apparatus (Model SR-6R, Narishige, Tokyo, Japan), a craniotomy is performed using a dental drill to open a hole above the ACC according to the stereotaxic coordinates. A tungsten electrode (impedance, 10 MΩ, A-M systems, Sequim, WA) was placed into the ACC, and conventional extracellular recordings were obtained as shown previously [39] with an AC differential amplifier (DAM 80, World Precision Instruments, Sarasota, FL). Data were filtered (300 to 5 kHz) and digitized (10 kHz). Unit firings were sorted with Offline Sorter software (version 3, Plexon, Dallas, TX). Putative pyramidal neurons in the ACC were identified based on their waveforms as shown previously [40]. In the case of drug microinjection into the ACC, a cannula was inserted through the same hole, and the tip was placed in the vicinity (approximately 1 mm) of the recording electrode. Drugs (noradrenaline, 50 mg/0.5 mL; phenylephrine, 5 mg/0.5 mL; Isoproterenol, 37 mg/0.5 mL) diluted in normal saline were microinjected over a 5 min period. For the LC electrical stimulation, a hole was further opened on the skull above the cerebellum ipsilateral to the recording site, and a combined bipolar stimulating electrode-tungsten extracellular recording electrode (1 MΩ, A-M systems, Sequim, WA) was stereotaxically inserted into the floor of the fourth ventricle above the LC. The electrode was then lowered and placed into the LC if neuronal activity is considered to be obtained from LC neurons. LC neurons were identified based on their characteristic spontaneous firing and responses to contralateral cutaneous noxious stimulation as described previously [41, 42]. Trains of electrical pulses (duration 200 ms, 50 times, 4 ms interval) were then applied with an interval of 2 s for 1-2 min.
Immunohistochemistry
Immunostaining procedure was applied as described previously [15]. In brief, mice were perfused with 0.1 M PBS and 4% paraformaldehyde. After fixation, brainstem containing LC and brain containing ACC were removed, cryoprotected and serially cut into transverse slices with 30 mm thickness. The sections were then rinsed in PBS with 0.3% Triton X-100 and 1% normal goat serum (NGS) for 0.5 hr. LC sections were dual-immunostained with GFP and TH. Sections were incubated with mouse anti-GFP antibodies (1:500, Abcam, ab183734) and rabbit anti-TH (1:500, Abcam, ab112) for 24 hrs, following with Alex594 conjugated anti-rabbit (1:1000, Jackson) and Alexa488 conjugated anti-mouse (1:1000, Jackson) antibodies overnight at room temperature. ACC sections were dual-immunostained for GFP/RFP or GFP/GAD. One set of series sections were incubated with mouse anti-GFP antibodies (1:500, Abcam) and rabbit anti-RFP antibody (1:500, Abcam, ab62341) for 24 hrs, following with Alex594 conjugated anti-rabbit (1:1000, Jackson) and Alexa488 conjugated anti-mouse (1:1000, Jackson) antibodies overnight at room temperature. Another set of series sections were incubated with mouse anti-GFP antibodies (1:500) and rabbit anti-GAD antibody (1:200, Abcam, ab203063) for 24 hrs, following with Alex594 conjugated anti-rabbit (1:1000) and Alexa488 conjugated anti-mouse (1:1000) antibodies overnight at room temperature. Sections were then rinsed in PBS, counterstained with DAPI antibodies for 5-10 mins and mounted onto glass slides. The signals were visualized under confocal microscope under appropriate filter.
For the electron microscopic staining and observation, mice were deeply anesthetized and then transcardially perfused with normal saline, followed by 0.1 M PB containing 4% paraformaldehyde, 0.1% glutaraldehyde and 15% picric acid. Sections from the brain containing ACC were generated using a vibratome at a 50-µm thickness. Sections were then incubated overnight with mouse anti-GFP antibodies (1:200, Abcam) and rabbit anti-RFP antibody (1:200, Abcam), followed by anti-rabbit IgG conjugated to 5-nm gold particles (1:100, Nanoprobes) and biotinylated anti-mouse IgG (1:200, Abcam) for 4 hr. Silver enhancement was performed with HQ Silver Kit (Nanoprobes) for visualization of RFP (mCherry) immunoreactivity. Then sections were incubated in the avidin-biotin peroxidase complex for 45 min and then incubated with diaminobenzidine (DAB) solution. Immunolabelled sections were then fixed with 0.5% osmium tetroxide in 0.1 M PB for 1 hr, dehydrated in graded ethanol series and then in propylene oxide, and finally flat-embedded in Epon 812 between sheets of plastic. After polymerization, acrylic sheets were peeled from the polymerized resin, and flat-embedded sections were examined under the light microscope. Three to four sections containing GFP and RFP immunoreactivity were selected from each brain, trimmed under a stereomicroscope, and glued onto blank resin stubs. Serial ultrathin sections were cut with an Ultramicrotome using a diamond knife and mounted on formvar-coated mesh grids. They were then counterstained with uranyl acetate and lead citrate, and observed under a JEM-1230 electron microscope.
Behavioural tests
Four -five weeks after AAV2/9- hEF1a-DIO-ChR2(H134R)-eYFP or AAV2/9-hEF1a - DIO-eYFP injection, mice were anesthetized with an intraperitoneal injection of ketaminexylazine (0.1 mg per gram body weight ketamine, 0.01 mg per gram body weight xylazine) and the head was fixed in a stereotaxic apparatus. A small craniotomy was performed and a hole were drilled. The optic cannula (MFC_200/230-0.39_ 2mm_ZF1.25_FLT, Doric Lenses., Quebec, Canada) was implanted in the middle line of ACC (0.98 mm anterior to Bregma and 1.0 mm deep from skull surface). The optic cannula was then fixed with dental cement [15].
One week after optic cannula implantation, mice with ChR2-eYFP or eYFP infection were placed into Lucite cubicles with a plain pedestal to observe the basal behavior. After acclimation for 5 min, optostimulation at 5 or 20 Hz (3 min) were applied through the implanted fibers by Master 8 automatically. The whole experiment process was video captured and analyzed by observers. The bouts and duration of scratching and wiping behaviors were collected and averaged per min before, during and after optostimulation. For testing the paw withdrawal thresholds, mice were places into Lucite cubicles over a wire mesh with von Frey filaments applied to their left and right hind paws. After acclimation for 5 min, series of filaments (0.008, 0.02, 0.04, 0.16, 0.4, 0.6, 1, 1.4, 2 g) with various bending forces (according to 0.078, 0.196, 0.392, 1.568, 3.92, 5.88, 9.8, 13.72, 19.6 mN) were manually applied to the plantar surface of the hindpaw until the mice withdrew from the stimulus. The lowest force at which a withdrawal response obtained was considered as the paw withdrawal threshold. The paw withdrawal thresholds before and after optostimulation (5 or 20 Hz, 3 min) were tested. In all experiments, observers were blind to animal grouping.
After behavior test, brains containing ACC were fixed and cut into transverse slices (30 mm). The sections were immunostained with rabbit-Fos antibodies (1:300, Abcam, ab190289), following with Alex594 conjugated anti-rabbit (1:1000, Jackson). ACC areas were then observed under confocal microscope for checking the Fos and LC-ACC projecting fiber expression.
Optogenetics
AAV2/9- hEF1a-DIO-ChR2(H134R)-eYFP and AAV2/9-hEF1a-DIO-eYFP were purchased from Taitool Bioscience Co. Ltd. (ShangHai, China) and AAV2/9-CaMKIIa-mCherry was purchased from BrainVTA Co. Ltd. (WuHan, China). AAV2/9-DIO-ChR2-eYFP or AAV2/9-DIO-eYFP (1x 1013, 200 nL per site) were injected into the dual sites of LC and AAV- CaMKIIa-mCherry (3x 1012, 200 nL per site) were injected into the dual sites of the ACC in DBH-cre mice. After one month, the immunostaining, whole cell patch recording or in vivo behavioural experiments were carried out, respectively. For performing whole-cell patch recording or in vivo behavioural experiments, optostimulation was applied with 5 or 20 Hz blue light (470 nm, 5 ms pulse width in an intensity of 15 mW/mm2 at the optic fiber tip) [15, 43-45].
Statistics
Statistical comparisons were made using the Student t-test, one-way or two-way ANOVA as appropriate. Significance between groups was tested with a Holm-Sidak or Tukey tests to adjust for multiple comparisons. All data were presented as the mean ± S.E.M. In all cases, P < 0.05 was considered statistically significant.