We usedtransgenic mice carryingtTA under the control of a DBHorTPH2 promoter (DBH-tTA mice or TPH2-tTA mice, respectively) [22, 23, 24] (Fig. 1a);10–14-week-old male mice were usedin this experiment. Rearing was carried outin standard conditions,with lights on at 7:00 AM and off at 7:00 PM, atemperature of 24 ± 1°C, and food and water adlibitum. We made efforts to minimize animal affliction and reduce the number of animals used.All experimentalprocedures were carried out in accordance with the National Institute of Health Guide for the Care and Use of laboratory Animals and approved by the Institutional Animal Use Committee of Kagoshima University (MD17105).
Stereotaxic AAV injection
Adeno-associated virus (AAV) vectors were produced using the AAV Helper-Free system (Agilent Technologies, Inc., Santa Clara, CA, USA) and purified as described previously. AAV-TetO(3G)-G-CaMP6 (Serotype:DJ; 0.3μl/injection, 4×1013copies/ml) and AAV-Tet(3G)-mCherry (Serotype:DJ; 0.3μl/injection, 6×1012copies/ml)were produced using the Tet system (Fig. 1b). We slowly sucked up AAV into the glass micropipette (1B150F-3, World Precision Instruments, Inc., Sarasota, FL, USA) connectedto an injection manipulator (I-200J, Narishige, Tokyo, Japan) linked toa nitrogen pressure source through polyethylene tubing. Under 2-3% isoflurane anesthetic conditions, mice were fixed witha stereotaxic instrument (ST-7, Narishige) with the help of supportive ear bar (EB-6, Narishige). To minimize suffering, the surfaces of their ears were covered with local anesthetic jelly (lidocaine, 2%xylocaine),and both eyes were preserved with vaseline. Head hair was shaved using an electric shaver and the cranial dura mater was cut open withsmall scissors. AAV was unilaterally injected into thetarget sites(A5: bregma -5.52mm, lateral +1.4mm, and ventral -5.30mm from the cranium; A7: bregma -4.96mm, lateral +1.88 mm, and ventral -3.10 mm from the surface of the brain; B2: bregma -7.56mm, and ventral -3.93mm from the cranium) (Fig. 1c)and the glass microtube was let sitin the sites for 10minutes before being withdrawn.Postoperative antibiotic administration was carried out by asubcutaneous injection (penicillin G, 40,000 U kg-1) to prevent postoperative infections. After the operation, mice were maintained in normal rearing conditions (see adobe) for 14 days (twoweeks) to recover from thedamage and to set aside a period allowing for the G-CaMP6/mCherry fluorescent protein to be fully expressed prior to the experimental sessions.
After the experiment, we performed immunohistochemistry to confirm the AAV-induced site-specific expression of G-CaMP6 and mCherry in mice. We transcardially perfused mice with 20ml of phosphate-buffered saline (PBS) and 20 ml of a 4% paraformaldehyde (PFA) solution under anesthesia with urethane (1.6 g/kg, ip). The brain was removed and post-fixed with 4% PFA and soaked in 30% sucrose in PBS for two days.We created a series of 30 µm slices including the target sites with the cryostat (Cryotome FSE, Thermo Scientific, Yokohama, Japan) and immersed the sections in PBS for 24 hours at 4℃.Every third section was usedfor analysis. We washed the sections with PBS three times and incubated the sections with primary antibodies overnight. We diluted primary antibodies in blocking buffer and set the concentration as follows: A5/A7; anti-Tyrosine Hydroxylase raised in rabbit antibody (AB152, EMD Millipore Corp., Temecula, CA, USA) at 1:500, B2; anti-TPH antibody (AB1541, EMD Millipore Corp.) at 1:1000. The next day, we washed the sections with PBS three times and incubated the sections with secondary antibodies at room temperature for two hours. We diluted secondary antibodies in PBS and set the concentration as follows;A5/A7: CF647 donkey anti-rabbit (20047, Biotium Inc., Fremont, CA, USA) in PBS at 1:200;B2: CF647 donkey anti-sheep (20284, Biotium Inc.) in PBS at 1:200.Next, we washed the sections with PBS once and mounted them onmicroscope slides (PRO-02, Matsunami, Osaka, Japan) and covered them with a micro cover glass (C024601, Matsunami). We observed the sections undera fluorescence microscope (BZ-X700, Keyence, Osaka, Japan), and created imagesusingAdobe Photoshop CC software (Adobe Systems Inc., San Jose, CA, USA).
In vivo fiber photometry system
In this study, we usedthe same fiber photometry system with two channels that we usedin previous papers [22, 23, 24, 25]. We portray the scheme of the fiber photometry system (Fig. 2a). In brief, a high-power LED driver (LEDD1B/M470F3, Thorlabs Inc., Newton, NJ, USA) generates anexcitation blue light (470nm, 0.5mW at the tip of the silica fiber) or yellow light (590nm) and the blue or yellow lights pass the excitation bandpass filter (blue light: pass 475±12.5nm; yellow light; 590 ± 12.5nm) and are reflected by a dichroic mirror-1 and joined into the single silica fiber (diameter: 400µm, numerical aperture = 0.6). Blue/yellow lights emitted from the tip of the silica fiber reflect G-CaMP6/mCherry fluorescent proteins andgreen/red fluorescence signalsare detected and transmitted to the same tube. The signals pass the dichroic mirror-1 and are reflected by a dichroic mirror-2 and pass the bandpass emission filter (green: pass 510±12.5nm; red: 607±12.5nm). At the endof the line, the signalsare guided to a photomultiplier tube (PMT) (PMTH-S1-1P28, Zolix Instruments, Beijing, China). The signals were digitized by an A/D converter (PowerLab8/35, ADInstruments Inc., Dunedin, New Zealand) and recorded by Labchart version-7 software (ADInstruments Inc.).
In this study, we divided experimental mice into three groups: the A5, A7, and B2 groups. Fourteen days prior torecording, AAV was unilaterally injected into A5/A7 regions in DBH-tTA mice and B2 regions in TPH2-tTA mice(Fig. 1c). Each mouse was individually kept in normal breeding conditions for 14 days after the operation. In this study, we recorded the G-CaMP6/mCherry green/red fluorescence intensityof A5/A7 NA neuronal cell bodies and B2 5-HT neuronal cell bodiesin response to acute nociceptive stimuli. Each of the four stimuli was presentedonce to each mouse(Fig. 2b). We applied an acute tail pinch stimulus at aforce of 400 gusing a pinch meter (PM-201, Soshin-Medic, Chiba, Japan) and an acute heat stimulus at a temperature of 55°Cusing a heating probe (5R7-570, Oven Industries Inc., Mechanicsburg, PA, USA) as per previous reports [23, 25]. As noninvasive control stimuli, we applied a gentle touch using a cotton stick and a low temperature stimulus corresponding to a temperature of 25℃ using the same heating probe (5R7-570, Oven Industries Inc.).
We carried out the recording according to the following procedure. Each mouse was anesthetized with 2–3% isoflurane using a vaporizer and fixed with a stereotaxic instrument (ST-7, Narishige)using a supportive ear bar (EB-6, Narishige). Surfaces of the ears werecovered with a local anesthetic jelly (lidocaine, 2% xylocaine) to minimize suffering. We then carried out the silica fiber implantation operation. The silica fiber was placed just above the A5 site (bregma -5.52mm, lateral +1.4mm, and ventral -5.30mm from the cranium), A7 site (bregma -4.96mm, lateral +1.88 mm, and ventral -3.10 mm from the surface of the brain) and B2 site (bregma -7.56mm, and ventral -3.93mm from the cranium). We implanted the silica fiber slowly while monitoring the fluorescence signal intensity and confirmed that the fluorescence intensity increased rapidly when the optical position approached the target sites(Fig. 2c). After implantation, we ceased the administration of anesthesia. We waited for two hours after ceasinganesthesia until the first stimulus to reduce any possible effects of anesthesia. To reduce any possible effects of a previous stimulus, we set the inter-stimulus interval to30 minutes and the order of stimuli as follows: the first was thelow heat stimulus (25℃), the second was thegentle touch, the thirdwas the heat stimulus(55℃) and the last was thepinch stimulus(Fig. 2b).We usedthe pinch meter (PM-201, Soshin-Medic) for the tail pinch stimulus and attached the apparatus to the root of the tail for three seconds with a force of 400g.We alsoused aheating probe (5R7-570, Oven Industries, Inc.) for the heat stimulus set to55°C and attached the probe to the root of the tail for three seconds.For noninvasive control stimuli, we used alow heat stimulus using the same heat probe by touching the root of the tail for three seconds and by gentlytouching the mouse using a cotton stick at the root of the tail for three seconds.
We defined the neuronal activity characteristic index as follows: F: averaged fluorescence signal intensity value for three seconds immediately prior toeach stimulus and defined as 100%; ΔF: maximum fluorescence signal intensity value during each stimulus – F; onset latency: time from the start of the stimulus to the time when the fluorescence signal intensity exceeded the maximum value during the baseline period; peak latency: time from the start of the stimulus to the maximum peak signal intensity.
Statisticalanalyses were conductedusing atwo-way analysis of variance (ANOVA) with theSidak’s test for post hoc analyses. The twofactors ofΔF/F were the stimulus style (mechanical vs thermal) and stimulus intensity (nociceptive vs control). The twofactors ofonset/peak latency were the stimulus style (pinch vs heat) and target sites (A5 vs A7 vs B2).Data values wereexpressed as the mean ± standard error of the mean (S.E.M). Probability values of p< 0.05 were considered statically significant. Analyses were performed using GraphPad Prism version 7 (GraphPad software, San Diego, CA, USA).