Animals
The tryptophan hydroxylase-2 tetracycline-controlled transactivator (TPH2-tTA) transgenic mice were used [18, 23, 24] (Fig. 1a). We have already confirmed the specificity of TPH2-tTA expression in the previous report [24]. Ten to fourteen-week-old mice were used in this study. All mice were kept on a condition of 12 h light/dark cycle (7:00 AM to 7:00 PM), the temperature of 24 ± 1 °C, food and water were available ad libitum. All efforts were made to minimize animal suffering and discomfort; to reduce the number of animals used. All experimental procedures were performed 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 (MD17090).
Stereotaxic AAV injection
AAV vector production was performed by AAV Helper-Free system (Agilent Technologies, Inc., Santa Clara, CA, USA); their purification was as previously described [17, 25, 26]. Mice were anesthetized with 2–3% isoflurane using a vaporizer for small animals and fixed with stereotaxic instrument (ST-7, Narishige, Tokyo, Japan) with an aid of supportive ear bar (EB-6, Narishige) of which touching surfaces to the animal were covered with local anesthetic jelly (lidocaine, 2% Xylocaine AstraZeneca). Both eyes were preserved with vaseline, head hair was shaved using an electric hair shaver, and cranial dura mater was cut open with small scissors. We slowly sucked up AAV into a glass micropipette (1B150F-3, World Precision Instrument, Inc., Sarasota, FL, USA), which was connected to a nitrogen pressure source through polyethylene tubing and to an injection manipulator (I-200 J, Narishige). In this study, AAV-TetO(3G)-G-CaMP6 (Serotype: DJ; 1 μl/injection, 4 × 1013 copies/ml) and AAV-TetO (3G)-mCherry (Serotype: DJ; 1 μl/injection, 6 × 1012 copies/ml) (Ohkura et al., 2012) (Fig. 1a) were unilaterally injected into B9 site (Injection site was from bregma − 4.36 mm, lateral + 0.38 mm left side, and ventral − 5.08 mm from the cranium) (Fig. 1b). After AAV injection, the micropipette was left in place for 10 min before being slowly withdrawn; mice were given an antibiotic, penicillin G (40,000 U kg-1) through subcutaneous injection. After operation, each mouse was individually kept for 14 days (2 weeks) in normal breeding conditions (as mentioned in Animals section) because it is needed for mice to recover and it takes about 2 weeks for G-CaMP6 or mCherry to fully express (Fig. 1a).
In vivo fiber photometry system
We showed fiber photometry system in previous reports [17, 18, 25, 26]. In this study, we adopted the fiber photometry system with two channels (Fig. 2a). In the first channel setting, the high-power LED driver (LEDD1B/M470F3, Thorlabs, Inc., Newton, NJ, USA) continuously produces blue excitation light (470 nm, 0.5 mW at the tip of the silica fiber) and the light passes through the excitation bandpass filter (475 ± 12.5 nm) and reflected by dichroic mirror-1; into a silica fiber (diameter: 400 μm, numerical aperture = 0.6). The same fiber detects and collects the green fluorescent signal of G-CaMP6. The signal passes through dichroic mirror-1 and reflected by dichroic mirror-2 and passes through the bandpass emission filter (510 ± 12.5 nm) and guided to a photomultiplier tube (PMTH-S1-1P28, Zolix Instrument, Beijing, China). At the second channel setting, the high-power LED driver continuously produces yellow excitation light (590 nm) and the light passes through the excitation bandpass filter (590 ± 12.5 nm) and goes forward as well. The same fiber detects and collects the red fluorescent signal of mCherry. The signal was forward and passed through the bandpass emission filter (607 ± 12.5 nm) and guided to another photomultiplier tube. First channel was adopted for detecting the neuronal activity and the second channel was used as an indicator of total stability of the fiber photometry system because mCherry fluorescence doesn’t reflect neuronal activity [26]. Both signals were digitized by an A/D converter (PowerLab8/35, ADInstruments Inc., Dunedin, New Zealand) and recorded by Labchart version-7 software (ADInstruments Inc.). Signals were collected at a sampling frequency of 100 Hz.
Immunohistochemistry
To confirm AAV-induced expression of G-CaMP6 and mCherry in 5-HT neurons, after the experiments, mice were processed for immunostaining. Mice were deeply anesthetized with urethane (1.6 g/kg, i.p.) and transcardially perfused with 20 ml of phosphate buffered saline (PBS) and 20 ml of 4%-paraformaldehyde in PBS (Nacalai Tesque Inc., Kyoto, Japan). The brain was removed and post fixed in the same paraformaldehyde solution and soaked in 30% sucrose in PBS for 2 days. We formed serial 30 μm coronal sections including target sites (B9, LC, and VTA) with the cryostat (Cryotome FSE, Thermo Scientific, Yokohama, Japan). Every third section was adopted and floating immunohistochemical staining was performed. The sections were soaked in blocking solution (PBS containing 1% normal horse serum and 0.3% Triton-X) for 1 h at room temperature and incubated with anti-TPH antibody (AB1541, raised in sheep, EMD Millipore Corp., 1:1000) or anti-serotonin transporter (SERT) antibody (HTT-GP Af1400, raised in guinea pig, Frontier Institute, Hokkaido, Japan, 1:200) in blocking solution for overnight. The next day, the sections were washed three times with PBS and incubated with CF647 donkey anti-sheep IgG (20,284, Biotium, Inc., Fremont, CA, USA, 1:200) or CF647 donkey anti-guinea pig IgG (20,837, Biotium, 1:200) in PBS for 2 h in a dark box. In some sections in LC, VTA, B9 were treated with anti-tyrosine hydroxylase (TH) antibody (AB152, Millipore, raised in rabbit, 1:500) and visualized with CF647 donkey anti-rabbit IgG (20,047, Biotium).
After incubation, the sections were washed once with PBS and mounted on microscope slides (PRO-02, Matsunami, Osaka, Japan) and covered with microcover glasses (C024601, Matsunami). We observed and imaged the sections under the fluorescence microscope (BZ-X700, Keyence, Osaka, Japan), and analyzed the images with Adobe Photoshop CC (Adobe Systems, Inc., San Jose, CA, USA). G-CaMP6 and mCherry were visible without immunostaining.
Acute nociceptive test
We applied two types of acute stress test showed in previous reports [17, 18]. We applied acute tail pinch stimulus using a pinch meter (PM-201, Soshin-Medic, Chiba, Japan) and acute heat stimulus using a heating probe (5R7–570, Oven Industries, Inc., Mechanicsburg, PA, USA). Pinch stimulus was attached to the root of the tail for three seconds with a force of 400 g and heat probe set at 55 °C was attached to the root of the tail for three seconds. We applied two noninvasive stimuli in the control group: gentle touch using a cotton stick and low temperature heat stimulus set at 25 °C using the same heating probe (5R7–570).
Experimental protocol
In this study, we recorded G-CaMP6/mCherry green/red fluorescence intensity of B9 5-HT neuronal cell body and the axon located in LC/VTA (projecting sites of B9 5-HT neurons, Fig. 2b) to acute nociceptive stimuli. Each mouse was individually kept for at least 14 days after injection of AAV (Fig. 1a). The mouse was again anesthetized with 2–3% isoflurane using a vaporizer for small animals and fixed with stereotaxic instrument (ST-7) with an aid of supportive bar (EB-6) of which touching surfaces to the animal was covered with local anesthetic jelly (AstraZeneca). Following experiments were conducted in head-fixed condition. The head hair was shaved using electrical hair shaver; cranial dura mater was cut open with small scissors. A silica fiber was slowly implanted into the places just above B9 (bregma − 4.36 mm, lateral + 0.38 mm left, and ventral − 5.08 mm from the cranium), LC (bregma − 5.34 mm, lateral + 0.80 mm left, and ventral − 2.60 mm from the surface of the brain) and VTA (bregma − 3.15 mm, lateral + 0.50 mm left, and ventral − 4.15 mm from the surface of the brain) (Fig. 2b). We monitored the fluorescence signal intensity throughout fiber implantation and confirmed that the fluorescence signal intensity increased abruptly when the optimal position of the fiber tip was placed just above the target site (Fig. 2c). Open space around the optic fiber was covered with an ointment to avoid possible drying. After the fiber was fixed and positioned to the optimal position, anesthesia was turned off; each mouse recovered from anesthesia. We set 3 h before the beginning of experimental sessions so that anesthesia would not affect experimental sessions. The mice were divided into three groups; B9 group (n = 6), LC group (n = 6) and VTA group (n = 6). Experimental sessions of each mouse consist of two kinds of acute nociceptive stimuli and two kinds of noninvasive control stimuli (total four stimuli). To reduce the effect from the previous stimulus, we set inter-stimulus intervals at 30 min; their stimuli were set in order of weaker to stronger stimulus as follows; the first is low temperature heat stimulus at 25 °C, the second is a gentle touch, the third is acute heat stimulus at 55 °C; the last is acute mechanical tail pinch stimulus at the force of 400 g (Fig. 2d). After experiments, mice were euthanized and processed for immunostaining.
The definition of neuronal activity characteristic index was set as follows: F: averaged fluorescent signal intensity value for three seconds just before each stimulus and defined as 100%; ΔF: (maximum fluorescent signal intensity value during each stimulus) – F; onset latency: time from the start of stimulus to the time when the fluorescence signal intensity exceeded the maximum value during the baseline period; peak latency: time from the start of stimulus to the time when the fluorescence signal intensity reach the maximum value.
Statistical analysis
Data analysis was conducted by two-way analysis of variance (ANOVA) with Sidak’s test for post hoc analysis. Two factors on ∆F/F were modality (mechanical vs. thermal) and intensity (nociceptive vs. gentle control). Factors on latency were modality (mechanical vs. thermal) and brain area (B9, LC, or VTA). Values are expressed as the mean ± standard error of the mean (S.E.M). Probability values less than (p < 0.05) were considered statistically significant. The analyses were performed using GraphPad Prism version 7 (GraphPad software, San Diego, CA, USA).