神经损伤与修复

    Involvement of A5/A7 noradrenergic neurons and B2 serotonergic neurons in nociceptive processing: a fiber photometry study
  • Figure 1|NA or 5-HT neuron-specific expression patterns of G-CaMP6 or mCherry fluorescent proteins adopting the Tet system. 

    We used transgenic mice carrying tetracycline-controlled transactivator transgene (tTA) under the control of a dopamine β-hydroxylase (DBH) (24.0 ± 1.0 g, n = 12) or a tryptophan hydroxylase-2 (TPH2) promoters (23.8 ± 1.1 g, n = 6) (DBH-tTA mice or TPH2-tTA mice, respectively, from Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan) (Moriya et al., 2018, 2019, 2020a) (Figure 1A); 10–14-week-old male mice were used in this experiment). Rearing was carried out in standard conditions, with lights on at 7:00 a.m. and off at 7:00 p.m., a temperature of 24 ± 1°C, and food and water available ad libitum. We made efforts to minimize animal suffering and reduce the number of animals used. All experimental procedures were performed following 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) on February 22, 2018. 
    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 (Inutsuka et al., 2016). AAV-TetO(3G)-G-CaMP6 (Serotype: DJ; 0.3 μL/injection, 4 × 1013 copies/mL) and AAV-Tet(3G)-mCherry (Serotype: DJ; 0.3 μL/injection, 6 × 1012 copies/mL) were produced using the Tet system (Figure 1B). The specific sequences of AAV vectors are provided (Additional file 1). We slowly siphoned AAV into a glass micropipette (1B150F-3, World Precision Instruments, Inc., Sarasota, FL, USA) connected to an injection manipulator (I-200J, Narishige, Tokyo, Japan) linked to a nitrogen pressure source through a polyethylene tubing. Under the influence of 2–3% of isoflurane (Pfizer Inc., New York, NY, USA) as the inhalation anesthetic, the mice were fixed with a stereotaxic instrument (ST-7, Narishige) with the help of a supportive ear bar (EB-6, Narishige). To minimize suffering, the surfaces of their ears were covered with a local anesthetic jelly (lidocaine, 2% xylocaine [Fujifilm Wako Pure Chemical Inc., Osaka, Japan]) and both eyes were preserved with Vaseline. Head hair was shaved using an electric shaver and the cranial dura mater was cut open with small scissors. We chose target coordinates according to Paxinos and Flanklin’s the Mouse Brain in Stereotaxic Coordinates, Fifth Edition (Paxinos and Franklin, 2019). AAV was unilaterally injected into the target sites (A5: bregma –5.52 mm, lateral +1.4 mm, and ventral –5.30 mm from the cranium; A7: bregma –4.96 mm, lateral +1.88 mm, and ventral –3.10 mm from the surface of the brain; B2: bregma –7.56 mm and ventral –3.93 mm from the cranium) (Figure 1C) and the glass microtube was left in the sites for 10 minutes before being withdrawn. Postoperative antibiotic administration was carried out by subcutaneous injection (penicillin G: 40,000 U/kg) to prevent postoperative infections. After operation, the mice were maintained at standard rearing conditions (as described above) for 14 days to recover from the surgery and to allow the G-CaMP6/mCherry fluorescent protein to be fully expressed before the experimental sessions

    Figure 2|Experimental procedures. 

    In this study, we used the same fiber photometry system with two channels that was used in previous studies (Moriya et al., 2018, 2019, 2020a, b). The scheme of the fiber photometry system is illustrated in Figure 2A. In brief, a high-power LED driver (LEDD1B/M470F3, Thorlabs Inc., Newton, NJ, USA) generated an excitation blue light (470 nm) or yellow light (590 nm) of power 0.1 mW at the tip of the silica fiber; the blue or yellow light passed the excitation bandpass filter (passband—blue light: 475 ± 12.5 nm; yellow light: 590 ± 12.5 nm), was reflected by a dichroic mirror-1, and joined the single silica fiber (diameter: 400 μm; numerical aperture: 0.6). The blue/yellow light emitted from the tip of the silica fiber reflected the G-CaMP6/mCherry fluorescent proteins; the reflected green/red fluorescence signals were detected and transmitted to the same tube. The signals passed the dichroic mirror-1, were reflected by dichroic mirror-2, and passed the bandpass emission filter (passband—green: 510 ± 12.5 nm; red: 607 ± 12.5 nm). Finally, the signals were guided to a photomultiplier tube (PMTH-S1-1P28, Zolix Instruments, Beijing, China). The signals were digitized by an A/D converter (PowerLab8/35, AD Instruments Inc., Dunedin, New Zealand) and recorded with the LabChart version-7 software (AD Instruments Inc.). 
    In this study, we divided the experimental mice into three groups: A5, A7, and B2 groups. Fourteen days prior to recording, AAV was unilaterally injected into the A5/A7 regions in DBH-tTA mice and B2 region in TPH2-tTA mice (Figure 1C). Each mouse was individually maintained in standard breeding conditions for 14 days after the operation. In this study, we recorded the G-CaMP6/mCherry green/red fluorescence intensities of A5/A7 NA and B2 5-HT neuronal cell bodies in response to acute nociceptive stimuli. 
    We carried out the recording according to the following procedure. Each mouse was anesthetized using 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 were smeared 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 –5.52 mm, lateral +1.4 mm, and ventral –5.30 mm from the cranium), A7 site (bregma –4.96 mm, lateral +1.88 mm, and ventral –3.10 mm from the surface of the brain), and B2 site (bregma –7.56 mm and ventral –3.93 mm from the cranium). The coordinates were confirmed according to Paxinos and Flanklin’s the Mouse Brain in Stereotaxic Coordinates, Fifth edition (Paxinos and Franklin, 2019). We implanted the silica fiber slowly while monitoring the fluorescence signal intensity; the fluorescence intensity was increased rapidly when the optical position approached the target sites (Figure 2B). After implantation, we stopped administering anesthesia. We waited for 2 hours after ceasing anesthesia for the first stimulus when the possible effects of anesthesia were reduced. To reduce any possible effects of a previous stimulus, we set the inter-stimulus interval to 30 minutes; the order of stimuli was as follows: first was the low heat stimulus (25°C), second gentle touch, third the heat stimulus (55°C), and last the pinch stimulus (Figure 2C). We used the pinch meter (PM-201, Soshin-Medic, Chiba, Japan) for the tail pinch stimulus and attached the apparatus to the root of the tail for three seconds with a force of 400 × g. We also used a heating probe (5R7-570, Oven Industries, Inc., Mechanicsburg, PA, USA), for the heat stimulus, set at 55°C, and attached the probe to the root of the tail for three seconds. For non-noxious control stimuli, we used the same heat probe for the low heat stimulus and a cotton stick for the gentle touch; these stimuli were attached to the root of the tail for three seconds.


    Figure 3|Expression of AAV-induced G-CaMP6/mCherry. 

    The specific expression patterns of G-CaMP6/mCherry were confirmed in the A5/A7 NA (Figure 3A and B) and B2 5-HT neuronal soma (Figure 3C). G-CaMP6/mCherry-positive neurons were rarely observed outside the A5, A7, and B2 areas. TH-positive cells were found in the A5 and A7 areas. A large proportion of them expressed G-CaMP6 (A5 area: 90.6%, A7 area: 90.9%; n = 6, each; Figure 3D and E). All G-CaMP6 positive cells expressed mCherry, and a large proportion of them expressed TH (A5 area: 97.0%, A7 area: 85.7%; Figure 3D and E). TPH-positive cells were found in the B2 area. A large proportion of them expressed G-CaMP6 (91.8%; n = 6; Figure 3F), and all G-CaMP6-positive cells expressed mCherry, while 94.3% expressed TPH (Figure 3F). Therefore, green/red fluorescence was confirmed to be derived from specific A5/A7/B2 sites. 

    Figure 4|Averaged traces of G-CaMP6/mCherry (green/red) fluorescence intensity in response to stimuli.  

    We histologically confirmed that the fiber was properly positioned (Additional Figure 1). G-CaMP6/mCherry fluorescence intensity was abruptly increased when the optical position was just above the target sites (Figure 2C). Figure 4 shows the averaged traces of G-CaMP6/mCherry fluorescence intensities in response to acute nociceptive stimuli or a non-noxious control test (n = 6, each). ANOVA revealed that the G-CaMP6 fluorescence intensity was significantly different across different stimulus intensities (A5 group: pinch vs. touch: F(1, 5) = 36.84, P = 0.0018, heat vs. low heat: F(1, 5) = 24.28, P = 0.0044; A7 group: pinch vs. touch: F(1, 5) = 12.36, P = 0.0170, heat vs low heat: F(1, 5) = 11.48, P = 0.0195; B2 group: pinch vs touch: F(1, 5) = 50.10, P = 0.0009, heat vs. low heat: F(1, 5) = 23.81, P = 0.0046; Figure 5A). ANOVA revealed no significant differences in onset/peak latency values across stimulus intensities (onset latency: pinch vs. touch: F(2, 10) = 0.3465, P = 0.7153; heat vs low heat: F(2, 10) = 0.1266, P = 0.8825; peak latency: pinch vs. touch: F(2, 10) = 0.4141, P = 0.6718; heat vs. low heat: F(2, 10) = 0.1148, P = 0.8927). ANOVA revealed that the mCherry fluorescence intensity was not significantly different across stimulus intensities (A5 group: pinch vs. touch: F(1, 5) = 0.3742, P = 0.5675, heat vs. low heat: F(1, 5) = 1.0350, P = 0.3557; A7 group: pinch vs. touch: F(1, 5) = 0.3585, P = 0.5755, heat vs. low heat: F(1, 5) = 0.0225, P = 0.8867; B2 group: pinch vs. touch: F(1, 5) = 0.0014, P = 0.9719, heat vs. low heat: F(1, 5) = 0.3598, P = 0.5747).


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  • 发布日期: 2021-10-18  浏览: 500
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