the integrative role of orexin/hypocretin neurons in nociceptive perception and analgesic regulation
Induced Analgesia supports the survival of animals through rapid defense responses to predators or competitors.
Studies have shown that orexin peptides have an analgesic effect.
However, orexin neurons contain not only orexin, but also other
Nervous transmission factors such as strong nor, neurotensin and monosodium glutamate.
Therefore, the physiological importance of orexin neuron activity in injury perception is unknown.
Here we show the adults
Stage Selective Ablation of Orexin neurons enhances pain
The related behavior, while the drug genetic activation of orexin neurons induced pain relief.
In addition, we found that during the injury sensation, the optical fiber metering recording of orexin neurons in conscious animals was used, and the related activation of orexin neurons.
These findings suggest an integrated role of orexin neurons in the regulation of injury sensation and pain.
All experimental procedures involving animals are approved by the Institute of Environmental Medicine, Nagoya University, Committee on Animal Care and Use.
All experiments were conducted in accordance with the regulations on animal facilities at Nagoya University.
Mice are maintained under a strict 12-hour light/dark cycle (
Lamp period: 8: 00-20: 00;
Dark Period: 20: 00-8: 00)
Control Room (22u2009°C).
Food and water are supplied at any time, and every effort is made to reduce the pain and discomfort of animals and to reduce the number of animals used.
Mice expressing the cross-activation factor of chlorin (tTA)
Orexin neurons only under the control of the human pre-Reproductive system
Orexin promoter bred in mice (B6. Cg-Tg(tetO DTA)
Jackson Lab 1Gfi/J)
In these double-genetically modified mice, there is no (DOX).
Both mice and mice are in the genetic background. DOX-
Including weeks (DOX chow)
By adding 10% ethanol powder (
Normal week (Mr. Niang-StokeNosan, Japan)
The final concentration was 100 mg/kg.
Mating pairs of mice and mice fed with DOX
Including weeks (DOX(+)condition)
From the date of mating
Before and early postpartum, ethanol was supplied through maternal blood circulation or lactation, respectively.
Feeding DOX to mice after weaning (+)
Until the day of the experiment.
Start ablation, DOX (+)
Chow was replaced by Mr. stock (DOX(−)condition).
Sensitivity to mechanical stimulation using self-testing
I made von fridges.
Place the mice individually in a plastic container with perforated metal floors (
Varese Ugo Basile 37450, Italy)
, And adapt to at least 30 minutes before testing.
10 Silk Series (
Bending force 0. 2~6. 8u2009g in quasi-
Logarithmic order, 0.
Applied to the middle surface of the foot of the left hind legpaw.
We first apply the weakest filament, then apply the filament twice every 1 minute.
The threshold was determined to be the minimum force that caused at least one paw explicit exit reaction in two trials.
Thermal sensitivity to harmful heat was tested using Hargreaves equipment (
Varese Ugo Basile 7371, Italy).
30 minutes before the experiment, put the mouse into a separate plastic cage with a glass floor.
Exit delay in response to infrared beam measurement (Strength: IR40)
Applied to the foot surface of the left hind leg-paw. The cut-
In order to avoid heat damage to the tissue, the turn off delay is set to 20 m/s.
The damage perception threshold of cold stimulation was measured by cold plate test.
Place the mouse on a cold stainless steel plate surrounded by transparent plexiglass (12x20x10 cm).
By circulating water containing 20% ethylene glycol under the plate, the temperature of the cold plate is continuously monitored and maintained at-5 °c. Pain-
Hind-and other related acts
Observe and video the lifting, licking and jumping of the claws.
Total duration of pain
Relevant behavior was measured during the observation period of 3 min.
After the domestication period of at least 30 min, 10 μm μ l 5% formaldehyde was dissolved in 0.
Subcutaneous injection of 01 µm PBS onto the plantar surface of the left hind leg-paw with a 30-gauge needle.
Put the mouse back in the plastic box immediately after injection.
Total duration of pain
Related Behavior (i. e.
Lick and bite after injection-paw)
Measured every 5 minutes after injection, up to 50 minutes.
We identified and analyzed the two stages of pain.
Related Behaviors: initial acute phase (
Stage 1, 0-5 min after injection)
Followed by a long period of replenishment (
Phase II, 10-45 min after injection).
All AAV carriers are generated using the AAV assistantFree System (
Agilent Technology Co. , Ltd.
Santa Clara, California, USA)
And purified according to the published method.
To put it simply, a pAAV vector containing the genes of interest, pHelper and pAAV-was used to transfer the cells intoRC (serotype DJ;
Buy from Cell Biolabs Inc. in San Diego, California, USA)
The standard calcium phosphate method is used.
After 3 days, the cells were collected and suspended in the artificial spinal fluid (aCSF;
Report of 124 mM sodium chloride, potassium chloride 3 mM, 26 mM sodium bicarbonate, 2 mM calcium chloride, 1 mM magnesium sulfate and final accounts.
KHPO 25mm, D-10mmGlucose). After 4 freeze-
After thawing the cycle, the cell lysate was treated with benzonase nuclease (
Damstadt Merck, Germany)
Centrifuge for 15 minutes at 45 °c, 2 times at 16,000 °c, and 10 minutes.
Cultured cells are used as viruses
To determine the titer of the purified virus, the supernatant was dissolved in artificial CSF.
Quantitative PCR was performed using the following primer pairs to measure virus titer: a post-transcription regulator of the groundhog hepatitis virus (WPRE), WPRE-Forward: 5′-
Actgtttgctgaccaac-3′, WPRE-Reverse: 5′-
PolyA, human growth hormoneForward: 5′-
3 \', polyA, human growth hormone-Reverse: 5′-
The AAV carrier is stored at-80 °c with a small equal sample until the day of the experiment. The pAAV-hSyn-FLEX-hM3Dq-
Vector mCherry purchased from Addgene (ID: 44361).
AA v injection surgery under anesthesia (50u2009mg/kg, i. p. )
And isofluoride (2%, inhalation)
Using a stereo instrument (
David Kopf instruments, Tujunga, California, United States of America).
Restructuring AAVhSyn-FLEX-hM3Dq-mCherry (serotype: DJ;
600nl nl/injection, 3 µx 10 copies/ml)
Stereo orientation and bilateral injection into LHA in mice.
Glass micromobile with pull pul (
Novato, CA, USA, Passat instrument)
The diameter of the tip filled with AAV is 100 μm.
Air jet system (
World precision instruments
Sarasota, Florida, USA)
Connect with polyethylene tube to glass microtransfer tube. Air pressure (10–20u2009psi)
For injection of AAV.
The injection site is as follows: from bregma-1.
4 u2009 mm lateral u2009 ± u2009 0.
7mm, abdomen-5. 0u2009mm for AAV-hSyn-FLEX-hM3Dq-mCherry.
After three weeks of AA v injection, the mice were tested for behavior.
Deep anesthesia of mice with infusions, heart perfusion with 20 ml of frozen saline, and then 10% formaldehyde solution with 20 ml of frozen (
Weike Pure Chemical Co. , Ltd. , Osaka, Japan).
The brain is removed.
Fix overnight in 10% formaldehyde solution at 4 °c and immerse in 30% sucrose for at least 2 days in PBS at 4 °c.
Obtain a series of sections of 40 μm with a low temperature thermostat (Leica CM3050 S;
Wetzlar Leica Microsystems, Germany).
For staining, immerse the coronary brain slices in the blocking buffer (1% BSA and 0. 25% Triton-X in PBS)
And then incubated overnight with one antibody at 4 °c.
Wash the slices with closed buffer, and then incubate with secondary antibody for 1 hour at RT.
Install and examine brain slices with a fluorescence microscope (BZ-
9000, Keyence, Osaka, Japan or IX71, Olympus, Tokyo, Japan).
Dilute the primary and secondary antibodies in the closed buffer as shown below:
Goat antibody orexin (
Santa Cruz Biotechnology Co. , Ltd.
Dallas, Texas, USA)
In 1:2000, reverse
Rabbit antibody in Maternal and Child Health HospitalSigma-Aldrich, St. Louis, MO, USA)
In 1:2000, reverse
Antibody to Prodynorphin guinea pig
Empirical mode memory wave of damstadt, Germany)
CF488-1 1000Combinedmouse or anti-
Rabbit antibody (Biotium Inc.
Hayward, California, USA)
CF594-1 1000Combinedrabbit or anti-goat antibody (Biotium)
At 1:1000 and CF647-Combinedgoat antibody (Biotium)at 1:1000.
We did not use the daffodil to enhance the peptide signal.
We used NIH image j software to manually count positive cells and calculate the relative percentage of positive neurons in DOX (−)
The average number of mice by using DOX-positive neurons (+)
Mouse as a reference.
The number of absolute cell counts is as follows: orexin in DOX (+)
: 648 Vivo ± 47 47 cells, in DOX (−)
: Maternal and Child Health Hospital of 8 ÷ ± 4. 1. 4 cells (+)
: 1108 Vivo ± 9 9 cells, in DOX (−)
: 1153 cells, strong cells (LHA)in DOX(+)
: 495 ÷ ± 32 32 cells in DOX (−)
: 135 cells, strong cells (CeA)in DOX(+)
: 189 ÷ ± 15 cells, in DOX (−)
: 198 cells.
To count, we analyzed one of every four coronary brain slices.
Both male and female mice, 3-8 months old, were used for electrophysiological recording and calcium imaging.
These mice were injected with AAV-TetO(3G)G-CaMP6 (serotype: DJ;
Price: 1 × 10 copies/ml; volume: 1. 2u2009μl/injection)in the LHA (from bregma −1.
4 u2009 mm lateral u2009 ± u2009 0.
8mm, abdomen-5. 0u2009mm).
At least three weeks later.
After the injection, the mouse was deeply anesthesia with infusions and the head was broken.
Then, the brain is quickly isolated and frozen in the ice.
Cold Cut liquid (in mM: 110 K-
Glucose acid, 15-1 potassium chloride, 0.
05 EGTA, 5 HEPES, 26.
2 NaHCO, 25 glucose, 3. 3 MgCl and 0. 0015 (±)-3-(2-
Bubbling with 95% O and 5% CO
Use the vibration group to cut the brain into a coronary slice with a thickness of 300 μm (VTA-1200S; Leica, Germany)
Transfer the slices containing the LHA region to the incubation room containing bath liquid (
In mM: 124 of nac1, 3 of potassium chloride, 2 of MgCl, 2 of CaCl, 1.
23 NaHPO, 26 NaHCO, 25 glucose)
Bubble for 30 minutes in a water bath at 35 °c with 95% O and 5% CO.
The brain slices were then incubated at room temperature for 30-60 minutes in the same chamber.
The solution was modified based on the Presler method. .
Transfer brain slices to the recording room (RC-27L;
Warner instruments, USA)
On the fluorescent microscope (BX51WI;
Olympus, Tokyo, Japan)
Super Fusion with bubbling (95% O and 5% CO)
The Bath rate of 0.
8 ml/minute use of the worm pump (
Dynamax in Rainin, United States).
Infrared camera (C2741-79;
Hamamatsu, Hamamatsu, Japan)
Installed in a fluorescent microscope with charge
Camera equipment (
Evolution 512 Delta;
American photographic survey
The two images are displayed on the display separately.
A trace liquid transfer device (P-1000;
For the preparation of patch liquid transfer tubes (GD150-
Harvard instruments, USA
5-8mm Ω resistor.
A kind of potassium is filled in the patch pipettes-
Based on internal solutions (
Participate in mM: 145 kg 1 MgCl, 10 HEPES, 1.
1 EGTA, 2 MgATP, 0. 5 NaGTP; pH 7. 3 with KOH)
The penetration concentration of the solution was confirmed to be 280-290 mOsm.
Identification of orexin neurons by green fluorescence of G-CaMP6.
Positive pressure was introduced in the patch transfer tube and moved to the cell.
After touching the battery, the pressure is released and giga is emitted-seal was made.
Repair the rupture of the membrane by suction to form a whole
Monitoring membrane potential with Axopatch 200B amplifier (
Axon Instruments, USA).
Orexin neurons are hyperpolarized by an amplifier through negative current injection of about 70-80 mv to stop spontaneous discharge.
In order to produce a faithful ignition, a depolarized rectangular current of about 50 pa, 10 mmsec in width, 5, 10, 20, and 50 hz was applied using an electrical stimulusSEN-3301;
And applied to the cell by recording the transfer tube.
Low output signal-
Filtered at 5 khz, digitized at a 10 khz sampling rate.
The patch data was recorded through simulationto-
Digital converter (Digidata 1322A;
American Molecular Devices, Axon Instruments)
Use pClamp month. 2 software (
American Molecular Devices.
Transfer brain slices to the recording room to identify orexin neurons with G-green fluorescenceCaMP6.
Super Fusion of slices and Bubbles (95% O and 5% CO)
The bath is at a speed of 0. 8u2009ml/min.
Excitation light of G-
CaMP6 is emitted from a light source (
Spectral light engine;
Beaverton or lumense in the United States)
Controlled by Metamorph software (
Molecular Devices, Sunnyvale, California, USA).
Guide the light to the microscope with a liquid fiber with a diameter of 1 cm.
The brain was irradiated with a blue light of 475 u2009 ± 17.
Wavelength of 5 nm and 9.
7 mw power was obtained by objective lens of fluorescence microscope. G-
The CaMP6 fluorescence intensity was continuously recorded at 2 hz using the Metamorph software with an exposure time of 100 mmsec.
To synchronize calcium imaging and patch clamp recording, the pClamp software is triggered by the TTL output of the Metamorph software.
By setting the area of interest (ROI)on G-
CaMP6 and Δf/F expressing orexin neurons are calculated based on the average intensity of ROI.
Optical fiber metering system (COME2-
Japan tsukubo Lucille FTR/OPT)
Used to record the activity of orexin neurons in conscious mice.
The system provides excitation light using a single quartz fiber and detects from G-
Stimulate blue light (465u2009nm, 0.
5 mw quartz fiber tip)
Is by a high
Power LED System (
PlexonBright OPT/LED blue _ tt_fc, Plexon, Dallas, TX, United States of America).
The excitation blue light emitted from the LED is reflected by a two-color mirror and coupled to a 400 u2009 μm quartz fiber via an excitation pass filter (
Path 472 u2009 ± u2009 35 u2009 nm). G-
The CaMP6 fluorescence is collected and guided to the photoelectric multiplication tube by the same silica fiber (PMTH-S1M1-
CR131, zulix instrument company, Beijing, China)
Transmit the filter through the pass band (
Path 525 u2009 ± u2009 25 u2009 nm).
Digitize the signal using A/D converter (Micro1401)
And record through Spike2 software (
Cambridge Electronic Design Co. , ltd. uk).
The signal is collected at a sampling frequency of 100 hz, and the software samples every 10 on average to minimize fluctuations and noise.
In order to transfer the blue light to orexin-
Neurons, implanted silicon fiber into LHA.
At the beginning of the operation, the mouse was anesthesia (2. 5%, inhalation)
And placed on the stereo positioning frame of small animals (
David Kopf 963).
Restructuring AAVTetO (3G)G-CaMP6 (
S. swine disease: DJ, 600 µnl/injection molding, 7*10 Copy/ml)
Injection of LHA in the single aspect (from bregma −1.
5 u2009 mm lateral u2009 ± u2009 0.
8mm, abdomen-5. 0u2009mm)
Use a glass microtransfer tube and a pneumatic syringe system (
World precision instruments
Sarasota, Florida, USA).
At least 3 weeks after the virus injection, the mice were implanted with silica gel fibers by surgery for recording orexin neurons.
The fiber is placed directly above the LHA (from bregma −1.
5 u2009 mm lateral u2009 ± u2009 0.
8mm, abdomen-5. 0u2009mm).
Once the mice recovered from anesthesia, the record began.
Tweezers with powerful sensors (PIS-
2001, manufactured by Aizawa S.
University of Medicine, Arts and Sciences, Tokyo, Japan)
Used for mechanical stimulation.
Pinch stimulation with tweezers is applied to the tail in conscious mice or anesthesia mice with a force of 100g, 200g and 300g.
In addition, heat stimulation is performed using a heating probe with a thermal sensor. A ramp-
At 2 °c/sec, the harmful thermal stimulation of the shape gradually increased from 30 °c to 50 °c or 60 °c, applied to the left rear-
Claws using a thermal stimulus with a circular probe (
Diameter: 1mm, Cross
Intercross 2000 N, Tokyo, Japan).
Then, cool the probe with circulating water with a cooling gradient of about 1. 5u2009°C/sec. Clozapine N-oxide (CNO)
Life Sciences from Enzo (Tokyo, Japan).
CNO is dissolved in water with a stock solution of 10 mg/ml and diluted to 100 μg/ml solution with saline before use. I. p.
Injection of CNO (1u2009mg/kg)
Finish at 12: 00 in plenty of light (
Zeitgeber Time = Gears 4)
The biggest effect of observation on waking.
Statistical analysis using ORIGIN 9 software (LightStone).
The Student\'s t-HomMed made a simple comparison of means and SEMtest.
Multiple comparisons of mean and SEM by one-one
The variance analysis was performed after the test of Tukey or the multiple comparison test of Dunn.
P value less than 0.
05 is considered important in these studies.