a retinoraphe projection regulates serotonergic activity and looming-evoked defensive behaviour - led projector

Animals promote their survival by avoiding quick access to objects that indicate threats.
On the mouse.
The induced defense response is triggered by the Upper Hill (SC)
It receives direct retina input.
However, the specific neural circuits that start with the retina and regulate this important behavior are still unclear.
Here, we identified a subset of the retinal node cells (RGCs)
Control the mouse to faint
Induced defense response to the dorsal nucleus of the middle seam through the axial cable (DRN)and SC.
The hidden signal of DRN-transmission
The projected RGCs activate DRN gabaenergy neurons, which in turn inhibit 5-amino energy neurons.
In addition, the activation of DRN serum energy neurons decreased.
Defensive behavior caused
Therefore, a group of dedicated RGCs signals, quickly approaching visual threats, and inputting them into DRN, controlling a group of serum-capable self-
Regulate the gating mechanism of innate defense response.
Our study provides new insights into how DRN and SC work together to extract visual threats and translate them into defensive behavior responses.
All the experiments were approved by the animal conservation and use Committee of the Southern University of Economics. Adult male (6–8 weeks)C57BL/6, Sert-Cre mice (strain name B6. Cg-Tg(Slc6a4-Cre)ET33Gsat;
Davis MMRRC, California, USA)and vGAT-Cre mice (strain name B6. FVB-Tg(Slc32a1-cre)2. 1Hzo/FrkJ;
Jackson Laboratories, Hong Kong, United States)
Used in this study.
Animals are kept in the light and dark cycle of 12 h: 12 light h (
7:00 turn on the light)
Food and water are provided.
Animals were randomly divided into experimental group and control group.
The experimenter is blind to the experimental group, and the Test order is balanced in the behavior experiment.
Anesthesia in mice (
On average, 13 μ l g, I. p. )
Placed in a stereo instrument (
Rear wheel drive, Shenzhen, China.
Prevent corneal dryness and heat pad with erythromycin eye ointment (
Rear wheel drive, Shenzhen, China
Used to keep the body temperature at 37 °c.
A small open bone hole was made with a dental drill (
OmniDrill35, Shiping)
And a trace liquid transfer device connected to a typical stereo syringe (
Ille, Wooddale, Storting, United States)
And its controller (Micro4;
Sarasota WPI, USA)
For injection.
CTB fluorine binding of Alexa (CTB-488, C-3,477; CTB-594, C-34,777; CTB-647, C-34,778; Invitrogen Inc.
Grand Island, New York, USA)
Injected into DRN (0.
2 μ l per injection, CTB-488 or CTB-647; : −5. 2u2009mm; : ±0u2009mm; : −2.
7mm with an angle of 15 °);
Intermediate layer of SC (0.
4 μ l per injection, CTB-594; : −3. 7u2009mm; : ±0. 6u2009mm; : −1. 85u2009mm), Followed by 0. 03u2009μl oil (sesame oil; Sigma-Aldrich Corp. )
Limit the diffusion of CTB tracers. For cell-type-
Specific tracking, total transaction volume 0.
8 u2009 μ l containing equal volume aa v-EF1α-DIO-EGFP-TVA (
2x10 capsules per ml)and AAV-EF1α-DIO-RVG (
2x10 capsules per ml)
In DRN (eight Sert-
Cre mice and eight vGAT-Cre mice)(
Coordinates:-5. 2u2009mm; : ±0u2009mm; : −2.
7mm with an angle of 15 °).
Place the transfer tube for 10 minutes and take it out slowly. Twenty-
After 1 day, 0. 4u2009μl of SAD-ΔG-DsRed (EnvA)(
2x10 capsules per ml)
Injected into the same place. For di-
Synaptic tracking retina → SC → lp pathway, 0.
8 μ l auxiliary virus (rAAV-hSyn-GFP-2a-TVA-2a-RVG-WPRE-pA)(
2x10 capsules per ml)
Injected into SC (
6 mice/6 mice)(: −3. 7u2009mm; : ±0. 6u2009mm; : −1. 85u2009mm).
Place the transfer tube for 10 minutes and take it out slowly. Twenty-
After 1 day, 0. 4u2009μl of SAD-ΔG-DsRed (EnvA)(
2x10 capsules per ml)
Injected into LP (: −2. 4u2009mm; : ±1. 5u2009mm; : −2. 2u2009mm)and 0.
Inject 2 μ l CTB488 into DRN.
A similar procedure for injecting CTB was used to inject 0. 4u2009μl AAV-DIO-GCaMP6m, AAV-DIO-eYFP, AAV-DIO-ChR2-mCherry, AAV-DIO-eNpHR3. 0-MCherry and AAV-DIO-
MCherry nuclear (Virus titer: 4.
35 × 10 capsules per ml, 10 inserts-
Cre mice and 11 vGAT-
Use Cre mice for AAV-DIO-
Injection of GCaMP6m; 3 Sert-
Cre mice and 3 vGAT-
Use Cre mice for AAV-DIO-eYFP injection; 8 Sert-
Cre mice and 7 vGAT-
Use Cre mice for AAV-DIO-ChR2-
MCherry injection; 6 vGAT-
Use Cre mice for AAV-DIO-eNpHR3. 0-
MCherry injection; 10 Sert-
Cre mice and 5 vGAT-
Use Cre mice for AAV-DIO-
MCherry injection molding).
A similar procedure for injecting CTB was used to inject 0. 4u2009μl AAV-DIO-hM3D-mCherry or AAV-DIO-hM4D-
MCherry nuclear (Virus titer: 2.
7 × 10 particles per ml, 35 inserts-
Use Cre mice for AAV-DIO-hM3D-
MCherry injection; 18 vGAT-
Use Cre mice for AAV-DIO-hM4D-
MCherry injection molding).
Suture the wound after injection and use antibiotics (
Bacterial peptide and netinin)
Applied to surgical wounds and ketone acids respectively (5u2009mgu2009kg)
Subcutaneous injection;
Animals are allowed to recover from anesthesia under a heating lamp.
As mentioned earlier, the optical fiber metering system (
Nanjing thinker technology)
Used to record 5-from gene identification-
3) HT and
To put it simply, a week after AAVDIO-
Optical fiber GCaMP6m virus injection (230u2009mm OD, 0.
37 numerical aperture (NA);
Shanghai fiber laser)
Placed in a ceramic sleeve and inserted into the DRN by bone opening (
Coordinates:-5. 2u2009mm; : ±0u2009mm; : −2. 65u2009mm).
The ceramic core is supported by a skull.
Penetrate M1 screws and dental acrylic resin.
Mice were raised separately and allowed to recover for at least a week.
In order to record the fluorescent signal, from 488-nm laser (OBIS 488LS; Coherent)
Reflected by a two-color mirror (MD498; Thorlabs)
Focus with x 10 lens (NA=0. 3; Olympus)
Then coupled to the optical converter (Doric Lenses).
A fiber (Mm OD, NA = 0. 37, 2-m long)
The light between the guide and the injected fiber.
Adjust the laser power at the fiber tip to a low level of 0. 01–0.
02 u2009 mW to minimize bleaching.
GCaMP6m fluorescence is filtered through the band (MF525-39, Thorlabs)
Collected by a photoelectric multiplier tube (
R3896 binsong). An amplifier (
Binsong c73 19)
Used to convert the photoelectric multiplier tube current output to a voltage signal and further filter through low levelpass filter (40u2009Hz cut-off; Brownlee 440).
Analog voltage signals are digitized at 500 hz and recorded by Power 1401 digitizer and Spike2 software (
(Cambridge, UK).
After heart perfusion, 0.
9% psychological saline is followed by more than 4% formaldehyde in 0.
1 m PBS, after taking out the brain
Fix overnight with more than 4% formaldehyde at 4 °c, then transfer to 30% sucrose until sliced with a low temperature thermostat (
USA, IL, Bannockburn, Leica Microsystem, CM1900).
A series of 40 μm slices were collected to verify the injection site;
Tissue was examined under apparent fluorescence using Leica DM6000B microscope.
As mentioned earlier, CTB-was accepted in the B/6 mice-488 or CTB-647 (
For optical fiber test)(0. 2u2009μl peranimal)
Stored in DRN.
Three days later, the mice received a bilateral eyeball injection that was customized to bind the immune toxin (2u2009μg per eye)(=10)
In saporin (IT-27-
Advanced aiming system 100)
And affinity-purified anti-CTB antibody (
Meridian Life Sciences C86204M), or anti-Melanopsin-saproin (=8)(2u2009μg per eye)(IT-
44. advanced aiming system). Mice (=10)
The control group received bilateral intra-eyeball injection with equal volume 0. 1u2009M PBS.
After the injection, the mice were put back into the cage for 14 days. DRN-
As mentioned earlier, the prominent RGCs are recorded.
To put it simply, animals are under anesthesia (
On average, 13 μ l g, I. p. )
Under the dim red light, the eyes were removed.
Carefully remove with a pair of thin lenses and glass body filmforceps.
The eye mask is flat.
Install directly at the bottom of the recording room and over-melt by filling oxygen (95% O/5% CO)Ames medium (Sigma-
Aldridge, St. Louis, Missouri, USA)
At a fixed rate (5u2009mlu2009min)
At room temperature (22–24u2009°C).
By programming the psychological physics toolbox in Matlab shown on the Samsung mini LED projector, an imminent stimulus is generated (Samsung SP-
P3 10me, Samsung Electronics Co. , Ltd. , won city, South Korea)
Take the first photo
Mirrors and lenses (
Edmond science, Barrington, New Jersey, United States of America)
On the film plane of the microscope camera port.
Measure the brightness level of the projector with a digital radiator (
S370 radiator, UDT instrument, San Diego, California, United States of America)
Use water X 40-
Immersion goals (
Carl Zeiss, Thornwood, New York, United States of America).
The irradiance is further reduced using a neutral density filter (Oriel Corp.
Strafu, CT, USA).
Hidden response of DRN
The prominent RGCs are recorded using a glass micro-electrode and amplified with a patch amplifier (Multi-lights group (700 B)and digitized (Digidata 1,440;
Axon Instruments Co. , Ltd.
Forest City, California, USA).
RGCs marked retroactively by CTB are the target of the record.
After the physiological analysis was completed, the recorded RGCs were filled with neurobiodin in the cells for morphological evaluation.
Further analysis of the physiological data obtainedline (pCLAMP9; Axon Corp. , CA, USA). AAV-DIO-ChR2-mCherry or AAV-DIO-eNpHR3. 0-
Inject the mCherry virus into the DRN region of vGAT-Cre or Sert-
Cre mice and leave for 2-3 weeks.
In the preparation of brain slices, E. obarbital (100u2009mgu2009kg i. p. )
The thickness of the coronary brain is 250 μm.
Restore the slice 30 minutes before recording. Whole-
Cell patch clamp recording was performed on ChR2-
New cell body identified under fluorescence microscope.
Overall internal solution-
Cell recording pipettes (3–5u2009MΩ)contained (in mM)
: 135 KMeSO, 10 potassium chloride, 10 HEPES, 10 Na-
8 phosphate ester of 4 MgATP, 0. 3 NaGTP (pH 7. 2–7. 4);
Or CsMeSO 130, nac10, EGTA 10, MgATP 4, NaGTP 0. 3, HEPES 10 (pH 7. 2–7. 4). 0.
5% the neuropol was included in some records
Record staining. Voltage-
Clamp and current-
Fixture recording using a multi-lamp 700 B amplifier (
Molecular Devices. For voltage-
The neuron is kept at-60 mv.
Very low marks
Filter at 2 khz and digitize at 10 khz (
DigiData 1,550, molecular device).
Get and analyze data using Clampfit 10. 0 software (
Molecular Devices.
Light stimulation, 470-nm LED or 590-nm LED (Thorlabs)
For transmitting optical pulses (
Blue light: 4 kbps ms × 5 pulse at 20 hz;
Yellow light: 1 s)
Digital Command via Digidata 1440.
The recorded in-cell-filled neuropol was used for morphological evaluation.
Anesthesia in all animals (
On average, 13 μ l g, I. p. )
Heart perfusion 0.
Add 9% salt water to PBS, then add more than 4% formaldehyde.
Cut off the brain and eyes.
Used to detect black sight, shopping cart and SMI-
32 retina expressing RGCs were separated and cleaned 0.
3 times 1 µm PBS (10u2009min each)
Before hatching at 0.
1 µm PBS containing 10% normal goat serum (
Bollingame Vector Laboratory, California, USA)and 0. 3% Trition-X-100 (T8787, Sigma-
Aldridge, St. Louis, Missouri, USA)for 1u2009h.
Then resist the retina with the rabbit at 4 °c-
Antibody to black sight (AB-
N38, early targeting system; 1:600)or anti-CART antibody (H-003-
Phoenix pharmaceutical 621:1,000)or anti-SMI32 antibody
801703, Biolegend; 1:1,000).
Subsequently, six rinses were carried out in 0.
1 m PBS, and then with secondary (
Dylight 488 or Dylight 549goat-anti-rabbit IgG (
Vector lab 1:400)
For 6 hours at room temperature.
The retina of RGCs in the cell was filled with neuropol and fixed in more than 4% formaldehyde for 1 h, at 0.
1 m PBS at room temperature, at 0.
3 times 1 µm PBS (10u2009min each)
Put in 10% normal goat serum containing 2% nutrientsX-
48 h at 4 °c is 100
Then incubated the retina in the chain mildew resistanceAlexa Fuor 488 (
S32354, Life Technology, 1:100)
Working at 4 ° C 48 ° h
Then rinse three times in 0. 1u2009M PBS.
To mark the cholinergic class without long-Turkic cells, the retina was rinsed in 0.
3 times 1 µm PBS (10u2009minu2009time)
Put in 10% normal goat serum containing 2% nutrientsX-
100 lasts for 1 hour at room temperature.
Then culture the retina in the goatanti-ChAT antibody (
AB144P with Millipore; 1:200)
Working at 4 ° C 48 ° h
Subsequently, six rinses were carried out in 0.
1 m PBS, then incubated with secondary antibody Alexa Fluor 594 donkeygoat IgG (1:400, A-
Molecular probe 11058)
For 6 hours at room temperature.
Finally, rinse all retina in 0.
1 m PBS and lid-In the opposite
Fading water-based installation medium (
PA Hatfield EMS, USA). For c-
Fos markers containing 40 μm low temperature thermostat slices of DRN, SC, LP, BLA or gray around the back Guide water pipe are placed in a closed solution for 1 µh and then incubated with anti-c-Fos (rabbit, 1:500;
PC38T, Calbiochem)(36u2009h at 4u2009°C).
Then, at room temperature, incubated with the corresponding secondary antibody at a dilution of 1:400 for 6 u2009 h: goat antibody
Rabbit 488 sand (
Jackson immune research 107909).
Program used in C-
In addition to an anti-substitution, Fos markers were usedTPH (mouse, TPH; 1:1,000; T8575, Sigma-Aldrich)or anti-GABA (mouse, 1:1,000; A0310, Sigma-Aldrich)
And to fight the goat-
Alexa 594 mouse (115-587-
Jackson immunization study 003)or goat anti-
Alexa 647 mouse (115-587-
Jackson immunization study 003).
Finally, rinse all brain slices with 0.
1 m PBS and lid-In the opposite
DAPI for fading water-based solids (
PA Hatfield EMS, USA).
The retina and sections were imaged with a Zeiss 700 with a X 5 or X 20 target with a total focus microscope or a x 40 oil immersion target. For three-
3D reconstruction of RGCs marked by injection or virus, optical slices were collected at 0. 2u2009μm intervals.
Each stack of optical slices covers 325 of the retina. 75 × 325. 75u2009mm (
1,024x1,024 pixels).
Use Image J and Photoshop CS5 (Adobe Corp.
San Jose, California)
, Monte-slice each stack of optics and project to a plane of 0 ° X-Y and a plane of 90 ° Y-Z to obtain three
Three dimensional reconstruction of cells.
Three details.
The dimensional reconstruction and co-focus calibration procedures are described elsewhere.
Adjusted contrast and brightness, red-
The green image has been converted to magenta-green.
The total soma and branch-like field size of each filled cell were analyzed.
Calculate the area of the branch crystal field by drawing a convex polygon connecting the end of the branch crystal.
Then calculate the area of the branch crystal field, the diameter is expressed as the diameter of the circle with equal area.
An imminent stimulus test was conducted in a closed arena of 50 × 37 cm with or without shelter.
An LED display is embedded in the ceiling to show the upcoming stimulus.
An imminent stimulus consisting of an expanded black disc appears in 0 at a angle of 2 ° to 20 ° in diameter.
25 u2009 s, and presented once in 0.
15 times in 25 u2009 s or 10. 75u2009s.
When the mouse is in the center of the arena, the experimenter manually triggers the stimulus.
Optical fiber metering for recording Ca signals from DRN 5-for gene identification
Connect the implanted fiber to the optical fiber metering system and record the Ca signal throughout the test process (
15 immediate stimuli per trial). For chemico-
Gene experiments. Cre or vGAT-
The Cre mice were given normal saline or psychological saline N-oxide (
CNO, ab141704, abcam)
30 minutes before the arena test with or without a shelter (
15 immediate stimuli per trial).
For the photogenerated activation experiment, connect the implanted fiber to 463-
Nm Blu-ray laser or 590-
At the optical fiber tip, the optical power of the nm yellow laser is set to about 10 mw.
The duration of neuron activation fully incorporates the duration of the upcoming stimulus.
Optomotor reaction of animals in VEH (=6 animals)and anti-CTB-SAP (=6 animals)
Determination described by group. khaled. .
Simply put, the mouse is placed on the platform in the form of a grid (
Diameter 12 cm, 19.
0 cm above the bottom of the drum)
Surrounded by motor drums (29. 0u2009cm diameter)
Can rotate the clock-
The wisdom of two revolutions per minute or counter-clockwise.
After adapting to the dark for 10 minutes, present the animals with vertical black and white stripes that define the frequency of space.
These stripes rotate alternately clockwise and counter-clockwise for 2 minutes in each direction, and the interval between the two rotations is 30 minutes.
Various spatial frequencies tend to 0. 03, 0. 13, 0. 26, 0. 52 and 1.
25 cycles were tested in random order on different days.
The animals were recorded with an infrared digital camera in order to score the head tracking action.
The procedure for measuring the optomotor reaction under open-light conditions is similar to dark-light conditions, except that the animal accepts 400 Lux within 5 min to suit the light.
The experimenter who turned a blind eye to the experimental conditions analyzed the data. One-
Use ANOVA to quantify the duration from start to end, peak speed during and after near stimulation, c-
Fos cells and iprgc.
The data is displayed as an average. e. of the mean (s. e. m. ).
The statistical significance is