TITLE: Fiber Photometry during Sleep
AUTHORS: daniel.dautan daniel, Per Svenningsson

[DESCRIPTION]
Measurement of photometry during sleep using AAVs

[STEPS]
SECTION: Fiber Photometry- Virus Injection
1. Preparation:
SECTION: Fiber Photometry- Virus Injection
4. Injection Site Determination: 
SECTION: Fiber Photometry- Virus Injection
3. Surgical Site Preparation: 
SECTION: Fiber Photometry- Virus Injection
2. Anesthesia and Animal Preparation:
SECTION: Fiber Photometry- Virus Injection
5. Syringe Preparation: 
SECTION: Fiber Photometry- Virus Injection
6. Injection Procedure:
SECTION: Fiber Photometry- Virus Injection
7. Post-injection Care: 
SECTION: Fiber Photometry- Virus Injection
8. Monitoring and Follow-up:
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
9. Preparation: 
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
10. Anesthesia and Animal Preparation: 
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
12. Craniotomy: 
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
13. Implantation of Optic Fiber: 
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
14. Postoperative Care: 
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
15. Monitoring and Follow-up: 
SECTION: Fiber photometry- Habituation and Calibration
16. Day 1: Habituation
Preparation: 
SECTION: Fiber photometry- Habituation and Calibration
17. Mouse Handling: 
SECTION: Fiber photometry- Habituation and Calibration
18. Fiber Connection: 
SECTION: Fiber photometry- Habituation and Calibration
19. Monitoring: 
SECTION: Fiber photometry- Habituation and Calibration
22. Following this three-day habituation and calibration protocol will help ensure that the mice are acclimated to the experimental environment and that the fiber photometry system is properly calibrated for accurate data collection during subsequent experiments.
SECTION: Fiber photometry- Habituation and Calibration
21. Post-session Handling: 
SECTION: Fiber photometry- Habituation and Calibration
20. Data Logging: 
SECTION: Fiber Photometry- Recording
23. On the day of the recording, it is VERY IMPORTANT to proceed as early as possible to record as much sleep as possible. 
We record the animals ~1h after the switch from dark to light phase. We record bouts of 30 minutes and repeat the recording as many times as necessary.
SECTION: Fiber Photometry- Recording
24. Day 4: Post-Experimental Recording and Analysis
Post-Experimental Recording:
SECTION: Fiber Photometry- Recording
25. Data Analysis: 
SECTION: Fiber Photometry- Virus Injection
1.1. Prepare the adenovirus solution at the desired concentration.
We use a dopamine and acetylcholine sensor (AAV-Ach-SnFR addgene #137955 and AAV-rGRAB-DA addgene #140557) with a ratio of 1:1.
SECTION: Fiber Photometry- Virus Injection
1.2. Set up the surgical area in a clean and sterile environment. 
We recommend using a quartz beads sterilizer for all procedures.
SECTION: Fiber Photometry- Virus Injection
1.3. Prepare the anesthesia setup according to institutional guidelines.
SECTION: Fiber Photometry- Virus Injection
2.1. Induce anesthesia using isoflurane in an induction chamber.
We recommend using between 2-3% Isoflurane in 02.
SECTION: Fiber Photometry- Virus Injection
2.2. Confirm the depth of anesthesia by checking the pedal reflex.
SECTION: Fiber Photometry- Virus Injection
2.3. Once adequately anesthetized, transfer the mouse to a stereotaxic frame and secure its head using ear bars. 
We use the digital control Kopf stereotaxic instrument (69100 series).
SECTION: Fiber Photometry- Virus Injection
2.4. Place the heating pad or lamp to maintain the mouse's body temperature throughout the procedure.
SECTION: Fiber Photometry- Virus Injection
3.1. Shave the scalp area over the skull to expose the surgical site. 
We use epilation cream to provide better recovery.
SECTION: Fiber Photometry- Virus Injection
3.2. Clean the exposed area with alternating iodine (50% in water).
SECTION: Fiber Photometry- Virus Injection
4.1. Use stereotaxic coordinates to determine the injection site in the brain based on the desired region. 
Here inject a total of 500nl of viral mix in the dorsomedial striatum (coordinates from the Bregma: AP 0.5mm, ML 1.1, DV 3.0mm).
SECTION: Fiber Photometry- Virus Injection
4.2. Mark the coordinates on the skull surface using a surgical marker.
SECTION: Fiber Photometry- Virus Injection
5.1. Load the Neuros7001 syringe with the adenovirus solution. As there is no dead volume, it is recommended to load only 5-10% more than the required volume.
SECTION: Fiber Photometry- Virus Injection
5.2. Ensure there are no air bubbles in the syringe.
SECTION: Fiber Photometry- Virus Injection
6.1. Attach the syringe to the stereotaxic arm, ensuring stability and precision. 
SECTION: Fiber Photometry- Virus Injection
6.2. Lower the needle to the marked coordinates on the skull surface. 
SECTION: Fiber Photometry- Virus Injection
6.3. Slowly insert the needle into the brain to the desired depth.
SECTION: Fiber Photometry- Virus Injection
6.4. Inject the adenovirus solution at a controlled rate using a microinjector. 
We use 50-75 nl/min.
SECTION: Fiber Photometry- Virus Injection
6.5. Wait for a few minutes to allow for diffusion of the solution. It is recommended to wait at least 50% of the injection time (i.e. Injection 10min, diffusion at least 5 minutes).
SECTION: Fiber Photometry- Virus Injection
6.6. Slowly retract the needle from the brain.
SECTION: Fiber Photometry- Virus Injection
7.1. Apply pressure to the injection site with sterile gauze to minimize bleeding and leakage. 
SECTION: Fiber Photometry- Virus Injection
7.2. Clean the surgical site with sterile saline solution. 
SECTION: Fiber Photometry- Virus Injection
7.3. Close the incision with sutures. 
We use 4.0 Vicryl suture with PGA to allow better recovery.
SECTION: Fiber Photometry- Virus Injection
7.4. Monitor the mouse closely during recovery from anesthesia.
SECTION: Fiber Photometry- Virus Injection
7.5. Return the mouse to its cage once fully recovered.
SECTION: Fiber Photometry- Virus Injection
8.1. Monitor the mice regularly for any signs of distress or complications. 
SECTION: Fiber Photometry- Virus Injection
8.2. Provide appropriate postoperative care, including analgesics if needed. 
SECTION: Fiber Photometry- Virus Injection
8.3. Follow institutional guidelines for postoperative monitoring and euthanasia procedures if necessary.
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
9.1. Prepare the surgical area in a clean and sterile environment.
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
9.2. Prepare the anesthesia setup according to institutional guidelines. 
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
9.3. Ensure all surgical tools are sterilized and ready for use.
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
10.1. Induce anesthesia using isoflurane in an induction chamber.
We recommend using between 2-3% Isoflurane in 02.
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
10.2. Confirm the depth of anesthesia by checking the pedal reflex.
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
10.3. Once adequately anesthetized, transfer the mouse to a stereotaxic frame and secure its head using ear bars. 
We use the digital control Kopf stereotaxic instrument (69100 series).
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
10.4. Place the heating pad or lamp to maintain the mouse's body temperature throughout the procedure.
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
12.1. Use stereotaxic coordinates to determine the craniotomy site relative to the injection site. 
As the implantation is 2-3 weeks after injection if the previous hole from AAV injection are visible and clean, you can ignore these steps.
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
12.2. Mark the coordinates on the skull surface using a surgical marker. 
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
12.3. Make an incision in the scalp along the marked coordinates. 
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
12.4. Use a micro drill or dental drill to create a small craniotomy window above the intended implantation site, being careful not to damage the dura.
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
13.1. Place the ferrule from the optic fiber on the holder. 
We use a 0.50NA, 400µm, 4mm long, Thorlabs.
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
13.2. At various places, insert surgery screws (Agntho’s #MCS1X2).
We recommend using at least 2 screws. Here, we are measuring sleep combined with photometry, the 2 anchor screws are for the sleep telemetry devices. Surgery is similar as the one for sleep, with the exception of the implantation of the fiber photometry.
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
13.3. Lower the optic fiber implant to the target depth using stereotaxic coordinates, ensuring it is positioned approximately 100-200 µm above the injection site.
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
13.4. Secure the optic fiber in place using dental cement or adhesive applied around the base of the fiber. 
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
13.5. Allow the cement to dry completely.
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
14.1. Apply topical antibiotic ointment to the surgical site to prevent infection.
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
14.2. Close the scalp incision using surgical adhesive or sutures. 
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
14.3. Monitor the mouse closely during recovery from anesthesia. 
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
14.4.  Return the mouse to its cage once fully recovered.
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
15.1. Monitor the mice regularly for signs of distress or complications. 
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
15.2. Provide appropriate postoperative care, including analgesics if needed. 
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
15.3. Ensure proper functioning of the optic fiber implant and patch cable setup before commencing experiments.
SECTION: Fiber photometry- Habituation and Calibration
16.1. Set up the experimental room in a quiet, dimly lit area with minimal distractions. 
SECTION: Fiber photometry- Habituation and Calibration
16.2. Prepare the fiber photometry setup, ensuring all components are properly connected and functional. 
SECTION: Fiber photometry- Habituation and Calibration
16.3. Ensure the mice are acclimatized to the room for at least 1h before beginning the habituation.
SECTION: Fiber photometry- Habituation and Calibration
17.1. Gently handle each mouse to minimize stress and transfer it to the experimental cage. 

SECTION: Fiber photometry- Habituation and Calibration
17.2. Allow the mouse to freely explore the open lids cage for a designated period to habituate to the new environment.
SECTION: Fiber photometry- Habituation and Calibration
18.1. After habituation, connect the implanted optic fiber to the fiber photometry system without activating the light source. 
We are using the Doric lenses FPS_2S_GCAMP+RedFluo system that allow acquisition of GCamP and RCamP signal combined with 405nm isosbestic. Connection is done with ceramic sleeves. The system collects 3 channels isosbestic channel (405nm), GCAMP (460-490), and RCAMP (555-570) and collect isosbestic, Ach-SnFr, and rGRAB-DA signals at 1017Hz.
SECTION: Fiber photometry- Habituation and Calibration
18.2. Ensure the connection is secure but does not restrict the mouse's movement.
SECTION: Fiber photometry- Habituation and Calibration
19.1. Observe the mouse for any signs of distress or discomfort during the habituation period. 
SECTION: Fiber photometry- Habituation and Calibration
19.2. Note any abnormal behaviors or reactions.
It is recommended to leave the mice alone in the room for 10-20min until the mousee seems to be sleeping, we are using webcam to monitor the animal behavior.
SECTION: Fiber photometry- Habituation and Calibration
20.1. Begin data logging on the fiber photometry system to capture baseline activity. Progressively increase each LED power until calcium spikes are visible. When one LED is set, turn OFF the LED and proceed to the next one. You need to see signals only in the dedicated signal to avoid cross talks.
SECTION: Fiber photometry- Habituation and Calibration
20.2. After calibration, decrease progressively each LED power until no calcium spikes are visible, yet calcium oscillation is still present. This will avoid having strong and high amplitude calcium spike that might alter the analyses. For each mouse, note the signal amplitude as well as their dedicated LED power. In average we decrease for each channel the excitation power by 10%.
SECTION: Fiber photometry- Habituation and Calibration
21.1. After the habituation session, return the mouse to its home cage. 
SECTION: Fiber photometry- Habituation and Calibration
21.2. Clean the experimental cage and any equipment used with appropriate disinfectants. Repeat the experiment for 2-3 days.
SECTION: Fiber Photometry- Recording
24.1. Repeat the setup process as on previous days. This time turn ON the EMG/EEG device at the same time as the photometry system. We are using the computer clock to synchronize both systems.

SECTION: Fiber Photometry- Recording
24.2. Record sleep parameters and fluorescence signals to assess post-experimental recovery and baseline levels.
SECTION: Fiber Photometry- Recording
25.1. Analyze recorded data from both sleep recordings and fiber photometry to extract relevant parameters. 
SECTION: Fiber Photometry- Recording
25.2. Compare sleep patterns with neural activity recorded via fiber photometry to investigate correlations or effects of experimental manipulations.
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
11. Surgical Site Preparation: 
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
11.1. Shave the scalp area over the skull to expose the surgical site. 
We use epilation cream to provide better recovery.
SECTION: Fiber photometry- Implantation (2-3 weeks after AAV injection)
11.2. Clean the exposed area with alternating iodine (50% in water).
SECTION: Fiber Photometry- Recording
25.3. Nota bene for analyses:
We processed sleep scoring as described, by extracting timestamps of Wake, REM, NREM, and RBD events. Using a custom-made Matlab script, photometry data were downsampled to 500Hz (average smooth window function), then aligned with the sleep scoring. For each 1-minute window, each channel of the photometry was normalized using the z-score function and then averaged over the 20s window corresponding to the sleep scoring. For each animal, photometry data were then averaged for specific sleep stages and compared across groups.