An Integrated Method for Crafting Flexible and Convenient Electrophysiological Optrodes for Multi-Region In Vivo Recording

This protocol received approval from the Animal Care and Use Committee at Fudan University and was conducted following the guidelines and regulations designed by the National Institutes of Health Guide for Care and Use of Laboratory Animals. All possible measures were implemented to minimize the number of animals utilized in this study. The time required to perform each step is included in the respective steps.

1. Preparation of electrodes (Figure 1)

1. Cut an appropriate length of tungsten wire (~20 mm) and burn off the insulation layer at one end to expose the tungsten wire (1.6-2 mm). Bend the other end of the wire into an "L" shape (see Figure 1A, ~1 min)
2. Carefully place the "L" end of the tungsten wire onto the optical fiber (see Figure 1B, ~1 min).
   NOTE: The tungsten wire should be 0.2-0.4 mm longer than the tip of optical fiber.
3. Dip a drop of adhesive and apply it to the interface of the optical fiber and the tungsten wire (see Figure 1C, ~1 min). Due to the surface tension of the liquid, the tungsten wire will attach to the optical fiber.
4. Apply the adhesive once and allow it to dry. Then, apply a second coat. This will further tighten the bond between the tungsten wire and the optical fiber (see Figure 1C, ~1 min).
   NOTE: During the application process, care should be taken to prevent the adhesive from flowing to the tip of the optical fiber, as this may affect its transmittance.
5. At the corner of the "L" shape, use adhesive to closely bond the base of the optical fiber and the tungsten wire together (~1 min).
   NOTE: The increased contact area and the spherical solidification of the adhesive droplet ensure a firm bond between the optical fiber and the tungsten wire.
6. Use soldering to establish a connection between the tungsten wire and the pin of the female connector (see Figure 1D, ~3 min for each)
   NOTE: Since tungsten wire is not sensitive to heat, the success rate of soldering can be increased to nearly 100% by a metal sleeve fashioned by cutting the pin slot location to physically unite the two components. Subsequently, solder filling the sleeve ensures a stable connection between the inserted tungsten wire and the pin.
7. According to the experimental design, solder the required number of optrodes (see Figure 1E, ~10 min)
   NOTE: Three optrodes were utilized in this study.
8. Cut the enameled wire to the appropriate length (~30 mm) and burn off the insulation on both ends. Wrap one end of the enameled wire around the base of the screw and solder it. Connect the other end of the enameled wire to the pins of the female connector via a soldering iron, serving as the EEG recording and ground connection for the skull electrode (see Figure 1F, ~5 min)
   NOTE: Due to the flexibility of the enameled wire, it is convenient to connect the skull and the pins of the female connector. Soldering after winding the enameled wire around a screw is more reliable than surface-mount soldering.
9. Apply hot-melt adhesive to the female connector's pin array to encapsulate the female connector and ensure the solder joints are fully enveloped. During the solidification of the hot-melt adhesive, shape the adhesive using a plastic board to minimize space occupancy during implantation (see Figure 1F, ~3 min)
   NOTE: Due to the potential burn risks associated with using hot-melt glue, AB adhesive is considered a safer alternative.
10. Next, use a light intensity meter and digital multimeter to measure luminousness and conductivity. Upon passing these tests, the setup is ready for use (see Figure 1G, ~2 min)
    NOTE: When working with lasers, always wear protective eyewear suitable for the specific wavelength and power of the laser light. High-quality fibers are required to transmit at least 80% of the light from the end of the patch cord to the tip of the implantable fiber. Additionally, the resistance from the tip of the tungsten wire to the pin header slot was measured with an acceptable range of 1-5 Ω.
11. Before use, immerse the prepared electrodes in 75% alcohol for 15 min, then transfer them to sterile saline (~16 min).
    NOTE: The total weight of the encapsulated electrode device (0.6701 ± 0.01123 g, n = 4) does not affect the activity of the mice.

2. Protocol for electrode implantation (Figure 2)

1. Anesthetize the mice with isoflurane (1%-1.5%) and secure them in a stereotaxic apparatus (see Figure 2A, ~2 min). Use eye ointment on mouse eyes to prevent dryness while under anesthesia.
   NOTE: Proper anesthetization is confirmed through the absence of the righting reflex and the loss of response to toe pinch. For prolonged surgical procedures, using a liquid anesthetic is more appropriate.
2. After securing the animal in the stereotaxic apparatus, trim away the scalp along the edge of the skull and wipe the skull surface with 75% alcohol to expose the positions of the bregma and lambda (see Figure 2B, ~3 min).
3. Level the brain and drill holes (~0.8 mm) above the regions of interest (ROI) using a microdrill. Inject the relevant viruses with a syringe pump (see Figure 2C – F, ~20 min).
   NOTE: Detailed information about the viruses and ROIs in this study is shown in the Table of Materials and Figure 3.
4. Hold the optrodes with a holder and move their tip to the bregma point, setting this position as 'zero'. Using the bregma point as the reference coordinate, move the optrodes to the target regions (see Figure 2G, ~3 min). Position the optrodes 0.3 mm above the virus injection site.
   NOTE: In the vertical implantation strategy, the minimum distance between the two electrodes is approximately 2 mm, depending on the thickness of the fiber optic sleeve and the outer diameter of the ferrule. An angled insertion method can be used for simultaneous recording from closely located regions.
5. After inserting the optic fibers into the corresponding brain areas, apply a small amount of tissue adhesive around them to cover the exposed tissue. Then, sequentially apply additional adhesive and a little dental cement. After the dental cement solidifies, release the holder carefully (see Figure 2H, ~5 min).
6. Before each new optrode implantation, perform recalibration, which involves repeating step 2.4 (see Figure 2I).
   NOTE: For each implantation, position the optrode at bregma as the 'zero' point.
7. After implanting all optrodes, drill the holes behind the lambda point for 'Ground' and above the neocortex for 'EEG' using a microdrill (~1 min).
   NOTE: The size of the drilled hole (~1.1 mm) needs to match the diameter of the screw.
8. Insert skull screws into the hole above the cortex and behind the lambda point (see Figure 2J, ~5 min).
   NOTE: Skull screws are used for EEG recording and grounding, respectively.
9. Organize the tungsten wires and enameled wires so they are positioned above the implantation area and not exposed outside the skull. Secure the position of the female connector using a holder (see Figure 2K, ~2 min).
10. Fill the implantation site with dental cement to ensure a stable connection between the electrode device and the skull. Encapsulate the internal wires, leaving the female connector's insertion holes exposed for connection to the headstage (see Figure 2L, ~6 min).
11. After dental cement hardening, apply iodine to the wound and then place the animal on a warming pad (~1 min).
    NOTE: Ensure that the animal is not left unattended until it has regained sufficient consciousness to maintain sternal recumbency. Sterile conditions should be maintained throughout the survival surgery.
12. Administer antibiotics and analgesics to support post-surgical recovery.
    NOTE: Animals that have undergone surgery are not reintroduced to the company of other animals until they have fully recovered.Animals were administered intraperitoneal injections of ceftriaxone sodium (180 mg/kg), and lidocaine gel was applied to the surgical wounds daily for 3 days.

3. Protocol for recording in vivo brain

1. Before conducting signal recording and behavioral testing (open field test)¹⁵, ensure that the mouse undergoes a 3-day period of gentle handling to acclimatize them to the environment.
   NOTE: In this study, the mice were placed in an open field (42 cm × 42 cm) to move around freely, allowing us to observe their seizure behavior.
2. After anesthetizing the mice with isoflurane (1%-1.5%), connect the optical fibers sequentially in the order of the recording site before inserting the headstage.
3. Set the 635 nm light pulse with a 10% duty cycle at 20 Hz. Set up the light stimulation for 10 s (200 pulses) during recording. Set the intensity at the optical fiber tips at 3 mW.
4. Use a fiber photometry system to record Gcamp6m signals behavioral testing period at a sampling rate of 30 Hz by 470 nm LED channel.
5. Simultaneously record calcium signals, LFPs, and EEG under optogenetic stimulation, along with corresponding behavioral performances (see Figure 3).
6. Employ transistor-transistor logic (TTL) signal synchronization tagging to ensure temporal consistency in recorded data.

4. Data processing

1. Load photometry data to MATLAB software. Then, use custom coding (Supplementary File 1) to detrend the calcium signal.
2. Define the changes in fluorescence intensity with equation (1):
   ΔF/F = (F_t - F₀)/ F₀    (1)
   Where F_t represents the real-time calcium signal fluorescence values, and F₀ denotes the averaged baseline fluorescence intensity before the event.
3. Import the electrophysiological data, sampled rate of 1000 Hz, into MATLAB (Supplementary File 2).
4. Coordinate the display of calcium signaling and electrophysiological data in alignment with synchronized markers.