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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of the Integrative Micro-Drive Array
- Create a micro-drive array for cortical local field potential (LFP) recordings as described elsewhere. Leave at least 6 metal holes open on an electrode interface board (EIB) for use as electrocardiography/electromyography/breathing rate (ECG/EMG/BR) channels that are connected to bioflex wires as described in 1.2.
- Cut a bioflex wire into 6 pieces with lengths of 5.0 cm. Peel off the polytetrafluoroethylene (PTFE) coating of both ends of all the wire pieces with lengths of ~5.0 mm. Connect one end of each of the wire pieces to one of the open metallic holes (ECG/EMG/BR channels) on the EIB with a gold pin.
- Cut an enamel wire into two 5.0-cm pieces. Solder one end of each of these wires to the ground/reference (g/r) channels on the EIB (Figure 1).
- For the preparation of ECG electrodes, cut a bioflex wire into two 16-cm pieces. Peel off the PTFE coating of the ends of these wire pieces at lengths of ~5.0 mm on one end (short end) and ~15 mm on the other end (long end).
- Form a wire ring with a diameter of 2.0 mm by bending the long end of the wire and fixing the shape of the ring by soldering.
- For the preparation of EMG electrodes, cut a bioflex wire into 2 pieces with lengths of 8 cm. Peel off the PTFE coating from both ends of these wire pieces with lengths of ~5.0 mm.
- For the preparation of BR electrodes, cut a bioflex wire into 2 pieces with lengths of 6.0 cm. Peel off the enamel coating of both ends of these wire pieces with lengths of ~5.0 mm. Solder one end of each of these wire pieces to the head of a stainless- steel screw (stem diameter: 1.0 mm, stem length: 4.0 mm).
- For the preparation of ground/reference (gr) electrodes, cut an enamel wire into 2 pieces with lengths of 6.0 cm. Peel off the enamel coating of both ends of these wire pieces with lengths of ~5.0 mm. Solder one end of each of these wire pieces to the head of a stainless- steel screw (stem diameter: 1.4 mm, stem length: 3.0 mm).
- Gas sterilizes all electrodes and stainless screws and keeps these in a clean space.
2. Implantation of the Integrative Micro-Drive Array and the BR Electrodes
- Fix the rat on a stereotaxic device. Make an incision of ~3.0 cm on the head along the midline from the point between the eyes to the neck area. Expose the skull.
- Make two circular craniotomies with diameters of 0.7-1.0 mm above the olfactory bulb 11.0 mm anterior and 1 mm bilateral to bregma with a high-speed drill. Implant two BR electrodes in the skull until the tips of the screw stems are attached to the brain surface.
- Make two circular craniotomies with diameters of 0.7-1.0 mm above the frontal cortex 2.7 mm anterior and 2.7 mm bilateral to bregma. Implant two g/r electrodes in the skull until the tip of the screw stem is attached to the brain surface.
- Make six to eight holes with diameters of 1.0 mm in the area surrounding the large craniotomy. Implant anchor screws (stem diameter: 1.4 mm, stem length: 3.0 mm) in the skull.
- Make a large circular craniotomy with a diameter of ~2.0 mm above the hippocampus 3.8 mm posterior and 2.5 mm bilateral to bregma. Place the integrative micro-drive array such that the cannula tip of the drive array is located above the large craniotomy
- Fill the gap space between the cannula tip and the brain surface with ~100 µL of two solutions, i.e., 0.5% (by mass) sodium alginate and 10% (by mass) calcium chloride.
NOTE: This process forms a transparent gel in ~5 min, after the two solutions are mixed on the skull.
- Cover the cannula, BR electrodes, g/r electrodes, and anchor screws with dental cement with a thickness of 0.5 cm. Be careful NOT to cover the open ends of BR and g/r electrodes with the cement at this step.
- Solder the open ends of the ECG, EMG, BR and g/r electrodes to the individual wire tips that were previously connected to the EIB (see the steps 1.2 and 1.3).
- Cover the bottom part of the integrative micro-drive array, and all electrode wires, with dental cement. Ensure that all the electrode wires are completely covered so that the rat cannot scratch them out after the implantation.
- After regaining sufficient consciousness to maintain sternal recumbency, return the animal to its transparent Plexiglas home cage, and keep it on its own with free access to water and food. After the surgery, treat the animal with antibiotics (gentamicin).
3. In Vivo Recordings
NOTE: All signals are amplified, sampled at 2 kHz, and band-pass filtered (0.1 - 500 Hz) except for unit activities (sampled at 30 kHz and bandpass filtered (500 - 6 kHz)).
- Connect the EIB of the integrative micro-drive array to the headstage of a recording device.
- Advance the tetrodes by turning the screws for a few weeks after surgery. Once the tetrodes are adjacent to the target brain areas, settle the tetrodes into the areas over a period of several days for stable recordings.
- Monitor the electrical signals while the animal freely moves in a recording chamber.
