Implanting a Wireless Bidirectional Microelectrode System in a Rat Brain

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Animals

1. House male adult Wistar rats (200-250 g) in groups of 3-4 under standard laboratory conditions for at least one week before surgery to allow acclimatization.
2. Two days after surgery, house rats in pairs. Cover single cages with high acrylic lids. Avoid conventional lids made of metal grid since implants can get stuck, increasing the risk that they become damaged and/or unstable over time.

2. Stereotactic surgery

1. Before starting the surgery, organize and prepare the following equipment and materials:​
   1. Obtain sterile surgical equipment consisting of sterile scissors, blunt-end forceps, spatulas, surgical clippers, dental drill, and cotton buds.
   2. Obtain drugs and chemicals including isoflurane, xylocaine, tramadol hydrochloride, dexpantenol eye salve, 3% hydrogen peroxide, povidone iodine and 70% ethanol.
   3. Obtain fixation material including stainless steel screws, acrylic resin, ultraviolet glue, and cap protector.
   4. Obtain a microelectrode unit, consisting of (i) a recording single electrode (quartz glass insulated platinum tungsten microelectrode, with conical tip shape, outer diameter: 80 µm, conical tip, impedance at 1 kHz: 500 kOhm) or a tetrode (quartz glass insulated platinum/tungsten 4 cores microelectrode, outer diameter: 100 µm, conical tip, impedance at 1 kHz: 500-800 kOhm); (ii) a stimulation electrode (platinum/iridium wire (90% platinum, 10% iridium), core diameter 125µm, outer diameter 150 µm, impedance <10 kOhm) connected to a contact plate and (iii) a platinum wire reference electrode (shaft diameter, 100 µm; Figure 1A).
   5. Obtain an electrode holder glued with water-soluble glue to the microelectrode unit and test for functionality at least 2 h in advance (Figure 1B).
   6. Obtain a conventional tethered system consisting of a differential preamplifier, a main amplifier, and a bandpass filter amplifier for recordings.
   7. Obtain additional material, such as gloves, heating pad, syringes, and physiological saline.
   8. Obtain home cages (L x W x H: 42 cm x 26 cm x 38 cm).
2. Prosedür
   NOTE: Electrode implantation is performed during a conventional stereotactic surgery under isoflurane anesthesia.
   1. Ensure that the experimenter is wearing gloves, a surgical mask, and a lab coat.
   2. Initiate anesthesia by placing the animal in an induction chamber (isoflurane 4-5%, oxygen flow 1 L/min, duration ~5 min).
   3. Test for loss of reflexes (tail and toe reflexes) with forceps to confirm deep anesthesia.
   4. Place the animal's head in an anesthesia mask fixed around the upper incisor bar of the stereotactic frame and adjust the anesthesia (isoflurane 2-3%, oxygen flow 0.7-0.8 L/min).
   5. Fix and horizontally align the animal's head in the stereotactic apparatus using ear bars and upper incisor bar.
   6. Shave the surgical field using surgical clippers or a scissor and sterilize with povidone-iodine.
   7. Place the animal on a heating pad to prevent hypothermia and treat the eyes with dexpanthenol eye salve to prevent them from drying.
   8. Inject xylocaine (0.3-0.4 mL, subcutaneously, s.c.) in the center of the surgical field.
   9. Test for loss of reflexes again.
   10. Make a small incision (1.5 cm) with a scalpel in the middle of the surgical field to expose the skull. Separate the skin gently and remove residual tissue using forceps, scissors, and a spatula.
   11. Carefully clean the skull using hydrogen peroxide-coated cotton buds.
   12. Drill 4-5 small holes (4.7 mm) in the skull for fixation of stainless steel screws.
   13. Connect the microelectrode unit/electrode holder to the preamplifier and attach it to the stereotactic micromanipulator (Figure 1B and 1C).
   14. Drill a hole (approximately 7 mm) in the skull above the target area using coordinates from a brain atlas according to the animal used. In the present study, position the electrode tips aimed at the inferior colliculus using the following coordinates, with the bregma serving as the reference: anterior/posterior, −8.8 mm; medial/lateral, 1.5 mm; and dorsal/ventral, 3.5 mm.
   15. Absorb any blood with cotton buds.
   16. Vertically introduce the microelectrode unit until the electrode tips reach the target area.
   17. Position the ground cable along the stainless-steel screws and under the skin.
   18. Monitor spike activity and carefully adjust electrode position with micromanipulator until reaching a zone of active neurons in the target structure and detect neural activity with a signal-to-noise ratio suited for spike sorting.
   19. Fix the microelectrode unit to the skull with ultraviolet glue and cover the contact plate and screws with acrylic resin.
   20. Inject physiological saline (1 mL i.p.) and tramadol (25 mg/kg, s.c.) to prevent dehydration and ensure post-operative analgesia, respectively.
   21. Disconnect the microelectrode unit from the electrode holder using a brush soaked in water.
   22. Stop anesthesia, and carefully remove the rat from the stereotactic frame. Disconnect the preamplifier from the microelectrode unit.
   23. Connect the cap protection on the microelectrode unit implanted and disconnect it only during the experimental procedures.