In Vivo Recording of Intracellular Signals from a Single Neuron

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

1. Surgical Preparation

Note: All incision and pressure points should be repeatedly infiltrated with local anesthetic (lidocaine or bupivacaine). The present procedure is terminal, if an aseptic preparation is required several modifications should be implemented.

1. Anesthetize a rat with sodium pentobarbital (40 mg/kg) and ketamine (50 mg/kg) in two locally separated intraperitoneal (IP) injections.
2. Let the animal go under general anesthesia and repeatedly verify that a surgical plane of anesthesia is attained (no reaction to toe pinching). Place the rat on a feedback heating blanket and insert a rectal probe to maintain a core temperature of around 37 °C.
3. Place a catheter in the peritoneal cavity to facilitate subsequent injection of anesthetic agents and to avoid perforating organs by repeated needle punctures.
   1. Clip the hair over a small (~2 – 3 mm) area above a region located within the stomach's lower right or left quadrant.
      Note: Depilatory cream can also be used.
   2. Make a 2 – 3 mm incision on the skin with sharp scissors or a scalpel. Using blunt dissection, remove fat and muscle layers until the peritoneal cavity is observed.
   3. Insert about 1 – 2 cm of a small catheter in the cavity and close the wound with surgical glue.
      Note: The diameter and the total length of the catheter should be minimal (e.g., 2 French – corresponding to 0.043 mm inside diameter) to reduce dead volumes. Polyurethane tubes are most suited.
   4. Make a loop and suture the catheter to the skin to secure it in place.
4. Install a tracheal tube to control ventilation during artificial respiration.
5. Install the animal in a stereotaxic frame and carefully monitor the following physiological variables: ECoG, SpO₂, EtCO₂, heart rate (via an electrocardiogram, ECG) and internal temperature. Monitor and adjust these variables to keep a proper depth of anesthesia and physiological state. Specifically, supplement anesthesia with a small dose (10 mg/kg) of sodium pentobarbital if necessary.
6. Apply eye ointment on both eyes to avoid desiccation. Clip the hair over the scalp, make a longitudinal incision (~2 cm), and respect the connective tissues overlying the skull using a scalpel or a curette.
7. Make a small (~1.5 mm diameter) craniotomy over the region of interest with a dental drill.
   Note: Here, the barrel field of the primary somatosensory cortex is targeted ( 7 – 8 mm anterior to the interaural line, 4.5 – 5.5 mm lateral to the midline). Rinse repeatedly to dissipate heat.
8. Use extra fine forceps to gently make a small hole in the dura. Reserve a ~0.5 mm region within the cranial trepanation to place the ECoG electrode (see next step). Permanently keep the cortex moist with 0.9% Sodium chloride or NaCl solution (or artificial cerebro-spinal fluid).
9. Place a low impedance (~60 kΩ) silver electrode (the ECoG electrode) on the dura, avoiding the cortical region not covered by the meninges, and place the reference electrode on a scalp muscle on the other side of the head.
10. At this stage (30 min after the last sodium pentobarbital injection), maintain the anesthesia by repeated injections of sodium pentobarbital (10-15 mg/kg/h) or fentanyl (3-6 µg/kg/h) via the IP catheter. The former will result in a slow oscillatory, sleep-like, ECoG pattern whereas the latter will result in a desynchronized, waking-like, cortical profile.
11. Adjust the artificial ventilation system so that the respiratory frequency and volume are similar to those of the rat's spontaneous breathing (normal range 70 – 115 breaths/min). Then, connect the mechanical ventilation to the tracheal tube and verify the proper thoracic cage inflation (on both sides). If not, adjust the position of the ventilation tubing in the tracheal axis. If necessary, suck up the secretion present in the trachea with a catheter connected to a syringe or a vacuum pump.
12. If all physiological variables and ECoG patterns reflect a stable surgical plane of anesthesia, do an intramuscular injection of gallamine triethiodide in each leg to paralyze the rat, 40 mg/kg for the first injection and then 20 mg/kg, every 2hr.

2. Intracellular Recordings

1. Pull a glass micropipette (sharp microelectrode) with a ~0.2 µm tip such that its resistance ranges between 50 and 80 MΩ once filled with 2 M potassium acetate (KAc).
2. Place the pipette in a specific holder with a silver/silver-chloride (Ag/AgCl) wire to connect the pipette solution to an intracellular amplifier (via a head stage). The holder should be attached to a micromanipulator. Place an Ag/AgCl reference electrode on the rat's neck muscles.
3. Slowly insert the pipette in the brain down to the region of interest and verify its resistance by monitoring the voltage drops in response to current steps. Use the buzz (or zap) button of the amplifier to clear the pipette if needed.
4. Use cotton swabs or synthetic absorption triangles to dry the craniotomy {be careful not to touch the ElectroCorticography (ECoG) or intracellular electrodes} before covering it with silicone elastomer or 4% agarose to reduce brain movements.
5. Lower the pipette in 1 – 2 µm steps until its resistance increases when approaching a cell. Then use the buzz function of the amplifier to penetrate into the neuron.