Recording Gamma Band Oscillations From Pedunculopontine Nucleus Neurons in a Brain Slice

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

1. Preparation of Standard-artificial Cerebrospinal Fluid (aCSF)

1. Preparation of Stock Solution A
   1. Add 700 ml of distilled water to a clean 1 L beaker before adding chemicals.
   2. While continuously stirring a volume of 500 ml, add 136.75 g of NaCl, 6.99 g of KCl, 2.89 g of MgSO₄, and 2.83 g of NaH₂PO₄.
   3. Add more distilled water to reach a final volume of 1 L. After dilution, the final concentration of each compound is 117 mM NaCl, 4.69 mM KCl, 1.2 mM MgSO₄, and 1.18 mM NaH₂PO₄.
   4. Keep refrigerated at 4 °C for up to 2 weeks.
2. Preparation of Stock Solution B
   1. Add 700 ml of distilled water to a clean 1L beaker before adding chemicals.
   2. While continuously stirring a volume of 500 ml, add 41.45 g of D-glucose and 41.84 g of NaHCO₃.
   3. Add more distilled water to reach a final volume of 1 L. After dilution, the final concentration of each compound is 11.5 mM D-glucose and 24.9 mM NaHCO₃.
   4. Keep refrigerated at 4 °C for up to 2 weeks.
   5. Before each experiment mix 50 ml of stock A with 50 ml stock B and bring to 1 L with distilled water to obtain final concentration aCSF solution and leave at RT while continuously oxygenating it with carbogen (95% O₂ – 5% CO₂ mix) for at least 30 min. Prepare final concentration aCSF only at the beginning of each experiment and discard at the end of the day.

2. Preparation of Sucrose-artificial Cerebrospinal Fluid (Sucrose-aCSF)

1. Preparation of Stock Solution C
   1. Add 700 ml of distilled water to a clean 1 L beaker before adding chemicals.
   2. While continuously stirring 500 ml of distilled water, add 240 g of sucrose, 6.55 g of NaHCO₃, 0.671 g of KCl, 4.88 g of MgCl₂, 0.22 g of CaCl_2, and 0.21 g of ascorbic acid.
   3. Add more distilled water to reach a final volume of 1 L. After dilution, the final concentration of each compound is 701.1 mM sucrose, 78 mM NaHCO₃, 9 mM KCl, 24 mM MgCl₂, 1.5 mM CaCl_2, and 1.2 mM ascorbic acid.
   4. Keep stock solution C at 4 °C for up to one week.
   5. Before each experiment, mix 300 ml of 50 ml of stock C with 600 ml distilled water to obtain the sucrose-aCSF solution at the final concentration. Leave at RT while continuously oxygenating it with carbogen (95% O₂ – 5% CO₂ mix) for at least 30 min.

3. Slice Preparation

1. Place a clean beaker with 100 ml sucrose-aCSF solution in ice while oxygenating it with carbogen and fix the pH at 7.4 using a pH meter while adding a few drops of 0.1 M NaOH solution (when pH < 7.4) or 0.1 M HCl solution (when pH > 7.4).
2. Fill up a cutting chamber of a vibratome with sucrose-aCSF and oxygenate it. Turn on the glycerol-based refrigerating system coupled to the cutting chamber and wait 15 min to allow it to cool down to 0 – 4 °C.
3. Anesthetize rat pups (aged 9 to 12 from adult timed-pregnant Sprague-Dawley rats) with Ketamine (70 mg/kg, i.p.; using < 50 µl final volume). When the pup is calm, double-check that the tail pinch reflex is absent.
4. Decapitate pups.
   1. Cut the head skin longitudinally from the front to back using a carbon steel scalpel blade, and pull the skin to the sides using forceps. Cut the bone covering the brain, move the scissors laterally, and totally remove it to expose the brain.
   2. Then, rapidly remove the brain using a spatula (initially placed under the olfactory bulb to gently push it out from the most rostral towards the most caudal areas). Gently push the brain into ice-cooled oxygenated sucrose-artificial cerebrospinal fluid (sucrose-aCSF).
5. Make a parasagittal cut on the right hemisphere (removing approximately one-third of the hemisphere), and glue the trimmed side of the brain onto a metallic disk that will be magnetically fixed to the cutting chamber of a vibroslicer to cut sagittal 400 µm sections containing the pedunculopontine nucleus (PPN). Keep PPN slices at RT for 45 minutes prior to whole-cell patch-clamp recordings.

4. Recordings of Gamma-band Oscillations in PPN Slices

1. Preparation of Potassium-gluconate Intracellular Solution (High-K+ Solution)
   1. Place in ice a clean beaker with 10 ml distilled water. While continuously stirring add: K+-gluconate, 90.68 mg phosphocreatine di Tris salt, 47.66 mg HEPES, 1.5 mg EGTA, 40.58 mg Mg²⁺-ATP, and 4 mg Na⁺₂-GTP.
   2. Adjust pH to 7.3 with KOH (100 mM in distilled water). Add distilled water to reach a final volume of 20 mL. If needed, adjust osmolarity with sucrose (100 mM in distilled water) to be 280 – 290 mOsm. After dilution, the final concentration of each compound is 124 mM K+-gluconate, 10 mM phosphocreatine di Tris salt, 10 mM HEPES, 0.2 mM EGTA, 4 mM Mg²⁺-ATP, and 0.3 mM Na⁺₂-GTP.
   3. Aliquot intracellular solutions in 1 ml tubes and freeze at -20 °C. Use one aliquot per day and keep at 4 °C during experiments.
2. Whole-cell Patch Clamp Recordings
   1. Place slices in an immersion chamber and perfuse them (1.5 ml/min) with oxygenated (95% O₂ -  5% CO₂) aCSF containing the following receptor antagonists: selective NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV, 40 μM), competitive AMPA/Kainate glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10μM), glycine receptor antagonist strychnine (STR, 10 μM), the specific GABA_A receptor antagonist gabazine (GBZ, 10 μM), and sodium channel blocker tetrodotoxin (TTX, 3μM)
      ​Fill the recording patch pipettes (Resistance 2-7 MΩ; made from regular, commercially available thick wall borosilicate glass capillaries of 1.0 mm outer diameter and 0.6 mm inner diameter) with intracellular high-K+ solution using commercially available patch-pipette fillers with a solution filter. Insert the pipette in its amplifier's holder. Apply a small positive pressure using a 1 ml syringe connected to the pipette holder using a silicon tube connected to a three-way valve. Connect the back of the pipette holder to a patch clamp amplifier.
   2. Move the recording pipette using a mechanical micromanipulator near the PPN nucleus using a 4X objective combined to near-infrared differential interference contrast optics.
      NOTE: PPN nucleus can be observed in slices dorsal to the superior cerebellar peduncle (SCP; observable as a thick bundle of axons). Recording pipettes were located in the PPN pars compacta, which is located immediately dorsal to the posterior end of the peduncle.
   3. Bring the recording pipette in contact with a PPN neuron while visualized using a 40X water immersion lens, and rapidly apply negative suction to form a seal with the cell.
   4. Use voltage-clamp seal software to monitor pipette resistance during negative suction using the manufacturer's protocol.
      1. When negative suction is slowly increased, and resistance values read by the patch-clamp monitor at the tip of the pipette reach 80 – 100 MOhm, rapidly change the holding potential to -50 mV and release the negative pressure. Start continuously applying negative suction until rupturing the neuron's membrane and electrical access is achieved in the whole-cell configuration.
      2. If access resistance values measured by the voltage-clamp seal software are 10 MOhm or higher, then continue applying negative suction in smaller amounts.
   5. Compensate capacitance (i.e., slow and fast transients observed after rupturing the membrane of the cell) and series resistance in voltage-clamp mode. Switch recording mode to the current clamp, and rapidly compensate bridge values (e.g., move the amplifier knob or click on the automatic compensation menu using the computer´s mouse).
      1. Continuously monitor resting membrane potential of PPN neuron being recorded using manufacturer's protocol. If resting membrane potential shift towards depolarizing or hyperpolarizing values, then use small amounts of direct current (up to 100 pA) to keep the optimum -50 mV final value.
         NOTE: Keep only PPN neurons with a stable resting membrane potential (RMP) of -48 mV or more hyperpolarized (i.e., RMP < -48 mV).