Establishing a Whole-Cell Voltage-Clamp Configuration for Electrophysiological Recordings in Brain Slices

Protocollo

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

1. Solutions

1. Slicing solution​
   1. Refer to Table 1 for the composition of the slicing solution.
   2. Prepare a 20x stock solution in advance and store it at 4 °C for up to 1 month.
   3. For the 1x slicing solution, dissolve NaHCO₃, glucose, and sucrose in ddH₂O and add the 20x stock. Ensure the osmolarity is between 315 and 320 mOsm and store the solution for no more than 1 week at 4 °C.
   4. Fill two beakers with 100 mL of slicing solution and cover them with parafilm. Chill the solution in a freezer until it becomes partially frozen (approximately 20 min in a -80 °C freezer). Using a gas dispersion tube, bubble both beakers of slicing solution with 95% O2/5% CO2 for 20 min on ice.
2. aCSF(Artificial cerebrospinal fluid), for slice recovery and maintenance
   1. Refer to Table 1 for the composition of the aCSF.
   2. Prepare a 20x stock solution in advance and store it at 4 °C for up to 1 month.
   3. For 1x aCSF, dissolve NaHCO3 and glucose in ddH2O and add the 20x stock. Ensure the osmolarity is between 298-300 mOsm. Use the solution within 1 day.
3. aCSF (low Ca²⁺ for recording)
   1. Refer to Table 1 for the composition of the low Ca²⁺ aCSF.
   2. Prepare a 20x stock solution in advance and store it at 4 °C for up to 1 month.
   3. For 1x low Ca²⁺ aCSF, dissolve NaHCO3 and glucose in ddH2O and add 20x (CaCl2 and MgCl2 free) stock, CaCl2 and MgCl2 to specified concentrations. Ensure the osmolarity is between 298-300. Use the solution within 1 day.
4. aCSF (Sr²⁺ for recording)
   1. Refer to Table 1 for the composition of the Sr²⁺ aCSF.
   2. Prepare a 20x stock solution in advance and store it at 4 °C for up to 1 month.
   3. For 1x Sr²⁺ aCSF, dissolve NaHCO3 and glucose in ddH2O and add 20x (CaCl2 and MgCl2 free) stock, SrCl2 and MgCl2 to specified concentrations. Ensure the osmolarity is between 298-300. Use the solution within 1 day.
5. Internal solution
   1. Refer to Table 1 for the composition of the K-gluconate-based internal solution.
   2. To make 20 mL of internal solution, add 15 mL of molecular biology-grade water to a 50 mL tube. Then, perform the subsequent steps on ice.
   3. Prepare the following solutions ahead of time to 1 M stock concentrations in molecular biology grade water. Add (in mL): 2.32 K-gluconate, 0.24 Na-gluconate, 0.20 HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 0.16 KCl, 0.05 K₂-EGTA (Potassium ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid), 0.04 MgCl2 to the 50 mL tube.
   4. Add 100 µL of 0.3 M Na₃GTP (Guanosine 5′-triphosphate sodium salt)
   5. Weigh 44.08 mg of K2 ATP (Adenosine 5′-triphosphate dipotassium salt) in a 2 mL microcentrifuge tube and add 1 mL of molecular biology grade water, then add to a 50 mL tube.
   6. Adjust the pH to 7.2-7.4 with 1 M KOH. Ensure the osmolarity is between 283-289 mOsm.

2. Slice Preparation

1. Prepare tools
   1. Add 200 mL of aCSF to the recovery chamber (constructed from a 250 mL beaker with 4 wells and netting) and place the recovery chamber in a water bath (35 °C).
   2. Cover the chamber with a paraffin film and constantly bubble the aCSF with 95% O2/5% CO2 using a glass dispersion tube for at least 20 min.
   3. Set up the dissection tools (scalpel, angled fine scissors, forceps, fine paintbrush, plastic spoon).
   4. Fill a 60 mL syringe with approximately 15 mL of the ice-cold slicing solution from step 1.1.4.
   5. Prepare the dissection platform by placing filter paper on the lid of a well plate.
   6. Prepare the slicing chamber by placing it in the ice tray and filling the tray with ice.
   7. Set up the vibratome by securing a disposable blade in the blade holder.
   8. To make a transfer pipette, break the tip of a Pasteur pipette and place a rubber bulb over the broken end.
2. Dissect mouse brain
   1. Anesthetize the animal in a chamber saturated with 4% isoflurane until spinal reflexes are absent.
   2. Decapitate the animal using a guillotine and quickly remove the brain.
      1. Make a midline incision with a No. 22 scalpel blade from rostral to caudal.
      2. Laterally, peel the scalp on each side of the head.
      3. Use fine scissors to cut the skull on one side from caudal to rostral (including the side of the frontal bones), using caution not to damage the brain.
      4. Use forceps to lift the skull piece off the brain and quickly cool the brain with 15 mL of ice-cold slicing solution using the syringe from step 2.1.4.
      5. Lift the brain out of the skull.
      6. Place the brain in one of the beakers filled with ice-cold slicing solution (from step 1.1.4) bubbled with 95% O2/5% CO2.

3. Prepare slices of mouse hypothalamus.

1. Block the brain for the desired brain area and cut angle (e.g., for coronal hypothalamic slices, trim off the tissue rostral to the optic chiasm and caudal to the pons using a blade and ensure the caudal block has a flat surface perpendicular to the base of the brain).
2. Using a cut piece of filter paper, pick up the brain from the anterior side and glue the posterior side to the holding plate using instant glue.
3. Quickly place the holding plate into the slicing chamber and fill it with the slicing solution from the second beaker in step 1.1.4.
4. Secure the slicing chamber and ice tray on the vibratome.
5. Define the slicing area (anterior and posterior to the brain) and begin slicing 250 µm thickness coronal slices. Recommended parameters: speed 0.10 mm/s, amplitude 2 mm.
6. Trim the slices to the appropriate size for the desired brain area.
7. Recover the slices at 35 °C for 30-45 min. Then, remove the recovery chamber from the warming bath and allow the slices to recover at room temperature for an additional 30 min. Keep slices at room temperature for the rest of the day and continue to bubble the bath constantly with 95% O2/5% CO2.

3. Whole-cell Patch ClampRecording

1. Pull the patch pipettes.​
   1. Using the suggested parameters for the whole-cell recording from the pipette puller’s manual, pull patch pipettes from thick-walled glass to a pipette resistance of 3-5 MΩ.
   2. Using a microsyringe (commercial or homemade), fill a pipette tip with a filtered internal solution. To make a micro syringe, burn the tip of a 1 mL syringe and allow the tip to fall, creating a long, fine tip.
2. Obtain the whole-cell configuration.
   1. Place the recording pipette just above the slice and offset the pipette current in the voltage clamp mode. Apply slight positive pressure to the pipette and lock the stopcock.
   2. Select a healthy cell with an intact membrane and approach it with the pipette. The positive pressure should cause a slight disturbance in the tissue (i.e., a slow wave in the tissue when entering).
   3. Slowly continue to bring the pipette closer to the cell using a diagonal motion until the pipette forms a small dimple on the cell surface.
   4. Release the positive pressure lock. The cell will begin to form a seal, and the resistance will increase above 1 GΩ. In the voltage clamp, hold the cell at -68 mV.
   5. Slightly pull away from the cell diagonally to remove excess pressure from the cell.
   6. Compensate for the fast and slow pipette capacitance.
   7. Apply a brief suction through the tube connected to the pipette holder to break through the cell and obtain a whole-cell configuration.
   8. Switch to Cell mode on the membrane test window in an electrophysiology Data acquisition and analysis software (e.g., Clampex).
   9. Before each voltage clamp recording, perform a membrane test using the same software and record the relevant parameters in a lab book (membrane resistance, access resistance, and capacitance).
   10. Maintain the temperature of the recording bath at 27–30 °C and the flow rate at 1.5–2.0 mL/min for subsequent experiments.

Table 1: The composition of various solutions.

	Solution Concentrations (mM)
	Slicing	Normal aCSF	Low Ca2+ aCSF	Sr+ aCSF	Pipette/Internal
NaCl	87	126	126	126	-
KCl	2.5	2.5	2.5	2.5	8
CaCl2	0.5	2.5	0.5	-	-
SrCl2	-	-	-	2.5	-
MgCl2	7	1.5	2.5	1.5	2
NaH2PO4	1.25	1.25	1.25	1.25	-
NaHCO3	25	26	26	26	-
Glucose	25	10	10	10	-
Sucrose	75	-	-	-	-
K-gluconate	-	-	-	-	116
Na-gluconate	-	-	-	-	12
HEPES	-	-	-	-	10
K2-EGTA	-	-	-	-	1
K2ATP	-	-	-	-	4
Na3GTP	-	-	-	-	0.3

Materiali

Name	Company	Catalog Number	Comments
1 ml syringe	BD	309659
10 blade	Fisher Scientific/others	35698
22 blade	VWR/others	21909-626
22 uM syringe filters	Milipore	09-719-000
Adson foreceps	Harvard Instruments	72-8547
Angled sharp scissors	Harvard Instruments	72-8437
Clampex	Molecular Devices	pClamp 10
Double edge blade	VWR	74-0002
Filter paper	Sigma/others	1001090
Fine paintbrush	Fisher/various	15-183-35/various
Gas Dispersion Tube	VWR	LG-8680-120
Isoflurane	Fresenius Kabi/others	M60303
Krazy glue	various	various
Mini analysis	Synaptosoft	MiniAnalysis 6
Osmomoter	Wescor Inc	Model 5600
Parafilm	Sigma	PM-996
Pasteur pipette	VWR	14672-200
ph meter	Mettler Toledo	FE20-ATC
Rubber bulb	VWR	82024-550
Scalpel handle No. 3	Harvard Instruments	72-8350
Scalpel handle No. 4	Harvard Instruments	72-8356
Single edge blade	VWR	55411-050
Vibratome slicer	Leica	VT1200S
Water Purification System	Millipore	Milli-Q Academic A10
Well plate lid	Fisher/various	07-201-590/various
CaCl2 2H2O	Sigma	C7902
CdCl2	sigma	202908
EGTA	Sigma	E3889
glucose	Sigma	G5767
HEPES	Sigma	H3375
K2-ATP	Sigma	A8937
KCl	Sigma	P9333
K-gluconate	Sigma	G4500
MgCl2 6H2O	Sigma	M2670
Molecular biology grade water	Sigma	W4502-1L
Na3GTP	Sigma	G8877
NaCl	Bioshop	SOD001.1
Na-gluconate	Sigma	S2054
NaH2PO4	Sigma	71504
NaHCO3	Sigma	S6014
Picrotoxin	sigma	P1675
SrCl	Sigma	255521
sucrose	Bioshop	SUC507.1