Whole-Cell Patch Clamp Recording in a Substantia Gelatinosa Neuron of a Spinal Cord Slice

Abstract

Source: Zhu, M., et al. Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons. J. Vis. Exp. (2019)

This video demonstrates the procedure for performing electrophysiological recordings in substantia gelatinosa (SG) neurons of spinal cord slices, using whole-cell patch-clamp techniques to analyze neuron firing patterns. This method allows the study of neural responses to stimuli in SG neurons of the spinal cord.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review.

1. Preparation of Solutions and Materials

1. Solutions​
   1. Prepare artificial cerebrospinal fluid (ACSF) (in mM): 117 sodium chloride (NaCl), 3.6 potassium chloride (KCl), 1.2 sodium dihydrogen phosphate dihydrate (NaH₂PO₄·2H₂O), 2.5 calcium chloride dihydrate (CaCl₂·2H₂O), 1.2 magnesium chloride hexahydrate (MgCl₂·6H₂O), 25 sodium bicarbonate (NaHCO₃), 11 D-glucose, 0.4 ascorbic acid, and 2 sodium pyruvate. See Table 1.
   2. Prepare K⁺-based intracellular solution (in mM): 130 K-gluconate, 5 KCl, 10 Na₂-phosphocreatine, 0.5 ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 10 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 4 magnesium adenosine triphosphate (Mg-ATP), and 0.3 lithium guanosine triphosphate (Li-GTP). See Table 2.
   3. Prepare Cesium (Cs⁺)-based intracellular solution (in mM): 92 cesium methane sulfonate (CsMeSO₄), 43 cesium chloride (CsCl), 10 Na₂-phosphocreatine, 5 tetraethylammonium (TEA)-Cl, 0.5 EGTA, 10 HEPES, 4 Mg-ATP, and 0.3 Li-GTP. See Table 3.
      Note: All solutions must be prepared using distilled water. ACSF and sucrose-ACSF should be carbogenated (95% O₂ and 5% CO₂ mixture) prior to use to maintain an optimal pH of approximately 7.4, and the osmolality of these two solutions should be adjusted to 300–310 milliosmols (mOsm). Because ascorbic acid could affect calcium channels, this agent must be omitted if one would like to record calcium currents. The osmolality and pH of intracellular solutions should be measured and adjusted to 290–300 mOsm and 7.2–7.3, respectively. It is recommended to filter the intracellular solutions with 0.2 µm filters and store the solutions as 1 mL aliquots at -20 °C. Cs⁺ and TEA are applied in Cs⁺-based intracellular solution to block the potassium channel, which is conducive to using the amplifier to hold the membrane potential steady at 0 mV when recording inhibitory postsynaptic currents (IPSCs).
2. Instruments
   1. For a typical electrophysiological system, use an upright microscope equipped with infrared differential interference contrast (IR-DIC) and a high-resolution water-immersion objective, a CCD/CMOS camera, a patch-clamp amplifier, a micropipette holder, and a micromanipulator allowing fine adjustment of the pipette position. An XY stage is also needed to move the microscope.
   2. Mount all equipment on a vibration isolation table surrounded by a Faraday cage. Connect a video monitor to the video camera to observe the neurons and visualize the micropipettes.
3. Micropipettes
   1. Make recording electrodes from borosilicate glass capillaries using a micropipette puller. The typical pipette resistance ranges from 3-6 MΩ when filled with intracellular solution.

2. Whole-cell Patch-clamp Recordings

1. To conduct the whole-cell patch-clamp recordings from substantia gelatinosa (SG) neurons, use K⁺-based intracellular solution for most recording cases while applying Cs⁺-based solution only for the recording of inhibitory postsynaptic currents.
2. Gently move a spinal cord slice to the recording chamber, and then maintain it with a U-shaped platinum wire attached with nylon threads firmly for optimal slice stability. Steadily perfuse the slice with bubbled ACSF at RT through a gravity system and set the perfusion rate at 2–4 mL/min to achieve sufficient oxygenation.
3. Identify the region of SG (a translucent band) using a low-resolution microscope lens, choose a healthy neuron by using the high-resolution objective as the target cell, and adjust it to the center of the video monitor screen.
4. Fill a micropipette with an appropriate volume of K⁺-based or Cs⁺-based intracellular solution as needed, insert the micropipette into the electrode holder, and ensure that the intracellular solution contacts the silver wire inside the holder.
5. Bring the micropipette into focus, immerse it into the ACSF using a micromanipulator, and then apply a mild positive pressure (~1 psi when measured with a manometer) to force the micropipette away from any dirt and debris.
6. Move the micropipette towards the targeted neuron gradually. Release the positive pressure once the pipette approaches the neuron, and a very small dimple forms on the neuronal membrane to form a gigaseal.
7. Alter the holding potential to -70 mV, which is close to the physiological resting membrane potential (RMP) of a cell. Then, apply a transient and gentle suction to the micropipette to rupture the membrane and create a good whole-cell configuration.
   Note: After transferring the slice into the recording chamber, ensure steady perfusion for at least 5 min to clear the debris on the slice surface. It is worth noting that the ability to distinguish between healthy and unhealthy/dead neurons is of paramount importance for good sealing and stable recording. An unhealthy/dead neuron has a swollen or shrunken appearance, together with a visible large nucleus, while a healthy neuron is characterized by a 3-dimensional (3D) shape with a bright and smooth membrane, and its nucleus is invisible. To achieve a whole-cell configuration, it is essential to compensate for fast or slow capacitance step-by-step when necessary. At room temperature (RT), the liquid junction potential is calculated to be 15.1–15.2 millivolts (mV) and 4.3–4.4 mV in K⁺-based and Cs⁺-based intracellular solutions, respectively. In our studies, the recorded data were not corrected for liquid junction potential.
8. Recordings of intrinsic membrane properties
   1. Record firing properties: Test the firing pattern of each neuron in the current clamp with a series of 1 s depolarizing current pulses (25–150 picoamps (pA) with 25 pA increment) at RMP. Measure the threshold, amplitude, and half-width of a single action potential offline.

Table 1: Recipe for ACSF

Component	Molecular Weight	Concentration (mM)	g/L
NaCl	58.5	117	6.84
KCl	74.5	3.6	0.27
NaH2PO4·2H2O	156	1.2	0.19
CaCl2·2H2O	147	2.5	0.37
MgCl2·6H2O	203	1.2	0.24
NaHCO3	84	25	2.1
D-Glucose	180	11	1.98
Ascorbic acid	198.11	0.4	0.08
Sodium pyruvate	110	2	0.22

Table 2: Recipe for K⁺-based intracellular solution

Component	Molecular Weight	Concentration (mM)	mg/100 mL
K-gluconate	234.2	130	3044.6
KCl	74.5	5	37.28
Na2-Phosphocreatine	453.38	10	453.38
EGTA	380.35	0.5	19.02
HEPES	238.31	10	238.3
Mg-ATP	507.18	4	202.9
Li-GTP	523.18	0.3	15.7

Table 3: Recipe for Cs⁺-based intracellular solution

Component	Molecular Weight	Concentration (mM)	mg/100 mL
CsMeSO4	228	92	2097.6
CsCl	168.36	43	723.95
Na2-Phosphocreatine	453.38	10	453.38
TEA-Cl	165.71	5	82.86
EGTA	380.35	0.5	19.02
HEPES	238.31	10	238.3
Mg-ATP	507.18	4	202.9
Li-GTP	523.18	0.3	15.7

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
NaCl	Sigma	S7653	Used for the preparation of ACSF and PBS
KCl	Sigma	60130	Used for the preparation of ACSF, sucrose-ACSF, and K+-based intracellular solution
NaH2PO4·2H2O	Sigma	71500	Used for the preparation of ACSF, sucrose-ACSF and PBS
CaCl2·2H2O	Sigma	C5080	Used for the preparation of ACSF and sucrose-ACSF
MgCl2·6H2O	Sigma	M2670	Used for the preparation of ACSF and sucrose-ACSF
NaHCO3	Sigma	S5761	Used for the preparation of ACSF and sucrose-ACSF
D-Glucose	Sigma	G7021	Used for the preparation of ACSF
Ascorbic acid	Sigma	P5280	Used for the preparation of ACSF and sucrose-ACSF
Sodium pyruvate	Sigma	A7631	Used for the preparation of ACSF and sucrose-ACSF
Sucrose	Sigma	S7903	Used for the preparation of sucrose-ACSF
K-gluconate	Wako	169-11835	Used for the preparation of K+-based intracellular solution
Na2-Phosphocreatine	Sigma	P1937	Used for the preparation of intracellular solution
EGTA	Sigma	E3889	Used for the preparation of intracellular solution
HEPES	Sigma	H4034	Used for the preparation of intracellular solution
Mg-ATP	Sigma	A9187	Used for the preparation of intracellular solution
Li-GTP	Sigma	G5884	Used for the preparation of intracellular solution
CsMeSO4	Sigma	C1426	Used for the preparation of Cs+-based intracellular solution
CsCl	Sigma	C3011	Used for the preparation of Cs+-based intracellular solution
TEA-Cl	Sigma	T2265	Used for the preparation of Cs+-based intracellular solution
Na2HPO4	Hengxing Chemical Reagents		Used for the preparation of PBS
Borosilicate glass capillaries	World Precision Instruments	TW150F-4	1.5 mm OD, 1.12 mm ID
Micropipette puller	Sutter Instrument	P-97	Used for the preparation of micropipettes
Infrared CCD camera	Dage-MIT	IR-1000
Patch-clamp amplifier	HEKA	EPC-10
Micromanipulator	Sutter Instrument	MP-285
X-Y stage	Burleigh	GIBRALTAR X-Y
Upright microscope	Olympus	BX51WI
Osmometer	Advanced	FISKE 210
PH meter	Mettler Toledo	FE20