Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Podsumowanie

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

Streszczenie

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms underlying sensory transmission, nociceptive regulation, and chronic pain or itch development. Implementations of electrophysiological recordings together with morphological studies based on the utility of acute spinal cord slices have further improved our understanding of neuronal properties and the composition of local circuitry in SG. Here, we present a detailed and practical guide for the preparation of spinal cord slices and show representative whole-cell recording and morphological results. This protocol permits ideal neuronal preservation and can mimic in vivo conditions to a certain extent. In summary, the ability to obtain an in vitro preparation of spinal cord slices enables stable current- and voltage-clamp recordings and could thus facilitate detailed investigations into the intrinsic membrane properties, local circuitry and neuronal structure using diverse experimental approaches.

Wprowadzenie

The substantia gelatinosa (SG, lamina II of the spinal dorsal horn) is an indisputably important relay center for transmitting and regulating sensory information. It is composed of excitatory and inhibitory interneurons, which receive inputs from the primary afferent fibers, local interneurons, and the endogenous descending inhibitory system¹. In recent decades, the development of acute spinal cord slice preparation and the advent of whole-cell patch-clamp recording have enabled various studies on the intrinsic electrophysiological and morphological properties of SG neurons^2,3,4 as well as studies of the local circuitry in SG^5,6. In addition, by using the in vitro spinal cord slice preparation, researchers can interpret the changes in neuronal excitabilities^7,8, the function of ion channels^9,10, and synaptic activities^11,12 under various pathological conditions. These studies have deepened our understanding of the role that SG neurons play in the development and maintenance of chronic pain and neuropathic itch.

Essentially, the key prerequisite to achieve a clear visualization of neuronal soma and ideal whole-cell patching using acute spinal cord slices is to ensure the excellent quality of slices so healthy and patchable neurons can be obtained. However, preparing spinal cord slices involves several steps, such as performing a ventral laminectomy and removing the pia-arachnoid membrane, which may be obstacles in obtaining healthy slices. Although it is not easy to prepare spinal cord slices, performing recordings in vitro on spinal cord slices has several advantages. Compared to cell culture preparations, spinal cord slices can partially preserve inherent synaptic connections that are in a physiologically relevant condition. In addition, whole-cell patch-clamp recording using spinal cord slices could be combined with other techniques, such as double patch clamp^13,14, morphological studies^15,16 and single-cell RT-PCR¹⁷. Therefore, this technique provides more information on characterizing the anatomical and genetic diversities within a specific region and allows for investigation of the composition of local circuitry.

Here, we provide a basic and detailed description of our method for preparing acute spinal cord slices and acquiring whole-cell patch-clamp recordings from SG neurons.

Protokół

All experimental protocols described were approved by the Animal Ethics Committee of Nanchang University (Nanchang, PR China, Ethical No.2017-010). All efforts were made to minimize the stress and pain of the experimental animals. The electrophysiological recordings performed here were carried out at room temperature (RT, 22–25 °C).

1. Animals

1. Use Sprague-Dawley rats (3–5 weeks old) of either sex. House the animals under a 12 h light-dark cycle and give them ad libitum access to adequate food and water.

2. Preparation of Solutions and Materials

1. Solutions
   1. Prepare artificial cerebrospinal fluid (ACSF) (in mM): 117 NaCl, 3.6 KCl, 1.2 NaH₂PO₄·2H₂O, 2.5 CaCl₂·2H₂O, 1.2 MgCl₂·6H₂O, 25 NaHCO₃, 11 D-glucose, 0.4 ascorbic acid, and 2 sodium pyruvate. See Table 1.
   2. Prepare sucrose-ACSF (in mM): 240 Sucrose, 2.5 KCl, 1.25 NaH₂PO₄·2H₂O, 0.5 CaCl₂·2H₂O, 3.5 MgCl₂·6H₂O, 25 NaHCO₃, 0.4 Ascorbic acid, and 2 sodium pyruvate. See Table 2.
   3. Prepare K⁺-based intracellular solution (in mM): 130 K-gluconate, 5 KCl, 10 Na₂-phosphocreatine, 0.5 EGTA, 10 HEPES, 4 Mg-ATP, and 0.3 Li-GTP. See Table 3.
   4. Prepare Cs⁺-based intracellular solution (in mM): 92 CsMeSO₄, 43 CsCl, 10 Na₂-phosphocreatine, 5 tetraethylammonium (TEA)-Cl, 0.5 EGTA, 10 HEPES, 4 Mg-ATP, and 0.3 Li-GTP. See Table 4.
      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 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 potassium channel, which is conducive to using the amplifier to hold the membrane potential steady at 0 mV when recording inhibitory postsynaptic currents (IPSCs).
   5. Prepare the 0.05% neurobiotin 488 solution. Dissolve 2 mg of neurobiotin 488 in 4 mL of K⁺-based intracellular solution and adjust the osmolality to 290–300 mOsm by using distilled water or sucrose if needed.
      Note: 1 mM of sucrose increases osmolarity by 1 mOsm.
   6. Prepare 3% agar as a block for spinal cord. Dissolve 7.5 g of agar in 250 mL of purified water in a glass beaker, and then use a microwave to heat it until boiling and clear. Swirl the solution and pour the mixture into a 17.5 cm x 10.5 cm x 1.8 cm plastic box for solidification afterwards. Keep the agar at 4 °C prior to use.
   7. Prepare 4% paraformaldehyde (PFA) for immunohistochemical processing. Mix 40 g of PFA powder to ~800 mL of heated (approximately 60 °C) 1x PBS solution (130 mM NaCl, 7 mM Na₂HPO₄, 3 mM NaH₂PO₄). Slowly adjust the pH by adding 1 N NaOH drops until PFA powder is completely dissolved. Once the solution has become clear, adjust the total volume to 1 L with 1x PBS. Readjust the pH to 7.2–7.4 using 1 N HCl when needed, then filter the 4% PFA solution and store it at -20 °C until use.
      Note: PFA is toxic, so it is needed to wear masks, gloves as well as safety glasses. Conduct the process of preparing 4% PFA inside a ventilated hood. PFA powder can be completely dissolved at a pH of approximately 9–10.
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.
4. Agar block
   1. Prepare a 1.2 cm x 1.5 cm x 2.0 cm agar block. Trim the block into the shapes shown in Figure 1 as required.

3. Acute Spinal Cord Slice Preparation

Note: Transverse or parasagittal spinal cord slices are prepared as previously described^18,19,20.

1. Prior to transcardial perfusion and spinal cord extraction, prepare ~500 mL sucrose-ACSF equilibrated with 95% O₂ and 5% CO₂ and cool the solution to ice-cold (0–4 °C).
2. Cool down all the dissection tools (e.g., dissecting scissors, iris scissors, toothed forceps, fine forceps, curved forceps) on the ice. The diagram of the preparation of acute spinal cord slice is shown in Figure 1.
3. Transcardial perfusion
   1. After a single injection (intraperitoneal, i.p.) of urethane (1.5 g/kg), wait for 2–3 min, and assess the anesthesia depth of rats by testing toe or tail pinch responses. Once a surgical plane of anesthesia is maintained, place the rat on crushed ice in the supine position.
      Note: For producing profound anesthesia sufficient for non-perception of pain during thoracotomy, follow your local IACUC guidelines.
   2. Make an incision (3–4 cm) through the skin from the xiphoid process to the clavicle, and then make a transverse incision beneath the level of the xiphoid process.
   3. Grasp and raise the xiphoid process with a pair of curved forceps to expose the diaphragm completely. Make a transverse incision through the diaphragm, and then cut through the ribcage between the breastbone and ribs bilaterally to open the chest cavity. Use the curved forceps to grasp the xiphoid process so the ribcage is fixed, and the heart is sufficiently exposed.
   4. Hold the heart with another curved forceps gently, and then insert a 22 G needle through the left ventricle to the base of the aortic arch.
   5. Cut the right atrium with fine scissors immediately, and then start the perfusion of ice-cold, oxygenated sucrose-ACSF through a gravity system.
      Note: Rapid and sufficient transcardial perfusion with ice-cold sucrose-ACSF can facilitate the rapid cooling of the spinal cord, while low sodium and calcium concentration may help alleviate excitatory toxicity and protect neuronal function. In addition, clearing red blood cells would be beneficial for reducing the background staining of biocytin when performing a morphological study. The perfusion is considered sufficient and satisfied as long as the fluid exiting the right atrium is clear and the color of the rat’s liver and paws is pale. The tip of the 22 G needle should be blunt to avoid rupturing the heart and aortic arch.
4. Spinal cord dissection and slicing
   1. Make a longitudinal incision (5 cm) on the back skin from caudal to rostral, then cut through the ribcage between the spine and ribs on either side in the state of perfusion.
   2. Make a cut at the caudal end of the spine, use scissors to cut away surrounding tissues, and isolate the lumbosacral segment of the spine rapidly.
   3. Transfer the lumbosacral segment to a glass dish containing ice-cold sucrose-based ACSF. With the ventral side upward, use fine scissors to cut through the vertebral pedicle bilaterally and expose the spinal cord carefully. Isolate a 2-cm long spinal cord with lumbosacral enlargement (L1–S3) and transfer the spinal section to another glass dish filled with cold sucrose-ACSF.
   4. Remove the meninges and the pia-arachnoid membrane under a dissecting microscope. Cut all the ventral and dorsal roots away as quickly as possible.
   5. Place the spinal cord on a previously trimmed agar block. To prepare transverse slices, attach the ventral side of the spinal cord to the agar and let the dorsal side toward the blade. To prepare parasagittal slices, attach the ventral side with superglue to the agar in a vertical direction as shown in Figure 1. Then, mount the agar block to a platform of a vibratome with superglue. Prepare 300–500 µm transverse or parasagittal slices with an advance speed of 0.025 mm/s and a vibration frequency of 80 Hz.
   6. Use a plastic-trimmed pipette to transfer the slices onto nylon mesh in a storage chamber containing continuously oxygenated ACSF at 32 °C for at least 30 min prior to recording.
      Note: Take care to avoid injury to the spinal cord, especially the dorsal horn, when removing the meninges and spinal roots. The spinal cord should be sliced dorsal-ventrally. For the best results, the slices should be prepared rapidly (within 15–20 min). The thickness of a spinal slice should be no more than 600 µm to satisfy cell visibility. In addition, the technique described above could be used to obtain horizontal spinal slices.

4. Whole-cell Patch-clamp Recordings

1. To conduct the whole-cell patch-clamp recordings from 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 is contacting the silver wire inside the holder.
5. Bring the micropipette into focus and 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 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 RT, the liquid junction potential is calculated to be 15.1–15.2 mV and 4.3–4.4 mV in K⁺-based and Cs⁺-based intracellular solution, respectively. In our studies, the recorded data were not corrected for liquid junction potential.
8. Recordings of intrinsic membrane properties
   1. Record passive intrinsic membrane properties: Record RMP immediately (within 20 s) after break in. Determine the neuronal input resistance by measuring the voltage response to a depolarizing current (10 pA, 500 ms) at RMP in current-clamp mode.
   2. Record firing properties: Test the firing pattern of each neuron in current-clamp with a series of 1 s depolarizing current pulses (25–150 pA with 25 pA increment) at RMP. Measure the threshold, amplitude and half-width of a single action potential offline.
   3. Record subthreshold current: To assess the somatic subthreshold currents, hold the membrane potential at -50 mV in voltage-clamp mode. Then, apply a series of hyperpolarizing voltage pulses of 1 s duration from -60 mV to -120 mV, with a 10-mV decrement.
   4. Record excitatory postsynaptic currents (EPSCs): Record EPSCs with the K⁺-based intracellular solution in voltage-clamp mode at a holding potential of -70 mV.
   5. Record IPSCs: Apply Cs⁺-based intracellular solution for recording IPSCs. Once the whole-cell configuration is established, hold the membrane potential at -70 mV for ~5 min, and then change the holding potential to 0 mV gradually. Wait a few minutes for stabilization, and then start to record the IPSC events.
      Note: Only neurons with an RMP less than -50 mV and showing overshoot should be selected for further study. The series resistances in our study are typically 10-30 MΩ, and a recording should be excluded once the series resistance changes by more than 20%. EPSCs could be confirmed with a bath application of 50 µM APV and 20 µM CNQX, while IPSCs could be confirmed with 10 µM bicuculline and 1 µM strychnine.

5. Morphological study

1. For morphological experiments, use a K⁺-based intracellular solution containing 0.05% neurobiotin 488.
2. After maintaining a stable electrophysiological recording for at least 20 min, slowly remove the micropipette in the upward direction to allow the cell membrane to reseal and transfer the spinal cord slice to a container filled with 4% PFA. Fix the slices in 4% PFA at RT for 1 h and then at 4°C overnight.
3. Rinse the slices in PBS, and then immerse them in 50% ethanol for 30 min. After another three washes in PBS, mount the slices in their original thickness onto slides with a mounting medium.
4. Use a confocal microscope (see Table of Materials) for image acquisition and neuronal 3D reconstruction. Scan neurons through a 20X lens with a z-stack of 1.5 µm.

Wyniki

Acute spinal cord slices were prepared according to the diagram shown in Figure 1. After slicing and recovery, a spinal cord slice was transferred to the recording chamber. Healthy neurons were identified based on soma appearance using IR-DIC microscopy. Next, the action potentials of SG neurons were elicited by a series of depolarizing current pulses (1 s duration) when neurons were held at RMP. As shown in Figure 2, the firing ...

Dyskusje

This protocol details the steps for preparing spinal cord slices, which we have used successfully when performing whole-cell patch-clamp experiments on SG neurons^18,19,20,21. By implementing this method, we recently reported that minocycline, a second generation of tetracycline, could markedly enhance inhibitory synaptic transmission through a presynaptic mechanism in SG neurons

Materiały

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
Neurobiotin 488	Vector	SP-1145	0.05% neurobiotin 488 could be used for morphological studies
Agar	Sigma	A7002	3% agar block was used in our protocol
Paraformaldehyde	Sigma	P6148	4% paraformaldehyde was used for immunohistochemical processing
Na2HPO4	Hengxing Chemical Reagents		Used for the preparation of PBS
Mount Coverslipping Medium	Polyscience	18606
Urethan	National Institute for Food and Drug Control	30191228	1.5 g/kg, i.p.
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
Vibratome	Leica	VT1000S
Vibration isolation table	Technical Manufacturing Corporation	63544
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
Confocol microscope	Zeiss	LSM 700