This work was supported by grants from the National Natural Science Foundation of China (No. 81560198, 31660289).

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&#8211;25 &#176;C).
1. Animals
<ol>
	<li>Use Sprague-Dawley rats (3&#8211;5 weeks old) of either sex. House the animals under a 12 h light-dark cycle and give them ad...

<ol>
	<li>Todd, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+circuitry+for+pain+processing+in+the+dorsal+horn.">Neuronal circuitry for pain processing in the dorsal horn.</a> Nature Reviews Neuroscience. 11 (12), 823-836 (2010).</li><li>Yoshimura, M., Nishi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blind+patch-clamp+recordings+from+substantia+gelatinosa+neurons+in+adult+rat+spinal+cord+slices:+pharmacological+properties+of+synaptic+currents.">Blind patch-clamp recordings from substantia gelatinosa neurons in adult rat spinal cord slices: pharmacological properties of synaptic currents.</a> Neuroscience. 53 (2), 519-526 (1993).</li><li>Maxwell, D. J., Belle, M. 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H., Hu, T., Liu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minocycline+enhances+inhibitory+transmission+to+substantia+gelatinosa+neurons+of+the+rat+spinal+dorsal+horn.">Minocycline enhances inhibitory transmission to substantia gelatinosa neurons of the rat spinal dorsal horn.</a> Neuroscience. 319, 183-193 (2016).</li><li>Peng, S. 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This protocol details the steps for preparing spinal cord slices, which we have used successfully when performing whole-cell patch-clamp experiments on SG neurons18,19,20,21. 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<sup class="xref...

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 neurons2,3,4 as well as studies of the local circuitry in SG5,6. In addition, by using the in vitro spinal cord slice preparation, researchers can interpret the changes in neuronal excitabilities7,8, the function of ion channels9,10, and synaptic activities11,12 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 clamp13,14, morphological studies15,16 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.

<table>
	<tbody>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td>Used for the preparation of ACSF and PBS</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>60130</td>
			<td>Used for the preparation of ACSF, sucrose-ACSF, and K+-based intracellular solution</td>
		</tr>
		<tr>
			<td>NaH2PO4&#183;2H2O</td>
			<td>Sigma</td>
			<td>71500</td>
			<td>Used for the preparation of ACSF, sucrose-ACSF and PBS</td>
		</tr>
		<tr>
			<td>CaCl2&#183;2H2O</td>
			<td>Sigma</td>
			<td>C5080</td>
			<td>Used for the preparation of ACSF and sucrose-ACSF</td>
		</tr>
		<tr>
			<td>MgCl2&#183;6H2O</td>
			<td>Sigma</td>
			<td>M2670</td>
			<td>Used for the preparation of ACSF and sucrose-ACSF</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td>Used for the preparation of ACSF and sucrose-ACSF</td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma</td>
			<td>G7021</td>
			<td>Used for the preparation of ACSF</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma</td>
			<td>P5280</td>
			<td>Used for the preparation of ACSF and sucrose-ACSF</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma</td>
			<td>A7631</td>
			<td>Used for the preparation of ACSF and sucrose-ACSF</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S7903</td>
			<td>Used for the preparation of sucrose-ACSF</td>
		</tr>
		<tr>
			<td>K-gluconate</td>
			<td>Wako</td>
			<td>169-11835</td>
			<td>Used for the preparation of K+-based intracellular solution</td>
		</tr>
		<tr>
			<td>Na2-Phosphocreatine</td>
			<td>Sigma</td>
			<td>P1937</td>
			<td>Used for the preparation of intracellular solution</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E3889</td>
			<td>Used for the preparation of intracellular solution</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td>Used for the preparation of intracellular solution</td>
		</tr>
		<tr>
			<td>Mg-ATP</td>
			<td>Sigma</td>
			<td>A9187</td>
			<td>Used for the preparation of intracellular solution</td>
		</tr>
		<tr>
			<td>Li-GTP</td>
			<td>Sigma</td>
			<td>G5884</td>
			<td>Used for the preparation of intracellular solution</td>
		</tr>
		<tr>
			<td>CsMeSO4</td>
			<td>Sigma</td>
			<td>C1426</td>
			<td>Used for the preparation of Cs+-based intracellular solution</td>
		</tr>
		<tr>
			<td>CsCl</td>
			<td>Sigma</td>
			<td>C3011</td>
			<td>Used for the preparation of Cs+-based intracellular solution</td>
		</tr>
		<tr>
			<td>TEA-Cl</td>
			<td>Sigma</td>
			<td>T2265</td>
			<td>Used for the preparation of Cs+-based intracellular solution</td>
		</tr>
		<tr>
			<td>Neurobiotin 488</td>
			<td>Vector</td>
			<td>SP-1145</td>
			<td>0.05% neurobiotin 488 could be used for morphological studies</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma</td>
			<td>A7002</td>
			<td>3% agar block was used in our protocol</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td>4% paraformaldehyde was used for immunohistochemical processing</td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Hengxing Chemical Reagents</td>
			<td></td>
			<td>Used for the preparation of PBS</td>
		</tr>
		<tr>
			<td>Mount Coverslipping Medium</td>
			<td>Polyscience</td>
			<td>18606</td>
			<td></td>
		</tr>
		<tr>
			<td>Urethan</td>
			<td>National Institute for Food and Drug Control</td>
			<td>30191228</td>
			<td>1.5 g/kg, i.p.</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries</td>
			<td>World Precision Instruments</td>
			<td>TW150F-4</td>
			<td>1.5 mm OD, 1.12 mm ID</td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instrument</td>
			<td>P-97</td>
			<td>Used for the preparation of micropipettes</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1000S</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibration isolation table</td>
			<td>Technical Manufacturing Corporation</td>
			<td>63544</td>
			<td></td>
		</tr>
		<tr>
			<td>Infrared CCD camera</td>
			<td>Dage-MIT</td>
			<td>IR-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Patch-clamp amplifier</td>
			<td>HEKA</td>
			<td>EPC-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Sutter Instrument</td>
			<td>MP-285</td>
			<td></td>
		</tr>
		<tr>
			<td>X-Y stage</td>
			<td>Burleigh</td>
			<td>GIBRALTAR X-Y</td>
			<td></td>
		</tr>
		<tr>
			<td>Upright microscope</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmometer</td>
			<td>Advanced</td>
			<td>FISKE 210</td>
			<td></td>
		</tr>
		<tr>
			<td>PH meter</td>
			<td>Mettler Toledo</td>
			<td>FE20</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocol microscope</td>
			<td>Zeiss</td>
			<td>LSM 700</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of acute spinal cord slices for whole-cell patch-clamp recording in substantia gelatinosa neurons

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.

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 ...

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

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.

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...

This method can help clarify the spinal mechanisms underlying sensory transmission, nociceptive regulation and chronic pain or ache development The main advantage of this technique is that it can permit ideal neuronal preservation and it can mimic in-vivo conditions to a certain extent. Start by pulling recording electrodes from borosilicate glass microcapillaries using a capillary puller. Then, fill a mold with molten agar to prepare a 1.2 centimeter by 1.5 centimeter by 2.0 centimeter agar block.Trim the block to have a central channel if planning transverse sections. Or a channel flesh with the edge of the block for parasagittal sections. Prior to transcardial profusion and spinal cord extraction, prepare 500 milliliters of sucrose based ACSF.Cool the solution to ice cold and equilibrate with 95%O2 and 5%CO2. Cool all the dissection tools on ice. Make a longitudinal five centimeter incision on the back skin from caudal to rostral.Then, cut through the rib cage between the spine and ribs on either side. Use scissors to made a cut at the caudal end of the spine. Then cut away the surrounding tissues and quickly isolate the lumbosacral segment of the spine.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 two centimeter long section of spinal cord with the lumbosacral enlargement and transfer the spinal section into another glass dish filled with cold sucrose ACSF.Work under a dissection microscope to remove the meninges and the pia arachnoid membrane. Cut all the ventral and dorsal roots away as quickly as possible. Take care to avoid the injuring to the spinal cord, especially the dorsal horn when removing the meninges and the spinal roots.Place the spinal cord on the previously trimmed agar block. To prepare transverse slices, attach the ventral side of the spinal cord to the agar with the dorsal side toward the blade. To prepare parasagittal slices, attach the ventral side with superglue to the agar in a vertical direction.Then, mount the agar block to a platform of a vibratome with superglue. And prepare 300 to 500 micron transverse or parasagittal slices with an advanced speed of 0.025 millimeters per second and a vibration frequency of 80 Hertz. The spinal cord should be sliced dorso-ventrally.For the best results, the slice should be prepared within 15 to 20 minutes. The thickness of the spinal slice should be no more than 600 microns to satisfy cell visibility. Use a plastic trimmed pipette to transfer the slices onto nylon mesh in a storage chamber containing continuously oxygenated ACSF at 32 degrees Celsius.To conduct whole-cell patch clamp recordings from substantia gelatinosa or SG neurons, use potassium ion based intracellular solutions for recording while applying caesium cation based solution only for the recording of inhibitory post-synaptic currents. Gently move the spinal cord slice to the recording chamber. And then stabilize it firmly with a U shaped platinum wire with nylon threads for optimal slice stability.Steadily profuse the slice with bubbled ACSF at room temperature and set the profusion rate at two to four milliliters per minute to achieve sufficient oxygenation. Then, using a low resolution microscope lens, identify the region of SG neurons as a translucent band. Choose a healthy neuron, characterized by a three dimensional shape with a bright and smooth membrane as the target cell using the high resolution objective and adjust it to the center of the video monitor screen.Fill a micro pipette with an appropriate volume of potassium ion based or caesium ion based intracellular solution. Then insert the micro-pipette into the electrode holder and ensure that the intracellular solution is contacting the silver wire inside the holder. Bring the micro pipette into focus and use the micromanipulator to immerse it into the ACSF.Apply a mild positive pressure to force away any dirt and debris. Gradually move the micro pipette towards the targeted neuron. Once the pipette approaches the neuron and a very small dimple forms on the neuronal membrane, release the positive pressure to form a gigaseal.Alter the holding potential to minus 70 millivolts, which is close to the physiological resting membrane potential of a cell. Then, apply a transient and gentle suction to the micro pipette to rupture the membrane and create a good whole-cell configuration. Record firing properties by testing the firing pattern of each neuron in current clamp with a series of one second depolarizing current pulses of 25 to 150 picoamps with 25 picoamp increments at resting membrane potential.To record somatic subthreshold currents, hold the membrane potential at minus 50 millivolts in voltage clamp mode then apply a series of hyperpolarising voltage pulses of one second duration from minus 60 millivolts to minus 120 millivolts with a 10 millivolt decrement. Record excitatory post-synaptic currents with the potassium ion based intracellular solution in voltage clamp mode at a holding potential of minus 70 millivolts. Record IPSC's using caesium ion based intracellular solution for recording IPSC's.After holding the membrane potential at minus 70 millivolts for approximately five minutes, gradually change the holding potential to zero millivolts. Wait a few minutes for stabilization and then start to record the IPSC events. Firing patterns determined by injecting a series of one second depolarizing current pulses into an SG neuron at resting membrane potential may be classified as tonic-firing, delayed-firing, gap-firing, initial-burst, phasic-bursting, single-spike and reluctant-firing.This image shows the evoking protocol for subthreshold currents in voltage clamp. These representative traces show the response to hyperpolarizing current injection classified as IH, IA and IT.This is a representative trace of SEPSC's recorded from SG neurons at minus 70 millivolts in the absence and presence of 50 micromolar APV and 20 micromolar CNQX. This trace shows the recorded SEPC's before the addition of APV and CNQX on an expanded timescale.Following this procedure are the methods like paired patch clamp recordings or optogenetics can be performed to answer additional questions like characterizing specific neuronal microcircuits in substantia gelatinosa.

preparation-acute-spinal-cord-slices-for-whole-cell-patch-clamp

Research

JoVE Journal

Neuroscience

13.7K visualizações.  First Affiliated Hospital of Nanchang University. Este método pode ajudar a esclarecer os mecanismos espinhais subjacentes à transmissão sensorial, regulação nociceptiva e desenvolvimento de dor crônica ou dor A principal vantagem dessa técnica é que ela pode permitir a preservação neuronal ideal e pode imitar condições in vivo até certo ponto. Comece puxando eletrodos de gravação de microcapillarias de vidro borossilicato usando um puxador capilar. Em seguida, encha um molde com ágar derretido para preparar um 1,2 centímetr...