Name: preparation of acute spinal cord slices for whole-cell patch-clamp recording in substantia gelatinosa neurons
Uploaded: 2019-01-18T12:30:00
Description: Watch this Scientific Journal Video about Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons at JoVE.com

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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Y., Hughes, D. I., Riddell, J. S., 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=Populations+of+inhibitory+and+excitatory+interneurons+in+lamina+II+of+the+adult+rat+spinal+dorsal+horn+revealed+by+a+combined+electrophysiological+and+anatomical+approach.">Populations of inhibitory and excitatory interneurons in lamina II of the adult rat spinal dorsal horn revealed by a combined electrophysiological and anatomical approach.</a> Pain. 151 (2), 475-488 (2010).</li><li>Yin, H., Park, S. A., Han, S. K., Park, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+5-hydroxytryptamine+on+substantia+gelatinosa+neurons+of+the+trigeminal+subnucleus+caudalis+in+immature+mice.">Effects of 5-hydroxytryptamine on substantia gelatinosa neurons of the trigeminal subnucleus caudalis in immature mice.</a> Brain Research. 1368, 91-101 (2011).</li><li>Hu, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lidocaine+Inhibits+HCN+Currents+in+Rat+Spinal+Substantia+Gelatinosa+Neurons.">Lidocaine Inhibits HCN Currents in Rat Spinal Substantia Gelatinosa Neurons.</a> Anesthesia and Analgesia. 122 (4), 1048-1059 (2016).</li><li>Peng, H. Z., Ma, L. X., Lv, M. 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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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+whole-cell+recording+from+visually+preselected+neurons+of+perirhinal+cortex+in+brain+slices+from+young+and+aging+rats.">Methods for whole-cell recording from visually preselected neurons of perirhinal cortex in brain slices from young and aging rats.</a> Journal of Neuroscience Methods. 86 (1), 35-54 (1998).</li><li>Rothman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurotoxicity+of+excitatory+amino+acids+is+produced+by+passive+chloride+influx.">The neurotoxicity of excitatory amino acids is produced by passive chloride influx.</a> The Journal of Neuroscience. 5 (6), 1483-1489 (1985).</li><li>Rice, M. 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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 ...

Watch this Scientific Journal Video about Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons at JoVE.com

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 ...

Research

JoVE Journal

Neuroscience

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