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

Summary

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.

Abstract

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.

Introduction

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,

Protocol

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 <....

Results

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 .......

Discussion

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

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
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