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

Mengye Zhu; Daying Zhang; Sicong Peng; Nana Liu; Jing Wu; Haixia Kuang; Tao Liu

doi:10.3791/58479

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelati...

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JoVE Journal Neuroscience

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

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08:30 min

January 18, 2019

DOI: 10.3791/58479-v

Mengye Zhu¹, Daying Zhang¹, Sicong Peng², Nana Liu², Jing Wu², Haixia Kuang², Tao Liu^2,3

¹Department of Pain,First Affiliated Hospital of Nanchang University, ²Department of Pediatrics,First Affiliated Hospital of Nanchang University, ³Center for Laboratory Medicine,First Affiliated Hospital of Nanchang University

Overview

This study outlines the procedures for conducting whole-cell patch-clamp recordings from substantia gelatinosa (SG) neurons within in vitro spinal cord slices. By employing this method, researchers are able to investigate intrinsic membrane properties, synaptic transmission, and the morphological characteristics of SG neurons.

Key Study Components

Area of Science

Neuroscience
Electrophysiology
Cell Biology

Background

The substantia gelatinosa is critical for understanding sensory processing and pain regulation.
Whole-cell patch-clamp can elucidate the electrophysiological properties of these neurons.
This method enhances neuronal preservation and simulates in vivo conditions.
Understanding SG neurons can provide insights into chronic pain mechanisms.

Purpose of Study

To describe the methodology for patch-clamp recordings in spinal cord slices.
To investigate the inherent properties of SG neurons.
To explore synaptic transmission in relation to pain mechanisms.

Methods Used

Whole-cell patch-clamp recordings performed on in vitro spinal cord slices.
The biological model focuses on substantia gelatinosa neurons.
No multiomics workflow is mentioned in the text.
The critical steps include preparing a sucrose-based ACSF, isolating the spinal cord segment, and slicing the tissue within specific time constraints.
Recording techniques include adjusting holding potentials and identifying neuronal properties under a microscope.

Main Results

The study presents a protocol for identifying and recording from SG neurons, allowing detailed characterization of their electrophysiological properties.
Electrophysiological changes and firing patterns can be analyzed to gain insights into neuronal excitability and synaptic dynamics.
Key biological responses include characterizations of tonic-firing and other firing patterns based on experimental conditions.
The method facilitates the exploration of pain modulation mechanisms based on synaptic activities.

Conclusions

The described methodology enables the investigation of SG neuron functionality and is crucial for understanding pain pathways.
The method's precision and preservation of neurons enhance its applicability in neuroscience research.
Findings contribute to a better understanding of the mechanisms underlying chronic pain and sensory transmission.

Frequently Asked Questions

What advantages does the whole-cell patch-clamp method provide?

This technique offers ideal neuronal preservation, mimicking in vivo conditions and allowing for detailed electrophysiological analyses of neurons.

How is the spinal cord segment isolated for recording?

The lumbosacral segment of the spinal cord is quickly isolated after making careful incisions to avoid damaging surrounding tissues, ensuring optimal conditions for recordings.

What types of data are obtained during the recordings?

Data includes intrinsic membrane properties and synaptic responses, with classification of firing patterns and recording of excitatory/inhibitory post-synaptic currents.

Can this method be adapted for other neuronal types?

Yes, while this protocol focuses on SG neurons, similar techniques can be tailored for other neuronal types in various regions of the nervous system.

Are there any limitations associated with this method?

Handling could damage delicate tissues if not done with precision; also, the method requires careful timing to ensure slice viability and quality.

What specific experimental conditions must be maintained?

Maintaining oxygenation of the ACSF, controlling temperature, and preparing slices within a specific time frame are critical for successful recordings.

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.

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.

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