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Abstract

Introduction

Protocol

Representative Results

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Acknowledgements

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References

Neuroscience

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Published: October 13th, 2016

DOI:

10.3791/54525

1Centre National de la Recherche Scientifique (UMR 8119), Centre de Neurophysique, Physiologie et Pathologie, Université Paris Descartes

We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots.

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to network properties. We show how to prepare viable oblique slices of the spinal cord of young mice (P2 - P11). In this preparation, the motoneurons retain their axons coming out from the ventral roots of the spinal cord. Stimulation of these axons elicits back-propagating action potentials invading the motoneuron somas and exciting the motoneuron collaterals within the spinal cord. Recording of antidromic action potentials is an immediate, definitive and elegant way to characterize motoneuron identity, which surpasses other identification methods. Furthermore, stimulating the motoneuron collaterals is a simple and reliable way to excite the collateral targets of the motoneurons within the spinal cord, such as other motoneurons or Renshaw cells. In this protocol, we present antidromic recordings from the motoneuron somas as well as Renshaw cell excitation, resulting from ventral root stimulation.

Historically, motoneuron recordings using sharp-electrode were conducted in vivo on large animals such as cats or rats1 or on an isolated whole spinal cord in mice2. The emergence of the patch-clamp recording technique during the 1980s, called for direct access to the motoneuron somas as sealing needed to be achieved under visual guidance. Thus, spinal cord slice preparation has been readily achieved since the early 1990s3. However, early slice preparation often did not allow for the stimulation of the ventral roots. To the best of our knowledge, only two studies have reported successful stimulation of the ventral ro....

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 The experiments were performed in accordance with European directives (86/609/CEE and 2010-63-UE) and French legislation, and were approved by the Paris Descartes University ethics committee.

1. Spinal Cord Slice Preparation

  1. Prepare the following solutions daily or one day in advance. If kept overnight, bubble with 95% O2 and 5% CO2 and keep refrigerated in tightly closed bottles.
    1. Prepare Low Na + artificial cerebrospi.......

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Confirmation of Motoneuron Identity Using Antidromic Action Potentials

Cell targeting

Motoneurons are found in the ventral horn (visible in red in Figure 2C). Start from the bundle of axons forming the ventral root and go up until the bundle disperses fully and one starts seeing large cells (long soma axis, above 20.......

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Oblique slicing of the spinal cord is important since it allows for unilateral stimulation of motoneuron pools and Renshaw cells at a single vertebral segment in a reliable, comprehensive and specific way. Furthermore, it allows for a quick, elegant and non-ambiguous identification of recorded motoneurons. Next, we will highlight the advantages of this technique compared to other slice preparation methods, and then we will stress out the most common pitfalls to avoid while performing this procedu.......

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The authors thank Marin Manuel and Olivia Goldman-Szwajkajzer for their help in taking the photographs. The authors also thank Arjun Masukar and Tobias Bock for proofreading the manuscript. Financial supports were provided by the Agence Nationale pour la Recherche (HYPER-MND, ANR-2010-BLAN-1429-01), the NIH-NINDS (R01NS077863), the Thierry Latran Foundation (OHEX Project), the French association for myopathy (grant number 16026) and Target ALS are gratefully acknowledged. Felix Leroy was the recipient of a "Contrat Doctoral" from the Ecole Normale Supérieure, Cachan.

....

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Name Company Catalog Number Comments
Na-kynurenate ABCAM ab120256 dissolves better then other brands
KCl Sigma P3911
NaH2PO4 Sigma P5655
sucrose  Sigma S9378
NaHCO3  Sigma S6014
CaCl2  G Biosciences R040
MgCl2  Quality Biological 351-033-721
glucose  Sigma G5767
ascorbic acid  Sigma A5960
Na-pyruvate  Sigma P2250
K-gluconate  Sigma P1847
EGTA  Sigma E3889
HEPES  Sigma H4034
NaCl Sigma S9888
Agar Sigma A9799
QX-314 Alomone Q150
Mg-ATP Sigma A9187
CsOH Sigma 232041
Na-GTP Sigma 51120
gluconic acid Sigma G1951
Cesium hydroxide solution Sigma 232041
KOH Sigma P5958
Vannas Spring Scissors - 2.5mm  FST 15000-08 only use for cutting the dura, might get damaged if cutting bones
Stimulator A-M Systems Isolated Pulse Stimulator Model 2100
Vibratome Campden Vibrating Microtome 7000 - Model 7000smz-2

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