De Voorbereiding van de Oblique Spinal Cord Slices voor Ventral Root Stimulation

Summary

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

Abstract

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.

Introduction

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 roots in transverse slices, and none was obtained from mice^4,5.

In this article we present a technique to achieve viable spinal cord slices of neonatal mice (P2 – P11) in which the motoneuron pool retains its ventral root departing axons. Ventral root stimulation triggers antidromic action potential back into the somas of the motoneuron pool exiting from the same ventral root. It also excites the motoneuron collateral targets, other motoneurons^6-10 and the Renshaw cells^11-13. Since only motoneurons send their axons down the ventral roots, we use the recording of antidromic action potentials as a simple and definitive way to physiologicaly identify motoneurons¹⁰.

In addition to using potentially non-inclusive or misleading electrophysiological and morphological criterions to confirm the motoneuron identity, recent studies on spinal cord motoneurons also relied on tedious and time-consuming post hoc stainings¹⁶. Such identification is usually performed only on a sample of the recorded cells. Other identification strategies rely on mouse lines in which the motoneurons express endogenous fluorescence^17-19. However, using genetically encoded markers may be difficult at a young age when marker expression is still variable or if the study already requires using a transgenic mouse line. Alternatively, antidromic action potential recordings can be performed routinely on all mice from the onset of cell recording. Experimenters working on intact spinal cord preparations in the cat, rat and mouse, have reliably used such identification techniques since the 1950's^1,2,20,21. In optimal conditions, we were able to elicit antidromic action potentials from virtually all of the recorded motoneurons.

Furthermore, ventral root stimulation can be used to reliably excite other motoneurons^22,23 or their targets. the Renshaw cells^10,24,25. We present here applications of the ventral root stimulation in the form of antidromic action potential recordings from motoneuron somas, as well as excitation of Renshaw cells.

Protocol

 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 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. Prepare Low Na + artificial cerebrospi…

Representative Results

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…

Discussion

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…

Materials

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