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Method Article

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

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DOI:

10.3791/54525

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October 13th, 2016

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

In This Article

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 mice4,5.

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 motoneurons6-10 and the Renshaw cells11-13. 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 fluorescence17-19. 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's1,2,20,21. 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 motoneurons22,23 or their targets. the Renshaw cells10,24,25. 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.

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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

  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 cerebrospinal fluid (ACSF): 3 mM KCl, 1 mM NaH2PO4, 230 mM sucrose, 26 mM NaHCO3, 0.8 mM CaCl2, 8 mM MgCl2, 25 mM glucose, 0.4 mM ascorbic acid, 1 mM Na-kynurenate, 2 mM Na-pyruvate. Bubble with 95% O2 and 5% CO2 (pH 7.4). Since Na-kynurenate is often hard to dissolve, make sure to purchase the one listed in the materials table.
    2. Prepare K-gluconate solution: 130 mM K-gluconate, 15 mM KCl, 0.05 mM EGTA, 20 mM HEPES, 25 mM glucose, 1 mM Na-kynurenate, 2 mM Na-pyruvate, adjusted to pH 7.4 with KOH.
    3. Prepare ACSF: 130 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 1 mM NaH2PO4, 26 mM NaHCO3, 25 mM glucose, 0.4 mM ascorbic acid, 2 mM Na-pyruvate. Bubble with 95% O2 and 5% CO2 (pH 7.4).
    4. Prior to the beginning of the dissection, dissolve 2% agar in 80 ml of the K-gluconate solution, and keep warm at 60 °C.
  2. Intracardiac perfusion
    1. Perform this preparation on female and male mice, ranging in age from P2 to P11.
    2. Anesthetize the mouse with an intraperitoneal injection of 0.1 ml of 25 mM pentobarbital sodium (50 mg/kg).
    3. Using needles or tape, immobilize the mouse on its back on a large petri dish filled with silicon. Use a dissecting microscope for the rest of the dissection.
    4. Holding the tip of the sternum, lift the chest and cut the diaphragm using fine scissors. Then open the chest on both sides by cutting through the ribs to expose the heart.
    5. Cut the right atrium before puncturing the left ventricle with a 27G needle.
    6. Perfuse with ice-cold low Na+ ACSF. After 30 sec, low Na+ ACSF should be seen flowing out of the atrium. Low amounts of sodium prevent the cells from spiking and reduce cellular death during dissection.
    7. Keep holding the needle in the heart under the dissecting microscope until the liver turns yellow when the blood is drained.
  3. Spinal cord dissection
    1. Decapitate the animal and put it on its stomach.
    2. Quickly remove the skin of the back (Figure 1A1). Make two cuts through the shoulders and going down the chest cage (Figure 1A2). Then cut the cord as low as possible in the caudal section in order to isolate the vertebral column with the beginning of the ribs from the lower part of the animal. Flip the animal again and remove the viscera still attached to the ribs.
    3. Transfer the vertebral column to another, smaller silicon-filled petri dish and use 4 insect pins to hold it dorsal side up (Figure 1A).
    4. Continuously perfuse the animal with carbogen-bubbled ACSF (at about 4 °C) while performing a laminectomy of the dorsal side, and exposing the spinal cord from the rostral end (Figure 1B). To do so, insert the tip of fine scissors between the bone and the spinal cord and cut the bone little by little from the rostral end, making sure to stay away from the white matter. Alternate on each side while using tweezers to keep away the band of bone already cut (Figure 1B1).
    5. Using the smallest available spring scissors and tweezers, lift the dura and cut on both sides while holding the loose part of the dura in order to avoid damaging the spinal cord with the scissors. Cut along the rostro-caudal axis.
      NOTE: The dura is a semi transparent continuous membrane; at this age the pia mater is too fragile and will come apart during the dissection and slicing (Figure 1B2).
    6. Once the dura is removed use a blunt glass or plastic tip to gently push the cord on the left side of the groove formed by the half-cut spinal column, and cut the ventral and dorsal roots on the right side, starting from the rostral side, furthest from where they enter the cord (a few mm at least, Figure 1B2).
    7. Repeat the operation on the left side, always going from rostral to caudal. If left-handed, start from the left side and then move to the right side.
  4. Embedding in agar
    1. Slip the cord out of the vertebral column. Use a smaller insect pin to pin down the cord with the dorsal surface up and delicately remove any piece of membrane still attached to it (Figure 1C1).
    2. Once cleaned, trim both ends (Figure 1C2). Insert a bended insect pin in the anterior part of the cord to manipulate the cord and note its orientation (arrow in Figure 1C2). Then transfer the spinal cord to an ice-cold K-gluconate solution. 
      NOTE: This solution mimics the intracellular composition of the CSF and will prevent the cells from dying of osmotic shock once the motoneurons will be cut26.
    3. Once the spinal cord is within the intra-cellular solution, take the beaker with the agar out of the dry bath and cool it down on a mixture of ice and water.
    4. Keep stirring while measuring the temperature. When the temperature reaches 38 °C immerse the spinal cord, holding it by the insect pin and place it rostral side down. Make sure the spinal cord is as straight as possible, away from the walls, caudal part slightly upward (Figure 1D1).
    5. Leave the beaker in the mixture of ice and water to allow the agar to solidify as quickly as possible. Make sure it stays in place and that the cord is as straight as possible (Figure 1D1).
  5. Slicing
    1. After solidification, cut the agar block containing the spinal cord in such a way that the base of the block is at a 35° angle with the lumbar part of the cord (arrow in Figure 1D2). The dorsal surface should be facing away from the base (Figure 1D2).
      NOTE: This is a critical step in the procedure, for maintaining the continuity of the motoneuron pools to the ventral roots from which they exit.
    2. Glue the block into the chamber of the vibratome using cyanoacrylate glue. Immerse it in K-gluconate solution and add slushed frozen K-gluconate solution to maintain the bath chilled (below 2 °C).
    3. Cut 350 - 400 µm thick slices of the lumbar region (identifiable by its curvature and larger diameter). In its proper orientation (see 1.4.1.), use the blade to cut from the dorsal to the ventral surface and make slices continuously more rostral. Typically, there are 4-5 suitable slices with ventral roots extending 2 mm or more. Use the following parameters: 10° angle, 70 Hz vibration frequency and 10 mm/min speed of slicing.
  6. Incubation
    1. Transfer the slices to ACSF at 34 °C. After approximately 30 min, cool the slices down to RT and begin the recording session.

Dissection process and tissue preparation, series of experimental images, biological research.
Figure 1. Dissection
A1: Removal of the skin of the back to expose the dorsal column. A2: Cutting of the shoulders and ribs to free the dorsal column. B1: Vertebral column pinned onto the silicon-filled petri dish (dorsal side up, caudal side left). B2: Same with the spinal cord exposed and dissected. C1: Spinal cord isolated (rostral side left). C2: Spinal cord ready to be embedded (ventral side up, rostral side left). Note the smaller insect pin on the rostral side. D1: Spinal cord in the agar beaker (rostral side dow, ventral side facing the bottom). D2: Cut agar block with the embedded spinal cord. Note the 35° angle the spinal cord forms with the base of the block and the lumbar enlargement (Arrow). Scale bars 1 cm. Please click here to view a larger version of this figure.

2. Placing the Slice into the Chamber

NOTE: Ventral roots are of variable size.

  1. Prepare a box of various pipettes with tip diameters ranging from 40 to 170 µm in advance. To prepare suction pipettes, prepare many pipettes with a long taper. Using a diamond knife, make a cut at different positions. Then under a dissecting microscope, break it by hitting the tip with tweezers.
  2. Remove the chamber from the recording microscope and place it under a dissecting microscope.
  3. Select a slice that contains a ventral root of sufficient length (2 mm or more) to be mounted on a suction stimulation electrode. Select the proper orientation of the slice with the ventral roots upward (Figure 2A) and delicately cut the agar around the ventral roots while leaving the rest of the agar around the slice (Figure 2B).
    NOTE: Because the agar is firmer than the slice, this will allow the threads of the slice's anchor to rest on the agar rather than on the slice and thus the anchor will not damage the tissue. Make sure the threads of the slice's anchor are above the motoneuron pool (shown in red in Figure 2C).
  4. Mount the chamber back onto the microscope and continuously perfuse the recording chamber with ACSF at a rate of 1 - 2 ml/min, at RT. Using a glass pipette filled with ACSF and connected to a syringe, suck one of the ventral root (arrow in Figure 2C). In order to achieve good stimulation of the ventral root, the pipette tip needs to be tight around the ventral root. One pole should be in the stimulating electrode and the other one in the bath (or connected to the patch-clamping electrode reference).
  5. Achieve patch-clamp recording of the desired cell-type and record the effect of the ventral root stimulation as described previsouly10.
    1. Here, use an amplifier for data acquisition. Filter whole-cell recordings at 3 kHz. Digitize at 10 kHz. Compensate bridge resistance in current-clamp mode.

Histological dye penetration, microscopic images of tissue cross-sections showing staining techniques.
Figure 2. Ventral Root Preparation
A: Lumbar spinal cord slice embedded in agar with the ventral root facing up. B: Lumbar spinal cord slice with the ventral roots freed from the agar. C: Lumbar spinal cord slice with a stimulating electrode tightly placed around the ventral roots (arrow). Note the location of the Renshaw cells expressing red fluorescence in the chrna2-Cre mouse28 crossed with the mouse reporter R26Tom 17. Scale bars 1 mm. Please click here to view a larger version of this figure.

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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 µm)....

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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...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

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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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Na-kynurenateABCAMab120256dissolves better then other brands
KClSigmaP3911
NaH2PO4SigmaP5655
sucrose SigmaS9378
NaHCO3 SigmaS6014
CaCl2 G BiosciencesR040
MgCl2 Quality Biological351-033-721
glucose SigmaG5767
ascorbic acid SigmaA5960
Na-pyruvate SigmaP2250
K-gluconate SigmaP1847
EGTA SigmaE3889
HEPES SigmaH4034
NaClSigmaS9888
AgarSigmaA9799
QX-314AlomoneQ150
Mg-ATPSigmaA9187
CsOHSigma232041
Na-GTPSigma51120
gluconic acidSigmaG1951
Cesium hydroxide solutionSigma232041
KOHSigmaP5958
Vannas Spring Scissors - 2.5mm FST15000-08only use for cutting the dura, might get damaged if cutting bones
StimulatorA-M SystemsIsolated Pulse Stimulator Model 2100
VibratomeCampdenVibrating Microtome 7000 - Model 7000smz-2

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Oblique Slice PreparationMotoneuron IdentificationRenshaw Cell ExcitationAntidromic RecordingElectrophysiological RecordingsVibratome SectioningAgar Embedding TechniqueLow Sodium ACSF Perfusion
The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation
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