Name: The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation (Video) | JoVE Journal
Uploaded: 2016-10-13T13:00:00.000+00:00
Duration: 9 min 10 s
Description: Learn to prepare oblique spinal cord slices from young mice for ventral root stimulation. This method enables reliable identification of motoneurons and study of ventral cells innervated by axon collaterals.

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5 minutes

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 &#34;Contrat Doctoral&#34; from the Ecole Normale Sup&#233;rieure, Cachan.

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
<ol>
	<li>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.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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).</li>
		<li>Prior to the beginning of the dissection, dissolve 2% agar in 80 ml of the K-gluconate solution, and keep warm at 60 °C.</li>
	</ol>
	</li>
	<li>Intracardiac perfusion
	<ol>
		<li>Perform this preparation on female and male mice, ranging in age from P2 to P11.</li>
		<li>Anesthetize the mouse with an intraperitoneal injection of 0.1 ml of 25 mM pentobarbital sodium (50 mg/kg).</li>
		<li>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.</li>
		<li>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.</li>
		<li>Cut the right atrium before puncturing the left ventricle with a 27G needle.</li>
		<li>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.</li>
		<li>Keep holding the needle in the heart under the dissecting microscope until the liver turns yellow when the blood is drained.</li>
	</ol>
	</li>
	<li>Spinal cord dissection
	<ol>
		<li>Decapitate the animal and put it on its stomach.</li>
		<li>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.</li>
		<li>Transfer the vertebral column to another, smaller silicon-filled petri dish and use 4 insect pins to hold it dorsal side up (Figure 1A).</li>
		<li>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).</li>
		<li>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).</li>
		<li>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).</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Embedding in agar
	<ol>
		<li>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).</li>
		<li>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.</li>
		<li>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.</li>
		<li>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).</li>
		<li>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).</li>
	</ol>
	</li>
	<li>Slicing
	<ol>
		<li>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.</li>
		<li>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).</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Incubation
	<ol>
		<li>Transfer the slices to ACSF at 34 °C. After approximately 30 min, cool the slices down to RT and begin the recording session.</li>
	</ol>
	</li>
</ol>
<img alt="Dissection process and tissue preparation, series of experimental images, biological research." src="/files/ftp_upload/54525/54525fig1.jpg" data-altupdatedat="1788235205000" data-alt="Figure 1"> 
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. <a href="https://www.jove.com/files/ftp_upload/54525/54525fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Placing the Slice into the Chamber
NOTE: Ventral roots are of variable size.
<ol>
	<li>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.</li>
	<li>Remove the chamber from the recording microscope and place it under a dissecting microscope.</li>
	<li>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).</li>
	<li>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).</li>
	<li>Achieve patch-clamp recording of the desired cell-type and record the effect of the ventral root stimulation as described previsouly10.
	<ol>
		<li>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.</li>
	</ol>
	</li>
</ol>
<img alt="Histological dye penetration, microscopic images of tissue cross-sections showing staining techniques." src="/files/ftp_upload/54525/54525fig2.jpg" data-altupdatedat="1788235205000" data-alt="Figure 2"> 
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. <a href="https://www.jove.com/files/ftp_upload/54525/54525fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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<li>Morin, F., Schwartz, H. G., O'Leary, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Experimental+study+of+the+spinothalamic+and+related+tracts%5BTitle%5D)+AND+%22Acta+Psychiatr+Neurol+Scand%22%5BJournal%5D)">Experimental study of the spinothalamic and related tracts.</a> Acta Psychiatr Neurol Scand. 26 (3-4), 371-396 (1951).</li>
<li>Sengul, G., Fu, Y., Yu, Y., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spinal+cord+projections+to+the+cerebellum+in+the+mouse%5BTitle%5D)+AND+%22Brain+Struct+Funct%22%5BJournal%5D)">Spinal cord projections to the cerebellum in the mouse.</a> Brain Struct Funct. 220 (5), 2997-3009 (2015).</li>
<li>Russier, M., Carlier, E., Ankri, N., Fronzaroli, L., Debanne, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A-%2C+T-%2C+and+H-type+currents+shape+intrinsic+firing+of+developing+rat+abducens+motoneurons%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">A-, T-, and H-type currents shape intrinsic firing of developing rat abducens motoneurons.</a> J Physiol. 549 (Pt 1), 21-36 (2003).</li>
<li>Dourado, M., Sargent, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Properties+of+nicotinic+receptors+underlying+Renshaw+cell+excitation+by+alpha-motor+neurons+in+neonatal+rat+spinal+cord%29%5BTitle%5D)+AND+%22J+Neurophysiol%22%5BJournal%5D)">Properties of nicotinic receptors underlying Renshaw cell excitation by alpha-motor neurons in neonatal rat spinal cord).</a> J Neurophysiol. 87 (6), 3117-3125 (2002).</li>
<li>Mitra, P., Brownstone, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+in+vitro+spinal+cord+slice+preparation+for+recording+from+lumbar+motoneurons+of+the+adult+mouse%5BTitle%5D)+AND+%22J+Neurophysiol%22%5BJournal%5D)">An in vitro spinal cord slice preparation for recording from lumbar motoneurons of the adult mouse.</a> J Neurophysiol. 107 (2), 728-741 (2012).</li>
<li>Rothman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+neurotoxicity+of+excitatory+amino+acids+is+produced+by+passive+chloride+influx%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">The neurotoxicity of excitatory amino acids is produced by passive chloride influx.</a> J Neurosci. 5 (6), 1483-1489 (1985).</li>
<li>Olney, J. W., Price, M. T., Samson, L., Labruyere, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+specific+ions+in+glutamate+neurotoxicity%5BTitle%5D)+AND+%22Neurosci+Lett%22%5BJournal%5D)">The role of specific ions in glutamate neurotoxicity.</a> Neurosci Lett. 65 (1), 65-71 (1986).</li>
</ol>

The authors have nothing to disclose.

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&#160;and specific way. Furthermore, it allows for a quick, elegant&#160;and non-ambiguous identification of recorded motoneurons. Next, we will highlight&#160;the advantages of this&#160;technique compared to other slice preparation methods, and then we will stress out&#160;the most common pitfalls to avoid while performing this procedu...

Historically, motoneuron recordings using sharp-electrode were conducted in vivo on large animals such as&#160;cats or rats1 or on an isolated whole spinal cord in&#160;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&#160;pool&#160;retains its&#160;ventral root departing&#160;axons. Ventral root stimulation triggers antidromic action potential back into the somas of the motoneuron&#160;pool&#160;exiting from the same ventral root. It also excites the motoneuron collateral targets,&#160;other motoneurons6-10 and the Renshaw cells11-13. Since only motoneurons&#160;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&#160;criterions to confirm the motoneuron&#160;identity, recent studies on spinal cord motoneurons also relied on tedious and&#160;time-consuming post&#160;hoc stainings16. Such identification is usually performed only on a sample of the recorded cells. Other identification strategies rely&#160;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&#39;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&#160;or their targets. the&#160;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.

<table><tbody><tr><td>Na-kynurenate</td><td>ABCAM</td><td>ab120256</td><td>dissolves better then other brands</td></tr><tr><td>KCl</td><td>Sigma</td><td>P3911</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P5655</td><td></td></tr><tr><td>sucrose&amp;nbsp;</td><td>Sigma</td><td>S9378</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;&amp;nbsp;</td><td>Sigma</td><td>S6014</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>G Biosciences</td><td>R040</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>Quality Biological</td><td>351-033-721</td><td></td></tr><tr><td>glucose&amp;nbsp;</td><td>Sigma</td><td>G5767</td><td></td></tr><tr><td>ascorbic acid&amp;nbsp;</td><td>Sigma</td><td>A5960</td><td></td></tr><tr><td>Na-pyruvate&amp;nbsp;</td><td>Sigma</td><td>P2250</td><td></td></tr><tr><td>K-gluconate&amp;nbsp;</td><td>Sigma</td><td>P1847</td><td></td></tr><tr><td>EGTA&amp;nbsp;</td><td>Sigma</td><td>E3889</td><td></td></tr><tr><td>HEPES&amp;nbsp;</td><td>Sigma</td><td>H4034</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888</td><td></td></tr><tr><td>Agar</td><td>Sigma</td><td>A9799</td><td></td></tr><tr><td>QX-314</td><td>Alomone</td><td>Q150</td><td></td></tr><tr><td>Mg-ATP</td><td>Sigma</td><td>A9187</td><td></td></tr><tr><td>CsOH</td><td>Sigma</td><td>232041</td><td></td></tr><tr><td>Na-GTP</td><td>Sigma</td><td>51120</td><td></td></tr><tr><td>gluconic acid</td><td>Sigma</td><td>G1951</td><td></td></tr><tr><td>Cesium hydroxide solution</td><td>Sigma</td><td>232041</td><td></td></tr><tr><td>KOH</td><td>Sigma</td><td>P5958</td><td></td></tr><tr><td>Vannas Spring Scissors - 2.5mm&amp;nbsp;</td><td>FST</td><td>15000-08</td><td>only use for cutting the dura, might get damaged if cutting bones</td></tr><tr><td>Stimulator</td><td>A-M Systems</td><td>Isolated Pulse Stimulator Model 2100</td><td></td></tr><tr><td>Vibratome</td><td>Campden</td><td>Vibrating Microtome 7000 - Model 7000smz-2</td><td></td></tr></tbody></table>

the preparation of oblique spinal cord slices for ventral root stimulation

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

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&#160;their axons coming out from the ventral roots of the&#160;spinal cord. Stimulation of these axons elicits back-propagating action potentials invading the motoneuron&#160;somas and exciting the motoneuron&#160;collaterals within the spinal cord. Recording of antidromic action potentials is an immediate, definitive and elegant way to characterize motoneuron identity, which surpasses&#160;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.

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)....

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 ...

The overall goal of this procedure is to prepare oblique slices of the spinal cord that allow for the stimulation of the ventral roots. The main advantage of this technique is that it allows motoneural identifications for ventral-root stimulation and to study the ventral cells enervated by motoneural axon collaterals. We first had the idea for this method when needing a reliable way to stimulate ventral cells.Individuals new to this method will struggle because of the difficulty to properly execute the different steps of the dissection. Begin by perfusing a P2 to P11 mouse with low-sodium ACSF. After decapitating the mouse and removing the skin on the back, make two cuts through the shoulders and down the rib cage.Then, cut the spinal cord as low as possible in the caudal section to isolate the vertebral column and ribs from the lower part of the animal. Flip the vertebral column and remove the viscera still attached to the ribs. Transfer the vertebral column to another smaller, silicon-filled Petri dish.Add cold ACSF and bubble with carbogen and use four insect pins to hold the tissue dorsal-side up. Then perform a laminectomy of the dorsal side by first inserting the tip of fine scissors between the bone and the spinal cord and cutting the bone from the rostral end, making sure to stay away from the white matter. Alternate the cuts on each side while using tweezers to hold the band of bone already cut away.Next, use small tweezers to lift the dura and small scissors to cut along the rostral caudal access on both sides. Removal of the dura is a critical step. It is well worth spending a few extra minutes to do this thoroughly.Once the dura is removed, use a blunt glass tip to gently push cord towards the left side of the groove formed by half-cut spinal column. Then cut the ventral and dorsal roots on the bright side as far as possible from where they enter the cord. Repeat the operation on the left side, always going from rostral to caudal.After dissection, slip the spinal cord out of the vertebral column. Then use a small insect pin to secure the cord for the dorsal surface-up. Delicately remove any pieces of attached membrane.Once cleaned, trim both ends of the cord and insert a bent insect pin into the anterior part of the cord to mark its orientation. Then transfer the spinal cord to ice-cold intracellular solution. Next, cool the previously prepared molten agar on a mixture of ice and water.Stir the agar while measuring the temperature. When the temperature of the agar reaches 38 degrees Celsius, hold the spinal cord by the insect pin and immerse it in the agar, rostral-side down. Make sure the spinal cord is as straight as possible, away from the walls, with the caudal part slightly upward.Leave the beaker in the mixture of ice and water to allow the agar to solidify around the cord as quickly as possible. After solidification, cut the agar block containing the spinal cord in such a way that the base of the block is at a 35-degree angle with the lumbar part of the cord. The dorsal surface should be facing away from the base.Glue the block into the chamber of the vibratome, using cyanoacrylate glue. It is critical to mount the block correctly to maintain the continuity of the motor pools with the ventral roots from which they exit. Immerse the block in potassium gluconate solution.Set the vibratome parameters. Next, cut 350 400 micron-thick slices of the lumbar region, which is identifiable by its curvature and larger diameter. Transfer the suitable slices to ACSF at 34 degrees Celsius.Typically, there are four to five suitable slices with ventral roots extending two millimeters or more. After approximately 30 minutes at 34 degrees Celsius, cool the slices to room temperature, then begin the recording session. Prepare a box of various pipettes with tip diameters ranging from 40 to 170 microns in advance.To prepare the section pipettes, select pipettes with a long tapure. Then, using a diamond knife, make cuts at different positions and under a dissecting microscope, break each pipette by hitting the tip with tweezers. Next, remove the chamber from the recording microscope and place it under a dissecting microscope.Select a slice that contains a ventral root of sufficient length to be mounted on a suction-stimulation electrode. Mount the slice, orienting it with the ventral roots upward, then delicately cut the agar around the ventral roots while leaving the rest of the agar around the slice and place the grid to hold the sample down. Mount the chamber back onto the microscope and continuously perfuse the recording chamber with ACSF at a rate of one to two milliliters per minute at room temperature.Using a glass pipette filled with ACSF and connected to a syringe, suck one of the ventral roots. In order to achieve good stimulation of the ventral root, the pipette tip needs to be tight around the ventral root. Achieve patch-clamp recording of the desired cell type and record the effect of the ventral root stimulation as described previously.This image shows the results from voltage-clamp recording. The recordings of the upper panels are taken from different motoneurons than the ones in the bottom panels. The upper panel shows the antidromic response of a motoneuron to a one-volt stimulation in red, and a five-volt stimulation in black.The lower trace shows the orthodromic action potential response to a 20-volt stimulation in red, and a 30-volt stimulation in black. Black dots indicate stimulus timing. The double arrow heads emphasize the difference in latencies between ortho-and antidromic spikes.Here, current clamp recordings are shown. The upper panel shows the antidromic response to 10-volt and 15-volt stimulations in red and black, respectively. The lower panel shows the orthodromic response to 25-and 40-volt stimulations in red and black, respectively.Again, the stimulus timing and differences in latencies are indicated. This image of the Renshaw cell pool was acquired using an oblique condenser and a 20x objective. Note the motoneuron axons merging into the ventral root as shown by the arrow.This image of the same region was taken using a 40x objective. Putative Renshaw cells are indicated with arrow heads. Shown here is a voltage-clamp recording of a Renshaw cell following stimulation of the ventral root, indicated by the black dot.QX-314 in the intracellular solution prevented the cell from firing and glycine and gaba responses were blocked by 3 micromolar gabazine and one micromolar strychnine, respectively. The red trace is the average of the gray ones. Once mastered, this technique can be performed in 20 or 30 minutes.While attempting this procedure, it's important to remember to keep the preparation in cold medium at all time. After watching this video, you should have a good understanding of how to prepare oblique slices of spinal cord suitable for ventral-root stimulation. Ventral-root stimulation can be used to easily confirm the motoneural identity, and more importantly, to reliably stimulate the ventral cells.

the-preparation-oblique-spinal-cord-slices-for-ventral-root

Research

JoVE Journal

Neuroscience

We describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral axis of the medial entorhinal cortex (MEC). Because neural encoding of location follows a dorsal-ventral organization within the MEC, these procedures facilitate investigation of cellular mechanisms important for navigation and memory.

Preparation of Parasagittal Slices for the Investigation of Dorsal-ventral Organization of the Rodent Medial Entorhinal Cortex

This video describes the in vivo intracellular recording of rat motoneurons under trans-spinal direct current stimulation. The protocol describes how to measure membrane properties and record the rhythmic firing of motoneurons before, during, and after anodal or cathodal polarization of the spinal cord.

Trans-Spinal Direct Current Stimulation of a Rat&#39;s Spinal Motoneuron

This video demonstrates a microelectrode array-based assay for studying neuronal network activity in mouse spinal cord sections. First, the electrophysiological activity from the superficial dorsal horn (SDH) of the slice is recorded. Then, a potassium channel inhibitor is introduced to prolong depolarization, resulting in synchronous rhythmic activity across the neuronal network.

Microelectrode Array-Based Assessment of Neuronal Networks in Mouse Spinal Cord Slices

Recording Ca2+ currents at the presynaptic release face membrane is key to a precise understanding of Ca2+ entry and neurotransmitter release. We present an acute dissociation of the lamprey spinal cord that yields functional isolated reticulospinal axons, permitting recording directly from the release face membrane of individual presynaptic terminals.

Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals

The efficacy of intramuscular uptake and retrograde transport of molecules to corresponding motor neurons depends on the location of the injection sites with respect to the motor end plates (MEPs). Here, we describe how to locate MEPs on skeletal muscles to optimise retrograde transport of tracers into motor neurons.

Intramuscular Injections Along the Motor End Plates: A Minimally Invasive Approach to Shuttle Tracers Directly into Motor Neurons

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.

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

This protocol both visually communicates the brainstem-spinal cord preparation and clarifies the preparation of brainstem transverse slices in a comprehensive step-by-step manner. It was designed to increase reproducibility and enhance the likelihood of obtaining viable, long lasting, rhythmically-active slices for recording neural output from the respiratory regions of the brainstem.

Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

This protocol describes in vivo intracellular recording of rat lumbar motoneurons with simultaneous trans-spinal direct current stimulation. The method enables us to measure membrane properties and to record rhythmic firing of motoneurons before, during and after anodal or cathodal polarization of the spinal cord.

In Vivo Intracellular Recording of Type-Identified Rat Spinal Motoneurons During Trans-Spinal Direct Current Stimulation

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

The combined use of microelectrode array technology and 4-aminopyridine-induced chemical stimulation for investigating network-level nociceptive activity in the spinal cord dorsal horn is outlined.

Recording Network Activity in Spinal Nociceptive Circuits Using Microelectrode Arrays

This protocol describes a method using a patch-clamp to study the electrical responses of motor neurons to spinal cord stimulation (SCS) with high spatiotemporal resolution, which can help researchers improve their skills in separating the spinal cord and maintaining cell viability simultaneously.

The Ex vivo Preparation of Spinal Cord Slice for the Whole-Cell Patch-Clamp Recording in Motor Neurons During Spinal Cord Stimulation

Learn to prepare oblique spinal cord slices from young mice for ventral root stimulation. This method enables reliable identification of motoneurons and study of ventral cells innervated by axon collaterals.

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

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