Isolation Procedure for Spinal Motor Neurons

Overview: The procedure begins with isopycnic centrifugation to obtain a crude fraction of MN cell bodies on a discontinuous sucrose gradient. From the crude fraction, individual cell bodies are collected with a glass pipette, using mouth suction, and rinsed in fresh sucrose-containing buffer to obtain several hundred purified cell bodies. The figure below summarizes the method used successfully for grass frog (R. pipiens), mouse, rat and human MNs. Human MNs are dissociated through a larger pore nylon bolting cloth (351 x 351 um), while MNs from smaller animals are dissociated using 202 x 202 um pore size.

Procedure: When spinal tissue is frozen and thawed, proteins are released from cells as ice crystals damage the cell membrane during the thaw cycle. In order to maximize protein retention, small pieces of spinal tissue are first exposed to ice-cold buffered, ethylene glycol for 1.5h, which cryoprotects the MNs and improves cell and protein/cell yields (Sinicropi et al., Anal. Biochem. 180: 286-290, 1989). Low ionic strength, low pH buffers are also used throughout the procedure to improve the retention of soluble proteins by the isolated cell bodies (Weil et al., J. Neurochem. 29: 847-852, 1977). These buffers and the cryoprotection step can be omitted if one does not need to maximize retention of soluble proteins.

Lumbar or cervical spinal cords are cleaned of meninges and roots are removed. With human or bovine spinal cord, ventral gray matter trimmed of much of its surrounding white matter can be dissected before or after freezing the tissue at –80 °C. If cryoprotection is used, the unfrozen tissue is first minced with a razor blade into 1-2 mm 3 pieces and suspended in approximately 5 ml of ice-cold 70% ethylene glycol-30% 0.9 M sucrose-CG.   (Note: CG = 1.7 mM citrate-NaOH, pH 5.0 and 15 mM glucose). Tissue is then allowed to remain on ice for 90 minutes before -80 °C storage or immediate use.

Before use, cryoprotected tissue pieces are brought to ice temperature and ethylene glycol solution is removed by filtration through nylon bolting cloth, using 0.9M sucrose-CG to rinse off remaining ethylene glycol from the tissue, which remains on top of the nylon. We use Nitex 202 (pore size: 202 um x 202 um) for rodent and frog motoneurons and Nitex 351 (351 um x 351 um) for human motoneurons. Tissue pieces are then expressed through the Nitex with a spatula and washed through with 0.9 M sucrose-CG, keeping to total volume below that which can be accomodated in the centrifuge tubes used for the succeeding spin.

A sucrose gradient is formed by sequential addition of 2 ml of 2.1M sucrose-CG followed by 2 ml of 1.5 M sucrose-CG in a 15ml Corex glass centrifuge tube. The tissue homogenate, whose volume in 0.9 M sucrose-CG will vary, is layered atop the 1.5 M sucrose-CG and centrifuged at 7000 rpm (9400 x g) for 40 minutes at 4 °C. For large preps, these volumes may be scaled upward and centrifuge tubes changed to accommodate larger volumes. Important: When isolating human MNs, one should substitute 1.2M sucrose for 1.5M sucrose.

Motoneuron cell bodies migrate to the interface between 1.5 and 2.1M sucrose. Myelin and other large debris particles float on the surface of 0.9M sucrose.   Most free nuclei will pellet through the 2.1M sucrose.   It is useful to check for cell bodies at the interface between 1.5 (or 1.2M) sucrose and 0.9M sucrose, particularly with human MNs, which are often also found at that interface.

Total number of cells from 3 frogs (lumbar cord only) ranges from 100-800 with typical yields in the range of 200-300.   Much higher yields are obtained for mouse or human MNs, because of their larger number of MNs in typical starting material. The higher the concentration of cell bodies in the crude fraction, the faster the collection rate during the purification steps, because less time is required to locate cell bodies. The final total volume of the crude cell body fraction will be several mls, with small volumes and high cell body densities preferred.

For cell body purification, hand-pulled micropipettes, covalently coated with bovine serum albumin (see procedure below), are used to minimize cells sticking to the glass pipette. The micropipette is mounted on a micromanipulator and connected to 3-4 feet of rubber tubing, equipped with a plastic mouthpiece. Mouth suction was found to be superior to automated devices for picking up and manipulating individual MN cell bodies.

Fifty to 100 ul aliquots of the crude cell body suspension are transferred to a glass cover slip (No. 1, 22 x 22 mm ) which is place on a custom-built brass cover slip holder, and viewed by phase contrast on Diavert microscope using a 6.3 objective. Conventional microscopic slides are too thick for use with our configuration of this microscope. To minimize neuronal adhesion to the glass, about 50 ul of 2.1M sucrose is first used to coat the top of the cover slip, before adding the crude cell body suspension. MN cell bodies have distinctive sizes and appearances and are easily identified in frog, mouse and human preparations. Gamma MNs are absent in the frog and interneurons are much less frequently found than larger MNs. The criteria for identifying MN cell bodies have been discussed in many of our laboratory’s publications. The distinctive appearance of neurons, with large, pale nuclei and dark, single, spherical nucleoli, along with their large size, facilitate decisions about which cell bodies to pick up, and which to leave behind. Use of methylene blue to stain the neurons makes their identification easier in motoneurons that contain little lipofuscin.

Ten to 20 MN cell bodies are drawn into the pipette by gentle mouth suction in a minimal volume of medium, (usually 1-2 ul) and delivered below the surface of a wash solution containing 100 ul of 2.1M sucrose-CG spread onto a second cover slip. Cell bodies are distributed over about one-third of the wash “puddle,” and fresh solution from the remaining two-thirds of the puddle” is introduced around the cell bodies, dispersing small contaminants around the MNs. The partially washed cell bodies are then picked up and transferred to a second cover slip on which 100 ul of 2.1M sucrose-CG has been spread and washed a second time in the same manner. After two such washes, cell bodies that are virtually free of visible contaminants can be delivered into an tube appropriate for the analyses planned for the experiment (often a conical 400 ul polyethylene microfuge tube). Controls (“blanks’) are taken from sucrose medium surrounding cells in the second wash step, when micro Lowry analyses to be performed.

MN cell bodies from the grass frog contain an average of 4.35 ng protein each, so that preparations containing 200 frog cell bodies contain about 870 ng of protein, which is easily measure by the micro Lowry method and which will display as many as 250 spots on a conventional 2-D gel. Human MN cell bodies contain an average of 13.1 ng protein each or about 1.3 ug per 100 isolated cell bodies. We have not quantified protein in isolated mouse MN cell bodies, but would expect, from their size, a value closer to frog MNs.

BSA-Coated Micropipettes:

Protocol adapted from Aplin, J.D. and Hughes, R.C.   Protein-derivatized glass coverslips for the study of cell-to-cell substratum adhesion.   Anal. Biochem. 113: 144-148. 1981

Reagent Preparation:

200mM Sodium Phosphate, pH 7.2

36 ml of 0.2M Na2HPO4

14 ml of 0.2M NaH2PO4

Phosphate Buffered Saline (PBS)

5ml of 0.2M Na2HPO4, pH 7.2

0.9 g of NaCl.   Add H20 to make 100 ml volume

2.5% Glutaraldehyde in PBS

300ul of 25% glutaraldehyde; 3ml of PBS

BSA Solution

100ul of 10% BSA;   100ul of 0.2M Sodium Phosphate, pH 7.2

Coupling procedure:

 1)   Clean pipette with Chromerge/H2SO4, water, 0.1N NaOH, water.

 2)   Dry inside of pipette by suction or with stream of N 2.

 3)   Fill the pipette with 3-aminopropyltriethoxysilane. Leave pipette tip down in test tube containing a small amount of the silane in the bottom

     for 4 minutes at room temp. (Silane available from Sigma)

4)    Rinse the pipette 8x with water and 3x with PBS.

5)    Fill the pipette with 2.5% glutaraldehyde in PBS.   Leave tip down for 30 minutes at room temp.

6)    Rinse the pipette 6x with PBS.   Dry with nitrogen.

7)    Fill the pipette with BSA solution.   Leave tip down for 1 hr. at room temp.

8)    Rinse pipette with PBS 12x.

9)    Fill the pipette with 2.1M sucrose-CG, pH 5 and store at 4 ° C.

Sucrose Solutions:

0.9 M SUCROSE-CG, pH 5.0

Sucrose (MW = 342.31 g/mol) Weigh out 616.2 grams. [final] = 0.9M

Citrate (MW = 192.13 g/mol) Weigh out 653.2 milligrams. [final] = 1.7mM

Glucose (MW = 180.16 g/mol) Weigh out 5.40 grams. [final] =15mM

Start with 1 liter of deionized water.   Add citrate and glucose and adjust the pH to 5.0.   Then add sucrose and adjust final volume to 2 liters.   Adjust pH to 5.0.

2.1 M SUCROSE-CG, pH 5.0

Sucrose (MW = 342.31 g/mol) Weighed out 1,078.35 grams. [final] = 2.1M

Citrate (MW = 192.13 g/mol) Weighed out 0.48993 grams. [final] = 1.7mM

Glucose (MW = 180.16 g/mol) Weigh out 4.05 grams. [final] =15mM

Add citrate and glucose to a volume of 400 ml. Adjust pH to 5.0 and then add   sucrose.   Final volume = 1.5 liters; final pH = 5.1.