Growing and Working with Spinal Motor Neurons

Dorn Spinal Therapy

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Thomas B. Kuhn

Department of Pharmaceutical Sciences University of Montana Missoula, Montana 59812

I. Introduction

II. Incubation of Fertilized Chicken Eggs

A. Materials

B. Procedure

III. Preparation of Chick Embryo

A. Materials

B. Procedures

IV. Dissection of Intact Spinal Cords and Isolation of Ventral Halves

A. Materials

B. Procedures

V. Enzymatic and Mechanical Dissociation of Intact Spinal Cords or Ventral Halves

A. Materials

B. Procedure

VI. Plating Motor Neurons or Spinal Cord Neurons

A. Materials

B. Procedure

VII. Motor Neuron Enrichment by Density Gradient Centrifugation

A. Materials

B. Procedure

VIII. Experimental Use of Motor Neuron Cultures and Spinal Cord Cultures IX. Preparation of Solutions, Culture Dishes, Media, and Media Supplements References

The chick embryo has a long tradition as a model organism in developmental biology as well as embryology. A year-round supply of fertilized eggs, accessibility to all stages of development, and the ease of manipulation of the embryo all

METHODS IN CELL BIOLOGY, VOL. 71 Copyright 2003, Elsevier Science (USA). All rights reserved. 0091-679X/03 $35.00

contribute to the advantages of investigations using chick embryos. A plethora of culture systems have been developed over the past century allowing to culture intact embryos from as early as 2 days of development. Other culture systems include whole embryo slices, organotypic cultures, tissue explants, and dissociated cultures. Studies utilizing the chick embryo, and in particular spinal motor neurons, were crucial for our present knowledge of the development but also adult physiology, injury, and disease of the nervous system. Extensive studies on spinal motor neurons revealed many molecular mechanisms underlying fundamental events, such as neural induction, axon guidance, programmed cell death, and neuron-target interaction. Cultures of dissociated spinal motor neurons represent one important experimental paradigm. This chapter describes two alternative procedures to establish dissociated spinal motor neuron cultures with virtually no contamination by nonneuronal cells.

I. Introduction

The common domestic chick (Gallus gallus) has a long history as one of the most intensely studied model organisms in embryology and developmental biology, dating as far back as Aristotle (384-323 b.c.e.). William Harvey (1578-1657) resumed Aristotle's work, followed by many great scientists such as Marcello Malphigi (1628-1694, first microscopic study of chick development), Kaspar Friedrich Wolff (1733-1794), Karl Ernst von Baer (1792-1876), Frank Rattray Lillie (1870-1947), and Viktor Hamburger (1900-2001). Motor neurons of the chick spinal cord represent the best characterized population of central nervous system (CNS) neurons. Extensive studies have covered every aspect of CNS development, ranging from neural induction, cell fate, axon guidance, trophic factors, and target interactions all the way to function, disease, and injury in the adult organism.

The spatial and temporal induction of motor neurons in the chick spinal chord under the regulation of the notochord, the axial mesoderm organizing center, represents one of the best understood systems of neurogenesis during early embryonic development (Tanabe and Jessell, 1996; Pituello, 1997). The pivotal role of sonic hedgehog, a prototypic morphogen, was recognized in the induction and organization of the first postmitotic motor neurons (Roelink et al., 1995). Fundamental principles of axon guidance and target interaction were derived from studies on motor neurons (Yaginuma et al., 1994; deLapeyriere and Henderson, 1997). For instance, distinct subsets of LIM homeodomain transcription factors are crucial for the establishment of the motor neuron subpopulation with distinct innervation patterns (Pfaff et al., 1996). Axon guidance studies on motor neurons revealed the simultaneous integration of attractive and repulsive guidance cues as diverse as target derived, deposited in the extracellular matrix, exposed on the surface of other neurons or nonneuronal cells, or soluble. Other studies have addressed autocrine, paracrine, and even systemic effects of growth factors and androgens and underlying synergisms at different stages during development (Oppenheim, 1996; Gould et al., 1999; Bibel and Barde, 2000). Formation of the neuromuscular junction is by far the best understood neuron-target interaction in the CNS (Lance-Jones, 1988). Investigations on the naturally occurring cell death of motor neurons during development have contributed significantly to our understanding of programmed cell death (Hamburger, 1975; Houenou et al., 1994). As many as 50% of all motor neurons are eliminated during spinal cord development in vertebrates. Motor neurons are also the study object for various injury models, as well as diseases (Camu and Henderson, 1994; Guale and Burrows, 1997; Bar, 2000). The ease of manipulation of the developing chick embryo contributes significantly to the understanding of the complexity of spinal cord injury (Keirstead et al., 1995; Lu et al., 2000). Also, many inherited and sporadic diseases derive from the selective vulnerability of different motor neuron populations reflected by dysfunction, atrophy, degeneration, and cell death (e.g., amyotrophic lateral sclerosis, juvenile spinal muscle atrophy, Kennedy's disease).

Numerous experimental paradigms, such as dissociated neurons, organotypic culture system, and even in ovo experiments, have been utilized to study the aspects of motor neuron development, adult physiology, injury, and disease (Gaehwiler et al., 1997; Kuhn et al., 1999). The homogeneity of a neuronal population is often critical for understanding neuronal responses to extrinsic cues or insults. For motor neurons, two principal approaches achieve this goal:

(1) retrograde labeling of motor neurons in the living chick embryo followed by selective isolation (Calof and Reichardt, 1984; Honig and Hume, 1986) and

(2) enrichment of motor neurons based on their unique characteristics (Masuko et al., 1979; Juurlink et al., 1990; Kuhn et al., 1998). In retrograde labeling, spinal cord tissue is dissociated and labeled motor neurons are isolated by cell sorting. Although this approach results in a highly purified, homogeneous population of motor neurons, the yields are low and thus pose a significant limitation. The latter approach takes advantage either of the defined anatomy of motor neurons residing in the ventral spinal cord or the unique buoyant density of motor neurons in conjunction with a virtual absence of nonneuronal cells at early developmental stages. The enrichment of motor neurons based on their restricted localization in the ventral spinal cord or their unique buoyant density are technically simple, result in high yields, and achieve considerable enrichment of motor neurons (approximately 95% motor neurons) with very little to no contamination with other neuronal or nonneuronal cell types. These procedures are described in detail. Protocols for the preparation of solutions, media, sera, culture dishes, and more are summarized in Section IX.

II. Incubation of Fertilized Chicken Eggs

Fertilized chicken eggs can be obtained from many different breeds of chicken, but white leghorns are the most common source for research. Fertilized white leghorn eggs can be purchased from local farms or from several commercial suppliers, e.g., Carolina Biological (www.carolina.com). Chicken breed independent of seasons and thus fertilized eggs are available year round and in almost any quantity. A good start to finding local suppliers of fertilized eggs, incubators, and anything related to breeding various avian species can be found on the web site of the Poultry Connection (http://www.poultryconnection.com).

A. Materials

Humidified incubator (Carolina Biological Supply Company, Burlington, NC; Humidaire Incubator Company, New Madison, OH; G.F.Q. Manufacturing Company, Savannah, CA)

Fertilized white leghorn eggs

Egg candler

Refrigerator

Controlled parameters for the incubation of fertilized eggs comprise correct humidity, stable temperature, and regular rotation of the eggs to prevent sticking of the embryo and are imperative for proper embryonic development. Numerous models of incubators are available, ranging from inexpensive to very expensive, and several texts provide useful information regarding egg incubation (Stromberg, 1975; Demming and Ferguson, 1992; Mason, 1999). Fertilized eggs can be stored for up to 14 days in a refrigerator (10 ± 1° C); however, fertility declines pro-portional to storage length.

B. Procedure

It is best not to wash or rinse eggs prior to incubation to avoid clogging of the tiny pores in the eggshell, which are required for proper gas exchange between the embryo and its environment. Although immediate incubation is recommended, fertilized eggs can be stored at 10 ± 1° C (refrigerator) for up to 14 days. Longer storage should be avoided as fertility declines significantly. Place fertilized eggs into egg cartons or egg trays blunt end up and mark the set date on the egg with a pencil. Incubation should be performed in a forced-air, humidified incubator at 37.5-38° C and with 50-60% relative humidity. Eggs will tolerate incubation temperatures from 35° to 40° C but will affect the time course of embryonic development. Humidity is maintained easily utilizing an open water reservoir, which has to be filled daily. During incubation, eggs should be turned several times a day to a 45° angle off the vertical. Turning is important as this prevents the embryo from becoming stuck to the eggshell.

To test whether development occurs, eggs can be candled using a specific egg candler or an improvised light source inside a box containing an egg-sized opening. While moving the egg around, it is held against the light source. Victor Hamburger has established a detailed atlas of defined stages of chick embryo development according to several morphological criteria (Hamburger and Hamilton, 1951). Correct incubation time can be determined according to this atlas. Other excellent texts also refer to chick embryo development (Romanoff, 1960; Freeman and Vince, 1974; Stern, 1994; Bellairs and Osmond, 1998). Motor neurons are obtained from stage 30 embryos (approximately 6-day-old chick embryos). As a rule of thumb, the number of days of incubation taken from the set time correlates well with later stages of embryonic development. Chick embryos older than 6 days are not well suited due to significant motor neuron cell death between embryonic day 6 and 9 and an increased proliferation of nonneuronal cells. Note that chickens hatch at 21 days after incubation.

III. Preparation of Chick Embryo

Spinal cord tissue containing motor neurons is obtained from 6-day-old chick embryos. Removing the chick embryo from the egg requires some practice as at this developmental stage, the embryo is very fragile. To avoid any contamination, all procedures should be carried out in a laminar flow hood. Stainless steel dissection tools can be obtained from several suppliers, including Fine Science Tools (www.finescience.com), World Precision Instruments (www.wpiinc.com), Stoelting (www.stoeltingco.com), Carolina Biological (www.carolina.com), and others. The lifetime of dissection tools can be extended greatly by carefully protecting tips and blades with covers. Bent tips of forceps and scissors can be straightened and sharpened using a sharpening stone.

A. Materials

Fertilized eggs incubated for 6 days Egg carton

70% ethanol in a spray bottle

Waste receptacle lined with a plastic bag

Box of tissue paper

Dissection instruments (sterile): one straight forceps, one curved forceps, one fine scissors, and one spring scissors Hank's balanced salt solution (HBSS, Gibco) Pasteur pipette cotton plugged with a small rubber bulb (sterile) 100-mm glass petri dishes (sterile) 50-ml glass beaker containing 70% ethanol

Dissecting instruments are sterilized either by autoclaving or by immersing into 70% ethanol and very brief flaming in a gas burner. Do not hold instruments into the flame for extended periods of time, as overheating will turn the fine tips of forceps and scissors brittle and useless. A typical setup for the dissection of embryonic chick spinal motor neurons is shown in Fig. 1.

Fig. 1 Setup for spinal motor neuron dissection. Initial setup for the dissection of motor neurons. (A) A stereo zoom microscope (M) is placed in a laminar flow and wiped with 70% alcohol. Dissection tools (T) are flamed with 70% alcohol. A beaker with 70% alcohol (not shown) is used to sterilize tools during the dissection. (B) An ice bucket (I) carries a 60-mm glass petri dish containing HBSS for the collection of spinal cord ventral halves. Six eggs incubated for 6 days have been placed in an egg carton (C) and are rinsed with 70% alcohol. A wastebasket (W) is ready for the disposal of eggs and remains of embryos after dissection. (C) The most important dissection tools include two forceps (#5) and spring scissors (SS). The tips of these instruments have to be sharp and straight. (See Color Insert.)

Fig. 1 Setup for spinal motor neuron dissection. Initial setup for the dissection of motor neurons. (A) A stereo zoom microscope (M) is placed in a laminar flow and wiped with 70% alcohol. Dissection tools (T) are flamed with 70% alcohol. A beaker with 70% alcohol (not shown) is used to sterilize tools during the dissection. (B) An ice bucket (I) carries a 60-mm glass petri dish containing HBSS for the collection of spinal cord ventral halves. Six eggs incubated for 6 days have been placed in an egg carton (C) and are rinsed with 70% alcohol. A wastebasket (W) is ready for the disposal of eggs and remains of embryos after dissection. (C) The most important dissection tools include two forceps (#5) and spring scissors (SS). The tips of these instruments have to be sharp and straight. (See Color Insert.)

B. Procedures

1. Accessing the Chick Embryo

Remove fertilized eggs from the incubator after 6 days of incubation and put, blunt end up, into an egg holder (part of an egg carton). Do not remove more than six eggs at one time. Briefly spray eggs with 70% alcohol, which cleans the shell sufficiently but does not sterilize it completely. Using the straight forceps, punch a hole through the blunt end of the shell (Figs. 2A and 2B), which is where the air space of the egg is located. Insert one tip of the forceps into the hole and remove the eggshell by breaking off pieces around the entire air space (Fig. 2C). At this time, the embryo is hidden under the inner shell membrane, a thick white membrane. It is the easiest to do this right over the waste receptacle. Resterilize the straight forceps by soaking in the beaker filled with 70% ethanol.

2. Isolation of Chick Embryo

The following procedure allows a quick removal of the chick embryo from the egg and minimizes both stress and discomfort of the animal. All methods adhere to the guidelines set by the International Animal Care and Use Committee and a report on euthanasia ( Journal of the American Veterinary Medical Association, Vol. 202, No. 2, pp. 229-249, 1993). Using the bent forceps, peel the inner shell membrane off; the embryo becomes visible enclosed in the highly vascularized, chorioallantoic membrane. Pierce this membrane with the bent forceps or make an incision with the fine scissors to create an opening. Hook the embryo around the neck and lift it out very gently (Fig. 2D). All the extraembryonic membranes, including amnion, yolk sac, allantois, and chorion, are still connected to the embryo. Lifting the embryo out too quickly will inevitably rupture its neck. Place the embryo on its side in the dissecting dish and remove the head immediately by

Fig. 2 Removal of the chick embryo from the egg. (A) Schematic drawing of a chicken egg incubated for 6 days. A robust eggshell (S) protects the embryo. The outer shell membrane (O) and the inner shell membrane (I) enclose the embryo (E), albumen, and yolk. The blunt end of the egg contains the air space (A), which lies between the outer and the inner shell membranes. (B) Using forceps (straight or bent types work well), a hole is punched through the blunt end of the egg. (C) The eggshell is removed piece by piece all around the entire air space. After peeling off the thick, white inner shell membrane, the embryo is readily visible. (D) The highly vascularized chorioallantoic membrane, which encloses the embryo, is pierced with #5 forceps or cut with fine scissors, and the embryo is lifted out at its neck using the bent forceps. If the embryo is lifted out too quickly, its neck will rupture. (See Color Insert.)

Fig. 2 Removal of the chick embryo from the egg. (A) Schematic drawing of a chicken egg incubated for 6 days. A robust eggshell (S) protects the embryo. The outer shell membrane (O) and the inner shell membrane (I) enclose the embryo (E), albumen, and yolk. The blunt end of the egg contains the air space (A), which lies between the outer and the inner shell membranes. (B) Using forceps (straight or bent types work well), a hole is punched through the blunt end of the egg. (C) The eggshell is removed piece by piece all around the entire air space. After peeling off the thick, white inner shell membrane, the embryo is readily visible. (D) The highly vascularized chorioallantoic membrane, which encloses the embryo, is pierced with #5 forceps or cut with fine scissors, and the embryo is lifted out at its neck using the bent forceps. If the embryo is lifted out too quickly, its neck will rupture. (See Color Insert.)

either using fine scissors or firmly grasping the neck just caudal of the head with the bent forceps and squeezing. Finally, using the fine scissors, remove all the extraembryonic membranes and rinse the embryo thoroughly with HBSS.

IV. Dissection of Intact Spinal Cords and Isolation of Ventral Halves

The enrichment of motor neurons by isolating the ventral halves of the spinal cords takes advantage of the unique anatomy of motor neurons, their exclusive residence in the ventral hemichords (Masuko et al., 1979; Kuhn et al., 1998). With a little practice, it is surprisingly easy to isolate the intact spinal cord from 6-day-old chick embryos.

A. Materials

Dissection instruments (sterile): two #5 forceps, one fine scissors, one spring scissors, and one scalpel (blade #10, handle #3).

Dissecting microscope (a stereo zoom microscope with incident and/or transmitted light works well)

HBSS (Gibco)

Pasteur pipette cotton plugged with a small rubber bulb (sterile)

60-mm glass petri dish (sterile, kept on ice) filled with 3 ml ice-cold HBSS

Ice bucket with ice

70% ethanol in a spray bottle

Box of tissue paper

50-ml glass beaker containing 70% ethanol

Prior to dissecting, wipe the stereomicroscope with tissues soaked in 70% alcohol. Sterilize all dissecting instruments by rinsing with 70% alcohol and flaming briefly. Instruments can be resterilized during the dissection by soaking in a beaker containing 70% alcohol. Always keep the embryo moist during the dissection using ice-cold HBSS. As general rules for microscopy, adjust your optics properly for each eye, use low light levels, and work with the least magnification. These precautions all reduce stress on your eyes.

B. Procedures

1. Dissection of Intact Spinal Cords

At this point, the head and all the extraembryonic membranes of the embryo have been removed. To eviscerate the embryo, cut along the ventral side ofthe spine without touching the spine from neck to tail, (spring scissors) remove all internal organs, cut off limb buds and wing buds (Fig. 3A). Hold the embryo in place by grabbing the skin on the back with #5 forceps. Turn the remainder of the embryo onto the ventral side. Starting at the neck, remove the epidermis on the back by pulling to the sides using one #5 forceps in each hand and clip the very end of the tail with the spring scissors. Care should be taken not to puncture or break the spine. Now the two dorsal halves of the spinal cord are visible as two solid, light strands separated by a thin, dark midline. Make a deep incision into the spinal cord along the midline (Fig. 3B). Starting at the tail, insert one tip of the spring scissors into the central canal of the spinal cord and cut (Fig. 4A). Continue along the midline toward the neck while holding the embryo in place using #5 forceps. This incision should separate the dorsal hemicordes, yet leave the ventral halves intact. Gently push each side of the cut apart from each other (use #5 forceps closed), both

Fig. 3 Preparation of the embryo for spinal cord dissection. (A) After the embryo is lifted out of the egg, it is placed on its side in a 100-mm glass petri dish and the head is removed immediately. The dashed line along the ventral side of the spine indicates where the incision is made using fine scissors to eviscerate the embryo. Anterior end or neck (A). Posterior end or tail (P). Wing buds (W). Limb buds (L). (B) After the embryo has been eviscerated, the back containing the spine and all surrounding tissue is turned onto the ventral side. The dashed line indicates the midline along which the incision is made. (See Color Insert.)

Fig. 3 Preparation of the embryo for spinal cord dissection. (A) After the embryo is lifted out of the egg, it is placed on its side in a 100-mm glass petri dish and the head is removed immediately. The dashed line along the ventral side of the spine indicates where the incision is made using fine scissors to eviscerate the embryo. Anterior end or neck (A). Posterior end or tail (P). Wing buds (W). Limb buds (L). (B) After the embryo has been eviscerated, the back containing the spine and all surrounding tissue is turned onto the ventral side. The dashed line indicates the midline along which the incision is made. (See Color Insert.)

hemicordes of the spinal cord are now clearly visible (Fig. 4B). Insert #5 forceps (keep closed) between the spinal cord and the surrounding tissue and gently move along each lateral surface of the spinal cord to separate the spinal cord from meninges and dorsal root ganglia (Fig. 4C). Rinse and cover the preparation well with HBSS. Lift the spinal cord carefully out of its cavity by inserting closed #5 forceps between the spinal cord and underlying tissue starting from the neck (Fig. 4D). If enough HBSS is used, the spinal cord will actually float, which facilitates this procedure greatly. Isolate the entire spinal cord without breaking or twisting. To establish dissociated neuronal cultures from whole spinal cords or when enriching for motor neuron utilizing density gradient centrifugation, transfer the intact spinal cord to a 60-mm petri dish containing ice-cold HBSS (use a Pasteur pipette) and continue with Section V. Otherwise proceed with isolating ventral halves.

2. Isolation of Ventral Halves of Spinal Cords

The intact isolated spinal cord now lies flat with the dorsal halves facing outward and the two adjacent ventral halves (open-book preparation). It is

Fig. 4 Isolation of the ventral spinal cord. (A) After the skin is removed, an incision is made along the midline using spring scissors. This incision reaches all the way into the central canal of the spinal cord, thus separating the dorsal halves but leaving the ventral halves connected. (B) With closed #5 forceps, the incision is enlarged by pushing each side outward. (C) The meninges and the nerve fiber bundles of the dorsal root and ventral root are severed by gently moving closed #5 forceps anterior to posterior along the surface of the spinal cord. (D) With #5 forceps, the spinal cord is separated carefully from the underlying tissue and lifted out. Plenty of HBSS facilitates this step greatly, as the loosened spinal cord will float. (E) Once the entire spinal cord is isolated (open-book preparation), the dorsal halves (dotted areas) are removed from the ventral halves (white area) using a scalpel. Each spinal cord hemisphere displays characteristic morphologies and is easily distinguished. (F) Ventral halves are collected in ice-cold HBSS.

Fig. 4 Isolation of the ventral spinal cord. (A) After the skin is removed, an incision is made along the midline using spring scissors. This incision reaches all the way into the central canal of the spinal cord, thus separating the dorsal halves but leaving the ventral halves connected. (B) With closed #5 forceps, the incision is enlarged by pushing each side outward. (C) The meninges and the nerve fiber bundles of the dorsal root and ventral root are severed by gently moving closed #5 forceps anterior to posterior along the surface of the spinal cord. (D) With #5 forceps, the spinal cord is separated carefully from the underlying tissue and lifted out. Plenty of HBSS facilitates this step greatly, as the loosened spinal cord will float. (E) Once the entire spinal cord is isolated (open-book preparation), the dorsal halves (dotted areas) are removed from the ventral halves (white area) using a scalpel. Each spinal cord hemisphere displays characteristic morphologies and is easily distinguished. (F) Ventral halves are collected in ice-cold HBSS.

imperative to keep the intact isolated spinal cord always covered with ice-cold HBSS. Any attached meninges or dorsal root ganglia can be removed using #5 forceps. Using the scalpel, separate the dorsal halves by pushing down between dorsal and ventral halves, moving stepwise from anterior to posterior (Figs. 4E

and 4F). Slicing will inevitably rupture the spinal cord. The two halves of the spinal cord exhibit distinct morphology and are easily distinguishable. While dorsal halves display an opaque, coarse morphology, the ventral halves are homogeneous and almost transparent. Transfer ventral spinal cord halves into a 60-mm petri dish containing ice-cold maintenance solution using a Pasteur pipette. Isolated ventral halves or whole spinal cords can be kept on ice in maintenance solution for up to 4 h.

V. Enzymatic and Mechanical Dissociation of Intact Spinal Cords or Ventral Halves

Chelating free Ca2+ ions in combination with tryptic digestion significantly weakens many cell-cell and cell-matrix adhesion interactions and facilitates the mechanical dissociation by shear forces. Neuronal viability is compromised by prolonged exposure to trypsin.

A. Materials

15-ml conical centrifuge tubes (sterile)

Pasteur pipette cotton plugged with a small rubber bulb (sterile) 5-ml serological pipettes (sterile)

Clinical centrifuge (swing-out rotor up to 2000 x gmax is sufficient) Ice

Cell dissociation solution (Gibco; 10 x stock aliquots)

2% DNase I (Roche Diagnostics Corp.; 100 x sterile stock solution)

Horse serum (HS), sterile and heat inactivated (Hyclone)

Fetal bovine serum (FBS), sterile and heat inactivated (Hyclone)

Water bath heated to 37° C

Dulbecco's modified Eagle medium (DMEM) high glucose (Gibco) DMEM/10% FBS

B. Procedure

Transfer intact spinal cords or ventral halves to a 15-ml conical tube and rinse twice with HBSS by gently swirling and removing the supernatant after tissue has settled. Adjust volume with HBSS using 1 ml HBSS for each four ventral halves or whole spinal cords. Add cell dissociation solution (1/10 of final volume) and DNase I (1/100 of final volume) and incubate for 10min at 37° C (water bath). Extensive tryptic digestion will result in considerable cell debris and significant cell death. Add horse serum (10% final concentration) and swirl the tissue gently.

Remove the supernatant after tissue has settled and resuspend in DMEM. Mechanically dissociate tissue (triturate) by pushing tissue through a Pasteur pipette pressed against the bottom of a conical tube. The resulting shearing forces will dissociate the tissue quickly, resulting in a slightly pale cell suspension. Allow any remaining fragments to settle before collecting the cell suspension in a 15-ml conical tube. Pellet cells (200 x gmax, 2min)andresuspendin2mlDMEM/10%FBS for each four ventral or whole spinal cords. Section VII describes the enrichment of spinal motor neurons by density gradient centrifugation utilizing cell suspensions obtained from intact spinal cords.

VI. Plating Motor Neurons or Spinal Cord Neurons

Motor neurons or spinal cord neurons can be grown in either serum-containing or serum-free medium. Long-term cultures (5 days or longer) require the presence of serum. It is recommended to adjust cell density prior to plating by counting cells in a hemacytometer. Motor neurons or spinal cord neurons exhibit robust neurite outgrowth and excellent survival when plated on laminin-coated dishes.

A. Materials

Pasteur pipette cotton plugged with a small rubber bulb (sterile) 5-ml serological pipettes (sterile) Pipettors with tips Hemacytometer

Clinical centrifuge (swing-out rotor up to 2000 x gmax) 15-ml conical tubes

Drilled tissue culture dishes (35 mm) equipped with a laminin-coated glass coverslip

Laminin-coated glass coverslips inserted into 35-mm tissue culture dishes Laminin-coated 35- or 60-mm tissue culture dishes Spinal cord medium (see Section VIII) Defined medium (see Section VIII.)

Plate motor neurons or spinal cord neurons into either of the tissue culture dishes listed earlier depending on the purpose of the experiment. Drilled dishes (see Section XI) are ideal for live video observations at high magnification, whereas glass coverslips inserted into 35-mm dishes work well for immunocyto-chemical staining. For biochemical assays, plate cells directly into 35- or 60-mm tissue culture dishes. Although laminin is suggested as a substrate for growing motor neurons or spinal cord neurons, other substrates can be substituted, such as fibronectin or poly-D-lysine (Section XI).

B. Procedure

Place cell suspensions of dissociated ventral halves or whole spinal cords in a 60-mm tissue culture dish (four ventral halves of whole spinal cord, 2 ml DMEM/ 10% FBS) and preplate for 1 h (CO2 incubator, 37° C, 5% CO2). This preplating step depletes nonneuronal cells due to their preferential adhesion to tissue culture plastic while neuronal cells remain in suspension.

Carefully recover the supernatant, pellet cells (200 x gmax for 5min), and resuspend the cell pellet in spinal cord medium or defined medium using 1 ml medium for four spinal cords or ventral halves. Determine the cell density using a hemacytometer. One intact spinal cord yields approximately 1 x 106 neurons, whereas ventral halves of one spinal cord should yield around 1-2 x 105 motor neurons. For live video observation, of individual neurons, plate cells at 75,000 cells/ml (plating volume 500 yl) onto a laminin-coated glass coverslip glued over a 1-cm hole drilled into the bottom of a 35-mm tissue culture dish. For immunocytochemical staining, plate cells at 1.5 x 105 cells/ml (plating volume 500 yl) onto laminin-coated glass coverslips inserted into 35-mm tissue culture dish. For biochemical assays, cells are best plated into laminin-coated 35-mm tissue culture dishes (0.5-1 x 106 cells/ml, plating volume 2ml) or into 60mm tissue culture dishes (0.5-1 x 106 cells/ml, plating volume 4ml).

VII. Motor Neuron Enrichment by Density Gradient Centrifugation

Density gradient centrifugation takes advantage of the buoyant density of large motor neurons. Although this technique does not require isolation of the intact spinal cord, the yield of motor neuron enrichment is slightly less compared to the isolation of ventral halves. The technique described in this section is adapted with modifications (Juurlink, 1992).

A. Materials

6.4% metrizamide

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