Dmem

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

Pasteur pipette cotton plugged with small rubber bulb (sterile)

5-ml serological pipettes (sterile)

B. Procedure

After enzymatic and mechanical dissociation of whole spinal cords (see Section V), centrifuge the resulting cell suspension for 5min (200 x gmax). Resuspend the cell pellet in 5 ml DMEM (15-ml centrifuge tube), carefully layer on top of a 5-ml cushion of 3% BSA, and spin for 10min at 200 x gmax. The BSA cushion traps much of the small debris. Remove the entire supernatant and resuspend the cell pellet in 5 ml DMEM. Layer the cell suspension onto 5 ml 6.4% metrizamide. Add the cell suspension slowly along the side of the tube slightly above the meniscus and avoid considerable intermixing of the cell suspension and metrizamide. After centrifugation at 500 x gmax for 30min, a band of cells becomes visible at the DMEM/metrizamide interface consisting mostly of motor neurons. Aspirate this fraction, which contains the motor neurons (Pasteur pipette), dilute with twice the volume of DMEM, and centrifuge at 200 x gmax for 10min. Resuspend the cell pellet in spinal cord medium or defined medium using 1 ml per four spinal cords (ventral halves or intact). No preplating is necessary, and plate recovered motor neurons as described in Section VI.

VIII. Experimental Use of Motor Neuron Cultures and Spinal Cord Cultures

Using the conditions described previously, motor neurons, as well as spinal cord neurons, extend neuronal processes within 8-10 h after plating. Neurite outgrowth on laminin is very vigorous with little bifurcations. In contrast, motor neurons on fibronectin or poly-D-lysine grow slower and display more bifurcations. The differentiation of neuronal processes into axons and dendrites can be detected after 3 days in vitro using double indirect immunocytochemical staining. Axons can be revealed using anti-Tau, whereas dendrites are identified using anti-MAP2 (Kuhn et al., 1998). Neurons will survive for over 14 days with regular changes of serum-containing medium every 3 days. Inclusion of 5-fluoro-2'-deoxyruridine, an irreversible inhibitor of thymidylate synthase, interferes with the proper DNA replication of mitotically active nonneuronal cells, such as astrocytes and fibroblasts, which account for less than 5% of total cells in motor neuron cultures (Fig. 5A). Low-density motor neuron cultures allow for investigating the responses of individual neurons or even individual neuronal growth cones to extrinsic cues (Fig. 5B). Preferentially, motor neurons are cultured in a defined, serum-free medium. However, at very low densities, motor neurons survive for about 4 to 6 days. The covalent coupling of extrinsic cues to small polystyrene beads and their presentation to advancing growth cones represents an elegant assay system used to elucidate growth cone responses in detail. For this assay, motor neurons are best cultured on drilled dishes, which allow the use of high magnification objectives for both visible light (phase contrast, DIC) and fluorescence observations. For instance, upon contacting laminin-coated beads, the growth cones of dorsal root ganglia neurons turn toward beads and exhibit increased rates of advance for a considerable time period after passing the beads (Kuhn et al., 1995). Moreover, growth cones are repelled by beads coated with semaphorin3A, and collateral branching results

Chick Forebrain Neurons

Fig. 5 Cultures of spinal motor neurons. (A) A typical high-density culture of spinal motor neurons obtained from 6-day-old chick embryos. Motor neurons were grown for 5 days on laminin in spinal cord medium containing 10% FBS. Preplating and incorporation of FdUr in the medium virtually depletes these cultures of any contaminating nonneuronal cells, such as fibroblasts and astrocytes. (B) Low-density cultures provide an excellent assay system to investigate individual neurons, their processes, and growth cones. A motor neuron after 1 day in culture on laminin extends a neuronal process (most likely an axon). Bifurcation is readily visible by two individual growth cones. Scale bars: 100 ^m.

Fig. 5 Cultures of spinal motor neurons. (A) A typical high-density culture of spinal motor neurons obtained from 6-day-old chick embryos. Motor neurons were grown for 5 days on laminin in spinal cord medium containing 10% FBS. Preplating and incorporation of FdUr in the medium virtually depletes these cultures of any contaminating nonneuronal cells, such as fibroblasts and astrocytes. (B) Low-density cultures provide an excellent assay system to investigate individual neurons, their processes, and growth cones. A motor neuron after 1 day in culture on laminin extends a neuronal process (most likely an axon). Bifurcation is readily visible by two individual growth cones. Scale bars: 100 ^m.

from contact to nerve growth factor-coated beads (Fan and Raper, 1995; Gallo and Letourneau, 1998). As shown in Fig. 6, a motor neuron growth cone encounters a bead coupled covalently with tumor necrosis factor (TNF) a, a proinflammatory cytokine. Over a time period of 30min, the growth cone displays a series of morphological changes and ultimately loses its morphology and ceases advance, i.e., the typical behavioral pattern of growth cone collapse. It is possible that the failure of motor neurons to reestablish neuronal processes following acute injury results partially from the growth inhibitory properties of proinflammatory cytokines such as TNF a . Live video observation of motor

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