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1 ml

Transferrin

5 ml

Insulin

1 ml

Tri-iodothyronine

50 ^l

Progesterone

50 ^l

Corticosterone

50 ^l

^-Estradiol

0.5 ml

aA 20x prestock solution of SeO2 is prepared at 0.75 mg/ml and then diluted in PBS.

Indianapolis, IN), and (c) 10% by volume of a supplement mixture we call N9 (Table II). This supplement mixture was developed from the basic N2 supplement (Bottenstein and Sato, 1979), but several component concentrations have been changed, and we have added tri-iodothyronine, estradiol, and corticosterone (Akuzawa and Wakabayashi, 1985; Ferreira and Caceres, 1991). The final concentrations of N9 components (all from Sigma) in the medium are bovine serum albumin (20 ^g/ml), corticosterone (200 ng/ml), ^-estradiol (100 ng/ml), insulin (20 ^g/ml), progesterone (63 ^g/ml), putrescine dihydrochloride (32 ,ug/ml), selenium dioxide (75 ng/ml), partially iron-saturated human holo-transferrin (not apo-transferrin) (100 ^g/ml), and tri-iodothyronine (20 ng/ml). As outlined in Table II these supplements are prepared by making and freezing concentrated stocks of each component in appropriate solvents. These are then used to mix 10 x concentrated supplement in unsupplemented L15+ medium, which in turn is frozen in aliquots for addition to the basic medium. This supplement was substantially better at supporting axonal development of chick forebrain neurons than commercially available N2 or B27 supplements used according to the supplier's directions (Life Technologies Inc.). Needless to say, we have not tried every potential growth supplement; we found that the addition of ascorbic acid or its metabolites (Makar et al., 1994) or pyruvate (Bottenstein and Sato, 1979) to the supplement mixture did not improve neuronal development. The addition of gangliosides to synthetic medium has been reported to improve axonal outgrowth (Ferrari et al., 1983; Cestelli et al., 1985), and our original formulation for N9 (Heidemann et al., 2001) included a crude mixture of bovine brain gangliosides (Sigma). However, this particular product is no longer available, and we found that higher purity ganglioside preparations, which are expensive, inhibited growth. Insofar as the addition of the original ganglioside preparation improved growth only marginally, we have not pursued gangliosides further. The chick embryo extract, both commercial and laboratory prepared, did not improve the development of forebrain neurons at 0.5% concentration and inhibited development at higher concentrations.

Many brain neurons in culture benefit from medium containing high [K+] (Franklin and Johnson, 1992), but not chick forebrain neurons. Added at 12, 20, or 25 mM in either L15+- or medium 199-supplemented media, K+ reduced the frequency of cells developing axons to approximately half that seen in control cultures lacking added K+, and by the fifth day nearly all cells were dead.

As we have described, chick forebrain neurons share several growth characteristics with rat hippocampal neurons. The latter are usually cultured with glia or with glia-conditioned medium. Curiously, our experience coculturing chick forebrain neurons with either rat or chick glia has been uniformly negative. Far from helping to support development, in our hands, the addition of glia on an overlying coverslip or growth in glia-conditioned medium substantially inhibited axonal development and hastened cell death even during the first day of culture. It should be noted that Pettman et al. (1979) emphasized the lack of need for glia to support development in chick forebrain neurons.

C. Plating Efficiency and Culture Density

As mentioned earlier, chick forebrain neurons will not develop axons at the very low densities (1000 cells/cm2) of which hippocampal neurons are capable (Goslin and Banker, 1991). We have conducted most of our work with 8-10 x 103 cells/cm2 as an actual density of cells on the dish or coverslip surface. This is low enough to allow for most axons to be clearly associated with a particular cell body but high enough to support axonal development by >50% of cells. In general, the apparent health of the culture increases with increasing density, and we get little or no axonal development at densities below 5 x 103 cells/cm2.

As with most primary cell cultures, there is a substantial loss of cells at early times after plating. After allowing 2-4 h for cell settling and attachment, only 60-70% of neurons added to the dish can be accounted for on the dish surface. By the third day after plating, attrition has reduced the cell number to approximately 50% of those plated. Thus, we typically add twice the number of cells that would otherwise be expected to achieve the desired plating density. After day 4 or 5, we find that those cells that have not yet elaborated an axon begin to die and lose adhesion to the culture surface. Thus, cell density decreases while the fraction of cells in stage 3 increases at longer times of culture, particularly in Medium 199 in which stage 3 neurons remain healthy for about 10 days if the medium is changed every other day. After the elaboration of axons, the fasiculation of axons from different neurons occurs to varying extents. In some cultures, cell bodies also have a tendency to clump after 5 days in culture. Based on anecdotal observations from time-lapse video, this is due in part to neurites contacting other cell bodies, and subsequent contraction of the neurite pulls the cell bodies together.

D. Improvements for the Future

The economy, ease, and simplicity of obtaining freshly prepared chick forebrain neurons would appear to be nearly optimal. However, it is hoped that improvements in culture conditions can be developed for this preparation that would increase their resemblance to rat hippocampal neurons in culture, particularly at longer times. That is, hippocampal neurons cocultured with astrocytic glia can be routinely maintained for 3-5 weeks (Goslin and Banker, 1991), whereas chick forebrain neurons remain healthy only for 10 days in the conditions described earlier. Hippocampal neurons develop axons at a very low plating density and also develop dendrites after about a week in culture, although this is less routine than axonal development (Goslin and Banker, 1991; G. Ruthel, personal communication). These growth characteristics of cultured hippocampal neurons are highly dependent on coculture with rat glial cells. In the absence of glia, hippocampal neurons develop much less well than chick sensory neurons. For this reason, we were particularly disappointed by our lack of success in improving culture conditions for chick forebrain neurons using the glial coculture (Section IV,B). In our own work, the limitations of chick forebrain neurons compared to rat hippocampal neurons have not been serious. For this reason, we have made several, but by no means exhaustive, efforts to improve the culture of these cells. It is hoped in the future that additional growth supplements and/or culture conditions can be identified that improve the development and health of chick forebrain neurons. As noted in the introduction, the advantages of these neurons seem to us to make improvements in their culture very worthwhile, and cooperation among users would be most welcome. Thus, we would be grateful to workers who use chick embryonic neurons if they would share their experiences with us and, for our part, we would be pleased to answer queries from scientists regarding our experiences with these neurons.

Acknowledgment

Our work on chick forebrain neurons was supported by NSF Grant IBN 9603640.

References

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Bottenstein, J. E., and Sato, G. H. (1979). Growth of rat neuroblastoma cell line in serum-free supplemented medium. Proc. Natl. Acad. Sci. USA 76, 514-517.

Bray, D. (1991). Isolated chick neurons for the study of axonal growth. In "Culturing Nerve Cells'' (G. Banker and K. Goslin, eds.), pp. 119-135. MIT Press, Cambridge, MA.

Cestelli, A., Savettieri, G., Ferraro, G., and Vitale, F. (1985). Formulation of a novel synthetic medium for selectively culturing rat CNS neurons. Dev. Brain Res. 22, 219-227.

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Ferreira, A., and Caceres, A. (1991). Estrogen-enhanced neurite growth: Evidence for a selective induction of tau and stable microtubules. J. Neurosci. 11, 392-400.

Fischer, M., Kaech, S., Knutti, D., and Matus, A. (1998). Rapid actin-based plasticity in dendritic spines. Neuron 20, 847-854.

Franklin, J. L., and Johnson, E. M., Jr. (1992). Suppression of programmed neuronal death by sustained elevation of cytoplasmic calcium. Trends Neurosci. 15, 501-508.

Goslin, K., and Banker, G. (1991). Rat hippocampal neurons in low-density culture. In "Culturing Nerve Cells'' (G. Banker and K. Goslin, eds.), pp. 251-281. MIT Press, Cambridge, MA.

Heidemann, S. R., Lamoureux, P., and Atchison, W. D. (2001). Inhibition of axonal morphogenesis by nonlethal, submicromolar concentrations of methylmercury. Toxicol. Appl. Pharmacol. 174, 49-59.

Makar, T. K., Nedergaard, M., Preuss, A., Gebard, A. S., Perumal, A. S., and Cooper, A. J. L. (1994). Vitamin E, ascorbate, glutathione, glutathione disulfide, and enzymes of oxidative metabolism in cultures of chick astrocytes and neurons: Evidence that astrocytes play an important role in oxidative processes in the brain. J. Neurochem. 62, 45-53.

Morgan, J. F., Campbell, M. E., and Morton, H. J. (1955). The nutrition of animal tissues cultivated in vitro. I. A survey of natural materials as supplements to synthetic Medium 199. J. Natl. Cancer Inst. 16, 557-566.

National Research Council. (2000). "Toxicological Effects of Methylmercury," National Academy Press, Washington, DC.

North, M. O., and Bell, D. D. (1990). In "Commercial Chicken Production Manual,'' 4th Ed., pp. 96-100. Van Nostrand Reinhold, New York.

Pettmann, B., Louis, J. C., and Sensenbrenner, M. (1979). Morphological and biochemical maturation of neurones cultured in the absence of glial cells. Nature 281, 378-380.

Ruthel, G., and Banker, G. (1998). Actin-dependent anterograde movement of growth-cone-like structures along growing hippocampal axons: A novel form of axonal transport? Cell Motil. Cytoskel. 40, 160-173.

Ruthel, G., and Banker, G. (1999). Role of moving growth cone-like "wave" structures in the outgrowth of cultured hippocampal axons and dendrites. J. Neurobiol. 39, 97-106.

Tsai, H. M., Garber, B. B., and Larramendi, L. M. H. (1981). 3H-thymidine autoradiographic analysis of telencephalic histogenesis in the chick embryo. I. Neuronal birthdates of telencephalic compartments in situ. J. Comp. Neurol. 198, 275-292.

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