Immortal rat hippocampal cell lines exhibit neuronal and ... - NCBI - NIH

Proc. Natl. Acad. Sci. USA Vol. 89, pp. 4373-4377, May 1992 Neurobiology

Immortal rat hippocampal cell lines exhibit neuronal and glial lineages and neurotrophin gene expression (brain-derived neurotrophic factor/nerve growth factor/neurotrophin 3/neuronal differentiation/neural trophic interactions)

EVA M. EVES*, MARCY S. TUCKER*t, JOHN D. ROBACKO, MARTHA DOWNEN*, MARSHA RICH ROSNER*t, AND BRUCE H. WAINER*t§ Departments of *Pharmacological and Physiological Sciences and of VPathology, tThe Ben May Institute, and §The Committee on Neurobiology, The University of Chicago, Chicago, IL 60637

Communicated by Daniel E. Koshland, Jr., January 13, 1992

conditional for growth, and several lines exhibit morphological and biochemical phenotypes after induction indicative of neuronal, glial, or bipotential lineage. Several of the lines show differential patterns of transcript expression for the neurotrophin genes, nerve growth factor (NGF), brainderived growth factor (BDNF), and neurotrophin 3 (NT-3). In some lines, the relative levels of BDNF and NT-3 message may reflect the reciprocal expression of these genes in the developing hippocampus (9) or the differential expression observed for adult hippocampal regions (10). In addition, one line of putative glial lineage secretes trophic factors that support the maturation of primary septal neurons in culture. These cell lines are distinguished from previously developed hippocampal lines in that they are not derived from transformed cells and they do express, in culture, markers indicative of commitment to glial and neuronal lineages.

Clonal cell lines of rat embryonic hippocamABSTRACT pal orin have been developed by using retroviral transduction of temperature-sensitive simian virus 40 large tumor antigens. The cell lines undergo morphological differentiation at the nonpermissive temperature and in response to differentiating agents. Immunocytochemical analysis indicates that various lines are derived from progenitors of neuronal, glial, and bipotential lineages. Selected neuronal lines differentiate in response to diffusible factors released by primary glia, and one line of glial lineage supports the maturation of primary neurons in culture. Selected cell lines exhibit different patterns of neurotrophin gene expression that change after differentiation. In some lines, the relative levels of neurotrophin 3 and brainderived neurotrophic factor message expression may reflect the developmental or regional differential expression seen for these genes in the hippocampus in situ. These hippocampal cell lines, which express markers indicative of commitment to neuronal or glial lineages, are valuable for studies of development and plasticity in these lineages, as well as for studies of the regulation of neural trophic interactions.

MATERIALS AND METHODS Vectors. Two i2 NIH 3T3 lines that produce replicationdefective retrovirus carrying the neo gene (producing resistance to G418) and either the tsA58 or the U19tsa temperaturesensitive mutation of the simian virus 40 large tumor antigen gene were obtained from P. Jat (Ludwig Institute for Cancer Research) (11). The mutant large tumor antigens are functional at 33°C and defective at 37°-39°C. Primary Cells, Infection, and Selection. Hippocampi were dissected from embryonic day 17 (E17) Holtzman and E18 Wistar-Kyoto rat embryos. Cells were dissociated with 0.25% trypsin and plated in DMEM-complete (Dulbecco's modified Eagle medium/10%o fetal bovine serum (FBS)/2 mM L-glutamine/penicillin at 50 units/ml/streptomycin at 50 ,Ag/ml) in tissue-culture dishes coated with polyornithine (15 ,ug/ml). Retroviral infection was done the following day as described (11), except that the cells were not subcultured before selection with G418 at 250 ,ug/ml. Single clones were isolated 2-4 weeks after infection. Initial Screening. One day after plating in DMEM-complete at 33°C, the culture medium was replaced with low-serum (1% FBS) DMEM containing 20 nM hydrocortisone, 0.3 nM triiodothyronine, 0.1 mM putrescine, 20 nM progesterone, 1 pM estradiol, 30 nM NaSeO3, transferrin at 1 ,ug/ml, and insulin at 5 ,ug/ml (lsDMEM), and the cultures were shifted to 39°C (nonpermissive temperature). When used, the differentiation agent 10 nM phorbol 12,13-dibutyrate (PBt2) was added on day 3. Growth arrest and morphological changes were microscopically assessed on day 6 or 7.

A major challenge in neurobiology is to understand the mechanisms mediating the formation and maintenance of synaptic connections. Current hypotheses include internal cell programs as well as chemical signals, such as cell adhesion molecules (1), neurotransmitters (2), and neural

trophic factors (3). To study such mechanisms in detail it is necessary to examine the properties of individual cell types. Although primary culture techniques can enrich for particular cell types of interest (4, 5), they do not easily produce sufficient material for biochemical analyses. An alternative approach, the use of tumor cell lines with neural phenotypes, is limited by their malignant nature and lack of cell-lineage specificity. For these reasons, it would be advantageous to have available cell lines of known brain-region origin that express phenotypes of particular subsets of cells. Of the immortalization methods available (6), retroviral-mediated oncogene transduction [e.g., temperature-sensitive (ts) large tumor antigen or myc under the control of a glucocorticoid-inducible promoter] has several clear advantages. The immortalizing gene function can be conditional to allow control of growth, the cells to be immortalized can be preselected by anatomical (7) or molecular (8) criteria, and the presence of a selectable marker in the vector allows transduced cells to be selected over spontaneous transformants and dividing primary cells. The present study reports the use of two ts alleles of the simian virus 40 large tumor antigen to produce immortal cell lines from embryonic rat hippocampus. These lines are

Abbreviations: PBt2, phorbol 12,13-dibutyrate; NFP, neurofilament protein; GFAP, glial fibrillary acidic protein; MAP-2, microtubuleassociated protein 2; GAP-43, 43-kDa growth-associated phosphoprotein; NGF, nerve growth factor; BDNF, brain-derived neurotrophic factor; NT-3, neurotrophin 3; E (followed by number), embryonic day; FBS, fetal bovine serum; ts, temperature sensitive.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 4373

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(3HJThymidine Incorporation. Cells were plated at 104 cells per well in polylysine-coated 12-well culture plates. Parallel wells were either maintained at 330C in DMEM-complete or subjected to the differentiation protocol described above. Then the medium was replaced with fresh lsDMEM containing [3H]thymidine at 2 ACi/ml (1 Ci = 37 GBq) for 24 hr. The cells were then fixed in cold 5% (vol/vol) trichloroacetic acid for 20 min and washed with 100% ethanol. The acid-insoluble precipitate was solubilized with 400 Ald of 0.1 M NaOH for 30 min at 650C, neutralized with 27 Al of H3PO4, and added to 8 ml of scintillation fluid. Immunocytochemistry. Antibodies recognizing neurofilament protein (NFP) subunits and glial fibrillary acidic protein (GFAP) were from V. Lee (University of Pennsylvania) (12-14). Antibody to microtubule-associated protein 2 (MAP-2) (15). was obtained from L. Binder (University of Alabama). Antibody to the 43-kDa growth-associated phosphoprotein (GAP43) (16) was obtained from D. Schreyer (Stanford University). Low-affinity NGF receptor was detected with monoclonal antibody 192 IgG (17). Choline acetyltransferase was detected with monoclonal antibody AB8 (18). Cells were fixed in 3% (wt/vol) paraformaldehyde/0.1% glutaraldehyde in 0.1 M phosphate (pH 7.4), blocked with 5% (wt/vol) milk in Tris-buffered saline (TBS; 0.05 M Tris/0.14 M NaCI, pH 7.4) (30 min), and incubated at 4°C with primary (16 hr) and secondary (16 hr) antibodies and peroxidaseantiperoxidase (4 hr). Immunoreactivity was visualized with diaminobenzidine at 0.5 mg/ml/0.01% peroxide in TBS. mRNA Analysis. RNA isolation, blotting, hybridization, and analysis were done as described (19). The probes were a 0.9-kilobase (kb) Pst I fragment of mouse NGF cDNA from L. Reichardt (University of California at San Francisco) (20), a 1.1-kb rat BDNF cDNA clone from G. Yancopoulos (Regeneron Pharmaceuticals) (21), and a 1-kb NT-3 PCR fragment from rat genomic DNA (T. Large, Case Western Reserve University). Biaminar Cultures. Bilaminar cultures were set up as described by Banker and Cowan (4, 5) and modified by Scholz and Palfrey (22). Primary glial layers were from 2-day-old rat cortices and were used directly or passaged once. The neuronal layer was prepared from freshly dissociated cells from specific embryonic brain regions (E16 septa). Two days after plating the neuronal layer, antimitotic (15 AM cytosine ,-D-arabinofuranoside) was added to prevent glial overgrowth and progenitor cell proliferation. RESULTS AND DISCUSSION Production. We immortalized, via retroviral transduction of the tsA58 and U19tsa simian virus 40 large tumor antigens, 16 clones from E17 Holtzman and six cell lines from E18 Wistar-Kyoto rat hippocampi. In general, at 33°C, the cells are polygonal and quite flat with prominent nuclei when viewed with phase optics (Fig. 1A). Some of the clones exhibit morphological heterogeneity at 33°C, which persists after subcloning and may be attributable to the immortalization of multipotential progenitors (23). Population-doubling times were estimated by counting cells in discrete clones on successive days after plating. For four lines the doubling times range from 22 to 31 hr. Initial Characterization. Two properties essential to the lines sought are conditional proliferation and a capacity for differentiation after cessation of division. The lines were evaluated for changes in growth rate upon a shift from 33°C to 39°C. Twenty of 22 lines slow division at 39°C, reflecting expression of the ts large tumor antigens. Cell lines that did not display any obvious reduction in growth rate at 39°C were not included in further characterization studies. For cell lines of particular interest, conditional proliferation was confirmed by [3H]thymidine incorporation studies. After differentiation (see Materials and Methods), the lines tested have 10-fold or

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FIG. 1. Effects of temperature and medium on the morphology of a neuronal hippocampal cell line. Photomicrographs of parallel cultures of H19-7 cells at 33TC in DMEM-complete (A), at 39TC in DMEM-complete (B), at 39TC in IsDMEM (C), and at 39TC in IsDMEM and 10 nM PBt2 (D). (E) H19-7 cells plated on coverslips and grown over primary glia at 3TC. During differentiation cells that were flat and polygonal became elongated, cell bodies thickened, and multiple processes were extended. Note the longer, more complex processes on cells over glia (E) compared with those cultured alone (C and D). (Scale bar = 50 pum.)

greater reductions in the amount of [3H]thymidine incorporated compared with undifferentiated cultures (Fig. 2). When 10%o FBS is added to differentiated cells and the cultures are maintained at 390C, the cells reacquire the flat, spreading morphology of cells at 39TC in high serum (Fig. 1B) but do not incorporate [3H]thymidine (data not shown), indicating that differentiated cells are viable and plastic with respect to morphology but are not returned to proliferation by the mitogenic factors in FBS. Neural differentiation in the hippocampal lines was first evaluated at the level of morphology. Those lines in which some proportion of the cells elaborate processes after serum reduction at 39"C, with or without PBt2 addition (see MatecD

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Neurobiology: Eves et A Table 1. Immunostaining for neuronal and glial markers after differentiation Marker GAP-43 MAP-2 GFAP NFP Line + + + _ H19-7 + + H583-8 + + WH19-2 + + WH19-3 + + + WH19-4 + + + WH19-5 + + WH19-6 + + WH583-1 + + H19-6 + + + H583-4 + ND + ND H583-5 + H19-1 + ND ND H19-5 +, Estimated 20%o or more of cells stain for marker; ±, a few cells stein; -, staining was not detected for marker; ND, not done. Holtzman E17-derived clones are designated H19-x or H583-x, and Wistar-Kyoto E18 clones are designated WH19-x or WH583-x, reflecting their species derivation and immortalizing large tumor antigen.

rials and Methods and Fig. 1 C and D), are considered capable of differentiation. Several agents including dibutyryl cAMP PBt2, and a mixture (dibutyryl cAMP/isobutylmethylxanthine/NGF) used by Ronnett et al. to induce morphological differentiation in a spontaneous human cortical line (24) elicit differentiation in these cells (E.M.E., M.R.R. and

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FIG. 3. Immunostaining for NFP and GFAP. Immunoreactivity in two lines H19-7 (A and B) and WH19-4 (C and D) with antibodies against the middle- (A and B) or high- (C and D) molecular-weight NFP. H19-7 cells exhibited no immunoreactivity in the proliferating state (33°C, DMEM-complete) (A), but most cells stained moderately after differentiation at 39°C in lsDMEM with PBt2 (B). WH19-4 cells exhibited immunostaining before (C) and after (D) subjection to the differentiation protocol. Line H19-5 stained by using an antibody to GFAP (E) but shows no immunoreactivity with anti-NFP antibodies (F), indicating a glial, nonneuronal lineage. In F cells were photographed with differential interference-contrast optics to visualize nonstaining cells. (Scale bar = 100 am.)

FIG. 4. Primary septal neurons from E16 rats grown in bilaminar cultures. Cells were maintained over primary glial cell layers (A, C, E, and G) or over H19-5 cells (B, D, F, and H). After 7 days in culture the neurons were stained by using antibodies to NFP (A and B), GAP-43 (C and D), low-affinity NGF receptor (E and F), and choline acetyltransferase (G and H). (Scale bar = 100 ,um.)

B.H.W., unpublished work). PBt2 was chosen initially as the differentiating agent because it induces morphological differentiation in many of the lines after a single low-dose exposure (10 nM), and less toxicity was observed. Agents that have no evident effect on morphology include sodium butyrate, retinoic acid, and NGF alone or in combination with basic fibroblast growth factor (E.M.E., M.R.R. and B.H.W., unpublished work). Those lines in which at least half of the cells differentiate or in which some cells extend very long or elaborate processes were further characterized. Proliferating and differentiated cultures were stained immunochemically using antibodies to GFAP and NFPs (Table 1). The proportion of cells that stain positively and the staining intensity vary from line to line. For example, line H19-7 does not stain for NFP before induction; after induction most H19-7 cells stain positively for NFP. In contrast, most WH19-4 cells stain for NFP both before and after induction (Fig. 3). The results indicate that cells of neuronal lineage (NFP+ GFAP-) and of glial lineage (NFP- GFAP+), as well as uncommitted progenitors (NFP+ GFAP+), have been immortalized. For the latter, whether all the cells are bipotential or a mixture of different lineages remains to be determined. NFP-positive lines were further characterized by using antibodies to MAP-2 and GAP-43. In mature neurons MAP-2 is found predominantly in cell bodies and dendrites (25), and GAP-43 is found predominantly in cell bodies and axons (16),

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t. FIG. 5. Neurotrophin gene expression in hippocampal lines. Four lines were screened for neurotrophin mRNA. On the autoradiographs, the first lane in each panel contains RNA from adult rat cortex used here as a tissue standard. In each set of three lanes, the first is total RNA from cells growing at 330C; the second is from cells maintained at 390C in lsDMEM for 5 days; for the third lane the cells were at 390C in lsDMEM for 5 days with PBt2 for the last 3 days (39P). The histograms at right were constructed after densitometry and normalization for poly(dT)-hybridizing RNA. Asterisks in BDNF panels indicate a previously unreported BDNF RNA band and the portion of the total BDNF signal in that lane attributable to this band. Two independent sets of cultures produced similar expression patterns.

whereas in neurons undergoing maturation in culture, both markers are initially distributed throughout the cell (26, 27). All NFP+ lines also express MAP-2. Six of 10 NFP+ MAP 2+ lines express GAP-43. A NFP- GFAP+ line, H19-1, expresses neither marker (Table 1). None of the lines tested appears to exhibit segregation of MAP-2 versus GAP-43 immunoreactivity in a pattern indicative of dendritic and axonal differentiation. These findings suggest that the lines do not undergo complete cytochemical maturation under the conditions used. Variability among the lines for neuronalmarker expression could reflect immortalization of progenitors of different neuronal lineages or progenitors of the same lineage at different stages of differentiation. Differentiation in these cell lines depends both on the inactivation of the ts large tumor antigen and on environmental conditions. Culture at 390C produces an extremely flat cell morphology (Fig. 1B), but serum reduction is required for distinct process formation (Fig. 1C). Morphological differentiation after FBS withdrawal has been described for a number of permanent neuronal and glial lines including PC12 cells (28). In many of our lines the effect of reduced serum is potentiated by PBt2 treatment (Fig. 1D). Several NFP+ GFAP- hippocampal lines are responsive to factors secreted by primary glia. H19-7 is a line of particular interest as a good example of a ts neuronal progenitor; upon

induction it ceases DNA replication and cell division (Fig. 2) and exhibits a high degree of morphological differentiation (Fig. 1), its expression of neuronal markers is inducible (Fig. 3), and it does not express GFAP. When H19-7 is plated onto coverslips and placed at 370C, in IsDMEM, over a layer of glia treated with an antimitotic, the cells differentiate more rapidly and to a greater extent than when plated alone (Fig. lE). However, the H19-7 cells do not attain the morphology or NFP repertoire of primary rat hippocampal neurons undergoing maturation in the same culture system. The limited maturation of the H19-7 cells could be a consequence of progenitor immortalization or of the clonal nature of the line. Thus, other types of neurons or accessory cells or factors produced by such cells may be necessary for complete maturation. Another line of particular interest is H19-5, a GFAP+ NFPline (Fig. 3 E and F). H19-5 cells have been used to substitute for the glial layer in bilaminar cultures where they maintain the viability of and support the normal in vitro differentiation of E16 rat septal neurons (Fig. 4). Parallel 7-day cultures of septal neurons over glia or over H19-5 were stained immunochemically for NFP, GAP-43, low-affinity NGF receptor and choline acetyltransferase. No evident differences are detected in viability or the proportions of cells expressing these markers. In contrast, primary septal neurons plated over a mouse fibroblast line, NIH 3T3, die within 4 days. H19-5 cells also

Neurobiology: Eves et al. support the morphological differentiation of selected neuronal cell lines to the same degree as do primary glia. Neurotrophin Gene Expression. The ability of H19-5 cells to support primary neuronal survival and differentiation and the induction of neuronal markers in neuronal lines suggest that some lines may be producing neurotrophic factors normally expressed in the hippocampus and that such production might be developmentally regulated. H19-5 (glial lineage), H19-7 (inducible for neuronal markers), WH19-4 (constitutive for some neuronal markers), and H583-5 (bipotential in lineage) cells were analyzed for NGF, BDNF, and NT-3 transcripts. RNA was isolated from proliferating cells, from nonproliferating cells (39"C, lsDMEM), and from nonproliferating cells treated with 10 nM PBt2. All four lines express neurotrophins (Fig. 5). No two lines have identical patterns of expression, although in three lines NGF message increases, and in general BDNF and NT-3 messages decrease after the shift to 39"C. No consistent effect of PBt2 treatment on neurotrophin gene expression at 390C was seen. In some lines (e.g., H19-7 and WH194) the expression of the BDNF and NT-3 genes is reciprocal, in that lines that express higher levels of NT-3 message tend to express lower levels of BDNF message and vice versa. BDNF and NT-3 transcripts are reciprocally expressed in developing hippocampus (9) and differentially expressed in regions of the adult hippocampus (10). The differential expression of the neurotrophins in these lines suggests that they will be extremely valuable in studies of the regulatory mechanisms of these genes. Although neurotrophin protein production has not yet been quantified, these lines also have potential utility as producers of trophic factors. In summary, a series of cell lines have been generated from embryonic hippocampus using ts simian virus large tumor antigens. Previously, a cell line with some hippocampal neuronal characteristics has been described (29-31), and hippocampal somatic-cell hybrids that express characteristics of mature hippocampal neurons have been generated (32). However, the neuroblastoma derivations of those lines present problems with respect to lineage, growth control, normal maturation, and genomic integrity that are endemic to lines of tumor origin. A ts large tumor antigen-immortalized hippocampal line has been reported recently (33) that does not express neuronal- or glial-specific markers in culture but appears to take on both neuronal and glial morphologies when transplanted into either the hippocampus or cerebellum of neonatal rats. The lines reported here are derived from primary hippocampal cell cultures, morphologically differentiate to neuronal and/or glial phenotypes in culture, and express neural trophic factors known to be expressed in the hippocampus. In addition, several of the neuronal lines express the epidermal growth factor receptor, which is known to be expressed by a subset of hippocampal neurons (34), and the expression of epidermal growth factor receptor is developmentally regulated in these lines (M.S.T., E.M.E., B.H.W. and M.R.R., unpublished work). These cell lines are thus useful for studies of development, plasticity and commitment in hippocampal lineages and for studies of receptor function and intracellular signaling pathways in mitotic versus postmitotic cells. Finally, the cell lines will facilitate analysis of the development and regulation of hippocampal trophic interactions, an area of intense interest because the hippocampal formation expresses trophic signals that support the development and maintenance of synaptic inputs from the septal region (35). These inputs are critical for normal cognition and are selectively vulnerable in diseases such as Alzheimer disease. Note Added in Proof. Since the preparation of this manuscript, we have isolated a H19-7 subclone that differentiates morphologically after a 2-day exposure to bFGF at 10 ng/ml.


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M.R.R. and B.H.W. contributed equally to this work. We thank Xianyu Hou for retinoic acid screening and Linda Sherman and Steven Price for technical assistance. This work was supported by grants from the Brain Research Foundation of The University of Chicago, the Alzheimer's Society, the National Institutes of Health (NS25787 to B.H.W., GM07151 to M.S.T.), the International Life Sciences Institute, and the American Cancer Society (CD 401 to M.R.R.). 1. Jessell, T. M. (1988) Neuron 1, 3-13. 2. Kater, S. B. & Mills, L. R. (1991) J. Neurosci. 11, 891-899. 3. Hefti, F., Hartikka, J. & Knusel, B. (1989) Neurobiol. Aging 10, 515-533. 4. Banker, G. A. & Cowan, W. M. (1977) Brain Res. 126, 397425. 5. Banker, G. A. & Cowan, W. M. (1979) J. Comp. Neurol. 187, 469-494. 6. Lendahl, U. & McKay, R. D. G. (1990) Trends Neurol. Sci. 13, 132-137. 7. Frederiksen, K., Jat, P. S., Valtz, N., Levy, D. & McKay, R. (1988) Neuron 1, 439-448. 8. Birren, S. J. & Anderson, D. J. (1990) Neuron 4, 189-201. 9. Maisonpierre, P., Belluscio, L., Friedman, B., Alderson, R. F., Wiegand, S. J., Furth, M. E., Lindsay, R. M. & Yancopoulos, G. D. (1990) Neuron 5, 501-509. 10. Ernfors, P., Wetmore, C., Olson, L. & Persson, H. (1990) Neuron 5, 511-526. 11. Jat, P. S. & Sharp, P. A. (1989) Mol. Cell. Biol. 9, 1672-1681. 12. Lee, V. M.-Y., Page, C. D., Wu, H.-L. & Schlaepfer, W. W. (1984) J. Neurochem. 42, 25-32. 13. Lee, V. M.-Y., Carden, M. J., Schlaepfer, W. W. & Trojanowski, J. Q. (1987) J. Neurosci. 7, 3474-3488. 14. Trojanowski, J. Q., Kelsten, M. L. & Lee, V. M.-Y. (1989) Am. J. Pathol. 135, 747-758. 15. Binder, L., Frankfurter, A. & Rebhun, L. (1986) Ann. N.Y. Acad. Sci. 466, 145-166. 16. Goslin, K., Schreyer, D. J., Skene, J. H. & Banker, G. (1988) Nature (London) 336, 672-674. 17. Chandler, C. E., Parsons, L. M., Hosang, M. & Shooter, E. M. (1984) J. Biol. Chem. 259, 6882-6889. 18. Levey, A. I., Armstrong, D. M., Atweh, S. F., Terry, R. D. & Wainer, B. H. (1983) J. Neurosci. 3, 1-9. 19. Roback, J. D., Large, T. H., Otten, U. & Wgiier, B. H. (1990) Dev. Biol. 137, 451-455. 20. Large, T. H., Bodary, S. C., Clegg, D.A., Weskamp, G., Otten, U. & Reichardt, L. F. (1986) SciencE234, 352-355. 21. Maisonpierre, P. C., LeBeau, M. M., Espinosa, R., III, Ip, N. Y., Belluscio, L., De la Monte, S. M., Squinto, S., Furth, M. E. & Yancopoulos, G. D. (1991) Genomics 10, 558-568. 22. Scholz, W. K. & Palfrey, H. C. (1991) J. Neurosci. 11, 24222432. 23. Temple, S. (1989) Nature (London) 340, 471-473. 24. Ronnett, G. V., Hester, L. D., Nye, J. S., Connors, K. & Snyder, S. H. (1990) Science 248, 603-605. 25. Caceres, A., Banker, G., Steward, O., Binder, L. & Payne, M. (1984) Brain Res. 315, 314-318. 26. Caceres, A., Banker, G. A. & Binder, L. (1986) J. Neurosci. 6, 714-722. 27. Goslin, K., Schreyer, D. J., Skene, J. H. P. & Banker, G. (1990) J. Neurosci. 10, 588-602. 28. Bottenstein, J. E. (1985) in Cell Culture in the Neurosciences, eds. Bottenstein, J. E. & Sato, G. (Plenum, New York), pp. 3-43. 29. Whittemore, S. R., Holets, V. R., Keane, R. W., Levy, D. J. & McKay, R. D. G. (1991) J. Neurosci. Res. 28, 156-170. 30. Morimoto, B. H. & Koshland, D. E., Jr. (1990) Proc. Natl. Acad. Sci. USA 87, 3518-3521. 31. Morimoto, B. H. & Koshland, D. E., Jr. (1990) Neuron 5, 875-880. 32. Lee, H. J., Hammond, D. N., Large, T. H., Sim, J. A., Brown, D. A., Otten, U. H. & Wainer, B. H. (1990) J. Neurosci. 10, 1779-1787. 33. Renfranz, P. J., Cunningham, M. G. & McKay, R. D. G. (1991) Cell 66, 713-729. 34. Werner, M. H., Nanney, L. B., Stoscheck, C. M. & King, L. E. (1988) J. Histochem. Cytochem. 36, 81-88. 35. Hsiang, J., Wainer, B. H., Shalaby, I. A., Hoffmann, P. C., Heller, A. & Heller, B. R. (1987) Neuroscience 21, 333-343.