I~.YPERIhIENTAL

Culture

NEUROLOGY

Techniques

Department

48,

NO.

3, PART

and

2, 135-162

(1975)

Glial-Neuronal /n Vitro

of Biology, School Sun Diego,

of Mrdicirlc, Califontia

Interrelationships

Unizwsity 92037

of C’aliforrtia,

CONTENTS Introduction ...................................... Characteristic Traits of Neuronal and Glial Cells ...... Preparation of Purified Neuroual and Glial Populations ........................ Glial-Neuronal Interactions Axonal Growth ................................... Culture Applications to Selected Problems ............

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136 138 143 146 152 156

PARTICIPANTS Department of Anatomy, Washington University School of Medicine, Missouri 63110. RICHARD BUNGE, Department of Anatomy, Washington University School of Medicine, St. Louis, Missouri 63110. JEAN DE VELLIS, Department of Anatomy, Neuropsychiatric Institute, 48-173, University of California, Los Angeles, Los Angeles, California 90024. LLOYD GREENE, Department of Neuroscience, Children’s Hospital Medical Center, 300 Longwood Avenue, Boston, Massachusetts 0211.5. HARVEY HERSCHIMAN, Department of Biological Chemistry, Laboratory of Nuclear Medicine and Radiation Biology, 900 Veteran Avenue, Warren Hall, University of California, Los Angeles, Los Angeles, California 90024. ROBERT LASHER, Department of Anatomy, University of Colorado Medical School, Denver, Colorado 80220. MILTON SAIER, Department of Biology, University of California, San Diego, La Jolla, California 92037. DAVID SHUBERT, The Salk Institute for Biological Studies, P. 0. Box 1809, San Diego, California 92112.

MARY

BUNGE,

St. Louis,

1 This report is a summary of the proceedings of a workshop California, April 4-5, 1974, and chaired by Dr. Silvio Varon. 2 Requests for detailed information regarding specific comments participants should be addressed directly to the participants. 135 Copyright All rights

1975 by Academic Press, Inc. DB reproduction in any form reserved.

held by

in the

La

Jolla,

individual

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SEEDS, Department of Biophysics and Genetics, University of Colorado Medical Center, Denver, Colorado 80220. EUGENE STREICHER, Staff Scientist, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland 20014. DONALD B. TOWER, Director, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland 20014. SILVIO VARON, Department of Biology, University of California, San Diego, School of Medicine, La Jolla, California 92037. NICHOLAS

INTRODUCTION Regeneration in a damaged central neural system requires that neurons affected by pathological changes, imposed on them and/or their environment, be allowed to : (a) survive, (b) initiate and sustain axonal growth, (c) direct the elongating axons to the correct destinations across an often abnormal terrain, (d) meet the correct partner-neurons while these are still “receptive,” and (e) establish with them appropriate and functional synapses. The performance of each of these tasks must involve intrinsic capabilities of the regenerating neurons, extrinsic triggers and regulatory influences, and favorable (i.e., permissive) overall conditions. Progress in the field of neural regeneration, therefore, will depend on improved knowledge of neuronal characteristics and the way by which their individual expressions are affected by defined humoral and cellular environmental features. The validity and usefulness of in vitro techniques for the study of neural cells has been firmly established over the last decade. Several i~z vitro systems are currently investigated : explant cultures, aggregate cultures, monolayer cultures of mixed and/or purified cell populations, clonal cell cultured from neoplastic neurons or glia. In complementation with one another, these systems provide opportunities for the study of neuronspecific and glia-specific properties, and for the controlled manipulation of the humoral and cellular environments in which neurons and glia are asked to operate. A major concept emerging from such in vitro studies has been that in the normal tissue neurons and glia probably operate according to only a restricted portion of their potentialities, such portion being defined by the relatively invariant sets of extrinsic conditions characteristic of the in situ situation. The in vitro systems allow one to define and reproduce such sets and elucidate the critical determinants of “normal” neural cell behavior. On the other hand, analysis of different sets of extrinsic conditions, bringing forth extraordinary cell behavior, may instruct us on the critical determinants of pathological situations and open the way to their experimental manipulation. The in vitro techniques currently in use and their major contributions to date are the subject of a separate survey (77). The Workshop upon

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of investigators actively engaged itI the stutly of it& vi/ro neural systems, so as to draw ul~n their common experience :tild ljerspectives with particular regard to the following: (a) Conceptual and technical problems in relationships neural cultures, (b) survey and evaluation of glia-neuron ilr vitro, and (c) applications of culture approaches to selected aspects of neural regeneration. Not included were the topics of (i) directional guidance of axonal growth (and the role of glia in it), and (ii) synapse formation and specific synaptic connectivity, which are the subjects of separate reports (2, 74). The familiarity of all participants with the general literature in the field and with one another’s work made it possible to devote the one and onehalf days of this workshop to a general discussion rather than to the presentation of individual contributions. In the same spirit, this report seeks to provide a collective view of the problems examined and will identify individual participants only when specific sets of data or special viewpoints are described. Considerable liberties have been taken in editing the proceedings of this discussion to achieve an organized presentation. Omissions, amendments, insertions of background material, and addition of more recent information have been kindly reviewed by the participants, thereby increasing the already considerable debt that these reporters owe to them all. The following reconlmelldatiolls by the workshop participants stem from the recognition that the rapid advances achieved recently with neural cell cultures only begin to tap the potentialities of these approaches. All three main culture systems (explant and aggregate, monolayer. and clonal tumoral cultures) need to be vigorously developed, as each one offers distinct and complementary opportunities. The following lines of investigation appear to be best-or even uniquely-approachable by irk vitro techniques. (i) Nez~on-spcrific arm’ glia-specific traits. Definition and measurement of increasing numbers of cell-specific markers (with special efforts to be directed to surface antigens, receptors and enzymes) and determination of the extrinsic influences that regulate their expression. (ii) Purified fzeztronal and glial populations. Development of effective techniques for dissociation of neural tissues, segregation of different classes of neurons and glial cells, and quantitative evaluation of purity and viability. Acquisition of more clonal cell lines of either category, endowed with specific traits (including the ability to form interneuronal synapses). (iii) G&x-neuron “WOPld injhde~~ces. Screening systems to recognize morphological or biochemical responsesby one cell type to the presentation

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of intact cells, cell extracts or cell secretions of the other type. Definition of the and elucidation of the responses. Isolation and characterization active factors. (iv) Axonal growth. Growth cone activities and their mechanisms. New membrane addition, maturation, and specialization. Axoskeletal components and regulation of their assembly. Supportive cell activities (production and delivery systems). Extrinsic signals to initiate or stop axonal elongation. Cellular or cell-derived terrains favoring or hindering axonal progress (as well as guidance and ultimate synapse connections). (v) Culture applications. Experimental models for development, maintenance and aging of young and adult neural tissue, including myeliscreennation and demyelination processes. Systems for pharmacological ing and investigations, and for clinical diagnostic tests. Source materials for implantation in viva (explants, aggregates, cells or cell-derived materials), and development of techniques to recognize their evolution after implantation and/or their effects on host cells. CHARACTERISTIC

TRAITS OF GLIAL CELLS

NEURONAL

AND

In vitro studies have drawn attention to the need for a sharper definition of three elements involved in the display of differentiated traits by a cell. The cell must be programmed for such traits, i.e., must have undergone an intrinsic and stable change in the readout of gene-encoded information. Also, extrinsic cues may be required to modulate the expression of such programs (modulation, as contrasted with induction, implies an effect which is graded and persists only as long as the extrinsic influence is applied). Finally, the cell must operate under permissive conditions, that is, the expression of the program is not hindered by restrictions imposed from the outside on other, ancillary, cell activities. Thus, the display of a differentiated trait offers positive evidence for the occurrence of all three elements. Conversely, lack of such a display does not discriminate between the absence of the corresponding program and the occurrence of conditions that do not elicit, or actually oppose, its expression. These considerations raise the possibility that the programmed repertoires of normal neurons or glia be more extended than those suggested in situ, where expressions my be restricted by tissue-specific or age-specific environmental conditions. A traditional task for the investigators of neural systems in vitro has been to equate in vitro with in tivo properties of neural cells. It has become increasingly clear that this will be properly achieved only by first clarifying and extending our knowledge about which traits are specifically programmed in neurons and glial cells and what extrinsic influences elicit or limit their expressions.

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Normal neural cells, prior to their collection for in vitro studies, have undoubtedly acquired &t&sic pvogva%s for differentiated traits in the course of their development irz viva. Acquisition by the cells of additional programs during their life in culture has not been systematically investigated, and such a possibility is not to be ruled out at present. Several questions arise with respect to neural differentiation in viva or in vitro. One question is how early in its history does a neural cell acquire its differentiated programs. A related question is whether neuronal cells become programmed for all their differentiated traits before they lose their ability to replicate, even though the traits may be displayed sequentially with the later appearance of extrinsic conditions appropriate to their expression. No unequivocal information has yet been supplied on either question by in viva or in vitro studies. Use of clonal cell lines may provide an approach to the investigation of a third question, namely the extent to which differentiated traits in a given cell class are programmed together in one or more “packages.” Schubert and coworkers (67) have induced a high incidence of CNS tumors in rats, by transplacental administration of nitrosoethylurea. They have isolated clonal lines from several, independently arisen such tumors after their adaptation to cell culture. Beside their independent origin and their CNS derivation, many of these new linesunlike those from the Cl300 mouse neuroblastoma-are not far removed in numbers of generations from the original neoplastic cell, have nearly normal karyotypes, and appear to be genetically stable. The functional diversity exhibited by several such lines may reflect real genotypic differences and, thus, yield insights into the linked or independent programming of individual neuronal or glial traits. It remains possible, however, that some of these differences relate to differential requirements for extrinsic regulation. The extrinsic conditions that promote or oppose individual differentiated expressions are still very poorly understood, if at all, and will require the most extensive investigative efforts. At the current state of the art, characterization of neural cells Zn. vitro must continue to rely on their examination under a variety of culture conditions and the compilation of several morphological, bioelectric, biochemical, and immunochemical properties, whose display has been successfully elicited. Cell Morphology. Morphological identification of normal neural cells in monolayer cultures, or in the monolayer outgrowth from explant cultures, is relatively easy for neurons of large or medium size, and for glial cells that assume typical shapes or positions relative to neurons. It may be, however, highly problematic for small cells, and for replicating cell populations (neuroblasts and glia) which assume a variety of forms. Neuroblasts and small neurons are poorly identifiable even in situ, despite the availability of topographical references. Glial morphology is strongly influenced by

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culture conditions (attachment substrate, media, serum, cyclic AM I’, etc.) and the growth activities of the cell population. With neoplastic clonal lines, morphological diversity is even more common for both neuronal and glial elements, and is encountered within the same clonal population as much as across different clones. Some observations favor the view that normal and neoplastic cell diversity may largely reflect morphological modulations of the same cell type by yet undefined influences (cell stage, microenvironment, intercellular contacts), rather than a number of cell subclasses each with its own distinctive and stable morphology. Saier reported that C6 cells display differences within the same culture with respect to shape (fibroblastic vs epithelioid), pattern of colony formation (compact vs dispersed), SlOO production, and catecholamine-induced mortality. Subclones selected for one or another such characteristics exhibited spontaneous changes with a frequency of lP3/cell/generation, suggesting the re-establishment of some kind of an equilibrium among the different “options.” Varon described three distinct morphologies (typical neurilemmal, triangular, and flat elements) displayed by glial cells from dissociated dorsal root ganglia (newborn mouse). The same three morphologies were observed both in cultures derived from pure satellite cells and in the cell outgrowth from explanted fragments of peripheral nerve. Thus, all three culture systems appear to involve a single glial class which, in situ, might express itself as either satellite or Schwann (neurilemma) cells depending upon its topographical relationship to neurons. Seeds mentioned some observations made in M. Bornstein’s laboratory (27) on explant cultures of normal rat spinal cord. In this study, treatment with serum from animals with experimental allergic encephalomyelitis caused the disappearance of recognizable oligodendrocytes, and withdrawal of the agent was followed by the reappearance of such cells, indicating either that they had undergone morphological modulation or that oligodendroglia can arise from another class of glial cells. Finally, morphological “maturation” of cultured glial cells has been described to be favored by stationary conditions (82)) prenatal brain extracts (72), and a partially purified protein factor (46). Bioelectric Properties. The ability to generate action potentials upon electrical stimulation is characteristic of nerve and muscle cells in tivo and in vitro, and has never been observed in glial or fibroblastic elements. Thus, electrically excitable cells in a neural culture can be unequivocally identified as neurons or neuroblasts. On the other hand, the degree to which electric excitability may be subject to genetic or extrinsic regulations remains a debated issue (21, 51, 66)) and lack or abnormalities of a cellular response to electrical stimuli cannot be taken as definitive disproval of neuronal identity.

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Chemical excitability of neural ceils has not been investigated ilz vitro 51) ostensively as has electrical escitability, but it is also generally corlsidered a uniquely neuronal marker. Some recent information suggests that chemical excitability is programmed separately from electrical escitability, and that its expression may be under extrinsic regulation (51, 66, 67). One necessary component of chemical excitability is the occurrence of neurotransmitter receptors on the cell surface. It remains to be established, however, whether the presence of receptors is by itself sufficient to demonstrate a fully competent apparatus for the generation of postsynaptic potentials. In the case of acetylcholine, the presence and distribution of receptors on cultured neural cells can be readily studied by use of radiofaheled a-neurotoxiu (67) or a-bungarotoxin (32). Greene raised the possibility that restricted localization of toxin receptors be used as x marker for trophic if not synaptic connections between nerve fibers and target cells in mixed neural cultures. Lasher also suggested the use of affinity chromatography to attempt the isolation of GAB,4 presynaptic receptors. NczlrotrnllsllzittE1..s. The question whether a neuron carries programs for one or more than one type of neurotransmitters continues to be unresolved. With normal neural cultures, past experience has favored the view that neurons do not switch from one to another neurotransmitter with regard to production, synaptic use, or even reuptake mechanisms. On the other hand, provocative questions are raised by more recent studies with NGFsupported culture,: uf dissociated rat superior cervical ganglia (53, 55). These cultures synthesize, from the corresponding radioprecursors, considerable amounts of catecholamines but not histamine, serotonin, or GABA. They also synthesize small amounts of radioactive acetylcholine (from radiocholine) in the near absence of nonneuronal cells, and increase acetylcholine production and choline acetyltransferase activity several hundred times when supplied with ganglionic non-neurons (the nonneurons, by themselves, do not produce acetylcholine). In addition, the mixed cultures develop over several weeks a large number of functional, excitatory cholinergic synapses between ganglionic neurons (found in as many as 50% of the neuronal pairs tested intracellularly). One cannot rule out that the mixed culture conditions selectively promoted the synaptic activity of a small number of cholinergic neurons, already present in the original ganglia (the occurrence of cholinergic synapses in this type of culture has been confirmed by R. Bunge in some unpublished work with H. Burton and C.-P. I(O). However, the above observations clearly raise the possibility that ganglionic neurons may carry programs for both catecholamine and acetylcholine neurotransmitter systems, which could be susceptible to preferential regulation by different sets of environmental con-

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ditions in vitro or in &JO. A similar view is strongly encouraged by the study of new neuronal tumor lines in stationary cultures (67) where more than one neurotransmitter synthetic enzyme were detected in single clonal populations (40). Nemotransmitter synthetic enzymes were also displayed by new clonal lines of presumptive glial origin (67). Occurrence of neurotransmitter enzymes and neurotransmitter uptake mechanisms have been reported in several other glial populations both in tivo and in vitro (77). Schubert pointed out that the possibility of developmental interrelationships between neuronal and glial cell types may deserve much more attention than it has received in the past. In keeping with such possibility are the reports, from in viva studies, of an ability by neurilemma cells in neurotransmitter junctions to elicit miniature endplate potentials and to synthesize acetylcholine after nerve ending degeneration (6). Other Protein Markers. SlOO and 14-3-Z proteins are generally viewed as reliable markers for glia and neurons, respectively, although their absence does not deny such identities. In the new clonal lines (67), 14-3-2 was found in all neuronal lines and SlOO in many, though not all, glial ones. However, the two proteins were also found to coexist in either cell type, another example of the overlap in the biochemical programs between the two neural cell classes. Inducibility of glycerol phosphate dehydrogenase by cortisol, and that of lactic dehydrogenase by catecholamines (via cyclic AMP), however, continue to appear reliable glial markers. Several of such glial traits are not displayed until some time-dependent or density-dependent events have taken place in culture. C6 cells accumulate SlOO protein only in confluent monolayer cultures (Herschmann). Cortisol inducibility appears in C6 cells only two days after attachment to a culture surface and reaches a maximum at 8-10 days, when the cells are in a stationary phase; this rise in inducibility correlates with the increase in cytoplasmic concentration of glucocorticoid receptors (DeVellis) . Aggregate cultures of prenatal mouse brain cells become capable of responding to catecholamines with a rise in cyclic AMP only after a few days in vitro (Seeds). At least in this case, the delayed appearance i~z.vitro of a differentiated trait resemblesthe time pattern of its development in vivo (69)) raising the possibility that both situations involve similar mechanisms of extrinsic regulation. Surfa~ceAntigens. The investigation of neural cell-specific surface antigens has barely begun, but already holds considerable promise for rapid and important progress. Herschmann reviewed some work on antisera to neurite-growing and neurite-free neuroblastoma cell cultures, which revealed antigens specific to the neurite-growing neuroblastoma cells and shared by them only with normal brain tissue (1). These immune sera have not yet been tested on cultures of normal neurons (with or without

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neurite growth), but the sera have shown no reactivity to neoplastic C6 glial cells. Greene described attempts, just started in collaboration with M. Schachner, to produce neuron-specific antibodies by injection into rabbits of a purified, glutaraldehyde-fixed neuronal population from normal sympathetic ganglia. Seeds reported some preliminary success in obtaining region-specific antibrain immune sera, by using as immunogens aggregate cultures of selected brain cells (70). These sera cross-react with neuroblastoma but not glioma cells, suggesting a neuronal specificity. Schachner (60) has already reported the recognition of a glia-specific antigen (NS-1) through the use of antiserum against a chemically-induced glial tumor. Tower mentioned that immune sera against human glioma are already being tested in combined antitumor therapies. Several suggestions were made toward future progress of this important approach. Neural tissue from regions or species with highly biased ratios of neurons and glia could be used as selective antigen sources in the absence of purified populations of either class. Developmental changes in surface antigens are likely to occur and need to be taken into consideration, as well as investigated on their own merit. The use of whole cells as immunogens appears to be most effective in eliciting highly cytotoxic antisera. Mild fixation of the cells (glutaraldehyde, zinc) prior to their injection does not appear to alter their immunogenic competence and might help to preserve cell integrity during their preparation. Lastly, attention was drawn to recent reports (22) on the successful establishment of a cell line from SV40-transformed spleen cells. Such cloned cells had been obtained from hyperimmunized rabbits and remained capable of continued production in vitro of specific antibodies to the original antigen. PREPARATION

OF PURIFIED NEURONAL GLIAL POPULATIONS

AND

Availability of homotypic cell populations of normal neuronal or glial elements is rapidly becoming one of the most pressing issues for the ilz vitro investigation of neural problems. Such populations are required for the validation of information obtained from neoplastic clonal cell lines, as well as for the independent recognition of other cell-specific traits and regulatory mechanisms. They are needed as sources of immunogenic material for the production of antibodies specific to each class and, ultimately, to subcategories within the two classes. Furthermore, they are essential for a controlled establishment of heterotypic cell systems with which to study glia-neuron interactions at morphological and functional levels. Dissociation of peripheral and central neural tissues is likely to require different treatments. Enzymatic treatment (e.g., with weak solutions of trypsin or collagenase) of peripheral ganglia is usually very effective,

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presumably through the loosening of periganglionic and intraganghonic capsule cells, and no long-term consequences to the basic capabilities of the neurons have been recognized. R. Bunge pointed out that mechanical dissociation can also be successful with superior cervical ganglia (lZ), but no detailed comparison is available between the two approaches in terms of total and differential cell yields or subsequent behaviors of the cells in culture. On the other hand, use of trypsin or other enzymes for central neural tissue dissociation has given dubious results at best, particularly with regard to the yield and viability of the neurons. Seeds drew attention to the large variations in quality to be found in different batches of trypsin even from the same supplier. Varon raised the possibility that neutral pH and serum presence during the enzymatic treatment might reduce the damaging effects of trypsin without diminishing its dissociation effectiveness (80). Lasher suggested that the extent of cell damage imposed during dissociation, particularly by the use of enzymes, is also important for the tendency of initially dispersed cells to form clumps with one another. Clumping is a source of significant interference with subsequent uses of the cell dissociate, and the use of DNAse to limit clumping may no longer be required if such damage is minimized. It is also a common experience that dissociation has been much less successful with older than with younger neural tissues. Lasher believes that this, too, may be a matter of developing more appropriate modifications of current dissociation procedures, and reported satisfactory results even with three-week old postnatal rat cerebral tissue. Finally, cell breakage during dissociation may be expected to involve some cell types more than others and impose a population bias which could facilitate, or conversely, hinder subsequent attempts to fractionate the resulting dissociate. In general, investigators dealing with dissociated neural cells must be encouraged always to provide information on total cell yields, and preferably on differential cell yields as well. This would characterize the cell preparations and supply information toward future attempts to standardize and optimize dissociation procedures. Fractionation of neural cell dissociates poses different problems with regard to neuronal or glial populations because of their different replicative abilities. With neurons, a numerical ceiling is imposed by the yield achieved during dissociation, and even an initially modest contamination by other cells could become increasingly important if these cells subsequently proliferate. In contrast, subsequent proliferation of glial cells could help to build up an initially small population, while at the same time diluting out any minor neuronal contamination. Several approaches to neural cell fractionation were discussed : Use of a Selected Source. Small glial populations could be obtained without neuronal contamination, by using white matter regions, gliotic tissue, or optic or peripheral nerve. Amplification of the initial population

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could then be sought by developing culture conditions particularly suitable for glial proliferation. R. Bunge called attention to the possible importance, in this respect, of “mitogenic” inputs from neuronal elements. In cell cultures from sympathetic ganglia, neurilemma cells show little propensity to divide unless neuronal axons are present. In the latter situation, however, the neurilemma cells proliferate until they cover the network of neuronal elements, then stop without establishing a confluent layer over the entire plate (as C6 cells, for example, would do). Optimal conditions for glial growth would also be essential for a further segregation of glial cell subclasses via cloning procedures. Lasher reported some initial success, limited to only a few passages, with neurilemma cells from chick embryo dorsal root ganglia. Lasher also mentioned that germinal cells of the external granular layer of a two-day postnatal rat cerebellum will continue to divide for several days in culture, an observation that opens the way to the growth of neuronal populations in vitro. Sedimentation. This has been the choice approach for bulk preparations of neuronal and glial fractions (77), but cell viability was not a concern in these studies. Success on a small scale has been recently reported (4) for the isolation of cerebellar granule cells, but their survival capabilities have not been thoroughly investigated. A different approach of considerable promise has been reported by Schachner and Hammerling (61). Red blood cells are caused to form rosettes on selected cells via immunochemical (or other) specific ligands, the rosette-loaded cells are readily sedimented out and subsequently freed of the attached erythrocytes. Diffeventiul Cell Szlrface Propertirs. If cell-characteristic ligands are recognized-such as those involved in the rosette-loading approach-they could also be used to “derivatize” attachment surfaces such as glass or nylon fibers, or adsorption columns. Besides immune reagents, NGF and other hormones, as well as lectins, were mentioned as possible selective ligands. Selective adhesion properties operate in the attachment of dissociated cells to culture vessels, and successful collection of neuronal fractions has been achieved on such a base (e.g., 79, Sl, 53). It might be possible to increase the effectiveness of differential attachment as a fractionation approach by alterin g the suspension medium as well as the attachment surface. Lasher pointed out that with several CNS dissociates a collagen surface is needed when Ca” concentrations are below 0.2 mM, but neurons will attach directly to plastic with 1 mM Ca2+ levels. Attachment and maturation of neurons on plastic appear to be potentiated by a polylysine treatment of the plastic, but survival rarely extended beyond one week in the absence of a non-neuronal cell substrate (see page o&00). Ccl1 llljgrofion. Migration of cells out of an explant &en occ~~~-s with time patterns reflecting the differential motility of different cell classes: for example, fibroblasts usually migrate ahead of glial cells, and neurons

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migrate very little if at all. R. Bunge noted that this approach could yield cell fractions in substantial amounts (e.g., some 250 spinal cord explants can be obtained from one rat litter). Greene mentioned the possibility of using gradients of palladium, coating a plastic surface, to accumulate fibroblasts in the most adhesive regions (i.e., those with the highest palladium coat). Differential Cell Survival. Culture conditions usually favor survival and/ or growth of some cell classes over others. Serum supplementation of the media is well known to promote cell proliferation, and so does a CO2 atmosphere (e.g., 55). Conversely, antimitotic agents (aminopterin, cytosine arabinoside, 5-fluorodeoxyuridine) have been used to block growth of neoplastic neurons (51) or unwanted non-neuronal elements (26). High K+ concentrations are required for the survival of dissociated rat cerebellar neurons (44) and found beneficial for peripheral ganglionic neurons as well (19, 68). Ca2+ levels, besides their effects on cell attachment, may also influence cell survival, though no firm information in this respect is available (62). More specific agents could also be applied, or withheld, to eliminate selected cell populations. NGF is required for the survival of certain ganglionic neurons (cf. 78), and NGF-free media have been used to obtain ganglionic non-neuronal populations (81, 83), purified satellite cell cultures (83), or SIF (small, intensely fluorescent) neurons (36). Several drugs are available which will selectively destroy adrenergic neurons (35), and others might be tried for the selective destruction, rather than segregation, of the corresponding cell classes. GLIAL-NEURONAL

INTERACTIONS

The basic advantage of the in vitro approaches to the study of neural tissue lies in the possibility to manipulate, and ultimately control, the humoral and cellular environments in which neural cells are asked to operate. This has resulted in an increasing recognition of the importance of extrinsic influences for the expression of neuronal differentiated traits, hence for the functional performance of the neuron. In vivo, the local environment of a nerve cell is provided by the glia and the intercellular fluid, which in turn is subject to glial influences if not to their outright control. Thus, extrinsic regulation of neuronal function appears to be largely exerted by glial cells, through cell-cell surface contacts, modification of the intercellular fluid, and/or transfer of glial products to the nerve cell. Considerable information has been supplied by both in viva and in vitro studies on the likely involvement of the glia in regulating the humoral environment of the neurons with regard to nutrients, ionic composition, neurotransmitter levels, and, possibly, cyclic nucleotides (see 77).

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The roles that glial cells may play with respect to neuronal migration during development, directional guidance of axonal growth, and, possibly, selection of synaptic partners are examined in other reports in this volume (see 2, 74). In this workshop, the discussion was focused on the possible supply by glial cells of specific protein factors playing a critical role in the maintenance and/or general activities of their neighboring neurons. The notion that at least certain neuronal populations require a specific protein agent for their survival and functional performance in z&ro and possibly in Z&JO, has been firmly established with the discovery of Nerve Growth Factor (NGF) by Levi-Montalcini and Hamburger (45) some twenty years ago, and the extensive studies carried out on the NGF phenomenon in the subsequent years (see 78). An involvement of glial cells in the production, modification 0; delivery of NGF or NGF-like agents has been suggested by several recent lines of neural studies ipl Z&O. Glial Cells and NGF. Varon summarized the data obtained in his laboratory with respect to an NGF-like competence of ganglioxic glia (see also 78). NGF is essential for the survival in culture of most neurons dissociated from early embryonic dorsal root ganglia. A similar requirement for exogenous NGF was shown by neurons dissociated from postnatal mouse dorsal root ganglia when the dissociation had caused a considerable loss of ganglionic glial cells. But if the cultures were resupplemented with adequate numbers of homologous glial cells, the presence of exogenous NGF in the medium was no longer required and had no recognized effects. Similarly, the requirement for, and the effects of, NGF were considerably reduced when neurons from embryonic chick or neonatal rat dorsal root ganglia were supplemented with non-neurons from the same source (in those cases, a mixture of glial cells and fibroblasts) . Neurons from sympathetic ganglia of chick embryo or postnatal mouse and rat also require NGF for their survival in vitro, and such requirement was again removed by supplementation with homologous glial cells. Primary cells from sources other than dorsal root ganglia were unable to substitute for exogenous NGF in the support of dorsal root ganglia neurons (although they may help neuronal performance in the presence of NGF-42, 78), and non-neurons from dorsal root ganglia of different species or ages gave maximal support only to their strictly homologous neurons. Finally, mouse or chick embryo dorsal root ganglia neurons that were exclusively supported by their glial cells failed to attach and perform in the presence of antibody to mouse submaxillary NGF, or when the ganglionic glia provided to them had been pretreated with anti-NGF immune materials. Hased on such findings, Varon and coworkers have proposed that gangli(JTliC glia could be the indigenous source of an NGF-like support t(J their

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neurons in z&o as well as in vitro, with extra-ganglionic NGF being required (and, thus, recognizably effective) only when the glial support is inadequate. Neoplastic glial cells have also provided information in a similar direction. Monard et al. (50) have reported that monolayers of C6 cells, cultured in a serum-free medium, confer to the medium the ability to promote neurite production by mouse neuroblastoma cells. The activity of the “conditioned media” appeared to result from an actual release of materials from the C6 cells. However, no information was given on a possible relationship between the C6 cell materials and NGF nor on possible neuritepromoting capabilities of the CG-conditioned medium with respect to NGFdependent normal neurons. Varon mentioned that his own attempts to seed mouse dorsal root ganglia neurons on C6 cells instead of ganglionic glia had yielded inconclusive results, although a moderate degree of NGFlike competence by C6 cells was suggested in some experiments. More recently, Longo and Penhoet (47) have reported that extracts from a rat glioma grown in tivo elicit neurite production from both neuroblastoma cell cultures and embryonic explants of dorsal root ganglia. Gel electrophoresis of a partially purified glioma preparation indicated the presence of NGF-like proteins, and an immunochemical analysis suggested a 90% overlap in the amino acid sequences of the glioma-derived and the standard submaxillary NGF antigens. A direct production of NGF or NGF-like antigen by glial cells is strongly suggested by such experiments both with the normal and the neoplastic glia, but the evidence cannot yet be viewed as definitive. Production and/or storage of NGF antigen by neoplastic glia may reflect the tumoral, rather than the glial nature of these cells, in view of the historical association between NGF and tumoral tissue (78). Ganglionic glia could be responsible for the modification of an inactive NGF precursor (e.g.. from fibroblastic or serum sources) rather than the synthesis of a glial NGF protein. However, Johnson et al. (38) have reported that organ cultures of rat superior cervical ganglia release NGF antigens into the medium in amounts greater than those detectable in the initial system, suggesting actual synthesis of the antigen in vitro. Because the release occurred for a few days only and its decline was accompanied by degeneration of the ganglionic satellite cells, these investigators also raised the possibility that ganglionic glia synthesize NGF. A physiological action of NGF on CNS neurons has been suggested by the effects of exogenous NGF or its antiserum on axonal regeneration by catecholamine-containing nerve cells (9, lo), or on functional recovery after hypothalamic lesions (5). In view of the obstacle presumably posed by the blood-brain barrier to blood-borne proteins, such a central action by NGF would require local, i.e., glinl, synthesis of the factor.

On the assumption that NGF production does occur in glia cells (though by no means exclusively there-78), several questions were raised for future investigation : (i) Is NGF production a property of all glial cells, or only of some glial subclasses ? (ii) Is the glial production of NGF subject to extrinsic regulation, such that it may normally occur only in selected neural regions ? (iii) If all glial cells do not produce NGF, do they produce other protein factors with analogous roles with respect to their neurons ? (iv) Is the mode of delivery of such glial factors identical in all glia-neuron partnerships, yet one that permits a degree of intercellular specificity (as suggested by the dorsal root ganglion cross-test experiments) ? It was also pointed out that the concept of glial cells as sources of neuron-directed agents raises some points of caution at the experimental level. For example, the detection of active humoral agents derived from glial material may require a test system that is deliberately contrived to minimize direct glial support within the system itself. Also, other agents or conditions observed to be beneficial to cultured neurons might operate by enhancing the supportive activity of coexisting glial cells rather than by supporting the neurons directly. “Trophic” Effects of Glia on Neuvom. In monolayer cultures of embryonic or fetal central neural tissues, non-neuronal elements are often described as “flat” cells which rapidly cover the entire surface of the culture dish, or as “phase-dark” cells which overlie the flat cell layer together with more distinctive glial and neuronal elements (e.g., 63, 79). The flat or phase-dark cells occasionally assume more characteristic glial morphologies either “spontaneously” (82) or upon stimulation by selected agents (46. 72). In general, however, the presumptive identification of either cell category as glial cells or glial cell precursors only rests on the tissue source, the lack of fibroblast-like behavior, or the recognition of glin-characteristic biochemical traits (e.g., 39). It is a frequent observation, with monolayer central neural cultures, that those neurons fare much better which are sitting on flat cells or are associated with phase-dark cells, than do those in relative isolation. Lasher, for example, reported that seeding low density CNS dissociates on a fixed number of preattached non-neurons improved the quality of the cultures. Conversely, cerebellar neurons displayed a less healthy behavior when seeded after enrichment by selective plating out techniques. To proceed beyond a simple statement of positive influences by nonlleuronal cells on the behavic,r of neurons ij~ 7*i?t.0 (and ultini;itely ill viva) , it will be critical to (i) define neuronal performances in more

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precise and measurable parameters (such as numerical and temporal survival, selected morphological features, biochemical activities, etc.), and (ii) have available purified or at least enriched populations both of neuronal and of glial (or presumptive glial) cells. Schubert pointed out the contributions on both points that can be expected from the use of clonal lines, particularly as more of them will become available. With more defined parameters for neuronal performance and the capability to deliberately alter the cell composition of the cultures, several questions can be effectively addressed. Do neurons have an absolute requirement for other cells or cell products, or do they benefit in the extent to which they will perform general or specific tasks ? Are such services provided selectively by glial cell elements (specific glia-neuron interaction) or are they equally supplied by other cell populations (a “feeder-layer” type of involvement) ? Are the non-neuronal influences applied via cell-cell surface contact, via transfer of humoral agents from non-neurons to neurons, or through both of these modalities? Some information and several suggestions pertaining to these questions are already provided by a number of experimental observations. Sensenbrenner et al. (72) described effects of chick embryo cerebral extracts on monolayer cultures of early (5-7 day) chick embryo cerebral cells, in which neurons and glia display otherwise still poorly defined morphologies. They report that extracts from S-day cerebrum promoted the appearance and maturation of large multipolar, as well as of pyramidal neurons and the assumption by glial cells of typical oligodendrocyte and astrocyte morphologies. In contrast, extracts from 12-day cerebral tissue promoted maturation of bipolar neurons and favored astrocytic over oligodendrocytic maturation. Garber and Moscona (28) demonstrated that serum-free medium conditioned over cerebral cell cultures have aggregation-enhancing properties that appear tissue-specific, age-specific and (to a lesser extent) species-specific (see also 2). Seeds noted that these aggregate-promoting factors appear to enhance the rates of formation rather than affecting the pattern of the aggregates. While neither study provides information as to the cellular (glial or neuronal) sources of these promoting agents, both offer evidence for the humoral nature of neural cell-directed and neural cell-produced agents with considerable target specificity. Schubert noted that clonal glial cultures do secrete proteins into the medium and methods are available for their analysis (64). Additionally, there is evidence for the transfer from glia to neuronal axons of radioamino acids in leech (31) and radio-protein in squid (41). While no such direct evidence is yet available with higher animal systems, pinocytotic uptake of protein has been demonstrated for sympathetic nerve endings in viva (34) and in vitro (7, 17)) and at least suggested for dorsal root ganglia neurons in vitro (52). Another suggestion to be de-

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rivctl frown the ;111ove studies concerns the occurrence of humcml mtl possibly macromolecular signals from neurons to glia. Greene pointed out that this is a largely ignored area of investigation that should receive more attention. More explicit indications of a selective involvement of glial cells in altering neuronal performances are found in two other studies. Sensenbrenner and Mandel (73) report that embryonic cerebral neurons attached much sooner and underwent morphological specialization much more extensively when seeded over an astroblast layer than when seeded over plastic, meningeal, or fibroblast cell layers. In the already mentioned studies by Patterson and Chun (55) and O’Lague et al. (53), the development of acetylcholine synthesis and cholinergic synapses in sympathetic ganglionic cultures occurred only in the presence of non-neuronal cells, and was elicited by sympathetic non-neurons and C6 glial cells, but not by 3T3 fibroblasts. No information is yet available as to whether the biochemical and the synaptic responses were elicited by glial cells separately or consequential to one another. Seeds pointed out that a similar rise in choline acetyltransferase activity has been reported for coculture of spinal cord and muscle cells in association with the formation of functional neuromuscular junctions (30). Glial cells are undoubtedly critical determinants of the social patterns that nerve cells assume in viva as well as in explant, aggregate or monolayer cultures. In turn, the organization achieved by the heterotypic cell population may determine the extent to which differentiated traits will be expressed by either neurons and glia. While both cellular organization and biochemical cell modulation may occur exclusively through cell surface signals, it is also conceivable that transfer of materials from one to another cell may be particularly favored by the extent and the nature of the contacts established between them. Varon mentioned that, in monolayer cultures from spinal sensory or sympathetic ganglionic dissociates (76, Sl), the relative numbers of non-neurons and neurons appear to dictate whether or not aggregation of initially dispersed neurons will take place. Seeds reported that seeding high numbers of brain cells on “glial” layers results in aggregation and good biochemical activities, while seeding of low cell numbers leads to no clump formation and low differentiated activities. The best expression of differentiated traits appears to occur in aggregate cultures (69), where aggregation has been promoted directly from a cell suspension. In such systems, intercellular contacts are tridimensional, the non-neuronal population is prevented from overgrowing the system, and cell organization achieves organotypic patterns. Admittedly, however, the evaluation of many biochemical parameters remains a difficult task in mixed neural cultures. The higher expression of differentiated traits observed in aggregate relative to monolayer cultures may be

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due to actually improved performances at the itidivitlual cell level, 01’ a selection of the better performing cells. Alternatively, it might be due to the absence of an overgrowing population of other (nonperforming, or more poorly performing) cells, which could “dilute out” the expressed performance, interfere with performance-conducive cell-cell contacts, or cause humoral changes less favorable to differentiated expression. AXONAL

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Many neural culture studies have investigated the conditions under which neurite production takes place. However few of them have attempted to analyze temporal sequences of events and distinguish between initiation and sustenance of neurite growth. In the current view (see 77), sustained growth and maintenance of neurites in vitro require (a) the activity of growth cones, (b) stabilizing axoskeletal elements (tubules, filaments), and (c) continued supply and delivery of materials from the soma. Thus, conditions or agents noted in the past to promote or oppose neurite growth may have done so by acting at any one of these different levels. For example, only the resupply component appears involved in the dependence on protein synthesis observed for neurite production from normal neurons (20, 54) or neuroblastoma cells (59). Schubert noted that, in some new clonal lines, round cells do form long processes while protein synthesis is blocked, as Seeds had already reported in certain neuroblastoma cultures (71). Similarly, colchicine interference with microtubular structures opposes the stabilization of the neurite structure (85)) and, possibly, the functioning of microtubules in the axonal delivery system (cf. 33). An important direction for future efforts will be to re-examine neurite-promoting conditions and agents by ways that will recognize which of them actually act to initiate neurite growth. Initiation of neurite growth may require the triggering of a new train of events or the removal of extrinsic inhibitions preventing such events from taking place. With neuroblastoma cells, neurite growth (the so-called morphological differentiation) probably requires a triggering signal. Schubert proposes that an enhanced interaction between cell and culture surface may constitute such a signal. Cells that are not, or are poorly, attached to a surface will not grow neurites, and growth cones (also well documented in neuroblastoma cultures) are known to exhibit high adhesiveness (13). Herschmann’s observations (1) of different surface antigens in neuritegrowing neuroblastoma cells also point to the relevance of altered surface properties. Many of the conditions found to promote neurite production in neuroblastoma cells do so by enhancing cell adhesiveness. 5-bromodeoxyuridine causes a flattening of attached cells of several types (8, 43), and

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111~ al)jxarance of a surface glycoprotein i 1.5). ‘I’lic Ilcul-itc-l)roln( ~tiilg activity of conditioned media may also reflect an enhancement of cell adhesion by small molecular weight metabolites (65,) or special macromolecules (50). Conversely, proteins in the medium interfere with electrostatic surface interactions (65). Experimental systems providing greater resolution of the neurite-promoting situations are needed to verify whether surface changes do act as the primary signal for neurite initiation, Or as a necessary but not SuffiCietlt condition for it. Normal neurons, on the other hand, could be competent to grow neurites without a positive signal, if provided with permissive conditions or freed from inhibitory influences. As Tower pointed out, the appearance of sprouting and axonal regrowth upon spinal cord transection suggests the lift of some inhibitory control of asonal growth in the in VWO situation, which would also be removed upon transfer of neurons to an in vitro system. R. Bunge suggested that neurons may be subject to growth controls as are other cells, except that neuronal growth would occur as neurite production rather than cell replication. Thus, “contact-inhibition” of growth may be relevant as a signal for neurite initiation. R. Bunge noted that fasciculation may inhibit neurite growth (24). Greene raised the question whether neurites growing as pioneer fibers or as follower fibers (along prelaid neuronal processes) might differ in growth modalities, or even in the growth cones that they both exhibit. On the other hand, it is a common experience that cultured normal neurons, like the neoplastic ones, appear to require interaction with an appropriate surface or semisolid substrate. Lasher pointed out that pinocytotic activity, supposedly high in growth cones, may be involved in the intake of important extrinsic signals. Pinocytosis might be promoted by the right surface interactions, while hindered by cell-cell contacts. These considerations stress once again the importance, in vizto or in vitro, of the terrain in or on which neurites will grow. The terrain may be important with respect to the actual growth as well as its directionality (see also 2, 72). Interesting experimental models might be developed by presenting the neurons with an attachment matrix that contains gradients of presumptive neurite-promoting agents or that contains layers preformed with different cell types. Another observation reported by Seeds is pertinent to differential cell-cell adhesiveness. He investigated the ability of cultured aggregates to pick up new cells, using the technique developed by Roth’s group (3), and noted that the aggregate’s surface changes continually in this respect with a progressive loss of pickup ability. Few humoral agents have been tested for neurite-promotion activity on normal neurons. Nerve Growth Factor, classically described as such an agent, does affect membrane properties and enhance adhesiveness, but is

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also essential for the survival of its neurons (see 78). Of the treatments effective on neuroblastoma, cyclic AMP has been reported to promote neurite outgrowth (48, SS), but other agents have yet to be systematically examined. Since normal neurons that survive in culture generally will put out processes, evaluation of neurite-promoting activities will require the use of more detailed parameters (e.g., 13) than mere cell counts as well as the use of experimental systems capable of distinguishing between initiating and supportive actions. In monolayer cultures treated with cytochalasin B, withdrawal of the drug leads to the appearance of new growth cones from both soma and existing neurites and to resumption of growth within four hours (85). Varon suggested that such a system could be adapted to provide (i) a “synchonized” population of neurite growing cells, (ii) a test system for agents acting at the growth cone level right after drug withdrawal, or (iii) a test system for agents that stimulate synthesis of neurite materials, before the withdrawal of the drug. Similarly, a detailed study of the consequences of colchicine withdrawal at the appropriate time might provide a test system for agents that influence the assembly of microtubules. Seeds pointed out that much remains to be learned about tubulin polymerization, even in cell-free conditions. Does polymerization proceed from some initial “nucleus”? What are the energy requirements ? Which agents affect it ? Is assembly of neurite microtubules different from that of spindle ones. ? And, if so, could one select for clonal lines unable to assemble neurite microtubules? Increasing attention should also be directed to the involvement of calcium ions in neurite growth. Lasher’s observations on the effects of Ca2+ on neuroblast and neuronal adhesiveness have already been mentioned. Seeds had described the absolute requirement for Ca2+ and Mg” shown by neurite growth in neuroblastoma cultures (71). Ca2+ levels are known to be critical in the polymerization of microtubules, but also in actinmyosin interactions and, thus, conceivably in microfilament structures. R. Bunge has observed that, when neurites in culture are amputated, axonal degeneration appears to result from an influx of Ca2+ into the severed neurite (62). The growth, but not the persistance, of neurites was temporarily and reversibly blocked by 5-20 mM Ca2+ in NGF-supported ganglionic monolayers, suggesting an effect on growth cone activity rather than microtubular stability (49). NGF itself may be involved in regulating intracellular Ca2+ levels via changes in membrane permeation (see 78)) or other modalities (49). Work with fucoid eggs has encouraged a hypothesis that Ca*+ currents across a cell be the trigger for polarized growth at the entry site (37, 57). Finally, the role of Ca*+ in endocytotic and exocytotic activities suggests its possible involvement in the process of membrane accretion during neurite growth.

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M. Bunge discussed her investigations with Pfenninger (14, 16) on the insertion of new plasma membrane at the growth cone level, and the hypothesis that immature membrane-recognized by its low content of intramembranous particles-is packaged into vesicles at the Golgi apparatus, transported down the neurite to the growth cone, and added there to the plasma membrane, possibly in insertion areas which look like vesicle-packed bulges (“mounds”) of the cell surface (see 77). Lasher reported observing similar vesicle-packed bulges at the bulbous ends of parallel fibers from cerebellar granule cells. M. Bunge noted that similar bulges or mounds are occasionally seen also at the margin of the perikaryon, before or during neurite outgrowth, and may well represent initiation sites. Most of the evidence accumulated thus far implicates the mound area as a region of new membrane insertion, but more data are still needed to eliminate completely the possibility that some membrane intake is also occurring there. Freeze-fracture electron microscopy (56) has shown that growth cone membranes have about 85 intramembranous particles per pm2 (as compared with several hundreds in glial processes), and that the content of these particles increases nearly lo-fold as one proceeds from periphery to soma or upon aging of the neurons in vitro. These observations raise several questions for future investigation. What controls a shift from balanced insertion and removal of membrane (as may occur in a nongrowth situation, or in mobile cells) to the slight excess of the insertion component, that must be responsible for membrane accretion during neurite production ? What other markers can be detected for immature membrane (e.g., surface antigens, or receptors) ? What are the nature and the functions of intramembranous particles (e.g., glycoprotein? pumps?) ? Does the immature membrane acquire these particles from the axoplasm, or via relocation within a “fluid” membrane ? R. Lunge has looked for changes that might occur when a neurite growing out of a spinal cord esplant joins with a sympathetic neurons to form a synapse. Upon arrival at the target the nerve ending reorganizes its growth cone to assume presynaptic characteristics, even though the tip may continue growth elsewhere. Within 2-3 days, for example, the typical growth-cone morphology disappears and is replaced by typical synaptic vesicles. Also, the recipient cell shows reactions to the neurite’s approach. Its Golgi system produces an increased number of coated vesicles, and these reach the plasma membrane in greater numbers at the approach point, resulting in a local “thickening.” The synaptic material, the coated vesicles, and the Golgi apparatus can be stained with phosphotungstic acid (cf. 11). Q uantification of this coated vesicle response derives from comparison with other symapthetic neurons, within the same culture, which have not been approached for synaptic connection. These observa-

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the vast potential of such approaches to the problems of and possibly even to the basis for specific connectivrty

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PROBLEMS

As discussed in the preceeding sections (see also 77)) current work with neural culture is directed to the following main goals: (i) To define the intrinsic properties of neurons and glia, to recognize and control the extrinsic influences that regulate their behavior, and to learn how to keep neural cells alive and healthy in abnormal circumstances, (ii) to understand the relationships between glia and neurons and to provide for their optimal interactions ; and (iii) to define the conditions which permit and promote regrowth of axons and reconstitution of functional and appropriate synapses. However, neural cultures can also serve as testing grounds for applied problems and approaches, and as sources of surrogate material for clinical applications. Problems in neuropathology (e.g., demyelinating conditions) have been approached by use of neural cultures, largely through the efforts of M. B. Bornstein and his collaborators (cf. 18). On the other hand, surprisingly little advantage has yet been taken of the potentialities of in vitro neural systems for research in the field of neuropharmacology. However, rapid development in both of these directions should be expected as in vitro systems become available which can be reliably, reproducibly and quantitatively analyzed. Greene also pointed out that current culture work essentially uses perinatal tissue. A good culture model of adult neural tissue is not yet available for the study of maintenance, aging, and/or degeneration of mature neural systems. Attempts to provide such a system can proceed in two directions. As Lasher had noted, nonenzymatic dissociation of adult neural tissue is in fact possible and cultures could conceivably be started with older cells than is presently done. Alternatively, development could be allowed to proceed in vitro as recent work by Crain and Peterson (23) has illustrated (see also 77). Glial cells grown in culture could become sources of materials for clinical interventions. Glia-produced factors, with an identified role in the welfare of nerve cells, could be extracted and purified for in viva administration. Glial surface constituents may be used to line suitable inert materials and provide effective substrates for neural repair processes. Intact glial cells could be injected or implanted for remyelination purposes. Finally, cultured neurons could become a source of substitute, or adjuvant, materials for the repair of central neuron lesions. Such projections are, undoubtedly, beyond the present reach, but they can no longer be regarded as visionary. Research in these directions is already beginning to gather momentum.

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hl. llunge described work, started a Teu. years ago (e.g., 75), un the possibility of transplanting cultured spinal cord back into an animal. Longitudinal hemisections of fetal rat spinal cord, freed from their meninges, were first cultured as explants, under conditions designed to provide good maintenance and outgrowth of neurites within l-3 days. The initial purpose of this culture step was to verify the viability of the hemicord. Herschmann pointed out that an additional advantage would be the loss of transplantation antigens, believed to occur over the first 10 days in vitro. After about one week, cultured explants were implanted into adult rats, either in a longitudinal groove provided in the white matter of the dorsal column, or between cord and dura. Postoperative damage was not a problem. The implanted cord, by histological examination, was shown to survive and “mature” for as long as 56 days and, in fact, appeared to fare better than comparable specimensmaintained in vitro. The implanted cord became vascularized, grew neurites, became myelinated, and developed intraimplant synapses. However, nerve fibers from the host did not invade the implant. In contrast with such results, survival of the implant was not observed when the cultured cord was implanted over the dorsal margins of a lesion inflicted to the host cord. Discussion of future developments of approaches to transplantation centered mainly on how to deal with the region of the lesion and replace the obstacles of a potential “scar” tissue with a terrain conducive to ingrowth of axons from the implanted cord. R. Bunge mentioned that implantation of adult cerebellar explants, cultured on agar, has been reported to suppress scar formation while leaving no viable remnants (39). Destruction of intralesion material and interference with scar formation have been attempted with enzymes, cortisol and other chemicals (e.g., 53). R. Bunge noted that treatment of myelinated explants of spinal ganglia with trypsin results in damage to the myelin, alteration of the nonneuronal elements but no discernible effects on the neurons (86). Varon pointed out that culture systems could be designed for the specific investigation in vitro of such, or other, treatments. If a “gap” could be secured in the lesion, attempts could then be made to bridge it with a suitable terrain. R. Bunge proposed ependymal cells as the choice candidatesfor such a bridge, in view of M. Singer’s work on the role that they play in guiding and supporting axonal regeneration in the lizard spinal cord (e.g., 25). Varon advanced the suggestion of testing different cells and different conditions in vitro for the ability to favor elongation and/or selected directionality of neurites from cultured cord neurons. Lasher noted that choroid plexus and ependymal materials provide excellent sources of growing cells in z&a. The cultured ependymal (or other) cells could then be implanted inside the lesion in several forms: a cell pellet or an aggregate culture of the appropriate size (R. Bunge), sponge fragments

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on which the cells had been allowed to grow in a tridimensional matrix (Schubert), cells subjected to a mild glutaraldehyde fixation that might still preserve their surface qualities (Herschman) , cell-coated tubes of Millipore filters as already used in peripheral regeneration (R. Bunge). Lasher raised the possibility of also making use of cellulose acetate tubing (now commercially available) as perfusable “vessels.” Finally, the cultured cord specimen would be implanted, to take advantage of the new terrain. REFERENCES 1. AKESON, R., and H. R. HERSCHMAN. 1974. Modulation of cell-surface antigens of a murine neuroblastoma. Proc. Nat. Acad. Sci. USA 71: 187-191. 2. APPEL, S. 1975. Neuronal recognition and synaptogenesis. Exp. Neurol. 43 (No. 3Part 2) : 52-74. 3. BARBERA, A. J., R. B. MARCHASE, and S. ROTH. 1973. Adhesive recognition and retinotectal specificity. Proc. Nat. Acad. Sci. USA 70: 2482-2486. 4. BARKLEY, D. S., L. L. RAKIC, J. K. CHAFFEE, and D. L. WONC. 1973. Cell separation by velocity sedimentation of postnatal mouse cerebellum. J. Cell Physiol. 81: 271-280. 5. BERGER, B. D., C. B. WISE, and L. STEIN. 1973. Nerve Growth Factor: enhanced recovery of feeding after hypothalamic damage. Science 180: X16-508. 6. BEVAN, S., R. MILEDI, and W. GRAMPP. 1973. Induced transmitter release from Schwann cells and its suppression by actinomycin D. Nature New Biol. 241: 85-86. 7.

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BIRKS, R. I,, M. C. MACKEY, and P. R. WELDON. 1972. Organelle formation from pinocytotic elements in neurites of cultured sympathetic ganglia. J. Neurocytol. 1: 311-340. BISCHOFF, R., and H. HOLZER. 1970. Inhibition of myoblast fusion after one round of DNA synthesis in S-bromodeoxyuridine. J. Cell Biol. 44: 134-150. BJERRE, B., A. BJGRKLUND, and U. STENEVI. 1974. Inhibition of the regenerative growth of central noradrenergic neurons by intracerebrally administered antiNGF serum. Brain. Res. 74: 1-18. BJGRKLUND, A., and U. STENEVI. 1972. Nerve Growth Factor: stimulation of regenerative growth of central noradrenergic neurons. Science 175 : 1251-1253. BLOOM, F. E. 1970. Correlating structure and function of synaptic ultrastructure. In “The Neurosciences: Second Study Program,” pp. 729-746. F. 0. Schmitt, [Ed.-in-Chief]. Rockefeller University Press, New York. BRAY, D. 1970. Surface movements during the growth of single explanted neurons. Proc. Nat. Acad. Sci. USA 65: 905-910. BRAY, D. 1973. Branching patterns of isolated sympathetic neurons. 1. Cell Biol. 56 : 702-712. BRAY, D., and M. B. BUNGE. 1973. The growth cone in neurite extension. In “Locomotion of Tissue Cells,” pp. 195-209. Ciba Foundation Symp. 14, R. Porter and D. W. Fitzsimmons [Eds.]. Elsevier, Amsterdam (Holland). BROWN, J. C. 1971. Surface glycoprotein characteristic of the differential state of neuroblastoma Cl300 cells. Exp. Cell Res. 69: 440-443. BUNGE, M. B. 1973. Fine structure of nerve fibers and growth cones of isolated sympathetic neurons in culture. J. Cell Biol. 56: 713-735.

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17. BUNGE, M. B. 1973. Uptake of peroxydase by growth cones of cultured neurons. Amt. Rec. 175: 280. 18. BUNGE, R., and BUNGE, M. 1975. Tissue culture in the study of peripheral nerve pathology. Ift “Peripheral Neuropathy,” P. Dyck, P. Thomas, and E. Lambert [Eds.], W. B. Saunders Co., Philadelphia, Pa., in press. 19. BUNGE, R. P., R. REES, P. WOOD, H. BURTON, and C. P. Ko. 1974. Anatomical and physiological observations on synapses formed on isolated autonomic neurons in tissue culture. Brain Res. 66: 401-412. 20. BURNHAM, P. A., and S. VAKON. 1974. Biosynthetic activities of dorsal root ganglia in vitro and the influence of Nerve Growth Factor. Neurobiology 4: 57-70. 21. CHALAZONITIS, A., and L. A. GREENE. 1974. Enhancement in excitability properties of mouse neuroblastoma cells cultured in the presence of dibutyryl cyclic AMP. Brain Res. 72 : 340-345. 22. COLLINS, J. J., P. H. BLACK, A. D. STROSBERG, E. HABER, and K. S. BLOCH. 1974. Transformation by simian virus 40 of spleen cells from a hyperimmune rabbit: evidence for synthesis of immunoglobulin by the transformed cells. hoc. Nat. Acad. Sci. USA 71: 260-262. 23. GRAIN, S. M., and E. R. PETERSON. 1974. Development of neural connections in culture. Am. N. Y. Acad. Sci. 228: 6-34. 24. DUNN, G. A. 1971. Mutual contact inhibition of extension of chick sensory nerve fibers in vitro. /. Cornp. New. 143: 491-508. 25. EGAR, M., S. B. SIMPSON, and M. SINGER. 1970. The growth and differentiation of the regenerating spinal cord of the lizard, Allolis carolirzensis. I. Morph. 131: 131-1.52. 26. FISCHBACH, G. D. 1972. Synapse formation between dissociated nerve and muscle cells in low density cell cultures. Dezvl. Biol. 28: 407-429. 27. FRY, J. M., G. M. LEHRER, and M. B. BORNSTEIN. 1973. Experimental inhibition of myelination in spinal cord tissue cultures. J. Neurobiol. 4: 453459. 28. GARBER, B. B., and A. A. MOSCONA. 1972. Reconstruction of brain tissue from cell suspensions. II. Specific enhancement of aggregation of embryonic cells by supernatant from homologous cell cultures. Devel. Biol. 7: 235-243. 29. GILMAN, A. G., and B. K. SCHRIER. 1972. Cyclic AMP in fetal rat brain cell cultures. I. Effect of catecholamines. Mol. Pharmacol. 8: 410-416. 30. GILLER, E. L., B. K. SCHRIER, A. SHAINBERG, H. R. FISK, and P. G. NELSON. 1973. Choline acetyltransferase activity is increased in combined cultures of spinal cord and muscle cells from mice. Science 182: 588-589. 31. GLOBUS, A., H. D. Lux, and P. SCHUBERT. 1973. Transfer of amino acids between neuraglia cells and neurons in the leech ganglion. Exp. Neural. 40: 1OP 113. 32. GREENE, L. A. 1974. The use of a-bungarotoxin to probe acetylcholine receptors on sympathetic neurons in cell culture, pp. 765-771. In “The Nemosciences: Third Study Program,” F. 0. Schmitt and F. G. Worden [Eds.], MIT Press, Cambridge, Mass. 33. GUTH, L. 1974. Axonal regeneration and functional plasticity in the central nervous system. Exp. Neural. 45 : 610-658. 34. HENRY, I. A,, K. ST~CKEL, H. THOENEN, and L. L. IVEHSEN. 1974. The retrograde axonal transport of Nerve Growth Factor. Urczin Rrs. 68: 103-121. 35. HILL, C. E., G. E. MARK, 0. ER~~NK~, L. ER~CNKG, and G. BURNSTOCK. 1973. Use of tissue culture to examine the actions of guanethidine and 6-hydroxydopamine Eur. I. Pharmacol. 23 : 162-174.

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