I~SPERIhIENTAL

Nli~~ROl.0C.Y

48,

Neurons

NO.

3.

PART

and Glia

in Neural

SILVIO Dcpartmerl

t

of Biology,

School

San Diego,

2, 93-131 (1975)

Cultures

VARON

of Mediche,

Uniwrsity

of

CaliforGz

California 92037

CONTENTS Introduction ............................................... Neuronal and Glial Properties from in Viva Studies Neurons ............................................... Glia .................................................. Neuron-glia interactions ................................ Neural tissue proteins .................................. In Vitro Approaches and Techniques .......................... Explant cultures ....................................... Cell dissociates ......................................... Aggregate cultures ..................................... Monolayer cell cultures ................................. Purified cell fractions .................................... Neoplastic neural cells and derived clonal lines .............. Contribution of in Y&o Neural Studies ........................ Replication ............................................. Neurite growth ........................................ Electrical and chemical excitabilities ....................... Synaptic activity ....................................... Biochemical properties .................................. Cortisol induction of Glycerol Phosphate Dehydrogenase (GPDH) .......................................... Catecholamine induction of cyclic AMP and Lactic Dehydrogenase (LHD) ..................................... Other glial properties ................................... Coda .....................................................

93 95 96 98 99 100 102 103 104 105 105 106 107 108 109 113 115 118 121 122 123 124

INTRODUCTION Research in the neurosciences has traditionally aimed at defining characteristics and modes of operation of the nervous system as a finished, 93 Copyright All rights

1975 by Academic Press, Inc. o9 reproduction in any form reserved.

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stable entity. In recent years, increasing attention is being given to the dynamic features of the nervous system: how it develops, how it is maintained upon maturity, and what plasticity it still retains at that stage. Consideration of these problems requires a distinction between the inherent capabilities of the neural cells and the extent to which they are expressed under the complex influences of their humoral and cellular environments. In tivo studies of the adult nervous system provide information on neural performances within a stable context and, therefore, cannot distinguish between intrinsic and extrinsic contributions to such performances. Thus, they tend to encourage a view of fixed and irreversible capabilities of the cells even though the constancy in neural performance may only reflect an invariant, and continuously applied, environmental situation. The now classical studies of neuroembryology, on the other hand, offer a view of neuronal and glial cells operating in progressively changing contexts. They have yielded important information regarding the involvement of several morphogenetic activities : proliferation, migration, neurite elongation and modeling, “programmed” cell death, and specific interneuronal connections, as well as some information on the biochemical differentiation that overlaps or follows such events. Here too, however, the investigator is restricted to the observation of cellular changes within contexts, whose complexity is neither understood nor readily susceptible to experimental alterations. The problem of regeneration in the central and peripheral nervous systems of higher animals centers around the dynamic behavior of neural cells. In a regenerative situation, both neurons and glia are forced to operate in an abnormal context and, thus, in subjection to abnormal extrinsic influences. An understanding of favorable and unfavorable influences and of how they operate is essential if one wishes to attempt promoting the former and opposing the latter. At the cellular level, then, several basic questions require systematic investigation. (i) What are the characteristics of normal cells (neurons and glia) which belong to the cell itself and not to its involvement in a particular situation ? (ii) When and how are these cell-specific traits irreversibly acquired (true differentiation), and how is this expression subsequently affected by the external circumstances (modulation) ? (iii) How are interneuronal connections established and then maintained (specific connectivity, synaptogenesis, possibly synapse “activation” and plasticity) ? (iv) What roles do glial cells play in the survival, fiber elongation and guidance, synaptic connections and general maintenance of the neurons ?

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The study of neural tissue by in vitro techniques provides ways to investigate neural cell performances in contexts that are different from, and much simpler than, the normal ones. Furthermore, the humoral environments can be extensively manipulated, and so can the cellular environments, i.e., the composition and organization of the neural cell societies to be studied. Thus, information can be acquired both on distinctive properties of neurons and glia, and on the extent and nature of glial-neuronal interactions. The inherent abnormality of the i~t vitro situations has raised considerable criticism as to their meaningfulness to neural behavior in situ. Mainly, the untested assumptions underlying such criticism were that cells transferred out of their natural habitat would permanently lose their specialized traits (dedifferentiation), or would at least ceaseto proceed on their expected specialization (arrested development). By now, however, the evidence is overwhelming that neither assumption was correct and aberrant behavior need not occur in z&o. Furthermore, an appreciation has developed of the opportunities, rather than the liabilities, provided by in vitro situations where neural cells will not conform to the in vivo behavior. It is in such situations that complex performances can be dissected, the conditions controlling them analyzed, and corrective alterations attempted to provide insights into what controls the “normal” behaviors themselves. The validation of the iw vitro approaches to neural studies and the considerable progress already made in the field are attracting increasing numbers of investigators, and the literature on both mixed cell societies and purified neuronal and glial populations is rapidly expanding. The present review will attempt to cover a considerable portion of such literature, either directly or through referenced papers, but cannot lay claim to being complete. Nor does it intend to minimize the relevance of those studies that could not be quoted for lack of space or becauseof the intended orientation of the review. The review is arranged in three parts. First, a summary is given of neuronal and glial properties as revealed by in vie~o studies. Secondly, different in vitro techniques are described, with some discussion of their respective advantages and difficulties. Finally. contributions of in vitro studies are reviewed with regard to neuronal and glial properties and to to heterotypical cell interactions. The review is also meant to serve as additional background for the proceedings of a workshop on Culture Techniques and Glial-Neuronal Interrelationships ilr rrif,-(1 (we 185). NEURONAT,

AND GLIAL PROPERTTES IN V/Y0 STUDIES

FROM

Netirons. The bulk of research in neuroscience has dealt with the neuron, and neuronal properties are extensively described throughout the

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literature and in several textbooks. Here, it appears appropriate to merely list general neuronal characteristics which might serve as tentative markers for neuronal identification in the aberrant, in vitro situations. They are: (a) Failure to replicate : in compliance with the Boulder Convention (19)) the term neuron will be restricted to postmitotic elements, while the term neuroblast will designate neuronal precursors still capable of replication ; (b) Morphology: sharply defined body contours and processes,cytoplasmic Nissl substance, large and clear nucleus; (c) Neurites: ability to grow and maintain dendrites and axon with extended microtubule and neurofilament apparatus ; (d) Electrical excitability : elicitation by an electrical stimulus of an all-or-none action potential (voltage-dependent and time-dependent sequence of ionic conductance changes) ; (e) Chemical excitability ; elicitation by a chemical stimulus (neurotransmitter) of a graded, concentration-dependent alteration of ionic conductances, mimicking a post-synaptic potential ; (f) Receptor sites: specific to a given neurotransmitter and present on the surface membrane (of dendrites, soma and/or axon) to mediate the chemical excitability ; (g) Neurotransmitter apparatus : ability to synthesize, store, release and possibly take up a given neurotransmitter ; (h) Synaptic connectivity : ability to form a synapse (presynaptic component) with another neuron or peripheral effector cell, and/or to accept a synapse (postsynaptic component) from another neuron or a primary sensory receptor cell. Glia. In contrast with the neurons, glial cells have had a very limited coverage. Textbooks and neurocytological reviews (e.g., 16) usually devote less than ten percent, if that much, of their spaceto the glia. Furthermore, information in the research literature has been greatly fragmented, generally reflecting the unsystematic use of different source tissues and techniques, as well as the early difficulties in recognizing and defining different glial classes.The following survey is restricted to the macroglia, i.e., astrocytes, oligodendrocytes and related cell types, and has drawn considerably on an excellent review by Bunge (29). As for the microglia, the question is still unsettled whether it originates from mesenchymal rather than neuroectodermal elements (cf. 188), and little is known about what other functions it may have beside its phagocytic activities (cf. 189). Astrocytes are relatively small cells by ordinary microscopy, but have several processes which electron microscopy has revealed to be greatly extended and convoluted. The earlier distinction in fibrous and protoplasmic astrocytes is currently believed to reflect differences in location

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(white vs grey matter) rather than a real distinction in cell types. The cytoplasm of both is characterized by extensive fibrillar material (hence, the often used term of “skeletal” glia) . Astrocytes contribute the “surface” glia (glial and ependymal linings) and a large portion of the parenchymal neuropil and, thus, constitute an exchange zone between neurons and extraneural fluids (cerebrospinal fluid, blood). The role of astrocytes in the regulation of such exchanges is a generally accepted concept, but the modalities of astroglial involvement in their regulation are still debated. The extensive investment of blood capillaries by astrocytic “end-feet” has prompted the view prevalent in the early 1960s that astrocytes constitute the anatomical counterpart of the blood-brain-barrier, with blood-borne substances reaching the neurons (and vice versa) essentially through trans-glial pathways. More recently (99)) recognition of the continuity of intercellular spaces across pial and ependymal linings and within the neural parenchyma, as well as a number of experimental observations (29), have strongly proposed that the exchange pathway be predominantly intercellular. In the latter case, the astroglia would act as a monitoring system rather than a physical barrier (1.57)) and the exchange substances wo~~lcl “run a gauntlet” (28; between contiguous glial surfaces. The composition of such an intercellular flow would be regulated by unselective adsorption or differential binding to surface membranes, as well as uptake into glial cells, and by the release of glial products or of trans-glially conveyed materials. Furthermore, the intercellular space should be viewed as a dynamic one, undergoing considerable alterations according to the physical state of cell surface materials and the biochemical changes imposed on the latter by the activities of the glial cells. Oligodendrocytes are characterized by a more granular cytoplasm and, because of this greater ribosomal content, they are occasionally described as “producer” glia. They also entertain a closer, more direct relationship to the somata or the axons of the neurons (satellite or sheath cells, respectively). It is still unresolved whether satellite and sheath cells are different subtypes of glia, or the same cell operating in different situations. Analogous cells occur in the peripheral nervous system where they are called satellite and Schwann (neurilemma) cells. The intimate association between neurons and oligodendroglia or its peripheral counterpart strongly suggeststhat these glial cells play more direct roles in supporting neuronal performance. One well-established role of sheath oligocytes and neurilemma cells is that of myelin formation (29). Individual glial cells provide a single process to one axon (PSN) or several processeseach to different axons (CNS) to form the spiral compaction of glial membranes which constitutes each myelin segment. Like the astrocytes, therefore, sheath glia is involved in the production of large amounts of plasma membrane. As Bunge (29) points out, the myelin membrane is a live structure

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that requires maintenance from the parent glial cell. Therefore, pathological defects of myelin could conceivably result from an altered metabolism of the parent cell, or from a defective intracellular transport of materials from the production site. Neuron-Glia Interactions. A functional coupling between glia and neurons has been suggested by several observations, even though it is rarely specified whether it involves the astroglial or the oligodendroglial cells, or both. Repetitive impulses in the optic nerve of mud puppy and frog consistently lead to a slow and protracted depolarization of the adjacent glia, due to the intercellular accumulation of K+ released by the firing axons (136). In the leech, the glial membrane displays a high electrical resistance and a large membrane potential, and is more susceptible than neuronal membrane to being depolarized by an increase in extracellular K+ ( 127). The glial cells, therefore, could serve as a spatial buffer by taking up the excess K+. In addition, they could redistribute it to neighboring regions via a trans-glial pathway, as suggested by the glial surface potentials recorded extracellularly (127). A direct glial contribution to slow potential changes in neural tissue has often been suggested, as has the possibility that glial cells act as storage of weak signals or as modulators of neuronal impedance (e.g., 60). Glial cells may also be involved in the removal of neurotransmitters released from presynaptic sites. Although glial components do not participate in the physical structure of a normal synapse, it has been noted that synaptic regions are often bordered by glial cells (137). Acetylcholine removal depends on its hydrolysis by acetylcholinesterase, and this enzymatic activity has been reported in the glial cap overlying neuromuscular junctions in the frog (154), glial sheaths of the cockroach’s CNS (173) and, possibly, neurilemma cells on the squid axon (87). GABA removal occurs via re-uptake into presynaptic terminals. Glial cells take up GABA even more extensively than do neurons at neuromuscular junctions of the lobster (135), and radio-labeled GABA was found localized in glial cells of rat cerebellum (86, 158), rat retina (124), and rat and cat dorsal root ganglia (64). Uptake of neurotransmitters by glial cells could be relevant both as a component of the glial regulation of neuronal environment, and as a mode of communication from neuron to glia. Neuronal signals directing glial actions may be involved in several other situations. The release of K+ upon firing may serve as a trigger for the release by glial cells of materials useful to the neuron (99). Initiation of myelin formation after a glial process has contacted an axon appears to require a triggering signal from the axon itself. Axonal degeneration leads to “reactive” gliosis, i.e., proliferation and/or influx of

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glial cells that assume active phagocytic functions (cf. 189). In the neuromuscular junction of the frog (13). miniature end-plate potentials are abolished by transection of the motor fiber but reappear after a few days. They are reportedly induced by quanta1 releases of neurotransmitter from neurilemma cells that have come to occupy the nerve ending position. Since reappearance of such potentials requires new RNA synthesis, the investigators have proposed that glial transcription must be instructed by the degenerating nerve terminals. The ability of glial cells to take up protein from the intercellular space (15, 26) provides a possible mechanism for their reception of special neuronal signals. Also reported is an opposite traffic, from glia to neurons, of amino acids in the leech ganglion (63) and protein in the squid (101). One must note that no specialized junctions have been observed between glia and glia (29). HydPn (e.g., 91) has examined a number of biochemical parameters in individual neurons and their surrounding glia, hand-dissected separately from each other, from rabbits that had been submitted to various functional situations. He noted several inverse changes taking place in the two cell types, for example after rotation of the animal, at different stages of sleep, during nerve regeneration, or on treatment with RNA-stimulating drugs. Hydtn suggested (i) that neurons and glia are energetically coupled (with neurons being the dominant users), and (ii) that RNA may be produced in glial cell for transfer to the partner neuron. From these, and other, observations emerges the concept of a neuronglia unit (16, 1.57) operating at anatomical, biochemical and functional levels. Each partner might depend on the other for the execution or the facilitation of certain functions, or even for life-sustaining operationsa truly symbiotic relationship. Neural Tissue Proteins. As the most diversified and specific molecular effecters, proteins should provide important markers to characterize neural vs non-neural tissue, and neuronal versus glial cells. The latter problem faces the difficulty that neural tissues comprise different cell types comixed with one another. Past attempts to circumvent this difficulty have had to rely on correlations between the amounts of a given constituent and the relative distribution of different cell types in different regions, developmental stages or pathological situations. Neuron-specific proteins : Several of the enzymes involved in synthesis and degradation of neurotransmitters have appeared to be located in the corresponding neurons. In addition, the soluble acidic protein 14-3-2, isolated from brain extracts (120), was found to occur primarily in the nervous system of most vertebrates. The protein appears relatively late in development and its distribution has suggested a neuronal localization. Antigenic cross-reactivity among different species indicates some evolutionary changes, but no function has been yet even suggested.

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Glia-specific proteins: Carbonic anhydrase of neural tissues is generally taken to be predominantly, if not exclusively, localized in the neuroglia (61). Myelin-characteristic proteins have been isolated, such as the basic encephalitogenic protein and derived peptides (74) from central myelin and the Pr and Pa basic proteins (27) from peripheral myelin, which upon injection induce experimental allergic encephalomyelitis or neuritis, respectively. Also characteristic of myelin appears to be the enzyme 2’, 3’ cyclic nucleotide-3’ phosphohydroIase (CNPase) , which occurs at 25-fold higher levels in neural than in other tissues (100). A glial fibrillary acidic protein (GFA) has been extracted from multiple sclerotic plaques and gliotic CNS areas, and immunologically recognized in normal brain astrocytes of several species, as well as in human astrocytomas (14, 178). Considerable information has been gathered on the SlOO protein, a small, very acidic, highly soluble protein isolated from brain extracts (119), which is believed to be restricted to neural tissue and occurs in all vertebrates and several invertebrates with a high evolutionary stability. Its glial localization is proposed by the usual correlative criteria, and demonstrated in hand-dissected glial cells as well as in the nuclei of their partner neurons (92). The function of SlOO is not known. Its ability to bind Ca2+ selectively has suggested a role in Ca?+ regulation within the neural tissue, and SlOO-induced changes in the translocation of monovalent cations across lipid membrane have been reported (37, 38). However, S-100 is not the Ca2+-binding brain protein reported to activate cyclic nucleotide phosphodiesterase (193). Brain-specific membrane antigens : These have been described without selective attribution to particular classes of neural cells. Regional antigenie specificities are suggested by early studies ( 116)) according to which antisera to caudate nucleus or hippocampal region, injected intraventricularly or intracerebrally, altered the electrical activity and related behavior from the corresponding, but not the other, brain region. More recently (lOS), a membrane antigen has been solubilized and isolated from mouse brain particulate matter, and antiserum to it has failed to detect its presence in several non-neural tissues. Other proteins: Although not specific to neural tissue, other proteins have nevertheless received considerable attention because of their relative abundance in the nervous system and their presumptive importance in axonal properties. Such are tub&n (the protein subunit of microtubules, a possible neurofilament subunit mitotic spindles, cilia and flagella), protein (89), actin (21, 155, 160), and myosin (1, 12). IN

VITRO

APPROACHES

AND

TECHNIQUES

Several in vitro experimental systems have been explored (76, 179) which diverge in varying degrees from the in situ situation. They range

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from the organ culture that retains the closest similarity to the original tissue to the extreme case of clonal cell lines derived from neoplastic neuronal or glial elements. In all cases, these esperimental systems have the advantage of involving a restricted set of cell types and a limited humoral environment. The humoral environment can be manipulated at will and selected agents (nutrients, ions, hormones, drugs) can be brought to act on the cells without mediation by other tissues and to reach them without selective barriers. Furthermore, the cells become directly accessible in their living state to visual observation (phase contrast microscopy, time lapse cinematography) and electrophysiological or biochemical investigations. Finally, data obtained from the live cells can be correlated on an individual base with data obtained after their fixation (cytochemical stains, autoradiography, electron microscopy). While the several in vitro systems share many properties with one another, each one also has distinctive advantages and disadvantages and can complement other systems with regard to both confirmation and further definition of individual findings. Normal (or primary) neural cultures, for example, provide the option of choosing tissue sources of the desired species, region and developmental age. The source material will have acquired different sets of properties during its in vivo differentiation, whose disappearance, maintenance, restoration or alterations can be examined in the absence of the complex and obscure influences of the whole organism. Neoplastic ceI1 cultures, on the other hand, have the considerable advantages of a greater bulk availability and the homotypic composition inherent to their derivations. They suffer, however-at least for the present mouse and human lines-, from three major disadvantages: their substantial aneuploidy (abnormal genetic content), considerable instability of their karyotype, and the very limited choice of the available cell lines. A problem central to all in zritro systems is the search for a physical and biochemical milieu that optimizes neural cell performances. The problem is further compounded for the heterocellular in vitro systems by the likelihood that different cell types (and the same cell type at different development stages) may require different “optimal” conditions. One must stress that the search for optimal environments is not merely a technical investigation, but a crucial component in the study of cell properties and extrinsic regulatory influences underlying neuronal and glial performances. In general, defined media (balanced salt solutions enriched with glucose, amino acids, vitamins and, optionally, antibiotics) fail to support neural cultures unIess supplemented with more complex and stil1 poorly defined biological fluids (various fetal or adult sera, embryonic extracts, ascitic fluid, etc.). The role of these biological supplements is to provide not only greater nutritional support but also some special agents necessary to the attachment, survival, proliferation and/or expression of differentiated properties of the cultured cells. One classical example of such agents, be-

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side the more traditional hormones, is the Nerve Growth Factor discovered several years ago by Levi-Montalcini and Hamburger (103, 104 ; see also 181). At present, different laboratories involved in culturing the same or different neural materials often use media that differ to varying extents from one another in the composition of balanced salt solutions, the choice and amounts of serum, and the selection of special supplements. While all these media appear to serve the limited ambition to keep cells alive and, presumably, well, their choice is undoubtedly not a trivial one with regard to several cellular performances, as many studies have explicitly shown. Clearly, the ultimate goal is to achieve more elaborate but still fully definable media, each with an explicit composition fitting best a given cell population and/or the desired choice of a particular set of cellular performances. Explant Cultures. Small organs (e.g., a peripheral ganglion) or small can be fragments of a tissue (organ” and “tissue” cultures, respectively) isolated and “explanted” into a culture vessel. The size of each explant must be small enough (usually, 1 mm or less) to allow for adequate exchanges with the medium. Even so, centrally located cells are frequently nourished less effectively and may become necrotic. The piece of tissue could be left free-floating and studied biochemically (e.g., brain slices). However, for more detailed investigations, the explant must be anchored to a surface, either by adhesion to suitably coated surfaces or by entrapment against it within a gel matrix (e.g., plasma clot). While some damage is undoubtedly inflicted by the dissection (cut edges, severed axons), the explant retains the original cell composition and an organotypic relationship among component cells. By the same token, cells wthin it are not readily visualized nor selectively approachable. Both features, however, are temporary. Some cells die within the explant and many others migrate out along the attachment surface until the explant becomes flattened and resolved well enough to permit a better view of, and approach to, the cells remaining within its confines. Outward migration does not occur equally for all cell types, with cells engaging in successive waves of migration and others hardly participating at all. Neurons, for example, usually display little active mobility, although they may be passively pulled around by other cells. The explant approach was the first one undertaken in the field of neural cultures, and has dominated the scene for the first fifty years since the pioneering experiments of Harrison (73). This period involved mainly morphological investigations of different neural cell types and the dynamics of cell movement and neurite growth, and owes the greatest debt to the work of Paul Weiss, Charles Pomerat and Margaret Murray. An extensive review of such studies is available (122 ; see also 76 j . In the 1950s

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ai~d lWJ.s, neural explants became the subject of electro~~l~ysiological investigations, largely to the credit of Stanley Crain (45), and electrun microscopy was also brought to bear on such preparations. Cell Dissociates. The next step toward removing in vivo constraints requires a complete resolution of the neural tissue structure, that is dissociation into component cells. The dissected tissue may be first cut into small pieces and predisposed to a more effective disruption by exposure to selected media (Ca’+, Mg’+-free solutions, trypsin or other enzymes). A cell suspension is then obtained by mechanical disruption through one or several small pore systems (narrow bore pipets, nylon bolting cloth, stainless steel screens or other filters). Attendant to the dissociation are problems pertaining to the choice of source tissue, amount of cells recovered, identification and numerical assessment of different cell types, and viability of the dissociated cells. Source tissue: Different neural tissues, or tissue of different developmental ages, vary considerably in cohesion and intercellular complexity. Thus, their dissociation may meet with varying degrees of success, even though minor or substantial alterations of the general procedure have been applied. Thus far, satisfactory results have been mainly restricted to perinatal neural tissues. Cell yield: Relatively few studies have been directed to maximize cell recovery (e.g., 183). Tl le dissociated cells can be readily counted (hemocytometer chamber under phase microscopy, Coulter counter) as long as they are adequately dispersed. Cell compositions : Upon isolation from their source tissue, cells tend to round up and lose their tissue-characteristic morphology (as well as their context references). Thus, recognition of different cell classes and differential cell counts in the dissociate can only be made on grossly different physical attributes (size, birefringence, nuclear appearance, etc.). Finer distinctions and more definitive identifications will requre additional treatments, some of which are discussed further on. Cell viability: Viability can be evaluated directly in the dissociate, for the total cell population or the distinguishable cell classes, by making use of the ability of live cells to exclude certain dyes (e.g., trypan blue) or selectively take up certain others (e.g., vital red), and differentially counting dye-positive and dye-negative cells in a sample of the dissociate. Subsequent uses of the dissociated cells may provide additional, and more critical, assessments (e.g., plating efficiency and survival in monolayer cultures, or selected metabolic activities). The current state of the art of neural dissociation is still far from satisfactory, and some of the options concerning the above aspects nlay have to be sacrificed in favor of others, depending on the use planned for the neural dissociates. There are several such uses. The cell suspension can

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be directly examined for biochemical or metabolic parameters. The dissociated cells can be allowed to reaggregate and aggregate cultures developed from them. The still dispersed cells can be plated out in culture dishes or flasks to former monolayer cultures. Or, cell fractionation procedures can be applied to obtain enriched or purified subpopulations of different cell types. In all cases, it is important to recognize several consequences of the dissociation process, which will affect cell performance in ways and to degrees yet to be fully understood : (i) the original tissue organization no longer exists, (ii) cell-cell contacts are abolished, (iii) intercellular distances are increased depending on the volume of fluid in which the cells are dispersed, (iv) numerical balances among different cell types are changed, and (v) individual cells are altered to varying extents depending on type, source and procedure. Aggregate Cultures. Cells dispersed in a liquid medium will tend to come together by random collision, and stay together. As Moscona and other investigators have shown over several years (121), this aggregation is a property of all animal cells, but the degree to which aggregates will form and persist depends on the origin, age and state of the cells, as well as the ambient conditions. Moscona’s method, which has been more or less generally adopted (e.g., 165)) is to submit the suspension to a gentle rotation (e.g., 70 rpm in a gyratory shaker), which causes a vortex and, thus, increases cell collisions; faster rotation would increase shearing forces and limit the size (though increase the number) of the aggregates. Aggregation, however, is not a purely mechanical event. Bonds must be established among colliding cells, which require Ca2+, are facilitated by serum proteins and special protein factors, and depend on sufficient competence of the cell surfaces. This last requirement is a dynamic one, as shown by the fact that aggregation occurs optimally at 37-38 C, and will be totally prevented at 15 C or under blocked protein synthesis. Once aggregates are optimally formed (usually, in less than 24 hr), they can be transferred to a stationary system and maintained as aggregate cultures in a chosen medium for several weeks to months. Very early in the cultured aggregates a second process takes place (again, a universal property of animal cells), namely migration of the cells within the aggregate to assume selective positions and orientations. If the aggregate comprises different cell types, this will result in a sorting-out of the different classes and a histiotypic reorganization largely mimicking the original tissue architecture. Sorting out reflects differences in the adhesive properties of different cells. There is still debate as to whether such differences need to be qualitative (involving different cell smface materials) or could be only quantitative (175). In aggregate cultures, cells are no more accessible to direct observation and approach than in the early explant cultures, and their morphological

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esamiuation requires histological techniques as does normal tissue. However, this experimental system provides several new opportunities. It permits the study of cell surface properties relevant to histogenesis. It examines the histogenetic process itself (patterns, sequences, influences), while proving its dependence on intrinsic cell properties rather than a preexisting in situ matrix. Finally, it reconstructs the closest analog to origconditions for further developinal tissue and may provide “normotypic” ment and/or function of the reorganized neural cells. Aggregate cultures from several neural sources have been studied, e.g. retina (121, 174), whole brain (167)) hipp ocampal region (50)) cerebellum (51) . Monolayer Cell Cultures. If a dispersed cell suspension is “seeded” (or plated”) on an attachment surface, cells will settle and attach individually. The cells will then undergo shape changes, move along the surface, make contact with other cells and establish population patterns that will occur essentially in a two-dimensional field (monolayers or, more limitedly, plurilayers). Attachment, shape changes, motility, contacts and patterns vary with the properties of the attachment surface and culture conditions. Because the cells are at all times accessible to observation (phase contrast microscopy), they can be characterized early and in great detail with refeatures and classification. Dynamic gard to number, morphological changes (shapes, positions, collective patterns) can be followed by time lapse cinematography, and bioelectric properties examined under visual control. Finally, the entire plate can be fixed, stained or autoradiographed, and single sections (parallel to the attachment surface) may include individual cells in much of their entirety-soma, neurites and intercellular contacts. Thus, losses in tissue-like attributes may be offset by gains in resolution and independent analysis of different properties. In addition, the total accessibility of each cell permits direct, uniform and immediate interactions between cell and experimental medium constituents. Purijed Cell Fractions. Availability of purified neural cell subpopulations is becoming increasingly important in several directions : Chemical characterization of different neural cell types requires bulk amounts of homotypic cells, uncontaminated by materials from cells of other types. Viability is not essential, as long as the purified cells are viewed merely as a record of events or activities that have occurred in situ. Bulk preparation of cell fractions for such purposes has been attempted in various ways, and a few procedures have emerged that are now being increasingly utilized (147). They all involve segregation of different cells by centrifugal force on step gradients (sucrose, ficoll, etc.) or continuous gradients in zonal rotors. Important technical aspects are the choice of media minimizing cell breakage (usually by inclusion of high molecular weight substances), and a thorough elimination of any contaminating cell debris. Since the cells are segregated on the base of physical properties,

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adequate identification of each cell class is a critical problem for a correct attribution of the chemical findings. Currently, identification still relies mainly on morphological traits, which are not always entirely dependable. Such reliance will be considerably reduced as more biochemical markers for neurons, glial cells and their subclasses will be firmly established through the combined contributions of the several in vitro systems. With the increasingly perceived importance of surface membranes for intercellular communications (e.g., 121)) much research is currently directed to the recognition and study of surface antigens. In the nervous system, migration, guidance of axonal growth, synaptic connectivity and glia/neuron interactions, among other processes, are most likely to occur via surface interactions (see 5, 171). Furthermore, surface antigens may reveal fine differences not only between neurons and glia but among different cell species within each major class. Purified cell fractions can be used as immunogenic material to elicit the production of cell-specific antibodies, or as antigenic material to select such antibodies out of antisera directed to the whole tissue. The main requirement is again for adequate amounts, rather than viability, of the purified cell populations. Purified cell populations that have retained viability, on the other hand, are essential to determine distinctive properties of their behaviors in vitro. In the absence of heterotypic cells, these populations could be examined with respect to survival requirements, morphological and morphodynamic performances, metabolic activities, and the regulatory influences to which individual cell-specific traits may be subjected. Recombined with selected partner populations, and in deliberately varied proportions, they can provide information-both in aggregate and in monolayer cultures-on heterotypic cell influences and interactions, as well as their cell-related specificities. Thus far, viable neural cell fractions have been obtained only in a few, and still imperfect, cases. Some have used physical methods similar to those developed for bulk preparation of cell fractions (e.g., 9). A less traumatic, and equally promising, approach has been the exploitation of differential adhesion properties of viable cells-that is, the propensity of some cell types (usually, glial and fibroblastic elements) to attach more readily to appropriately selected surfaces (131, 182, 184, 186, 187). Further help may be gained by selectively suppressing growth and/or survival of the residual undesirable cells (20, 109). Neoplmtic Neural Cells and Derived Clonal Cell Lines. An alternative source of bulk amounts of neuronal or glial materials is potentially provided by neoplastic tissue of either derivation (115). Two advances in recent years have considerably enhanced the interest in such sources : (i) several tumors of neural as well as non-neural origin were shown to retain at least some of the differentiated characteristics of their normal counterparts, and (ii) neoplastic cells were successfully adapted to grow in

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vitro and still exhibit some such characteristics. Furthermore, in these cultures, single cells can be allowed to form individual and separate colonies (clones), which can be passed to another vessel and grown there as a clonal cell line, transmitting the same genotype through many generations. Clonal cell lines obtained from the same or from similar tumors may be different and can be compared to investigate the genetic control of specified phenotypes. Finally (e.g., llS), hybrid cells can be produced by fusion (e.g., with inactivated Sendai virus) of two cells from different clonal lines, or of altogether different origin (including one normal partner). The obvious liability of these approaches rests with the aberrant nature of the material, related to its neoplastic origin. However, to the extent that the neoplastic cells do exhibit differentiated traits grossly comparable to their normal counterparts, they can be used for the investigation of such traits, as has been successfully demonstrated in several non-neural systems (endocrine, hepatic, endoreticular cells, etc.). Detailed findings will have to be subsequently compared with whatever related information is available for the normal cell, to verify the extent to which they are compatible and directly applicable to the normal expression of the differentiated trait. The obtention of useful clonal material involves several steps (e.g., 144) : (i) recognition of, and development of adequate assays for, organspecific functions expressed at the cell level, preferably in biochemical or immunochemical terms ; (ii) selection, or induction, of tumors with the chosen traits, and adaptation of the tumor to cell cultures retaining them ; (iii) development of clonal lines from the tumoral cell cultures. Clonal cell cultures have the same technical advantages as monolayer cultures from normal material. In addition, they are already homotypic and can be readily grown in bulk amounts. Most of the studies carried out, thus far, have involved mainly two neoplastic neural sources: the mouse neuroblastoma Cl300 (6, 98, 162), and the C6 cell strain from a rat astrocytoma (IO). Recent, and extensive, reviews of such studies are already available (e.g., 115). CONTRIBUTION

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Exploration of the several in vitro systems is still in its infancy. Nevertheless, the contributions already made by these approaches is very considerable (76, 77, 122, 179). Three general points have been solidly established: (a) All general properties of neurons and glia described in wivo have been observed in one or more of the in vitro systems. Thus, the capability of in vitro systems to provide information on “normal” neural behaviors is no longer in question. In addition, this demonstrates that once such neuronal and glial traits are established, they become intrinsic

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properties of the cells and no longer require the overall in viva intluence. Lastly, some of these properties are not only retained outside the organism, but can actually develop in the iut vitro situation. (b) Additional properties of neural cells, previously unsuspected or barely suggested from in viva studies. can be identified and characterized in vitro. Following the i~ vitro leads, several such properties have been subsequently confirmed to apply to the tissue in situ. (c) Neuron-specific programs and expressions are not incompatible with a retained capability for cell replication-perhaps the most startling contribution of the clonal cell approach. This raises the possibility that at least some differentiated traits of the postmitotic neurons are already programmed in the normal neuroblast. This section will mainly examine, from the in vitro perspective, a number of neuronal and glial properties, and some aspects of their interactions. The study of the in vitro neural systems, however, has made fundamental or preliminary contributions in several other areas of neurobiology, which will not be reviewed here. Some examples are: the Nerve Growth Factor phenomenon (see Ml), neural morphogenesis in normal and mutant animals (50, 51, 121, 150)) early differentiation of neural cells (128, 191). Other aspects are reviewed elsewhere (5, 171). Replication. In clonal cell cultures of neuronal origin (e.g., C-1300 neuroblastoma), the neuronal elements are replicating cells, whose growth rate is subject to extrinsic, and reversible regulation. Proliferation of neuroblastoma cells can be turned off by a variety of means, such as high cell density in suspension, cell-cell contact in confluent monolayers, serum deficiency, certain conditioned media, hypertonic media and various other agents (115, 125, 153, 163). The ability of these cells to express several differentiated neuronal traits despite the retained ability to replicate demonstrates that differentiation programs and replication programs need not be mutually exclusive. However, performance of differentiated functions is usually found associated with a stationary phase of these cultured cells, and mutual exclusion-or at least mutual interference-may well occur at the level of program expression rather than program retention. Normal neurons, on the other hand, are defined in vivo as postmitotic permanent elements (19). Upon dissociation from the source tissue and maintenance in culture, neurons continue to display an inability to divide. Their nonreplicability in situ, therefore, is not due to extrinsic inhibitory influences, such as synaptic connections, other cell-cell contacts, or a “mature” humoral environment. Thus, the permanent postmitotic feature of the neuron is an intrinsic trait, acquired in early development and maintained outside the organism. The nature of this neuronal feature remains to be investigated. In vitro neural systems uniquely provide two potential directions for such investigation, the use of cultured normal neuroblasts and the analysis of purified neuronal preparations. Little attention has

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been paid, thus far, to normal neuroblasts in culture, but the feasibility of obtaining them has been established (9, lo- 3, 108). Using the inputs from for different neuexperimental neuroembryology on specific “birthdates” ronal subclasses (e.g., 4), it may be possible to examine whether neuroblasts will follow the same growth schedule in vitro as they do in tivo, and to look into how the transition takes place from replicating to nonreplicating cell. In the second direction, purified postmitotic neurons can be analyzed for the presence or absence of known components of the replicative machinery, or for the occurrence of intracellular inhibitors. Several observations offer suggestions for more specific approaches. Nuclei from nondividing, differentiated cells (including neurons) have been made to resume DNA synthesis when transferred to replicating cells on their cytoplasm (66, 70, 94, 151). Neuroblastoma growth is blocked by inhibitors of thymidylate synthetase (39). Brain particulate matter has been reported to contain an ATFase which decreasesDNA polymerase activity of the soluble fraction, and which increases during development (110). Non-neuronal cells will proliferate in monolayer cultures and the outgrowth zone of esplant cultures. There is usually a lag phase (attachment and flattening out, or outward migration, of the cellsj, a grossly exponential growth phase (16-24 hours doubling times), and a stationary phase reached at or after confluency (density-or contact-inhibition of growth, as exhibited by most cells in culture). Growth is promoted by serum and a COP atmosphere and inhibited by their absence or a variety of other agents. Growth rates, and possibly their susceptibility to different inhibitors, vary with different cell types and the numbers of viable cells seeded. As a result, mixed neural cultures will undergo not only an increase in their total non-neurons, but a shift in the relative proportions of different cell types-both changes having important consequenceson the neuronal performances. In contrast, in aggregate cultures “contact” inhibition of growth is established early in a tridimensional manner, and these systems are characterized by very low cell growth and a near retention of the initial cell balance (cf. 165). The ability of glial cells to grow in monolayer cultures can be exploited to amplify the initial population, and weed out accompanying neurons, over several successivepassages.However, the number of generations is usually limited, and conditions for optimal growth of primary glia remain to be worked out. Neurite Groeerth.Viable neurons from normal sources will grow neurites in explant, aggregate or monolayer cultures. A tentative distinction can often be made between axons and dendrites, but definitive identification usually requires electron microscopic examination. Neurite numbers, length and branching complexity vary with the source tissue, culture COW ditions and individual cells within the same culture. However, no correlation has yet been established among these several features. Similarly,

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neurite production can be observed to varying degrees in nearly all neuroblastoma clonal lines, and can be markedly enhanced by several means. Most of these neurite-promoting treatments also depress cell proliferation, but the latter event in itself is not sufficient to elicit neurite growth (153, 163). Under best promoting conditions, neuroblastoma neurites were hardly distinguishable from those of normal sympathetic neurons in culture. Normal, and to a less extent clonal, cultures systems have provided most of our current information on neurite growth (67, 171). Neurite extension requires an interaction of the neuronal membrane with other surfaces. Initiation of neurites occurs asynchronously in individual neurons of the same culture, but it remains unclear whether this reflects microdifferences in the attachment surface or different starting conditions of the cells themselves. Elongation proceeds in vitro at about the same rate as it does in viva (S-50 pm/hr). Several dynamic processes have been revealed by time lapse cinematography (e.g., 123, 149). Most impressive is the membrane activity at the fiber tip, with continuous outthrusts and retractions in several directions (rufflings, filopodia), as if the tip palpates and searches the surface around it. As most of the outthrusts retract, one stabilizes and appears to pull forward while drawing the neurite behind it. As neurites become extended, anchorage of the neuron appears to reside at the fiber tips, with soma and proximal fiber segments adhering only loosely if at all, indicating a high adhesiveness of the tip membrane (22). Extensive pinocytotic activity is also considered to take place at the fiber tip and distal portion, which may involve both intake and output of materials. Nuclear rotation has often been reported for neurons actively engaged in neurite production. Neurite production involves several cell activities: synthesis of new building materials (soma), delivery to the site of growth (axonal transport), assembly into neurite structures, motile activity of the tip. It is now generally accepted that elongation proceeds from the tip, rather than the base, of the neurite. The distal segment of a neurite, severed from its soma, may continue to elongate for hours (SS), just as it maintains a normal rate of axonal transport (130). In a growing neurite, the position of each branchpoint remains constant relative to the soma while the tip advances, and so do particles placed on the surface of the growing neurite (22). Thus, elongation is originated by tip activities, draws its materials from supplies already stored in the neurite, and requires somal production and delivery in order to be sustained rather than initiated. a. The growth cone: Growth cone is the term assigned by Ram6n and Cajal in the 1890s to bulbous expansions (or varicosities) observed in wivo at the tip of growing axons. While some recent electron microscopical studies (e.g., SS), have reported the general similarities between such structures and the growth cones examined in vitro, the in tivo situation

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is hardly conducive to detailed studies. Growth cones irz vitro display sheetlike projections, similar to those of many process-growing cells, and finger-like projections that appear characteristic of neuronal processes. These filopodia (or microspikes) are thin cylinders (0.2 pm), which may extend to a lo-20 pm length in less than a minute and wave about for five minutes before retracting or becoming stabilized. When encountering the process or the outthrusts of another cell, they may establish protracted contact and, through contractile activity, bring the two elements together. Bifurcation of a growing neurite also is preceded by a bifurcation of the growth cone. Thus, filopodial activity appears to underlie elongation, directionality and the ultimate shape of the total cell, as well as fasiculation (binding together of different neurites) . Yamada et al., (19.5) first described the fine structure of growth cones from central and peripheral neurons in monolayer cultures. Microtubules and neurofilaments (100 ;i) do not reach into the growth cone. Instead, a filamentous network (polygonal patterns of 50 A elements) appears to link the axoskeletal structures and the inner face of the plasma membrane, reaching into the very ends of the filopodia. M. Bunge (30) reported further details using relatively pure cultures of sympathetic neurons and a fixation technique believed to block instantaneously the membrane activities. She confirmed the distinctive “fine meshwork” filling the moving parts of the growth cone, and the exclusion of the usual organelles from them. However, the more central region of the growth cone displayed a considerable amount of agranular reticulum, as well as dense-core vesicles, multivesicular bodies, mitochondria and 100 L%filaments. The most proximal portion, like the neurite proper, contains microtubules and clusters of vesicles and vacuoles no different from those seen in other cells. b. Membrane production: Bray and M. Bunge (33) pointed out the resemblance between growth cones and ruffling membrane of motile cells, such as neurilemma cells and fibroblasts. All growth cones in a culture elongate at the same rates, despite vast differences in length, diameter or number of bifurcations of the neurite interposed between cone and soma (32). While growth cones are engaged in elongation, fibroblasts are engaged in migratory movement, yet both activities occur at the same rate (30-40 pm/hr). Both phenomena could occur, therefore, on a common molecular base, involving insertion of membrane at the leading edge and subsequent resorption of membrane at locations closer to the soma. Minimal changes in this balance could be responsible for the distinguishing feature of neurite growth, namely an ncrrctiorz of membrane rather than a net translocation of it. The large anlount of smooth membrane material, seen to fill the central portions of the growth cone, exists in arrays of anastomatic channels and clearly differs from the coated vesicles characteristic of pinocytotic activity.

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The bulk of this agranular reticulum did not become associated with exogenous peroxidase (31) suggesting its involvement in some other function. Some additional information on the insertion of new membrane comes from the work of Pfenninger and M. Bunge (146). Neuronal growth cones (as well as glial processes) exhibit moundlike protrusions, containing many vesicles (1000 A) that very occasionally fuse with the mound plasma membrane. The membrane of these vesicles and mounds lacks intramembranous particles (IMP), suggesting that this is immature membrane destined to become part of the plasma membrane. Clusters of IMPfree vesicles were also seen along the neurite and within the soma. The only other cytomembranes lacking IMPS are on the concave (maturing) faces of Golgi cisternae. These investigators suggest that immature (IMPfree) membrane arises in the Golgi apparatus, is shuttled down the neurite in the form of 1000 A vesicles to the mounds of the growth cones, is inserted there into the plasma membrane, and will subsequently “mature” through the acquisition of IMPS. It must be pointed out, however (M. Bunge, personal communication), that exogenous tracer (e.g., ferritin) may appear within mound vesicles through a route that is still under investigation. c. Tubules and filaments: Colchicine is known to bind to tubulin, and to prevent or disrupt microtubule assembly. Colchicine causes reversible, dose-dependent inhibition of neurite growth in monolayer cultures of normal neurons (49, ISO), as well as of neuroblastoma cells (166). Yamada et al. (195) showed that colchicine had no early effect on shape or activity of filopodia and growth cone. Rather, it first affected the neurite itself, causing its progressive shortening (while elongation still continued for 20-30 min) and its eventual retraction within the cell body. Quite distinct were the effects of cytochalasin B. This agent has been known to inhibit cytokinesis and morphogenetic movements of several cell types, apparently by disrupting their microfilament apparatus. When cytochalasin B was applied to monolayer cultures of normal neurons (195), all filopodia wilted and retracted within minutes, leaving the growth cones bare and immobilized. The filamentous meshwork was not disrupted but altered in shape and design. Minutes later, peristaltic waves were seen to course up the neurites, thinning them out progressively to an ultimate disappearance. Withdrawal of the agent induced the appearance of new growth cones and, within a few hours, resumption of growth or even reinitiation of neurite production from the soma. Resumption of neurite growth was not prevented by cycloheximide, confirming the independence of “primary” neurite growth from new protein synthesis. It appears, therefore, that microtubules are required for the stabilization of newly grown neurites, rather than for their elongation. Indeed, neuro-

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blastoma cells were shown to extend neurites 200-300 pm in length which exhibited no microtubules in semiserial electron microscope sections (163). In contrast, neurite growth requires the functional integrity of the microfilamentous meshwork in growth cone and filopodia, possibly anchored to microfilaments in the neurite. Several observations raise the possibility that microfilaments (and possible neurofilaments) are polymeric structures having acfin as their subunit. Actin, one of the two major functional components of the contractile machinery of myofbrils, has been recognized in many other cells and is relatively high in neural tissue. F-actin from muscle has the same diameter as microfilaments (93). Heavy meromyosin from muscle binds to microfilaments with the formation of arrowhead structures characteristic of the actin-myosin complex (93, 160). Contractile functions for microfilaments are suggested by their location in several cells (cf. 23), and the effects of cytochalasin R on general cell mobiIity (40, 194). Elccthcal and Chemical Excitabilities. Normal neurons have been shown to be electrically excitable in explant and monolayer cultures from a variety of peripheral and central neural tissues. One should recognize that, even in the best monolayer procedures, neuronal sampiing has been unavoidably selective by excluding cells not readily recognized as neurons. or unsuited for penetration because of size, fragility, or insufficent attachment. However, the detailed characteristics of the observed action potentials, as well as their occasional complexities, were similar to those recorded in viva. In some cases, distinctive responseshave been reported for neurons of different origins (e.g., 58, 59, 141). With monolayer cultures of ganglionic neurons, electrical excitability could be demonstrated routine11 within a few hours in vitro, and occasionally even in cells that had not yet attached to grow a neurite (177; Tyszka and Varon, unpublished). It appears, therefore, that neurons retain, or readily recover, their electrical excitability despite trypsin treatment and dissociation, and that they do not require the extension of neurites for expression of electrical activity. A similar independence of electrical excitability from extensive attachment and neurite growth has been documented with neuroblastoma cells from several clonal lines, grown for twenty generations in suspension {hence, with no prior history of morphological “differentiation*‘) and loosely attached under neurite-preventive conditions (163). Most studies with clonal cells, however, were carried out under neurite-promoting con‘ditions. Nearly all neuroblastoma lines exhibited electrical excitability to one degree or another, but responsesvaried considerably within the same clonal culture as well as across different clones. A question still under debate is whether electrical excitability is subject to intrinsic or extrinsic regulations. One position (163 j is that imperfect or even missing responses

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must be viewed as artifacts of the electrophysiological techniques used, or of inadequate test conditions. The other position (118, 125) is that such explanations are inadequate for all the observed variations, and that variation must reflect either different genetic programs or different stages of differentiation. In particular, analysis of electrical responses from neuroblastoma clonal lines and from cloned hybrids derived from them (e.g., neuroblastoma X L fibroblasts) suggests that voltage-dependent conductance changes for K+ and Na+ may be under independent genetic controls, and could be transmitted, or expressed, independently from each other (118). Chemical excitability has been investigated much less extensively, thus far. In normal cultures, only ganglionic neurons have been examined in this respect. In monolayer cultures from neonatal rat dorsal root ganglia, neurons could be depolarized and caused to generate action potentials by iontophoretic or perfusate administration of GABA, but displayed no responses to acetylcholine, norepinephrine, or glutamate (129). Neonatal rat sympathetic neurons (129) were similarly stimulated by GABA as well as acetylcholine, but not by norepinephrine, dopamine, or glutamate. Acetylcholine sensitivity was reversibly blocked by d-tubocurarin, but not affected by a-bungarotoxin or atropine, and appeared to be widely distributed over the neuronal surface. Chick embryo sympathetic neurons were also shown to be sensitive to acetylcholine (41). However, a separate study of these cultures (65) demonstrated binding sites for a-bungarotoxin on all neurons and over their entire surfaces. Binding sites for a-neurotoxin (another accepted marker for acetylcholine receptors) were also demonstrated on neuroblastoma cells, as well as on neuronal elements of new clonal lines derived from central neural tissue (164). Neuroblastoma cells were examined for electrical responsesupon iontophoretic administration of neurotransmitters (71, 125, 163). The mouse tumor itself, while exhibiting electrical excitability, failed to show acetylcholine sensitivity. So did cultured clonal cells that were growing no neurites. Acetylchohne sensitivity, however, could be demonstrated in neurite-growing cells and mapped in different regions of the cell surfaces. Sensitivity was either uniformly distributed, or restricted to the soma and the growth cone regions. Unexpectedly, acetylcholine could induce both excitatory and inhibitory responseson the same cell (125). Depolarizing (D) and hyperpolarizing (H) effects had different topographical distributions (D on soma, H on distal process) and different susceptibilities to inhibitors (D to curare, H to atropine). They were also subject to different, and independent, regulations, since shifting the culture to a different medium doubled the frequency of only the H effects. Thus, different acetylcholine receptors and/or ionic channels associatedwith them can be produced, and differentially distributed, within the same cell. Dopamine, but not norepi-

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nephrine or serotonin, was also found to elicit responses (only H type) in about one-third of the cells tested. Sympathetic ganglionic neurons normally receive two types of cholinergic synapses (both, however, excitatory), as well as a dopaminergic synapse (inhibitory) (cf. 75). The neuroblastoma clonal cell exhibited the right choices of transmitter and responded correctly to them in all but one case. The incorrect response appeared particularly sensitive to the medium composition. In addition, responsiveness was correlated with the presence of neurites, either because neurites were a prerequisite for it or because the conditions that promoted neurite growth were also conducive to an expression of transmitter sensitivity. Further investigation of the system, carefuIIy paralleled with studies of normal sympathetic cultures, could provide considerable insights into the ontogenesis of chemical excitability and its regulation. Synaptic Activity. Neuroblastoma cells have failed to exhibit, functionally or morphologically, synaptic connections with one another or with skeletal muscle clonal cells. On the other hand, electrotonic junctions have been repeatedly observed. Connnections with muscle cells (72) were very firm and often caused up to lOO@fold increases in acetylcholine sensitivity in the contacted muscle cell region-an attractive model to study trophic influences of “innervation” in the absence of functional connections. Failure to form synapses could reflect a real absence of the intrinsic genetic programs required for it, or culture conditions not suitable for their expression, or the presentation of the wrong (in fact, “nonphysiological”) partners. In recent studies (153 j, hypertonic media applied to neuroblastoma cultures induced the appearance of intercelluiar junctions accompanied by synaptic-like vesicles, as well as complete arrest of replication and extensive morphological “differentiation.” With normal neurons from a variety of sources, synapses have been repeatedly demonstrated by electron microscopy, electrophysiology, or both. a. Explants: Fetal rat cord, and rat or mouse cerebrum, explanted at a stage when no synapses had yet developed in GVO, displayed the progressive appearance of complex polysynaptic activities, paralleled with the appearance of morphologically mature synaptic structures (34, 45, 47). Throughout such development, and particularly in older explants, strychnine enhanced the synaptic activity, demonstrating a concomitant development of inhibitory as well as excitatory connections. Equally complex activities could be seen in cultures carried from the beginning in the presence of magnesium or xylocaine, once the drugs were withdrawn. Thus, the development of polysynaptic networks took place not only outside the organismic influences but even in the absence of local electrical activities. Cord-myotome preparations and cord-dorsal root ganglia preparations, dissected and explanted without severing the cord from its partner tissue,

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also demonstrated full competence for synaptic development, exhibiting not only the correct heterotypic synapses but the same time course of functional development as they would have in vivo (45) The occurrence of complex synaptic activity has been similarly demonstrated in explant cultures from spinal cord, brainstem, cerebrum, cerebellum, and hypothalamus of several animal species, in some cases including man. Individual explants, cultured side by side, grow fibers and invade the neighboring tissue. By stimulating within one explant and recording from the other, newly formed interexplant synapses can be examined, and the selectivity of these associations tested. Cord/cord, cord/brain stem, brain stem/cerebrum, cord/cerebrum, cord/brain stem/cerebrum, all give evidence of new synaptic connections in both directions (45). Formation of neuromuscular junctions has been obtained with cord/skeletal muscle (47). Electron microscopic evidence has also been provided for superior cervical ganglion/iris (172) and cord/superior cervical ganglion (134) preparations, but no synapses were provided by superior cervical ganglia to cord or by cerebrum to superior cervical ganglia (134). Crain and Peterson (48) allowed dorsal root ganglion/cord/muscle cocultures to establish themselves for 1 to 4 months in vitro. The ‘(matured” cord was then ablated and transferred to a new culture vessel. The transferred cord displayed shortly a profuse outgrowth of neurites. With time it developed myelination, of the peripheral type where the outgrown fibers had associated with glial (meningeal) cells and of the central type within the transplant. Extensive and complex synaptic discharges developed in 3-4 weeks, much as they had in the freshly explanted cord. The transplant interconnected well when cocultured with fresh fetal cord, but much less so when co-cultured with another “matured” transplant. An intriguing interpretation of these results would appear to be that in vitro aging altered the receptivity to new synapses, while leaving unaffected the ability to donate them. Experiments of this kind hold a vast potential for both the problem of aging and the problem of regeneration of central neural tissue. b. Aggregates : From the studies with explant cultures, one is concerned with the possibility that the ability to donate or receive synapses may depend not only on an appropriate partnership, but also on a correct, “organotypic” organization of the cells surrounding each of the two putative partners. In the aggregate culture systems, the original organization is lost through the process of dissociation, but a substitute one is acquired in the course of the culture. Aggregates cultured without attachment to a surface have not been examined by electrophysiology. However, electron microscopical evidence of newly formed synapses has been provided for retina (174) and whole brain (167). Aggregates generated in attached cell cultures from cord, brain stem or cerebrum were additionally shown by elec-

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trophysiological techniques to display both spontaneous and evoked, highly complex synaptic activities across, as well as within, individual aggregates (18, 46). c. Monolayers : In dissociated cell cultures, cellular organization occurs to varying degrees, but generally along patterns that are quite different from those of aggregates, or tissue. Nevertheless, new synapses are formed of both excitatory and inhibitory types. In some cases, synaptic parternship did not conform to the usual in Z&VO situation. In dorsal root ganglia, no synapses have been reported to occur in Z&JO. Nevertheless, synapses have been observed, at the electron microscopical level (117) and at the electrophysiological level (56), in dissociated cultures of such ganglia from the chick embryo, though not from the mouse (141). Perinatal rat superior cervical ganglia contain few, if any, noradrenergic synapses between principal neurons, although such synapses have been suggested in other autonomic ganglia. Neurons from these ganglia, maintained in monolayer cultures in the near absence of nonneuronal cells (with Nerve Growth Factor support), exhibited synapses characterized electron microscopically by dense core vesicles, but no spontaneous synaptic activity was noted (35). In another study (129) high concentrations of catecholamines did not elicit membrane potential changes in such neurons. When the same neurons were cocultured with a fetal rat thoracic cord explant (35), additional synapses, with vesicles typical of acetylcholine, appeared on them. The ganglionic neurons displayed synaptic activity, spontaneously and upon cord stimulation. Both the new type of synapses and the synaptic activity disappeared upon extirpation of the cord explant. Thus, neurons unquestionably isolated and deprived of their glia retained the capacity to receive functional innervation from their normal presynaptic source. although they may accept unusual synapses as well. Even more startlingly, it was recently reported (132) that the same neurons can receive large number of functional, cholinergic synapses from other ganglionic neurons in the culture, when glial cells are abundant and growing in it-a condition that also increases production and accumulation of acetylcholine (139). The implications for the donor neurons and the glial involvement are discussed elsewhere (see 185). Cocultures of dissociated cord and dorsal root ganglia were examined from both chick embryo (56) and fetal mouse (141). Spontaneous postsynaptic potentials, excitatory and inhibitory, were seen with high incidence in the cord neurons. Both could be evoked by extracellular or intracellular stimulation of other cord neurons or, much less frequently, by stimulation of ganglionic ones. Co-cultures of dissociated chick embryo cord and muscle cells also exhibited mature neuromuscular junctions by (58, 59, 140). both electron microscopy (169) and electrophysiology Finally, neurons in monolayer cultures from fetal mouse cerebellum were

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shown in an elegant study (126) to display both excitatory and inhibitory postsynaptic potentials, as well as complicated interneuronal functional relationships. In conclusion, the ability to connect synaptically is another property retained by normal neurons through dissociation and maintenance in an in vitro environment. Its expression does not require histiotypic organization, or even the presence of glial cells, not apparently the exercise of electrical activities. One feels justified in speculating, however, whether heterotypic cell interactions may not be critical in defining the specificity of synaptic partnerships, the nature of the transmitter used andif one were to extrapolate from the neuroblastoma studies-possibly even the type of postsynaptic response. Biochemical Properties. a. Neurotransmitter enzymes : Many, but not all, neuroblastoma clones were found to contain tyrosine hydroxylase (the starting enzyme for noradrenalin synthesis), with or without the presence of the other related enzymes (DOPA-decarboxylase, dopamine-p-hydroxylase, catechol-orthomethyltransferase) , as would befit their sympathetic origin. It remains unclear whether genetic or extrinsic factors are responsible for the differences noted among clones or within the same clone with regard to presence and/or levels of the several enzymes, and for the occasional occurrence of conflicting reports. Moreover, cultures with a recognized production of catecholamines fail to exhibit the corresponding fluorescence ( Falck-Hillarp technique), even though the Cl300 tumor does. The reasons for this behavior are also still unclear. The most readily observed neurotransmitter enzyme is acetylcholinesterase, in agreement with the sympathetic nature of the cells and the general parallel distributions of this enzyme and acetylcholine receptors. Acetylcholinesterase activity is described to increase considerably in stationary, neurite-growing cultures, but the relative importance of attachment versus growth is still debated (cf. 163) and there are reports denying relevance to both (7). In C6 glial cells, this enzyme is also present (though at a relatively low level), but the activity drops from growth to stationary phases. Unpredictably, neuroblastoma cells also exhibit biochemical properties of cholinergic neurons. Choline acetyltransferase, the synthetic enzyme for acetylcholine, has been found in several clonal lines and may also increase as growth rates drop ( 152). Choline uptake is the mechanism by which cholinergic neurons are presumed to remove from the synaptic cleft the hydrolytic product of acetylcholinesterase, and this too was observed to occur with neuroblastoma cells (111). Clonal rat nerve cell lines also can synthesize more than one neurotransmitter, and this synthesis appears to be regulated by the growth conditions of the cultures (97). Future investigations will have to determine what relationship there may be between

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these multiple sets of transmitter properties in clonal Iieolilnstic: nerve cells and the already mentioned report of acetylcholine production in normal sympathetic culture (139). Also to be noted is the finding of active uptake of GABA by neuroblastoma cells, as well as by C6 cells (90) and other clonal neuronal and glial lines (161) . In aggregate cultures of fetal mouse brain cells (165) neurite growth and histiotypic reorganization are followed by extensive synapse formation, as well as some moderate degree of myelination, over a period of weeks i~z vitro. The temporal development of transmitter enzymes pracSpecific activities of acetyltically coincides with that observed in tivo. cholinesterase and choline acetyltransferase increased 20 times while that of glutamic acid decarboxylase (the GABA-producing enzyme) increased 5-fold. Of the catecholamine-degrading enzymes, monoamine oxydase (also present in clonal cells and much higher in glial than in neuronal ones) increases, while catechol-orthomethyltransferase does not change, relative to other proteins. One important advantage of this system, as already noted, is that cell proliferation-hence, shifts in cell composition -is at a minimum and specific activity measurements do define a selective expression of differentiated traits. In contrast, monolayer cultures usually allow or promote proliferation of non-neuronal cells. Thus. besides the lack of histiotypic organization and of more stringent cell-cell contact inhibitions, growing cells progressively dilute the contributions of the stationary (or slower-growing) ones, and may affect performances of the latter elements by altering medium composition or even applying direct influences (186). This makes it still difficult to interpret biochemical data from heterocellular monolayer cultures. Seeds (165) noted that in this system the same fetal mouse brain cells exhibited an actual decline in specific activities of acetylcholinesterase, choline acetyltransferase and glutamic dehydrogenase-although enzyme-producing cells may well have developed in the same direction as they did in aggregate cultures. Shapiro and Schrier (168) showed that cell proliferation and enzyme activities were influenced by the choice of media and by the size of the initial cell seeding. In particular, specific activity of choline acetyltransferase in fetal rat brain cultures increased under conditions favoring growth, suggesting that fast growing cells either produced the enzyme or elicited better production of it by the more stationary cells (cf. 139). Differential effects may also be achieved by defined agents, such as monobutyryl-cyclic AMP which caused a decline in growth and choline acetyltransferase, but an increase in acetylcholinesterase and glutamic decarboxylase (159). Biochemical studies will gain immensely from an availability of purified populations of normal neurons and glia, and the possibility to study controlled recombination of the two cell classes. At present,

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only purified neurons from sympathetic ganglia have provided adequate cultures, yielding information on developmental, nutritional and kinetic aspects of catecholamine synthesis (109). Explant or organ cultures have not been the subject of systematic biochemical investigations (cf. 77). Most work involved the behavior of spinal sensory and sympathetic ganglia under the influence of Nerve Growth Factor, as reviewed elsewhere (181). Synthesis and metabolism of serotonin have been examined in raphe nuclei cultures from neonatal rat, showing an in vitro development parallel to that observed in wivo (68). Cortisol induction of the glial enzyme glycerol phosphate dehydrogenase (see further on, subsection c) has been also examined in rat brain explant cultures (25). b. Neural proteins: Neuroblastoma cells have been shown to contain actin (42), tubulin (133), 14-3-2 (78), but not SlOO (however, see 164, 18.5). Neurite-growing cells and neurite-free ones show qualitative differences in radio-protein electrophoretic patterns (163, 7), although such differences have yet to be investigated. Akeson and Herschman (2, 3) prepared antisera against neurite-positive and negative neuroblastoma cells, and examined the immune activities persisting after absorption on different tissues as well as on the opposite neuroblastoma population. Neurite-positive cells possess antigens, which are distinct from those shared with neurite-negative cells and are (at least in part) also present in brain, but not liver, spleen or kidney tissue. Appearance of the neurite-positive antigens correlated with neurite extension and also, to some extent, with attainment of confluency in monolayers. Morphological development appeared to precede the serological one. In C6 glial clonal cultures, SlOO accumulation increases rapidly after confluency is reached (SO). Expression of SlOO is not regulated by the rate of cell replication, but the establishment of extensive cell-cell contacts, which appear to be cell-specific. The increased accumulation reflects a stimulation of synthesis rather than reduced degradation. Pfeiffer (144) examined several cultures derived from new, chemically-induced tumors. SlOO was present in the cultures from virtually all peripheral neural tumors and most central neural tumors, but not from non-neural ones. CNPase occurred in both neural and non-neural materials but was 25 times higher in the former. One new clonal line, RN-2, was morphologically classified as a neurilemma cell tumoral population, contained the Pl encephalitogenic protein as well as SlOO and CNPase, and could also be shown to synthesize collagen (collagen presence in neurinomas has raised questions of the glial versus mesenchymal origin of these sheath cell tumors). Tumoral glial cells are also being investigated with regard to surface antigens. Pfeiffer et al. (145) reported that antiserum to C6 stationary cultures was more extensively absorbed by stationary monolayers than by

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growing suspensions of the same cells, but it remains to he determinctl whether confluency causes an increase of the shared antigens or an actual appearance of new ones (79j. Schachner (156~ grew a chemically induced mouse brain tumor (classified as an oligodendroglioma) through several subcutaneous or intraperitoneal passages, dissociated it by mechanical dispersion and used the irradiated cells to obtain mouse immune sera to them. Conlplement-dependent cytotoxicity tests with this antiserum reveal the occurence of a glia-specific antigen (NS-l), detected only in brain tissue of several species including man, more abundant in white than in grey matter, and increasing postnatally over the first 3-4 weeks. NS-1 was low in myelin-deficient mouse mutants, absent in Cl300 neuroblastoma, but present in 3 out of 4 gliomas examined. Studies of selected proteins in normal neural cultures have yet to be undertaken, with a few esceptions concerning actin (57) or tubulin (81). Seminal contributions, however, should be expected in the near future from the application to normal neural cultures of the immune materials being developed against tumoral neurons and glia. c. Responses to hormones: Another area which has yet to be systematically explored is the action of hormones on normal neural cells (8). Examples of past sporadic studies in vitro are the effects of thyroid hormones on myelinogenesis (69), ACTH on serotonin metabolism (68), insulin on ganglionic anabolism (36, 138). Little information has also been gathered, thus far, on neuroblastoma responses to hormones, except for cyclic nM1’ (e.g., 115, pp. 457460). H owever, two hormonal effects on glial cells were uncovered by use of clonal cultures and are currently under extensive investigations. Co&sol Inducfiolz of Glycerol Phosphate Dehydrogetzase (GPDH). GPDH is an NAD+-linked, cytoplasmic enzyme associated with energy metabolism and phospholipid synthesis. Hypophysectomy or adrenalectomy in rat reduces GPDH levels in the adult brain or stops its developmental rise. Both effects are reversed by cortisol injection. Slices and explant cultures showed that only neural tissue responded to cortisol with a rise in GPDH activity. DeVellis and coworkers (52, 54) discovered that monolayers of C6 cells also respond to cortisol with a massive rise (lo20 times) in GPDH activity. The effect was blocked by inhibitors of both protein and RNA synthesis, demonstrating the involvement of transcriptional mechanismsand new enzyme synthesis. The effect was specific for cortisol, and withdrawal of the agent interrupted the rise, or caused a slow reversion to basal levels. Subsequent studies (34) have confirmed the phenomenon with monolayer cultures of normal cerebrum, cerebellum and brainstem, regardless of their proliferative activity (suggesting the noninvolvement of fast-growing cells), and noted no differences in the size of the responsesfrom corresponding aggregate, or explant, cultures (25).

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Histochemical csamination of normal neural tissue has also localized GPDH in the glia, but not the neuronal cells. Thus, cortisol-inducible GPDH appears to be a new, and reliable, glial marker. Treatment of the C6 cells with BUdR, or hybridization with fibroblast cell lines, totally abolished GPDH inducibility. Other effects of cortisol on C6 cells were also reported (55). Brain GPDH has recently been purified and characterized (113) and the identity between basal and cortisol-induced enzymes demonstrated by immunochemical methods ( 114). Catecholamine Induction of Cyclic AMP and Lactic Dehydrogenase. (LDH) . DeVellis and his collaborators (52, 55) also found that catecholamine treatment of C6 cells causes, among several other effects, a rise in LDH accompanied by a shift in subunit composition. The induction of LDH was highly specific relative to general protein and other enzymes, as well as to the nature of the inducer. LDH increase started only after a few hours, progressed to a plateau over the next 20 hours and persisted for several hours even after withdrawal of the hormone. It involved both transcriptional events and the synthesis of new enzyme. However, induction of LDH is but a consequence of an induction by catecholamine of cyclic AMP production. Catecholamines act extracellularly on p-adrenergic receptors on the C6 cell surface, causing within minutes a lo-fold increase in intracellular cyclic AMP. This in turn triggers transcriptional events over the next two hours, which lead eventually to the rise in LDH. Withdrawal of the hormone at any time reverses the cyclic AMP response within minutes, but prevents the LDH response only if applied over the first two hours, namely when the LDH-related transcriptional events need to take place (as seen by the same time restriction on the effects of actinomycin D). Catecholamine induction in C6 cells of both cyclic AMP and LDH are abolished partially by treatment wth BUdR or totally by hydridization with fibroblast lines, as already seen in the case of cortisol inducibility. A loss of p-adrenergic receptors upon hybridization has also been reported (118). Catecholamine induction of cyclic AMP has subsequently been confirmed in a human astrocytoma clonal line (44) and in brain slices. Mouse brain develops such inducibility after 4 days from birth, and aggregate cultures from fetal brain cells were similarly found to exhibit it after several days in vitro (165). Monolayer brain cell cultures responded much more extensively than aggregate ones, presumably as a reflection of the growing number of their glial elements (165). The high response of glial cells in monolayer cultures (62, 165) could also reflect a “supersensitivity” resulting from the lack of prior exposure to catecholamines. Recent observations with C6 cultures (53) have indicated that CAMP accumulation by C6 cells in response to catecholamines is under the control of a rapidly turning over protein, which is itself inducible by cyclic AMP and

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c;tu~c’s the cells to become refractory to a second catecholnn~ine adiiiinisti-ation. These findings raise intriguing possibilities wth regard to neuronto-glia regulatory mechanisms. The converse relationship, a glial regulation of neuronal activities, is also proposed by another observation, namely that the catecholamine-induced production of cyclic AMP in C6 cells appears to be accompanied by active secretion of the cyclic neucleotide into the medium (M. Saier, personal communication). 0 they Glial Prop&es. Time-lapse cinematographic studies of neural explant cultures have revealed pulsatile activities of glial cells. Oligodendrocytes undergo rthythmic (5 min) alternations of contraction and relaxation (107), and similar behaviors were observed with neurilemma cells (148). Tonic or spastic contractions of oligodendroglia could be and selectively blocked by serotonin reversibly induced by serotonin, antagonists (11). Astrocytes were also noted to undergo spontaneous, slow contractions (82). Electrical stimulation of astrocytes in cerebellar or midbrain explants led to graded membrane depolarization, whose reversal was a lOOO-fold slower than is observed with neurons (83, 84) and similar electrical responses were noted in viva (176). Finally, electrical stimulation of cultured astrocytes led to a very weak contraction, also graded with stimulus strength and followed by a very slow reversal (43). Myelin formation has been examined mainly in explant cultures (cf. 32, 122). Mvelination in monolayer cultures has been reported only with i regard to spinal sensory ganglia, and only when neurilemma cells were conspicuously abundant ( 106) and the neuronal somata were heavily invested by satellite cells (17Oj. Behavior and relevance of satellite, neurilemma and other sheath cells for the sequential progress of myelination were described in detail in explant cultures of spinal sensory ganglia from chick embryo (142) and fetal rat (112). Myelination in explant cultures has subsequently been repeatedly reported for spinal cord (33, 96, 143), cerebellum (192), and many other central neural tissues. The involvement of glial cells in demyelination processes is well known, and ipa vitro approaches will play an increasing part in its understanding. Weiss and Wang (190) first showed that neurilemma cells, upon migration out of an explanted nerve fragment, could assume morphology and activities typical of a macrophage. Kaprowsky and Fernandez (95) obtained lymph nodes from rats immunized with guinea pig spinal cord, added them to a monolayer culture of puppy brain cells, and observed that the lymphocytes aggregated around glial cells to cause their destruction. Bornstein and Crain (17) demonstrated that antiserum to the basic encephalitogenic protein prevented myelination in cord explants or caused demyelination. Either effect was reversible upon withdrawal of the agent, suggesting no permanent damage to the axons, even though electrical excitability and synaptic activities were abolished. The same effects were

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elicited with serum from animals with experimental allergic CXlCephil~~Jmyelitis or, most importantly, with scra from human patients with multiple sclerosis. Several subsequent studies, largely in association with M. Bornstein, have vigorously pursued the in vitro investigation of demyelinating conditions (cf. 33). CODA It must be apparent, from the preceding review, that cultures of neural cells have only started to supply the neuroscientist with the information and the tools which they are potentially capable of providing. Much remains to be developed, particularly with regard to biochemical performances by neurons and glia, quantitative in vitro systems for their analysis, purified populations of each cell category, and the definition of their functional interactions. A more detailed discussion of some of these problems is presented in the Proceedings of a Workshop on Culture Techniques and Glial-Neuronal Interrelationships 1.n Vitro (185). REFERENCES 1. ADELSTEIN, R. S., T. D. POLLARD, and W. M. KUEHL. 1971. Isolation and characterization of myosin and two myosin fragments from human blood platelets. Proc.

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2. AKESON, R., and H. R. HERSCHMAN. 1974. Modulation of cell-surface antigens of a murine neuroblastoma. Proc. Nat. Acad. Sci. USA 71: 187-191. 3. AKESON, R., and H. R. HERSCHMAN. 1974. Neural antigens of morphologically differentiated neuroblastoma cells. Nature 249 : 620-623. 4. ANGEVINE, J. R., JR. 1970. Critical cellular events in the shaping of neural centers. 11) “The Neurosciences : : Second Study Program,” pp. 62-72. F. 0. Schmitt [Ed.-in-Chief] Rockefeller University Press, New York. 5. APPEL, S. 1975. Neuronal recognition and synaptogenesis. Exp. Nczhrol., Supplement 6 : xxx--xxx. 6. AUGUSTI-TO~CO, G., and G. SATO. 1969. Establishment of functional clonal lines of neurons from mouse neuroblastoma. Proc. Nat. Acad. Sci. USA 64: 311315. 7. AUGUSTI-TOCCO, G., E. PARISI, and F. Zucco. 1973. Biochemical characterization of a clonal line in neuroblastoma, Pp. 87-106. In “Tissue Culture of the Nervous System,” G. Sato [Ed.]. Plenum Press, New York. 8. BALASZ, R. 1974. Influence of metabolic factors on brain development. B&t. Med. Bull. 30 : 126-134. 9. BARKLEY, D. S., L. L. RAKIC, J. K. CHAFFEE, and D. L. WONG. 1973. Cell separation by velocity sedimentation of postnatal mouse cerebellum. J. Cell Phyvsiol. 81: 271-280. 10. BENDA, P., J. LIGHTBODY, G. SATO, L. LEVINE, and W. SWEET. 1968. Differentiated rat glial strain in tissue culture. Scie+zce 161 : 370-371. 11. BENITEZ, H. H., M. R. MURRAY, and D. W. WOOLLEY. 1955. Effects of serotonin and certain of its antagonists upon oligodendroglia cells in vitro. Excerpta

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12. BERL, S., and S. PUSZKIN. 1970. Mg2t - Caa+-activated adenosine triphosphatase system isolated from mammalian brain. Biochemistry 9: 2058-2067.

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