Neulvchemical Research, VoL 17, No. 12, 1992, pp. 1163-1180

Overview

Neuronal Cell Cultures: A Tool for Investigations in Developmental Neurobiology Alessandro Cestelli 1,3, Giovanni Savettieri 2, Giuseppe Salemi 2, and Italia Di Liegro I (Accepted May 14, 1992)

The aim of this review is to describe environmental requirements for survival of neuronal cells in culture, and secondly to survey the complex interplay between hormones, neurotrophic factors, transport- and extracellular matrix- proteins, which characterize the developmental program of differentiating neurons. An overall reconsideration of the literature in this vast field is above the limits of the present paper; since progress and refinement in the techniques of neuronal cell cultures have paralleled the advancement in Developmental Neurobiology, we will run instead through the main steps which form the conceptual framework of neuronal cell cultures. KEY WORDS: Neuronal cell cultures; hormone supplemented-serum free-media.

HISTORICAL BACKGROUND

neurons and nerve fibers, undertaken by Harrison himself (120) and by Lewis and Lewis (175) among others. These authors showed that process outgrowth could occur at the interface between a solid substratum and liquid medium, only if neuronal fibers were in contact with a surface such as fibrin. In 1933 Ramon y Cajal published a very detailed study in which the most significant data (many of which were obtained by the pioneers of neuronal cell culture) were critically collected in favour of the Neuronal Doctrine (246). Some of the results reported were considered with diffidence by the contemporaries of the Spanish neuroscientist because culture conditions were then quite crude: cells did not survive in culture for more than a few days. Only the income of new technologies in the mid 50s, such as electron microscopy and intracellular microelectrode recording, paved the way to the complete acceptance of the Neurone Theory (reviewed in 146). In the mean time neuronal cell cultures enjoyed the refinement of the conditions in which animal cells were kept in culture. Since the late 30s the poorly enriched

In the beginning of our century Harrison was among the first scientists to succeed in maintaining cells and tissues outside the living body. Harrison's experiments, carried out with frog neural tube fragments covered by a drop of clotted lymph, showed without any doubt that the naked axonal processes sprouting from the original explant were not syncytial in origin, but the products of neuronal units (118,119). This observation also confirmed the validity of the Neuronal Theory by Ramon y Cajal and His, and stands even nowadays as a keystone of Developmental Neurobiology. Harrison was aware that this methodology could be applied to "the study of the influences which act upon the growing nerve" (118). The next step was the study of locomotion of young Ddpartimento di Biologia Cellulare e dello Sviluppo "Alberto Monroy" 2 Clinica Neurologica, University of Palermo, Palermo, Italy. 3 To whom correspondence should be addressed at: Dipartimento di Biologia Cellulare e dello Sviluppo "Alberto Monroy", via Archirafi 22, 90123 Palermo, Italy.

1163 0364-3190/92/1200-1163506.50/09 1992 PlenumPublishingCorporation

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saline solutions of the pioneers were gradually enriched at first by cerebrospinal fluid and then by placental and fetal serum (reviewed in 212). This brought about a better understanding of the concept that a living system requires a complex mixture of nutrients for development, and also a lengthening of the life time of neuronal cultures. The primitive conditions of cell culturing in simple thermostats evolved gradually toward the use of more faithful and sophisticated incubators where some of the most important environmental conditions, such as temperature, CO2 and humidity were carefully checked and maintained as close as possible to the needs of specific cell types (reviewed in 159). By the late 40s the addition of antibiotics to the culture media drastically reduced the threat of bacterial contamination (reviewed in 199). A better understanding of neuronal physiology in vitro was obtained by Cavanaugh (48) who firstly cultivated dissociated neurons from chick embryo spinal cord. The discovery which ideally closes the formative period of neuronal cell culture methodology came in the early 50s. Levi-Montalcini et al. had found that sensory and sympathetic ganglia explanted from 8-day chick embryos in a semi-solid medium in proximity but not in contact with fragments of a mouse sarcoma, produced within 24 hours a halo of nerve fibers with maximal density on the side facing the tumor (174). This observation later led to the identification of NGF and opened a new era in the comprehension of the complex mechanisms of action of neurotrophic factors (see 296 for a comprehensive discussion).

CHOICE OF THE TISSUE The choice of the tissue for cell culture mainly depends on the particular aspect of neuronal biology to be studied. In general the most favourable time for surgical removal of a given area of the nervous system strictly depends on the maturation schedule of that district. It is generally acknowledged that the best cultures derive from tissues taken from fetal or newborn animals. If the tissue is too immature, neurons fail to undergo a complete differentiation in culture (292). If, on the contrary, complete maturation has already been reached, the disruptive procedure of tissue disaggregation brings neurons to death and the ensuing culture contains mainly glial cells (34). A few scientists however claim to be able to maintain in culture nerve cells from adult tissue (154).

Cestelli, Savettieri, Salemi, and Di Liegro

The most suitable moment for the commencement of a culture varies from species to species, and from district to district of the nervous system: for example, even if duration of the embryogenesis is the same for chick and rodents (i.e. 21 days), the best age for the commencement of a neuronal cell culture from cerebellum is the first postnatal week in the rat (206), but the llth embryonal day in the chick (213); cerebral hemispheres from a 8-day chick embryo (238) are at the same developmental stage as those from a mouse embryo of 15 days (126); and the developmental stage of the retina from a 12-day chick embryo corresponds to that of the mouse 9-days post-partum (213).

DEVELOPMENT OF SERUM-FREE MEDIA

The traditional technique of nourishing cultured cells with a medium enriched with serum hampers the establishment of long-lasting neuronal cultures because of the growth-stimulating activities present in the serum itself (114,286): in a heterogeneous system, such as a CNS primary cell culture adhering to a surface, serum elicits the growth of the most aggressive cell types (i.e. glial cells) that, in the lapse of a few generations, outnumber neurons (307). This picture gets more complicated by considering that in serum are present not only growth stimulating agents, but also factors deleterious for cell growth and functioning (14,90). Furthermore the complexity of serum components, many of which await still to be discovered, causes serious problems at the level of experimental reproducibility. It has been demonstrated, in fact, that commercial sera contain varying amounts of hormones, vitamins and other constituents (83,134,225): for example, in serum-fed cerebellar monolayer cultures from mouse, the behaviour of cells varies significantly, not only from lot to lot of serum used, but even from preparation to preparation when the same lot is used (205). Finally, it is noteworthy that cells in the organism do never come into direct contact with serum, except in case of injury and that cells cultured in the presence of pure serum usually die within a few days. Many of the components which are normally present in serum do never reach most cells of the body (105). On the basis of these considerations many laboratories have been involved in formulating defined combinations of hormones, growth factors, nutrients and transport proteins able to substitute for traditional serum-supplemented media (see: 20-22,159 and 254 for comprehensive reviews, and 196 for a methodological approach). A wide spectrum of cell types are now cultured in the complete absence of serum (23). As far as only neu-

Neuronal Cell Cultures

ronal cells are concerned, the number of different hormones needed and the variability of cell responses are amazing. The intricate picture now emerging sheds light on the great complexity of the differentiative program of neurons.

COMPONENTS OF SYNTHETIC MEDIA FOR NEURONAL CELLS

As already mentioned in the previous section, neurons, unlike glial cells, do not divide in vitro. This property has been exploited by the early experimenters (36) who formulated serum-free synthetic media able to sustain survival and differentiation of these cells in vitro. Insulin and Insulin-Like Growth Factors. Among the various components of synthetic media, insulin is the most fundamental and in most formulations is present at extraordinarily high concentrations (5 jxg/ml). In general, this hormone exerts a pleiotropic effect on cell metabolism and stimulates cell growth especially during fetal development (86,151). In mammalian blood serum, insulin levels fluctuate in the range of ng/ml. In some brain regions these levels can be 10-100 fold higher (123); accordingly, insulin receptors are highly concentrated in the CNS (122,297) and are present both on neuronal and glial cells in culture (244). Further, the brain appears to be capable to synthesize insulin (308). Unexpectedly, however, insulin fails to increase glucose uptake in neuronal cultures (37,103). The actual role of this hormone in the brain awaits, therefore, to be elucidated, even if in the last few years growing evidence has been accumulating in favour of the hypothesis that insulin might function as a neuromodulator and a neurotransmitter (37,302,304). Incubation of chick embryo retinal explants with insulin has been recently demonstrated to result in a pronounced inhibition of cell proliferation, thus suggesting a role of this hormone on retinal development (287). The requirement of high concentrations of insulin in most synthetic media is rationalized by the following facts: i) there is evidence that stimulatory contaminants exist in insulin preparations (115); ii) at supraphysiological concentration, insulin competes for the binding to receptors for insulin-like growth factor I (IGF I) and could mimic its effects (95; for review, see 255,309); iii) insulin is quickly inactivated at 37~ in cysteinecontaining F12 media: we have reported, in fact, that it is possible to lower significantly (100 fold) the titer of insulin both by lowering the F12 component in the culture medium and by doubling cell plating density (52). As far as IGFs I and II are concerned, they both

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are synthesized in mammalian brain during fetal development (266,267). IGFs I and II stimulate RNA and DNA synthesis in primary cultures of neuronal precursor cells from fetal rat brain (221), thus suggesting that they act as autocrine and paracrine factors during brain development. In contrast to the canonic neurotrophic factors (296,299), IGFs I and II appear to exert more generalized functions, as they stimulate both proliferation and differentiation of astroglial, oligodendroglial and neuronal precursor cells in culture (11,42,201,278). Transport Proteins. In interstitial fluids serum albumin contributes to establish colloid osmotic pressure and to stabilize pH. In culture, this compound acts also as a carrier for fatty acids (250) and keeps the concentrations of T3 (259) and steroid hormones (71) within the physiological range. It also binds trace elements and other compounds having one or more electrically charged groups, such as aminoacids and vitamins, and serves as detoxifying factor by binding residual detergents, endotoxins and toxic metals (113). Transferrin is the major iron-carrying protein in the circulation. During embryogenesis most of the transferfin needed for cell growth is synthesized initially by the egg yolk and later by the liver (2), with minor sites of synthesis spread in many tissues (202): multiple sites for transferrin synthesis are supposed to be required to meet the needs for this factor by rapidly growing tissues. In the adult, however, transferrin mRNA levels in nonhepatic tissues remain significantly elevated only in testis and brain (142), that is in districts outside an efficient blood-tissue barrier. In rat brain, transferrin is predominantly synthesized by oligodendrocytes (56,57) and by the choroid plexus (68), but it is also supplemented by uptake from the blood, since it has been localized immunocytochemically in brain capillary endothelial cells and transferrin receptors have been demonstrated on the luminal surface of these cells (147). In primary cultures transferrin is synthesized and secreted both by oligodendrocytes and astrocytes but not by neurons (84). The real functions of transferrin in the nervous system await still to be understood (however, for a thorough discussion, see 204). Indirect evidence from immunohistochemical staining of transferrin-receptors suggests that iron accumulates at synapses (127,208). According to this hypothesis neurons from both chick brains (5) and sympathetic ganglia (275) in culture become strictly transferrin-dependent at a time when they are presumably engaged in stabilizing synaptic contacts. Steroid and Thyroid Hormones. Steroid and thyroid hormones act on target tissues mostly by interacting with DNA-binding receptors which belong to the c erb A superfamily of proteins (70,85,224,226). This interac-

1166 tion is a key event in regulating tissue-specific gene expression and is accompanied by changes in the structural organization of chromatin, including 5'-flanking regulatory sequences of the target genes (for review, see: 27,38,66,96,306). In addition to the pattern outlined above, steroids may also affect brain function through a non-genomic mechanism (for review, see: 185,198,270) by binding to neuronal membranes (230,245). It has been demonstrated that steroid hormones rapidly influence the excitability of neuronal cells in vitro by changing the ion conductances (94,216), the release of neurotransmitters (71-73), and the characteristics of the neurotransmitter receptors (136). The finding that biosynthesis of "'neurosteroids" from cholesterol occurs in brain (25) has led to the interesting possibility that steroids formed entirely within the brain influence some neumtransmitter receptors (240). For example, an influence on the GABA-activated C1currents has been reported for rat hippocampal neurons in primary cultures (117,186) and in human embryonic kidney cells transfected with the genes encoding the subunits of the GABAA receptor (239). It is believed that sex differences in behaviour reflect differences in specific neural circuits, which in turn are responsible for sex-specific patterns of morphology and function in specific areas of CNS, such as hypothalamus and limbic system (102,139,193-195,217, 222,235). The most accepted hypothesis states that sexual differentiation of the brain is solely due to the epigenetic action of gonadal steroids during a critical period which varies from species to species (3,9,106,197,303). Several groups of researchers (88 among others) have used primary neuronal cell cultures to study these phenomena. In particular, a new experimental approach has been recently adopted to distinguish the effects of gonadal hormones from that of the genotype itself on neural cells: gender-specific dissociated cultures were run in fact only after recognition of the sex of the single individuals by inspection of the gonads (reviewed in 249). These neurons, which are mainly dopaminergic, noradrenergic and serotoninergic, were cultured in the absence or in the presence of sex steroids. Surprisingly, the main region-specific, morphological and functional sexual differences were observed in dopaminergic cultures, independently of the presence of sex steroids (30,158,248). Moreover, noradrenergic neurons in vitro respond to 13estradiol or testosterone treatment with an enhanced outgrowth of neurites in a sex- and phase-specific manner (reviewed in 249), thus indicating the existence of a gender-specific time window in the sensitivity to steroids.

Cestelli, Savettieri, Salemi, and Di Liegro Corticosteroids have been shown to reduce transmitter-evoked excitability in slices from hippocampus (149), presumably via a receptor-mediated genomic action. Such reciprocal control may be a general principle by which transmitters and steroid hormones can modulate neuronal excitability. Glucocorticoids seem to exacerbate the vulnerability of hippocampus to degenerative insults, like hypoxiaischemia, hypoglycemia, antimetabolites, oxygen radical generators, epilepsy, chronic aIcoholism, and aging (157,261-263,265). Because of the obvious clinical implications, it is of the utmost importance to understand the mechanism by which these hormones compromise neuronal viability; by using a suitable cell culture system it has been found that glucocorticoids, but not non-glucocorticoid steroids, act synergistically with various insults as previously reported in vivo, on primary hippocampal neuronal cultures (264), while no effect is exerted on cerebellar or hypothalamic neurons in vitro (232). One of the best characterized examples of neuronal differentiation modulated by glucocorticoids, is the developmental fate of neural crest cells. The neural crestderived precursors of sympathoadrenal lineage differentiate alternatively as sympathetic neurons or pheochromocytes depending on environmental cues. In cultured neural crest cells, expression of the A2B5 antigen (a ganglioside characteristic of neurons) precedes the appearance of adrenergic traits (i.e. expression of tyrosine hydroxylase). Glucocorticoid treatment downregulates neuronal traits (A2B5 and process-bearing morphology) of tyrosine hydroxylase positive neural crest cells (295) and could be the "environmental cue" normally encountered by the sympathoadrenal precursors. As concerning thyroid hormones (TH), their pleiotropic influences on mammalian brain development and maturation are well known (for review, see 75,269). Triiodothyronine (T3) is however unnecessary for neuronal survival in culture (4,52,97,242): instead it seems to be involved in the final part of the neuronal differentiative program (269). This critical period is characterized by an overall rearrangement of neuronal chromatin, which implies that the average length of nucleosomes scales down from 200 to about 165 base pairs (reviewed in 269). This rearrangement requires in vitro the presence of T3 (50) and is accompanied by a drastic change in the turnover of chromosomal proteins (49,51). TH-nuclear receptors, which were recognized as the products of the cellular protooncogenes erb A, have been classified into two subtypes (a and 13), that differ both in primary sequence and chromosomal localization of the corresponding genes (for review, see 70,85,224,226).

Neuronal Cell Cultures

From both e~ and 13 genes derive different proteins by alternative splicing of identical primary transcripts. The existence of multiple erb A isoforms suggests that they could be, at least in part, tissue-specific (92,129,289); they might have different ligand affinity or different abilities in activating target genes; finally, their activity could be differentially modulated by interaction with other tissue-specific cellular factors (211), including receptors for other hormones and morphogens (for review, see 100,191). The a2 variant lacks the hormone-binding domain and functions in transfection experiments as an inhibitor of erb A a l - or 13-induced activation of target genes (145,156). This form is predominant in the brain (289): in vivo a2-mRNA accumulates maximally at birth (47,285); in glial-free neuronal cultures, on the contrary, it keeps accumulating continuously, thus suggesting its predominant Iocalization in neurons (47). The great majority of acetylcholinesterase positive neurons from fetal brain hemispheres in culture are immunoreactive for T3-nuclear receptor: the addition of T3 to the culture medium increases significantly the neurite length (98). At a more detailed level, the expression of cholinergic enzymes is regulated synergistically by T3 and NGF (99). A similar T3-dependent maturation effect has been observed for hypothalamic neurons (242). Mesencephalic neurons, on the contrary, respond to TH with an increased size of perikarya, without any enhancement of neurite density (243). The modulatory role of T3 in orchestrating neuronal maturation has been confirmed in a recent paper in which T3 is reported to produce an earlier onset in neuronspecific enolase mRNA expression and a mor,~ precocious drop of nonneuronal enolase mRNA level (69). Further, T3 accelerates synthesis of synapsin I in developing neurons from rat brain cortices (258). TH are able to induce also the formation of neurite-like extensions in N2A neuroblastoma cells by increasing the synthesis of the microtubule-associated protein MAP 1B (116). T3 seems to be required during a narrow temporal window for brain maturation (for review, see 146,269): an initial two day input is indeed sufficient to induce in culture the rearrangement of neuronal chromatin two weeks later (50); moreover, an 8-fold increase in choline acetyltransferase can be monitored in cultured telencephalic neurons if T3 is present in the medium either continuously or during the first two weeks of culture (133). Neuronal Growth Factors. In general, neuronal growth factors do not appear to stimulate neuronal cell division, probably because they have been assayed almost exclusively on postmitotic neurons. An adequate supply of these factors regulates survival of neurons during the stage of target field innervation: this observation

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is at the basis of the Neurotrophic Theory (241,288). This theory is a keystone for understanding some essential points of Developmental Neurobiology, including the question of why, during development, approximately 50% of postmitotic neurons degenerate and die by a process of naturally occurring cell death (for a comprehensive discussion, see 228,229). Two lines of evidence led to the proposal that neurons compete for some compound that is supplied by the targets in limiting amount (for review, see 60): i) the temporal coincidence between normal cell loss and the establishment of synaptic contacts; ii) the demonstration that manipuIations of the availability of putative synaptic targets alter the number of surviving neurons. Cell cultures have greatly supported the advancement of knowledge in this field: in an elegant study, Thoenen and coworkers (8,276) demonstrated that pM concentrations of ciliary neurotrophic factor (CNTF) promotes survival of motoneurons in culture. Some facts are emerging: 1) a single factor has not yet been discovered which promotes survival and differentiation of neurons from both the whole CNS and PNS; 2) on the contrary, each factor exerts its activity on a unique combination of neuronal districts; 3) the effects of several factors are often redundant: i.e. survival and differentiation of a given neuronal population depends on the orchestrated activity of more than one trophic factor, which act often in parallel, but not always synergistically, with each other; 4) in some cases several factors act in series at different stages of neuronal development. Neurotrophins. NGF is a physiologically important neurotrophic factor in the development of cholinergic neurons of the basal forebrain, sympathetic postganglionic neurons, and sensory ganglion cells (for review, see 17,61,171). A recent paper by Levi-Montalcini and Aloe (172) describes a protocol for the quantitative bioassay of NGF in vitro. The biological effects of NGF include: i) maintenance of neuronal survival; ii) stimulation of neurite outgrowth; iii) induction of the enzymes that synthesize neurotransmitters. These trophic influences derive from the target organ, which synthesize NGF upon arrival of nerve fibers (62). These fibers exhibit on their surface high affinity receptors which, upon binding the factor, are first internalized by a mechanism substantially identical to receptor-mediated endocytosis, and then transported retrogradally to the cell body (271). The effects of NGF-receptor complexes are not exerted directly on the genome, but require the intermediate of a ras p21 protein. In PC12 cells, in fact, the injection of NGF into the cytoplasm does not produce any effect (274), while an immediate neurite outgrowth

1168 is stimulated by the injection of Ha-ras p21 protein (24), or by transfection with N-ras gene (108). Since NGF supports a limited set of neuronal populations, the existence of additional neurotrophic factors has been only postulated for decades (see 294 for a foreseeing discussion). While there is now growing evidence that these factors do exist, their extremely low abundance has greatly delayed their molecular characterization. Two of these factors, namely brain derived neurotrophic factor (BDNF) and neurotrophin (NT)-3, show a significant enough structural homology with NGF to be regarded as members of a gene family (163,184). BDNF was originally purified from pig brain (19). The corresponding messenger has been found predominantly in the spinal cord and the superior colliculus: these districts contain the target cells of neurons previously shown to be BDNF-dependent (primary sensory neurons: 63,180, and retinal ganglion cells: 150). This factor supports survival of, and stimulates outgrowth from, 60-70% dorsal root ganglia (DRG) neurons from chick embryo at the 6th day and approximately 40% from 12 day chick embryos (180): this means that BDNF exerts its maximal effect at the time when these embryonic neurons contact their targets in the CNS. This observation strengthens the hypothesis that BDNF modulates neurite growth from specific populations of peripheral sensory neurons toward CNS. These neurons are postulated to require two sources of neurotrophic factors for survival and growth, the first (i.e. BDNF) from the CNS, and the second (such as NGF) from the periphery (18). Further, unlike NGF, BDNF stimulates neurite outgrowth and supports the survival of sensory neurons derived from the ectodermal placodes, namely nodose ganglion neurons (130). BDNF also induces pluripotent neural crest cells to differentiate in vitro into primary sensory neurons (279). NT-3 is more widespread than BDNF (184): its mRNA is in fact expressed not only in the CNS, but also in many peripheral tissues, such as skeletal muscle, liver and intestine (131,183). Proprioceptive sensory trigeminal mesencephalic neurons, which respond to BDNF (17), also respond to NT-3; BDNF and NT-3 exert an additive effect on neurons of the nodose ganglion (131,184). Thus NT-3 seems to be the trophic factor of proprioceptive neurons projecting to the skeletal muscle and of the somatosensory fibers of the nodose ganglion neurons: this confirms previous observations that these neurons are supported in culture by muscle or liver extracts (59). However, the expression levels of these three neurotrophic factors in the developing nervous system are

Cestelli, Savettieri, Salemi, and Di Liegro significantly different. NT-3 is by far the most highly expressed in immature regions of the CNS: at the time when proliferation, migration and differentiation of neuronal precursors is ongoing. NT-3 expression decreases with maturation of these regions. By contrast, BDNF expression is low in developing regions of the CNS and increases as these regions mature. NGF expression varies during the development of discrete CNS regions, but not in any consistent manner compared with NT-3 and BDNF (183). Two new members of this family, NT-4 and NT5, have been recently identified by DNA homology screening. NT-4, which was isolated from Xenopus and Viper, elicits in culture neurite outgrowth from explanted dorsal root ganglia, while it has no and a lower activity on sympathetic and nodose ganglia respectively (112). A subsequent study aimed to identify additional neurotrophins resulted in the cloning of an equivalent gene from rat and human. The product of this gene, which was named NT-5, shares a high level of homology with Xenopus NT-4 but shows a different pattern of neurotrophic action: it, in fact, promotes the survival of peripheral sensory and sympathetic neurons and induces differentiation of PC12 cells (29). Other Neurotrophic Factors. CNTF, which is also involved in the control of type 2 astrocyte differentiation (7,138,177), promotes the survival in culture of motoneurons (8,182) and neurons from embryonic parasympathetic, sympathetic and sensory ganglia (15,188). It also promotes the differentiation of sympathetic neuroblasts by inhibiting their proliferation and inducing the expression of cholinergic (257) and vasoactive intestinal peptide synthesizing enzymes (82). Recently it has been purified and cloned (178,179,284) and has been shown to be totally different in sequence from the neurotrophins described above: it lacks a conventional signal sequence for secretion; thus, like fibroblast growth factors (FGF), CNTF is either secreted by an unconventional mechanism (58) or is released after cell damage. CNTF receptor is expressed exclusively within the nervous system and skeletal muscle (64): its structure is unrelated to that of receptors for the NGF family of neurotrophic factors, but instead resembles the receptor for interleukin-6 (IL6); this similarity suggests that CNTF-receptor, like IL6-receptor, requires a different transducing component. A protocol describing a procedure for the preparation of low density microcultures for their routine use in monitoring the neuronal cell survival-promoting activity of CNTF has been recently published (189). Cholinergic differentiation factor (CDF), a protein isolated from skeletal muscle, rescues motoneurons during the period of naturally occurring cell death and in-

Neuronal Cell Cultures

creases the level of choline acetyltransferase activity in motoneurons-enriched cultures of rat spinal cord (200). Structural analysis and biological assays demonstrated that this protein is identical to leukemia inhibitory factor (LIF), which regulates the growth and differentiation of embryonic stem cells (305). S100b, which is present in brain extracts and is an astroglial specific protein (for review, see 74), has been purified and shown to stimulate neurite outgrowth from chick telencephalic neurons in serum-free cell culture (155). Recently S100b has been identified as CNS serotonergic factor for cultured mesencephalic neurons (10). Lymphokines. The idea that the immune and nervous systems are somehow integrated and that among these two systems there is an intense exchange of signals through peptide hormones and factors and lymphokines has fascinated a great number of investigators for decades (for review, see 173). This hypothesis led Blalock and Smith to suggest that "certain cells of the immune system may serve as free floating nerve cells. Perhaps collectively such cells represent a mobile brain" (32). Immune cells appear to express membrane antigens similar to those of neural cells (for review, see 219). The concept that CNS modulate immune functions implies that the immune system feeds back information to the CNS: in fact interleukins (IL) exert neuroendocrine functions both when theY are produced at the periphery by the immune cells, and at the CNS level by glial cells. Further, it has been demonstrated that IL are able to cross the blood-brain barrier (13). Data from neuronal cell cultures gave strong support to this hypothesis. Neuroleukin (NLK), a T cell-derived growth factor for B cells (110), is also produced by skeletal muscle to stimulate neurite outgrowth from spinal cord motoneurons (109). This factor is also trophic for sensory cells from DRG, and neurons from septum and hippocampus (109). The most striking observation about NLK is however its homology with the HIV-1 gp 120 coat protein, which suggests that it might play a role in the pathogenesis of AIDS dementia: in bioassays with DRG neurons in culture, the HIV-1 coat protein, or peptides from the homologous region, were potent inhibitors of NLK bioactivity (162). IL-6, an essential cytokine for the differentiation and growth of B cells (128), is also effective in PC12 cells (268). Further, IL-6, which in the CNS is produced by astrocytes and microglial cells, may also stimulate the local production of other neurotrophic factors (93). IL-3 can promote neurite outgrowth and elevate the cholinergic phenotype of cultured neurons from the septum (153). IL-2 (for a comprehensive review, see 223), which

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stimulates proliferation and maturation of oligodendrocytes (28), enhances the number and length of neurites of cultured sympathetic, but not sensory neurons, without having a significant effect on cell survival (121). On the basis of structural considerations it has been demonstrated a significant homology between the two neurotrophic factors CNTF and CDF/LIF and a group of hematopoietic cytokines, namely IL-6, granulocyte colony stimulating factor, myelomonocytic growth factor, IL-3, oncostatin 1',I, and interferons c~/13.This structural homology, together with a similar pattern of exon junctions, has suggested a common evolutionary origin of the members of this "neuropoietic cytokine family" (26,234). Mitogenic Factors. In the last few years the number of molecules found to exert a neurotrophic effect has been continuously growing, and a picture is now emerging which comprehends an ever expanding list of hormones and trophic factors which were traditionally considered to be involved at various levels in cellular growth and differentiation of nonneuronal cells. Basic (b) and acidic (a) FGF are polypeptides with potent effects on the growth and differentiation of many celt types (for review, see 43). Compared to other tissues, FGFs are found at relatively high concentrations both in developing and in mature mammalian brain (44). As already mentioned, FGF molecules lack a conventional signal sequence, and yet they find their way to leave the ceils (33, 58). Once outside the cells, these factors bind strongly to extracellular matrix, presumably by virtue of their strong affinity for heparin-like molecules (251). In serum-free cell cultures both bFGF and aFGF support survival of neurons from peripheral and central nervous system (89,293,298,300). Both factors stimulate differentiation of neonatal chromaffin cells but, unlike NGF, neither of them is capable of supporting the survival of these cells, once they have undergone a complete differentiation (55,283). These studies were confirmed by Bitten and Anderson (31), who demonstrated that FGF triggers the differentiation of an immortalized sympathoadrenal precursor cell into a neuronal ceil. This cell subsequently expresses the receptor for NGF and becomes dependent on NGF, and not on FGF, for continued survival. As we have already mentioned, the expression of NGF receptors by postsynaptic cells in vivo occurs only at the time of synapse formation (59). In the light of these observations, FGFs might act on neurons by eliciting neurite outgrowth and cell differentiation before NGF becomes available to the ceils via their synaptic contacts. Epidermal growth factor (EGF), a peptide originally purified from mouse submaxillary gland, is a po-

1170 tent mitogen for epidermal cells (for review, see 101). Recently, its mitogenic activity has also been demonstrated for neuronal and glial progenitor cells of rat retina in vitro (6). This suggests that EGF is involved in the regulation of neuron production in the histogenesis of CNS; perhaps mitogenic growth factors favour the emergence of particular classes of neurons or glial ceils, at the same way as cytokines mediate activation of specific cell lineages in the hemopoietic system (for review, see 207). EGF also promotes neurite outgrowth and cell survival in neurons from the subcortical telencephalon (210). Beside having opposite effects on cell proliferation, NGF and EGF also are subjected to reciprocal regulation: pretreatment of PC12 cells with NGF decreases, in fact, within a few minutes EGF-binding capacity (39). Moreover, some of the effects elicited by the two factors in PC12 cells are similar: the induction of ornithine decarboxylase (137), the transient increase in cmyc and c-fos transcription (107), and the rise of the ribosomal protein $6 phosphorylation (215). However, the different phosphorylation patterns produced by the two growth factors suggest that the two functional states of the $6 protein are important in the functional state of the cells themselves (214). From this complex picture it emerges that polypeptide growth factors both regulate neuronal complement during development of the nervous system, and act on cell survival and differentiation.

Choice of Substratum The traditional surface-adhering cultures restrict development of three-dimensional intercellular interactions that are at the basis of constructing a nervous system (for review, see 132,273). Actually, beyond a critical cell-plating density, most neurons tend to gather together rather than adhering to the surface of the culture dish. Reaggregating cell cultures, which take advantage of this neuronal tendency, produce, on the other hand, a threedimensional neuronal network, but have several disadvantages: i) formation of too large aggregates causes neuronal degeneration and death because of unsufficient metabolite exchange with medium; ii) periodical removal and substitution of the medium by centrifugation of cell suspensions causes mechanical stress to neurons. The formation of a polycation film over the plastic surface of the culture dish strongly promotes adhesion of cells to substrate (167,307). One reason why plastic or glass have been demonstrated to be poor substrates for neuronal cells is in fact their negative charge that repels neuronal surface which is also highly negative. To enhance cell adhesion, polymers of the L-isomers of

Cestelli, Savettieri, Salemi, and Di Liegro lysine and ornithine are commonly utilized; some papers have reported however the use of D-isomers (4,52), because they are not hydrolyzed by proteases released by cells. The wide range of concentrations used (from 2 txg/ml up to 1 mg/ml) arises from the variety of the biological responses that the experimenters try to manage. Low concentrations of polycation favour clumping of neuronal bodies into islets and fasciculation of neurites (4,52,169,307). Higher concentrations ensure instead a rapid and long-lasting attachment of single neuronal cells to the substrate (104,124). The first approach is preferred in studies on "social behaviour" of neurons: in this case neurons are plated at higher densities, thus obtaining higher levels of neurotrophic factors (16). Dispersed low-density neuronal ceil cultures permit, on the other hand, both electrophysiological and immunocytochemical analyses at the single cell level (166), and a detailed characterization of the requirement for neurotrophic factors which, in this case, have to be added by the experimenter (187,189). The most obvious advantage of using a polycation as a culture dish coat stems from the easiness of using it. Such a coat is not, of course, a natural substrate; it works quite well with neurons from the CNS, but it has been reported to be less suitable for PNS neurons (for a thorough discussion, see 12). In the last decade a more detailed knowledge on the composition of the extracellular matrix (ECM) of CNS and PNS (for review, see: 65,247,260,291), has led many scientists to turn their choices toward more natural substrata. Polylysine is still widely used, especially by groups who are not interested in the study of interactions between neurons and ECM. The use of cell cultures has been fundamental for the identification of ECM molecules which act on survival and differentiation of neurons. The main constituents initially identified in ECM included collagens, noncollagenous glycoproteins and proteoglycans. Recent researches revealed however an unpredictable diversity of these molecules: the number of distinct collagens has now exceeded a dozen (41), and the amount of known species of adhesive glycoproteins is ever-expanding (77,148). This extraordinary wealth of ECM proteins stems, at least in part, from alternative splicing of primary transcripts encoded by one (as is the case of fibronectin: 141) or more genes (e.g. laminin: 79; thrombospondin: 35). Laminin (LN) is one of the most powerful neuriteoutgrowth promoting molecules for many types of cultured neurons from both the CNS and PNS (for review, see 203,277); in addition, it selectively modulates axonal but not dendritic growth in sympathetic neurons in

Neuronal Cell Cultures

culture (164). It also regulates the activity of neurotrophic factors (76) and the expression of transmitter enzymes (1). In vitro, LN is also mitogenic (233): this unpredicted property is shared by thrombospondin and tenascin: all of these ECM proteins contain multiple repeats with some homology to EGF (78). LN supports also the survival of both cultured sympathetic (80) and sensory neurons (81) at a precocious stage of development when these cells do not depend on any trophic factor. When, at later stages, both these neuronal populations acquire trophic factor-dependence, some sympathetic neurons remain LN-dependent, while others, although able to survive without LN, require a significantly lower amount of NGF if maintained on LN. In addition, it is worth noting that embryonic sensory neurons, when separated from the spinal cord in vivo, can be rescued from cell death by BDNF only in combination with LN (152). In general, LN expression reaches the highest levels during the periods of axonal growth (168) and decreases later in development, with two significant exceptions: namely, the basal lamina of mammalian peripheral nerve (54) and the optic nerve of goldfish (135), which represent two favorable sites in the adult for either a continued axonal growth or nerve regeneration. The carboxyl-terminal end of LN (fragment E8) is fundamental in promoting cell attachment and neuriteoutgrowth; by the use of subfragments derived from this region, it has been possible to identify the two distinct sites responsible for each of these functions (67). Fibronectin (FN), one of the best characterized ECMproteins, plays a major role in a great variety of biological processes, including cell differentiation and malignant transformation, maintenance of cytoskeletal organization and normal cell morphology (for a comprehensive review, see 141). From the alternative splicing of a single primary transcript, multiple FN isoforms are generated in a cell type-specific manner. FN heterogeneity is further amplified by the combination of FN subunits that are assembled into disulfide-bonded dimers. Each monomer is organized into three types of repeating modules (I, II and III) arranged into domains with sites for binding to other ECM molecules, such as collagen and heparin, cell surface proteins, such as integrins, and blood proteins such as fibrin (for review, see 272). Peripheral neurons in culture are stimulated to extend neurites by interacting with two FN domains (140): the first one contains the canonic RGDS cell-recognition sequence, while the second is an alternatively spliced type III module. On the contrary, neurons from CNS interact poorly with the first module, but very efficiently with the second one (252): these different responses probably

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reflect differences in integrin subunit expression by different neuronal populations. The distribution of FN in developing nervous system substantiate the observations on PNS and CNS neurons in culture. Neural crest cells start to migrate over FN- and LN-enriched paths (170,220,236): their migration, both in vivo and in vitro, is in fact strongly impaired by RGDS-containing peptides, which function as competitors of the main cell binding domain in FN (reviewed in 247). After migration some neural crest cells differentiate into neurons and glia of sensory and sympathetic ganglia: the nerve fibers that emerge from these ganglia grow along paths which contain FN and other ECM proteins (reviewed in 260). In contrast, immunohistochemical characterization of FN in the CNS has been controversial (for review, see 253), and interestingly, the different responses of PNS and CNS neurons in culture to this protein reflect faithfully the differential exposures of these cells to FN in vivo. In addition, both LN and FN receptors are developmentally regulated: chick retinal neurons lose functional FN and LN receptors between E6 and E12, i.e. at the time of the establishment of functional contacts between these neurons and their natural target, the optic rectum (111). Tenascin, a large homo-hexameric glycoprotein, which is expressed transiently by astrocytes in the CNS and by Schwann cells in the PNS (53), is characterized by discrete adhesive and anti-adhesive domains (281). This unusual property renders arduous to interpret the data on a molecule with such an ambiguous feature. Substrate-bound tenascin mediates growth cone and neurite elongation by both CNS (87) and PNS neurons (301), only if the neuronal cell body has got the chance to be anchored to an adhesive (i.e. polycation coated) substrate: this condition which induces aggregation of neurons and fasciculation of neurites, is indicative of a reduction in cell-substrate interactions and a concomitant preponderance of cell-cell adhesion forces. On the contrary, soluble tenascin inhibits neurite outgrowth by neurons plated on different substrate-bound ECM molecules, including tenascin itself (181), thus strengthening the hypothesis that neurite-outgrowth-promoting activity and anti-spreading effect may be exerted by two distinct functional domains of the molecule. Discrete transient astroglial expression of tenascin has been observed in many areas of developing CNS, at the time of the formation of fiber tracts and axonal projections (282). Interestingly astrocytes, which secrete this protein and participate in the compartimentalization of axonal terminal arborization in developing CNS, do not appear to recognize the inhibitor site of tenascin (87), thus sug-

1172 gesting a role for these glial cells in patterning neuronal structures. Moreover, a tenascin-specific label of the astrocyte processes which abut nodes of Ranvier in the adult rat optic nerve (91) has suggested that this glycoprotein is also involved in the maintenance of the exquisite cytoarchitecture of this important structure in the CNS. Two other components of the J1/tenascin family of ECM proteins, J1-160 and J1-180, are expressed by mature oligodendrocytes and appear late during brain development (237). In culture, these proteins mediate a transient interaction between these cells and CNS neurons, but at later stages become repulsive substrates only for neurons, while eliciting an opposite effect on astrocytes (209). Vitronectin and thrombospondin, initially studied for their role in platelet physiology (161), are now known to be expressed in the nervous system. Thrombospondin is expressed in the cerebellum where its presence is required for granule cell migration from the external granule layer in cerebellar slices (231). The potential role of both these proteins in retinogenesis is supported by their presence in developing neural retina (218): the two proteins, when allowed to adhere to the substrate, support both neuron attachment and neurite outgrowth by retinal cells (218). In PNS, collagen IV serves as a structural component of the endoneurial basement membrane, providing tensile strength as well as a scaffold with which other basement membrane components associate (for review, see 144,290). This molecule, which is synthesized by Schwann cells (46), is present both in fiber tracts and ganglia (40). In vitro it promotes neurite outgrowth from selected neuronal cell types (45,111). Like LN, also collagen IV elicits axonal but not dendritic outgrowth in cultured sympathetic ganglia, although through an alternative mechanism: both the growth rate and the initial number of axons are lower in the presence of collagen; moreover, neuritic outgrowth on collagen IV, but not LN, requires protein synthesis; and lastly, immunologically distinct integrins mediate the response of peripheral neurons to collagen IV and LN (165). In contrast to the stimulatory effects exerted by LN and Collagen IV on axonal growth, an unidentified molecule secreted by cultured astrocytes promotes in vitro dendritic outgrowth by mesencephalic neurons: this condition correlates with an increased adhesiveness of neurons to the substrate (256). This could mean that neuronal polarity (i.e. the choice of elaborating axons and dendrites) is dependent, at least in vitro, on the differential adhesion properties of the environment around developing neurons.

Cestelli, Savettieri, Salemi, and Di Liegro A large proportion of extracellular matrix glycoproteins, cell surface adhesion molecules, polypeptide growth factors, extracellular proteases and protease inhibitors exhibit binding to glycosaminoglycans (GAG) and/or proteoglycans (PG) (for review, see: 160,190,192,299). During early stages of brain development it is likely that extracellular glycosaminoglycans provide a highly hydrated and easily penetrable matrix through which migrating neurons may find their way by a dynamic process of removal and/or relocalization of the matrix components themselves (176). Recently in rat brain twenty five putative core PG proteins have been identified which are differentially expressed during development (125). Two types of sulfated PG (chondroitin sulfate PG and keratan sulfate PG) have been found associated with regions of the nervous system which act as barriers for axonal extension during development. The GAG portion of these PG is avoided by cultured sensory neurons if allowed to extend neurites on alternative substrates (280). In addition, the core protein of chondroitin sulfate PG has been shown to exert inhibitory (227) or stimulating (143) effect on neurite outgrowth depending on the type of cultured neurons: this paradoxical effect could only be explained by the presence of distinct receptors on the different neuronal types.

CONCLUSIONS In summary, regulation of cell fate depends on intimately coordinated cellular and extracellular events. Cells are, in fact, able to transduce signals from the environment to their own genome, which in turn responds by modifying cell activity and ability to transduce signals. Because of this complex network of reciprocal relationships, it may be difficult to distinguish in vivo the direct effect of a given signal, such as a hormone, from the indirect effects exerted by the same hormone via other endocrine and/or paracrine organs. This problem is even more complicated when studying maturation of the nervous system, which is by far the district where epigenetic cues are most important in defining cell identity and functioning. As we have reviewed, culture systems are quite versatile due to the possibility of choosing culture conditions different from time to time, depending on the specific aspect of cell behaviour one is interested in. Primary cultures offer the unique opportunity to probe cause-effect relationships simply by testing the direct effect of a given compound on metabolism and behaviour of specific cell types. They can be also used to probe the effects of different substrates in promoting cell adhe-

N e u r o n a l Cell C u l t u r e s

sion and migration, process outgrowth and synapse formation. As a c o n c l u d i n g remark, it must be r e m e m b e r e d that cell cultures are not an o r g a n i s m and that a n y f i n d i n g in culture is o n l y instrumental to understand what actually happens in vivo.

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ACKNOWLEDGMENT 19. D u r i n g the writing of this paper the authors were supported b y the C N R target project " B i o t e c h n o l o g y and B i o i n s t r u m e n t a t i o n " .

20. 21. 22.

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