The influence of laminin (LN) and fibronectin (FN) on the differentiation of individual neurones from the embryonic rat central nervous system was studied in vitro. In control cultures or in the presence of soluble FN, most neurones had several dendrite-like and one axon-like processes. On substratum-bound LN, multipolar and unipolar cells were present. Soluble LN and bound FN induced a very simple neuronal morphology, most neurones having only one axon-like neurite as defined by morphological and immunocytochemical characteristics. The significant reduction of neuronal adhesion and spreading in conditions leading to the growth inhibition of dendrite-like processes suggests that, contrary to that of axons, dendrite growth strongly depends on neuronal adhesion. We propose a model in which the different dependency of axonal and dendritic outgrowth towards adhesion and spreading is explained by the respective physical properties of the two types of neurites.

Neuronal process outgrowth in vitro is regulated by numerous chemical and physical factors. Among the chemical factors, NGF and basic FGF are known to stimulate the elongation of neurites in specific peripheral and central neuronal populations (Levi-Montalcini, 1982; Korshing et al. 1985; Morrisson et al. 1986; Walicke et al. 1986; Unsicker et al. 1987). The importance of physical parameters, such as tension and adhesion, has also been emphasized (Letourneau, 1975; Fraser, 1980; Bray, 1984). In addition, the cellular substrate and the extracellular matrix (ECM) proteins influence neuronal process outgrowth. The role of these proteins has been studied mainly in the peripheral nervous system (PNS) where they have been shown to promote the migration of neural crest derivatives, the outgrowth and guidance of neurites and the induction of proteins involved in neurotransmitter metabolism (Baron-van Evercooren et al. 1982; Thiery et al. 1982; Edgar et al. 1984; Smalheiser et al. 1984; Davis et al. 1985; Lander et al. 1985; Hammarback et al. 1985; Acheson et al. 1986; Millaruelo et al. 1988). In the central nervous system (CNS), ECM proteins and in particular laminin (LN) and fibronectin (FN) are likely to play a role in neuronal differentiation and regeneration and in the process of neurite elongation (Manthorpe et al. 1983; Smalheiser et al. 1984; Faivre-Bauman et al. 1984; Carri et al. 1988). LN and FN are synthesized in the CNS during development and after brain injury as well as by astrocytes in culture (Liesi et al. 1983, 1984; Price & Hynes, 1985). Further, LN and FN receptors are present on neurones in culture (Bozyczko & Horwitz, 1986).

In a previous series of studies, we examined the regulation of neuronal process outgrowth by CNS neurones in vitro from a different point of view. We demonstrated that dendritic and axonal growth are separate events under distinct regulatory controls. Using short-term in vitro cocultures of neurones and astrocytes from two regions of the brain (mesencephalon and striatum), we observed vigorous dendrite growth in neuroastroglial cocultures of neurones and astrocytes derived from the same region (homotopic cocultures). In comparison, neurones grown on heterotopic astrocytes had a poorly developed dendritic tree and exhibited a long and thin axon-like process (Denis-Donini et al. 1984; Chamak et al. 1987; Autillo-Touati et al. 1988). This finding that regulation of dendritic and axonal growth is differentiable has been confirmed by experiments with other neuronal populations (Bruckenstein & Higgins, 1988a). Furthermore, Bruckenstein & Higgins (1988b) have demonstrated that a purified fraction of serum stimulated dendritic outgrowth by sympathetic neurones in culture.

In a recent study, we have started to characterize the factors regulating dendrite outgrowth in rat mesencephalic neurones. We found that molecules able to stimulate or inhibit the growth of dendrites were present in astrocyte-conditioned media (Rousselet et al. 1988). Preliminary analysis of these media revealed that they contain large amounts of different isoforms of LN and FN (Rousselet et al. in preparation).

Therefore, in order to set up a model system we have started to analyse the effects of LN and FN on the expression of neuronal polarity in CNS neurone cultures. In this report, we demonstrate that LN and FN do regulate dendritic and axonal extension but that the physical form (soluble or sustrate bound) of these molecules is a critical factor in the differential growth of the two neuronal compartments.

Cell cultures

Dissociated mesencephalic neurones were prepared from 14-day-old rat embryos (IFFA CREDO, France), plated at low density (104 cells cm-2 ) on polyornithine (1·5μg ml-1, Sigma) -coated culture dishes (16 mm diameter wells) and cultured for 2 days in serum-free medium (SFM) consisting of DMEM/F12 (1:1, Gibco), 33mm-D-glucose, 2mm-L-glutamine, 3mm-NaHCO3, 5-mm-Hepes pH7·4, 5i.u.ml-1 penicillin and 5μg ml-1 streptomycin. This SFM was supplemented with 25μg ml-1 insulin, 100 μg ml-1 transferrin, 2×10−8M-progesterone, 6×10−5M-putrescine, 3×10−8M-selenium and 0·1% ovalbumin (chemically defined medium, CDM). In some cases, dissociated brain stem and striatal neurones were prepared from 14- and 16-day-old rat embryos, respectively.

Laminin (Bethesda Research Laboratories or provided by Dr N. Brunet de Carvalho) was isolated from an Engelbreth-Holm-Swarm (EHS) sarcoma, a transplantable mouse tumour. Fibronectin (Collaborative Research Incorporated) was obtained from the fibrinogen fraction of human plasma. In order to obtain the concentrations of bound LN (bLN) and bound FN (bFN) reported in Table 1, culture dishes were incubated overnight at 37°C with 50 μg ml-1 (bFN) or 1μgml-1 (bLN) of the matrix molecules. They were washed three times with serum-free medium before neuronal plating. Iodination of the matrix molecules was achieved with lodogen (Pierce), according to the manufacturers’ instructions. Soluble fibronectin or laminin diluted in SFM at the indicated concentrations were added to the medium 1 h after neuronal plating on polyomithine-coated (1·5μg ml-1) culture dishes. 48 h later (or more when indicated), cultures were fixed for 1 h at 4 °C with 2·5% glutaraldehyde in phosphate-buffered saline (PBS), washed three times with PBS and incubated for 5 to 10min with 0·5% toluidine blue in 2·5% Na2CO33. Stained cells were washed with distilled water and air dried. For reversion experiments, the cells were detached after 24 h by a brief (5 min) trypsin treatment (0·25 % in SFM) at room temperature and replated as indicated in the Results section.

Table 1.

Amounts of soluble and bound matrixmolecules in the different culture conditions

Amounts of soluble and bound matrixmolecules in the different culture conditions
Amounts of soluble and bound matrixmolecules in the different culture conditions

Immunocytochemistry

For immunocytochemistry cells were grown on 16 mm diameter polyomithine-coated glass coverslips, fixed for 1 h at 4°C with freshly prepared 4% paraformaldehyde in PBS, and washed three times with Tris-buffered saline (TBS) containing 10 % fetal calf serum and 0·1% Triton × 100 (buffer A). Incubations with the first antibodies were for 1 h at 37 °C, the cells were washed three times with buffer A, further incubated for 1h at 37°C with fluorescein- or rhodamine-linked second antibodies (Byosis) diluted 100-fold in buffer A, washed three times and mounted in PBS: glycerol (5:95) for microscopic observation. The polyclonal anti-MAP2 antibody was provided by Dr A. Fellous (Chamak et al. 1987) and the polyclonal anti Glial Fibrillary Acidic Protein (GFAP) were purchased from Dakopatts. The axon-specific monoclonal antibodies recognizing phosphorylated isoforms of the 160 and 200 neurofilament proteins (Foster et al. 1987) were characterized and generously provided by Dr P. Levitt or by Dr R. Liem.

Adhesion assays

Cells were seeded on plastic culture dishes, with or without polyornithine. After 2 days in culture, the plates were shaken at 300 revs min-1 for 1 h. Attached cells were fixed with 2·5 % glutaraldehyde in PBS, washed, stained with toluidine blue and counted in 10 fields chosen at random.

Analysis of neuronal morphology

The number of cells was determined by counting 50 independent fields. Cell survival was calculated as a fraction of the number of cells extruding trypan blue 2h after plating. In order to analyse neuronal morphology, 50 to 100 neurones taken at random were drawn with the help of a drawing tube. In order not to introduce a bias in the choice of the neurones, we proceeded to a double blind analysis, starting at a given joint of the culture dish and drawing all isolated neurones encountered, field by field, until the desired number of cells was obtained. In the culture conditions used for this study, most neurones were clearly isolated and only a few aggregates had to be eliminated from the statistics. For each neurone, the number of primary neurites and branching points was counted. From the drawings, neunte length and cell body surfaces were calculated using the Jandel Scientific SigmaScan computer program. For each parameter studied (neuritic length, number of primary neuntes, number of branch points), the neurones were separated into several classes and the percentage distributions were calculated. In some cases, extreme classes had to be pooled in order to obtain the minimum number of neurones per class necessary for the validity of the statistical analysis. Differences between distributions were analysed with a test of homogeneity (Chisquare).

Cellular survival and characterization

Mesencephalic cells were plated at low density and 2 h later non-attached cells were washed away. A 40% plating efficiency (number of cells extruding trypan blue) was calculated. After 2 days, the number of live attached cells had decreased by 40% in the control medium or with soluble extracellular matrix proteins and by 25% when bound LN or FN were used. Immunostaining of the cells after 2 days in vitro demonstrated that more than 98% of them could be recognized by the neurone-specific anti-neurofilament triplet antiserum, the remaining 2 % being stained by the anti-GFAP antibody. This clearly demonstrated the neuronal nature of the majority of the cells present at the time of morphological analysis.

The amounts (in μg/well) of soluble fibronectin (sFN), bound fibronectin (bFN), soluble laminin (sLN) and bound laminin (bLN), were calculated with the help of 125I-iodinated matrix molecules. They are presented in Table 1. This table also gives the amounts of matrix molecules which attach to, or detach from, the substratum during the time of culture, as well as the ratios between bound and soluble forms in the different culture conditions.

Effects of matrix molecules on neuronal morphology

The presence of matrix proteins had a strong influence on the number of cells bearing neurites longer than two cell diameters. As shown in Table 2, compared to control medium, bFN and bLN increased the number of cells with processes whereas sFN and sLN had no effect. Fig. 1 illustrates the typical morphologies of the mesencephalic neurones in the different culture conditions. In control medium or in sFN, the cell bodies had several cytoplasmic extensions (Fig. 1A). On bFN or with sLN most of the neurones were unipolar (Fig. IB and 1C) whereas on bLN both multipolar and unipolar neurones were observed (Fig. ID). The presence of these two morphologies on bLN was probably due to the solubilization of 4% of bound laminin (Table 1). In fact adding sLN to bLN increased dramatically the percentage of unipolar neurones (not shown). Similar results were obtained with neuronal populations from the striatum and the hindbrain (not shown).

Table 2.

Percentage of neurite-bearing cells in different culture conditions

Percentage of neurite-bearing cells in different culture conditions
Percentage of neurite-bearing cells in different culture conditions
Fig. 1.

Typical morphologies of mesencephalic neurones cultured for 48h in control medium (A), on bound FN (B), with soluble LN (C) and on bound LN (D). The morphology of neurones in presence of soluble FN was similar to that of the control culture (not shown).

Fig. 1.

Typical morphologies of mesencephalic neurones cultured for 48h in control medium (A), on bound FN (B), with soluble LN (C) and on bound LN (D). The morphology of neurones in presence of soluble FN was similar to that of the control culture (not shown).

Quantitative morphological analysis was done on all neurite-bearing cells. A typical experiment in which the effects of soluble and bound ECM molecules were compared is illustrated in Fig. 2. The large majority of neurones in control medium and in the presence of sFN were multipolar. In contrast with sFN, which permitted the extension of several neurites (Fig. 2A), bFN strongly reduced the number of multipolar neurones (Fig. 2B). The same unipolar morphology was also observed in the presence of sLN (Fig. 2C). In the presence of bLN, both unipolar and multipolar neurones were present (Fig. 2D).

Fig. 2.

Drawings of mesencephalic neurones grown for 48 h in different culture conditions. Neurones were chosen at random and drawn with the help of a light chamber. (A) Soluble FN; (B) bound FN; (C) soluble LN; (D) bound LN.

Fig. 2.

Drawings of mesencephalic neurones grown for 48 h in different culture conditions. Neurones were chosen at random and drawn with the help of a light chamber. (A) Soluble FN; (B) bound FN; (C) soluble LN; (D) bound LN.

In order to verify that the unipolar neurones obtained with sLN did not reflect the selective loss of multipolar cells, reversion experiments were performed. The result of such an experiment is shown in Fig. 3. Mesencephalic neurones were cultured 48 h either in control conditions (Fig. 3A) or in the presence of sLN (Fig. 3B). Neurones were cultured in sister wells for 24 h in sLN where they adopted the simple unipolar phenotype. They were then trypsinized, replated on polyomithine and cultured for another 24 h. The multipolar morphology of the cells shown in Fig. 3C demonstrates the complete phenotypic reversal of the unipolar neurones.

Fig. 3.

Drawings of mesencephalic neurones cultured 48 h in control medium (A) or in presence of soluble LN (B). (C) Neurones cultured for 24 h in soluble LN, trypsinized, replated on polyomithine in control medium and left for another 24 h. (D) Neurones cultured for 24 h in control medium and supplemented for another 24 h with soluble LN, note the presence of longer neurites (compare with A) indicated by arrows.

Fig. 3.

Drawings of mesencephalic neurones cultured 48 h in control medium (A) or in presence of soluble LN (B). (C) Neurones cultured for 24 h in soluble LN, trypsinized, replated on polyomithine in control medium and left for another 24 h. (D) Neurones cultured for 24 h in control medium and supplemented for another 24 h with soluble LN, note the presence of longer neurites (compare with A) indicated by arrows.

In another experiment, mesencephalic neurones were cultured for 24 h in control conditions and sLN was then added for another 24h. In these conditions, we observed a small population of unipolar and a large population of multipolar neurones (Fig. 3D). The unipolar neurones were present in control conditions after 24 h (not shown), and the effect of adding LN was only to increase the length of their single neurite. Multipolar neurones differed from those seen in control medium alone (after 48 h). They now had an asym-metrical shape, one neurite elongating much faster than the others (arrowheads).

From these experiments, we could first conclude that the unipolar population seen in sLN (and also in bFN) did not reflect the selective loss of a multipolar population, since they could be induced to adopt the multipolar phenotype. Second it appeared that among the several neurites present in the control condition one of them started to elongate at a faster rate when sLN was added to the culture medium. These conclusions are partially summarized in the scheme presented in Fig. 4.

Fig. 4.

Schematic representation of the results of the experiments presented in Figs 2 and 3 and of the treatments leading to the different morphologies.

Fig. 4.

Schematic representation of the results of the experiments presented in Figs 2 and 3 and of the treatments leading to the different morphologies.

Better quantification and characterization of neuronal morphologies were obtained from a statistical analysis in which we included the following parameter: number of primary neurites, number of branch points, total neuritic length and length of the longest neurite. Fig. 5 shows that sFN had no effect on the number of primary neurites and branch points. On the contrary, sLN and bFN reduced the mean number of primary neurites from 5 to 1 and 1·5, respectively. The number of branch points was also significantly reduced in these two culture conditions. Laminin adsorbed to the substratum had an intermediate effect easily explained by the presence of the two neuronal populations presenting different morphological traits. As indicated in

Fig. 5.

Number of primary neurites and branch points of neurones cultured in different conditions. 50 neurones chosen at random were studied in each condition. The significance of the differences in neuronal distributions between the control (no matrix proteins added) and the experimental conditions were calculated using the Chisquare analysis and are indicated as follows: ***, P<0·001; n.s., not significant; bFN, bound fibronectin; sFN, soluble fibronectin; bLN, bound laminin; sLN, soluble laminin.

Fig. 5.

Number of primary neurites and branch points of neurones cultured in different conditions. 50 neurones chosen at random were studied in each condition. The significance of the differences in neuronal distributions between the control (no matrix proteins added) and the experimental conditions were calculated using the Chisquare analysis and are indicated as follows: ***, P<0·001; n.s., not significant; bFN, bound fibronectin; sFN, soluble fibronectin; bLN, bound laminin; sLN, soluble laminin.

Table 3, total neuritic length per neurone was identical in control medium and in the presence of sFN but decreased in the presence of bLN, bFN and sLN. Decrease in total length was particularly striking with bFN and sLN, conditions favouring the unipolar phenol-type. However, the length of the longest neurite was greatly enhanced in these latter conditions (bFN and sLN), some of the neurites reaching 900, μm after 48h. The intermediate values found on bLN are explained by the presence of two populations with different morphologies (Fig. 2).

Table 3.

Influence of matrix molecules on neurite length

Influence of matrix molecules on neurite length
Influence of matrix molecules on neurite length

Identification of ‘dendrite-like’ and ‘axon-like’ neurites

The single long neurite observed in bFN and sLN had the morphological aspect of an axon suggesting that, in these conditions, axonal growth only was permitted. On the contrary, several neurites present in the control cultures and with sFN or bLN had a dendritic morphology. In order to confirm these morphological characterizations, we used immunocytochemical markers preferentially staining the dendritic or axonal compartments of the neurones. Fig. 6 illustrates the anti-MAP2 staining of the dendrites and cell bodies after 2 days in culture. In this figure, the same negatives were exposed either normally (Fig. 6A, C, E) or were overexposed (Fig. 6B, D, F). This later condition permitted visualization of some neurites which were only faintly immunostained. In control medium alone (Fig. 6A, B) or with sFN (not shown), neurones extended several short neurites stained by the antiserum. In sLN, the single ‘axon-like’ neurite was strongly stained only in the region proximal to the cell body (Fig. 6C, D). The staining decreased in the most distal part and its visualization was made possible only by overexposure. The same patterns of staining were obtained in sLN and on bFN (not shown). The anti-MAP2 staining of neurones grown on bLN is illustrated in Fig. 6E and F. Several dendrites are strongly labelled and the long putative axon is faintly stained, as clearly demonstrated in the overexposed condition (Fig. 6F).

Fig. 6.

Anti-MAP2 staining of mesencephalic neurones after 2 days in culture. (A,B) Control medium; (C,D) soluble laminin; (E,F) bound laminin. In B, D and F the negatives were overexposed in order to visualize the axons. Strong staining was limited to cell bodies and dendrites.

Fig. 6.

Anti-MAP2 staining of mesencephalic neurones after 2 days in culture. (A,B) Control medium; (C,D) soluble laminin; (E,F) bound laminin. In B, D and F the negatives were overexposed in order to visualize the axons. Strong staining was limited to cell bodies and dendrites.

In order to better characterize the ‘axon-like’ neuntes, the cells were cultured for 2 or 5 days and stained with a monoclonal antibody directed against a 200×103Mr phosphorylated neurofilament isoform specifically expressed in the immature axons (Liem, personal communication). Identical results were obtained with a monoclonal antibody directed against axon-specific phosphorylated 160 and 200 × 105Mr proteins (Levitt et al. in preparation). After 2 days, only one neurite in multipolar cells and the single neurite in monopolar cells were stained by the axon-specific antibodies. This staining pattern was even more striking after 5 days (Fig. 7). From this figure, it is also clear that the stained neurites bear axonal morphological characters: they are long, unbranched in their proximal segment and their thin diameter does not taper as the distance from the cell body increases. In addition, the pearly pattern of the staining indicated that, at this stage, the phosphorylated isoforms were not uniformly distributed along the ‘axon-like’ shaft. It can therefore be proposed that axon-like and dendrite-like neurites are present in the control conditions and in sFN but that in sLN and bFN the neuritic arborization is almost entirely restricted to the axon-like compartment.

Fig. 7.

Staining by the axon-specific antibody of mesencephalic neurones cultured for 5 days in different conditions. (A) Control medium; (B) bound FN; (C) soluble LN; (D) bound LN. Note that in all conditions, only the longest neurite is stained by the antibody.

Fig. 7.

Staining by the axon-specific antibody of mesencephalic neurones cultured for 5 days in different conditions. (A) Control medium; (B) bound FN; (C) soluble LN; (D) bound LN. Note that in all conditions, only the longest neurite is stained by the antibody.

Cell body surfaces and adhesion

The morphology of the cell bodies in the different conditions suggested a decreased spreading of the soma in conditions in which only axon-like neurites were present. In order to verify this point, 100 cell bodies of process-bearing neurones were chosen at random, drawn and their surfaces were calculated using the SigmaScan digitizing program. Results of one of these experiments are shown in Table 4. Compared to polyornithine (PORN) alone, the presence of bound or soluble matrix proteins decreased the apparent surfaces of the neurones. However, these surfaces were significantly reduced in the two conditions leading to the growth of a unique ‘axon-like’ process.

Table 4.

Influence of matrix molecules on the size of the cell body surface

Influence of matrix molecules on the size of the cell body surface
Influence of matrix molecules on the size of the cell body surface

We hypothesized that the apparent surface of the cell body was an index of cellular adhesion and spreading. Thus, in order to verify if adhesion was decreased in the two conditions inhibiting dendritic growth and favouring axonal elongation (sLN and bFN), adhesion assays were performed. After 2 days in culture, the plates were shaken for 1h at 300revsmin-1, and the number of attached cells was determined. As shown in Table 5, the number of attached cells was significantly decreased in sLN and bFN conditions, even in the presence of the adhesive substratum of polyornithine. In the absence of PORN, the effect of sLN and bFN on cellular adhesion was even more dramatic, and the few attached cells, if left to develop for 48 h, exhibited the expected unipolar phenotype. From these observations, it was concluded that, compared to axon-like neuntes, the growth of the dendrite-like arborization was strongly dependent upon adhesion.

Table 5.

Adhesion assay

Adhesion assay
Adhesion assay

In this article, we report the effects of LN and FN on neurite outgrowth and on the establishment of neuronal polarity. The results obtained with mesencephalic neurones were reproduced with striatal and brain stem neurones (not shown). They therefore present a certain degree of generality.

LN and FN had different effects depending on their physical (soluble or attached) form. These differences principally concerned two parameters: the number of process-bearing neurones and neuronal morphology. Compared to control medium, bFN and bLN increased the number of process-bearing neurones. This confirmed the observation of several authors on the influence of matrix substrata on neuritic extension (Manthorpe et al. 1983; Smalheiser et al. 1984; Faivre-Bauman et al. 1984; Carri et al. 1988).

However, the precise examination of neurite-bearing cells allowed by the sparse culture conditions led us to demonstrate that, in control medium and sFN, most neurones were multipolar whereas in sLN and bFN they adopted a unipolar morphology. On bLN the two morphological types were present, a result which can be explained by the partial solubilization of the bound molecule, as calculated from experiments with 125I-iodinated LN. This was validated by the fact that adding sLN to bLN increased the number of unipolar neurones (not shown). The latter experiment also confirmed the theory that, in sLN alone, the simple morphological type was indeed due to the presence of the molecule in its soluble state.

In the present study, most analysis was done after 2 days in culture and with neurones plated at low density in a chemically defined medium. These culture conditions allowed a good morphological description of individual neurones and prevented the conditioning of the medium by the cells. Indeed neurone-conditioned medium has been shown to modify neuronal morphology (Bartlett & Banker, 1984; Chamak et al. 1987). The choice of a short-term culture was dictated by our interest in the initial steps in neurite formation. How-ever, experiments performed after 5 days in culture led to the same conclusions regarding the developmental patterns created by the different forms of matrix molecules.

A special difficulty was encountered because of the neuronal death observed in the conditions of low neuronal density. Indeed the unipolar or multipolar morphologies observed in the different conditions could have resulted from the differential loss of specific neuronal subpopulations. This possibility was ruled out by reversion experiments in which unipolar neurones, trypsinized and replated in control medium, were still able to adopt a multipolar morphology. Conversely, the addition of sLN to multipolar neurones promoted the elongation of only one of the several neurites.

Classification of the neurites was based on morphological and immunocytochemical criteria. The neurites of multipolar neurones (on average 4 to 5) were thick and branched in the region proximal to the cell body, as expected for dendrites. The neurite of unipolar neurones conformed to axonal criteria, they were rather long, did not taper and showed a limited number of branch points after two days in culture. After 5 days, some branching was observed in regions distal to the cell body.

Immunocytochemical methods were used to confirm the morphological characterization of axon-like and dendrite-like neurites. We first used an antibody directed against MAP2, a protein highly enriched in the dendritic compartment of most neurones (Matus et al. 1981; Caceres et al. 1984; De Camilli et al. 1984; Caceres et al. 1986). The cell bodies and the short neurites exhibiting dendrite-like morphology were strongly stained with the anti-MAP2 antiserum. However, we also observed a strong MAP2 staining in the proximal portion and in the varicosities of some axon-like neurites, a staining pattern already observed in immature axons by Caceres et al. (1986).

Neurite characterization was confirmed with the help of antibodies directed against phosphorylated forms of 160 and 200 ×103Mr neurofilament proteins which are specifically enriched in the axonal compartment (Stemberger & Stemberger, 1983; Shaw et al. 1985; Dahl & Bignami, 1985; Peng et al. 1986; Foster et al. 1987; Levitt, personal communication; Liem, personal communication). In all conditions, only the neurites with axonal morphological traits were stained by the two different axon-specific antibodies and the dendrite-like neurites never showed any immunoreactivity.

Therefore we proposed that bFN and sLN induced axonal outgrowth and inhibited dendritic development, whereas bLN, sFN and medium without ECM proteins permitted both axonal and dendritic growth.

This interpretation of an inhibiting effect of sLN and bFN on dendritic growth was further substantiated by the comparative analysis of neuritic lengths in the different culture conditions. Clearly the accelerated outgrowth of the axon in bFN and sLN was associated with an almost complete absence of dendritic growth. A possible interpretation of this result is that, in a defined metabolic state, neurones synthesize a given amount of membrane and cytoskeleton but that external conditions dictate in which compartments (axonal or axonal and dendritic) this neurite-building material is dispatched. One must underline the possible physiological importance of a mechanism (dendrite growth inhibition) by which all neurite-building elements are dispatched into the axonal compartments, reducing thus the time necessary for the axonal growth cone to reach its target.

In the two culture conditions favouring axonal out-growth (sLN and bFN), quantitative measurements of the apparent cell body surfaces demonstrated a decreased spreading of the neuronal soma. This observation is similar to that of Bruckenstein & Higgins (1988a) on sympathetic neurones. The decrease in neuronal spreading suggests a simple physical explanation for the separate regulation of dendrite and axon outgrowth. This hypothesis draws on D’Arcy Thompson’s and Plateau’s calculations for fluids mechanic (1917). In high-surface-tension conditions (low adhesion), process outgrowth strongly depends on neuritic viscosity. In fact, in our experiments (Table 5), dendritic outgrowth was observed in all conditions permitting strong neuronal adhesion (possibly impheating the low viscosity of the dendroplasm) whereas axons were able to grow in weak adhesion conditions, probably because of their high viscosity, easily explained by their large amount of fasciculated microtubules (Autillo-Touati et al. 1988; Chamak et al. in preparation).

In order to understand the direct effect of LN and FN on neuronal morphology and polarity, we must assume the existence of neuronal receptors for these molecules. Bozycko & Horwitz (1986) have already demonstrated the participation of a putative cell surface receptor for LN and FN in peripheral neurite extension. We are presently studying the presence of ECM protein receptors on CNS neurones in culture. Specific staining of such neurones with an antibody directed against the integrin ß subunit has already been observed (not shown).

We have not yet investigated the mechanism implied in the decreased adhesion observed in conditions leading to the unipolar morphology. We speculate that sLN occupies LN-binding sites on the cell surface, decreasing the number of receptors available for the formation of adhesion plaques between cells and substrate. Conversely, it is possible that bFN occupies important cellular sites for adhesion without leading to the intracellular changes required for adhesion and spreading. In fact, the numerous matrix molecules, matrix receptors and adhesion molecules involved in cell-cell or cell-substratum adhesion indicate that several mechanisms could be involved (Bozyczko & Horwitz, 1986; Cohen et al. 1987; Bixby et al. 1988; Tomaselli et al. 1988; Werz & Schachner, 1988). Among these mechanisms, a direct trophic activity of the matrix molecules cannot be excluded (Panayotou et al. 1989).

In our previous studies, we showed that mesencephalic and striatal neurones develop their dendritic arborization much faster on homotopic than on heterotopic astrocytes (Chamak et al. 1987). This finding can now be interpreted in term of preferential adhesion of the neuronal cells on astrocytes derived from the same region of the brain. Such a region-specific preferential adhesion of neurones to astrocytes could be explained in several ways. First, one can speculate on the existence of region-specific cell adhesion molecule isoforms created through post-translational modifications and alternative splicing (Rutishauser & Goridis, 1986). Second, homotopic situations could induce a faster neuronal maturation, due to the existence of regionspecific astrocyte-derived factors which, in turn, accelerate the transition from the embryonic sialylated N-CAM to the more adhesive adult form (Edelman, 1986).

Finally, the study presented here raises the possibility that the equilibrium between substrate-attached and soluble LN or FN plays an important role in the separate regulation of axonal and dendritic growth. The region specificity of astrocyte-induced dendrite growth therefore suggests the presence of different isoforms of matrix molecules and of their receptors during development in the different regions of the brain. We are presently investigating this hypothesis of a role of matrix receptors as positional markers in the nervous tissue. It is, however, noteworthy that such a positional marker role for the high-affinity LN receptor and for the a subunit of the integrin heterodimer has been demonstrated in completely different systems (Leptin et al. 1987; Rabacchi et al. 1988).

We thank Dr J. Glowinski for his useful advices and constant support. We are also grateful to Drs N. Brunet de Carvalho, A. Rousselet, F. Lafont and M. Vigny. This work was supported by grants from INSERM, Rhône-Poulenc Santé and DRET.

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