Neurite outgrowth patterns in cerebellar microexplant cultures are affected by antibodies to the cell surface glycoprotein L1

To probe for the role of the L1 cell surface glycoprotein during neurite outgrowth and fasciculation in the early postnatal mouse cerebellar cortex, a microexplant culture system was used. Fasciculation of neurites was reduced in the presence of antigen-binding fragments (Fab) of poly- and monoclonal L1 antibodies, as compared to untreated controls. In addition, speed of neurite outgrowth was enhanced in the presence of antibodies. Migration of cell bodies of small neurons was also significantly increased. Very similar effects on these outgrowth parameters were observed with Fab fragments from poly- and monoclonal neural cell adhesion molecule (N-CAM) antibodies. Antibodies from preimmune sera had no effect. These findings suggest that L1 antigen not only plays a role in adhesion of isolated neural cell bodies and migration of granule cell neurons in the early postnatal mouse cerebellar cortex (Lindner et al., 1983; Rathjen and Schachner, 1984), but also in neurite outgrowth and fasciculation.     

Developmental expression of neural cell adhesion molecules in the mouse neocortex and olfactory bulb.

Polyclonal antibodies to N-CAM and L1 and monoclonal antibodies to epitopes of N-CAM (designated 12F11, 8A2, and 12F8) were used to investigate the spatial and temporal distribution of these neural cell adhesion molecules during the development of mouse cortex and olfactory bulb. The aim of the study was to correlate developmental events such as cell migration, dendritic and axonal outgrowth, and synaptogenesis with the appearance and disappearance of specific molecules involved in cell-cell interactions. Western transfer studies indicated that 12F8 antibody recognized polysialic acid found on embryonic N-CAM; 8A2 antibody primarily recognized the 140 kD component of N-CAM while the 12F11 antibody recognized the 180 and the 140 kD forms. The study demonstrates a high degree of cell surface molecular specialization of different compartments in developing neocortex and olfactory bulb. L1 is found on a variety of unmyelinated fiber tracts including thalamocortical fibers, olfactory nerve, and inner plexiform layer of the olfactory bulb. In contrast, N-CAM epitope recognized by 12F11 antibody is present on olfactory nerve fibers but appears later and is much weaker than L1 on thalamocortical fibers and is absent from the olfactory lobe inner plexiform layer. Dendritic regions are best labeled by 12F8 antibody; the epitope becomes faint in adult cortex but remains strongly expressed in olfactory bulb. This study reveals that widespread N-CAM expression in the central nervous system is constituted by a diversity of local expression of different molecular forms of N-CAM; their different anatomical distributions suggest they may each have unique roles.     

Fasciculation of Granule Cell Neurites is Responsible for the Perpendicular Orientation of Small Inhibitory Interneurons in Mouse Cerebellar Microexplant Cultures In Vitro

To study the cellular and molecular mechanisms involved in the perpendicular orientation of stellate and/or basket cells to the direction of fasciculating granule cell neurites, we have used cultures of microexplants from early postnatal mouse cerebellum that show this cellular behaviour in vitro. When these cultures were maintained in the presence of antibodies to the neural cell adhesion molecules L1 and N-CAM or immunoglobulin-like domains I, II and IV of N-CAM, the resulting decrease or increase in the fasciculation of granule cell neurites changed the perpendicular orientation and morphology of the small inhibitory interneurons. Additives which did not perturb fasciculation did not affect the perpendicular orientation and morphology of stellate and/or basket cells. Furthermore, when perturbation of fasciculation was prevented, neither L1 nor N-CAM antibodies modified the positioning or morphology of interneurons. These observations indicate that ordered fasciculation of granule cell neurites is an important parameter in the perpendicular orientation and elaboration of the typical morphology of the small cerebellar inhibitory interneurons.     

Evidence for Cis Interaction and Cooperative Signalling by the Heat-stable Antigen Nectadrin (murine CD24) and the Cell Adhesion Molecule L1 in Neurons

L1 is a transmembranal hornophilic cell adhesion molecule of the immunoglobulin superfamily expressed by neural and lymphoid cells. The heat-stable antigen (HSA, murine CD24) nectadrin is a highly and heterogeneously glycosylated glycophosphatidylinositol-linked differentiation antigen of haematopoietic and neural cells. L1 and nectadrin have been shown to mediate cell adhesion and intracellular Ca²⁺ signals in neurons and B lymphoblasts, respectively. Here we show that nectadrin is co-expressed with L1 in murine cerebellar granule cell neurons and neuroblastoma N2A cells. Purified nectadrin bound to L1 with an apparent binding ratio of five nectadrin molecules to one L1 molecule at saturation. Binding between nectadrin and purified N-CAM was not observed. In co-capping experiments nectadrin co-redistributed with L1 and N-CAM. Since in these cells N-CAM and L1 cohere by cis binding nectadrin appears to join the L1-N-CAM complex through binding to L1. Antibodies to each L1 and nectadrin evoked small increases in the intracellular Ca²⁺ concentration. However, when both antibodies were added together or in tandem to the cells, a strong intracellular Ca²⁺ signal was measured that was at least 6- and 10-fold stronger than the signal separately induced by L1 and nectadrin antibodies respectively. Such a cooperative effect was not observed in B lymphoblasts, using the same antibodies, or in neurons, using a combination of L1 and Thy-1 antibodies. Both the weak Ca²⁺ signal mediated by L1 alone and the enhanced signal jointly triggered by antibodies to L1 and nectadrin were inhibited by phorbol 12-myristate 13-acetate and were not significantly affected by Ni²⁺ and Cd²⁺ cations, suggesting that they are related to one another and involve recruitment of intracellular Ca²⁺. Nectadrin therefore appears to join a functional complex of neuronal adhesion molecules and to potentiate the signal transduction pathway of L1, possibly in response to neuron-neuron contact formation.     

Regional distribution of neural cell adhesion molecule (N-CAM) and L1 in human and rodent hippocampus

Cell surface adhesion molecules N-CAM and L 1 are implicated in central nervous system (CNS) cell migration and axon outgrowth in in vitro and in vivo developmental studies. These molecules show a differential distribution during CNS development, thus suggesting that they subserve different roles in process outgrowth and tissue organization. A variety of N-CAM isoforms are known, and individual N-CAMs undergo posttranslational modification. Such changes and the potential for generating numerous molecules may mediate development of specific neural cell contacts and circuitry. We evaluated immunohistochemical staining of polyclonal antibodies to L 1 and N-CAM, as well as monoclonal antibodies directed against embryonic N-CAM and the 140 and 180 kDa species of N-CAM in human, rat, and mouse hippocampus. Staining patterns in the three species were qualitatively similar, but staining in the mouse hippocampus was quantitatively greater for some epitopes. A distinctive pattern of staining was found, corresponding to the known anatomy of the structure. Total N-CAM staining was intense in the hilus and inner molecular layer (ML) of the dentate gyrus with lighter staining in the dentate outer ML. The mossy fiber tract (MFT), comprising axons traveling from the dentate granule cells to CA3 pyramidal cells, was strongly stained by polyclonal antibody to N-CAM. There was abundant staining of the stratum radiatum (SR) and stratum oriens (SO) of CA1, but stratum lacunosum moleculare (LM) showed very little staining. The monoclonal antibody 12F11, which recognizes the 140 and 180 kDa forms of N-CAM, intensely stained the MFT, hilus, and inner ML. With 12F11, SO and SR stained uniformly throughout CA1, with much reduced staining in CA2 and CA3. There was little staining in LM. L1 staining was more evenly distributed throughout the hippocampus and dentate, with light hilar and MFT staining, and even staining through the SR and SO. Stratum LM stained intensely for L1, with a narrow clear zone between SR and LM. The ML of the dentate gyrus stained intensely with anti-L1, with a discrete clear zone separating the inner and outer ML. Embryonic N-CAM had little staining in CA1 but strong hilar and MFT staining; thus, this “embryonic” determinant continues to be expressed in limited regions in the adult. In contrast to the adult rodent hippocampus, human hippocampus exhibited embryonic N-CAM in the outer two-thirds of the ML, but showed very little staining in the hilar region. These distinctive patterns suggest that the distribution of the N-CAM and L1 molecular species have clear-cut structural and functional roles in the laminated circuitry of the hippocampus. (c) 1993 Wiley-Liss, Inc.     

Immunocytological localization of cell adhesion molecules L1 and N-CAM and the shared carbohydrate epitope L2 during development of the mouse neocortex

The expression of the two adhesion molecules L1 and N-CAM and their shared carbohydrate epitope recognized by monoclonal antibody L2, was studied during development of the embryonic mouse neocortex by immunohistology at light- and electron-microscopic levels between embryonic days 9 and 18. Throughout this time period N-CAM is expressed in all layers of the telencephalic anlage. L1 antigen shows a more restricted expression than N-CAM. It is not detectable at day 9. From day 10 onward it is expressed on young neurons in the marginal zone, but not in the ventricular layer. At embryonic day 13 L1 antigen appears also in the intermediate zone on afferent fibers from subcortical structures and on migrating neurons. Neuronal cell bodies in the cortical plate and subplate express L1 antigen only transiently on embryonic days 13-16. These observations suggest that L1 antigen does not play a prominent role in the initiation of neuronal migration in the ventricular zone, but could be functional during later stages of migration and in the aggregation of neuronal cell bodies at their final position in the cortical plate. The L2 epitope also shows a more restricted expression than N-CAM during the time period studied. Similar to L1 antigen, it first appears at embryonic day 10 in the marginal zone and remains undetectable in the ventricular layer also at later stages. In the marginal zone the L2 epitope is strongly expressed on neuroepithelial endfeet at the basal lamina. The basal lamina itself is L2 epitope-negative. From embryonic day 10 onward the L2 epitope is most strongly expressed in the marginal zone and subplate and more weakly in the cortical plate and intermediate zone. In the subplate it is not only associated with the surface membrane, but also with the extracellular matrix. These observations support previous biochemical data which show that the L2 epitope is not present on all N-CAM molecules of the embryonic or adult forms and suggest that the independent regulation or L2 epitope expression may have functional implications during development.     

Molecular specificity of L2 monoclonal antibodies that bind to carbohydrate determinants of neural cell adhesion molecules and their resemblance to other monoclonal antibodies recognizing the myelin-associated glycoprotein

L2 monoclonal antibodies and HNK-1 have been shown to bind to related carbohydrate determinants in the myelin-associated glycoprotein (MAG) and several adhesion molecules of the nervous system including neural cell adhesion molecule (N-CAM), L1 and J1. It is shown here that MAG is the principal component in human white matter binding the L2 antibodies, but the most prominent antigens with the L2 epitopes in human gray matter are of higher Mr. It is also shown that the L2 antibodies resemble HNK-1 in binding to some 19-28 kDa glycoproteins and some sulfated, glucuronic acid-containing sphingoglycolipids of the peripheral nervous system (PNS). In addition, monoclonal and polyclonal antibodies raised to human MAG are shown to cross react with bovine N-CAM due to the presence of common carbohydrate constituents. The results further emphasize the shared antigenicity between MAG, N-CAM and other adhesion molecules. In addition, they demonstrate that the L2 antibodies belong to a family of monoclonal antibodies (including HNK-1, human IgM paraproteins associated with neuropathy, and others) that are characterized by reactivity against carbohydrate determinants shared by human MAG, the 19-28 kDa glycoproteins of the PNS and the sulfated, glucuronic acid-containing sphingoglycolipids of the PNS.     

Cell Type-specific Effects of the Neural Adhesion Molecules L1 and N-CAM on Diverse Second Messenger Systems

We have previously shown that the neural adhesion molecules L1 and N-CAM influence second messenger systems when triggered with specific antibodies at the surface of the phaeochromocytoma PC12 cell line (Schuch et al., Neuron, 3, 13 - 20, 1989). To determine whether the two molecules are linked to the same intracellular signalling cascades, independent of the cell type expressing them, or whether different neural cell types respond with different signal transduction mechanisms, we have investigated the effects of antibodies to L1 and N-CAM, and the isolated molecules themselves, on second messenger systems in different neural cell types. We have investigated cultures of cerebellar and dorsal root ganglion neurons and transformed Schwann cells and related these results to those obtained with the PC12 cell line. Here we show that addition of L1 and N-CAM antibodies and the isolated molecules themselves elicit cell type-specific responses that can be modulated by the substrate on which the cells are maintained. Depending on the cell type, cells respond to the triggering of L1 and N-CAM with antibodies, or addition of the purified molecules, by either up-regulation or down-regulation of inositol phosphate turnover, by a rise in intracellular Ca2+ levels dependent on or independent of the opening of voltage-gated Ca2+ channels, or by an increase or decrease in intracellular pH. Moreover, cerebellar neurons expressing N-CAM respond to addition N-CAM, but not to N-CAM antibodies, in contrast to the other neural cell types studied, which respond to both triggers. Furthermore, cerebellar neurons were the only cells to show a rise in cAMP levels in response to any of the ligands tested. This stimulation of cAMP production by L1 antibodies depended on the cross-linking of L1 molecules at the cell surface, whereas the other responses did not depend on clustering of L1. Simultaneous addition of L1 and N-CAM antibodies either elicited an additive or more than additive effect on the intracellular responses which, for cerebellar neurons, depends on the substrate on which the cells are maintained. These observations indicate that L1 and N-CAM or their antibodies activate cell type-specific intracellular signalling systems and that the two molecules can act interdependently or independently of each other.     

The L2/HNK-1 Carbohydrate Mediates Adhesion of Neural Cells to Laminin

The L2/HNK-1 carbohydrate epitope shared by several neural adhesion molecules has been implicated in cell-to-cell and cell-to-laminin adhesion (Keilhauer et al., Nature , 316 , 728–730, 1985; Künemund et al., J. Cell Biol. , 106 , 213–223, 1988). As demonstrated previously for chicken retinal ganglion cells (Cole et al., Neurosci. Lett. , 93 , 170–175, 1988), cerebral cortex astrocytes or cerebellar neurons could not be shown to adhere to the substrate-bound L2/HNK-1 carbohydrate. The cell-bound L2/HNK-1 carbohydrate, however, was a potent mediator of astrocytic and neuronal cell adhesion to laminin, which was strongly reduced in the presence of the L2/HNK-1 carbohydrate-carrying glycolipids or Fab fragments of a monoclonal antibody against it. Inhibition of adhesion could not be observed in the presence of the negatively charged gangliosides or sulphatide, but in the presence of heparin. To investigate whether the L2/HNK-1 carbohydrate and heparin use the same or different binding sites on laminin, adhesion of cells to laminin was determined in the presence of heparin and Fab fragments of a monoclonal L2 antibody, which gave an additive value of inhibition as compared to the inhibition caused by the single compounds. This result, as well as studies of the binding of the L2/HNK-1 glycolipids to laminin in the presence of heparin, indicates that the L2/HNK-1 carbohydrate and heparin are implicated in different aspects of neural cell adhesion to laminin.     

Immunoelectron-microscopic localization of the 180 kD component of the neural cell adhesion molecule N-CAM in postsynaptic membranes

In order to investigate the expression of cell adhesion molecules in synapses, we have studied the localization of the neural cell adhesion molecule N-CAM in the cerebellum and hippocampus of adult mice by immunocytological and immunochemical methods. Of the three molecular components of N-CAM with relative molecular masses (Mr) of 120, 140, and 180 kD, N-CAM 120 is not detectable in synaptosomal membranes, whereas N-CAM 140 is expressed on both pre- and postsynaptic membranes and N-CAM 180 is restricted to postsynaptic sites, with localization of the N-CAM 180-specific epitope in postsynaptic densities. Specificity of immunoreactivity is indicated by the observation that antibodies to the neural cell adhesion molecule L1 do not label synaptic membranes, whereas antibodies to two major components of postsynaptic densities, actin and erythrocyte spectrin, react with synaptic structures. Interestingly, N-CAM 180 is only detectable in subpopulations of synapses in the intact tissue. Isolated synaptosomes, opened for unimpeded accessibility of antibody by hypoosmotic treatment, also reveal a partial expression of N-CAM 180 in that 67% are labeled by antibodies to N-CAM 180, while antibodies to actin and erythrocyte spectrin react with 95% and 88% of all synaptosomes, respectively. N-CAM 180 does not appear to be differentially expressed in synapses of a particular morphological type, but is detectable in all types of synapses in the cerebellum and hippocampus, except for mossy fiber synapses and synapses between basket and Purkinje cells, which are generally N-CAM 180-negative. Since N-CAM 180 has been shown to be characteristic of stabilized or stabilizing cell contacts, possibly by its association with the cytoskeleton-membrane linker protein spectrin (Pollerberg et al.: J. Cell Biol. 101:1921-1929, '85; Nature 324:462-465, '86; Cell Tissue Res. 250:227-236, '87), we would like to suggest N-CAM 180 plays an important role in determining the stability of contacts between pre- and postsynaptic membranes and state of synaptic activity.     

Molecular Association of the Neural Adhesion Molecules L1 and N-CAM in the Surface Membrane of Neuroblastoma Cells is Shown by Chemical Cross-linking

The two neural cell adhesion molecules L1 and N-CAM could be shown to be associated in the surface membrane of cultured neuroblastoma cells by chemical cross-linking with 3,3'-dithiobis(sulphosuccinimidyl-propionate) and subsequent immunopurification and precipitation using antibodies to L1 and N-CAM. Glycoproteins recognized in neuroblastoma cells by antibodies to mouse liver membranes were not chemically cross-linked to L1 or N-CAM. These observations suggest that a molecular association between the two molecules may be the basis for their functional cooperativity (Kadmon et al., 1990a,b, J. Cell Biol., 110, 193-208; 209-218).     

Accumulation of N-CAM 180 at Contact Sites Between Neuroblastoma Cells and Latex Beads Coated with Extracellular Matrix Molecules

Neuronal cells expressing neural cell adhesion molecule (N-CAM) accumulate the largest N-CAM component (N-CAM 180) at cell - cell contact sites. To test whether this accumulation is induced by interactions at the surface membrane, latex beads coated with several purified adhesion molecules or extracellular matrix (ECM) components were co-cultured with neuroblastoma cells. Beads coated with L1, N-CAM, the L2/HNK-1 carbohydrate epitope-carrying molecules from adult mouse brain or laminin from Engelbreth-Holm-Swarm (EHS) sarcoma did not induce an accumulation of N-CAM 180 or L1 at sites of contact suggesting that these molecules are not directly involved in N-CAM 180 accumulation or that their mobility is required for this process. Beads coated with ECM components of the PF-HR9 cell line induced accumulation of N-CAM 180 at sites of contact with neuroblastoma cells. Accumulation was seen at cell bodies of undifferentiated and differentiated neuroblastoma cells, as well as on neurites and growth cones of differentiated neuroblastoma cells. Accumulation of the neural adhesion molecule L1 was also seen, but less prominently and reproducibly. These observations suggest that molecules of the ECM can directly or indirectly, e.g. via molecules linked to N-CAM 180 on the cell surface, induce accumulation of N-CAM 180.     

Adhesion of neural cells to extracellular matrix constituents. Involvement of glycosaminoglycans and cell adhesion molecules

Single cell suspensions of early postnatal mouse cerebellum adhere to substrate-bound culture supernatants of the teratocarcinoma cell line PF-HR9 and can be inhibited to adhere by antibodies to the neural cell adhesion molecules L1 and N-CAM. Adhesion can also be inhibited by the glycosaminoglycans heparin and heparan sulfate, and less by chondroitin sulfate or hyaluronic acid. Heparinase treatment of cells, but not of HR9 substrate, reduces adhesion. Adhesion does not appear to be mediated by laminin, a constituent of HR9 extracellular matrix, since L1 and N-CAM antibodies do not interfere with cell adhesion on EHS sarcoma laminin as substrate and since antibodies to EHS sarcoma laminin partially inhibit adhesion to HR9 extracellular matrix which contains laminin. Of the other extracellular matrix constituents analysed in HR9 culture supernatants (collagen type IV, a heparan sulfate proteoglycan and fibronectin) none could be shown to promote adhesion, when coated as substrate, suggesting that yet unidentified compounds are responsible for L1- or N-CAM-mediated cell adhesion. These experiments show for the first time that extracellular matrix constituents can act as binding partners for the neural cell adhesion molecules L1 and N-CAM.     

The adhesion molecule on glia (AMOG) incorporated into lipid vesicles binds to subpopulations of neurons

To investigate the functional role of the novel adhesion molecule on glia (AMOG) in cell surface interactions, immunoaffinity-purified AMOG was incorporated into liposomes and measured for its ability to bind to cells in monolayer cultures. AMOG could be incorporated into liposomes in functionally active form after solubilization from membranes in 1% cholate buffer containing soybean lecithin, elution from the AMOG monoclonal antibody column with 4 M MgCl2, containing 1% octylglucoside, and removal of detergent for liposome incorporation by gel filtration. AMOG-containing liposomes bound to neurons, but not to oligodendrocytes, astrocytes, or fibroblasts in early postnatal cerebellar cultures. AMOG-containing liposomes also bound to the pheochromocytoma cell line PC12, but not to neurons in cultures of spinal cord and dorsal root ganglia after various times in vitro. Fab fragments of monoclonal AMOG antibodies, but not of L3 monoclonal antibodies directed against a carbohydrate structure on AMOG, inhibited binding of liposomes. Liposome binding was not reduced by preincubation of cerebellar cells with antibodies to AMOG, to the neuron adhesion molecule L1, the neural cell adhesion molecule N-CAM, or the L3 carbohydrate structure, nor with 2 monoclonal antibodies reacting with neuronal cell surface glycoproteins related to the L2/HNK-1 family. These results show that AMOG is indeed a ligand in adhesion and binds to particular subpopulations of neurons in L1- and N-CAM-independent mechanisms.     

Structure, expression, and function of Ng-CAM, a member of the immunoglobulin superfamily involved in neuron-neuron and neuron-glia adhesion.

The neuron-glia cell adhesion molecule (Ng-CAM) mediates neuron-neuron adhesion by a homophilic mechanism and neuron-astrocyte adhesion by a heterophilic mechanism. The protein is expressed on neurons and Schwann cells but not on astrocytes. It is most prevalent during development on cell bodies of migrating neurons and on axons during formation of nerves. Ng-CAM expression is greatly increased following nerve injury. Anti-Ng-CAM antibodies inhibited migration of granule cells along Bergmann glia in cerebellar explants and fasciculation of neurites in outgrowths from explants of dorsal root ganglia. The combined results indicate that Ng-CAM on neurons binds to Ng-CAM on adjacent neurons and to as yet unidentified ligands on astrocytes. Ng-CAM is synthesized in chicken neurons from a 6 kb mRNA as Mr approximately 200,000 forms which are cleaved to yield two components of Mr 135,000 and 80,000. It is glycosylated and can be phosphorylated. Amino acid sequence analysis indicates that it contains six immunoglobulin domains, five fibronectin type III repeats, a transmembrane domain and a cytoplasmic region. Structural analyses indicate that Ng-CAM is most closely related to the mammalian glycoprotein L1 but significant differences between them strongly suggest that they are not equivalent molecules. The recent identification of another structurally related molecule in the chicken called Nr-CAM underscores the notion that these molecules are members of a subfamily of neural cell adhesion molecules within the immunoglobulin superfamily that have related or complementary functions in the nervous system.     

Monozygotic twins discordant for schizophrenia are discordant for N-CAM and L1 in CSF

While schizophrenia has a genetic component, its pathogenesis is unknown. Abnormal concentrations of two cell recognition molecules (CRMs), neural-cell adhesion molecule (N-CAM) and L1 antigen have been described in the cerebrospinal fluid (CSF) of patients with schizophrenia. Studies of monozygotic twins discordant for schizophrenia may help separate genetic and environmental contributions to the disease. In the present study of monozygotic twins discordant for schizophrenia, the affected twins had increased N-CAM and decreased L1 antigen in their CSF. Non-affected twins were not different from normals. Although processes related to genetic instability cannot be entirely ruled out, these results suggest that these abnormalities are not a part of the genetic predisposition to become schizophrenic. Thus the changes in N-CAM and L1 antigen may reflect either the events which precipitated the onset of schizophrenia, or events which are associated with the experience of having the disease.© 1997 Elsevier Science B.V. All rights reserved.     

The Neural Cell Adhesion Molecule (N-CAM) Modulates K+ Channels in Cultured Glial Precursor Cells

Application of antibodies against the neural cell adhesion molecule (N-CAM) to O4-positive murine glial precursor cells in vitro results in a reduction of two distinct K+ currents measured using the whole cell patch clamp technique. Both the A-type and delayed rectifier K+ currents are reduced in amplitude within a few minutes of the application of poly- or monoclonal antibodies against N-CAM. This effect is not due to the binding of any antibody to the surface of the glial precursor cells because monoclonal antibody directed against the O4 surface antigen, or polyclonal antibodies directed against liver cell membranes (which also bind to the surface of glial precursor cells), do not affect membrane currents. Activators of protein kinase C, such as phorbol esters or diacylglycerol, also induce changes in potassium currents that appear, both in magnitude and kinetics, to be similar to those induced by antibodies against N-CAM. In contrast, activation of G proteins upregulates K+ currents. Glial precursor cells thus respond to triggering of N-CAM by altering channel properties. These observations suggest that adhesive events between neural cells can influence the intracellular ionic milieu.     

Immunohistological localization of the adhesion molecules L1, N-CAM, and MAG in the developing and adult optic nerve of mice

The localization of the cell adhesion molecules L1, neural cell adhesion molecule (N-CAM), and myelin-associated glycoprotein (MAG) was studied immunohistologically at the light and electron microscopic levels and immunochemically in the developing and adult mouse optic nerve and retina. The neural adhesion molecule L1 is strongly expressed on the shafts of fasciculating unmyelinated axons at all ages studied from embryonic day 15 through adulthood. Growth cones of retinal ganglion cell axons were weakly L1-positive or L1-negative when contacting glial cells. Unmyelinated axons were not only L1-positive when contacting each other but also when contacting glia, whereas contacts between glial cells were L1-negative at all developmental unmyelinated retinal nerve fiber layer or in the unmyelinated optic nerve head became L1-negative when enwrapped by myelin in the optic nerve proper. At all stages of development N-CAM showed profuse labeling on fasciculating axons, growth cones, and their contact sites with glial cells as well as contacts between glial cells. In contrast to L1, axons remained N-CAM-positive when becoming myelinated. Sometimes, N-CAM was found in compact myelin. However, N-CAM was absent from glial surfaces contacting basement membranes at the interface to meninges, blood vessels, and the vitreous body of the eye. MAG was first detectable intracellularly in oligodendrocytes associated with the endoplasmic reticulum and Golgi apparatus before it became apparent at the cell surface. There it was present on oligodendrocytes prior and during the first stages of ensheathment of axons, both on cell body and processes. After formation of compact myelin MAG remained strongly expressed periaxonally and was only weakly detectable in noncompacted myelin including inner mesaxon and paranodal loops. None of the adhesion molecules was detectable on extracellular matrix, in the meninges, or on endothelial cells. Immunochemical analysis of antigen expression at different developmental stages was in agreement with the immunohistological data. We infer from these observations that L1 is involved in stabilization not only of axon-axon, but also axon-glia contacts, while the more dynamic structure of the growth cone generally expresses less L1. A differential expression of L1 along the course of an axon--being present on its unmyelinated, but absent on its myelinated part--further supports the notion that L1 may be involved in the stabilization of axonal fascicles but not of axon-myelin contacts.(ABSTRACT TRUNCATED AT 400 WORDS)     

The cellular neurobiology of neuronal development: the cerebellar granule cell

Cerebellar granule cells in vivo and in vitro have been widely used in the study of the cellular neurobiology of neuronal development. We have described the basic neuroanatomical data on the granule cell in the developing and mature cerebellum. The importance of the cytoskeleton in determining the morphology of the granule cell and in process outgrowth and cell migration has been described. Extensive information is now available on the composition of the granule cell cytoskeleton. Cell surface glycoproteins are thought to be involved in the control of cell adhesion and cellular interactions during development. A number of surface molecules belonging to either the N-CAM or the Ng-CAM groups of glycoproteins have been studied in detail in the cerebellum. The role of these proteins in cell adhesion and in granule cell-astroglial interactions during granule cell migration has been reviewed. The survival and differentiation of neurones is controlled by soluble trophic factors. Several factors have been described which act as trophic factors for granule cells in vitro and may do the same in vivo. The numerous studies that have been carried out on the cerebellar granule cell have allowed us to describe certain aspects of the cellular neurobiology of this class of neurones as an example with general significance for the understanding of neuronal differentiation and function.     

Expression of the Neural Recognition Molecule L1 by Cultured Neural Cells is influenced by K+ and the Glutamate Receptor Agonist NMDA

To investigate the influence of neuronal activity on the expression of neural recognition molecules, cultures of neural cell lines and dissociated cells of early postnatal mouse cerebellum were maintained in the presence of elevated concentrations of K+ and the glutamate agonist N-methyl-d-aspartate (NMDA). Levels of expression of the neural adhesion molecules L1 and N-CAM at the cell surface were measured by an enzyme-linked immunosorbent assay. Expression of L1 was up-regulated in neuroblastoma N2A cells after 1 day of maintenance in 40 and 60 mM K+, but not in phaeochromocytoma PC12 cells. Expression levels of N-CAM and antigens recognized by the monoclonal antibody A2B5 or by polyclonal antibodies to crude membrane fractions of liver were not significantly altered by elevated K+ concentrations in these two cell lines. In monolayer cultures of early postnatal mouse cerebellum, an increase of 60% in expression of L1, but not N-CAM or A2B5, was seen at 20 and 40 mM K+. This increase in L1 expression was specifically inhibitable by the Ca2+ channel blocker nicardipine. NMDA at a concentration of 100 microM increased levels of L1, but not of N-CAM. This increase was inhibitable by the NMDA antagonists 2-amino-5-phosphonovalerate and MK-801, but not significantly by the kainate/quisqualate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. The increase in L1 expression at higher K+ concentrations was not inhibitable by the NMDA antagonists, indicating that the K+-mediated increase in L1 expression is not due to release of glutamate by cerebellar neurons. These observations indicate that compounds influencing neuronal membrane properties, and thus neuronal excitability, are capable of regulating the expression of L1. In a more general context, these findings suggest that previously observed changes in synaptic connectivity in situ, resulting from activity-dependent fine tuning of neuronal morphology, may be mediated by alterations in the expression of recognition molecules.