Olfactory neurons express a unique glycosylated form of the neural cell adhesion molecule (N-CAM)

mAb-based approaches were used to identify cell surface components involved in the development and function of the frog olfactory system. We describe here a 205-kD cell surface glycoprotein on olfactory receptor neurons that was detected with three mAbs: 9-OE, 5-OE, and 13-OE. mAb 9-OE immunoreactivity, unlike mAbs 5-OE and 13-OE, was restricted to only the axons and terminations of the primary sensory olfactory neurons in the frog nervous system. The 9-OE polypeptide(s) were immunoprecipitated and tested for cross-reactivity with known neural cell surface components including HNK-1, the cell adhesion molecule L1, and the neural cell adhesion molecule (N-CAM). These experiments revealed that 9-OE-reactive molecules were not L1 related but were a subset of the 200-kD isoforms of N-CAM. mAb 9-OE recognized epitopes associated with N-linked carbohydrate residues that were distinct from the polysialic acid chains present on the embryonic form of N-CAM. Moreover, 9-OE N-CAM was a heterogeneous population consisting of subsets both with and without the HNK-1 epitope. Thus, combined immunohistochemical and immunoprecipitation experiments have revealed a new glycosylated form of N-CAM unique to the olfactory system. The restricted spatial expression pattern of this N-CAM glycoform suggests a possible role in the unusual regenerative properties of this sensory system.     

Presence of novel N-CAM glycoforms in the rat olfactory system

The functional activity of the neural cell adhesion molecule N-CAM can be modulated by posttranslational modifications such as glycosylation. For instance, the long polysialic acid side chains of N-CAM alter the adhesion properties of the protein backbone. In the present study, we identified two novel carbohydrates present on N-CAM, NOC-3 and NOC-4. Both carbohydrates were detected on N-CAM glycoforms expressed by subpopulations of primary sensory olfactory neurons in the rat olfactory system. Based on the expression of NOC-3 and NOC-4 and the olfactory marker protein (OMP), four independent subpopulations of primary sensory olfactory neurons were characterized. These neurons expressed: both NOC-3 and NOC-4 but not OMP; both NOC-4 and OMP but not NOC-3; NOC-3, NOC-4, and OMP together; and OMP alone. The NOC-3- and NOC-4-expressing neurons were widely dispersed in the olfactory neuroepithelium lining the nasal cavity. The axons of NOC-4 expressing neurons innervated all glomeruli in the olfactory bulb, whereas the NOC-3 expressing axons terminated in a discrete subset of glomeruli scattered throughout the whole olfactory bulb. We propose that both NOC-3 and NOC-4 are part of a chemical code of olfactory neurons which is used in establishing the topography of connections between the olfactory neuroepithelium and the olfactory bulb. © 1997 John Wiley & Sons, Inc. J Neurobiol 32 : 659–670, 1997     

The central pathway of primary olfactory axons is abnormal in mice lacking the N-CAM-180 isoform

Although N-CAM has previously been implicated in the growth and fasciculation of axons, the development of axon tracts in transgenic mice with a targeted deletion of the 180-kD isoform of the neural cell adhesion molecule (N-CAM-180) appears grossly normal in comparison to wild-type mice. We examined the organization of the olfactory nerve projection from the olfactory neuroepithelium to glomeruli in the olfactory bulb of postnatal N-CAM-180 null mutant mice. Immunostaining for olfactory marker protein revealed the normal presence of fully mature primary olfactory neurons within the olfactory neuroepithelium of mutant mice. The axons of these neurons form an olfactory nerve, enter the nerve fiber layer of the olfactory bulb, and terminate in olfactory glomeruli as in wild-type control animals. The olfactory bulb is smaller and the nerve fiber layer is relatively thicker in mutants than in wild-type mice. Previous studies have revealed that the plant lectin Dolichos biflorus agglutinin (DBA) clearly stains the perikarya and axons of a subpopulation of primary olfactory neurons. Thus, DBA staining enabled the morphology of the olfactory nerve pathway to be examined at higher resolution in both control and mutant animals. Despite a normal spatial pattern of DBA-stained neurons within the nasal cavity, there was a distorted axonal projection of these neurons onto the surface of the olfactory bulb in N-CAM-180 null mutants. In particular, DBA-stained axons formed fewer and smaller glomeruli in the olfactory bulbs of mutants in comparison to wild-type mice. Many primary olfactory axons failed to exit the nerve fiber layer and contribute to glomerular formation. These results indicate that N-CAM-180 plays an important role in the growth and fasciculation of primary olfactory axons and is essential for normal development of olfactory glomeruli. © 1997 John Wiley & Sons, Inc. J Neurobiol 32 : 643–658, 1997     

Selective expression of the 180-kD component of the neural cell adhesion molecule N-CAM during development

The rodent neural cell adhesion molecule (N-CAM) consists of three glycoprotein chains of 180, 140, and 120 kD in their adult forms. Although the proportions of the three components are known to change during development and differ between brain regions, their individual distribution and function are unknown. Here we report studies carried out with a monoclonal antibody that specifically recognizes the 180-kD component of mouse N-CAM (N-CAM180) in its highly sialylated embryonic and less glycosylated adult forms. In primary cerebellar cell cultures, N-CAM180 antibody reacts intracellularly with all types of neural cells including astrocytes, oligodendrocytes, and neurons. During cerebellar, telencephalic, and retinal development N-CAM180 is detectable by indirect immunohistology in differentiated neural cells, but, in contrast to total N-CAM, not in their proliferating precursors in the ventricular zone and primordial and early postnatal external granular layer. In monolayer cultures of C1300 neuroblastoma cells, N-CAM180 appears by immunofluorescence more concentrated at contact points between adjacent cells, while N-CAM comprising the 180- and 140-kD component shows a more uniform distribution at the plasma membrane. Treatment of neuroblastoma cells with dimethylsulfoxide, which promotes differentiation, induces a shift toward the predominant expression of N- CAM180. These observations support the notion that N-CAM180 is expressed selectively in more differentiated neural cells and suggest a differential role of N-CAM180 in the stabilization of cell contacts.     

Topography of N-CAM structural and functional determinants. I. Classification of monoclonal antibody epitopes

12 distinct neural cell adhesion molecule (N-CAM) epitopes, each recognized by a different monoclonal antibody (mAb), have been characterized in terms of the major structural and functional features of the molecule. Seven antibodies, each recognizing the amino-terminal region of the molecule, altered the rate of N-CAM-mediated adhesion. Four of these were inhibitors, two of which also recognized a heparin-binding N-CAM fragment. The other three antibodies specifically enhanced the rate of N-CAM-mediated adhesion. Three epitopes, one polypeptide- and two carbohydrate-dependent, were associated with the sialic acid-rich central portion of the molecule. The remaining two antibodies were found to react with intracellular determinants, and are specific for the largest of the three major N-CAM polypeptide forms. Studies on the ability of one antibody to hinder recognition of native N-CAM by another antibody suggested that the epitopes associated with N-CAM binding functions are in close proximity compared with the other determinants. The classification of these mAb epitopes has allowed the topographical placement of key N-CAM features, as described in the following paper, and provides valuable probes for analysis of both the structure and function of N-CAM.     

Expression of L1 and N-CAM cell adhesion molecules during development of the mouse olfactory system

The expression of the neural adhesion molecules L1 and N-CAM has been studied in the embryonic and early postnatal olfactory system of the mouse in order to gain insight into the function of these molecules during development of a neural structure which retains neuronal turnover capacities throughout adulthood. N-CAM was slightly expressed and L1 was not significantly expressed in the olfactory placode on Embryonic Day 9, the earliest stage tested. Rather, N-CAM was strongly expressed in the mesenchyme underlying the olfactory placode. In the developing nasal pit, L1 and N-CAM were detectable in the developing olfactory epithelium, but not in regions developing into the respiratory epithelium. At early developmental stages, expression of the so-called embryonic form of N-CAM (E-N-CAM) coincides with the expression of N-CAM, whereas at later developmental stages and in the adult it is restricted to a smaller number of sensory cell bodies and axons, suggesting that the less adhesive embryonic form is characteristic of morphogenetically dynamic neuronal structures. Moreover, E-N-CAM is highly expressed at contact sites between olfactory axons and their target cells in the glomeruli of the olfactory bulb. L1 and N-CAM 180, the component of N-CAM that accumulates at cell contacts by interaction with the cytoskeleton are detectable as early as the first axons extend toward the primordial olfactory bulb. L1 remains prominent throughout development on axonal processes, both at contacts with other axons and with ensheathing cells. Contrary to N-CAM 180 which remains detectable on differentiating sensory neuronal cell bodies, L1 is only transiently expressed on these and is no longer detectable on primary olfactory neuronal cell bodies in the adult. Furthermore, whereas throughout development L1 has a molecular form similar to that seen in other parts of the developing and adult central nervous systems, N-CAM and, in particular, N-CAM 180 retain their highly sialylated form at least partially throughout all ages studied. These observations suggest that E-N-CAM and N-CAM 180 are characteristic of developmentally active structures and L1 may not only be involved in neurite outgrowth, but also in stabilization of contacts among fasciculating axons and between axons and ensheathing cells, as it has previously been found in the developing peripheral nervous system.     

Analysis of high PSA N-CAM expression during mammalian spinal cord and peripheral nervous system development.

Using a monoclonal antibody that recognizes specifically a high polysialylated form of N-CAM (high PSA N-CAM), the temporal and spatial expression of this molecule was studied in developing spinal cord and neural crest derivatives of mouse truncal region. Temporal expression was analyzed on immunoblots of spinal cord and dorsal root ganglia (DRGs) extracts microdissected at different developmental stages. Analysis of the ratio of high PSA N-CAM to total N-CAM indicated that sialylation and desialylation are independently regulated from the expression of polypeptide chains of N-CAM. Motoneurons, dorsal root ganglia cells and commissural neurons present a homogeneous distribution of high PSA N-CAMs on both their cell bodies and their neurites. Sialylation of N-CAM can occur in neurons after their aggregation in peripheral ganglia as demonstrated for dorsal root ganglia at E12. Furthermore, peripheral ganglia express different levels of high PSA N-CAM. With in vitro models using mouse neural crest cells, we found that expression of high PSA N-CAM was restricted to cells presenting an early neuronal phenotype, suggesting a common regulation for the expression of high PSA N-CAM molecules, neurofilament proteins and sodium channels. Using perturbation experiments with endoneuraminidase, we confirmed that high PSA N-CAM molecules are involved in fasciculation and neuritic growth when neurons derived from neural crest grow on collagen substrata. However, we demonstrated that these two parameters do not appear to depend on high PSA N-CAM molecules when cells were grown on a fibronectin substratum, indicating the existence of a hierarchy among adhesion molecules.     

Expression sequences and distribution of two primary cell adhesion molecules during embryonic development of Xenopus laevis

Studies of chicken embryos have demonstrated that cell adhesion molecules are important in embryonic induction and are expressed in defined sequences during embryogenesis and histogenesis. To extend these observations and to provide comparable evidence for heterochronic changes in such sequences during evolution, the local distributions of the neural cell adhesion molecule (N-CAM) and of the liver cell adhesion molecule (L-CAM) were examined in Xenopus laevis embryos by immunohistochemical and biochemical techniques. Because of the technical difficulties presented by the existence of multiple polypeptide forms of CAMs and by autofluorescence of yolk-containing cells, special care was taken in choosing and characterizing antibodies, fluorophores, and embedding procedures. Both N-CAM and L-CAM were found at low levels in pregastrulation embryos. During gastrulation, N-CAM levels increased in the presumptive neural epithelium and decreased in the endoderm, but L-CAM continued to be expressed in all cells including endodermal cells. During neurulation, the level of N-CAM expression in the neural ectoderm increased considerably, while remaining constant in non-neural ectoderm and diminishing in the somites; in the notochord, N-CAM was expressed transiently. Prevalence modulation was also seen at all sites of secondary induction: both CAMs increased in the sensory layer of the ectoderm during condensation of the placodes. During organogenesis, the expression of L-CAM gradually diminished in the nervous system while N-CAM expression remained high. In all other organs examined, the amount of one or the other CAM decreased, so that by stage 50 these two molecules were expressed in non-overlapping territories. Embryonic and adult tissues were compared to search for concordance of CAM expression at later stages. With few exceptions, the tissue distributions of N-CAM and L-CAM were similar in the frog and in the chicken from early times of development. In contrast to previous observations in the chicken and in the mouse, N-CAM expression was found to be high in the adult liver of Xenopus, whereas L-CAM expression was low. In the adult brain, N-CAM was expressed as three components of apparent molecular mass 180, 140, and 120 kD, respectively; in earlier stages of development only the 140-kD component could be detected. In the liver, a single N-CAM band appears at 160 kD, raising the possibility that this band represents an unusual N-CAM polypeptide. L-CAM appeared at all stages as a 124-kD molecule.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cell Adhesion Molecules and the Migration of LHRH Neurons during Development

During embryogenesis, LHRH neurons arise in the olfactory epithelium, migrate along the olfactory nerve, and enter the forebrain. We have examined the distribution of several cell adhesion molecules (CAMs) in the developing chick olfactory system and brain to determine whether differential distributions of these adhesion molecules might be important in pathway choices made by migrating LHRH neurons. Single- and double-label immunocytochemical studies indicated that high levels of N-CAM and N-cadherin were expressed throughout the olfactory epithelium and not restricted to the medial half of the olfactory epithelium where most of the LHRH neurons originate. Further, high levels of N-CAM, Ng-CAM, and N-cadherin were uniformly expressed throughout the entire olfactory nerve while migrating LHRH neurons were confined to the medial half of the nerve. However, once LHRH neurons reach the brain, they migrate dorsally and caudally, tangential to the medial surface of the forebrain, along a region enriched in N-CAM and Ng-CAM. After this first stage of migration within the brain, LHRH neurons migrate laterally. At this stage, there is no correlation between the intensity of N-CAM and Ng-CAM immunostaining and the location of LHRH neurons. These results suggest that N-CAM, Ng-CAM, and N-cadherin do not play a guiding role in LHRH neuronal migration through the olfactory epithelium and olfactory nerve but that migrating LHRH neurons may follow a "CAM-trail" of N-CAM and Ng-CAM along the medial surface of the forebrain.     

Expression of cell adhesion molecules in the olfactory system of the adult mouse: presence of the embryonic form of N-CAM

The expression of the neural cell adhesion molecules N-CAM and L1 was investigated in the olfactory system of the mouse using immunocytochemical and immunochemical techniques. In the olfactory epithelium, globose basal cells and olfactory neurons were stained by the polyclonal N-CAM antibody reacting with all three components of N-CAM (N-CAM total) in their adult and embryonic states. Dark basal cells and supporting cells were not found positive for N-CAM total. The embryonic form of N-CAM (E-N-CAM) was only observed on the majority of globose basal cells, the precursor cells of olfactory neurons, and some neuronal elements, probably immature neurons, since they were localized adjacent to the basal cell layer. Differentiated neurons in the olfactory epithelium did not express E-N-CAM. In contrast to N-CAM total, the 180-kDa component of N-CAM (N-CAM180) and E-N-CAM, L1 was not detectable on cell bodies in the olfactory epithelium. L1 and N-CAM180 were strongly expressed on axons leaving the olfactory epithelium. Olfactory axons were also labeled by antibodies to N-CAM180 and L1 in the lamina propria and the nerve fiber and glomerular layers of the olfactory bulb, but only some axons showed a positive immunoreaction for E-N-CAM. Ensheathing cells in the olfactory nerve were observed to bear some labeling for N-CAM total, L1, and N-CAM180, but not E-N-CAM. In the olfactory bulb, L1 was not present on glial cells. In contrast, N-CAM180 was detectable on some glia and N-CAM total on virtually all glia. Glia in the nerve fiber layer were labeled by E-N-CAM antibody only at the external glial limiting membrane. In the glomerular layer, E-N-CAM expression was particularly pronounced at contacts between olfactory axons and target cells. The presence of E-N-CAM in the adult olfactory epithelium and bulb was confirmed by Western blot analysis. The continued presence of E-N-CAM in adulthood on neuronal precursor cells, a subpopulation of olfactory axons, glial cells at the glia limitans, and contacts between olfactory axons and their target cells indicates the retention of embryonic features in the mammalian olfactory system, which may underlie its remarkable regenerative capacity.     

Biochemical and functional characterization of a novel neuron-glia adhesion molecule that is involved in neuronal migration

Adhesion molecule on glia (AMOG) is a novel neural cell adhesion molecule that mediates neuron-astrocyte interaction in vitro. In situ AMOG is expressed in the cerebellum by glial cells at the critical developmental stages of granule neuron migration. Granule neuron migration that is guided by surface contacts between migrating neurons and astroglial processes is inhibited by monoclonal AMOG antibody, probably by disturbing neuron-glia adhesion. AMOG is an integral cell surface glycoprotein of 45-50-kD molecular weight with a carbohydrate content of at least 30%. It does not belong to the L2/HNK-1 family of neural cell adhesion molecules but expresses another carbohydrate epitope that is shared with the adhesion molecules L1 and myelin-associated glycoprotein, but is not present on N-CAM or J1.     

Glycoconjugates are stage- and position-specific cell surface molecules in the developing olfactory system, 2: Unique carbohydrate antigens are topographic markers for selective projection patterns of olfactory axons.

Three monoclonal antibodies specific for different carbohydrate antigens were used to analyze the development of the olfactory system in rats. CC2 antibodies react with a subset of main olfactory neurons, their axons, and terminals in the olfactory bulb. CC2 antigens are expressed on dorsomedial neurons in the olfactory epithelium (OE) from embryonic (E) day 15 to adults. In the olfactory bulb (OB), only dorsomedially located glomeruli express CC2 glycoconjugates from postnatal day (P) 2 to adults. Thus CC2 defines a dorsomedially organized projection that is established early in embryonic development and continues in adults. P-Path antibodies react with antigens that are expressed on the olfactory nerve in embryos, and are also detected on cell bodies in the neuroepithelium and in glomeruli of the OB at P2. At P14, P-Path staining is weaker, but remains present on many cells in the epithelium and in many glomeruli in the bulb. Postnatally, P-Path immunostaining continues to decrease in most regions of the OE and OB. At P35 and afterwards, only a few P-Path-positive neuronal cells can be detected in the OE. Furthermore, after P35 only two groups of glomeruli in the OB are P-Path immunoreactive. One is situated adjacent to the accessory olfactory bulb (AOB) at the dorsocaudal surface of the OB. The other is adjacent to the AOB at the ventrocaudal surface of the OB. Thus, in adults, P-Path glycoconjugates are expressed in neurons and axons that project only to a specific subset of caudal glomeruli of the OB. Monoclonal antibody 1B2, reacts with beta-galactose-terminating glycolipids and glycoproteins. At P2, 1B2 immunoreactivity is seen on a subset of cell bodies that are distributed throughout the OE and is expressed in most glomeruli in the OB at this age. By P35 and in adults, 1B2 continues to be expressed on a subset of neurons in the OE that project to only a small subset of glomeruli in the OB. Unlike CC2 and P-Path antigens that define specific groups of glomeruli, 1B2-immunoreactive glomeruli do not have a detectable spatial pattern. It is more likely that 1B2 antigens define a specific stage in the maturation of connections between the OE and OB.     

Glycoconjugates are stage- and position-specific cell surface molecules in the developing olfactory system, 2: Unique carbohydrate antigens are topographic markers for selective projection patterns of olfactory axons

Three monoclonal antibodies specific for different carbohydrate antigens were used to analyze the development of the olfactory system in rats. CC2 antibodies react with a subset of main olfactory neurons, their axons, and terminals in the olfactory bulb. CC2 antigens are expressed on dorsomedial neurons in the olfactory epithelium (OE) from embryonic (E) day 15 to adults. In the olfactory bulb (OB), only dorsomedially located glomeruli express CC2 glycoconjugates from postnatal day (P) 2 to adults. Thus CC2 defines a dorsomedially organized projection that is established early in embryonic development and continues in adults. P-Path antibodies react with antigens that are expressed on the olfactory nerve in embryos, and are also detected on cell bodies in the neuroepithelium and in glomeruli of the OB at P2. At P14, P-Path staining is weaker, but remains present on many cells in the epithelium and in many glomeruli in the bulb. Postnatally, P-Path immunostaining continues to decrease in most regions of the OE and OB. At P35 and afterwards, only a few P-Path-positive neuronal cells can be detected in the OE. Furthermore, after P35 only two groups of glomeruli in the OB are P-Path immunoreactive. One is situated adjacent to the accessory olfactory bulb (AOB) at the dorsocaudal surface of the OB. The other is adjacent to the AOB at the ventrocaudal surface of the OB. Thus, in adults, P-Path glycoconjugates are expressed in neurons and axons that project only to a specific subset of caudal glomeruli of the OB. Monoclonal antibody 1B2, reacts with β-galactose-terminating glycolipids and glycoproteins. At P2, 1B2 immunoreactivity is seen on a subset of cell bodies that are distributed throughout the OE and is expressed in most glomeruli in the OB at this age. By P35 and in adults, 1B2 continues to be expressed on a subset of neurons in the OE that project to only a small subset of glomeruli in the OB. Unlike CC2 and P-Path antigens that define specific groups of glomeruli, 1B2-immunoreactive glomeruli do not have a detectable spatial pattern. It is more likely that 1B2 antigens define a specific stage in the maturation of connections between the OE and OB.     

Heterogeneity in olfactory neurons in mouse revealed by differential expression of glycoconjugates

Cell surface glycoconjugates have been implicated in the growth and guidance of subpopulations of primary olfactory axons. While subpopulations of primary olfactory neurons have been identified by differential expression of carbohydrates in the rat there are few reports of similar subpopulations in the mouse. We have examined the spatiotemporal expression pattern of glycoconjugates recognized by the lectin from Wisteria floribunda (WFA) in the mouse olfactory system. In the developing olfactory neuroepithelium lining the nasal cavity, WFA stained a subpopulation of primary olfactory neurons and the fascicles of axons projecting to the target tissue, the olfactory bulb. Within the developing olfactory bulb, WFA stained the synaptic neuropil of the glomerular and external plexiform layers. In adults, strong expression of WFA ligands was observed in second-order olfactory neurons as well as in neurons in several higher order olfactory processing centres in the brain. Similar, although distinct, staining of neurons in the olfactory pathway was detected with Dolichos biflorus agglutinin. These results demonstrate that unique subpopulations of olfactory neurons are chemically coded by the expression of glycoconjugates. The conserved expression of these carbohydrates across species suggests they play an important role in the functional organization of this region of the nervous system.     

Characterization of epithelial domains in the nasal passages of chick embryos: spatial and temporal mapping of a range of extracellular matrix and cell surface molecules during development of the nasal placode

The formation of the nasal passages involves complex morphogenesis and their lining develops a spatially ordered pattern of differentiation, with distinct domains of olfactory and respiratory epithelium. Using antibodies to the neural cell adhesion molecule (N-CAM), keratan sulphate and heparan sulphate proteoglycan (HSPG) and a panel of lectins (agglutinins of Canavalia ensiformis (ConA), Dolichos biflorus (DBA), peanut (PNA), Ricinis communis (RCA1), soybean (SBA), Ulex europaeus (UEA1), and wheatgerm (WGA], we have documented cell surface characteristics of each epithelial domain. Binding of antibodies to N-CAM and to keratan sulphate, and the lectins ConA, PNA, RCA1, SBA and WGA marks the olfactory epithelial domain only. The restriction of N-CAM to the sensory region of the epithelium has also been reported in the developing ear. This striking similarity is consistent with the idea that N-CAM may be involved in the division of functionally and histologically distinct cell groups within an epithelium. We traced the olfactory-specific cell markers during development to gain insights into the origin of the epithelial lining of the nasal passages. All reagents bind at early stages to the thickened nasal placode and surrounding head ectoderm and then become progressively restricted to the olfactory domain. The expression of these characteristics appears to be modulated during development rather than being cell autonomous. The distribution of keratan sulphate was compared with collagen type II in relation to the specification of the chondrocranium. Keratan sulphate and collagen type II are only colocalized at the epithelial-mesenchymal interface during early nasal development. At later stages, only collagen type II is expressed at the interface throughout the nasal passages, whereas keratan sulphate is absent beneath the respiratory epithelium.     

Polysialic acid is associated with sodium channels and the neural cell adhesion molecule N-CAM in adult rat brain.

We have studied alpha 2,8-linked polysialic acid (polySia) and the neural cell adhesion molecule (N-CAM) in the adult rat brain by immunohistochemistry and Western blot analysis. Both molecules were widely distributed but not ubiquitous. Various brain regions showed colocalization of polySia and N-CAM. Strong immunoreactivity for polySia was seen in regions which were negative for N-CAM, such as the main and accessory olfactory bulbs. Immunohistochemical evidence for the heterogeneity of polySia expression in different brain regions was confirmed by immunoblotting. We present evidence that N-CAM is not the only polySia bearing protein in adult rat brain. Specifically, immunoprecipitation using the polySia-specific monoclonal antibody mAb 735 precipitated not only N-CAM isoforms carrying polySia, but also the sodium channel alpha subunit. Immunoblotting using sodium channel alpha subunit antibody (SP20) revealed a smear from 250 kDa upwards. PolySia removal using an endoneuraminidase specific for alpha 2,8-linked polysialic acid of 8 or more residues long, reduced this smear to a single band at 250 kDa. Thus both N-CAM and sodium channels carry homopolymers of alpha 2,8-linked polysialic acid in adult rat brain.     

Individual neural cell types express immunologically distinct N-CAM forms

The neural cell adhesion molecules, or N-CAMs, are a group of structurally and immunologically related glycoproteins found in vertebrate neural tissues. Adult brain N-CAMs have apparent molecular weights of 180,000, 140,000, and 120,000. In this article we identify, using monoclonal antibody (Mab) 3G6.41, an immunologically distinct adult rat N-CAM form and show that this form is selectively expressed by some clonal neural cell lines. Consecutive immunoprecipitation experiments indicate that rabbit anti-N-CAM can remove from solubilized cerebellar neuron primary cultures all 180,000- and 140,000-mol-wt N-CAM molecules that react with Mab 3G6.41. However Mab 3G6.41 cannot remove all N-CAM molecules that react with rabbit anti-N-CAM. Rabbit anti-N-CAM binds to and immunoprecipitates N-CAM forms from the rat neuronal cell lines B35, B65, and B104, the glial lines B12 and C6, and L6 myoblasts. Mab 3G6.41 does not bind to or immunoprecipitate N-CAM from the B12 and B65 lines but does react with the other four lines by both criteria. Many cells in primary cultures of postnatal rat that express glial fibrillary acidic protein also bind Mab 3G6.41. Thus a unique form of rat N-CAM recognized by Mab 3G6.41 is found on some but not all neuronal, glial, and muscle cells.     

Ultrastructural evidence for multiple mucous domains in frog olfactory epithelium

Summary This study showed that the olfactory mucus is a highly structured extracellular matrix. Several olfactory epithelial glycoconjugates in the frog Rana pipiens were localized ultrastructurally using rapid-freeze, freeze-substitution and post-embedding (Lowicryl K11M) immunocytochemistry. Two of these conjugates were obtained from membrane preparations of olfactory cilia, the glycoproteins gp95 and olfactomedin. The other conjugates have a carbohydrate group which in the olfactory bulb appears to be mostly on neural cell-adhesion molecules (N-CAMs); in the olfactory epithelium this carbohydrate is present on more molecules. Localization of the latter conjugates was determined with monoclonal antibodies 9-OE and 5-OE. Ultrastructurally all antigens localized in secretory granules of apical regions of frog olfactory supporting cells and in the mucus overlying the epithelial surface, where they all had different, but partly overlapping, distributions. Monoclonal antibody 18.1, to gp95, labeled the mucus throughout, whereas poly- and monoclonal anti-olfactomedin labeled a deep mucous layer surrounding dendritic endings, proximal parts of cilia, and supporting cell microvilli. Labeling was absent in the superficial mucous layer, which contained the distal parts of the olfactory cilia. Monoclonal antibody 9-OE labeled rather distinct areas of mucus. These areas sometimes surrounded dendritic endings and olfactory cilia. Monoclonal antibody 5-OE labeled membranes of dendritic endings and cilia, and their glycocalyces, and also dendritic membranes.