Coexpression of lactosyl and gangliotetraosyl gangliosides in rat cerebellar radial glial cells in culture

The expression of gangliosides of the lactosylceramide (LC) and of the gangliotetraosylceramide (GTC) series on the surface of cells from rat embryonic cerebellar tissue was investigated by double-color indirect immunofluorescence. GD3 was assumed to be representative of LC and was detected using a specific monoclonal antibody. GM1 was assumed to be representative of GTC and was detected using the binding of cholera toxin followed by the binding of cholera toxin antibodies. The expression of polysialosylated GTC (polysialosyl-GTC) was detected using the cholera toxin-cholera toxin antibody experimental approach after conversion of polysialosyl-GTC to GM1 by treatment of the cells with neuraminidase. To distinguish the major neural cell types present in the cultures the expression of the following cell type-specific markers was investigated: neuron-specific enolase and microtubule-associated protein-2 (MAP-2) as probes for neuronal cells and the intermediate filament protein glial fibrillar acidic protein (GFAP) as a probe for astroglial cells. More than 80% of cells dissociated from cerebellar tissue of 15-day-old rat embryos (E15) are positive for the expression of GD3 and about 50% for the expression of GM1 and polysialosyl-GTC, but most are negative for the expression of neuron-specific enolase, MAP-2, and GFAP. After culturing for 4 days (E15 + 4) most cells that show characteristics of neuronal cells are positive for the expression of polysialosyl-GTC and "inactivate" the expression of GD3. Most cells with characteristics of radial and stellate glial cells are also positive for the expression of polysialosyl-GTC, but unlike neuron-like cells, they do not "inactivate" the expression of GD3.     

Neuroimmunology of gangliosides in human neurons and glial cells in culture

Gangliosides (sialic-acid-bearing glycolipids) have received attention in recent years because of their role in cell recognition phenomena, synaptic transmission, memory generation, and nerve regeneration in the fields of neurosciences. It is suggested that each brain region or each neural cell type may contain a specific and characteristic set of gangliosides. We have investigated the immunocytochemical localization of several classes of gangliosides that include GM1, GM4, GD3, and GQ gangliosides on the cell surface of various cell types found in human neural cell cultures with antibodies specific for these gangliosides. Cell cultures were obtained from adult human brains and fetal human dorsal root ganglia and spinal cord and cultured in vitro for the period up to 6 months and utilized for the ganglioside immunocytochemistry. It was demonstrated that GM1 ganglioside was present in all galactocerebroside-positive oligodendrocytes and most of glial fibrillary acid protein (GFAP)-positive astrocytes (80%), most of neurofilament-positive neurons (80%), 50-70% of Schwann cells, and 5-10% of fibronectin-positive fibroblasts; GM4 ganglioside could be detected in all oligodendrocytes, 80% of astrocytes, and 50% of Schwann cells, while no staining was found in neurons or fibroblasts; GD3 ganglioside was present in all oligodendrocytes and 5-10% of astrocytes but not in neurons, Schwann cells, or fibroblasts; and all of fetal CNS neurons and approximately 80-90% of fetal dorsal root ganglia (DRG) neurons and a small percentage of astrocytes (10-20% in fetal and less than 1% in adult astrocytes) was labeled by A2B5 antibody which is specific for GQ ganglioside, while this antibody did not stain cell surface of oligodendrocytes, Schwann cells, or fibroblasts. Three classes of gangliosides, GM1, GM4, and GD3 were found to be definite components of fetal and adult human oligodendroglial plasma membrane, while GM1 and GM4 gangliosides were detected on the surface of most astrocytes. Only a minor population of astrocytes from both fetal and adult human CNS contained GD3 and GQ gangliosides. Two classes of gangliosides, GM1 and GQ, were detected on the surface of fetal human neurons. More than half of fetal Schwann cells reacted to GM1 and GM4 antibodies but did not to GD3 or GQ antibodies. We recognized the presence of a specific and characteristic set of gangliosides on the cell surface of different human neural cell types and these findings should facilitate further investigation of the precise biological activity of these gangliosides.     

Reversibility of ganglioside effects on astrocyte morphology

The B-subunit of cholera toxin (BCT) induces a morphological change in cultured rat cerebral astrocytes from flat (epithelioid) to stellate (process-bearing). This stellation is reversed by the gangliosides GM1 and GD1a at concentrations of 10 microM or higher. Upon changing to a ganglioside-free medium, the flat astrocytes reacquire the stellate morphology within 3 hr, indicating that the antistellation effect of gangliosides is reversible. The possibility that this reversibility was due to a loss of exogenously acquired gangliosides from the cell membrane can be ruled out since pretreatment with GM1, but not GD1a, which does not bind BCT, results in an increased responsiveness to BCT, which was identical whether measured immediately after withdrawal of the ganglioside or 3 hr later. Asialo-GM1, which neither binds BCT nor reverses BCT-induced stellation by itself, prevents the return to stellation after withdrawal of the gangliosides. These data suggest that while gangliosides remain associated with the cell, their effect on astrocytes can change from opposing to permitting the stellate morphology.     

Correlation of gangliotetraose gangliosides with neurite forming potential of neuroblastoma cells.

Gangliosides of 11 different neuroblastoma cell lines, grown to confluence, were extracted and quantified with respect to: (a) total lipid-bound sialic acid, (b) total gangliotetraose family, and (c) GM1 content. The cultured cells were induced to grow neurites in 3 ways: (a) serum reduction, (b) exogenous ganglioside, and (c) retinoic acid. Neurite outgrowth was quantified in terms of % of cells bearing neurites and average number of neurites per cell. No correlation was observed between neurite outgrowth and total ganglioside concentration, but a reasonably good correlation was observed with respect to neuritogenesis and gangliotetraose content. When exogenous ganglioside was the stimulant the best correlation was with GM1, whereas retinoic acid-stimulated outgrowth was approximately proportional to GD1a content. The 'neurite minus' N1A-103 line, which had the lowest level of GM1, GD1a, and total gangliotetraose gangliosides, showed little if any response to any of the stimuli.     

EGF-induced neuritogenesis and correlated synthesis of plasma membrane gangliosides in cultured embryonic chick CNS neurons

Epidermal growth factor (EGF), over a low range of concentrations (165-825 pM), induced neuritogenesis in post-mitotic chick CNS precursor neurons cultured in a serum-free medium, without the addition of other growth factors. Antibody to EGF blocks the neurite-promoting activity of EGF. Similarly, neuritogenesis of cultured chick CNS neurons in medium supplemented with 20% fetal bovine serum is blocked by antibody to EGF, even though serum may contain other neuronotrophic bioactive proteins and steroids. Quantitatively, the only major gangliosides of the undifferentiated post-mitotic neurons are GD3 and GD2. GD3 as well as its biosynthetic precursor, GM3, undergo active biosynthesis in serum-free medium as evidenced by their vigorous labeling by radioactive galactose supplied in the culture medium. When the undifferentiated neurons in serum-free medium are exposed to EGF, the ensuing generation of neurite plasma membrane coincides with initiation of biosynthesis of the sialosyl gangliotetraosyl ceramide species of gangliosides (GD1A, GD1B, GT1B, GQ1B). Antibody to EGF simultaneously inhibits biosynthesis of these gangliosides as well as inhibition of neuritogenesis. These findings indicate that EGF may be a primary neurite-inducing growth factor for post-mitotic embryonic CNS neurons and that gangliosides, particularly those of the sialosyl gangliotetraosyl ceramide species, characterize the plasma membrane of CNS neurons during neuritogenesis.     

Neutral glycolipid and ganglioside composition of type-1 and type-2 astrocytes from rat cerebral hemisphere.

We reported previously that the major gangliosides in primary mixed-type astrocyte cultures are GM3 and GD3. To obtain more information regarding the exact distribution of glycosphingolipids in different types of astrocytes, we established a line of type-1 astrocytes that are characterized by a Ran-2 positive, broad flat morphology, and by the absence of binding to A2B5 antibodies. We also purified O-2A progenitor cells by immunopanning and cultured them in the presence of 10% newborn calf serum. They differentiated into type-2 astrocytes that were identified by immunostaining for each of GD3, A2B5, and GFAP. Using these cell cultures, we demonstrate that the major gangliosides were GM3 in type-1 astrocytes and GM3 and GD3 in type-2 astrocytes. In addition, a set of neutral glycolipids was identified based on the HP-TLC migration properties of CMH, CDH, CTH, and Glob, but the component distribution of these glycolipids is related to that of glycolipids of astrocytes. A marked increase in the expression of CTH and Glob was shown in type-2 astrocytes. The amount of neutral glycolipid-sugar was higher in the type-2 astrocytes than in the type-1 astrocytes. These results suggest that the increase in the total glycosphingolipid content and the change in the neutral glycolipid composition produced by type-2 astrocytes may be related to their biological functions and the cellular compositions.     

Expression of gangliosides on glial and neuronal cells in normal and pathological adult human brain

Few studies have assessed the glycolipid phenotype of glial cells in the human central nervous system (CNS) in situ. We investigated by immunohistochemistry the expression and cellular distribution of a panel of gangliosides (GM1, GM2, acetyl-GM3, GD1a, GD1b, GD2, GD3, GT1b, GQ1b and the A2B5 antibody) in adult, human normal and pathological brain, namely multiple sclerosis (MS) and other neurological diseases (OND). In normal conditions, we found diffuse expression in the white matter of most gangliosides tested, with the exception of acetyl-GM3, GT1b and GQ1b. By double immunofluorescence with phenotypic markers, GM1 and GD1b were preferentially expressed on GFAP+ astrocytes, GD1a on NG2+ oligodendrocyte precursors, A2B5 immunostained both populations, while GD2 was selectively present on mature oligodendrocytes. In the gray matter, only GM1, GD2 and A2B5 were present on neuronal cells. Interestingly, those gangliosides present on astrocytes in normal conditions were preferentially expressed on NG2+ cells in chronic MS lesions and in OND. Selective expression of GT1b upon astrocytes and NG2+ cells was instead observed in MS lesions, but not in OND. The definition of the glycolipid phenotype of CNS glial cells may be useful to identify distinct biological glial subsets and provide insights on the potential autoantigenic role of gangliosides in CNS autoimmune diseases.     

Morphological modulation of cultured rat brain astroglial cells: antagonism by ganglioside GM1

Secondary cultures of neonatal rat astroglial cells, maintained in a serum-free, chemically defined medium were treated with several agents thought to activate cyclic AMP-synthesizing systems. Dibutyryl cyclic AMP (dBcAMP), forskolin and cholera toxin promoted, within 2 h, the near-complete conversion of 1-day-old (D1) astroglial cells from a flat, epithelioid morphology to a stellate (star-shaped) morphology. With all 3 agents, cell susceptibility to morphological change declined with culture age, 5-day-old cultures failing to respond altogether. D1 cultures, after 48 h of treatment, had reverted to the flat morphology. Gangliosides reported to stimulate adenylate cyclase were also tested, using purified GM1 X GM1 failed to stimulate the conversion to stellate morphologies. GM1, however, did affect these astroglial cells by causing a block or reversal of their morphological response to dBcAMP, forskolin or cholera toxin. The GM1 response was specific for the intact ganglioside molecule, asialo GM1 and sialic acid having no effect. Gangliosides GD1a, GD1b and GT1b were also active, being effective at ca. 4-fold lower concentrations. The response to GM1 appeared to involve a direct interaction with the astroglial cell, rather than influencing either substratum or medium components.     

Ganglioside potentiation of NGF-independent conditioned medium enhancement of neuritic outgrowth from spinal cord and ciliary ganglia explants

Culture medium conditioned (CM) by embryonic chick skeletal muscle or RN22 Schwannoma cells enhanced dramatically the neuritic development of chick embryonic spinal cord slices explanted onto a collagen substratum. The addition of a mixture of bovine brain gangliosides (BBG) or the monosialoganglioside GM1 to this medium potentiated the nerve growth factor (NGF)-independent CM-mediated neuritogenesis. A 3-4 fold increase in spinal cord outgrowth was due to increased neurite number, length and branching. The ability of the gangliosides to potentiate the positive neuritogenic action of CM was not limited solely to spinal cord cultures since similar results were obtained in parallel studies employing organized cultures of embryonic chick ciliary ganglia. These studies demonstrate the ability of gangliosides to enhance the trophic action of factor(s) present in CM. They suggest further that gangliosides may play a modulatory role in the development of the nervous system.     

Immunostaining of ganglioside GD1b, GD3 and GM1 in rat cerebellum: cellular layer and cell type specific associations

We have studied the cellular distribution of gangliosides GD1b, GD3 and GM1 in rat cerebellum by immunostaining, using monoclonal antibodies and confocal microscopy. Antibodies against astroglial, neuronal and synaptic vesicle associated molecules were used for colocalization analyses. In the gray matter, the anti-GD1b antibody stained thin strands in the molecular layer (ML), interpreted as Bergman glia fibers based on colocalized staining with anti-glial fibrillary acidic protein (GFAP). The neuropil in the granule (GL) and Purkinje (PL) cell layers was also anti-GD1b positive. The anti-GD3 antibody stained the ML, the neuropil in the GL and PL and also the granule and Purkinje cell bodies, appearing intracytoplasmically and vesicle associated. Anti-GD1b and anti-GD3 staining in the GL glomeruli were colocalized with anti-synaptophysin staining. The anti-GM1 antibody stained cell bodies in the ML but they could not be characterized in colocalization experiments. The GL and PL were not stained with the anti-GM1 antibody. In the white matter, different staining patterns were seen for the gangliosides, the anti-GM1 staining being the most intense. This study shows cellular layer and cell type specific associations of the investigated gangliosides and localization of GD1b and GD3 at synaptic sites, warranting further studies on their role in synaptic mechanisms.     

Influence of monensin on ganglioside anabolism and neurite stability in cultured chick neurons

We have examined the effects of monensin, a monovalent cationophore that disrupts exo- and endocytosis of membrane vesicles and diminishes Golgi anabolic function, on the incorporation of [3H]-galactose into glycosphingolipids in neurited primary cultures of chick embryo central nervous system neurons. A linear rate of incorporation into all ganglioside species from extracellular-labeled galactose was observed. Specific activity of anabolic labeling was markedly lower in GT1b and GQ1b than in the other major gangliosides of the embryonic neuron (GM3, GD3; GM2, GD2; GM1, GD1a, GD1b). With 100 nM monensin in the extracellular medium, the rate of labeling of GT1b diminished markedly to 20% of control; GD1a, GD1b, and GD2, to 35%; GQ1B to 48%; GD3 to 60%. Vigorous incorporation of label into GM3 was entirely undiminished by monensin. From these findings, it is suggested that ganglioside biosynthesis is compartmentalized in the cytodifferentiating embryonic neuron, with GM3 entirely, and GD3 and GQ1b partially, an extra-Golgi product. Extensive loss of neurites that occurred after several hours of exposure of the neurons to monensin could not be correlated directly with decreased ganglioside anabolism.     

Gangliosides alter morphology and growth of astrocytes and increase the activity of choline acetyltransferase in cultures of dissociated septal cells

Administration of gangliosides has been reported to stimulate regeneration of motoneurons and of central dopaminergic and cholinergic neurons. To shed light on the mechanism by which gangliosides mediate the effects on cholinergic neurons, we studied their actions on cultures of cells dissociated from the septal area of fetal rat brains. These cultures contain cholinergic neurons, which, in vivo, give rise to the cholinergic septo-hippocampal pathway. Gangliosides produced prominent changes in the morphological appearance of the cultures. In contrast to control cultures, which contained many process-bearing cells and a confluent layer of flat cells, there were no flat cells in cultures grown in the presence of gangliosides (0.2 to 0.8 mg/ml of medium). Using immunocytochemical visualization of the astrocytic marker glial fibrillary acid protein, it was shown that all astrocytes in cultures grown in the presence of gangliosides exhibited the morphology of process-bearing cells, whereas in control cultures astrocytes represented the majority of the flat cells. Furthermore, gangliosides attenuated astrocytic proliferation. The effects of gangliosides apparently were not mediated by cAMP, since they could be differentiated from actions of forskolin, an activator of adenylate cyclase. Astrocytic growth and morphology were affected by ganglioside mixtures of various sources and composition and also by the pure gangliosides GM1 and GD1a, whereas lipid and carbohydrate components of gangliosides were ineffective. In contrast to the prominent effects on astrocytes, gangliosides failed to significantly alter survival or fiber growth of cholinergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibodies to GM1(NeuGc) in Guillain-Barré syndrome after ganglioside therapy

N-Glycolylneuraminic acid-containing GM1 [GM1(Gc)] is a molecule for serum antibodies in patients with Guillain-Barré syndrome (GBS). To clarify the pathogenesis of GBS after treatment with bovine brain ganglioside, we investigated the presence of anti-GM1(Gc) antibody in patients who developed GBS after ganglioside injection. Serum samples were taken from nine Italian patients with GBS after ganglioside therapy as well as from untreated Italian (n=30) and Japanese (n=131) GBS patients. Bovine brain gangliosides fractionated in a column were used as antigens, and binding of serum IgG or IgM was examined. An absorption study of IgG anti-GM1(Gc) antibody was made with GM1, asialo-GM1, GM2, GD1a, and GD1b. Four of the nine patients who developed GBS after being administered gangliosides had IgG anti-GM1(Gc) antibodies. Anti-GM1(Gc) IgG antibody frequencies were higher in patients with GBS after ganglioside therapy than in those who were untreated. Rates of absorption of IgG anti-GM1(Gc) antibodies by GM1 were significantly higher (except for asialo-GM1 and GD1b) than by GM2 and GD1a. The presence of GM1(Gc) was confirmed in bovine brain immunochemically using cholera toxin and Hanganutziu-Deicher antibody. Secondary ion mass spectra showed that the structure of the ganglioside was consistent with that of GM1(Gc). GM1(Gc) was recognized more frequently in sera from patients who developed GBS after ganglioside therapy than in sera from untreated GBS patients. Because N-glycolylneuraminic acid-containing gangliosides seem to be highly immunogenic in humans, GM1(Gc) may act as an immunogen in some patients who develop GBS following ganglioside therapy.     

GD3 ganglioside is prevalent in fully differentiated neurons from rat retina

Adult mammalian retinas contain unusually high amounts of GD3, a ganglioside of the lactosylceramide series. In this respect, they differ from adult avian retina and other regions of the adult avian and mammalian brain, where GD3 is a minor ganglioside and gangliosides of the gangliotetraosylceramide series (GM1, GD1a, GD1b, GT1b) are the predominant ones. We compare here the ganglioside patterns of rat, human, horse, and guinea pig retinas, which are known to differ in the degree of vascularization and astrocytic cell content. All these retinas showed a prevalence of pathway "b" gangliosides over pathway "a" gangliosides but showed no correlation between GD3 content and the degree of vascularization and astrocytic cell content. Immunostaining of rat retina sections showed the presence of GD3 in the inner and outer plexiform layers and also in the ganglion cell and inner nuclear layers. About 60% of the cells dissociated from rat retina showed immuno-colocalization of GD3 and the neuronal marker class III beta tubulin isotype or cholera toxin binding. All morphologically identifiable glial Muller cells coexpress GD3 and gangliotetraosylgangliosides. GD3 was a minor ganglioside among these axonally transported by ganglion cells in rats and guinea pigs, suggesting that it is either not synthesized by ganglion cells or, if so, it is restricted to the cell soma and/or dendritic tree. Our results demonstrate that, unlike neurons from avian retina and other regions of avian and mammalian brain, neurons from mammalian retina not only contain gangliosides of the gangliotetraosylceramide series but also keep a prevalence of gangliosides of the lactosylceramide series (GD3) when they are fully differentiated.     

Intracellular sulfatide expression in a subpopulation of astrocytes in primary cultures

A highly specific antibody against sulfatide, a myelin-associated glycolipid, has been investigated using indirect double immunocytochemistry in rat primary astroglial cultures from cerebral cortex. Sulfatide was expressed in a selected subpopulation of astrocytes (2–3%) and was found to be exclusively located intracellularly. The sulfatide-positive cells appeared in two different morphologies: flat and stellate. Immunolabeling of the astroglial cultures showed that sulfatide always co-existed with GFAP or S-100, and in some cells with GD3 (flat 90% and stellate 50%) or A2B5 (1%) antibody. The sulfatide-positive cells did not bind the O1 antibody, which is used as a marker for oligodendrocytes. Glial cultures from other regions and mixed cultures, with both neurons and glial cells, were examined and showed similar results. Biochemical analysis by TLC-ELISA verified the presence of sulfatide in the astroglial culture and showed decreasing amounts of sulfatide with days in vitro; 0.05 nmol/mg protein at day 10 and 0.01 nmol/mg protein at day 17. This analysis also showed that neither sulpholactosylceramide nor seminolipid was present, each of which also has affinity for the sulfatide antibody. This selective and intracellular expression encourages further identification of the astrocytes expressing sulfatide and the biological role of sulfatide in these cells. J. Neurosci. Res. 52:559–568, 1998. © 1998 Wiley-Liss, Inc.     

The glioma-associated gangliosides 3'-isoLM1, GD3 and GM2 show selective area expression in human glioblastoma xenografts in nude rat brains

This work describes the in vivo expression and distribution of glioma-associated gangliosides (GD3, GM2, 3'-isoLM1) in a novel human brain tumour nude rat xenograft model. In this model, the tumours, which are established directly from human glioblastoma biopsies, show extensive infiltrative growth within the rat brain. This model therefore provides an opportunity to study ganglioside expression not only within the macroscopic tumour, but also in brain areas with tumour cell infiltration. The ganglioside expression was studied by confocal microscopy of immunostained brain sections using antiganglioside monoclonal antibodies. Xenografts from four human glioblastoma multiformes were established in rats and the brains removed after 3-4 months. Ganglioside GD3 was expressed in the tumour parenchyma while ganglioside 3'-isoLM1 was more abundantly expressed in the periphery of the tumour associated with areas of tumour cell invasion. GM2 expression was only seen in one tumour, where it was located within the main tumour mass. Double staining with a pan antihuman monoclonal antibody (3B4) and the antiganglioside monoclonal antibodies confirmed that the ganglioside expression was associated with tumour cells. This work supports the concept of different biological roles for individual gangliosides and indicates that antibodies or ligands directed against GD3 and 3'-isoLM1 might be complementary when applied in the treatment of human glioblastomas.     

Astrocytes cultured from mature brain derive from glial precursor cells

We have previously shown that enriched preparations of oligodendrocytes from either mature bovine brain or 30-d-old rat brain, when cultured in serum-free medium, yield mixed cultures of oligodendrocytes and astrocytes even though no GFAP+ cells were present after 24 hr in culture (Norton et al., 1986, 1988). To test the possibility that the astrocytes in these cultures arose from glial precursor cells, we followed the expression of ganglioside GD3, galactosylceramide (GC), glial fibrillary acidic protein (GFAP), and vimentin in the cultures. GD3 has already been shown to be a marker of immature neuroectodermal cells, which in the postnatal brain are glial progenitor cells (Goldman et al., 1984, 1986). The cultures from both species contained at 1 DIV only two populations of cells; 90-95% GC+/GD3- oligodendrocytes and 4-10% GD3+/GC- small, round cells. With time, the oligodendrocytes remained GD3-/GFAP-/vimentin-. The kinetics of antigen expression of the GD3+ cells could best be interpreted by the following sequence: (sequence; see text) We interpret these results to show that the astrocytes arose from a small population of GD3+ glial precursor cells present in the brain that were co-isolated with oligodendroglia. No evidence was obtained that these GD3+ cells could also differentiate into oligodendrocytes.     

Immunocytochemical analysis of gangliosides in rat primary cerebellar cultures using specific monoclonal antibodies

We studied the expression of ganglioside antigens in primary cultures of rat cerebellum using an immunocytochemical technique with mouse monoclonal antibodies (MAbs) specific for various gangliosides. Twelve MAbs that specifically recognize each ganglioside were used. Our study revealed that there is a cell type-specific expression of ganglioside antigens in the primary cultures. A number of b-series gangliosides were detected in the granule cells, whereas a-series gangliosides were not intensely expressed. GD1b was detected in the granule cells. GD2 appeared to be present in a subset of the granule cells or a type of small neurons. GD3 was associated not only with the granule cells, but also with both astrocytes and oligodendrocytes. An O-Ac-disialoganglioside, which was suggested to be O-Ac-LD1, was restrictedly detected in Purkinje cells. The other gangliosides were not detected clearly in these cells. These results suggest that several gangliosides may be useful markers for identifying cells in primary cultures of the rat cerebellum; particularly b-series gangliosides such as GD2 and GD1b for the granule cells and O-Ac-LD1 for Purkinje cells.     

Developmental changes of ganglioside expressions in postnatal rat cerebellar cortex

We previously described the differential distribution of gangliosides in adult rat brain as detected by specific antibodies. We report here the distribution of gangliosides during the development of postnatal rat cerebellum by an immunofluorescence technique with mouse monoclonal antibodies (mAbs). Eleven mAbs that specifically recognize each ganglioside were used. Our study revealed that the expression of each ganglioside changed dramatically during the development. GD3 and O-Ac-GD3 were expressed intensely in the external granular layer at 1, 5, and 10 days, whereas GD2 was firstly detected in the internal granular layer at 5 days and GD1b was diffusely detected throughout all layers of the cerebellar cortex at early postnatal days. GD2 and GD1b were more intensely expressed in the granular layer at 20, 30, and 80 days, suggesting that premature granule cells express GD3 and its derivative, O-Ac-GD3, whereas mature granule cells express GD2 and GD1b intensely. On the other hand, GM1 was exclusively detected in the external granular layer and the molecular layer at 1 and 5 days. The staining sites spread gradually from these outer layers into the internal granular layer and the white matter after 10 days. The positive cells in the external granular layer and the molecular layer appeared to be Bergmann glial cells and their radially ascending cytoplasmic processes. The intensity of the staining in these specialized astroglial cells decreased gradually during postnatal days. In contrast, the expression of GQ1b was very faint at birth, but gradually increased during the development and was detected intensely in the internal granular layer, particularly in the cerebellar glomeruli in adulthood, suggesting that GQ1b expression may be associated with synapse-related structures. The developmental changes of the expression of other gangliosides were also recognized in the postnatal rat cerebellum. These results suggest that specific gangliosides may play an important role in regulating the early events responsible for the orderly formation of the cerebellar cortex.