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.     

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.     

Uniform distribution and similar turnover rates of individual gangliosides along axons of retinal ganglion cells in the chicken

In 5-month-old chickens, an intracranial injection of N-[³H]acetylmannosamine led to a labelling of all optic lobe ganglioside species in a fashion parallelling the relative ganglioside distribution. In contrast, after an intraocular injection of the same precursor, the optic nerve and the optic lobe connected to the injected eye, possessed an exceptionally high labelling of GD1a (in comparison with GD1a-sialic acid), and only negligible incorporation of radioactivity into the myelin-specific GM4 and into a fraction migrating close to GM1. Subtracting both these very low labelling fractions from the total gave a percentage distribution of ganglioside sialic acid which now corresponded well to the distribution of radioactivity along the whole optic nerve, including the region of nerve terminals in the optic lobe. This pattern of ganglioside labelling, which indicates that GD1a carries about 60% of total ganglioside sialic acid of retinal ganglion cell axons, did not change remarkably during post-hatching development up to 5 months. Long-time incorporation studies revealed similar turnover rates of the main retinal ganglion cell gangliosides. The average half-lives were 34 (GD1a), 35 (GQ1b), 36.3 (GT1b) and 38.5 days (GD3). The findings suggest that the retinal ganglion cell axons and their presynaptic terminals possess a similar ganglioside pattern, characterized by a high content of GD1a.     

Effect of Purkinje cell loss on cerebellar gangliosides in nervous mutant mice

The distribution of cerebellar gangliosides was studied in adult (73 ± 2 days) nervous (nr/nr) mutant mice which lose 50–90% of their Purkinje cells. This neuronal loss is associated with significant reductions in cerebellar weight and ganglioside concentration. The cerebellar dry weights (mg) and the ganglioside concentrations (μg N-acetylneuraminic acid per 100 mg dry weight) in nr/nr mice and age-matched normal littermates (+/?) are 7.4 ± 0.3 mg and 13.2 ± 0.4 mg; and 411.7 ± 4.8 μg and 438.5 ± 2.1 μg, respectively. Abnormalities were also observed for the concentration of certain ganglioside species. Most notably, GT1a is significantly reduced by 42%, and GD3 is significantly increased by 29% in the nr/nr mice compared to the +/? mice. The nr/nr mice also express a slight but significant reduction in GT1b. No ganglioside abnormalities were observed between the nr/nr and +/? mice in cerebral cortex. We previously found reduced cerebellar GT1a content in other mutants that also lose Purkinje cells, i.e., sg/sg, pcd/pcd, and Lc/+. GT1a is not reduced, however, in wv/wv mice that lose mostly granule cells. The findings in nr/nr mice are therefore consistent with our hypothesis that GT1a is enriched in Purkinje cells. GD1a, which is enriched in mature granule cells, is not reduced in the nr/nr mice. Since we previously found that GD3 is a good marker for reactive glia in neurological disease, the elevated GD3 concentration in the nr/nr mice indicates a mild gliosis. Our findings with nr/nr and the other neurological mutants indicate that gangliosides can be useful as cell-surface markers for monitoring changes in the cytoarchitecture of the mouse cerebellum.     

A gradient molecule in developing rat retina: expression of 9-O-acetyl GD3 in relation to cell type, developmental age, and GD3 ganglioside

In the embryonic and postnatal rat retina a cell surface antigen that is detected by monoclonal antibody JONES is distributed in a dorsoventral gradient. Biochemical analysis has determined that the antigen is a modified ganglioside, 9-O-acetyl GD3. In the present study, the distributions of 9-O-acetyl GD3 and its possible precursor GD3 in developing rat retina were compared immunocytochemically using specific monoclonal antibodies JONES and R24. On embryonic day 13 (E13) immunoreactivity to JONES was localized to central retina; however, R24 stained throughout the retinal epithelium. By E20, when JONES binding was distributed in a gradient along the dorsoventral axis, R24 again stained dorsal and ventral regions with uniform intensity. Analysis of freshly dissociated retinal cells further revealed that GD3 and 9-O-acetyl GD3 expressions do not necessarily coincide. At E15 and postnatal day 2 (PN2), the majority of cells (78 and 92%, respectively) were immunolabeled by antibody to GD3, while between E15 and PN2 the percentage of cells immunolabeled by antibody to 9-O-acetyl GD3 rose from 19 to 68%. By PN4, labeling decreased for both molecules; however, the rate of decline in 9-O-acetyl GD3 labeling was more pronounced. Regulation of the numbers of JONES-positive cells does not appear to depend on interaction with the extraretinal environment, for in neural retina explanted at E15 the proportions of 9-O-acetyl GD3-bearing cells was found to be similar to the percentage observed in neural retina developing to an equivalent age in vivo. Experiments to identify the retinal cell types bearing 9-O-acetyl GD3 revealed that it is expressed by both neurons and glia growing in monolayer cultures of rat perinatal neural retina. Examination of freshly dissociated retinal cells following simultaneous labeling for some specific cell types and 9-O-acetyl GD3 demonstrated that the latter determinant is present on photoreceptor, amacrine, and ganglion cells. For each neuronal cell type, however, not all of the cells were immunoreactive with JONES. We conclude that the differences in the percentages of JONES- and R24-positive cells, and in particular the different rates at which JONES and R24 staining declined with age, indicate that the expression of the JONES epitope is regulated with some independence from parent ganglioside GD3.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differentiation of oligodendrocytes cultured from developing rat brain is enhanced by exogenous GM3 ganglioside

Cultures consisting primarily of O-2A progenitor cells and immature oligodendrocytes with a few microglia and astrocytes were obtained by shaking primary cultures from neonatal rat brain after 12--14 days in vitro. Addition of 50 μg/ml exogenous Neu-NAcα2-3Galβ1-1′ ceramide (GM3 ganglioside) to the cultures resulted in an increase in the number and thickness of cell processes that stained intensely for sulfatide and galactocerebroside (galC) in comparison to control cultures without added GM3. The treated cultures also contained fewer astrocytes than control cultures as revealed by immunostaining for glial fibrillary acidic protein (GFAP). Cells that immunostained for both GFAP and sulfatide/galC were very rare in control cultures but were frequently seen in the GM3-treated cultures, suggesting that these may represent cells changing their direction of differentiation away from type II astrocytes toward oligodendrocytes under the influence of GM3. These effects on the developing rat oligodendrocytes were specific for GM3 ganglioside and were not produced by adding GM1, GM2, GD3, or GD1a to the cultures. Lactosyl ceramide and neuraminyl lactose were also ineffective. When control cultures were initially plated on polylysine and incubated with [¹⁴C]galactose, GD3 was the principal labeled ganglioside. However, as the control cells differentiated over time in culture without the addition of exogenous GM3 and produced increasing amounts of myelin-related components, the incorporation of [¹⁴C]galactose into endogenous GM3 increased to become the predominant labeled ganglioside by 6 days after plating. Metabolic labeling of the GM3-treated oligodendrocytes with [¹⁴C]galactose revealed increased incorporation into galC and sulfatide in comparison to control cultures, but a decreased labeling of endogenous GM3. Similarly, incorporation of an amino acid precursor into the myelin-associated glycoprotein (MAG) was increased by GM3 treatment, but incorporation into myelin basic protein (MBP) was not affected. Although the overall effect of added GM3 was to decrease the phosphorylation of most proteins in the oligodendrocytes, including MBP, GM3 enhanced the phosphorylation of MAG. These findings indicate that GM3 ganglioside has an important role in the differentiation of cells of the O-2A lineage toward myelin production, since differentiation is associated with increased metabolic labeling of endogenous GM3 in control cultures and is enhanced by the addition of exogenous GM3. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Neuritogenic and metabolic effects of individual gangliosides and their interaction with nerve growth factor in cultures of neuroblastoma and pheochromocytoma

The 4 major ganglioside species, GM1, GD1a, GD1b and GT1b (200 micrograms/ml), were tested individually for the ability to stimulate neuronal trophic responses. The growth parameters measured were: morphologic changes, quantitated by computer-assisted morphometry of neurite length and number per soma, and metabolic changes, indicated by alterations in ornithine decarboxylase activity (ODC). In addition, the interaction of each ganglioside with nerve growth factor (NGF) was investigated with an NGF-responsive pheochromocytoma PC12 cell line and NGF-insensitive neuroblastoma Neuro-2a cultures. PC12 cells responded to gangliosides only in the presence of NGF (20 micrograms/ml): GM1 produced the greatest morphologic response, but did not alter metabolic levels; GT1b increased both parameters. The presence (5 micrograms/ml) or absence of NGF did not have an effect on the ganglioside-mediated morphologic responses of Neuro-2a cells to each species: GD1b elicited the greatest increase in neurite length, while GD1a and GT1b stimulated both length and number. In contrast, while GT1b alone was able to elevate ODC activity independently of NGF, the simultaneous exposure of Neuro-2a cultures to NGF and GM1 or GD1a resulted in a stimulation of cellular metabolism. These results indicate that each ganglioside species has a specific target action in the stimulation of different trophic responses and that performance in one category is not a predictor of the result in another. In addition, it is possible to confer a sensitivity to NGF by simultaneous treatment with specific gangliosides. This indicates that membrane gangliosides may modulate the actions of neurotrophic factors.     

Glycosphingolipids in the cerebrospinal fluid of patients with multiple sclerosis

Glycosphingolipids in cerebrospinal fluid (CSF) of individual patients with multiple sclerosis (MS) were analyzed using a glycolipid-overlay technique. The ganglioside composition of CSF of non-MS patients was characterized by an abundance of polysialo species, including GT1b and GQ1b. This pattern is completely different from that of human white or gray matter, in which mono- and disialogangliosides predominate. Increased levels of GM1, either associated with or without increases of other gangliosides, such as GD1a, were observed in 16% of the patients with MS (6 of 37 cases: 1 of 15 progressive progressive stage, 4 of 16 progressive stationary stage, and 1 of 6 relapsing stage). The concentration of GD3 was increased in 23% (3 of 13 cases), whereas 1 of 13 cases (8%) showed a dramatic increase of sulfoglucuronyl paragloboside (SGPG) associated with a high level of GD3. These changes may reflect the cellular changes associated with the known pathological lesions in MS, which are characterized by demyelination, gliosis, and/or remyelination with oligodendrocytic proliferation.     

Antibodies in sera from patients with inflammatory demyelinating polyradiculoneuropathy react with ganglioside LM1 and sulphatide of peripheral nerve myelin

Summary Sera from 23 patients with acute Guillain Barré syndrome (GBS), 15 patients with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and from 40 age-matched blood donors were analysed for antibodies to acidic glycosphingolipids from human brain and peripheral nerve. Antibodies to ganglioside LM1, the major ganglioside of peripheral nerve myelin, were found in 43% of GBS and in 67% of CIDP patients' sera, and in 20% of the blood donors. However, anti-sulphatide antibodies were detected in 65% and 87% of the sera from GBS and CIDP patients, respectively, but only in 15% of the control sera. Sulphatide is the major acidic glycosphingolipid in myelin and its concentration in peripheral nerve myelin is 100 times higher than that of LM1. The high frequency of LM1 and, in particular of sulphatide antibodies, might thus be relevant to the pathogenesis of the GBS and CIDP. Abbreviations: The ganglioside nomenclature used according to Svennerholm [24]. LM1, IV³NeuAc-nLcOse₄Cer, GM1, II³NeuAcGgOse₄Cer; GD1a, IV³NeAc,II³NeuAc-GgOse₄Cer; GD1b, II³(NeuAc)₂-GgOse₄Cer; GT1b, IV³NeuAc,II³(NeuAc)₂-GgOs₄Cer; LU1, sulphate-3-glucuronyl paragloboside; sulphatide, 3-sulphogalacto-sylceramide     

Gangliosides in Neuroblastomas

Neuroblastomas from children presenting with tumors at various ages and different primary sites (abdominal, adrenals, pelvic, and thoracic) were studied. Analysis of the ganglioside patterns of 53 tumors indicated that patients who were either disease positive 2 yr following surgery or dead of disease, had significantly (p<0.005) less GT1b plus GD1b than tumors from patients that were disease free 2 yr post surgery. The presence of GD2 in 45 of the tumors correlates well with the suggestion that it can be used as a marker in neuroblastoma diagnosis. Children with thoracic neuroblastomas have a significantly better prognosis than children with tumors in other anatomic sites. Analysis of the ganglioside composition of these tumors only, indicated that they had a significantly higher (p<0.005) concentration of GT1b and GD1b and a significantly lower concentration (p<0.025) of monosialogangliosides than those patients who were dead of disease or had persistent disease. These results suggest that low levels of GT1b and GD1b correlate with a poor prognosis. The thoracic neuroblastomas may be comprised of more “differentiated” neuroblastoma cells (ganglioside patterns more similar to the CNS), and this may contribute to the fact that about 85% of children with thoracic neuroblastoma recover. To understand why the ganglioside pattern may serve as a prognostic indicator for neuroblastoma, it is necessary to know whether gangliosides have specific roles in neuronal differentiation. Our approach to this question is to compare the effect(s) of added ganglioside or the corresponding oligosaccharide on neuroblastoma cells. Results obtained suggest that the oligosaccharide from GM1 is able to enhance neuritogenesis by S20Y murine neuroblastoma cells to the same extent that GM1 does.     

Effect of colchicine on ganglioside composition of rat primary culture neurons.

Developmental changes in gangliosides in the course of neurite outgrowth were examined in dissociated fetal rat cerebral neurons in culture. About a 2-fold increase in ganglioside levels was seen with the progression of neurite formation for up to 24 h in predominantly neuronal cultures. Ganglioside patterns appeared to be unchanged during the first 24 h, subsequently consisted of higher amounts of GD3 and b-series gangliosides (such as GD1b, GT1b, and GQ1b), and lower amounts of a-series gangliosides (GM1 and GD1a). Although the addition of colchicine to the cell growth medium inhibited neurite outgrowth in developing neurons, little if any differences in ganglioside patterns were found between control and colchicine-treated cells. Ganglioside levels decreased slightly in colchicine-treated cells in agreement with the decrease in cell attachment to culture dishes. Although colchicine treatment 8 h after plating caused complete retraction of formed neurites, the ganglioside level of the cells continued to increase during the following 16-hour incubation. Thus, the data suggest that ganglioside synthesis in differentiating neurons does not primarily accompany the expansion in cell surfaces due to neurite formation, and raises the possibility that a large proportion of gangliosides is retained in intracellular compartments.     

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.     

N-acetylgalactosaminyl GD1a is a target molecule for serum antibody in Guillain-Barré syndrome

Serum antibodies against such major glycolipids as GM1, GD1b, and LM1 have been reported in patients in the acute phase of Guillain-Barré syndrome (GBS). Because minor unidentified glycolipids also may be targets of antibodies in GBS sera, we assayed serum antibody against a crude ganglioside fraction using thin-layer chromatogram immunostaining. Antibody activity was detected against a band that migrated just below GD1a in 6 of the 50 patients with GBS tested. Antibody titer, as determined by enzyme-linked immunosorbent assay, decreased during the course of the disease. All 6 patients had suffered gastrointestinal infection before the neurological onset of GBS and showed low amplitudes for the compound muscle action potentials and normal or only slightly decreased nerve conduction velocities. Thin-layer chromatogram immunostaining did not show this antibody activity in any of the 16 normal and 119 disease controls. The unidentified glycolipid was isolated by DEAE–Sephadex A-25 column chromatography, sialidase treatment, and Iatrobeads column chromatography. Fast atom bombardment-mass spectra showed it to be N-acetyl-galactosaminyl GD1a.     

Disialoganglioside GD2 in human neuroectodermal tumor cell lines and gliomas

Summary Monoclonal antibodies (mAbs) recognizing the disialoganglioside II³(NeuAc)₂GgOse₃Cer (GD2) were produced by immunizing mice with the GD2-expressing neuroblastoma cell line LAN-1 and a prefusion boost with purified GD2 coupled to Salmonella minnesota. Two IgM mAbs were isolated which demonstrated high levels of reactivity (binding ratios in excess of 100) with GD2 by solid-phase radioimmunoassay and positivity in high-performance thin-layer chromatography (HPTLC) immunostain; only one (DMAb-20) was subsequently shown by analysis with a panel of defined ganglioside species to be specific for the minimum epitope of GD2, GalNAc1-4(NeuAc2-8NeuAc2-3)Gal-. DMAb-20 was used to evaluate the expression of GD2 by malignant glioma and medulloblastoma cell lines using cell surface radioimmunoassay, indirect membrane immunofluorescence, HPTLC immunostain, and densitometric analysis of extracted gangliosides from selected cell lines. Sixteen of 20 (80%) malignant glioma and 5 of 5 medulloblastoma cell lines reacted with DMAb-20; in agreement with previous studies, 5 of 5 neuroblastoma and 2 of 3 melanoma cell lines also reacted with DMAb-20. GD2 was proportionally increased in the glioma and medulloblastoma cell lines relative to levels in normal brain, as determined by densitometric analysis. In a phenotypic survey of malignant glioma biopsies, tumor cells in 24 of 30 (80%) cases stained positively with DMAb-20. Reactive astrocytes, both within and adjacent to tumors, were frequently intensely stained. Among the morphological variants of glioblastoma examined, the most intense staining with DMAb-20 was observed in neoplastic gemistocytes, with the weakest or absent staining in small cell glioblastomas. As GD2 is a commonly expressed surface antigen of gliomas and medulloblastomas, expression of which is retained in tissue culture, DMAb-20 will be useful in determining the functional role of GD2 in cell-cell interaction, adhesion, and invasion, and in defining altered growth control mechanisms of central nervous system neoplasms in in vitro models.Abbreviations xxGangliosides have been designated according to CBN recommendations [22] and to the coding system of Svennerholm [35] GD2 II³(NeuAc)₂GgOse₃Cer - GM3 II³NeuAc-LacCer - GD3 II³(NeuAc)₂-LacCer - GM2 II³NeuAcGgOse₃Cer - GM1 II³NeuAcGgOse₄Cer - GD1a II³NeuAcIV³NeuAcGgOse₄Cer - GD1b II³(NeuAc)₂GgOse₄Cer - GT1b IV³NeuAcII³(NeuAc)₂GgOse₄Cer - GQ1b IV³(NeuAc)₂II³(NeuAc)₂GgOse₄Cer - 3,8-LD1 IV³(NeuAc)₂nLeOse₄Cer Supported in part by NIH Grants R37 CA 11898, NS 20023, CA 32672, and T32-NS 07304, and by a grant from the Swedish Medical Research Council (Project 03X-627). Dr. Longee is an Association of Medical School Pediatric Department Chairmen, Inc., Pediatric Scientist Training Program Fellow supported by St. Jude Children's Research Hospital     

Brain gangliosides in birds with different types of postnatal development (nidifugous and nidicolous type)

Developmental profiles of 14 different brain gangliosides were followed from the first day after hatching to the adult stage in two bird species representing different strategies of posthatch development: the nidifugous type (leaving the nest directly post-hatch, e.g. quail) and the nidicolous type (remaining for longer period in the nest, e.g. finch). In the zebra finch, parallel with a striking increase in ganglioside concentration, two main postnatal changes in the ganglioside composition occurred: after hatching, concomittantly to an increased outgrowth of nerve fibers and synaptogenesis, the polysialogangliosides GQ1b and GP decreased in favour of the less polar fractions GD1b, GD1a and GT1b. The second period of changes started with the onset of myelination and was characterized by an increase of GM1 and GM1'. The results obtained for quails were in close agreement with those of chicken, showing only slight postnatal changes due to the nearly completed morphological differentiation. These data show that gangliosides are useful biochemical markers for brain development, indicating successive periods of brain maturation by means of preferential biosynthesis of specific fractions regardless of the type of development.     

Antiganglioside antibodies in the pathogenesis of autoimmune neuropathies]

Antiganglioside antibodies are frequently detected in sera from patients with autoimmune neuropathies, such as Guillain-Barré syndrome, Miller Fisher syndrome, IgM paraproteinemic neuropathy, chronic inflammatory demyelinating polyneuropathy, and multifocal motor neuropathy. In the acute phase sera from GBS patients, antiganglioside antibodies are detected in 60-70%. Ganglioside antigens recognized by serum antibodies are varied from case to case. IgG antibody against GQ1b ganglioside is specifically raised in sera from patients with Miller Fisher syndrome and Guillain-Barré syndrome with ophthalmoplegia. That antibody may bind to the paranodal myelin of oculomotor, trochlear and abducens nerves, where GQ1b ganglioside is specifically localized, to cause ophthalmoplegia. IgM M-protein which recognizes the disialosyl residue of GD1b is specifically associated with sensory ataxic neuropathy. The IgM M-protein may bind to the primary sensory neurons, where GD1b ganglioside is localized, to cause sensory disturbance. After we confirmed the localization of GD1b in the rabbit primary sensory neurons, we sensitized rabbits with GD1b and induced sensory ataxic neuropathy in them. This is the first established animal model of autoimmune neuropathy induced by sensitization with ganglioside. Some antiganglioside antibodies may determine the clinical phenotype of neuropathy by binding specifically to the ganglioside antigens which have unique localization.     

Elevated Potassium Enhances Glutamate Vulnerability of Dopaminergic Neurons Developing in Mesencephalic Cell Cultures

This study examines the effects of high K⁺concentration on the growth and development of mesencephalic cells and their glutamate vulnerability. Mesencephalic cell cultures obtained from Wistar rat embryos on the 14th gestational day were maintained for 14 days in medium with either normal (4.2 mM) or elevated (24.2 mM) potassium concentration. There was no significant difference due to various K⁺concentration in cell growth and survival up to dayin vitro(DIV) 13–15. In order to test the glutamate (Glu) vulnerability, cultures were treated with 100 μMGlu for 15 min in salt solution on the DIV 3, 6, 8, and 13. Glu-induced neuronal damage was estimated 24 h later by measuring the neuron-specific enolase (NSE) content in the culture medium and by counting the number of tyrosine hydroxylase-immunoreactive (TH-IR) neurons. Glu had no damaging effect on the cells on DIV 3, but became pronounced beyond DIV 6. Elevated potassium concentration (24.2 mM) in the culture medium during development significantly increases neuronal vulnerability to Glu treatment, indicated by a higher increase of NSE content in the medium and by a more pronounced Glu-induced decrease of the number of TH-IR cells. The Glu-induced decrease of the number of TH-IR cells and of NSE-IR cells let us conclude that dopaminergic neurons are more vulnerable to glutamate than other neurons from mesencephalic culture.     

Gangliosides in lesion-induced synaptogenesis: studies in the hippocampus of the rat brain

Changes in ganglioside composition, biosynthesis and individual distribution were studied in hippocampal regions after unilateral destruction of the entorhinal cortex. After 1 and 3 days postlesion (dpl), a decrease in ganglioside content was detected in area dentata (AD) and pyramidal cell regions CA1-CA3 (CA), both ipsilateral and contralateral to the lesion. By 5 dpl all the values had returned to control values, except in AD which showed a dramatic increase in total ganglioside content reaching a maximum at 12 dpl. By 30 dpl this area also showed control content. A significant increase in biosynthesis of gangliosides was observed at 5 and 8 dpl in the hippocampus ipsilateral to the lesion without changes in the contralateral counterpart. Individual ganglioside distribution showed a pronounced change in GM1 and GQ1b with small changes in the other major gangliosides. Significant differences were observed in the distribution of gangliosides between the two hippocampal regions studied in unoperated control animals. GD1a was more concentrated in AD, whereas GQ1b, GT1b and GD1b predominated in CA. The data presented here indicate that important modifications in ganglioside content as well as pattern occur in the deafferented hippocampus a phenomenon that could be related with the known effect of gangliosides on neuritogenesis observed in cell culture studies.