Inhibition of neurite outgrowth of neuroblastoma neuro-2a cells by cholera toxin B-subunit and anti-GM1 antibody

The role of cell surface GM1 ganglioside in neurite outgrowth of Neuro-2a neuroblastoma cells was investigated by application of anti-GM1 antibody and the B subunit of cholera toxin (cholera B) to cultured cells stimulated to grow neurites in various ways. When the cells were simultaneously treated with stimulatory agent and cholera B, inhibition, as measured by percent of neurite-bearing cells, was observed with most stimuli: neuraminidase; GD1a ganglioside, retinoic acid, and low serum. However, with dibutyryl cyclic AMP the small reduction observed was not statistically significant. The inhibitory effect of cholera B on neurite outgrowth induced by low serum was dose-dependent, reaching a maximum at 200 ng/mL; 48 h after washout of cholera B the cells were released from inhibition and regrew neurites at nearly the previous rate in the presence of low serum. When the cells were exposed to stimulus for 6 h or more the inhibitory effect of subsequent addition of cholera B was reduced or eliminated; inhibition thus occurs during an early stage of neurite initiation. Anti-GM1 anti-body at dilutions of 1∶100–1∶400 had the same inhibitory effect as cholera B with cells stimulated by GD1a or retinoic acid, whereas anti-GM2 antibody had no effect at 1∶200 or 1∶400; inhibition by the latter antibody at 1∶100 dilution was similar to that attained with control ascites fluid. These results point to a pivotal role for cell surface GM1 in Neuro-2a differentiation induced by many (but not all) neuritogenic agents.     

Inhibition of neurite outgrowth of neuroblastoma neuro-2a cells by cholera toxin B-subunit and anti-GM1 antibody

The role of cell surface GM1 ganglioside in neurite outgrowth of Neuro-2a neuroblastoma cells was investigated by application of anti-GM1 antibody and the B subunit of cholera toxin (cholera B) to cultured cells stimulated to grow neurites in various ways. When the cells were simultaneously treated with stimulatory agent and cholera B, inhibition, as measured by percent of neurite-bearing cells, was observed with most stimuli: neuraminidase; GD1a ganglioside, retinoic acid, and low serum. However, with dibutyryl cyclic AMP the small reduction observed was not statistically significant. The inhibitory effect of cholera B on neurite outgrowth induced by low serum was dose-dependent, reaching a maximum at 200 ng/mL; 48 h after washout of cholera B the cells were released from inhibition and regrew neurites at nearly the previous rate in the presence of low serum. When the cells were exposed to stimulus for 6 h or more the inhibitory effect of subsequent addition of cholera B was reduced or eliminated; inhibition thus occurs during an early stage of neurite initiation. Anti-GM1 anti-body at dilutions of 1∶100–1∶400 had the same inhibitory effect as cholera B with cells stimulated by GD1a or retinoic acid, whereas anti-GM2 antibody had no effect at 1∶200 or 1∶400; inhibition by the latter antibody at 1∶100 dilution was similar to that attained with control ascites fluid. These results point to a pivotal role for cell surface GM1 in Neuro-2a differentiation induced by many (but not all) neuritogenic agents. ganglioside nomenclature follows that of Svennerholm (1963).     

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.     

Gangliosides of cultured astroglia

Cultured astrocytes prepared from newborn rat brain and 13-day-old chick embryonic brain were analyzed qualitatively and quantitatively for ganglioside content. All preparations contained approximately the same total level: 2.4-3.4 micrograms N-acetylneuraminic acid (NeuAc)/mg protein. In contrast, the value for primary cultures of neurons from chick embryonic brain was 5.9. The non-hexosamine-containing species, GM3 and GD3, comprised 75-85% of the total in astroglial cultures, the remainder consisting mainly of structural types other than the gangliotetraose series; choleragenoid assay revealed the latter to be virtually absent or to comprise at most a few percent. Deficiency of gangliotetraose synthesizing ability was indicated by the very low level of UDP-GalNac:GM3 N-acetylgalactosaminyltransferase detected in the cells. Treatment of cultured astrocytes with astroglial growth factor 2 or dibutyryl cyclic AMP caused little if any change in quantity or pattern of gangliosides. The large majority of cells stained in a manner characteristic of astrocytes: positive for glial fibrillary acidic protein, negative for galactosyl ceramides. Staining with cholera toxin and anti-GM1 antibody was essentially negative, as was that with tetanus toxin, A2B5 monoclonal antibody, and antibody to GD3. All evidence thus points to cultured astrocytes of rat and chick brain containing appreciable gangliosides, most of which are GM3 and GD3 with the majority of the remainder comprising structures other than the gangliotetraose type.     

Axonogenesis in neuro-2a cells correlates with GM1 upregulation in the nuclear and plasma membranes.

GM1 ganglioside was previously shown to occur in the nuclear membrane, as well as the plasma membrane, of central nervous system (CNS) and peripheral nervous system (PNS) neurons undergoing morphological differentiation in culture. NG108-15 neuroblastoma cells showed the same phenomenon when induced to extend axon-like but not dendrite-like processes, although in both cases terminal differentiation was evidenced by failure of extended neurites to retract following washout of neuritogenic agent. The present study of Neuro-2a neuroblastoma cells subjected to similar treatments has revealed both similarities and differences compared to NG108-15 cells. Similar to the latter, Neuro-2a cells responded to neuraminidase and ionomycin with axon-like outgrowth together with upregulation of nuclear GM1, and to three other agents (retinoic acid, dibutyryl cyclic AMP, exogenous GM1) with dendrite-like outgrowth that was unaccompanied by nuclear GM1 increase. Although both cell types responded to low serum by extending neurites of mixed axonal-dendritic properties, Neuro-2a, in keeping with its greater tendency to extend some neurites of axonal character in low serum, showed elevated nuclear GM1 in a significant number of such differentiated cells. All three axonogenic agents induced parallel upregulation of GM1 in plasma-, nuclear-, and Golgi membranes, and these increases were stable to washout. Neurites generated in Neuro-2a cells by the three dendritogenic agents lacked stability, unlike those produced by the same agents in NG108-15 cells. This study also amplified the differences in response triggered by exogenous GM1 compared to that resulting from enzyme-mediated elevation of endogenous GM1.     

Upregulation of nuclear GM1 accompanies axon‐like,but not dendrite‐like,outgrowth in NG108–15 cells

Recent work has demonstrated that induced neurite outgrowth in neuroblastoma cells and spontaneous differentiation of primary neurons in culture are accompanied by upregulation of GM1 ganglioside in the nuclear envelope. Previous reports have depicted morphological variations in the nature of stimulated neurites resulting from different neuritogenic agents, and a recent study by this laboratory demonstrated that such stimulants could be divided into two categories: those which induce axon‐like neurites (group I) as opposed to those that stimulate dendrite‐like outgrowths (group II). The former includes KCl, ionomycin, neuraminidase, and cholera toxin B subunit (all agents which elevate intracellular Ca²⁺), while the latter group is comprised of retinoic acid, dibutyryl cAMP, exogenous GM1, and low serum treatment. The present study was undertaken to determine whether differences in neuritic phenotype could be correlated with upregulation of nuclear GM1. The neuroblastoma × glioma NG108–15 cell line was employed because of its ability to respond robustly to a variety of neuritogenic stimuli. It was found that although both groups of stimulants are capable of inducing stable neurites (terminal differentiation) in this cell line, nuclear GM1 is elevated only in the presence of group I stimulants. Thus, a correlation is indicated between axonogenesis and upregulation of GM1 in the nuclear envelope. Additionally, these two events appear to coincide with elevation of intracellular Ca²⁺. Conversion of cells to the differentiated phenotype, with or without nuclear GM1 elevation, was found to depend in some cases on concentration of stimulant and duration of treatment. J. Neurosci. Res. 55:107–118, 1999. © 1999 Wiley‐Liss, Inc.     

Gangliosides of human cerebrospinal fluid in various neurologic diseases.

Simultaneous profile determination and quantification of human cerebrospinal fluid (CSF) gangliosides in various neurologic diseases (n = 71) was examined. Gangliosides were extracted with methanol/chloroform from clinically available amounts of CSF (4-5 ml), then separated and quantified by high-performance thin-layer chromatography (HPTLC) and direct densitometry. Based on chromatographic comparison with standards, the percentage of lipid-bound NeuAc positive fractions in 'normal' CSF samples were: GM1 (II3 NeuAc-GgOse4Cer) (3%); GD3 (II3 NeuAc2-Lac-Cer) (4%); GD1a (IV3 NeuAc, II3 NeuAc-GgOse4 Cer) (15%); X1 (3%); GD1b (II3(NeuAc)2-GgOse4 Cer) (16%); X2 (4%); GT1b (IV3 NeuAc, II3(NeuAc)2-GgOse4-Cer) (40%); and GQ1b (IV3(NeuAc)2, II3(NeuAc)2-GgOse4-Cer (15%). Similarity between CSF and CSF and human cerebellar cortex, particularly in proportion of "b" series gangliosides (GQ1b, GT1b, GD1b), could be observed. A higher proportion of GD1a ganglioside, with decreased GQ1b was found in infancy. The total ganglioside content (mean +/- 2 SD) varied between 645-894 micrograms/l. Significant alterations of the CSF ganglioside profile, with an increase in less polar gangliosides, GM3 and GD3, correlated with the blood-brain barrier dysfunction (CSF hemorrhages, compressive syndrome), or some malignant processes (metastatic brain melanoma). A statistically significant increase in the content of total CSF gangliosides was found in the following groups of patients as compared to controls: (1) ischemic cerebrovascular accident (CVI) with good outcome (P less than 0.02); (2) peripheral neuropathy and polyneuropathy (P less than 0.001) and (3) intravertebral discopathy (P less than 0.05). A significant decrease in the content of total CSF gangliosides was found in CVI group with lethal outcome (P less than 0.05).     

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.     

Ganglioside lateralization in the brain of female rats

The ganglioside composition of the cerebral hemispheres of young and adult rats of either sex has been herein assessed for the first time. In females, the total ganglioside content at any age, the content of GM1, GD1a, and GD1b at 8 days, and the content of GM1, GD1b, GT1b, and GQ1b at 60 days were higher in the right than in the left hemisphere. In males, no difference was observed. Concerning the ceramide moiety, a difference was displayed by C18:1 long-chain base in GD1a, whose proportion was higher in the left than in the right hemisphere of females aged 8 days. The comparison between homolateral hemispheres of rats of different sex revealed several differences. On average, in 8-day-old animals, the content of gangliosides was higher in females than in males. At 60 days the amount of gangliosides was on average lower in females than in males, even if with some exception. The data obtained with the current investigation show the existence of a ganglioside lateralization in rat brain, exclusively in females, and almost entirely at charge of the oligosaccharide portion. Moreover, age-dependent changes of ganglioside pattern and content show a dependence on brain lateralization. J. Neurosci. Res. 50:643–648, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Ganglioside complexes as new target antigens in Guillain-Barré syndrome

Antibodies specific for a complex of gangliosides GD1a and GD1b (GD1a/GD1b) were found in sera from eight of 100 patients with Guillain-Barre syndrome (GBS) by the use of enzyme-linked immunosorbent assay and thin-layer chromatogram immunostaining. Those sera also had antibody activities to such ganglioside complexes as GD1a/GM1, GD1b/GT1b, and GM1/GT1b but had little or no reactivity to the each isolated antigen. Clustered epitopes of the ganglioside complex in the plasma membrane may be targeted by such an antibody, and interaction between the antibody and ganglioside complex may induce the neuropathy.     

A novel ganglioside, 9-O-acetyl GD1b, is recognized by serum antibodies in Guillain-Barré syndrome

A hitherto undescribed ganglioside was detected in a crude ganglioside fraction of bovine brain using an IgM M-protein binding to Ga1β 1,3Ga1NAc residue. We purified and identified it as 9-O-acetyl GD1b based on results of alkali treatment that yielded GD1b and results of fast atom bombardment-mass and gas chromatography-mass spectrometries. 9-O-acetyl GD1b was also found to be present in human peripheral nerve tissue. The reactivities of the serum antibodies from patients with Guillain-Barré syndrome to 9-O-acetyl GD1b, GD1b, and GM1 were determined by ELISA and TLC immunostaining. Nineteen of 85 serum samples from Guillain-Barré syndrome patients had antibodies that bound to 9-O-acetyl GD1b: 14 of the positive samples also reacted with GM1 and GD1b, three reacted with GM1 but not with GD1b, one with GD1b but not with GM1, and one with neither GM1 nor GD1b. These results show that a subset of patients with Guillain-Barré syndrome had antibodies that react with 9-O-acetyl GD1b; therefore, this ganglioside can serve as a target antigen against the antibodies present in Guillain-Barré syndrome.     

Effects of exogenous GM1 and GD1a on S20Y neuroblastoma cells

The effects of exogenous GM1 and GD1a on S20Y murine neuroblastoma cells were assessed by monitoring morphology, tumorigenicity, mitotic index, and plating efficiency. S20Y cells were seeded at a density equivalent to 5 X 10(4) cells per 35-mm tissue culture dish; 38-42 hr after seeding (preconfluent stage) the cells were treated for 12 hr with 100 micrograms of ganglioside per ml of medium in which the serum content was reduced from 10% to 0.5%. Analysis of the cell lipids indicated that added ganglioside became tightly associated with the membrane during the 12-hr exposure. GM1 treatment resulted in increased projections on the cell surface and fine structures projecting from the cell processes. GD1a treatment resulted in a reduction in the cellular mitotic index. Plating efficiency was reduced by both GM1 and GD1a. Neither ganglioside affected tumorigenicity of the S20Y cells. Twelve hours after removal of the added ganglioside and exposure of the cells to normal medium, the ganglioside composition of the membranes from treated cells approached that of the controls, and the ganglioside-induced effects had been reversed. These results suggest that addition of specific gangliosides induces different cellular responses and that these changes are dependent upon the continued presence of the ganglioside.     

Association of antibodies to ganglioside complexes and conduction blocks in axonal Guillain‐Barré syndrome presenting as acute motor conduction block neuropathy

A close relationship between acute motor conduction block neuropathy and antibodies against the complex of GM1 and GalNAc‐GD1a has been reported. This study investigates the hypothesis that conduction block at the early phase of axonal Guillain‐Barré syndrome (GBS) is also associated with such ganglioside complexes. Sera were obtained from seven French patients with initial evidence of isolated conduction blocks that resolved or progressed to acute motor axonal neuropathy. Serum IgG to asialo‐GM1 and gangliosides of LM1, GM1, GM1b, GD1a, GalNAc‐GD1a, GD1b, GT1a, GT1b, and GQ1b as well as their complexes were measured. Five of seven patients progressed within the first month of disease to AMAN. One patient had IgG antibodies against the complex of asialo‐GM1 and each of the other ganglioside antigens. Another patient carried IgG antibodies against GM1 complex with GM1b, GD1a, and GT1a as well as asialo‐GM1 complex with GD1a and GT1a. None had IgG antibodies against GM1/GalNAc‐GD1a complex. Six patients had IgG against single antigens GM1, GD1a, GalNAc‐GD1a, GD1b, and asialo‐GM1. In three patients, a reduced reaction against GM1/GalNAc‐GD1a complex was observed. The presence of conduction block in axonal GBS is not always associated with anti‐GM1/GalNAc‐GD1a complex antibodies.     

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.     

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.     

Ganglioside patterns in neuroepithelial tumors of childhood

To elucidate a relationship between neuronal anaplasia, tumor proliferation, and ganglioside contents, we quantified gangliosides by HPTLC in tumors of neuroepithelial tissues at different grade, i. e. peripheral primitive neuroectodermal tumor (PPNET, grade IV), ependymoma (EPEN, grade III), neuroblastoma (NB, grade IV), and dysembryoplastic neuroepithelial tumor (DNT, grade I). PPNET, the most undifferentiated tumor examined had lowest concentration of total lipid-bound sialic acid. GM3 was the major ganglioside in all undifferentiated tumors (46-72.7% of total lipid-bound sialic acid). GD3 was an another component in PPNET and EPEN (7.2-17.3%). Concentration of a complex gangliosides GM1 was decreased in all tumor tissues and those of GT1a, GD1b and GT1b were decreased in tumors of high grade. The results suggest that the composition of gangliosides could be of considerable value in refining the classification of neuroepithelial tumors in infancy and childhood.     

Brief treatment of sensory ganglion neurons with GM1 ganglioside enhances the efficacy of opioid excitatory effects on the action potential

In previous studies, we showed that low (nM) concentrations of opioid prolong the action potential duration (APD) of many mouse dorsal root ganglion (DRG) neurons via Gs-linked excitatory opioid receptors, whereas micromolar opioid levels shorten the APD via Gi/Go-linked inhibitory receptors. In addition, cholera toxin-B subunit (CTX-B) selectively blocks opioid- but not forskolin-induced prolongation of the APD in DRG neurons. Since CTX-B binds with selective high affinity to GM1 ganglioside located on the cell surface, the results suggest that GM1 plays an essential role in regulating excitatory opioid receptor functions. This hypothesis was tested by treating DRG neurons in mouse DRG-cord explants with exogenous gangliosides and determining whether the efficacy of opioid agonists in prolonging the APD is enhanced. The threshold concentration of the opioids, dynorphin(1-13) and morphine required to prolong the APD in many DRG neurons was markedly decreased from nM to fM levels after bath exposure to 10 nM to 1 microM GM1 ganglioside for less than 5 min. In contrast, GM2 and GM3 gangliosides and asialo-GM1 ganglioside were ineffective, even when DRG neurons were exposed to high concentrations (1-10 microM) for periods greater than 1 h. Although GD1a, GD1b and GQ1b gangliosides appeared to be as effective as GM1 when tested at microM concentrations for 15 min, tests at lower concentrations, shorter periods, and/or at lower temperature (24 degrees vs 34 degrees C), showed that they were significantly less effective than GM1.(ABSTRACT TRUNCATED AT 250 WORDS)