Evidence that ruthenium red disturbs the synaptic transmission in the rat hippocampal slices through interacting with sialic acid residues

Ruthenium red (RR) at a concentration of 0.71 mM selectively blocked synaptic transmission in hippocampal slices. Antidromically evoked potentials and fibre potentials were only little affected. The action of RR was reversible by washout, but only following shorter (40-50 min) times of incubation. After longer incubation times (hours), the abolished population spike did not recover after washout but could be restored by facilitation of the calcium transport into the nerve terminal with 3,4-diaminopyridine. Partial liberation of sialic acid with neuraminidase from Vibrio Cholerae markedly increased the time after which the potential was abolished by RR. Exogenously added gangliosides and sialic acid also delayed the action of RR. Calcium at a concentration of 13.2 mM prevented or reduced the RR effect. It is concluded that RR binds to sialic acid residues, interfering with neurotransmission by disturbing the calcium transport into the cell.     

The role of monosialoganglioside GM1 in the synaptic plasticity: in vitro study on rat hippocampal slices

Rat hippocampal slices were incubated with neuraminidase from Vibrio Cholerae. This enzyme liberates sialic acid from polysialogangliosides converting them into monosialoganglioside GM1. Thus, the tissue is enriched in GM1 content. Another set of slices was incubated with GM1 itself. Both treatments increased the magnitude of potentiation of synaptic response recorded from pyramidal cell layer following high frequency stimulation of Schaffer collateral-commissural fibers. It is concluded that enrichment of synaptic membranes in GM1 enhances the ability of these nerve endings to be potentiated.     

Effect of low glucose concentration on synaptic transmission in the rat hippocampal slice

Severe hypoglycemia in vivo is known to slow down the EEG, then to produce complete electrical silence in the brain. To find out why low glucose concentrations reduce electrical activity, synaptic transmission from Schaffer collateral/commissural fibers to CA1 pyramidal cells in the submerged rat hippocampal slice was investigated using extracellular recording techniques. Superfusion for 30 min with 1 mM glucose reversibly reduced population spike amplitude, without affecting the size of the presynaptic volley and the slope of the field EPSP. Lower glucose concentrations also affected the EPSP, although to a lesser extent than the population spike. Antidromic population spikes were not decreased by low glucose. Depolarization with 8-10 mM K+ reduced both presynaptic volley amplitude and EPSP, but enhanced the population spike, an effect clearly different from that of low glucose. The slope of the input/output curve between presynaptic volley and EPSP remained unaltered in 1 mM glucose but the slope between EPSP and population spike was reduced by about 50%. Results suggest that low glucose concentrations interrupt synaptic transmission by reducing, but not abolishing, the excitability of pyramidal cells.     

The composition of glycosphingolipids and the effect of neuraminidase on cultured glioma cell lines

The composition of glycosphingolipid on human cultured glioma cell line U 251 and rat glioma cell line C6 was analysed by high performance thin layer chromatography. As a result, the major gangliosides were simple gangliosides such as GM3 (U 251: 7.7%, C6: 84.3%), GM2 (U 251: 32.6%) and SPG (U 251: 30.0%) on glioma cells whereas the major neutral glycosphingolipids were CDH, CTH and globoside. After treatment with neuraminidase 2.92 nmol/mg dry weight and 3.73 nmol/mg dry weight of sialic acid were freed from U 251 cells and C6 cell, but only 8.11% (U 251 cell) and 11.24% (C 6 cell) of these sialic acids originated from glycolipid, and thus the major part of sialic acid might be released from glycoprotein of the cells. The gangliosides that react to neuraminidase are SPG, GD1a and GD1b in U 251 cells and are GM1a and little GM3 in C 6 cells. The biolabelling study using N-acetyl-14C-mannosamine as a precursor of sialic acid demonstrated that the precursor was mainly incorporated into both or either of GM3 and SPG in the acidic glycolipid fraction. In addition, no significant change on proliferation and morphology of glioma cells after neuraminidase treatment was observed in this study.     

Total ganglioside ablation at mouse motor nerve terminals alters neurotransmitter release level

Neuronal membrane gangliosides, forming a large family of sialylated glycosphingolipids, have been hypothesized to play important roles in synaptic transmission. We studied the ex vivo electrophysiological function of neuromuscular junctions of GM2/GD2‐synthase*GD3‐synthase compound null‐mutant mice after acute removal of GM3, the only remaining ganglioside in this mouse, by in vitro treatment with neuraminidase. We found 16% enhancement of the acetylcholine release per nerve impulse at low‐rate (0.3 Hz) nerve stimulation. Conversely, the treatment reduced the acetylcholine release evoked by high‐rate (40 Hz) nerve stimulation. Also, 25 ms paired‐pulse facilitation of endplate potentials was reduced by the neuraminidase‐treatment. These effects may indicate a modest modulatory influence of the negative electrical charges carried by the sialic acid molecules of gangliosides on the function of presynaptic Ca_v2.1 channels, affecting the magnitude and kinetics of the Ca²⁺ influx that induces neurotransmitter release from the motor nerve terminal. Our results show that gangliosides are to some extent involved in neurotransmission at the neuromuscular junction, but that their presence is not an absolute requirement in this process. Synapse 64:335–338, 2010. © 2009 Wiley‐Liss, Inc.     

Gangliosides and sialoglycoproteins in normal and denervated rat diaphragm muscle

Ganglioside- and glycoprotein-bound sialic acid in endplate and non-endplate regions of rat diaphragm muscles were assessed 1–16 days after phrenic nerve transection. Sialic acid in glycoproteins (90% of total) and gangliosides (10% of total) from intact hemidiaphragms was distributed uniformly throughout endplate and nonendplate regions. After denervation, the total sialic acid per hemidiaphragm increased (days 1–4) and reached a maximum level (days 8–12) which remained constant (days 12–16). These sialic acid changes reflected increments in gangliosides (40% over control), which occurred simultaneously in endplate and nonendplate regions (days 1–8), and increments in sialoglycoproteins (70%–80% over control), which took place much earlier in endplate (days 1–8) than in nonendplate (days 8–14) regions. The results of this study, together with previous data, favor the hypothesis that maintenance of both sialoglycoproteins and gangliosides depends upon an intact innervation, probably through separate mechanisms involving different neurogenic factors.     

Ganglioside changes in the chicken optic lobes as biochemical indicators of brain development and maturation

The development profiles of 16 different gangliosides of the optic lobes of the chicken were followed from the sixth day of incubation to the tenth posthatching week and correlated to known morphological development. Several, previously undetected novel fractions occurred between the sixth and tenth embryonic days. According to their migration rates on TLC-plates 4 of them may be GT3, GT2, GT1c, GQ1c. Three even more slowly migrating fractions represent penta-, hexa-, and septa-sialogangliosides. At the sixth day of incubation, characterized by maximal proliferation of neuro-epithelial cells, the optic lobes contained predominantly GD3.Up to the eleventh day of incubation, parallel to decreased mitotic activity, maximal cell migration and neuron differentiation, GD3, GD2, and GT3 decreased in favor of newly detected polysialogangliosides. Thereafter, up to hatching, parallel to increased growth and arborization of dendrites and axons as well as synaptogenesis, the newly detected polysialogangliosides decreased in favor of GD1b, GT1b, GQ1b, and GD1a.At hatching the myelin-specific GM4 appeared, reaching about 8% of total ganglioside sialic acid after 10 weeks. Likewise a fraction, migrating somewhat faster than GM1,increased. This band, named GM1', is suggested to be also myelin-associated. The other monosialogangliosides were always minor fractions, none exceeding 4% of total ganglioside sialic acid.     

Kainic acid and penicillin: Differential effects on excitatory and inhibitory interactions in the CA1 region of the hippocampal slice

The effects of kainic acid (KA, 0.05-1.0 microM), and penicillin (PN, 3.4 mM) were studied in the CA1 region of rat hippocampal slices. Three components of the overall input/output function were taken: (1) the amplitude of the presynaptic compound action potential (prevolley) vs stimulation current applied to Schaffer collaterals, (2) the magnitude of the focally recorded synaptic potential (population EPSP) vs prevolley amplitude; and (3) the amplitude of the focally recorded population spike vs population EPSP magnitude. Recurrent inhibition was measured using the antidromic-orthodromic paired pulse method. KA caused a significant and reversible enhancement of all 3 component input/output functions while having no effect on paired pulse inhibition. PN caused a left shift in the EPSP-population spike relationship and decreased or abolished paired pulse inhibition; the other two measures of excitability were not changed. These results suggest that PN and KA differ fundamentally in the mechanisms by which they produce seizures: PN by removing inhibition while not affecting neuronal excitability per se; KA by exerting a generalized excitatory effect on neural membranes and on synaptic function while leaving recurrent inhibition unchanged.     

The effects of moderate changes of extracellular K+ and Ca2+ on synaptic and neural function in the CA1 region of the hippocampal slice

The effects of moderate changes of the concentration of ions on the function of mammalian central nervous tissue have not exactly been determined. We placed tissue slices from rat hippocampal formation in an interface chamber for study in vitro. Extracellular potentials were recorded in stratum radiatum and stratum pyramidale in response to stimuli of varying intensity applied to the Schaffer collateral bundle. The overall input-output relationship of excitatory synaptic transmission was gauged by expressing postsynaptic population spike amplitude as a function of presynaptic volley amplitude. The components of the transmission process were also examined by plotting the maximal rate of rise (slope) of the focally recorded synaptic potential (fEPSP) as a function of presynaptic volley amplitude, and the population spike amplitude as a function of the fEPSP slope. Raising the concentration of K+ from the normal level of 3.5 mM to 5 mM caused an average increase of 48% in the population spike evoked by a given presynaptic volley. This was due to an increased electrical excitability of pyramidal cells, as indicated by an increase of the population spike evoked by a given magnitude of fEPSP. Conversely, lowering [K+]o from 3.5 to 2 mM caused a decrease of the population spike relative to a given magnitude of either the presynaptic volley or the fEPSP. Changing [K+]o within these limits caused no significant change of the fEPSP evoked by a given presynaptic volley. Raising [Ca2+]o from 1.2 to 1.8 mM caused a 35% increase in both the fEPSP and the population spike evoked by a given presynaptic volley, and lowering [Ca2+]o to 0.8 mM caused a decrease of both these functions. The amplitude of the population spikes evoked by given fEPSPs changed surprisingly little (but consistently) when [Ca2+]o was varied within these limits. We conclude that moderate changes of [K+]o influence mainly the electric excitability of hippocampal pyramidal cells, with little effect on transmitter release or on the response of the postsynaptic membrane to transmitter, while moderate changes of [Ca2+]o affect the release of excitatory synaptic transmitter more than they affect postsynaptic membrane function.     

The effects of moderate changes of extracellular K and Ca on synaptic and neural function in the CA1 region of the hippocampal slice

The effects of moderate changes of the concentration of ions on the function of mammalian central nervous tissue have not exactly been determined. We placed tissue slices from rat hippocampal formation in an interface chamber for study in vitro. Extracellular potentials were recorded in stratum radiatum and stratum pyramidale in response to stimuli of varying intensity applied to the Schaffer collateral bundle. The overall input-output relationship of excitatory synaptic transmission was gauged by expressing postsynaptic population spike amplitude as a function of presynaptic volley amplitude. The components of the transmission process were also examined by plotting (1) the maximal rate of rise (slope) of the focally recorded synaptic potential (fEPSP) as a function of presynaptic volley amplitude, and (2) the population spike amplitude as a function of the fEPSP slope. Raising the concentration of K⁺ from the normal level of 3.5 mM to 5 mM caused an average increase of 48% in the population spike evoked by a given presynaptic volley. This was due to an increased electrical excitability of pyramidal cells, as indicated by an increase of the population spike evoked by a given magnitude of fEPSP. Conversely, lowering [K⁺]_o from 3.5 to 2 mM caused a decrease of the population spike relative to a given magnitude of either the presynaptic volley or the fEPSP. Changing [K⁺]_o within these limits caused no significant change of the fEPSP evoked by a given presynaptic volley. Raising [Ca²⁺]_o from 1.2 to 1.8 mM caused a 35% increase in both the fEPSP and the population spike evoked by a given presynaptic volley, and lowering [Ca²⁺]_o to 0.8 mM caused a decrease of both these functions. The amplitude of the population spikes evoked by given fEPSPs changed surprisingly little (but consistently) when [Ca²⁺]_o was varied within these limits. We conclude that moderate changes of [K⁺]_o influence mainly the electric excitability of hippocampal pyramidal cells, with little effect on transmitter release or on the response of the postsynaptic membrane to transmitter, while moderate changes of [Ca²⁺]_o affect the release of excitatory synaptic transmitter more than they affect postsynaptic membrane function.     

Strontium supports synaptic transmission and long-lasting potentiation in the hippocampus

(1) Synaptic transmission was studied in isolated transverse hippocampal slices from guinea pigs. Extracellular evoked potentials were recorded in the region CA1. (2) Changing the normal perfusion solution (containing 2 mM Ca2+) to calcium-free Ringer abolished synaptic transmission which was again restored by adding strontium. A synaptic efficacy of 25--50% ofn normal was obtained for 10 mM Sr2+. (3) Two different synaptic inputs to CA1 pyramidal cells were tested with respect to their ability to produce long-lasting synaptic potentiation after tetanization in strontium Ringer. Following a brief tetanus the field EPSP and, especially, the population spike were greatly enhanced. (4) The potentiation so produced was similar to the long-lasting potentiation seen in the normal slice, because it (i) had a very long duration (hours), (ii) was specific for the tetanized pathway, (iii) showed potentiation of both 'volley-EPSP' and 'EPSP-spike' relations, and (iv) was accompanied by short-lasting (less than 5 min) generalized depression.     

Primed-burst potentiation occludes the potentiation phenomenon and enhances the epileptiform activity induced by transient pentylenetetrazol in the CA1 region of rat hippocampal slices

The effects of pentylenetetrazol (PTZ) following induction of long-term potentiation (LTP) on population spikes in CA1 of hippocampal slices were investigated. Population spikes were evoked by activation of Schaffer collaterals with a range of stimulation intensities. LTP was induced using θ-pattern primed burst tetanic stimulation. Changes in the population spike amplitude and number of population spikes were used as indices to quantify the effects of PTZ exposure in the control (non-tetanized) and LTP (tetanized) conditions. The amplitude of population spike was measured 20 min before, during 20 min chemical application (3 mM), and also after 30 or 60 min washout period. In non-tetanized slices, the population spike input-output curve was significantly increased 20 min after PTZ application and persisted at least for 60 min. Multiple population spikes or after potentials also appeared, but did not persist. When PTZ was applied on tetanized slices, 60 min after LTP induction, the amplitude increase produced by PTZ was smaller than the increase seen in the control condition. Also LTP induction preceding PTZ exposure increased the number of population spikes evoked by stimulation of Schaffer collaterals. It is concluded that a transient PTZ application produces a long-lasting increase in population spike amplitude. Primed burst LTP occludes PTZ-induced potentiation while also increasing the epileptogenic effect of PTZ.     

c-pathway polysialogangliosides in the nervous tissue of vertebrates, reacting with the monoclonal antibody Q211.

The mouse monoclonal antibody Q211, previously shown to recognize a common epitope of chicken brain GP1c and of two other polysialogangliosides containing 4 and 6 sialic acid residues respectively, is demonstrated to bind to gangliosides with identical thin-layer chromatography (TLC) migration in the brain of representatives of boney fish, rays, reptiles and mammals, including man. In the boney fish brains, the Q211 binding gangliosides were found to be alkali-labile, the Q211 epitope, however, is alkali-stable. After alkaline treatment, the cichlid fish contained at least 4 Q211-binding gangliosides, migrating as GT1c, GQ1c, GP1c and 'GH'. In the trout brain only one Q211 antigenic fraction was found, migrating as GQ1c. In the brains of ray, turtle and embryonic chicken an identical pattern of Q211-binding gangliosides (GQ1c, GP1c, 'GH') occurred. In the embryonic rat and human brain, the content of Q211-binding gangliosides was much lower as compared to the other vertebrate species. The epitope was found in two fractions, migrating like GQ1c (human and rat) and GP1c (rat). The presence of Q211 epitope in all species was confirmed by immunohistochemistry. These data confirm that the Q211-epitope contains a complete c-ganglio-tetraose structure, carrying 3 sialic residues at the inner galactose. They furthermore demonstrate that the expression of c-pathway polysialogangliosides is a general feature of the vertebrate nervous tissue, either during whole life (fish, reptiles) or more or less transient during embryonic development (birds, mammals).     

Possible role of myelin-associated neuraminidase in membrane adhesion

Previous studies from our laboratory have demonstrated the presence of a specific interaction between myelin-associated neuraminidase and GM1 (Saito and Yu, J. Neurochem 47:632–641, 1986). In the present study, we further characterized this neuraminidase-GM1 interaction and examined its role in the adhesion of rat oligodendroglial cells to GM1. Hydrolysis of N-acetylneuramin-lactitol by the enzyme was inhibited by GM1 in a competitive manner; GM1 itself was not hydrolyzed, suggesting that GM1 may serve as a competitive inhibitor of the enzyme. Asialo-GM1 had no inhibitory effect. When a soluble enzyme preparation was applied to a GM1-linked affinity column, the enzyme activity was retained on the column and was recovered from the column only by elution with a buffer containing 5 mM 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (Neu2en5Ac), a competitive inhibitor of neuraminidase. A binding study with ⁵¹ Cr-labeled rat oligodendroglial cells showed that oligodendroglial cells bound preferentially to GM1 developed on a thin-layer plate, but not to other gangliosides such as GM3, GD1a, GD1b, and GT1b. The binding reaction to GM1 was inhibited by Neu2en5Ac (5 mM). These results suggest that myelin-associated neuraminidase specifically interacts with GM1 and may be involved in adhesion of oligodendroglial cells to GM1. This neuraminidase-GM1 interaction may play an important role in the formation and stabilization of the multilamellar structure of the myelin sheath. © 1993 Wiley-Liss, Inc.     

Neuraminidase inhibitor, oseltamivir blocks GM1 ganglioside-regulated excitatory opioid receptor-mediated hyperalgesia, enhances opioid analgesia and attenuates tolerance in mice

The endogenous glycolipid GM1 ganglioside plays a critical role in nociceptive neurons in regulating opioid receptor excitatory signaling demonstrated to mediate "paradoxical" morphine hyperalgesia and to contribute to opioid tolerance/dependence. Neuraminidase (sialidase) increases levels of GM1, a monosialoganglioside, in these neurons by enzymatic removal of sialic acid from abundant polysialylated gangliosides. In this study, acute treatment of mice with the neuraminidase inhibitor, oseltamivir enhanced morphine analgesia. Acute oseltamivir also reversed "paradoxical" hyperalgesia induced by an extremely low dose of morphine, unmasking potent analgesia. In chronic studies, co-administration of oseltamivir with morphine prevented and reversed the hyperalgesia associated with morphine tolerance. These results provide the first evidence indicating that treatment with a neuraminidase inhibitor, oseltamivir, blocks morphine's hyperalgesic effects by decreasing neuronal levels of GM1. The present study further implicates GM1 in modulating morphine analgesia and tolerance, via its effects on the underlying excitatory signaling of Gs-coupled opioid receptors. Finally, this work suggests a remarkable, previously unrecognized effect of oseltamivir-which is widely used clinically as an antiviral agent against influenza-on glycolipid regulation of opioid excitability functions in nociceptive neurons.     

Glycolipid sialosyltransferase activity in synaptosomes exhibits a product specificity for (2-8)disialosyl lactosyl ceramide (ganglioside GD3)

Intact synaptosomes prepared from 28-day-old rat brains were incubated with CMP-N-acetyl-(14C) neuraminic acid in Krebs-Henseleit buffer in an atmosphere of 95% O2: 5% CO2, at 37 degrees C. The activity of CMP-NANA:ganglioside sialosyltransferase using endogenous acceptors was 0.84 pmoles NANA transferred/mg synaptosomal protein/hr. Analysis of the distribution of labeled sialic acid revealed that GD3 ganglioside (alpha 2----8 disialosyl, alpha 2----3 galactosyl, beta 1----4 glucosyl, beta 1----1-ceramide) was the major product in the membrane carrying 32% of the total lipid bound label. Treatment of the reaction products with Clostridium neuraminidase liberated labeled sialic acid from GD3 and yielded labeled GM3, then unlabeled lactosyl ceramide. Lac-cer and GM3 are present in small amounts in synaptosomes, and GD3 represents less than 2% of the total ganglioside. Our findings indicate that the sialosyltransferase activity of synaptosomes exhibits a preferential product specificity for the small pool of synaptosomal membrane GD3 ganglioside that may be formed in situ, via sialosylation of its precursor (GM3 or lactosyl ceramide) which pre-exists in the synaptosomal plasma membrane. The second major labeled product quantitatively was GD1a whose precursor substrate, GM1, is quite abundant in the membrane, so that the conversion rate of GM1 to GD1a was low in comparison with GD3 formation. Sialosylation of other synaptosomal membrane gangliosides was negligible.     

Long-term potentiation in the hippocampus involves activation of N-methyl-D-aspartate receptors

A series of ω-phosphono-α-car?ylic acids were tested as antagonists of excitatory amino acid depolarizations and long-term potentiation (LTP) in region CA1 of rat hippocampal slices. The 5- and 7-phosphono compounds (±AP5and±AP7) blocked N-methyl-D-aspartate (NMDA) depolarizations and prevented the induction of LTP of the synaptic field potential and population spike components of the Schaffer collateral response.±AP5and±AP7 did not reduce kainate or quisqualate depolarizations and did not affect unpoten synaptic response amplitude.±AP5, ±AP6and±AP8 did not block amino acid excitant responses or LTP.These results demonstrate that NMDA receptors present in hippocampal region CA1 are not necessary for normal synaptic transmission, but are involved in the initiation of long-term synaptic plasticity.     

Stimulation-dependent release of adenosine triphosphate from hippocampal slices

Schaffer collaterals of rat and mouse hippocampal slices were stimulated with bursts of pulses (300 Hz for 50 ms, 2-s intervals) for 30-s which caused a stable increase in the size of the population spike known as long-term potentiation. The release of adenosine triphosphate (ATP) was measured with a luciferase-luciferine system and the light emitted was recorded with a photomultiplier placed beneath a modified slice chamber. ATP release was observed shortly after the start of stimulation and was quantified by comparison with the response of standard solutions of ATP. No ATP release was observed in a Ca2+ free solution or after low frequency stimulation (1 Hz). Glutamate (2 mM), applied without electrical stimulation, did not evoke ATP release. Also, the glutamate receptor blocker, kynurenic acid (10 mM), did not block ATP release. It is concluded that ATP is released from electrically stimulated hippocampal slices from presynaptic nerve terminals in a calcium-dependent fashion and may play a role in the modulation of synaptic efficiency.     

The effect of acidic amino acid antagonists on synaptic transmission in the hippocampal formation in vitro

The effects on synaptic efficacy of the putative acidic amino acid antagonists, 2-amino-4-phosphonobutyric acid (APB), 2-amino-3-phosphonopropionic acid (APP), 1-hydroxy-3-amino-pyrrolidone-2 (HA-966) and glutamic acid diethyl ester (GDEE), were tested by bath application to the hippocampal slice preparation. On the basis of previous work, we hypothesized that APB, HA-966 and GDEE might antagonize synaptic responses to either glutamate or aspartate, but APP should antagonize only synaptic responses to aspartate.APB and HA-966 reduced the amplitude of the extracellular EPSP recorded during stimulation of the perforant path fibers, but APP and GDEE were without effect. APB, APP and HA-966, but not GDEE, consistently inhibited transmission at Schaffer collateral and commissural synapses. The mossy fiber evoked extracellular EPSP was unaffected by these agents. At the concentrations used in this study (usually 2.5 mM) none of these drugs affected the amplitude of presynaptic fiber potentials or antidromic responses, indicating that they probably acted at synapses.The spontaneous activity of hippocampal pyramidal cells, but not of dentate granule cells, increased in the presence of 2.5 mM APB. The amplitude of the population spike generated by Schaffer commissural stimulation initially increased following introduction of APB into the medium and then declined in parallel with the extracellular EPSP. In addition, APB reduced the duration of recurrent inhibition during the period when pyramidal cell firing was enhanced. These results can be explained by an antagonism at the synapse between pyramidal cell and inhibitory interneuron.The actions of these antagonists support previous suggestions that glutamate serves as transmitter of the hippocampal perforant path fibers, that asparate and possibly also glutamate mediate transmission at Schaffer commissural synapses and that neither amino acid serves as transmitter of the mossy fibers.     

Gangliosides modulate Schwann cell proliferation and morphology

We examined the effect of gangliosides on Schwann cell cultures isolated from neonatal rat sciatic nerves. Addition of gangliosides (GM1, GM3, and ganglioside mixture) at concentrations between 0.25 and 2 mg/ml significantly diminished both the baseline rate of proliferation of the Schwann cells and their response to two types of mitogens, the axolemmal fragments and derivatives of adenosine 3'-5'-monophosphate (cAMP). Gangliosides, the sialic acid residue of which had been removed, were highly toxic to the Schwann cells, which went to indicate that sialic acid is necessary to produce the inhibitory effects. Gangliosides also produced prominent changes in the morphological appearance of the Schwann cells. Most of the Schwann cells treated with gangliosides had an elongated shape with long processes and an alignment of end-to-end or side-by-side cell adhesion. These effects of gangliosides apparently were not mediated by cAMP, since intracellular cyclic adenosine monophosphate (cAMP) of Schwann cells at a basal- and forskolin-stimulated level was not altered by the exogenous gangliosides. These findings indicate that the direct effect of gangliosides on Schwann cells should also be considered as a background mechanism of ganglioside-induced facilitation of neuronal regeneration.