Neuritic Varicosities Research Articles

SARS-CoV-2 S1 Protein Induces Endolysosome Dysfunction and Neuritic Dystrophy.

SARS-CoV-2 is the viral cause of the COVID-19 pandemic. Increasingly, significant neurological disorders have been associated with COVID-19. However, the pathogenesis of these neurological disorders remains unclear especially because only low or undetectable levels of SARS-CoV-2 have been reported in human brain specimens. Because SARS-CoV-2 S1 protein can be released from viral membranes, can cross the blood-brain barrier, and is present in brain cells including neurons, we tested the hypothesis that SARS-CoV-2 S1 protein can directly induce neuronal injury. Incubation of primary human cortical neurons with SARS-CoV-2 S1 protein resulted in accumulation of the S1 protein in endolysosomes as well as endolysosome de-acidification. Further, SARS-CoV-2 S1 protein induced aberrant endolysosome morphology and neuritic varicosities. Our findings suggest that SARS-CoV-2 S1 protein directly induces neuritic dystrophy, which could contribute to the high incidence of neurological disorders associated with COVID-19.

Three-dimensional fine structure of the organization of microtubules in neurite varicosities by ultra-high voltage electron microscope tomography.

Neurite varicosities are highly specialized compartments that are involved in neurotransmitter/ neuromodulator release and provide a physiological platform for neural functions. However, it remains unclear how microtubule organization contributes to the form of varicosity. Here, we examine the three-dimensional structure of microtubules in varicosities of a differentiated PC12 neural cell line using ultra-high voltage electron microscope tomography. Three-dimensional imaging showed that a part of the varicosities contained an accumulation of organelles that were separated from parallel microtubule arrays. Further detailed analysis using serial sections and whole-mount tomography revealed microtubules running in a spindle shape of swelling in some other types of varicosities. These electron tomographic results showed that the structural diversity and heterogeneity of microtubule organization supported the form of varicosities, suggesting that a different distribution pattern of microtubules in varicosities is crucial to the regulation of varicosities development.

Reelin immunoreactivity in neuritic varicosities in the human hippocampal formation of non-demented subjects and Alzheimer’s disease patients

BackgroundReelin and its downstream signaling members are important modulators of actin and microtubule cytoskeleton dynamics, a fundamental prerequisite for proper neurodevelopment and adult neuronal functions. Reductions in Reelin levels have been suggested to contribute to Alzheimer’s disease (AD) pathophysiology. We have previously reported an age-related reduction in Reelin levels and its accumulation in neuritic varicosities along the olfactory-limbic tracts, which correlated with cognitive impairments in aged mice. Here, we aimed to investigate whether a similar Reelin-associated neuropathology is observed in the aged human hippocampus and whether it correlated with dementia status.ResultsOur immunohistochemical stainings revealed the presence of N- and C-terminus-containing Reelin fragments in corpora amylacea (CAm), aging-associated spherical deposits. The density of these deposits was increased in the molecular layer of the subiculum of AD compared to non-demented individuals. Despite the limitation of a small sample size, our evaluation of several neuronal and glial markers indicates that the presence of Reelin in CAm might be related to aging-associated impairments in neuronal transport leading to accumulation of organelles and protein metabolites in neuritic varicosities, as previously suggested by the findings and discussions in rodents and primates.ConclusionsOur results indicate that aging- and disease-associated changes in Reelin levels and proteolytic processing might play a role in the formation of CAm by altering cytoskeletal dynamics. However, its presence may also be an indicator of a degenerative state of neuritic compartments.

Three-dimensional distribution of TrkA neurotrophin receptors in neurite varicosities of differentiated PC12 cells treated with NGF determined by immunoelectron tomography

Nerve growth factor (NGF) initiates the activation of TrkA tyrosine kinase receptors and numerous subsequent signaling cascades. However, the dynamics of the process including the translocation of TrkA is still unclear. In this study, the effect of NGF or membrane depolarization on the endocytic process and TrkA localization in the neuronal cell line PC12 was analyzed by live-cell imaging and immunoelectron tomography using an ultra-high voltage electron microscope (UHVEM). Both NGF re-stimulation and high potassium-induced depolarization enhanced the endocytic uptake of the fluorescent indicator into acidic organelles within varicosities as well as cell bodies. However, the transition of uptake differed completely. NGF also significantly increased the number of TrkA-containing varicosities. Immunoelectron tomography in whole-mounted cells showed that NGF induced the recruitment of TrkA to the surface membrane of neurite varicosities as well as the multivesicular bodies (MVBs) and lysosomal complexes inside the varicosities. Three-dimensional analysis revealed that invagination pits and intralumenal vesicles of MVBs contained TrkA immunoreactivity. In addition, TrkA immunoreactivity was scattered in the lysosomal matrices after NGF treatment. These results suggest that the neurite varicosities are intensely active in intracellular membrane trafficking, and play an important role in the degradation and accumulation of the NGF receptor, TrkA, after ligand stimulation.

Notch activation induces neurite remodeling and functional modifications in SH‐SY5Y neuronal cells

Notch proteins are definitely recognized as key regulators of the neuronal fate during embryo development, but their function in the adult brain is still largely unknown. We have previously demonstrated that Notch pathway stimulation increases microtubules stability followed by the remodeling of neuronal morphology with neurite varicosities loss, thicker neuritis, and enlarged growth cones. Here we show that the neurite remodeling is a dynamic event, dependent on transcription and translation, and with functional implications. Exposure of differentiated human SH-SY5Y neuroblastoma cells to the Notch ligand Jagged1 induces varicosities loss all along the neurites, accompanied by the redistribution of presynaptic vesicles and the decrease in neurotransmitters release. As evaluated by time lapse digital imaging, dynamic changes in neurite morphology were rapidly reversible and dependent on the activation of the Notch signaling pathway. In fact, it was prevented by the inhibition of the proteolytic gamma-secretase enzyme or the transcription machinery, and was mimicked by the transfection of the intracellular domain of Notch. One hour after treatment with Jagged1, several genes were downregulated. Many of these genes encode proteins that are known to be involved in protein synthesis. These data suggest that in adult neurons, Notch pathway activates a transcriptional program that regulates the equilibrium between varicosities formation and varicosities loss in the neuronal presynaptic compartment involving the expression and redistribution of both structural and functional proteins.

TorsinA negatively controls neurite outgrowth of SH-SY5Y human neuronal cell line

Early onset generalized dystonia is a severe form of primary dystonia linked to a mutation of the DYT1(TOR1A) gene on chromosome 9q34. DYT1 gene codifies for human torsinA, an AAA+ ATPase associated with the membranes of endoplasmic reticulum (ER) and the synaptic vesicles and proposed to be involved in trafficking of tubular–vesicular membrane through neuronal processes. In this study, the presence and the intracellular distribution of torsinA protein in SH-SY5Y neuroblastoma cells were evaluated by immunofluorescence and Western blot analysis following differentiation with retinoic acid and BDNF. Protein expression was then inhibited by transient antisense transfection and the possible effect on neurite outgrowth was observed. In SH-SY5Y cells torsinA, with an apparent MW of 38 kDa, is endogenously present and distributed, with a punctate pattern, in the cytosol and along the neurites. The protein showed high intensity of immunoreactivity in the neurite varicosities and was partially co-localized with vesicles markers. Terminally differentiated cells showed an increase of protein expression. Oligonucleotide antisense treatment induced a significant response to differentiating stimuli, lead to sprouting of longer neurites and increase in growth cone areas. A relationship between torsinA and tau protein, which is involved in axon elongation and establishment of neuronal polarity, was demonstrated by co-immunoprecipitation experiments. These findings suggest that torsinA, throughout the interaction with microtubule associated proteins, may contribute to control neurite outgrowth and could be involved in maintaining cell polarity.

TorsinA negatively controls neurite outgrowth of SH-SY5Y human neuronal cell line

Early onset generalized dystonia is a severe form of primary dystonia linked to a mutation of the DYT1(TOR1A) gene on chromosome 9q34. DYT1 gene codifies for human torsinA, an AAA+ ATPase associated with the membranes of endoplasmic reticulum (ER) and the synaptic vesicles and proposed to be involved in trafficking of tubular–vesicular membrane through neuronal processes. In this study, the presence and the intracellular distribution of torsinA protein in SH-SY5Y neuroblastoma cells were evaluated by immunofluorescence and Western blot analysis following differentiation with retinoic acid and BDNF. Protein expression was then inhibited by transient antisense transfection and the possible effect on neurite outgrowth was observed. In SH-SY5Y cells torsinA, with an apparent MW of 38 kDa, is endogenously present and distributed, with a punctate pattern, in the cytosol and along the neurites. The protein showed high intensity of immunoreactivity in the neurite varicosities and was partially co-localized with vesicles markers. Terminally differentiated cells showed an increase of protein expression. Oligonucleotide antisense treatment induced a significant response to differentiating stimuli, lead to sprouting of longer neurites and increase in growth cone areas. A relationship between torsinA and tau protein, which is involved in axon elongation and establishment of neuronal polarity, was demonstrated by co-immunoprecipitation experiments. These findings suggest that torsinA, throughout the interaction with microtubule associated proteins, may contribute to control neurite outgrowth and could be involved in maintaining cell polarity.

Non-synaptic exocytosis enhanced in rat cerebellar granule neurons cultured under survival-promoting conditions

Cultured rat cerebellar granule neurons (CGNs) are often used to analyze activity-dependent neuronal selection occurring during brain development. The CGNs survive long only when the culture medium contains a depolarizing agent. However, it is argued whether the depolarization critical for survival is of presynaptic or postsynaptic compartment. Since CGNs form no synapses among them, it is generally assumed that the latter would be the case. But it is possible that the depolarization would induce exocytosis of survival-promoting substances whether or not CGNs form synapses. Here we directly examined the exocytotic activities of CGNs under survival-promoting and survival-limiting conditions by electron microscopy to support this possibility. CGNs possessed clusters of synaptic vesicle-like vesicles (SVVs) in neuritic varicosities. CGNs cultured in high-KCl medium had significantly smaller SVV clusters than those cultured in low-KCl medium. The number of SVVs increased when the high KCl-cultured CGNs were transferred to low-KCl medium, indicating a sustained high rate of exocytosis in high-KCl medium. The majority of the varicosities containing SVVs were not apposed to definite postsynaptic structures, indicating that exocytosis occurs from a non-synaptic surface. Fluorescence Ca 2+ imaging revealed that the high KCl-cultured CGNs had spots of high Ca 2+ along their neurites, corresponding to the varicosities.

A novel function of synapsin II in neurotransmitter release

Although synapsin has been localized to presynaptic structures, its function remains poorly understood. In the present study, we investigated the presynaptic function of synapsin II using a synaptic vesicle recycling process using synapsin-II-overexpressing NG108-15 cells. Western blot analysis with antibodies for synaptic-vesicle-associated protein indicated that the number of synaptic vesicles was approximately doubled in synapsin II transfectants as reported previously. In differentiated synapsin-II-overexpressing and control cells, the application of high potassium induced strong intracellular calcium elevation along neurites and varicosities after differentiation and a weak calcium rise in the cell bodies. The uptake and release of the fluorescent dye FM1-43 revealed that synaptic vesicle recycling in synapsin-II-transfected cells occurred with the same kinetics in the cell body and neuritic varicosities. Furthermore, the area labeled with FM1-43 fluorescence in the synapsin-II-transfected cells was approximately twice as much as in control cells after stimulation, and ATP released after synaptic vesicle fusion with the plasma membrane in synapsin-II-expressing cells was significantly elevated relative to controls. The number of synaptic vesicles paralleled the amount of transmitter released from the cells leading to the conclusion that the number of releasable synaptic vesicles were increased by synapsin II transfection into NG108-15 cells, suggesting that synapsin II may have a role in the regulation of synaptic vesicle number in presynapse-like structures in NG108-15 cells.

In vitro synaptotrophic effects of Cerebrolysin in NT2N cells.

Recent studies have shown that Cerebrolysin can enhance synaptic function and ameliorate synaptodendritic alterations in animal models of neurodegeneration, suggesting a synaptotrophic effect. We hypothesize that Cerebrolysin might exert this effect, in part, by regulating the expression of amyloid precursor protein (APP). We studied the patterns of expression of synaptic proteins during differentiation of human teratocarcinoma cell line NTera 2 (NT2) in the presence or absence of Cerebrolysin. This study showed that the terminally differentiated neurons (NT2N) expressed a wide variety of synaptic markers and that expression of these synaptic-associated proteins coincided with the shift in expression from APP770/751 to APP695. Furthermore, APP immunoreactivity was colocalized with synaptophysin-immunoreactive neuritic varicosities in NT2N neurites, and Cerebrolysin treatment of NT2N cells resulted in an augmented and earlier expression of synaptic-associated proteins. This increased synaptic protein expression coincided with an increase in APP695 over APP770/751. These results support the possibility that synaptotrophic effects of Cerebrolysin might be mediated via regulation of APP expression.

Neuronal cytoskeletal alterations evoked by a platelet-activating factor (PAF) analogue.

Platelet-activating factor (PAF), a phospholipid signaling molecule found in brain, modulates several neural functions and is implicated in the human developmental brain disorder Miller-Dieker Lissencephaly (MDL). Exposure to PAF, and a non-hydrolyzable analogue, methyl carbamyl PAF (mc-PAF), produces the following rapid, reversible effects upon cultured hippocampal neurites: growth cone collapse, neurite retraction, and neurite varicosity formation. In this study, the cytoskeletal alterations that mediate these shape changes were investigated by comparing the effects of mc-PAF with other cytoskeletal-altering drugs, through the fluorescent labeling of cytoskeletal proteins and mitochondria, and by electron microscopy. Results indicate that rearrangements of microtubules (MTs), F-actin, and mitochondria underlie the neurite shape changes produced by mc-PAF. Evidence for MT alteration was obtained by comparing the effects of mc-PAF with nocodozole and taxol. Exposure to nocodazole, a MT-depolymerizing agent, produced growth cone collapse and neurite varicosity formation similar to mc-PAF, whereas pre-incubation of neurites in taxol, a MT-stabilizing drug, was effective in blocking mc-PAF-induced neurite effects. Immunofluorescent labeling and EM revealed MT splaying and unbundling within neurite varicosities following mc-PAF treatment. Immunofluorescent labeling also revealed that F-actin shifted from concentration in the growth cone to a diffuse distribution along the neurite shaft following mc-PAF exposure. Fluorescent labeling and EM also revealed retrograde movement and morphological alterations of mitochondria following mc-PAF exposure, resulting in mitochondrial aggregates within neurite varicosities. These cytoskeletal rearrangements may provide insights into the mechanisms by which PAF influences neuronal activity, and could have important implications for the impairment of neuronal motility observed in MDL.

Calpain I activation in rat hippocampal neurons in culture is NMDA receptor selective and not essential for excitotoxic cell death

Administration of glutamate (100 μM) to primary cultures of rat hippocampal neurons for 1 h led to calpain I activation as determined by monitoring the extent of spectrin breakdown with the antibodies designed to specifically recognize the calpain I-mediated spectrin breakdown products. Based on the studies with subtype selective antagonists of glutamate receptors, glutamate caused calpain I activation specifically through the activation of the NMDA receptor. In parallel experiments, the magnitude and the temporal profiles of Ca 2+ rise were determined by Fura-2 microfluorimetry. Ca 2+ influx through voltage-sensitive Ca 2+ channels, even though leading to substantial Ca 2+ rise, did not by itself activate calpain I. These results indicate that for calpain I activation, the source of Ca 2+ influx is more important than the magnitude of Ca 2+ rise. Glutamate-mediated calpain I activation was fully blocked by preincubation (30 min) of the cultures with calpain inhibitor I, calpain inhibitor II, or calpeptin (all 10 μM). The presence of calpain inhibitors did not, however, in any way ameliorate the massive excitotoxicity resulting from 16 h exposure to glutamate, indicating that calpain I activation and excitotoxicity are not causally related events. Similarly, preincubation with any of the tested calpain inhibitors did not modulate the toxicity resulting from a 10-min exposure to glutamate. Additionally, the presence of calpain inhibitors was detrimental to the clearance of neuritic varicosities resulting from a short-term sublethal exposure to glutamate, suggesting that a physiological level of calpain I activation might actually play an important homeostatic role in the restoration of normal cytoskeletal organization.

Neurites of period-expressing PDH cells in the fly's optic lobe exhibit circadian oscillations in morphology.

Circadian rhythms have been shown both in the expression of the period (per) gene in 'lateral neurons' and in cells of the outermost neuropil, or lamina, of the fly's optic lobe. Some lateral neurons also exhibit PDH peptide-like immunoreactivity, arborizing widely throughout the optic lobe. Using confocal microscopy in the housefly, we analysed the size and spacing of PDH neurite varicosities, sites of possible peptide release exhibiting circadian rhythmicity. During the subjective day in constant darkness, there were fewer, larger varicosities than during subjective night. The endogenous rhythm was masked by the light exposure that occurred under a day-night cycle and continuous light conditions. Our findings indicate that PDH neurites convey circadian information out from the pacemaker, where they could regulate the circadian rhythms that have been described in the lamina, possibly via cyclical release of their peptide.

Differentiation of Y79 cells induced by prolonged exposure to insulin.

Y79 human retinoblastoma cells are known to contain receptors for both insulin and insulin-like growth factors (IGFs), to produce these cytokines and release them in the culture medium. Previously we have demonstrated that IGFs and insulin stimulate Y79 cell proliferation through the involvement of type I IGF receptor and Insulin Receptor Substrate 1 (IRS-1). This paper studies the effect of prolonged exposure to insulin on Y79 cells. Cells grown for 10 days in the presence of insulin were reseeded and incubated once more with insulin. In the reseeded cells proliferation lowered and morphological changes appeared. After 10 days of reseeding, cells stopped proliferating and showed long ramifying neurite processes and varicosities consistent with neuronal differentiation. Morphological differentiation was accompanied by a marked increase in the content of total protein and in that of tubulin, the major protein constituent of microtubules, a marked increase in the content of specialized protein markers of dopaminergic and cholinergic differentiation (dopamine beta-hydroxylase and choline acetyltransferase activities, respectively); a contemporaneous decrease in the content of glial fibrillary acidic protein (GFAP), a specific marker of glial cells, was also observed. Our results demonstrate that prolonged exposure to insulin induces Y79 cells to differentiate into a neuronal-like phenotype. At this moment it is not possible to establish the mechanism by which insulin induces this differentiative effect.

Laminin and neuropeptide Y are increased by synapsin transfection in cultured NG108-15 neuroblastoma/glioma hybrid cells.

We have investigated the presence and expression of laminin and neuropeptide Y (NPY) in several NG108-15 cell lines transfected with synapsin Ib, IIa, or IIb. The content of laminin, a basal membrane glycoprotein that promotes adhesion and induces neurite out-growth and neuronal differentiation, was increased in all transfected cell lines examined. In cells that were chemically differentiated with prostaglandin E1 plus 3-isobutyl-1-methylxanthine, laminin levels were increased even further. The content of NPY, suggested to be a neurotransmitter/neuromodulator in peripheral sympathetic neurons as well as in central neurons, was also increased in all transfected cell lines examined. Immunohistochemical analysis combined with confocal laser microscopy showed that NPY staining was granular and very often enriched in neuritic varicosities. The distribution and the staining pattern of NPY were consistent with storage of NPY in large dense-cored vesicles. The results indicate that, in differentiated neurons, the synapsins increase the levels of a neuropeptide transmitter stored in large dense-cored vesicles and of an extracellular matrix protein associated with neuronal maturation.

Resting membrane potentials and excitability at different regions of rat dorsal root ganglion neurons in culture

To study the role of electrical membrane processes in neuronal regeneration and growth, resting membrane potentials and action potentials of sensory (dorsal root ganglion) neurons growing in culture were measured at the soma, neurite and growth cone using the whole-cell patch-clamp technique. Our results show that resting membrane potentials measured at the soma (−56.8 ±8.8 mV), neurite varicosity (−55.8 ± 5.2 mV) and growth cone (−57.2 ± 4.1 mV) of growing neurons were not statisti cally different. The membrane resistance measured around the resting membrane potential at the neurite varicosity (160 ± 70 MΩ) was smaller than those at the soma (687 ± 540 MΩ) and growth cone (922 ± 825 MΩ). The resting membrane potential measured at the soma using a perforated patch (− 60.3 ± 4.4 mV) was not different from that measured in the normal whole cell. In both configurations, isotonic KC1 (140 mM) depolarized the membrane potential to above OmV. The K + channel blockers quinine, Cs +, 4-aminopyridine and tetraethylammonium depolarized the membrane potential by 10–40 mV, while Na +-free extracellular solution hyperpolarized it by about 10 mV. Extracellularly applied ouabain, intracellular Na +-free or low Cl −-containing solutions did not affect the resting membrane potential. Similar results were obtained for growth cones. Action potentials could be evoked by current pulses in 81% of somata and in all growth cones, but not in neurite varicosities. Current-induced repetitive firing was found in 19% of somata and in 65% of growth cones. The present results indicate identical resting potentials over the whole plasma membrane of process forming dorsal root ganglion neurons in culture, but a variable expression of excitability at the soma, neurite and growth cone.

Aluminum induces neurite elongation and sprouting in cultured hippocampal neurons.

It has been reported that the aluminum content in the human brain increases with age, and it is particularly high in those with Alzheimer's disease. In this study, we found that a low aluminum concentration (100 mumol/L) in the culture media of opossum hippocampal neurons can induce extensive neurite outgrowth (ie, elongation and branching of neurites) and sprouting (ie, outgrowth of filiform processes from neurite varicosities) within 48 hours. Such changes in neurite morphology were remarkably similar to those described in the aged or Alzheimer's disease brain. Neurites that responded to aluminum varied greatly in length, thickness, and branching pattern. Many neurites appeared to have no clear directional growth pattern because they frequently changed their course and formed a meshwork of neurites with others originating from the same cell body. Sprouting neurites varied in length, thickness, and branching pattern, but they always originated from a globular enlargement of neurites along the neurite shaft or at the terminal end. Such growth pattern and extensive sprouting of neurites did not fit the growth pattern displayed by the control neurons. Our findings suggest that aluminum may be involved in the neuronal remodeling characteristic of aging and Alzheimer's disease.

Development of glycine receptor alpha subunit in cultivated rat spinal neurons: An immunocytochemical study

Ontogenesis of the inhibitory glycine receptor was studied up to 12 days in vitro in spinal neurons placed previously in culture at embryonic day 14. The alpha subunit of the receptor was detected using standard and confocal immunofluorescence and a specific monoclonal antibody. The immunostaining was compared to that of synaptophysin, a synaptic vesicle antigen, which was taken as an index of synaptic maturity. Glycine receptors could be detected intracellularly, and not at the cellular surface in some cells as early as 2–3 days in vitro (DIV) prior to any synaptic contact. At 4–5 DIV, the number of cells which expressed the immunoreactivity and the fluorescence intensity increased. At this stage, spherical fluorescent blobs started to migrate in the neurites. From 6 DIV, the glycine receptor alpha subunit was detected at the neuronal surface and was organized in clusters whose number increased progressively with time. From 7 DIV, the intrasomatic immunoreactivity decreased, and at day 12, the pattern of labelling was similar to that observed in the adult spinal cord. A diffuse presence of the receptor at the surface of neurons could never be visualised, and when detected, the glycine receptors were always clustered. Thus, the increasing expression of clusters of glycine receptors at the neuronal surface was paralleled by that of synaptophysin in neuritic varicosities. These data suggest that transport of glycine receptors to the plasmamembrane and the formation of aggregates occurs simultaneously to synaptogenesis.

Induction of formation of presynaptic terminals in neuroblastoma cells by synapsin IIb

The synapsins are a family of closely related phosphoproteins (termed synapsins Ia, Ib, IIa and IIb) associated with synaptic vesicles and implicated in the short-term regulation of neurotransmitter release from nerve endings. During development, expression of the synapsins correlates temporally with synapse formation, but there has been no direct evidence that they are involved in synaptogenesis. Here we report that overexpression of synapsin IIb in the neuroblastoma x glioma hybrid clonal cell line NG108-15 leads, during cell differentiation, to marked increases in the number of neuritic varicosities and in the numbers of small clear vesicles and large dense core vesicles per varicosity, as well as to the appearance of synapse-like cell-cell contacts. Thus, synapsin IIb may be involved in the regulation of synapse formation and, as a result, in long-term neuronal signalling.

Calcium regulation of neuronal differentiation: the role of calcium in GM1-mediated neuritogenesis

Cultures of mouse Neuro-2a neuroblastoma cells treated with 3–6 mM extracellular Ca 2+ exhibited enhanced neurite extension characterized by increased neurite numbers and lengths. The ganglioside GM1 potentiated the effect of extracellular Ca 2+ by increasing further the number and length of the neurites formed in response to exogenous Ca 2+. Maximal neuritic numbers were achieved with 4 mM Ca 2+ while the longest neurites were observed in medium containing 4–6 mM Ca 2+. Stimulation of the Ca 2+ influx with the ionophore A23187 or the amino acid taurine also enhanced neurite formation and GM1 potentiated these actions. Transmission electron microscopy revealed numerous microtubules and neurofilaments in neurites and microfilaments within the spine-like processes along fine neuritic branches and in the filopodia of growth cones. Neuritic varicosities and growth cones contained a variety of vesicles. All of these structures were increased in the presence of GM1 and were increased further by extracellular Ca 2+ or A23187. The ability of GM1 to enhance neuritogenesis was diminished by EGTA or Ruthenium red. Similarly, the effect of GM1 was diminished or abolished by Ca 2+ channel blockers such as CdCl 2 or LaCl 3. X-ray microprobe analysis revealed that GM1 alone enhanced intracellular levels of total ionic and membrane bound Ca 2+, perhaps accounting for the increased neuritogenesis observed under conditions in which Ca 2+ was manipulated. The present study suggests that the neuritogenic action of GM1 is Ca 2+ dependent.