The effect of tau antisense oligonucleotides on neurite formation of cultured cerebellar macroneurons

Tau, a microtubule-associated protein (MAP) enriched in axons, may have a role in the generation and maintenance of an axonal morphology. Neurons from embryonic day 15 rat cerebellum in culture elaborate two morphologically distinct neurite populations--one with nontapering, elongated axonlike neurites and the other with tapered dendritelike neurites that branch frequently and are selectively stained with antibodies to MAP2. Tau antisense oligonucleotides were utilized in two ways: (1) continuous application of antisense every 24 hr for variable periods of time or (2) application of antisense that was delayed until neurite differentiation was underway. In both cases, 24 hr after the administration of the antisense, tau protein was not detected immunocytochemically. When the antisense was given continuously directly after plating, the neurites persisted as simple minor outgrowths. When antisense was added 72 hr after plating, axonlike neurites were lost, while the remaining neurites continued to grow and increase in complexity. We concluded that the initial establishment of an elongated axonlike neurite is a prerequisite for further neurite maturation; however, once the axon is established, the remaining neurites are able to grow independently and assume a tapered dendritelike appearance.     

Sequential effects of astroglial-derived factors on neurite outgrowth: initiation by protease inhibitors and potentiation by extracellular matrix components.

Astroglial-conditioned medium (GCM) induced two distinct, but intimately related, phases of neuritogenesis in NB2a/d1 neuroblastoma cells--a "rapid-outgrowth," unstable phase, and a delayed, relatively stable phase, which are apparently regulated by glial-derived protease inhibitors and laminin, respectively. The initial rapid outgrowth (less than 4 hr) may be mediated by inhibition of a thrombin-like protease, present as a serum component and/or adsorbed to the outer neuronal surface, since (1) a similar effect was obtained by serum removal or by adding the specific thrombin inhibitor, hirudin; (2) exogenous thrombin inhibited the rapid outgrowth of neurites by GCM; and (3) cell-free enzyme assays confirmed the presence of thrombin-inhibitory activity in GCM. Although neurites induced by removal of serum removal or hirudin addition are rapidly resorbed following serum replenishment or hirudin depletion, GCM-induced neurites continued to elongate after GCM removal, indicating that GCM contained additional neurite-promoting factors. Anti-laminin antiserum did not inhibit the initial elaboration of neurites by GCM but prevented their continued elongation. Anti-laminin antiserum had no affect on neurite outgrowth induced by serum deprivation. The more protracted, second phase of neurite outgrowth could also be achieved by the addition of soluble purified laminin to undifferentiated cells. Unlike neurites at 4 hr, neurites at 24 hr were no longer dependent on the protease inhibitors in GCM, since exogenous thrombin no longer caused them to retract. Simultaneous addition of thrombin and anti-laminin antiserum with GCM had identical inhibitory effects on continued neurite elaboration at 24 hr as did anti-laminin antiserum without thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of mature tau isoforms in the stabilization of neurites in PC12 cells.

Tau microtubule-associated proteins are believed to play a role in regulation of the growth of neuronal processes. In order to study the function of tau protein in vivo, we examined the inhibition of tau expression in PC12 cells by exposing the cells to tau antisense oligodeoxynucleotides. A specific retraction of neurites was observed after 3-4 days of incubation with nerve growth factor (NGF) and the antisense oligodeoxynucleotides. This is different from the previously described retraction of neurites at the initiation step following exposure to tubulin antisense oligodeoxynucleotides, indicating that tau proteins are involved at later stages of neurite outgrowth. Analysis of tau protein isoforms in NGF-induced PC12 cells showed a transition from immature to mature tau isoforms, thus relating the appearance of the latter with the stabilization step of neurite outgrowth. Use of an RNase-protection assay demonstrated a similar switch from immature to mature tau mRNA species. The transition to stable microtubules was verified by the appearance of microtubule bundles and their stability to colchicine treatment. Both phenomena occurred between 2 and 4 days of NGF induction. These results indicate that in vivo only mature tau isoforms are involved in the transition from unstable to stable neurites, which is a key step in neuronal development.     

Role of vimentin in early stages of neuritogenesis in cultured hippocampal neurons

Vimentin is expressed initially by nearly all neuronal precursors in vivo, and is replaced by neurofilaments shortly after the immature neurons become post-mitotic. Moreover, both vimentin and neurofilaments can be detected transiently within the same neurite, leaving open the possibility that vimentin may play a role in the early stages of neuritogenesis. In the present study, cultured hippocampal neurons, which transiently express vimentin in culture, were treated with sense- and antisense-oriented deoxyoligonucleotides encoding regions of the vimentin sequence that overlap the translation initiation codon. Antisense oligonucleotide treatment reduced vimentin-immunoreactivity to background levels. Moreover, while 90–100% of cultured hippocampal neurons elaborated neurites within the first 24 hr following plating, only 24–30% did so in the presence of vimentin antisense oligonucleotides. Inhibition of neurite outgrowth was reversible following removal of antisense oligonucleotide. These findings substantiate earlier studies in neuroblastoma cells, indicating a possible role for vimentin in the initiation of neurite outgrowth.     

Depletion of a fatty acid-binding protein impairs neurite outgrowth in PC12 cells

Epithelial fatty acid-binding protein (E-FABP) is up-regulated in rat dorsal root ganglia after sciatic nerve crush and in differentiating neurons during development. The present study investigates the role of E-FABP during nerve growth factor (NGF)-mediated neurite outgrowth in PC12 cells. Undifferentiated PC12 cells express low levels of E-FABP, while NGF triggers a 6- and 8-fold induction of E-FABP mRNA and protein, respectively. Up-regulation of E-FABP mRNA occurs as early as 24 h after NGF treatment and remains highly expressed over the course of several days, corresponding to NGF-mediated neurite outgrowth. Withdrawal of NGF leads to down-regulation of E-FABP mRNA and retraction of neurites. Immunofluorescence microscopy reveals E-FABP immunoreactivity in the perinuclear cytoplasm, neurites and growth cones of NGF-differentiated cells. To examine the role of E-FABP during neurite outgrowth, PC12 cells were transfected with a constitutive antisense E-FABP vector to create the E-FABP-deficient line PC12-AS. By morphometric analysis, PC12-AS cells treated for 2, 4, and 7 days with NGF exhibited significantly decreased neurite expression relative to control (mock-transfected) cells. Taken together, these data indicate that E-FABP is important in normal NGF-mediated neurite outgrowth in PC12 cells, a finding that is consistent with a potential role in axonal development and regeneration.     

Suppression of an actin-binding protein, drebrin, by antisense transfection attenuates neurite outgrowth in neuroblastoma B104 cells.

Drebrins, actin-binding proteins, are dominantly expressed during embryogenesis and accumulated in neurite processes of postmigratory neurons. While the cytoskeletal proteins are the important factors for regulating neurite outgrowth, the cellular mechanism in neurons is still unclear. To address the role of drebrins in the neurite outgrowth, we have studied the effect of suppression of drebrin on a rat neuroblastoma B104 cell line, which constitutively expresses drebrin. Deprivation of serum or addition of gangliosides in the culture medium induced remarkable neurite outgrowth of B104 cells. We transfected B104 cells with an antisense construct of human drebrin E cDNA and found that the drebrin expression was significantly reduced in the stable antisense cell lines. In response to serum deprivation and gangliosides treatment, their ability to extend neurite processes was significantly attenuated. In contrast, the cell proliferation of the antisense transfectants was arrested by serum deprivation similar to control B104 cells. These data suggest that the drebrins are required for neurite outgrowth in neuronal cells.     

The protein phosphatase inhibitor okadaic acid increases axonal neurofilaments and neurite caliber,and decreases axonal microtubules in NB2a/d1 Cells

When cells were treated with dbcAMP for 3 days to induce the outgrowth of axonal neurites, the addition of the phosphatase inhibitor okadaic acid (0A; 5 nM) for the last 24 hr markedly increased neurofilament subunit immunoreactivity including phosphate-dependent NF-H epitopes in axonal neurites, increased axonal neurite caliber by approximately 30 %, but did not increase neurite contour length. Ultrastructural analysis demonstrated a >2-fold increase in neurofilaments and indicated that neurofilaments were phosphorylated to a similar extent in the presence and absence of OA. Vimentin immunoreactivity, which undergoes down-regulation during dbcAMP-mediated differentiation, was not increased by OA. OA did not induce the precocious appearance of delayed phosphate-dependent neurofilament epitopes suggesting that it did not induce the activation of additional neurofilament kinases. NF-H subunits from cytoskeletons of OA-treated cells were less susceptible to degradation by an endogenous calcium-dependent protease, providing a possible mechanism for neuro filament accumulation during OA treatment. By contrast, OA decreased axonal neurite microtubules, and eliminated stabilized (acetylated) axonal microtubules. OA treatment at earlier times prevented and reversed neurite outgrowth. Despite increased deposition of phosphorylated neurofilaments, OA did not hasten the development of colchicine resistance to neurites, suggesting that stabilization of the axonal cytoskeletal lattice requires neurofilament-microtubule interaction. © 1993 Wiley-Liss, Inc.     

Reexpression of vimentin in differentiated neuroblastoma cells enhances elongation of axonal neurites

Vimentin (Vm) is initially expressed by early neuronal precursors in situ and in culture. Vm is essential for neuritogenesis at least in culture and is gradually replaced by neurofilaments (NFs) because of down-regulation of Vm expression. This period is accompanied by a slowing of axonal elongation. We examined whether continued expression of Vm could foster continued axonal elongation. NB2a/d1 cells differentiated with dibutyryl cAMP were transfected with constructs expressing Vm or the middle-molecular-weight NF subunit (NF-M) each conjugated to green fluorescent protein (GFP). Axonal neurites of cells expressing GFP-Vm were 30% longer than those of nonexpressing cells, or cells expressing GFP-M, and exhibited a decrease in neurite caliber. Expression of GFP-M did not enhance axonal neurite length but significantly increased caliber. These findings provide further evidence of a role for Vm in axonal outgrowth. Culturing of nontransfected cells on laminin increased neurite length, but cells expressing GFP-Vm demonstrated an equivalent increase whether cultured on laminin or culture plastic. Axonal neurites of cells expressing GFP-Vm turned to avoid a nonfavorable substrate (nitrocellulose), but culturing of these cells on nitrocellulose did not impair axonal outgrowth. These latter findings indicate that the more robust outgrowth following reexpression of Vm is independent of a favorable or nonfavorable substrate but that axonal neurites of these cells still interact with the substrate to the extent that the substrate can influence directionality.     

Exogenous BDNF, NT-3 and NT-4 differentially regulate neurite outgrowth in cultured hippocampal neurons

Multiple growth factors contribute to the differentiation of dendritic and axonal processes by a neuron. Cultured hippocampal cells elaborate dendritic and axonal processes following well-defined steps. We used this culture system to determine the specific effects of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) on dendritic and axonal differentiation in hippocampal pyramidal neurons. We demonstrated that each of these neurotrophins exert distinct effects on neurite outgrowth. Both BDNF and NT-3 had positive effects on the outgrowth of undifferentiated neurites, called minor neurites, and on the axonal process of hippocampal pyramidal neurons. However, the effect of NT-3 was more important than that of BDNF. On the other hand, NT-4 did not enhance axonal outgrowth but had only an effect on the outgrowth of minor neurites. Since cytoskeletal proteins play crucial roles in promoting neurite outgrowth, we examined the protein levels of some of these proteins that are associated with neurite outgrowth: beta-actin, gamma-actin, alpha-tubulin, MAP2 and tau. Surprisingly, we did not detect any change in their protein levels. Taken together, our results show that BDNF, NT-3 and NT-4 exert distinct effects on the neuritic compartments of hippocampal neurons.     

Extracellular matrix derived from astrocytes stimulates neuritic outgrowth from PC12 cells in vitro

The ability of astrocyte extracellular matrix to stimulate axonal elongation was examined using an in vitro model system. Extracellular matrix (ECM) was derived from primary cultures of rat astrocytes or meningeal cells, or from a cell line of bovine aortic endothelial cells. The cells were grown in 35-mm tissue culture dishes for 24 h and then removed non-enzymatically, leaving ECM attached to the surface of the culture dishes. Subsequently, PC12 pheochromocytoma cells were seeded onto the ECM and de novo neurite outgrowth was measured. Within 24 h, the PC12 cells exhibited profuse neuritic outgrowth on ECM derived from astrocytes and endothelial cells, without addition of exogenous nerve growth factor. Over a period of 4 days, the neurites continued to elongate and branched extensively. Little or no neuritic outgrowth was observed from PC12 cells grown on uncoated culture dishes or on culture dishes treated with astrocyte-conditioned medium. Only a slight stimulation of neurite outgrowth was observed on meningeal cell-derived ECM. These results indicate that astrocyte ECM, as well as endothelial cell ECM, possesses one or more molecular factors that can stimulate and maintain de novo axonal elongation from PC12 cells. It is suggested that immature astrocytes secrete neurite-promoting factors as a component of the ECM which act to stimulate and possibly guide the growth of axons during in vivo development.     

Sabeluzole accelerates neurite outgrowth in different neuronal cell lines

The outgrowth of neurites in neuronal cell cultures reflects the intrinsic capacity for neurite regeneration and morphological rearrangements after axotomy and in plasticity. The role of fast axonal transport in these neurite outgrowth responses has not been investigated. We have recently shown that sabeluzole (R58735), a new neuro-active compound increases fast axonal transport in cultures of hippocampal neurons. In rat hippocampal neurons, N4 neuroblastoma cells and adult rat dorsal root ganglion cultures, incubation with sabeluzole at an optimal concentration of between 0.1 μM and 0.5 μM enhances neurite outgrowth between 10 and 30%. The relative number of cells with neurite length greater than twice the cell body, is also increased dose-dependently. Time-dependent studies further indicate that the rate of neurite elongation is markedly enhanced during the first 24-48 h. This neurite enhancing effect of sabeluzole is discussed in relation to the enhancement of fast axonal transport.     

Opposing influences of protein kinase activities on neurite outgrowth in human neuroblastoma cells: initiation by kinase A and restriction by kinase C.

The respective roles of cAMP-dependent protein kinase (protein kinase A [PKA]) and protein kinase C (PKC) in the early stages of neurite outgrowth were examined in SH-SY-5Y human neuroblastoma cells. Forskolin or dbcAMP, agents that increase intracellular cAMP levels, and intracellular delivery of PKA catalytic subunit induced neurite outgrowth. The PKA inhibitor, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004), prevented the increases, and decreased further the percentage of cells possessing short, filopodia-like neurites in the absence of inducers. In contrast to effects on PKA activation, PKC activation by 12-0-tetradecanoylphorbol-13-acetate (TPA) reduced the percentage of filopodia-like neurites elaborated by otherwise untreated cells, and prevented neurite outgrowth induced by PKA activators. PKC inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7), staurosporine, and sphingosine induced neurite outgrowth. Neurites induced by PKA activation contained higher levels of tubulin immunoreactivity than those induced by PKC inhibition. Furthermore, PKA-induced neurites rapidly retracted in the presence of colchicine, while those elaborated following PKC inhibition were more resistant. These data suggest that neurites elaborated in response to PKA activation are dependent upon microtubule polymerization, and that neurite induction following PKC inhibition is mediated by a different mechanism. PKA activators and PKC inhibitors exerted additive effects on neurite outgrowth, suggesting that the distinct pathways regulated by these two kinases function cooperatively during neuritogenesis.     

Neurite regeneration by Aplysia neurons in dissociated cell culture: modulation by Aplysia hemolymph and the presence of the initial axonal segment

Neurons from the abdominal ganglion of the mollusc Aplysia californica regenerate neurite processes in dissociated cell culture. Both the nature of neurite outgrowth and the morphology of the cells are influenced by the presence of adult Aplysia hemolymph in the growth medium and the presence of a portion of a cell's original axonal process. Aplysia hemolymph enhances cell survival, the initiation of neurite outgrowth from multiple sites on the cell body surface, the linear growth of the processes, and the amount of branching by those processes. Hemolymph also decreases the diameter of the outgrowing neurite fascicles and the diameter of the individual neurites within the fascicles. The presence of a cell's original axon reduces the time required for the initiation of neurite outgrowth and restricts the formation of multipolar processes. In addition, the presence of an initial axonal segment is essential for neurite regeneration from large adult neurons.     

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.     

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.     

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.     

Characterization of a Neurite Outgrowth Inhibitor Expressed After CNS Injury

Reactive gliosis, a general response to injury in the central nervous system grey and white matter, represents a serious obstacle to axonal regeneration in mammals. In culture, myelin-free plasma membranes from normal rat brain tissue promoted neurite outgrowth, whereas myelin-free membranes purified from injured tissue were inhibitory. The inhibitory activity could be solubilized by detergent, was sensible to glycosaminoglycan lyase digestion and eluted with an apparent molecular weight of 160 – 220 kDa in gel filtration chromatography. When presented as a surface-bound molecule, the inhibitor prevented neurite initiation; when added in a soluble form to growing neurites, it induced their retraction. These results provide cellular and molecular evidence supporting the classical view that, in the mammalian central nervous system, damage-evoked gliosis correlates with the expression of molecules capable of preventing neurite outgrowth.     

Neuro-2a neuroblastoma cells form neurites in the presence of taxol and cytochalasin D

We have examined the role of microtubules and microfilaments in neurite outgrowth by chemically modifying their interaction in Neuro-2a neuroblastoma cells. Cells exposed to taxol (1 microM), an agent that promotes microtubule polymerization and stabilization, did not form neurites over a 24 h period. Similarly, cells exposed to cytochalasin D (4 microM), an agent which promotes microfilament depolymerization, did not develop neurites. However, cells treated simultaneously with taxol (1 microM) and cytochalasin D (4 microM) produced long (50 microns) thin, unbranched neurites. Neurites formed during this simultaneous treatment grew in a circular pattern, lacked typical growth cones, were packed densely with microtubules and were deficient in microfilaments. Untreated cells maintained in control medium for 24 h formed short (15 microns), thick, highly branched neurites containing a dense meshwork of microtubules, microfilaments and neurofilaments. These results demonstrate that taxol does not block neurite outgrowth from Neuro-2a cells maintained under microfilament-limiting conditions. They suggest further that microtubules may provide the major cytoskeletal framework for neurite elongation.     

Selective stabilization of microtubules within the proximal region of developing axonal neurites

This study examined the distribution of labile and stable microtubules (MTs) during axonal neurite elaboration in NB2a/d1 cells using immunocytochemical markers of unmodified (tyrosinated; Tyr), modified (detyrosinated [Glu] and acetylated [Acet]) and total tubulin. Prominent total and Tyr tubulin immunoreactivity was relatively evenly distributed throughout axonal neurites. By contrast, Acet or Glu immunoreactivity was relatively concentrated within the proximal region of the neurite. Ultrastructural analyses demonstrated an array of longitudinal MTs that apparently span the entire neurite length. The observed differential localization of modified tubulin subunits in axonal neurites of these cells may therefore derive from selective stabilization of proximal regions of full-length axonal MTs. This was substantiated by the observation of Acet immunoreactivity on 30-50% of MTs within the most proximal axonal region, along with a proximal-distal decline to < or =5% of Acet immunoreactive MTs, in immunoelectron microscopy (immuno-EM) analyses. Microinjected biotinylated subunits were initially detected in assembled form within soma and proximal neurites, indicative of ongoing tubulin subunit incorporation into MTs within, and/or MT translocation into, proximal neurites. Because acetylation and detyrosination are functions of MT age, their concentration in this region despite deposition and/or transport of biotinylated tubulin suggests that a subset of axonal MTs undergoes subunit turnover and/or translocation at rates vastly slower than that of the majority of axonal MTs. Selective stabilization of the proximal region of a subset of axonal MTs may serve to construct a relatively stationary scaffold against which other axonal elements could translocate to more distal axonal regions for continued axonal outgrowth.     

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).