Inhibition of protein synthesis during CNS myelination produces focal accumulations of membrane vesicles in oligodendrocytes

Summary Optic nerves ofXenopus tadpoles were exposed to cycloheximide to identify changes that occur during CNS myelin membrane formation when protein synthesis is inhibited. Groups of stage 51–56 tadpoles were immersed in either 10 or 20 g ml^–1 cycloheximide, and at specified times between 12 and 18 h after initial immersion tadpoles were killed and their optic nerves prepared for ultrastructural analysis. As early as 12h there were alterations in oligodendrocytes from treated animals compared with control animals. The number of polyribosomes in the perikarya and cell processes was greatly reduced and the rough endoplasmic reticulum was disorganized. Mitochondria and microtubules were normal in appearance. Many oligodendroglial tongue processes at the inner margin of the myelin sheath were enlarged, occasionally indented the axon and were filled with vesicular profiles. Vesicles were noted in other cytoplasmic regions of oligodendrocytes and focal changes in the lamellar structure of myelin were found in paranodal regions. The internodal portions of the myelin sheath, axons and astrocytes appeared normal. Polyacrylamide gels of optic nerves showed that the incorporation of³⁵S-methionine into polypeptides had been almost completely inhibited by treatment with cycloheximide. These observations suggest that cycloheximide, by inhibiting synthesis of myelin proteins, alters the ability of oligodendrocytes to incorporate membrane components into CNS myelin sheaths.     

Novel Dynein DYNC1H1 Neck and Motor Domain Mutations Link Distal Spinal Muscular Atrophy and Abnormal Cortical Development

DYNC1H1 encodes the heavy chain of cytoplasmic dynein 1, a motor protein complex implicated in retrograde axonal transport, neuronal migration, and other intracellular motility functions. Mutations in DYNC1H1 have been described in autosomal‐dominant Charcot–Marie–Tooth type 2 and in families with distal spinal muscular atrophy (SMA) predominantly affecting the legs (SMA‐LED). Recently, defects of cytoplasmic dynein 1 were also associated with a form of mental retardation and neuronal migration disorders. Here, we describe two unrelated patients presenting a combined phenotype of congenital motor neuron disease associated with focal areas of cortical malformation. In each patient, we identified a novel de novo mutation in DYNC1H1: c.3581A>G (p.Gln1194Arg) in one case and c.9142G>A (p.Glu3048Lys) in the other. The mutations lie in different domains of the dynein heavy chain, and are deleterious to protein function as indicated by assays for Golgi recovery after nocodazole washout in patient fibroblasts. Our results expand the set of pathological mutations in DYNC1H1, reinforce the role of cytoplasmic dynein in disorders of neuronal migration, and provide evidence for a syndrome including spinal nerve degeneration and brain developmental problems.     

Myelin repair in vivo is increased by targeting oligodendrocyte precursor cells with nanoparticles encapsulating leukaemia inhibitory factor (LIF)

Multiple sclerosis (MS) is a progressive demyelinating disease of the central nervous system (CNS). Many nerve axons are insulated by a myelin sheath and their demyelination not only prevents saltatory electrical signal conduction along the axons but also removes their metabolic support leading to irreversible neurodegeneration, which currently is untreatable. There is much interest in potential therapeutics that promote remyelination and here we explore use of leukaemia inhibitory factor (LIF), a cytokine known to play a key regulatory role in self-tolerant immunity and recently identified as a pro-myelination factor. In this study, we tested a nanoparticle-based strategy for targeted delivery of LIF to oligodendrocyte precursor cells (OPC) to promote their differentiation into mature oligodendrocytes able to repair myelin. Poly(lactic-co-glycolic acid)-based nanoparticles of ∼120 nm diameter were constructed with LIF as cargo (LIF-NP) with surface antibodies against NG-2 chondroitin sulfate proteoglycan, expressed on OPC. In vitro, NG2-targeted LIF-NP bound to OPCs, activated pSTAT-3 signalling and induced OPC differentiation into mature oligodendrocytes. In vivo, using a model of focal CNS demyelination, we show that NG2-targeted LIF-NP increased myelin repair, both at the level of increased number of myelinated axons, and increased thickness of myelin per axon. Potency was high: a single NP dose delivering picomolar quantities of LIF is sufficient to increase remyelination.Impact statementNanotherapy-based delivery of leukaemia inhibitory factor (LIF) directly to OPCs proved to be highly potent in promoting myelin repair in vivo: this delivery strategy introduces a novel approach to delivering drugs or biologics targeted to myelin repair in diseases such as MS.     

Leukodystrophy caused by plasmalogen deficiency rescued by glyceryl 1‐myristyl ether treatment

Plasmalogens are the most abundant form of ether phospholipids in myelin and their deficiency causes Rhizomelic Chondrodysplasia Punctata (RCDP), a severe developmental disorder. Using the Gnpat‐knockout (KO) mouse as a model of RCDP, we determined the consequences of a plasmalogen deficiency during myelination and myelin homeostasis in the central nervous system (CNS). We unraveled that the lack of plasmalogens causes a generalized hypomyelination in several CNS regions including the optic nerve, corpus callosum and spinal cord. The defect in myelin content evolved to a progressive demyelination concomitant with generalized astrocytosis and white matter‐selective microgliosis. Oligodendrocyte precursor cells (OPC) and mature oligodendrocytes were abundant in the CNS of Gnpat KO mice during the active period of demyelination. Axonal loss was minimal in plasmalogen‐deficient mice, although axonal damage was observed in spinal cords from aged Gnpat KO mice. Characterization of the plasmalogen‐deficient myelin identified myelin basic protein and septin 7 as early markers of dysmyelination, whereas myelin‐associated glycoprotein was associated with the active demyelination phase. Using in vitro myelination assays, we unraveled that the intrinsic capacity of oligodendrocytes to ensheath and initiate membrane wrapping requires plasmalogens. The defect in plasmalogens was rescued with glyceryl 1‐myristyl ether [1‐O‐tetradecyl glycerol (1‐O‐TDG)], a novel alternative precursor in the plasmalogen biosynthesis pathway. 1‐O‐TDG treatment rescued myelination in plasmalogen‐deficient oligodendrocytes and in mutant mice. Our results demonstrate the importance of plasmalogens for oligodendrocyte function and myelin assembly, and identified a novel strategy to promote myelination in nervous tissue.     

Microfluidic compartmentalized co-culture platform for CNS axon myelination research

This paper presents a circular microfluidic compartmentalized co-culture platform that can be used for central nervous system (CNS) axon myelination research. The microfluidic platform is composed of a soma compartment and an axon/glia compartment connected through arrays of axon-guiding microchannels. Myelin-producing glia, oligodendrocytes (OLs), placed in the axon/glia compartment, interact with only axons but not with neuronal somata confined to the soma compartment, reminiscent to in vivo situation where many axon fibres are myelinated by OLs at distance away from neuronal cell bodies. Primary forebrain neurons from embryonic day 16–18 rats were cultured inside the soma compartment for two weeks to allow them to mature and form extensive axon networks. OL progenitors, isolated from postnatal day 1-2 rat brains, were then added to the axon/glia compartment and co-cultured with neurons for an additional two weeks. The microdevice showed fluidic isolation between the two compartments and successfully isolated neuronal cell bodies and dendrites from axons growing through the arrays of axon-guiding microchannels into the axon/glia compartment. The circular co-culture device developed here showed excellent cell loading characteristics where significant numbers of cells were positioned near the axon-guiding microchannels. This significantly increased the probability of axons crossing these microchannels as demonstrated by the more than 51 % of the area of the axon/glia compartment covered with axons two weeks after cell seeding. OL progenitors co-cultured with axons inside the axon/glia compartment successfully differentiated into mature OLs. These results indicate that this device can be used as an excellent in vitro co-culture platform for studying localized axon-glia interaction and signalling.     

The Nogo receptor,its ligands and axonal regeneration in the spinal cord; a review

At least three proteins present in CNS myelin, Nogo, MAG and OMgp are capable of causing growth cone collapse and inhibiting neurite outgrowth in vitro. Surprisingly, Nogo and OMgp are also strongly expressed by many neurons (including neocortical projection cells). Nogo expression is increased by some cells at the borders of CNS lesion sites and by cells in injured peripheral nerves, but Nogo and CNS myelin are largely absent from spinal cord injury sites, which are none the less strongly inhibitory to axonal regeneration. Nogo is found on growing axons during development, suggesting possible functions for neuronal Nogo in axon guidance. Although Nogo, MAG and OMgp lack sequence homologies, they all bind to the Nogo receptor (NgR), a GPI-linked cell surface molecule which, in turn, binds p75 to activate RhoA. NgR is strongly expressed by cerebral cortical neurons but many other neurons express NgR weakly or not at all. Some neurons, such as DRG cells, respond to Nogo and CNS myelin in vitro although they express little or no NgR in vivo which, with other data, indicates that other receptors are available for NgR ligands. NgR expression is unaffected by injury to the nervous system, and there is no clear correlation between NgR expression by neurons and lack of regenerative ability. In the injured spinal cord, interactions between NgR and its ligands are most likely to be important for limiting regeneration of corticospinal and some other descending tracts; other receptors may be more important for ascending tracts. Antibodies to Nogo, mainly the poorly-characterised IN-1 or its derivatives, have been shown to enhance recovery from partial transections of the spinal cord. They induce considerable plasticity from the axons of corticospinal neurons, including sprouting across the midline and, to a limited extent, regeneration around the lesion. Regeneration of corticospinal axons induced by Nogo antibodies has not yet been demonstrated after complete transections or contusion injuries of the spinal cord. It is not clear whether antibodies against Nogo act on oligodendrocytes/myelin or by binding to neuronal Nogo, or whether they can stimulate regeneration of ascending axons in the spinal cord, most of which express little or no NgR. Despite these uncertainties, however, NgR and its ligands offer important new targets for enhancing plasticity and regeneration in the nervous system.An erratum to this article can be found at     

The taiep rat: A myelin mutant with an associated oligodendrocyte microtubular defect

Summary This report describes a new inherited disorder of myelination in the rat, namedtaiep, in which failure of normal myelination of the CNS and subsequent demyelinatiori result in a progressive neurological disturbance. At two months of age, myelin is present throughout the spinal cord, but is immature in the fasciculus gracilis and corticospinal tracts despite the presence of abundant oligodendrocytes. By 12 months, myelin has largely been lost in these spinal cord tracts and also in more rostral parts of the CNS, such as the cerebellum and optic nerves. Other funiculi of the spinal cord show a more diffuse lack of myelin. Oligodendrocytes develop a unique cellular abnormality, most obviously in older rats, which is characterized by the accumulation of microtubules throughout their cytoplasm. As the mutant rats age, there is a continued protracted breakdown of myelin throughout the CNS, with evidence suggesting either persistent hypomyelination or attempts at remyelination of affected axons. It is proposed that the microtubular defect in oligodendrocytes results in a disruption of the normal myelination process in certain areas of the CNS of this mutant, and eventually leads to failure of maintenance of the myelin sheath.     

Dynein motors transport activated Trks to promote survival of target-dependent neurons

Mutations that alter dynein function are associated with neurodegenerative diseases, but it is not known why defects in dynein-dependent transport impair neuronal survival. Here we show that dynein function in axons is selectively required for the survival of neurons that depend on target-derived neurotrophins. Stimulation of axon terminals with neurotrophins causes internalization of neurotrophin receptors (Trks). Using real-time imaging of fluorescently tagged Trks, we show that dynein is required for rapid transport of internalized, activated receptors from axon terminals to remote cell bodies. When dynein-based transport is inhibited, neurotrophin stimulation of axon terminals does not support survival. These studies indicate that defects in dynein-based transport reduce trafficking of activated Trks and thereby obstruct the prosurvival effect of target-derived trophic factors, leading to degeneration of target-dependent neurons.     

The temporal progression of the myelination defect in the taiep rat

The Sprague Dawley myelin mutant, the taiep rat, demonstrates a defect in CNS myelination which worsens with age and which is associated with abnormal accumulations of microtubules in oligodendrocytes. Quantitative and qualitative electron microscopic studies of myelin development and oligodendrocyte morphology were used to describe the temporal development of the defect in this mutant, in three regions of the CNS. The results indicate that the time of onset of myelination is similar in mutant and control rats, however the amount of myelin formed is reduced in the mutant, compared to controls, and there is a loss of myelin from the taiep CNS as the animals age. Thus the myelination defect in taiep has features of both hypomyelination and demyelination. Oligodendrocyte microtubule abnormalities were noted in each region of the taiep CNS at the time of onset of myelination. The earliest changes seen were close associations of oligodendrocyte microtubules with endoplasmic reticulum, with marked accumulations of microtubules filling the cytoplasm of oligodendrocytes from older taiep rats. These findings suggest that the microtubule abnormality in the taiep mutant inhibits both the initial formation and the long-term maintenance of myelin by the oligodendrocyte. In addition, there is also evidence to suggest that although the microtubule abnormality is present in oligodendrocytes throughout the taiep CNS, it results in a more marked defect in the myelination of axons of small diameter.     

Myelination and axonal regeneration in the central nervous system of mice deficient in the myelin-associated glycoprotein

Summary The myelin-associated glycoprotein, a member of the immunoglobulin superfamily, has been implicated in the formation and maintenance of myelin sheaths. In addition, recent studies have demonstrated that myelin-associated glycoprotein is inhibitory for neurite elongationin vitro and it has therefore been suggested that myelin-associated glycoprotein prevents axonal regeneration in lesioned nervous tissue. The generation of mice deficient in the expression of myelin-associated glycoprotein by targeted disruption of themag gene via homologous recombination in embryonic stem cells has allowed the study of the functional role of this moleculein vivo. This review summarizes experiments aimed at answering the following questions: (i) is myelin-associated glycoprotein involved in the formation and maintenance of myelin in the CNS? and (ii) does myelin-associated glycoprotein restrict axonal regeneration in the adult mammalian CNS? Analysis of optic nerves from mutant mice revealed a delay in myelination when compared to optic nerves of wild-type animals, a lack of a periaxonal cytoplasmic collar from most myelin sheaths, and the presence of some doubly and multiply myelinated axons. Axonal regeneration in the CNS of adult myelin-associated glycoprotein deficient mice was not improved when compared to wild-type animals. These observations indicate that myelin-associated glycoprotein is functionally involved in the recognition of axons by oligodendrocytes and in the morphological maturation of myelin sheaths. However, results do not support a role of myelin-associated glycoprotein as a potent inhibitor of axonal regeneration in the adult mammalian CNS.     

Cytoplasmic dynein subunit heterogeneity: implications for axonal transport

The formation and maintenance of neuronal synapses is dependent on the active transport of material between the cell body and the axon terminal. Cytoplasmic dynein is one motor for microtubule-based axonal transport. Two pools of cytoplasmic dynein have been identified in the axon. They are distinguished by their intermediate and light intermediate chain subunits. Each pool is transported at different rates down the axon in association with different proteins or organelles. This review presents several models to discuss the potential functional roles of these different pools of cytoplasmic dynein during axonal transport.     

The early phenotype associated with the jimpy mutation of the proteolipid protein gene

The jimpy mutation of the X-linked proteolipid protein (Plp) gene causes dysmyelination and premature death of the mice. The established phenotype is characterised by severe hypomyelination, increased numbers of dead oligodendrocytes and astrocytosis. The purpose of this study was to define the earliest cellular abnormalities in the cervical spinal cord. We find that on the first and third postnatal days the amount of myelin in jimpy spinal cord is approximately 20% of wild-type. However, the total glial cell density, the number of dead glial cells and the number and distribution of Plp-positive cells, as assessed by in situ hybridization, are similar to wild-type during the first week of life. Immunostaining of cryosections has identified that jimpy spinal cords express on schedule, a variety of antigens associated with mature oligodendrocytes. Dissociated oligodendrocytes, cultured for 18 hours to reflect their in vivo differentiation, express MBP and surface myelin-associated glycoprotein at the same frequency as wild-type. By comparison, the proportion of jimpy oligodendrocytes expressing surface myelin/oligodendrocyte glycoprotein is reduced by approximately 34%. In vivo, however, only a small minority of axons is surrounded by a collar of myelin-associated glycoprotein, suggesting that the majority of jimpy oligodendrocytes fail to make appropriate ensheathment of axons. Although the DM20 isoform is expressed in the embryonic CNS prior to myelin formation, the cellular abnormalities appear to correspond to the time at which the Plp isoform becomes predominant. The results suggest that the primary abnormality in jimpy is the inability of oligodendrocytes to properly associate with, and then ensheath, axons and that oligodendrocyte death compounds, rather than initiates, the established phenotype.     

Novel neuronal proteolipid protein isoforms encoded by the human myelin proteolipid protein 1 gene

The human myelin proteolipid protein 1 gene (hPLP1), which encodes the major structural myelin proteins of the central nervous system (CNS), is classically described as expressed in the oligodendrocytes, the CNS myelinating cells. We identified two new exons in the intron 1 of the hPLP1 gene that lead to the expression of additional mRNA and protein isoforms mainly expressed in neurons instead of oligodendrocytes. Those novel neuronal PLP isoforms are detected as soon as human fetal development and their concomitant expression is specific of the human species. As classical PLP proteins, the novel protein isoforms seem to be addressed to the plasma membrane. These results suggest for the first time that PLP may have functions in humans not only in oligodendrocytes but also in neurons and could be implicated in axono-glial communication. Moreover, this neuronal expression of the hPLP1 gene might explain the neuronal dysfunctions in patients carrying hPLP1 gene mutations.     

Oligodendrocytes and their myelin-plasma membrane connections in JHM mouse hepatitis virus encephalomyelitis.

Mice infected with JHM strain of mouse hepatitis virus develop a demyelinating encephalomyelitis. Myelin sheaths are stripped off axons by invading macrophages after degeneration of the infected oligodendrocytes. The derivation of the virus from granular cytoplasmic particles that condense around and bud into endoplasmic reticulum is demonstrated. The infected oligodendrocytes undergo hypertrophic changes prior to degeneration. Hypertrophic cells are characterized by abundant microtubules, filaments, mitochondria, aggregates of electron-dense particles, and numerous, unusual plasma membrane connections to myelin lamellae. Vacular and hydropic changes are prominent in degenerating cells. The significance of finding infected oligodendrocytes with altered myelin-plasma membrane connections is discussed with reference to the pathogenesis of recurrent, postinfectious demyelination known to develop subsequent to acute virus infections.     

A monoclonal antibody (IN-1) which neutralizes neurite growth inhibitory proteins in the rat CNS recognizes antigens localized in CNS myelin

Summary In previous studies two neurite growth inhibiting protein fractions of 35 and 250 kDa were identified in myelin preparations of the rat CNS. These activities were not found in the myelin of PNS. A monoclonal antibody (mAb IN-1) was raised against the 250 kDa protein fraction and selected for its ability to neutralize the inhibitory effect of CNS myelin and of both isolated protein fractions. IN-1 has been shown bothin vitro andin vivo to neutralize the inhibitory effect of differentiated oligodendrocytes and CNS white matter. In the present study, the antigens of IN-1 were localized by immunohistochemistry on cryostat sections of the adult rat nervous system. The staining pattern of IN-1 was compared to that of mAbs specific for proteins found in CNS and PNS myelin. These proteins include myelin basic protein, myelin oligodendrocyte glycoprotein, and myelin associated glycoprotein. IN-1 stained white matter and myelinated fibre tracts in the CNS on sections of fresh frozen tissue fixed with 95% ethanol: 5% acetic acid (Clark's solution). Sciatic nerve myelin and spinal roots remained unstained. The staining pattern of IN-1 corresponded most closely to that of a mAb against myelin oligodendrocyte glycoprotein, a protein which occurs exclusively in CNS myelin and on differentiated oligodendrocytes.     

东方对虾有髓神经纤维的个体发生的电镜观察

用电镜技术观察了东方对虾（Ｐｅｎａｅｕｓｏｒｉｅｎｔａｌｉｓ）有髓神经纤维的髓鞘化，以及位于其髓鞘与轴突间被命名为髓鞘下间隙和微管鞘这两个侍有结构的个体发生过程。结果如下：（１）在无节幼体（ｎａｕｐｌｉｕｓ）已有由神经历细胞和神经胶质母细胞构成的神经成分出现；（２）在状幼体（ｚｏａｅａ）神经细胞发生裸露的突起，其直径不等，走向不一；（３）在糠虾幼体阶段（ｍｙｓｉｓｓｔａｇｅ）已形成了与成体虾相似的腹神经索，其中的一对内侧巨大纤维已出现，并开始髓鞘化。在此发育阶段后期这两根巨大纤维的髓鞘开始脱离轴突，形成间隙，并在轴突周围开始出现由微管束构成的鞘。在此发育阶段虽已可辨认出一对外侧巨大纤维，但通常尚未髓鞘化；（４）在仔虾（ｐｏｓｔ－ｌａｒｖａ）阶段神经索中许多较粗神经纤维开始髓鞘化，也是由新生的髓鞘直接包绕轴突，然后与之脱离形成髓鞘下间隙和形成直接复盖轴突的微管鞘；（５）髓鞘的形成不是如在脊椎动物髓鞘通常所见到的那样，由一许旺细胞的浆膜片以螺旋方式包绕轴突而形成的，对虾髓鞘的形成则是由许旺氏细胞向两侧伸出许多浆膜片以同心方式包绕轴突，当两侧的浆膜片在某处相遇时，均形成内含２至数根微管的终扣（ｔｅｒｍｉｎａｌｌｏ?     

Kinesin motors and microtubule-based organelle transport in Dictyostelium discoideum

Movement of membrane cargoes and chromosomes is driven by kinesin and dynein motors in most eukaryotic cells. In this review, we describe the known kinesin and dynein genes in Dictyostelium. Dictyostelium primarily utilizes two conventional kinesins, an Unc104/KIF1 kinesin, and cytoplasmic dynein to transport membrane organelles within its cytoplasm. We describe how the biological functions of these motors has been dissected through a combination of biochemical to genetic approaches.     

Relation between axons and oligodendroglial cells during initial myelination I. The glial unit

Summary The morphology of oligodendroglial-axon units was examined by electron microscopy during ensheathment and initial myelination in developing feline spinal cord and corpus callosum white matter. In addition to a qualitative examination of single sections from many stages of development, a morphological analysis of spinal cord and corpus callosum units was made on the basis of serial sections from a few stages. The results show that myelination commences around embryonic/fetal day 40 and the 20th postnatal day in the spinal cord and corpus callosum areas, respectively. In both areas immature glial cells, lacking the cytological features of typical oligodendrocytes, initially associate with several axons and provide them with cytoplasmic sheaths. Serial section analysis of units, which have begun formation of compact myelin, indicates that individual cells are associated with single myelin sheaths in the spinal cord area, in a way principally similar to the Schwann cell-myelin units in developing peripheral nerves. This suggests the possibility that early spinal cord oligodendrocytes might shift from a polyaxonal to a monoaxonal association after initial ensheathment and before formation of compact myelin. In the corpus callosum area the examined serially-sectioned cells were found to be connected to several myelin sheaths through long thin processes. The myelin sheaths related to one cell are relatively uniform in terms of number of myelin lamellae and axon diameter, but the clockwise/counter-clockwise course of the myelin spiral varies randomly. Units containing both homogeneously uncompacted (cytoplasmic) and fully compacted (myelin) sheaths have not been found. In both areas the ensheathing cells achieve an oligodendrocyte-like cytology during formation of the first layers of compact myelin. These observations support the view that oligodendrocytes are structurally heterogeneous: those myelinating prospective large axons seem to differ from those myelinating axons destined to remain small. The possible functional and pathophysiological implications of this heterogeneity remain to be elucidated.     

Minisegments of newborn rat optic nerves in vitro: gliogenesis and myelination

Summary The question of whether the development of CNS glial cells requires the presence of axons or not can be studied with in vitro systems. In order to compare the differentiation of glial cells during development in vitro with that in situ, we have selected the optic nerve, which is anatomically as well as histotypically a well defined structure. For the in vitro investigations, small explants, called minisegments, of newborn rat optic nerves were cultivated taking four major conditions into account: (1) the regular size of the minisegments should guarantee a permanent exchange of the culture medium in order to avoid cell death, (2) neither mechanical nor enzymatic dissociation of the tissue were applied, (3) the minisegments were explanted into flasks without substrate for cell adhesion and (4) the minisegments were under constant gyratory agitation. The following in situ results were obtained: optic nerves of newborn rats are morphologically characterized by the presence of naked axons, astrocytes, glial precursors, and the absence of both differentiated oligodendrocytes and myelin. At postnatal day 5 myelin sheaths are still absent. Two weeks after birth, differentiated oligodendrocytes and microglial cells are present and numerous axons are surrounded by compact myelin. The in vitro experiments show the following main results, which were obtained after 14 h, 2 d, 5 d and 14 d in culture: during time in culture, the shape of minisegment of newborn rat optic nerves undergoes drastic changes, which indicate high cellular dynamics. After 14 h in vitro, axonal profiles, cells with pyknotic nuclei as well as clusters of astrocytes and glial precursors are present. After 2 days in culture the axonal profiles disappeared and the number of degenerating cells decreased drastically. Many large cells, probably phagocytes containing inclusions and more cells are differentiated. At the stage of 5 d in vitro 4 major types of cells can be distinguished: differentiated oligodendrocytes, which form compact and loose myelin, astrocytes, large and small glioblasts and phagocytes. Immunoprecipitates for myelin basic protein and/or myelin associated glycoprotein were found in oligodendrocytes, in their processes and associated to the myelin. Processes of some astrocytes showed immunoreactive products of glial fibrillary acidic protein. After two weeks in culture, the minisegments were mostly composed of astrocytes, whereas oligodendrocytes became rare and phagocytes disappeared. It can be concluded that CNS glial cells can attain their structural and immunocytochemical characteristics in the total absence of neuronal cell bodies and axons. However, it can be speculated that neurons (or neuronal factors) could regulate the number of astrocytes and oligodendrocytes and keep these glial cells in a physiological equilibrium.