CHANGES IN AMOUNT OF COLCHICINE-BINDING PROTEIN (TUBULIN) IN RABBIT BRAIN DURING DEVELOPMENT1

Colchicine binding was used as a measure of the levels of microtubule protein (tubulin) in several regions of rabbit brain during postnatal development. All regions studied showed a decrease in tubulin per mg of total protein; however, each region showed an increase in total tubulin from 1 day of age to adulthood. The net change in tubulin during development coincided with a proliferation of dendrites (which are rich in microtubules) and a decrease in microtubules from spindle apparatus, axons and astrocytes. We suggest that the total amount of tubulin changes in response to demands of the maturing brain cell for microtubules with different functional roles.     

Immunohistochemical Localization of α-Mannosidase During Postnatal Development of the Rat Cerebellum

The localization of alpha-D-mannosidase in the rat cerebellum was studied by using indirect immunohistochemistry at both optical and electron microscopic levels. In the adult the enzyme is particularly concentrated in the dendrites and cell bodies of Purkinje cells, basket cells, and Golgi neurons in the cerebellar cortex and in the cytoplasm and dendrites of deep nuclei neurons. The cytoplasm of granule cells is poorly stained, whereas parallel fibers, white matter, Bergman fibers, and Golgi epitheloid cell perikarya show virtually no staining. Electron microscopy suggests that most of the staining is found in the cytosol, although some staining is found in the postsynaptic densities of the synapses between parallel fibers and Purkinje dendrites. The pattern of staining was followed throughout the postnatal development of the rat cerebellum. At bith an intense and diffuse staining is found in all cells except those of the external germinative layer. At the 6th postnatal day, Purkinje cell bodies and apical cones are strongly labeled. From the 13th day on the pattern is very similar to that found in the adult. However, at the 18th postnatal day (when compared with the other structures), the staining of Purkinje cell dendrites seems to be higher than at all other ages. These data are correlated with biochemical studies and discussed in relation to the possible role of this enzyme during the postnatal development of the rat cerebellum.     

Electron microscopic localization of neuron-specific enolase in rat and mouse brain

The cellular distribution and intracellular localization of neuron-specific enolase (NSE) has been studied by electron microscopic immunocytochemistry in the brain of the rat and of the mouse. Although the intensity of staining was less in the mouse, the same structures were positive in both species. In the cerebrum, the neuronal perikarya and dendrites were intensely stained, but staining was almost entirely absent in the presynaptic terminals. The deep neurons of the brain stem were also positive. In the cerebellum, perikarya, axons, and parallel fibers of the granule cell neurons were stained as were the synaptic vesicles and presynaptic membranes of the synapses between the parallel fibers and the Purkinje cell dendrites. Golgi cell dendrites, basket cells and their axons, and mossy fibers were also positive. In contrast, the Purkinje cells including their dendrites, and the climbing fibers that formed synapses with the Purkinje cell dendrites were not stained. The majority of the myelinated axons in both the cerebrum and the cerebellum did not stain, but the fibrillary astrocytic processes between myelinated axons in the white matter did. Oligodendroglia, protoplasmic astrocytes, Bergmann glia, astrocytes investing capillaries, and vascular endothelial cells were negative for reaction product. In the positively staining cells and their processes, the positivity was dispersed throughout the cytoplasm and corresponded most closely to the distribution of ribosomes, the granular endoplasmic reticulum, and microtubules. Nuclei, mitochondria, the cisternae of the Golgi complex, myelin lamellae, and most membranes were not stained.     

Microtubule-associated protein 2 within axons of spinal motor neurons: associations with microtubules and neurofilaments in normal and beta,beta'-iminodipropionitrile-treated axons

We have examined the distribution of microtubule-associated protein 2 (MAP2) in the lumbar segment of spinal cord, ventral and dorsal roots, and dorsal root ganglia of control and beta,beta'-iminodipropionitrile- treated rats. The peroxidase-antiperoxidase technique was used for light and electron microscopic immunohistochemical studies with two monoclonal antibodies directed against different epitopes of Chinese hamster brain MAP2, designated AP9 and AP13. MAP2 immunoreactivity was present in axons of spinal motor neurons, but was not detected in axons of white matter tracts of spinal cord and in the majority of axons of the dorsal root. A gradient of staining intensity among dendrites, cell bodies, and axons of spinal motor neurons was present, with dendrites staining most intensely and axons the least. While dendrites and cell bodies of all neurons in the spinal cord were intensely positive, neurons of the dorsal root ganglia were variably stained. The axons of labeled dorsal root ganglion cells were intensely labeled up to their bifurcation; beyond this point, while only occasional central processes in dorsal roots were weakly stained, the majority of peripheral processes in spinal nerves were positive. beta,beta'- Iminodipropionitrile produced segregation of microtubules and membranous organelles from neurofilaments in the peripheral nervous system portion and accumulation of neurofilaments in the central nervous system portion of spinal motor axons. While both anti-MAP2 hybridoma antibodies co-localized with microtubules in the central nervous system portion, only one co-localized with microtubules in the peripheral nervous system portion of spinal motor axons, while the other antibody co-localized with neurofilaments and did not stain the central region of the axon which contained microtubules. These findings suggest that (a) MAP2 is present in axons of spinal motor neurons, albeit in a lower concentration or in a different form than is present in dendrites, and (b) the MAP2 in axons interacts with both microtubules and neurofilaments.     

VIP induces the elongation of dendrites and axons in cultured hippocampal neurons: role of microtubules

In neurons from rat hippocampus, VIP induces the elongation of dendrites. In the present study, we have investigated in cultured hippocampal neurons whether VIP changed the actin and tubulin cytoskeleton in dendrites. VIP caused the elongation of dendrites and induced the outgrowth of microtubules, so that they extended up to the tips. In contrast, VIP reduced the F-actin content measured as total pixel after phalloidin staining in dendritic tips. These results suggest that VIP causes dendrite elongation by facilitating the outgrowth of microtubules into the newly formed extensions.     

A composite model for establishing the microtubule arrays of the neuron

Neurons generate two distinct types of processes, termed axons and dendrites, both of which rely on a highly organized array of microtubules for their growth and maintenance. Axonal microtubules are uniformly oriented with their plus ends distal to the cell body, whereas dendritic microtubules are nonuniformly oriented. In neither case are the microtubules attached to the centrosome or any detectable structure that could establish their distinct patterns of polarity orientation. Studies from our laboratory over the past few years have led us to propose the following model for the establishment of the axonal and dendritic microtubule arrays. Microtubules destined for these processes are nucleated at the centrosome within the cell body of the neuron and rapidly released. The released microtubules are then transported into developing axons and dendrites to support their growth. Early in neuronal development, the microtubules are transported with their plus ends leading into immature processes that are the common progenitors of both axons and dendrites. This sets up a uniformly plus-end-distal pattern of polarity orientation, which is preserved in the developing axon. In the case of the dendrite, the plus-end-distal microtubules are joined by another population of microtubules that are transported into these processes with their minus-ends leading. Implicit in this model is that neurons have specialized machinery for regulating the release of microtubules from the centrosome and for transporting them with great specificity.     

MAP3: characterization of a novel microtubule-associated protein

Using monoclonal antibodies we have characterized a brain protein that copurifies with microtubules. We identify it as a microtubule-associated protein (MAP) by the following criteria: it copolymerizes with tubulin through repeated cycles of microtubule assembly in vitro; it is not associated with any brain subcellular fraction other than microtubules; in double-label immunofluorescence experiments antibodies against this protein stain the same fibrous elements in cultured cells as are stained by antitubulin; and this fibrous staining pattern is dispersed when cytoplasmic microtubules are disrupted by colchicine. Because it is distinct from previously described MAPs we designate this novel species MAP3. The MAP3 protein consists of a closely spaced pair of polypeptides on SDS gels, Mr 180,000, which are present in both glial (glioma C6) and neuronal (neuroblastoma B104) cell lines. In brain the MAP3 antigen is present in both neurons and glia. In nerve cells its distribution is strikingly restricted: anti-MAP3 staining is detectable only in neurofilament-rich axons. It is not, however, a component of isolated brain intermediate filaments.     

Biochemical and immunochemical analysis of rat brain dynamin interaction with microtubules and organelles in vivo and in vitro

We have purified a 100-kD rat brain protein that has microtubule cross- linking activity in vitro, and have determined that it is dynamin, a putative microtubule-associated motility protein. We find that dynamin appears to be specific to neuronal tissue where it is present in both soluble and particulate tissue fractions. In the cytosol it is abundant, representing as much as 1.5% of the total extractable protein. Dynamin appears to be in particulate material due to association with a distinct subcellular membrane fraction. Surprisingly, by immunofluorescence analysis of PC12 cells we find that dynamin is distributed uniformly throughout the cytoplasm with no apparent microtubule association in either interphase, mitotic, or taxol-treated cells. Upon nerve growth factor (NGF) induction of PC12 cell differentiation into neurons, dynamin levels increase approximately twofold. In the cell body, the distribution of dynamin again remains clearly distinct from that of tubulin, and in axons, where microtubules are numerous and ordered into bundles, dynamin staining is sparse and punctate. On the other hand, in the most distal domain of growth cones, where there are relatively few microtubules, dynamin is particularly abundant. The dynamin staining of neurites is abolished by extraction of the cells with detergent under conditions that preserve microtubules, suggesting that dynamin in neurites is associated with membranes. We conclude that dynamin is a neuronal protein that is specifically associated with as yet unidentified vesicles. It is possible, but unproven, that it may link vesicles to microtubules for transport in differentiated axons.     

Immunocytochemical localization of microtubule-associated protein 1 in rat cerebellum using monoclonal antibodies

Immunohistochemical staining with monoclonal antibodies showed that microtubule-associated protein 1 (MAP1) has a restricted cellular distribution in the rat cerebellum. Anti-MAP1 staining was found only in neurons, where it was much stronger in dendrites than in axons. There were striking variations in the apparent concentration of MAP1 in different classes of neurons. Purkinje cells were the most strongly labeled, while granule cell neurons gave a faint, threshold-level reaction with the antibody. The reaction of Golgi neurons was intermediate between these two extremes. Equivalent results were obtained using two different methods of tissue preparation. Thus MAP1 appears to be a neuron-specific protein that is highly concentrated in dendrites and occurs at markedly different levels in different types of neurons. These observations provide further indications of heterogeneity among brain microtubules.     

Effects of kinesin-5 inhibition on dendritic architecture and microtubule organization

Kinesin-5 is a slow homotetrameric motor protein best known for its essential role in the mitotic spindle, where it limits the rate at which faster motors can move microtubules. In neurons, experimental suppression of kinesin-5 causes the axon to grow faster by increasing the mobility of microtubules in the axonal shaft and the invasion of microtubules into the growth cone. Does kinesin-5 act differently in dendrites, given that they have a population of minus end–distal microtubules not present in axons? Using rodent primary neurons in culture, we found that inhibition of kinesin-5 during various windows of time produces changes in dendritic morphology and microtubule organization. Specifically, dendrites became shorter and thinner and contained a greater proportion of minus end–distal microtubules, suggesting that kinesin-5 acting normally restrains the number of minus end–distal microtubules that are transported into dendrites. Additional data indicate that, in neurons, CDK5 is the kinase responsible for phosphorylating kinesin-5 at Thr-926, which is important for kinesin-5 to associate with microtubules. We also found that kinesin-5 associates preferentially with microtubules rich in tyrosinated tubulin. This is consistent with an observed accumulation of kinesin-5 on dendritic microtubules, as they are known to be less detyrosinated than axonal microtubules.     

Dysmyelination, demyelination and reactive astrogliosis in the optic nerve of the taiep rat

Taiep is an autosomal recessive mutant rat that shows a highly hypomyelinated central nervous system (CNS). Oligodendrocytes accumulate microtubules (MTs) in association with endoplasmic reticulum (ER) membranes forming MT-ER complexes. The microtubular defect in oligodendrocytes, the abnormal formation of CNS myelin and the astrocytic reaction were characterized by immunocytochemical and ultrastructural methods during the first year of life. Optic nerves of both control and taiep rats were processed by the immunoperoxidase method using antibodies against tubulin, myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP). Taiep oligodendrocytes are strongly immunoreactive against tubulin, indicative of a significant accumulation of microtubules. Early differentiated oligodendrocytes observed with electron microscopy show that MT-ER complexes are mainly present in the cell body. This defect increases during the first year of life; oligodendrocytes show large MT-ER complexes projected within oligodendrocyte processes. Using anti-MBP, there was a progressive reduction of immunolabeling in the myelin sheaths as taiep rats grew older. Ultrastructural analysis revealed severely dysmyelinated axons with a frequently collapsed periaxonal collar. However, through age the myelin sheath became gradually infiltrated by MTs, suggesting their contribution to premature loss of myelin in the taiep rat. Axons of one-year-old taiep rats were severely demyelinated. Modifications in astrocytes revealed by the GFAP antibody showed a strong hypertrophy with increased immunostaining in their processes. As demyelination of axons progressed, taiep rats developed a strong astrogliosis. The present findings suggest that in taiep rats the early abnormal myelination of axons affects the adequate maintenance of myelin, leading to a progressive loss of myelin components and severe astrogliosis, features that should be considered in the pathogenesis of dysmyelinating diseases.     

Specific labeling of climbing fibers shows early synaptic interactions with immature Purkinje cells in the prenatal cerebellum

During development, growing axons must locate target cells to form synapses. This is not easy, since target cells are also growing and even actively migrating. In some brain regions, such axons have been reported to wait for the timing when target cells become mature, without invading their target region. However, in the cerebellum climbing fibers (CFs), major afferent axons, arrive near their target neurons, Purkinje cells, when the neurons are still actively migrating. We, therefore, examined whether synaptic contacts are established at such early stages. To specifically label CFs, we introduced by in utero electroporation a mixture of genes encoding for Ptf1a‐enhancer‐driven Cre recombinase and Cre‐dependent fluorescent protein into the mouse hindbrain at embryonic day (E) 10.5 and observed them during development. The earliest stages at which labeled CFs were observed in the cerebellar primordium were E15.5–E16.5. These fibers were fasciculated in the dorsal region and entered the cerebellar primordium. Some fibers defasciculated and reached the caudal region. At E17.5 and E18.5, fasciculated fibers were also found in the mantle region, and some grew toward the surface of the primordium to penetrate a mass of Purkinje cells. Interestingly, as early as E16.5, labeled fibers were found to run in close apposition to Purkinje cell dendrites and to express a presynaptic marker. These observations suggest that CFs form synapses with Purkinje cells as soon as the fibers enter the cerebellum. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 927–934, 2015     

Formation of new synaptic contacts by Purkinje axon collaterals in the granular layer of deafferented cerebellar cortex of adult rat

In addition to (i) mossy terminals, (ii) Golgi axons, (iii) granule cell dendrites and (iv), occasionally, Golgi cell dendrites, a third axonal profile identified by morphological criteria as the collateral of Purkinje axons, has been found in 2% of all cerebellar glomeruli. These infrequent components of a few glomeruli, however, were never seen in normal cerebellar cortex to establish specialized synaptic contact with glomerular dendrites. Two to four weeks after surgical isolation of the cerebellar cortex, i.e. following the destruction of both efferent and afferent fibres, the number of glomeruli containing (hypertrophic) axonal branches of Purkinje cells has increased to 13% of all surveyed glomeruli. In addition, the Purkinje axon terminals in the mossy fibre-deprived glomeruli were observed to establish numerous Gray II-type synaptic contacts with surrounding granule cell dendrites. It is suggested that the development of heterologous synapses between hypertrophic, or even intact, Purkinje axon collaterals on the one hand and the mossy fibre-vacated granule cell dendrites on the other, is a compensatory, reactive process to the synaptic "desaturation" of granule neurons, which demonstrate a dormant potential of Purkinje cells to form new synaptic contacts in the adult cerebellum.     

Dynamics of outgrowth in a continuum model of neurite elongation

Neurite outgrowth (dendrites and axons) should be a stable, but easily regulated process to enable a neuron to make its appropriate network connections during development. We explore the dynamics of outgrowth in a mathematical continuum model of neurite elongation. The model describes the construction of the internal microtubule cytoskeleton, which results from the production and transport of tubulin dimers and their assembly into microtubules at the growing neurite tip. Tubulin is assumed to be largely synthesised in the cell body from where it is transported by active mechanisms and by diffusion along the neurite. It is argued that this construction process is a fundamental limiting factor in neurite elongation. In the model, elongation is highly stable when tubulin transport is dominated by either active transport or diffusion, but oscillations in length may occur when both active transport and diffusion contribute. Autoregulation of tubulin production can eliminate these oscillations. In all cases a stable steady-state length is reached, provided there is intrinsic decay of tubulin. Small changes in growth parameters, such as the tubulin production rate, can lead to large changes in length. Thus cytoskeleton construction can be both stable and easily regulated, as seems necessary for neurite outgrowth during nervous system development. Action Editor: Upinder Bhalla     

A role of microtubules during the formation of cell processes in neuronal and non-neuronal cells

This review discusses the role of microtubules in the formation of processes from neuronal and non-neuronal cells. In elongating axons of the neuron, tubulin molecules are transported toward the end of pre-existing microtubules, which may be nucleated at the centrosome, via a mechanism called slow axonal flow. Two different hypotheses are presented to explain this mechanism; the transport of soluble monomers and/or oligomers versus the transport of polymerized microtubules. The majority of tubulin seems to be transported as small oligomers as shown by the data presented so far. Alternatively, an active transport of polymerized microtubules driven by microtubule-based motor proteins is postulated as being responsible for the non-uniform polarity of microtubule bundles in dendrites of the neuron. Microtubule-associated proteins (MAPs) play a crucial role in stabilizing the microtubular arrays, whereas the non-uniform polarity of microtubules may be established with the aid of microtubule-based motor proteins. The signals activating centrosomal proteins and MAPs, resulting in process formation, include phosphorylation and dephosphorylation of these proteins. Not only neuronal cells, but also renal glomerular podocytes develop prominent cell processes equipped with well-organized microtubular cytoskeletons, and intermediate and actin filaments. A novel cell culture system for podocytes, in which process formation can be induced, should provide further evidence that microtubules play a pivotal role in process formation of non-neuronal cells.     

A Histochemical Study of the Distribution of β-Hydroxybutyrate Dehydrogenase in Developing Rat Cerebellum

The distribution of beta-hydroxybutyrate dehydrogenase (3-hydroxybutyrate dehydrogenase, EC 1.1.1.30) in the developing rat cerebellum has been determined using a histochemical method. Staining of Purkinje cells, particularly the soma, was seen at all ages examined. Intense staining of the proximal portions of Purkinje dendrites was noted at 8-11 days postnatally, with less prominent staining of Purkinje dendrites and surrounding structures of the molecular layer seen at later times. Development of glomeruli in the granule cell layer could also be observed due to the intense staining of these structures. (Although noncerebellar structures were not the focus of this study, intense staining of the choroid plexus of the fourth ventricle was also noted.) the transient external germinal layer of the cerebellum did not show appreciable staining. Since beta-hydroxybutyrate dehydrogenase is required for ketone body metabolism, the apparent low level of this enzyme in the external germinal layer suggests that the cells of this layer are not particularly well adapted for utilization of ketone bodies. Thus these results do not provide support for the suggestion that ketone bodies may serve as major substrates for energy metabolism in the external germinal layer of the developing cerebellum. Indeed, the rather restricted distribution of this enzyme in both developing and mature cerebellum (and presumably elsewhere in brain) suggests that ketone body metabolism may be largely confined to relatively few specific cellular compartments.     

MAP2 and tau bind longitudinally along the outer ridges of microtubule protofilaments

MAP2 and tau exhibit microtubule-stabilizing activities that are implicated in the development and maintenance of neuronal axons and dendrites. The proteins share a homologous COOH-terminal domain, composed of three or four microtubule binding repeats separated by inter-repeats (IRs). To investigate how MAP2 and tau stabilize microtubules, we calculated 3D maps of microtubules fully decorated with MAP2c or tau using cryo-EM and helical image analysis. Comparing these maps with an undecorated microtubule map revealed additional densities along protofilament ridges on the microtubule exterior, indicating that MAP2c and tau form an ordered structure when they bind microtubules. Localization of undecagold attached to the second IR of MAP2c showed that IRs also lie along the ridges, not between protofilaments. The densities attributable to the microtubule-associated proteins lie in close proximity to helices 11 and 12 and the COOH terminus of tubulin. Our data further suggest that the evolutionarily maintained differences observed in the repeat domain may be important for the specific targeting of different repeats to either alpha or beta tubulin. These results provide strong evidence suggesting that MAP2c and tau stabilize microtubules by binding along individual protofilaments, possibly by bridging the tubulin interfaces.     

Growth cones: the mechanism of neurite advance

Growth cones are the highly motile structures found at the tips of growing axons and dendrites (neurites), which extend from neurones, during the development of the nervous system. They function both as detectors and transducers of extrinsic guidance cues and as regions where the neurite assembly, advance cannot occur. Assembly of the neurite cytoskeleton in growing neurites chiefly involves microtubule assembly at the growth cone. Some of the factors that may influence microtubule assembly in growth cones are becoming apparent and include post-translational modification of tubulin itself and microtubule associated proteins, particularly tau and MAP1B.     

Localization of 4.1 related proteins in cerebellar neurons

Localization of 4.1 related proteins in neurons was studied with immunofluorescence microscopy and with immunoelectron microscopy on ultrathin cryosections. In rat cerebellum, 4.1 immunoreactive proteins were demonstrated in Purkinje cell bodies, dendrites and other neurons in the cerebellar cortex. Some glial cells showed staining, but no labeling was found in myelinated axons of the white matter and of the glomeruli in the granule cell layer. At the ultrastructural level, the 4.1 related proteins were localized mainly in the cytoplasmic matrix, while some labeling was found underneath the plasma membrane. To determine whether 4.1 related proteins in neuronal cytoplasm exist as part of the cytoskeleton or not, PC12 cells cultured in the presence of nerve growth factor were stained with the anti-4.1 antibody. Since cytoplasmic staining was retained after detergent treatment, the 4.1 related proteins seem to exist as a component of the neural cell cytoskeleton. Localization of 4.1 related proteins during the postnatal development of the cerebellum was also studied. In Purkinje cells, localization of 4.1 related proteins changed according to the stages of the postnatal development. The present data suggest that 4.1 related proteins in neurons localized mainly in the cytoplasm and may play some role in organizing cytoskeletal networks in the cytomatrix. Their distribution is developmentally regulated in some neurons, possibly in relationship to their maturation in the cytoskeleton.