Effect of the weaver (wv) mutation on cerebellar neuron differentiation. II. Quantitation of neuron behavior in culture

Quantitative measurements of neuron behavior from time-lapse microcinematography of dissociated cultures of normal (+/+), heterozygous weaver (+/wv), and homozygous weaver (wv/wv) 7-day-old mouse cerebellum were performed to identify dose-dependent expressions of the mutant allele. Impaired neurite growth by granule cell neurons is a direct result of a dose-dependent increased frequency of neurite retraction and decreased rate of growth cone advancement. The number of retractions per neurite is 0.2, 1.0, and 2.0 for +/+, +/wv, and wv/wv neurites, respectively. Maximal rates of growth cone advancement are 1041, 443, and 250 micron/day for +/+, +/wv, and wv/wv granule cell neurites, respectively. Neurite initiation is actually increased in wv/wv cultures, though the neurites are not well sustained. The frequency of neurite initiation is 1.0, 1.7, and 2.2 for +/+, +/wv, and wv/wv neurons, respectively. Measurements of oscillations of somal position revealed that the cell center moves increasing distances over short times in proportion to the number of mutant genes. Nuclear translocation, the mode of somal migration in vivo and in vitro, occurs at the same frequency and rate in normal and mutant cultures. Weaver gene expression induces a cytopathology affecting various morphogenetic events rather than producing a block at a specific stage in granule cell differentiation. It is hypothesized that the dose-dependent impairments of cell motility reflect weaver gene action at the cell surface or cytoskeleton.     

Neuronal differentiation in cultures of weaver (wv) mutant mouse cerebellum

In the present study we report for the first time a weaver (wv) gene dose effect on neuron survival and neurite formation in vitro. Dissociated cerebellar cells from postnatal 7- and 8-day-old normal ( + / + ), heterozygous weaver ( + /wv) and homozygous weaver (wv/wv) mice were cultured as monolayers on poly-L-lysine coated glass. Cell death occurred rapidly in wv/wv cultures. Cell counts showed that less than 20% of the total neurons and neuronal precursors (identified by “birthday” radiolabeling techniques) survived by Day 3. Cell death was less extensive in + /wv cultures with 65% of the total neurons and 80% of the precursors surviving by Day 3. In contrast to wv/wv cultures, younger neurons survive better than the total population in + /wv cultures. The impairment of neurite formation over the first week is also proportional to the number of mutant genes as shown by quantitation of (a) the percentage of cells with neurites; (b) the percentage of cells with neurites of a given length class with time; (c) the lengths of the longest processes formed per cell. The mean longest neurite lengths obtained by computer digitization at 6 days in vitro were 41.8, 26.8, and 9.0 μm for + / +, + /wv, and wv/wv granule cells, respectively.     

Weaver granule neurons are rescued by calcium channel antagonists and antibodies against a neurite outgrowth domain of the B2 chain of laminin

The weaver mutation impairs migration of the cerebellar granular neurons and induces neuronal death during the first two weeks of postnatal life. To elucidate the molecular mechanisms for the impaired neuronal migration, we investigated the rescue mechanisms of the weaver (wv/wv) granule neurons in vitro. We found that Fab2 fragments of antibodies against a neurite outgrowth domain of the B2 chain of laminin enhanced neurite outgrowth and neuronal migration of the weaver granule neurons on a laminin substratum and in the established cable culture system. The rescue of the weaver granule neurons by antibodies against the B2 chain of laminin may result from the neutralizing effect of these antibodies against the elevated B2 chain levels of the weaver brain. The L-type calcium channel blocker, verapamil (1-5 microM), also rescued the weaver granule neurons. High concentrations of MK-801 (10- 20 microM), a glutamate receptor antagonist and voltage-gated calcium channel blocker, rescued the weaver granule neurons similar to verapamil, but low concentrations of MK-801 (1 microM) had no rescue effect. Simultaneous patch-clamp studies indicated that the weaver granule neurons did not express functional N-methyl-D-aspartate receptors further indicating that the rescue of the weaver granule neurons by MK-801 resulted from its known inhibition of voltage-gated calcium channels. The present results indicate that antibodies against the B2 chain of laminin, verapamil, and high concentrations of MK-801 protect the weaver granule neurons from the otherwise destructive action of the weaver gene. Thus, both the laminin system and calcium channel function contribute to the migration deficiency of the weaver granule neurons.     

Cellular localization of gangliosides in the developing mouse cerebellum: analysis using the weaver mutant

Abstract: The distribution of gangliosides was studied in the weaver ( wv/wv ) mutant mouse, where the vast majority of postmitotic granule cell neurons die prior to their differentiation. The wv mutation also shows a dosage effect, as granule cell migration is slowed or retarded in the + /wv heterozygotes. By correlating changes in ganglioside composition with the well-documented histological events that occur during cerebellar development in the normal (+/+), heterozygous ( +/wv ), and weaver ( wv/ wv ) mutant mice, information was obtained on the cellular localization and function of gangliosides. Ganglioside G_M1 may be enriched in granule cell growth cones and play an important role in neurite outgrowth. A striking accumulation of G_M1, which may result from altered metabolism, occurred in the adult wvlwv mice. G_D3 was heavily concentrated in undifferentiated granule cells, but was rapidly displaced by the more complex gangliosides during differentiation. G_D1a became enriched in granule cells during formation of synaptic and dendritic membranes, whereas G_T1a appeared enriched in Purkinje cell synaptic spines. A possible fucose-containing ganglioside was quantitated only in the wvlwv mice. Ganglioside G_T1b became enriched in granule cells during synaptogenesis, whereas G_Q1b became enriched in these cells after synaptogenesis. The concentrations of G_T1b and especially G_Q1b increased continuously with age. Our results provide further evidence for a differential cellular enrichment of gangliosides in the mouse cerebellum and also suggest that certain gangliosides may be differentially distributed within the membranes of these cells at various stages of development.     

Defective interfering particles modulate VSV infection of dissociated neuron cultures.

Infection of dissociated neuron cultures of mice with VSV and its defective particle DI-T was studied using fluorescent light microscopy as well as transmission and scanning electron microscopy. When cultures are infected with wild virus, VSV replicates selectively in neurons, producing cell death within 24-48 hr. Sensory and immature neurons express viral antigen most rapidly. Viral antigen and viral budding sites are detected along the neuron soma and dendrites. When large amounts of DI-T particles are added to the wild virus inoculum, viral growth is completely suppressed in mature neurons, the cell killing effects of VSV are considerably delayed and co-infected cultures survive for 5-16 days. Viral antigen accumulates in cytoplasmic inclusions and on the membrane of neuron cell somas and dendrites in the virtual absence of viral assembly. Identical modulation of VSV infection in mature neuron cultures is obtained when DI-T particles are added before or after the wild virus, but ultraviolet inactivation of DIs completely abolishes their protective effect. Immature neurons or Vero cells cannot be protected from acute cytopathic changes by an equivalent amount of DI particles. Thus DIs interfere with replication and assembly of the wild virus and attenuate cell killing effects in mature neurons in vitro.     

The weaver granuloprival phenotype is due to intrinsic action of the mutant locus in granule cells: evidence from homozygous weaver chimeras

The weaver mutation (wv) causes a near total loss of midline granule cells in the mouse cerebellum. The cellular site of mutant locus action leading to the granuloprival phenotype was examined with experimental intraspecific and interspecific homozygous weaver chimeras. It was found that the granule cells which survived and successfully migrated to the internal granular layer of the chimeric cerebellum were all of the wild-type (non-wv) genotype. Using interspecies chimeras, it was determined that the genotype of Purkinje cells and Bergmann glia cells was apparently irrelevant to the survival of granule cells. It is concluded that granule cell death is most likely due to the wv locus acting intrinsically to the weaver granule cells, and not to another cellular site of gene action.     

Migration Behavior of Granule Cell Neurons in Cerebellar Cultures. II. An Electron Microscopic Study

We examined the fine structure of migrating granule cell neurons in cerebellar microexplant cultures. Radially migrating bipolar cells extended microspikes or small filopodia from their soma and processes and frequently made contact with neighboring cells. These microspikes contained microfilaments but no microtubules. At the later phase of the migration, in which they had symmetrical bipolar long processes, filopodia extending from perikarial region of cells contained microtubules, suggesting that they are precursors of the future thick perpendicular processes. When cell bodies changed orientation from radial to perpendicular, microtubules that were nucleated from perinuclear centrioles frequently extended into both thick radial and perpendicular processes from the perikarial region. Bundles of 10nm intermediate filaments also appeared in these processes. During migration by the perpendicular contact guidance, many filopodia extending from both the thick leading processes and thin trailing processes made close contacts with the radial parallel neurite. These findings suggest that; 1) The direct contact of the filopodia from both the growth cones and their processes of the granule cells to the neurite bundle plays roles in both the parallel and perpendicular contact guidances. 2) The spacial and temporal changes of cytoskeletons and the association of microtubules with perinuclear centrioles are important for the formation of perpendicular processes and initiation of the perpendicular contact guidance.     

Specificity of a target cell-derived stop signal for afferent axonal growth.

With a novel model culture system in which afferents are co-cultured with purified populations of target neurons, we have demonstrated that a target cell within the central nervous system (CNS), the cerebellar granule neuron, poses a "stop-growing signal" for its appropriate afferents, the mossy fibers. To ask whether this stop signal is afferent specific, we co-cultured granule neurons with another cerebellar afferent system, the climbing fibers from the inferior olivary nuclei, which normally contact Purkinje neurons, and with retinal ganglion cell afferents, which never enter the cerebellum. Granule neurons do not pose a stop signal to either of these afferents. In contrast to pontine mossy afferents that grow well on laminin and showed reduced outgrowth on granule neurons, both olivary and retinal fibers displayed similar growth on laminin alone or on granule neurons. In addition, each afferent showed different degrees of fasciculation and growth cone morphology on laminin. Thus, the growth arrest signal sent by granule neurons is specifically recognized by their appropriate afferents. Moreover, these three types of afferents exhibit varying growth patterns on the same noncellular and cellular substrates, implicating distinct molecular characteristics of growth regulation for different classes of neurons that would contribute to specificity of synapse formation.     

Inhibition of caspases protects cerebellar granule cells of the weaver mouse from apoptosis and improves behavioral phenotype

The homozygous mouse mutant weaver exhibits a massive loss of cerebellar granule neurons postnatally. The death of these cells is associated with a single amino acid mutation in the G protein-activated inwardly rectifying potassium channel, Girk2. Evidence suggests that both the mutated Girk2 channel and the calcium channel-associated N-methyl-d-aspartate receptor play important roles in the apoptotic death of weaver cerebellar granule cells, but the downstream events associated with this process are unknown. In this study, we demonstrate that the consequences of the mutation result in caspase activation. In addition, our results show that caspase inhibition in vivo decreases caspase activation and granule cell apoptosis and significantly improves behavioral deficits associated with the weaver's phenotype.     

Specificity of a target cell-derived stop signal for afferent axonal growth

With a novel model culture system in which afferents are co-cultured with purified populations of target neurons, we have demonstrated that a target cell within the central nervous system (CNS), the cerebellar granule neuron, poses a ?stop-growing signal”? for its appropriate afferents, the mossy fibers. To ask whether this stop signal is afferent specific, we co-cultured granule neurons with an other cerebellar afferent system, the climbing fibers from the inferior olivary nuclei, which normally contact Purkinje neurons, and with retinal ganglion cell afferents, which never enter the cerebellum. Granule neurons do not pose a stop signal to either of these afferents. In contrast to pontine mossy afferents that grow well on laminin and showed reduced outgrowth on granule neurons, both olivary and retinal fibers displayed similar growth on laminin alone or on granule neurons. In addition, each afferent showed different degrees of fasciculation and growth cone morphology on laminin. Thus, the growth arrest signal sent by granule neurons is specifically recognized by their appropriate afferents. Moreover, these three types of afferents exhibit varying growth patterns on the same noncellular and cellular substrates, implicating distinct molecular characteristics of growth regulation for different classes of neurons that would contribute to specificity of synapse formation. © 1992 John Wiley & Sons, Inc.     

The weaver gene encodes a nonautonomous signal for CNS neuronal differentiation.

In the neurological mutant mouse weaver, CNS precursor cells in the external germinal layer (EGL) of the cerebellar cortex proliferate normally, but fail to differentiate and die in the proliferative zone. To examine the autonomy of expression of the weaver gene, we carried out cell-mixing experiments in vitro. In homotypic, reaggregate cultures, weaver EGL precursor cells expressed the general neuronal markers N-CAM, L1, and MAP2, but failed to express the late neuronal antigens TAG-1 and astrotactin, to extend neurites or to migrate on glial fibers. After reaggregation with wild-type EGL precursor cells, weaver precursor cells extended neurites equivalent in length to wild-type cells, migrated along astroglial fibers, and expressed TAG-1 and astrotactin. Rescue of neurite production was also achieved by the addition of membranes from, but not by medium conditioned by wild-type cells. These findings suggest that the weaver gene acts non-autonomously, encoding a membrane-associated ligand that induces EGL neuronal differentiation.     

Axon initiation and growth cone regeneration in cultured motor neurons

Axon initiation in cultured neurons from embryonic ciliary ganglia involves a process in which cell surface motile activity gradually becomes restricted to sites of growth cone formation. Once frank growth cones have commenced to move outward, away from the soma, the broad connecting isthmus of cytoplasm connecting the growth cone to the soma rounds up to form the base of the definitive axon. Motile activity usually does not occur along the sides of axons or of somas. When axons are cut using sharp blades, ruffling and microspike activity are seen on both proximal and distal stumps within times as short as 3–10 min. On rare occasions, portions of the somal surface may also display ruffling and motile activity. It is concluded that the capacity to generate new growth cones and cell surface movements characteristic of locomotion is widely distributed through axoplasm and the neuron.     

Sodium channel beta1 subunits promote neurite outgrowth in cerebellar granule neurons

Many immunoglobulin superfamily members are integral in development through regulation of processes such as growth cone guidance, cell migration, and neurite outgrowth. We demonstrate that homophilic interactions between voltage-gated sodium channel beta1 subunits promote neurite extension in cerebellar granule neurons. Neurons isolated from wild-type or beta1(-/-) mice were plated on top of parental, mock-, or beta1-transfected fibroblasts. Wild-type neurons consistently showed increased neurite length when grown on beta1-transfected monolayers, whereas beta1(-/-) neurons showed no increase compared with control conditions. beta1-mediated neurite extension was mimicked using a soluble beta1 extracellular domain and was blocked by antibodies directed against the beta1 extracellular domain. Immunohistochemical analysis suggests that the beta1 and beta4 subunits, but not beta2 and beta3, are expressed in cerebellar Bergmann glia as well as granule neurons. These results suggest a novel role for beta1 during neuronal development and are the first demonstration of a functional role for sodium channel beta subunit-mediated cell adhesive interactions.     

Distribution of Glutamate Receptors of the NMDA Subtype in Brains of Heterozygous and Homozygous Weaver Mutant Mice

In weaver mice, mutation of an G-protein inwardly rectifying K⁺ channel leads to a cerebellar developmental anomaly characterized by granule and Purkinje cell loss and, in addition, degeneration of dopaminergic neurons. To evaluate other deficits, glutamate receptors sensitive to N-methyl-d-aspartate (NMDA) were examined by autoradiography with [³H]MK-801 in 36 brain regions from heterozygous (wv/+) and homozygous (wv/wv) weaver mutants, and compared to wild type (+/+) mice. In wv/+ and wv/wv mutants labelling decreased in cortical regions, septum, hippocampus, subiculum, neostriatum, nucleus accumbens, superior colliculus and in the cerebellar granular layer. The reductions in [³H]MK-801 binding were particularly specific in the cerebellar granular layer of wv/wv mutants, but an ubiquitous altered NMDA receptor topology was revealed in other brain regions. Abnormal developmental signals, or aberrant cellular responses, may underlie widespread NMDA receptor reductions, while in cerebellar cortex they could be lacking due to the massive loss of cerebellar granule cells.     

Apoptosis and DNA degradation induced by 1-methyl-4-phenylpyridinium in neurons.

Apoptosis is a prominent mechanism of programmed cell death in lymphocytes and in cancer cells not previously found in neurons. We have identified apoptosis and internucleosomal DNA degradation in cultures of cerebellar granule neurons. 1-methyl-4-phenylpyridinium, a selective neurotoxin that destroys the dopaminergic nigrostriatal pathway and results in a parkinsonian syndrome, increases the rate of apoptosis and kills cerebellar granule cells in culture via induction of programmed cell death. Inhibition of gene expression in granule cells with cycloheximide prevents the MPP(+)-induced apoptosis and the DNA fragmentation. Our findings demonstrate a new pathway of neuron death and suggest the possibility that neurodegenerative diseases may result from the inappropriate activation of programmed cell death by apoptosis.     

Rodent CNS neuroblasts exhibit both perpendicular and parallel contact guidance on the aligned parallel neurite bundle.

Mouse cerebellar granule cells showed two types of migration behavior in microexplant cultures. They first migrated along their neurites, showing the typical contact guidance, and then oriented themselves at right angles to the parallel neurites, thus exhibiting the 'perpendicular contact guidance' (Nakatsuji, N. and Nagata, I. 1989 Development, 106, 441-447). To study whether other neurons have the capacity to show similar 'perpendicular contact guidance', we cultured dissociated neuroblasts from various parts of CNS or PNS on parallel neurite bundles. The PNS neuroblasts always extended their processes parallel to the neurite bundle. In contrast, almost all kinds of CNS neuroblasts tested oriented their processes both perpendicular and parallel to the neurite bundles that were all free of glia. Time-lapse video recording revealed that neuroblasts migrated in both directions. Thus, CNS neuroblasts possess the capacity to migrate and extend their processes at right angles to the substratum of heterotypic neurite bundles, which may play an important role in histogenesis of the CNS during development.     

BDNF and NT4/5 promote survival and neurite outgrowth of pontocerebellar mossy fiber neurons.

The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), and NT4/5 are all found in the developing cerebellum. Granule cells, the major target neurons of mossy fibers, express BDNF during mossy fiber synaptogenesis. To determine whether neurotrophins contribute to the development of cerebellar afferent axons, we characterized the effects of neurotrophins on the growth of mossy fiber neurons from mice and rats in vitro. For a mossy fiber source, we used the basilar pontine nuclei (BPN), the major source of cerebellar mossy fibers in mammals. BDNF and NT4/5 increased BPN neuron survival, neurite outgrowth, growth cone size, and elongation rate, while neither NT3 nor NGF increased survival or outgrowth. In addition, BDNF and NT4/5 reduced the size of neurite bundles. Consistent with these effects, in situ hybridization on cultured basilar pontine neurons revealed the presence of mRNA encoding the TrkB receptor which binds both BDNF and NT4/5 with high affinity. We detected little or no message encoding the TrkC receptor which preferentially binds NT3. BDNF and NT4/5 also increased TrkB mRNA levels in BPN neurons. In addition to previously established functions as an autocrine/paracrine trophic factor for granule cells, the present results indicate that cerebellar BDNF may also act as a target-derived trophic factor for basilar pontine mossy fibers.     

Purkinje-cell-derived Sonic hedgehog regulates granule neuron precursor cell proliferation in the developing mouse cerebellum

Purkinje cells (PCs) are the projection neurons of the cerebellar cortex. They receive two major types of synaptic input - that from the inferior olive via climbing fibres and that from the granule neurons via parallel fibres. The precursors of granule neurons proliferate at the surface of the developing cerebellumin the external granule layer (EGL), which persists until postnatal day 14 in the mouse [1]. PCs are thought to provide trophic support for granule neurons [2][3] and to stimulate the proliferation of cells in the EGL [4], but the signalling molecules that mediate these cell-cell interactions have not been identified. I show here that PCs in the developing mouse cerebellum express the gene encoding the morphogen Sonic hedgehog (Shh) and that dividing cells in the EGL express Patched (Ptc) and Gli1, two target genes of which expression is upregulated in response to Hedgehog signalling (see [5] and references therein). Treatment of developing mice with hybridoma cells that secrete neutralizing anti-Shh antibodies [6] disrupted cerebellar development and reduced bromodeoxyuridine (BrdU) incorporation in the EGL of neonatal mice, whereas treatment of dissociated granule neuron cultures with recombinant Shh stimulated BrdU incorporation. These results suggest that PC-derived Shh normally promotes the proliferation of granule neuron precursors in the EGL.     

Geometry of isolated sensory neurons in culture. Effects of embryonic age and culture substratum

Sensory neurons were dissociated from lumbar dorsal root ganglia of embryonic chick and put into culture, either directly or after removing non-neuronal cells by density gradient centrifugation. The cells were grown on culture substrata of various kinds in medium containing nerve growth factor (NGF). After 24 h the cultures were fixed, mounted and analysed. Lengths of neurites were measured, and the numbers of primary processes formed at the cell body and of growth cones were counted. From these values, the rates of growth cone advance and frequency of growth cone branching were calculated. Neuronal outgrowths increased strikingly in length and complexity with embryonic age; there was a 3.5-fold increase in total neurite length and a 3-fold increase in the number of growth cones when neurons from 15-day embryos (E15) were compared with those from 8-day embryos (E8) grown on the same substratum (glass). Growth was markedly greater on surfaces prepared with laminin or conditioned medium compared with plain glass or air-dried collagen. When E15 neurons grown on glass were compared with those grown on laminin, for example, a 2.5-fold increase in total neurite length and a 3-fold increase in the number of growth cones was observed. Calculations showed that a major factor in these changes was an increase in the frequency of growth cone branching. The number of initial processes emanating from the cell body changed with age, but not with the different substrata tested. Non-neuronal cells when present in low numbers and in contact with neurons did not appear to influence neuronal geometry in a systematic way. Our results document the fact that both external factors (in this case, the nature of the culture substratum) and intrinsic factors (stage of development of the neuron) can influence the geometry of neurite outgrowth.     

Presence of protein I, a phosphoprotein associated with synaptic vesicles, in cerebellar granule cells

Abstract: The cerebellar levels of Protein I, a synapse-specific neuronal phosphoprotein, have been investigated in the cerebellar mouse mutants staggerer ( sg ), weaver ( wv ), nervous ( nr ), and Purkinje cell degeneration ( pcd ). The Protein I concentration was reduced by about 66% in sg and wv mutants, representing a 90% loss of Protein I per cerebellum. A heterozygote effect was observed in the wv mutant. These results indicate that a great majority of Protein I in the normal cerebellum may be present in the granule cells. in nr mutants the cerebellar Protein I concentration was reduced by only 12% in 62-day-old mice, suggesting that Purkinje cells contribute little to cerebellar Protein I. However, a greater reduction was observed in pcd mutants, which may reflect on the nature of the pcd mutation.