Effects of 5-bromodeoxyuridine on development of Mauthner''s neuron and neural retina of Xenopus laevis embryos

Bromodeoxyuridine (BrUdR) injected into Xenopus embryos at stages before, during or after the last period of DNA synthesis in Mauthner's neurons had no serious effect on differentiation of Maunthner's neurons. However, the BrUdR caused widespread cell degeneration in the central nervous system.

The action of BrUdR on retinal neuronal differentiation depended on dose and stage of injection. At all doses (0.005–0.9 μg per injection), and all stages (22–29), the main effect was retinal cell degeneration. The drug produced complete absence of differentiated retinal neurons only when given at early stages (22–24) and at the highest doses (0.5–0.9 μg) that enable the embryo to survive to stage 44. At stages 22–24 at lower doses (0.005 or 0.05 μg per injection) and at stages 26–29 at all doses, some retinal neurons differentiated, although retinal cell degeneration was the main effect.

In these two system BrUdR did not selectively inhibit neuronal differentiation.     

Neural precursor differentiation following transplantation into neocortex is dependent on intrinsic developmental state and receptor competence.

Reconstruction of neocortical circuitry by transplantation of neural precursors, or by manipulation of endogenous precursors, may depend critically upon both local microenvironmental control signals and the intrinsic competence of populations of precursors to appropriately respond to external molecular controls. Dependence on the developmental state of donor or endogenous precursor cells in achieving appropriate differentiation, integration, and connectivity is not clearly understood. Recent studies have demonstrated the ability to generate expandable, often clonal neural precursors at various stages of development. Transplantation of a variety of these precursors suggests that precursor differentiation and integration within the central nervous system (CNS) may depend directly on the level of cellular maturation, with less differentiated, earlier stage precursors offering more flexible but less efficient integration and more differentiated, later stage precursors offering more efficient differentiation to specific phenotypes. To further investigate this hypothesis within neocortex, we used the relatively immature HiB5 multipotent neural precursor cell line derived from embryonic day 16 hippocampus, which is less mature than precursor types that have demonstrated neuronal differentiation in adult neocortex. HiB5 cells labeled fluorescently, radioactively, and genetically were transplanted into murine neocortex under three different conditions expected to offer varying levels of instructive and permissive microenvironmental signals: (1) the developing cortex in utero; (2) regions of adult neocortex undergoing targeted pyramidal neuronal degeneration in which developmental signals are upregulated and in which later stage precursors and immature neurons undergo directed pyramidal neuron differentiation; or (3) the intact adult neocortex. Differentiation and integration of transplanted cells were examined histologically and immunocytochemically by morphology and using neuronal- and glial-specific markers. We found that these precursors underwent differentiation toward cortical neuron phenotypes with characteristic morphologies when transplanted in utero, but failed to do so under either of the adult conditions. HiB5 precursors demonstrated highly immature characteristics in vitro, consistently expressing neuroepithelial but not glial or neuronal markers. Under all conditions, donor cells survived and migrated 1-2 mm from the injection track 2 to 4 weeks after transplantation. HiB5 neural precursors transplanted into the developing cortex of embryonic mice in utero migrated within the cortex, integrated well into the host parenchyma, and differentiated toward morphologically diverse, neuronal phenotypes. HiB5 cells transplanted into the intact cortex of adult mice survived, but did not show neuronal differentiation. In contrast to slightly later stage neural precursors and embryonic neurons used in previous transplantation studies, the HiB5 cells also failed to undergo neuronal differentiation after transplantation into regions undergoing induced apoptotic neuronal degeneration in adult cortex. These results suggested that these early hippocampal-derived precursors might not be fully competent to respond to later stage differentiation and/or survival signals important in neocortex and known to be upregulated in regions undergoing targeted neuronal apoptosis, including the TrkB neurotrophin receptor ligands BDNF and NT-4/5. We investigated this hypothesis and found that undifferentiated HiB5 cells lack catalytic trkB neurotrophin receptors at the mRNA and protein levels, while confirming that they express trkC receptors under the same conditions. Taken together, these findings support a progressive sequence of neural precursor differentiation and a spectrum of competence by precursors to respond to instructive microenvironmental signals. (ABSTRACT TRUNCATED)     

Expression of cell cycle-related proteins in developing and adult mouse hippocampus

Developmental structuring of brain is the result of a strictly coordinated process that involves controlled cell division, neuronal migration and terminal differentiation. Neurogenesis occurs generally during embryonic and early postnatal stages and will be finished in the mature brain. Once differentiated, neurons are incapable of further division but retain the capability of structural and functional plasticity. However, there are distinct regions in the adult brain of mammals that generate neurons continuously throughout life. Among them, the hippocampus, which is known as a region with a high degree of neuroplasticity, is of particular interest in the context of adult neurogenesis. In general, progression through cell cycle phases is regulated by the sequential expression and activation of regulatory proteins like cyclin dependent kinases (cdk), cyclins, or cdk inhibitors (cdki). In postmitotic and terminally differentiated neurons, cell cycle activity is arrested by enrichment of cdkis. The timing of cell cycle exit and neuronal differentiation is likely to be regulated in part by cell cycle regulatory proteins. However, the expression of cell cycle markers in the postnatal or adult brain is still a matter of controversial debate. In the present study, we examined the expression of cdks, cyclins and cdkis within the mouse hippocampus at different developmental stages (embryonic days 17, 19; postnatal day 11 and adult) using immunohistochemical methods. During the prenatal development, cell cycle proteins were localized predominantly in nuclei of all presumptive neuronal populations but expression was not restricted to proliferative cells. With developmental progression, the subcellular localization of most markers was increasingly shifted from nuclear to the cytoplasmic compartment. However, even in the adult, cell cycle-related proteins were found in terminally differentiated pyramidal and granule neurons. Here, they were mainly localized in the perikaryal cytoplasm but only sporadically in neuronal nuclei. Occasionally, immunoreactivity was also found in dendrites and mossy fibers. The present results suggest that cell cycle arrest and terminal differentiation is not necessarily incompatible with the expression of cell cycle-related markers. Thus, they may have supplementary functions in differentiated neurons that might be associated with neuronal plasticity.     

Pigment epithelium-derived factor protects cultured retinal neurons against hydrogen peroxide-induced cell death.

Pigment epithelium-derived factor (PEDF) is a neurotrophic protein synthesized and secreted by retinal pigment epithelial (RPE) cells in early embryogenesis and has been shown to be present in the extracellular matrix between the RPE cells and the neural retina. It induces neuronal differentiation and promotes survival of neurons of the central nervous system from degeneration caused by serum withdrawal or glutamate cytotoxicity. Because the role of PEDF in the retina is still unknown, we examined its ability to protect cultured retinal neurons against hydrogen peroxide (H(2)O(2))-induced cell death. Retinas of 0-2-day-old Sprague-Dawley rats were isolated and dissociated, and the neurons were maintained for 2 weeks in a synthetic serum-free medium. Immunocytochemical labeling showed that 50-60% of the cultured cells were rod photoreceptors. Treatment with H(2)O(2) induced significant death of retinal neurons in a dose- and time-dependent manner. Pretreatment with PEDF prior to insult greatly attenuated H(2)O(2)-induced cytotoxicity, and its effect was shown to be dose dependent. Cytotoxicity was determined by 3,(4,5-dimethylthiazol-2-yl)2, 5-diphenyl-tetrazolium bromide and lactate dehydrogenase assays, and apoptotic cell death was evaluated by the TdT-mediated digoxigenin-dUTP nick-end labeling assay. The present study also showed that H(2)O(2)-induced retinal neuron death was by apoptosis that could be inhibited by PEDF. Combination of PEDF with basic fibroblast growth factor, brain-derived neurotrophic factor, or ciliary neurotrophic factor improves the protection. These data strongly suggest that PEDF is a potential neuroprotective agent in the treatment of retinal degeneration.     

Differentiation and maturation of embryonal carcinoma-derived neurons in cell culture

We have previously shown that retinoic acid-treated cultures of the P19 line of embryonal carcinoma cells differentiate into neurons, glia, and fibroblast-like cells (Jones-Villeneuve et al., 1982). We report here that the monoclonal antibody HNK-1 reacts with the neurons at a very early stage of their differentiation and is, therefore, an early marker of the neuronal lineage. Cells in differentiated P 19 cultures synthesized acetylcholine but not catecholamines, suggesting that at least some of the neurons are cholinergic. The neurons also carry high-affinity uptake sites for GABA but not for serotonin. In long-term cultures, neuronal processes differentiated into axons and dendrites, which formed synapses. This biological system should prove valuable for examining the development and maturation of cholinergic neurons, since their differentiation occurs in cell culture.     

Expression and distribution of microtubule-associated protein 2 (MAP2) in neuroblastoma and primary neuronal cells

We examined the expression and distribution of microtubule-associated protein 2 (MAP2) during the differentiation in culture of both mouse NB2a neuroblastoma and primary embryonic rat neurons. The differentiation of NB2a cells was induced with retinoic acid (RA) which stimulated the extension of a highly branched neuritic network and dibutyryl cAMP which stimulated the outgrowth of long bipolar or monopolar processes. We found that although monoclonal antibodies to MAP2 stained the cell bodies of control and differentiated cells, only the RA-induced neurites were positive for this antigen. These data support our ultrastructural studies indicating that the RA-induced neurites were dendrite-like and that the dibutyryl cAMP-induced processes were axon-like. Studies on the biosynthesis of MAP2 indicated that RA induced a 2-3-fold increase in MAP2 synthesis in 24 h; however, this effect was transient, with the synthesis of MAP2 in RA-treated cells returning to control level by 72 h. Although biosynthetic studies suggested the synthesis of species at 250-300 kdalton, the major molecular weight form in the neuroblastoma cells was 230 kdalton. Immunocytochemical analysis of primary neurons showed staining of neuronal cell bodies and of short processes, but virtually no staining of the long axon-like processes. The staining of neuronal cell bodies and processes was evident at all stages of cell differentiation. This finding was corroborated by immunoblots which showed significant amounts of MAP2 throughout cell development. The molecular weight of the immunoreactive material was ca. 300 kdalton in both primary neurons and rat brain. Immunoblots also revealed that embryonic neurons expressed only MAP2B as they differentiated in culture for 14 days. Biosynthesis studies suggested that early in culture there was a modest increase in MAP2 synthesis, but no detectable change was observed thereafter. We concluded therefore that both neuroblastoma cells and primary neurons can differentiate neuritic processes, which show dendritic properties in terms of morphology and preferential distribution of MAP2.     

Morphology and quantitative changes of transient NPY-ir neuronal populations during early postnatal development of the cat visual cortex

The early postnatal development of neuropeptide Y-containing neurons in the visual cortex of the cat was analyzed. Immunohistochemistry reveals several stages of morphological differentiation and degeneration. Completely undifferentiated neurons have very small somata with nuclei surrounded by a thin rim of cytoplasm and processes unclearly differentiated into dendrites and axons. Processes bear growth cones. Differentiating neurons show an increase in soma size and complexity of processes. Axons are recognizable. Fully differentiated neurons have well-defined axonal and dendritic patterns. Degenerating neurons are identified by thick, heavily beaded processes covered by hairy appendages and vacuolar inclusions in the somata. Cell death is expressed by shrunken somata and lysed, fragmented processes. According to their postnatal time course of differentiation and/or degeneration, NPY-immunoreactive neurons, which form several morphologically distinct cell types, are grouped into 3 neuronal populations. (1) Pseudopyramidal cells, bitufted "rectangular" cells with wide dendritic fields, unitufted cells, and small multipolar cells are located in the gray matter and have a rather primitive morphology resembling cell types found in lower vertebrate cortex and tectum. They constitute a first transient neuronal population, because all neurons are fully differentiated at birth and become largely eliminated by postnatal day (P) 12. (2) Axonal loop cells are mainly located in the white matter. Their most prominent feature is an often long hairpin loop formed by either the main axon itself or by a major collateral. The axonal branches pass through the cortex to connect the white matter and layer I. Axons do not form local plexusses and terminal elements in the gray matter. Neurons differentiate perinatally, form a first peak from P6 to P10, followed by a decrease in cell number and innervation density at P12, followed by a second peak from P15 to P20. After P20 the number of axonal loop cells steadily decreases, and they become eliminated by P48. (3) A third population consists of neurons with a higher degree of axonal ramification and a variety of axonal patterns. Early members are located mainly at the layer VI/white matter border, differentiate during the first postnatal week, and give rise to a diffuse innervation of the gray matter without forming specific terminal elements. Some of the early axonal patterns persist into adulthood, whereas others are not found in the adult brain.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fibroblast growth factor stimulates photoreceptor differentiation in vitro.

Dissociated newborn rat retinal cells were maintained in monolayer culture for periods of up to 11 d. When grown in the absence of exogenous growth factors, 1-2% of the total neuronal population expressed opsin (the photopigment that is specific for maturing photoreceptors). Addition of a single dose of 10 ng/ml basic fibroblast growth factor (bFGF) to the culture medium induced an average increase of sixfold in the numbers of neurons expressing opsin. This supplementation had little effect on the total number of differentiated neurons or of glial cells when measured at the same time points. Furthermore, another specific class of retinal neurons, the amacrine cells, showed no changes following exposure to this growth factor. Two other growth factors known to exert neurotrophic effects, epidermal and nerve growth factor, were without effect. The effect of bFGF was dose dependent, with highly significant differences being observed with as little as 100 pg/ml, and with 700 pg/ml eliciting half-maximal stimulation; maximal effects were observed at 10 ng/ml. Induction of opsin expression by low concentrations of bFGF was blocked completely by an antiserum directed specifically against bFGF, but not by preimmune serum immunoglobulins. This increase in the number of photoreceptors expressing opsin following exposure to bFGF could have been due to either increased cell survival, increased proliferation of progenitor cells, or increased differentiation of immature photoreceptors. There was no increase in overall cell survival under the experimental conditions used, and double labeling immunocytochemistry combined with autoradiographic analysis of 3H-thymidine uptake showed that proliferation of neuronal precursors was not enhanced by the addition of bFGF. In contrast to these observations, cultures established from older (postnatal day 3) retina revealed large numbers of opsin-expressing photoreceptors in all culture plates, with or without added growth factors. This reduction in the stimulatory effects of bFGF with increasing postnatal age is consistent with the period of sensitivity being limited to the cycling of neuronal precursors. It is possible that a bFGF-like molecule is secreted by neighboring cells such as the retinal pigmented epithelium, to participate in retinal development and differentiation. To our understanding, this molecule is the first protein identified to influence specifically the differentiation of photoreceptor cells.     

Memantine protects against secondary neuronal degeneration in lateral geniculate nucleus and superior colliculus after retinal damage in mice

The purpose of this study, on mice, was to determine whether memantine, a glutamate-receptor antagonist of the N -methyl- _d -aspartate (NMDA) subtype, protects against neuronal degeneration in the dorsal lateral geniculate nucleus (dLGN) and superior colliculus (SC) after the induction of retinal damage by intravitreal injection of NMDA.
NMDA (20 mM/2 μl) was injected into the vitreous body of the left eye in mice (day 0). To evaluate the neuroprotective effect of memantine, mice were assigned to one of two memantine-treated groups: receiving a daily administration of memantine at 30 mg/kg/day, p.o. either from day 0 (administered at 1 h before NMDA injection) to day 90 (pretreated group) or from day 7 to day 90 (post-treated group).
The pretreated group exhibited significant suppression of the retinal damage induced by intravitreal injection of NMDA and significant prevention of transsynaptic neuronal degeneration in the dLGN and SC on the contralateral side. Although the mice of the post-treated group displayed no reversion of such retinal damage, they did exhibit protection against neuronal degeneration in the LGN and SC on the contralateral side. These data indicate that memantine can protect against transsynaptic neuronal degeneration in the murine brain (LGN and SC) even if treatment is begun after retinal ganglion cell (RGC) death has started.
Memantine protects against the secondary neuronal degeneration in brain regions in the visual pathway that occurs after retinal damage in mice.     

Differentiation renders susceptibility to excitotoxicity in HT22 neurons

HT22 is an immortalized mouse hippocampal neuronal cell line that does not express cholinergic and glutamate receptors like mature hippocampal neurons in vivo.This in part prevents its use as a model for mature hippocampal neurons in memory-related studies.We now report that HT22 cells were appropriately induced to differentiate and possess properties similar to those of mature hippocampal neurons in vivo,such as becoming more glutamate-receptive and excitatory.Results showed that sensitivity of HT22 cells to glutamate-induced toxicity changed dramatically when comparing undifferentiated with differentiated cells,with the half-effective concentration for differentiated cells reducing approximately two orders of magnitude.Moreover,glutamate-induced toxicity in differentiated cells,but not undifferentiated cells,was inhibited by the N-methyl-Daspartate receptor antagonists MK-801 and memantine.Evidently,differentiated HT22 cells expressed N-methyl-D-aspartate receptors,while undifferentiated cells did not.Our experimental findings indicated that differentiation is important for immortalized cell lines to render post-mitotic neuronal properties,and that differentiated HT22 neurons represent a better model of hippocampal neurons than undifferentiated cells.     

The Relationship of Microglial Cells to Dying Neurons During Natural Neuronal Cell Death and Axotomy-induced Degeneration of the Rat Retina

The interactions between dying neurons and phagocytotic cells within the developing and injured retina remain controversial. The present work explored the role of microglia and investigated whether so-called resident microglial cells are permanently responsible for removing cell debris whenever it is produced. As a first goal, I characterized some quantitative and morphometric features of the small ipsilateral retinocollicular projections and analysed the permanent function of phagocytosing microglia with these projections as a paradigm. To achieve this, I combined the fluorescent dyes Dil and 4Di-10ASP, both of which persist in the labelled ganglion cells after injection into the superior colliculus (SC), and retrograde labelling. After neuronal degradation, the dyes accompany the degradation products, become interiorized and then persist within the phagocytosing microglia. Consequently, early labelling of microglial cells can be assessed by injecting one dye into the SC during the first postnatal day of life, that is, prior to advanced natural neuronal cell death. Labelling of the remaining ipsilaterally projecting neurons with the second dye following intraorbital axotomy in adulthood and during subsequent neuronal death would therefore result in double labelling of some microglial cells, if these were involved in phagocytosis during both the natural and the induced phases of neuronal degradation. The ganglion cells which survived natural neuronal cell death remained fluorescent for 3 months after labelling with either dye on the day of the animal's birth, indicating that both fluorescent probes persisted within neurons. Quantitatively, 1770+/-220 ganglion cells/mm2 were labelled within the contralateral retina and a total population of 1442+/-120 cells/retina were observed within the periphery of the inferior/temporal quadrant of the ipsilateral retina. A smaller, ipsilateral projection of 150+/-24 cells/retina was uniformly scattered throughout the rest of the retinal surface. Transient projections of ganglion cells to either the contralateral or the ipsilateral colliculi and death of labelled ganglion cells during the first postnatal days resulted in labelling of 210+/-36 microglial cells/mm2 within the contralateral retina and a total number of 800+/-120 cells/retina within the inferior/temporal and 200+/-22 cells/retina within the rest of the retina. These labelled microglial cells were observed in adulthood and indicated that after taking away the neuronal cell debris they persisted within the retinal tissue. The small number of prelabelled ganglion cells which formed persistent ipsilateral projections until adulthood were axotomized by transecting the optic nerve, and resulted in additional labelling of microglial cells with the second fluorescent dye as well. Double-labelled microglia were observed within the inferior/temporal quadrant (3500+/-240 cells/retina) and to a lesser extent (340+/-40 cells/retina) scattered over the entire retinal surface. The chronotopological sequence of microglial labelling paralleled that of ganglion cell degeneration. Injection of protease inhibitors into the vitreous body during optic nerve transection retarded retrograde glial cell degeneration, probably by blocking microglial proteases. The results directly proved that the same microglial cells which remove neuronal cell debris in the postnatal retina were reactivated later in life to proteolytically degrade and then phagocytose neurons which had altered because of the axotomy.     

Development of substance P-like immunoreactivity in Xenopus embryos

The development of substance P-like immunoreactivity (SPLI) was studied in the Xenopus embryonic nervous system in order to determine in which neuronal populations and at what developmental times SPLI is expressed. Although Rohon-Beard neurons initially were thought to be the only substance P-immunoreactive cells in the embryonic frog spinal cord, we have demonstrated that several neuronal phenotypes are immunoreactive. The earliest evidence of SPLI was seen at stage 28 (Nieuwkoop and Faber, '67), at which time only some trigeminal ganglion cells, their axons in the ophthalmic nerve, and axons in the lateral tracts of the hindbrain showed SPLI. In the embryonic brain at stages 29/30, 37/38, and 42, SPLI was seen in the hypothalamus, trigeminal ganglion cells and their peripheral axons, the sensory roots of cranial nerve IX/X, and axons in the hindbrain lateral tracts. At premetamorphic stages, SPLI was found in several populations that are immunoreactive in adult amphibia. In the embryonic spinal cord, Rohon-Beard neurons were labeled consistently with reaction product; there was a rostrocaudal time gradient of immunoreactivity with increasing development. The Rohon-Beard neurons were not immunoreactive at developmental stages in which axonal outgrowth was beginning (stage 21), but were strongly immunoreactive at stages in which target cells had been contacted (stage 29). Several types of interneurons in the spinal cord (as classified by Roberts and Clarke, '82) showed SPLI during embryonic stages. At premetamorphic stages the Rohon-Beard neurons began to disappear and the immunoreactive interneurons were distributed similarly to those reported in the adult. Dorsal root ganglia differentiated during these stages, and at this time some of the neurons belonging to these ganglia exhibited substance P-like immunoreactivity.     

Expression, activity and distribution of Na,K-ATPase subunits during in vitro neuronal induction

The expression pattern of the alpha and beta isoforms and the gamma subunit of the Na,K-ATPase was investigated during in vitro induction of pluripotent murine embryonic stem (ES) cells into neuronal cells. alpha1 protein was expressed in undifferentiated ES (UES) cells and throughout all stages studied. In contrast, alpha3 protein was prominent only when neuronal cells have reached full differentiation. In this model, neuron-depleted cultures did not express the alpha3 isoform, indicating its specificity for mature neuronal cells. UES possessed Na,K-ATPase activity consistent with a single isoform (alpha1), whereas in fully mature neuronal cells a ouabain-sensitive isoform (alpha3) accounted for 27+/-4% of the activity, and a ouabain-resistant isoform (alpha1) 66+/-3%. Immunocytochemistry of mature neuronal cells for alpha1 and alpha3 proteins showed a similar distribution, including cell soma and processes, without evidence of polarization. beta1 protein was expressed in uninduced ES, embryonic bodies (EB) and neuronal cells. While proteins of the beta2 and beta3 isoforms were not detected by immunoblots (except for beta2 in UES), their mRNAs were detected in UES and EB (beta2 and beta3), and in immature and fully differentiated neuronal cells (beta3). Message for the beta2 isoform, however, was not present in neuronal cells. gamma subunit mRNA and protein were undetectable at any stage. These results provide further characterization of neuron-like cells obtained by induction of ES cells in vitro, and establish a model for the expression of isoforms of the Na,K-ATPase during neuronal differentiation. The relation to other aspects of neuronal cell development and relevance to a specialised function for the alpha3 subunit in neurons are discussed.     

Dopamine cell degeneration induced by intraventricular administration of 6-hydroxydopamine in the rat: similarities with cell loss in parkinson's disease

In an attempt to find a convenient rat model to study cell vulnerability in Parkinson's disease, we have investigated the cell-loss profile in different midbrain dopaminergic nuclei and subnuclei of rats injected with 6-hydroxydopamine (6-OHDA) in the third ventricle. Following administration of different doses (5-1000 microgram) of 6-OHDA, motor behavior was evaluated and tyrosine hydroxylase-immunostained neurons were counted in the A8 group and different subdivisions of A9 and A10 groups. Animals developed hypokinesia, repetitive chewing movements, and catalepsia. Signs of cell degeneration were evident from the first day after injection, reaching the definitive pattern at the end of the first week. There was a similar degeneration in both brain sides, the A9 group showing the highest degree of cell-loss, followed by A8 and A10 groups. In the A9 group, the degeneration mostly affected those subgroups located in its ventral, lateral, and posterior regions. In the A10 group the degeneration mainly affected the parabrachial pigmented nucleus, the paranigral nucleus and the ventral tegmental area. This topographic pattern of degeneration is very similar to that previously described in Parkinson's disease, suggesting that this model may be a useful tool in the study of the cell vulnerability mechanisms in this neurodegenerative disorder. In addition, our results also showed that small dopaminergic neurons are more resistant to degeneration than the large ones. In some DA subgroups, the cells that contained calbindin but not calretinin were less vulnerable to the neurotoxic effect of 6-OHDA.     

Cell death of motoneurons in the chick embryo spinal cord. III. The differentiation of motoneurons prior to their induced degeneration following limb-bud removal

The differentiation of motoneurons following early limb-bud ablation was studied in chick embryos from four days to hatching. Following the removal of the normal targets of these cells about 90% of the neurons in the lateral motor column (LMC) of segments 23–29 (lumbar) were found to disappear. By counting degenerating cells it was shown that virtually all of the cell loss could be accounted for by cell death, rather than impaired proliferation or enhanced migration away from the LMC. Quantitative comparisons of cell death between the peripherally deprived and the control, non-deprived side demonstrated that limb-bud removal not only enhanced the 50% natural cell death known to occur in this system, but also greatly accelerated the whole process. By stage 30 (6.5-7 days) 75% of the final cell loss had occurred on the deprived side, whereas only 40% of the final cell loss had occurred on the control side. In both cases, however, cell death was confined to the period of limb innervation. Axon counts of the peripherally deprived ventral root showed that all the deprived neurons initially had sent an axon out of the spinal cord. Most of these, however, became caught in a neuroma before reaching the site of limb attachment. Though no synapses were found in the neuroma the axons were shown to be able to transport HRP back to the spinal cord. Before they began to degenerate, the deprived LMC motoneurons developed dendritic processes and these were able to form synapses with axons in the prospective lateral white matter. In early stages, frequent axo-glial “synapses” were observed in the prospective lateral white matter of both deprived and control sides of the spinal cord. Since by stage 36 (day 10) these had virtually all disappeared, it was suggested that synapse formation in this region of the spinal cord may initially be under few constraints. In late stages (i.e., after day 8) it was noted that there were frequently signs of axonal degeneration in the lateral white matter on both sides of the spinal cord, suggesting a retrograde transneuronal degeneration initiated by the earlier cell death of motoneurons. Electron microscopic examination of the deprived LMC cells at different stages prior to degeneration failed to uncover any obvious differences between them and control cells on the non-deprived side of the spinal cord. By histochemical and neurochemical methods the cholinergic enzymes acetylcholinesterase and choline acetyltransferase were found to develop normally up until the onset of frank degeneration in the deprived motoneurons, on day 5 or 6. After this the enzymes decreased at a rate comparableto the morphological loss of motoneurons by cell death. On the basis of these various lines of evidence, it is argued that all the motoneurons in the LMC have a remarkable intrinsic capacity to initiate differentiation and that neurons experimentallydeprived of their normal target are no different in this respect.     

Abrupt loss of dependence of retinopetal neurons on their target cells, as shown by intraocular injections of kainate in chick embryos

We have examined the capacity of neurons in the chick isthmo-optic nucleus (ION) to survive when their target neurons in the contralateral retinal are destroyed by intraocular injections of kainate (KA) at different stages in development. The retinal vulnerability to KA builds up progressively from embryonic day 10 (E10) until a plateau is reached at E15 (see accompanying paper); and the effects on the ION increase in parallel, almost all the ION neurons being rapidly lost after the E15 injections. KA injection before E15 lesioned only part of the retina and caused degeneration only in the topographically corresponding region of the ION. Near the end of the natural cell death period in the ION (E17), this initial dependence on the target cells is rapidly lost. Already at E16 the injections kill less ION neurons, and by E19 they kill none of them. The ION neurons have become completely insensitive to the KA injections and appear normal more than 4 months later, although axotomy (by eye removal) at a similar age would by then have killed them. The ectopic ION neurons, scattered outside the ION but projecting to the retina, are never affected by KA injections at any age.     

Calpain I activation is specifically related to excitatory amino acid induction of hippocampal damage

Sustained stimulation of receptors for excitatory amino acids leads to both activation of the calcium-dependent cysteine protease calpain I and to the death of receptive neurons. Here, we have examined the relationship between the calpain I activation and neurodegeneration. Calpain I activation was manifested as increased levels of the major proteolytic fragments of the calpain substrate spectrin, detected and quantified by immunoblotting. Intraventricular administration of the excitatory amino acids kainate or N-methyl-D-aspartate (NMDA) produced calpain I-mediated spectrin degradation and hippocampal neuronal loss. The NMDA antagonist 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid selectively blocked NMDA- but not kainate-induced protease activation and hippocampal damage. Temporally, spectrin degradation preceded the onset of pyramidal cell degeneration monitored by silver-impregnation histochemistry. Only those doses of kainate (0.15-1 microgram) or NMDA (40-80 micrograms) sufficient to cause hippocampal damage markedly increased spectrin breakdown. Both the neuronal damage and calpain I activation induced by kainate occurred primarily in area CA3. Degeneration of hippocampal neurons evoked by colchicine was not accompanied by calpain activation, indicating that proteolysis is not stimulated simply as a secondary response to neuronal destruction. Thus, a close correspondence exists between excitatory amino acid induction of neuronal degeneration and of calpain I-mediated spectrin degradation. The results suggest that calpain I may be an intracellular mediator of excitatory amino acid action, and further, they support the hypothesis that calcium influx and calpain I activation are obligatory events in the initiation of excitatory amino acid neurotoxicity.     

Initial cholinergic differentiation in embryonic chick retina is responsive to insulin and cell-cell interactions

Previous work [Kyriakis et al., Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 7463-7467] had shown that insulin, when added during a window of binding from embryonic days 9-11, stimulates the normal developmental increase in choline acetyltransferase (ChAT) activity (a marker for cholinergic differentiation) in cultured embryonic chick retinal neurons. Here, we investigated the effect of insulin and IGF 1 on embryonic chick retinal neurons at the stage of development (embryonic day 6) when ChAT activity is first expressed. We investigated insulin peptide effects in retinal tissue developing in vitro as well as in cultures of retinal cells. We show that insulin also stimulated the initial embryonic increase in ChAT activity but had no stimulatory effect on glutamic acid decarboxylase activity (a marker for GABAergic differentiation), an enzyme whose activity also increases developmentally in the same retinal neurons. In fact, insulin inhibited the expression of GAD activity in the retina. The insulin-mediated increase in ChAT activity was independent of normal cell-cell interactions but could not replace them. Insulin also stimulated choline uptake but only after a two day delay, suggesting that the normal program for cholinergic differentiation in the chick retina was induced by insulin. IGF 1 did not have any effect on either cholinergic or GABAergic differentiation. We conclude that cholinergic differentiation in chick embryo retinal neurons is dependent on both insulin- and cell contact-mediated signals.     

Dopamine induces phenotypic differentiation or apoptosis in a dose-dependent fashion: involvement of the dopamine transporter and p53

The effect of dopamine on the growth, phenotypes (morphological and biochemical) and programmed cell death (apoptosis) of the human neuronal NMB cell line was examined. Exposure to 20-50 microM of dopamine decreased cell growth, induced an apparent differentiated cell morphology and increased (3)H-dopamine uptake. At higher concentrations (100-300 microM) dopamine was neurotoxic and induced apoptosis, as reported previously. The observed effects of both low and high doses of dopamine were blocked by cocaine, which suggested involvement of dopamine transporters. Indeed, several experiments demonstrated the relationship between dopamine uptake of cells and their vulnerability to the toxic effect of dopamine. High concentrations of dopamine, which induced apoptosis, also increased p53 levels, detected by RT-PCR analysis and immunoblotting, whereas lower dopamine concentrations, which induced a differentiated phenotype, did not increase p53 immunoblotting. Dibutyryl-cAMP and dimethyl sulfoxide, which induced differentiation but not apoptosis of the NMB cells, did not increase p53 expression. These findings provide an insight into the role of dopamine, dopamine transporters and p53 in the differentiation and apoptosis of dopaminergic neurons, which will further our understanding of neuronal development and neurodegenerative diseases.     

Expression of gangliosides in neuronal development of P19 embryonal carcinoma stem cells

Gangliosides are constituents of the cell membrane and are known to have important functions in neuronal differentiation. We employed an embryonal carcinoma stem cell line P19 as an in vitro model to investigate the expression of gangliosides during neuronal development. After treatment with retinoic acid, these cells differentiate synchronously into neuron-like cells by a series of well-defined events of development. We examined several aspects of ganglioside metabolism, including the changes of ganglioside pattern, the activities and gene expression of several enzymes at different stages of differentiation, and the distribution of gangliosides in differentiating neurons. Undifferentiated P19 cells express mainly GM3 and GD3. After P19 cells were committed to differentiation, the synthesis of complex gangliosides was elevated more than 20-fold, coinciding with the stage of neurite outgrowth. During the maturation of differentiated cells, the expression of c-series gangliosides was downregulated concomitantly with upregulation of the expression of a- and b-series gangliosides. We also examined the distribution of gangliosides in differentiating neurons by confocal and transmission electron microscopy after cholera toxin B subunit and sialidase treatment. Confocal microscopic studies showed that gangliosides were distributed on the growth cones and exhibited a punctate localization on neurites and soma. Electron microscopic studies indicated that they also are enriched on the plasma membranes of neurites and the filopodia as well as on the lamellipodia of growth cones during the early stage of neurite outgrowth. Our data demonstrate that the expression of gangliosides in P19 cells during RA-induced neuronal differentiation resembles that of the in vivo development of the vertebrate brain, and hence validates it as an in vitro model for investigating the function of gangliosides in neuronal development.