Marker Of Postmitotic Neurons Research Articles

Constitutive cell proliferation and neurogenesis in the organum vasculosum lamina terminalis and subfornical organ of adult rats.

Neurogenesis continues throughout adulthood in the subventricular zone, hippocampal subgranular zone, and the hypothalamic median eminence (ME) and the adjacent medio-basal hypothalamus. The ME is one of the circumventricular organs (CVO), which are specialized brain areas characterized by an incomplete blood-brain barrier and, thus, are involved in mediating communication between the central nervous system and the periphery. Additional CVOs include the organum vasculosum laminae terminalis (OVLT) and the subfornical organs (SFO). Previous studies have demonstrated that the ME contains neural stem cells (NSCs) capable of generating new neurons and glia in the adult brain. However, it remains unclear whether the OVLT and SFO also contain proliferating cells, the identity of these cells, and their ability to differentiate into mature neurons. Here we show that glial and mural subtypes exhibit NSC characteristics, expressing the endogenous mitotic maker Ki67, and incorporating the exogenous mitotic marker BrdU in the OVLT and SFO of adult rats. Glial cells constitutively proliferating in the SFO comprise NG2 glia, while in the OVLT, both NG2 glia and tanycytes appear to constitute the NSC pool. Furthermore, pericytes, which are mural cells associated with capillaries, also contribute to the pool of cells constitutively proliferating in the OVLT and SFO of adult rats. In addition to these glial and mural cells, a fraction of NSCs containing proliferation markers Ki67 and BrdU also expresses the early postmitotic neuronal marker doublecortin, suggesting that these CVOs comprise newborn neurons. Notably, these neurons can differentiate and express the mature neuronal marker NeuN. These findings establish the sensory CVOs OVLT and SFO as additional neurogenic niches, where the generation of new neurons and glia persists in the adult brain.

NeuN distribution in brain structures of normal and Zika-infected suckling mice.

Microcephaly is the more severe brain malformation because of Zika virus infection. Increased vulnerability of neural stem and progenitor cells to Zika infection during prenatal neurodevelopment impairs the complete formation of cortical layers. Normal development of cerebellum is also affected. However, the follow-up of apparently healthy children born to Zika exposed mothers during pregnancy has revealed other neurological sequelae. This suggests Zika infection susceptibility remains in nervous tissue after neurogenesis end, when differentiated neuronal populations predominate. The neuronal nuclear protein (NeuN) is an exclusive marker of postmitotic neurons. Changes in NeuN expression are associated with neuronal degeneration. We have evaluated immunohistochemical expression of NeuN protein in cerebral cortex, hippocampus, and cerebellum of normal and Zika-infected neonatal Balb/c mice. The highest NeuN immunoreactivity was found mainly in neurons of all cortical layers, pyramidal layer of hippocampus, granular layer of dentate gyrus and in internal granular layer of cerebellum. Viral infection caused marked loss of NeuN immunostaining in all these brain areas. This suggests neurodegenerative effects of Zika virus infection during postmitotic neuron maturation and contribute to interpretation of neuropathogenic mechanisms of Zika.

Neurogenetic Heterochrony in Chick, Lizard, and Rat Mapped with Wholemount Acetylcholinesterase and the Prosomeric Model

In the developing brain, the phenomenon of neurogenesis is manifested heterotopically, that is, much the same neurogenetic steps occur at different places with a different timetable. This is due apparently to early molecular regionalization of the neural tube wall in the anteroposterior and dorsoventral dimensions, in a checkerboard pattern of more or less deformed quadrangular histogenetic areas. Their respective fate is apparently specified by a locally specific combination of active/repressed genes known as “molecular profile.” This leads to position-dependent differential control of proliferation, neurogenesis, differentiation, and other aspects, eventually in a heterochronic manner across adjacent areal units with sufficiently different molecular profiles. It is not known how fixed these heterochronic patterns are. We reexamined here comparatively early patterns of forebrain and hindbrain neurogenesis in a lizard (Lacerta gallotia galloti), a bird (the chick), and a mammal (the rat), as demonstrated by activation of acetylcholinesterase (AChE). This is an early marker of postmitotic neurons, which leaves unlabeled the neuroepithelial ventricular cells, so that we can examine cleared wholemounts of the reacted brains to have a birds-eye view of the emergent neuronal pattern at each stage. There is overall heterochrony between the basal and alar plates of the brain, a known fact, but, remarkably, heterochrony occurs even within the precocious basal plate among its final anteroposterior neuromeric subdivisions and their internal microzonal subdivisions. Some neuromeric units or microzones are precocious, while others follow suit without any specific spatial order or gradient; other similar neuromeric units remain retarded in the midst of quite advanced neighbors, though they do produce similar neurogenetic patterns at later stages. It was found that some details of such neuromeric heterochrony are species-specific, possibly related to differential morphogenetic properties. Given the molecular causal underpinning of the updated prosomeric model used here for interpretation, we comment on the close correlation between some genetic patterns and the observed AChE differentiation patterns.

Environmental enrichment applied with sensory components prevents age-related decline in synaptic dynamics: Evidence from the zebrafish model organism.

Progression of cognitive decline with or without neurodegeneration varies among elderly subjects. The main aim of the current study was to illuminate the molecular mechanisms that promote and retain successful aging in the context of factors such as environment and gender, both of which alter the resilience of the aging brain. Environmental enrichment (EE) is one intervention that may lead to the maintenance of cognitive processing at older ages in both humans and animal subjects. EE is easily applied to different model organisms, including zebrafish, which show similar age-related molecular and behavioral changes as humans. Global changes in cellular and synaptic markers with respect to age, gender and 4-weeks of EE applied with sensory stimulation were investigated using the zebrafish model organism. Results indicated that EE increases brain weight in an age-dependent manner without affecting general body parameters like body mass index (BMI). Age-related declines in the presynaptic protein synaptophysin, AMPA-type glutamate receptor subunits and a post-mitotic neuronal marker were observed and short-term EE prevents these changes in aged animals, as well as elevates levels of the inhibitory scaffolding protein, gephyrin. Gender-driven alterations were observed in the levels of the glutamate receptor subunits. Oxidative stress markers were significantly increased in the old animals, while exposure to EE did not alter this pattern. These data suggest that EE with sensory stimulation exerts its effects mainly on age-related changes in synaptic dynamics, which likely increase brain resilience through specific cellular mechanisms.

Nwd1 Regulates Neuronal Differentiation and Migration through Purinosome Formation in the Developing Cerebral Cortex.

SummaryEngagement of neural stem/progenitor cells (NSPCs) into proper neuronal differentiation requires the spatiotemporally regulated generation of metabolites. Purines are essential building blocks for many signaling molecules. Enzymes that catalyze de novo purine synthesis are assembled as a huge multienzyme complex called “purinosome.” However, there is no evidence of the formation or physiological function of the purinosome in the brain. Here, we showed that a signal transduction ATPases with numerous domains (STAND) protein, NACHT and WD repeat domain-containing 1 (Nwd1), interacted with Paics, a purine-synthesizing enzyme, to regulate purinosome assembly in NSPCs. Altered Nwd1 expression affected purinosome formation and induced the mitotic exit and premature differentiation of NSPCs, repressing neuronal migration and periventricular heterotopia. Overexpression/knockdown of Paics or Fgams, other purinosome enzymes, in the developing brain resulted in a phenocopy of Nwd1 defects. These findings indicate that strict regulation of purinosome assembly/disassembly is crucial for maintaining NSPCs and corticogenesis.

Exposure to the cannabinoid agonist WIN 55, 212–2 in adolescent rats causes sleep alterations that persist until adulthood

Cannabis and, to a lesser extent, synthetic cannabinoids are used during adolescence, a period in which multiple brain areas are still undergoing development. Among such areas is the hypothalamus, which is implicated in the control of sleep-wake cycle. In the present report, we show that exposing adolescent rats to the cannabinoid receptor agonist WIN 55, 212–2 (0.1, 0.3 or 1.0 mg/kg, i.p) for 14 days during adolescence (i.e., from post-natal day 30–44) resulted in significant sleep disturbances when the animals became adult (post-natal day 80). These included decreased wakefulness and enhanced rapid eye movement sleep. Furthermore, we found that labeling for NeuN, a marker of postmitotic neurons, was significantly increased the dorsomedial hypothalamic nucleus of rats treated with WIN 55, 212–2. The results suggest that excessive cannabinoid receptor activation during adolescence can persistently influence sleep patterns and neuronal activity later in life.

NeuN immunoreactivity in the brain of Xenopus laevis.

Neuronal nuclear antigen (NeuN), discovered in mice brain cell nuclei by Mullen et al. (1992), is used as an excellent marker of post-mitotic neurons in vertebrates. In this study, the expression pattern of NeuN was examined in the Xenopus brain to explore phylogenetic differences in NeuN expression. Anti-NeuN antibody showed selective staining in mouse and Xenopus brain extracts, but the number and molecular weight of the bands differed in Western blotting analysis. In immunostaining, anti-NeuN antibody showed selective staining of neurons, but not glial cells, in the Xenopus brain. Most neurons, including olfactory bulb mitral cells and cerebellar Purkinjie cells, which show no immunoreactivity in birds/mammals, showed NeuN immunoreactivity in Xenopus. This study revealed that anti-NeuN antibody is a useful marker of post-mitotic neurons in amphibians, but it also stains neurons that show no reactivity in more derived animals.

Increased Neuronal DNA/RNA Oxidation in the Frontal Cortex of Mice Subjected to Unpredictable Chronic Mild Stress

Background Chronic stress is implicated in the development of various psychiatric illnesses including major depressive disorder. Previous reports suggest that patients with major depressive disorder have increased levels of oxidative stress, including higher levels of DNA/RNA oxidation found in postmortem studies, especially within brain regions responsible for the cognitive and emotional processes disrupted in the disorder. Here, we aimed to investigate whether unpredictable chronic mild stress in mice induces neuronal DNA/RNA oxidation in the prelimbic, infralimbic, and cingulate cortices of the frontal cortex and the basolateral amygdala and to explore potential associations with depressive-like behaviors. We expected that animals subjected to unpredictable chronic mild stress will present higher levels of DNA/RNA oxidation, which will be associated with anxiety-/depressive-like behaviors. Methods C57BL/6J mice were assigned to unpredictable chronic mild stress or nonstress conditions (n = 10/group, 50% females). Following five weeks of unpredictable chronic mild stress exposure, mice were tested in a series of behavioral tests measuring anxiety- and depressive-like behaviors. Frontal cortex and amygdala sections were then immunolabeled for neuronal nuclei, a marker of post-mitotic neurons and anti-8-hydroxy-2-deoxyguanosine/8-oxo-7,8-dihydroguanosine, which reflects both DNA and RNA oxidation. Results Levels of neuronal DNA/RNA oxidation were increased in the frontal cortex of mice subjected to unpredictable chronic mild stress (p = 0.0207). Levels of neuronal DNA/RNA oxidation in the frontal cortex were positively correlated with z-emotionality scores for latency to feed in the novelty-suppressed feeding test (p = 0.0031). Statistically significant differences were not detected in basolateral amygdala levels of neuronal DNA/RNA oxidation between nonstress- and unpredictable chronic mild stress-exposed mice, nor were correlations found with behavioral performances for this region. Conclusion Our results demonstrate that unpredictable chronic mild stress induces a significant increase in neuronal DNA/RNA oxidation in the frontal cortex that correlate with behavioral readouts of the stress response. A lack of DNA/RNA oxidation alterations in the basolateral amygdala suggests greater vulnerability of frontal cortex neurons to DNA/RNA oxidation in response to unpredictable chronic mild stress. These findings add support to the hypothesis that chronic stress-induced damage to DNA/RNA may be an additional molecular mechanism underlying cellular dysfunctions associated with chronic stress and present in stress-related disorders.

Transforming Growth Factor-β-Induced RBFOX3 Inhibition Promotes Epithelial-Mesenchymal Transition of Lung Cancer Cells.

The RNA-binding protein Rbfox3 is a well-known splicing regulator that is used as a marker for post-mitotic neurons in various vertebrate species. Although recent studies indicate a variable expression of Rbfox3 in non-neuronal tissues, including lung tissue, its cellular function in lung cancer remains largely unknown. Here, we report that the number of RBFOX3-positive cells in tumorous lung tissue is lower than that in normal lung tissue. As the transforming growth factor-β (TGF-β) signaling pathway is important in cancer progression, we investigated its role in RBFOX3 expression in A549 lung adenocarcinoma cells. TGF-β1 treatment inhibited RBFOX3 expression at the transcriptional level. Further, RBFOX3 depletion led to a change in the expression levels of a subset of proteins related to epithelial-mesenchymal transition (EMT), such as E-cadherin and Claudin-1, during TGF-β1-induced EMT. In immunofluorescence microscopic analysis, mesenchymal morphology was more prominent in RBFOX3-depleted cells than in control cells. These findings show that TGF-β-induced RBFOX3 inhibition plays an important role in EMT and propose a novel role for RBFOX3 in cancer progression.

Dynamic expression of Notch-dependent neurogenic markers in the chick embryonic nervous system.

The establishment of a functional nervous system requires a highly orchestrated process of neural proliferation and differentiation. The evolutionary conserved Notch signaling pathway is a key regulator of this process, regulating basic helix-loop-helix (bHLH) transcriptional repressors and proneural genes. However, little is known about downstream Notch targets and subsequently genes required for neuronal specification. In this report, the expression pattern of Transgelin 3 (Tagln3), Chromogranin A (Chga) and Contactin 2 (Cntn2) was described in detail during early chick embryogenesis. Expression of these genes was largely restricted to the nervous system including the early axon scaffold populations, cranial ganglia and spinal motor neurons. Their temporal and spatial expression were compared with the neuronal markers Nescient Helix-Loop-Helix 1 (Nhlh1), Stathmin 2 (Stmn2) and HuC/D. We show that Tagln3 is an early marker for post-mitotic neurons whereas Chga and Cntn2 are expressed in mature neurons. We demonstrate that inhibition of Notch signaling during spinal cord neurogenesis enhances expression of these markers. This data demonstrates that Tagln3, Chga and Cntn2 represent strong new candidates to contribute to the sequential progression of vertebrate neurogenesis.

Differentiation patterns of mouse embryonic stem cells and induced pluripotent stem cells into neurons.

Mouse embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have the ability to differentiate in vitro into various cell lineages including neurons. The differentiation of these cells into neurons has potential applications in regenerative medicine. Previously, we reported that a chick dorsal root ganglion (DRG)-conditioned medium (CM) promoted the differentiation of mouse ES and iPS cells into neurons. Here, we used real-time PCR to investigate the differentiation patterns of ES and iPS cells into neurons when DRG-CM was added. DRG-CM promoted the expression levels of βIII-tubulin gene (a marker of postmitotic neurons) in ES and iPS cells. ES cells differentiated into neurons faster than iPS cells, and the maximum peaks of gene expression involved in motor, sensory, and dopaminergic neurons were different. Rho kinase (ROCK) inhibitors could be very valuable at numerous stages in the production and use of stem cells in basic research and eventual cell-based therapies. Thus, we investigated whether the addition of a ROCK inhibitor Y-27632 and DRG-CM on the basis of the differentiation patterns promotes the neuronal differentiation of ES cells. When the ROCK inhibitor was added to the culture medium at the initial stages of cultivation, it stimulated the neuronal differentiation of ES cells more strongly than that stimulated by DRG-CM. Moreover, the combination of the ROCK inhibitor and DRG-CM promoted the neuronal differentiation of ES cells when the ROCK inhibitor was added to the culture medium at day 3. The ROCK inhibitor may be useful for promoting neuronal differentiation of ES cells.

Integration of neuronally predifferentiated human dental pulp stem cells into rat brain in vivo

Pluripotency and their neural crest origin make dental pulp stem cells (DPSCs) an attractive donor source for neuronal cell replacement. Despite recent encouraging results in this field, little is known about the integration of transplanted DPSC derived neuronal pecursors into the central nervous system. To address this issue, neuronally predifferentiated DPSCs, labeled with a vital cell dye Vybrant DiD were introduced into postnatal rat brain. DPSCs were transplanted into the cerebrospinal fluid of 3-day-old male Wistar rats. Cortical lesion was induced by touching a cold (−60 °C) metal stamp to the calvaria over the forelimb motor cortex. Four weeks later cell localization was detected by fluorescent microscopy and neuronal cell markers were studied by immunohistochemistry. To investigate electrophysiological properties of engrafted, fluorescently labeled DPSCs, 300 μm-thick horizontal brain slices were prepared and the presence of voltage-dependent sodium and potassium channels were recorded by patch clamping. Predifferentiated donor DPSCs injected into the cerebrospinal fluid of newborn rats migrated as single cells into a variety of brain regions. Most of the cells were localized in the normal neural progenitor zones of the brain, the subventricular zone (SVZ), subgranular zone (SGZ) and subcallosal zone (SCZ). Immunohistochemical analysis revealed that transplanted DPSCs expressed the early neuronal marker N-tubulin, the neuronal specific intermediate filament protein NF-M, the postmitotic neuronal marker NeuN, and glial GFAP. Moreover, the cells displayed TTX sensitive voltage dependent (VD) sodium currents (I Na) and TEA sensitive delayed rectifier potassium currents (K DR). Four weeks after injury, fluorescently labeled cells were detected in the lesioned cortex. Neurospecific marker expression was increased in DPSCs found in the area of the cortical lesions compared to that in fluorescent cells of uninjured brain. TTX sensitive VD sodium currents and TEA sensitive K DR significantly increased in labeled cells of the cortically injured area. In conclusion, our data demonstrate that engrafted DPSC-derived cells integrate into the host brain and show neuronal properties not only by expressing neuron-specific markers but also by exhibiting voltage dependent sodium and potassium channels. This proof of concept study reveals that predifferentiated hDPSCs may serve as useful sources of neuro- and gliogenesis in vivo, especially when the brain is injured.

The synapsin gene family in basal chordates: evolutionary perspectives in metazoans

BackgroundSynapsins are neuronal phosphoproteins involved in several functions correlated with both neurotransmitter release and synaptogenesis. The comprehension of the basal role of the synapsin family is hampered in vertebrates by the existence of multiple synapsin genes. Therefore, studying homologous genes in basal chordates, devoid of genome duplication, could help to achieve a better understanding of the complex functions of these proteins.ResultsIn this study we report the cloning and characterization of the Ciona intestinalis and amphioxus Branchiostoma floridae synapsin transcripts and the definition of their gene structure using available C. intestinalis and B. floridae genomic sequences. We demonstrate the occurrence, in both model organisms, of a single member of the synapsin gene family. Full-length synapsin genes were identified in the recently sequenced genomes of phylogenetically diverse metazoans. Comparative genome analysis reveals extensive conservation of the SYN locus in several metazoans. Moreover, developmental expression studies underline that synapsin is a neuronal-specific marker in basal chordates and is expressed in several cell types of PNS and in many, if not all, CNS neurons.ConclusionOur study demonstrates that synapsin genes are metazoan genes present in a single copy per genome, except for vertebrates. Moreover, we hypothesize that, during the evolution of synapsin proteins, new domains are added at different stages probably to cope up with the increased complexity in the nervous system organization. Finally, we demonstrate that protochordate synapsin is restricted to the post-mitotic phase of CNS development and thereby is a good marker of postmitotic neurons.

Impaired hippocampal plasticity and altered neurogenesis in adult Ube3a maternal deficient mouse model for Angelman syndrome

Angelman syndrome (AS) is a severe neurodevelopmental disorder characterized by mental retardation, seizures and sleep disturbances. It results from lack of the functional maternal allele of UBE3A gene. Ube3a maternal-deficient mice (Ube3a m- /p+), animal models for AS, are impaired in hippocampal-dependent learning tasks as compared with control ( Ube3a m+/p+) mice. We first examined the basal expression of immediate early genes which expression is required for synaptic plasticity and memory formation. We found that basal expression of c-fos and Arc genes is reduced in the DG of Ube3a maternal deficient mice compared to their non-transgenic littermates. We then examined whether adult hippocampal neurogenesis, which likely serves as a mechanism toward brain plasticity, is altered in these transgenic mice. Neurogenesis occurs throughout life in mammalian dentate gyrus (DG) and recent findings suggest that newborn granule cells are involved in some forms of learning and memory. Whether maternal Ube3a deletion is detrimental on hippocampal neurogenesis is unclear. Herein, we show, using the mitotic marker Ki67, the birthdating marker 5-bromo-2′-dexoyuridine (BrdU) and the marker doublecortin (DCX) to respectively label cell proliferation, cell survival or young neuron production, that the Ube3a maternal deletion does not affect the proliferation nor the survival of newborn cells in the hippocampus. In contrast, using the postmitotic neuronal marker (NeuN), we show that Ube3a maternal deletion is associated with a lower fraction of BrdU+/NeuN+ newborn neurons among the population of surviving new cells in the hippocampus. Collectively, these findings suggest that some aspects of adult neurogenesis and plasticity are affected by Ube3a deletion and may contribute to the hippocampal dysfunction observed in AS mice.

Is There a Role for P63 in Neural Differentiation?

Event Abstract Back to Event Is There a Role for P63 in Neural Differentiation? Benedita V. Fonseca1, Ana F. Nunes1 and Cecilia M. Rodrigues1* 1 University of Lisbon, iMed.UL- Faculty of Pharmacy, Portugal The transcription factor p63, member of the p53 family, is crucial for epithelial development [1]. In addition, p63 is an essential proapoptotic protein during neural development [2]. The focus of our study is to investigate the role of p63 in neural differentiation, characterizing p63 null mice phenotype with regard to neurogenesis and dissecting the molecular signals triggered by p63 in the process of neural differentiation. We investigated protein levels of the different p63 isoforms in differentiating neural stem cell cultures from wild-type mice. The TAp63 isoform was readily detected and increased during differentiation. In addition, we have analyzed the levels of several proteins in differentiating neural stem cell cultures from wild-type mice in an attempt to unravel p63 targets during the process of neural differentiation. The results identified ERK5 as a potential target of p63. Additional p63 targets will be further investigated. Finally, we are currently evaluating neurogenesis in the brain of wild-type and p63 null mice. Brain sections of embryonic mice were immunostained for β-III-Tubulin (marker of immature neurons), and NeuN (marker of post-mitotic neurons). The results suggest that p63 null mice display impairment in neuronal development; however, further investigation and confirmation by additional approaches are warranted. We expect to show that apoptotic factors play an essential role in neuronal differentiation without, however, leading to effective apoptotic cell death.Supported by grant PTDC/BIA-BCM/67922/2006 (to CMPR), and fellowships SFRH/BD/43959/2008 (to MBF), and SFRH/BPD/34603/2007 (to AFN) from FCT and FEDER.

Widespread immunoreactivity for neuronal nuclei in cultured human and rodent astrocytes

The monoclonal antibody (mAb) neuronal nuclei (NeuN) labels the nuclei of mature neurons in vivo in vertebrates. NeuN has also been used to define post-mitotic neurons or differentiating neuronal precursors in vitro. In this study, we demonstrate that the NeuN mAb labels the nuclei of astrocytes cultured from fetal and adult human, newborn rat, and embryonic mouse brain tissue. A non-neuronal fibroblast cell line (3T3) also displayed NeuN immunoreactivity. We confirmed that NeuN labels neurons but not astrocytes in sections of P10 rat brain. Western blot analysis of NeuN immunoreactive species revealed a distribution of bands in nucleus-enriched fractions derived from the different cell lines that was similar, but not identical to adult rat brain homogenates. We then examined the hypothesis that the glial fibrillary acidic protein/NeuN-double positive population of cells might correspond to neuronal precursors. Although the NeuN-positive astrocytes were proliferating, no evidence of neurogenesis was detected. Furthermore, expression of additional neuronal precursor markers was not detected. Our results indicate that primary astrocytes derived from mouse, rat, and human brain express NeuN. Our findings are consistent with NeuN being a selective marker of neurons in vivo, but indicate that studies utilizing NeuN-immunoreactivity as a definitive marker of post-mitotic neurons in vitro should be interpreted with caution.

Expression and regulation of Xenopus CRMP-4 in the developing nervous system

The collaspin response mediator proteins (CRMPs) are a family of cytosolic phosphoproteins which play a critical role in the establishment of neuronal polarity and growth cone guidance. Here, we describe the temporal and spatial expression of CRMP-4 during early Xenopus embryogenesis. CRMP-4 transcripts were first detected by whole mount in situ hybridization at the end of gastrulation in the prospective neuroectoderm. During open neural plate stages, CRMP-4 was expressed broadly throughout the anterior neural plate and in the three bilateral stripes of the posterior neural plate where primary neurons arise. The expression in the territories of primary neurogenesis prefigures that of the post-mitotic neuronal marker N-tubulin. At tadpole stages, expression was maintained throughout the central nervous system and in the retina of the eye. Consistent with the observed expression, CRMP-4 transcripts are positively regulated by X-ngnr-1 and negatively by Notch signaling. The observed expression and regulation of CRMP-4 differ from that of the CRMP-2, which is induced by the events of neural induction.

Neurogenesis and gliogenesis in the spinal cord of turtles.

A 5-bromo-3'-deoxyuridine (BrdU) pulse administered to juvenile turtles resulted in cell labeling throughout the gray matter (GM) and white matter (WM) of the spinal cord. One and twenty-four hours postinjection, larger densities of BrdU-labeled nuclei (LN) occurred within the GM, with a density peak localized in the central region (CR). Seven days later, density differences between GM and WM disappeared, accompanying a more uniform distribution of LN in the GM (absence of the central peak). Multiple injection experiments also showed similar evolution in the distribution of LN. Morphometric studies revealed that the size of LN had undergone time-related increments: Larger nuclei appeared at protracted fixation time points. Double-labeling experiments indicated that BrdU-labeled cells expressed neuroactive substances, such as gamma-aminobutyric acid (GABA), neuron-specific nuclear protein (NeuN), and the cytoplasmic early postmitotic neuronal marker (TUC-4). Other BrdU-labeled cells expressed the glial-specific protein (GFAP). GABA-BrdU, TUC-4-BrdU, and GFAP-BrdU double-labeled cells were recognized 6 days after the first BrdU injection. NeuN-BrdU double-labeled cells were found at 50 days postinjection. Three-dimensional transmission electron microscopy revealed the presence of synapses and typical kinocilia in putative immature nerve cells. Kinocilia were also found in putative immature glial cells. In consideration of the scattered distribution pattern of BrdU-labeled cells, in animals fixed 1 hour postinjection, the existence of a single proliferating center was discarded. The CR, including the ependymal epithelium, showed the highest density of LN.

A zebrafish homologue of deleted in colorectal cancer (zdcc) is expressed in the first neuronal clusters of the developing brain.

DCC (deleted in colon cancer), Neogenin and UNC-5 are all members of the immunoglobulin superfamily of transmembrane receptors which are believed to play a role in axon guidance by binding to their ligands, the Netrin/UNC-40 family of secreted molecules (Cell. Mol. Life Sci. 56 (1999) 62; Curr. Opin. Genet. Dev. 7 (1997) 87). Although zebrafish homologues of the Netrin family of secreted molecules have been reported, to date there has been no published description of zebrafish DCC homologues (Mol. Cell. Neurosci. 9 (1997) 293; Mol. Cell. Neurosci. 11 (1998) 194; Mech. Dev. 62 (1997) 147). We report here the expression pattern of a zebrafish dcc (zdcc) homologue during the initial period of neurogenesis and axon tract formation within the developing central nervous system. Between 12 and 33 h post-fertilisation zdcc is expressed in a dynamic spatiotemporal pattern in all major subdivisions of the central nervous system. Double-labelling for zdcc and the post-mitotic neuronal marker HNK-1 revealed that subpopulations of neurons within the first nuclei of the zebrafish brain express zdcc. These results support our previous observation that patterning of neuronal clusters in the zebrafish brain occurs early in development (Dev. Biol. 229 (2001) 271).

N-methyl-D-aspartate receptor-mediated increase of neurogenesis in adult rat dentate gyrus following stroke.

Neurogenesis in the adult rat dentate gyrus was studied following focal ischemic insults produced by middle cerebral artery occlusion (MCAO). Animals were subjected to either 30 min of MCAO, which causes damage confined to the striatum, or 2 h of MCAO, which leads to both striatal and cortical infarction. When compared to sham-operated rats, MCAO-rats showed a marked increase of the number of cells double-labelled for 5-bromo-2'-deoxyuridine-5'-monophosphate (BrdU; injected during 4-6 days postischemia) and neuronal-specific antigen (NeuN; a marker of postmitotic neurons) in the ipsilateral dentate granule cell layer and subgranular zone at 5 weeks following the 2 h insult. Only a modest and variable increase of BrdU-labelled cells was found after 30 min of MCAO. The enhanced neurogenesis was not dependent on cell death in the hippocampus, and its magnitude was not correlated to the degree of cortical damage. Systemic administration of the N-methyl-D-aspartate (NMDA) receptor blocker dizocilpine maleate (MK-801) completely suppressed the elevated neurogenesis following 2 h of MCAO. Our findings indicate that stroke leads to increased neurogenesis in the adult rat dentate gyrus through glutamatergic mechanisms acting on NMDA receptors. This modulatory effect may be mediated through changes in the levels of several growth factors, which occur after stroke, and could influence various regulatory steps of neurogenesis.