Doublecortin is a microtubule-associated protein and is expressed widely by migrating neurons.

Doublecortin (DCX) is required for normal migration of neurons into the cerebral cortex, since mutations in the human gene cause a disruption of cortical neuronal migration. To date, little is known about the distribution of DCX protein or its function. Here, we demonstrate that DCX is expressed in migrating neurons throughout the central and peripheral nervous system during embryonic and postnatal development. DCX protein localization overlaps with microtubules in cultured primary cortical neurons, and this overlapping expression is disrupted by microtubule depolymerization. DCX coassembles with brain microtubules, and recombinant DCX stimulates the polymerization of purified tubulin. Finally, overexpression of DCX in heterologous cells leads to a dramatic microtubule phenotype that is resistant to depolymerization. Therefore, DCX likely directs neuronal migration by regulating the organization and stability of microtubules.     

Effects of corticosterone and DHEA on doublecortin immunoreactivity in the song control system and hippocampus of adult song sparrows

Adult neuroplasticity is strongly influenced by steroids. In particular, corticosterone (CORT) and dehydroepiandrosterone (DHEA) can have opposing effects, where CORT reduces while DHEA increases neurogenesis and neuron recruitment. It has been previously shown that in adult male song sparrows, DHEA treatment increases neuron recruitment throughout the telencephalon, including the lateral ventricular zone, while the effect of CORT treatment is restricted to HVC, one of the song control regions. These data suggest that the two steroids may differentially affect proliferation, migration, differentiation, and/or survival of new neurons. To determine if CORT or DHEA alters the migration and differentiation of young neurons, we examined an endogenous marker of migrating immature neurons, doublecortin (DCX), in HVC and hippocampus of adult male song sparrows that were treated with CORT and/or DHEA for 28 days. In HVC, DHEA increased the number of DCX‐labeled round cells, while CORT had no main effect on the number of DCX‐labeled cells. Furthermore, DHEA increased the area covered by DCX immunoreactivity in HVC, regardless of CORT treatment. In the hippocampus, neither DHEA nor CORT affected DCX immunoreactivity. These results suggest that DHEA enhances migration and differentiation of young neurons into HVC while CORT does not affect the process, whether in the presence of DHEA or not. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 52–62, 2014     

New GABAergic interneurons in the adult neocortex and striatum are generated from different precursors

Ongoing neurogenesis in the adult mammalian dentate gyrus and olfactory bulb is generally accepted, but its existence in other adult brain regions is highly controversial. We labeled newly born cells in adult rats with the S-phase marker bromodeoxyuridine (BrdU) and used neuronal markers to characterize new cells at different time points after cell division. In the neocortex and striatum, we found BrdU-labeled cells that expressed each of the eight neuronal markers. Their size as well as staining for gamma-aminobutyric acid (GABA), glutamic acid decarboxylase 67, calretinin and/or calbindin, suggest that new neurons in both regions are GABAergic interneurons. BrdU and doublecortin-immunoreactive (BrdU+/DCX+) cells were seen within the striatum, suggesting migration of immature neurons from the subventricular zone. Surprisingly, no DCX+ cells were found within the neocortex. NG2 immunoreactivity in some new neocortical neurons suggested that they may instead be generated from the NG2+ precursors that reside within the cortex itself.     

Enhanced neurogenesis and cell migration following focal ischemia and peripheral stimulation in mice

Peripheral stimulation and physical therapy can promote neurovascular plasticity and functional recovery after CNS disorders such as ischemic stroke. Using a rodent model of whisker-barrel cortex stroke, we have previously demonstrated that whisker activity promotes angiogenesis in the penumbra of the ischemic barrel cortex. This study explored the potential of increased peripheral activity to promote neurogenesis and neural progenitor migration toward the ischemic barrel cortex. Three days after focal barrel cortex ischemia in adult mice, whiskers were manually stimulated (15 min x 3 times/day) to enhance afferent signals to the ischemic barrel cortex. 5-Bromo-2'-deoxyuridine (BrdU, i.p.) was administered once daily to label newborn cells. At 14 days after stroke, whisker stimulation significantly increased vascular endothelial growth factor and stromal-derived factor-1 expression in the penumbra. The whisker stimulation animals showed increased doublecortin (DCX) positive and DCX/BrdU-positive cells in the ipsilateral corpus of the white matter but no increase in BrdU-positive cells in the subventricular zone, suggesting a selective effect on neuroblast migration. Neurogenesis indicated by neuronal nuclear protein and BrdU double staining was also enhanced by whisker stimulation in the penumbra at 30 days after stroke. Local cerebral blood flow was better recovered in mice that received whisker stimulation. It is suggested that the enriched microenvironment created by specific peripheral stimulation increases regenerative responses in the postischemic brain and may benefit long-term functional recovery from ischemic stroke.     

DCXs Phosphorylation by Not Just aNother Kinase (JNK)

The mammalian cortex is generally subdivided into six organized layers, which are formed during development in an organized fashion. This organized cortical layering is disrupted in case of mutations in the doublecortin (DCX) gene. DCX is a Microtubule Associated Protein (MAP). However, besides stabilization of microtubules, it may be involved in additional functions. The participation of this molecule in signal transduction is beginning to emerge via discovery of interacting molecules and its regulation by phosphorylation using several different kinases. We raise the hypothesis, that the combinatorial phosphorylation of DCX by different kinases and at different sites may be a molecular regulatory switch in the transition of a migrating neuron through multiple phases of migration. Our recent research has suggested the involvement of DCX in the JNK (Jun-N-terminal Kinase) pathway. The JNK pathway is linked to the reelin pathway, known to regulate cortical layering. Positioning of DCX in this signaling pathway opens up additional possibilities of understanding how migrating neurons are controlled.     

Expression of alpha2A adrenoceptors during rat neocortical development

Norepinephrine has been suggested to play a neurotrophic role during development and is present in the brain as early as embryonic day (E) 12. We have recently demonstrated that the alpha2A adrenoceptor subtype is widely expressed during times of neuronal migration and differentiation throughout the developing brain. Here, we report the temporal and spatial expression pattern of alpha2A adrenoceptors in neocortex during late embryonic and early postnatal development using in situ hybridization and receptor autoradiography. Functional alpha2 receptors in embryonic rat cortex were also detected using agonist stimulated [35S]GTPgammaS autoradiography. Both alpha2A mRNA and protein expression were strongly increased by E19 and E20, respectively. The increased expression was in the cortical plate and intermediate and subventricular zones, corresponding to tiers of migrating and differentiating neurons. This transient up-regulation of alpha2A adrenoceptors was restricted to the lateral neocortex. At E20, functional alpha2 adrenoceptors were also detected in deep layers of lateral neocortex. During the first week of postnatal development, the expression of alpha2A mRNA and protein changed markedly, giving rise to a more mature pattern of anatomical distribution. The temporal and spatial distribution of alpha2A adrenoceptors in developing neocortex is consistent with expression of functional proteins on migrating and differentiating layer IV to II neurons. These findings suggest that alpha2A receptors may mediate a neurotrophic effect of norepinephrine during fetal cortical development. The early delineation of the lateral neocortex, which will develop into somatosensory and auditory cortices, suggests an intrinsic regulation of alpha2A mRNA expression.     

Immunohistological markers for proliferative events,gliogenesis, and neurogenesis within the adult hippocampus

Biologists long believed that, once development is completed, no new neurons are produced in the forebrain. However, as is now firmly established, new neurons can be produced at least in two specific forebrain areas: the subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampal formation. Neurogenesis within the adult DG occurs constitutively throughout postnatal life, and the rate of neurogenesis within the DG can be altered under various physiological and pathophysiological conditions. The process of adult neurogenesis within the DG is a multi-step process (proliferation, differentiation, migration, targeting, and synaptic integration) that ends with the formation of a post-mitotic functionally integrated new neuron. Various markers are expressed during specific stages of adult neurogenesis. The availability of such markers allows the time-course and fate of newly born cells to be followed within the DG in a detailed and precise fashion. Several of the available markers (e.g., PCNA, Ki-67, PH3, MCM2) are markers for proliferative events, whereas others are more specific for early phases of neurogenesis and gliogenesis within the adult DG (e.g., nestin, GFAP, Sox2, Pax6). In addition, markers are available allowing events to be distinguished that are related to later steps of gliogenesis (e.g., vimentin, BLBP, S100beta) or neurogenesis (e.g., NeuroD, PSA-NCAM, DCX).     

Identification of Cortexin: A Novel, Neuron-Specific, 82-Residue Membrane Protein Enriched in Rodent Cerebral Cortex

Abstract: Nucleotide sequence analysis of a cDNA clone of a rat cortex-enriched mRNA identifies a novel integral membrane protein of 82 amino acids. The encoded protein is named cortexin to reflect its enriched expression in cortex. The amino acid sequence of rat cortexin and its mouse homologue are nearly identical (98% similarity), and both contain a conserved single membrane-spanning region in the middle of each sequence. Northern blot analysis shows that cortexin mRNA is brain-specific, cortexenriched, and present at significant levels in fetal brain, with peak expression in postnatal rodent brain. In situ hybridization studies detect cortexin mRNA primarily in neurons of rodent cerebral cortex, but not in cells of the hindbrain or white matter regions. The function of cortexin may be particularly important to neurons of both the developing and adult cerebral cortex.     

Early specification of GAD67 subventricular derived olfactory interneurons

Olfactory bulb interneurons are continuously generated in the subventricular zone (SVZ) and migrate along the rostral migratory stream (RMS) into the olfactory bulb (OB) where the majority becomes local GABAergic interneurons. We previously showed that SVZ-derived progenitor cells expressed glutamic acid decarboxylase 65 kDa (GAD65) very early in the migratory pathway. However, only approximately half of OB GABAergic interneurons use GAD65, an equal number express the 67 kDa GAD enzyme. To investigate the differentiation of these GABAergic interneurons we examined their migration in a transgenic mouse expressing green fluorescent protein (GFP) under the control of the GAD67 promoter. In adult, GFP was expressed by a subpopulation of migratory cells in the SVZ and along the RMS. Using Doublecortin (DCX) as a marker of migrating neuroblasts and bromodeoxyuridine (BrdU) incorporation, we show that these GAD67-GFP neurons co-express DCX and incorporate BrdU indicating they are newly born migratory neuroblasts. This is similar to GAD65 transgene expression, and in contrast to dopaminergic interneuron transgene expression which occurs only after cells reach the olfactory bulb. Although the GAD65/67 transgenes are expressed early in migration, there is minimal protein production in the cells prior to reaching the OB. These results suggest that migrating SVZ-derived neuroblasts acquire GABAergic identity prior to reaching their final location in the olfactory bulb.     

Doublecortin and a tale of two serines

Doublecortin (DCX) is a microtubule-associated protein that interacts with and regulates the microtubule cytoskeleton and is required for neuronal migration in the cortex. Two papers in this issue of Neuron (Schaar et al. and Tanaka et al.) demonstrate a role for phosphorylation in the regulation of Doublecortin. Together with recent results showing that Doublecortin may play a role regulating the morphology of migrating neurons, these findings provide new insight into the mechanisms governing neuronal migration.     

Visualization of Rostral Migratory Stream in the Developing Rat Brain by In Vivo Electroporation

Interneurons in the olfactory bulb (OB) are generated from neuronal precursor cells migrating from anterior subventricular zone (SVZa) not only in the developing embryo but also throughout the postnatal life of mammals. In the present study, we established an in vivo electroporation assay to label SVZa cells of rat both at embryonic and postnatal ages, and traced SVZa progenitors and followed their migration pathway and differentiation. We found that labeled cells displayed high motility. Interestingly, the postnatal cells migrated faster than the embryonic cells after applying this assay at different ages of brain development. Furthermore, based on brain slice culture and time-lapse imaging, we analyzed the detail migratory properties of these labeled precursor neurons. Finally, tissue transplantation experiments revealed that cells already migrated in subependymal zone of OB were transplanted back into rostral migratory stream (RMS), and these cells could still migrate out tangentially along RMS to OB. Taken together, these findings provide an in vivo labeling assay to follow and trace migrating cells in the RMS, their maturation and integration into OB neuron network, and unrecognized phenomena that postnatal SVZa progenitor cells with higher motility than embryonic cells, and their migration was affected by extrinsic environments.     

Cdk5 is required for multipolar-to-bipolar transition during radial neuronal migration and proper dendrite development of pyramidal neurons in the cerebral cortex

The mammalian cerebral cortex consists of six layers that are generated via coordinated neuronal migration during the embryonic period. Recent studies identified specific phases of radial migration of cortical neurons. After the final division, neurons transform from a multipolar to a bipolar shape within the subventricular zone-intermediate zone (SVZ-IZ) and then migrate along radial glial fibres. Mice lacking Cdk5 exhibit abnormal corticogenesis owing to neuronal migration defects. When we introduced GFP into migrating neurons at E14.5 by in utero electroporation, we observed migrating neurons in wild-type but not in Cdk5(-/-) embryos after 3-4 days. Introduction of the dominant-negative form of Cdk5 into the wild-type migrating neurons confirmed specific impairment of the multipolar-to-bipolar transition within the SVZ-IZ in a cell-autonomous manner. Cortex-specific Cdk5 conditional knockout mice showed inverted layering of the cerebral cortex and the layer V and callosal neurons, but not layer VI neurons, had severely impaired dendritic morphology. The amount of the dendritic protein Map2 was decreased in the cerebral cortex of Cdk5-deficient mice, and the axonal trajectory of cortical neurons within the cortex was also abnormal. These results indicate that Cdk5 is required for proper multipolar-to-bipolar transition, and a deficiency of Cdk5 results in abnormal morphology of pyramidal neurons. In addition, proper radial neuronal migration generates an inside-out pattern of cerebral cortex formation and normal axonal trajectories of cortical pyramidal neurons.     

Semaphorin-3A and its receptor neuropilin-1 are predominantly expressed in endothelial cells along the rostral migratory stream of young and adult mice

In the adult brain, neuroblasts originating in the subventricular zone migrate through the rostral migratory stream to the olfactory bulb. While migrating, neuroblasts undergo progressive differentiation until reaching their final locations and fates. Because molecules involved in migration may also exert differentiating effects on young neurons, the identification of factors that support migration could also shed light on the processes of adult neuroblast differentiation. This is the case for members of the family of semaphorins and of its cognate receptors, the neuropilins. Here, we have evaluated the presence of semaphorin-3A and of its receptor neuropilin-1 along the rostral migratory stream in young and adult mice by using immunocytochemical, histochemical, and in situ hybridization techniques. Our morphological studies show that semaphorin-3A and neuropilin-1 are both mainly expressed on endothelial cells along the rostral migratory stream during postnatal development. Our results suggest that endothelial cells constitute the primary source and target of semaphorin-3A along the rostral migratory stream. Moreover, the present work outlines the potential role of blood vessels on neuroblast migration in the postnatal rostral migratory stream.     

利用激光显微切割与microarray 技术对恒河猴脑中白质与 灰质基因表达的差异性的研究

目的:利用激光显微切割技术和microarray技术比较恒河猴脑组织中前额叶皮质(prefontal cortex,PFC)与小脑皮质(cere-bellar cortex,CBC)的灰质与白质基因表达的差异。方法:利用激光显微切割技术(laser capture dissection,LCM)与microarray技术的有效结合,提取恒河猴PFC与CBC的白质与灰质,分别提取RNA,合成cDNA文库。最后利用GeneChip 1.0 ST芯片技术,分析得出大脑与小脑中灰质与白质的表达差异性。结果:无论是灰质还是白质,在PFC中的高表达基因都要远远多于在CBC中的高表达基因。结论:使用LCM可以提取单一的细胞群,从而用于要求更为精确的实验当中。     

Expression of α2A adrenoceptors during rat neocortical development

Norepinephrine has been suggested to play a neurotrophic role during development and is present in the brain as early as embryonic day (E) 12. We have recently demonstrated that the α_2A adrenoceptor subtype is widely expressed during times of neuronal migration and differentiation throughout the developing brain. Here, we report the temporal and spatial expression pattern of α_2A adrenoceptors in neocortex during late embryonic and early postnatal development using in situ hybridization and receptor autoradiography. Functional α₂ receptors in embryonic rat cortex were also detected using agonist stimulated [³⁵S]GTPγS autoradiography. Both α_2A mRNA and protein expression were strongly increased by E19 and E20, respectively. The increased expression was in the cortical plate and intermediate and subventricular zones, corresponding to tiers of migrating and differentiating neurons. This transient up‐regulation of α_2A adrenoceptors was restricted to the lateral neocortex. At E20, functional α₂ adrenoceptors were also detected in deep layers of lateral neocortex. During the first week of postnatal development, the expression of α_2A mRNA and protein changed markedly, giving rise to a more mature pattern of anatomical distribution. The temporal and spatial distribution of α_2A adrenoceptors in developing neocortex is consistent with expression of functional proteins on migrating and differentiating layer IV to II neurons. These findings suggest that α_2A receptors may mediate a neurotrophic effect of norepinephrine during fetal cortical development. The early delineation of the lateral neocortex, which will develop into somatosensory and auditory cortices, suggests an intrinsic regulation of α_2A mRNA expression. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 259–269, 1999     

Demyelination-Induced Inflammation Attracts Newly Born Neurons to the White Matter

There is compelling evidence that microglial activation negatively impacts neurogenesis. However, microglia have also been shown to promote recruitment of newly born neurons to injured areas of the gray matter. In the present study, we explored whether demyelination-triggered inflammation alters the process of neurogenesis in the white matter. A 2-μl solution of 0.04 % ethidium bromide was stereotaxically injected into the corpus callosum of adult male rats. Brain inflammation was dampened by daily injections of progesterone (5 mg/kg, s.c.) for 14 days. Control rats received oil (s.c.). Newly born neurons (DCX and Tbr2), microglia (Iba-1), astrocytes (vimentin or GFAP), oligodendrocyte progenitor cells (OPCs; NG2), and mature oligodendrocytes (CC-1) were monitored in the vicinity of demyelination site using immunofluorescent staining. Western blot was used to explore microglial polarization using M1 (iNOS) and M2 (arginase-1) markers. Focal demyelination elicited strong microglial and astroglial activation and reduced the number of OPCs at the site of demyelination. This inflammatory response was associated with enhanced number of newly born neurons in the white matter and the subventricular zone (SVZ). A proportion of newly born neurons within the white matter showed features of OPCs. Interestingly, blunting brain inflammation led to reduced neurogenesis around the demyelination area and in the SVZ. These data suggest that the white matter inflammation creates a conducive environment for the recruitment of newly born neurons. The fact that a sizable fraction of these newly born neurons adopt OPC features suggests that they could contribute to the remyelination process.     

Localization of tissue plasminogen activator mRNA in the developing rat cerebellum and effects of inhibiting tissue plasminogen activator on granule cell migration

Tissue plasminogen activator (tPA) mRNA was localized in the developing cerebellum and the potentials role of tPA in migration of cerebellar granule cells was investigated. Proteolytic assays and Northern blots showed little variation in levels of tPA proteolytic activity or tPA mRNA expression in the developing cerebellum. The distribution of cerebellar tPA mRNA at different ages was visualized by in situ hybridization histochemistry. At postnatal day 7 (P7), most labeled cells were in the internal granule layer or developing white matter, and very few if any premigratory granule cells contained tPA mRNA. Although the molecular layer contained labeled cells at all ages, cell counts indicated that a greater percentage of cells in the molecular layer contained tPA mRNA during adulthood than during the period of granule cell migration. The most striking change in tPA mRNA expression was in Purkinje neurons, most of which began to express tPA mRNA between P7 and P14. The potential role of tPA in granule cell migration was investigated by performing migration assays in cerebellar slice explants in the presence or absence of protease inhibitors. The presence of inhibitors did not affect the distance that granule cells migrated. Data in the present study do not support a role for tPA in granule neuron migration; however, they do indicate that tPA is both spatially and temporally regulated during cerebellar development. Possible functions of tPA in the cerebellum are discussed. © 1995 John Wiley & Sons, Inc.     

Postnatal expression of Doublecortin (Dcx) in the developing cerebellar cortex of mouse

We have investigated the expression of Doublecortin (Dcx) protein in the developing cerebellum of mouse from postnatal 2nd day to postnatal 22nd day and in young adults by immunohistochemistry. Strong expression of Dcx was present in the inner zone of the external granule cell layer, and remained strong while postmitotic granule cell precursors were present in this transitory layer. Descending granule cell precursors exhibited Dcx immunostaining not only while migrating but for a short time also after their settlement. Dcx-immunostained cells appeared in deep cerebellocortical territories and in the cerebellar white matter during the first postnatal week. These bipolar cells were arranged in the sagittal plane and built up transitory migratory streams during the second postnatal week and their number gradually decreased during the third postnatal week. Upward migration of bipolar cells was observed while leaving the migratory streams, penetrating the internal granule cell layer and the molecular layer. These cells were considered as precursors of late migrating molecular layer interneurons. However, a proportion of Dcx-immunostained cells underwent a bipolar-to-multipolar dendritic remodellation and - on the basis of strong morphological similarities - was taken for "multipotent progenitor cells", described recently in the neocortex of adult rat.     

Glial progenitors of the neonatal subventricular zone differentiate asynchronously, leading to spatial dispersion of glial clones and to the persistence of immature glia in the adult mammalian CNS

The subventricular zone (SVZ) of the developing mammalian forebrain gives rise to astrocytes and oligodendrocytes in the neocortex and white matter, and neurons in the olfactory bulb in perinatal life. We have examined the developmental fates and spatial distributions of the descendants of single SVZ cells by infecting them in vivo at postnatal day 0-1 (P0-1) with a retroviral "library". In most cases, individual SVZ cells gave rise to either oligodendrocytes or astrocytes, but some generated both types of glia. Members of glial clones can disperse widely through the gray and white matter. Progenitors continued to divide after stopping migration, generating clusters of related cells. However, the progeny of a single SVZ cell does not differentiate synchronously: individual clones contained both mature and less mature glia after short or long intervals. For example, progenitors that settled in the white matter generated three types of clonal oligodendrocyte clusters: those composed of only myelinating oligodendrocytes, of both myelinating oligodendrocytes and non-myelinating oligodendrocytes, or of only non-myelinating cells of the oligodendrocyte lineage. Thus, some progenitors do not fully differentiate, but remain immature and may continue to cycle well into adult life.