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.     

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.     

Identification and characterization of neuroblasts in the subventricular zone and rostral migratory stream of the adult human brain

It is of great interest to identify new neurons in the adult human brain, but the persistence of neurogenesis in the subventricular zone (SVZ) and the existence of the rostral migratory stream (RMS)-like pathway in the adult human forebrain remain highly controversial. In the present study, we have described the general configuration of the RMS in adult monkey, fetal human and adult human brains. We provide evidence that neuroblasts exist continuously in the anterior ventral SVZ and RMS of the adult human brain. The neuroblasts appear singly or in pairs without forming chains; they exhibit migratory morphologies and co-express the immature neuronal markers doublecortin, polysialylated neural cell adhesion molecule and βIII-tubulin. Few of these neuroblasts appear to be actively proliferating in the anterior ventral SVZ but none in the RMS, indicating that neuroblasts distributed along the RMS are most likely derived from the ventral SVZ. Interestingly, no neuroblasts are found in the adult human olfactory bulb. Taken together, our data suggest that the SVZ maintains the ability to produce neuroblasts in the adult human brain.     

Neurogenesis and neuronal migration in the neonatal rat forebrain anterior subventricular zone do not require GFAP-positive astrocytes

A prolific neuronal progenitor cell population in the anterior portion of the neonatal rat forebrain subventricular zone, the SVZa, is specialized for the production of olfactory bulb interneurons. At all ages, SVZa-derived cells traverse a tangential migratory pathway, the rostral migratory stream (RMS), while en route to the olfactory bulb. Unlike other neuronal progenitor cells of the forebrain, migrating progeny of SVZa progenitors express neuronal-specific proteins and continue to divide into adulthood. Recent studies indicate that in the adult, migrating SVZa-derived cells are ensheathed by astrocytes, although the function of these astrocytes has not been determined. To explore the possible role(s) of astrocytes in the rat SVZa and RMS, we examined the expression of astroglial-specific genes in the postnatal SVZa and RMS using RT-PCR, in situ hybridization, and immunohistochemistry during (Postnatal Days 1-10) and after the period of peak olfactory bulb interneuron generation. We also examined the expression of neuronal-specific genes throughout the rostral-caudal extent of the postnatal subventricular zone to determine if differential cell type-specific gene expression could distinguish the neurogenic SVZa as a region distinct from the remainder of the SVZ. We found little to no astrocyte-specific gene expression in the P0-P7 SVZa, although the neuron-specific isoforms of tubulin (T alpha 1 and beta-III tubulin) were expressed abundantly in the SVZa and RMS. In contrast, astrocyte-specific genes were strongly expressed in the SVZ posterior to the SVZa. GFAP expressions begins to appear in some restricted areas of the rostral migratory stream after the first postnatal week. These data suggest that astroglia are not involved in the generation or migration of most olfactory bulb interneurons. Moreover, the scarcity of glial markers in the neonatal SVZa indicates that the forebrain subventricular zone includes a distinct neurogenic anterior region containing predominantly committed neuronal progenitor cells.     

Targeted Deletion of the ERK5 MAP Kinase Impairs Neuronal Differentiation,Migration, and Survival during Adult Neurogenesis in the Olfactory Bulb

Recent studies have led to the exciting idea that adult-born neurons in the olfactory bulb (OB) may be critical for complex forms of olfactory behavior in mice. However, signaling mechanisms regulating adult OB neurogenesis are not well defined. We recently reported that extracellular signal-regulated kinase (ERK) 5, a MAP kinase, is specifically expressed in neurogenic regions within the adult brain. This pattern of expression suggests a role for ERK5 in the regulation of adult OB neurogenesis. Indeed, we previously reported that conditional deletion of erk5 in adult neurogenic regions impairs several forms of olfactory behavior in mice. Thus, it is important to understand how ERK5 regulates adult neurogenesis in the OB. Here we present evidence that shRNA suppression of ERK5 in adult neural stem/progenitor cells isolated from the subventricular zone (SVZ) reduces neurogenesis in culture. By contrast, ectopic activation of endogenous ERK5 signaling via expression of constitutive active MEK5, an upstream activating kinase for ERK5, stimulates neurogenesis. Furthermore, inducible and conditional deletion of erk5 specifically in the neurogenic regions of the adult mouse brain interferes with cell cycle exit of neuroblasts, impairs chain migration along the rostral migratory stream and radial migration into the OB. It also inhibits neuronal differentiation and survival. These data suggest that ERK5 regulates multiple aspects of adult OB neurogenesis and provide new insights concerning signaling mechanisms governing adult neurogenesis in the SVZ-OB axis.     

Rostral migratory stream neuroblasts turn and change directions in stereotypic patterns

Neuroblasts generated in the adult subventricular zone (SVZ) migrate through the rostral migratory stream (RMS) to the olfactory bulb (OB). Previous work uncovered motility ranging from straight to complex, but it was unclear if directional changes were stochastic or exhibited stereotypical patterns. Here, we provide the first in-depth two-photon time-lapse microscopy study of morphological and dynamic features that accompany turning and direction reversals in the RMS. We identified three specific kinds of turning (30–90 degrees): bending of the leading process proximal to the cell body (P-bending 47% of cases), bending of the distal leading process (D-bending 30%) or branching of the leading process or lamellipodium (23%). Bending and branching angles were remarkably constrained and were significantly different from one another. Cells reversed direction (>90 degrees) through D-bendings (54%), branching (11%) or de novo growth of processes from the soma (23%), but not P-bending. Direction reversal was often composed of several iterations of D-bending or branching as opposed to novel modalities. Individual neuroblasts could turn or change direction in multiple patterns suggesting that the patterns are not specific for different lineages. These findings show that neuroblasts in the RMS use a limited number of distinct and constrained modalities to turn or reverse direction.Key words: neurogenesis, subventricular zone, migration, direction, motility     

Neuroblasts of the postnatal mammalian forebrain: Their phenotype and fate

The subventricular zone (SVZ) is the only germinal zone of the developing mammalian forebrain to persist postnatally. Although the SVZ has been known to give rise to most of the glial cells of the forebrain, several studies over the past few years have shown that the cells of the neonatal and adult SVZ can also generate neurons. Recent studies have demonstrated that a discrete region of the anterior part of the neonatal SVZ is composed exclusively of neuronal progenitor cells, whose progeny become interneurons of the olfactory bulb. This review will explore the properties that distinguish this anterior segment of the neonatal subventricular zone (SVZa) from the more posterior, gliogenic region. The cells of the SVZa, as well as its anterior extension forming the rostral migratory stream that enters the middle of the olfactory bulb, have antigenic characteristics of a neuronal phenotype, yet continue to divide during migration. In vitro, SVZa progenitor cells also retain a neuronal phenotype despite persistent division. Intriguingly, SVZa cells and their progeny migrate long distances along a highly stereotypical pathway. To better understand the guidance cues used by SVZa-derived cells during migration, both homotopic and heterotopic transplantation experiments have been conducted. SVZa cells homotopically transplanted into another animal's SVZa migrate with the recipient's endogenous SVZa cells in an indistinguishable manner, whereas those from the embryonic telencephalic ventricular zone, normally destined to follow radial glia to the cerebral cortex, fail to migrate following transplantation to the SVZa. SVZa cells transplanted heterotopically into the neonatal and adult striatum were able to disperse from their site of implantation. Thus, SVZa cells are special proliferating cells for which the rostral migratory stream is a particularly permissive pathway. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 221–233, 1998     

GABA modulation of SVZ‐derived progenitor ventral cell migration

The subventricular zone (SVZ) is a proliferative region that provides neurons to olfactory bulb throughout life. The new neurons undergo cell migration from SVZ and travel until they reach their final destination. We previously showed in the early postnatal mouse a ventral migratory subpopulation from SVZ targets the Islands of Calleja (ICC) in the basal forebrain. However, unlike the well‐characterized rostral migratory stream, little is known about the guidance mechanisms operating in the ventrally directed migratory pathway. In this study, we examined the role of neurotransmitter γ‐aminobutyric acid (GABA) in SVZ‐derived progenitor ventral migration and the involvement of this neurotransmitter in the cytoarchitectual organization of dispersed cells into the tight clusters of the ICC. Our results show that the ventral SVZ cell migration rate was enhanced by GABA acting through a GABA_A receptor and that GABA acts as a directional guidance cue for ventral migrating cells. Furthermore, disruption of GABA signaling inhibited the formation of Island clusters in vitro. Taken together, these data suggest that GABA is an important guidance and organizational cue for the Island of Calleja. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 791–804, 2015     

Migration and differentiation of neural precursors derived from human embryonic stem cells in the rat brain

Human embryonic stem (hES) cells provide a potentially unlimited cell source for regenerative medicine. Recently, differentiation strategies were developed to direct hES cells towards neural fates in vitro. However, the interaction of hES cell progeny with the adult brain environment remains unexplored. Here we report that hES cell-derived neural precursors differentiate into neurons, astrocytes and oligodendrocytes in the normal and lesioned brain of young adult rats and migrate extensively along white matter tracts. The differentiation and migration behavior of hES cell progeny was region specific. The hES cell-derived neural precursors integrated into the endogenous precursor pool in the subventricular zone, a site of persistent neurogenesis. Like adult neural stem cells, hES cell-derived precursors traveled along the rostral migratory stream to the olfactory bulb, where they contributed to neurogenesis. We found no evidence of cell fusion, suggesting that hES cell progeny are capable of responding appropriately to host cues in the subventricular zone.     

Retinoic acid regulates postnatal neurogenesis in the murine subventricular zone-olfactory bulb pathway

Neurogenesis persists throughout life in the rodent subventricular zone (SVZ)-olfactory bulb pathway. The molecular regulation of this neurogenic circuit is poorly understood. Because the components for retinoid signaling are present in this pathway, we examined the influence of retinoic acid (RA) on postnatal SVZ-olfactory bulb neurogenesis. Using both SVZ neurosphere stem cell and parasagittal brain slice cultures derived from postnatal mouse, we found that RA exposure increased neurogenesis by enhancing the proliferation and neuronal differentiation of forebrain SVZ neuroblasts. The RA precursor retinol had a similar effect, which was reversed by treating cultures with the RA synthesis inhibitor disulfiram. Electroporation of dominant-negative retinoid receptors into the SVZ of slice cultures also blocked neuroblast migration to the olfactory bulb and altered the morphology of the progenitors. Moreover, the administration of disulfiram to neonatal mice decreased in vivo cell proliferation in the striatal SVZ. These results indicate that RA is a potent mitogen for SVZ neuroblasts and is required for their migration to the olfactory bulb. The regulation of multiple steps in the SVZ-olfactory bulb neurogenic pathway by RA suggests that manipulation of retinoid signaling is a potential therapeutic strategy to augment neurogenesis after brain injury.     

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.     

Drebrin Regulates Neuroblast Migration in the Postnatal Mammalian Brain

After birth, stem cells in the subventricular zone (SVZ) generate neuroblasts that migrate along the rostral migratory stream (RMS) to become interneurons in the olfactory bulb (OB). This migration is crucial for the proper integration of newborn neurons in a pre-existing synaptic network and is believed to play a key role in infant human brain development. Many regulators of neuroblast migration have been identified; however, still very little is known about the intracellular molecular mechanisms controlling this process. Here, we have investigated the function of drebrin, an actin-binding protein highly expressed in the RMS of the postnatal mammalian brain. Neuroblast migration was monitored both in culture and in brain slices obtained from electroporated mice by time-lapse spinning disk confocal microscopy. Depletion of drebrin using distinct RNAi approaches in early postnatal mice affects neuroblast morphology and impairs neuroblast migration and orientation in vitro and in vivo. Overexpression of drebrin also impairs migration along the RMS and affects the distribution of neuroblasts at their final destination, the OB. Drebrin phosphorylation on Ser142 by Cyclin-dependent kinase 5 (Cdk5) has been recently shown to regulate F-actin-microtubule coupling in neuronal growth cones. We also investigated the functional significance of this phosphorylation in RMS neuroblasts using in vivo postnatal electroporation of phosphomimetic (S142D) or non-phosphorylatable (S142A) drebrin in the SVZ of mouse pups. Preventing or mimicking phosphorylation of S142 in vivo caused similar effects on neuroblast dynamics, leading to aberrant neuroblast branching. We conclude that drebrin is necessary for efficient migration of SVZ-derived neuroblasts and propose that regulated phosphorylation of drebrin on S142 maintains leading process stability for polarized migration along the RMS, thus ensuring proper neurogenesis.     

Thrombospondin-1 binds to ApoER2 and VLDL receptor and functions in postnatal neuronal migration

Apolipoprotein E receptor 2 (ApoER2), very low-density lipoprotein receptor (VLDLR), and Dab1 are the main components of the Reelin signalling cascade. Reelin is the sole ligand defined so far in signalling through this pathway. Postnatal migration of neuronal precursors from the subventricular zone (SVZ) to the olfactory bulb (OB), however, depends on ApoER2 and Dab1, but functions independently of Reelin. Here, we show that thrombospondin-1 (THBS-1) is a novel physiological ligand for ApoER2 and VLDLR. THBS-1 is present in the SVZ and along the entire rostral migratory stream (RMS). It binds to ApoER2 and VLDLR and induces phosphorylation of Dab1. In contrast to Reelin, it does not induce Dab1 degradation or Akt phosphorylation, but stabilizes neuronal precursor chains derived from subventricular explants. Lack of THBS-1 results in anatomical abnormalities of the RMS and leads to a reduction of postnatal neuronal precursors entering the OB.     

Paced-mating increases the number of adult new born cells in the internal cellular (granular) layer of the accessory olfactory bulb

The continuous production and addition of new neurons during life in the olfactory bulb is well accepted and has been extensively studied in rodents. This process could allow the animals to adapt to a changing environment. Olfactory neurogenesis begins in the subventricular zone where stem cells proliferate and give rise to young undifferentiated neuroblasts that migrate along the rostral migratory stream to the olfactory bulb (OB). Olfaction is crucial for the expression of sexual behavior in rodents. In female rats, the ability to control the rate of sexual interactions (pacing) has important physiological and behavioral consequences. In the present experiment we evaluated if pacing behavior modifies the rate of new cells that reach the main and accessory olfactory bulb. The BrdU marker was injected before and after different behavioral tests which included: females placed in a mating cage (control), females allowed to pace the sexual interaction, females that mated but were not able to control the rate of the sexual interaction and females exposed to a sexually active male. Subjects were sacrificed fifteen days after the behavioral test. We observed a significant increase in the density of BrdU positive cells in the internal cellular layer of the accessory olfactory bulb when females paced the sexual interaction in comparison to the other 3 groups. No differences in the cell density in the main olfactory bulb were found. These results suggest that pacing behavior promotes an increase in density of the new cells in the accessory olfactory bulb.     

Rostral migratory stream neuroblasts turn and change directions in stereotypic patterns

Neuroblasts generated in the adult subventricular zone (SVZ) migrate through the rostral migratory stream (RMS) to the olfactory bulb (OB). Previous work uncovered motility ranging from straight to complex, but it was unclear if directional changes were stochastic or exhibited stereotypical patterns. Here, we provide the first in-depth two-photon time-lapse microscopy study of morphological and dynamic features that accompany turning and direction reversals in the RMS. We identified three specific kinds of turning (30-90 degrees): bending of the leading process proximal to the cell body (P-bending 47% of cases), bending of the distal leading process (D-bending 30%) or branching of the leading process or lamellipodium (23%). Bending and branching angles were remarkably constrained and were significantly different from one another. Cells reversed direction (>90 degrees) through D-bendings (54%), branching (11%) or de novo growth of processes from the soma (23%), but not P-bending. Direction reversal was often composed of several iterations of D-bending or branching as opposed to novel modalities. Individual neuroblasts could turn or change direction in multiple patterns suggesting that the patterns are not specific for different lineages. These findings show that neuroblasts in the RMS use a limited number of distinct and constrained modalities to turn or reverse direction.     

Neural stem cells and the regulation of adult neurogenesis

Presumably, the 'hard-wired' neuronal circuitry of the adult brain dissuades addition of new neurons, which could potentially disrupt existing circuits. This is borne out by the fact that, in general, new neurons are not produced in the mature brain. However, recent studies have established that the adult brain does maintain discrete regions of neurogenesis from which new neurons migrate and become incorporated into the functional circuitry of the brain. These neurogenic zones appear to be vestiges of the original developmental program that initiates brain formation. The largest of these germinal regions in the adult brain is the subventricular zone (SVZ), which lines the lateral walls of the lateral ventricles. Neural stem cells produce neuroblasts that migrate from the SVZ along a discrete pathway, the rostral migratory stream, into the olfactory bulb where they form mature neurons involved in the sense of smell. The subgranular layer (SGL) of the hippocampal dentate gyrus is another neurogenic region; new SGL neurons migrate only a short distance and differentiate into hippocampal granule cells. Here, we discuss the surprising finding of neural stem cells in the adult brain and the molecular mechanisms that regulate adult neurogenesis.