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Human brain development proceeds via a sequentially transforming stem cell population in the ventricular-subventricular zone (V-SVZ). An essential, but understudied, contributor to V-SVZ stem cell niche health is the multi-ciliated ependymal epithelium, which replaces stem cells at the ventricular surface during development. However, reorganization of the V-SVZ stem cell niche and its relationship to ependymogenesis has not been characterized in the human brain. Based on comprehensive comparative spatiotemporal analyses of cytoarchitectural changes along the mouse and human ventricle surface, we uncovered a distinctive stem cell retention pattern in humans as ependymal cells populate the ventricle surface in an occipital-to-frontal wave. During perinatal development ventricle-contacting stem cells are reduced. By 7-months few stem cells are detected, paralleling neurogenesis decline. In adolescence and adulthood, stem cells and neurogenesis are not observed along the lateral wall. Volume, surface area, and curvature of the lateral ventricles all significantly change during fetal development but stabilize after 1-year, corresponding with the wave of ependymogenesis and stem cell reduction. These findings reveal normal human V-SVZ development, highlighting the consequences of disease pathologies such as congenital hydrocephalus.

Significant migration cues are required to guide and contain newly generated rodent subventricular zone (SVZ) neuroblasts as they transit along the lateral ventricles and then through the anterior forebrain to their ultimate site of differentiation in the olfactory bulbs (OBs). These cues enforce strict neuroblast spatial boundaries within the dense astroglial meshwork of the SVZ and rostral migratory stream (RMS), yet are permissive to large-scale neuroblast migration. Therefore, the molecular mechanisms that define these cues and control dynamic interactions between migratory neuroblasts and surrounding astrocytes are of particular interest. We found that deletion of EphA4 and specifically ablation of EphA4 kinase activity resulted in misaligned neuroblasts and disorganized astrocytes in the RMS/SVZ, linking EphA4 forward signaling to SVZ and RMS spatial organization, orientation, and regulation. In addition, within a 3 week period, there was a significant reduction in the number of neuroblasts that reached the OB and integrated into the periglomerular layer, revealing a crucial role for EphA4 in facilitating efficient neuroblast migration to the OB. Single-cell analysis revealed thatEPHA4and itsEFNbinding partners are expressed by subpopulations of neuroblasts and astrocytes within the SVZ/RMS/OB system resulting in a cell-specific mosaic, suggesting complex EphA4 signaling involving both homotypic and heterotypic cell–cell interactions. Together, our studies reveal a novel molecular mechanism involving EphA4 signaling that functions in stem cell niche organization and ultimately neuroblast migration in the anterior forebrain.SIGNIFICANCE STATEMENTThe subventricular zone neurogenic stem cell niche generates highly migratory neuroblasts that transit the anterior forebrain along a defined pathway to the olfactory bulb. Postnatal and adult brain organization dictates strict adherence to a narrow migration corridor. Subventricular zone neuroblasts are aligned in tightly bundled chains within a meshwork of astrocytes; however, the cell–cell cues that organize this unique, cell-dense migration pathway are largely unknown. Our studies show that forward signaling through the EphA4 tyrosine kinase receptor, mediated by ephrins expressed by subpopulations of neuroblasts and astrocytes, is required for compact, directional organization of neuroblasts and astrocytes within the pathway and efficient transit of neuroblasts through the anterior forebrain to the olfactory bulb.

Age-associated ventriculomegaly is typically attributed to neurodegeneration; however, additional factors might initiate or contribute to progressive ventricular expansion. By directly linking postmortem human MRI sequences with histological features of periventricular tissue, we show that substantial lateral ventricle surface gliosis is associated with ventriculomegaly. To examine whether loss of ependymal cell coverage resulting in ventricle surface glial scarring can lead directly to ventricle enlargement independent of any other injury or degenerative loss, we modeled in mice the glial scarring found along the lateral ventricle surface in aged humans. Neuraminidase, which cleaves glycosidic linkages of apical adherens junction proteins, was administered intracerebroventricularly to denude areas of ependymal cells. Substantial ependymal cell loss resulted in reactive gliosis rather than stem cell-mediated regenerative repair of the ventricle lining, and the gliotic regions showed morphologic and phenotypic characteristics similar to those found in aged humans. Increased levels of aquaporin-4, indicative of edema, observed in regions of periventricular gliosis in human tissue were also replicated in our mouse model. 3D modeling together with volume measurements revealed that mice with ventricle surface scarring developed expanded ventricles, independent of neurodegeneration. Through a comprehensive, comparative analysis of the lateral ventricles and associated periventricular tissue in aged humans and mouse, followed by modeling of surface gliosis in mice, we have demonstrated a direct link between lateral ventricle surface gliosis and ventricle enlargement. These studies highlight the importance of maintaining an intact ependymal cell lining throughout aging.

The two major stem cell niches that support neurogenesis in the adult mammalian brain are the ventricular-subventricular zone that lies along the lateral wall of the lateral ventricles and the subgranular zone found within the dentate gyrus of the hippocampus. Recent advances reveal that adult neurogenesis and integration of newborn neurons within the brain's neural circuitry provides a level of neural plasticity that had previously gone unrecognized. Investigations into the cytoarchitectural organization and molecular controls that regulate the developing embryonic and mature adult neuronal stem cell niches can thus inspire and influence strategies for future regenerative and replacement therapies in cases of brain aging, injury, or disease. Equally important, studies of the brain's stem cell niches can define normal developmental mechanisms and reveal etiologies linked to cognitive deficits and other neurological pathologies. Here, we define the development of the brain's neuronal stem cell niches and identify key characteristics and molecular regulators that help support the mammalian brain's two neurogenic stem cell niches and the molecular pathways that regulate these niches.

Through adulthood, the rodent subventricular zone (SVZ) stem cell niche generates new olfactory bulb interneurons. We had previously reported that the number of new neurons produced in the SVZ declines through aging; however, age-related changes attributable specifically to the SVZ neural stem cell (NSC) population have not been fully characterized. Here, we conducted a spatiotemporal evaluation of adult SVZ NSCs. We assessed ventricle-contacting NSCs, which together with ependymal cells form regenerative units (pinwheels) along the lateral wall of the lateral ventricle. Based on their apical GFAP-expressing process, individual NSCs were identified across the ventricle surface using serial reconstruction of the SVZ. We observed an 86% decline in total NSCs/mm2of intact ependyma in 2-year old versus 3-month-old mice, with fewer NSC processes within each aged pinwheel. This resulted in an associated 78% decline in total pinwheel units/mm2. Regional analysis along the lateral ventricle surface revealed that the age-dependent decline of NSCs and pinwheels is spatially uniform and ultimately maintains the conserved ratio of olfactory bulb interneuron subtypes generated in young mice. However, the overall neurogenic output of the aged SVZ is reduced. Surprisingly, we found no significant change in the number of actively proliferating NSCs per mm2of ventricle surface. Instead, our data reveal that, although the total NSC number, pinwheel units and NSCs per pinwheel decline with age, the percentage of actively, mitotic NSCs increases, indicating that age-related declines in SVZ-mediated olfactory bulb neurogenesis occur downstream of NSC proliferation.

Nucleus accumbens is involved in several aspects of instrumental behavior, motivation and learning. Recent studies showed that dopamine (DA) release in the accumbens shell was significantly increased on the first day of training on a fixed ratio (FR) 5 schedule (i.e. the transition from FR1 to FR5) compared with those rats that continued FR1 training, even though the rats on their first day of FR5 training received less food reinforcement than rats continuing on the FR1 schedule. Additionally, the second day of FR5 responding was marked by a significant increase in DA release in accumbens core. The present studies employed immunohistochemical methods to characterize the changes in cellular markers of accumbens and neostriatal neural activity that occur during various stages of food-reinforced FR5 training. c-Fos and DARPP-32 immunoreactivity in accumbens shell was significantly increased on the first day of FR5 training, while core c-Fos and DARPP-32 expression showed large increases on the second day of FR5 training. Additional studies showed that c-Fos and DARPP-32 expression in neostriatum increased after more extensive training. Double-labeling studies with immunofluorescence methods indicated that increases in accumbens c-Fos and DARPP-32 expression were primarily seen in substance-P-positive neurons. These increases in accumbens c-Fos and DARPP-32 immunoreactivity seen during the initial phases of FR training may reflect several factors, including novelty, learning, stress or the presentation of a work-related challenge to the organism. Moreover, it appears that the separate subregions of the striatal complex are differentially activated at distinct phases of instrumental training.

Coordinated regulation of the adult neurogenic subventricular zone (SVZ) is accomplished by a myriad of intrinsic and extrinsic factors. The neurotransmitter dopamine is one regulatory molecule implicated in SVZ function. Nigrostriatal and ventral tegmental area (VTA) midbrain dopamine neurons innervate regions adjacent to the SVZ, and dopamine synapses are found on SVZ cells. Cell division within the SVZ is decreased in humans with Parkinson's disease and in animal models of Parkinson's disease following exposure to toxins that selectively remove nigrostriatal neurons, suggesting that dopamine is critical for SVZ function and nigrostriatal neurons are the main suppliers of SVZ dopamine. However, when we examined theaphakiamouse, which is deficient in nigrostriatal neurons, we found no detrimental effect to SVZ proliferation or organization. Instead, dopamine innervation of the SVZ tracked to neurons at the ventrolateral boundary of the VTA. This same dopaminergic neuron population also innervated the SVZ of control mice. Characterization of these neurons revealed expression of proteins indicative of VTA neurons. Furthermore, exposure to the neurotoxin MPTP depleted neurons in the ventrolateral VTA and resulted in decreased SVZ proliferation. Together, these results reveal that dopamine signaling in the SVZ originates from a population of midbrain neurons more typically associated with motivational and reward processing.

We present optimal perfusion conditions for the growth of primary mouse embryonic fibroblasts (mEFs) and mouse embryonic stem cells (mESCs) using a microfluidic perfusion culture system. In an effort to balance nutrient renewal while ensuring the presence of cell secreted factors, we found that the optimal perfusion rate for culturing primary embryonic fibroblasts (mEFs) in our experimental setting is 10 nL/min with an average flow velocity 0.55 microm/s in the microchannel. Primary mEFs may have a greater dependence on cell secreted factors when compared to their immortalized counterpart 3T3 fibroblasts cultured under similar conditions. Both the seeding density and the perfusion rate are critical for the proliferation of primary cells. A week long cultivation of mEFs and mESCs using the microculture system exhibited similar morphology and viability to those grown in a petri dish. Both mEFs and mESCs were analyzed using fluorescence immunoassays to determine their proliferative status and protein expression. Our results demonstrate that a perfusion-based microculture environment is capable of supporting the highly proliferative status of pluripotent embryonic stem cells.

Somatic cell nuclear transfer enables the generation of embryonic stem cells (ESCs) that genetically match the donor and can be used to treat disease through cell replacement therapies or to recapitulate patient-specific disease via in vitro differentiation. We performed a "proof-of-principle" study using tail tip fibroblasts from a mouse model of Parkinson's disease (Aphakia) as the donor cell nuclei for nuclear transfer and derived "customized" ESCs for in vitro analysis. Aphakia mice contain deletions in the pitx3 gene and show selective loss of dopamine neurons of the substantia nigra, specifically the neuron population susceptible to degeneration in Parkinson's disease. Using electrofusion nuclear transfer, we produced cloned Aphakia oocytes at rates similar to those for control, cloned oocytes. Aphakia ESCs were isolated and live mice were generated using tetraploid embryo complementation. In vitro differentiation of Aphakia ESCs to dopaminergic neurons revealed significantly fewer TH+ neurons that expressed MAP2, DAT, synaptophysin, VMAT2, and AHD2 compared to control nuclear transfer ESC cultures, supporting a role for Pitx3 in mesodiencephalic dopamine neuron maturation. Taken together, our studies define a customized in vitro ESC culture system used to analyze gene-specific contribution to dopamine neuron generation, maturation, and susceptibility to degeneration.

The subventricular zone (SVZ) of the adult mouse brain is a narrow stem cell niche that lies along the length of the lateral wall of the lateral ventricles. The SVZ supports neurogenesis throughout adulthood; however, with increasing age, the ventral SVZ deteriorates and only the dorsolateral SVZ remains neurogenic. Associated with the elderly dorsolateral SVZ, we reported previously an increased number of astrocytes interposed within the adjacent ependymal lining. Here, we show that astrocytes integrated within the ependyma are dividing, BrdU-labeled astrocytes that share cellular adherens with neighboring ependymal cells. By tracking BrdU-labeled astrocytes over time, we observed that, as they incorporated within the ependyma, they took on antigenic and morphologic characteristics of ependymal cells, suggesting a novel form of SVZ-supported “regenerative” repair in the aging brain. A similar form of SVZ-mediated ependyma repair was also observed in young mice after mild ependymal cell denudation with low dosages of neuraminidase. Together, this work identifies a novel non-neuronal mechanism of regenerative repair by the adult SVZ.

The ventricular system carries and circulates cerebral spinal fluid (CSF) and facilitates clearance of solutes and toxins from the brain. The functional units of the ventricles are ciliated epithelial cells termed ependymal cells, which line the ventricles and through ciliary action are capable of generating laminar flow of CSF at the ventricle surface. This monolayer of ependymal cells also provides barrier and filtration functions that promote exchange between brain interstitial fluids (ISF) and circulating CSF. Biochemical changes in the brain are thereby reflected in the composition of the CSF and destruction of the ependyma can disrupt the delicate balance of CSF and ISF exchange. In humans there is a strong correlation between lateral ventricle expansion and aging. Age-associated ventriculomegaly can occur even in the absence of dementia or obstruction of CSF flow. The exact cause and progression of ventriculomegaly is often unknown; however, enlarged ventricles can show regional and, often, extensive loss of ependymal cell coverage with ventricle surface astrogliosis and associated periventricular edema replacing the functional ependymal cell monolayer. Using MRI scans together with postmortem human brain tissue, we describe how to prepare, image and compile 3D renderings of lateral ventricle volumes, calculate lateral ventricle volumes, and characterize periventricular tissue through immunohistochemical analysis of en face lateral ventricle wall tissue preparations. Corresponding analyses of mouse brain tissue are also presented supporting the use of mouse models as a means to evaluate changes to the lateral ventricles and periventricular tissue found in human aging and disease. Together, these protocols allow investigations into the cause and effect of ventriculomegaly and highlight techniques to study ventricular system health and its important barrier and filtration functions within the brain.

The generation of mice lacking specific neurotrophins permits evaluation of the trophic requirements of particular neuronal populations throughout development. In the present study, we examined the developing vestibulo-cochlear system to determine the time course of neurotrophin dependence and to determine whether competition occurred among developing cochlear or vestibular neurons for available amounts of either brain-derived neurotrophic factor (BDNF) or neurotrophin-4/5 (NT-4/5). Both cochlear and vestibular neurons were present in mice lacking NT-4/5. In contrast, vestibular neurons decreased in number beginning at mid-stages of inner ear development, in mice lacking BDNF. Early in development (E12.5-13), the size of the vestibular ganglion was normal in bdnf−/− mice. Decreased innervation to vestibular sensory epithelia was detected at E13.5-15, when progressive loss of all afferent innervation to the semicircular canals and reduced innervation to the utricle and saccule were observed. At E16.5-17, there was a reduction in the number of vestibular neurons in bdnf−/− mice. A further decrease in vestibular neurons was observed at P1 and P15. Compared to bdnf−/− mice, mice heterozygous for the BDNF null mutation (bdnf+/−) showed an intermediate decrease in the number of vestibular neurons from E16.5-P15. These data indicate a late developmental requirement of vestibular neurons for BDNF and suggest competition among these neurons for limited supplies of this factor.

Ciliary neurotrophic factor (CNTF) supports motor neuron survival in vitro and in mouse models of motor neuron degeneration and was considered a candidate for the muscle-derived neurotrophic activity that regulates motor neuron survival during development. However, CNTF expression is very low in the embryo, and CNTF gene mutations in mice or human do not result in notable abnormalities of the developing nervous system. We have generated and directly compared mice containing null mutations in the genes encoding CNTF or its receptor (CNTFR alpha). Unlike mice lacking CNTF, mice lacking CNTFR alpha die perinatally and display severe motor neuron deficits. Thus, CNTFR alpha is critical for the developing nervous system, most likely by serving as a receptor for a second, developmentally important, CNTF-like ligand.

NERVE growth factor and other neurotrophins signal to neurons through the Trk family of receptor tyrosine kinases1–6. TrkB is relatively promiscuous in vitro, acting as a receptor for brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT4) and, to a lesser extent, NT3 (refs 3–5). Mice lacking TrkB7 show a more severe phenotype than mice lacking BDNF8,9, suggesting that TrkB may act as a receptor for additional ligands in vivo. To explore this possibility, we generated mice lacking NT4 or BDNF as well as mice lacking both neurotrophins. Unlike mice lacking other Trks7,10,11 or neurotrophins8,9,12–14, NT4-deficient mice are long-lived and show no obvious neurological defects. Analysis of mutant phenotypes revealed distinct neuronal populations with different neurotrophin requirements. Thus vestibular and trigemi-nal sensory neurons require BDNF but not NT4, whereas nodose-petrosal sensory neurons require both BDNF and NT4. Motor neurons, whose numbers are drastically reduced in mice lacking TrkB, are not affected even in mice lacking both BDNF and NT4. These results suggest that another ligand, perhaps NT3, does indeed act on TrkB in vivo.

Nucleus accumbens is involved in several aspects of instrumental behavior, motivation and learning. Recent studies showed that dopamine (DA) release in the accumbens shell was significantly increased on the first day of training on a fixed ratio (FR) 5 schedule (i.e. the transition from FR1 to FR5) compared with those rats that continued FR1 training, even though the rats on their first day of FR5 training received less food reinforcement than rats continuing on the FR1 schedule. Additionally, the second day of FR5 responding was marked by a significant increase in DA release in accumbens core. The present studies employed immunohistochemical methods to characterize the changes in cellular markers of accumbens and neostriatal neural activity that occur during various stages of food-reinforced FR5 training. c-Fos and DARPP-32 immunoreactivity in accumbens shell was significantly increased on the first day of FR5 training, while core c-Fos and DARPP-32 expression showed large increases on the second day of FR5 training. Additional studies showed that c-Fos and DARPP-32 expression in neostriatum increased after more extensive training. Double-labeling studies with immunofluorescence methods indicated that increases in accumbens c-Fos and DARPP-32 expression were primarily seen in substance-P-positive neurons. These increases in accumbens c-Fos and DARPP-32 immunoreactivity seen during the initial phases of FR training may reflect several factors, including novelty, learning, stress or the presentation of a work-related challenge to the organism. Moreover, it appears that the separate subregions of the striatal complex are differentially activated at distinct phases of instrumental training.

The persistence of an active subventricular zone neural stem cell niche in the adult mammalian forebrain supports its continued role in the production of new neurons and in generating cells to function in repair through adulthood. Unfortunately, with increasing age the niche begins to deteriorate, compromising these functions. The reasons for this decline are not clear. Studies are beginning to define the molecular and physiologic changes in the microenvironment of the aging subventricular zone niche. New revelations from aging studies will allow for a more thorough understanding of which reparative functions are lost in the aged brain, the progression of niche demise and the possibility for therauptic intervention to improve aging brain function.

The transcription factor Pitx3 is expressed exclusively by mesodiencephalic dopaminergic neurons; however, ablation of Pitx3 results in selective degeneration of primarily dopaminergic neurons of the substantia nigra pars compacta, the neuronal population that is most vulnerable in Parkinson's disease. Although the exact molecular mechanisms of the action of Pitx3 are unclear, roles in both terminal maturation and/or survival of substantia nigra dopaminergic neurons have been suggested. To investigate the connection between Pitx3 and selective neurodegeneration, we generated embryonic stem cells from a Pitx3-deficient mouse (aphakia) for in-vitro differentiation to dopaminergic neurons. This 'loss of function'in-vitro system allowed us to examine characteristic features in dopaminergic neuron development and to assess the role that Pitx3 plays in the differentiation/maturation process. We found that aphakia embryonic stem cells generated 50% fewer tyrosine hydroxylase-positive/microtubule-associated protein (Map)2-positive mature neurons compared with control cultures. The expression of dopamine transport regulators and vesicle release proteins was reduced and dopamine release was unregulated in the Pitx3-deficient tyrosine hydroxylase-positive neurons generated. Treatment of aphakia embryonic stem cell cultures with retinoic acid resulted in a significant increase in mesodiencephalic tyrosine hydroxylase-positive neurons, providing further support for the role of Pitx3 in dopaminergic neuron specification through the retinoic acid pathway. Our study, using Pitx3-deficient embryonic stem cells in an in-vitro differentiation culture system, allowed us to assess the role of Pitx3 in the specification and final maturation of dopaminergic neurons.

Primary mouse embryonic fibroblasts (mEFs) were cultured in a microfluidic system for a period of 8 days. The perfusion flow rate for these cells is different from those used for 3T3 fibroblasts and human adult fibroblasts due to the strong dependence on cell secreted factors. Primary mouse embryonic fibroblasts have not been cultured in a microfluidic system before. Our results have potential applications for these cells reprogrammed to a pluripotent state and important implications for primary cell culture using microdevices.

The adult subventricular zone (SVZ) supports a population of cells that display the hallmarks of stem cells: they are self-renewing and multipotent-capable of generating neurons, oligodendrocytes, and astrocytes. In vivo, these adult neural stem cells (aNSCs) are fated primarily for a gamma-amino butyric acid (GABA)-ergic lineage of olfactory bulb interneurons, a small subpopulation of which is dopaminergic. Here, we investigate the plasticity of aNSCs in vitro, in particular, their ability to generate a specific neuronal lineage, midbrain dopamine neurons. Previous work using mouse embryonic stem (ES) cells showed that introduction of early developmental inductive cues, sonic hedgehog (SHH) and fibroblast growth factor-8 (FGF-8), directed ES cell-derived neuroepithelial cells to generate midbrain dopaminergic neurons, those lost in Parkinson's disease. Placing aNSCs under similar culture conditions, immunocytochemistry and RT-PCR analysis revealed early dopaminergic neuron specification. However, aNSC-derived neurons remained morphologically immature, exhibiting concurrent nestin and tyrosine hydroxylase (TH) expression, with cell death occurring in the final differentiation stage. High-performance liquid chromatography (HPLC) analysis revealed that while aNSC-derived neurons released dopamine, release was not significantly increased following depolarization with K+. In contrast, ES cell-generated TH+ neurons expressed the mature markers MAP2 and NeuN and showed K+-evoked release of dopamine. Reduced culture time of aNSC-derived nestin+ progenitors in FGF-2-containing medium improved survival of TH+ neurons. However, these neurons exhibited characteristics of forebrain dopamine neurons and also expressed low levels of midbrain transcription factors. Together, our data indicate that when presented with in vitro conditions that promote midbrain-specific dopamine neuron specification, aNSCs instead generate forebrain-like dopamine neurons, demonstrating their restricted and prescribed nature.

The neural stem cell niche defines a zone in which stem cells are retained after embryonic development for the production of new cells of the nervous system. This continual supply of new neurons and glia then provides the postnatal and adult brain with an added capacity for cellular plasticity, albeit one that is restricted to a few specific zones within the brain. Critical to the maintenance of the stem cell niche are microenvironmental cues and cell-cell interactions that act to balance stem cell quiescence with proliferation and to direct neurogenesis versus gliogenesis lineage decisions. Ultimately, based on the location of the niche, stem cells of the adult brain support regeneration in the dentate gyrus of the hippocampus and the olfactory bulb through neuron replacement. Here, we provide a summary of the current understanding of the organization and control mechanisms of the neural stem cell niche.

The MRL mouse is unique in its capacity for regenerative healing of wounds. This regenerative ability includes complete closure, with little scarring, of wounds to the ear pinna and repair of cardiac muscle, without fibrosis, following cryoinjury. Here, we examine whether neurogenic zones within the MRL brain show enhanced regenerative capacity. The largest neurogenic zone in the adult brain, the subventricular zone (SVZ), lies adjacent to the lateral wall of the lateral ventricle and is responsible for replacement of interneuron populations within the olfactory bulb. Initial gross observation of the anterior forebrain in MRL mice revealed enlarged lateral ventricles; however, little neurodegeneration was detected within the SVZ or surrounding tissues. Instead, increased proliferation within the SVZ was observed, based on incorporation of the thymidine analogue bromodeoxyuridine. Closer examination using electron microscopy revealed that a significant number of SVZ astrocytes interpolated within the ependyma and established contact with the ventricle. In addition, subependymal, protuberant nests of cells, consisting primarily of neuroblasts, were found along the anterior SVZ of MRL mice. Whole mounts of the lateral wall of the lateral ventricle stained for the neuroblast marker doublecortin revealed normal formation of chains of migratory neuroblasts along the entire wall and introduction of enhanced green fluorescent protein-tagged retrovirus into the lateral ventricles confirmed that newly generated neuroblasts were able to track into the olfactory bulb. J. Comp. Neurol. 498: 747‐761, 2006. © 2006 Wiley-Liss, Inc. Indexing terms: neurogenesis; neural stem cell; neuroblasts; regeneration; migration

In the adult mouse brain, the subventricular zone (SVZ) is a neurogenic stem cell niche only 4-5 cell diameters thick. Within this narrow zone, a unique microenvironment supports stem cell self-renewal, gliogenesis or neurogenesis lineage decisions and tangential migration of newly generated neurons out of the SVZ and into the olfactory bulb. However, with aging, SVZ neurogenesis declines. Here, we examine the dynamic interplay between SVZ cytoarchitecture and neurogenesis through aging. Assembly of high-resolution electron microscopy images of corresponding coronal sections from 2-, 10- and 22-month-old mice into photomontages reveal a thinning of the SVZ with age. Following a 2-h BrdU pulse, we detect a significant decrease in cell proliferation from 2 to 22 months. Neuroblast numbers decrease with age, as do transitory amplifying progenitor cells, while both SVZ astrocytes and adjacent ependymal cells remain relatively constant. At 22 months, only residual pockets of neurogenesis remain and neuroblasts become restricted to the anterior dorsolateral horn of the SVZ. Within this dorsolateral zone many key components of the younger neurogenic niche are maintained; however, in the aged SVZ, increased numbers of SVZ astrocytes are found interposed within the ependyma. These astrocytes co-label with markers to ependymal cells and astrocytes, form intercellular adherens junctions with neighboring ependymal cells, and some possess multiple basal bodies of cilia within their cytoplasm. Together, these data reveal an age-related, progressive restriction of SVZ neurogenesis to the dorsolateral aspect of the lateral ventricle with increased numbers of SVZ astrocytes interpolated within the ependyma.

The subventricular zone (SVZ), which lines the lateral walls of the lateral ventricle, persists as a neurogenic zone into adulthood and functions as the largest site of neurogenesis in the adult brain. In recent years, with the acceptance of the concept of postembryonic mammalian neurogenesis, neurogenesis in the adult SVZ has been an area of active research. With the rapid accumulation of new information on the SVZ, some of which is contradictory, summarizing existing knowledge on the SVZ and outlining future research directions in this area become important. In this review, we will cover recent molecular and cellular investigations that characterize the SVZ niche, SVZ neurogenesis, and SVZ cell migration within the adult brain.

The subventricular zone (SVZ) of the lateral ventricles, the largest remaining germinal zone of the adult mammalian brain, contains an extensive network of neuroblasts migrating rostrally to the olfactory bulb. Little is known about the endogenous proliferation signals for SVZ neural stem cells or guidance cues along the migration pathway. Here we show that the receptor tyrosine kinases EphB1-3 and EphA4 and their transmembrane ligands, ephrins-B2/3, are expressed by cells of the SVZ. Electron microscopy revealed ephrin-B ligands associated with SVZ astrocytes, which function as stem cells in this germinal zone. A three-day infusion of the ectodomain of either EphB2 or ephrin-B2 into the lateral ventricle disrupted migration of neuroblasts and increased cell proliferation. These results suggest that Eph/ephrin signaling is involved in the migration of neuroblasts in the adult SVZ and in either direct or indirect regulation of cell proliferation.

The neurotrophins, NGF, BDNF, NT3 and NT4, are one family in a growing repertoire of neurotrophic factors. The neurotrophins have long been implicated in neuronal survival and recent studies from mice with targeted disruptions of the neurotrophin genes confirm this role, but also reveal that the action of the neurotrophins is more complex, and in some instances more interactive, than originally envisaged. Lack of functional NGF, BDNF and NT3 genes results in severe neuronal deficits and an early postnatal death. However, NT4 is unique among the neurotrophins and while the absence of NT4 does result in limited sensory neuron loss these mice do not die early, suggesting that NT4-dependent neurons are not critical for survival. Phenotypic analyses of mice lacking neurotrophin receptors, TrkA, B and C, confirm that TrkA is the functional receptor for NGF, TrkB acts as the primary receptor for BDNF and NT4, and NT3 signals primarily through TrkC. However, the finding that TrkC mutant mice have a less dramatic phenotype than their NT3 counterparts implicates NT3 in signaling via receptors other than TrkC. Further studies, using combinatorial Trk and neurotrophin deletions, reveal that while BDNF and NT4 subserve distinct neuron populations in most cases, other neuron sub-populations can be supported by either BDNF or NT4, providing evidence for compensatory actions between neurotrophins. As a mechanism to explain programmed cell death that occurs in the developing nervous system, recent studies examining neurotrophin gene-dosage effects suggest that the availability of neurotrophins, NGF, BDNF and NT3, may be limiting for some neuron populations. In addition, the proposed switch in neurotrophin dependency for some neuron populations is now being determined using neurotrophin mutant mice. We discuss these and other recent findings on neurotrophin requirements for the developing nervous system.

The neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT4) act via the TrkB receptor and support survival of primary somatic and visceral sensory neurons. The major visceral sensory population, the nodose–petrosal ganglion complex (NPG), requires BDNFandNT4 for survival of a full complement of neurons, providing a unique opportunity to compare gene dosage effects between the two TrkB ligands and to explore the possibility that one ligand can compensate for loss of the other. Analysis of newborn transgenic mice lacking BDNF or NT4, or BDNFandNT4, revealed that survival of many NPG afferents is proportional to the number of functional BDNF alleles, whereas onlyonefunctional NT4 allele is required to support survival of all NT4-dependent neurons. In addition, subpopulation analysis revealed that BDNF and NT4 can compensate for the loss of the other to support a subset of dopaminergic ganglion cells. Together, these data demonstrate that the pattern of neuronal dependencies on BDNF and NT4in vivois far more heterogeneous than predicted from previous studies in culture. Moreover, BDNF knockout animals lack a subset of afferents involved in ventilatory control and exhibit severe respiratory abnormalities characterized by depressed and irregular breathing and reduced chemosensory drive. BDNF is therefore required for expression of normal respiratory behavior in newborn animals.

During the initial phase of their development, sensory neurons of the dorsal root ganglion (DRG) require target-derived trophic support for their survival, but as they mature they lose this requirement. Because many of these neurons express BDNF (brain-derived neurotrophic factor) messenger RNA, we hypothesized that BDNF might act as an autocrine survival factor in adult DRG neurons, thus explaining their lack of dependence on exogenous growth factors. When cultured adult DRG cells were treated with antisense oligonucleotides to BDNF, expression of BDNF protein was reduced by 80%, and neuronal survival was reduced by 35%. These neurons could be rescued by exogenous BDNF or neurotrophin-3, but not by other growth factors. Similar results were obtained with single-neuron microcultures, whereas microcultures derived from mutant mice lacking BDNF were unaffected by antisense oligonucleotides. Our results strongly support an autocrine role for BDNF in mediating the survival of a subpopulation of adult DRG neurons.

Ciliary neurotrophic factor was discovered based on its ability to support the survival of ciliary neurons, and is now known to act on a variety of neuronal and glial populations. Two distant relatives of ciliary neurotrophic factor, leukemia inhibitory factor and oncostatin M, mimic ciliary neurotrophic factor with respect to its actions on cells of the nervous system. In contrast to ciliary neurotrophic factor, leukemia inhibitory factor and oncostatin M also display a broad array of actions on cells outside of the nervous system. The overlapping activities of leukemia inhibitory factor, oncostatin M and ciliary neurotrophic factor can be attributed to shared receptor components. The specificity of ciliary neurotrophic factor for cells of the nervous system results from the restricted expression of the alpha component of the ciliary neurotrophic factor receptor complex, which is required to convert a functional leukemia inhibitory factor/oncostatin M receptor complex into a ciliary neurotrophic factor receptor complex. The recent observation that the alpha component of the ciliary neurotrophic factor receptor complex is expressed by very early neuronal precursors suggested that ciliary neurotrophic factor may act on even earlier precursors, particularly on cells previously thought to be targets for leukemia inhibitory factor action. Here we show the first example of ciliary neurotrophic factor responsiveness in cells residing outside of the nervous system by demonstrating that embryonic stem cells express a functional ciliary neurotrophic factor receptor complex, and that ciliary neurotrophic factor is similar to leukemia inhibitory factor in its ability to maintain the pluripotentiality of these cells.

The respective amino acid sequences of mature brain-derived neurotrophic factor (BDNF) and of mature neurotrophin-3 (NT-3) are identical among mammals, making these among the structurally conserved factors known. Here we show that only a single conservative amino acid substitution distinguishes the chicken mature NT-3 protein from its mammalian counterpart. Chicken mature BDNF shows slightly more variation, differing from mammalian BDNF at several positions. We also note the presence of amino acid sequence motifs in the precursor protein sequences of chicken BDNF and NT-3 that are universally conserved among all known mammalian neurotrophin precursors and have been demonstrated to play a crucial role in promoting correct processing of the pro-proteins.

Transcriptional activation of the embryonic genome occurs during the two-cell stage in the mouse embryo and is marked by the synthesis of a set of α-amanitin-sensitive proteins of Mr 73,000, 70,000, and 68,000. We have characterized these three proteins by two-dimensional gel electrophoresis of [35S]methionine radiolabeled two-cell embryos. Their isoelectric points range from 6.2 to 6.8 and their synthesis, which can constitute 5–10% of total protein synthesis, is restricted to the two-cell stage. These proteins are not heat shock proteins that have previously been reported as major products of transcriptional activation. Peptide mapping by limited proteolysis indicates that these three proteins are highly related to one another and the results of pulse-chase experiments indicate that they are likely to be degraded by the eight-cell stage. These proteins are nuclear-associated and insoluble in 2% Triton X-100/0.3 M KCl. Although these proteins share some features with somatic lamins—they exhibit solubility properties similar to somatic lamins—they do not cross-react with polyclonal antibodies to either lamins A C or B, nor do they comigrate with somatic lamins on two-dimensional gels. Additional evidence that these proteins are not lamins is that although treatment of two-cell embryos with okadaic acid, which is an inhibitor of protein phosphatases 1 and 2A, results in precocious nuclear envelope breakdown, the proteins remain insoluble in 2% Triton X-100/0.3 M KCl.

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.

In contrast to Whitten's medium, CZB medium, which lacks glucose but is supplemented with glutamine, supports development of 1-cell embryos beyond the 2-cell stage for embryos obtained from mice that exhibit the 2-cell block. The molecular basis for this effect, however, is not known. We report that correlated with the ability of CZB medium to support development beyond the 2-cell stage is an enhanced rate of total protein synthesis and higher level of synthesis of proteins that reflect activation of transcription of the embryonic genome when compared to embryos cultured in Whitten's medium +/- ethylenediaminetetraacetate (EDTA). The overall patterns of protein synthesis of embryos cultured in CZB or Whitten's medium +/- EDTA are similar, although the synthesis of the transcription-dependent proteins is markedly delayed in embryos cultured in Whitten's-EDTA. Last, there are no significant differences in the adenosine 5'-triphosphate (ATP) levels in 2-cell embryos cultured from the 1-cell stage in each of the media.

Failure of epididymal spermatozoa from T/t mutant mice, but not from t/t individuals, to fertilize oocytes in vitro was partially overcome by opening a small aperture in the zona pellucida with acidified Tyrode's solution to permit direct access of the spermatozoon to the vitellus. This study provides a model system to evaluate requirements for successful zona drilling in the treatment of human infertility and further insights into the effects of the t complex on sperm fertility.

The degree and normality of nuclear maturation were assessed with the fluorochrome Hoechst 33342 in two groups of inseminated human oocytes that had failed to undergo fertilization. Group 1 consisted of 67 oocytes from 27 patients, each of whom had at least two other oocytes that had been fertilized and had cleaved. Group 2 consisted of 65 oocytes from 14 patients, none of whose oocytes had been fertilized. In group 1, 52.3% of the oocytes were found to be immature (germinal vesicle stage or metaphase-telophase l), whereas in group 2 only 26% were found to be immature. Thus oocyte nuclear immaturity was the major cause of fertilization failure when companion oocytes were fertilized. When no oocytes of a patient were fertilized, most cocytes were found to be mature, so other factors, such as sperm dysfunction or zona binding abnormalities, must account for most of the fertilization failure in this group of patients.