The adult mouse dentate gyrus contains populations of committed progenitor cells that are distinct from subependymal zone neural stem cells

There is currently a debate as to whether or not a neural stem cell (NSC) exists in the adult mammalian hippocampus. Clonal colony-forming assays allow single cells to cells to be evaluated for stem cell properties: self-renewal and multipotentiality. In these in vitro assays, single cells from the subependymal zone (SEZ) of the adult lateral ventricle yield large colonies which self-renew and are multipotential, while single cells from the adult dentate gyrus (DG) produce small, unipotent, and nonself-renewing colonies. We find that multipotential and long-term self-renewing colonies can be isolated only from the early embryonic hippocampus, before the formation of the DG. No movement of progenitors from the postnatal SEZ to the newly forming DG subgranular zone is detected and adult DG colonies in vitro originate from the embryonic hippocampal primordium. These data support a model where embryonic hippocampal NSCs change their properties as the organism ages. When adult DG spheres are cocultured with embryonic brain slices, self-renewal (but not multipotentiality) is restored and maintained for several passages off of slices. Adult clonal DG spheres grown on embryonic brain slices or transplanted into brains of neonatal mice do not give rise to neurons. Neurons arise from separate, small clones that are approximately 10 times more frequent than sphere colonies in vitro and may be responsible for maintaining neurogenesis in the adult in vivo. We propose that there are separate glial and neuronal clones in the adult hippocampus, with glial progenitors being the most proliferative in culture.     

Effect of canonical Wnt inhibition in the neurogenic cortex, hippocampus, and premigratory dentate gyrus progenitor pool.

Canonical Wnt signaling is crucial for the correct development of both cortical and hippocampal structures in the dorsal telencephalon. In this study, we examined the role of the canonical Wnt signaling in the dorsal telencephalon of mouse embryos at defined time periods by inhibition of the pathway with ectopic expression of Dkk1. Transgenic mice with the D6-driven Dkk1 gene exhibited reduced canonical Wnt signaling in the cortex and hippocampus. As a result, all hippocampal fields were reduced in size. Neurogenesis in the dentate gyrus was severely reduced both in the premigratory and migratory progenitor pool. The lower number of progenitors in the dentate gyrus was not rescued after migration to the subgranular zone and thus the dentate gyrus lacked the entire internal blade and a part of the external blade from postnatal to adult stages.     

The persistent expression of a highly polysialylated NCAM in the dentate gyrus of the adult rat.

The expression of a highly polysialylated form of the neural cell adhesion molecule (NCAM-H), often termed 'embryonic NCAM', has been investigated in the hippocampal formation of developing and adult rats. To determine the immunohistochemical localization of NCAM-H, a monoclonal antibody that reacts with the polysialic acid portion of NCAM-H was used. In the late embryonic and early postnatal periods, immunoreactivity for NCAM-H was found throughout the hippocampal formation, except for the ventricular layer. Thereafter, the immunoreactivity gradually decreased and almost vanished in most parts in the adult. However, a strong immunoreactivity remained on a number of cells in the dentate gyrus of adult rats, particularly in the deepest part of the granular layer. The immunoreactive arborized dendrites, mostly arising from the primary apical pole of the granule cells, were found to enter the molecular layer. The mossy fibers also were positive. Electron-microscopic examination of the hilus portion showed that the immunoreactivity was detected on the plasma membrane of some axons in the mossy fiber bundles. Since postnatal neurogenesis is known to continue into adulthood in the deepest part of the granule cell layer of the dentate gyrus, these results suggest that, in the adult dentate gyrus, NCAM-H is expressed by newly generated granule cells, and that the NCAM-H-expressing new cells may participate in the formation of new neural circuits.     

Characterization of primary cilia and Hedgehog signaling during development of the human pancreas and in human pancreatic duct cancer cell lines

Hedgehog (Hh) signaling controls pancreatic development and homeostasis; aberrant Hh signaling is associated with several pancreatic diseases. Here we investigated the link between Hh signaling and primary cilia in the human developing pancreatic ducts and in cultures of human pancreatic duct adenocarcinoma cell lines, PANC-1 and CFPAC-1. We show that the onset of Hh signaling from human embryogenesis to fetal development is associated with accumulation of Hh signaling components Smo and Gli2 in duct primary cilia and a reduction of Gli3 in the duct epithelium. Smo, Ptc, and Gli2 localized to primary cilia of PANC-1 and CFPAC-1 cells, which may maintain high levels of nonstimulated Hh pathway activity. These findings indicate that primary cilia are involved in pancreatic development and postnatal tissue homeostasis.     

Neuron type-selective effects of activin on development of the hippocampus

Activin is a member of the transforming growth factor-β superfamily and affects the viability of hippocampal neurons during postnatal neurogenesis. We used primary hippocampal neuron to study the actions of activin on developing neurons. Continuous treatment of hippocampal cultures with activin suppressed the emergence of GAD67⁺ neurons, which are a subtype of GABAergic interneurons, and increased the percentage of Prox1⁺ neurons, which are dentate granule cells. The effects of activin were abolished by co-treatment with follistatin, which is a direct inhibitor of activin. In contrast, follistatin treatment alone increased the percentage of GAD67⁺ neurons and decreased the percentage of Prox1⁺ neurons. These results indicate that changes in activin signaling during postnatal neural development alter the composition of the neural circuitry and suggest that alterations in the ratio of excitatory to inhibitory neurons may be responsible for changes in the spontaneous and evoked-reactivity of these neurons to other neural inputs.     

Primary cilium and brain aging: role in neural stem cells,neurodegenerative diseases and glioblastoma

Brain aging is characterized by a progressive loss of tissue integrity and function as a consequence of impaired homeostasis and regeneration capacities. The primary cilium is a highly conserved organelle that projects from the cell surface in a single copy in virtually all mammalian cell types including neural stem/progenitors cells and neurons. Increasing evidence in the last decade points out that primary cilium could be a relevant mediator of neural stem cell activity, neurogenesis, neuronal maturation and maintenance, and brain tumorigenesis. In this review, we summarize the current knowledge about primary cilia roles in these processes. There is currently sufficient background to propose that defective primary cilia contribute to age-related cognitive decline and brain tumor development due to their critical roles in cell cycle control and signaling transduction. This might have potential applications on therapy against age-associated brain diseases.     

Cell Cycle Regulation During Neurogenesis in the Embryonic and Adult Brain

Throughout the development of the central nervous system, neural crest cells and the primary neural stem cells originate several non-neuronal and neuronal cell types. Undifferentiated stem cells exist in the adult brain, mainly in the dentate gyrus of the hippocampus and in the subventricular zone of the lateral ventricles, and can produce new neurons, participating in brain plasticity and tissue regeneration. Neurogenesis in the embryonic and adult brain occurs under the control of intrinsic and extrinsic factors. However, the mechanisms, by which cell cycle components control neural stem cell proliferation and consequently neurogenesis, still lack further investigation. We discuss here recent knowledge obtained on cell cycle components as key regulators of neural stem and progenitor cell proliferation and differentiation in the embryonic and adult brain.     

Development of full-length Trk B-immunoreactive structures in the hippocampal formation of the macaque monkey

Distribution and morphological changes of cells containing the signal transducing neurotrophin receptor, full-length Trk B (fl-Trk B), were investigated in the hippocampal formation of the macaque monkey between embryonic day 140 and the adult stage. Western blot analysis showed that one main protein band, which migrated at 141 kDa, was detected in both the embryonic and adult hippocampal formation. In the pyramidal cells in CA1 and CA3 subfields, the subiculum, and the entorhinal cortex, fl-Trk B-immunoreactive dendrites were observable in the embryonic stage. In contrast, in the granule cells of the dentate gyrus, few dendrites were immunoreactive during embryonic and early developmental stages. This difference may be due to the later growth of the granule cells of the dentate gyrus. The existence of fl-Trk B immunoreactivity in the cell body and dendrites in the embryonic hippocampal neurons, suggests that BDNF and/or NT4/5 act on the hippocampal cells by autocrine/paracrine mechanisms. In the entorhinal cortex, fl-Trk B immunoreactivity became localized in the stellate cells in layer II and the pyramidal cells in layers III, V and VI in adulthood. This indicates that BDNF and/or NT4/5 are important for the maintenance of the projection neurons in the entorhinal cortex at the adult stage. The strongest fl-Trk B immunoreactivity in the hippocampal neurons occurred at postnatal month 4, corresponding to the period of greatest synapse production in the monkey hippocampus, suggesting that BDNF and/or NT4/5 with fl-Trk B may play a role in synapse formation in the monkey hippocampus.     

Selective upregulation of 3-phosphoglycerate dehydrogenase (Phgdh) expression in adult subventricular zone neurogenic niche

In the adult rodent brain, constitutive neurogenesis occurs in two restricted regions, the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus, where multipotent neural stem/progenitor cells generate new neurons. Using Western blotting and immunohistochemistry for established markers, we demonstrated that the expression of 3-phosphoglycerate dehydrogenase (Phgdh), an enzyme involved in de novo synthesis of l-serine, was upregulated in the SVZ. The expression was selective to cells having morphological features and expressing markers of astrocyte-like primary neural stem cells (type B cells) and their progeny, actively proliferating progenitors (type C cells). By contrast, Phgdh protein expression was virtually absent in committed neuronal precursors (type A cells) derived from type C cells. High levels of Phgdh were also expressed by glial tube cells located in the rostral migratory stream (RMS). Interestingly, ensheathment of type A cells by these Phgdh-expressing cells was persistent in the SVZ and RMS, suggesting that l-serine mediates trophic support for type A cells via these glial cells. In vitro neurosphere assays confirmed that growth-factor-responsive, transient amplifying neural progenitors in the SVZ, but not differentiated neurons, expressed Phgdh. In the aged brain, a decline in Phgdh expression was evident in type B and C cells of the SVZ. These observations support the notion that availability of l-serine within neural stem/progenitor cells may be a critical factor for neurogenesis in developing and adult brain.     

Transient expression of phospholipase D1 in developing rat hippocampus

We investigated the distribution of phospholipase D1 (PLD1) protein in the developing rat hippocampus using an affinity-purified peptide antibody against PLD1. Immunoreactivity for PLD1 was first seen in some scattered cells in the hippocampus at embryonic day 18. At postnatal day 1 (P1), many PLD1 immunoreactive cells were observed in the CA1 and CA3 sectors, subiculum and the hilus of the dentate gyrus. During the first postnatal week, there was an abrupt increase of immunoreactive neurons in the hippocampus, and their number and intensity peaked at P7. During the second postnatal week, there was an abrupt decrease in the number of immunoreactive hippocampal neurons. By P14, no significant labeling was found in the hippocampus. These results corresponded well with those from Western blot analysis, suggesting that PLD1 may regulate the developmental processes of hippocampal neurons.     

Increased dentate neurogenesis after grafting of glial restricted progenitors or neural stem cells in the aging hippocampus

Neurogenesis in the dentate gyrus (DG) declines severely by middle age, potentially because of age-related changes in the DG microenvironment. We hypothesize that providing fresh glial restricted progenitors (GRPs) or neural stem cells (NSCs) to the aging hippocampus via grafting enriches the DG microenvironment and thereby stimulates the production of new granule cells from endogenous NSCs. The GRPs isolated from the spinal cords of embryonic day 13.5 transgenic F344 rats expressing human alkaline phosphatase gene and NSCs isolated from embryonic day 9 caudal neural tubes of Sox-2:EGFP transgenic mice were expanded in vitro and grafted into the hippocampi of middle-aged (12 months old) F344 rats. Both types of grafts survived well, and grafted NSCs in addition migrated to all layers of the hippocampus. Phenotypic characterization revealed that both GRPs and NSCs differentiated predominantly into astrocytes and oligodendrocytic progenitors. Neuronal differentiation of graft-derived cells was mostly absent except in the dentate subgranular zone (SGZ), where some of the migrated NSCs but not GRPs differentiated into neurons. Analyses of the numbers of newly born neurons in the DG using 5'-bromodeoxyuridine and/or doublecortin assays, however, demonstrated considerably increased dentate neurogenesis in animals receiving grafts of GRPs or NSCs in comparison with both na?ve controls and animals receiving sham-grafting surgery. Thus, both GRPs and NSCs survive well, differentiate predominantly into glia, and stimulate the endogenous NSCs in the SGZ to produce more new dentate granule cells following grafting into the aging hippocampus. Grafting of GRPs or NSCs therefore provides an attractive approach for improving neurogenesis in the aging hippocampus. Disclosure of potential conflicts of interest is found at the end of this article.     

Characterization of adult neural stem cells and their relation to brain tumors

The adult mammalian brain contains neural stem cells that are capable of generating new neurons and glia over the course of a lifetime. Neural stem cells reside in 2 germinal niches, the subventricular zone (SVZ) and the dentate gyrus subgranular zone. These primary progenitors have been identified in their niche in vivo; these cells have characteristics of astrocytes. Recent studies have shown that adult SVZ stem cells are derived from radial glia, the stem cells in the developing brain, which in turn are derived from the neuroepithelum, the earliest brain progenitors. Thus, SVZ stem cells are a continuum from neuroepithelium to radial glia to astrocytes, and are contained within what has been considered the lineage for astrocytes. However, it seems that only a small subset of the astrocytes present in the adult brain have stem cell properties. Recent findings have shown that SVZ stem cell astrocytes express a receptor for platelet-derived growth factor (PDGF), suggesting that the ability to respond to specific growth factor stimuli, such as PDGF, epidermal growth factor and others, may be unique to these stem cell astrocytes. Intriguingly, activation of these same signaling pathways is widely implicated in brain tumor formation. Since the adult brain has very few proliferating cells capable of accumulating the numerous mutations required for transformation, the adult neural stem and/or progenitor cells may be likely candidates for the brain tumor cell of origin. Indeed, activation of the PDGF or epidermal growth factor pathways in adult neural stem or progenitor cells confers tumor-like properties on these cells, lending support to this hypothesis.     

GFAP-expressing progenitors are the principal source of constitutive neurogenesis in adult mouse forebrain

Establishing the cellular identity in vivo of adult multipotent neural progenitors is fundamental to understanding their biology. We used two transgenic strategies to determine the relative contribution of glial fibrillary acidic protein (GFAP)-expressing progenitors to constitutive neurogenesis in the adult forebrain. Transgenically targeted ablation of dividing GFAP-expressing cells in the adult mouse subependymal and subgranular zones stopped the generation of immunohistochemically identified neuroblasts and new neurons in the olfactory bulb and the hippocampal dentate gyrus. Transgenically targeted cell fate mapping showed that essentially all neuroblasts and neurons newly generated in the adult mouse forebrain in vivo, and in adult multipotent neurospheres in vitro, derived from progenitors that expressed GFAP. Constitutively dividing GFAP-expressing progenitors showed predominantly bipolar or unipolar morphologies with significantly fewer processes than non-neurogenic multipolar astrocytes. These findings identify morphologically distinctive GFAP-expressing progenitor cells as the predominant sources of constitutive adult neurogenesis, and provide new methods for manipulating and investigating these cells.     

Cell formation in the human hippocampal formation from mid-gestation to the late postnatal period

In the present study cell formation was studied in the human hippocampal formation from the 24th gestational week until the end of the first postnatal year. Proliferating cells were detected with the monoclonal antibody MIB-1.The cytoarchitectonic layers of Ammon's horn are formed before the 24th gestational week. In harmony with this observation, cell proliferation in the hippocampal ventricular zone is minimal after the 24th week. In addition, local cell multiplication in Ammon's horn is occasional and the proliferating cells are glial or endothelial cells. In contrast, cell formation continues in the hilar region of the dentate gyrus even after birth. Immature cells accumulate in the hilus, and at the border between the hilus and the granule cell layer throughout the first eight postnatal months. The subgranular zone of the dentate gyrus becomes a cell sparse area at about the 11th postnatal month, indicating that immature cells from the hilus have already migrated to the granule cell layer and differentiated into granule cells. There is an increase in glial cell proliferation both in Ammon's horn and the dentate gyrus at the 11.5th postnatal month suggesting the onset of myelination by the end of the first year.Our findings indicate that most pyramidal neurons of Ammon's horn are generated in the first half of pregnancy and no pyramidal neurons are formed after the 24th gestational week. In contrast, granule cells of the dentate gyrus proliferate in a decreasing rate during the second half of pregnancy and after birth. Proliferating neuronal precursors occur in a low percentage in the dentate gyrus of 3-, 5- and 11.5-month-old children.     

Aging and neuronal replacement

Neural stem cells contribute to neurogenesis in both the embryonic and adult brain. However, while adult neural stem cells produce new neurons that populate the olfactory bulb and the granule cell layer of the hippocampus, they do not normally participate in reparative neurogenesis following injury or disease affecting regions distant from the subventricular zone or the dentate gyrus. Here we review differences between neural stem cells found in the embryo and the adult, and describe factors that enhance neuronal output from these cells in vivo. Additionally, we review evidence that neural stem cells can be transplanted into injured regions of the adult brain to enhance compensatory neurogenesis from endogenous precursors. Pre-differentiation of neural stem cells into immature neurons prior to transplantation can also aid in functional recovery following injury or disease.     

Forebrain-specific promoter/enhancer D6 derived from the mouse Dach1 gene controls expression in neural stem cells

Drosophila dachshund is involved in development of eye and limbs and in the development of mushroom bodies, a brain structure required for learning and memory in flies. Its mouse homologue mDach1 is expressed in various embryonic tissues, including limbs, the eye, the dorsal spinal cord and the forebrain. We have isolated a forebrain-specific 2.5-kb enhancer element termed D6 from the mouse mDach1 gene and created D6-LacZ and D6-green fluorescent protein (GFP) reporter gene mouse lines. In embryonic stages, the D6 enhancer activity is first detected at embryonic day 10.5 in scattered cells of the outbuldging cortical vesicles. By embryonic day 12.5, D6 activity expands throughout the developing neocortex and the hippocampus. In the adult mouse brain, D6 enhancer is active in neurons of the cortical plate, in the CA1 layer of the hippocampus and in cells of the subventricular zone and the ventricular ependymal zone. Adult mice also show D6 activity in the olfactory bulb and in the mamillary nucleus. Cultured D6-positive cells, which were derived from embryonic and postnatal brains, show characteristics of neural stem cells. They form primary and secondary neurospheres that differentiate into neurons and astrocytes as examined by cell-specific markers.Our results show that D6 enhancer exerts highly tissue-specific activity in the neurons of the neocortex and hippocampus and in neural stem cells. Moreover, the fluorescence cell sorting of D6-GFP cells from embryonic and postnatal stages allows specific selection of primary neural progenitors and their analysis.     

Adult neurogenesis and the olfactory system

Though initially described in the early 1960s, it is only within the past decade that the concept of continuing adult neurogenesis has gained widespread acceptance. Neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) into the olfactory bulb, where they differentiate into interneurons. Neuroblasts from the subgranular zone (SGZ) of the hippocampal formation show relatively little migratory behavior, and differentiate into dentate gyrus granule cells. In sharp contrast to embryonic and perinatal development, these newly differentiated neurons must integrate into a fully functional circuit, without disrupting ongoing performance. Here, after a brief historical overview and introduction to olfactory circuitry, we review recent advances in the biology of neural stem cells, mechanisms of migration in the RMS and olfactory bulb, differentiation and survival of new neurons, and finally mechanisms of synaptic integration. Our primary focus is on the olfactory system, but we also contrast the events occurring there with those in the hippocampal formation. Although both SVZ and SGZ neurogenesis are involved in some types of learning, their full functional significance remains unclear. Since both systems offer models of integration of new neuroblasts, there is immense interest in using neural stem cells to replace neurons lost in injury or disease. Though many questions remain unanswered, new insights appear daily about adult neurogenesis, regulatory mechanisms, and the fates of the progeny. We discuss here some of the central features of these advances, as well as speculate on future research directions.     

Monoaminergic regulation of Sonic hedgehog signaling cascade expression in the adult rat hippocampus

Monoamines are implicated in the modulation of adult hippocampal neurogenesis in depression models and following chronic antidepressant treatment. Given the key role of Sonic hedgehog (Shh) in adult neurogenesis, we examined whether monoaminergic perturbations regulate the expression of Shh or its co-receptors Smoothened (Smo) and Patched (Ptc). Combined depletion of both serotonin and norepinephrine with para-chlorophenylalanine (PCPA) resulted in a significant decrease in Smo and Ptc mRNA within the dentate gyrus subfield of the hippocampus. However, selective depletion of serotonin, using the serotonergic neurotoxin 5,7-dihyrdroxytryptamine (5,7-DHT), or norepinephrine, using the noradrenergic neurotoxin DSP-4, did not alter expression of Shh and its co-receptors, Smo and Ptc. Acute treatment with the monoamine releasing agent, para-chloroamphetamine (PCA) significantly upregulated Smo mRNA within the dentate gyrus. However, acute or chronic treatment with pharmacological antidepressants that modulate monoaminergic neurotransmission did not regulate Shh cascade expression. These results indicate that robust changes in monoamine levels can regulate the expression of the Shh signaling cascade in the adult rodent brain.     

A role for primary cilia in glutamatergic synaptic integration of adult-born neurons

The sequential synaptic integration of adult-born neurons has been widely examined in rodents, but the mechanisms regulating the integration remain largely unknown. The primary cilium, a microtubule-based signaling center, is essential for vertebrate development, including the development of the CNS. We examined the assembly and function of the primary cilium in the synaptic integration of adult-born mouse hippocampal neurons. Primary cilia were absent in young adult-born neurons, but assembled precisely at the stage when newborn neurons approach their final destination, further extend dendrites and form synapses with entorhinal cortical projections. Conditional deletion of cilia from adult-born neurons induced severe defects in dendritic refinement and synapse formation. Deletion of primary cilia led to enhanced Wnt and β-catenin signaling, which may account for these developmental defects. Taken together, our findings identify the assembly of primary cilia as a critical regulatory event in the dendritic refinement and synaptic integration of adult-born neurons.