Ultrastructure of the subventricular zone in Macaca fascicularis and evidence of a mouse‐like migratory stream

Recent publications have shown that the lateral wall of the lateral ventricles in the Macaca fascicularis brain, in particular the subventricular zone (SVZ), contains neural stem cells throughout adulthood that migrate through a migratory pathway (RMS) to the olfactory bulb (OB). To date, a detailed and systematic cytoarchitectural and ultrastructural study of the monkey SVZ and RMS has not been done. We found that the organization of the SVZ was similar to that of humans, with the ependymal layer surrounding the lateral ventricles, a hypocellular GAP layer formed by astrocytic and ependymal expansions, and the astrocyte ribbon, composed of astrocytic bodies. We found no cells corresponding to the type C proliferating precursor of the rodent brain. Instead, proliferating cells, expressed as Ki‐67 immunoreactivity, were predominantly young neurons concentrated in the anterior regions, and occasional astrocytes of the ribbon. We observed displaced ependymal cells of still unknown significance. New neurons tended to organize in chain‐like structures, which were surrounded by astrocytes. This pattern was highly reminiscent of that observed in rodent RMS, but not in humans. These chains spread from the frontal SVZ along a GAP‐like layer, uniquely composed of astrocytic expansions, to the olfactory bulb (OB). The number of neuronal chains and the number of chain‐forming cells decreased gradually upon reaching the OB. The purpose of this work is to provide a reference for future studies in the field of adult neurogenesis that may lead to an understanding of the fate and functionality of newborn neurons in primates, and ultimately in humans. J. Comp. Neurol. 514:533–554, 2009. © 2009 Wiley‐Liss, Inc.     

NG2 cells are distinct from neurogenic cells in the postnatal mouse subventricular zone

NG2 cells express the chondroitin sulfate proteoglycan NG2 and are a fourth type of glia distinct from astrocytes, oligodendrocytes, and microglia. NG2 cells generate oligodendrocytes but have also been reported to represent neuronal progenitor cells in the postnatal mouse subventricular zone (SVZ). We performed a detailed immunohistochemical analysis of NG2 cells in the mouse SVZ, rostral migratory stream (RMS), and olfactory bulb granule cell layer (OB GCL), which constitute a neurogenic niche in the postnatal forebrain. NG2 cells in the SVZ and RMS expressed the oligodendrocyte precursor cell antigen platelet‐derived growth factor receptor‐α but did not express antigens known to be expressed by neuronogenic cells in the SVZ, such as doublecortin, PSA‐NCAM, beta‐tubulin, Dlx2, or GFAP. More than 99.5% of the proliferating cells in the SVZ were NG2 negative. In the olfactory bulb, NG2 cells were found to generate primarily oligodendrocytes and a small number of astrocytes but not neurons. In the SVZ and RMS, NG2 cells were sparse and made up a much smaller fraction of the cells compared with the surrounding nonneurogenic parenchyma. Parenchymal NG2 cells were often located along the border of the SVZ and RMS. The abundance of NG2 cells increased in the distal parts of the RMS and especially in the OB GCL, where NG2 cell processes were seen in close proximity to many maturing interneurons. Our findings indicate that NG2 cells do not represent neuronal progenitor cells in the postnatal SVZ but are likely to be oligodendrocyte precursor cells. J. Comp. Neurol. 512:702–716, 2009. © 2008 Wiley‐Liss, Inc.     

Calorie restriction activates new adult born olfactory‐bulb neurones in a ghrelin‐dependent manner but acyl‐ghrelin does not enhance subventricular zone neurogenesis

The ageing and degenerating brain show deficits in neural stem/progenitor cell (NSPC) plasticity that are accompanied by impairments in olfactory discrimination. Emerging evidence suggests that the gut hormone ghrelin plays an important role in protecting neurones, promoting synaptic plasticity and increasing hippocampal neurogenesis in the adult brain. In the present study, we investigated the role of ghrelin with respect to modulating adult subventricular zone (SVZ) NSPCs that give rise to new olfactory bulb (OB) neurones. We characterised the expression of the ghrelin receptor, growth hormone secretagogue receptor (GHSR), using an immunohistochemical approach in GHSR‐eGFP reporter mice to show that GHSR is expressed in several regions, including the OB but not in the SVZ of the lateral ventricle. These data suggest that acyl‐ghrelin does not mediate a direct effect on NSPC in the SVZ. Consistent with these findings, treatment with acyl‐ghrelin or genetic silencing of GHSR did not alter NSPC proliferation within the SVZ. Similarly, using a bromodeoxyuridine pulse‐chase approach, we show that peripheral treatment of adult rats with acyl‐ghrelin did not increase the number of new adult‐born neurones in the granule cell layer of the OB. These data demonstrate that acyl‐ghrelin does not increase adult OB neurogenesis. Finally, we investigated whether elevating ghrelin indirectly, via calorie restriction (CR), regulated the activity of new adult‐born cells in the OB. Overnight CR induced c‐Fos expression in new adult‐born OB cells but not in developmentally born cells, whereas neuronal activity was absent following re‐feeding. These effects were not present in ghrelin^?/? mice, suggesting that adult‐born cells are uniquely sensitive to changes in ghrelin mediated by fasting and re‐feeding. In summary, ghrelin does not promote neurogenesis in the SVZ and OB; however, new adult‐born OB cells are activated by CR in a ghrelin‐dependent manner.     

Thrombospondin 4 deficiency in mouse impairs neuronal migration in the early postnatal and adult brain

In the post-natal rodent brain, neuronal precursors originating from the sub-ventricular zone (SVZ) migrate over a long distance along the rostral migratory stream (RMS) to eventually integrate the olfactory bulb neuronal circuitry. In order to identify new genes specifically expressed in the RMS, we have screened the Allen Brain Atlas Database. We focused our attention on Thrombospondin 4 (Thbs4), one of the 5 members of the Thrombospondin family of large, multidomain, extracellular matrix proteins. In post-natal and adult brain Thbs4 mRNA and protein are specifically expressed in the neurogenic regions, including the SVZ and along the entire RMS. RMS cells expressing Thbs4 are GFAP (Glial Fibrillary Acidic Protein) positive astrocytes. Histological analysis in both wild-type and Thbs4 knock-out mice revealed no major abnormality in the general morphology of these neurogenic regions. Nevertheless, immunostaining for doublecortin demonstrates that in Thbs4-KO, migration of newly formed neurons along the RMS is somehow impaired, with several neurons migrating out of the RMS. This is further supported by a Bromodeoxyuridine-based in vivo approach showing a decrease in the number of newly born neuronal precursors reaching the olfactory bulb, while proliferation in the SVZ is not affected compared to wild-type, both in young animals (P15) and in adults (8 to 12 weeks of age). Corroborating this observation, the number of Parvalbumin- and Calbindin-immunoreactive interneurons in the olfactory bulb is also reduced in Thbs4-KO. Together, these observations support a role for the astrocyte-secreted protein Thbs4 in the migration of newly form neurons within the RMS to the olfactory bulb.     

An Fgfr3‐iCreERT2 transgenic mouse line for studies of neural stem cells and astrocytes

The lack of markers for astrocytes, particularly gray matter astrocytes, significantly hinders research into their development and physiological properties. We previously reported that fibroblast growth factor receptor 3 (Fgfr3) is expressed by radial precursors in the ventricular zone of the embryonic neural tube and subsequently by differentiated astrocytes in gray and white matter. Here, we describe an Fgfr3‐iCreER^T2 phage artificial chromosome transgenic mouse line that allows efficient tamoxifen‐induced Cre recombination in Fgfr3‐expressing cells, including radial glial cells in the embryonic neural tube and both fibrous and protoplasmic astrocytes in the mature central nervous system. This mouse strain will therefore be useful for studies of normal astrocyte biology and their responses to CNS injury or disease. In addition, Fgfr3‐iCreER^T2 drives Cre recombination in all neurosphere‐forming stem cells in the adult spinal cord and at least 90% of those in the adult forebrain subventricular zone. We made use of this to show that there is continuous accumulation of all major interneuron subtypes in the olfactory bulb (OB) from postnatal day 50 (P50) until at least P230 (∼8 months of age). It therefore seems likely that adult‐born interneurons integrate into existing circuitry and perform long‐term functions in the adult OB. © 2010 Wiley‐Liss, Inc.     

Origin and function of olfactory bulb interneuron diversity

In adult rodents, subventricular zone (SVZ) astrocytes (B cells) function as primary progenitors in the generation of new neurons that migrate to the olfactory bulb (OB), where they differentiate into multiple types of interneurons. It has been generally considered that individual adult SVZ stem cells are capable of generating different types of neurons and glial cells. However, recent studies indicate that these adult SVZ primary progenitors are heterogeneous and predetermined to generate specific types of neurons. Surprisingly, OB interneurons are generated by stem cells not only in the walls of the lateral ventricle facing the striatum but also in the rostral migratory stream and walls of the lateral ventricle facing the cortex and the septum. SVZ B cells in different locations within this extensive germinal region generate different kinds of interneurons. General physiological characteristics of major classes of OB interneurons have begun to emerge, but the functional contribution of each subtype remains unknown. The mosaic organization of the SVZ offers a unique opportunity to understand the origin of interneuron diversity and how this assortment of neurons contributes to plasticity of postnatal olfactory circuits.     

Characterization of the Netrin/RGMa receptor neogenin in neurogenic regions of the mouse and human adult forebrain

In the adult rodent forebrain, astrocyte‐like neural stem cells reside within the subventricular zone (SVZ) and give rise to progenitors and neuroblasts, which then undergo chain migration along the rostral migratory stream (RMS) to the olfactory bulb, where they mature into fully functional interneurons. Neurogenesis also occurs in the adult human SVZ, where neural precursors similar to the rodent astrocyte‐like stem cell and neuroblast have been identified. A migratory pathway equivalent to the rodent RMS has also recently been described for the human forebrain. In the embryo, the guidance receptor neogenin and its ligands netrin‐1 and RGMa regulate important neurogenic processes, including differentiation and migration. We show in this study that neogenin is expressed on neural stem cells (B cells), progenitor cells (C cells), and neuroblasts (A cells) in the adult mouse SVZ and RMS. We also show that netrin‐1 and RGMa are ideally placed within the neurogenic niche to activate neogenin function. Moreover, we find that neogenin and RGMa are also present in the neurogenic regions of the human adult forebrain. We show that neogenin is localized to cells displaying stem cell (B cell)‐like characteristics within the adult human SVZ and RMS and that RGMa is expressed by the same or a closely apposed cell population. This study supports the hypothesis that, as in the embryo, neogenin regulates fundamental signalling pathways important for neurogenesis in the adult mouse and human forebrain. J. Comp. Neurol. 518:3237–3253, 2010. © 2010 Wiley‐Liss, Inc.     

The PROK2/PROKR2 signaling pathway is required for the migration of most olfactory bulb interneurons

Neural stem cells in the subventricular zone (SVZ) of the lateral ventricle generate new interneurons, which migrate tangentially through the rostral migratory stream (RMS) to the olfactory bulb (OB). The PROK2 (prokineticin 2) and PROKR2 (prokineticin receptor 2) signaling pathway has been identified to cause human Kallmann syndrome, a developmental disease that associates hypogonadism with anosmia (OB developmental defects). However, the identities and properties of PROK2⁺ and PROKR2⁺ cells in the SVZ-RMS-OB remain largely unknown. Here we examine the expression patterns of Prok2 and Prokr2 in the SVZ-RMS-OB using Prok2^EGFP transgenic and Prokr2^LacZ/+ knockin mice. Our results show that Prokr2 is expressed in postmitotic immature interneurons in the SVZ-RMS-OB. Prok2 is not expressed in the SVZ, but a few PROK2⁺ cells are found in the medial part of the RMS; they are not neural progenitors or migrating neuroblasts. In the OB, Prok2 is expressed in a subset of granule cells and tufted cells, but no coexpression of Prok2 and Prokr2 in the OB cells is observed. In Prok2 and Prokr2 mutant mice, severe tangential and radial migration defects of neuroblasts in the SVZ-RMS-OB result in loss of ~75% of GABAergic interneurons in the OB. These analyses demonstrate that PROK2/PROKR2 signaling is crucial for the tangential and radial migration of OB interneurons.     

Adult subventricular zone neuronal precursors continue to proliferate and migrate in the absence of the olfactory bulb.

Neurons continue to be born in the subventricular zone (SVZ) of the lateral ventricles of adult mice. These cells migrate as a network of chains through the SVZ and the rostral migratory stream (RMS) into the olfactory bulb (OB), where they differentiate into mature neurons. The OB is the only known target for these neuronal precursors. Here, we show that, after elimination of the OB, the SVZ and RMS persist and become dramatically larger. The proportion of dividing [bromodeoxyuridine (BrdU)-labeled] or dying (pyknotic or terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end-labeled) cells in the RMS was not significantly affected at 3 d or 3 weeks after bulbectomy (OBX). However, by 3 months after OBX, the percentage of BrdU-labeled cells in the RMS decreased by half and that of dying cells doubled. Surprisingly, the rostral migration of precursors continued along the RMS after OBX. This was demonstrated by focal microinjections of BrdU and grafts of SVZ cells carrying LacZ under the control of a neuron-specific promoter gene. Results indicate that the OB is not essential for proliferation and the directional migration of SVZ precursors.     

Different astroglia permissivity controls the migration of olfactory bulb interneuron precursors

The rostral migratory stream (RMS) is a well defined migratory pathway for precursors of olfactory bulb (OB) interneurons. Throughout the RMS an intense astroglial matrix surrounds the migratory cells. However, it is not clear to what extent the astroglial matrix participates in migration. Here, we have analyzed the migratory behavior of neuroblasts cultured on monolayers of astrocytes isolated from areas that are permissive (RMS and OB) and nonpermissive (cortex and adjacent cortical areas) to migration. Our results demonstrate robust neuroblast migration when RMS‐explants are cultured on OB or RMS‐astrocytes, in contrast to their behavior on astroglia derived from nonpermissive areas. These differences, mediated by astrocyte‐derived nonsoluble factors, are related to the overexpression of extracellular matrix and cell adhesion molecules, as revealed by real‐time qRT‐PCR. Our results show that astroglia heterogeneity could play a significant role in migration within the RMS and in cell detachment in the OB. © 2009 Wiley‐Liss, Inc.     

Notch signaling in astrocytes and neuroblasts of the adult subventricular zone in health and after cortical injury

The postnatal subventricular zone (SVZ) is a niche for continuous neurogenesis in the adult brain and likely plays a fundamental role in self-repair responses in neurodegenerative conditions. Maintenance of the pool of neural stem cells within this area depends on cell-cell communication such as that provided by the Notch signaling pathway. Notch1 receptor mRNA has been found distributed in different areas of the postnatal brain including the SVZ. Although the identity of Notch1-expressing cells has been established in the majority of these areas, it is still unclear what cell types within the SVZ are expressing components of this pathway. Here we demonstrate that most of expression of Notch1 in the adult SVZ occurs in polysialylated neural cell adhesion molecule (PSA-NCAM)-positive neural precursors and in glial fibrillary acidic protein-positive SVZ astrocytes. Notch1 was also found in PSA-NCAM-positive neuroblasts located within the rostral migratory stream (RMS) but much less in those that have reached the olfactory bulb. We show that two of the naturally occurring Notch1 activators, Jagged1 and Delta1, are also expressed in the SVZ and within the RMS in the adult mouse brain. Finally, using a model of cortical stab wound, we show that the astrogliogenic response of the SVZ to injury is accompanied by activation of the Notch pathway.     

Fasting induces astroglial plasticity in the olfactory bulb glomeruli of rats

The detection of food odors by the olfactory system, which plays a key role in regulating food intake and elaborating the hedonic value of food, is reciprocally influenced by the metabolic state. Fasting increases olfactory performance, notably by increasing the activity of olfactory bulb (OB) neurons. The glutamatergic synapses between olfactory sensory neurons and mitral cells in the OB glomeruli are regulated by astrocytes, periglomerular neurons, and centrifugal afferents. We compared the expansion of astroglial processes by quantifying GFAP‐labeled areas in fed and fasted rats to see whether OB glomerular astrocytes are involved in the metabolic sensing and adaptation of the olfactory system. Glomerular astroglial spreading was much greater in all OB regions of rats fasted for 17 hr than in controls. Intra‐peritoneal administration of the anorexigenic peptide PYY_3‐36 or glucose in 17 hr‐fasted rats respectively decreased their food intake or restored their glycemia, and reversed the fasting‐induced astroglial spreading. Direct application of the orexigenic peptides ghrelin or NPY to OB slices increased astroglial spreading, whereas PYY_3‐36 resulted in astroglial retraction, in agreement with the in vivo effects of fasting and satiety on glomerular astrocytes. Thus the morphological plasticity of OB glomerular astrocytes depends on the metabolic state of the rats and is influenced by peptides that regulate food intake. This plasticity may be part of the mechanism by which the olfactory system adapts to food intake.     

Temporal profile of stem cell division, migration, and differentiation from subventricular zone to olfactory bulb after transient forebrain ischemia in gerbils.

The stage of neurogenesis can be divided into three steps: proliferation, migration, and differentiation. To elucidate their detailed relations after ischemia, the three steps were comprehensively evaluated, in the subventricular zone (SVZ) through the rostral migratory stream (RMS) to the olfactory bulb (OB), in adult gerbil brain after 5 minutes of transient forebrain ischemia. Bromodeoxyuridine (BrdU), highly polysialylated neural cell adhesion molecule (PSA-NCAM), neuronal nuclear antigen (NeuN), and glial fibrillary acidic protein (GFAP) were used as markers for proliferation, migration, and differentiation, respectively. The number of BrdU-labeled cells that coexpressed PSA-NCAM and the size of PSA-NCAM-positive cell colony increased in the SVZ with a peak at 10 d after transient ischemia. In the RMS, the number of BrdU-labeled cells that coexpressed PSA-NCAM increased, with a delayed peak at 30 d, when the size of RMS itself became larger and the number of surrounding GFAP-positive cells increased. In the OB, BrdU + NeuN double positive cells were detected at 30 and 60 d. NeuN staining and terminal deoxynucleotidyl dUTP nick-end labeling staining showed no neuronal cell loss around the SVZ, and in the RMS and the OB after transient ischemia. These findings indicate that transient forebrain ischemia enhances neural stem cell proliferation in the SVZ without evident neuronal cell loss, and has potential neuronal precursor migration with activation of GFAP-positive cells through the RMS to the OB.     

Astrocytes control the development of the migration-promoting vasculature scaffold in the postnatal brain via VEGF signaling

New neurons are constantly being generated in the postnatal subventricular zone. They have to migrate long distances via the rostral migratory stream (RMS) to reach their final destination in the olfactory bulb (OB). In adults, these neuronal precursors migrate in chains, ensheathed by astrocytic processes, and travel toward the OB along blood vessels (BVs) that topographically outline the RMS. The molecular and cellular mechanisms leading to the development of the RMS and the formation of the migration-promoting vasculature scaffold in the adult mice remain unclear. We now reveal that astrocytes orchestrate the formation and structural reorganization of the vasculature scaffold in the RMS and, during early developmental stages, the RMS contains only a few BVs oriented randomly with respect to the migrating neuroblasts. The first parallel BVs appeared at the outer border of the RMS, where vascular endothelial growth factor (VEGF)-expressing astrocytes are located. Gain-of-function and loss-of-function experiments revealed that astrocyte-derived VEGF plays a crucial role in the formation and growth of new BVs. Real-time videoimaging also showed that the migration of neuronal precursors in the developing RMS differs substantially from neuronal displacement in the adult migratory stream partially because of not yet fully developed vasculature scaffold. The downregulation of VEGF in vivo, specifically in the astrocytes of the developing RMS, affected the development of the vasculature scaffold and led to alterations in neuroblast migration. Altogether, our results demonstrate that astrocytes orchestrate the formation and growth of parallel BVs, crucial migration-promoting scaffolds in the adult migratory stream, via VEGF signaling.