Marla B. Luskin

Infusion of Brain-Derived Neurotrophic Factor into the Lateral Ventricle of the Adult Rat Leads to New Neurons in the Parenchyma of the Striatum, Septum, Thalamus, and Hypothalamus

The findings that brain-derived neurotrophic factor (BDNF) promotes in vitro the survival and/or differentiation of postnatal subventricular zone (SVZ) progenitor cells and increasesin vivo the number of the newly generated neurons in the adult rostral migratory stream and olfactory bulb prompted us to investigate whether the infusion of BDNF influences the proliferation and/or differentiation of cells in other regions of the adult forebrain. We examined the distribution and phenotype of newly generated cells in the adult rat forebrain 16 d after intraventricular administration of BDNF in conjunction with the cell proliferation marker bromodeoxyuridine (BrdU) for 12 d. BDNF infusion resulted in numerous BrdU+ cells, not only in the SVZ lining the infused lateral ventricle, but moreover, in specific parenchymal structures lining the lateral and third ventricles, including the striatum and septum, as well as the thalamus and hypothalamus, in which neurogenesis had never been demonstrated previously during adulthood. In each region, newly generated cells expressed the neuronal marker microtubule-associated protein-2, or neuron-specific tubulin, identified by the antibody TuJ1. The percentage of the newly generated cells expressing TuJ1 ranged from 27 to 42%, suggesting that the adult forebrain has a more profound capacity to produce neurons than recognized previously. The extent of cell proliferation after BDNF infusion was correlated with the level of expression of full-length TrkB, the high-affinity receptor for BDNF, despite the fact that the BrdU+ cells were not themselves TrkB+. Collectively, our results demonstrate that the adult brain parenchyma may recruit and/or generate new neurons, which could replace those lost as a result of injury or disease.

Intraventricular Administration of BDNF Increases the Number of Newly Generated Neurons in the Adult Olfactory Bulb

We have previously demonstrated that the most rostral part of the subventricular zone (SVZ) is a source of neuronal progenitor cells whose progeny are destined to become interneurons of the olfactory bulb. To determine whether the number of newly generated neurons in the adult olfactory bulb could be increased by the administration of an exogenous factor, brain-derived neurotrophic factor (BDNF) was infused for 12 days into the right lateral ventricle of adult rat brains. The production of new cells was monitored by either the intraventricular infusion or intraperitoneal injection of the cell proliferation marker BrdU. In both experimental paradigms we observed significantly more BrdU-labeled cells in the olfactory bulbs on the BDNF-infused side than in the olfactory bulb of PBS-infused animals. Analysis of the BDNF-infused brains of animals injected intraperitoneally with BrdU demonstrated a 100% increase in the number of BrdU-labeled cells in the bulb, the preponderance ( approximately 90%) of which were double-labeled with a neuron-specific antibody. These results demonstrate that the generation and/or survival of new neurons in the adult brain can be increased substantially by an exogenous factor. Furthermore, the SVZ, and in particular the rostral part, may constitute a reserve pool of progenitor cells available for neuronal replacement in the diseased or damaged brain.

The Division of Neuronal Progenitor Cells during Migration in the Neonatal Mammalian Forebrain

In the mammalian forebrain most neurons originate from proliferating cells in the ventricular zone lining the lateral ventricles. These neurons become postmitotic before they undergo migration to their final destinations. In this study we examined the proliferative and migratory properties of cells destined for the olfactory bulb that arise postnatally from progenitor cells situated at the anterior extent of the subventricular zone (SVZa). The SVZa-derived cells migrate along a stereotypical pathway to the olfactory bulb where they become interneurons. Using lineage tracers and the cell proliferation marker BrdU, we have demonstrated that SVZa-derived cells in the rat retain the capacity for division after migrating away from their initial site of generation. These cells also express a neuron-specific tubulin, recognized by the antibody TuJ1. These results suggest that, unlike other immature neurons, these SVZa-derived cells have made a commitment to become neurons before becoming postmitotic.

Adult olfactory epithelium contains multipotent progenitors that give rise to neurons and non-neural cells

We have infused replication‐incompetent retroviral vectors into the nasal cavity of adult rats 1 day after exposure to the olfactotoxic gas methyl bromide (MeBr) to assess the lineage relationships of cells in the regenerating olfactory epithelium. The vast majority of the retrovirus‐labeled clones fall into three broad categories: clones that invariably contain globose basal cells (GBCs) and/or neurons, clones that always include cells in the ducts of Bowmans glands, and clones that are composed of sustentacular cells only. Many of the GBC‐related clones contain sustentacular cells and horizontal basal cells as well. Most of the duct‐related clones contain gland cells, and some also include sustentacular cells. Thus, the destruction of both neurons and non‐neuronal cells that is caused by MeBr activates two distinct types of multipotent cells. The multipotent progenitor that gives rise to neurons and non‐neuronal cells is a basal cell, whereas the progenitor that gives rise to duct, gland, and sustentacular cells resides within the ducts, based on the pattern of sparing after lesion and the analysis of early regeneration by using cell type‐specific markers. We conclude that the balance between multipotency and selective neuropotency, which is characteristic of globose basal cells in the normal olfactory epithelium, is determined by which cell types have been depleted and need to be replenished rapidly. J. Comp. Neurol. 400:469–486, 1998.

Neuroblasts of the postnatal mammalian forebrain: Their phenotype and fate

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

Neuronal Progenitor Cells Derived from the Anterior Subventricular Zone of the Neonatal Rat Forebrain Continue to Proliferatein Vitroand Express a Neuronal Phenotype

A discrete area of the anterior part of the subventricular zone, or SVZa, of the postnatal forebrain is composed of progenitor cells that are dissimilar to those elsewhere in the CNS. In vivo SVZa progenitor cells retain the ability for division, even though they are phenotypically neurons. To characterize further the properties of SVZa cells, we have analyzed their characteristics in vitro using cell-type specific antibodies and their proliferative capacity by the incorporation of bromodeoxyuridine. At 2 h in vitro, as well as after 1 day in vitro, virtually all SVZa cells isolated from the neonatal forebrain express TuJ1, an antibody that recognizes neuron-specific tubulin, and are GFAP-negative. Likewise, the preponderance of SVZa cells express the neuron-specific markers N-CAM and MAP-2 when examined after 1 day in culture. The majority of SVZa cells cultured for as long as 8 days also possessed a neuronal phenotype. In addition, process-bearing TuJ1-positive SVZa cells continued to proliferate throughout the entire culture period. Thus, the neuronal progenitor cells of the SVZa constitute a unique cell population with characteristics distinct from the cells of other germinal zones.

Dopaminergic and GABAergic interneurons of the olfactory bulb are derived from the neonatal subventricular zone.

Earlier studies in our laboratory have demonstrated that a discrete region of the anterior part of the neonatal subventricular zone (SVZa) contains exclusively neuronal progenitor cells. The descendants of the SVZa progenitor cells are destined for the granule cell and glomerular layers of the olfactory bulb, where they differentiate into granule and periglomerular cells, the interneurons of the olfactory bulb, respectively. In the present set of experiments we examined the neurotransmitter phenotype of the SVZa‐derived cells. In order to label SVZa‐derived cells, the cell proliferation marker bromodeoxyuridine (BrdU) was injected into the SVZa of postnatal day 2 (P2) rats. After 3 weeks, by which time most of the SVZ‐aderived cells have migrated to their final destination in the bulb, the animals were perfused and their brains processed for immunohistochemistry. To identify the neurotransmitter phenotype of the SVZa‐derived cells, sagittal sections of the forebrain, including the olfactory bulb, were double‐labeled with an antibody to BrdU in conjunction with an antibody to γ‐amino‐butyric acid (GABA) or tyrosine hydroxylase (TH), the rate limiting enzyme in the synthesis of dopamine. Using simultaneous indirect immunofluorescence to detect the presence of single‐ and double‐labeled cells, we found that 59% and 51% of the BrdU‐positive cells were immunoreactive for GABA in the granule cell and glomerular layers, respectively. In addition, 10% of the BrdU‐positive periglomerular cells were immunoreactive for TH. The presence of double‐labeled (BrdU‐positive/GABA‐positive and BrdU‐positive/TH‐positive) cells in the olfactory bulb, demonstrates that the SVZa is a source of the GABAergic and dopaminergic interneurons of the olfactory bulb during postnatal development.

Strategies utilized by migrating neurons of the postnatal vertebrate forebrain

Structural brain repair has become a possibility with the identification and characterization of persistent neuronal progenitor cells in both the neonatal and adult brain. However, despite recent advances in the identification, propagation and expansion of these cells, they will not be useful therapeutically until methods are available for directing or delivering them to sites of need. As a result, the natural history and induction of neuronal migration into adult brain tissue has assumed new importance in clinical neurobiology. In this review we consider the cellular and molecular bases of neuronal migration into the postnatal forebrain. In particular, we discuss two natural paradigms of postnatal neuronal recruitment: radial-cell-directed neuronal migration to the songbird neostriatum, and neurophilic migration to the rodent olfactory bulb. In each, we will focus on the dynamic interactions between the migrants, their cellular guides and the local environment, and the effect of those interactions on migrational success.

Increased Number of BrdU-Labeled Neurons in the Rostral Migratory Stream of the Estrous Prairie Vole

In the mammalian forebrain, most neurons originate from proliferating cells in the ventricular zone lining the lateral ventricles, including a discrete area of the subventricular zone in which neurogenesis continues into adulthood. The majority of the cells generated in the anterior portion of the subventricular zone (SVZa) are neuronal precursors with progeny that migrate to the olfactory bulb (OB) along a pathway known as the rostral migratory stream (RMS). The list of factors that influence the proliferation and survival of neurons in the adult brain remains incomplete, but previous studies have implicated neurotrophins in mammals and estrogen in birds. This study examined the effect of estrus induction on the proliferation of SVZa neurons in female prairie voles. Prairie voles, unlike many other rodents, are induced into estrus by chemosensory cues from a male. This olfactory-mediated process results in an increase in serum estrogen levels and the consequent induction of behavioral estrus (sexual receptivity). Female prairie voles induced into estrus by male exposure had a 92% increase in BrdU-labeled cells in the SVZa compared to females exposed to a female. Double-label immunocytochemical studies demonstrated that 80% of the BrdU-labeled cells in the RMS displayed a neuronal phenotype. Ovariectomized females exposed to a male did not show an increase in serum estrogen or BrdU labeling in the RMS. Conversely, ovariectomized females injected with estrogen were sexually receptive and had more BrdU-labeled cells in the RMS than oil-injected females. These data suggest that, in female prairie voles, estrus induction is associated with increased numbers of dividing cells in the RMS, possibly via an estrogen-mediated process.

Embryonic-derived glial-restricted precursor cells (GRP cells) can differentiate into astrocytes and oligodendrocytes in vivo

We have isolated and characterized a unique glial-restricted precursor cell (GRP) from the embryonic spinal cord. Clonal analysis demonstrated that these cells are able to generate oligodendrocytes and two distinct type of astrocytes (type 1 and type 2) when exposed to appropriate signals in vitro. We now show that many aspects of these cells are retained in vivo. GRP cells are restricted to the glial lineage in vivo as they seem to be unable to generate neuronal phenotypes in an in vivo neurogenic environment. GRP cells survive and migrate in the neonatal and adult brain. Transplanted GRP cells differentiate into myelin-forming oligodendrocytes in a myelin-deficient background and also generate immature oligodendrocytes in the normal neonatal brain. In addition, GRP cells also consistently generated glial fibrillary protein-expressing cells in the neonatal and adult brain, a property not consistently expressed by other glial precursor cells like the O-2A/OPC cells. We suggest that the lineage restriction of GRP cells and their ability to generate both oligodendrocytes and astrocytes in vivo together with their embryonic character that allows for extensive in vitro expansion of the population makes the cell useful for clinical application.