Neural progenitor cells of the neonatal rat anterior subventricular zone express functional GABA(A) receptors.

The interneurons of the olfactory bulb arise from precursor cells in the anterior part of the neonatal subventricular zone, the SVZa, and are distinctive in that they possess a neuronal phenotype and yet undergo cell division. To characterize the differentiation of neonatal SVZa progenitor cells, we analyzed the complement of ionotropic neurotransmitter receptors that they express in vitro. For this analysis, we tested the sensitivity of SVZa progenitor cells to gamma-amino-n-butyric acid (GABA), adenosine triphosphate (ATP), kainate, N-methyl-D-aspartate (NMDA), and acetylcholine (ACh) after 1 day in vitro. SVZa progenitor cells had chloride currents activated by GABA and muscimol, the GABA(A) receptor-specific agonist, but were insensitive to ATP, kainate, NMDA, and ACh. In addition, GABA- or muscimol-activated chloride currents were blocked nearly completely by 30 microM bicuculline, the GABA(A) receptor-specific antagonist, suggesting that GABA(B) and GABA(C) receptors are absent. Measurements of the chloride reversal potential by gramicidin-perforated patch clamp revealed that currents generated by activation of GABA(A) receptors were inward, and thus, depolarizing. A set of complementary experiments was undertaken to determine by reverse transcription and polymerase chain reaction (RT-PCR) whether SVZa progenitor cells express the messenger RNA (mRNA) coding for glutamic acid decarboxylase 67 (GAD67), used in the synthesis of GABA and for GABA(A) receptor subunits. Both postnatal day (P0) SVZa and olfactory bulb possessed detectable mRNA coding for GAD67. In P0 SVZa, the GABA(A) receptor subunits detected with RT-PCR included alpha 2-4, beta 1-3, and gamma 2S (short form). By comparison, the P0 olfactory bulb expressed all of the subunits detectable in the SVZa and additional subunit mRNAs: alpha 1, alpha 5, gamma 1, gamma 2L (long form), gamma 3, and delta subunit mRNAs. Antibodies recognizing GABA, GAD, and various GABA(A) receptor subunits were used to label SVZa cells harvested from P0-1 rats and cultured for 1 day. The cells were immunoreactive for GABA, GAD, and the GABA(A) receptor subunits alpha 2-5, beta 1-3, and gamma 2. To relate the characteristics of GABA(A) receptors in cultured SVZa precursor cells to particular combinations of subunits, the open reading frames of the dominant subunits detected by RT-PCR (alpha 2-4, beta 3, and gamma 2S) were cloned into a mammalian cell expression vector and different combinations were transfected into Chinese hamster ovary-K1 (CHO-K1) cells. A comparison of the sensitivity to inhibition by zinc of GABA(A) receptors in SVZa precursor cells and in CHO-K1 cells expressing various combinations of recombinant GABA(A) receptor subunits suggested that the gamma 2S subunit was present and functional in the GABA(A) receptor chloride channel complex. Thus, SVZa precursor cells are GABAergic and a subset of the GABA(A) receptor subunits detected in the olfactory bulb was found in the SVZa, as might be expected because SVZa progenitor cells migrate to the bulb as they differentiate.

Neurogenesis in the subventricular zone and rostral migratory stream of the neonatal and adult primate forebrain.

Throughout life, the anterior part of the postnatal rodent subventricular zone (SVZa), surrounding the lateral ventricles, contains a prolific source of neuronal progenitor cells that retain their capacity to concurrently generate neurons and migrate along the rostral migratory stream (RMS) to the olfactory bulb, where they differentiate into interneurons. This study was designed to determine whether the SVZ and RMS of the postnatal primate also harbor a specialized population of neuronal progenitors with the capacity to divide while they migrate. In order to reveal the spatial-temporal changes in the distribution and composition of the neuronal progenitor cells in the primate SVZ and RMS, seven rhesus monkeys, ranging in age from 2 days to 8 years, were given a single injection of the cell proliferation marker bromodeoxyuridine (BrdU) 3 h before they were perfused. The phenotypic identity of the BrdU(+) cells was revealed by double labeling sagittal sections with cell type-specific markers. From birth onward the distribution of BrdU(+) cells with a neuronal phenotype is extensive and largely overlapping with that of the rodent. Similar to the rodent brain the neuronal progenitors are most numerous in neonates. The BrdU(+) neurons in the primate forebrain extend lateral and ventral to the lateral ventricle and all along the RMS. The cytoarchitectonic arrangement and appearance of the neuronal progenitor cells is quite varied in the primate compared to the rodent; in some locations the cells are aligned in parallel arrays resembling the neuronal chains of the adult rodent RMS, whereas in other positions the cells have a homogeneous "honeycomb" arrangement. The chains are progressively more pervasive in older primates. Akin to the RMS of adult rodents, in the primate SVZ and RMS the astrocytes often form long tubes enveloping the chains of neuronal progenitors. Our study demonstrates that the primate forebrain, similar to the rodent forebrain, harbors a specialized population of mitotically active neuronal progenitor cells that undergo extensive rearrangements while continuing to proliferate throughout life.

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 increases in 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.

The expression pattern of the cell cycle inhibitor p19(INK4d) by progenitor cells of the rat embryonic telencephalon and neonatal anterior subventricular zone.

In this study we investigated whether the pattern of expression of the cyclin-dependent kinase inhibitor p19(INK4d) by the unique progenitor cells of the neonatal anterior subventricular zone (SVZa) can account for their ability to divide even though they express phenotypic characteristics of differentiated neurons. p19(INK4d) was chosen for analysis because it usually acts to block permanently the cell cycle at the G(1) phase. p19(INK4d) immunoreactivity and the incorporation of bromodeoxyuridine (BrdU) by SVZa cells were compared with that of the more typical progenitor cells of the prenatal telencephalic ventricular zone. In the developing telencephalon, p19(INK4d) is expressed by postmitotic cells and has a characteristic perinuclear distribution depending on the laminar position and state of differentiation of a cell. Moreover, the laminar-specific staining of the developing cerebral cortex revealed that the ventricular zone (VZ) is divided into p19(INK4d)(+) and p19(INK4d)(-) sublaminae, indicating that the VZ has a previously unrecognized level of functional organization. Furthermore, the rostral migratory stream, traversed by the SVZa-derived cells, exhibits an anterior(high)-posterior(low) gradient of p19(INK4d) expression. On the basis of the p19(INK4d) immunoreactivity and BrdU incorporation, SVZa-derived cells appear to exit and reenter the cell cycle successively. Thus, in contrast to telencephalic VZ cells, SVZa cells continue to undergo multiple rounds of division and differentiation before becoming postmitotic.

Retroviral manipulation of the expression of bone morphogenetic protein receptor Ia by SVZa progenitor cells leads to changes in their p19(INK4d) expression but not in their neuronal commitment.

Bone morphogenetic proteins (BMPs), a group of cytokines in the TGF-beta superfamily, have complex regulatory roles in the control of neural proliferation and cell fate decision. In this study, we analyzed the potential role(s) of BMP signaling on the regulation of the proliferation and differentiation of the unique progenitor cells of the neonatal anterior subventricular zone (SVZa). Unlike other progenitor cells of the brain, SVZa progenitor cells have the capacity to divide even though they express a neuronal phenotype. In order to augment or inhibit endogenous BMP signaling, we injected into the neonatal rat SVZa replication-deficient retroviruses encoding for either the wild-type BMP receptor subtype Ia (wt-BMPR-Ia) or a mutated dominant-negative version of BMPR-Ia (dn-BMPR-Ia) in conjunction with a reporter gene, human alkaline phosphatase (AP) and perfused the pups 1, 4 and 7 days post injection. We analyzed whether changing the expression of BMPR-Ia has an effect on the spatial-temporal expression pattern of the cyclin dependent kinase inhibitor, p19(INK4d), or on the phenotype of SVZa derived cells. The results of our study confirmed and extended our previous findings that in control (non injected) animals, the rostral migratory stream (RMS), traversed by the SVZa-derived cells en route to the olfactory bulb, exhibits an anterior(high)-posterior(low) gradient of p19(INK4d) expression; p19(INK4d) expression is essentially absent in the SVZa and highest in the subependymal zone in the middle of the olfactory bulb. However, SVZa progenitor cells encoding the wt-BMPR-Ia gene express p19(INK4d) within the SVZa, suggesting that the BMPs induce SVZa cells to ectopically undergo cell cycle exit within the SVZa. Furthermore, unlike striatal SVZ progenitor cells, which acquire an astrocytic phenotype when exposed to BMPs, SVZa progenitor cells retain their neuronal commitment under augmented BMP signaling.

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.

Region-specific differentiation of neural tube-derived neuronal restricted progenitor cells after heterotopic transplantation.

Spinal cord neuronal restricted progenitor (NRP) cells, when transplanted into the neonatal anterior forebrain subventricular zone, migrate to distinct regions throughout the forebrain including the olfactory bulb, frontal cortex, and occipital cortex but not to the hippocampus. Their migration pattern and differentiation potential is distinct from anterior forebrain subventricular zone NRPs. Irrespective of their final destination, NRP cells do not differentiate into glia. Rather they synthesize neurotransmitters, acquire region-specific phenotypes, and receive synapses from host neurons after transplantation. Spinal cord NRPs express choline acetyl transferase even in regions where host neurons do not express this marker. The restricted distribution of transplanted spinal cord NRP cells and their acquisition of varied region-specific phenotypes suggest that their ultimate fate and phenotype is dictated by a combination of intrinsic properties and extrinsic cues from the host.

The properties of hNT cells following transplantation into the subventricular zone of the neonatal forebrain.

Neurons derived from the human teratocarcinoma cell line (hNT) establish structural polarity and a fully mature phenotype following transplantation into the rodent brain. Here we describe the transplantation of hNT cells into the anterior part of neonatal subventricular zone (SVZa), which is a prolific region of neuronal progenitor cells. Ordinarily, the progeny of endogenous or homotopically transplanted SVZa cells migrate to the olfactory bulb (OB) along a restricted pathway, the rostral migratory stream (RMS), and differentiate into interneurons. To compare the phenotype of cultured hNT cells to their transplanted cohorts, hNT cells labeled by the fluorescent dye PKH26 were cultured for 1 day and stained with cell-type-specific antibodies. Clusters as well as individual hNT cells were immunoreactive for TuJ1, an antibody that recognizes neuron-specific class III beta-tubulin. The distribution and phenotype of the transplanted hNT cells were examined. The majority of transplanted PKH26-labeled hNT cells were found at their site of implantation in the SVZa, while a small proportion of the transplanted hNT cells was situated in the migratory pathway leading to the OB and in the subependymal zone and granule cell layer of the olfactory bulb. Many of the transplanted hNT cells, both within the SVZa and within the RMS, revealed a neuronal phenotype. Collectively, these results reveal the capacity of hNT cells to respond, at least partially, to cues that ordinarily govern the migration of SVZa-derived cells and maintain their neuronal identity.

Characterization and distribution of a new cell surface marker of neuronal precursors.

In a screen for novel cell surface markers of neuronal progenitors, we recently identified mAb 2F7 that recognizes an epitope present on both progenitor cells and postmitotic neurons, in the developing CNS and PNS. In the embryonic rat telencephalon, the mAb 2F7 epitope is expressed by migratory and postmigratory neurons in the developing cerebral cortex, as well as by presumptive neuronal progenitor cells of the ventricular zone. In the neonatal forebrain mAb 2F7 labels postmitotic neurons, including those of the developing cerebral cortex and olfactory bulb, as well as the axons of the corpus callosum. While mAb 2F7 immunoreactivity is present on only a low density of the neuronal progenitor cells situated in the anterior part of the subventricular zone, a progressively higher proportion of cells forming the rostral migratory stream express this epitope. mAb 2F7 labels the surfaces of neurons and neuronal precursors, but not mature oligodendrocytes and astrocytes in primary cultures derived from the rat neural tube. In vivo, migrating neural crest cells, motor neurons, and axonal projections associated with the spinal cord express the mAb 2F7 epitope. Immunoblot analyses reveal that the mAb 2F7 epitope resides on several high-molecular-weight, membrane-associated proteins, and is likely to be composed of N-linked carbohydrate. These findings suggest that mAb 2F7 recognizes a novel epitope that is present on progenitor cells and postmitotic, differentiating neurons in the developing mammalian nervous system.

Potassium currents in precursor cells isolated from the anterior subventricular zone of the neonatal rat forebrain.

The progenitor cells from the anterior part of the neonatal subventricular zone, the SVZa, are unusual in that, although they undergo division, they have a neuronal phenotype. To characterize the electrophysiological properties of the SVZa precursor cells, recordings were made of potassium and sodium currents from SVZa cells that were removed from postnatal day 0-1 rats and cultured for 1 day. The properties of the delayed rectifier and A-type potassium currents were described by classical Hodgkin and Huxley analyses of activation and inactivation. In addition, cells were assessed under current clamp for their ability to generate action potentials. The A-type potassium current (IK(A)) was completely inactivated at a holding potential of -50 mV. The remaining potassium current resembled the delayed rectifier current (IK(DR)) in that it was blocked by tetraethylammonium (TEA; IC50 4.1 mM) and activated and inactivated slowly compared with IK(A). The conductance-voltage (G-V) curve revealed that G increased continuously from 0.2 nS at -40 mV to a peak of 2.6 nS at +10 or +20 mV, and then decreased for voltages above +30 mV. Activation time constants were largest at -40 mV ( approximately 11 ms) and smallest at 100 mV ( approximately 1.5 ms). The properties of IK(A) were studied in the presence of 20 mM TEA, to block IK(DR), and from a holding potential of -15 mV, to inactivate both IK(DR) and IK(A). IK(A) was then allowed to recover from inactivation to negative potentials during 200- to 800-ms pulses. Recovery from inactivation was fastest at -130 mV ( approximately 21 ms) and slowest at -90 mV ( approximately 135 ms). Inactivation was voltage independent from -60 to +60 mV with a time constant of approximately 15 ms. At steady state, IK(A) was half inactivated at -90 mV. GK(A) increased from 0.2 nS at -60 mV to a peak of 2.4 nS at +40 mV. Finally, the activation time constants ranged from approximately 1.9 ms at -50 mV to 0.7 ms at +60 mV. The properties of IK(A) resembled those of IK(A) found in differentiating cerebellar granule neurons. Most SVZa cells had sodium currents (28/32 cells). However, in current clamp 11 of 12 cells were incapable of generating action potentials from voltages of -30 to -100 mV, suggesting that the available current densities were too low to support excitability.

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

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

Transforming growth factor-alpha expands progenitor cells of the basal forebrain, but does not promote cholinergic differentiation.

Transforming growth factor-alpha (TGF-alpha), a member of the epidermal growth factor (EGF) family, binds to the EGF-receptor (EGF-R). The early expression and widespread distribution of TGF-alpha and EGF-R in the developing central nervous system (CNS) suggest that TGF-alpha may play a role in the developing CNS. To study possible effects of TGF-alpha on cholinergic differentiation in the basal forebrain, we cultured septal nuclei with adjacent basal forebrain from embryonic rat brain in the presence and absence of TGF-alpha. At the highest dose of TGF-alpha used (100 ng/mL), activity of choline acetyltransferase (ChAT; EC 2.3.1.6) and the number of cholinergic neurons doubled. However, because protein levels tripled, specific ChAT activity actually declined. To determine the mechanism accounting for the increase in ChAT, we labeled dividing precursors present in the cultures with a replication-deficient retrovirus expressing beta-galactosidase in the presence and absence of TGF-alpha. By staining the cultures for both LacZ and ChAT, we determined that the precursor population expanded in size (individually labeled clones contained more cells), but the percentage of cholinergic neurons present in the clones was unchanged. Therefore, while TGF-alpha expands the precursor pool, it does not promote cholinergic differentiation. Interleukin-9, included to prompt neuronal differentiation, did not by itself increase ChAT activity, nor did it enhance the action of TGF-alpha. This was true even when basic fibroblast growth factor was included.

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 Bowman's 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.

Cell cycle length of olfactory bulb neuronal progenitors in the rostral migratory stream.

The anterior portion of the neonatal telencephalic subventricular zone (SVZa) contains proliferating cells that generate an immense number of neurons destined to become the granule and periglomerular cells of the olfactory bulb. In contrast to other immature neurons in the central nervous system, cells arising in the SVZa maintain the ability to divide as they traverse the rostral migratory stream to their final destinations despite expressing an antigenic marker of differentiated neurons (Menezes et al. [1995] Molec. Cell. Neurosci. 6:496-508). Because of their considerable proliferative capacities and unusual mitotic behavior, we decided to determine the cell cycle length of proliferating cells within the SVZa and within the migratory pathway used by SVZa-derived cells. Following the methodology of Nowakowski et al. [1989](J. Neurocytol. 18:311-318), postnatal day 2 rat pups were exposed to 5'-bromo-2'deoxyuridine (BrdU) for increasing periods of time before perfusion. By plotting the percentage of nuclei undergoing DNA synthesis in the SVZa at each time versus the BrdU labeling interval, we determined that approximately 15% of the SVZa population is actively dividing and that these cells have a cycle length of approximately 14 hr, significantly less than the 18.6 hr determined to be the cycle length of dividing cells in more posterior, glia-generating regions of the subventricular zone (Thomaidou et al. [1997] J. Neurosci. 17:1075-1085). The cycle length of cells dividing in the mid portion of the rostral migratory stream, however, is considerably longer: 17.3 hr. This may reflect the need for these cells to coordinate the processes of migration and division. Our studies also suggest that there may be regional differences in the types of descendants produced by the proliferating cells. Retroviral lineage tracing studies showed that those cells that divide within the rostral migratory stream, like proliferating cells within the SVZa, make cells destined for the olfactory bulb. Unlike the progenitors that divide within the SVZa and generate more granule cells than periglomerular cells, the proliferating cells within the migratory pathway generate more periglomerular cells than granule cells. Collectively the proliferating cells of the SVZa and migratory pathway produce a large number of olfactory bulb interneurons. Our work suggests that this may be achieved in part by the relatively rapid divisions of progenitor cells within the SVZa and in part by the ongoing division of migrating cells en route to the olfactory bulb.

Neuroblasts of the postnatal mammalian forebrain: their phenotype and fate.

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

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.

Neuronal progenitor cells of the neonatal subventricular zone differentiate and disperse following transplantation into the adult rat striatum.

We have investigated the suitability of a recently identified and characterized population of neuronal progenitor cells for their potential use in the replacement of degenerating or damaged neurons in the mammalian brain. The unique population of neuronal progenitor cells is situated in a well-delineated region of the anterior part of the neonatal subventricular zone (referred to as SVZa). This region can be separated from the remaining proliferative, gliogenic, subventricular zone encircling the lateral ventricles of the forebrain. Because the neurons arising from the highly enriched neurogenic progenitor cell population of the SVZa ordinarily migrate considerable distances and ultimately express the neurotransmitters GABA and dopamine, we have examined whether they could serve as an alternative source of tissue for neural transplantation. SVZa cells from postnatal day 0-2 rats, prelabeled by intraperitoneal injections of the cell proliferation marker BrdU, were implanted into the striatum of adult rats approximately 1 mo after unilateral denervation by 6-OHDA. To examine the spatio-temporal distribution and phenotype of the transplanted SVZa cells, the experimental recipients were perfused at short (less than 1 wk), intermediate (2-3 wk) and long (5 mo) postimplantation times. The host brains were sectioned and stained with an antibody to BrdU and one of several cell-type specific markers to determine the phenotypic characteristics of the transplanted SVZa cells. To identify neurons we used the neuron-specific antibody TuJ1, or antimembrane-associated protein 2 (MAP-2), and anti-GFAP was used to identify astrocytic glia. At all studied intervals the majority of the surviving SVZa cells exhibited a neuronal phenotype. Moreover, morphologically they could be distinguished from the cells of the host striatum because they resembled the intrinsic granule cells of the olfactory bulb, their usual fate. At longer times, a greater number of the transplanted SVZa cells had migrated from their site of implantation, often towards an outlying blood vessel, and the density of cells within the core of the transplant was reduced. Furthermore, there were rarely signs of transplant rejection or a glial scar surrounding the transplant. In the core of the transplant there were low numbers of GFAP-positive cells, indicating that the transplanted SVZa cells, predominantly TuJ1-positive/MAP2-positive, express a neuronal phenotype. Collectively, the propensity of the SVZa cells to express a neuronal phenotype and to survive and integrate in the striatal environment suggest that they may be useful in the reconstruction of the brain following CNS injury or disease.

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.

The neuronal progenitor cells of the forebrain subventricular zone: intrinsic properties in vitro and following transplantation.

During the development of the central nervous system, progenitor cells, located within distinct germinal zones, produce presumptive neurons that migrate to their destinations and differentiate. Recent studies have demonstrated that a discrete region of the anterior part of the postnatal subventricular zone (SVZa) comprises neuronal progenitor cells whose progeny are fated to become the interneurons of the olfactory bulb. The SVZa is of particular interest because it is one of few germinal zones to persist postnatally and may be the only postnatal germinal zone to give rise exclusively to neurons. To the extent that the SVZa is unique among proliferative zones, the SVZa progeny are unique among neurons. First, unlike most cortical neurons, the SVZa-derived cells do not rely on radial glia-assisted migration when traveling to their target region. Second, the SVZa progeny continue to proliferate as they migrate to their target region. And third, the SVZa progeny express early neuron-specific antigens prior to their final division and, therefore, prior to reaching their destination where they will terminally differentiate. To better understand the capacity of the SVZa progeny to concurrently proliferate, migrate, and differentiate, we studied the cells in vitro and following transplantation into the neonatal SVZa and adult striatum. In each setting, we found that the SVZa cells continue both to proliferate and to differentiate into neurons. In addition, after homotopic and heterotopic transplantation, we found that the SVZa cells maintain their ability to migrate. These results suggest that the unique features of the SVZa progeny are specified intrinsically rather than by their extrinsic environment.