[Usefulness of MALDI-TOF and REP-PCR against PFGE for the epidemiological study of Acinetobacter baumannii]. / Utilidad de MALDI-TOF y REP-PCR frente a PFGE para el estudio epidemiológico de Acinetobacter baumannii.

OBJECTIVE: To evaluate the ability of MALDI-TOF MS and rep-PCR to discriminate Acinetobacter baumannii clones. METHODS: A total of 21 strains of A. baumannii with different epidemiological and phenotipycal characteristics were included in the study. All isolates were analyzed in parallel by MALDI-TOF MS and rep-PCR and the spectra obtained were compared with each other and with the results obtained by pulsed field gel electrophoresis (PFGE). Isolates with a similarity equal to or greater than 87% were considered to be part of the same clonal group. RESULTS: The analysis of the 21 isolates included in the study, resulted in 8 clonal groups using PFGE, 3 groups by MALDI-TOF MS and 7 groups by rep-PCR analysis. The isolates that formed the different groups by the 3 techniques used were totally different, so it can be concluded that there is no equivalence between the results obtained with the three typing methods used. CONCLUSIONS: Despite its simplicity, neither MALDI-TOF MS nor rep-PCR can at this time replace PFGE for the epidemiological study of A. baumannii.

The heterogeneity of adult neural stem cells and the emerging complexity of their niche.

Neural stem cells persist in the adult mammalian brain in a neurogenic niche known as the subventricular zone (SVZ). SVZ neural stem cells (NSCs) can self-renew and are multipotent in culture. In rodents, adult NSCs correspond to SVZ astrocytes (type B cells) that are derived from radial glia, the NSCs of the embryonic and early postnatal brain. Type B cells generate transit-amplifying (type C) cells that give rise to young neurons (type A cells) and oligodendrocytes. Young neurons are born throughout the adult neurogenic niche and migrate tangentially through a complex network of chains that merge into the rostral migratory stream (RMS), a major pathway that leads into the olfactory bulb (OB). Within the OB, young neurons differentiate into multiple types of interneurons. The SVZ was thought to be limited to the lateral wall of the lateral ventricle, but recent work shows that the adult neurogenic niche is significantly more extensive and includes portions of the medial and dorsal walls of the lateral ventricle and the RMS itself. Furthermore, several recent studies explain why young OB neurons are generated in such an extensive region. Type B cells in different regions of the SVZ, although able to self-renew and generate both neurons and glial cells in vitro, are heterogeneous and committed to producing defined neuronal subtypes in vivo. The adult SVZ therefore provides a rich system to study not only neural replacement, but also the cellular and molecular mechanisms underlying regionalization and cell-fate specification.

Primary cilia are required for cerebellar development and Shh-dependent expansion of progenitor pool.

Cerebellar granule cell precursors (GCPs), which give rise to the most abundant neuronal type in the mammalian brain, arise from a restricted pool of primary progenitors in the rhombic lip (RL). Sonic hedgehog (Shh) secreted by developing Purkinje cells is essential for the expansion of GCPs and for cerebellar morphogenesis. Recent studies have shown that the primary cilium concentrates components of Shh signaling and that this structure is required for Shh signaling. GCPs have a primary cilium on their surface [Del Cerro, M.P., Snider, R.S. (1972). Studies on the developing cerebellum. II. The ultrastructure of the external granular layer. J Comp Neurol 144, 131-64.]. Here, we show that 1) this cilium can be conditionally ablated by crossing Kif3a(fl/-) mice with hGFAP-Cre mice, 2) removal of Kif3a from GCPs disrupts cerebellar development, and 3) these defects are due to a drastic reduction in Shh-dependent expansion of GCPs. A similar phenotype is observed when Smoothened (Smo), an essential transducer of Shh signaling, is removed from the same population of GCPs. Interestingly, Kif3a-Smo double conditional mutants show that Kif3a is epistatic to Smo. This work shows that Kif3a is essential for Shh-dependent expansion of cerebellar progenitors. Dysfunctional cilia are associated with diverse human disorders including Bardet-Biedl and Joubert syndromes. Cerebellar abnormalities observed in these patients could be explained by defects in Shh-induced GCP expansion.

Unsupervised learning and adaptation in a model of adult neurogenesis.

Adult neurogenesis has long been documented in the vertebrate brain and recently even in humans. Although it has been conjectured for many years that its functional role is related to the renewing of memories, no clear mechanism as to how this can be achieved has been proposed. Using the mammalian olfactory bulb as a paradigm, we present a scheme in which incorporation of new neurons proceeds at a constant rate, while their survival is activity-dependent and thus contingent on new neurons establishing suitable connections. We show that a simple mathematical model following these rules organizes its activity so as to maximize the difference between its responses and can adapt to changing environmental conditions in unsupervised fashion, in agreement with current neurophysiological data.

In utero fate mapping reveals distinct migratory pathways and fates of neurons born in the mammalian basal forebrain.

Recent studies suggest that neurons born in the developing basal forebrain migrate long distances perpendicularly to radial glia and that many of these cells reach the developing neocortex. This form of tangential migration, however, has not been demonstrated in vivo, and the sites of origin, pathways of migration and final destinations of these neurons in the postnatal brain are not fully understood. Using ultrasound-guided transplantation in utero, we have mapped the migratory pathways and fates of cells born in the lateral and medial ganglionic eminences (LGE and MGE) in 13.5-day-old mouse embryos. We demonstrate that LGE and MGE cells migrate along different routes to populate distinct regions in the developing brain. We show that LGE cells migrate ventrally and anteriorly, and give rise to the projecting medium spiny neurons in the striatum, nucleus accumbens and olfactory tubercle, and to granule and periglomerular cells in the olfactory bulb. By contrast, we show that the MGE is a major source of neurons migrating dorsally and invading the developing neocortex. MGE cells migrate into the neocortex via the neocortical subventricular zone and differentiate into the transient subpial granule neurons in the marginal zone and into a stable population of GABA-, parvalbumin- or somatostatin-expressing interneurons throughout the cortical plate.

Astrocytes give rise to new neurons in the adult mammalian hippocampus.

Neurogenesis in the dentate gyrus of the hippocampus persists throughout life in many vertebrates, including humans. The progenitors of these new neurons reside in the subgranular layer (SGL) of the dentate gyrus. Although stem cells that can self-renew and generate new neurons and glia have been cultured from the adult mammalian hippocampus, the in vivo primary precursors for the formation of new neurons have not been identified. Here we show that SGL cells, which express glial fibrillary acidic protein and have the characteristics of astrocytes, divide and generate new neurons under normal conditions or after the chemical removal of actively dividing cells. We also describe a population of small electron-dense SGL cells, which we call type D cells and are derived from the astrocytes and probably function as a transient precursor in the formation of new neurons. These results reveal the origins of new neurons in the adult hippocampus.

A unified hypothesis on the lineage of neural stem cells.

For many years, it was assumed that neurons and glia in the central nervous system were produced from two distinct precursor pools that diverged early during embryonic development. This theory was partially based on the idea that neurogenesis and gliogenesis occurred during different periods of development, and that neurogenesis ceased perinatally. However, there is now abundant evidence that neural stem cells persist in the adult brain and support ongoing neurogenesis in restricted regions of the central nervous system. Surprisingly, these stem cells have the characteristics of fully differentiated glia. Neuroepithelial stem cells in the embryonic neural tube do not show glial characteristics, raising questions about the putative lineage from embryonic to adult stem cells. In the developing brain, radial glia have long been known to produce cortical astrocytes, but recent data indicate that radial glia might also divide asymmetrically to produce cortical neurons. Here we review these new developments and propose that the stem cells in the central nervous system are contained within the neuroepithelial --> radial glia --> astrocyte lineage.

Disruption of Eph/ephrin signaling affects migration and proliferation in the adult subventricular zone.

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

The subventricular zone: source of neuronal precursors for brain repair.

The subventricular zone (SVZ) is a major germinal zone which persists in the adult brain. The SVZ contains cells that self renew and continuously produce new neurons and glia. In this chapter we discuss the development, architecture and function of the adult SVZ, as well as the fate of SVZ cells after transplantation. We focus on identification of neural stem cells, factors which regulate neurogenesis and mechanisms for neuronal migration through the adult brain. Detailed understanding of these processes is necessary to utilize the SVZ as a source of neuronal and glial precursors for genetic manipulation, transplantation or brain self repair.

Noggin antagonizes BMP signaling to create a niche for adult neurogenesis.

Large numbers of new neurons are born continuously in the adult subventricular zone (SVZ). The molecular niche of SVZ stem cells is poorly understood. Here, we show that the bone morphogenetic protein (BMP) antagonist Noggin is expressed by ependymal cells adjacent to the SVZ. SVZ cells were found to express BMPs as well as their cognate receptors. BMPs potently inhibited neurogenesis both in vitro and in vivo. BMP signaling cell-autonomously blocked the production of neurons by SVZ precursors by directing glial differentiation. Purified mouse Noggin protein promoted neurogenesis in vitro and inhibited glial cell differentiation. Ectopic Noggin promoted neuronal differentiation of SVZ cells grafted to the striatum. We thus propose that ependymal Noggin production creates a neurogenic environment in the adjacent SVZ by blocking endogenous BMP signaling.

Adult-derived neural precursors transplanted into multiple regions in the adult brain.

Neural stem cells persist in the adult brain subventricular zone (SVZ). These cells generate a large number of new neurons that migrate to the olfactory bulb, where they complete their differentiation. Here, we transplanted cells carrying beta-galactosidase under the control of neuron-specific enolase promoter (NSE::LacZ) from the SVZ of adult mice into the striatum cortex and olfactory bulb, with or without an excitotoxin lesion. Between 2 and 8 weeks after transplantation, grafted cells were present in the recipient regions, but extensive migration and differentiation into mature neurons of grafted cells were only observed in the olfactory bulb. Clusters of graft-derived neuroblasts forming chain-like structures were observed within or close to the grated sites in the cortex and striatum; electron microscopy confirmed that graft-derived cells in the olfactory bulb and a small number in the striatum were neurons. Surprisingly, most of the cells expressing NSE::LacZ outside the olfactory bulb were astrocytes. We conclude that primary precursors from the SVZ migrate and differentiate effectively only within the environment of the olfactory bulb. Only limited survival and differentiation were observed in other brain regions studied.

Regeneration of a germinal layer in the adult mammalian brain.

Neuronal precursors reside in the subventricular zone (SVZ) of adult mammals. This region is composed of a network of chains of migrating neuroblasts ensheathed by astrocytes and juxtaposed by clusters of immature precursors (type C cells). Here we show that after antimitotic treatment with cytosine-beta-D-arabinofuranoside, neuroblasts and type C cells are eliminated but some astrocytes remain. Remarkably, the SVZ network rapidly regenerates. Soon after cytosine-beta-D-arabinofuranoside treatment astrocytes divide. Two days later, type C cells reappear, followed at 4.5 days by migrating neuroblasts. By 10 days the SVZ network is fully regenerated, and the orientation and organization of chains of migrating neuroblasts resemble that of normal mice. This regeneration reveals an unexpected plasticity in the adult central nervous system and should provide a model system to study the early stages of neurogenesis in the adult brain.

Fate of new neurons in adult canary high vocal center during the first 30 days after their formation.

Projection neurons are added to the high vocal center (HVC) of adult songbirds. Here we report on events associated with their initial arrival in HVC. Neurons formed in adult canaries were labeled with [(3)H]-thymidine and examined 8, 15, 22, and 31 days later. By 8 days, some [(3)H]-labeled cells with the nuclear profile of postmigratory neurons were already present in HVC but could not be retrogradely labeled by Fluoro-Gold injections in the robust nucleus of the archistriatum (RA); 7 days later, a few such cells could be backfilled from RA. Thus, new neurons may arrive in HVC as much as 1 week prior to establishing connections with RA. By 31 days, 43% of the [(3)H]-labeled neurons could be backfilled from RA. In no case were new neurons backfilled by tracer injections into Area X, suggesting that newly formed HVC cells do not establish a transient connection with this region. At all survival times, the somata of new neurons were often clustered tightly together with other HVC neurons that differed in age and projection. Between days 15 and 25 after their birth, half of the new HVC neurons disappeared. We conclude: (1) that neurons arrive in HVC earlier than previously thought, (2) that soon after their arrival they become part of cell clusters in HVC, and (3) that in addition to the previously described death of new neurons that occurs over a period of months, there is an early wave of death that occurs soon after new neurons adopt a postmigratory phenotype.

Interaction between astrocytes and adult subventricular zone precursors stimulates neurogenesis.

Neurogenesis continues in the mammalian subventricular zone (SVZ) throughout life. However, the signaling and cell-cell interactions required for adult SVZ neurogenesis are not known. In vivo, migratory neuroblasts (type A cells) and putative precursors (type C cells) are in intimate contact with astrocytes (type B cells). Type B cells also contact each other. We reconstituted SVZ cell-cell interactions in a culture system free of serum or exogenous growth factors. Culturing dissociated postnatal or adult SVZ cells on astrocyte monolayers-but not other substrates-supported extensive neurogenesis similar to that observed in vivo. SVZ precursors proliferated rapidly on astrocytes to form colonies containing up to 100 type A neuroblasts. By fractionating the SVZ cell dissociates with differential adhesion to immobilized polylysine, we show that neuronal colony-forming precursors were concentrated in a fraction enriched for type B and C cells. Pure type A cells could migrate in chains but did not give rise to neuronal colonies. Because astrocyte-conditioned medium alone was not sufficient to support SVZ neurogenesis, direct cell-cell contact between astrocytes and SVZ neuronal precursors may be necessary for the production of type A cells.

Subventricular zone astrocytes are neural stem cells in the adult mammalian brain.

Neural stem cells reside in the subventricular zone (SVZ) of the adult mammalian brain. This germinal region, which continually generates new neurons destined for the olfactory bulb, is composed of four cell types: migrating neuroblasts, immature precursors, astrocytes, and ependymal cells. Here we show that SVZ astrocytes, and not ependymal cells, remain labeled with proliferation markers after long survivals in adult mice. After elimination of immature precursors and neuroblasts by an antimitotic treatment, SVZ astrocytes divide to generate immature precursors and neuroblasts. Furthermore, in untreated mice, SVZ astrocytes specifically infected with a retrovirus give rise to new neurons in the olfactory bulb. Finally, we show that SVZ astrocytes give rise to cells that grow into multipotent neurospheres in vitro. We conclude that SVZ astrocytes act as neural stem cells in both the normal and regenerating brain.

Young neurons from medial ganglionic eminence disperse in adult and embryonic brain.

In this study, we identified neuronal precursors that can disperse through adult mammalian brain tissue. Transplanted neuronal precursors from embryonic medial ganglionic eminence (MGE), but not from lateral ganglionic eminence (LGE) or neocortex, dispersed and differentiated into neurons in multiple adult brain regions. In contrast, only LGE cells were able to migrate efficiently from the adult subventricular zone to the olfactory bulb. In embryonic brain slices, MGE cells migrated extensively toward cortex. Our results demonstrate that cells in different germinal regions have unique migratory potentials, and that adult mammalian brain can support widespread dispersion of specific populations of neuronal precursors. These findings could be useful in repair of diffuse brain damage.

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.

Stem cells in the adult mammalian central nervous system.

Over the past year, evidence has accrued that adult CNS stem cells are a widespread progenitor cell type. These cells may normally replace neurons and/or glia in the adult brain and spinal cord. Advances have been made in understanding the signals that regulate stem cell proliferation and differentiation. A deeper understanding of the structure of germinal zones has helped us move towards identifying stem cells in vivo. Recent studies suggest that the fate of stem cell progeny in vivo may be linked to the complexity of the animal's environment.

BDNF mediates the effects of testosterone on the survival of new neurons in an adult brain.

New neurons are incorporated into the high vocal center (HVC), a nucleus of the adult canary (Serinus canaria) brain that plays a critical role in the acquisition and production of learned song. Recruitment of new neurons in the HVC is seasonally regulated and depends upon testosterone levels. We show here that brain-derived neurotrophic factor (BDNF) is present in the HVC of adult males but is not detectable in that of females, though the HVC of both sexes has BDNF receptors (TrkB). Testosterone treatment increases the levels of BDNF protein in the female HVC, and BDNF infused into the HVC of adult females triples the number of new neurons. Infusion of a neutralizing antibody to BDNF blocks the testosterone-induced increase in new neurons. Our results demonstrate that BDNF is involved in the regulation of neuronal replacement in the adult canary brain and suggest that the effects of testosterone are mediated through BDNF.