Differentiation of oligodendroglial progenitors derived from cortical multipotent cells requires extrinsic signals including activation of gp130/LIFbeta receptors.

We have previously isolated epidermal growth factor (EGF)-responsive multipotent progenitor cells from the early postnatal rodent cerebral cortex independent of generative zones. In this study we have examined the mechanisms regulating the generation of differentiated oligodendrocytes (OLs) from these multipotent cells. Although cultures of primary cortical OL progenitor cells propagated at clonal density spontaneously gave rise to differentiated OLs in defined medium, cultures of multipotent progenitors isolated from identical regions supported the elaboration of OL progenitors but not differentiated OLs. These observations indicate that the terminal maturation of OL progenitors derived from multipotent cells is dependent on signals present within the cellular environment. Application of cytokines such as basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), or neurotrophin 3 (NT3) to clonal density cultures of cortical multipotent progenitors increased the proportion of OL progenitors but failed to support the generation of differentiated OLs. By contrast, application of factors that activate gp130/leukemia inhibitory factor beta (LIFbeta) heterodimeric receptors, such as ciliary neurotrophic factor (CNTF), activated signal transducers and activators of transcription-3 in these OL progenitor cells and promoted the generation of differentiated OLs. Clonal analysis also demonstrated that CNTF directly targets OL progenitors derived from the multipotent cells. These observations suggest that two distinct progenitor cell pathways contribute to the generation of differentiated OLs during postnatal cortical gliogenesis. Although oligodendroglial maturation of classical OL progenitor cells is driven by cell autonomous mechanisms, our findings demonstrate that the generation of differentiated OLs from cortical multipotent progenitor cells is dependent on environmental cues, including activation of gp130/LIFbeta receptors.

Isolation and developmental characterization of cerebral cortical multipotent progenitors.

Multipotent neural progenitor species present within developing and adult periventricular generative zones can give rise to all of the major cellular elements of the brain. Although lineage specification during development has been thought to be restricted to these generative zones, we have utilized quantitative immunoselection techniques to isolate an enriched population of multipotent neural progenitor cells that express polysialylated neural cell adhesion molecule (PSA-NCAM) from postnatal day 2 cerebral cortex independent of generative zones. This population of cerebral cortical progenitor cells exhibited robust proliferation in response to epidermal growth factor and subsequently gave rise to clonally derived neurons, astrocytes, and oligodendrocytes. Quantitative regional analysis further demonstrated that while the multipotent cells derived from the cerebral cortex uniformly expressed PSA-NCAM, multipotent cells derived from generative zones contained equal proportions of PSA-NCAM-positive and -negative multipotent progenitor cells. The generation of individual cellular lineages from cortical multipotent progenitors could be enhanced by specific cytokines that are expressed within the cerebral cortex. Further, while oligodendroglial progenitor cells derived from cortical multipotent progenitors exhibited responsiveness to platelet-derived growth factor (PDGF) and neurotrophin-3 (NT-3), primary cultures of cortical oligodendroglial progenitors were responsive to PDGF but not to NT-3. These observations suggest that in addition to glial progenitors that commit to a specific lineage prior to migration from generative zones, there is within the cerebral cortex a separate pool of multipotent cells that are capable of generating mature glial progeny in response to specific environmental cues. Therapeutic interventions aimed at differentiation of endogenous cerebral pools of multipotent progenitors may provide a novel strategy for amelioration of the sequelae of environmental and genetic insults to the postnatal cerebrum.

Paracrine regulation of colony-stimulating factor-1 in medulloblastoma: implications for pathogenesis and therapeutic interventions.

OBJECTIVE: Colony-stimulating factor (CSF)-1, a chemotactic and mitogenic factor for macrophages and microglia, is expressed in a variety of nervous system tumors and when present in nonneural malignancies, is associated with marked inflammatory infiltrates, dissemination, and poorer prognosis. This study investigated the paracrine effects of CSF-1 production by medulloblastoma cells on the macrophage/microglial lineage. METHODS: A recurrent metastatic desmoplastic medulloblastoma was isolated from a 26-year-old man and propagated in tissue culture. Cellular phenotype and proliferation were assessed by immunocytochemical techniques; transcript expression for CSF-1, granulocyte macrophage-CSF, interleukin-3, and c-fms (the receptor for CSF-1) was examined with reverse transcriptase-polymerase chain reaction; and conditioned media and coculture paradigms were used to study cytokine effects on cellular proliferation. RESULTS: Serially passaged cells were uniformly immunoreactive for two lineage-independent neuroepithelial markers, nestin and vimentin. A subpopulation of cells with morphological characteristics of early differentiation stained for neurofilament 66 (7%) and microtubule-associated protein (5%) (markers of early neuronal precursors and postmitotic neurons, respectively) and for the Yp subunit of glutathione-S-transferase (3%) (a marker of early oligodendroglial progenitors). Tumor cells expressed transcripts for CSF-1, but not for granulocyte macrophage-CSF, interleukin-3, or c-fms. Treatment of microglia with serum-free medulloblastoma-conditioned media significantly increased proliferation (P < 0.001), suggesting the secretion of CSF-1. Coculture of medulloblastoma cells and microglia significantly increased proliferation of both cell types (each condition, P < 0.01). CONCLUSION: These observations suggest that CSF-1 mediates important paracrine interactions between transformed cells and the immune system, resulting in increased growth rate and metastatic potential. Future therapeutic goals need to include immunotherapeutic protocols to modulate this interaction.

Bone morphogenetic proteins induce astroglial differentiation of oligodendroglial-astroglial progenitor cells.

We have used bipotent postnatal cortical oligodendroglial-astroglial progenitor cells (O-2As) to examine the role of inductive signals in astroglial lineage commitment. O-2A progenitor cells undergo progressive oligodendroglial differentiation when cultured in serum-free medium, but differentiate into astrocytes in medium supplemented with FBS. We now report that the bone morphogenetic proteins (BMPs), a major subclass of the transforming growth factor beta (TGFbeta) superfamily, promote the selective, dose-dependent differentiation of O-2As into astrocytes with concurrent suppression of oligodendroglial differentiation. This astroglial-inductive action is not sanctioned by other members of the TGFbeta superfamily. Astroglial differentiation requires only very brief initial exposure to the BMPs and is accompanied by increased cellular survival and accelerated exit from cell cycle. Dual-label immunofluorescence microscopy documents that O-2A progenitor cells express a complement of BMP type I and type II receptor subunits required for signal transduction. Furthermore, expression of BMP2 in vivo reaches maximal levels during the period of gliogenesis. These results suggest that the BMPs act as potent inductive factors in postnatal glial lineage commitment that initiate a stable program of astroglial differentiation.

Nerve growth factor and neurotrophin-3 differentially regulate the proliferation and survival of developing rat brain oligodendrocytes.

There is increasing evidence that the neurotrophins, particularly nerve growth factor (NGF) and neurotrophin-3 (NT-3), play a role in the regulation of glial development in the CNS. Recent studies have shown that the proliferation of optic nerve-derived O2A progenitors (OLPs) is potentiated by NT-3 in combination with platelet-derived growth factor, whereas NT-3 alone supports the survival of their differentiated progeny (Barres et al., 1994). In this study, we have examined the expression of the high-affinity neurotrophin receptors (trks) and the low-affinity nerve growth factor receptor p75 in developing oligodendrocytes (OLs). In addition, we have examined the effects of NGF and NT-3 on proliferation and survival of OLPs and OLs, respectively. TrkC, the high-affinity NT-3 receptor, and trkA, the high-affinity NGF receptor, are both expressed from the early OLP through the mature OL stage. The truncated form of trkB, lacking the tyrosine kinase domain, and the low-affinity neurotrophin receptor p75 are expressed at low levels in OLPs and are upregulated in mature OLs. NGF and NT-3 both induced the phosphorylation of mitogen-activated protein kinase (MAPK) in OLPs and in OLs. In both OLPs and OLs, NT-3 sustained the activation of MAPK more than NGF. NT-3 enhanced the proliferation of OLPs and supported the survival of OLs. By contrast, unless coadministered with FGF-2, NGF did not exhibit mitogenic effects on OLPs but did enhance the survival of differentiated OLs. Our data demonstrate the presence of functional trkA and trkC in developing OLs and indicate that both NGF and NT-3 have a broad spectrum of developmental actions on cells of the OL lineage.

Microglial lineage species are expressed in mammalian epidermal growth factor-generated embryonic neurospheres.

The epigenetic signals and progenitor cell species involved in progressive neural maturation in the mammalian brain are poorly understood. Although these complex developmental issues can be examined in cultures of generative zone progenitor cells, analysis of signaling relationships in complex progenitor cell systems requires the meticulous definition of the cellular complement at each developmental stage. The presence of microglia within the generative zone cultures would further complicate these developmental analyses. Utilizing the microglial markers Griffonia simplicifolia B4 isolectin, carbocyanine dye-acetylated low density lipoprotein, F4/80, and Mac-1 we now report the presence of microglia within cultures of late embryonic murine epidermal growth factor-derived generative zone progenitor cells. Cytokine treatment of serially passaged epidermal growth factor-generated neurospheres altered the phenotype of the microglia in culture. Macrophage colony-stimulating factor treatment promoted the expression of spindle-shaped microglia, whereas granulocytemacrophage colony-stimulating factor treatment promoted the elaboration of flat and amoeboid microglia. Treatment with microglial-conditioned medium or 10% non-heat inactivated fetal calf serum led to an increased complement of both phenotypes. Microglia could be generated from single isolated neurospheres, and there were differences in the number of microglial lineage species obtained from distinct oligopotent progenitor cells, raising the possibility that a complement of this cellular lineage may be derived from a progenitor cell present within the generative zones. These observations indicate that microglia are present within the generative zone progenitor cell system, and this system thus represents an important experimental resource to examine the progenitor cell maturation and the origin of the microglial lineage.

Systemic cocaine challenge after chronic cocaine treatment reveals sensitization of extracellular dopamine levels in nucleus accumbens but direct cocaine perfusion into nucleus accumbens does not: implications for the neural locus of cocaine sensitization.

Rats were treated chronically with 20 mg/kg/day cocaine (by intraperitoneal injection) for 16 days, followed by 7 days of cocaine wash-out. On the next day, rats were challenged with an acute dose of cocaine administered by one of two routes (systemic or intracranial), and extra-cellular dopamine (DA) in the nucleus accumbens (Acb) was measured by in vivo microdialysis. Rats acutely challenged systemically with 20 mg/kg cocaine showed enhanced Acb extracellular DA levels (compared to control rats that had not previously received chronic cocaine). However, rats acutely challenged with intracranial cocaine by perfusion of 10(-5) M cocaine directly into the Acb did not. It is suggested that both the development and triggering of cocaine sensitization, as manifested by enhanced Acb DA content to subsequent acute cocaine challenge, may involve more than just neural mechanisms occurring locally within the Acb.

Cytokines regulate the cellular phenotype of developing neural lineage species.

The patterns and mechanisms of action of inductive signals that orchestrate neural lineage commitment and differentiation in the mammalian brain are incompletely understood. To examine these developmental issues, we have utilized several culture systems including conditionally immortalized cell lines, subventricular zone progenitor cells and primary neuronal cultures. A neural stem and progenitor cell line (MK31) was established from murine embryonic hippocampus by retroviral transduction of temperature-sensitive alleles of the simian virus 40 large tumor antigen. At the non-permissive temperature for antigen expression (39 degrees C) in serum-free media, the neural stem cells give rise to a series of increasingly mature neuronal progenitor and differentiated cellular forms under the influence of a subset of hematolymphopoietic cytokines (interleukins 5, 7, 9 and 11), when individually co-applied with transforming growth factor alpha, after pretreatment with basic fibroblast growth factor. These cellular forms elaborated a series of progressively more mature neurofilament proteins, a sequential pattern of ligand-gated channels, and inward currents and generation of action potentials with mature physiological properties. Because the factors regulating the development of central nervous system astrocytes have been so difficult to define, we have chosen to focus, in this manuscript, on the elaboration of this cell type. At 39 degrees C, application of a subfamily of bone morphogenetic proteins of the transforming growth factor beta superfamily of growth factors sanctioned the selective expression of astrocytic progenitor cells and mature astrocytes, as defined by sequential elaboration of the Yb subunit of glutathione-S-transferase and glial fibrillary acidic protein. These lineage-specific cytokine inductive relationships were verified using subventricular zone neural progenitor cells generated by the application of epidermal growth factor, alone or in combination with basic fibroblast growth factor, to dissociated cellular cultures derived from early embryonic murine brain, a normal non-transformed developmental population. Finally, application of a different series of cytokines from five distinct factor classes (basic fibroblast growth factor, platelet-derived growth factor-AA, insulin-like growth factor 1, neurotrophin 3 and representative gp130 receptor subunit-related ligands) caused the elaboration of oligodendroglial progenitor species and post-mitotic oligodendrocytes, defined by progressive morphological maturation and the expression of increasingly advanced oligodendroglial and oligodendrocyte lineage markers. In addition, seven different gp130-associated neuropoietic (ciliary neurotrophic factor, leukemia inhibitory factor, oncostatin-M) and hematopoietic (interleukins 6, 11, 12, granulocyte-colony stimulating factor) cytokines exhibited differential trophic effects on oligodendroglial lineage maturation and factor class interactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Ventral tegmental microinjection of delta 9-tetrahydrocannabinol enhances ventral tegmental somatodendritic dopamine levels but not forebrain dopamine levels: evidence for local neural action by marijuana's psychoactive ingredient.

Basal extracellular levels of dopamine (DA) and its metabolites in both ventral tegmental area (VTA) and nucleus accumbens (Acb) were simultaneously monitored by in vivo brain microdialysis in laboratory rats. Microinjection of 12 micrograms or 24 micrograms delta 9-tetrahydrocannabinol (delta 9-THC), the psychoactive ingredient in marijuana and hashish, into the VTA produced a dose-dependent increase in somatodendritic DA levels in VTA but failed to produce any simultaneous change in extracellular DA in Acb. Direct delta 9-THC perfusion (5 x 10(-5) and 2 x 10(-4) M) into Acb via the microdialysis probes caused a significant increase in extracellular DA levels in Acb. These findings suggest that (1) delta 9-THC has a facilitating effect on somatodendritic DA efflux, and (2) the elevation of Acb DA levels seen in our previous studies with systemic administration of delta 9-THC may result from local actions of delta 9-THC at or near the DA terminal projections in Acb.

Effects of the antiglucocorticoid RU 38486 on the induction of learned helpless behavior in Sprague-Dawley rats.

Learned helplessness (LH) is induced by exposure to an inescapable or uncontrollable stressor which results in an inability to escape or avoid the same stressor when subsequently presented in a different context. In order to understand which central mechanisms may influence the expression of the learned helpless phenotype, we have pursued an experimental approach that seeks to elucidate the behavioral effects of glucocorticoid (GC) hormones in this animal model of depression. We have previously shown that the induction of LH behavior is enhanced by adrenalectomy, an effect that is reversed by corticosterone. In this study, our aim was to attempt to locate CNS sites responsible for the observed effects of glucocorticoids on learned helpless behavior by introducing the type II GC receptor antagonist, RU 38486 to discrete brain regions. We did not observe a significant effect in LH with acute systemic, acute dentate gyrus or intracerebroventricular injection of RU 38486 in contrast to previous studies using the Porsolt swim test, another animal model of depression. However, we were able to observe a significant change upon chronic administration to the dentate gyrus. These findings suggest that glucocorticoids exert a long-term influence on stress-induced behavior, presumably by affecting glucocorticoid responsive genes in the dentate gyrus.

Prognosis of motor development and joint hypermobility.

In a study of 59 infants aged 18 months there were 20 with joint hypermobility and delayed motor development, 19 with joint hypermobility and normal motor development, and 20 normal controls. They were reassessed for motor function 3.5 years later at the age of 5 years. Both gross and fine motor performance were significantly delayed in the group of children who exhibited joint hypermobility and motor delay in infancy. No significant delay was evident in those with joint hypermobility only. Joint hypermobility resolved more frequently in children who presented normal motor development at age 18 months. Infants with joint hypermobility and motor delay are a subgroup associated with a less favourable motor outcome and careful follow up is indicated.