Identification of Urop11, a novel leptin-modulated gene that is upregulated in the hypothalamus of mice with virus-induced obesity.

Obesity results from disturbances of tightly regulated interactions between the nervous, endocrine, and metabolic systems that can be caused by external factors, such as viral infections. A mouse model of obesity induced by brain infection with a morbillivirus, canine distemper virus, allowed us to identify obesity-related genes. Using a subtractive library for the hypothalamus, the main brain structure regulating energy homeostasis, we identified a new gene on mouse chromosome 19 which we named upregulated obese product (Urop) 11 and, which has no homology with any known mRNA. A step-by-step molecular approach allowed us to isolate the full-length mRNA, predict the protein sequence, and identify consensus sites. Urop11 was mainly detected in the hypothalamus and adipocytes, and was dramatically upregulated in these central and peripheral structures in obese mice. Urop11 was also expressed in human neural and lymphoid samples and its expression seemed to be regulated by the state of lymphocyte activation. Interestingly, Urop11 expression was strongly upregulated both in vivo in mouse hypothalamus and in vitro in mouse neural cell lines, after leptin treatment. Taken together, our data show that Urop11 is a target of leptin, the satiety factor produced by adipocytes, in physiological and pathological conditions, including obesity. This new gene can be considered a key molecule in the hypothalamic integration pathway and demonstrates the importance of Urop11 as a target of leptin action.

Bone marrow CD34+/B220+ progenitors target the inflamed brain and display in vitro differentiation potential toward microglia.

Recent evidence indicates that microglial cells may not derive from blood circulating mature monocytes as they express features of myeloid progenitors. Here, we observed that a subpopulation of microglial cells expressed CD34 and B220 antigens during brain development. We thus hypothesized that microglia, or a subset of microglial cells, originate from blood circulating CD34+/B220+ myeloid progenitors, which could target the brain under developmental or neuroinflammatory conditions. Using experimental allergic encephalomyelitis (EAE) as a model of chronic neuroinflammation, we found that a discrete population of CD34+/B220+ cells expands in both blood and brain of diseased animals. In EAE mice, intravenous transfer experiments showed that macrophage-colony stimulating factor (M-CSF) -expanded CD34+ myeloid progenitors target the inflamed central nervous system (CNS) while keeping their immature phenotype. Based on these results, we then assessed whether CD34+/B220+ cells display in vitro differentiation potential toward microglia. For this purpose, CD34+/B220+ cells were sorted from M-CSF-stimulated bone marrow (BM) cultures and exposed to a glial cell conditioned medium. Under these experimental conditions, CD34+/B220+ cells were able to differentiate into microglial-like cells showing the morphological and phenotypic features of native microglia. Overall, our data suggest that under developmental or neuroinflammatory conditions, a subpopulation of microglial cells derive from CNS-invading CD34+/B220+ myeloid progenitors.

Extracellular matrix-remodeling metalloproteinases and infection of the central nervous system with retrovirus human T-lymphotropic virus type I (HTLV-I).

Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are involved in physiological processes and contribute to the phenotype of several pathological conditions associated with uncontrolled tissue degradation. In the central nervous system (CNS), MMPs are thought to play a role in cell migration and synaptic plasticity. We have investigated the expression, regulation and possible role of MMPs and TIMPs during infection of glial cells with human T-lymphotropic virus type I (HTLV-I), the causative agent of a progressive chronic myelopathy, TSP/HAM. The major alteration consists in a high increase in MMP-9 secretion and TIMP-2 mRNA expression. Cytokines TNF alpha and IL1 alpha, induced in glial cells during HTLV-I infection, promote the upregulation of MMP-9. In addition, cerebrospinal fluid from TSP/HAM patients contain high MMP-9 level. The exact role of dysregulated MMPs/TIMPs in the pathogenesis of TSP/HAM is not known; however, functions of these proteases in physiological processes should provide valuable clues. MMPs can affect the blood-brain barrier and the intercellular connectivity by degrading the extracellular matrix of endothelial and neural cells. They can be involved in autoimmunity by generating preformed specific peptides from myelin components. Finally, they can direct and prolong TNF activity in the CNS by converting its inactive precursor into active molecules.

[Dendritic cells and gliomas: a hope in immunotherapy?]. / Cellules dendritiques et gliomes: un espoir en immunothérapie?

Immunotherapy has been explored for several decades to try to improve the prognosis of gliomas, but until recently no therapeutic benefit has been achieved. The discovery of dendritic cells, the most potent professional antigen presenting cells to initiate specific immune response, and the possibility of producing them ex vivo gave rise to new protocols of active immunotherapy. In oncology, promising experimental and clinical therapeutic results were obtained using these dendritic cells loaded with tumor antigen. Patients bearing gliomas have deficit antigen presentation making this approach rational. In several experimental glioma models, independent research teams have showed specific antitumor responses using these dendritic cells. Phase I/II clinical trials have demonstrated the feasibility and the tolerance of this immunotherapeutic approach. In neuro-oncology, the efficiency of such an approach remains to be established, similarly in oncology where positive phase III studies are missing. Nevertheless, dendritic cells comprise a complex network which is only partially understood and capable of generating either immunotolerance or immune response. Numerous parameters remain to be explored before any definitive conclusion about their utility as an anticancer weapon can be drawn. It seems however logical that immunotherapy with dendritic cells could prevent or delay tumor recurrence in patients with minor active disease. A review on glioma and dendritic cells is presented.

Human primitive neuroectodermal tumour cells behave as multipotent neural precursors in response to FGF2.

Primitive neuroectodermal tumours (PNET) are thought to derive from the malignant transformation of pluripotent CNS precursors although this hypothesis has yet to be tested in vitro. Here we show that cells of a human PNET cell line 'Dev' express functional fibroblast growth factor (FGF) receptors (FGFR) and respond to FGF2 as multipotent CNS precursors. FGF2 induces tyrosine phosphorylation of FGFR-1 and FGFR-2 and many cellular substrates including MAP kinases and stimulates proliferation. Cells detach from plastic substrates and proliferate to form large clusters of undifferentiated cells. After adhesion to polylysine, cells migrate out from the clusters and differentiate. The majority of differentiated cells express neuronal phenotypes but distinct subpopulations express oligodendrocytic and astrocytic markers. Mature neural differentiation markers are not otherwise detected in Dev cells in defined medium. Identical results were obtained with 12 monoclonal subclones as well as the parent cell line, confirming that Dev cells are multipotent. This extends evidence that multipotent CNS precursors are the cellular substrate from which certain PNET develop and shows that FGF2 is a potent proliferation and differentiation inducer for PNET cells in vitro, suggesting that FGF2 may also modulate the evolution of PNET in vivo. Finally our results suggest that PNET cell lines may provide models to elucidate the biology of human multipotent CNS precursors.

Spatiotemporal expression patterns of metalloproteinases and their inhibitors in the postnatal developing rat cerebellum.

Matrix metalloproteinases (MMPs) are proteolytic enzymes that degrade the components of the extracellular matrix (ECM). The balance between MMPs and their inhibitors [tissue inhibitors of metalloproteinases (TIMPs)] in the pericellular environment determines the most significant proteolytic events in tissue remodeling. In vitro evidence is accumulating that these molecules may be crucial in the maturation of neural cells. Here, we investigated the in vivo expression of MMPs 2, 3, and 9 and TIMPs 1, 2, and 3 in the developing and adult rat cerebellum using immunohistochemistry and in situ hybridization. During postnatal development, all Purkinje (PK) cell somata expressed all the MMPs and TIMPs studied, whereas their growing dendritic trees expressed only MMP 3 and TIMP 3. In the adult, MMP 3 was confined to PK cell bodies, whereas TIMP 3 was expressed in PK cell somata and processes. Irrespective of the developmental stage, Bergmann glial processes contained only MMP 9, but their somata contained both TIMP 1 and MMP 9. In granular cells, MMPs 3 and 9 and TIMPs 1, 2, and 3 were chiefly detected at a time when migration is known to be maximal; except for that of TIMP 1, their expression persisted in the internal granular layer in the adult. The functional relevance of MMP expression was verified by gelatin zymography. MMP 9 activity was maximal on postnatal day 10 (P10) and was detectable at a low level on P15 and in the adult, whereas MMP 2 activity remained similar throughout postnatal development. Regional and cell-specific expression of MMPs and TIMPs closely reflects the successive stages of cerebellar development, thereby suggesting a pivotal role for ECM proteolysis in brain development and plasticity.

Isolation and expression pattern of human Unc-33-like phosphoprotein 6/collapsin response mediator protein 5 (Ulip6/CRMP5): coexistence with Ulip2/CRMP2 in Sema3a- sensitive oligodendrocytes.

The Unc-33-like phosphoprotein/collapsin response mediator protein (Ulip/CRMP) family consists of four homologous phosphoproteins considered crucial for brain development. Autoantibodies produced against member(s) of this family by patients with paraneoplastic neurological diseases have made it possible to clone a fifth human Ulip/CRMP and characterize its cellular and anatomical distribution in developing brain. This protein, referred to as Ulip6/CRMP5, is highly expressed during rat brain development in postmitotic neural precursors and in the fasciculi of fibers, suggesting its involvement in neuronal migration/differentiation and axonal growth. In the adult, Ulip6/CRMP5 is still expressed in some neurons, namely in areas that retain neurogenesis and in oligodendrocytes in the midbrain, hindbrain, and spinal cord. Ulip2/CRMP2 and Ulip6/CRMP5 are coexpressed in postmitotic neural precursors at certain times during development and in oligodendrocytes in the adult. Because Ulip2/CRMP2 has been reported to mediate semaphorin-3A (Sema3A) signal in developing neurons, in studies to understand the function of Ulip6/CRMP5 and Ulip2/CRMP2 in the adult, purified adult rat brain oligodendrocytes were cultured in a Sema3A-conditioned medium. Oligodendrocytes were found to have Sema3A binding sites and to express neuropilin-1, the major Sema3A receptor component. In the presence of Sema3A, these oligodendrocytes displayed a dramatic reduction in process extension, which was reversed by removal of Sema3A and prevented by anti-neuropilin-1, anti-Ulip6/CRMP5, anti-Ulip2/CRMP2 antibodies, or VEGF-165, another neuropilin-1 ligand. These results indicate the existence in the adult brain of a Sema3A signaling pathway that modulates oligodendrocyte process extension mediated by neuropilin-1, Ulip6/CRMP5, and Ulip2/CRMP2, and they open new fields of investigation of neuron/oligodendrocyte interactions in the normal and pathological brain.

Semaphorin 3A-vascular endothelial growth factor-165 balance mediates migration and apoptosis of neural progenitor cells by the recruitment of shared receptor.

The dynamic and coordinated interaction between cells and their microenvironment controls cell migration, proliferation, and apoptosis, mediated by different cell surface molecules. We have studied the response of a neuroectodermal progenitor cell line, Dev, to a guidance molecule, semaphorin 3A (Sema3A), described previously as a repellent-collapsing signal for axons, and we have shown that Sema3A acts as a repellent guidance cue for migrating progenitor cells and, on prolonged application, induces apoptosis. Both repulsion and induction of cell death are mediated by neuropilin-1, the ligand-binding component of the Sema3A receptor. The vascular endothelial growth factor, VEGF165, antagonizes Sema3A-induced apoptosis and promotes cell survival, migration, and proliferation. Surprisingly, repulsion by Sema3A also depends on expression of VEGFR1, a VEGF165 receptor, expressed in Dev cells. Moreover, we found that these repulsive effects of Sema3A require tyrosine kinase activity, which can be attributed to VEGFR1. These results indicate that the balance between guidance molecules and angiogenic factors can modulate the migration, apoptosis (or survival), and proliferation of neural progenitor cells through shared receptors.

Retroviral vectors for the expression of two genes in human multipotent neural precursors and their differentiated neuronal and glial progeny.

Retroviral vectors allow stable integration of exogenous DNA into genomic DNA and therefore gene transmission to progeny. Multipotent neural precursors and immortal cell lines prepared from them have been demonstrated to integrate into either adult or developing brain in a nontumorigenic, functional manner, without interfering with normal neurobiological processes. These cells thus appear to provide a Trojan horse ideally adapted to directing the expression of transgenes appropriately in a host brain. Here we investigated and optimized the transduction capacity of MuLV-based retroviral vectors in which internal ribosomal entry segments (IRESs) drive coexpression of two heterologous gene products from a single bicistronic mRNA in a human multipotent neural precursor cell line, "Dev," which was prepared from a medulloblastoma. For this, two vectors containing two different combinations of three viral IRESs were used and the capacity of different pseudotyped vectors to permit an efficient and stable transduction of Dev cells was compared. Our data show that (1) the best recombinant vectors for Dev cell transduction are those pseudotyped with the 10A1 MuLV envelope (40% of transduction) and (2) the initial coexpression of neo and plap, observed in transduced undifferentiated Dev cells, is maintained in differentiated Dev cells with a neuronal or glial phenotype. Therefore, these double-IRES vectors may provide an efficient means of transducing the coexpression of two proteins in undifferentiated human neural precursors that is maintained in their differentiated progeny. These data suggest that the double-IRES strategy is well adapted to potential therapeutic situations when coexpression of two different transgenes may be required in the same cell.

Inhibition of tyrosine hydroxylase expression within the substantia nigra of mice infected with canine distemper virus.

Experimental infection of mouse brain with a neuroadapted strain of canine distemper virus (CDV) leads to early acute encephalitis, followed by late neurological diseases such as motor pathologies (paralysis and turning behavior) or obesity syndrome. We have previously shown that, during the early stage of infection, CDV replicates transiently in selective structures of the brain including the substantia nigra, a structure known to play a critical role in motor control. In this study we demonstrate that CDV replication in the substantia nigra induces an early decrease in transcript level of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. The CDV infection of neuroblastoma cell culture, constitutively expressing TH, results in downregulation of TH transcription in the absence of cell death. In the few surviving mice with motor deficiencies, a pronounced decrease in TH expression is associated with a loss of dopaminergic cell bodies in the absence of any viral transcripts and proteins, suggesting that the initial CDV infection was sufficient to trigger irreversible neurodegenerative processes.

Retroviral infection (HTLV-I) induces cytokine-regulated immunomodulation and cytotoxicity of medulloblastoma cells.

Primitive neuroectodermal tumors are thought to result from disturbed differentiation of neuroepithelial stem cells. These tumor cells retain the capacity to differentiate toward the neuron or glia phenotype under extrinsic stimuli. Previously, we have developed a model for the differentiation of a medulloblastoma cell line (Dev cells) induced by infection with the human retrovirus HTLV-I. This virus delivers signals which trigger the Dev cells to differentiate toward an astrocytic lineage. The aim of this study was to characterize the time course of viral infection, to identify the soluble factors released and to analyze their effects on Dev cells. The early phase of viral replication is followed by latent infection. Viral infection induces glial differentiation in a subpopulation of cells and results in the death of others. The inflammatory cytokines TNF alpha, IL1 alpha and IL6 were detected in medium conditioned by infected Dev cells. TNF alpha was cytotoxic and cytostatic for subpopulations of Dev cells. Furthermore, TNF alpha treatment reproduced the modulation of expression of the major histocompatibility complex antigens (MHC class I) observed in infected Dev cells. These observations support the view that HTLV-I infection, which triggers glial differentiation of medulloblastoma Dev cells, also causes the release of soluble factors capable of downregulating proliferation of dividing tumor cells and of modifying their recognition by cellular immune effectors.

POP66, a paraneoplastic encephalomyelitis-related antigen, is a marker of adult oligodendrocytes.

Paraneoplastic neurological diseases are disorders of the central nervous system, associated with neuronal degeneration in patients with systemic cancer, but are not a direct result of the tumor mass or metastasis. The biological diagnosis of these syndromes is based mainly on the detection, in the patient's serum and cerebrospinal fluid, of autoantibodies (anti-Hu, anti-Yo, for example), suggesting an autoimmune origin for these disorders. Recently, we described novel autoantibodies (anti-CV2 autoantibodies) associated with paraneoplastic neurological disease, which recognize a 66 kDa brain protein. We named this antigen POP66, for Paraneoplastic Oligodendrocyte Protein of 66 kDa molecular weight, as in the adult human, rat, and mouse brain, it is specifically expressed by a subpopulation of oligodendrocytes. This cell type specificity was surprising given the fact that the cell loss in the brains of patients with anti-CV2 autoantibodies is neuronal. POP66-expressing oligodendrocytes are distributed along the longitudinal axis of the brain according to an increasing rostro-caudal gradient, with no positive oligodendrocytes being found in the forebrain and the greatest number found in the spinal cord. In addition, in transverse sections of the spinal cord, the distribution of POP66-positive oligodendrocytes follows an increasing dorsal-to-ventral gradient, which may be related to different oligodendrocyte precursor pools. In addition, the neuronal loss without demyelination seen in the brains of patients with anti-CV2 autoantibodies, together with the exclusive oligodendroglial expression of POP66 in the adult brain, raises the question of the possible involvement of POP66 in neuron survival via neuron/oligodendrocyte interactions.

Brain structures selectively targeted by canine distemper virus in a mouse model infection.

Paramyxoviruses such as measles virus or canine distemper virus are etiological agents for acute and chronic encephalitis (measles inclusion body encephalitis, subacute sclerosing panencephalitis and chronic distemper encephalitis or old dog encephalitis). The mechanisms by which viral injury leads to neurological diseases have not yet been fully elucidated. We have developed an experimental model in mice in order to analyze the spatial and temporal distribution of canine distemper virus in the central nervous system. Cerebral target structures for viral replication were examined for the presence of viral material (proteins and mRNA) during the two stages of the biphasic disease. During the acute stage of infection all target areas could be identified by day 6 with a similar anatomical distribution in all the animals examined, which were either intracranially or intracerebroventricularly infected. Viral mRNA and proteins were selectively localized in certain brain structures such as the thalamus, hypothalamus, substantia nigra (pars compacta), locus ceruleus and raphe nuclei (dorsalis and centralis), and limbic system (hippocampus, septum, entorhinal and cingulate cortex, amygdala). The virus was apparently unable to replicate in cerebellum, striatum, a large part of cortex, or endothelial cells. During the subacute disease, viral material was no longer detectable except in a few structures such as hypothalamus up to 4-6 weeks after inoculation. After this time, all target structures were devoid of any labeling in spite of the occurrence of pathology (obesity, paralysis) during this viral quiescent phase. These results suggest that after the initial viral exposure, expression of viral genes in defined structures might disrupt central homeostasis and finally may lead to neurological or neuroendocrine diseases, even in the absence of the hallmarks of the virus.

Collapsin response mediator protein-3/unc-33-like protein-4 gene: organization, chromosomal mapping and expression in the developing mouse brain.

CRMPs (collapsin response mediator proteins)/ULIPs (unc-33-like proteins) are a family of intracytoplasmic proteins that are expressed mainly in the brain. The involvement of CRMP/ULIP members in neuronal differentiation, growth cone motility and axonal collapse has been suggested. We recently found that a member of this family, CRMP3/ULIP4, corresponds to POP66 (paraneoplastic oligodendrocyte protein of 66 kDa), a protein which may be associated with auto-immune induced-neuronal degeneration in paraneoplastic neurological syndromes. However, the physiological functions of these proteins remain to be elucidated. Further studies, including the generation of cell lines and of animals with modified/disrupted CRMP/ULIP gene expression, are necessary to explore the functions of this protein. We have cloned and determined the organization and chromosomal localization of the mouse gene encoding CRMP3/ULIP4. The gene is composed of 14 exons and spans more than 20 kb. We assigned the mouse CRMP3/ULIP4 gene to the distal end of chromosome 7. In mouse brain, in situ hybridization showed that CRMP3/ULIP4 mRNA is expressed mainly in the dentate gyrus of hippocampus, in the granular layers of cerebellum and in the inferior olive of the pons, the nucleus which controls movement and posture, and adjusts the major output of descending motor system.

Specific potentialities of embryonic rat serotonergic neurons to innervate different periventricular targets in the adult brain.

During the development of the central nervous system, neurons are directed by both genetic and environmental factors to differentiate and form connections with their targets. We took advantage of the abundant homogeneous serotonergic innervations of the ependyma forming the supra- and subependymal plexuses to investigate possible commitment of embryonic neurons to innervate specific targets during axogenesis in the rat. The origin of the supraependymal innervation was determined by retrograde transport of cholera toxin (CT) from the ventricles. The supraependymal plexuses of the fourth ventricle mainly originated from neurons in the dorsocaudal region of the raphe dorsalis (DRN), while the rostral DRN and raphe centralis (CRN) contained perikarya projecting into the third ventricle. This suggested the existence, along the rostrocaudal axis of the raphe, of different neuronal subsets able to form distinct supraependymal plexuses in the third or fourth ventricle. To determine whether serotonergic neurons were committed to innervate specific areas of the ependyma, different embryonic metencephalic segments (rostral, median, or caudal) from 14-day-old rat embryos were independently grafted into the third or fourth ventricle of an adult brain in which the serotonergic neurons had been previously destroyed. The distinctive patterns of re-innervation specific to each of grafted segments indicate that subsets of embryonic serotonergic neurons are indeed committed to innervate certain restricted ependymal areas of the adult brain, presumably in response to different neurotropic and/or neurotrophic cues.

Autoantibodies to glutamate decarboxylase in a patient with cerebellar cortical atrophy, peripheral neuropathy, and slow eye movements.

OBJECTIVE: To study the existence of autoimmunity against the cerebellum in patients with sporadic cortical cerebellar atrophy. DESIGN: The presence of autoantibodies against the cerebellum in the serum and cerebrospinal fluid samples that were obtained from patients with sporadic cortical cerebellar atrophy and control patients was investigated by using immunohistochemical techniques. SETTING: University hospital and research laboratory in Lyons, France. PATIENTS: Eight patients with cortical cerebellar atrophy that was associated with or without other neurological symptoms; 350 patients with various neurological diseases; and 33 normal, healthy subjects. OUTCOME MEASURES: Serum and cerebrospinal fluid anti-cerebellar autoantibodies were investigated by using indirect immunofluorescence techniques in rat cerebellum. To characterize antigen labeled by patient's serum, we used an immunotrapping enzyme activity assay of glutamate decarboxylase. RESULTS: Serum and cerebrospinal fluid samples that were taken from one patient with sporadic cortical cerebellar atrophy associated with peripheral neuropathy and slow eye movements contained anti-glutamate decarboxylase autoantibodies. CONCLUSIONS: These results suggest a participation of autoimmunity in the pathogenesis of some cases of sporadic cerebellar cortical atrophy and the involvement of the cerebellar gamma-aminobutyric acid-ergic system in the pathogenesis of this disease.

Cytokines are increased in the rat hippocampus after serotonergic neuron degeneration and upregulate the expression of GDH, an enzyme involved in glutamate detoxification.

The degeneration of serotonergic neurons increases the expression of glutamate dehydrogenase (GDH) in hippocampal astrocytes. This process was demonstrated to be independent of the serotonin level. At the same time, upregulation of tumor necrosis factor (TNF) alpha and interleukin (IL)-1 alpha mRNA were observed, whereas levels of transforming growth factor (TGF) beta 1 mRNA remained unchanged. The level of GDH mRNA was increased in primary cultures of hippocampal astrocytes treated with TNF alpha and IL-1 alpha suggesting that these cytokines act on the GDH metabolism. TNF alpha and IL-1 alpha induced an increase in GDH promoter activity in C8S (an astrocytic cell line) transfected with constructs containing 5' flanking genomic sequences of GDH driving the expression of a reporter gene. These observations suggest that cytokines may be signals that upregulate the astrocytic GDH expression in response to the degeneration of serotonergic terminals in the hippocampus.

Selective induction of cytokines in mouse brain infected with canine distemper virus: structural, cellular and temporal expression.

We have previously shown that, in experimentally inoculated mice, canine distemper virus (CDV), a neurotropic virus, selectively infects certain brain structures (hypothalamus, hippocampus, monoaminergic nuclei, etc). Here we demonstrate that tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta and IL-6 transcripts are selectively expressed in these CDV-targeted structures, except in the dentate gyrus, where cytokines are induced without prior CDV replication. The time-course of TNF-alpha expression vs. viral replication in the hypothalamus was different from that in hippocampus. In addition, we show that a substantial number of neurons express TNF-alpha and IL-6. These findings provide new insights into the possible participation of cytokines in the neurological disorders triggered by CDV infection.

Differentiation of a medulloblastoma cell line towards an astrocytic lineage using the human T lymphotropic retrovirus-1.

Constituent cells of medulloblastoma, the most common brain tumor occurring in childhood, resemble the primitive neuroepithelial cells normally found in the developing nervous system. However, mutational events prevent their further differentiation. We used the human T cell lymphotrophic virus type 1 to activate these deregulated immature cells by means of its transactivating protein Tax. Concomitant with viral infection was a decrease in cell proliferation characterized by inhibition of [3H]thymidine incorporation and in the number of cells in the G2/M phase of the cell cycle. Morphological changes suggested that medulloblastoma cells differentiated along the astrocytic lineage. The glial phenotype was confirmed by the induction of the glial fibrillary acidic protein and the glial enzyme glutamine synthetase. A direct viral effect and/or secondary effects to viral infection via paracrine/autocrine pathways could counterbalance the maturational defect in these medulloblastoma cells.

gamma-Aminobutyric acid and 5-hydroxytryptamine interrelationship in the rat nucleus raphe dorsalis: combination of radioautographic and immunocytochemical techniques at light and electron microscopy levels.

Serotonin and gamma-aminobutyric acid (GABA) neurons in the nucleus raphe dorsalis were identified by immunocytochemistry using antibodies to 5-hydroxytryptamine or GABA. The pattern of the 5-hydroxytryptamine and GABA immunostaining presented similar features: 5-hydroxytryptamine or GABA immunoreactive somata were fusiform or ovoid (15-20 micron) and positive dendritic profiles were found either without any connection with other nerve elements or in contact with one or several terminals. In addition, some 5-hydroxytryptamine nerve endings were apposed to 5-hydroxytryptamine immunoreactive cell bodies or dendrites; also some GABA-immunopositive terminals were in contact with GABA-immunopositive nerve cell bodies. On the other hand, GABA and 5-hydroxytryptamine patterns may be differentiated in several respects: the 5-hydroxytryptamine-reactive nerve cell bodies were more numerous than the GABA ones. Some small, round (8-10 micron) nerve cell bodies were reactive with GABA antiserum, but no neurons of this type were reactive with a 5-hydroxytryptamine antiserum; finally, GABA nerve terminals were more numerous than 5-hydroxytryptamine ones. In order to understand the relationship between GABA and 5-hydroxytryptamine neurons, radioautographic and immunocytochemical procedures were combined: 5-hydroxytryptamine and GABA immunocytochemistry was combined with radioautography of [3H]GABA and [3H]5-hydroxytryptamine uptake, respectively. Some nerve cell bodies, dendrites or terminals, which were 5-hydroxytryptamine-immunopositive, were also capable of accumulating [3H]GABA and, conversely, some GABA-immunopositive elements were capable of accumulating [3H]5-hydroxytryptamine. Moreover, several nerve elements were reactive with both glutamate decarboxylase and 5-hydroxytryptamine antisera. These data confirm in electron microscopy previous studies suggesting the coexistence of both GABA and 5-hydroxytryptamine in the same neurons. The presence of uptake mechanisms for GABA and 5-hydroxytryptamine may indicate the action of both neurotransmitters in the same neuron. On the other hand, the [3H]GABA-labelled nerve endings in contact with 5-hydroxytryptamine-positive dendrites or nerve cell bodies indicate the possibility of a GABAergic control of the activity of some 5-hydroxytryptamine neurons; this corroborates biochemical and electrophysiological studies whereby a trans-synaptic control of the 5-hydroxytryptamine neurons by GABA may be envisaged.