RESUMEN
Proteoglycans are abundant in the developing brain and there is much circumstantial evidence for their roles in directional neuronal movements such as cell body migration and axonal growth. We have developed an in vitro model of astrocyte cultures of the lateral and medial sectors of the embryonic mouse midbrain, that differ in their ability to support neuritic growth of young midbrain neurons, and we have searched for the role of interactive proteins and proteoglycans in this model. Neurite production in co-cultures reveals that, irrespective of the previous location of neurons in the midbrain, medial astrocytes exert an inhibitory or nonpermissive effect on neuritic growth that is correlated to a higher content of both heparan and chondroitin sulfates (HS and CS). Treatment of astrocytes with chondroitinase ABC revealed a growth-promoting effect of CS on lateral glia but treatment with exogenous CS-4 indicated a U-shaped dose-response curve for CS. In contrast, the growth-inhibitory action of medial astrocytes was reversed by exogenous CS-4. Treatment of astrocytes with heparitinase indicated that the growth-inhibitory action of medial astrocytes may depend heavily on HS by an as yet unknown mechanism. The results are discussed in terms of available knowledge on the binding of HS proteoglycans to interactive proteins, with emphasis on the importance of unraveling the physiological functions of glial glycoconjugates for a better understanding of neuron-glial interactions
Asunto(s)
Animales , Axones , Sulfatos de Condroitina , Heparitina Sulfato , Mesencéfalo , Neuronas , Astrocitos , División Celular , Células Cultivadas , Mesencéfalo , NeuroglíaRESUMEN
Selective pressures from polluted environments have led to the development of resistance systems in aquatic organisms. Using different techniques, this study examined a cadmium defense mechanism of the freshwater unicellular protozoa Euglena gracilis, and found it to be an efflux pump similar to the multidrug resistance P-glycoprotein. Cd(2+)-treated E. gracilis were able to extrude Rhodamine 123 at 21 degrees C, but not at 4 degrees C. Furthermore, verapamil, a P-glycoprotein modulator, partially blocked the efflux process (at 21 degrees C), and enhanced the Cd(2+) toxic effects on these cells. Western immunoblots of cell lysates, using the anti-P-glycoprotein antibody JSB-1, revealed a 120-KDa protein, which was expressed, in high amounts on Cd(2+)-exposed cells (74% above the control values). Moreover, cells treated with JSB-1 became more sensitive to the harmful effects of cadmium, showing a decreased survival rate. Taken together, these results suggest that a MDR phenotype has evolved in Euglena as one of the mechanisms for cadmium detoxification.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/fisiología , Cadmio/farmacología , Euglena gracilis/fisiología , Subfamilia B de Transportador de Casetes de Unión a ATP/análisis , Subfamilia B de Transportador de Casetes de Unión a ATP/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Western Blotting , Cadmio/toxicidad , División Celular/efectos de los fármacos , División Celular/fisiología , Relación Dosis-Respuesta a Droga , Resistencia a Medicamentos , Euglena gracilis/efectos de los fármacos , Proteínas de Transporte de Membrana/fisiología , Microscopía Fluorescente , Rodamina 123/metabolismo , Rodamina 123/farmacología , Temperatura , Verapamilo/farmacologíaRESUMEN
Proteoglycans are abundant in the developing brain and there is much circumstantial evidence for their roles in directional neuronal movements such as cell body migration and axonal growth. We have developed an in vitro model of astrocyte cultures of the lateral and medial sectors of the embryonic mouse midbrain, that differ in their ability to support neuritic growth of young midbrain neurons, and we have searched for the role of interactive proteins and proteoglycans in this model. Neurite production in co-cultures reveals that, irrespective of the previous location of neurons in the midbrain, medial astrocytes exert an inhibitory or nonpermissive effect on neuritic growth that is correlated to a higher content of both heparan and chondroitin sulfates (HS and CS). Treatment of astrocytes with chondroitinase ABC revealed a growth-promoting effect of CS on lateral glia but treatment with exogenous CS-4 indicated a U-shaped dose-response curve for CS. In contrast, the growth-inhibitory action of medial astrocytes was reversed by exogenous CS-4. Treatment of astrocytes with heparitinase indicated that the growth-inhibitory action of medial astrocytes may depend heavily on HS by an as yet unknown mechanism. The results are discussed in terms of available knowledge on the binding of HS proteoglycans to interactive proteins, with emphasis on the importance of unraveling the physiological functions of glial glycoconjugates for a better understanding of neuron-glial interactions.
Asunto(s)
Axones/fisiología , Sulfatos de Condroitina/fisiología , Heparitina Sulfato/fisiología , Mesencéfalo/embriología , Neuronas/fisiología , Agrecanos , Animales , Astrocitos/efectos de los fármacos , Astrocitos/fisiología , División Celular/fisiología , Movimiento Celular , Células Cultivadas , Proteoglicanos de Heparán Sulfato/fisiología , Mesencéfalo/citología , Ratones , Neuroglía/fisiología , Polisacárido Liasas/farmacología , Proteoglicanos/fisiologíaAsunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Hipocampo/efectos de los fármacos , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Nitrofenoles/farmacología , Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/química , Animales , Células Cultivadas , Hipocampo/citología , Hipocampo/metabolismo , Humanos , Microinyecciones , Neuronas/citología , Neuronas/metabolismo , Estructura Terciaria de Proteína , RatasRESUMEN
Astroglial cells derived from lateral and medial midbrain sectors differ in their abilities to support neuritic growth of midbrain neurons in cocultures. These different properties of the two types of cells may be related to the composition of their extracellular matrix. We have studied the synthesis and secretion of sulfated glycosaminoglycans (GAGs) by the two cell types under control conditions and beta-D-xyloside-stimulated conditions, that stimulate the ability to synthesize and release GAGs. We have confirmed that both cell types synthesize and secrete heparan sulfate and chondroitin sulfate. Only slight differences were observed between the proportions of the two GAGs produced by the two types of cells after a 24-h labeling period. However, a marked difference was observed between the GAGs produced by the astroglial cells derived from lateral and medial midbrain sectors. The medial cells, which contain derivatives of the tectal and tegmental midline radial glia, synthesized and secreted approximately 2.3 times more chondroitin sulfate than lateral cells. The synthesis of heparan sulfate was only slightly modified by the addition of beta-D-xyloside. Overall, these results indicate that astroglial cells derived from the two midbrain sectors have marked differences in their capacity to synthesize chondroitin sulfate. Under in vivo conditions or a long period of in vitro culture, they may produce extracellular matrix at concentrations which may differentially affect neuritic growth.
Asunto(s)
Astrocitos/metabolismo , Glicosaminoglicanos/biosíntesis , Mesencéfalo/metabolismo , Sulfatos/metabolismo , Animales , Técnicas de Cultivo de Célula , Sulfatos de Condroitina/biosíntesis , Sulfatos de Condroitina/metabolismo , Electroforesis en Gel de Agar , Glicosaminoglicanos/metabolismo , Heparitina Sulfato/biosíntesis , Heparitina Sulfato/metabolismo , Mesencéfalo/citología , RatonesRESUMEN
Astroglial cells derived from lateral and medial midbrain sectors differ in their abilities to support neuritic growth of midbrain neurons in cocultures. These different properties of the two types of cells may be related to the composition of their extracellular matrix. We have studied the synthesis and secretion of sulfated glycosaminoglycans (GAGs) by the two cell types under control conditions and ß-D-xyloside-stimulated conditions, that stimulate the ability to synthesize and release GAGs. We have confirmed that both cell types synthesize and secrete heparan sulfate and chondroitin sulfate. Only slight differences were observed between the proportions of the two GAGs produced by the two types of cells after a 24-h labeling period. However, a marked difference was observed between the GAGs produced by the astroglial cells derived from lateral and medial midbrain sectors. The medial cells, which contain derivatives of the tectal and tegmental midline radial glia, synthesized and secreted ~2.3 times more chondroitin sulfate than lateral cells. The synthesis of heparan sulfate was only slightly modified by the addition of ß-D-xyloside. Overall, these results indicate that astroglial cells derived from the two midbrain sectors have marked differences in their capacity to synthesize chondroitin sulfate. Under in vivo conditions or a long period of in vitro culture, they may produce extracellular matrix at concentrations which may differentially affect neuritic growth
Asunto(s)
Animales , Ratones , Astrocitos/metabolismo , Glicosaminoglicanos/biosíntesis , Mesencéfalo/citología , Sulfatos/metabolismo , Ésteres del Ácido Sulfúrico , Astrocitos/metabolismo , Técnicas de Cultivo de Célula , Sulfatos de Condroitina/biosíntesis , Sulfatos de Condroitina/metabolismo , Electroforesis en Gel de Agar , Glicosaminoglicanos/metabolismo , Heparitina Sulfato/biosíntesis , Heparitina Sulfato/metabolismoRESUMEN
Lateral (L) and medial (M) midbrain astrocytes differ in their ability to support neuritic growth (L, permissive; M, non-permissive) with properties of M glia depending on heparan sulfate (HS). Here we show by atomic force microscopy that the surfaces of formaldehyde-fixed astrocytes differ by conspicuous 250 nm protrusions in L and by a HS-dependent fibrillar network in M glia, thus, demonstrating correlations between cell surface morphology and functional properties.
Asunto(s)
Astrocitos/citología , Astrocitos/fisiología , Microscopía de Fuerza Atómica , Neuritas/fisiología , Animales , Astrocitos/efectos de los fármacos , Membrana Celular/ultraestructura , Fijadores , Formaldehído , Heparitina Sulfato/farmacología , Mesencéfalo/citología , Ratones/embriología , Red Nerviosa/efectos de los fármacos , Red Nerviosa/fisiología , Neurofibrillas/fisiologíaRESUMEN
Radial glial cells and astrocytes are heterogeneous with respect to morphology, cytoskeletal- and membrane-associated molecules and intercellular interactions. Astrocytes derived from lateral (L) and medial (M) midbrain sectors differ in their abilities to support neuritic growth of midbrain neurons in coculture (Garcia-Abreu et al. J Neurosci Res 40:471, 1995). There is a correlation between these abilities and the differential patterns of laminin (LN) organization that is fibrillar in growth-permissive L astrocytes and punctate in the non-permissive M astroglia (Garcia-Abreu et al. NeuroReport 6:761, 1995). There are also differences in the production of glycosaminoglycans (GAGs) by L and M midbrain astrocytes (Garcia-Abreu et al. Glia 17:339, 1996). We show that the relative amounts of the glycoproteins laminin LN, fibronectin (FN) and tenascin (TN) are virtually identical in L and M glia, thus, confirming that an abundant content of LN is not sufficient to promote neurite growth. To further analyze the role of GAGs in the properties of M and L glia, we employed enzymatic degradation of the GAGs chondroitin sulfate (CS) and heparan sulfate (HS). Treatment with chondroitinase has little effect on the non-permissive properties of M glia but reduces the growth-supporting ability of L glia. By contrast, heparitinase I produces no significant changes on L glia but leads to neurite growth promotion by M glia. Taken together, these results suggest that glial CS helps to promote neurite growth and, more importantly, they indicate that a HS proteoglycan is, at least, partially responsible for the non-permissive role of the midline glia to the growth of midbrain neurites.
Asunto(s)
Heparitina Sulfato/fisiología , Mesencéfalo/fisiología , Neuritas/fisiología , Neuroglía/fisiología , Animales , Astrocitos/metabolismo , Astrocitos/fisiología , Células Cultivadas , Embrión de Mamíferos , Fibronectinas/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Glicosaminoglicanos/metabolismo , Immunoblotting , Laminina/metabolismo , Mesencéfalo/citología , Ratones , Tenascina/metabolismoRESUMEN
As a result of recent investigations, the cytoskeleton can be viewed as a cytoplasmic system of interconnected filaments with three major integrative levels: self-assembling macromolecules, filamentous polymers, e.g., microtubules, intermediate filaments and actin filaments, and supramolecular structures formed by bundles of these filaments or networks resulting from cross-bridges between these major cytoskeletal polymers. The organization of this biological structure appears to be sensitive to fine spatially and temporally dependent regulatory signals. In differentiating neurons, regulation of cytoskeleton organization is particularly relevant, and the microtubule-associated protein (MAP) tau appears to play roles in the extension of large neuritic processes and axons as well as in the stabilization of microtubular polymers along these processes. Within this context, tau is directly involved in defining neuronal polarity as well as in the generation of neuronal growth cones. There is increasing evidence that elements of the extracellular matrix contribute to the control of cytoskeleton organization in differentiating neurons, and that these regulations could be mediated by changes in MAP activity. In this brief review, we discuss the possible roles of tau in mediating the effects of extracellular matrix components on the internal cytoskeletal arrays and its organization in growing neurons.
Asunto(s)
Matriz Extracelular/fisiología , Proteínas Asociadas a Microtúbulos/fisiología , Neuronas/fisiología , Proteínas tau/fisiología , Proteínas del Citoesqueleto , Regulación del Desarrollo de la Expresión Génica , Morfogénesis , Factores de Crecimiento Nervioso , NeuroglíaRESUMEN
Gap-junctional communication between neurons and astrocytes dissociated from rat brain was identified in culture by using dye-transfer assays and electrophysiological measurements. Cell types were identified by using antibodies against beta-tubulin III, glial fibrillary acidic protein, and 2',3'-cyclic-nucleotide phosphohydrolase, which are antigenic determinants of neurons, astroglia, and oligodendrocytes, respectively. Dye coupling was examined as a function of time after dissociated embryonic brain cells were plated onto confluent monolayers of postnatal astrocytes by intracellularly injecting the fluorochrome Lucifer yellow. Coupling of neurons to the astrocytic monolayer was most frequent between 48 h and 72 h in culture and declined over the next 4 days. This gradual uncoupling was accompanied by progressive neuronal maturation, as indicated by morphological measurements in camera lucida drawings. Dye spread was abolished reversibly by octanol, an agent that blocks gap junction channels in other systems. Double whole-cell voltage-clamp measurements confirmed the presence of heterocellular electrical coupling in these cocultures. Coupling was also seen between neurons and astrocytes in cocultures of cells dissociated from embryonic cerebral hemispheres but was rarely detectable in cocultures of postnatal brain cells. These data strongly suggest that junctional communication may provide metabolic and electrotonic interconnections between neuronal and astrocytic networks at early stages of neural development and that such interactions are weakened as differentiation progresses.
Asunto(s)
Astrocitos/citología , Sistema Nervioso Central/citología , Uniones Comunicantes/fisiología , Neuronas/citología , Animales , Astrocitos/fisiología , Comunicación Celular/fisiología , Células Cultivadas , Sistema Nervioso Central/fisiología , Técnicas de Cocultivo , Colorantes Fluorescentes , Isoquinolinas , Neuronas/fisiología , Ratas , Ratas WistarRESUMEN
In order to investigate the influence of neuron-glia interaction on astrocyte differentiation, we used a transgenic mouse bearing part of the gene promoter of the astrocytic maturation marker GFAP linked to the beta-galactosidase (beta-gal) reporter gene. Addition of embryonic cerebral hemisphere (CH) neurons to transgenic CH astrocyte monolayers increased by 50-60% beta-gal positive cell number. Such event was dependent on the brain regional origin of the neurons and was followed by an arrest of astrocytes from the cell cycle and induction of glial differentiation. Time-course assays demonstrated that maximum effect was observed after 24 h of coculture. Addition of conditioned medium (CM) derived from CH neurons also increased beta-gal positive CH astrocytic cell number. However, such CM had no effect on midbrain and cerebellum astroglia. Together, these data suggest that neurons secrete brain region-specific soluble factors which induce GFAP gene promoter, as measured by beta-gal expression, thus suggesting that neuron-glia interaction might induce the astrocytic differentiation program.
Asunto(s)
Astrocitos/fisiología , Proteína Ácida Fibrilar de la Glía/genética , Neuronas/fisiología , Regiones Promotoras Genéticas/fisiología , Animales , Encéfalo/citología , Encéfalo/metabolismo , Encéfalo/fisiología , Comunicación Celular/fisiología , Ciclo Celular/fisiología , Células Cultivadas , Difusión , Expresión Génica/fisiología , Regulación de la Expresión Génica/fisiología , Operón Lac/fisiología , Ratones , Ratones Transgénicos/genética , Neuroglía/fisiología , Neuronas/metabolismo , Factores de TiempoRESUMEN
As a result of recent investigations, the cytoskeleton can be viewed as a cytoplasmic system of interconnected filaments with three major integrative levels: self-assembling macromolecules, filamentous polymers, e.g., microtubules, intermediate filaments and actin filaments, and supramolecular structures formed by bundles of these filaments or networks resulting from cross-bridges between these major cytoskeletal polymers. The organization of this biological structure appears to be sensitive to fine spatially and temporally dependent regulatory signals. In differentiating neurons, regulation of cytoskeleton organization is particularly relevant, and the microtubule-associated protein (MAP) tau appears to play roles in the extension of large neuritic processes and axons as well as in the stabilization of microtubular polymers along these processes. Within this context, tau is directly involved in defining neuronal polarity as well as in the generation of neuronal growth cones. There is increasing evidence that elements of the extracellular matrix contribute to the control of cytoskeleton organization in differentiating neurons, and that these regulations could be mediated by changes in MAP activity. In this brief review, we discuss the possible roles of tau in mediating the effects of extracellular matrix components on the internal cytoskeletal arrays and its organization in growing neurons
Asunto(s)
Matriz Extracelular/fisiología , Proteínas Asociadas a Microtúbulos/fisiología , Neuronas/fisiología , Proteínas tau/fisiología , Proteínas del Citoesqueleto , Regulación del Desarrollo de la Expresión Génica , Morfogénesis , Factores de Crecimiento Nervioso , NeuroglíaRESUMEN
Prions, the etiological agents for infectious degenerative encephalopathies, act by entering the cell and inducing conformational changes in PrPC (a normal cell membrane sialoglycoprotein), which result in cell death. A specific cell-surface receptor to mediate PrPC and prion endocytosis has been predicted. Complementary hydropathy let us generate a hypothetical peptide mimicking the receptor binding site. Antibodies raised against this peptide stain the surface of mouse neurons and recognize a 66-kDa membrane protein that binds PrPC both in vitro and in vivo. Furthermore, both the complementary prion peptide and antiserum against it inhibit the toxicity of a prion-derived peptide toward neuronal cells in culture. Such reagents might therefore have therapeutic applications.
Asunto(s)
Proteínas PrPC/metabolismo , Receptores de Superficie Celular/análisis , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Células Cultivadas , Técnicas Genéticas , Humanos , Ratones , Datos de Secuencia Molecular , Neuronas/citología , Proteínas PrPC/inmunología , Proteínas PrPC/toxicidad , Ratas , Receptores de Superficie Celular/química , Células Tumorales CultivadasRESUMEN
In the developing mammalian midbrain, radial glial cells are divided into median formations and lateral radial systems with differential properties including rate and timing of cell proliferation, expression of cytoskeletal and calcium-binding proteins, storage of glycogen and relations to afferent fiber systems. To test the hypothesis that radial glial cells of median and lateral midbrain sectors and/or their derivatives are heterogeneous in their relations with local neurons, an in vitro system has been developed and has also been characterized in terms of extracellular matrix (ECM) components. Confluent astrocyte cultures, derived from median (M) or lateral (L) embryonic mouse midbrain sectors, were used as substrates for culturing dissociated cells from median (m) or lateral (l) sectors of embryonic midbrains. In spite of the morphological invariance of glial substrates at confluency, cells that were plated onto these substrates and that were immunoreactive for neuronal markers (MAP2, polysialylated N-CAM or beta III tubulin) showed differences in the aggregation of somata and in the length, caliber and branching of neurites. These differences, which depend mostly on the sector of origin of astrocytes (L: permissive, M: non-permissive for neuronal growth), suggest that the substrates may differ in adhesiveness and/or their carrying of growth-promoting vs. growth-interfering molecules. Indeed, L and M cultures differ in laminin deposition patterns (L: fibrillar, M: punctate pattern). Furthermore, sulfated glycosaminoglycans (s-GAGs) isolated from the pericellular (P), intracellular (I) and extracellular (E) compartments of these sectoral cultures also showed correlations with the ability to support neurite growth. The total amount of s-GAGs in M cultures was twice that in L cultures and was particularly high in the P compartment, with about 3 times as much heparan sulfate (HS) and about 15 times as much chondroitin sulfate (CS) in this fraction of M than in the corresponding compartment of L glia. Our results indicate that cultured astrocytes have heterogeneous properties including different organization of their extracellular matrix that reflect the roles played by their parent radial glia in regions favorable to axonal growth or barrier regions of the developing brain.
Asunto(s)
Astrocitos/fisiología , Axones/metabolismo , Matriz Extracelular/metabolismo , Mesencéfalo/fisiología , Neuroglía/fisiología , Animales , MamíferosRESUMEN
Thyroid hormones have profound effects on growth and development. In the brain L-3,5,3'-triiodothyronine (T3), the bioactive hormone, is involved with the harmonious development acting in neuronal and glial cell differentiation. T3 acts on the cells by interacting with nuclear receptors that can regulate the expression of several genes. Astrocytes also show receptors to the hormone. We reported herein data on the effects of T3 on astrocytes. We have verified that T3 has a morphological effect on cultured cortical astrocytes with rearrangement of GFAP filaments, and induces proliferation in the cultured cerebellar astrocytes of newborn rats. We discuss here the effects of T3 on astrocytes, considering the possibility that thyroid hormone prepares the astrocytes to interact with neurons.
Asunto(s)
Astrocitos/fisiología , Neuroglía/fisiología , Triyodotironina/fisiología , Animales , RatasRESUMEN
The central nervous system (CNS) midline plays an important role in growth and guidance of axons. At the midline, a multiplicity of cell types establish boundaries that control the navigation of crossed and uncrossed axonal fibers. The extracellular matrix (ECM) molecules of the resident neuroepithelial or committed neuronal or glial cells could be involved in the control of axon growth and axon guidance. This review reports the recent advances in the study of the structure and functional role of the ECM at the midline locus of the CNS. In vivo and in vitro approaches are considered to provide new clues in the understanding of processes involved in the cellular decisions of the CNS midline.
Asunto(s)
Matriz Extracelular/fisiología , Mesencéfalo/crecimiento & desarrollo , Neuritas/fisiología , Neuroglía/fisiología , Células Cultivadas , Colágeno/fisiología , Fibronectinas/fisiología , Humanos , Laminina/fisiología , Tenascina/fisiologíaRESUMEN
The central nervous system (CNS) midline plays an important role in growth and guidance of axons. At the midline, a multiplicity of cell types establish boundaries that control the navigation of crossed and uncrossed axonal fibers. The extracellular matrix (ECM) molecules of the resident neuroepithelial or committed neuronal of glial cells could be involved in the control of axon growth and axon guidance. This review reports the recent advances in the study of the structure and functional role of the ECM at the midline locus of the CNS. In vivo and in vitro approaches are considered to provide new clues in the understanding of processes involved in the cellular decisions of the CNS midline.
Asunto(s)
Humanos , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Técnicas In Vitro , Laminina/metabolismo , Mesencéfalo/citología , Neuritas/ultraestructura , Neuroglía/metabolismo , Tenascina/metabolismo , Sistema Nervioso Central/citología , Mesencéfalo/crecimiento & desarrolloRESUMEN
Sulfated glycosaminoglycans (S-GAGs) were isolated from the pericellular (P), intracellular (I), and extracellular (E) compartments of astrocytes cultures from lateral (L) and medial (M) sectors of embryonic mouse midbrain; these sectors differ in their ability to support neurite growth (L, permissive, M, non-permissive for growth) and laminin deposition patterns (L, fibrillar; M, punctate pattern). The total amount of S-GAGs in M cultures was twice that in L cultures and was particularly high in the P compartment of M glia. Both glial cultures showed heparan sulfate (HS) in the three cellular compartments but chondroitin sulfate (CS) GAGs were vestigial in I and P compartments of L glia. Our results suggest that M and L astrocytes are heterogeneous concerning the ability to synthesize GAGs and distribute them among the different cellular compartments. Together with other data (Garcia-Abreu et al: J Neurosci Res 40:471, 1995; Garcia-Abreu et al: Neuroreport 6:761, 1995), the present results suggest that this heterogeneous features might be at least partially responsible for the differential effects of L and M glial cultures on the growth of midbrain neurons and may also be involved in complex ways in the guidance of axons at the brain midline.
Asunto(s)
Astrocitos/metabolismo , Glicosaminoglicanos/metabolismo , Mesencéfalo/metabolismo , Animales , Células Cultivadas , Glicosaminoglicanos/química , Mesencéfalo/citología , Ratones/embriología , Ratones Endogámicos , Distribución TisularRESUMEN
Regional astrocyte cultures were obtained by dissecting and dissociating medial and lateral sectors of the midbrain from 14-day Swiss mouse embryos. Once confluent, these cultures were tested by glial fibrillary acidic protein (GFAP) immunocytochemistry to confirm their astrocyte composition and for 2'-3' cyclic nucleotide 3'-phosphohydrolase (CNPase) and microtubule-associated protein 2 (MAP2) immunocytochemistry to rule out oligodendroglial and neuronal components, respectively. In confluent astrocyte cultures from either sector, virtually all cells were GFAP-positive elements, most of which were flat cells accompanied by smaller numbers of flat cells with processes. Confluent astrocyte cultures, derived from medial (M) or lateral (L) sectors, were used as substrata for culturing dissociated cells from medial (m) or lateral (l) sectors of 14-day embryonic midbrains. Fixed cocultures (Ll, Lm, Mm, Ml) were stained with an anti-MAP2 antibody to verify neuronal aggregation and neuritic morphology. In spite of the morphological constancy of glial substrata at plating, MAP2-positive cells in cocultures showed differences in the aggregation of somata and in the length, caliber, and branching of neurites. These differences, which depend mostly on the sector of origin of astrocytes, suggest that the substrata may differ in adhesiveness and/or growth-promoting vs. growth-interfering properties. Together with evidence for sectorial heterogeneity in brainstem radial glia, the present results raise the possibility that cultured astrocytes have properties that reflect the roles played by their parent radial glia in the developing brain.
Asunto(s)
Mesencéfalo/fisiología , Neuroglía/fisiología , Neuronas/fisiología , Animales , Astrocitos/fisiología , Recuento de Células , Células Cultivadas , Inmunohistoquímica , RatonesRESUMEN
An analysis of the extra cellular matrix (ECM) in regionally heterogeneous midbrain glia has been started. Immunoreactivity to laminin has been tested in confluent glial cultures from lateral (L) and medial (M) sectors of 14 days mouse embryos (E14) and in neurone-glia cocultures kept for 48 hours after plating of E14 midbrain freshly-dissociated neurones. Laminin is present in both types of glial cultures, but its distribution assumes a punctate pattern in glia that is not permissive for neurite growth (M-glia) and a fibrillar configuration in a favourable glial substrate (L-glia). Moreover, laminin expression is dramatically upregulated in co-cultures although fibrillar and punctate patterns are maintained.