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1.
Ann N Y Acad Sci ; 971: 135-8, 2002 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-12438103

RESUMEN

Although the adult brain contains nicotinic acetylcholine (ACh) receptors vital to cortical function, little is known about the assembly of embryonic receptor subunits into functional receptors or their role in fetal brain development. We now report the first evidence of functional nicotinic ACh receptors on stem and progenitor cells of fetal mouse cerebral cortex as early as embryonic day 10.


Asunto(s)
Encéfalo/embriología , Corteza Cerebral/embriología , Receptores Nicotínicos/biosíntesis , Células Madre/metabolismo , Animales , Calcio/metabolismo , Diferenciación Celular , Electrofisiología , Ratones , Factores de Tiempo , Receptor Nicotínico de Acetilcolina alfa 7
2.
Dev Biol ; 240(1): 143-56, 2001 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-11784052

RESUMEN

The adult cerebral cortex contains nicotinic acetylcholine (ACh) receptors vital to cortical function. However, little is known about the assembly of embryonic nicotinic receptor subunits into functional receptors or whether they play an active role in cortical development. We now report evidence of functional nicotinic acetylcholine receptor channels in fetal mouse cerebral cortex as early as embryonic day 10 (E10), when the cortex consists of dividing stem and progenitor cells. Patch-clamp electrophysiological measurements indicate that nicotine and ACh evoke sizable inward currents characteristic of nicotinic receptors, that are strongly rectifying with a reversal potential near 0 mV. Three different nicotinic agonists, ACh, nicotine, and dimethylphenylpiperazinium, evoked cytosolic Ca(2+) signals. Agonist-evoked Ca(2+) signals and electrophysiological responses were found in greater than 70% of all E10-E11 cells tested and were blocked by nicotinic receptor antagonists. The Ca(2+) response to nicotinic agonists was markedly prolonged in cells from early embryonic stages relative to later stages of development. alpha3, alpha4, and alpha7 receptor subunit proteins were detected immunocytochemically in cortical cells from E10 to birth. The incidence of each subunit declined with embryonic age, suggesting a role in early development. We discuss the possible function of nicotinic receptors in early cortical development and their role as a target for nicotine in the developmental pathologies associated with the fetal tobacco syndrome.


Asunto(s)
Corteza Cerebral/embriología , Receptores Nicotínicos/metabolismo , Células Madre/metabolismo , Acetilcolina/farmacología , Animales , Calcio/metabolismo , Señalización del Calcio , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Yoduro de Dimetilfenilpiperazina/farmacología , Femenino , Técnica del Anticuerpo Fluorescente , Ratones , Nicotina/farmacología , Técnicas de Placa-Clamp , Embarazo , Células Madre/efectos de los fármacos
3.
Neuron ; 28(1): 69-80, 2000 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-11086984

RESUMEN

Multipotent stem cells that generate both neurons and glia are widespread components of the early neuroepithelium. During CNS development, neurogenesis largely precedes gliogenesis: how is this timing achieved? Using clonal cell culture combined with long-term time-lapse video microscopy, we show that isolated stem cells from the embryonic mouse cerebral cortex exhibit a distinct order of cell-type production: neuroblasts first and glioblasts later. This is accompanied by changes in their capacity to make neurons versus glia and in their response to the mitogen EGF. Hence, multipotent stem cells alter their properties over time and undergo distinct phases of development that play a key role in scheduling production of diverse CNS cells.


Asunto(s)
Diferenciación Celular , Corteza Cerebral/citología , Neuroglía/citología , Neuronas/citología , Células Madre/citología , Animales , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula , Separación Celular , Células Cultivadas , Corteza Cerebral/embriología , Corteza Cerebral/crecimiento & desarrollo , Células Clonales/citología , Células Clonales/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Ratones , Microscopía por Video , Células Madre/efectos de los fármacos , Factores de Tiempo
4.
Development ; 125(16): 3143-52, 1998 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-9671587

RESUMEN

Using long-term, time-lapse video-microscopy, we investigated how single progenitor cells isolated from the early embryonic cerebral cortex produce neurons and glia over time. Clones of 10 cells or less were produced by short symmetric or asymmetric division patterns, commonly terminating in a 'pair progenitor' for two morphologically identical neurons. Larger trees were composites of these short sub-lineages: more prolific neuroblasts underwent repeated asymmetric divisions, each producing a minor neuroblast that typically made (3/4)10 progeny, and a sister cell capable of generating more progeny. Particular division patterns were seen repeatedly. In contrast, glioblasts underwent a prolonged series of symmetric divisions. These patterned lineage trees were generated from isolated cells growing on plastic, suggesting they are largely intrinsically programmed. Our data demonstrate for the first time that CNS progenitor cells have stereotyped division patterns, and suggest that as in invertebrates, these may play a role in neural development.


Asunto(s)
Sistema Nervioso Central/crecimiento & desarrollo , Células Madre/metabolismo , Animales , División Celular/fisiología , Células Clonales/citología , Inmunohistoquímica , Ratones , Microscopía por Video , Neuronas/citología
5.
Neuron ; 18(1): 81-93, 1997 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-9010207

RESUMEN

The embryonic cerebral cortex contains a population of stem-like founder cells capable of generating large, mixed clones of neurons and glia in vitro. We report that the default state of early cortical stem cells is neuronal, and that stem cells are heterogeneous in the number of neurons that they generate. In low fibroblast growth factor (FGF2) concentrations, most maintain this specification, generating solely neuronal progeny. Oligodendroglial production within these clones is stimulated by a higher, threshold level of FGF2, and astrocyte production requires additional environmental factors. Because most cortical neurons are born before glia in vivo, these data support a model in which the scheduled production of cortical cells involves an intrinsic neuronal program in the early stem cells and exposure to environmental, glia-inducing signals.


Asunto(s)
Corteza Cerebral/embriología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Neuroglía/citología , Neuronas/citología , Células Madre/citología , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Células Clonales , Cartilla de ADN , Desarrollo Embrionario y Fetal , Femenino , Proteína Ácida Fibrilar de la Glía/análisis , Ratones , Neuroglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Oligodendroglía/citología , Oligodendroglía/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Embarazo , Receptores de Factores de Crecimiento de Fibroblastos/biosíntesis , Células Madre/efectos de los fármacos , Células Madre/fisiología
6.
Brain Res ; 622(1-2): 237-42, 1993 Sep 17.
Artículo en Inglés | MEDLINE | ID: mdl-8242361

RESUMEN

Continuous perfusion methods are widely used to monitor release of substances, particularly transmitters, from brain cell cultures growing as monolayers. However, if stimuli used to produce release also cause loss or lysis of cells, the appearance of label in the perfusate due to such effects will be indistinguishable from release. Using a perfusion method we have studied release of preloaded, radiolabelled amino acids from primary astrocyte cultures due to a variety of stimuli; hypotonic or high K+ media, activation of beta-receptors or swelling-induced release due to isosmotic ethanol. In this study primary astrocyte cultures were simultaneously labelled with Na2(51)CrO4 and allowed to take up radiolabelled D-aspartate or taurine. It was found that while all of the above methods caused release of radiolabelled amino acids none caused release of 51Cr into the perfusion fluid. In contrast, perfusion with 0.05% (v/v) Triton X-100 did lead to release of 51Cr. Thus a variety of means of inducing swelling or shape changes in astrocytes causes true release of radiolabelled amino acids and simultaneously monitoring 51Cr release seems a convenient means of distinguishing such release from cell loss or lysis.


Asunto(s)
Aminoácidos/metabolismo , Astrocitos/metabolismo , Radioisótopos de Cromo , Ácido Aspártico/metabolismo , Astrocitos/patología , Supervivencia Celular/fisiología , Células Cultivadas , Detergentes , Etanol/farmacología , Soluciones Hipotónicas , Norepinefrina/farmacología , Octoxinol , Concentración Osmolar , Potasio/farmacología , Taurina/metabolismo
7.
J Neurochem ; 60(5): 1682-9, 1993 May.
Artículo en Inglés | MEDLINE | ID: mdl-8473890

RESUMEN

Exposure of primary astrocyte cultures to isosmotic ethanol from 10-100 mM led to both swelling of the cells and release of [3H]taurine and D-[3H]aspartate. Exposure to hyperosmotic ethanol, in the same concentration range, caused neither swelling nor release. Release was inhibited by the anion transport blocker L-644,711, already shown to inhibit amino acid release evoked by hypoosmotic or high-potassium medium, conditions that also cause astrocytic swelling. Ethanol-induced release generally showed a decline in response to successive exposures to ethanol, and release was not dependent on extracellular calcium. Thus, the characteristics of swelling-induced release of amino acids by isosmotic ethanol seem to correspond to those of swelling-induced release from astrocytes due to exposure to hypotonic or high-K+ media. We discuss whether such effects may contribute to CNS damage after head injury and stroke.


Asunto(s)
Ácido Aspártico/metabolismo , Astrocitos/metabolismo , Etanol/farmacología , Taurina/metabolismo , Animales , Proteínas de Transporte de Anión , Astrocitos/citología , Astrocitos/efectos de los fármacos , Calcio/farmacología , Proteínas Portadoras/antagonistas & inhibidores , Células Cultivadas , Concentración Osmolar , Ratas , Sacarosa/farmacología
8.
Brain Res ; 602(1): 41-4, 1993 Jan 29.
Artículo en Inglés | MEDLINE | ID: mdl-8448657

RESUMEN

We have previously reported that primary astrocyte cultures prepared from neonatal rat brains show Na(+)-dependent, tricyclic antidepressant-sensitive, high-affinity uptake of [3H]norepinephrine ([3H]NE). Other workers, however, using primary astrocyte cultures from neonatal mice, have failed to find such uptake. This prompted us to examine possible reasons for the variability of the uptake in primary astrocyte cultures such as growth conditions and the effect of ascorbic acid. The presence of ascorbic acid increased the Na(+)-dependent uptake of NE by inhibiting the Na(+)-independent component. Na(+)-dependent uptake in rat cultures occurs when either fetal bovine or horse serum are present in the growth media, but not in a serum-free growth medium. Other workers have shown a species difference such that, even under optimal uptake conditions where rat astrocyte cultures exhibit Na(+)-dependent [3H]NE uptake, mouse astrocyte cultures do not.


Asunto(s)
Animales Recién Nacidos/metabolismo , Ácido Ascórbico/farmacología , Astrocitos/efectos de los fármacos , Corteza Cerebral/efectos de los fármacos , Norepinefrina/metabolismo , Sodio/farmacología , Animales , Astrocitos/metabolismo , Fenómenos Fisiológicos Sanguíneos , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Medios de Cultivo , Ratas , Tritio
10.
Glia ; 6(1): 9-18, 1992.
Artículo en Inglés | MEDLINE | ID: mdl-1355075

RESUMEN

Regional astrocyte cultures were derived by dissecting six regions; brain stem, cerebellum, mesencephalon, basal ganglia plus diencephalon, cerebral cortex, and hippocampus, from 3 to 4-day-old neonatal rat brains. Glial fibrillary acidic protein (GFAP) immunocytochemistry was used to confirm the astrocyte composition of the cultures. The percentage of GFAP (+) cells between regions varied from 75% to 100%. Once confluent these cultures were incubated with radiolabeled serotonin or glutamate for uptake and autoradiographic studies. For the different brain regions Na(+)-dependent, [3H] L-glutamate, and fluoxetine-sensitive [3H] 5-HT uptake varied markedly. The relative order of uptake for [3H] 5-HT was MS (mesencephalon) greater than CC (cerebral cortex) greater than BG + DI (basal ganglia + diencephalon) greater than HP (hippocampus) greater than BS (brain stem) greater than CB (cerebellum). For [3H] L-glutamate the order was HP greater than CC greater than BG + DI greater than MS = BS greater than CB. For [3H] 5-HT this essentially corresponds to the reported order of binding in situ of the [3H] 5-HT-specific uptake ligand [3H] citalopram. For [3H] L-glutamate regional variation of the uptake for the different cultures corresponds to the regional uptake reported for different regions of rat brain. Double-label studies with GFAP and radiolabeled neurotransmitters were also used to study uptake into GFAP(+) astrocytes by autoradiography. Flat GFAP cells with or without processes comprised 65-98% of the cultures and represented most of the uptake. The percentage of all GFAP(+) cells that were positive for uptake of ARG varied from 50% to 90% and also showed differences in grain density both intra- and inter-regionally. These differences in transmitter uptake by GFAP(+) astrocytes in primary culture, which are dependent on the region of origin and correspond to regional differences in situ, suggest that such uptake in vitro may reflect uptake by astrocytes in vivo. Implied in this is that uptake by astrocytes represents a significant component of serotonin uptake in vivo.


Asunto(s)
Astrocitos/metabolismo , Encéfalo/citología , Glutamatos/metabolismo , Serotonina/metabolismo , Animales , Animales Recién Nacidos , Autorradiografía , Encéfalo/crecimiento & desarrollo , Proteína Ácida Fibrilar de la Glía/inmunología , Proteína Ácida Fibrilar de la Glía/metabolismo , Ácido Glutámico , Ligandos , Neuronas/metabolismo , Ratas , Serotonina/fisiología , Sodio/metabolismo
11.
Glia ; 6(1): 1-8, 1992.
Artículo en Inglés | MEDLINE | ID: mdl-1355074

RESUMEN

Na(+)-dependent, fluoxetine-sensitive high-affinity uptake of serotonin and Na(+)-dependent uptake of glutamate were studied in primary astrocyte cultures from 1-day-old rat neocortex. This uptake was independent of time in culture from 1 to 6 weeks. High-affinity serotonin uptake was decreased when cells were grown in horse serum as compared to fetal bovine serum and was almost absent when cells were grown in chemically defined medium. In contrast, glutamate uptake was unaffected by the composition of the medium in which the cultures were grown. The serum effect on serotonin uptake was not due to the greater level of serotonin in the fetal bovine serum and was only reversed by a change of serum over a time period of days.


Asunto(s)
Astrocitos/metabolismo , Glutamatos/metabolismo , Serotonina/metabolismo , Animales , Animales Recién Nacidos/fisiología , Autorradiografía , Bovinos , Células Cultivadas , Medios de Cultivo , Femenino , Técnica del Anticuerpo Fluorescente , Proteína Ácida Fibrilar de la Glía/inmunología , Proteína Ácida Fibrilar de la Glía/metabolismo , Ácido Glutámico , Caballos , Inmunohistoquímica , Embarazo , Ratas
12.
J Neurosci ; 10(5): 1583-91, 1990 May.
Artículo en Inglés | MEDLINE | ID: mdl-1970603

RESUMEN

Swelling of primary astrocyte cultures by exposing them to hypotonic media caused release of label after the cells had been allowed to accumulate 3H-L-glutamate, 3H-D-aspartate, or 3H-taurine. Comparable release of endogenous L-glutamate or taurine, as measured by high-pressure liquid chromatography (HPLC), was also found. Release of label was not affected by treating the cells with cytochalasin B, indicating that microfilament polymerization was not significantly involved. Hypotonic-induced release did not appear to principally involve reversal of the Na(+)-dependent uptake system since increasing external K+ to depolarize the cells by replacement of external Na+, thus maintaining isotonic conditions, increased release to a lesser extent. Threo beta-hydroxyaspartate, a potent 3H-L-glutamate uptake blocker, added externally stimulated efflux of 3H-L-glutamate independently of the swelling-induced efflux. Upon restoration of swollen cells to isotonic medium they showed an unimpaired ability to take up 3H-L-glutamate. The swelling-induced release of label was inhibited by a number of anion transport inhibitors, one of which has been shown to significantly improve outcome in an experimental brain trauma/hypoxia model in which astrocyte swelling is an early event.


Asunto(s)
Ácido Aspártico/metabolismo , Astrocitos/metabolismo , Glutamatos/metabolismo , Taurina/metabolismo , Aminoácidos/metabolismo , Animales , Aniones/antagonistas & inhibidores , Aniones/metabolismo , Astrocitos/citología , Astrocitos/efectos de los fármacos , Transporte Biológico , Supervivencia Celular , Células Cultivadas , Medios de Cultivo , Citocalasina B/farmacología , Ácido Glutámico , Soluciones Hipotónicas/farmacología , Sodio/farmacología
13.
J Cell Physiol ; 133(2): 389-94, 1987 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-3680396

RESUMEN

We examined the effects of alterations in endothelial cell volume on transendothelial albumin permeability. Studies were done using a confluent monolayer of bovine pulmonary artery endothelial cells grown on gelatinized microporous filters. When endothelial cells were exposed to media made hypertonic with 200 mM mannitol, the intracellular volume (measured with 14C-urea) decreased twofold and remained decreased over a 30-minute time-span, thus showing no significant regulatory volume increase (RVI) within this time period. When endothelial cells were exposed to hypotonic media, intracellular volume rapidly doubled within 2 minutes, and then decreased to baseline values within 10 minutes in spite of the sustained hypotonic environment, a process known as regulatory volume decrease (RVD). We also measured the transendothelial flux of 125I-albumin with the cells exposed to the same osmotic changes. We observed that only under hypertonic conditions was there a significant change in the 125I-albumin permeability. These results indicate that the pulmonary artery endothelial cells in culture alter their cell volume when exposed to variations in the osmotic environment, and also show RVD in response to hypotonic conditions but no RVI within 40 minutes after exposure to hypertonic conditions. The transendothelial albumin permeability did not change under hypotonic conditions but increased under hypertonic conditions. Thus, endothelial cells shrinkage may be an important mechanism of increased endothelial macromolecule permeability. These volume changes may occur in endothelial cells in situ and have a role in inducing alterations in the transendothelial permeability to proteins.


Asunto(s)
Albúminas/metabolismo , Endotelio Vascular/citología , Animales , Permeabilidad Capilar , Bovinos , Medios de Cultivo , Endotelio Vascular/metabolismo , Soluciones Hipertónicas , Soluciones Hipotónicas , Radioisótopos de Yodo , Arteria Pulmonar
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