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Astrocyte cell lineage. V. Similarity of astrocytes that form in the presence of dBcAMP in cultures to reactive astrocytes in vivo.

The relationship between astrocytes forming in the presence of dibutyryl cyclic AMP (dBcAMP) in culture and reactive astrocytes responding to a cerebral cortex stab wound was investigated using computerized image analysis (Zeiss IBAS 1) and immunocytochemical staining. The diameters of the nuclei of astrocytes in primary cultures of newborn mouse neopallial cells were compared to those of the nuclei of normal and reactive astrocytes in histological sections of mouse cerebral cortex. We found that the nuclei of astrocytes that formed in the presence of dBcAMP in cultures are significantly larger than those of spontaneously occurring small stellate astrocytes in culture and of normal astrocytes of the cerebral cortex in vivo but corresponded more closely to the nuclei of reactive astrocytes in the area surrounding a stab wound in the cerebral cortex. Large stellate cells formed in the presence of dBcAMP had vimentin and an increase in GFP-containing intermediate filaments. Formation of reactive astrocytes in vivo is also associated with an increase in both vimentin and GFP-containing intermediate filaments. These observations indicate a closer relationship of astrocytes formed in the presence of dBcAMP in cultures to the reactive astrocytes in the cerebral cortex than to normal astrocytes. We propose, therefore, that the large stellate astrocytes that form in the presence of dBcAMP be referred to as reactive astrocytes in culture.

Animals↗

Astrocyte cell lineage. II. Mouse fibrous astrocytes and reactive astrocytes in cultures have vimentin- and GFP-containing intermediate filaments.

When cells from mouse neopallium are grown in colony cultures for 10-12 days, small cells with many processes, resembling normal fibrous astrocytes, form on top of the astrocyte precursor cells independently of the presence of dBcAMP in the culture medium. These cells are distinctly different from the much larger, previously described reactive astrocytes which also form in colony cultures and whose maturation is greatly enhanced by the presence of dBcAMP in the culture medium. Immunofluorescence studies showed that both vimentin-containing and glial filament protein (GFP)-containing intermediate filaments (IF) are present in the small normal fibrous astrocytes as well as in the larger reactive astrocytes. The vimentin-containing IF are assembled first in astrocyte precursor cells, whereas GFP-containing IF are assembled later toward the final stages of astrocyte differentiation both in vivo and in vitro. Thus in respect to the expression of the two types of IF, astrocyte differentiation in vitro closely resembles that in vivo. Parallel studies by electron microscopy showed that the vimentin-positive but GFP-negative astrocyte precursor cells contain single IF or small groups of IF, whereas in the more differentiated normal fibrous astrocytes and reactive astrocytes which are also GFP-positive, additional IF arranged in large bundles are present.

Animals↗

ATP-dependent astrocyte-endothelial calcium signaling following mechanical damage to a single astrocyte in astrocyte-endothelial co-cultures.

In the brain, endfeet of perivascular astrocytes make close contact with capillary endothelial cells that form the blood-brain barrier. The aim of the present work was to investigate whether and how calcium signals can be communicated from astrocytes to endothelial cells following acute mechanical cell damage. The experiments were performed on astrocyte-endothelial co-cultures prepared from primary rat brain astrocytes and an endothelial cell line (ECV304). A single astrocyte was acutely damaged by mechanical stimulation of sufficient strength with a micropipette, and the resulting cytoplasmic calcium changes were monitored using fura-2 and digital calcium imaging. Mechanical damage to a single astrocyte triggered a large intercellular calcium wave that propagated to surrounding astrocytes and also to even remotely located (several hundred micrometers) endothelial cells. Astrocyte-endothelial calcium waves induced by mechanical cell damage were largely deflected by fast superfusion, were able to cross a cell-free lane, were dose-dependently inhibited by suramin, a P2-purinoceptor blocker, and were largely reduced in size in the presence of the ATP-degrading enzyme apyrase. Our results indicate that mechanical damage to a single astrocyte can produce far reaching calcium signals that are propagated by the release of a calcium mobilizing P2-purinergic agonist and that can be communicated to endothelial cells. As endothelial cytoplasmic calcium is an important factor in the regulation of blood-brain barrier permeability and transport, mechanical cell damage-induced astrocyte-endothelial calcium signals are hypothesized to play a role in the initiation of brain edema and the stimulation of brain glucose uptake.

Adenosine Triphosphate↗

Differential inhibition of neurone-neurone, neurone-astrocyte and astrocyte-astrocyte adhesion by L1, L2 and N-CAM antibodies.

The cell adhesion molecules L1, N-CAM and Ng-CAM have been implicated in cell-cell interactions among developing neural cells. L1 and N-CAM are structurally and functionally distinct molecular entities and act synergistically in mediating Ca2+-independent adhesion between re-aggregating early postnatal cerebellar cells. N-CAM has been reported to be neurone-specific in the chicken and to mediate fasciculation of neurites and of nerve-muscle interactions. L1, which in the central nervous system has been found only on post-mitotic neurones, mediates migration of granule cell neurones in the mouse cerebellar cortex. In view of the molecules' distinct effects on cell interactions, we wondered whether different neural cell types are involved in the actions of each molecule. Here we report that L1 antigen promotes neurone-neurone adhesion. N-CAM, which is expressed on both neurones and glia, mediates neurone-neurone, neurone-astrocyte and astrocyte-astrocyte adhesion. The L2 carbohydrate epitope shared between the two adhesion molecules seems to be involved in neurone-astrocyte and astrocyte-astrocyte adhesion and acts in a more than additive manner in N-CAM-mediated neurone-neurone adhesion.

Animals↗

Fibrous astrocytes and reactive astrocyte-like cells in transplants of cultured astrocyte precursor cells.

Mouse neopallium was disaggregated at 3 developmental stages (E15, E18, PO) and grown in colony cultures for 7 days. On the seventh day of culturing the colonies of cells were transplanted into the cerebellums of neonatal mice. After 3 weeks the astrocytes within the transplants were identified with GFAP immunoperoxidase staining and by morphometric nuclear measurements of the GFAP positive cells. Cultures of E15 and E18 disaggregated neopallium gave rise to typical fibrous astrocytes in the transplants which were similar to fibrous astrocytes in the cerebral white matter of adult mice. Cultures of PO disaggregated neopallium gave rise to reactive astrocyte-like cells in the transplants which stained intensely for GFAP and had nuclei significantly larger than the astrocytes in the cerebral white matter of adult mice and in transplants of the E15 and E18 cultures.

Animals↗

Voltage gated ionic channels in rat cultured astrocytes, reactive astrocytes and an astrocyte-oligodendrocyte progenitor cell.

Astrocytes (both type 1 and type 2), cultured from the central nervous system of newborn or 7 day old rats show voltage gated sodium and potassium channels that are activated when the membrane is depolarized to greater than -40 mV. The sodium channels in these cells have an h-infinity curve similar to that of nodal membranes but the activation (peak current-voltage) curves are shifted along the voltage axis by about +30 mV. These sodium currents are blocked only by high concentrations of tetrodotoxin. The voltage activated potassium currents in both types of astrocyte show at least two components; an inactivating component that is suppressed at holding potentials of greater than -40 mV and a persistent, non-inactivating current. Several types of single channel currents were observed in outside-out membrane patches from type 2 astrocytes. One type of potassium channel showed inactivation on depolarization and may contribute to the whole-cell inactivating current. In contrast, oligodendrocytes showed no obvious voltage gated membrane channels. The properties of the type 2 astrocyte-oligodendrocyte progenitor cell were investigated in two ways: 1) by examination of cells just beginning to differentiate along the "electrically silent" oligodendrocyte pathway or 2) by recording from progenitor cells cultured for 24 hours in the presence of cycloheximide to block the appearance of new membrane channels. In both cases, voltage gated inward (sodium) and outward (potassium) currents were noted. The outward current response showed both an inactivating and a non-inactivating component. Similar voltage activated inward and outward membrane currents were noted in reactive astrocytes freshly isolated (3-6 hours) from lesioned areas of adult rat brains.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Distribution of astrocytic plaques in the corticobasal degeneration brain and comparison with tuft-shaped astrocytes in the progressive supranuclear palsy brain.

Corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP) have some clinical and pathological features in common. Each, however, has been shown to have specific astrocytic inclusions. CBD is characterized by astrocytic plaques, and PSP is characterized by tuft-shaped astrocytes. To clarify differences between these inclusions, we investigated intracerebral distribution of astrocytic plaques and tuft-shaped astrocytes in autopsied brains of patients with either CBD or PSP. Specimens from ten patients with CBD and five patients with PSP were stained by the Gallyas-Braak method. Densities of the astrocytic plaques and tuft-shaped astrocytes were determined for 11 cerebral cortical regions, 6 subcortical nuclei, 5 brain stem regions, the cerebellar cortex and the dentate nucleus. Astrocytic plaques were most abundant in the prefrontal and premotor areas in the cerebral cortex of CBD brains, whereas tuft-shaped astrocytes were most prominent in the precentral gyrus and premotor area of PSP brains. Many astrocytic plaques were observed in the caudate nucleus of CBD brains, whereas tuft-shaped astrocytes were abundant in both the caudate and putamen and moderate in number in the globus pallidus, subthalamic nucleus and thalamus in PSP brains. Very slight accumulation of astrocytic plaques was seen in the brain stem of CBD brains, whereas numerous tuft-shaped astrocytes were found in the red nucleus and superior colliculus of PSP brains. Distribution of the astrocytic plaques and tuft-shaped astrocytes differed greatly. Thus, CBD and PSP can be considered different entities.

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Maturation of astrocyte morphology and the establishment of astrocyte domains during postnatal hippocampal development.

Mature protoplasmic astrocytes exhibit an extremely dense ramification of fine processes, yielding a 'spongiform' morphology. This complex morphology enables protoplasmic astrocytes to maintain intimate relationships with many elements of the brain parenchyma, most notably synapses. Recently, it has been demonstrated that astrocytes establish individual cellular-level domains within the neuropil, with limited overlap occurring between the extents of neighboring astrocytes. The highly ramified nature of protoplasmic astrocytes is closely associated with their ability to create such domains. This study was an attempt to characterize the development of spongiform processes and the establishment of astrocyte domains. A combination of immunolabeling for the astrocyte-specific markers glial fibrillary acidic protein and S100beta with intracellular dye labeling in fixed tissue slices allowed for the identification of immature astrocytes and the elucidation of their complete, well-preserved morphologies. We find that during the first two postnatal weeks astrocytes extend stringy, filopodial processes. Fine, spongiform processes appear during the third week. Protoplasmic astrocytes are quite heterogeneous in morphology at 1-week postnatum, but there is a remarkable consistency in morphology by 2 weeks of age. Finally, protoplasmic astrocytes initially extend long, overlapping processes during the first two postnatal weeks. The subsequent elaboration of spongiform processes results in the development of boundaries between neighboring astrocyte domains. Stray processes that encroach on neighboring domains are eventually pruned by 1 month of age. These observations suggest that domain formation is largely the consequence of competition between astrocyte processes, similar to the well-studied competitive interactions between certain neuronal dendritic fields.

Animals↗

Upregulation of protease-activated receptor-1 in astrocytes in Parkinson disease: astrocyte-mediated neuroprotection through increased levels of glutathione peroxidase.

In the present study, we investigated the expression of protease-activated receptors (PARs), receptors for thrombin, in substantia nigra pars compacta (SNpc) of Parkinson disease (PD) brains and cultures of human neurons, astrocytes, oligodendrocytes, and microglia as determined by immunocytochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). Expression of PAR-1 was demonstrated only in glial fibrillary acidic protein-positive astrocytes in SNpc, and the number of astrocytes expressing PAR-1 increased in SNpc of PD as compared with nonneurologic control brain. Immunoreactivity for thrombin and prothrombin was stronger in astrocytes and the vessel walls in SNpc of PD brains. PAR-1 was expressed in human astrocytes and neurons, but not in oligodendrocytes or microglia as determined by RT-PCR. We investigated thrombin-mediated activation of human astrocytes. Thrombin treatment activates human astrocytes and induces morphologic change and a marked increase in proliferation of astrocytes. Increased expression of glial cell line-derived growth factor and glutathione peroxidase (GPx) but no change in the expression of nerve growth factor and inflammatory cytokines/chemokine (IL-1beta, IL-6, IL-8, MCP-1) was found in thrombin/PAR-activated astrocytes. Next, we studied the neuroprotective effect exerted by thrombin-activated astrocytes in human cerebral neuron x human neuroblastoma hybrid neurons. Although thrombin showed neurotoxicity against human hybrid neurons in a dose-dependent manner, the conditioned media derived from thrombin-pretreated astrocyte cultures promoted the survival of human hybrid neurons. The protective effect was completely inhibited with a GPx inhibitor, mercaptosuccinic acid, indicating that GPx released from thrombin/PAR-activated astrocytes is responsible for neuroprotection of hybrid neurons against thrombin cytotoxicity. The present study suggests that the increased expression of PAR-1 in astrocytes in SNpc of PD brain is the restorative move taken by the brain to provide neuroprotection against neuronal degeneration and cell death of dopaminergic neurons caused by noxious insults during the progression of PD pathology.

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Astrocyte heterogeneity: endogenous amino acid levels and release evoked by non-N-methyl-D-aspartate receptor agonists and by potassium-induced swelling in type-1 and type-2 astrocytes.

The aim of the present study was to determine whether endogenous amino acids are released from type-1 and type-2 astrocytes following non-N-methyl-D-aspartate (NMDA) receptor activation and whether such release is related to cell swelling. Amino acid levels and release were measured by HPLC in secondary cultures from neonatal rat cortex, highly enriched in type-1 or type-2 astrocytes. The following observations were made. (a) The endogenous level of several amino acids (glutamate, alanine, glutamine, asparagine, taurine, serine, and threonine) was substantially higher in type-1 than in type-2 astrocytes. (b) The spontaneous release of glutamine and taurine was higher in type-1 than in type-2 astrocytes; that of other amino acids was similar. (c) Exposure of type-2 astrocyte cultures to 50 microM kainate or quisqualate doubled the release of glutamate and caused a lower, but significant increase in that of aspartate, glycine, taurine, alanine, serine (only in the case of kainate), and glutamine (only in the case of quisqualate). These effects were reversed by the antagonist CNQX. (d) Exposure of type-1 astrocyte cultures to 50-200 microM kainate or 50 microM quisqualate did not affect endogenous amino acid release, even after treating the cultures with dibutyryl cyclic AMP. (e) Exposure of type-1 or type-2 astrocyte cultures to 50 mM KCl (replacing an equimolar concentration of NaCl) enhanced the release of taurine greater than glutamate greater than aspartate. The effect was somewhat more pronounced in type-2 than in type-1 astrocytes. Veratridine (50 microM) did not cause any increase in amino acid release. (f) The release of amino acids induced by high [K+] appeared to be related to cell swelling, in both type-1 and type-2 astrocytes. Swelling and K(+)-induced release were somewhat higher in type-2 than in type-1 astrocytes. In contrast, neither kainate nor quisqualate caused any appreciable increase in cell volume. It is concluded that non-NMDA receptor agonists stimulate the release of several endogenous amino acids (some of which are neuroactive) from type-2 but not from type-1 astrocytes. The effect does not seem to be related to cell swelling, which causes a different release profile in both type-1 and type-2 astrocytes. The absence of kainate- and quisqualate-evoked release in type-1 astrocytes suggests that the density of non-NMDA receptors in this cell type is very low.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

Astrocytes promote process outgrowth by adult human oligodendrocytes in vitro through interaction between bFGF and astrocyte extracellular matrix.

Cell-cell interactions regulate many important functions within the central nervous system. In this report, we demonstrate that process outgrowth by adult human oligodendrocytes (OLs) in vitro, an early event of myelinogenesis in vivo, is promoted by astrocytes. To elucidate the mechanisms by which astrocytes might exert this effect, we tested several growth factors known to be produced by astrocytes and found that only basic fibroblast growth factor (bFGF) could enhance process extension by the OL. In correspondence, the treatment of astrocytes with a neutralizing antibody to bFGF decreased their effects in promoting oligodendroglial process outgrowth. The potency of bFGF, however, was only one-third that of astrocytes, and since bFGF did not synergize with other soluble growth factors, we investigated the potential facilitatory role of the extracellular matrix (ECM) deposited by astrocytes. The astrocyte ECM was found to be a promoter of oligodendroglial process extension, and significantly, bFGF synergized with astrocyte ECM to match the potency of live astrocytes. The astrocyte ECM was found in Western blot analyses to contain fibronectin, vitronectin, and laminin. These purified ECM components, as well as heparan sulfate proteoglycan, did not promote oligodendroglial process extension by themselves, although laminin and fibronectin potentiated the effects of bFGF. We conclude that process outgrowth by OLs is guided by astrocytes; the mechanism of the astrocyte effect appears to be due to the combination of bFGF and an unidentified ECM component.

Astrocytes↗

Factors determining the morphology and distribution of astrocytes in the cat retina: a 'contact-spacing' model of astrocyte interaction.

The retina provides a valuable opportunity to examine the interaction of astrocytes with neurones and vasculature, in adult tissue and in vivo. We have studied astrocytes in cat retina to delineate the interactions that determine their morphology and distribution. Their morphology varied with their interaction with surrounding cells, from a classic stellate shape to an elongated bipolar form associated with axon bundles. Evidence is presented that the distribution of astrocytes across the retina is determined by their morphology and by a previously unrecognised interaction between astrocytes, which we term 'contact-spacing,' in which astrocytes maintain contact with their neighbours through their processes, but keep their somas apart. Evidence is also presented that astrocytes are not influenced in their distribution by surrounding neurones, and the influence of developmental mechanisms is identified. These observations are summarised in a contact-spacing model of astrocyte distribution, and four predictions of the model are tested. The concentration of astrocytes along axon bundles dispersed when the axons degenerate but not when vessels were prevented from forming. Further, when both axons and vessels were eliminated, the concentrations of astrocytes dispersed and they became stellate in form. Finally, in the retina of the rat, in which astrocytes show no affinity for axons, the distribution of astrocytes is essentially uniform. We suggest that the contact-spacing interaction among astrocytes provides the anatomical basis of a functional glial network extending across the retina and throughout the central nervous system.

Animals↗

Astrocytes promote or impair the survival and function of embryonic ventral mesencephalon co-grafts: effects of astrocyte age and expression of recombinant brain-derived neurotrophic factor.

Intrastriatal grafting of dopamine-rich embryonic ventral mesencephalon (VM) is a potential therapeutic treatment for Parkinson's disease. However, it has been suggested that the efficacy of this procedure might be improved by enhancing the survival and/or degree of neurite outgrowth by the grafted VM, since these parameters are currently suboptimal. In the present study, we tested the ability of astrocytes retrovirally transduced to produce recombinant brain-derived neurotrophic factor (BDNF) to enhance the survival and/or function of embryonic VM in the unilateral 6-hydroxydopamine (6-OHDA) lesioned rat, a well-characterized rodent model of Parkinson's disease. In culture, primary astrocytes derived from Postnatal Day 0 (P0) rat striatum and transduced with the BDNF vector increased the survival of Embryonic Day 15 (E15) dopaminergic VM neurons by approximately threefold and reduced the loss of dopaminergic neurons following 6-OHDA treatment by approximately 20%. The cultured astrocytes were then mixed 1:1 with freshly dissociated E15 VM and co-grafted into the dopamine-denervated striatum. Unexpectedly, the control nontransduced astrocytes reduced the survival of dopaminergic neurons by 60% and restricted the pattern of neurite outgrowth by the co-grafted VM, compared to grafts of VM alone at 7 weeks postgrafting. These effects were paralleled by an attenuated rate and degree of behavioral recovery. The detrimental effects of the control astrocytes were partially reversed when the astrocytes were transduced to express BDNF, although dopaminergic neuron survival was still reduced by 30% compared to that within VM-only grafts. To begin to assess whether the detrimental effects of the astrocytes were related to the maturational state of the cultured astrocytes, astrocytes were obtained from E18 striatum and maintained in short-term culture (9 days vs several weeks for P0 cultures) prior to co-grafting with VM. Interestingly, the younger astrocytes did not reduce graft survival and allowed for better graft integration. These results suggest that primary astrocytes maintained in long-term culture are detrimental to embryonic neural grafts, an effect that is not completely overcome by expression of recombinant BDNF, and that astrocyte age may be an important consideration in the use of these cells as CNS gene delivery vehicles.

Animals↗

Regulation of glutamate transporters in astrocytes: evidence for a relationship between transporter expression and astrocytic phenotype.

The astrocytic glutamate transporters, EAAT1 and EAAT2, remove released L-glutamate from the synaptic milieu thereby maintaining normal excitatory transmission. EAAT dysfunction during the excitotoxicity and oxidative stress of neurological insults may involve homoeostatic mechanisms associated with astrocytic function. We investigated aspects of EAAT function and expression in concert with astrocytic phenotype in primary cultures of cortical astrocytes and mixed cells of the spinal cord. In spinal cord mixed cultures, hydrogen peroxide (300 microM) reduced both EAAT activity and cellular viability to half of their basal values at 24 h post-treatment, but at 2 h EAAT activity was unaltered, while cellular viability was significantly decreased, suggestive of a mechanism for the maintenance of EAAT activity. Cytochemistry for MAP2, GFAP and propidium iodide revealed that neurons and astrocytes were damaged in a time-dependent manner. A change in astrocyte morphology was observed, with astrocyte cell bodies becoming larger and processes becoming more stellate and often shorter in length. EAAT1 immunoreactivity was reduced at 24 h post-treatment and a re-distribution of the protein was noted after 2 h treatment. In pure astrocytes, lipopolysaccharide (1 microg/ml, 3 d) increased [3H]D-aspartate uptake by 90%, as well EAAT1 immunoreactivity and astrocyte stellation, as shown by immunofluorescent labelling for GFAP. In both culture systems, prominent changes were noted in EAAT function and localization in conjunction with altered astrocytic phenotype. Our findings are indicative of a relationship between astrocytic phenotype and the level of EAAT activity that may be a vital component of astrocytic homeostatic responses in brain injury.

Animals↗

Type-2 astrocyte development in rat brain cultures is initiated by a CNTF-like protein produced by type-1 astrocytes.

O-2A progenitor cells are bipotential glial precursors that give rise to both oligodendrocytes and type-2 astrocytes on a precise schedule in the rat CNS. Studies in culture suggest that oligodendrocyte differentiation occurs constitutively, while type-2 astrocyte differentiation requires an exogenous inducer such as fetal calf serum. Here we describe a rat brain cell culture system in which type-2 astrocytes develop on schedule in the absence of exogenous inducers. Coincident with type-2-astrocyte development, the cultures produce an approximately 20 kd type-2-astrocyte-inducing factor(s). Purified cultures of type-1 astrocytes can produce a similar factor(s). Under conditions where they produce type-2-astrocyte-inducing factor(s), both brain and type-1 astrocyte cultures produce a factor(s) with ciliary neurotrophic (CNTF)-like activity. Purified CNTF, like the inducers from brain and type-1 astrocyte cultures, prematurely induces type-2 astrocyte differentiation in brain cultures. These findings suggest that type-2 astrocyte development is initiated by a CNTF-like protein produced by type-1 astrocytes.

Aging↗

Laser microdissection of immunolabeled astrocytes allows quantification of astrocytic gene expression.

Astrocytes represent the major glial cell population within the central nervous system. In order to elucidate the function of astrocytes under physiological conditions and during the course of neurological disease, astrocytic gene expression profiling is necessary. However, since astrocytes form an intimately connected network with neurons and other cell types in the brain, gene expression analysis of astrocytes with a sufficient degree of cellular specificity is difficult. Here we are presenting a rapid and, thus, RNA preserving immunostaining protocol for the detection of astrocytes in rodent brain. This protocol can readily be combined with laser microdissection (Leica AS LMD platform) and quantitative RT-PCR (qPCR). Employing this method, we studied changes in glial fibrillary acidic protein (GFAP) expression in astrocytes of mouse entorhinal cortex following entorhinal cortex lesion. Using laser microdissection, astrocytes (n = 60) were collected in the tissue surrounding the lesion, the entorhinal cortex contralateral to the lesion, and in unlesioned control animals. Changes in GFAP mRNA were quantified using qPCR. GFAP mRNA levels were 82-fold higher in astrocytes of lesioned animals at the site of the lesion compared to GFAP mRNA levels in entorhinal cortex astrocytes of control mice. GFAP mRNA levels were only slightly elevated at the contralateral side (lesioned animals). This optimized protocol for immunolabeling and laser microdissection of astrocytes followed by qPCR allows quantification of astrocytic gene expression levels with a high degree of cellular specificity. It may similarly be employed in different settings where other cell types need to be identified and microdissected for gene expression profiling.

Animals↗

Differentiation and migration of astrocyte precursor cells and astrocytes in human fetal retina: relevance to optic nerve coloboma.

The presence of astrocyte precursor cells (APCs) and time course and topography of astrocyte differentiation during development were investigated by triple-label immunohistochemistry with intact fetal and adult human retinas. Throughout retinal development and adulthood, expression of Pax2 was restricted to cells of the astrocytic lineage. Three distinct stages of astrocytic differentiation were identified during development: i) Pax2+/vimentin+/GFAP- APCs; ii) Pax2+/vimentin+/GFAP+ immature perinatal astrocytes; and iii) Pax2+/vimentin-/GFAP+ mature perinatal astrocytes. In adult, cells with the antigenic phenotype of mature perinatal astrocytes were restricted to a region surrounding the optic nerve head (ONH), whereas cells at a fourth stage of differentiation, adult astrocytes (Pax2-/vimentin-/GFAP+), were apparent throughout the vascularized retina. APC appearance was centered around the ONH and preceded the appearance of perinatal astrocytes. A cluster of Pax2+ somas was also present in a small region surrounding the ONH at the ventricular surface of the developing retina, which suggests the existence of two distinct sites of astrocytic differentiation. The coincidence in the location of APCs and perinatal astrocytes at the ventricular zone with that of optic nerve colobomas, together with the association of Pax2 gene mutations with this condition, suggests that coloboma formation may result from impaired astrocyte differentiation during development.

Adult↗

Extracellular matrix proteins expressed by human adult astrocytes in vivo and in vitro: an astrocyte surface protein containing the CS1 domain contributes to binding of lymphoblasts.

Primary cultures of human astrocytes, expressing glial fibrillary acidic protein (GFAP), were obtained from postmortem brain tissue samples. These cultured astrocytes produced an extracellular matrix (ECM), containing laminin (Ln) and fibronectin (Fn), as shown with specific antibodies. The perinuclear staining observed in these cells indicated that these proteins were de novo synthesized. Monoclonal antibody (mAb) 90.45, which recognizes the CS1 sequence found in an alternatively spliced form of Fn, also stained cultured astrocytes. Immunohistochemical analysis of normal human brain tissue showed positive staining for the CS1 domain, both on protoplasmic and fibrous astrocytes located in the gray and white matter. In contrast to cultured astrocytes, no immunoreactivity for Ln or Fn was found on astrocytes in normal human brain tissue. These in situ data indicate that the CS1 domain expressed by astrocytes is not part of a splicing variant of Fn. Western blot analysis confirmed that the CS1 domain expressed by cultured human astrocytes is part of an astrocyte protein which is different from human Fn. The CS1 domain is a known ligand for the adhesion receptor alpha4beta1 (VLA-4). We found that the human lymphoma cell lines Jurkat and Ramos, which express alpha4beta1, bound to cultured human astrocytes, and that this interaction could be partly blocked by mAb 90.45 or a synthetic CS1 peptide. Thus, the novel CS1-containing surface protein expressed by astrocytes in vitro and in vivo, contributes to binding of lymphoblasts, and therefore may be a relevant adhesion molecule for the recruitment of alpha4-integrin expressing leukocytes into the central nervous system (CNS).

Aged↗