bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation: A singular craft
Authors: Yin Robert K.
Published: January 2005

This chapter identifies four commonalities between qualitative and quantitative research, thereby creating a new synthesis to diminish the divisiveness between the two camps.
The role of single evaluation courses in evaluation training
Authors: Morris Michael
Published: January 2005

The single course continues to make a significant contribution to the evaluation field, but the nature of this contribution appears to have changed over time.
The changing role of assessment in evaluating science education reform
Authors: Harmon Maryellen
Published: January 2005

This chapter reviews the qualities of existing assessments and their role in the past in evaluating students, programs, and reform efforts, and discusses the shifts in that role with the emergence of new forms of assessment.
Evaluators as collaborators in science education reform
Authors: McColskey Wendy , Parke Helen Meriwether , Harman Patrick , Elliott Rita M.
Published: January 2005

If science education reforms represent a journey toward a vision, then external evaluators play an important, collaborative role in keeping the journey on track.
The perinodal astrocyte
Authors: Black Joel A. , Waxman Stephen G.
Published: January 2005

Several studies have demonstrated the presence of perinodal astrocyte processes at nodes of Ranvier in the central nervous system, suggesting that, in addition to the axon and oligodendrocyte, astrocytes participate in the formation of mature central nodes. The specific association between perinodal astrocyte processes and nodal membrane develops at the time of, or soon after, the appearance of relatively differentiated nodes of Ranvier. This interaction is likely to be mediated by cell adhesion molecules. J1 is a member of a family of glycoproteins that share a common carbohydrate epitope, designated L2/HNK‐1, and that have been implicated in cell‐cell interactions. This glycoprotein is concentrated at the interface between perinodal astrocyte processes and the nodal region of the axon. Moreover, N‐CAM, which is a member of the same family as J1, and cytotactin, an extracellular matrix component produced by glia, are localized at the interface between the axon and perinodal astrocyte processes at nodes of Ranvier. The association of perinodal astrocyte processes with nodal membrane in the central nervous system is similar to that exhibited by perinodal Schwann cell processes at peripheral nodes, and similar functional properties have been suggested for these two glial cell processes, including production of nodal gap substance, buffering of perinodal extracellular ion concentration, and development and/or maintenance of nodal specializations in the axon membrane. Perinodal astrocyte and Schwann cell processes may also function as extraneuronal sites for the synthesis of voltage‐sensitive sodium channels, to complement neuronal perikaryal synthesis and axonal transport. Ultrastructural studies on specialized patches of axon membrane within some unmyelinated, demyelinated, and dysmyelinated axons support the hypothesis of a specific role for perinodal astrocyte processes in the assembly, stabilization, and/or maintenance of axolemma with nodal characteristics. These observations suggest a multiplicity of functions for perinodal astrocyte processes at central nodes and implicate the astrocyte as an important component of the node of Ranvier.
Potassium conductance in Müller cells of fish
Authors: Newman Eric A.
Published: January 2005

The distribution of potassium conductance across the surface of retinal glial (Müller) cells was determined in three species of fishes: two teleosts, the goldfish (Carassius auratus) and the alewife (Alosa pseudoharengus), and an elasmobranch, the spiny dogfish (Squalus acanthias). Potassium conductance was measured by monitoring cell depolarizations evoked by focal ejections of a 15 mEq/L K+ solution onto the surface of freshly dissociated cells. The K+ conductance distributions observed in these three species resembled those found previously in other animals with avascular retinas. In both alewife and dogfish, K+ conductance was highest in the endfoot; K+ conductance in the distal half of these cells ranged from 7.0 to 22.9% of the endfoot conductance. In goldfish, in contrast, K+ conductance was highest in the proximal region of the proximal process (114% of the endfoot conductance). As in the two other species, however, K+ conductance in goldfish was low in the distal half of the cell (7.6 to 40.1% of endfoot conductance). Mean input resistance values of isolated cells were as follows: goldfish, 12.5 MΩ; alewife, 26.4 MΩ; dogfish, 38.0 MΩ. The high resistance of dogfish Müller cells lacking their endfeet (749 MΩ) indicates that 95% of the cell membrane conductance is located in or near the endfoot in this species.
Stimulation of nerve growth factor mRNA content in C6 glioma cells by a β‐adrenergic receptor and by cyclic AMP
Authors: Schwartz Joan P.
Published: January 2005

Authentic β‐nerve growth factor mRNA, ∼1.35 kb in size, has been detected by Northern blot analysis in C6 glioma cells. Exposure of the cells to the β‐adrenergic agonist isoproterenol leads to a three‐ to fourfold increase in NGF mRNA, which reaches a peak by 2 hr. The EC50 for this effect of isoproterenol is ∼2nM. The effect can be blocked by the β‐blocker propranolol but not by the α‐blocker phenoxybenzamine. Treatment of the cells with forskolin also increases NGF mRNA three‐ to fourfold, with a maximal effect by 2 hr. The stimulation of NGF mRNA by maximal concentrations of forskolin and isoproterenol is not additive; similarly, the two drugs have a nonadditive effect on cyclic AMP content. The results suggest that NGF gene transcription can be stimulated via increases in intracellular cyclic AMP and that regulation of NGF production by glial cells may occur via activation of cell‐surface neurotransmitter receptors such as the β‐adrenergic receptor.
Characterization of glial subpopulations in cultures of the ovine central nervous system
Authors: Elder Gregory A. , Potts Barbara J. , Sawyer Mary
Published: January 2005

The cellular composition and in vitro development of glial cultures derived from the rat CNS has been well studied. However, less information is available on similar cultures from other species, particularly higher mammals. To study ovine glial development in vitro, cultures from 50‐day fetal to adult animals were characterized with various immunocytochemical markers, which are frequently used to define neural cell subsets in rat cultures. As in rats, both A2B5+ and A2B5− astrocytes can be identified in ovine cultures. However, ovine A2B5+ and A2B5− could not be reliably differentiated by their morphology, which was more influenced by whether the cells were in serum‐free or serum‐containing media than by their A2B5‐positive or ‐negative status. In addtion, ovine A2B5 + astrocytes were present in cultures from early fetal brain before the development of identifiable oligodendrocytes, unlike rat type II astrocytes, which develop only after the appearance of oligodendrocytes. An A2B5 + cell, morphologically similar to the rat 02‐A cell, can be found in cultures from fetal ovine cerebrum or cerebellum. A2B5 +/ glial fibrillry acidic protein (GFAP)– cells in cultures from 100‐ to 115‐day ovine cerebellum appeared to differentiate into A2B5 + astrocytes in serum‐containing media. However, in serum‐free media, although the A2B5 + cells assumed a more “oligodendroglial‐like” morphology, they did not express galactocerebroside or myelin basic protein, suggesting that these cells may not be bipotential as is the rat 02‐A cell. Oligodendroglial differentiation was not induced by treatment with dibutyryl cyclic AMP or insulin‐like growth factor I. Many cells in cultures from a variety of fetal ages did not label with any of the immunocytochemical markers used, suggesting the need for more cell‐type‐specific markers to identify neural cell subsets in higher mammals.
Astrocytes regulate GFAP mRNA levels by cyclic AMP and protein kinase C‐dependent mechanisms
Authors: Shafit‐Zagardo Bridget , Kume‐Iwaki Akiko , Goldman James E.
Published: January 2005

Glial fibrillary acidic protein (GFAP) mRNA and protein levels in rat astrocyte cultures and in the human astrocytoma line U‐373MG were examined in order to determine the effects of agents that regulate cAMP‐dependent kinase and protein kinase C. Treatment of cells with dibutyryl cAMP or forskolin and 3‐isobutyl‐1‐methylxan‐thine increased steady‐state GFAP mRNA levels. Short‐term treatment of cells with phorbol‐12‐myristate‐13‐acetate (PMA) increased GFAP mRNA levels, but prolonged treatment of cells with PMA or 1‐oleoyl‐2‐acetyl‐rac‐glycerol produced a dramatic decrease in GFAP mRNA; 4‐beta‐phorbol had no effect. Thus, both cAMP‐dependent kinase and protein kinase C may exert regulatory roles in determining GFAP mRNA levels. Nuclear run‐off studies showed no change in GFAP mRNA synthesis after cAMP or PMA treatment, suggesting post‐transcriptional mechanisms. Western blot analysis revealed that the effect of PMA on U‐373MG cells shows specificity in that GFA protein levels decline, while those of other major cytoskeletal proteins were unaltered.
Functions of glial cells in the retina of the honeybee drone
Authors: Coles Jonathan A.
Published: January 2005

In the retina of the honey bee drone, Apis mellifera ♂, physiological interactins between glial cells and neurons (the photoreceptors) are exceptionally clearcut and amenable to investigation. The principal glia (outer pigment cells) contribute to the homeostasis of extracellular [K+] and [Na+] by (1) spatial buffering of K+ and (2) net uptake fo K+ and Cl−. The glia supply carbohydrate metabolic substrate to the neurons; only the glia take up and phosphorylate glucose. Neuronal activity (1) modifies glycogen metabolism in the glia, and (2) can be signalled to the glia in the absence of elevated extracellular [K+].
Release of taurine from astrocytes during potassium‐evoked swelling
Authors: Pasantes‐morales Herminia , Schousboe Arne
Published: January 2005

Cultured astrocytes superfused with isosmotic solutions containing high concentrations of potassium, i.e., 25, 56, 75, and 100 mM, showed a proportional increase in cell volume corresponding to 25, 36, 57, and 75% greater than the cell volume in physiological solutions. This volume increase was abolished in low chloride or hypertonic solutions.

The release of 3H‐taurine previously accumulated by astrocytes was stimulated by potassium at all concentrations examined. During 4‐minute exposure to 25, 56, 75, or 100 mM of potassium, cells released 13.5, 15.6, 20.2, or 36.2%, respectively, of the total labeled taurine accumulated during the preloading period. The potassium‐stimulated release of 3H‐taurine was calcium‐independent and insensitive to BaCl2 and bumetanide. Substitution of chloride by gluconate to concentrations necessary to maintain the K+ × Cl− product constant abolished the potassium‐stimulated release of 3H‐taurine. Superfusion with solutions made hypertonic with sucrose also decreased the potassium‐elicited efflux of 3H‐taurine. In both conditions, the depolarizing effect of potassium measured with 3H‐TPP+ was unchanged. High potassium concentrations and hyposmotic solutions released 3H‐taurine by a nonadditive mechanism. These results indicate that in cultured astrocytes high concentrations of potassium produce both swelling and depolarization, but only swelling elicits the release of taurine. These observations suggest an involvement of taurine in cell‐volume regulation in astrocytes.
Vulnerability of rat and mouse brain cells to murine hepatitis virus (JHM‐strain): Studies in vivo and in vitro
Authors: van Berlo M.F. , Warringa R. , Wolswijk G. , Lopes‐Cardozo M.
Published: January 2005

The pathogenicity and cell tropism of mouse hepatitis virus (MHV‐JHM‐strain) in the developing mouse (Balb/c) and rat (Wistar and Lewis) brain were analysed. Intracranial infection of Balb/c mice at postnatal day 5 induced a lethal encephalitis in all animals. Of Wistar rats infected at day 2 or 5 after birth, 30 to 70%, respectively, survived. The distribution of viral antigen was studied in frozen brain sections of animals that died after infection; astrocytes were found to be the major virus‐infected cell type throughout the central nervous system. More than 75% of the surviving rat pups developed paralysis, but viral antigen was detected in only few brain cells and not in astrocytes. The cell tropism of MHV‐JHM was examined further in virus‐infected glial cell cultures derived from brains of rats or mice. In the glial cultures derived from Wistar rats, only oligodendrocytes were infected, whereas in cultures derived from mouse or Lewis rat brain viral antigen was detected in both astrocytes and oligodendrocytes. Infection of astrocytes led to the formation of syncytia and degradation of the cytoskeleton. Infected rat oligodendrocytes gradually disappeared from the cultures because of cell death. These phenomena indicate that, besides an indirect autoimmune response triggered by infected astrocytes, direct virus‐induced injury to astrocytes or to oligodendrocytes can have a dominant role in the neuropathogenicity of mouse hepatitis virus. The present results underscore the importance of species and developmental stage of experimental animals in the neurotropism and pathogenicity of MHV‐JHM.
Evidence that glutamate mediates Axon‐to‐Schwann cell signaling in the squid
Authors: Lieberman Edward M. , Abbott N. Joan , Hassan Sami
Published: January 2005

High‐frequency stimulation (100 Hz) of isolated giant axons of the small squid Alloteuthis subulata and the large squid Loligo forbesi caused the periaxonal Schwann cell resting potential (Em = ‐40 mV) to hyperpolarize up to 11 mV in direct proportion to train duration and action potential amplitude. In both species, the Schwann cell also hyperpolarized up to 17 m V with the application of L‐glutamate (10−9 to 10−6 M), in a dose‐dependent manner. By contrast, in the presence of 10−8 M d‐tubocurarine (d‐TC) to block the cholinergic component of the Schwann cell response, Schwann cells depolarized 8–9 mV during electrical stimulation of the axon or application of L‐glutamate. In the presence of 10−5 M 2‐amino‐4‐phosphonobutyrate (2‐APB), the hyperpolarization to glutamate and to axon stimulation was blocked, whereas the cholinergic (carbacholinduced) hyperpolarization was unaffected. In experiments with Alloteuthis, L‐aspartate (10−7 M) also caused a Schwann cell hyperpolarization, but this was not blocked by 2‐APB. In tests with glutamate receptor agonists and antagonists, quisqualate (10−5 M) produced a hyperpolarization blocked by 10−4 M L‐glutamic acid diethylester (GDEE), which also blocked the response to axonal stimulation. Kainic acid (10−4 M) also caused a hyperpolarization, but n‐methyl‐D‐aspartate (NMDA; 10−4 M), ibotenate (10−5 M), α‐amino‐3 hydroxy‐5‐methyl‐isoxazole proprionate (AMPA; 10−4M), and isethionate (10−5M) had no effect. The results suggest that glutamate is a mediator of communication between the active axon and its surrounding Schwann cells and, by acting on quisqualate/kainate (i.e., non‐NMDA) glutamate receptors, causes depolarization of the Schwann cell membrane. This depolarization in turn appears to activate cholinergic mechanisms in the Schwann cell that result in a secondary, long‐lasting Schwann cell hyperpolarization. The function of the hyperpolarization is presently not well understood, but a contribution to regulation of the [K+] in the periaxonal microenvironment is proposed.
Activation of substance P receptors leads to membrane potential responses in cultured astrocytes
Authors: Wienrich Marion , Kettenmann H.
Published: January 2005

Cultured astrocytes from rat cortex and spinal cord responded with different types of membrane potential changes upon brief (10 seconds) applications of the natural neurokinin agonists substance P and neurokinin A. The most prominent type of response was a long‐lasting membrane depolarization. In some cells, an initial rapid depolarization followed by a partial repolarization preceded the slow depolarizing event. Few astrocytes responded with a hyperpolarization of the membrane. Selective agonists at the NK‐1 receptive site, substance P‐methyl ester (SP‐OME) and septide, mimicked the response to the natural neurokinins as did DiMe‐C7, a selective NK‐3 receptor agonist. A putative neurokinin antagonist, (D‐Arg1,D‐Pro2,D‐Trp7,9,Leu11)SP (DADPDT) partially blocked membrane potential responses induced by substance P, SP‐OME, septide, DiMe‐C7, and NKA. The authors conclude that astrocytes express NK‐1 and NK‐3 receptors, which upon activation affect the electrical properties of these cells.
An endogenous lectin found in rat astrocyte cultures has a role in cell adhesion but not in cell proliferation
Authors: Kuchler Sabine , Perraud Frédéric , Sensenbrenner Monique , Vincendon Guy , Zanetta Jean‐Pierre
Published: January 2005

The presence of an endogenous cerebellar soluble lectin (CSL) has been demonstrated in cultured rat astrocytes by using immunocytochemical techniques. In these cells, the location of lectin CSL was found intracellularly as well as on the external surface of the plasma membrane of the cell bodies and processes, especially in the zones of contact between cells. This suggested that CSL could have a role in adhesion of astrocytes to sister cells. Kinetics of adhesion of astrocytes to culture dishes precoated with CSL showed a rapid binding of these cells. In confluent astrocyte cultures, anti‐CSL Fab fragments affected the shape and organization of astrocytes (retraction of the cytoplasm), but they did not detach cells from the substratum. These results indicated that CSL has adhesive properties for astroglial cells and is probably involved (1) in adhesion of astrocytes to sister cells; (2) in binding of protoplasmic regions of astrocyte membrane to the substratum. Further support for these roles came from demonstration of the presence in cultures of glycoprotein ligands recognized by this lectin. The problem of the mitogenic properties of the lectin was also questioned. The addition of CSL to confluent astroglial cultures was able to stimulate only by 40% the proliferation of these cells at an optimal concentration of 5 μg CSL lectin/ml of culture medium. This indicated that CSL is not a powerful growth factor for astrocytes.
Electrical coupling, without dye coupling, between mammalian astrocytes and oligodendrocytes in cell culture
Authors: Ransom B. R. , Kettenmann H.
Published: January 2005

Evidence of electrical and dye coupling between oligodendrocytes and astrocytes was sought in cultures of mouse spinal cord. Cell identity was verified using cell specific antigenic markers. In most experiments current was injected into oligodendrocytes while recording voltage in nearby astrocytes. Nine of 17 oligodendrocyteastrocyte cell pairs showed weak electrical coupling; the average estimated coupling ratio was 0.03 ± 0.06 (cf. 0.11 for oligodendrocyte‐oligodendrocyte and 0.44 for astrocyteastrocyte pairs; Kettenmann and Ransom: Glia, 1: 64–73, 1988). Application of 0.5 mM BaCl2 or 44.6 mM CsCl depolarized astrocytes and oligodendrocytes and was estimated to increase the coupling ratio between these cells 3–5‐fold; these effects were rapid in onset and completely reversible. In 5 of 7 cases, oligodendrocyte‐astrocyte pairs that appeared uncoupled in normal solution exhibited coupling during Ba++ or Cs+ exposure. The actions of these cations are believed to be mediated by blockade of glial K+ channels. Depolarization, per se, as induced by increasing [K+]o, did not increase coupling ratio. The fluorescent dye lucifer yellow (LY) was injected into 10 oligodendrocytes, 8 of which were electrically coupled to nearby astrocytes, and never passed into astrocytes in detectable quantities. Likewise, astrocytes injected with LY stained other astrocytes, but never oligodendrocytes. These findings document the presence of weak electrical coupling between astrocytes and oligodendrocytes, in the absence of dye coupling. Weak coupling of this sort could subserve metabolic interactions between these cells mediated by the passage of small but important molecules such as cyclic AMP, but would not allow strong electrical interactions. If such coupling among glial cells is widespread, it would constitute a “metabolic syncytium” that could serve to coordinate glial behavior.
Experimental inhibition of peroxisomal β‐oxidation in rats: Influence on brain myelination
Authors: Van Den Branden Christiane , Leeman Jan , Dacremont Georges , Collumbien Robert , Roels Frank
Published: January 2005

Oral administration of thioridazine, an inhibitor of peroxisomal β‐oxidation, to normal rats from weaning till day 60 causes a small increase of the very long chain fatty acid C26 in brain lipids. Myelination in the brain is decreased. In the genu of the corpus callosum the ratio of non‐myelinated/myelinated axons is increased. In the commissura anterior the myelin sheaths of the axons are significantly thinner in treated than in control animals. Undernourishment caused by the drug is minimal in this experiment. Area and total DNA of glial nuclei are unaltered in both the genu and the commissura anterior of treated rats. The distribution of chromatin (texture), however, shows small differences in the corpus callosum.
Isoproterenol selectively releases endogenous and [14C]‐labelled taurine from a single cytosolic compartment in astroglial cells
Authors: Waniewski Robert A. , Martin David L. , Shain William
Published: January 2005

Taurine is stored in and released from astroglial cells. We have investigated whether taurine is stored in multiple subcellular compartments and whether taurine is released from a distinct pool. Taurine compartmentation was examined by determining the subcellular distribution of labelled taurine and by comparing the specific radioactivities of taurine in cells with that of the taurine released into the superfusion medium during receptor stimulation. Three observations indicate that taurine is found in a single subcellular pool. First, labelled taurine was localized exclusively within a freely exchangeable pool and not sequestered in membrane‐bound compartments. Second, endogenous and newly acquired [14C]taurine was released identically by the β‐adrenergic agonist isoproterenol (IPR). Third, measurements of the specific radioactivity of taurine indicate that accumulated [14C]taurine mixes homogeneously with endogenous taurine and both are simultaneously released from the same pool. Chemical analysis revealed net uptake of taurine from the medium during loading and a net loss during IPR‐stimulated release. Taurine was selectively released by IPR; five other amino acids found in the superfusion medium were unaffected. One possible mechanism capable of explaining these results is that receptor‐mediated taurine release from astroglia occurs by a selective transporter moving taurine from a single cytoplasmic pool to the extracellular space.
Accumulation of extracellular glutamate and neuronal death in astrocyte‐poor cortical cultures exposed to glutamine
Authors: Rosenberg Paul A.
Published: January 2005

The function of astrocytes in cerebral cortex may be studied by comparing the properties of conventional, astrocyte‐rich cultures with astrocyte‐poor cultures in which astrocyte proliferation has been stringently suppressed. Exposure of astrocytepoor, but not astrocyte‐rich, cultures to fresh medium containing 2 mM glutamine resulted in the death of most neurons within 24 h. This study was undertaken to understand the basis for the apparent toxicity of glutamine in astrocyte‐poor cultures. The toxicity of glutamine was found to be mediated by glutamate, which demonstrated an LD50 as a neurotoxin in astrocyte‐poor cultures of 2 μM. Exposure of astrocyte‐poor (but not astrocyte‐rich) cultures to fresh medium containing glutamine for 17.5–24 h resulted in the accumulation of substantial quantities of glutamate (255 ± 158 μM; mean ± standard deviation) coincident with the death of neurons in the cultures. Exposure of astrocyte‐poor cultures to glutamate in the absence of glutamine did not result in the accumulation of extracellular glutamate. Both the neuronal death and the extracellular glutamate accumulation in astrocyte‐poor cultures exposed to glutamine could be blocked by N‐methyl‐D‐aspartate (NMDA) antagonists. These observations suggest that astrocytes as well as glutamine may play an important role in the pathogenesis of glutamate neurotoxicity in the central nervous system.
Division of astroblasts and oligodendroblasts in postnatal rodent brain: Evidence for separate astrocyte and oligodendrocyte lineages
Authors: Skoff Robert P. , Knapp Pamela E.
Published: January 2005

What precursor cells are the source of the macroglia generated during postnatal development? In order to answer this question, we studied the expression of glial specific antigens in proliferating neuroglia in postnatal rodent brain and optic nerve. Immunocytochemistry using antibodies to oligodendrocyte (OL) specific markers (sulfatide and galactocerebroside) and an astrocyte (AS) specific marker (glial fibrillary acidic protein) was combined with thymidine autoradiography. During the first week of postnatal development when most ASs are being generated, one third to one half of the proliferating cells in the optic system are positive for glial fibrillary acidic protein after a 1 h injection of thymidine (Skoff, Dev. Biol., 139:149–168, 1990). During the second postnatal week when OLs are being generated, 30 to 100% of the proliferating cells in presumptive white matter tracts are sulfatide positive and at least 10% are galactocerebroside positive. This finding demonstrates that ASs and OLs divide during postnatal development. These results confirm previous electron microscopic autoradiographic studies showing that the vast majority of proliferating cells in postnatal rat optic nerve have the morphologic characteristics of differentiating ASs or OLs (Skoff, J. Comp. Neurol., 169:291–312, 1976). Since proliferating ASs (astroblasts) and OLs (oligodendroblasts) constitute the majority of the dividing cells at the time that ASs and OLs are being generated, these glioblasts must be the major source for the macroglia generated postnatally.

The findings strongly suggest that separate lineages exist for ASs and OLs during postnatal development. There is no compelling in vivo evidence for a bipotential progenitor cell that generates the majority of OLs and certain ASs in postnatal rodent brain. There may, of course, be distinct lineages for the subtypes of ASs and possibly even for subtypes of OLs. We review the concepts of commitment and plasticity and apply these terms to glial differentiation. In situ, the presence of oligodendrobalsts and astroblasts demonstrates the COMMITMENT of proliferating cells to a specific glial lineage during normal development. Culture conditions may provide an environment that permits proliferating glial cells to vacillate in their selection of a specific lineage. This situation demonstrates developmental PLASTICITY and the ability of glia to adapt to an altered environment. Whether committed glial cells in situ can be induced to switch their lineage when normal CNS conditions are altered is an intriguing question that remains to be answered.
Characterization of glutamate uptake systems in astrocyte primary cultures from rat brain
Authors: Flott Berenike , Seifert Wilfried
Published: January 2005

The dependence of 3[H]‐L‐glutamate uptake on the presence of sodium, chloride, or calcium ions or on a combination of the three was investigated in astrocyte primary cultures. A stimulating effect on glutamate uptake by each of the ions tested was found. In addition to the comparably small effect by calcium alone, calcium exhibits a synergistic effect on the sodium‐ and chloride‐dependent uptake. The sodium‐dependent transport accumulates the glutamate analogue D‐aspartate as well as L‐glutamate. L‐aspartate is taken up by about 50% of the values observed for L‐glutamate transport. Sodium‐dependent glutamate uptake is strongly inhibited by aspartate‐beta‐hydrox‐amate (AβH) and threo‐beta‐hydroxyasparatate (TβH). Quisqualate is less potent in inhibiting this uptake.

In contrast, the chloride‐ and the calcium‐dependent uptake systems do not handle D‐ and L‐aspartate as substrates. AβH and TβH are only poor inhibitors of these transporters while quisqualate reduces glutamate uptake almost completely. Kinetic data of all uptake systems were estimated. High and low affinity components of each individual system are demonstrated by Eadie‐Hofstee analysis. Hill plots indicate that high and low affinity uptake may be due either to two respective uptake sites for Na+‐, Cl−‐, and Ca++‐dependent glutamate transport, or to two glutamate binding sites for each single transport system with negative cooperativity.
Contact spacing among astrocytes in the central nervous system: An hypothesis of their structural role
Authors: Distler Claudia , Dreher Zofia , Stone Jonathan
Published: January 2005

Astrocytes are found throughout the central nervous system. They interact closely with surrounding structures, their processes contributing to the glia limitans of the neural tube, and to the glial investment of blood vessels, and of the somas, axons, and synaptic structures of neurones. This paper presents evidence that astrocytes in the central nervous system also interact with each other in a dual way, adhering to their neighbours via their processes, and repelling the somas of those neighbours. We suggest that this interaction, which has been termed contact spacing, distributes astrocytes through the central nervous system, and forms the basis of their structural role.
Immunolocalization of ciliary neuronotrophic factor in adult rat sciatic nerve
Authors: Rende Mario , Muir David , Ruoslahti Erkki , Hagg Theo , Varon Silvio , Manthorpe Marston
Published: January 2005

Two rabbit polyclonal antibodies were raised against synthetic peptides corresponding to residue numbers 45–59 and 181–200 of rat ciliary neuronotrophic factor (CNTF). The resulting antibodies were purified by affinity chromatography and both purified antibodies reacted by enzyme‐linked immunoassay (ELISA) and immunoblotting with rat sciatic nerve CNTF. The anti‐CNTF peptide antibodies were used to immunostain sections of adult rat sciatic nerve, previously known as the richest tissue source of CNTF. By light microscopy both antibodies appeared to stain exclusively Schwann cells and axons and both did so with the same pattern of specific staining. Immunostaining was eliminated by absorption of the anti‐peptide antibodies with either their corresponding peptide or with purified rat nerve CNTF or by using purified nonspecific IgG. Schwann cells were stained and in semi‐thin sections this staining appeared to be in the Schwann cell cytoplasm. Axons could be stained in addition to Schwann cells providing higher concentrations of antibodies were used. Epineurial, endoneurial and endothelial cells appeared unstained. Since all Schwann cells and axons appear to contain CNTF and since CNTF is known to act in vitro to support sensory and sympathetic ganglionic and motor neurons, we suggest that Schwann cells may normally provide CNTF to those neurons contributing axons to the peripheral nerve.
Reperfusion paradox: A novel mode of glial cell injury
Authors: Kim‐Lee Myung H. , Stokes Bradford T. , Yates Allan J.
Published: January 2005

We have attempted to reconstruct in vitro the events that may occur in vivo during reperfusion injury after ischemia in the central nervous system. The phenomenon is induced by previous exposure to low calcium solutions (“calcium paradox”) before the reperfusion episode. Intracellular calcium alterations during reperfusion of human astrocytoma U1242MG cells have been investigated with microspectrofluorimetry using the calcium‐sensitive dye fura‐2. Cells were perfused in calcium‐free buffer solution for 30 min and then re‐exposed to the control buffer solution (1.5 mM CaCl2). [Ca2+]i increased up to 3.5 times control levels during the reperfusion period. The mechanism of the increase was also investigated. Addition of TTX (2 μM) or choline chloride sodium substitution during perfusion with low calcium prevented the [Ca2+]i increase during reperfusion. Reperfusion increases in [Ca2+]i were exacerbated by low potassium in the perfusion medium, but unaltered by the calcium channel blockers cadmium (100 μM) and nickel (100 μM). In a similar manner, flunarizine (10 μM) and cadmium (100 μM) were unable to modify reperfusion [Ca2+]i alterations. Low sodium in the reperfusion medium produced significant increases in [Ca2+]i if preceded by low potassium and calcium perfusion. The viability of cells after 24 h of incubation after the insult produced by exposure to Ca2+‐free media for 30 min was also investigated. Compared with control groups, the groups treated with Ca2+‐free media for 30 min had a decreased number of surviving cells and morphological alterations indicative of cell pathology. The relative number of cytotoxic cells was increased by maneuvers (low potassium perfusion) that presumably blocked the Na/K ATPase. These data support the hypothesis that calcium entry during reperfusion is via the Na/Ca exchanger and suggest a mechanism by which glia could become irreversibly injured in vivo. Such delayed pathological processes could exacerbate already described mechanisms of cell death in the central nervous system.
Characterization of Gsα mRNA transcripts in primary cultures of rat brain astrocytes
Authors: Feinstein Douglas L. , Mumby Susanne M. , Milner Robert J.
Published: January 2005

A cDNA clone encoding a stimulatory G‐protein α subunit (Gsα) was isolated from a cDNA library derived from cultured rat astrocytes. The nucleotide sequence of the cDNA indicated that it corresponds to the Gsα‐2 form og Gsα mRNA, one of four Gsα mRNAs known to be derived by alternative splicing from the human Gsα gene. A ribonuclease protection assay using cRNA from this clone allowed distinction between the Gsα‐1 and Gsα‐2 mRNAs, which encode the 52‐kDa (Gs‐L) forms of Gsα. Astrocytes express relatively high amounts of Gsα‐1 mRNA, much lower amounts of the Gsα‐2 mRNA, and no detectable amounts of the mRNAs (Gsα‐3 and Gsα‐4) encoding the two 45‐kDa forms of Gsα (Gsα‐S). Similar results were obtained with RNA samples isolated from whole brain. The 45‐kDa form of Gsα protein was not detectable by immunoblot analysis of a membrane preparation from rat cerebral corted (the source of the astrocyte cultures). These results indicate that the expression of Gsα forms in astrocytes is similar to that found in whole brain.
Migration of grafted rat astrocytes: Dependence on source/target organ
Authors: Hatton James D. , Garcia Ruel , Sangu Hoi
Published: January 2005

Neonatal rat cortical astrocytes migrate extensively after transplantation into the brains of adult hosts. However, the effects of cues provided by different sources of donor astrocytes and by different target sites of implantation on this migration is unknown. In order to investigate the significance of regional influences on glial migration, we established primary cultures of astrocytes derived from 1–3 day old rat cerebral cortex, hippocampus, and hypothalamus. After in vitro labelling with either Fast Blue or fluorescein‐labelled latex beads, these astrocytes were inoculated into different target regions of the adult rat brain by stereotaxic injection with a Hamilton syringe. Astrocytes implanted into the cerebral cortex imgrated extensively throughout the adult brain regardless of their donor source. These implanted cells were intimately associated with the ventricular wall, glial limitans, vasculature, and fiber bundles. Astrocytes homografted into the hippocampus and the hypothalamus migrated primarily within and around the respective homotopic target organs. Migration of astrocytes derived from cerebral cortex was also limited when injections were made into these two regions. In these latter cases, migration appeared to be less guided by other cellular or regional cues than was migration after implantation into the cerebral cortex. These results suggest that the migration patterns of astrocytes grafted after tissue culture are more dependent on the target implantation site than on the donor organ. © 1992 Wiley‐Liss, Inc.
Reactive astrocytes in myelin‐deficient rat optic nerve reveal an altered distribution of orthogonal arrays of particles (OAP)
Authors: Rohlmann Astrid , Gocht Andreas , Wolburg Hartwig
Published: January 2005

Reactive astrocytes are a common feature of various pathological conditions within the CNS. Morphological changes of reactive astrocytes include an altered nucleus‐cytoplasm relationship, nuclear indentations, an increased amount of intermediate filaments, and an immunologically immature phenotype. Additionally, the number of orthogonal arrays of particles (OAP) was found to be increased within parenchymal membranes of reactive astrocytes. This observation prompted us to investigate the distribution of astroglial OAP in the amyelinated CNS of the myelin‐deficient (md) rat in which reactive astrocytes prevail. In the present freeze‐fracture study, astroglial OAP were determined within endfoot and nonendfoot (parenchymal) membranes in the developing optic nerve of md rats and normal littermates at the age of 12, 23, 40, and 64 days postnatally (dpn).

The endfoot OAP density in md astrocytes remained constant during the entire period of investigation. In myelinated littermates, OAP densities continuously increased up to adult values. In contrast, the parenchymal OAP density in md astrocytes increased during the entire period of investigation. In normal littermates, OAP densities remained constant during the first 40 dpn and thereafter increased rapidly. These observations suggest that the absence of myelinogenesis in the md mutant may be a stimulus for parenchymal membranes of reactive astrocytes to insert OAP or to assemble OAP subunits into complete arrays. © 1992 Wiley‐Liss, Inc.
Heme oxygenase is a heat shock protein and PEST protein in rat astroglial cells
Authors: Dwyer Barney E. , Nishimura Robert N. , De Vellis Jean , Yoshida Tadashi
Published: January 2005

Cultured rat forebrain astrocytes contained significant amounts of immunostainable heme oxygenase‐1 (HO‐1) isozyme, whereas HO‐1 was undetectable in spontaneously transformed rat astroglial cells (ATs). HO‐1 was inducible in both cell types by heat shock and by submicromolar amounts of H2O2. Inhibition of RNA synthesis with actinomycin D or protein synthesis with cycloheximide resulted in the rapid loss of immunostainable heme oxygenase in astrocytes. Analysis of the primary structure of heme oxygenase suggests that it is a PEST protein, i.e., targeted for rapid turnover. © 1992 Wiley‐Liss, Inc.
Sodium channel expression in optic nerve astrocytes chronically deprived of axonal contact
Authors: Minturn J. E. , Sontheimer H. , Black J. A. , Ranson B. R. , Waxman S. G.
Published: January 2005

Immunocytochemical and electrophysiological methods were used to examine the effect of retinal ablation on the expression of sodium channels within optic nerve astrocytes in situ and in vitro. Enucleation was performed at postanatal day 3 (P3), and electron microscopy of the enucleated optic nerves at P28–P40 revealed complete degeneration of retinal ganglion axons, resulting in optic nerves composed predominantly of astrocytes. In contrast to control (non‐enucleated) optic nerve astrocytes, which exhibited distinct sodium channel immunoreactivity following immunostaining with antibody 7493, the astrocytes in enucleated optic nerves did not display sodium channel immunoreactivity in situ. Cultures obtained from enucleated optic nerves consisted principally ( > 90%) of glial fibrillary acidic protein (GFAP)+/A2B5− (“type‐1”) astrocytes, as determined by indirect immunofluorescence; GFAP+/A2B5+ (“type‐2”) astrocytes were not present, nor were GFAP−/A2B5+ (O‐2A) progenitor cells. Sodium channel immunoreactivity was not present in GFAP+/A2B5− astrocytes obtained from enucleated optic nerves; in contrast, GFAP+/A2B5− astrocytes from control optic nerves exhibited 7493 immunostaining for the first 4–6 days in culture. Sodium current expression, studied using whole‐cell patch‐clamp recording, was attenuated in cultured astrocytes derived from enucleated optic nerves. Whereas 39 of 50 type‐1 astrocytes cultured from intact optic nerves showed measurable sodium currents at 1–7 days in vitro, sodium currents were present in only 6 of 38 astrocytes cultured from enucleated optic nerves. Mean sodium current densities in astrocytes from the enucleated optic nerves (0.66 ± 0.3 pA/pF) were significantly smaller than in astrocytes from control optic nerves (7.15 ± 1.1 pA/pF). The hχ‐curves of sodium currents were similar in A2B5− astrocytes from enucleated and control rat optic nerves. These results suggest that there is neuronal modulation of sodium channel expression in type‐1 optic nerve astrocytes, and that, following chronic loss of axonal association in vivo, sodium channel expression is down‐regulated in this population of optic nerve astrocytes. © 1992 Wiley‐Liss, Inc.
Response of microglial cells to experimental rat glioma
Authors: Morioka Takato , Baba Takehiko , Black Keith L. , Streit Wolfgang J.
Published: January 2005

The response of indigenous CNS microglia to an experimentally induced glioma has been studied in rat brain using lectin histochemistry with the Griffonia simplicifolia B4‐isolectin. The study was undertaken 2 weeks after tumor cell injection when tumor size was near maximal. Reactive microglial cells formed a dense band that surrounded most of the well‐circumscribed tumor mass, and extended along the corpus callosum into the contralateral cerebral hemisphere. From the periphery inward, reactive microglia extended into the tumor tissue, where large numbers of them were found to be present as microglia‐derived macrophages. The lectin stain, which also labels endo thelial cells, revealed a highly vascularized tumor with ongoing neovascularization apparent as vascular sprouts. Moderate numbers of lectin‐stained blood monocytes were localized primarily inside the vessel lumina. Our results show that microglial cells react to brain tumors; however, it remains to be determined whether the microglial response represents an active antitumor defense mechanism that could be manipulated during immunotherapeutic approaches. © 1992 Wiley‐Liss, Inc.
Determination of the origin and nature of brain macrophages and microglial cells in mouse central nervous system, using non‐radioactive in situ hybridization and immunoperoxidase techniques
Authors: De Groot Corline J. A. , Huppes Wim , Sminia Taede , Kraal Georg , Dijkstra Christine D.
Published: January 2005

The origin and nature of brain macrophages and microglial cells in the mouse central nervous system (CNS) were investigated. First, the expression and localization of determinants recognized by the different monoclonal antibodies (mAbs) MOMA‐1, Mac‐1‐α, and F4/80 (raised against cells of the mononuclear phagocyte system) were immunohistochemically studied in the developing and adult mouse brain. In order to clarify the origin of brain macrophages and microglial cells, we used bacteriophage λ transgenic mice as donors for bone marrow transplantations in recipient mice of different ages.

During ontogeny, numerous MOMA‐1‐, Mac‐1‐α‐, and F4/80‐positive blood monocytederived brain macrophages (amoeboid microglia) infiltrated the CNS parenchyma. These brain macrophages gradually disappeared from the brain parenchyma at postnatal day 7 (P7). From P17 on, Mac‐1‐α‐ and F4/80‐positive cells were detected within the brain parenchyma with the morphology of resting microglial cells. Transitional forms between brain macrophages and “resting” microglia were not observed in the developing brain.

Combined non‐radioactive in situ hybridization and immunohistochemistry revealed many MOMA‐1‐positive bone marrow‐derived brain macrophages that were located in the leptomeninges, the ventricles, and occasionally the blood vessel walls. These results show that brain macrophages are of bone marrow origin. Many “resting” microglial cells were detected in the brain, mainly in the white matter. It appeared that about 10% of these cells displayed the transgenic signal. This result indicates that the majority of “resting” microglial cells are of local, presumably neuroectodermal, origin. © 1992 Wiley‐Liss, Inc.
Mechanisms of intercellular calcium signaling in glial cells studied with dantrolene and thapsigargin
Authors: Charles Andrew C. , Dirksen Ellen R. , Merrill Jean E. , Sanderson Michael J.
Published: January 2005

Mechanical stimulation of a single cell in a primary mixed glial cell culture induced a wave of increased intracellular calcium concentration ([Ca2+]i) that was communicated to surrounding cells. Following propagation of the Ca2+ wave, many cells showed asynchronous oscillations in [Ca2+]i. Dantrolene sodium (10 μM) inhibited the increase in [Ca2+]i associated with this Ca2+ wave by 60‐80%, and prevented subsequent Ca2+ oscillations. Despite the markedly decreased magnitude of the increase in [Ca2+]i, the rate of propagation and the extent of communication of the Ca2+ wave were similar to those prior to the addition of dantrolene. Thapsigargin (10 nM to 1 μM) induced an initial increase in [Ca2+]i ranging from 100 nM to 500 nM in all cells that was followed by a recovery of [Ca2+]i to near resting levels in most cells. Transient exposure to thapsigargin for 2 min irreversibly blocked communication of a Ca2+ wave from the stimulated cell to adjacent cells. Glutamate (50 μM) induced an initial increase in [Ca2+]i in most cells that was followed by sustained oscillations in [Ca2+]i in some cells. Dantrolene (10 μM) inhibited this initial [Ca2+]i increase caused by glutamate by 65‐90% and abolished subsequent oscillations. Thapsigargin (10 nM to 1 μm) abolished the response to glutamate in over 99% of cells. These results suggest that while both dantrolene and thapsigargin inhibit intracellular Ca2+ release, only thapsigargin affects the mechanism that mediates intercellular communication of Ca2+ waves. These findings are consistent with the hypothesis that inositol trisphosphate (IP3) mediates the propagation of Ca2+ waves whereas Ca2+ ‐induced Ca2+ release amplifies Ca2+ waves and generates subsequent Ca2+ oscillations.