摘要:

AbstractWe have shown by immunohistochemical methods that the gap junction protein connexin43 is heterogeneously distributed in rat brain (Yamamoto et al: J Comp Neurol 302:853, 1990). Here we have compared quantitatively the relative amount of connexin43 detected on Western blots of seven central nervous system (CNS) regions with the density of connexin43‐immunoperoxidase reactivity in these regions. As has been observed on Western blots of several cell types, homogenates of these CNS regions contained two forms of connexin43, its dephospho form with an apparent mobility of ∼41 kDa and its ∼3 kDa phosphorylated form. While the relative quantities of connexin43 varied considerably among the brain regions, the ratio of the 43/41 kDa forms, 0.71, was relatively uniform (correlation coefficient, r = 0.92). Sections of brain processed for connexin43‐immunolocalization by the peroxidase–antiperoxidase (PAP) method showed that chromogen deposition was linear with incubation time in reaction medium. Optical density of tissue connexin43‐immunoreactivity in each of the seven areas plotted against the density of connexin43 bands on Western blots gave a correlation coefficient of r = 0.90. Connexin43‐immunoreactivity had a similar appearance in sections processed by PAP or immunofluorescense procedures and consisted of isolated or aggregates of puncta. For the brain regions examined we conclude: (1) that although total levels of connexin43 vary widely, the levels of its phosphorylated and non‐phosphorylated forms are held in fairly constant proportion; (2) that there is a high correlation between the relative amounts of connexin43 detected biochemically and the density of connexin43‐immunostaining in tissue sections; and (3) that immunohistochemical staining for connexin43 reflects the relative amounts of connexin43 and possibly the numbers of gap junctions present in indiv

ISSN:0894-1491    

DOI:10.1002/glia.440050102

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractMale and female hamsters aged 1, 4, and 10 postnatal weeks were used to study the distribution of vimentin and glial fibrillary acidic protein (GFAP) in the cerebellum. Vimentin immunoreactivity exceeded that of GFAP during the first postnatal week, although GFAP was also observed in all cerebellar layers. Immunoperoxidase analysis revealed that by the fourth postnatal week vimentin was only detected in Bergmann fibers and the very scarce fibrous astrocytes located in the inner white matter. The Purkinje cell bodies were only coated with GFAP‐immunopositive processes. At 10 weeks, vimentin immunoreactivity was reduced to thin Bergmann glial processes, whereas GFAP immunoreactivity had greatly increased in the whole cerebellum.The GFAP immunostaining was denser in males than in females; however, in females, the Bergmann fibers were heavily immunostained with anti‐vimentin in contrast to the males. The results described in the present paper indicate a sex difference in vimentin and GFAP immunoreactivities in the cerebellar astrocytes at 4 weeks of age, which persisted in the oldest hamsters in this study. The existence of sexual dimorphism might suggest that the expression of both gliofilament proteins could be influenced by circulating sex stero

ISSN:0894-1491    

DOI:10.1002/glia.440050103

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractPotassium uptake, possibly together with chloride, is one of the presumed functions of Schwann cells in the peripheral nervous system. However, the presence of chloride channels has not been demonstrated in adult Schwann cells. We present here a new method which allows single channel recordings to be made from Schwann cells in situ without enzymatic treatment. Isolated rat spinal roots were split mechanically into several bundles. Within about 30 min after this procedure small belb‐like vesicles (∼20–30 μm in diameter) with a clean surface appeared at the edges of the fibre bundles. Immunofluorescence microscopy with a surface marker for Schwann cell membranes (monoclonal antibody O4) revealed that the vesicles originate from Schwann cells. In standard patch clamp recordings with symmetrical bath and pipette solutions (excised inside‐out configuration) an anion channel with the following characteristics was mainly observed: (1) single channel slope conductance of 337 ± 5 pS in 125 mM KCl and 209 ± 6 pS in 125 mM K+methylsulphate; (2) ion permeability ratio: PCl/PK/Pgluconate= 1/0.12/0.06; (3) linear current‐voltage relationship (range ± 60 mV) and (4) voltage‐ and time‐dependent inactivation (the channel was most active at potentials ± 20mV). Pharmacologically, the channel was completely blocked with zinc (1 mM) and barium (10 mM). A similar anion channel, showing characteristics 1 – (4), has been described in cultured Schwann cells of newborn rats (Gray et al., 1984). We now demonstrate that this channel is also present in adult S

ISSN:0894-1491    

DOI:10.1002/glia.440050104

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractTwo 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 perip

ISSN:0894-1491    

DOI:10.1002/glia.440050105

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractOf the three classes of sulphated proteoglycans produced by type 1 astrocytes in vitro and released into conditioned medium, only heparan sulphate (HS) was associated with enhanced neurite growth by sensory neurons following pretreatment of a laminin substratum. Astrocyte‐conditioned medium (ACM) produced in the presence of certain inflammatory mediators had reduced titers of neurite‐promoting activity. The low activity ACM contained inhibitors of neurite growth. Heparan sulphate proteoglycans may modulate neurite growth when complexed to constituents of the extracellular milieu either directly or by interacting with other growth‐promoting or growth‐inhibitory

ISSN:0894-1491    

DOI:10.1002/glia.440050106

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractDouble‐barrelled pH‐sensitive microelectrodes were used to measure the intracellular pH (pHi) of neuropil glial cells and the pH of extracellular spaces (pHe) within isolated, intact ganglia of the leechHirude medicinalis. By application of a weak acid (propionate, 40 mM) or a weak base (ammonium, 20 mM) the total buffer capacity was estimated by changes in glial pHiand in pHe. The buffering power of glial cells and in the extracellular spaces was increased by up to threefold in the presence of CO2/bicarbonate. The anion exchange inhibitor 4,4‐diisothiocyanatostillbene‐2,2′‐disulphonic acid (DIDS, 0.3–0.5 mM) reversed this increase in buffering powerbothin the glial cellsandin the extracellular spaces. Inhibitors of the carbonic anhydrases reduced the CO2/bicarbonate‐dependent increase in extracellular buffering power. The results suggest that extracellular H+buffering dependent upon the availability of bicarbonate is linked to DIDS‐sensitive bicarbonate transport across t

ISSN:0894-1491    

DOI:10.1002/glia.440050107

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractDopaminergic neurons are a primary target for 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) neurotoxicity. However, the conversion of MPTP to its neurotoxic 1‐methyl‐4‐phenylpyridinium metabolite (MPP+) is likely to occur in astrocytes via the monoamine oxidase (MAO)‐dependent formation of the 1‐methyl‐4‐phenyl‐2,3‐dihydropyridinium intermediate (MPDP+). The main purpose of this study was to characterize the molecular mechanism(s) by which MPP+, once generated by astrocytes, may reach the extracellular space to become available for the active accumulation into dopaminergic neurons. Primary cultures of mouse astrocytes were used as an in vitro model system. After the addition of MPTP, levels of MPP+ were found to increase at constant rates both intracellularly at time points when no sign of cytotoxicity was evident. In contrast, MPDP+ levels remained quite stable during 4 days of incubation in the presence of MPTP. Finally, when astrocytes were allowed to accumulate MPP+ by pretreatment with either MPTP or MPP+ and then were incubated in fresh medium not containing MPTP or MPP+, intracellular levels of MPP+ rapidly declined and corresponding amounts of this compound were found in the incubation medium. Results of this study are compatible with the following conclusions: (1) the MPP+ accumulated in the extracellular compartment during incubations with MPTP is not released from astrocytes as a consequence of its own cytotoxic effects; (2) MPP+ can be formed extracellularly presumably via autoxidation of MPDP+ after this latter compound has been generated within astrocytes and has crossed astrocyte membranes; and (3) despite its charged chemical structure, MPP+ can cross the plasma membrane toward the extracellular space after bei

ISSN:0894-1491    

DOI:10.1002/glia.440050108

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractWe 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+]iincreased 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+]iincrease during reperfusion. Reperfusion increases in [Ca2+]iwere 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+]ialterations. Low sodium in the reperfusion medium produced significant increases in [Ca2+]iif 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 ce

ISSN:0894-1491    

DOI:10.1002/glia.440050109

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractWe have investigated the effect of prenatal exposure to ethanol on the extent of binding and surface distribution of the lectin concanavalin A (con A) on rat cortical astrocytes during the periods of proliferation and differentiation in primary culture. The enzymatic activity of the plasma membrane glycoprotein 5′‐nucleotidase was also assessed. The cells were obtained from control fetuses (no exposure to ethanol) and from fetuses prenatally exposed to ethanol.The main findings were: (1) both proliferating and differentiating control astrocytes showed two distinct types of surface con A receptors that could correspond to high‐ and low‐affinity binding sites; (2) the extent of con A binding was greater in mature than in proliferating control cells; (3) the distribution of con A on cell surface components changed with differentiation; (4) the activity of 5′‐nucleotidase showed a substantial increment during the period of differentiation; and (5) prenatal exposure to ethanol clearly decreased the ability of astrocytes to bind con A, altered the surface distribution of the receptors for this lectin, and decreased the activity of 5′‐nucleotidase. These effects were more marked in proliferating cells.In conclusion, it is shown that the extent of con A labeling and the activity of 5′‐nucleotidase in astrocytes are dependent on the stage of cell differentiation and that prenatal exposure to ethanol alters the plasma membrane structure of these cells

ISSN:0894-1491    

DOI:10.1002/glia.440050110

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY

摘要:

AbstractAstrocytes have been prepared from adult rat cortex, cerebellum, and striatum, using a modification of the McCarthy‐DeVellis (J Cell Biol 85:890, 1980)method. The cultures consist of 99% type 1 polygonal astrocytes, which divide more slowly than cells from newborn animals. One day after preparing the cultures, 90% of the cells are glial fibrillary acidic protein (GFAP)‐positive and 80% are tin‐positive by immunohistochemical staining, suggesting that they are present de novo and not derived from precursor cells. The astrocytes from adult brain respond to an elevation of intracel‐lular cyclic AMP, following treatment with forskolin, by becoming more stellate in shape and putting out fine ramified processes. They contain the same amount of GFAP per mg protein, measured by immunoblot, as cells from newborn animals. These cultures thus offer the possibility of comparing the biochemical properties of astrocytes derived from adult animals with those from newborn animals, or with cultures of reactive astrocytes isolated from lesione

ISSN:0894-1491    

DOI:10.1002/glia.440050111

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1992

数据来源: WILEY