Structural organization of astrocytes in the rat hippocampus in the post-ischemic period

The aim of the present work was to study the location and structural organization of astrocytes in the rat hippocampus, which contain immunoreactive glial fibrillary acid protein (GFAP) after ischemic damage to the brain after intracerebroventricular administration of the neuroprotective agent creatine and without treatment. Light microscopy and immunocytochemical methods were used to study the brains of 26 adult male Sprague-Dawley (Koltushi) rats, some of which were subjected to total cerebral ischemia (12 min) under anesthesia with subsequent reperfusion (seven days). Creatine was given to 11 animals intracerebroventricularly using an osmotic pump (Alzet Osmotic Mini-Pump). The results showed that GFAP-immunoreactive hippocampal astrocytes were concentrated in two main zones (the stratum lacunosummoleculare of field CA1 and the stratum polymorphae of the dentate fascia). The neuroprotective effect of creatine had the result that moderate ischemic damage to the hippocampus did not lead to changes in the zones containing activated astrocytes. The redistribution of GFAP-positive astrocytes in the post-ischemic period was associated with loss of pyramidal neurons in cytoarchitectonic field CA1. Complete loss of pyramidal neurons in this area of the hippocampus leads to a qualitatively new level of astrocyte activation - proliferation.Translated from Morfologiya, Vol. 125, No. 2, pp. 19–21, March–April, 2004.     

Structural and quantitative analysis of astrocytes in the mouse hippocampus

We revealed the structural features of astrocytes by means of light microscopy, confocal laser scanning microscopy and high voltage electron microscopy, and estimated their numerical densities in the mouse hippocampus. The high voltage electron microscope examinations of Golgi-impregnated astrocytes clearly disclosed their fine leaflet-like processes in the masses occupied by individual astrocytes. The intracellular injection of two different fluorescent tracers into two neighboring astrocytes revealed that each astrocyte occupied a discrete area with a limited overlap only at its peripheral portion. In a quantitative analysis using an optical dissector, the numerical densities of astrocytes identified as S100-immunoreactive cells were only slightly different in their areal and laminar distributions. The numerical densities were higher in the stratum lacunosum-moleculare and dentate hilus, while they were slightly lower in the principal cell layers than the average (24.2 x 10(3) mm(-3)) in whole hippocampal regions. As for the dorsoventral difference, the numerical densities were significantly larger at the ventral level in the dentate gyrus, whereas such tendency was not apparent in the hippocampus proper. The projection area of the astrocytes estimated from Golgi-impregnated samples was roughly in inverse relation to the numerical densities; the areas in the stratum lacunosum-moleculare were somewhat smaller than the other layers, where the numerical densities were high. The present study indicates that astrocytes are distributed rather evenly without any prominent areal or laminar differences and that the individual astrocytes have their own domains; the periphery of the domain of a given astrocyte is interdigitated intricately with the processes of adjacent astrocytes whereas its inner core portion is not penetrated by them.     

Relationship between Na+ current expression and cell-cell coupling in astrocytes cultured from rat hippocampus

1. Cell-cell coupling between hippocampal astrocytes in culture was studied by following the intracellular spread of the low molecular weight fluorescent dye Lucifer yellow (LY). Dye coupling appeared as early as 24 h after plating, at which time approximately 20% of all astrocytes that physically contacted neighboring cells showed dye coupling. 2. The percentage of coupled cells increased with time in culture and peaked after 10 days in vitro (DIV) when approximately 50% of astrocytes showed coupling. Further time in culture, up to 20 DIV, did not increase the percentage of coupled cells. Thus, coupled and noncoupled astrocytes coexist in hippocampal cultures in approximately equal numbers. 3. Na+ currents were expressed in a subpopulation of hippocampal astrocytes and changed characteristics during in vitro development. A "neuronal type" of Na+ current, so called because of an h alpha curve that had a midpoint near -60 mV, was observed within the first 5 days post-plating. A "glial type" of Na+ current, characterized by a -25 mV shift in its h alpha curve, was only expressed after 6 days in culture. 4. Na+ current expression was restricted to hippocampal astrocytes that did not exhibit dye coupling; astrocytes that exhibited dye coupling (n = 39) did not show measurable Na+ currents. 5. The failure to see Na+ currents in coupled astrocytes cannot be explained by insufficient space-clamp since astrocytes acutely uncoupled with octanol (10 microM) did not reveal Na+ current expression. Control experiments showed that low concentrations of octanol (i.e., 10-100 microM) did not block Na+ currents; blockage of Na+ currents by octanol was only observed at high concentrations (e.g., 50-fold the concentration used for uncoupling). These observations support the idea that Na(+)-channel expression was restricted to noncoupled astrocytes. 6. The time courses for the development of cell coupling and Na+ current expression appeared to be inversely correlated and suggested a gradual increase in cell coupling in concert with a loss in Na+ current expression with time in culture.     

Glial fibrillary acidic protein immunoreactive astrocytes in developing rat hippocampus.

The developmental pattern of glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes was investigated in the hippocampus (subfields CA1, CA3 and CA4) and in the dentate gyrus of male and female rats aged 11, 16, 30, 90 and 150 days by immunohistochemistry associated with image analysis. Analysis was centred on stratum radiatum, a hippocampal area rich in GFAP-immunoreactive astrocytes. The volume of different portions of hippocampus, the number and the size of astrocytes, the intensity of cell body GFAP immunostaining as well as the extension of astrocyte were assessed. A maturation pattern consisting in higher cellular expression of GFAP, an increase in overall cell size and expanding arborisation from the 11th to the 30th postnatal day, followed by stabilisation of these parameters until the 90th day of life, and a subsequent decrease in the oldest age group studied was found. A sex-related different temporal pattern of astrocytes maturation in size and GFAP content was observed in the CA1 subfield only. The increase of GFAP content during pre-weaning ages was less pronounced in females than in males as well as the decrease between the 90th and the 150th day of age. Moreover, the size of astrocytes was larger in females than in males at the 11th and 150th days of life. These findings suggest that hippocampal astrocytes undergo rapid maturation in the 1st month of postnatal life, followed by a slow consolidation of this process until the 3rd month of life. At 5 months of age, there are still dynamic changes in the mature astrocytes, which become slender and thinner probably as a response to the increased volume of hippocampus noticeable at this age.     

点燃模型大鼠海马和皮层星形胶质细胞变化的动态观察

目的　探讨星形胶质细胞在戊四唑 (PTZ)化学点燃模型癫痫发病中的作用。方法　用 30只Wistar大鼠随机分为对照组和模型组。采用免疫组织化学方法和图像分析技术 ,对PTZ点燃各发作级别大鼠海马结构和颞叶皮质部位的胶质纤维酸性蛋白 (GFAP)的变化作动态观察。结果　GFAP值改变见于点燃后Ⅲ级组开始增加 ,Ⅴ级组达到高峰 ,Ⅴ级后 2 4h组仍有持续增加。结论　戊四唑点燃癫痫模型中 ,随着点燃级别的进展 ,GFAP免疫表达逐渐增加。提示星形胶质细胞GFAP含量的增加可能是PTZ点燃模型癫痫发病的原因之一。     

Reactive astrocytes in the kainic acid-damaged hippocampus have the phenotypic features of type-2 astrocytes

Summary Kainic acid treatment, a model of temporal lobe epilepsy, induces Ammon's horn sclerosis characterized by degeneration of CA3 pyramidal neurons and reactive gliosis. We now report that in kainic acid treated rats, reactive astrocytes in the hippocampus are A2B5 immunopositive and express GAP-43 immunoreactivity. A2B5 is a cell surface ganglioside selectively expressed in the glial O-2A lineage (oligodendrocytes and type-2 astrocytesin vitro). Since A2B5-positive cells were also GFAP immunoreactive, our observations suggest that hippocampal-reactive astrocytes in the epileptic process are type-2 astrocytes.GAP-43 is a membrane-associated phosphoprotein involved in neurite outgrowth.In vitro analysis showed that the glial O-2A lineage may express this phosphoprotein. In this study, we found that GAP-43 was coexpressed in astrocytes with A2B5 suggesting thatin vivo asin vitro type-2 astrocytes express GAP-43.     

Soluble factors from neocortical astrocytes enhance neuronal differentiation of neural progenitor cells from adult rat hippocampus on micropatterned polymer substrates

Rat adult hippocampal progenitor cells (AHPCs) are self-renewing, multipotent neural progenitors that have the ability to differentiate into neurons and glia. Previously, we demonstrated that coculture of AHPCs with postnatal day 2, type 1 cortical astrocytes on laminin-coated micropatterned polymer substrates facilitates selective neuronal differentiation of the AHPCs (Recknor et al., Biomaterials 2006;27:4098-4108). Under this condition, multidimensional cell-cell and/or cell-extracellular matrix interactions, as well as possible soluble factors released from astrocytes provided spatial and temporal control selectively enhancing neuronal differentiation and neurite alignment on topographically different regions of the same substrate. To investigate the potential role of astrocyte-derived soluble factors as cues involved in neuronal differentiation, a noncontact coculture system was used. Under control conditions, approximately 14% of the AHPCs were immunoreactive (IR) for the neuronal marker, class III beta-tubulin (TUJ1-IR). When cocultured in physical contact with astrocytes, neuronal differentiation increased significantly to about 25%, consistent with our previous results. Moreover, under noncontact coculture conditions using Transwell insert cultures, neuronal differentiation was dramatically increased to approximately 64%. Furthermore, neurite outgrowth from neuronal cell bodies was considerably greater on the patterned substrate when compared with the nonpatterned planar substrate under noncontact coculture conditions. Taken together, our results demonstrate that astrocyte-derived soluble factors provide cues for specific neuronal differentiation of AHPCs cultured on micropatterned substrates. In addition, a suppressive influence on neuronal differentiation appears to be mediated by contact with cocultured astrocytes. These results provide important insights into mechanisms for controlling neural progenitor/stem cell differentiation and facilitate development of strategies for CNS repair.     

Development of GLAST(+) astrocytes and NG2(+) glia in rat hippocampus CA1: mature astrocytes are electrophysiologically passive

Glia show marked heterogeneity in terms of electrophysiology in the developing brain, and two major types can be identified based on GFAP or NG2 expression. However, it remains to be determined if such an electrophysiological diversity holds for the adult brain and how GFAP and NG2 lineage glia are associated with different electrophysiological phenotypes during the course of development. To address these fundamental questions, we performed in situ whole cell recording from morphologically identified glia from the rat hippocampal CA1 region from postnatal (P) days 1-106 and double-stained postrecorded cells with GLAST and NG2 antibodies. We found glia express mostly voltage-gated outward K(+) currents and also have inward Na(+) currents in the newborn (P1-P3), but these are no longer present after P22. They consist equally of GLAST(+) and NG2(+) cells in the newborn, but are mainly NG2(+) in juvenile animals (P4-P21). Glia showing voltage-gated outward and inward K(+) currents are also present at P1, peak at P5 and decline to a stationary level of approximately 10% in the adult. They are GLAST(+) astrocytes from newborn to juvenile but NG2(+) glia in the adult. Electrophysiologically passive glia first appear at P4 and increase to 91% in adults, of which 85% are GLAST(+). These results indicate that glial electrophysiological diversity occurs predominantly in the developing brain. While most glia in the NG2 lineage preserve a certain amount of voltage-gated ion conductances, mature GLAST(+) astrocytes are electrophysiologically passive.     

Cryopreservation of rat astrocytes from primary cultures

Summary A deep-freezing procedure that makes possible a reproducible recovery of astrocytes for subsequent culture, after several months of cryopreservation, is described. The use of undiluted fetal bovine serum containing 10% dimethyl sulfoxide resulted in very high cell viability and survival. A simple and easy three-step (2 h at –20° C, 4 h at –70° C and storage in liquid N₂ at –196° C) cooling procedure has been shown to be adequate to yield very high cell viabilities. Cell viability, after a freezing-thawing cycle was about 94 to 97%, comparable to that of the astrocyte suspension obtained from the primary culture before freezing (95 to 100%). Three well-accepted markers of the astrocyte differentiation were examined in 7-day astrocytes, both primary cultured and cultured after thawing: glial fibrillary acidic protein, and the glutamine synthetase, and buthyl-cholinesterase activities.     

Direct evidence for mutual interactions between perineuronal astrocytes and interneurons in the CA1 region of the rat hippocampus

Recent studies have demonstrated that astrocytes express a variety of ion channels and neurotransmitter receptors and can modulate the activity of neurons. Since a single astrocyte makes tight contacts with many neighboring neuronal cells, they can provide efficient and wide modulation of neuronal networks. Here, we provide direct evidence for mutual interactions between perineuronal astrocytes and interneurons in the stratum radiatum of the rat hippocampus. Direct depolarization of a perineuronal astrocyte suppressed the excitatory postsynaptic currents in an adjacent interneuron and increased the paired-pulse ratio, indicating that perineuronal astrocytes have a suppressive effect on presynaptic elements. Moreover, perineuronal astrocyte activation modulated the directly induced firing pattern of the interneuron, with initial facilitation and subsequent suppression. Conversely, direct firing of the interneuron depolarized the membrane potential and reduced the input resistance of the perineuronal astrocyte. These results directly demonstrate the existence of bidirectional interactions between neurons and perineuronal astrocytes.     

雷公藤多甙对大鼠海马内注射β-淀粉样蛋白后星形胶质细胞增生的影响

目的：探讨大鼠海马内注射v淀粉样蛋白1-40(Aβ1-40)后海马星形胶质细胞表达GFAP的变化及雷公藤多甙(TWP)对其的影响。方法：在大鼠左侧海马定向注射Aβ1-40作为Aβ组,注射生理盐水作为对照组,雷公藤多甙腹腔注射治疗海马内注入了Aβ1-40的大鼠为TWP组。用免疫组织化学方法观察各组大鼠海马星形胶质细胞GFAP的表达。结果：Aβ组海马星形胶质细胞增生、肥大,GFAP阳性细胞数增多,细胞截面积明显增大。TWP组星形胶质细胞数较Aβ组减少,并且细胞截面积明显缩小。结论：雷公藤多甙能抑制Aβ诱导的海马星形胶质细胞的反应性增生。     

Potassium currents in primary cultured astrocytes from the rat corpus callosum

The corpus callosum (CC) is the main white matter tract in the brain and is involved in interhemispheric communication. Using the whole-cell voltage-clamp technique, a study was made of K⁺-currents in primary cultured astrocytes from the CC of newborn rats. These cells were positive to glial fibrillary acidic protein after culturing in Dulbecco’s Modified Eagle Medium (> 95% of cells) or in serum-free neurobasal medium with G5 supplement (> 99% of cells). Astrocytes cultured in either medium displayed similar voltage-activated ion currents. In 81% of astrocytes, the current had a transient component and a sustained component, which were blocked by 4-aminopyridine and tetraethylammonium, respectively; and both had a reversal potential of −66 mV, indicating that they were carried by K⁺ ions. Based on the Ba²⁺-sensitivity and activation kinetics of the K⁺-current, two groups of astrocytes were discerned. One group (55% of cells) displayed a strong Ba²⁺ blockade of the K⁺-current whose activation kinetics, time course of decay, and the current-voltage relationship were modified by Ba²⁺. This current was greatly blocked (52%) by Ba²⁺ in a voltage-dependent way. Another group (45% of cells) presented weak Ba²⁺-blockade, which was only blocked 24% by Ba²⁺. The activation kinetics and time course of decay of this current component were unaffected by Ba²⁺. These results may help to understand better the roles of voltage-activated K⁺-currents in astrocytes from the rat CC in particular and glial cells in general.     

Acquisition of vimentin in astrocytes cultured from postnatal rat brain

Summary Vimentin and glial fibrillary acidic protein (GFAP) represent the principal constituents of intermediate filaments found in astrocytes. In contrast to vimentin—GFAP transition which occurs during glial developmentin situ, vimentin coexists with GFAP in cortical astrocytes allowed to differentiate in culture. To examine whether culture conditions or proliferative activity of the cells is responsible for the expression of vimentin, we generated cultures of GFAP-positive, vimentin-negative astrocytes isolated from 26-day postnatal rat brain cortices. Isolated astrocytes are characterized by a very thin rim of perinuclear cytoplasm and by numerous processes. Antiserum to GFAP labelled major processes and cell somata of some astrocytes, especially those with relatively short and large processes. Within 3 days in culture, all astrocytes accumulated GFAP in hypertrophic cell bodies and many began to express vimentin. Vimentin appeared primarily close to nuclei, and filaments of vimentin extended into proximal segments of the cell processes. In some astrocytes, however, vimentin was always absent. Combined double immunolabelling and histoautoradiography experiments demonstrated that the acquisition of vimentin was independent of the ability of astrocytes to incorporate tritiated thymidine. The results indicate that astrocytes isolated from 26-day postnatal rat brain are heterogeneous with respect to their ability to express vimentin and that vimentin synthesis is not correlated with the growth state of the cells as had been previously suspected.     

Freshly isolated astrocytes from rat hippocampus show two distinct current patterns and different [K(+)](o) uptake capabilities

Whether astrocytes predominantly express ohmic K(+) channels in vivo, and how expression of different K(+) channels affects [K(+)](o) homeostasis in the CNS have been long-standing questions for how astrocytes function. In the present study, we have addressed some of these questions in glial fibrillary acidic protein [GFAP(+)], freshly isolated astrocytes (FIAs) from CA1 and CA3 regions of P7-15 rat hippocampus. As isolated, these astrocytes were uncoupled allowing a higher resolution of electrophysiological study. FIAs showed two distinct ion current profiles, with neither showing a purely linear I-V relationship. One population of astrocytes had a combined expression of outward potassium currents (I(Ka), I(Kd)) and inward sodium currents (I(Na)). We term these outwardly rectifying astrocytes (ORA). Another population of astrocytes is characterized by a relatively symmetric potassium current pattern, comprising outward I(Kdr), I(Ka), and abundant inward potassium currents (I(Kin)), and a larger membrane capacitance (C(m)) and more negative resting membrane potential (RMP) than ORAs. We term these variably rectifying astrocytes (VRA). The I(Kin) in 70% of the VRAs was essentially insensitive to Cs(+), while I(Kin) in the remaining 30% of VRAs was sensitive. The I(Ka) of VRAs was most sensitive to 4-aminopyridine (4-AP), while I(Kdr) of ORAs was more sensitive to tetraethylammonium (TEA). ORAs and VRAs occurred approximately equally in FIAs isolated from the CA1 region (52% ORAs versus 48% VRAs), but ORAs were enriched in FIAs isolated from the CA3 region (71% ORAs versus 29% VRAs), suggesting an anatomical segregation of these two types of astrocytes within the hippocampus. VRAs, but not ORAs, showed robust inward currents in response to an increase in extracellular K(+) from 5 to 10 mM. As VRAs showed a similar current pattern and other passive membrane properties (e.g., RMP, R(in)) to "passive astrocytes"in situ (i.e., these showing linear I-V curves), such passive astrocytes possibly represent VRAs influenced by extensive gap-junction coupling in situ. Thus, our data suggest that, at least in CA1 and CA3 regions from P7-15 rats, there are two classes of GFAP(+) astrocytes which possess different K(+) currents. Only VRAs seem suited to uptake of extracellular K(+) via I(Kin) channels at physiological membrane potentials and increases of [K(+)](o). ORAs show abundant outward potassium currents with more depolarized RMP. Thus VRAs and ORAs may cooperate in vivo for uptake and release of K(+), respectively.     

Heme oxgenase-1 is expressed in viable astrocytes and microglia but in degenerating pyramidal neurons in the kainate-lesioned rat hippocampus

The present study aimed to elucidate the distribution of heme oxygenase-1 (HO-1) in the hippocampus after intracerebroventricular injections of kainate. Very little or no staining of HO-1 was observed in the normal CA1, whilst moderate staining of dentate hilar neurons was observed in the dentate gyrus, in the normal hippocampus. At postinjection day 1, a slight increase in immunoreactivity in the neuropil of the lesioned CA fields and a marked increase in HO-1 immunoreactivity in glial cells of the stratum lacunosum moleculare of CA fields and the stratum moleculare of the dentate gyrus was observed. Electron microscopy showed that the glial cells had features of viable astrocytes. At postinjection day 3, glial cells in the dentate gyrus continued to express HO-1, whilst pyramidal neurons in the degenerating CA fields started to express intense HO-1 immunoreactivity in their cell bodies. At postinjection weeks 1–3, HO-1 was observed in glial cells in the center of the lesion, but also in neurons at the perifocal region of the glial scar. The glial cells were found to have features of viable astrocytes and microglia, whilst the neurons contained discontinuous cell membranes and nuclear outlines, and had features of degenerating neurons. Intense immunoreactivity was observed in the cytoplasm of the degenerating neurons. The density of staining was greater than that observed in astrocytes or microglia. Recent in vitro results on fibroblasts transfected with HO-1 cDNA showed that, despite cytoprotection with low (less than fivefold compared with untransfected cells) HO-1 activity, high levels of HO-1 expression (more than 15-fold) were associated with significant oxygen toxicity. These and the present observations suggest a destructive effect of increased expression of HO-1 in neurons, and possible novel therapeutic approaches involving overexpression of HO-1 must therefore be approached with caution. Electronic Publication     

Serotonin-immunoreactive projections to the hippocampus from the interpeduncular nucleus in the rat

The possibility of a serotonergic projection from the interpeduncular nucleus (IPN) to the hippocampus was examined using a combined immunohistochemical-fluorescence retrograde tracing technique. About one third of the cells in the apical subnucleus of the IPN which projected to the hippocampus could be shown to contain serotonin immunoreactivity, whereas double-labeled cells were only rarely encountered in the lateral subnuclei. These findings suggest that the IPN sends both serotonergic and non-serotonergic projections to the hippocampus and that the serotonergic projections arise primarily from the apical subnucleus.