Serum influences the differentiation of membrane structure in cultured astrocytes

The membranes of mammalian astrocytic processes apposed to blood vessels or forming the surface of the brain contain high concentrations of a characteristic intramembrane particle aggregate, termed "assemblies." In order to identify developmental processes which contribute to this remarkable regional specialization of membrane structure, we have devised culture conditions which support the differentiation of assemblies in secondary cultures of astrocytes derived from neonatal rat forebrain. We report here that different lots of fetal calf serum vary dramatically in their capacity to support the differentiation of assemblies. Fetal calf serum thus appears to exert two distinct influences on astrocyte development: it promotes the differentiation of type 2 astrocytes from bipotential precursor cells, as shown by others, and it influences the density of assemblies in type 1, flat, GFAP-immunoreactive astrocytes in our secondary cultures. Horse serum and defined media also support the appearance of assemblies in flat, GFAP-immunoreactive astrocytes. The separate effects of serum supplementation upon cell lineage and membrane differentiation have to be carefully considered in studies designed to examine factors influencing astrocytic development in vitro.     

Electrical coupling and gap junctions between neurosecretory cells in an insect

Intracellular injections of Lucifer yellow into single neurosecretory cells in the posterior (glandular) lobe of the corpus cardiacum of locusts resulted in dye transfer between these cells. Passage of hyperpolarizing or depolarizing current into one cell resulted in changes in the membrane potential of other cells. Freeze-fracture replicas of these cells revealed gap junctions which probably mediate the electrical and dye coupling. It seems apparent from these data that coupling may synchronize the neurosecretory cells' activity.     

Cell membrane structure of human giant-celled glioblastoma

Summary A giant-cell glioblastoma was examined by electron microscopy and by the freeze-fracture technique. The cell membranes bordering the extensive extracellular space often showed complicated undulations and peripheral vacuoles as well as occasional microvilli or filopodia. The undulations were mainly composed of plasmalemmal vesicles as well as of large (400–800 nm in diameter) and small (30–50 nm in diameter) localized protrusions and invaginations of the cell membrane. Deep invaginations of the cell membrane apparently resulted in two separate cytoplasmic portions. Locking of protruded cytoplasmic tongues and adherens junctions were sometimes seen in closely approximated cell membranes. The average number of membrane particles per m² was 630±130 on the P face and 180±30 on the E face. The membrane particles were occasionally aggregated to form clusters about 30 to 150 m in diameter. Gap junctions were occasionally found, but there were no tight junctions. Large particles about 30 nm in diameter were found in places.     

Freeze fracture studies of muscle plasma membrane in human muscular dystrophy

Summary Freeze fracture analysis of intramembranous particle density in skeletal muscle plasma membrane from 7 patients with Duchenne muscular dystrophy (DMD), 5 patients with facioscapulohumeral muscular dystrophy (FSH) and 5 patients with myotonic dystrophy (MyD) were carried out. Marked deplction of intramembranous particles including orthogonal arrays were significantly decreased in FSH. No abnormalities were noted in MyD.This work was supported by a Research Center Grant from the Muscular Dystrophy Association of America and by grants NS-08075, NS-14471 and 5 M01 RR00040 from the U.S. Public Health Service     

Changes in the structure of synaptic junctions during climbing fiber synaptogenesis

Synaptogenesis between climbing fiber axons and Purkinje cells involves both an orderly translocation of synaptic junctions over the Purkinje cell surface and an elimination of all but one innervating axon. We used thin-section and freeze-fracture electron microscopic techniques to study structural changes in synaptic junctions during this interval of synapse translocation and elimination. In freeze-fractured preparations, virtually all climbing fiber synaptic junctions with the perisomatic processes and somatic spines lacked the particle aggregates that characterized the extracellular half of the postsynaptic membrane of mature synaptic junctions with dendritic spines. Some climbing fiber junctions with the dendritic shaft in the second postnatal week were associated with such aggregates, despite the fact that these junctions are transient. Thus, during the interval when Purkinje cells initially were innervated by multiple climbing fibers, and subsequently denervated of all but one climbing fiber afferent per cell, only a few of the transient synaptic junctions on the cell body and proximal dendrites have associated particles. The presence of a particle aggregate at a synaptic junction does not appear to be correlated with the permanence of that junction and probably is not correlated with the capacity to support synaptic transmission. The particle aggregates might be indicative of relatively long-lived junctions, or might occur only at junctions formed by the climbing fiber that will persist in synaptic contact with the mature Purkinje cell.     

Effects of volatile anaesthetics on the membrane potential and ion channels of cultured neocortical astrocytes

Volatile anaesthetics cause changes in the membrane resting potential of central neurons. This effect probably arises from actions on neuronal ion channels, but may also involve alterations in the ion composition of the extracellular space. Since glial cells play a key role in regulating the extracellular ion composition in the brains of mammals, we analyzed the effects of halothane, isoflurane and enflurane on the membrane conductances and ion channels of cultured cortical astrocytes. Astrocytes were dissociated from the neocortex of 0–2-day old rats and grown in culture for 3–4 weeks. Anaesthetic-induced changes in the membrane potential were recorded in the whole cell current-clamp configuration of the patch-clamp technique. We further studied the effects of halothane and enflurane on single ion channels in excised membrane patches. At concentrations corresponding to 1–2 MAC (1 MAC induces general anaesthesia in 50% of the patients and rats), membrane potentials recorded in the presence of enflurane, isoflurane and halothane did not differ significantly from the control values. At higher concentrations, effects of enflurane and halothane, but not of isoflurane, were statistically significant. Single-channel recordings revealed that halothane and enflurane activated a high conductance anion channel, which possibly mediated the effects observed during whole cell recordings. In less than 10% of the membrane patches, volatile anaesthetics either increased or decreased the mean open time of K⁺-selective ion channels without altering single-channel conductances. In summary, it seems unlikely that the actions of volatile anaesthetics described here are involved in the state of general anaesthesia. Statistically significant effects occurred at concentrations ten times higher than those required to cause half-maximal depression of action potential firing of neocortical neurons in cultured brain slices. However, it cannot be excluded that the changes observed in the membrane conductance of cortical astrocytes disturb the physiological function of these cells, thereby influencing the membrane resting potential of neurons.     

Small size of orthogonal array in muscle plasma membrane of Fukuyama type congenital muscular dystrophy

Summary Freeze fracture analysis was carried out on the density of orthogonal array subunit particles in the plasma membrane of skeletal muscle of six patients with Fukuyama type congenital muscular dystrophy and seven control cases. The group mean density of orthogonal array subunit particles per one orthogonal array was significantly lower in the plasma membrane of Fukuyama patients. The results suggested the possible impairment of orthogonal array function in the plasma membrane of muscle fibers in congenital muscular dystrophy of the Fukuyama type.Supported in part by grant from the National Center for Nervous, Mental and Muscular Disorders of the Ministry of Health and Welfare (No. 85-04-34), Japan     

上皮膜蛋白1在实验性糖尿病大鼠模型中额叶皮质血—脑屏障上的表达

目的测定不同时间点实验性糖尿病大鼠额叶皮质血—脑屏障(BBB)上皮膜蛋白1(EMP1)的表达。方法选成年健康雄性Sprague-Dawley大鼠随机分为链脲佐菌素(STZ)诱导糖尿病组(STZ组)和对照组(CON组),每组分为2w、4w、8w 3个亚组,采用免疫组织化学法检测EMP1的表达。结果STZ大鼠与CON大鼠比较,4w组中STZ大鼠上EMP1的表达显著增多(P0.01),2w组和8w组中差异无统计学意义;EMP1的表达在STZ大鼠各亚组之间及CON大鼠各亚组之间比较差异无统计学意义(P0.05)。结论 EMP1在实验性糖尿病大鼠额叶皮质BBB上的表达改变,在模型建立成功后4w时表达显著增加。     

甘珀酸治疗蛛网膜下腔出血后脑血管痉挛的体内实验研究

目的 探讨缝隙连接抑制剂甘珀酸对实验性蛛网膜下腔出血后脑血管痉挛的治疗作用.方法 建立兔蛛网膜下腔二次出血模型,脑池及静脉分别给与缝隙连接抑制剂甘珀酸,脑血管造影及光镜观察分析基底动脉的直径及形态学变化,并应用Western blotting检测基底动脉Cx43蛋白的表达变化.结果 给与甘珀酸后,基底动脉狭窄程度及光镜下形态学变化显著减轻:单纯注血组(65.7±10.3)%,脑池处理组(91.2±6.4)%,静脉处理组(96.4±11.0)%,腑池预处理组(89.7±12.8)%;同时也显著抑制了痉孪后Cx43蛋白表达水平的升高:单纯注血组(57.2±2.8)%,脑池处理组(10.0±5.3)%,静脉处理组(15.2±1.7)%.结论 缝隙连接抑制剂甘珀酸可能对蛛网膜下腔出血后脑血管痉挛起到预防和治疗作用.     

Gap junctions couple astrocytes but not neurons in dissociated cultures of rat suprachiasmatic nucleus

Individual neurons dissociated from rat suprachiasmatic nucleus can express independently phased circadian firing rhythms in culture. The phases of these rhythms are unperturbed by reversible blockade of neuronal firing lasting 2.5 days, indicating that multiple circadian clocks continue to operate in the absence of conventional synaptic transmission. The possibility remains, however, that these circadian rhythms might depend on some other form of intercellular communication. In the present study, a potential role for gap junctional coupling in SCN cultures was evaluated by introduction of the tracer molecule Neurobiotin into both neurons (n = 98) and astrocytes (n = 10), as well as by immunolabeling for specific connexins, the molecular components of gap junctions. Astrocytes were extensively coupled to each other by connexin43-positive gap junctions, but no evidence was found for coupling of neurons to each other or to astrocytes. These data support the hypothesis that neurons expressing independently phased circadian rhythms in SCN cultures (‘clock cells’) are autonomous, single cell circadian oscillators, but do not exclude a role for glia in synchronizing neuronal clock cells in vivo.     

Plasma membrane of cultured oligodendrocytes: II. Possible structural and functional domains

An oligodendrocyte plasma membrane-rich fraction, F2.2, was resolved by equilibrium density centrifugation on a linear sucrose gradient from 0.5 M to 1.3 M into three fractions, F2.2a, F.2.2b, F2.2c, and a pellet F2.2p. F2.2a and F.2.2b were enriched 1.5-fold relative to F2.2 in plasma membrane markers at the expense of F2.2c and F2.2p, which became correspondingly impoverished. This gave F2.2a and F2.2b a 42-fold and 37-fold enrichment, respectively, in plasma membrane markers relative to the initial cell homogenate. F2.2c had a sevenfold enrichment in a Golgi marker; together with F2.2p, they contained all the Golgi marker initially present in F2.2. Preliminary data indicated that the F2.2-subfractions differed from one another in their molar ratios of cholesterol to phospholipids and protein to lipids but had similar protein profiles when examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Their content of fucosylated glycoproteins appeared also to be different. Morphologically, F2.2a and F2.2b were very similar: they contained large membrane vesicles, membrane sheets, and vesicles entrapped within other vesicles. Membrane-membrane interaction was apparent in these fractions. F2.2c had many of the same elements, but most of the membrane structures contained amorphous material. F2.2p differed morphologically from the other fractions in that it had principally electron-dense structures. It is postulated that F2.2a, F2.2b, and perhaps F2.2c represent different domains of oligodendrocyte plasma membrane. Alternatively, these fractions might correspond to the plasma membrane of oligodendrocyte subtypes.     

辛醇对红藻氨酸致痫大鼠的保护作用及其对海马组织连接蛋白43的影响

目的 研究辛醇对红藻氨酸(kainic acid,KA)致痫大鼠海马组织连接蛋白43(connexin43,CX43)表达的影响及其抗痫效应.方法 采用行为学评分评价KA致痫及辛醇腹腔注射后致痫大鼠的痫性发作程度和潜伏期,应用免疫组织化学方法检测海马CX43的表达.结果 KA组CX43的表达较正常对照组明显增高(P<0.01);辛醇组CX43的表达也较正常对照组明显增高(P<0.01),但较KA组明显下降.致痫后3h内辛醇组痫性发作评分较KA组明显下降(P<0.01),潜伏期明显延长(P<0.01).结论 反复痫性发作后CX43的表达增加.辛醇能够减少CX43表达,降低痫性发作的评分,延长痫性发作的潜伏期,具有较明显的抗癫痫效应.

Abstract：

Objective To study the effects of octanol on the expression of connexin 43 in the brain of epilepsy-rats induced by kainic acid and the anti-epileptic effect of octanol. Methods The epilepsy-rats induced by kainic acid and treated by octanol was assessed by behavior score. The expression of CX43 in the hippocampal tissue was measured by immuno-histochemistry. Results The expression of CX43 in the group of epilepsy-rats induced by kainic acid was higher than that in control group( P <0. 01) ;the expression of CX43 in the group treated by octanol was higher than that in control group( P <0. 01) ,but lower than the group of epilepsy-rats induced by kainic acid;the Patel score of the rats in the group treated by octanol was lower than the group only induced by kainic acid within 3 hours after the induction (P <0. 01) ,and the latency was longer (P <0.01). Conclusions After the epileptic seizure repeatedly, the expression of CX43 was upregulated. Octanol could reduce the expression of CX43, decrease the Patel score,and extend the latency of epileptic seizure. It has obviously anti-epileptic effect.     

Effect of denervation on regenerating muscle plasma membrane integrity: freeze-fracture and dystrophin immunostaining analyses

Summary We investigated time sequentially, the densities of intramembranous assembly orthogonal arrays of regenerating rat extensor digitorum longus muscle after bupivacaine-induced muscle injury. There was no evidence of orthogonal arrays at the early stage, but the densities of orthogonal arrays increased with the maturation of the innervated regenerating myofibers. In contrast, the orthogonal arrays were scarcely observed at any time point examined in denervated regenerating muscles. Therefore, the neural factor may have an important effect on the appearance of orthogonal arrays. Moreover, we studied the immunostainability of Duchenne muscular dystrophy gene product dystrophin in the regenerating muscle at the same time points. Positive immunostaining was observed in both innervated and denervated regenerating myofibers even from the early stage of regeneration. On the basis of these data, the relationship between orthogonal array and dystrophin are discussed.Supported in part by grants from the Ministry of Education and from the National Center for Nervous, Mental and Muscular Disorders of the Ministry of Health and Welfare (62A-2-20), Japan     

连接蛋白拟似肽对海人酸致痫大鼠脑电活动的影响

目的观察针对连接蛋白43(CX43)合成的特异性的缝隙连接阻断剂-连接蛋白拟似肽对海人酸致痫大鼠脑电活动的影响。方法建立18只大鼠癫痫动物模型,分连接蛋白拟似肽组、甘珀酸组和对照组(每组6只),在在体上分别局部给予连接蛋白似似肽、甘珀酸和生理盐水,用脑电图仪观测用药前后每组大鼠皮层脑电活动的变化情况。结果连接蛋白拟似肽组及甘珀酸组给药后癫痫的发作次数明显比给药前发作次数减少,癫痫波的平均振幅也明显变小,给药前后比较有显著性差异(P<0.01),生理盐水组给药前后癫痫的发作次数及平均振幅几乎没有变化。连接蛋白拟似肽组给药前后癫痫的发作次数和波幅的变化值与甘珀酸组给药前后癫痫的发作次数和波幅的变化值相比无显著性差异。结论针对CX43合成的连接蛋白拟似肽可以特异性地抑制癫痫的发作。     

Effects of barbiturates on energy and intermediary metabolism in cultured astrocytes

Effects of barbiturates on utilization of the two substrates, glucose and glutamate, were studied in astrocytes in primary cultures. Carbon dioxide formation from glucose was under ordinary conditions not affected by barbiturates but in the presence of 10 μM malate there, was a potassium-induced stimulation (20–25%) which was significantly (P < 0.001) inhibited (30–35%) by pentobarbital (0.5 mM). Glutamate oxidation was not enhanced by excess potassium but there was a distinct dose-dependent reduction in the presence of pentobarbital. In contrast, pentobarbital or phenobarbital had no effect on the formation of glutamine from glutamate.     

Freeze-fracture analysis of cholesterol in muscle plasma membrane of Fukuyama-type congenital muscular dystrophy

Summary We used digitonin in freeze-fracture analysis of the muscle plasma membrane cholesterol content in six patients with Fukuyama-type congenital muscular dystrophy and six control children. A significantly greater proportion of surface area was taken up by digitonin-cholesterol complexes in the patients (51.2%±4.7%) than in controls (31.9%±2.9%) (P<0.01). Since membrane cholesterol has a dynamic regulatory function in affecting the activity of membrane-bound proteins, the increased cholesterol content in the patients suggests a functional abnormality of the muscle plasma membrane in this disease.Supported by grant No. 86-02-34 from National Center of Neurology and Psychiatry (NCNP) of the Ministry of Health and Welfare, Japan     

Postnatal development of dye-coupling among astrocytes in rat visual cortex.

Intercellular coupling among astrocytes was studied in rat visual cortex slices from animals aged 1 week to 4 months. Cell coupling via gap junctions was determined by the dye spread of the low molecular weight dye Lucifer Yellow CH injected into electrophysiologically identified cells to adjacent cells. Coupling among glial cells was first detected at postnatal day 11 and was thereafter consistently observed until adulthood. Dye spread was observed up to 300 microns radially from the injected cell covering multiple cortical layers. Following dye injection into a single cell up to several hundred Lucifer Yellow-positive cells could be observed. Quantitative analysis revealed a similar extent of dye spread at different developmental stages including a quite constant number of dye-coupled astrocytes from the end of the second postnatal week to adulthood. Double labelling of Lucifer Yellow-filled cells with an antiserum against the glial fibrillary acidic protein confirmed the astrocytic nature of the injected and coupled cells. Comparison of the density of dye-coupled cells in a given area and the total number of astrocytes as revealed by immunocytochemical staining suggests that dye-coupling includes the entire local astrocytic population. It is concluded that coupling among astrocytes via gap junctions in rat visual cortex occurs shortly after birth and reflects one of the first steps in astroglial maturation.     

Connexins and gap junctions of astrocytes and oligodendrocytes in the CNS

This review article summarizes early and recent literature on the structure, distribution and composition of gap junctions between astrocytes and oligodendrocytes, and the differential expression of glial connexins in adult and developing mammalian CNS. In addition to an overview of the topic, discussion is focused on the organization of homologous gap junctional interactions between astrocytes and between oligodendrocytes as well as on heterologous junctional coupling between astrocytes and oligodendrocytes. The homotypic and heterotypic nature of these gap junctions is related to the connexins known to be produced by glial cells in the intact brain and spinal cord. Emphasis is placed on the ultrastructural level of analysis required to attribute gap junction and connexin deployment to particular cell types and subcellular locations. Our aim is to provide a firm basis for consideration of anticipated rapid advances in understanding of structural relationships of gap junctions and connexins within the glial gap junctional syncytium. Conclusions to date suggest that the glial syncytium is more complex than previously appreciated and that glial pathways of junctional communication may not only be determined by the presence of gap junctions, but also by the connexin composition and conductance regulation of junctional channels.     

Induction of presynaptic differentiation in cultured neurons by extracellular matrix components

Motoneurons reinnervating skeletal muscles form nerve terminals at sites of contact with a specialized basal lamina. To analyse the molecules and mechanisms that underly these responses, we introduce two systems in which basal lamina-derived components induce presynaptic differentiation of cultured neurons from chick ciliary ganglia in the absence of a postsynaptic cell. In one, ciliary neurites that contact substrates coated with a recombinant laminin beta2 fragment form varicosities that are rich in synaptic vesicle proteins, depleted of neurofilaments, and capable of depolarization-dependent exocytosis and endocytosis. Thus, a single molecule can trigger a complex, coordinated program of presynaptic differentiation. In a second system, neurites growing on cryostat sections of adult kidney form vesicle-rich, neurofilament-poor arbors on glomeruli. Glomerular basal lamina, like synaptic basal lamina, is rich in laminin beta2 and collagen (alpha3-5) IV. However, glomeruli from mutant mice lacking these proteins were capable of inducing differentiation, suggesting the glomerulus as a source of novel presynaptic organizing molecules.     

Activation of substance P receptors leads to membrane potential responses in cultured astrocytes

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.