Haloperidol and clozapine decrease S100B release from glial cells

Recent meta-analyses showed consistently elevated levels of S100B in serum and cerebrospinal fluid of schizophrenic patients. This finding has been attributed to glial pathology because S100B is produced by astrocytes and oligodendrocytes. However, S100B may be likewise associated with schizophrenia-related disturbances in glial cell as well as adipocyte energy supply and glucose metabolism. The influence of antipsychotic drugs on S100B levels remains unclear, and some studies have suggested that treatment with these drugs may actually contribute to the elevated S100B levels observed in schizophrenic patients. In this study, we explored the effects of the typical antipsychotic haloperidol and the atypical prototype drug clozapine on the release of S100B by astrocytic C6 cells and oligodendrocytic OLN-93 cells. Because of the association between schizophrenia and disturbances in energy metabolism, we assessed the effects of these drugs under basal condition (BC) compared to serum and glucose deprivation (SGD). We found that treatment of C6 and OLN-93 cells with haloperidol and clozapine reduced the release of S100B from C6 and OLN-93 cells under BC and SGD in vitro at a tissue concentration corresponding to the assumed therapeutic dose range of these drugs. These data suggest that elevated levels of S100B in bodily fluids of schizophrenic patients are normalized rather than increased by the effects of antipsychotic drugs on glial cells.     

A glial progenitor cell in the cerebral cortex of the adult rat

Summary A glial cell subtype, previously classified as a beta astrocyte on the basis of its ultrastructural and radiobiological characteristics, has now been shown to represent the most mitotically active component of the glial population in the grey matter of the cerebral cortex of the young adult rat. The labelling index of 0.83% was evaluated using semithin sections. A role for beta astrocytes as macroglial precursors is supported by the present observations. However, the mechanisms responsible for the intermediate radiosensitivity of these elements remain uncertain.     

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.     

A demonstration of glial filament distribution in astrocytes isolated from rat cerebral cortex

Astrocytes have been classically described based on morphological characteristics as either protoplasmic or fibrous. However, the appearance of astrocytes at the light microscopic level following immunoreaction with glial filament acidic protein antisera or Cajal's gold-chloride method is markedly different from the morphology of Golgi-impregnated or horseradish peroxidase-filled astrocytes. The present study combines immunohistochemistry using glial fibrillary acidic protein antisera and a cellular suspension of structurally intact astrocytes isolated from the rat cerebral cortex to demonstrate that the distribution of glial filaments is limited to major astrocytic processes. Because the sheet-like processes which decorate the major branches of protoplasmic astrocytes do not contain glial filaments, the entire protoplasmic astrocyte is not visible when viewed in situ in the gray matter of the cerebral cortex. Thus staining techniques that have a propensity for the glial filaments such as antisera to glial fibrillary acidic protein and the gold-chloride method provide only a skeletal outline of the major processes of the protoplasmic astrocyte. On the other hand, fibrous astrocytes which do not have feathery decorations on major processes are visible in the cerebral cortex in their structural entirety. The present investigation also offers additional evidence of the specificity of glial fibrillary acidic protein antisera for astrocytes.     

Strychnine protection against excitotoxic cell death in primary cultures of rat cerebral cortex

Strychnine-insensitive glycine receptors are known to modulate the toxicity of excitatory amino acids via an allosteric action at the N-methyl-D-aspartate receptor complex. To elucidate whether strychnine-sensitive glycine receptors may also influence excitotoxicity, the effect of strychnine on the excitotoxic cell death was examined in primary cultures of the rat cerebral cortex. To exclude any interference at the N-methyl-D-aspartate receptor complex, cell death was evoked by kainic acid. The release of lactic dehydrogenase (LDH) into the culture medium was taken as a quantitative measure of cell death. Strychnine reduced the excitotoxic cell death in a concentration-dependent fashion. This finding indicates that glycine may modulate the vulnerability of cortical cells to excitotoxic insults not only via the strychnine-insensitive population of glycine receptors within the N-methyl-D-aspartate receptor-complex, but also via strychnine-sensitive receptor channels.     

The beta astrocyte: a newly recognized radiosensitive glial cell type in the cerebral cortex

Summary A highly radiosensitive glial cell type is described. The cell displays ultrastructural characteristics intermediate between the normal astrocyte and the light oligodendrocyte. It disappears from the cerebral cortex shortly after irradiation with doses 20 Gy. One year after this treatment, the cell type had reappeared but its frequency was still significantly lower than in age-matched sham-treated controls. The numbers of oligodendrocytes and microglial cells were also reduced at this time. The new cell element, called here a beta astrocyte, is present in significant numbers in the normal rat brain and its role as a multipotential reserve glial cell is discussed.     

Identification of somatostatin-containing neurons in primary cultures of rat cerebral cortex

The expression of somatostatin-like immunoreactivity was investigated in primary cultures of dissociated cerebral cortical tissue from the rat. A subpopulation of neurons in the cortical cultures exhibited intense staining for somatostatin. These somatostatin-immunoreactive neurons corresponded to 1.25% of the total neuronal population. Stained neurons were typically small with a soma size of 10-20 micron. The majority of somatostatin-containing cells had stellate and bipolar morphology, with the bipolar class predominating.     

Ultrastructural organization of slice cultures from rat visual cortex

Summary We have been studying the fine structural organization of slice cultures prepared from the visual cortex of 6-day-old rats and cultured for 2 weeks using a roller culture technique. Neurons in culture exhibited the characteristic cytological differences between perikarya, axons and dendrites. Neuronal and glial processes formed a dense neuropil with minimal extracellular spaces, and within the neuropil there were numerous synaptic contacts. Both morphological types of cortical synapses, type I (asymmetrical) and type II (symmetrical) could be readily identified in slice cultures. The pattern of synaptic connections in culture was remarkably similar to that observed in normal cerebral cortex: asymmetrical synapses were usually found in contact with dendritic spines, less frequently with dendritic shafts, and never on perikarya, whereas symmetrical synapses were found mostly on perikarya, occasionally on dendritic shafts but never on dendritic spines. Synaptic morphology appeared mature after 2 weeksin vitro and did not show the immature features observed at the time of culture preparation. Taken together with our previous light microscopic studies, these results indicate that cortical slice cultures are organotypically organized and serve as a useful model to study mechanisms of cortical development and plasticity.     

Immunohistochemical identification of quinolinic acid phosphoribosyltransferase in glial cultures from rat brain

Glial cell cultures were shown to contain 3 identifiable classes of cells which could be specifically stained with antibodies directed against quinolinic acid phosphoribosyltransferase (QPRT), the catabolic enzyme of the endogenous excitotoxin quinolinic acid. Some, but not all, QPRT-positive cells also contained glial fibrillary acidic protein. These cultures may constitute an in vitro system in which cerebral quinolinic acid metabolism and function can be examined.     

In vitro formation of type 2 astrocytes derived from postnatal rat hypothalamus or cerebral cortex

The origin and function of type 2 astrocytes in the optic nerve are now well described, but there are few and controversial observations concerning their origin and functional significance in other regions of the mammalian brain. We here describe primary and highly enriched secondary glial cultures obtained from postnatal (P0-P6) rat hypothalami and cerebral cortices that included glial cells with morphological and immunocytochemical characteristics of type 2 astrocytes. The somata of such astrocytes were characteristically small and polygonal; they bore several processes with few branches. They were highly immunoreactive for glial fibrillary acidic protein and the surface antigens, A2B5 and NSP4; they were immunonegative for myelin basic protein and galactocerebroside. They grew on top of a continuous monolayer of much larger, flattened cells, that were glial fibrillary acidic protein-positive but A2B5- and NSP4-negative. In cultures derived from tissues younger than postnatal day 4, their appearance required the addition of adult (horse) serum to the culture milieu; they appeared spontaneously in cultures from older animals. Analysis of the origin of these cells, including experiments using tritiated thymidine incorporation, indicated that these astrocytes resulted from asymmetric divisions of the flat glial cells in the basal layer of the cultures. After their first appearance which varied according to the age of the source tissue, they were continuously generated, with a generation time no longer than 48 h; the life-span of individual cells was found to not exceed one week in neuron-free primary glial cultures. They displayed important process motility but did not show any significant migratory activity. The ready inducibility of glial cells showing many characteristics of type 2 astrocytes, in cultures derived from different brain areas, suggests that type 2-like astrocytes or their committed precursors are not restricted to particular neural structures, but are probably widely distributed within the mammalian brain. Their functional significance within the different brain areas remains to be determined.     

C型尼曼-匹克病小鼠小脑的胶质反应

探讨C型尼曼-匹克病(Nicmann-Pick disease type C,NPC)小脑胶质细胞的变化及其意义。应用组织蛋白酶D(Cat-D)和胶质原纤维酸性蛋白(GFAP)抗体以及免疫组织化学方法,观察生后不同年龄的NPC和正常小鼠小脑内小胶质细胞和星形胶质细胞的变化。结果显示随着年龄的增长,NPC小鼠小脑内Cat-D深染的小胶质细胞的数量呈进行性增加,细胞逐渐增大,尤以皮质分子层为甚。小脑髓质和颗粒层内GFAP阳性星形胶质细胞肿胀,数量增多;皮质内Bergmann胶质细胞逐渐增多,其放射状突起伸至分子层的表面。上述胶质细胞反应在小脑小结和蚓垂比较轻微。结果提示NPC小鼠小脑内小胶质细胞和星形胶质细胞显著激活,它们可能与浦肯野细胞等神经元的退行性变有关。     

Calretinin-immunoreactivity in organotypic cultures of the rat cerebral cortex: effects of serum deprivation

Calretinin, a calcium-binding protein, is expressed in a specific set of interneurons in the adult rat cortex. Although its role in development is not known with any degree of certainty, evidence in support of a neuroprotective function has been forthcoming. To test this hypothesis, we submitted organotypic cultures (interphase technique) of 4- to 6-day-old rat brain slices to nutritive stress by serum deprivation for 1–3 weeks. Cultures were immunolabelled either with an antiserum against calretinin or with an antibody against MAP2 (the latter being used to assess neuronal cell number). In control (serum-enriched) cultures, the pattern of development of calretinin immunoreactivity mimicked that evinced in vivo with respect to layer- and cell-type specificity, but the maturation process was retarded by about 1 week. In the experimental group, cultures were incubated for 1 week in the presence of serum and then transferred to serum-free medium for an additional 2 weeks. Tissue was characterized by necrotic foci, a marked decrease in neuronal cell number and a further retardation in the course of development of the calretinin immunoreactivity pattern. The proportion of calretinin-immunoreactive cells to total number of viable neurons was 16% in serum-free cultures as against 9% in serum-enriched ones, suggesting that cells expressing the calcium-binding protein exhibit a greater tenacity for survival under conditions of nutritive stress, and thereby supporting the contention that calretinin acts in a neuroprotective capacity.     

海马内注射LPS后诱导大鼠皮质星形胶质细胞活化和PirB的表达

目的:探讨海马内注射LPS(lipopolysaccharide,LPS)后,大鼠皮质神经元和星形胶质细胞PirB(paired-immunoglobulin-like receptor B)的表达变化。方法:成年SD大鼠,分为对照组和实验组,用免疫组织化学法检测大鼠脑皮质PirB的表达及星形胶质细胞GFAP的表达变化,免疫荧光双重标记技术,观察星形胶质细胞与PirB阳性细胞的共存。结果:PBS注射的对照组动物脑皮质Ⅴ层内可见PirB阳性神经元样细胞,呈圆形、卵圆形和锥体形,免疫反应产物呈棕色、主要位于细胞膜上;海马内注射LPS 30 d后,PirB阳性神经元样细胞和PirB阳性神经胶质样细胞主要分布于皮质Ⅳ、Ⅴ层,免疫反应产物位于胞质和突起内;另外,可见大鼠梨状皮质、内嗅皮质内GFAP阳性星形胶质细胞明显被激活,表现为细胞胞体相对增大,突起变粗。PirB分别和MAP-2、GFAP、CD11b免疫荧光双重标记染色显示:PirB和MAP-2阳性神经元的胞体存在共定位;PirB与GFAP阳性染色存在部分共定位,但未见PirB与CD11b阳性染色双标细胞。结论:LPS能诱导大鼠皮质星形胶质细胞活化和PirB蛋白表达上调,而且部分活化的星形胶质细胞表达PirB,PirB可能参与脑内炎症突触可塑性改变和学习记忆功能缺失的免疫调节。     

Cellular organization and development of slice cultures from rat visual cortex

Summary Slice cultures from the visual cortex of young rats were prepared using the roller culture technique (Gähwiler 1984). After 10 days in vitro the cortical cultures flattened to 1–3 cell layers, surviving for up to 12 weeks. The cultures were organotypically organized, the typical layered structure of the cortex was preserved. The neuronal composition of slice cultures was studied using intracellular staining, Golgi impregnation and GABA immunohistochemistry. Both pyramidal cells and several types of nonpyramidal cells were identified in the slice cultures. Electrophysiological recordings showed that the electrical properties of cells in culture were similar to those measured in acute slice preparations; for some cells, however, the spontaneous activity was higher. The maintained activity was strongly increased by application of the GABA antagonist bicuculline and decreased by GABA, suggesting that GABAergic inhibition is present in these preparations. We could observe the postnatal maturation of some characteristic morphological features in culture. For example, pyramidal cells in 6 day-old rats in situ have very short basal dendrites with growth-cones, and the dendrites are free of spines. After 2–3 weeks in culture growth-cones were no longer observed. Instead, the cells had developed a large basal dendritic field and the dendrites were covered with spines. Slice cultures therefore may provide a useful tool for physiological, anatomical, pharmacological and developmental studies of cortical neurons in an organotypical environment.     

脑缺血/再灌注损伤中产生IL-17A的神经细胞类型鉴定

目的在离体细胞水平鉴定脑缺血/再灌注损伤中产生白细胞介素-17A(IL-17A)的脑固有神经细胞类型。方法利用原代培养小鼠脑皮层神经元、小胶质细胞和星形胶质细胞1~4 h氧-糖剥夺/24 h复糖复氧(OGD/R)模拟细胞离体缺血/再灌注损伤模型,用免疫荧光双标、实时定量聚合酶链式反应(RT-qPCR)和蛋白免疫印迹(Western blot)等技术,确定产生IL-17A的神经细胞类型。结果三种神经细胞中,除NeuN阳性神经元外,小胶质细胞和星形胶质细胞在OGD/R处理后,均可表达IL-17A蛋白,且分别与其特定标志物Iba-1和GFAP存在共定位现象。星形胶质细胞经过1~6 hOGD/24 h R处理后,IL-17A mRNA表达水平随OGD时间延长显著增高,且在4 hOGD/R时达高峰[(2.74±2.48),P0.001,每组n=5]。此外,OGD/R可促进星形胶质细胞表达IL-17A蛋白[(3.17±0.91),P0.05,每组n=5]。结论脑缺血/再灌注损伤中星形胶质细胞可能是产生IL-17A的主要来源。     

Characterisation of the consequences of maternal immune activation on distinct cell populations in the developing rat spinal cord

Maternal immune activation (MIA) during gestation has been implicated in the development of neurological disorders such as schizophrenia and autism. Epidemiological studies have suggested that the effect of MIA may depend on the gestational timing of the immune challenge and the region of the central nervous system (CNS) in question. This study investigated the effects of MIA with 100 μg/kg lipopolysaccharide at either Embryonic days (E)12 or E16 on the oligodendrocytes, microglia and astrocytes of the offspring spinal cord. At E16, MIA decreased the number of olig2⁺ and Iba‐1⁺ cells in multiple grey and white matter regions of the developing spinal cord 5 h after injection. These decreases were not observed at postnatal day 14. In contrast, MIA at E12 did not alter Olig2⁺ or Iba‐1⁺ cell number in the developing spinal cord 5 h after injection, however, Olig2⁺ cell number was decreased in the ventral grey matter of the P14 spinal cord. No changes were observed in glial fibrillary acidic protein (GFAP) expression at P14 following MIA at either E12 or E16. These data suggest that E16 may be a window of immediate vulnerability to MIA during spinal cord development, however, the findings also suggest that the developmental process may be capable of compensation over time. Potential changes in P14 animals following the challenge at E12 are indicative of the complexity of the effects of MIA during the developmental process.     

Hundred-fold increase in neuronal vulnerability to glutamate toxicity in astrocyte-poor cultures of rat cerebral cortex

In cultures of rat cerebral cortex in which astrocyte proliferation was stringently suppressed, glutamate neurotoxicity occurred at glutamate concentrations similar to those which are normally found in the extracellular space in the hippocampus. Concentrations of glutamate one hundred-fold higher were required to produce neurotoxicity in the presence of abundant astrocytes. This suggests that the sensitivity of central neurons to glutamate toxicity may be dependent upon astrocyte function.     

Longterm stability and developmental changes in spontaneous network burst firing patterns in dissociated rat cerebral cortex cell cultures on multielectrode arrays

Spontaneous action potentials were recorded longitudinally for 4-7 weeks from dissociated rat occipital cortex cells cultured on planar multi-electrode plates, during their development from isolated neurons into synaptically connected neuronal networks. Activity typically consisted of generalized bursts lasting up to several seconds, separated by variable epochs of sporadic firing at some of the active sites. These network bursts displayed discharge patterns with age-dependent firing rate profiles, and durations significantly increasing in the 3rd week in vitro and decreasing after about 1 month in vitro, when they evolved into short events with prompt onsets. These findings indicate that after about a month in vitro these cultured neuronal networks have developed a degree of excitability that allows almost instantaneous triggering of generalized discharges. Individual neurons tend to fire in specific and persistent temporal relationships to one another within these network bursts, suggesting that network connectivity maintains a core topology during its development.