Composition of gangliosides and phospholipids of neuronal and glial cell enriched fractions

Abstract— Fractions enriched in neuronal cell bodies and in glial cells were isolated from rabbit cerebral cortex by discontinuous gradient centrifugation. The ratio of total lipid to protein was approx. 50 per cent higher in the glial fraction than in the neuronal fraction. The fatty acid composition for the major phosphoglycerides was with few exceptions, similar for neurons and glia. The ganglioside concentration was very low for both cell types, but was approx. twice as high in the glial cells as in the neurons. The pattern of individual gangliosides was, however, very similar for the glial and neuronal fractions and did not differ from that of unfractionated cerebral cortex, synaptosomes and mitochondria. The latter results are discussed in relation to the estimated amounts of plasma membrane in the neuronal and glial fractions.     

Topologic distribution of different types of neurons in the human putamen

OBJECTIVE: To test the assumption that the various types of neuron in the human putamen appear to be randomly distributed and to quantify the way in which they are arranged, stochastic geometry, multivariate analysis and the interactive evaluation technique were employed. STUDY DESIGN: Twenty-seven human putamina without demonstrable signs of neurologic change were dissected out, fixed in 4% formalin and embedded in paraffin. The 20-micron paraffin sections were stained in an aldehyde-fuchsin and cresyl-violet solution, which makes it possible to distinguish between seven different neuron populations in the putamen. The gravity centers, size and form factors of these neurons were determined morphometrically under a light microscope. The data obtained were used to calculate the spatial distribution of the neurons by interactive and structure analytical methods. RESULTS: Visual point field analysis revealed an irregular arrangement of the different types of neurons. Point process analysis detected a significant hard core process of type 1 and a cluster process of type 6 neurons. With nearest neighborhood analysis, significant differences were found between certain populations of neurons and Poisson processes. Comparison of the results of multivariate cluster analysis with the investigator-dependent results of visual point field analysis showed clear differences. CONCLUSION: By means of structure analytical methods, the arrangement of different populations of neurons can be demonstrated. Some neuronal distributions are detectable only by using one of these techniques. The question of random or nonrandom distribution of the neurons in the human putamen can now be answered definitively: arrangement of the different populations of neurons is structured.     

Oxidative events in neuronal and glial cell-enriched fractions of rat cerebral cortex

The aim of this work was to investigate how neurons and glial cells separated from rat brain cortex respond to “in vitro” oxidative stress induced by incubation of the cellular fractions in the presence of prooxidant mixtures; in addition, the endogenous enzymatic antioxidant capacity of the purified fractions was investigated. Neuronal and glial cell-enriched fractions were obtained from rat cerebral cortex following passages of the tissue through meshes and centrifugations. The following parameters were evaluated: antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx), and glucose-6-phosphate dehydrogenase (G6PDH); lipid peroxidation products (TBARS) prior to (basal) and after (iron-stimulated) incubation with a mixture of iron and ascorbic acid; intracellular production of reactive oxygen species (ROS) using a fluorescent probe, dichlorofluorescin-diacetate, in basal, iron-stimulated, and menadione stimulated conditions. SOD and GSHPx activities showed no significant changes between neurons and glia, whereas CAT and G6PDH activities were found to be significantly lower in glia than in neurons. TBARS levels were significantly lower in the glial fraction than in neurons, both in basal and iron-stimulated conditions. ROS production showed no differences between neurons and glia in both basal and menadione-stimulated conditions. Iron-stimulation produced a marked increase in ROS production, limited to the neuronal fraction, with the glial values being similar to the basal ones. Our conclusion is that glia and neurons isolated from rat cerebral cortex show a similar pattern of the most important antioxidant enzymes and of their basal ROS production, whereas glia is more resistant in “oxidative stress” conditions.     

Effect of Structure on Function in Model Nerve Nets

A theoretical analysis has been made on the effect of the pattern of interneuronal connectivity in model nerve nets on the activity of these nets. Two types of nets have been investigated: one in which the likelihood of a connection between a given neuron and any other element in the net is given by a Poisson probability distribution, and a second type in which the pattern of interconnection follows a Gaussian distribution. An analytical treatment is presented of the equations for noiseless nets in these two conditions. The principal result is that nets with Poisson connectivity law are activated by extraneous firing of a single neuron and continue in spontaneous activity indefinitely. On the other hand, similar nets in which the connections are, however, distributed according to a normal connectivity law, exhibit a definite threshold and produce spontaneous activity only subsequent to extraneous activation of a substantial fraction of the population. Moreover, spontaneous activity in Gaussian nets, but not in Poisson nets, becomes extinguished if the number of active neurons falls below the critical threshold. Some neuroanatomical implications are discussed which suggest that the pyramidal system of the cerebral cortex and other neuronal systems histologically characterized by large numbers of synapses per neuron may incorporate a Gaussian connectivity law, whereas a Poisson law may be characteristic of these cortical layers and nuclei primarily containing granule cells.     

The distribution of glutamate receptors in cultures of the motor area of the rat cerebral cortex studied by an immunoelectron microscopic method

The aim of the study was to analyse the distribution and localization of glutamate receptors in the cultured cells of the neonatal rat motor cortex, using immunoelectron microscopic technique, and monoclonal antibodies preliminary labeled with colloidal gold. Monoclonal antibodies against glutamate-binding proteins of the adult rat cerebral cortex were produced by means of hybridization of immune splenocytes with plasmocytoma cells. It was found that monoclonal antibodies labeled with colloidal gold could reveal selectively the localization of glutamate receptors on the membranes of neurons. Glutamate receptors were detected on differentiating neuron membranes only, being absent from the glia cell surface.     

Expression of Bcl-2 in Adult Human Brain Regions with Special Reference to Neurodegenerative Disorders

Abstract: The expression of the protooncogene bcl-2 , an inhibitor of apoptosis in various cells, was examined in the adult human brain. Several experimental criteria were used to verify its presence; mRNA was analyzed by northern blot with parallel experiments in mouse tissues, by RNase protection, and by in situ hybridization histochemistry. Bcl-2 protein was detected by western blot analysis and immunohistochemistry. Two bcl-2 mRNA species were identified in the human brain. The pattern of distribution of bcl-2 mRNA at the cellular level showed labeling in neurons but not glia. The in situ hybridization signal was stronger in the pyramidal neurons of the cerebral cortex and in the cholinergic neurons of the nucleus basalis of Meynert than in the Purkinje neurons of the cerebellum. Both melanized and nonmelanized neurons were labeled in the substantia nigra. In the striatum, bcl-2 mRNA was detected in some but not all neurons. In the regions examined for Bcl-2 protein, the expression pattern correlated with the mRNA results. In patients with Alzheimer's and Parkinson's diseases, quantification of bcl-2 mRNA in the nucleus basalis of Meynert and substantia nigra, respectively, showed that the expression was unaltered compared with controls, raising the possibility that the expression of other components of apoptosis is modulated.     

Morpho-chemical characteristics of the brain of rats genetically predisposed to catalepsy

Cell organization of the cerebral cortex and striatum has been studied by+light optics and enzymatic activity of neuromediators catabolism--histochemically. In layers III and V of the sensomotor cortex and in the nucleus caudatus the number of neurons per unit area is increased, and their size is decreased. Volume of cytoplasm and nuclei of neurons in both layers is decreased, the latter--to less extent. The total amount of the perineuronal glia in the nucleus caudatus is also decreased at the expense of astroglia. In the cerebral cortex it is equal, but the ratio between astro-++- and oligodendroglia is changed. Acetylcholinesterase activity in the layers III and V of the sensomotor cortex is lower than in the control, while monoamine oxidase activity is kept at the control level. The genetically determined anomalies++ of growth and development of the cerebral cells is supposed to be considered as a structural base of the cataleptic state.     

A radial glia-specific role of RhoA in double cortex formation

The positioning of neurons in the cerebral cortex is of crucial importance for its function as highlighted by the severe consequences of migrational disorders in patients. Here we show that genetic deletion of the small GTPase RhoA in the developing cerebral cortex results in two migrational disorders: subcortical band heterotopia (SBH), a heterotopic cortex underlying the normotopic cortex, and cobblestone lissencephaly, in which neurons protrude beyond layer I at the pial surface of the brain. Surprisingly, RhoA(-/-) neurons migrated normally when transplanted into wild-type cerebral cortex, whereas the converse was not the case. Alterations in the radial glia scaffold are demonstrated to cause these migrational defects through destabilization of both the actin and the microtubules cytoskeleton. These data not only demonstrate that RhoA is largely dispensable for migration in neurons but also showed that defects in radial glial cells, rather than neurons, can be sufficient to produce SBH.     

Methylmercury distribution, metabolism, and neurotoxicity in the mouse brain

Methylmercury distribution, biotransformation, and neurotoxicity in the brain of male Swiss albino mice were investigated. Mice were orally dosed with [203 Hg]methylmercury chloride (10 mg/kg) for 1 to 9 days. Methylmercury was evenly distributed among the posterior cerebral cortex, subcortex, brain stem, and cerebellum. The The anterior cerebral cortex had a significantly higher methylmercury concentration than the rest of the brain. The distribution of methylmercury's inorganic mercury metabolite was found to be uneven in the brain. The pattern of distribution was cerebellum greater than brain stem greater than subcortex greater than cerebral cortex. The order of the severity of histological damage was cerebral cortex greater than cerebellum greater than subcortex greater than brain stem. There was no correlation between methylmercury distribution in the brain and structural brain damage. However, there was a relationship between the distribution of methylmercury's inorganic mercury metabolite and structural damage in the anterior cerebral cortex (positive correlation) and the anterior subcortex (negative correlation). There was also a positive correlation between the fraction of methylmercury's metabolite of the total mercury present and structural brain damage in the anterior cerebral cortex. This study suggests that biotransformation may have a role in mediating methylmercury neurotoxicity.     

Neurons with choline acetyltransferase immunoreactivity and mRNA are present in the human cerebral cortex

 We examined the cerebral cortex of five autopsied individuals without neurological and psychiatric diseases by immunohistochemistry using an anti-human recombinant choline acetyltransferase (ChAT) polyclonal antibody and in situ hybridization with ³⁵S-labeled human ChAT riboprobes. The immunohistochemistry detected positive neurons which were medium-sized or large pyramidal neurons located predominantly in layers III and V. The density of such neurons was higher in the motor and secondary sensory areas than in other cortical areas; the immunoreactive neurons in layer V were more densely distributed in the motor area and those in layer III were distributed in the secondary sensory areas. Positively stained, non-pyramidal neurons were observed in the superficial layer of the cingulate gyrus and parahippocampus. No immunoreactive neurons were found in the primary sensory areas. The in situ hybridization detected some neurons with signals for ChAT mRNA in the cerebral cortex, most of which were distributed in layer V of the motor area and in layer III of the secondary visual area. These results indicate that the human cerebral cortex contains cholinergic neurons and displays regional and laminal variations in their distribution. Accepted: 17 November 1998     

Nonrandom spatial distribution by mammalian cells in culture

Quadrat analysis was used to investigate the spatial distribution of seven mammalian cell lines in culture. The number of cells in replicate unit areas of the culture was determined, and the variance to mean ratio used as an index of random and nonrandom spatial distribution. Only mouse SV3T3 cells distributed themselves randomly throughout the entire culture growth cycle. The remaining six lines all assumed a nonrandom distribution at some point in their growth cycles. Mouse L929 cells displayed avoidance behavior, and spaced themselves at regular intervals in a uniform spatial distribution. The five remaining lines (mouse S180, rat C6, hamster CHO, canine MDCK, and human BeWo) formed multicellular clusters, and were distributed aggregatively rather than randomly. Random walk migration can account for the random distribution of SV3T3 cells. Random walk combined with contact inhibition of movement provides a satisfactory explanation for the uniform distribution of L929 cells. Random walk and contact inhibition are incompatible with cell clustering, however. Thus other mechanisms of motility or adhesiveness must contribute to cell clustering. It is possible that random walk and contact inhibition may be less common components of cell movement than generally assumed.     

The distribution of cholinergic neurons in the human central nervous system

Choline acetyltransferase (ChAT), the enzyme responsible for the biosynthesis of acetylcholine, is presently the most specific marker for identifying cholinergic neurons in the central and peripheral nervous systems. The present article reviews immunohistochemical and in situ hybridization studies on the distribution of neurons expressing ChAT in the human central nervous system. Neurons with both immunoreactivity and in situ hybridization signals of ChAT are observed in the basal forebrain (diagonal band of Broca and nucleus basalis of Meynert), striatum (caudate nucleus, putamen and nucleus accumbens), cerebral cortex, mesopontine tegmental nuclei (pedunculopontine tegmental nucleus, laterodorsal tegmental nucleus and parabigeminal nucleus), cranial motor nuclei and spinal motor neurons. The cerebral cortex displays regional and laminal differences in the distribution of neurons with ChAT. The medial septal nucleus and medial habenular nucleus contain immunoreactive neurons for ChAT, which are devoid of ChAT mRNA signals. This is probably because there is a small number of cholinergic neurons with a low level of ChAT gene expression in these nuclei of human. Possible connections and speculated functions of these neurons are briefly summarized.     

Effect of audiogenic convulsions on the activity of n- and m-forms of lactate dehydrogenase in the neurons and neuroglia of different sections of central nervous system]

It was shown spectrophotometrically that in Krushinsky-Molodkina and Wistar rats the ratio of the activity of the aerobic H-forms of lactic dehydrogenase (LDH) to the activity of the anaerobic M-forms was higher in the neurons of the cerebral cortex and the Purkinje's cells of the cerebellum and in their glial cells-satellites than in the motor neurons of the anterior horns of the spinal cord and their perineuronal glia. In Krushinsky-Molodkina rats (with genetically-determined high sensitivity to audiogenic convulsions) epileptiform attacks under the effect of sound were accompanied by a marked activation of both the H- and the M-forms of LDH in the cortical neurons in the absence of any shifts in the perineuronal glia. On the contrary, the activity of all the forms of LDH was unchanged in the spinal motor neurons, whereas in the neuroglia cells surrounding these neurons there was a distinct increase in the activity of the H-forms of LDH. In the Purkinje's cells of the cerebellum an increase and in the glial cells- satellites -- a reduction of the activity of the M-forms of LDH in case of convulsions was seen.     

Radial glial cells. are they really glia?

During the development of the cerebral cortex, radial glia serve as a scaffold to support and direct neurons during their migration. This view is now changing in the light of emerging evidence showing that these cells have a much more dynamic and diverse role. A recent series of studies has provided strong support for their role as precursor cells in the ventricular zone that generate cortical neurons and glia, in addition to providing migration guidance.     

神经元缺氧复氧损伤时氧自由基的毒性作用及其机制

在原代分离培养Ｗｉｓｔａｒ乳鼠大脑皮质神经元上研究了缺氧复氧损伤（Ｈ／Ｒ）对神经细胞乳酸脱氢酶（ＬＤＨ）,漏出率,死亡率和脂质过氧化物含量的影响,并选用一氧化氮（ＮＯ）合酶抑制剂Ｌ－ＮＧ－硝基－精氨酸（Ｌ－ＮＮＡ）巯基供体Ｎ－乙酰半胱氨酸（ＮＡＣ）和超氧化物歧化酶（Ｃｕ,Ｚｎ－ＳＯＤ）三种自由基清除剂进行预保护等方法来探讨机制。结果表明 Ｈ／Ｒ损伤引起ＬＤＨ漏出率,细胞死亡率和脂过氧化物含量极显著     

Nogo-66 receptor at cerebellar cortical glia gap junctions in the rat

Nogo-A is a myelin inhibitor of neurite outgrowth that accounts for the difficulty in fiber regeneration in the central nervous system. Its 66-amino-acid extracellular domain (Nogo-66) contributes to the inhibitory activity of Nogo-A. The Nogo-66 receptor is widely distributed in neurons of the central nervous system, including the cerebellum. In our study on the distribution of Nogo-66 receptor in the cerebellar cortex in the rat, we unexpectedly found Nogo-66 receptor immunoreactivity in the glia cells, particularly abundant beneath the Purkinje cells. The presence of Nogo-66 receptor in glia cells has not been reported before. A detailed study was thus conducted. Immunoelectron microscopic investigation clearly demonstrated that the Nogo-66 receptor immunoreactivity could be ascertained at the gap junction between glia cells, indicating that the Nogo-66 receptor may modulate the communication between glia cells through gap junctions.     

Glia–Neuron Interactions in the Sensory-Motor Cortex of Warm-Blooded Animals (Guinea Pigs and Ground Squirrels) with Different Habitat Conditions and the M-Cholinergic Reaction of the Brain

The glia–neuron interactions were analyzed in the sensory-motor cortex of guinea pigs and ground squirrels (Spermophilus undulatus) during the active summer months. The glial cells were more concentrated in close proximity (15–25 μm) to neurons (38% in guinea pigs and 22.4% in ground squirrels). A more concentrated distribution of glial cells might be very necessary for spontaneous inactive nerve cells (37.2% in guinea pigs and 23% in ground squirrels), since these neurons are associated with the highest energy demand during their functioning and are most susceptible to disturbances of ion homeostasis. The network structure of glia and the close contact between glial cells and brain capillaries provide additional energy for neurons and stabilize the ion balance in the extracellular medium. Glial density in the sensory-motor cortex of ground squirrels is 3 times higher than that in the cortex of guinea pigs. The high content of glial cells in the ground-squirrel cortex is the most important protective factor for survival of animals during long-term hibernation, when the diffusion of K⁺ ions from nerve cells drastically increases due to the high temperature sensitivity of the M-cholinergic response.     

Protein synthesis in neurons and glial cells of the developing rat brain: an in vivo study

Abstract— A technique for the isolation of pure neuronal perikarya and intact glial cells from cerebral cortex has been developed for routine use. The yield of neuronal perikarya and glial cells was greater from highly immature (5–10 days) rat cerebral cortex than from the cortex of older rats (18–43 days). The perikarya/glia yield ratio decreased with age indicating that, as the glial population matured, the procedure succeeded in isolating a gradually smaller proportion of the existing neurons. The perikarya/glia ratio was highest for the 5-day-old cortex in which no mature glial cells could be identified. After a 10-min pulse in vivo of intrathecally injected [¹⁴C]phenylalanine, the specific radioactivity of the neuronal proteins was higher than that of the glial proteins in the 5-, 10- and 18-day-old rat but was lower in the 43-day-old rat. The values for absolute specific radioactivity of the ¹⁴C-labelled proteins in both cell types were greater, the younger the brain. The ¹⁴C-labelling of neuronal and glial proteins in the 18-day-old rat was assessed in vivo as a function of time by determining the incorporation of [¹⁴C]phenylalanine into such proteins at 5, 10, 20 and 45 min after administration of the amino acid. The rate of incorporation of [¹⁴C]phenylalanine into the glial cells was faster than into the neurons since higher specific radioactivities of the glial proteins could be achieved at earlier times. Also, a biphasic pattern of ¹⁴C-labelling of the glial proteins was noted, suggesting, perhaps, a sequential involvement of the oligodendrocytes and astrocytes. Homogenates of prelabelled neuronal perikarya were fractionated into the nuclear, mitochondrial microsomal and soluble cell sap fractions. In the 18-day-old cerebral cortex, the proteins of the microsomal fraction exhibited the highest specific radioactivity at the end of 10 min, whereas by 20 min proteins of the mitochondrial fraction were most highly labelled. The specific radioactivity of the nuclear proteins increased over the entire 45-min experimental period. On the contrary, the proteins of the soluble cell sap, in which the specific radioactivity was at all times by far the lowest, were maximally labelled by 5 min. Examination of the labelling of the neuronal subcellular fractions as a function of age revealed that at 10 min after administration of [¹⁴C]phenylalanine, the specific radioactivities of all ¹⁴C-labelled proteins were highest in the youngest (5-day-old) neurons. The proteins of the microsomal fraction were most rapidly labelled at all ages. During this interval the proteins of the soluble cell sap were only moderately labelled in the 5-day-old neurons and were totally unlabelled in the 43-day-old neurons, indicating age-dependent differences in the rate of utilization of the amino acid precursor by the neurons.     

植物空间分布格局中邻体距离的概率分布模型及参数估计

最近邻体法是一类有效的植物空间分布格局分析方法,邻体距离的概率分布模型用于描述邻体距离的统计特征,属于常用的最近邻体法之一。然而,聚集分布格局中邻体距离(个体到个体)的概率分布模型表达式复杂,参数估计的计算量大。根据该模型期望和方差的特性,提出了一种简化的参数估计方法,并利用遗传算法来实现参数优化,结果表明遗传算法可以有效地估计的该模型的两个参数。同时,利用该模型拟合了加拿大南温哥华岛3个寒温带树种的空间分布数据,结果显示:该概率分布模型可以很好地拟合美国花旗松(P.menziesii)和西部铁杉(T.heterophylla)的邻体距离分布,但由于西北红柏(T.plicata)存在高度聚集的团簇分布,拟合结果不理想;美国花旗松在样地中近似随机分布,空间聚集参数对空间尺度的依赖性不强,但西北红柏和西部铁杉空间聚集参数具有尺度依赖性,随邻体距离阶数增加而变大。最后,讨论了该模型以及参数估计方法的优势和限制。     

天山云杉种群空间格局与动态

天山云杉(Picea schrenkiana)林是天山林区的重要针叶林。研究调查了两块分别代表天山云杉不同演替阶段的标准地,应用Ripley’s K系数法研究了天山云杉种群空间格局与动态。结果表明：天山云杉是聚集型种群,天山云杉活立木、枯死木、天山桦(Betula tianschanica Rupr．)和天山柳(Salix tianschanica Rgl．)的空间格局均为聚集型;天山云杉枯死木的聚集强度大于天山云杉活立木的聚集强度;天山桦和天山柳的聚集强度大于天山云杉枯死木的聚集强度;天山桦和天山柳的空间格局随种群所处的演替阶段不同而不同,增长型种群在所有尺度上其空间格局都为聚集型,在大于70m尺度上表现为均匀格局,而天山云杉成熟林中的天山桦和天山柳只在小尺度(0～40m)上表现为聚集。随着天山云杉的生长,其聚集强度逐渐减小,聚集半径不断增大。从50a以下天山云杉到50～100a的天山云杉其聚集强度急速减小,这有利于天山云杉幼树获得足够的资源,是天山云杉幼树的一种生存策略或适应机制。150a以上天山云杉的聚集强度增加,这是由于天山云杉生长到150a左右时开始出现成熟后死亡,形成林窗有利于天山云杉林的更新。研究还对Ripley’s K系数法和频次检验法进行了比较,Ripley’s K系数法能够分析各种尺度下的种群空间格局,且不受样方大小的影响,结果清楚、直观。