大鼠大脑皮质星形胶质细胞条件培养液促进小脑皮质神经元的生长

本研究从大鼠大脑皮质分离、纯化星形胶质细胞,再经培养后收集星形胶质细胞的无血清条件培养液。用盖玻片培养法与快速自动比色微量分析法研究了星形胶质细胞条件培养液对小脑皮质神经元生存以及神经元活力的影响。发现星形胶质细胞条件培养液能够明显提高小脑皮质神经元的体外存活率,增强神经元的活力。表明星形胶质细胞具有神经营养性作用。     

CHARACTERIZATION AND LOCALIZATION OF ACID HYDROLASE ACTIVITY IN THE SYNAPTOSOMAL FRACTION FROM RAT CEREBRAL CORTEX

Synaptosomes were prepared from the cerebral cortex of adult rats by a rapid technique of centrifugation in a Ficoll-sucrose discontinuous gradient. The synaptosomal fraction contained 40 per cent of the total gradient activity of acid α-naphthyl phosphatase (EC 3.1.3.2). Quantitative electron microscopy of this fraction revealed rare, typical, extrasynaptosomal dense body lysosomes. pH-activity profiles of free and Triton X-100 (total) activities were prepared for α-naphthyl phosphatase, β-glucuronidase (EC 3.2.1.31), β-galactosidase (EC 3.2.1.23), arylsulfatase (EC 3.1.6.1) and N-acetylglucosaminidase (EC 3.2.1.30). The ratios of total to free activity varied in the order: arylsulfatase > β-galactosidase > β-glucuronidase > N-acetylglucosaminidase > acid phosphohydrolase. Incubation of synaptosomal fractions at pH 5 and 37°C produced significant activation of β-galactosidase and N-acetylglucosaminidase but no activation of cryptic lactate dehydrogenase (EC 1.1.1.27). Hyposmotic suspension and subfractionation of the synaptosomal fraction produced considerable solubilization of lactate dehydrogenase, arylsulfatase and β-galactosidase but only partial liberation of α-naphthyl phosphatase, the remainder being associated with synaptosomal membrane fragments. Incomplete equilibrium sedimentation of synaptosomes in a continuous sucrose gradient (0·55-1·5 M) provided a broad lactate dehydrogenase and Na + K ATPase (EC 3.6.1.4) peak (peak I) at low sucrose densities. β-Glucuronidase, β-glucosidase and α-naphthyl phosphatase were significantly present in peak I. Conversely, N-acetylglucosaminidase, arylsulphatase and β-galactosidase were predominantly located in denser particles sedimenting through 1·2 M sucrose (peak II). Electron microscopy confirmed the heterogeneity of this second peak and the presence of numerous extrasynapto-somal dense body lysosomes.     

METABOLISM OF HEXOSES IN RAT CEREBRAL CORTEX SLICES

Abstract—

• 1 The metabolism of two ¹⁴C-labelled hexoses and one hexose analogue, viz. mannose, fructose and glucosamine, has been compared with that of glucose for slices of rat cerebral cortex incubated in vitro.
• 2 The metabolism of [U-¹⁴C]mannose was essentially identical to that of glucose; oxygen consumption and CO₃ production were similar and maximal at a substrate concentration of 2·75 mM. Incorporation of label into lactate, aspartate, glutamate and GABA was similar for the two substrates at 5·5 mM substrate concentration.
• 3 With [U-¹⁴C]fructose, maximal oxygen consumption and CO₃ production were obtained at a substrate concentration of 11 mM. At 5·5 mM, incorporation into lactate was 5 per cent, into glutamate and GABA 30 per cent, into alanine 63 per cent and into aspartate 152 per cent of that from glucose. Increasing substrate concentration to 27·5 mm was without effect on incorporation into amino acids from glucose and raised incorporation from fructose into glutamate, GABA and alanine to a level similar to that found with glucose; at the higher substrate concentration aspartate incorporation from fructose was 200 per cent and lactate 42 per cent of that with glucose. Unlabelled fructose was without effect on incorporation of radioactivity from [3-¹⁴C]pyruvate into CO₂ or amino acids; it increased incorporation into lactate by 36 per cent. Unlabelled glucose diminished incorporation into CO₂ from [U-¹⁴C]fructose to 35 per cent; incorporation into lactate was stimulated 178 per cent at 5·5 mM fructose; at 27·5 mM it was diminished to 75 per cent.
• 4 By comparison with [1-¹⁴C]glucose, incorporation of radioactivity from [1-¹⁴C]-glucosamine into lactate, CO₂, alanine, GABA and glutamine was very low; incorporation into aspartate was similar to glucose. Thus the metabolism of glucosamine resembled that of fructose. Glucosamine-1-phosphate, glucosamine-6-phosphate, and an unidentified metabolite, all accumulated.

     

OXIDATIVE METABOLISM OF THE CEREBRAL CORTEX OF THE RAT IN SEVERE INSULIN-INDUCED HYPOGLYCAEMIA

—Measurements were made of organic phosphates, carbohydrate substrates, amino acids and ammonia in the cerebral cortex, as well as of cerebral blood flow and of cerebral metabolic rate for oxygen and glucose in rats that developed an isoelectric EEG pattern (‘coma’) during insulin-induced hypoglycaemia. The results were compared to those obtained in control animals, as well as in hypoglycaemic animals with an EEG pattern of slow waves and polyspikes. In animals with slow waves and polyspikes, there was a decrease in all citric acid cycle intermediates except succinate and oxaloacetate, and a decrease in the pool size of intermediates. In animals that had an isoelectric EEG for 5–15 min, there were further decreases in citrate, isocitrate, α-ketoglutarate, malate and fumarate, but since the concentration of succinate (and oxaloacetate) increased, the pool size remained the same. In isoelectric animals, the results revealed extensive utilization of amino acids by both transamination and deamination reactions. However, since glycogen had disappeared and the amino acid pattern was constant after the first 5 min of isoelectric EEG, further oxidation must have occurred at the expense of non-carbohydrate, non-amino acid substrates. There were two- to three-fold increases in cerebral blood flow in animals with slow waves and polyspikes and in animals with isoelectric EEG, and no decrease in the cerebral metabolic rate for oxygen. Since less than half of the oxygen consumption could be accounted for in terms of glucose extraction, the data indicate that severe hypoglycaemia is associated with extensive oxidation of endogenous substrates other than carbohydrates and free acids.     

癫痫大鼠大脑皮质及海马神经元NOS的变化

给大白鼠侧脑室注射马桑内酯（Ｃｏｒｉａｒｉａ Ｌａｃｔｏｎｅ, ＣＬ）（１７５×１０－ ２ｍ ｏｌ／Ｌ２μｌ）后可诱发癫痫,用ＮＡＤＰＨｄ 组织化学方法观察大脑皮质及海马ＮＯＳ阳性神经元的变化, 结果： 大脑皮质ＮＯＳ阳性神经元数目逐渐增加, 至２ｈ 达高峰, 与生理盐水组相比差异具有非常显著性意义（Ｐ＜ ００１）, 随着ＣＬ作用时间延长ＮＯＳ反应由弱变强;海马区ＮＯＳ阳性神经元２ｈ 时才出现染色明显加深。对体外培养的大脑皮质及海马神经元用ＣＬ （２５×１０－ ５ｍ ｏｌ／Ｌ） 作用１／２ｈ、１ｈ、２ｈ、４ｈ 后ＮＯＳ阳性神经元均未见明显增加。     

SUBCELLULAR LOCALIZATION OF NEWLY-FORMED [3H]ACETYLCHOLINE IN RAT CEREBRAL CORTEX IN VITRO

—Slices from rat brain cortex were incubated for either 5 or 60 min in a medium containing [³H]choline and 4·7 or 25 mm -KCl. Bioassayable ACh and labelled ACh were determined in the incubation medium, in the total tissue homogenate and in subcellular fractions. Raising the KCl concentration from 4·7 to 25 mm stimulated the release and synthesis of total and of labelled ACh. In medium containing 25 mm -KCl the amounts of ACh decreased in the tissue and in the nerve ending cytoplasm, but remained constant in the synaptic vesicles. After incubation in 25 mm -KCl medium the ACh in the vesicles was labelled to the same extent as the cytoplasmic ACh but after incubation in 4·7 mm -KCl medium vesicular ACh was labelled less than cytoplasmic ACh. During 5 min incubation in medium containing 25 mm -KCl the ratio of labelled to total ACh was much higher in the medium than in the homogenate, the vesicles or the cytoplasm. During the last 15 min of the 60 min incubation the ratio of labelled to total ACh in the medium was still higher than that in the tissue fractions, but less so than during the 5 min incubation. It is concluded that the vesicular and cytoplasmic fractions are not identical with the store in the tissue from which newly-synthesized ACh is preferentially released.     

DEVELOPMENTAL CHANGES IN GLYCOLYSIS IN RAT CEREBRAL CORTEX

Abstract— The ATP concentration in infant rat cerebral cortex slices which were incubated aerobically with glucose (5 m m ) as substrate was much higher than in those from the adult. The higher ATP concentration in slices from young rat was also obtained when they were incubated aerobically with pyruvate (10 m m ), dl -lactate (20 m m ) and dl -3-nydroxybutyrate (20 m m ) However, when the slices were incubated anaerobically with glucose, the ATP concentration was very low. Thus, the formation of ATP in the slices from the young rat was thought to be mainly due to their oxidative metabolism, as in those from the adult. The amounts of glycolytic key enzymes in rat cerebral cortex (hexokinase. phosphofructokinase and pyruvate kinase) increased with age. Glycolysis was actually shown to be less active in the cerebral slices from young rats than from the adult. In addition it is known that the tricarboxylic acid cycle enzymes in rat cerebrum also increase with age. Consequently, the activity with respect to ATP formation must be lower in the cerebral cortex slices from young rats than from the adult. The fact that ATP was nevertheless higher in the slices from young rats may be explained by a lower rate of degradation. Developmental increases in the amounts of Na⁺-K⁺-ATPase and Mg²⁺ -ATPase in rat cerebral cortex were greater than those of the glycolytic key enzymes. These are discussed in relation to the observation that the rate of aerobic glycolysis in slices from cerebral cortex of young rats was not increased by d -glutamate (5 m m ) and high potassium (50 m m ).     

THE SUBCELLULAR LOCALIZATION OF CARBAMYLCHOLINE-STIMULATED PHOSPHATIDYL INOSITOL TURNOVER IN RAT CEREBRAL CORTEX IN VIVO

Abstract— The intraventricular injection of 40 μCi of ³²P_i (carrier free) into adult rats resulted in maximum incorporation of ³²P_i into the phosphatidyl inositol of the whole cortex after 20 h. A further intraventricular injection of 2 nmol carbamylcholine plus 0.02 nmol eserine resulted in a 23% decrease in the specific activity of phosphatidyl inositol after 20 min. The specific radioactivities of phosphatidyl choline, phosphatidyl ethanolarmine and phosphatidyl serine were not changed. Cerebral cortex from rats treated in this way was subjected to an extensive subcellular fractionation. It was found that the specific radioactivity of the phosphatidyl inositol of the synaptic vesicle fraction showed a reduction of 60%. No other fractions showed effects of this magnitude.     

CYCLIC AMP IN THE RAT CEREBRAL CORTEX AFTER ACTIVATION OF NORADRENALINE NEURONS OF THE LOCUS COERULEUS

The levels of cyclic AMP in the rat brain were studied in vivo following destruction or stimulation of the noradrenergic pathway originating in the locus coeruleus. After chronic lesion of the locus coeruleus no alterations in cyclic AMP content were found. Electrical stimulation of the locus coeruleus produced an elevation of cyclic AMP in the cerebral cortex of chloral hydrate anaesthetized rats of 30%. Maximal increases were found after 15–60 s stimulation at a frequency of 30–100 Hz. This maximal response was slightly inhibited by phenoxybenzamine, an α-adrenergic blocking agent, and by the β-blocker propranolol. When the α and β blockers were administered together a highly significant decrease in cyclic AMP response was observed. Pretreatment of the rats with reserpinc +α methyl-p-tyrosine prevented the cyclic AMP response. In addition to the effect in the cerebral cortex, cyclic AMP-levels were also enhanced in the hippocampus, in the striatum and in the hypothalamus. These results suggest that the locus coeruleus regulates a small fraction of cerebral cyclic AMP levels, by both α- and β-adrenergic receptors.     

DEVELOPMENTAL TRANSITIONS IN UPTAKE OF AMINO ACIDS BY SYNAPTOSOMAL FRACTIONS ISOLATED FROM RAT CEREBRAL CORTEX

Developmental changes in mechanisms of synaptosomal amino acid transport have been studied in rat cerebral cortex. Well-defined changes over an age continuum could be observed in both the rates of amino acid accumulation and the effects of Na⁺ on the accumulation. The uptakes of five amino acids (threonine, serine and valine in Na⁺-free medium, aspartic acid and proline in Na⁺-containing medium) increased progressively with the age of the animal, whereas the uptakes of leucine and arginine (in Na⁺-free medium) decreased steadily. The uptake of serine or threonine by synaptosomal fractions prepared from newborn rats was markedly dependent on the presence of Na⁺in the incubation media. Na⁺exerted progressively less effect on the accumulation process with continuing postnatal development and to some extent inhibited uptake by fractions obtained from rats older than about 15 days. Na⁺significantly enhanced the accumulation of glycine in fractions from newborn and adult rats, but had only a slight effect in fractions prepared from 12 to 17-day old rats. A detailed study of the accumulation of glycine indicated that the synaptosomal transport of this amino acid proceeded by two independent systems, one of which was totally dependent on external Na⁺and the and adult animals than in fractions from 12 to 17-day-old rats, wheras the Na⁺-independent system was most active during this latter period of development. The decline in the Na⁺-independent accumulation of glycine from about the 15th day to adulthood was characterized by a decrease in the V_max. and an increase in the K_m.     

CATION UPTAKE BY INTACT NUCLEI FROM RAT CEREBRAL CORTEX

A method is described for determining the uptake of cations by intact nuclei from rat cerebral cortex. The divalent cations of magnesium, manganese and calcium were found to be concentrated in the nuclear pellet to levels well above the initial concentration of the medium, whereas little uptake of either potassium or sodium was observed. The binding of divalent cations to the nuclei will produce a cationic environment quite different from that of the external medium. Such localized differences may play a role in the control of nuclear activities.     

THE RELATIONSHIP BETWEEN ACETYLCHOLINE RELEASE FROM BRAIN SLICES AND THE ACETYLCHOLINE CONTENT OF SUBCELLULAR FRACTIONS PREPARED FROM BRAIN

Abstract— Acetylcholine (ACh) release from sliced cerebral cortex of rats was measured when the tissue was incubated in a high K⁺ (46 m m ) medium containing eserine. In the absence of hemicholinium (HC-3), ACh release was well maintained, but in the presence of HC-3, ACh release declined within 15–20 min. Subcellular fractions representing nerve-ending free (cytoplasmic) ACh and nerve-ending bound ACh were prepared from slices that had been stimulated to release ACh in the presence of HC-3. Both nerve-ending stores of ACh were depleted when their content was compared to tissue that had not been stimulated and there was no demonstrable difference in the rate of depletion of either of the two fractions. Stimulating slices with K⁺ in the absence of HC-3 also depleted cytoplasmic and vesicle-bound ACh. It is concluded that, under these experimental conditions, both nerve ending stores of ACh are available for release and that, in the absence of HC-3, ACh synthesis can maintain ACh release, but cannot maintain tissue ACh content.     

CONTROL OF THE REDOX STATE OF THE PYRIDINE NUCLEOTIDES IN THE RAT CEREBRAL CORTEX. EFFECT OF ELECTROSHOCK-INDUCED SEIZURES

—The concentrations of most of the intermediates of glycolysis and of the tricarboxylic acid cycle were determined in the cerebral cortex of rats, frozen 10 s after the induction of a generalized seizure by electroshock. The apparent equilibrium constant for the combined glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase and lactic dehydrogenase reactions, i.e. K_app= [Lactate] [3-Phosphoglycerate] [ATP]/[Pyruvate] [Glyceraldehyde-3-phosphate] [ADP] [HPO²₄], was evaluated and found to be similar to the value reported for the in vitro system at pH 7. During an estimated 4–5-fold increase in glycolytic flux imposed by the seizure, this system remained close to equilibrium. In control cortex the components of the aldolase reaction were deviated 80-fold from equilibrium but shifted slightly toward equilibrium during the seizure. The components of the aspartate aminotransferase reaction were maintained in equilibrium in both the control and the seizure states. Of 4 reactions used to assess the cytoplasmic and mitochondrial redox states, only the lactic dehydrogenase reaction was considered reliable in the acutely changing situation of the seizure, and yielded a calculated decrease in the cytoplasmic [NAD⁺]/[NADH] ratio. This change, coupled with an observed decrease in the [ATP]/[ADP] [HPO²₄] ratio during the seizure, supports the concept that in brain, as in liver (Krebs & Veech , 1969), the phosphate potential determines the redox state of the tissue.     

ISOLATION OF HYDROPHOBIC PROTEINS BINDING AMINO ACIDS: l-ASPARTIC ACID-BINDING PROTEIN FROM THE RAT CEREBRAL CORTEX

Abstract— Lyophilized rat cerebral cortex was treated with chloroform-methanol (2:1, v/v), and the extracted hydrophobic proteins (i.e. proteolipids) were separated by column chromatography on Sephadex LH-20. The first peak of protein, eluting with chloroform in the void volume, had high affinity binding for l -[¹⁴C]aspartic acid. The saturation of the binding showed three saturable sites with apparent dissociation constants of 0.2 μ m , 10 μ m and 50 μ m . The binding capacities of the three sites were 2.8, 132 and 617 nmol/mg of protein, respectively. There were 8.0 nmol of high affinity binding sites for l -aspartic acid and 1.53 nmol for l -glutamic acid per g of fresh tissue in the cerebral cortex of the rat. Differentiation between binding of l -aspartic and l -glutamic acid was clearly established by cross-binding and competition experiments with agonists and antagonists.
It is suggested that the isolated protein fraction may correspond to a synaptic receptor and not to the transport system. It is concluded that in the cerebral cortex there is a separate receptor for l -aspartic acid. This is further support to the possible role of this amino acid as a central excitatory transmitter.     

乙酰胆碱在刺激SⅡ区影响C类纤维传入诱发的SI区电反应中的作用

电刺激猫大脑皮层体感Ⅱ区(SⅡ)对隐神经C类纤维传入引起的体感Ⅰ区(SⅠ)诱发电位(C—CEP)有抑制和易化作用。在SⅠ区局部用阿托品能部分地阻断电刺激SⅡ区对C—CEP的抑制作用,但对易化作用的影响不明显;而用六烃季铵(C_6)能阻断易化作用,但对抑制作用的影响却不明显。在SⅠ区局部应用0.01％乙酰胆碱(ACh)对C—CEP有易化作用,用0.1％ACh则有抑制作用。结果提示,ACh可能参与SⅡ区对SⅠ区C—CEP的影响,通过SⅠ区的胆碱能M受体起抑制作用,通过N受体起易化作用。     

β-Ar-ACETYL D-GALACTOSAMINIDASE IN BULK SEPARATED NEURONS AND NEUROPIL FROM RAT CEREBRAL CORTEX

Abstract— β- N -Acetyl D-galactosaminidase was studied in isolated neuronal and neuropil fractions from cerebral cortex and subcellular fractions derived from them. Although the enzyme activity evinced some latency properties, its subcellular distribution pattern was broader than that observed with other acid hydrolases. By contrast with nine other acid hydrolases, it was more active in neuropil than neuronal fractions (neuronal/neuropil activity ratio 0.63). This ratio was preserved in lysosomal subfractions derived from the isolated cell fractions. The data is taken as further evidence for the microheterogeneity of lysosomal particles from the brain.