TOPOGRAPHICAL AND SUBCELLULAR DISTRIBUTION OF CHOLINE ACETYLTRANSFERASE AND GLUTAMATE DECARBOXYLASE IN PIGEON OPTIC TECTUM

Abstract— The distribution of choline acetyltransferase (ChAT) and glutamate decarboxylase (GAD) in different layers of the pigeon optic tectum and in some nuclei of the optic lobe have been investigated. About 40% of GAD and 25% of ChAT were found in the superficial part of tectum, but negligible activity was found in the stratum opticum. The highest GAD activity was found in layers 3-7 (according to the nomenclature of C ajal , 1911) with a peak in layer 4. ChAT activity peaked in layers 3, 5. 8 and 10/11. Its distribution correlated well with the staining pattern of AChE, particularly in the superficial part of the tectum. The distribution of ChAT and GAD did not change significantly 4 weeks after enucleation. ChAT and GAD activities were high in the nucleus isthmi, pars parvocellularis (Ipc). The activity of GAD was also high in the nucleus intercollicularis (ICo), the other nuclei showed less activity of both enzymes.     

POSTNATAL ALTERATIONS IN EFFECTS OF POTASSIUM ON UPTAKE AND RELEASE OF GLUTAMATE AND GABA IN RAT BRAIN CORTEX SLICES

The uptake and release of glutamate and of GABA, as well as the effect of high potassium concentrations (35 or 80 mM) hereupon, were studied by aid of ¹⁴C-labelled amino acids in brain cortex slices from rats of different ages between birth and adulthood. Both the extent of the uptake (i.e. the tissue/medium ratio of ¹⁴C at, or close to, equilibrium) and the rate of uptake (i.e. the tissue/ medium ratio of ¹⁴C after short (5 min) incubation periods) increased with age. Differences were, however, found between glutamate and GABA, and the extent of the GABA uptake had a distinct maximum during the second postnatal week. At all ages, high concentrations of potassium caused a decrease in the rate of GABA uptake but were without effect on the rate with which glutamate was taken up. The release of the two amino acids occurred with approximately the same half-time (50 min) in slices from animals of at least 14 days of age. Before that time the release of glutamate was somewhat faster, whereas that of GABA was much slower, especially during the first postnatal week (half-time 90 min). The ontogenetic alterations in the effect of excess potassium were complex and varied both between the two potassium concentrations used and between the two amino acids. The results are thus compatible with the existence of different transport systems for the two amino acids, They also suggest that glutamate may exert other functions in addition to its role as a putative transmitter.     

UPTAKE OF NEUROTRANSMITTER CANDIDATES BY PIGEON OPTIC TECTUM

—The kinetics of the uptake of various compounds into the crude mitochondrial fraction (P₂-fraction) of the pigeon optic tectum were studied. Aspartate, GABA, glutamate, glycine, proline and choline were taken up by high and low affinity systems. Only low affinity uptake was found for leucine and dopamine. The uptake for taurine was not saturable. The uptakes for all of the above compounds were temperature and sodium dependent. The high affinity system was more effected by sodium withdrawal than the low affinity system. Continuous sucrose gradients from 0.4 to 1.5 m were run with incubated P₂ fractions. Particle-bound radioactivity sedimented in a density range of 1.0 to 1.2 m -sucrose. Additional experiments with glutamate showed that its uptake is competitively inhibited by aspartate. The addition of a 1000-fold excess of glutamate to a P₂ fraction incubated at a concentration of 10^–6m -glutamate led to a massive decrease of particle-bound radioactivity, suggesting a coupled action of uptake and efflux.     

HIGH AFFINITY UPTAKE OF TRANSMITTERS: STUDIES ON THE UPTAKE OF l-ASPARTATE, GABA, l-GLUTAMATE AND GLYCINE IN CAT SPINAL CORD

Abstract— The uptake of l -aspartate, l -glutamate and glycine each appeared to be mediated by two kinetically distinct systems with apparent K_m's of the order of 10 ('high affinity') and 100 μM ('low affinity') in slices of cat spinal cord, whereas the uptake of GABA appeared to be mediated by a single system of high affinity. The high affinity uptake of these amino acids in slices of spinal grey matter was approximately 5 times faster than that in slices of spinal white matter. The high affinity uptake systems in the cord slices survived homogenisation of the tissue under conditions known to preserve nerve terminals. Subcellular fractionation studies indicated that osmotically-sensitive particles of equilibrium density equivalent to that of 1.0 m -sucrose were at least in part responsible for the uptake of these amino acids. Inhibition studies indicated that three structurally specific systems of high affinity transported these amino acids:

• 1 specific for glycine—not inhibited by GABA or any of the other depressant amino acids found in cat spinal cord;
• 2 specific for GABA—not inhibited by glycine, taurine, l -aspartate or l -glutamate and (3) specific for l -aspartate and l -glutamate—not inhibited by glycine or GABA but strongly inhibited by various acidic amino acids such as l -cysteate and l -cysteine sulphinate.

The high affinity uptake of these amino acids was not inhibited by any of the known antagonists of the postsynaptic actions of these amino acids—strychnine (glycine), bicuculline and benzyl penicillin (GABA), methioninesulphoximine and l -glutamate diethyl ester (l -aspartate and l -glutamate). p-Chloromercuriphenylsulphonate strongly inhibited the high affinity uptake of glycine and GABA but was much less effective as an inhibitor of l -aspartate/l -glutamate high affinity uptake. This is in good agreement with microelectrophoretic studies in which this mercurial was found to potentiate depression of neuronal firing induced by glycine and GABA much more readily than excitation induced by l -aspartate or l -glutamate. These findings suggest the importance of high affinity transport processes in the removal of amino acids from the synaptic environment.     

THE DISTRIBUTION OF GLYCINE, GABA, GLUTAMATE AND ASPARTATE IN RABBIT SPINAL CORD, CEREBELLUM AND HIPPOCAMPUS

The distribution of glycine, GABA, glutamate and aspartate was measured among about 60 subdivisions of rabbit spinal cord, and among the discrete layers of cerebellum, hippocampus and area dentata. A more detailed mapping for GABA was made within the tip of the dorsal horn of the spinal cord. Spinal ventral horn and dorsal root ganglion cell bodies were analyzed for the amino acids and for total lipid. The distribution of lipid and lipid-free dry weight per unit volume was also determined in spinal cord. Calculated on the basis of tissue water, glycine in the cord is highest in lateral and ventral white matter immediately adjacent to the ventral grey. The distribution of GABA is almost the inverse of that of glycine with highest level in the tip of dorsal horn. It is most highly concentrated in the central 75% of Rexed layers III and IV. Aspartate in the tip of ventral horn is 4-fold higher than in the tip of the dorsal horn and 3 times the average concentration in brain. Glutamate was much more evenly distributed and is relatively low in concentration with slightly higher levels in dorsal than in ventral grey matter. Large cell bodies in both ventral horn and dorsal root ganglion contained high levels of glycine. As reported by others, GABA was found to be high in cerebellar grey layers, area dentata, and regio inferior of hippocampus. Glycine was moderately high in cerebellar layers but moderate to low in hippocampus and area dentata.     

甘氨酸和γ-氨基丁酸对小鼠游离脊髓的初级传入终末兴奋性的作用

本工作在正常离体小鼠(天龄10—15d)脊髓进行。实验结果表明:电刺激邻近记录电极的背根,微电泳GABA及GABA的协同剂Thip、Thiomuscimol和甘氨酸(Glycine)均能引起小鼠脊髓单一初级传入纤维终末的兴奋阈值下降,兴奋性增高,说明终末发生了去极化的变化。同时电泳荷包牡丹碱(Bicuculline)能逆转GABA及其协同剂的去极化作用,但对Glycine的去极化作用无效。而士的宁(Strychnine)能逆转Glycine的去极化作用,对GABA的去极化作用无效。说明在小鼠脊髓初级传入终末存在GABA_A受体及Glycine受体,而且在传入终末区Glycine受体类型可能与脊髓内其它部位的相同。     

褪黑素对谷氨酸致痫大鼠海马内谷氨酸、GABA及其受体水平的影响

目的探讨褪黑素(melatonin,MT)对谷氨酸(glutamate,Glu)致痫大鼠海马内Glu及GluR2、γ-氨基丁酸(γ-aminobutyric-acid,GABA)及其受体GABRA1水平的影响,进而研究褪黑素的抑痫作用机制。方法随机将健康SD雄性大鼠40只分为A、B、C、D组,每组10只。A组:生理盐水组;B组:MT Glu组;C组:Glu致痫组;D组:Luzidole MT Glu组。观察并记录行为学变化,采用免疫组化法进行Glu、GluR2、GABA和GABRA1免疫组化染色和图像分析。结果行为学观察结果显示,C组和D组大鼠均有不同程度的癫痫发作,B组大鼠癫痫发作不明显,A组无发作;免疫组化结果显示,C组和D组海马内CA1-CA3区和齿状回Glu阳性反应较A组增强(P<0.05),GluR2、GABA和GABRA1均较A组减弱(P<0.05),B组Glu较C组和D组阳性反应有显著性减弱(P<0.05),GluR2、GABA和GABRA1阳性反应均较C组和D组有显著性增强(P<0.05),而B组与A组无明显差异性。结论MT通过增加GABA及其受体GABRA1和GluR2的作用和抑制Glu作用对Glu致痫大鼠癫痫发作发挥抑制作用。     

甾体激素对C6细胞摄取甘氨酸的快速作用

目的 :探讨甾体激素对C6细胞摄取甘氨酸快速作用的非基因组织机制。方法 :应用液体闪烁技术 ,通过检测C6细胞在加入了甾体激素和 /或其它试剂后摄入标记甘氨酸量的改变 ,确定甾体激素的作用。结果 :C6细胞高亲合力的甘氨酸依赖于钠离子和氯离子。皮质酮 ,孕酮 ,地塞米松可快速抑制这种摄取 ,雌二醇 ,脱氧皮质酮无显著的抑制作用 ,表明甾体激素作用有特异性。皮质酮的作用在 10 4 -8～ 10 -6 mmol/L范围内效应与浓度成正相关。皮质酮偶联牛血清蛋白后作用依然存在。RU38486 能部分阻断皮质酮的效应。细胞外液钙离子缺乏时皮质酮的作用基本消失。结论 :虽然皮质酮 ,孕酮 ,地塞米松神经胶质细胞和神经元摄取甘氨酸的快速作用不一样 ,但均是非基因组机制。     

家鸽血管活性肠肽阳性神经元在顶盖和峡核中的分布及形态特征

用免疫细胞化学技术研究了血管活性肠肽(VIP)阳性神经元在家鸽(Columba livia)视顶盖和峡核中的分布,实验结果表明.在顶盖中VIP阳性神经元主要分布在Ⅱ_1层.并有少数VIP阳性神经元位于Ⅱ_1和Ⅲ层,在峡核大细胞部(Ime)和峡核小细胞部(Ipc),VIP阳性神经元基本呈均匀分布,在Imc和Ipc内分别占细胞总数的80%和50%.     

兔孤束核腹外侧区微量注射谷氨酸钠及甘氨酸对膈神经放电的影响

实验在４０只麻醉、制动、断双侧颈迷走神经和人工通气的家兔上进行。在孤束核腹外侧区微量注射神经元胞体兴奋剂谷氨酸钠和抑制剂甘氨酸,探讨膈神经放电的变化。结果：微量注射谷氨酸钠,可使膈神经放电脉冲数明显增加,吸气时程延长,呼气时程缩短,呼吸频率变化不明显;微量注射甘氨酸,则膈神经放电脉冲数显著减少,甚至停止,吸气时程缩短,呼气时程不规则延长,呼吸频率降低。上述结果提示：孤束核腹外侧区对呼吸节律的形成具     

GABA UPTAKE IN RAT CENTRAL NERVOUS SYSTEM: COMPARISON OF UPTAKE IN SLICES AND HOMOGENATES AND THE EFFECTS OF SOME INHIBITORS

Abstract— Fifty-two substances were tested as inhibitors of the uptake of [³H]GABA in slices of rat cerebral cortex. Among GABA analogues tested, only the 2-fluoro, 3-hydroxy and 2-amino compounds had affinities for the uptake mechanism comparable to that of GABA. [³H]GABA uptake was also potently inhibited by p -chloromercuriphenylsulphonate, N -ethylmaleimide, chlorpromazine and haloperidol. No inhibitors were found to act in a competitive manner with respect to GABA. [³H]GABA uptake was also examined in homogenates of cerebral cortex and other regions of CNS. There was a rapid uptake of [³H]GABA into particles when homogenate samples were incubated with the labelled amino acid; this uptake had similar kinetic properties and inhibitor sensitivity to that observed in slices of intact tissue. Density gradient centrifugation experiments indicated that the particles responsible for the uptake of [³H]GABA in homogenates were probably synaptosomes. Uptake of [³H]GABA also occurred in slices and homogenates of rat spinal cord, and evidence was obtained by the simultaneous labelling of homogenates with [¹⁴C]glycine and [³H]GABA that these two amino acids were taken up by different nerve terminals in this region.     

EFFECTS OF POTASSIUM AND GLUTAMATE ON BRAIN CORTEX SLICES: UPTAKE AND RELEASE OF GLUTAMIC AND OTHER AMINO ACIDS

Abstract— Effects of an increased concentration of K⁺ (55 m m ) in the medium on fluxes of glutamate and other amino acids in the presence and absence of 10 m m -glutamate were studied. The following observations were made:
(1) The efflux of glutamate is slightly increased by excess K⁺. The glutamate efflux is smaller than the potassium fluxes.
(2) The K⁺-induced increase of glutamate efflux is enhanced under anoxia or in glutamate-containing media.
(3) The influx of glutamate is unaffected or slightly increased by excess K⁺.
(4) The efflux of GABA is increased by excess K⁺, both in the absence and in the presence of glutamate.
(5) Efflux of glutamine, leucine and lysine is increased by excess K⁺, but only provided that glutamate is also present in the medium.
(6) Efflux of glutamate and of GABA is increased by addition of 10 m m -glutamate.     

CHOLINE ACETYLTRANSFERASE AND THE HIGH AFFINITY UPTAKE OF CHOLINE IN CORPUS STRIATUM OF RESERPINISED RATS1

Rats treated with reserpine show increased V_max for the high affinity uptake of choline into small slices of corpus striatum. The choline acetyltransferase activity of whole homogenates of striatum is also increased. These changes are consistent with increased cholinergic neuronal activity in the striatum and seem likely to be adaptations mediating increased rates of synthesis of acetylcholine. The maximal increases found occurred concurrently, consistent with coupling of the high affinity uptake of choline and its acetylation in cholinergic nerve terminals of the rat. That increased high affinity uptake is accompanied by increased choline acetyltransferase activity, suggests the input of choline is not the sole determinant of rates of synthesis of acetylcholine, in spite of the large Vmas for striatal choline acetyltransferase, compared with that for high affinity uptake. These results seem best explained by kinetic coupling, in the rat, of the high affinity uptake of choline with a limited pool of choline acetyltransferase preferentially localised at the nerve terminal plasma membrane.     

EFFECTS OF DI-ISOPROPYLFLUOROPHOSPHATE ON THE METABOLISM OF CHOLINE AND PHOSPHATIDYLCHOLINE IN RAT BRAIN

—The subcutaneous administration of 2·0 mg DFP per kg to rats causes a diminution in the lysophosphatidylcholine content in the brain, which is followed by a decrease of glycerylphosphorylcholine concentration and by a reduced post mortem choline increase. This supports the hypothesis that a post mortem increase in choline is due to phosphatidylcholine breakdown. Since the amount of phosphatidylcholine in brains of di-isopropylfluorophosphate-treated rats increases, it is concluded that phospholipase A is inhibited by di-isopropylfluorophosphate, which corresponds to findings of other authors in vitro. The activity of glycerylphosphorylcholine diesterase (EC 3.1.4.2) is not altered.     

干旱、盐和低温胁迫对水稻幼苗脯氨酸含量的影响

以三叶期的水稻幼苗为材料,研究了干旱（－０．６ＭＰａ ＰＥＧ模拟）、盐（０．１５ｍｌ／Ｌ ＮａＣｌ）和低温（６℃）胁迫下,不同水稻品种脯氨酸积累的变化。结果表明,干旱、盐和低温胁迫下稻苗均可积累脯氨酸,且随着胁迫时间的处长而加剧。在同一胁迫条件下,耐性强的品种脯氨酸积累较少,而敏感品种脯氨酸积累则较多。脯氨酸的积累不宜作为稻苗抗逆性的筛选指标。     

MULTIPLE FORMS OF CHOLINE ACETYLTRANSFERASE AND THE HIGH AFFINITY UPTAKE OF CHOLINE IN BRAIN OF DEVELOPING AND ADULT RATS

The multiple molecular forms of choline acetyltransferase (ChAT) were analysed during the postnatal development of rat brain. Changes in the sodium-dependent, high affinity uptake of [³H]choline (HAUC) and in the efficiency of conversion of labelled choline into ACh in vitro were also examined. Both mature and 7-day old brain contained three molecular forms of ChAT, with isoelectric points of pH 7.3, 7.9 and 8.3, but the immature brain appeared to contain smaller concentrations of the most basic form of the enzyme (pI = 8.3). Of the total choline uptake measured in slices of frontal cortex, adult samples exhibited a greater proportion of HAUC than 7-day samples and appeared to acetylate more efficiently the [³H]choline accumulated by high affinity uptake. This evidence suggests a basic molecular form of ChAT, appearing in rat brain during postnatal development, might be responsible for the efficient coupling of the high affinity uptake and subsequent acetylation of choline in cholinergic nerve terminals.     

SYNAPTOSOMAL TRANSPORT: A CHLORIDE DEPENDENCE FOR CHOLINE, GABA, GLYCINE AND SEVERAL OTHER COMPOUNDS

Abstract— The synaptosomal transport of fourteen different neurotransmitters, neurotransmitter precursors and other amino acids was tested for a chloride dependence. Those compounds having sodium-dependent'high-affinity'transport systems exhibited a chloride dependence which was maintained over time and which was concentration dependent. Low chloride concentrations altered the transport kinetics of the compounds examined. The reduced accumulation of these compounds in chloride deficient media was not due to increased efflux or alterations in metabolism. The significance of these results for models of transport, methods in transport studies and for other factors are discussed.     

神经毒素ECMA对家鸽视网膜神经节单元反应特性的影响

从家鸽视差表层总共记录了１０１个视网膜神经节单元,并定量分析研究ＥＣＭＡ损伤对其反应特性的影响,在对照组中,神经节单元都没有自发放电,而需要视觉刺激才能引起反应,对闪光刺激的反应,分别为ＯＮ—ＯＦＦ,ＯＮ,ＯＦＦ三种,其反应均是瞬变的,而且也都对在感受野内运动的小条纹起反应。４２个单元中有１４个是方向选择性单元。其它的则为运动敏感单元。方向选择性单元的无效方向不是均等分布的,其中有８个单元的无效是从前向后的,但没有发现其无效方向是从后向前的单元。与对照组相比,经ＥＣＭＡ损伤后的实验组中只记录到ＯＮ－ＯＦＦ反应和ＯＮ反应单元,未能找到单独的ＯＦＦ反应单元。神经节单元的ＯＮ反应部分为持续成分。所有的单元对运动条纹刺激都失掉了方向选择性,这些现象的机理可能是由于ＥＣＭＡ去除了胆碱能无足细胞所致。     

ACETYLCHOLINE METABOLISM AND CHOLINE UPTAKE IN CORTICAL SLICES

Abstract— The uptake of [¹⁴C]choline was studied in cortical slices from rat brain after their incubation in a Krebs-Henseleit medium containing either 4.7 m m -KCl (low K), 25 m m -KCl (high K) or 25 m m -KCl without calcium (Ca free, high K). With 0.84 μ m -[¹⁴C]choline in the medium the uptake per gram of tissue was 0.62 nmol after incubation in low K medium, 1.13 nmol after incubation in high K medium and 0.78 nmol after incubation in a Ca free, high K medium. The differences caused by potassium were greater in fraction P₂ than in fractions P₁ and S₂. With 17 and 50 μ m -[¹⁴C]choline in the medium greater amounts of [¹⁴C]choline were taken up, but the effect of potassium on the uptake almost disappeared. The amount of radioactive material in fraction P₂ followed Michaelis-Menten kinetics with K _m values of 2.1 and 2.3 μ m after incubation in low and high K medium, respectively. Hemicholinium-3 only slightly inhibited choline uptake from a medium with 0.84 μ m -[¹⁴C]choline, but it abolished the extra-uptake induced by high K medium. The radioactivity in the slices consisted mainly of unchanged choline and little ACh was formed after incubation in low K medium, but after incubation in high K medium 50% of the choline taken up was converted into ACh. The hemicholinium-3 sensitive uptake of choline, the conversion of choline into ACh and the synthesis of total ACh, were stimulated about 7–8-fold by potassium. It is concluded that in cortical slices from rat brain all choline used for the synthesis of ACh is supplied by the high-affinity uptake system, of which the activity is geared to the rate of ACh synthesis.     

THE EFFECTS OF SOME NEWER ANAESTHETICS ON THE IN VITRO ACTIVITY OF GLUTAMATE DECARBOXYLASE AND GABA TRANSAMINASE IN CRUDE BRAIN EXTRACTS AND ON THE LEVELS OF AMINO ACIDS IN VIVO

—The effects of several anaesthetic and hypnotic compounds with well-defined excitatory side-effects on glutamate decarboxylase and γ-aminobutyric acid transaminase activity have been examined. The dissociative anaesthetics ketamine and γ-hydroxybutyric acid produced competitive inhibition of glutamate decarboxylase with respect to glutamate at concentrations which had no effect on GABA transaminase activity. The inhibitor constant (K_i) values were, ketamine: 13.3 mm , γ-hydroxybutyric acid; 8.8 mm . The steroid anaesthetic alphaxalone was also a potent competitive inhibitor of glutamate decarboxylase K_i= 4.1 mm ). Pentobarbitone, thiopentone and methohexitone non-competitively inhibited both glutamate decarboxylase and GABA-transaminase but only at high concentration (> 20 mm ). None of the drugs tested produced any significant change in brain GABA or glutamate levels following the injection of an hypnotic or anaesthetic dose. It is proposed that an alteration in the rate of GABA synthesis as a result of the inhibition of glutamate decarboxylase could explain the convulsive properties of the dissociative anaesthetics when given at high doses.