GABA FLUXES IN PRESYNAPTIC NERVE ENDINGS FROM IMMATURE RATS

Abstract— Several parameters of GABA Auxes across the synaptosomal membrane were studied using synaptosomes prepared from the brain of immature (8-day-old) rats. The following aspects of GABA carrier-mediated transport were similar in immature and mature synaptosomes: (1) magnitude of [³H]GABA accumulation; (2) GABA homoexchange in normal ionic conditions; (3) GABA homoexchange in the presence of cationic fluxes (Na⁺ and Ca²⁺ influx, K⁺ efflux) characteristic of physiological depolarization. As in adult synaptosomes (Levi & Raiteri , 1978), in these conditions the stoichiometry of GABA homoexchange was in the direction of net outward transport (efflux > influx). The essential differences between the behaviour of 8-day-old and adult synaptosomes were the following: (1) β-alanine (a glial uptake inhibitor) inhibited GABA uptake in immature synaptosomes (the inhibition being greater in crude than in purified preparations) and was without a significant effect in adult synaptosomes. DABA and ACHC (two neuronal uptake inhibitors) depressed GABA uptake more efficiently in purified than in crude immature synaptosomes, but were as effective in crude and purified nerve endings from adult animals. The data suggest a greater uptake of GABA in the‘gliosomes’contaminating the synaptosomal preparations from immature animals. (2) In immature synaptosomes prelabelled with [³H]GABA the specific radioactivity of the GABA released spontaneously or by heteroexchange (with 300 μm -OH-GABA) was the same as that present in synaptosomes, while in adult synaptosomes OH-GABA released GABA with increased specific radioactivity. The data suggest a homogeneous distribution of the [³H]GABA taken up within the endogenous GABA pool in immature, but not in mature synaptosomes. (3) In immature synaptosomes the release of GABA (radioactive and endogenous) induced by depolarization with high KC was not potentiated by Ca²⁺, unless the synaptosomes had been previously depleted of Na⁺ These data suggest that, although a Ca²⁺ sensitive pool of GABA may be present, this pool is not susceptible to being released in normal conditions, probably because the high intrasynaptosomal Na⁺ level prevents a sufficient depolarization. The possible significance of these findings in terms of functional activity of GABAergic neurotransmission in the immature brain is discussed.     

LEUCINE OXIDATION IN BRAIN SLICES AND NERVE ENDINGS1

—It is generally believed that leucine serves primarily as a precursor for protein synthesis in the central nervous system. However, leucine is also oxidized to CO₂ in brain. The present investigation compares leucine oxidation and incorporation into protein in brain slices and synaptosomes. In brain slices from adult rats, these processes were linear for 90min and ¹⁴CO₂ production from 0·1 mm -l -[l-¹⁴C]leucine was 23 times more rapid than incorporation into protein. The rate of oxidation increased further with greater leucine concentrations. Experiments with l -[U-¹⁴C]leucine suggested that all of the carbons from leucine were oxidized to CO₂ with very little incorporation into lipid. Oxidation of leucine also occurred in synaptosomes. In slices, leucine oxidation and incorporation into protein were inhibited by removal of glucose or Na⁺, or addition of ouabain. In synaptosomes, replacement of Na⁺ by choline also reduced leucine oxidation; and this effect did not appear to be due to inhibition of leucine transport. The rate of leucine oxidation did not change in brain slices prepared from fasted animals. Fasting, however, reduced the incorporation of leucine into protein in brain slices prepared from young but not from adult rats. These findings indicate that oxidation is the major metabolic fate of leucine in brain of fed and fasted animals.     

PURINE AND PYRIMIDINE NUCLEOTIDES IN THE BRAIN OF NORMAL AND CONVULSANT RATS

Abstract— Purine and pyrimidine nucleotides were measured in the brain of normal and electroshocked rats after chromatographic separation on ion-exchange resin of mono-, di- and tri-phosphorylated derivatives.
CMP, IMP and NAD did not show any significant quantitative change. Adenine nucleotides showed an abrupt change followed by a rapid return to the control value. GTP was the only purine nucleotide exhibiting a relatively slow return to its starting concentration. The greatest percentage increase after electroshock was observed in UMP, which returned to its control value only after 5 min; UDPCoenzymes (i.e. UDPA plus UDPG) showed a relatively small drop during the development of the seizure and the slowest return to the base line; UTP showed a late transistory increase above the normal level after an initial drop associated with convulsant activity.
Tritiated uridine was injected intracisternally to investigate the turnover of pyrimidine nucleotides. UTP showed the highest specific radioactivity at the earliest time, followed by UMP, UDPCoenzymes and CMP. It was found that convulsant activity is associated with dramatic changes in the specific radioactivity of pyrimidine nucleotides.     

A RAPID METHOD FOR SELECTIVE SILVER STAINING OF NERVE FIBRES AND NERVE ENDINGS IN MOUNTED PARAFFIN SECTIONS

Abstract The staining described in this paper fundamentally is performed by two manipulations, namely (1) the treatment of deparaffinized sections for about fifteen minutes in a solution containing 15 per cent silver nitrate, 10 per cent potassium nitrate and 0.05 per cent glycine, and (2) the reduction for one minute in a solution of 1 per cent pyrogallol, 55 per cent ethyl alcohol and a trace (0.002 per cent) of nitric acid. After toning, dehydrating and mounting in the usual manner the sections generally are ready for examinations within less than an hour. The method is available for specimens fixed in the ordinary fixatives but those containing oxidizing metal compounds. The procedure is discussed from a theoretical point of view and some results are shown in photomicrographs.     

GLYCOGEN AND GLYCOGEN PHOSPHORYLASE IN THE CEREBRAL CORTEX OF MICE UNDER THE INFLUENCE OF METHIONINE SULPHOXIMINE

Abstract— Glucose and glycogen levels in the mouse cerebral cortex in vivo were studied after recovery from methionine sulphoximine seizures. The animals appeared normal 24 h after methionine sulphoximine administration but both glucose and glycogen still persisted at higher levels 72 h after injection (by 64 and 275 per cent, respectively). When seizures were prevented by methionine, the increase in glucose and glycogen at the longer time intervals was significantly smaller than in animals treated with methionine sulphoximine only; glucose reached normal values at 48 or 72 h; the accumulation of glycogen was reduced by about three to five times, but after 72 h the levels were still significantly higher than in control animals (67 or 32 per cent increase, depending on the administered dose of methionine). In contrast to the considerable accumulation of glycogen after administration of methionine sulphoximine in vivo, it had no effect on the level of glycogen in brain cortex slices in vitro. After 3 h incubation in the absence of methionine sulphoximine, glycogen was resynthesized to a level of about 4 μmol/g wet tissue and this value was not significantly affected by the presence of various concentrations of methionine sulphoximine in the incubation medium (10^-5 to 10^-2 M). The total (a+b forms) phosphorylase activity of mouse cerebral cortex in vivo after methionine sulphoximine administration was not affected. The fraction of active phosphorylase was reduced by about 50 per cent at the time of seizures. When seizures were prevented by methionine, the decrease in active phosphorylase was also completely prevented. In the preconvulsive period (1-2 h) and after recovery from the seizures (48 h after methionine sulphoximine administration) active phosphorylase was normal. The possible mechanisms involved in the increased accumulation of glycogen after methionine sulphoximine administration are discussed.     

PLURIVESICULAR SECRETORY PROCESSES AND NERVE ENDINGS IN THE PINEAL GLAND OF THE RAT

The pineal body of white normal rats, 1.5 to 3 months old, was studied under the electron microscope. A single type of parenchymal cell—the pinealocyte—is recognized as the main component of the tissue, and some of the structural characteristics of the nucleus and cytoplasm are described. The main morphological characteristic of the pinealocytes is represented by club-shaped perivascular expansions connected to the cell by thin pedicles. They are found lying in a large, clear space surrounding the blood capillaries. The name plurivesicular secretory processes is proposed, to emphasize the main structural feature and the probable function of these cellular expansions. A tubulofibrillar component is mainly found in the pedicle, and within the expansion there are numerous small mitochondria and densily packed vesicles of about 425 A. Two types of vesicles, one with a homogeneous content and another with a very dense osmium deposit, are described. Between the two types there are intermediary forms. In these processes, mitochondria show profound changes which may lead to complete vacuolization. The significance of this plurivesicular secretory component is discussed in the light of recent work on the biogenic amines of the pineal body and preliminary experiments showing the release of the vesicles containing dense granules after treatment with reserpine. These vesicles are interpreted as the site of storage of some of the biogenic amines. Bundles of unmyelinated nerve fibers and endings on large blood vessels which also contain a plurivesicular content are described and tentatively interpreted as adrenergic nerve terminals.     

ULTRASTRUCTURAL AND BIOCHEMICAL CHANGES IN BROWN FAT IN COLD-EXPOSED RATS

During the first 3 days of exposure of rats to 5°C, the nitrogen concentration of interscapular brown fat increased by 50% and remained at this elevated level for the duration of the 8-wk observation period, while the mass of tissue increased fourfold. The concentration of both DNA and RNA per unit nitrogen reached a maximum after 3 days, then declined; however, the total quantity of each continued to rise. The concentration of various respiratory enzymes decreased during the first few days and then increased, but at different rates. The morphological changes in mature brown fat cells during cold acclimation were observed to be: a reduction in fat droplet size during the first 3 days, followed by a gradual increase in size through 6 wk in the cold; a continual increase in the amount of intermitochondrial ground substance during the first 3 wk, with increased granularity and glycogen content after 1 wk; initial disappearance of glycogen between mitochondria, followed by the reappearance of a few isolated particles in the intermitochondrial ground substance after 1 wk in the cold; initial increase in the density of intramitochondrial matrix for the first 3–4 days, followed by a gradual return to the control density; loss in integrity of mitochondrial outer membranes during the first 4 days, followed by gradual but incomplete restoration; temporary loss of the dense material in lipid droplets during the first 24 hr, with return after 1 wk in the cold; and a 40% increase in mitochondrial diameter within 1 day, followed by a decrease in diameter within 1 wk to a constant value about 15% larger than the controls.     

METABOLITES AND ENZYMES OF THE PENTOSE PHOSPHATE PATHWAY IN ISOLATED NERVE ENDINGS1

Abstract— The activities of each enzyme associated with the pentose phosphate pathway as well as the non-enzymatic intermediates in this pathway were measured in synaptosomes isolated from rat cerebral cortex. The specific activities of transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2) were significantly lower in synaptosomes than cerebral cortex; however, the specific activities of glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), ribosephosphate isomerase (EC 5.3.1.6) and ribulosephosphate epimerase (EC 5.1.3.1.) were comparable in homogenates of synaptosomal fractions and cerebral cortex. Concentrations of most intermediates of the pentose pathway were also similar in extracts of synaptosomes and brain homogenates. Six hours after treatment of rats with the nicotinamide analog, 6-aminonicotinamide (6-AN), 6-phosphogluconate levels in synaptosomes were increased 5-fold; however, glucose-6-phosphate levels remained unchanged. During a 30 min in uitro incubation 6-phosphogluconate levels increased approx 2-fold in synaptosomes obtained from 6-AN treated rats but did not change in synaptosomes from untreated rats. During the same period glucose-6-phosphate levels decreased in synaptosomes from both control and 6-AN treated rats. The conversion of both [1-¹⁴C]glucose and [6-¹⁴C]glucose to ¹⁴CO₂ was depressed in synaptosomes from 6-AN treated rats; however, the ratio of the two isotopes converted to ¹⁴CO₂ was essentially the same. It is concluded that the pentose phosphate pathway is active in nerve endings both in vivo and in vitro.     

人肾上腺内肽能神经的超微结构

应用抗生蛋白链菌素一生物素一过氧化物酶复合物的免疫电镜技术,在相邻切片中,分别观察人肾上腺内ＮＰＹ、ＶＩＰ、ＳＰ和ＣＧＲＰ免疫反应神经纤维的超微结构和免疫反应物的定位。ＮＰＹ和ＶＩＰ免疫反应物位于小颗粒羹泡和线粒体外膜,部分嗜铬细胞呈ＮＰＹ免疫反应阳性。ＳＰ和ＣＧＲＰ免疫反应物主要定位于大颗粒囊泡内。ＳＰ和ＣＧＲＰ免疫反应轴突与嗜铬细胞形成对称性或非对称性的轴体突轴。测量了这４种肽能神经末梢的直径,从而推测其来源,并讨论了该４种神经肽对人肾上腺的作用。     

A STUDY OF THE METABOLISM AND RELEASE OF DOPAMINE AND AMINO ACIDS FROM NERVE ENDINGS ISOLATED FROM SHEEP CORPUS STRIATUM

Abstract— Synaptosomes prepared from sheep corpus striatum showed a linear rate of respiration over a 90 min period of incubation in Krebs-bicarbonate medium containing glucose (10 mm ) and the rate of respiration was stimulated by electrical pulses. Dopamine was released from synaptosome beds to the medium by either electrical pulses or 56mm -K⁺ (10min), increasing 108% and 76% respectively above control levels of release. The presence of d- or 1-amphetamine (0.12mm ) in the incubation medium (40 min) increased the accumulation of dopamine in the medium by 310 and 275% respectively and 56mm -K⁺ also caused a significant increase in the release of glutamate, GABA and aspartate. Radioactively labelled dopamine was synthesized by the synaptosomes from l -[¹⁴C]tyrosine, l -DOPA or dl -DOPA, and electrical pulses caused a 35% increase in the rate of dopamine production from [U-¹⁴C] tyrosine. No increased release of [¹⁴C]dopamine in response to depolarizing stimuli was found to occur when synaptosome beds were transferred from medium containing radioactive precursors to fresh medium for further incubation (20 min). In the presence of 1- and d-amphetamine, accumulation of ¹⁴C-labelled doparnine in the incubation media was increased 129% and 380% respectively, the latter was partially depressed by absence of calcium from the medium. Three radioactively labelled metabolites formed by synaptosomes during incubation in dl -[2-¹⁴C]DOPA were detected; the major ones were dihydroxyphenylacetic acid and homovanillic acid and the third was unidentified. When the synaptosome beds were transferred to medium containing no radioactive precursors, it was found that labelled dihydroxyphenylacetic acid was 7 times more abundant than labelled dopamine in the incubation medium (20 min) and one-third as abundant in the synaptosomes. The dihydroxyphenylacetic acid n Ci/dopamine n Ci ratio was greatly affected by K⁺ stimulation, decreasing 52% and 34% in the incubation medium and synaptosomes respectively. A pathway of dihydroxyphenylacetic acid degradation was shown to occur through decarboxylation. These results are discussed in terms of the compartmentation of dopamine and its metabolism. It is proposed that one pool of dopamine is released by depolarizing agents and during the period of incubation it is replaced by synthesis from the endogenous tyrosine (19.5 nmol/100 mg protein) and not by the labelled dopamine in the synaptosome. The synaptosomal pool of dopamine which is radioactively labelled after pulse labelling with dl -[2-¹⁴C]DOPA appears to be prone to oxidation to DOPAC and homovanillic acid which are preferentially released from the synaptosomes.     

PHOSPHOINOSITIDE KINASES IN CHICK BRAIN AND SCIATIC NERVE, A DEVELOPMENTAL STUDY

Phosphatidylinositol kinase and diphosphoinositide kinase activities were measured in homogenates of brain and sciatic nerve of developing chick embryos and chicks. Characteristics of the chick nervous system enzymes were similar to those reported for rat brain. Diphosphoinositide kinase was inhibited by high concentrations of ATP and by low concentrations of triphosphoinositide. Both activities were greatly enhanced by the non-ionic detergent, Cutscum, and the ratio of detergent to protein in the reaction mixture was important. Optimum phosphatidylinositol kinase activity required a ratio of 7 : 1 for both tissues. The optimum ratio for diphosphoinositide kinase was 3:1 for nerve homogenates and 0.6:1 for brain. Cutscum increased the concentration of diphosphoinositide that is required for maximum diphosphoinositide kinase activity. Developmental changes were the same for both kinase activities, which were low in unmyelinated brain and sciatic nerve. The activities correlated with the concentration of polyphosphoinositides in chick brain where they increased 4-5 fold during the period of active myelination and remained high in the mature brain. The kinase activities correlated with the rate of triphosphoinositide deposition in sciatic nerve. Following a 2-3 fold increase during the initial phase of myelination the activities declined to values as low as those of embryonic nerve.     

ULTRASTRUCTURAL INTERRELATIONSHIPS OF NERVE PROCESSES AND SMOOTH MUSCLE CELLS IN THREE DIMENSIONS

The muscularis externa of the intestinal wall of frogs was fixed in osmium tetroxide, embedded in Vestopal-W, serially sectioned for electron microscopy, and stained with uranyl acetate. A method to obtain individually mounted and properly positioned serial sections is described. The three-dimensional techniques used during the course of this investigation demonstrate that it is possible to examine carefully relatively large areas of tissue on individual serial sections with the electron microscope and subsequently to construct montages of electron micrographs of pertinent areas from each section. Several carefully rendered interrelationships of nerve processes and smooth muscle cells in three dimensions are exhibited and described. Recent studies of other neuro-effector relationships are discussed in relation to the present status of the nature and organization of the autonomic nervous system in visceral organs.     

青年猫和老年猫视神经超微结构观察比较

目的探讨青年猫和老年猫视神经年龄相关的形态学变化及可能造成的生理影响。方法取4只青年猫(2-3岁,2-2.5kg)和4只老年猫(10-13岁,2.5-3.5kg)颅内相对应部分视神经,制作横向半薄切片和超薄切片,半薄切片用甲苯胺蓝硼砂溶液染色,光镜观察、测量视神经的直径(不含外层神经膜);超薄切片标本用醋酸和柠檬酸铅染色,电镜观察、计数视神经纤维密度、测量视神经纤维外径D(含髓鞘)和内径d(不含髓鞘),按一定分级范围算出各种直径的纤维及各种d/D比值的纤维所占百分比,分别画出直方图,对实验结果进行统计学分析并绘制纤维直径谱。结果与青年猫相比,老年猫视神经直径显著增大(P0.05);纤维数量显著下降(P0.05)。纤维直径谱分析结果显示,青、老年猫纤维直径分布范围相似,但老年猫纤维的峰直径及纤维平均直径比青年猫的显著减小(P0.05),老年猫视神经纤维的d/D比值亦明显降低。另外,老年猫视神经中部分轴突肿胀,髓鞘疏松、结构紊乱,板层脱离、空泡化,有的轴索髓鞘溶解。结论在衰老过程中,老年猫视神经纤维丢失,纤维直径减小,d/D比值下降,以及纤维髓鞘的松散解体,这些变化均可能导致视神经纤维对视觉信息的传导速度减慢,是老年个体视觉分析速度下降的重要原因。     

PHOSPHOINOSITIDES AND OTHER PHOSPHOLIPIDS IN SYMPATHETIC GANGLIA AND NERVE TRUNKS OF RATS

Abstract— Paired vagus nerves, phrenic nerves or superior cervical sympathetic ganglia from adult white rats were incubated for 4 h at 37°C in a bicarbonate-buffered physiological solution containing glucose and ³²P₁. At the end of incubation triphosphoinositide (TPI) contained more ³²P than any other lipid in the vagus nerves and was second only to phosphatidylcholine (PC) in the phrenic nerves. In the sympathetic ganglia phosphatidylinositol (PI) contained more ³²P than did TPI, but both had less than PC. Conducted nerve impulses, initiated by electrical stimulation during the final 3 h of incubation, caused a highly significant increase in the [32P]-labelling of PI in ganglia (as previously reported) probably decreased the labelling of TPI in the vagus nerves, and decreased the labelling of phosphatidylethanolamine (PE) in the ganglia. Addition to the incubation medium of §- or γ-hexachlorocyclohexane (analogs of inositol) reversibly blocked transmission through the sympathetic ganglia at concentrations less than 0·1 mM. The §-isomer also blocked conduction along axons at similar concentrations; only the γ-isomer (lindane) exerted a selective effect on synaptic transmission. In the ganglia, the §-isomer increased the [³²P]-labelling of PI and diphosphoinositide (DPI) relative to that of PC. The γ-isomer did not affect the relative labelling of PI in the ganglia, whereas it decreased that of TPI, but only at relatively high concentrations. Thus, various affects of the hexachlorocyclohexanes were not explicable by assuming that they acted as analog inhibitors of inositol metabolism. In the ganglia, the hexachlorocyclohexanes reduced the effect of neuronal activity on the labelling of PI in proportion to the extent by which they blocked transmission. This metabolic effect was therefore presumed to be secondary to a ganglionic blocking action.     

ULTRASTRUCTURAL TRANSFORMATION IN MITOCHONDRIA ISOLATED FROM KIDNEYS OF NORMAL AND LEAD-INTOXICATED RATS

Mitochondria isolated from kidneys of lead-intoxicated rats have been shown to have decreased oxidative and phosphorylative abilities. The purpose of this study was to determine whether these abnormal mitochondria would undergo ultrastructural transformation during controlled respiration in the absence of phosphate acceptor (State IV), as previously demonstrated for normal liver mitochondria. It was first shown that normal rat kidney mitochondria transforms from a condensed ultrastructural conformation to an orthodox conformation after 5 min of State IV respiration with pyruvate-malate substrate. Reversal to a condensed conformation follows stimulation of respiration with adenosine diphosphate (ADP). A large portion of kidney mitochondria from lead-poisoned rats do not change from condensed to orthodox conformation during State IV respiration. Other mitochondria do transform to the orthodox form but they rapidly degenerate. State IV respiration decreases as these few orthodox mitochondria disintegrate. The conclusion is that those mitochondria that do not undergo change in ultrastructure have impairment of electron transport, and that those that do become orthodox have increased membrane lability and undergo degeneration.