Nervous System-Specific Proteins in Developing Rat Cerebral Cells in Culture

Abstract: The nervous system-specific proteins: synaptin, D1, D2, D3, glial fibrillary acidic protein (GFA) and 14-3-2, were quantified in dissociated cerebral cells from the foetal rat brain at various times of growth in culture. By approximately 1 week in culture, the neuronal membrane markers synaptin, D1, D2, and D3 could all be demonstrated. A maximum concentration of 10–20% for synaptin, D1, and D3 and 160% for D2, in comparison with the levels in adult forebrain, was attained during the 2nd week in vitro. The astroglial gliofilament marker GFA increased continuously, reaching by 38 days of cultivation an 18-fold higher level than the concentration in adult forebrain. The neuronal cytoplasm marker 14-3-2 could be demonstrated in trace amounts, and only after more than 1 week in vitro. Neuronal cell bodies and processes stained by indirect immunofluorescence using an anti-D2 serum were strongly fluorescent after 1 week in vitro. Immunofluorescence staining for GFA revealed a cytoplasmatic filamentous network in perinuclear areas and processes of, presumably, astroblasts.     

BRAIN-SPECIFIC ANTIGENS IN THE QUAKING MOUSE DURING ONTOGENY

Abstract— By means of crossed Immunoelectrophoresis the concentrations of 7 brain-specific antigens have been investigated during the ontogenic development of normal and Quaking mice. Two proteins, the glial fibrillary acidic protein and the brain-specific membrane protein D5 were found to be strongly increased in mutant brains. The synaptosomal antigen synaptin (Cl), the 14-3-2 protein of neuronal cytoplasm, and the neuronal membrane antigens D1, D2 and D3 were all present at normal levels in mutant brains.     

Properties of phosphate activated glutaminase in astrocytes cultured from mouse brain

Astrocytes in primary cultures contain a relatively high activity, of phosphate activated glutaminase, although it is significantly lower than that of synaptosomal enriched preparations. The relatively high glutaminase activity in the astrocytes appears not to be caused by substrate induction, since a 10-fold variation in the glutamine concentration of the culture medium does not affect the activity. Of the reaction products, only glutamate inhibits astrocytic glutaminase whereas that of synaptosomal enriched preparations is inhibited by both glutamate and ammonia. Similar to the synaptosomal enzyme, glutaminase in astrocytes is inhibited about 50% by N-ethylmaleimide, indicating N-ethylmaleimide-sensitive and-insensitive compartments of the enzyme. Calcium activates glutaminase in astrocytes as in synaptosomes, by promoting phosphate activation. Except for the lower activity and the lack of effect of ammonia, the properties of the astroglial glutaminase has been found to be no different from that of the synaptosomal one. The relatively unrestrained astroglial glutaminase may, however, argue against the concept of a glutamine cycle operating in a stoichiometric manner.Abbreviations NEM N-ethylmaleimide - PAG Phosphate-activated glutaminase - PMB p-mercuribenzoate     

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.     

SUBCELLULAR DISTRIBUTION AND SOME PROPERTIES OF ACETYL-COENZYME A HYDROLASE IN THE BRAIN

—The detailed subcellular distribution and some properties of acetyl-CoA hydrolase were studied in the rat brain. The brain homogenate (S₁) hydrolysed acetyl-CoA at a rate of approx 2·3 nmol/min/mg of protein at 37°C. The total activity of acetyl-CoA hydrolase was distributed in the following order: soluble > mitochondrial > microsomal, synaptosomal > myelin fraction. The order of the specific activity of the enzyme was: soluble, microsomal > mitochondrial > synaptosomal > myelin fraction. The synaptic vesicle fraction (D) had relatively high specific activity among the intraterminal particulate fractions, having two or three times higher specific activity than that of the synaptic cytoplasmic membrane fraction (F or G). Attempts to de-occlude acetyl-CoA hydrolase in the particulate fraction showed that only the enzyme activity in the myelin fraction was increased markedly by the treatment with ether or Triton X-100. Lineweaver-Burk plots gave straight lines for each subcellular fraction and apparent K_m values for acetyl-CoA were between 0·1 and 0·2 mM. Neither diisopropyl fluorophosphate nor physostigmine at the concentration of 0·1 mm inhibited the enzyme activity.     

INCORPORATION OF AMINO ACIDS INTO PROTEINS IN NEURONAL AND NEUROPIL FRACTIONS OF RAT CEREBRAL CORTEX: PRESENCE OF A RAPIDLY LABELLING NEURONAL FRACTION

Abstract— The time course of incorporation of intraperitoneally injected [³H]lysine and [¹⁴C]phenylalanine into neuronal and neuropil proteins has been followed for up to 8 days. At short times after injection (<2 h) the specific activity of the neuronal fraction was higher than that of the neuropil. At longer time intervals, although the total brain specific activity continued to rise, neuronal perikaryal specific activity fell below that of neuropil. Thus the neuronal/neuropil incorporation ratio with [³H]lysine as substrate was 1·5 at 1 h, but by 4 h had fallen to 0·4, a ratio which was maintained for up to 8 days. A similar reversal occurred with phenylalanine as substrate. These changes were interpreted as evidence for the presence of a rapidly-labelling protein fraction in the neurons which is subsequently transported out. Subcellular fractionation showed that over the 4 h period the rapidly labelling fraction was not transported to the synaptosomes. Incubation of prelabelled cortex slices followed by cell fractionation showed that a differential transport of protein of higher than average specific activity from both neurons and neuropil fractions occurred; there is a tendency for preformed highly labelled protein to accumulate during the in vitro incubation in Fraction D, a pellet enriched in red cells, some large neuronal perikarya and cell nuclei. When cell fractions were prepared after in vitro incubation, the distribution of the material down the gradient differed from that when fresh tissue was fractionated, as demonstrated by microscopic examination and the distribution of β-galactosidase, a neuronal marker. Double-label experiments showed that this redistribution could not account for the preferential loss and accumulation of prelabelled protein. It was noted that in vivo incorporation into the rapidly labelling neuronal protein is suppressed under certain changed environmental conditions, such as dark rearing. This is interpreted as lending support to the concept of the state-dependence of neuronal and neuropil protein synthesis and their inter-relations.     

ACCELERATION OF CHOLINE UPTAKE AFTER DEPOLARIZATION-INDUCED ACETYLCHOLINE RELEASE IN RAT CORTICAL SYNAPTOSOMES

Abstract— Activation of nerve elements in vivo and in vitro is associated with an increased rate of choline uptake by a Na⁺-dependent high affinity transport system. Following the methodology of B arker (1976), rat cortical synaptosomes were depolarized (37°C, 10min) by 25mM-KCl in the presence of CaCl₂ (1 mM) or other divalent cations. After reisolation by centrifugation, the rate of ³H-choline uptake (1.25μM) was measured by Millipore filtration. KCl treatment alone failed to accelerate the rate of uptake in the reisolated synaptosomes. CaCl₂, BaC1₂ or SrCl₂ (but not MgCl₂ or MnCl₂) were necessary (1 mM) to observe the KCl induced acceleration. Moreover, RbCl, but not LiCl or CsCl, also produced the calcium-dependent rate enhancement in the reisolated synaptosomes. The conditions mediating the enhanced rate of choline uptake correlated strongly with those associated with neurotransmitter release. To test this possibility, synaptosomal acetylcholine content was measured in response to the various salt treatments. Treatment with KCI (25 mM) and CaCl₂ (1 mM), but not KCl alone, reduced the synaptosomal acetylcholine content from 154 to 113pmol/mg protein. The respective rates of choline uptake increased about 60%. The increased rate was reversed by incubation with 50 μM-choline followed by synaptosome reisolation. This procedure also normalized the acetylcholine content. In summary, the rate of choline uptake by the high affinity choline uptake system is inversely related to the synaptosomal acetylcholine content.     

UPTAKE OF [14C]ASPARTIC ACID AND [14C]GLUTAMIC ACID BY RETINAL SYNAPTOSOMAL FRACTIONS

Abstract— Uptake systems for [¹⁴C]aspartate and [¹⁴C]glutamate were characterized in two distinct synaptosomal fractions solated from rabbit retina. The P, synaptosomal fraction was highly enriched in large photoreceptor cell synaptosomes but contained very few conventional sized synaptosomes from amacrine, horizontal or bipolar cells. In contrast, the P₂ synaptosomal fraction contained numerous conventional sized synaptosomes and was virtually free of photoreceptor cell synaptosomes. Both synaptosomal fractions took up [¹⁴C]aspartate and [¹⁴C]glutamate with high affinity [ K _m= 1–2μM). Uptake characteristics were similar to those described for high affinity uptake systems in brain synaptosomes, i.e. saturation kinetics; temperature and Na⁺ dependence. Although the presence of a high affinity uptake system is not a definitive criterion for demonstration of functional neurotransmitter systems, it is an important and necessary prerequisite and can thus be considered as supportive evidence for the involvement of asparate and glutamate in neurotransmission in rabbit retina.     

Preparation of chick brain synaptosomes and synaptosomal membranes.

A method is described for the preparation of synaptosomes and synaptosomal membranes from chicken brain. Procedures for isolating rat synaptosomal membranes could not be used directly; several modifications of existing procedures are reported. Purity of the subcellular and subsynaptosomal fractions was monitored by electron microscopy and measurements of ferrocytochrome c: oxygen oxidoreductase (EC 1.9.3.)), monoamine: oxygen oxidoreductase (deaminating) EC 1.4.3.4), rotenone-insensitive NADH: cytochrome c oxidoreductase (EC 1.6.99.3), NADPH: cytochrome c oxidoreductase (EC 1.6.99.1), orthophosphoric monoester phosphohydrolase (EC 3.1.3.2), ATP phosphohydrolase (EC 3.6.1.4), and levels of RNA. Microsomes are the main contaminant of the synaptosomal membrane fraction. Mitochondrial and lysosomal enzymes occur in lesser amounts. No myelin contamination was observed. Marker enzymes for contaminants suggest that these synaptosomal membranes are as pure as membranes described by others, and the specific activity of a neuronal membrane marker, (Na+ -K+)-activated ATPase, is as high as other preparations. Levels of this enzyme in the membrane fraction are enriched 13-fold over homogenate ATPase levels.     

MEMBRANE AFFINITIES AND SUBCELLULAR DISTRIBUTION OF THE DIFFERENT MOLECULAR FORMS OF CHOLINE ACETYLTRANSFERASE FROM RAT

Choline acetyltransferase from rat brain is present in three different molecular forms with isoelectric points at pH 7·4-7.6, 7·7-7·9 and 8·3. The three forms were identified in a highly purified enzyme preparation, in a preparation of synaptosomes and in a cyto-plasmic preparation from disrupted axons and perikarya (fraction S₃). The three molecular forms differed in their affinities for synaptosome membranes and for a cation exchange resin (CM-Sephadex C-50). The positive surface charges of the different molecular forms and their affinities for membranes correlated well with their isoelectric points. The molecular form with jsoelectric point 8·3 had the largest positive surface charge and the highest membrane affinity. On isoelectric focusing of an extract from rat brain synaptosomes, the molecular form with isoelectric point 8·3 formed a complex with a negatively charged compound, presumably a protein. A method was developed to remove this compound by treatment with DEAE-Sephadex or by precipitation with vinblastine. These procedures are similar to methods known to remove the neurotubular protein. The complex formation did not occur in fraction S₃.     

EFFECTS OF ENVIRONMENTAL COMPLEXITY ON AMINO ACID INCORPORATION INTO RAT CORTEX AND HIPPOCAMPUS IN VIVO

Abstract— Amino acid incorporation in vivo was investigated in the cortex and hippocampus of rats raised in enriched and deprived environments for various periods of time following weaning. At early times after weaning (7 days), the incorporation of l -[³H]leucine into all sub-cellular fractions of both cortex and hippocampus was higher in enriched than in deprived rats. At 16 days, incorporation into synaptosomal sub-cellular fractions was higher in enriched than in deprived hippocampus, and lower in enriched than in deprived cortex; incorporation into perikaryal fractions of both brain regions was the same in the two groups of animals. Incorporation into subcortical nuclear protein fractions was higher in enriched rats at this time. At 35 days, the only difference between enriched and deprived rats was a lower incorporation into cortical synaptosomal sub-fractions in the former. Experiments involving double labelling and electrophoresis indicate that there is no stimulation or inhibition of the synthesis of any particular protein in hippocampal nuclear and synaptosomal sub-fractions of enriched rats. Synaptosomal proteins of cortex have a greater half-life in enriched than in deprived rats; proteins of perikaryal fractions of cortex, and of all fractions of hippocampus, are turning over at the same rate in enriched and deprived animals.     

Studies on the turnover of mouse brain synaptosomal proteins

(l) The half-lives of the proteins of various fractions of whole mouse brain increase with increasing insolubility; the supernatant and hypotonic-extractable proteins had half-lives of about 13 days, whereas the membrane proteins solubilized with Triton X-100 and SLS had half-lives of about 18 days. The proteins of the subfractions of synaptosomes had half-lives ranging from 15 to 19 days; those in the cytoplasm had a half-life of 18·3 days, in the membranes, about 17 days and in the synaptic vesicles, 15·6 days. (2) Although the half-life of the synaptic vesicles was not significantly different from that of other synaptosomal subfractions, the vesicles exhibited a different protein pattern on acrylamide gels, an observation which implies that the proteins of the vesicles are qualitatively different from those of other synaptic membranes. (3) The uptake of labelled lysine into the cytoplasm of the synaptosomes of youg mice in vivo was very rapid. (4) The data derived from the relative specific radioactivities of synaptosomal fractions compared with their whole brain analogs support the contention that a sizeable fraction of the synaptosomal cytoplasmic protein was transported to the synapse by axoplasmic flow. The relative specific radioactivities of synaptosomal membrane and synaptic vesicle proteins rose much more quickly than the comparable activities for the cytoplasmic material, and the alternate possibility of synthesis in situ is discussed.     

ACETYLCHOLINESTERASE TURNOVER IN BRAIN, CEREBROSPINAL FLUID AND PLASMA

—By assay of acetylcholine hydrolysis to measure total cholinesterase activity and acetyl-β-methylcholine hydrolysis to measure acetylcholinesterase (E.C 3.1.1.7) activity, patterns of regeneration of enzyme activity were measured in seven areas of brain, cerebrospinal fluid and plasma of cats after administration of an irreversible inhibitor. Halftimes of recovery of total cholinesterase in the brain tissues ranged from 0·9 to 3·8 days (av = 2·5 days) and acetylcholinesterase recovery halftimes ranged from 1·2 to 5·3 days (av = 3·6 days). Regeneration of total cholinesterase was also followed in subcellular fractions of guinea-pig and rat brains after similar inhibition. In both species, the fastest recovery occurred in the soluble fraction with halftimes of 1·8 and 1·6 days, while the synaptosomal fractions exhibited the slowest recoveries with halftimes of 8·3 and 4·1 days. Regeneration of activity in plasma and CSF most nearly resembled that of the soluble brain fraction.     

Isolation of astrocytes,neurons, and synaptosomes of rat brain cortex

A simplified method was developed for the bulk separation of neuronal perikarya and astroglial celis from adult rat brain without the involvement of density gradients. Activities of various enzymes involved in glutamate metabolism were estimated and compared with those of synaptosomes. The activities of glutamate dehydrogenase and aspartate aminotransferase were higher in synaptosomes than in neuronal perikarya or glia. Glutamine synthetase was distributed in all the three fractions while glutaminase activity was higher in astrocytes than in synaptosomes and was not detectable in neuronal perikarya. The significance of these results in relation to metabolic compartmentation was discussed.     

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.     

Variations in Membrane Sensitivity of Brain Region Synaptosomes to the Effects of Ethanol In Vitro and Chronic In Vivo Treatment

The effects of chronic ethanol treatment on the membrane order of synaptosomes from the cerebral cortex, striatum, cerebellum, brainstem, and hippocampus of rats were determined by measuring the fluorescence polarization of diphenylhexatriene (DPH) that had been incorporated into the synaptosomal membranes. Fischer-344 rats either were fed a nutritionally complete ethanol-containing liquid diet for 5 months or pair-fed with a diet that contained sucrose substituted isocalorically for ethanol. Polarization values for synaptosomes from all the brain regions studied were similar except for those from cerebral cortical synaptosomal membranes, which were significantly less ordered. Ethanol in vitro (30-500 mM) decreased the polarization values in synaptosomes from sucrose-control rats for all brain regions, although the sensitivity of cerebellar synaptosomes to the membrane disordering effects of ethanol in vitro was significantly greater that of synaptosomes from other brain regions. Chronic ethanol treatment did not alter baseline polarization for any brain region. Cerebellar and brainstem synaptosomes from the ethanol-fed rats were significantly less susceptible to the membrane disordering effects of ethanol in vitro compared to their sucrose controls, suggesting that chronic ethanol administration results in tolerance to ethanol's membrane effects. Striatal synaptosomes exhibited intermediate tolerance, whereas the sensitivities of cortical and hippocampal synaptosomes to membrane disordering by ethanol in vitro were not significantly affected by the chronic ethanol treatment. These results suggest that synaptosomal membranes have different membrane order requirements depending on the brain region from which they are prepared. Variations in brain regional neuronal membrane sensitivity to ethanol and differential tolerance development may contribute to some of the acute and chronic behavioral effects of ethanol.     

SODIUM-DEPENDENT EFFLUX AND EXCHANGE OF GABA IN SYNAPTOSOMES

Abstract— The influx and efflux of [³H]GABA were investigated in synaptosomes. Two efflux components were detected. The first, termed spontaneous efflux, was not affected by the external sodium chloride concentration. The second, termed GABA-stimulated efflux, was observed when low levels of GABA were added to the incubation medium and was found to require external sodium chloride. The rate of spontaneous efflux at 0°C was about 37 per cent of the rate at 27°C but both GABA-stimulated efflux and GABA influx were completely inhibited at 0°C. The stimulation of efflux by external GABA followed simple Michaelis–Menten kinetics with respect to external GABA. The concentration of external GABA required for half-maximal stimulation was 4·9 ± 1·4 μm and the V_max for efflux was 1·0 ± 0·6 nmol. min^-1.mg^-1 of protein. A similar stimulation of efflux was observed with GABA analogue l -2,4-diamino-butyric acid which is a competitive inhibitor of influx. The concentration of external l -2,4-diaminobutyric acid required for half-maximal stimulation of efflux was 51 ± 12 μm and the V_max for efflux was 0·8 ± 0·5 nmol.min^-1.mg^-1 of protein. Since the sodium-dependency, temperature sensitivity, and kinetic properties of the GABA-stimulated efflux system were similar to the influx system, GABA-stimulated efflux was attributed to carrier-mediated exchange diffusion. Measurement of efflux and influx in the same preparation showed there was a net efflux when total fluxes were considered and that the exchange ratio (influx to GABA-stimulated efflux) was 0·9 when carrier-mediated fluxes were considered. The effect of the temperature of the fluid used to rinse synaptosomes collected on filters in influx experiments was investigated. There was no detectable difference in measured values of influx between samples rinsed with cold fluid (0°C) and warm fluid (27°C). The endogenous GABA content of synaptosomes was found to be 20·3 ± 2·5 nmol GABA per mg of protein. From this value, the cytoplasmic concentration of GABA in synaptosomes was estimated to be a maximum of 40 mm . About 5 per cent of total cerebral cortical GABA was found in the synaptosomal fraction.     

ACTIVE UPTAKE OF [3H]5-HT BY SYNAPTIC VESICLES FROM RAT BRAIN

The question of whether synaptic vesicles accumulate [³H]5-HT by an active process was investigated in a mixed population of vesiclcs from whole rat brain. The temperature dependence and the effect of metabolic inhibitors were studied in synaptosomal suspensions and vesicular fractions. Arrhenius plots for synaptosomes differed from those for vesicles as did the temperature coefficients for these two fractions. For synaptosomes the Q₁₀ was 7 and for vesicles 1.6. However, if ATP was added to the incubation, the temperature dependence of vesicular amine accumulation became manifest; the Arrhenius plot resembled that of synaptosomes and the Q₁₀ was greater than 20 indicating strong temperature dependence. In the presence of ATP, vesicular uptake was stimulated approx 8-fold. Ouabain, dinitrophenol and NEM inhibited synaptosomal uptake but failed to affect [³H]5-HT accumulation by vesicles in the absence of ATP. When ATP was added, vesicular uptake was also blocked by NEM but was unaffected by either ouabain or DNP. Total observed uptake consisted of two components, one ATP-dependent and one nonsaturable and ATP-independent. The active process had a K_m= 1.25 × 10^?7 M and could be completely blocked by either 10^?3 M or 10^?7 M-reserpine. Active vesicular [³H]5-HT uptake was magnesium dependent and was inhibited by sodium and potassium. Cation effects on uptake were specific and could not be accounted for by either changes in osmotic pressure or ionic strength. It was concluded that synaptic vesicles from whole rat brain accumulate [³H]5-HT by an active process.     

Astroglial Cells Express Large Amounts of GABAA Receptor Proteins in Mature Brain

Abstract: GABA_A receptors were characterized in cellular fractions isolated from adult bovine brain. The fraction enriched in cortical astrocytes is very rich in high-affinity binding sites for [³H]flunitrazepam and other "central-type" benzodiazepine ligands. The amount of specific [³H]flunitrazepam binding was more than five times higher in the glial fraction than in synaptosomal and perikaryal fractions. [³H]Flunitrazepam was displaced by low concentrations of clonazepam and other specific ligands for central GABA_A receptors. Specific binding sites for GABA, flunitrazepam, barbiturates, and picrotoxin-like convulsants were characterized. Allosteric interactions between the different sites were typical of central-type GABA_A receptors. The presence of α-subunit(s), as revealed by [³H]flunitrazepam photoaffinity labeling, was demonstrated in all brain fractions at molecular mass 51–53 kDa. Photoaffinity labeling was highest in the glial fraction. However, in primary cultured astrocytes from neonate rat cortex, no photoaffinity labeling was detected. Information obtained from astrocytes in culture should thus be taken with caution when extrapolated to differentiated astroglial cells. Our results actually show that, in mature brain, most of the fully pharmacologically active GABA_A receptors are extrasynaptic and expressed in astroglia.     

Central Presynaptic Terminals Are Enriched in ATP but the Majority Lack Mitochondria

Synaptic neurotransmission is known to be an energy demanding process. At the presynapse, ATP is required for loading neurotransmitters into synaptic vesicles, for priming synaptic vesicles before release, and as a substrate for various kinases and ATPases. Although it is assumed that presynaptic sites usually harbor local mitochondria, which may serve as energy powerhouse to generate ATP as well as a presynaptic calcium depot, a clear role of presynaptic mitochondria in biochemical functioning of the presynapse is not well-defined. Besides a few synaptic subtypes like the mossy fibers and the Calyx of Held, most central presynaptic sites are either en passant or tiny axonal terminals that have little space to accommodate a large mitochondrion. Here, we have used imaging studies to demonstrate that mitochondrial antigens poorly co-localize with the synaptic vesicle clusters and active zone marker in the cerebral cortex, hippocampus and the cerebellum. Confocal imaging analysis on neuronal cultures revealed that most neuronal mitochondria are either somatic or distributed in the proximal part of major dendrites. A large number of synapses in culture are devoid of any mitochondria. Electron micrographs from neuronal cultures further confirm our finding that the majority of presynapses may not harbor resident mitochondria. We corroborated our ultrastructural findings using serial block face scanning electron microscopy (SBFSEM) and found that more than 60% of the presynaptic terminals lacked discernible mitochondria in the wild-type mice hippocampus. Biochemical fractionation of crude synaptosomes into mitochondria and pure synaptosomes also revealed a sparse presence of mitochondrial antigen at the presynaptic boutons. Despite a low abundance of mitochondria, the synaptosomal membranes were found to be highly enriched in ATP suggesting that the presynapse may possess alternative mechanism/s for concentrating ATP for its function. The potential mechanisms including local glycolysis and the possible roles of ATP-binding synaptic proteins such as synapsins, are discussed.