L-glutamate and phorbol ester stimulate the release of secretory amyloid precursor protein from rat cortical synaptosomes

Treatment of rat cortical synaptosomes with micromolar concentrations of L-glutamate stimulated the release of the secreted form of amyloid precursor protein in a concentration-dependent, however biphasic manner as assayed by semiquantitative Western blot analysis. The secreted amyloid precursor protein released from synaptosomes into the incubation medium was highest in the presence of 500 microM L-glutamate (about 64% over the level assayed in the incubation medium in the absence of any drug). In contrast, direct stimulation of protein kinase C by phorbol-12-myristate-13-acetate resulted in a concentration-independent increase in secretory amyloid precursor protein release by about 100% already detectable at a concentration of 0.1 microM but with no significant change at higher concentrations up to 10 microM. The presented data show that there is a constitutive release of secretory amyloid precursor protein from synaptosomes and suggest that (i) processing of amyloid precursor protein at the synaptic level is controlled by L-glutamate presumably via activation of protein kinase C, and (ii) isolated cortical synaptosomes represent a useful experimental approach to selectively study amyloid precursor protein metabolism at the synaptic level.     

PATTERNS OF AMINO ACID RELEASE FROM NERVE-ENDINGS ISOLATED FROM SPINAL CORD AND MEDULLA

The metabolic properties of synaptosomes prepared from the crude mitochondrial and crude nuclear fractions of the medulla/spinal cord were studied. They showed similar properties, glycine being enriched in the latter. The respiration and glycolysis rates were similar to the cortical synaptosomes previously studied. A major difference from cortical synaptosomes was the enrichment of glycine. Medulla/spinal cord synaptosome suspensions and beds responded metabolically to electrical pulses; respiration and lactate production increased by 50 and 25 per cent respectively. Differential release of glutamate, aspartate, GABA and glycine occurred during both electrical stimulation, and when potassium in the medium was increased. Omitting calcium and adding EGTA greatly reduced this response with both forms of stimulation. The electrically induced release of GABA was completely reversible whilst that of aspartate and glycine was only partially reversible. The electrically stimulated release of glycine and other amino acids was reduced in synaptosomes prepared from rats treated intramuscularly with tetanus toxin 15 hr before death. No action of the toxin was seen on synaptosomes incubated with tetanus toxin after preparation.     

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.     

METABOLISM OF GLUCOSE AND GLUTAMATE BY SYNAPTOSOMES FROM MAMMALIAN CEREBRAL CORTEX

—(1) Synaptosomes incubated in high sodium, low potassium media showed high linear respiration in the presence of glucose which was converted into lactate, aspartate, glutamate, glutamine, alanine and GABA during 1 hr incubation periods. (2) Total conversion of glucose into most of these substrates over the incubation period was similar in synaptosomes and cortex slices. Half the lactate and only a small fraction of the glutamine made by slices was formed by synaptosomes. (3) Pool sizes of amino acids in cortex slices after incubation with glucose were, in general, higher than in synaptosomes, glutamate and glutamine being four-fold higher in slices. (4) Most of the amino acids made from glucose by synaptosomes were contained within their structure and not lost to the medium. (5) Glutamate was actively metabolized by synaptosomes to aspartate, glutamine, alanine and GABA. The specific radioactivities of the amino acids (except glutamine) after 1 hr incubation, approached that of the glutamate. (6) Pyridoxal phosphate added to the incubation medium increased GABA production from glutamate but not from glucose.     

Presynaptic dendrites and dendro-dendritic synapses in the superior tubercles of the rat quadrigeminum]

The types of dendro-dendritic synapses and their participation in the synaptic, organization of superficial layers of the quadrigeminum superior tubercles were studied electron microscopically. In addition to simple forms of dendro-dentritic synapses the reciprocal dendro-dendritic synapses were revealed. Presynaptic dendrites formed the synaptic fields and glomerules of the superficial grey layer. The terminals of optical, cortical fibres from the visual cortex and other types of terminals terminated on presynaptic dendrites.     

A Comparative Study and Partial Characterization of Multi-Uptake Systems for γ-Aminobutyric Acid

Previous work by the authors had indicated that synaptosome-enriched preparations from the cerebral cortex of the rat contained a high-, a medium-, and a low-affinity uptake system for gamma-aminobutyric acid (GABA). The present study demonstrated that this phenomenon also prevailed in synaptosomes from rat diencephalon, mesencephalon, and cerebellum, although the Vmax values for the high- and medium-affinity systems in the cerebellum were very low relative to those of the other regions. When a different type of preparation containing nerve endings (glomeruli) was obtained from the cerebellum, it possessed a Vmax value for the high-affinity system that was more similar to that for the corresponding system in synaptosomes from the other brain regions. In contrast to the above situation, synaptosomes from rat olfactory bulb lacked the low-affinity uptake system, as did synaptosomes from dog olfactory bulb. The aspartate/glutamate uptake systems, as measured with D-aspartate, provided a regional pattern quite different from those of GABA uptake. Only two uptake systems, a high- and low-affinity system, were observed in all regions tested. All three GABA uptake systems were present in cortical synaptosomes from the mouse, hamster, and guinea pig, and all three systems were sodium dependent, energy dependent, temperature sensitive, and totally inhibited by nipecotic acid.     

Sodium-independent synaptosomal uptake of leucine

The uptake of leucine by rat brain synaptosomes was investigated in a Na+-free medium. It was found that leucine was transported into synaptosomes by 2 uptake processes which were both extremely sensitive to inhibition by 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid. These 2 components were distinguishable from each other by kinetic analysis and by their relative sensitivity to inhibition by selected amino acids.     

An inhibitor of dopamine uptake,LR5182, CIS-3-(3,4-dichlorophenyl)-2-N,N-dimethylaminomethyl-bicyclo-[2,2,2]-octane,hydrochloride

LR5182 inhibited the uptake of dopamine in rat striatal synaptosomes and the uptake of norepinephrine in cortical synaptosomes with inhibitor constants, Ki values, of 3nM and 58nM, respectively. It was only a week inhibitor of serotonin uptake in cortical synaptosomes with a Ki value of 1.7μM. The uptake of dopamine and norepinephrine were significantly lowered within an hour after an intraperitoneal injection of LR5182. Among known inhibitors of dopamine uptake in synaptosomes of rat brain, LR5182 is most effective and selective. The rigid structure of LR5182 (Figure 1) suggested a gauche conformation of dopamine to be favored by the striatal uptake of dopamine.     

Effects of secondary binding by activator and inhibitor peptides on covalent intermediates of pig pepsin.

1. Cerebral-cortex synaptosomes were shown to synthesize (14C)acetylcholine after incubation with (14C)choline, and 25mM-KCl released (14C)acetylcholine (but not (14C)choline) into the medium by a Ca2+-dependent and Mg2+-sensitive process. 2. The K+-stimulated release of (14C)acetylcholine was inhibited by more than 80% after preincubation of the synaptosomes with 10(5) mouse lethal doses of botulinum toxin/ml. (14C)choline uptake, (14C)acetylcholine synthesis, intrasynaptosomal K+ and occluded lactate dehydrogenase were unaffected by the toxin. It also failed to prevent the K+-stimulated release of (3H)noradrenaline and (14C)glycine from synaptosomes. 3. Fractionation of hypo-osmotically shocked synaptosomes revealed that more than 75% of the radioactive acetylcholine was in the cytoplasmic compartment, although the vesicle pellet contained more total acetylcholine than the cytoplasmic pool. Consequently the specific radioactivity of acetylcholine in the cytoplasmic pool was almost 5 times that of the vesicles. This distribution was unaffected by preincubation with botulinum toxin. It is concluded that the toxin acts directly on the release of acetylcholine, rather than influencing its storage. 4. After K+-stimulation, toxin-inhibited synaptosomes contained increased amounts of total acetylcholine, which suggests that its rate of synthesis is controlled by depolarization rather than release.     

Ultrastructure of synaptosomes from one-day old and adult rat brain stem

Summary The ultrastructure and protein content of the five subfractions of the crude mitochondrial fraction from the brain stem of the 1-day old and adult rat was examined. The morphological composition of the subfractions after fixation in glutaraldehyde and osmiumtetroxide in the adult rat brain stem resembled that previously reported for the whole brain; synaptosomes sedimented in a sucrose gradient in subfractions C and D. In the 1-day old rat, mature synaptosomes were found in subfractions A, B, C and D; E contained mainly free mitochondria. 80–95% of the processes in the adult and 10–30% in the 1-day old rat contained synaptic vesicles which were of four types: (1) small agranular vesicles (2) large dense core vesicles (3) large agranular vesicles (4) coated vesicles. Pre- and postsynaptic membrane thickenings were demonstrated in many nerve-ending particles. In the subfractions of the 1-day old rat the protein content was one half and the distribution resembled that in the adult. Evidently nerve endings develop faster in the brain stem than in cortical areas; a serotoninor adrenergic origin of the early synaptosomes is suggested.This study was supported by a grant from the Paulo Foundation.     

Effect of Adenosine, Adenosine Derivatives, and Caffeine on Acetylcholine Release from Brain Synaptosomes: Interaction with Muscarinic Autoregulatory Mechanisms

Synaptosomes, prepared from rat cerebral cortex and hippocampus, were preincubated with [methyl-3H]choline. The effect of adenosine, cyclohexyladenosine, N-ethylcarboxamide adenosine, 2'-deoxyadenosine, and oxotremorine on K+-evoked 3H efflux was investigated. High-voltage electrophoretic separation showed that in the presence of physostigmine, the K+-evoked 3H efflux from hippocampal synaptosomes was 90% [3H]acetylcholine and 10% [3H]choline. Adenosine (30 microM) and oxotremorine (100 microM) both decreased [3H]acetylcholine release from hippocampal synaptosomes. The effect was inversely proportional to the KCl concentration and disappeared at a KCl concentration of 50 mM. Cyclohexyladenosine was approximately 3,000 times more active than adenosine, whereas N-ethylcarboxamide adenosine and 2'-deoxyadenosine were inactive. This indicates that A1 adenosine receptors were involved in the inhibitory effect. Caffeine antagonized the adenosine effect, and at a concentration of 100 microM, it stimulated [3H]acetylcholine efflux. The inhibitory effect of oxotremorine was as great in cortical as in hippocampal synaptosomes. In contrast, adenosine was much less active in cortical than in hippocampal synaptosomes. When inhibitory concentrations of adenosine and oxotremorine were added together into the incubation medium, the effect of adenosine on [3H]acetylcholine release was consistently reduced. An interaction between muscarinic and A1 adenosine presynaptic receptors at a common site modulating acetylcholine release can be assumed.     

Uptake of L-glutamine into synaptosomes. Is the γ-glutamyl cycle involved?

Transport of L-glutamine into rat cortical synaptosomes has been investigated by (14C)L-glutamine uptake experiments. This amino acid enters synaptosomes both by an active carrier mediated system, which may be the result of gamma-glutamyl cycle activity and by a Na+-dependent transport system. This view is supported by the following observations: a) as demonstrated previously (10), glutamine inside synaptosomes reaches concentrations higher than those of the incubation medium, and initial rates of uptake approach saturation kinetics; b) the uptake of glutamine is inhibited by uncouplers; c) the uptake is inhibited by methionine sulfoximine, a suicide-inhibitor of an enzyme of the gamma-glutamyl cycle; d) the initial rate of uptake is lowered by decreasing the Na+-level of the incubation medium or by adding ouabain. The validity of this hypothesis is discussed.     

Release of Immunoreactive Insulin from Rat Brain Synaptosomes Under Depolarizing Conditions

Synaptosome preparations were utilized to characterize the release and compartmentalization of immunoreactive insulin (IRI) in the adult rat brain. Depolarization of synaptosomes by elevation of the external potassium ion concentration elicited release of IRI from the synaptosomes into the incubation medium. This release was reduced or eliminated under three conditions known to prevent depolarization-induced Ca2+ flux: elevating the external MgCl2, adding CoCl2, and eliminating external Ca2+ with EGTA. Depolarization of synaptosomes by veratridine also elicited release of synaptosomal IRI. This release was inhibited by tetrodotoxin. The amount of IRI released under depolarizing conditions represented 3-7% of that contained in the synaptosomes. High levels of IRI release also were observed upon removal of external Na+ to allow depolarization-independent influx of external Ca2+ into the synaptosomal compartment. The Ca2+ dependency of synaptosomal IRI release suggests IRI is stored in the adult rat brain in synaptic vesicles within nerve endings from which it can be mobilized by exocytosis in association with neural activity.     

Na+ Dependence of Tyrosine Transport Across the Synaptosomal Membrane Reflects Changes in the Morphology of Synaptosomes

The Na+ dependence of tyrosine uptake into rat brain synaptosomes and synaptosomal plasma membrane vesicles (SPMV) was examined in the present study. At low tyrosine concentrations, the isoosmotic substitution of Na+ by sucrose in the incubation medium led to an increase of tyrosine uptake in synaptosomes and to a decrease in SPMV. The removal of extracellular Ca2+ and Mg2+ and addition of isoosmotic sucrose completely prevented the augmented tyrosine uptake in Na+-free incubated synaptosomes. Morphological differences were found at the electron-microscopic level when synaptosomes were incubated in Na+-free and Na+-containing media. The internal volume measured for synaptosomes incubated in a Na+-free medium was almost half of that obtained in a Na+-containing medium, in good agreement with the observations made with the electron microscope. Also, the omission of Ca2+ and Mg2+ resulted in a specific swelling of only the synaptosomes incubated in Na+-free medium. When synaptosomes and SPMV were preloaded with several neutral amino acids, the tyrosine uptake rate was greatly increased, indicating fully operational exchange mechanisms for these amino acids. We propose that the enhancement of high-affinity synaptosomal tyrosine uptake observed in Na+-free medium is a consequence of a specific shrinkage of the synaptosomes and a parallel increase of the exchange rate with endogenous neutral amino acids.     

Synaptosomal Protein Kinase C Subspecies: A. Dynamic Changes in the Hippocampus and Cerebellar Cortex Concomitant with Synaptogenesis

The expression of protein kinase C (PKC) subspecies in synaptosomes prepared from a number of adult brain regions was compared. Cerebral cortical and thalamic/striatal synaptosomes were found to express three peaks of enzyme activity upon hydroxyapatite chromatography, corresponding to the type I(gamma), type II(beta), and type III(alpha) subspecies. Synaptosomes prepared from either the hippocampus or the cerebellar cortex, however, contained only two major peaks, corresponding to the alpha- and beta-subspecies, with barely detectable levels of the gamma-subspecies, even though these tissue areas were enriched in the latter enzyme. When the ontogenic pattern of hippocampal synaptosomal PKC subspecies was examined, it was found that at postnatal day 7, significant quantities of the gamma-subspecies were present and that this subspecies reached its peak levels at around postnatal day 14, before steadily declining to its adult level. Similar changes were observed also for the gamma-subspecies in cerebellar cortex synaptosomes. The dynamic changes in the synaptosomal PKC subspecies take place at a critical period in the development of the rat brain, concomitant with an active period of synaptogenesis, suggesting that it may play a role in synaptogenesis.     

Protein Methylation in Cerebellar Synaptosomes

Abstract: Synaptosomes from five regions of adult rat brain were isolated, analyzed for methyl acceptor proteins, and probed for methyltransferases by photoaffinity labeling. Methylated proteins of 17 and 35 kDa were observed in all regions, but cerebellar synaptosomes were enriched in a 21–26-kDa family of methyl acceptor proteins and contained a unique major methylated protein of 52 kDa and a protein of 50 kDa, which was methylated only in the presence of EGTA. When cerebellar and liver subcellular fractions were compared, the cytosolic fractions of each tissue contained methylated proteins of 17 and 35 kDa; liver membrane fractions contained few methylated proteins, whereas cerebellar microsomes had robust methylation of the 21–26-kDa group. Differential centrifugation of lysed cerebellar synaptosomes localized the 17- and 35-kDa methyl acceptor proteins to the synaptoplasm, the 21–26-kDa family to the synaptic membranes, and the 52-kDa to synaptic vesicles. The 21–26-kDa family was identified as GTP-binding proteins by [α-³²P]GTP overlay assay; these proteins contained a putative methylated carboxyl cysteine, based on the presence of volatile methyl esters and the inhibition of methylation by acetylfarnesylcysteine. The 52-kDa methylated protein also contained volatile methyl esters, but did not bind [α-³²P]GTP. When synaptosomes were screened for putative methyltransferases by S -adenosyl-L-[ methyl -³H]methionine photoaffinity labeling, a protein of 24 kDa was detected only in cerebellum, and this labeled protein was localized to synaptic membranes.     

Cell-type specific dendritic contacts between retinal ganglion cells during development.

The extent of a neuron's dendritic field defines the region within which information is processed. The dendritic fields of functionally distinct ON and OFF center retinal ganglion cells (RGCs) form separate mosaics across the retina. Within each mosaic, neighboring dendritic fields overlap by a constant amount, sampling the visual field with the appropriate coverage. Contact-mediated lateral inhibition between neighboring RGCs has long been thought to regulate both the extent and overlap of dendritic fields during development. Here we show that dendro-dendritic contact exists between developing RGCs and occurs in a manner that would regulate the formation of ON and OFF mosaics separately. Dye-filled neighboring ON and OFF ferret alpha RGCs were reconstructed using multiphoton microscopy. At all neonatal ages examined, we observed dendro-dendritic contacts between RGCs of the same sign (ON/ON; OFF/OFF), but never between cells of opposite signs (ON/OFF). Terminal dendrites of one cell often touched a dendrite of its neighbor as they intersected. In some instances, the distal dendrite of one cell formed a fascicle with the proximal process of its neighbor. Alpha cells did not form contacts with neighboring beta cells of the same sign. Together, these observations suggest that dendro-dendritic contact between RGCs is cell-type specific. Dendritic contacts were observed even before the alpha cell arbors were completely stratified, suggesting that cell-cell recognition may take place early in their development. For each cell type, the relative overlap of dendritic fields was constant with age, despite a two-fold increase in field area. We suggest that dendro-dendritic contacts may be sites of intercellular signaling that could regulate local extension of dendrites to maintain the relative overlap of RGCs within a mosaic during development.     

Effect of castration on the high affinity glutamate transporter in rat hypothalamic and cortical synaptosomes

High affinity transport of glutamic acid has been studied in cortical and hypothalamic synaptosomes from castrated male rats and compared to normal controls. For hypothalamic synaptosomes, both initial velocity of uptake and Va (apparent maximal velocity) were found to be about one-third lower in the castrated animals. Kt (glutamate concentration giving Va/2), however, was reduced by only 5%.Initial velocity of uptake in cortical synaptosomes was measured as a function of both sodium and glutamate concentration. Reductions in uptake subsequent to castration were found to be much less for cortical synaptosomes (2–15%) than for hypothalamic synaptosomes. Fit for these data to various models for the sodium dependence of transport resulted in the same minimal best fit model as that found for control animals. Thus castration does not alter the fundamental nature of the mechanism by which carrier, sodium, and glutamate interact in the process of transport. However quantitative changes were found to occur, as reflected in the best fit constants. These constants were used along with the rate equation for the minimal best fit model to calculate certain parameters which were then used to delineate the quantitative changes in the transporter following castration. A neuroregulatory role for glutamate in gonadotropin secretion has been recently proposed; the present study now provides additional information on the relationship between reproductive function and one aspect of glutamatergic synaptic function, namely, the high affinity transport system.     

Regulation of Norepinephrine Uptake by Adenine Nucleotides and Divalent Cations: Role for Extracellular Protein Phosphorylation

This study examined the hypothesis that ATP, released together with norepinephrine (NE) from brain noradrenergic nerve terminals, may serve as a cosubstrate for an extracellular protein phosphorylation system that regulates the reuptake of the transmitter, NE. The possible regulation of high-affinity uptake (uptake 1) of [3H]NE by divalent cations and ATP, both of which are involved in protein phosphorylation, was examined in rat cerebral cortical synaptosomes. A marked inhibition of uptake 1 by 5'-adenylylimidodiphosphate [App(NH)p], a nonhydrolyzable, competitive antagonist of ATP, was observed. A similar inhibition of uptake was observed when Ca2+ and Mg2+ were both omitted from the incubation medium. App(NH)p distinguished the actions of Ca2+ from those of Mg2+: Ca2+-stimulated uptake 1 was blocked by App(NH)p; Mg2+-stimulated uptake was not. In parallel experiments, the patterns of protein phosphorylation in crude and purified preparations of synaptosomes were examined under conditions similar to those used in uptake assays. A striking correlation was found between the inhibition of uptake 1, by either App(NH)p or Ca-omission, and inhibition of the phosphorylation of one specific, 39,000-dalton, Ca2+-dependent, protein component in synaptosomes. This 39K protein was distinct from the alpha subunit of pyruvate dehydrogenase, a mitochondrial protein of similar electrophoretic mobility. These findings are consistent with the possibility that an ectokinase on synaptosomes utilizes extracellular ATP and Ca2+ in phosphorylating a protein(s) associated with the regulation of NE uptake.     

A new experimental animal for the study of age-related changes in serotonergic neuronal activity in the brain cortex

Calcium uptake by the cortical synaptosomes in a rodent (Fischer rat) and an insectivore shrew (Suncus murinus) was detected as a parameter reflecting molecular dysfunction of the aging brain. The change in calcium uptake by the cortical synaptosomes in both species was concomitant which showed less than half the capacity at 24 months old animals compared with those at 8 months old. On the other hand, 5-hydroxytryptamine binding and imipramine binding to the membrane fraction of aging rat brain cortex was not altered in terms of binding capacity along with aging, while, in Suncus, the binding of both serotonergic ligands declined with aging. In order to elucidate decreased serotonergic activity in human demented aged brain, together with declining activity in neurotransmitting systems detectable as a function of calcium uptake by the cortical synaptosomes, Suncus may be an appropriate animal model for studying physiological aging processes in the mammalian brain cortex.Special Issue Dedicated to Dr. Abel Lajtha.