Effect of parathyroid hormone on the transport of 45Ca2+ and 3H-GABA in nerve endings isolated from the rat cerebral cortex

Parathyroid hormone (PTH) (0.1-10 ng/ml) evokes a dose-dependent increase in 45Ca2+ accumulation in synaptosomes isolated from the rat brain cortex. In the presence of PTH the fast (I sec) potential-dependent 45Ca2+ uptake was less than in the control. PTH had no effect on 3H-GABA uptake by synaptosomes (P2 fraction). Synaptosomes preincubated in the presence of PTH in Ca2+-free medium and transferred into Ca2+-containing normal medium released more 3H-GABA than control synaptosomes. In this case depolarization-evoked 3H-GABA release was diminished.     

Heterogeneity of presynaptic calcium channels revealed by species differences in the sensitivity of synaptosomal 45Ca entry to omega-conotoxin

The inhibition of K+-depolarization dependent Ca influx by omega-conotoxin GVIA was compared in the frog, chick, and rat brain synaptosomes. The toxin at concentrations greater than or equal to 0.3 microM completely inhibited Ca entry in the frog and chick preparations, but was only partly effective in blocking Ca influx in the rat brain synaptosomes. In chick synaptosomes the toxin's effect was biphasic: a small component (approximately equal to 15%) of total Ca influx was inhibited by the toxin with high affinity (I50 less than 0.002 microM); a major component (approximately equal to 80%) of Ca influx was inhibited with a moderate affinity (I50 approximately equal to 0.05 microM). In rat brain synaptosomes 40% of Ca influx was inhibited by the toxin with low affinity (I50 approximately equal to 0.3 microM), and 60% of Ca influx was unaffected by the toxin concentration of up to 10 microM. These data suggest a heterogeneity of voltage-sensitive Ca channels in vertebrate brain synaptosomes.     

Free Mitochondria and Synaptosomes from Single Rat Forebrain. A Comparison Between Two Known Subfractionation Techniques

Two published subcellular subfractionation techniques employing Ficoll-sucrose or sucrose-density gradient centrifugation, respectively, are evaluated for their capacity to yield fractions containing free mitochondria and synaptosomes from a single rat forebrain. The enzymes lactate dehydrogenase, acetylcholinesterase, NAD(P)H-cytochrome c reductase, and citrate synthase, markers of different subcellular components, were used to assess the purity and integrity of the fractions. Judged by the distribution of these specific enzymatic markers, the free mitochondria obtained by the Ficoll-sucrose gradient technique were less contaminated by synaptosomes and had greater biochemical integrity than those obtained by the sucrose-gradient technique. By contrast, the synaptosomes obtained by the Ficoll-sucrose gradient technique resulted in more contamination by microsomes than those prepared in a sucrose gradient.     

Changing Fatty Acid Content of Growth Cone Lipids Prior to Synaptogenesis

The developing mouse was used to assess biochemical changes in membrane lipids during the period when nerve growth cones become synapses. Growth cone particles and synaptosomes were simultaneously obtained from common brain homogenates. Incorporation of the essential fatty acid, docosahexaenoic acid (22:6 omega-3), was correlated with the developmental changes in endogenous fatty acid content of growth cones and synaptosomes. Analysis of endogenous lipid content indicated that, at all ages studied, the growth cones contained more arachidonoyl acyl chains (20:4 omega-6) than did synaptosomes. Before the onset of synaptogenesis, levels of arachidonoyl chains increased and levels of 22:6, oleoyl and linoleoyl chains decreased in synaptosomes. Although stearoyl and palmitoyl (16:0) remained stable in synaptosomes, 16:0 decreased in growth cones. With the exception of 16:0 and 20:4, endogenous fatty acyl content of growth cones and synaptosomes became similar by postnatal day 10, which coincides with the onset of synaptogenesis. When 5-day-old mouse pups were injected intraperitoneally with [3H]22:6, the incorporation into growth cone and synaptosome phospholipids was greatest in phosphatidylethanolamine, followed by phosphatidylserine and phosphatidylcholine. Nominal labeling was present in phosphatidic acid and phosphatidylinositol. Labeling in neutral lipids was less than that of phospholipids, with triacylglycerol incorporating most of the neutral lipid label, followed by diacylglycerol and free 22:6. Only the growth cone fraction contained detectable amounts of 22:6-labeled cholesterol esters. The distribution of 22:6 label in plasma 72 h after injection indicated that approximately 60% of the label was in phospholipids with approximately 40% in neutral lipids and less than 5% in free fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rat Brain Synaptosomes Prepared by Phase Partition

Synaptosomes from rat forebrain can easily be isolated by combining centrifugation with partition in an aqueous two-phase system composed of dextran T500 and polyethylene glycol 4000 in which synaptosomes have an extreme affinity for the upper phase. The fraction thus obtained has been characterized by electron microscopy and biochemical markers for synaptosomes and some other cell components. The contamination by microsomes, free mitochondria, and myelin was 4.4, 3.2, and 0.1%, respectively. The morphometric analysis of the electron micrographs shows that greater than 60% of the structures are synaptosomes. This preparation of the isolation procedure is remarkably short (less than 1 h), formance as assayed by their respiratory activities and ATP level in the absence and presence of depolarizing agents. Synaptosomes prepared by phase partition release the neurotransmitter glutamate in a Ca2(+)-dependent manner. The duration of the isolation procedure is remarkably short (less than 1 h), no ultracentrifuge is required, and the method can be applied for small- or large-scale preparations.     

Lipid composition of different preparations of brain Na+, K+-ATPase

The content and composition of phospholipids is determined in beef microsomal and synaptosomal fractions and also in these fractions preparations solubilized with triton X-100 (0.1%) and digitonin (0.2%). It is shown that the microsomal fraction is richer in phospholipids. The solubilized fragments of microsomes have less or the same amount of phospholipids per protein unit than the initial fraction of microsomes, and the solubilized fragments of synaptosomes contain a higher quantity of phospholipids than the initial fraction. The content of phospholipids in "the riton" fragments of synaptosomes is higher than in "those" of microsomes. Contrary to digitonin which solubilizes the active Na+, K+-ATPase complex of microsomes and synaptosomes, triton X-100 solubilizes the active enzyme of microsomes only. A higher total content of phospholipids in "the triton" extracts of synaptosomes does not probably correlate with the presence of Na+, K+-ATPase activity in them. But these extracts are found to contain less phosphatidylserine whose addition recovers Mg2+, Na+, K+-ATPase activity in them. The effect of phosphatidylserine is not strictly specific for "the triton" extracts of synaptosomes, this lipid activates to a definite extent the extracts of microsomes as well. It is shown that at the first stages of bull brain Na+, K+-ATPase purification the total content of phospholipids and cholesterol in the preparations increases but the composition of phospholipids remains unchanged.     

Effect of carbacholine and serotonin on the ATPase activity in synaptosomes of the projection and association areas of the feline cerebral cortex]

Na, K-ATPase and Mg-ATPase activities were measured in the synaptosomes of the temporal auditory projection area and the frontal association area. Moreover, the effects of carbacholine and serotonin on those activities were investigated. Na, K-ATPase activity in the synaptosomes of the association area was shown to be reliably higher that in the synaptosomes of the projection area (11.02 +/- 0.45 vs 8.40 +/- 0.55 microM Pi/mg of protein hr; P less than 0.05). Mg-ATPase activity was higher in the second case as compared to the first one (11.40 +/- 0.38 vs 9.04 +/- 0.35; p less than 0.05). Carbacholine and serotonin (10(-8)-10(-3) M) were found to induce equal inhibition of Na, K-ATPase activity in the synaptosomes of both cortices (1 max = 25-30%, 1C50 = 0.2-0.3 microM) which is blocked respectively with atropine (10(-6) M) and methysergide (10(-6) M) and enhanced in presence of GTP (5.10(-5) M). The enzyme activity is also inhibited by the non-hydrolysable guanine nucleotide, GTP gamma S (10(-8)-10(-4) M), in the absence of the antagonists (1 max = 35-40%, 1 C50 = 0.02 microM). In the methysergide-containing medium serotonin exerts a dose-dependent stimulatory effect on Na, K-ATPase which is more pronounced in the synaptosomes of the association area (A max = 25%, A C50 = 0.05 microM). Mg-ATPase activity of membrane preparations is liable to be stimulated by both serotonin and carbacholine, stimulation being more pronounced in the synaptosomes of the association cortex as well (A max = 35%, A C50 = 0.2-0.3 microM). This effect is insensitive either to the antagonists of the corresponding receptors or to GTP. GTP gamma S does not cause alterations in the enzymatic activity. Na, K-ATPase is suggested to be coupled to muscarine and serotonin receptors in the synaptic membranes of both projection and association cortical areas via a GTP-binding protein. At the same time, the agonists of receptors mentioned above are presumably also capable to effect Mg-ATPase activity by the receptor-independent way.     

A method for studying synaptosomal release of neurotransmitter candidates, as evaluated by studies on cortical cholecystokinin octapeptide release

A method for perfusing rat cortical synaptosomes for studying the regulation of cholecystokinin octapeptide (CCK-8) release has been developed and was found to have advantages over the static incubation system. Synaptosomes isolated from rat cortex were suspended in Biogel P2 columns and perfused with Krebs Ringer Bicarbonate buffer. One hundred mM KCl and 75 microM veratine stimulated CCK-8 release, which was Ca++-dependent. The synaptosomes were functionally viable for at least 135 min of incubation as indicated by multiple 100 mM KCl depolarizations and uptake of (3H)-norepinephrine and (14C)-choline. Dopamine and acetylcholine (10(-6)M) stimulated CCK-8 release while serotonin and norepinephrine were without effect. Approximately 20% of total occluded CCK-8 was released from synaptosomes by 100 mM KCl and degradation of CCK-8 was less than 10%. Perfusion of synaptosomes has several advantages over static incubation systems and allows systematic studies on the role of neurotransmitter in the regulation of neuropeptide secretion.     

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.     

Inhibitory effects of cis- and trans-resveratrol on noradrenaline and 5-hydroxytryptamine uptake and on monoamine oxidase activity

This study investigated for the first time the potential effects of cis- and trans-resveratrol (c-RESV and t-RESV) on noradrenaline (NA) and 5-hydroxytryptamine (5-HT) uptake by synaptosomes from rat brain, on 5-HT uptake by human platelets, and on monoamine oxidase (MAO) isoform activity. Both c-RESV and t-RESV (5-200 microM) concentration-dependently inhibited the uptake of [3H]NA and [3H]5-HT by synaptosomes from rat brain and the uptake of [3H]5-HT by human platelets. In both experimental models, t-RESV was slightly more efficient than c-RESV. Furthermore, in synaptosomes from rat brain, the RESV isomers were less selective against [3H]5-HT uptake than the reference drug fluoxetine (0.1-30 microM). On the other hand, both c-RESV and t-RESV (5-200 microM) concentration-dependently inhibited the enzymatic activity of commercial (human recombinant) MAO isoform (MAO-A and MAO-B) activity, c-RESV being slightly less effective than t-RESV. In addition, both RESV isomers were slight but significantly more selective against MAO-A than against MAO-B. Since the principal groups of drugs used in the treatment of depressive disorders are NA/5-HT uptake or MAO inhibitors, under the assumption that the RESV isomers exhibit a similar behaviour in humans in vivo, our results suggest that these natural polyphenols may be of value as structural templates for the design and development of new antidepressant drugs with two important biochemical activities combined in the same chemical structure: NA/5-HT uptake and MAO inhibitory activity.     

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.     

External calcium, intrasynaptosomal free calcium and neurotransmitter release

In a physiological medium the resting membrane potential of synaptosomes from guinea-pig cerebral cortex, estimated from rhodamine 6G fluorescence measurements, was nearly -50mV. This agreed with calculations using the Goldman-Hodgkin-Katz equation. With external [Ca2+] less than or equal to 3 mM veratridine depolarisation (to -30 mV) was accompanied by increases in intrasynaptosomal free calcium concentrations (monitored by entrapped quin2) and parallel increases in total acetylcholine release. With external [Ca2+] greater than 3 mM both intrasynaptosomal free calcium concentrations and transmitter release were paradoxically reduced, providing further evidence for a close correlation between the two events. To support an explanation of these findings based on divalent cation screening of membrane surface charge (increasing the voltage gradient within the membrane and closing voltage-inactivated channels) surface potential measurements were made on synaptic lipid liposomes by using a fluorescent surface-bound pH indicator. These experiments provided evidence for the presence of screenable surface charge on synaptosomes, and it was further shown in depolarised synaptosomes themselves that total external [Ca2+ + Mg2+], and not [Ca2+] alone, set the observed peak in intrasynaptosomal free calcium.     

ULTRASTRUCTURAL AND BIOCHEMICAL COMPARISONS OF DENDRODENDRITIC SYNAPTOSOMES FROM BOVINE SUPERIOR COLLICULI AND OLFACTORY BULBS WITH AXODENDRITIC AND AXOSOMATIC SYNAPTOSOMES

Abstract– The buoyant density, ultrastructure and protein composition of dendrodendritic (DD), axodendritic (AD) and axosomatic (AS) synaptosomes were compared. DD synaptosomes prepared from either bovine superior colliculi or olfactory bulbs sedimented in 0.32 m -sucrose at 1000 g whereas AD and AS synaptosomes from frontal and temporal lobe cortices sedimented at 10.000 g . In gradients of Renografin the DD contacts banded at a density of 1.12 whereas AD and AS banded at 1.07. The DD contacts appeared as large clusters of serial contacts, and each individual terminal had a mean surface area of 1.48 μm²; the mean area of AD and AS terminals was less than 0.35 μm². The protein compositions of DD, AD and AS synaptosomes were quite similar, with minor exceptions. These findings suggest that different synaptosmal types evolved from common membrane constituents.     

45Ca2+ and 3H-GABA transport in nerve endings separated from the cerebral cortex of rats with hypoparathyroidism

Ca2+ blood serum level was reduced by 34.5% in rats with hypoparathyroidism (HPT) on the 7th-12th day after the damage of parathyroid glands. Synaptosomes isolated from the brain cortex of rats during this period accumulated in a normal medium more 45Ca2+ than synaptosomes from healthy animals. In potassium depolarization, control and experimental synaptosomes accumulated more 45Ca2+, however in HPT the increment in 45Ca2+ uptake in high potassium medium was less temperature-dependent. In normal medium 3H-GABA uptake and release by synaptosomes from the brain of rats with HPT slightly differed from those in the control. On the contrary, 3H-GABA release induced by synaptosome depolarization was depressed in HPT. It is suggested that nerve terminal excretory function disturbances contribute to increased excitability of the central nervous system in hypoparathyroidism.     

Cholinergic location of delta-opioid receptors in canine atria and SA node

Delta-opioid receptors (DORs) are associated with ischemic preconditioning and vagal transmission in the sinoatrial (SA) node and atria. Although functional studies suggested that DORs are prejunctional on parasympathetic nerve terminals, their precise location remains unconfirmed. DORs were colocalized in tissue slices and synaptosomes from the canine right atrium and SA node along with cholinergic and adrenergic markers, vesicular acetylcholine transporter (VAChT), and tyrosine hydroxylase (TH). Synapsin I immunofluorescence verified the neural character of tissue structures and isolated synaptosomes. Acetylcholine and norepinephrine measurements suggested the presence of both cholinergic and adrenergic synaptosomes. Fluorescent analysis of VAChT and TH signals indicated that >80% of the synapsin-positive synaptosomes were of cholinergic origin and <8% were adrenergic. DORs colocalized 75-85% with synapsin in tissue slices from both atria and SA node. The colocalization was equally strong (85%) for nodal synaptosomes but less so for atrial synaptosomes (57%). Colocalization between DOR and VAChT was 75-85% regardless of the source. Overlap between DOR and TH was uniformly low, ranging from 8% to 17%. Western blots with synaptosomal extracts confirmed two DOR-positive bands at molecular masses corresponding to those reported for DOR monomers and dimers. The abundance of DOR was greater in nodal synaptosomes than in atrial synaptosomes, largely attributable to a greater abundance of monomers in the SA node. The abundant nodal and atrial DORs predominantly associated with cholinergic nerve terminals support the hypothesis that prejunctional DORs regulate vagal transmission locally within the heart.     

The regulation of cytosolic pH in isolated presynaptic nerve terminals from rat brain

Cytosolic pH (pHi) was measured in presynaptic nerve terminals isolated from rat brain (synaptosomes) using a fluorescent pH indicator, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The synaptosomes were loaded with BCECF by incubation with the membrane-permanent acetoxy-methyl ester derivative of BCECF, which is hydrolyzed by intracellular esterases to the parent compound. pHi was estimated by calibrating the fluorescence signal after permeabilizing the synaptosomal membrane by two different methods. Synaptosomes loaded with 15-90 microM BCECF were estimated to have a pHi of 6.94 +/- 0.02 (mean +/- standard error; n = 54) if the fluorescence signal was calibrated after permeabilizing with digitonin; a similar value was obtained using synaptosomes loaded with 10 times less BCECF (6.9 +/- 0.1; n = 5). When the fluorescence signal was calibrated by permeabilizing the synaptosomal membrane to H+ with gramicidin and nigericin, pHi was estimated to be 7.19 +/- 0.03 (n = 12). With the latter method, pHi = 6.95 +/- 0.09 (n = 14) when the synaptosomes were loaded with 10 times less BCECF. Thus, pHi in synaptosomes was approximately 7.0 and could be more precisely monitored using the digitonin calibration method at higher BCECF concentrations. When synaptosomes were incubated in medium containing 20 mM NH4Cl and then diluted into NH4Cl-free medium, pHi immediately acidified to a level of approximately 6.6. After the acidification, pHi recovered over a period of a few minutes. The buffering capacity of the synaptosomes was estimated to be approximately 50 mM/pH unit. Recovery was substantially slowed by incubation in an Na-free medium, by the addition of amiloride (KI = 3 microM), and by abolition of the Nao/Nai gradient. pHi and its recovery after acidification were not affected by incubation in an HCO3-containing medium; disulfonic stilbene anion transport inhibitors (SITS and DIDS, 1 mM) and replacement of Cl with methylsulfonate did not affect the rate of recovery of pHi. It appears that an Na+/H+ antiporter is the primary regulator of pHi in mammalian brain nerve terminals.     

Ultrastructural identification of monoaminergic synaptosomes from one day old rat brain

Summary Synaptosomes from one day old and adult rat brain were studied. Specific cytochemical methods for demonstrating monoaminergic (MA) nerve endings were used. Permanganate fixation after preincubation with 5-OHDA or -methyl-NA demonstrated MA synaptosomes. Their number was small in the adult (less than 1%) and still smaller in the one day old rat brain. The MA synaptosomes from developing rats were different from the adult ones. The large amount of endoplasmic reticulum in developing synaptosomes suggests that granular vesicles are formed from endoplasmic reticulum in nerve endings.     

Aging Is Associated with a Decrease in Synaptosomal Glutamate Uptake and an Increase in the Susceptibility of Synaptosomal Vitamin E to Oxidative Stress

We examined the influence of aging upon the uptake of glutamate by synaptosomes, and the oxidation of Synaptosomal vitamin E. Synaptosomes isolated from the cerebral hemispheres of Fischer 344 rats, 4 and 24 months old, were suspended at 37°C in buffer (pH 7.4) simulating extracellular fluid containing 10 mM glucose. The K_m for the high affinity uptake of tritium labeled glutamate was 10 M. The uptake of glutamate was lower in synaptosomes from older animals than those from younger animals for periods of up to 20 minutes. Upon incubation with a mixture of ferrous iron and ascorbate, more of the alpha tocopherol in synaptosomes derived from older rats was oxidized than in those derived from younger ones. Older animals may be more susceptible to excitotoxicity because: a) Synaptosomal reuptake of glutamate is less efficient and b) oxidative stress induced by various agents including glutamate may be higher in synaptosomes from the older animal.     

Changes in free-calcium levels and pH in synaptosomes during transmitter release

The free cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) in rat brain synaptosomes estimated by the indicator quin 2 is 104±8 nM (S.D.) in artificial cerebrospinal fluid (1.2 mM Ca²⁺), but decreases at lower Ca²⁺ concentrations in the medium. The presence of quin 2 in the synaptosomes does not affect either the spontaneous release of transmitter (γ-aminobutyric acid) or the release induced by K⁺ depolarisation. In quin 2-loaded synaptosomes, depolarisation by K⁺ causes an abrupt increase in [Ca]_i (less than 2-fold) that is approximately proportional to the extent of depolarisation, whereas depolarisation by veratrine alkaloids produces a slow rise in [Ca]_i. The increase in [Ca]_i produced by K⁺ depolarisation does not occur in the absence of Ca²⁺ in the medium. The data are consistent with a direct correlation between [Ca_i] and transmitter release in functional synaptosomes. The pH in synaptosomes estimated by the indicator quene 1 is 7.04±0.07 and is stable in media containing 5 mM bicarbonate. The pH in synaptosomes was decreased by protoveratrine but not by K⁺ depolarisation.     

Glucose and Synaptosomal Glutamate Metabolism: Studies with [15N]Glutamate

The metabolism of [15N]glutamate was studied with gas chromatography-mass spectrometry in rat brain synaptosomes incubated with and without glucose. [15N]Glutamate was taken up rapidly by the preparation, reaching a steady-state level in less than 5 min. 15N was incorporated predominantly into aspartate and, to a much lesser extent, into gamma-aminobutyrate. The amount of [15N]ammonia formed was very small, and the enrichment of 15N in alanine and glutamine was below the level of detection. Omission of glucose substantially increased the rate and amount of [15N]aspartate generated. It is proposed that in synaptosomes (a) the predominant route of glutamate nitrogen disposal is through the aspartate aminotransferase reaction; (b) the aspartate aminotransferase pathway generates 2-oxoglutarate, which then serves as the metabolic fuel needed to produce ATP; (c) utilization of glutamate via transamination to aspartate is greatly accelerated when flux through the tricarboxylic acid cycle is diminished by the omission of glucose; (d) the metabolism of glutamate via glutamate dehydrogenase in intact synaptosomes is slow, most likely reflecting restriction of enzyme activity by some unknown factor(s), which suggests that the glutamate dehydrogenase reaction may not be near equilibrium in neurons; and (e) the activities of alanine aminotransferase and glutamine synthetase in synaptosomes are very low.