Improved Method for Isolating Synaptosomes from 11 Regions of One Rat Brain: Electron Microscopic and Biochemical Characterization and Use in the Study of Drug Effects on Nerve Terminal γ-Aminobutyric Acid in Vivo

A procedure is described for the rapid preparation of nerve ending particles (synaptosomes) from 11 regions of one rat brain. The synaptosomal fractions have been characterized by electron microscopy and determination of four marker enzymes, i.e., glutamate decarboxylase (GAD), acetylcholinesterase, succinate dehydrogenase, and glycerol 3-phosphate dehydrogenase. Comparison with a much lengthier standard (Ficoll-sucrose) preparation showed that the synaptosomal yield of the new procedure was substantially better as judged by both morphological evaluation and protein recovery. The improved synaptosome preparation was used for determination of regional gamma-aminobutyric acid (GABA) levels in synaptosomal fractions. The postmortem increase in GABA level during removal and dissection of brain tissue and homogenization and fractionation procedures could be minimized by rapid processing of the tissue at low temperatures and inclusion of the GAD inhibitor 3-mercaptopropionic acid (3-MP; 1 mM) in the homogenizing medium. The addition of GABA (0.2 mM) to the homogenizing medium did not alter the GABA levels in the synaptosomes, indicating that no significant redistribution of GABA occurred during subcellular fractionation in sodium-free media. Synaptosomal GABA levels determined in the 11 rat brain areas showed the same regional distribution as the GABA-synthesizing enzyme GAD. On the basis of these findings, it was suggested that the synaptosome preparation could be used to evaluate the in vivo effects of drugs on nerve terminal GABA. Treatment of rats with a convulsant dose of 3-MP (50 mg/kg i.p.) 3 min before decapitation significantly lowered synaptosomal GABA levels in olfactory bulb, hippocampus, thalamus, tectum, and cerebellum. The 3-MP-induced seizures and reduction of GABA levels could be prevented by administration of valproic acid (200 mg/kg i.p.) 15 min before the 3-MP injection. The data indicate that the improved synaptosome preparation offers a convenient method of preparing highly purified synaptosomes from a large number of small tissue samples and can provide useful information on the in vivo effects of drugs on regional GABA levels in nerve terminals.     

Specific lysis of GABAergic synaptosomes by an antiserum to glutamate decarboxylase

An antiserum to pure glutamate decarboxylase (GAD) when incubated with rat cortical synaptosomes in the presence of complement caused release of 33-53% of lactate dehydrogenase (LDH) and 22-41% of total GAD. In addition most of the gamma-aminobutyrate (GABA) present was released. Anti-GAD antiserum alone, or complement alone, were without action. The antiserum plus complement had no effect on noradrenaline or choline uptake, and did not release choline acetylase (ChAT). Anti-ChAT serum plus complement released 30-37% of ChAT and 10-13% of LDH. It prevented choline uptake. This serum did not produce GAD release or prevent GABA, choline or noradrenaline uptake. When cortical synaptosomes were exposed to both antisera plus complement, their actions were strictly additive. The data indicate specific lysis of GABAergic and cholinergic synaptosomal sub-populations.     

Sodium channel activity in brain membrane fractions isolated from rats of different ages

The Na⁺ channel activity (tetrodotoxin sensitive ²²Na⁺ flux induced by veratridine and/or anemone toxin II) was studied in two fractions of brain cell plasma membranes, named A and B, isolated by the method of Gray and Whittaker ((1962) J. Anat. 96, 79–87) from rats 5, 10, 30 and 60 days old. The ²²Na⁺ flux was measured in membrane vesicles formed by the isolated membranes, in the absence of drugs (control), in the presence of veratridine, and in the presence of veratridine plus tetrodotoxin. Fraction A consists primarily of neuronal and glial membranes in rats of 5 and 10 days of age, while in the older rats this fraction becomes enriched in myelin. In Fraction A of 5-day-old and 10-day-old rats, veratridine (25 μM) increases the ²²Na⁺ flux 2.4- and 1.6-fold, respectively, and the increment continues to diminish with age, until it becomes negligible in the 60-day-old rats. Fraction B consists of synaptosomes and membrane vesicles, and at the four ages studied veratridine (25 μM) causes an increment of the ²²Na⁺ flux of about 2.5-fold. Fractions A and B from 10-day-old rats, and Fraction B from 60-day-old rats, which are sensitive to veratridine, also respond to anemone toxin II. When veratridine is used in presence of anemone toxin II (0.5 μM), the $\text{K}{}_{0.5}$ for veratridine is diminished and the maximum ²²Na⁺ flux is increased. The increments of ²²Na⁺ flux caused by veratridine and/or anemone toxin II in Fractions A and B are blocked by tetrodotoxin ($\text{K}{}_{0.5}$ approx. 5 nM). Fraction A from 60-day-old rats could be subfractionated by osmotic shock and sucrose gradient centrifugation to obtain three subfractions, two of which are enriched in axolemma and display Na⁺ chennel activity. The other subfraction is enriched in myelin and shows no Na⁺ channel actiivty. The plasma membrane preparations from young rats (up to 10 days) are devoid of myelin and are useful for studies of Na⁺ channel activity.     

Neuronal Glutamine Utilization: Glutamine/Glutamate Homeostasis in Synaptosomes

The synaptosomal metabolism of glutamine was studied under in vitro conditions that simulate depolarization in vivo. With [2-15N]glutamine as precursor, the [glutamine]i was diminished in the presence of veratridine or 50 mM KCl, but the total amounts of [15N]glutamate and [15N]aspartate formed were either equal to those of control incubations (veratridine) or higher (50 mM [KCl]). This suggests that depolarization decreases glutamine uptake and independently augments glutaminase activity. Omission of sodium from the medium was associated with low internal levels of glutamine which indicates that influx occurs as a charged Na(+)-amino acid complex. It is postulated that a reduction in membrane potential and a collapse of the Na+ gradient decrease the driving forces for glutamine accumulation and thus inhibit its uptake and enhance its release under depolarizing conditions. Inorganic phosphate stimulated glutaminase activity, particularly in the presence of calcium. At 2 mM or lower [phosphate] in the medium, calcium inhibited glutamine utilization and the production of glutamate, aspartate, and ammonia from glutamine. At a high (10 mM) medium [phosphate], calcium stimulated glutamine catabolism. It is suggested that a veratridine-induced increase in intrasynaptosomal inorganic phosphate is responsible for the enhancement of flux through glutaminase; calcium affects glutaminase indirectly by modulating the level of free intramitochondrial [phosphate]. Because phosphate also lowers the Km of glutaminase for glutamine, augmentation of the amino acid breakdown may occur even when depolarization lowers [glutamine]i. Reducing the intrasynaptosomal glutamate to 26 nmol/mg of protein had little effect on glutamine catabolism, but raising the pH to 7.9 markedly increased formation of glutamate and aspartate. It is concluded that phosphate and H+ are the major physiologic regulators of glutaminase activity.     

Studies on the Role of B-50 (GAP-43) in the Mechanism of Ca2+-Induced Noradrenaline Release: Lack of Involvement of Protein Kinase C After the Ca2+ Trigger

Abstract: The involvement of B-50, protein kinase C (PKC), and PKC-mediated B-50 phosphorylation in the mechanism of Ca²⁺-induced noradrenaline (NA) release was studied in highly purified rat cerebrocortical synaptosomes permeated with streptolysin-O. Under optimal permeation conditions, 12% of the total NA content (8.9 pmol of NA/mg of synaptosomal protein) was released in a largely (>60%) ATP-dependent manner as a result of an elevation of the free Ca²⁺ concentration from 10^?8 to 10^?5M Ca²⁺ The Ca²⁺ sensitivity in the micromolar range is identical for [³H]NA and endogenous NA release, indicating that Ca²⁺-induced [³H]NA release originates from vesicular pools in noradrenergic synaptosomes. Ca²⁺-induced NA release was inhibited by either N- or C-terminal-directed anti-B-50 antibodies, confirming a role of B-50 in the process of exocytosis. In addition, both anti-B-50 antibodies inhibited PKC-mediated B-50 phosphorylation with a similar difference in inhibitory potency as observed for NA release. However, in a number of experiments, evidence was obtained challenging a direct role of PKC and PKC-mediated B-50 phosphorylation in Ca²⁺-induced NA release. PKC pseudosubstrate PKC_19-36, which inhibited B-50 phosphorylation (IC₅₀ value, 10^?5M), failed to inhibit Ca²⁺-induced NA release, even when added before the Ca²⁺ trigger. Similar results were obtained with PKC inhibitor H-7, whereas polymyxin B inhibited B-50 phosphorylation as well as Ca²⁺-induced NA release. Concerning the Ca²⁺ sensitivity, we demonstrate that PKC-mediated B-50 phosphorylation is initiated at a slightly higher Ca²⁺ concentration than NA release. Moreover, phorbol ester-induced PKC down-regulation was not paralleled by a decrease in Ca²⁺-induced NA release from streptolysin-O-permeated synaptosomes. Finally, the Ca²⁺- and phorbol ester-induced NA release was found to be additive, suggesting that they stimulate release through different mechanisms. In summary, we show that B-50 is involved in Ca²⁺-induced NA release from streptolysin-O-permeated synaptosomes. Evidence is presented challenging a role of PKC-mediated B-50 phosphorylation in the mechanism of NA exocytosis after Ca²⁺ influx. An involvement of PKC or PKC-mediated B-50 phosphorylation before the Ca²⁺ trigger is not ruled out. We suggest that the degree of B-50 phosphorylation, rather than its phosphorylation after PKC activation itself, is important in the molecular cascade after the Ca²⁺ influx resulting in exocytosis of NA.     

Presynaptic α2 Adrenoceptors Inhibit Glutamate Release from Rat Spinal Cord Synaptosomes

Abstract: The presynaptic regulation of amino acid release from nerve terminals was investigated using synaptosomes prepared from the rat spinal cord. The basal releases of endogenous glutamate (Glu), aspartate (Asp), and γ-amino-butyric acid (GABA) were 34.6, 21.5, and 10.0 pmol/min/mg of protein, respectively. Exposure to a depolarizing concentration of KCl (30 m M ) evoked 2.7-, 1.5-, and 2.9-fold increases in Glu, Asp, and GABA release, respectively. Clonidine reduced the K⁺-evoked overflow of Glu to 56% of the control overflow with a potency (IC₅₀) of 17 n M , but it did not affect K⁺-evoked overflow of Asp, GABA, and their basal releases. Similarly, noradrenaline inhibited the K⁺-evoked overflow of Glu, although phenylephrine and isoproterenol showed no effect. The inhibitory effect of clonidine was counteracted by α₂-adrenoceptor antagonists, rauwolscine, yohimbine, and idazoxan, regardless of the imidazoline structures. Because Glu is considered a neurotransmitter of primary afferents that transmit both nociceptive and nonnociceptive stimuli in the spinal cord, these data suggest that part of Glu release may be regulated by the noradrenergic system through α₂ adrenoceptors localized on the primary afferent terminals.     

Effect of oxidative stress on excitatory amino acid release by cerebral cortical synaptosomes

Previous studies in our laboratory have suggested that an oxidation reaction is responsible for the actions of free radicals to decrease synaptic potentials. Recently we observed that free radicals both decreased depolarization-induced vesicular release and enhanced basal, nonvesicular release of the excitatory amino acid, [³H]L-glutamate. In order to evaluate the contribution of oxidative reactions to this latter effect, we evaluated the actions of the oxidizing agent chloramine-T on synaptosomal release of excitatory amino acids, using [³H]D-aspartate as the exogenous label. Basal and depolarization evoked [³H]D-aspartate release were calcium-independent and nonvesicular. Chloramine-T pretreatment significantly increased basal release, while having no effect on high K⁺-evoked release. These data suggest that an oxidative process can mimic the free radical increase of basal release, as well as the decrease in synaptic potentials. On the other hand, the calcium-independent-evoked release may involve a different mechanism. Our results demonstrate that under basal, nondepolarizing conditions, oxidative stress exerts an adverse effect on the presynaptic nerve terminal, resulting in an increased release of potentially damaging excitatory amino acid neurotransmitters.     

Persistent Enhancement of Sustained Calcium-Dependent Glutamate Release by Phorbol Esters: Role of Calmodulin-Independent Serine/Threonine Phosphorylation and Actin Disassembly

Abstract: The phorbol ester 4β-phorbol 12,13-dibutyrate increases the final extent of Ca²⁺-dependent glutamate release during the continuous depolarization of the synaptosomal plasma membrane. Based on this finding, we suggested that the sustained activation of protein kinase C has a positive influence on the efficiency of synaptic vesicle recycling in the presence of saturating concentrations of Ca²⁺. Previous work from our laboratory demonstrated that this 4β-phorbol 12,13-dibutyrate-dependent enhancement of synaptic vesicle recycling persists following the removal of 4β-phorbol 12,13-dibutyrate, requires localized Ca²⁺ entry through voltage-regulated channels, and is insensitive to the protein kinase inhibitor staurosporine. In the present study, we examined the possibility that the facilitation of glutamate release may be propagated through interactions between the protein kinase C- and multifunctional Ca²⁺/calmodulin-dependent protein kinase pathways. However, our data argue strongly against the involvement of such a mechanism in the persistent enhancement of sustained glutamate release. We observed that 4β-phorbol 12,13-dibutyrate did not increase the availability of cytosolic free calmodulin or the level of autonomous Ca²⁺/calmodulin-dependent protein kinase activity. In addition, we determined the effects of various serine/threonine kinase and phosphatase inhibitors on the phorbol ester-dependent enhancement of sustained glutamate release and found that protein kinase C increased the extent, but not the duration, of Ca²⁺-dependent glutamate release through a kinase-independent mechanism. Given our finding that the actin-depolymerizing agent cytochalasin D totally occluded the effect of 4β-phorbol 12,13-dibutyrate on release, we postulate that protein kinase C signals may be transduced through direct interactions between protein kinase C isoforms and cytoskeletal protein kinase C binding proteins.     

The effect of sodium on depolarization-induced calcium uptake and acetylcholine release by synaptosomes

The influence of external sodium concentration on potassium (depolarizing agent)-stimulated calcium uptake and Ca⁺-dependent acetylcholine release by rat cerebral cortex synaptosomes has been studied. It was found that increased sodium concentration decreases both the Ca²⁺ uptake and the acetylcholine release, whereas a low external sodium concentration is stimulatory.     

Characterization of the Release of Cholecystokinin-8 from Isolated Nerve Terminals and Comparison with Exocytosis of Classical Transmitters

In the present study, the release of the neuropeptide cholecystokinin-8 (CCK) from purified nerve terminals (synaptosomes) of the rat hippocampus was characterized with respect to the subcellular distribution, the release upon addition of various agents, the release kinetics, the Ca2+ and ATP dependence of release, and the relationship between CCK release and elevations of intraterminal free Ca2+ concentration ([Ca]i). These characteristics were compared with those for the release of classical transmitters in similar preparations. CCK-like immunoreactivity (CCK-LI) is enriched in the purified synaptosomal fraction of hippocampus homogenates and released in a strictly Ca2(+)-dependent manner upon chemical depolarization, addition of 4-aminopyridine, or stimulation with the Ca2+ ionophore ionomycin. The presence of Ca2+ in the medium significantly stimulates the basal efflux of CCK-LI from synaptosomes. The release upon stimulation develops gradually in time with no significant release in the first 10 s and levels off after 3 min of depolarization. At this time, a large amount of CCK-LI is still present inside the synaptosomes. A correlation exists between the release of CCK-LI and the elevations of [Ca]i. The release of CCK-LI is decreased, but not blocked, upon ATP depletion. These characteristics markedly differ from those for classical transmitters, which show a fast component of Ca2(+)-dependent (exocytotic) release, an absolute dependence on cellular ATP, and no marked stimulation of basal efflux in the presence of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)