THE EFFECT OF ELECTRICAL STIMULATION AND HIGH POTASSIUM CONCENTRATIONS ON THE EFFLUX OF [3H] γ-AMINOBUTYRIC ACID FROM BRAIN SLICES

Abstract— Brain slices were incubated with [³H]GABA in a medium containing aminooxyacetic acid to prevent metabolism of [³H]GABA by GABA-glutamate transaminase. The slices, which rapidly accumulated radioactivity, were then continuously perfused and the efflux of [³H]GABA from the tissue was measured. The spontaneous efflux of [³H]GABA consisted of an initial rapid phase followed by a much slower release of [³[H]GABA. After 40 min perfusion 90 per cent of the radioactivity remained in the tissue.
The slices were depolarized by electrical stimulation or by perfusion with a medium containing a high potassium concentration (40 mM). These procedures caused a striking increase in the efflux of [³H]GABA. The increased efflux produced by potassium, but not that produced by electrical stimulation, was dependent on calcium ions in the medium. The effect of electrical stimulation on [³H]GABA release was considerably reduced by a raised concentration (10 mM) of magnesium in the medium.
High potassium concentrations and electrical stimulation did not cause an increase in the efflux of [¹⁴C]urea, L-[³H]leucine or [¹⁴C]α-amino-isobutyric acid from brain slices. These results are consistent with the suggestion that GABA may be an inhibitory transmitter in the cerebral cortex.     

THE FORMATION OF CHOLINE AND OF ACETYLCHOLTNE BY BRAIN IN VITRO

Abstract— Free choline and acetylcholine (ACh) in mouse or rat brain were assayed biologically. The subcellular distribution of ACh in brain slices that had been incubated in the presence of eserine was compared to that in control brain; during incubation, the ACh outside nerve endings increased four-fold, the ACh released from synaptosomes by osmotic shock doubled but the ACh bound firmly within nerve endings did not increase. The two nerve ending stores of ACh were labelled to similar specific radioactivities when slices were incubated with [³H]choline, but the specific radioactivity of the ACh formed was much lower than that of the added choline. Tissue incubated in the presence of eserine released choline and ACh into the medium and the tissue levels of both substances increased. Brain tissue exposed to Na⁺-free medium lost 84 per cent of its ACh and 66 per cent of its free choline; the amounts of both substances returned towards control values during subsequent incubation in a normal-Na⁺ medium (choline-free). Both the ACh outside nerve endings and the ACh associated with synaptosomes were depleted when tissue was incubated in Na⁺-free medium.     

SYNTHESIS OF RADIOACTIVE ACETYLCHOLINE FROM [3H]CHOLINE AND ITS RELEASE FROM CEREBRAL CORTEX SLICES IN VITRO

Abstract— Guinea pig cerebral cortex slices were incubated for 60 min in a medium containing [³H]choline with or without the addition of 33 mM-KCl for the last 30 min. KC1 caused the release into the medium of large amounts of both bioassayable and radioactive ACh, while at the same time their concentrations in the tissue decreased. The specific activity (d.p.m./pmol) of the ACh released by KC1 was greater than that released in control incubations, indicating that it comes from a newly synthesized, more radioactive store. The amounts of [³H]choline, [³H]ACh and the specific activity of tissue acetylcholine reached a plateau in the tissue 30 min after the addition of isotope. However isotopic equilibrium was not achieved because the specific activity of the ACh released, with or without KC1 in the subsequent 30 min, was less than the specific activity of the ACh remaining in the tissue. This implies the existence of a pool of ACh in the tissue which is turning over very slowly or is being synthesized from a less radioactive pool of choline. This pool of ACh does not contribute substantially to that released by KC1. Levorphanol at 10⁻³ M, as well as the analgesically inactive stereoisomer, dextrorphan, blocked the KCl-stimulated release of both bioassayable and radioactive ACh. These drugs demonstrate the coupling of synthesis and release of ACh in cerebral cortex slices.     

Effect of Protein Kinase C Activation on the Release of [3H]Acetylcholine in the Presence of Vesamicol

Abstract: The present work tested whether pharmacological activation of protein kinase C (PKC) influences the release of [³H]-acetylcholine ([³H]ACh) synthesized in the presence of vesamicol, an inhibitor of the vesicular acetylcholine transporter (VAChT). Newly synthesized [³H]ACh was released from hippocampal slices by field stimulation (15 Hz) in the absence of vesamicol, but as expected [³H]ACh synthesized during exposure to vesamicol was not released significantly by stimulation. Treatment of slices with the PKC activator phorbol myristate acetate (PMA) decreased the inhibitory effect of vesamicol on [³H]ACh release. The effect of PMA was dose-dependent, was sensitive to calphostin C, a PKC-selective inhibitor, and could not be mimicked by α-PMA, an inactive phorbol ester. PMA did not alter the release of [³H]ACh in the absence of vesamicol, suggesting that the site of PKC action could be related to the VAChT. In agreement with this observation, immunoprecipitation of VAChT from ³²P-labeled synaptosomes showed that phosphorylation occurs and that incorporation of ³²P in the VAChT protein increases in the presence of PMA. We suggest that PKC alters the output of [³H]ACh formed in the presence of vesamicol and also provide circumstantial evidence for a role of phosphorylation of VAChT in this process.     

KINETICS OF [3H]ACETYLCHOLINE SYNTHESIS AND RELEASE IN PRIMARY CELL CULTURES FROM MAMMALIAN CNS

Abstract— The present study was undertaken to characterize the cholinergic system of primary cell cultures of mouse and rat CNS.
In confirmation of previous reports, primary cultures were found to contain choline acetyltransferase (ChAc). Furthermore they contain acetylcholine (ACh) as measured by two different bioassays. They also synthesize [³H]ACh from [³H]Choline offered to the cultures.
The formation of [³H]ACh is inhibited in the presence of hemicholinium-3 (10⁻⁶ m ) to 50% or ouabain (10⁻³ m ) to 20% of the values found in untreated cultures. Omission of Na ⁺ from the incubation solution also diminishes the [³H]ACh formation of the cells.
[³H]ACh is released upon depolarisation by K⁺ ions in a concentration dependent manner. The release can be prevented by lack of Ca²⁺ ions in the incubation solution.     

EFFLUX OF PHENYLALANINE FROM RAT CEREBRAL CORTEX SLICES AS INFLUENCED BY EXTRA- AND INTRACELLULAR AMINO ACIDS

Abstract— Effects of other amino acids on the efflux of l -[³H]phenylalanine from rat cerebral cortex slices were studied in a superfusion system. Extracellular large neutral amino acids caused a strong trans-stimulation of [³H]phenylalanine efflux. Some small neutral amino acids were less effective, whereas acidic and basic amino acids and the amino acids without an amino group in the α-position were ineffective. Any trans -inhibition was not detected. The stimulatory trans -effects of phenylalanine and tryptophan were additive, reversible and concentration-dependent. They were apparently mediated by the same mechanisms. The efflux of [³H]phenylalanine was much slower at 273 K than at 310 K, but the effects of unlabelled phenylalanine and tryptophan on it were qualitatively similar at both temperatures. Amino acids accumulated intracellularly at moderately high concentrations did not inhibit [³H]phenylalanine efflux, but phenylalanine, leucine, isoleucine and norleucine caused an enhancement. Spontaneous efflux of [³H]phenylalanine showed some similarities to physical diffusion, but its selective and specific modification by other amino acids strongly suggests the involvement of mediated processes.     

ACETYLCHOLINE TRANSLOCATION IN SYNAPTIC VESICLE GHOSTS IN VITRO

Abstract— Translocation of acetylcholine (ACh) into cholinergic synaptic vesicles depleted of ACh and ATP was studied by Sephadex gel filtration. The hypo-osmotically shocked vesicles become transiently leaky, but retain ACh under iso-osmotic conditions. Intravesicular accumulation of [³H]ACh is due to a simple diffusional equilibration. Addition of 2mM-ATP and Mg²⁺ to the incubation medium is without effect. When acetylcoenzyme A (AcCoA) and choline (Ch ⁺) are used in place of preformed ACh, the intravesicular concentration of ACh does not exceed that of the newly synthesized, extravesicular ACh. However, in the absence of Na ⁺ the quantity of [³H]ACh associated with the vesicles increased, presumably due to ACh binding to ion-exchanger sites in the vesicles.     

RELEASE OF [3H]GAMMA-AMINOBUTYRIC ACID FROM GLIAL CELLS IN RAT DORSAL ROOT GANGLIA.

Abstract— Desheathed rat dorsal root ganglia were incubated in a medium containing amino-oxyacetic acid and [³H]GABA. Under these conditions, [³H]GABA is taken up exclusively by the satellite glial cells in the ganglia. Efflux of [³H]GABA from the tissue was measured after passing the ganglia through a series of wash solutions. The spontaneous efflux of radioactivity, mostly [³H]GABA, was more rapid in the absence of amino-oxyacetic acid in the incubation and wash media.
Raising the potassium concentration in the wash media caused an increase in the efflux of [³H]GABA. This increase was sigmoidally related to the potassium concentration in the wash media, reaching a maximum at 64 m m -K⁺. The releasing effect of K⁺ was inhibited by removing calcium from the media. Reducing the calcium and raising the magnesium concentration in the wash solutions inhibited the increased efflux of [³H]GABA due to 64 m m -K⁺ by 48 per cent, while 5 mM-La³⁺ and diphenylhydantoin (0·005 and 0·5 m m ) had no effect on this increase.
Only a small increase in the efflux of [¹⁴C]glutamate was produced by 64 m m -K⁺ and it had no effect upon the effluxes of [³H]glycine, [³H]alanine or [³H]leucine. The efflux of lactate dehydrogenase was similarly unaffected by 64 mM-K⁺. The results suggest that glial cells in spinal ganglia can respond to depolarizing concentrations of potassium by releasing GABA in a calcium-dependent process.     

THE RELEASE OF AMINO ACIDS FROM THE HEMISECTED SPINAL CORD DURING STIMULATION

Abstract— Isolated frog or toad hemicords were incubated for 40 min with either [¹⁴C]glycine, [³H]GABA, l -[¹⁴C]glutamate. l -[¹⁴C]aspartate, l -[¹⁴C]serine, l [¹⁴C]threonine or l -[³H]leucine, and the release of these compounds from the cord was measured under resting conditions and during electrical stimulation. Stimulation of spinal roots produced no significant change in the efflux of any of the compounds tested. Direct stimulation of the rostral cord however, produced a large increase in the efflux of [¹⁴C]glycine, [³H]GABA, l -[¹⁴C]glutamate and l -[¹⁴C]aspartate. These increased effluxes were calcium dependent, the effects of stimulation being reduced in a calcium-free, or magnesium-supplemented (10 mM) medium. Stimulation failed to produce an increase in the efflux of l -[¹⁴C]serine, l -[¹⁴C]threonine, l -[¹⁴H]leucine, [¹⁴C]mannitol or [¹⁴C]urea. These results are consistent with the suggestions that glycine, GABA, glutamate and aspartate may be synaptic transmitters in the spinal cord.     

Platelet-Activating Factor: Diminished Acetylcholine Release from Rat Brain Slices Is Mediated by a Gi Protein

Abstract: The effect of platelet-activating factor (PAF) on neurotransmitter release from rat brain slices prelabeled with [³H]acetylcholine ([³H]ACh), [³H]norepinephrine ([³H]NE), or [³H]serotonin ([³H]5-HT) was studied. PAF inhibited K⁺ depolarization-induced [³H]ACh release in slices of brain cortex and hippocampus by up to 59% at 10 n M but did not inhibit [³H]ACh release in striatal slices. PAF did not affect 5-HT or NE release from cortical brain slices. The inhibition of K⁺-evoked [³H]ACh release induced by PAF was prevented by pretreating tissues with several structurally different PAF receptor antagonists. The effect of PAF was reversible and was not affected by pretreating brain slices with tetrodotoxin. PAF-induced inhibition of [³H]ACh release was blocked 90 ± 3 and 86 ± 2% by pertussis toxin and by anti-Gαi_1/2 antiserum incorporated into cortical synaptosomes, respectively. The results suggest that PAF inhibits depolarization-induced ACh release in brain slices via a Gαi_1/2 protein-mediated action and that PAF may serve as a neuromodulator of brain cholinergic system.     

Cholinergic Modulation of the Release of [3H]Acetylcholine from Synaptosomes of the Myenteric Plexus

Abstract: The purpose of these experiments was to determine if cholinergic agents affected the release of acetylcholine (ACh) from a synaptosomal preparation of the guinea pig ileum myenteric plexus. The synaptosomal preparation was first incubated with the precursor [³H]choline; subsequently, release of the stored [³H]ACh was measured. The release was decreased by oxotremorine or exogenous ACh plus hexamethonium and increased by exogenous ACh plus atropine. The nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) evoked release that was inhibited by nicotinic antagonists or muscarinic agonists. Release was stimulated half-maximally by approximately 2 μ m - and maximally by 10 μ m -DMPP. Either in the absence of calcium or at 0°C, DMPP was without effect. The effect of 10 μ m -DMPP was brief, a significant stimulation occurring only within the first 2 min at 37°C. Tetrodotoxin also inhibited excitation by DMPP but not completely. Thus, the release of [³H]ACh appears to be presynaptically modulated, negatively by muscarinic agonists and positively by nicotinic agonists.     

Dopamine Agonist-Mediated Inhibition of Acetylcholine Release in Rat Striatum Is Modified by Thyroid Hormone Status

Abstract: K⁺-evoked acetyl[³H]choline ([³H]ACh) release was inhibited in a concentration-dependent manner by apomorphine and the D₂ agonist quinpirole in striatal slices prepared from euthyroid and hypothyroid rats. However, there was a significant increase in the maximum inhibition observed with both agonists in the hypothyroid compared with the euthyroid group, which paralleled the increased D₂ agonist sensitivity reported for stereotyped behavior. The D₂ antagonist raclopride decreased, and the D, antagonist SCH 23390 increased, the inhibition of [³H]ACh release by apomorphine, confirming an inhibitory role for D₂ receptors and an opposing role for D₁ receptors. Because there is no difference in D₁ or D₂ receptor concentration between the euthyroid and hypothyroid groups, it is suggested that thyroid hormone modulation of D₂ receptor sensitivity affects a receptor-mediated event. Following intrastriatal injection of pertussis toxin (PTX), apomorphine no longer inhibited [³H]ACh release. In fact, increased [³H]- ACh release was observed, an effect reduced by SCH 23390, providing evidence that D₁ receptors enhance [³H]- ACh release, and confirming that a PTX-sensitive G protein mediates the D₂ response. As it has been reported that thyroid hormones modulate G protein expression, this mechanism may underlie their effect on dopamine agonist- mediated inhibition of ACh.     

EFFECT OF CHOLINE ON THE RATES OF SYNTHESIS and OF RELEASE OF ACETYLCHOLINE IN THE ELECTRIC ORGAN OF TORPEDO

Abstract— Slices of electric organ of Torpedo marmorata were chopped and incubated in a saline-urea-sucrose medium. This preparation of minced tissue exhibited a relative enrichment in ACh and nerve endings, which was attributed to a loss of electroplaque cytoplasm. Electron microscopic controls showed nerve endings of normal morphology, some of them forming 'chaplets' separated from electro-plaques. Miniature endplate potentials were recorded on sealed fragments also present in this preparation. ACh levels remained unchanged during incubation periods as long as 19 h. The time course of the incorporation of [1-¹⁴C]acetate of [2-¹⁴C]pyruvate into ACh pools was studied. These incorporations were similarly affected by the choline added to the medium. In the presence of increasing choline concentrations (up to 10^-4 m ), the incorporation of [¹⁴C]acetate or [¹⁴C]pyruvate into ACh increased. They both diminished when choline was added above 10^-4M. The ACh content of the tissue was not affected by added choline. From the constancy of ACh levels in the presence of various choline concentrations and from the steady state of our preparation, we can conclude that the release of transmitter varied in parallel to the incorporation rate of the precursor of the acetyl moiety of ACh. This fact was also found using the efflux of [¹⁴C]acetate as an evaluation of ACh release. The values of release calculated by this method were in good agreement with those determined from the incorporations of acetate and pyruvate into ACh. It is suggested that the primary action of choline is on its high affinity carrier system. This triggers a secondary action on the ACh release mechanisms.     

ACETYLCHOLINE METABOLISM AND CHOLINE UPTAKE IN CORTICAL SLICES

Abstract— The uptake of [¹⁴C]choline was studied in cortical slices from rat brain after their incubation in a Krebs-Henseleit medium containing either 4.7 m m -KCl (low K), 25 m m -KCl (high K) or 25 m m -KCl without calcium (Ca free, high K). With 0.84 μ m -[¹⁴C]choline in the medium the uptake per gram of tissue was 0.62 nmol after incubation in low K medium, 1.13 nmol after incubation in high K medium and 0.78 nmol after incubation in a Ca free, high K medium. The differences caused by potassium were greater in fraction P₂ than in fractions P₁ and S₂. With 17 and 50 μ m -[¹⁴C]choline in the medium greater amounts of [¹⁴C]choline were taken up, but the effect of potassium on the uptake almost disappeared. The amount of radioactive material in fraction P₂ followed Michaelis-Menten kinetics with K _m values of 2.1 and 2.3 μ m after incubation in low and high K medium, respectively. Hemicholinium-3 only slightly inhibited choline uptake from a medium with 0.84 μ m -[¹⁴C]choline, but it abolished the extra-uptake induced by high K medium. The radioactivity in the slices consisted mainly of unchanged choline and little ACh was formed after incubation in low K medium, but after incubation in high K medium 50% of the choline taken up was converted into ACh. The hemicholinium-3 sensitive uptake of choline, the conversion of choline into ACh and the synthesis of total ACh, were stimulated about 7–8-fold by potassium. It is concluded that in cortical slices from rat brain all choline used for the synthesis of ACh is supplied by the high-affinity uptake system, of which the activity is geared to the rate of ACh synthesis.     

Differential Labeling of Depot and Active Acetylcholine Pools in Nondepolarized and Potassium-Depolarized Rat Brain Synaptosomes

Abstract: To test the hypothesis that a pool of newly synthesized acetylcholine (ACh) turns over independently of preformed ACh, compartmentation and K⁺ -evoked release of ACh were examined in perfused synaptosomal beds intermittently stimulated by 50 m M K⁺. In resting synaptosomes, endogenous and labeled ACh was distributed between synaptic vesicles and the cytoplasm in a dynamic equilibrium ratio of 4:6. In the absence of new ACh synthesis, five sequential K⁺ -depolarizations caused a decremental release of preformed labeled ACh totaling 30% of the initial transmitter store. Further depolarization evoked little additional release, despite the fact that 60% of the labeled ACh remained in these preparations. Release of the preformed [¹⁴C]ACh was unaltered while new ACh was being synthesized from exogenous [³H]choline. Since the evoked release of [³H]ACh was maintained while that of [¹⁴C]ACh was decreasing, the [³H]ACh/[¹⁴C]ACh ratio in perfusate increased with each successive depolarization. This ratio was six to ten times higher than the corresponding ratio in vesicles or cytoplasm. These results indicate that the newly synthesized ACh did not equilibrate with either the depot vesicular or cytoplasmic ACh pools prior to release.     

VESICULAR STORAGE AND RELEASE OF ACETYLCHOLINE IN TORPEDO ELECTROPLAQUE SYNAPSES

Abstract— The disposition of newly synthesized ACh subsequent to depletion of vesicular endogenous ACh by stimulation was studied in the electromotor nerve terminals of Torpedo marmorata using [³H]acetate as a precursor of ACh. Little vesicular [³H]ACh could be isolated from tissue immediately after stimulation at 1 Hz. After 3 h post-stimulation recovery the newly synthesized [³H]ACh is found predominantly in a subpopulation of vesicles distinct from the vesicles containing most of the endogenous poorly labelled ACh. Restimulation of the tissue causes release of highly labelled ACh with a specific radioactivity (SRA) comparable to that of the newly synthesized [³H]ACh in the highly labelled subpopulation of vesicles and significantly greater than the SRA of ACh in the main vesicular pool or the total tissue.     

FACTORS AFFECTING THE SPONTANEOUS RELEASE OF [3H] γ-AMINOBUTYRIC ACID FROM THE FROG RETINA IN VITRO

Abstract— The spontaneous efflux of [³H]GABA and its metabolites from the frog retina has been studied. The efflux of radioactivity was multiphasic in the presence or absence of amino-oxyacetic acid (AOAA), an inhibitor of GABA metabolism, and was not affected by light or dark adaptation.
Strong retention of radioactivity was evident in the presence of AOAA, about 90% of the label remaining in the tissue after 4 h superfusion. Under these conditions, increases in the rate of release of radioactivity were evoked by electrical stimulation, 40 m m -potassium. unlabelled GABA (5 m m ), ouabain (5 × 10⁻⁵ m ) and the absence of calcium. The amount of [³H]GABA released by electrical stimulation was not markedly calcium dependent, whereas the response to 40 m m -potassium was reduced by 96% in the absence of calcium.     

MELATONIN: DEACETYLATION TO 5-METHOXYTRYPTAMINE BY LIVER BUT NOT BRAIN ARYL ACYLAMIDASE

Abstract— Rat liver and brain slices were incubated in vitro with [³H]melatonin. Liver slices synthesized small amounts of [³H]5-methoxyindoleacetic acid ([³H]5-MIAA) along with other melatonin metabolites including 6-hydroxymelatonin. Pretreatment of animals prior to killing with the irreversible monoamine oxidase inhibitor pargyline allowed [³H]5-methoxytryptamine ([³H]5-MT) to be recovered from the incubation. No [³H]5-MIAA or [³H]5-MT could be detected in incubations with hypothalamic slices or following intraventrieular injection of [³H]melatonin. The possibility that the deacetylase aryl acylamidase was in part responsible for the deacetylation occurring in liver slices was examined. Liver aryl acylamidase was able to utilize [³H]melatonin as substrate to produce [³H]5-MT. Furthermore, the liver enzyme was inhibited by melatonin ( K_i. 1 m m ) when tested with the alternate substrate o -nitroacetanalide. Brain aryl acylamidase did not generate any detectable [³H]5-MT nor was it inhibited by melatonin. These results suggest that 5-MT is not formed in brain from melatonin although trace amounts of 5-MT in the periphery could be derived from this precursor.     

Increase in Chloride-Dependent l-Glutamate Transport Activity in Synaptic Membrane After In Vitro Ischemic Treatment

Abstract: The effect of energy failure on Cl⁻-dependent l -glutamate ( l -Glu) transport was examined with an in vitro preparation. Rat brain slices were incubated in low oxygen and glucose-deprived medium (in vitro ischemia), and a synaptic membrane fraction was prepared from the slices. Cl⁻-dependent l -[³H]Glu uptake into vesicles increased about twofold after 20 min of in vitro ischemia. The increased l -[³H]Glu uptake was inhibited by l -Glu, dl -2-amino-4-phosphonobutyrate, l -homocysteic acid, l -cystine, 4,4'-diisothiocyano-2,2'-disulfonic stilbene, and removal of Cl⁻. Uptakes of Na⁺-dependent l -[³H]-Glu, [³H]GABA, and [³H]taurine were not changed by the in vitro ischemia. In vitro ischemia increased the V _max value without affecting the K _m value. The increased l -[³H]Glu uptake by in vitro ischemia was reduced by subsequent incubation in a normoxic glucose-containing solution. ATP content in brain slices decreased to <10% of control values by in vitro ischemia for 10 min. The decrease in ATP content was restored by subsequent incubation in normoxic glucose-containing solution. Treatment with veratrine, 2,4-dinitrophenol, carbonyl cyanide m -chlorophenylhydrazone, and NaCN in normoxic conditions increased l -[³H]Glu uptake with a concomitant decrease in ATP content in slices. These results suggest that Cl⁻-dependent l -Glu transport activity in synaptic membranes increases in ischemia- or hypoxia-induced brain energy failures.     

[3H]Imipramine Is Accumulated but Not Released from Slices of the Rabbit Caudate and Hypothalamus

Abstract: Slices of rabbit caudate and hypothalamus take up and accumulate [³H]imipramine. In superfused slices of both structures electrical stimulation or exposure to tyramine failed to release recently taken up [³H]imipramine. De-polarization by exposure to 30–60 mm-potassium caused only a small release of [³H]imipramine that was not concentration-dependent. The release of [³H]imipramine by high potassium was independent of the presence of calcium ions in the superfusion medium. These results contrasted with those obtained for the release of [³H]dopamine from the caudate and [³H]noradrenaline from the hypothalamus, where tyramine, electrical stimulation, and high potassium caused a significant release of the labeled neurotransmitters. The release of [³H]dopamine from the caudate and [³H]noradrenaline from the hypothalamus elicited by electrical stimulation or high potassium was entirely calcium-dependent. It is concluded that [³H]imipramine is taken up into the two brain regions and is accumulated in a nonvesicular site from which it is not released by calcium-dependent depolarizing stimuli.