The actin cytoskeleton and neurotransmitter release: an overview

Here we review evidence that actin and its binding partners are involved in the release of neurotransmitters at synapses. The spatial and temporal characteristics of neurotransmitter release are determined by the distribution of synaptic vesicles at the active zones, presynaptic sites of secretion. Synaptic vesicles accumulate near active zones in a readily releasable pool that is docked at the plasma membrane and ready to fuse in response to calcium entry and a secondary, reserve pool that is in the interior of the presynaptic terminal. A network of actin filaments associated with synaptic vesicles might play an important role in maintaining synaptic vesicles within the reserve pool. Actin and myosin also have been implicated in the translocation of vesicles from the reserve pool to the presynaptic plasma membrane. Refilling of the readily releasable vesicle pool during intense stimulation of neurotransmitter release also implicates synapsins as reversible links between synaptic vesicles and actin filaments. The diversity of actin binding partners in nerve terminals suggests that actin might have presynaptic functions beyond synaptic vesicle tethering or movement. Because most of these actin-binding proteins are regulated by calcium, actin might be a pivotal participant in calcium signaling inside presynaptic nerve terminals. However, there is no evidence that actin participates in fusion of synaptic vesicles.     

Independent Regulation of Basal Neurotransmitter Release Efficacy by Variable Ca2+ Influx and Bouton Size at Small Central Synapses

The efficacy of action potential evoked neurotransmitter release varies widely even among synapses supplied by the same axon, and the number of release-ready vesicles at each synapse is a major determinant of this heterogeneity. Here we identify a second, equally important, mechanism for release heterogeneity at small hippocampal synapses, the inter-synaptic variation of the exocytosis probability of release-ready vesicles. Using concurrent measurements of vesicular pool sizes, vesicular exocytosis rates, and presynaptic Ca²⁺ dynamics, in the same small hippocampal boutons, we show that the average fusion probability of release-ready vesicles varies among synapses supplied by the same axon with the size of the spike-evoked Ca²⁺ concentration transient. We further show that synapses with a high vesicular release probability exhibit a lower Ca²⁺ cooperativity, arguing that this is a direct consequence of increased Ca²⁺ influx at the active zone. We conclude that variability of neurotransmitter release under basal conditions at small central synapses is accounted for not only by the number of release-ready vesicles, but also by their fusion probabilities, which are set independently of bouton size by variable spike-evoked presynaptic Ca²⁺ influx.

Author Summary

Synaptic transmission underlies information transfer among neurons in the brain. The probability that a synapse will release neurotransmitter in response to an action potential varies widely, even among synapses supplied by the same axon. The molecular mechanisms underlying this heterogeneity remain poorly understood. At the level of single synapses, release efficacy is determined largely by two factors: (i) the number of neurotransmitter-containing vesicles ready to be released, and (ii) by the fusion probabilities of these vesicles. By using novel imaging techniques at individual hippocampal presynaptic boutons in culture, we distinguish two independent sources of variability of release probability in small central synapses. First, we find differences in the number of releasable vesicles, and second, we find differences in the exocytosis probability of individual vesicles. To our knowledge, this is the first direct experimental demonstration that the fusion probability of release-ready vesicles is variable among synapses supplied by a single axon, and contributes roughly as much to the overall variability in release probability as does the number of release-ready vesicles.     

Presynaptic active zones in invertebrates and vertebrates

The regulated release of neurotransmitter occurs via the fusion of synaptic vesicles (SVs) at specialized regions of the presynaptic membrane called active zones (AZs). These regions are defined by a cytoskeletal matrix assembled at AZs (CAZ), which functions to direct SVs toward docking and fusion sites and supports their maturation into the readily releasable pool. In addition, CAZ proteins localize voltage‐gated Ca²⁺ channels at SV release sites, bringing the fusion machinery in close proximity to the calcium source. Proteins of the CAZ therefore ensure that vesicle fusion is temporally and spatially organized, allowing for the precise and reliable release of neurotransmitter. Importantly, AZs are highly dynamic structures, supporting presynaptic remodeling, changes in neurotransmitter release efficacy, and thus presynaptic forms of plasticity. In this review, we discuss recent advances in the study of active zones, highlighting how the CAZ molecularly defines sites of neurotransmitter release, endocytic zones, and the integrity of synapses.     

Ouabain enhances exocytosis through the regulation of calcium handling by the endoplasmic reticulum of chromaffin cells

The augmentation of neurotransmitter and hormone release produced by ouabain inhibition of plasmalemmal Na⁺/K⁺-ATPase (NKA) is well established. However, the mechanism underlying this action is still controversial. Here we have shown that in bovine adrenal chromaffin cells ouabain diminished the mobility of chromaffin vesicles, an indication of greater number of docked vesicles at subplasmalemmal exocytotic sites. On the other hand, ouabain augmented the number of vesicles undergoing exocytosis in response to a K⁺ pulse, rather than the quantal size of single vesicles. Furthermore, ouabain produced a tiny and slow Ca²⁺ release from the endoplasmic reticulum (ER) and gradually augmented the transient elevations of the cytosolic Ca²⁺ concentrations ([Ca²⁺]_c) triggered by K⁺ pulses. These effects were paralleled by gradual increments of the transient catecholamine release responses triggered by sequential K⁺ pulses applied to chromaffin cell populations treated with ouabain. Both, the increases of K⁺-elicited [Ca²⁺]_c and secretion in ouabain-treated cells were blocked by thapsigargin (THAPSI), 2-aminoethoxydiphenyl borate (2-APB) and caffeine. These results are compatible with the view that ouabain may enhance the ER Ca²⁺ load and facilitate the Ca²⁺-induced-Ca²⁺ release (CICR) component of the [Ca²⁺]_c signal generated during K⁺ depolarisation. This could explain the potentiating effects of ouabain on exocytosis.     

The Usage of Plasmalemmal Vesicles Inverted by Brij 58 Treatment for Studying Processes,which Occur on the Cytosolic Membrane Surface

We studied the possible use of the detergent Brij 58 in physiological experiments for the reorientation of right-side-out plasmalemmal vesicles, which were isolated from wheat (Triticum aestivum L.) coleoptiles. The activities of K⁺, Mg²⁺-ATPase and the ATP-dependent H⁺-potential were higher in Brij 58-treated vesicles, whereas membrane permeability for K⁺ and Na⁺ remained unchanged. Brij 58 did not suppress the ATP-dependent IAA transport into vesicles and RNA polymerase II activation by IAA–protein plasmalemmal complexes in the system of isolated nuclei. The conclusion was that, using Brij 58, we could obtain the plasmalemmal fraction, which consisted almost completely of closed inverted vesicles. These vesicles can be applied for the in vitro study of the processes, which occur on the cytosolic plasmalemmal surface.     

Comparison between electrically evoked and potassium-induced 3H-noradrenaline release from rat neocortex slices: Role of calcium ions and transmitter pools

In this study ³H-noradrenaline (NA) release from rat neocortex slices evoked by electrical field-stimulation (1 Hz, 12 mA, 2 msec) was compared with that induced by K⁺-depolarization (13–30 mM K⁺) under similar experimental conditions, with a particular emphasis on the role of external Ca²⁺ and the releasable transmitter pool(s). Not only ³H-NA release evoked by electrical stimulation but also that induced by 13 mM K⁺ was almost completely blocked by 0.3 μM tetrodotoxin (TTX). Release induced by 20 mM K⁺ appeared to be less sensitive to TTX. Thus, under relatively mild stimulation conditions, the activation of sodium channels appears to be involved in ³H-NA release elicited by both stimuli.The electrically evoked ³H-NA release increased sigmoidally with the external Ca²⁺-concentration up to 1.2 mM. In contrast, ³H-NA release induced by 13–20 mM K⁺ reached a maximal value at 0.6–0.9 mM Ca²⁺ and gradually decreased at higher Ca²⁺-concentrations. The Ca²⁺-antagonist D-600 (1–30 μM) did not inhibit electrically evoked release, while K⁺-induced ³H-NA release was dose-dependently reduced. Upon repetitive K⁺-depolarization a strong depression of ³H-NA release could be demonstrated, while this phenomenon did not occur with repeated electrical stimulation. Moreover, a previous K⁺-induced (partial) depletion of ³H-NA stores did not affect the release evoked by electrical pulses and vice versa. Taken together these data are compatible with a much stronger activation of Ca²⁺-channels and a larger vesicle mobilizing capacity in case of electrical stimulation at physiological frequencies compared to sustained depolarization with moderate K⁺-concentrations.     

Assembly of SNARE core complexes prior to neurotransmitter release sets the readily releasable pool of synaptic vesicles

Precise regulation of neurotransmitter release is essential for the normal function of neural networks, but the mechanisms involved are largely unclear. Using superfused synaptosomes, we have studied the readily releasable pool of synaptic vesicles, measured as the amount of release triggered by hypertonic sucrose. We show that activation of presynaptic metabotropic glutamate receptors by dihydroxyphenylglycine and stimulation of protein kinase C by phorbol esters enhance the readily releasable pool of glutamate. Although the molecular nature of the readily releasable pool is unknown, one possibility is that during its generation, SNARE proteins form full core complexes, and that core complex formation occurs prior to neurotransmitter release. To test this possibility, we employed N-ethylmaleimide (NEM), an inhibitor of the ATPase N-ethylmaleimide-sensitive factor that dissociates core complexes, to study the relation of the readily releasable pool to core complex assembly in synaptosomes. NEM induced a dose-dependent increase in the readily releasable pool of neurotransmitters but by itself did not trigger release. Direct measurements of core complexes confirmed that NEM caused an increase in the levels of SNARE core complexes under these conditions. Our data suggest that in the readily releasable pool of synaptic vesicles, SNARE proteins are fully assembled into core complexes, and that SNARE complex assembly is a target of presynaptic regulation.     

On the Origin of Extracellular Glutamate Levels Monitored in the Basal Ganglia of the Rat by In Vivo Microdialysis

Abstract: Several putative neurotransmitters and metabolites were monitored simultaneously in the extracellular space of neostriatum, substantia nigra, and cortex and in subcutaneous tissue of the rat by in vivo microdialysis. Glutamate (Glu) and aspartate (Asp) were at submicromolar and γ-aminobutyric acid (GABA) was at nanomolar concentrations in all brain regions. The highest concentration of dopamine (DA) was in the neostriatum. Dynorphin B (Dyn B) was in the picomolar range in all brain regions. Although no GABA, DA, or Dyn B could be detected in subcutaneous tissue, Glu and Asp levels were ≈5 and ≈0.4 µM, respectively. Lactate and pyruvate concentrations were ≈200 and ≈10 µM in all regions. The following criteria were applied to ascertain the neuronal origin of substances quantified by microdialysis: sensitivity to (a) K⁺ depolarization, (b) Na⁺ channel blockade, (c) removal of extracellular Ca²⁺, and (d) depletion of presynaptic vesicles by local administration of α-latrotoxin. DA, Dyn B, and GABA largely satisfied all these criteria. In contrast, Glu and Asp levels were not greatly affected by K⁺ depolarization and were increased by perfusing with tetrodotoxin or with Ca²⁺-free medium, arguing against a neuronal origin. However, Glu and Asp, as well as DA and GABA, levels were decreased under both basal and K⁺-depolarizing conditions by α-latrotoxin. Because the effect of K⁺ depolarization on Glu and Asp could be masked by reuptake into nerve terminals and glial cells, the reuptake blocker dihydrokainic acid (DHKA) or l -trans-pyrrolidine-2,4-dicarboxylic acid (PDC) was included in the microdialysis perfusion medium. The effect of K⁺ depolarization on Glu and Asp levels was increased by DHKA, but GABA levels were also affected. In contrast, PDC increased only Glu levels. It is concluded that there is a pool of releasable Glu and Asp in the rat brain. However, extracellular levels of amino acids monitored by in vivo microdialysis reflect the balance between neuronal release and reuptake into surrounding nerve terminals and glial elements.     

Influence of hindlimb unloading on the modulation of neurotransmitter secretion through the autoreceptor system

In experiments on neuromuscular junctions of fast (m. extensor digitorum longus, EDL) and slow (m. soleus) muscles of rats under hindlimb unloading of varied duration, we compared the intensity of spontaneous quantal secretion of neurotransmitter in response to potassium depolarization and activation of presynaptic cholinoreceptors with a nonhydrolyzable acetylcholine analog. Secretion was assessed by the mean frequency of miniature endplate potentials. In the controls, carbachol raised this index by 363% in EDL and by 62% in soleus. Secretion in the fast muscle was also more sensitive to [K⁺]. Hindlimb unloading abolished the sensitivity to carbachol in EDL while in soleus it did not change. Preservation of the sensitivity of the fast muscle to potassium depolarization suggested that unloading reduced the number of functional presynaptic receptors.     

RIM‐binding proteins recruit BK‐channels to presynaptic release sites adjacent to voltage‐gated Ca2+‐channels

The active zone of presynaptic nerve terminals organizes the neurotransmitter release machinery, thereby enabling fast Ca²⁺‐triggered synaptic vesicle exocytosis. BK‐channels are Ca²⁺‐activated large‐conductance K⁺‐channels that require close proximity to Ca²⁺‐channels for activation and control Ca²⁺‐triggered neurotransmitter release by accelerating membrane repolarization during action potential firing. How BK‐channels are recruited to presynaptic Ca²⁺‐channels, however, is unknown. Here, we show that RBPs (for RIM‐binding proteins), which are evolutionarily conserved active zone proteins containing SH3‐ and FN3‐domains, directly bind to BK‐channels. We find that RBPs interact with RIMs and Ca²⁺‐channels via their SH3‐domains, but to BK‐channels via their FN3‐domains. Deletion of RBPs in calyx of Held synapses decreased and decelerated presynaptic BK‐currents and depleted BK‐channels from active zones. Our data suggest that RBPs recruit BK‐channels into a RIM‐based macromolecular active zone complex that includes Ca²⁺‐channels, synaptic vesicles, and the membrane fusion machinery, thereby enabling tight spatio‐temporal coupling of Ca²⁺‐influx to Ca²⁺‐triggered neurotransmitter release in a presynaptic terminal.     

Presynaptic Control of Glycine Transporter 2 (GlyT2) by Physical and Functional Association with Plasma Membrane Ca2+-ATPase (PMCA) and Na+-Ca2+ Exchanger (NCX)

Fast inhibitory glycinergic transmission occurs in spinal cord, brainstem, and retina to modulate the processing of motor and sensory information. After synaptic vesicle fusion, glycine is recovered back to the presynaptic terminal by the neuronal glycine transporter 2 (GlyT2) to maintain quantal glycine content in synaptic vesicles. The loss of presynaptic GlyT2 drastically impairs the refilling of glycinergic synaptic vesicles and severely disrupts neurotransmission. Indeed, mutations in the gene encoding GlyT2 are the main presynaptic cause of hyperekplexia in humans. Here, we show a novel endogenous regulatory mechanism that can modulate GlyT2 activity based on a compartmentalized interaction between GlyT2, neuronal plasma membrane Ca²⁺-ATPase (PMCA) isoforms 2 and 3, and Na⁺/Ca²⁺-exchanger 1 (NCX1). This GlyT2·PMCA2,3·NCX1 complex is found in lipid raft subdomains where GlyT2 has been previously found to be fully active. We show that endogenous PMCA and NCX activities are necessary for GlyT2 activity and that this modulation depends on lipid raft integrity. Besides, we propose a model in which GlyT2·PMCA2–3·NCX complex would help Na⁺/K⁺-ATPase in controlling local Na⁺ increases derived from GlyT2 activity after neurotransmitter release.     

A microdialysis study in rat brain of dihydrokainate,a glutamate uptake inhibitor

Microdialysis in neostriatum of anaesthetized rats was performed to study effects on amino acid efflux of the glutamate uptake-inhibitor dihydrokainate (DHK). Both basal and K⁺-evoked (100 mM) efflux of glutamate increased in the presence of DHK. The increase in the basal glutamate efflux occurred at lower DHK concentrations than during K⁺-depolarization (when the extracellular glutamate concentration was several-fold higher), confirming that DHK is a competitive inhibitor. The increase in basal efflux caused by DHK did not exhibit Ca²⁺-dependency, whereas ∼50% of the increase in glutamate efflux during K⁺-depolarization was Ca²⁺-dependent. The Ca²⁺-dependent efflux is related to transmitter release, whereas the Ca²⁺-independent efflux is probably due to metabolic events and/or transport of DHK into cells in exchange for glutamate. Taurine efflux in response to DHK increased both during basal conditions and K⁺-depolarization, probably secondary to the increase in glutamate concentration, whereas aspartate, GABA, glutamine and alanine effluxes did not change.     

Exocytotic dynamics and calcium cooperativity effects in the calyx of Held synapse: a modelling study

A stochastic computational approach to the study of secretory processes at the calyx of Held synapse is presented in this paper. The calyx of Held is a giant synapse located in the brainstem which is widely used for experimental recording of neurotransmitter release. We focus on the study of the exocytotic dynamics for a pool of readily releasable vesicles using a Monte Carlo simulation scheme that includes models for the P-type calcium channels, the kinetic reactions of endogenous and exogenous (mobile) buffers, the kinetic reactions for the secretory vesicles, as well as the microscopic diffusion of mobile buffers and calcium ions. The simulations are performed in a 3-D orthogonal grid which approximates a cylindrical domain representing an active zone of the presynaptic terminal of the calyx. For this domain, we quantify the release rates related to calcium currents in response to depolarizing voltage pulses. The influence on simulated pulse/action potential depolarization protocols of the kinetic scheme for the calcium sensor of vesicles and the geometry of calcium channels for the kinetic cooperativity for release, is analyzed at a microdomain level. Among other aspects, our results suggest that the spatial organization of Ca ^2 + channels could have measurable effects in the kinetic cooperativity which could reflect developing changes in the calyx of Held synapse.     

Rapid turn-over of plasma membrane sphingomyelin and cholesterol in baby hamster kidney cells after exposure to sphingomyelinase

Plasma membrane sphingomyelin in baby hamster kidney (BHK-21) cells was hydrolyzed with sphingomyelinase (Staphylococcus aureus) and the effects on membrane cholesterol translocation and the properties of membrane bound adenylate cyclase and Na+/K(+)-ATPase were determined. Exposure of confluent BHK-21 cells to 0.1 U/ml of sphingomyelinase led to the degradation (at 37 degrees C) of about 60% of cell sphingomyelin. No simultaneous hydrolysis of phosphatidylcholine occurred. The hydrolysis of sphingomyelin subsequently led to the translocation (within 40 min) of about 50-60% of cell [3H]cholesterol from a cholesterol oxidase susceptible pool to an oxidase resistant compartment. The translocation of [3H]cholesterol from the cell surface to intracellular membranes was accompanied by a paralleled increase in [3H]cholesterol ester formation. When cells were first exposed to sphingomyelinase (to degrade sphingomyelin) and then incubated without the enzyme in serum-free media, the mass of cell sphingomyelin decreased initially (by 60%), but then began to increase and reached control levels within 3-4 h. The rapid re-synthesis of sphingomyelin was accompanied by an equally rapid normalization of cell [3H]cholesterol distribution. The re-formation of cell sphingomyelin also led to a decreased content of cellular [3H]cholesterol esters, indicating that unesterified [3H]cholesterol was pulled out of the cholesterol ester cycle and transported to the cell surface. Exposure of BHK-21 cells to sphingomyelinase further led to a dramatically decreased activity of ouabain-sensitive Na+/K(+)-ATPase, whereas forskolin-stimulated adenylate cyclase activity was not affected. The activity of Na+/K(+)-ATPase returned to normal in parallel with the normalization of cell sphingomyelin mass and cholesterol distribution. We conclude that sphingomyelin has profound effects on the steady-state distribution of cell cholesterol, and that manipulations of cell sphingomyelin levels directly and reversibly affects the apparent distribution of cholesterol. Changes in the lipid composition of the plasma membrane also appears to selectively affect important metabolic reactions in that compartment.     

How to make a synaptic ribbon: RIBEYE deletion abolishes ribbons in retinal synapses and disrupts neurotransmitter release

Synaptic ribbons are large proteinaceous scaffolds at the active zone of ribbon synapses that are specialized for rapid sustained synaptic vesicles exocytosis. A single ribbon‐specific protein is known, RIBEYE, suggesting that ribbons may be constructed from RIBEYE protein. RIBEYE knockdown in zebrafish, however, only reduced but did not eliminate ribbons, indicating a more ancillary role. Here, we show in mice that full deletion of RIBEYE abolishes all presynaptic ribbons in retina synapses. Using paired recordings in acute retina slices, we demonstrate that deletion of RIBEYE severely impaired fast and sustained neurotransmitter release at bipolar neuron/AII amacrine cell synapses and rendered spontaneous miniature release sensitive to the slow Ca²⁺‐buffer EGTA, suggesting that synaptic ribbons mediate nano‐domain coupling of Ca²⁺ channels to synaptic vesicle exocytosis. Our results show that RIBEYE is essential for synaptic ribbons as such, and may organize presynaptic nano‐domains that position release‐ready synaptic vesicles adjacent to Ca²⁺ channels.     

Protein-protein interactions and protein modules in the control of neurotransmitter release

Information transfer among neurons is operated by neurotransmitters stored in synaptic vesicles and released to the extracellular space by an efficient process of regulated exocytosis. Synaptic vesicles are organized into two distinct functional pools, a large reserve pool in which vesicles are restrained by the actin-based cytoskeleton, and a quantitatively smaller releasable pool in which vesicles approach the presynaptic membrane and eventually fuse with it on stimulation. Both synaptic vesicle trafficking and neurotransmitter release depend on a precise sequence of events that include release from the reserve pool, targeting to the active zone, docking, priming, fusion and endocytotic retrieval of synaptic vesicles. These steps are mediated by a series of specific interactions among cytoskeletal, synaptic vesicle, presynaptic membrane and cytosolic proteins that, by acting in concert, promote the spatial and temporal regulation of the exocytotic machinery. The majority of these interactions are mediated by specific protein modules and domains that are found in many proteins and are involved in numerous intracellular processes. In this paper, the possible physiological role of these multiple protein-protein interactions is analysed, with ensuing updating and clarification of the present molecular model of the process of neurotransmitter release.     

Levels,uptake, and release of glycine and glutamate in the rat pontine reticular formation

In this work we have determined the levels of glycine, glutamate, and other amino acids in the rat pontine reticular formation (PRF), in addition to some properties of the uptake and release of labeled glycine and glutamate in slices of this region. Glutamate was the most concentrated amino acid in the PRF, although its content was about half that of the striatum. Surprisingly, glycine levels in the PRF were 3.2-fold higher than in the striatum, whereas GABA content was similar in both regions. The uptake of both glycine and glutamate by PRF slices was strictly Na⁺-dependent. Their release was stimulated by K⁺-depolarization, but only the release of glycine was Ca²⁺-dependent. These findings suggest that glycine is a strong candidate for a neurotransmitter role in the PRF and that glutamate might also play such a role in this region.Special issue dedicated to Dr. Morris H. Aprison     

Release of dopamine and serotonin from Limax ganglia in vitro

1. We wish to establish the kinetics of serotonin and dopamine release from Limax cerebral and buccal ganglia and find selective treatments to modify their release kinetics.2. The release of dopamine and serotonin from isolated ganglia was stimulated by high potassium exposure with and without prior treatment of ganglia with 6-hydroxydopamine (6-OHDA).3. Single ganglia release significant quantities of monoamines during a single 5 min high K⁺ exposure. Multiple high K⁺ exposures deplete a readily releasable transmitter store with little effect on storage pools.4. 6-OHDA exposure depletes readily releasable DA with little effect on total ganglion DA content or on serotonin.5. Feeding motor program responsiveness is suppressed reversibly by whole ganglion high K⁺ treatment.     

Neuronal dependence of extracellular dopamine,acetylcholine, glutamate,aspartate and gamma-aminobutyric acid (GABA) measured simultaneously from rat neostriatum using in vivo microdialysis: reciprocal interactions

Summary The neuronal origin of extracellular levels of dopamine (DA), acetylcholine (ACh), glutamate (Glu), aspartate (Asp) and gamma-aminobutyric acid (GABA) simultaneously collected from the neostriatum of halothane anaesthetized rats with in vivo microdialysis was studied. The following criteria were applied (1) sensitivity to K⁺-depolarization; (2) sensitivity to inhibition of synaptic inactivation mechanisms; (3) sensitivity to extracellular Ca²⁺; (4) neuroanatomical regionality; sensitivity to selective lesions and (5) sensitivity to chemical stimulation of the characterized pathways.It was found that: (1) Extracellular DA levels found in perfusates collected from the neostriatum fulfills all the above criteria and therefore the changes in extracellular DA levels measured with microdialysis reflect actual release from functionally active nerve terminals, and so reflect ongoing synaptic transmission. (2) Changes in neostriatal ACh levels reflect neuronal activity, provided that a ACh-esterase inhibitor is present in the perfusion medium. (3) Extracellular Glu, Asp and GABA could be measured in different perfusion media in the rat neostriatum and probably reflect metabolic as well as synaptic release. However, (4) the majority of the extracellular GABA levels found in perfusates collected from the neostriatum may reflect neuronal release, since GABA levels were increased, in a Ca²⁺-dependent manner, by K⁺-depolarization, and could be selectively decreased by an intrinsic neostriatal lesion. (5) It was not possible to clearly distinguish between the neuronal and the metabolic pools of Glu and Asp, since neostriatal Glu and Asp levels were only slightly increased by K⁺-depolarization, and no changes were seen after decortication. A blocker of Glu re-uptake, DHKA, had to be included in the perfusion medium in order to monitor the effect of K⁺-depolarization on Glu and Asp levels. Under this condition, it was found (6) that neostriatal Glu and Asp levels were significantly increased by K⁺-depolarization, although only increases in the Glu levels were sensitive to Ca²⁺ in the perfusion medium, suggesting that Glu but not Asp is released from vesicular pools. (7) Evidence is provided that selective stimulations of nigral DA cell bodies may lead to changes in release patterns from DA terminals in the ipsilateral neostriatum, which are in turn followed by discrete changes in extracellular levels of GABA and Glu in the same region. Finally, some methodological considerations are presented to clarify the contribution of neuronal release to extracellular levels of amino acid neurotransmitters in the rat neostriatum.     

Arachidonic acid release from K+-evoked depolarization of brain synaptosomes

Rat brain synaptosomes prelabeled with [¹⁴C]arachidonate in their phospholipids were superfused with well oxygenated Krebs-Ringer-bicarbonate solution containing 0.2% BSA and subsequently depolarized by elevating the K⁺ concentration in the superfusion medium from 5 to 55 mM. The efflux of labeled arachidonate at steady state was 0.19% (n = 12) of total radioactivity per min. In the presence of 2.5 mM Ca²⁺, high K⁺ (55 mM) in the medium elicited an increase in arachidonate efflux which amounted to 121.4% (n = 6) of control. Both Ca²⁺ and BSA were required for the stimulated efflux of arachidonate during K⁺-depolarization. Under the same condition, K⁺-stimulation also evoked the release of [³H]norepinephrine which was preloaded into the synaptosomes prior to superfusion. EGTA abolished the stimulated release of both arachidonate and norepinephrine during K⁺-depolarization. These results, together with the loss of labeled arachidonic acid from phospholipids (Majewska and Sun, 1982), indicate that deacylation of membrane lipids is involved in synaptic functions.