Matthijs Verhage

Differential release of amino acids, neuropeptides, and catecholamines from isolated nerve terminals

We have investigated transmitter release from small and large dense-core vesicles in nerve terminals isolated from guinea pig hippocampus. Small vesicles are found in clusters near the active zone, and large dense-core vesicles are located at ectopic sites. The abilities of Ca2+ channel activation and uniform elevation of Ca2+ concentration (with ionophores) to evoke secretion of representative amino acids, catecholamines, and neuropeptides were compared. For a given increase in Ca2+ concentration, ionophore was less effective than Ca2+ channel activation in releasing amino acids, but not in releasing cholecystokinin-8. Titration of the average Ca2+ concentration showed that the Ca2+ affinity for cholecystokinin-8 secretion was higher than that for amino acids. Catecholamine release showed intermediate behavior. It is concluded that neuropeptide release is triggered by small elevations in the Ca2+ concentration in the bulk cytoplasm, whereas secretion of amino acids requires higher elevations, as produced in the vicinity of Ca2+ channels.

Ca2+-Dependent Regulation of Presynaptic Stimulus-Secretion Coupling

Abstract: In the present study, we have investigated the role of Ca2+ in the coupling of membrane depolarization to neurotransmitter secretion. We have measured (a) intracellular free Ca2+ concentration ([Ca2+Ji) changes, (b) rapid 45Ca2+uptake, and (c) Ca2+‐dependent and ‐independent release of endogenous glutamate (Glu) and γ‐aminobutyric acid (GABA) as a function of stimulus intensity by elevating the extracellular [K+] to different levels in purified ijierve terminals (synaptosomes) from rat hippocampus. Duriijg stimulation, Percoll‐purined synaptosomes show an increased 45Ca2+ uptake, an elevated [Ca2+]i, and a Ca2+‐dependejnt as well as a Ca2+‐independent release of both Glu and GABA. With respect to both amino acids, synaptosomes respond on stimulation essentially in the same way, with maximally a fourfold increase in Ca2+‐dependent (exocytotic) release. Ca2+‐depen‐dent transmitter release as well as [Ca2+]; elevations show maximal stimulation at moderate depolarizations (30 mM K+). A correlation exists between Ca2+‐dependent release of both Glu and GABA and elevation of [Ca2+]i. C2+‐dependent release is maximally stimulated with an elevation of [Ca2+]Iof 60% above steady‐state levels, corresponding with an intracellular concentration of ∼400 nM, whereas elevations to 350 nM are ineffective in stimulating Ca2+‐dependent release of both Glu and GABA. In contrast, Ca2+‐independent release of both Glu and GABA shows roughly a linear rise with stimulus intensity up to 50 mM K+. 45Ca2+ uptake on stimulation also shows a continuous increase with stimulus intensity, although the relationship appears to be biphasic, with a plateau between 20 and 40 mM K+. These findings indicate that Ca2+‐dependent, exocytotic transmitter release is not simply enhanced by larger depolarizations of the plasma membrane and that a strong Ca2+‐dependent regulatory mechanism exists in synaptosomes for the trigger of exocytosis, operating at a mean [Ca2+]i of between 350 and 400 nM. Ca2+ transport and buffering mechanisms possibly involved in this regulation and the role of the membrane potential are discussed.

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

Abstract: 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+. Furthermore, the relationship between the volume average [Ca]i (measured with fura‐2) and the extent of release is more or less linear for the release of CCK‐LI, whereas this relationship is clearly nonlinear for the release of endogenous glutamate and γ‐aminobutyric acid in similar preparations. We hypothesize that these differences between the neuropeptide CCK‐8 and classical transmitters are caused by three differences: (a) vesicle type; (b) Ca2+ sensitivity of the release mechanism; and (c) release site.

Evaluation of the Ca2+ Concentration in Purified Nerve Terminals: Relationship Between Ca2+ Homeostasis and Synaptosomal Preparation

Abstract: The presynaptic Ca2+ concentration ([Ca]i) was evaluated by studying intracellular free Ca2+ with quin‐2 and fura‐2 in synaptosomal preparations. The synaptosomal preparations were purified with hyperosmotic (sucrose) and isoosmotic (Percoll) density gradient centrifugation. Synaptosomes are most viable in the heavier fractions of the density gradients. These synaptosomal fractions exhibit the lowest [Ca]i, [204 ± 2 nM for Percoll (C‐band) synaptosomes, loaded at 30°C with the acetoxymethyl ester of fura‐2 (fura‐2‐AM)], a high stability during prolonged incubations at 37°C, and a more potent response to membrane depolarization by elevated extracellular [K+]. [Ca]i measurement was critically dependent on dye loading, calibration, type of dye used, synaptosomal preparation, and incubation temperature (30° or 37°C). Loading quin‐2 in synaptosomes inserts a considerable buffer component in the synaptosomal [Ca]i regulation, and consequently there is a quin‐2 dependency of [Ca]i, independent of endogenous heavy metal ions. Use of fura‐2 is preferable in synaptosomes, although above a critical fura2‐AM/protein ratio during loading ester hydrolysis is not complete, giving rise to errors in [Ca]i determination. Ionomycin is a selective tool to detect the presence of partially hydrolyzed esters and saturate indicators in the cytosol with Ca2+ for calibration. Parallel studies on lactate dehydrogenase and fura‐2 fluorescence indicate that synaptosomal viability is very sensitive to prolonged incubations at 37°C. This study shows the applicability of measuring steady‐state [Ca]i and dynamic [Ca]i changes quantitatively in fura‐2‐loaded synaptosomes. The possible involvement of different synaptosomal pools to explain the divergence in [Ca]i between different preparations and the interpretation in physiological terms of [Ca]i measured in synaptosomes are discussed.

Kindling increases the K(+)-evoked Ca2(+)-dependent release of endogenous GABA in area CA1 of rat hippocampus.

The release of endogenous amino acids from hippocampal CA1 subslices under basal conditions and the release evoked by high potassium (50 mM K+) depolarization was studied during kindling epileptogenesis. Emphasis was put on the release of the amino acid neurotransmitters gamma-aminobutyric acid (GABA) and glutamate. Kindling was induced by tetanic stimulation of the Schaffer-collaterals/commissural fibers of the dorsal hippocampus of the rat. The calcium-dependent GABA release in the presence of high K+ was significantly increased (40-46%) in fully kindled animals, 24 h after the last seizure, in comparison to controls. At long-term, 28 days after the last seizure, the calcium-dependent GABA release was still significantly increased (45-49%). An increased release of GABA in kindled animals was still found when GABA uptake was blocked by nipecotic acid. In contrast, no significant alterations were encountered in the basal or high potassium induced release of the excitatory amino acids aspartate and glutamate. These results suggest that kindling epileptogenesis is accompanied by a specific and long-lasting enhancement of GABA exocytosis which may lead to a desensitization of the GABA receptor, and thus determine the increase of seizure sensitivity.

Endogenous Noradrenaline and Dopamine in Nerve Terminals of the Hippocampus: Differences in Levels and Release Kinetics

Abstract: The presence and release of endogenous catechol‐amines in rat and guinea pig hippocampal nerve terminals was studied by fluorimetric HPLC analysis. In isolated nerve terminals (synaptosomes) the levels and breakdown of endogenous catecholamines were determined and the release process was characterized with respect to its kinetics and Ca2+ and ATP dependence. Endogenous noradrenaline and dopamine, but not adrenaline, were detected in isolated hippocampal nerve terminals. For dopamine both the levels and the amounts released were more than 100‐fold lower than those for noradrenaline. In suspension, released endogenous catecholamines were rapidly broken down. This could effectively be blocked by monoamine oxidase inhibitors, Ca2+‐free conditions, and gluthatione. The release of both noradrenaline and dopamine was highly Ca2+ and ATP dependent. Marked differences were observed in the kinetics of release between the two catecholamines. Noradrenaline showed an initial burst of release within 10 s after K+ depolarization. The release of noradrenaline was terminated after approximately 3 min of K+ depolarization. In contrast, dopamine release was more gradual, without an initial burst and without clear termination of release within 5 min. It is concluded that both catecholamines are present in nerve terminals in the rat hippocampus and that their release from (isolated) nerve terminals is exocytotic. The characteristics of noradrenaline release show several similarities with those of other classical transmitters, whereas dopamine release characteristics resemble those of neuropeptide release in the hippocampus but not those of dopamine release in other brain areas. It is hypothesized that in the hippocampus dopamine is released from large, dense‐cored vesicles, probably colocalized with neuropeptides.

Perfusion of immobilized isolated nerve terminals as a model for the regulation of transmitter release: release of different, endogenous transmitters, repeated stimulation, and high time resolution.

Abstract: To study the release of neurotransmitters, i.e., the recruitment of transmitters for release and the regulation of the release process, isolated nerve terminals (synaptosomes) of the rat forebrain were immobilized in Sephadex gel inside a perfusion chamber. In this way, the following were achieved: (a) A very limited pressure stress was exerted on the synaptosomes, so that these remained viable for long periods (>30 min) inside the chamber and did not elute from the chamber, which allowed long‐term experiments with repeated stimulations; (b) estimation of the release of various endogenous transmitters, both in a Ca2+‐dependent (exocytotic) and Ca2+‐independent manner; (c) a step‐like stimulation with depolarizing agents (rise time, 3–4 s) and a high time resolution (600‐ms sampling); and (d) negligible reuptake of transmitter into the terminals or extracellular breakdown. It is concluded that this perfusion setup helps to provide new insights in the presynaptic stimulus–secretion coupling, co‐transmission, and the exo–endocytosis cycle.

Activity-dependent recruitment of endogenous glutamate for exocytosis

The Ca(2+)-dependent release of the neurotransmitter glutamate from purified nerve terminals (synaptosomes) of the rat hippocampus was studied in a rapid perfusion apparatus. The response of the terminals was investigated with respect to the kinetics and duration of the release of endogenous glutamate upon brief and sustained stimulation and upon repetitive stimulation. The synaptosomes were stimulated by sustained chemical depolarization (0.5-3 min 30 mM K+). The cellular levels of glutamate, free Ca2+ and ATP in the nerve terminals were measured. The Ca(2+)-dependent release of glutamate showed an immediate elevation upon K(+)-depolarization. When the stimulation was maintained, a prolonged phase of glutamate release was observed. After 3 min, the Ca(2+)-dependent release stopped, although K(+)-depolarization was still effective. When synaptosomes were stimulated again after a relatively short stimulation period (30 s), the second response was similar to the previous one. After a longer stimulation period, maintained until termination of release, the second response did not show the immediate initial elevation of Ca(2+)-dependent glutamate release. Only 30 s after stimulation the release developed with a time profile comparable to the first response. This initial lack of response was not due to low cytosolic levels of glutamate or ATP or to changes in cellular Ca(2+)-buffering. It can be concluded that the capacity to release glutamate after brief depolarizations is fully restored during the repolarization period. However, if stimulation periods are of long duration (until termination of release), this capacity is no longer fully restored, especially with respect to a fast component of release. New glutamate is recruited only during the subsequent depolarization and with a delay.(ABSTRACT TRUNCATED AT 250 WORDS)