Wim E. J. M. Ghijsen

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.

Altered hippocampal gene expression prior to the onset of spontaneous seizures in the rat post‐status epilepticus model

Neuronal loss, gliosis and axonal sprouting in the hippocampal formation are characteristics of the syndrome of mesial temporal sclerosis (MTS). In the post‐status epilepticus (SE) rat model of spontaneous seizures these features of the MTS syndrome can be reproduced. To get a global view of the changes in gene expression in the hippocampus we applied serial analysis of gene expression (SAGE) during the early phase of epileptogenesis (latent period), prior to the onset of the first spontaneous seizure. A total of 10 000 SAGE tags were analyzed per experimental group, resulting in 5053 (SE) and 5918 (control group) unique tags (genes), each representing a specific mRNA transcript. Of these, 92 genes were differentially expressed in the hippocampus of post‐SE rats in comparison to controls. These genes appeared to be mainly associated with ribosomal proteins, protein processing, axonal growth and glial proliferation proteins. Verification of two of the differentially expressed genes by in situ hybridization confirmed the changes found by SAGE. Histological analysis of hippocampal sections obtained 8 days after SE showed extensive cell loss, mossy fibre sprouting and gliosis in hippocampal sub regions. This study identifies new high‐abundant genes that may play an important role in post‐SE epileptogenesis.

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.

A presynaptic N-methyl-d-aspartate autoreceptor in rat hippocampus modulating amino acid release from a cytoplasmic pool

A possible role of the N‐methyl‐d‐aspartate receptor (NMDA‐R) as a presynaptic autoreceptor was investigated using Percoll‐purified hippocampus nerve terminals (synaptosomes). This preparation contained only a neglectable amount of postsynaptic structures. Two main effects of NMDA were observed. First, NMDA dose‐dependently (10–100 μm) and in the absence of Mg2+, stimulated basal release of aspartate and glutamate, but not of GABA. MK801 (10 μm), an open NMDA‐R‐channel blocker, reduced this effect even below control levels, indicating endogenous NMDA‐R activation. By superfusing synaptosomes, which prevents a tonic receptor occupation, also basal GABA release was stimulated by NMDA. The NMDA‐induced potentiation of amino acid superfusate levels was blocked both by MK801 and Mg2+ (1 m m), was slow in onset and returned to baseline after NMDA‐removal. The NMDA‐effect was also found in the absence of extracellular Ca2+, suggesting that amino acids were released from a non‐vesicular (cytoplasmic) pool. Secondly, in KCl‐depolarized synaptosomes exposed to 1 m m Mg2+, NMDA did not affect the release of the amino acids. MK801, however, reduced the KCl‐evoked Ca2+‐independent release of aspartate and glutamate, but not of GABA. l‐trans‐PDC, the selective inhibitor of the glutamate/aspartate transporter, prevented this MK801‐effect, suggesting a coupling between NMDA‐Rs and these transporters.

Dynamics of munc18-1 phosphorylation/dephosphorylation in rat brain nerve terminals

Munc18‐1 is a mammalian member of the SEC1 protein family implicated in neuronal secretion. Its sequence contains several consensus sites for phosphorylation by protein kinase C (PKC), a kinase known to enhance secretion. We have characterized the phosphorylation of the synaptic munc18‐1 pool by endogenous, presynaptic PKC‐isoforms. In isolated rat brain nerve terminals, munc18‐1 was almost completely nonphosphorylated. Its phosphorylation state increased by 250% on inhibition of endogenous phosphatases and by 1500% on additional, direct PKC activation using phorbol esters. K+‐evoked depolarization also increased munc18‐1 phosphorylation, by 50% within 5 s in a Ca2+‐dependent manner. Munc18‐1 phosphorylation in nerve terminals was blocked by PKC inhibitors. Activation of endogenous PKC in nerve terminals inhibited the interaction of synaptic munc18‐1 with its binding partner syntaxin‐1A by 50%. Munc18‐1 antisera precipitated 80% of native, brain‐derived munc18‐1 from salt solutions, but only 12% from synaptosomal lysates, together with 6% synaptic syntaxin‐1A/B; these amounts were not changed by PKC activation. In this 12%, the phosphate incorporation per mole of munc18 was four‐fold lower than the total pool. We conclude that the synaptic munc18‐1 pool can be readily and rapidly phosphorylated by endogenous presynaptic PKC isoforms. A high constitutive phosphatase activity keeps its basal phosphorylation state low so that PKC activation can increase the phosphorylation state dramatically. These phosphorylation dynamics and the effects on the interaction with syntaxin‐1A make munc18‐1 a prominent candidate to account for PKC‐dependent enhancement of secretion.

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.

Glutamate transporters alterations in the reorganizing dentate gyrus are associated with progressive seizure activity in chronic epileptic rats.

The expression of glial and neuronal glutamate transporter proteins was investigated in the hippocampal region at different time points after electrically induced status epilepticus (SE) in the rat. This experimental rat model for mesial temporal lobe epilepsy is characterized by cell loss, gliosis, synaptic reorganization, and chronic seizures after a latent period. Despite extensive gliosis, immunocytochemistry revealed only an up‐regulation of both glial transporters localized at the outer aspect of the inner molecular layer (iml) in chronic epileptic rats. The neuronal EAAC1 transporter was increased in many somata of individual CA1‐3 neurons and granule cells that had survived after SE; this up‐regulation was still present in the chronic epileptic phase. In contrast, a permanent decrease of EAAC1 immunoreactivity was observed in the iml of the dentate gyrus. This permanent decrease in EAAC1 expression, which was only observed in rats that experienced progressive spontaneous seizure activity, could lead to abnormal glutamate levels in the iml once new abnormal glutamatergic synaptic contacts are formed by means of sprouted mossy fibers. Considering the steady growth of reorganizing mossy fibers in the iml, the absence of a glutamate reuptake mechanism in this region could contribute to progression of spontaneous seizure activity, which occurs with a similar time course. J. Comp. Neurol. 442:365–377, 2002.

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.

Presynaptic plasticity: The regulation of Ca2+-dependent transmitter release

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