W.E.J.M. Ghijsen

Opposite Changes in GABAA Receptor Function in the CA1‐3 Area and Fascia Dentata of Kindled Rat Hippocampus

Abstract: Muscimol‐stimulated radiotracer 36Cl− uptake in synaptoneurosomes was used to investigate the function of the GABAA receptor complex in the CA1‐3 area and fascia dentata (granular and molecular layers and hilus) of rats kindled by stimulation, twice a day, of the Schaffer collateral fibers. Two kindled groups were studied: (a) 24 h after the last generalized tonic‐clonic seizure [fully kindled (FK) stage] and (b) 28 days after the last generalized seizure (long‐term stage). Synaptoneurosomes were prepared in parallel from subslices of the CA1‐3 area and fascia dentata. In FK animals, the muscimol‐stimulated 36Cl− uptake was significantly reduced by 21% in the CA1‐3 area in comparison with nonstimulated controls, whereas a significant increase of 29% was found in the fascia dentata. Significant changes were no longer present at 4 weeks after the last generalized seizure. The observed changes in muscimol‐stimulated 36Cl− uptake at the FK stage closely parallel the recently observed changes in [3H]muscimol binding in the CA1 area and fascia dentata. These results indicate that kindling causes a transiently decreased GABAA receptor‐mediated function in the CA1‐3, in contrast to an increased GABAA receptor‐mediated function in the fascia dentata.

Effects of uptake carrier blockers SK&F 89976-A and l-trans-PDC on in vivo release of amino acids in rat hippocampus

This report describes the in vivo effects of the uptake carrier blockers 1-(4,4-diphenyl-3-butenyl)-3-piperidine carboxylic acid hydrochloride (SK & F 89976-A) and L-trans-pyrrolidine-2,4-dicarboxylate (L-trans-PDC) on basal and K(+)-evoked extracellular levels of gamma-aminobutyric acid (GABA), glutamate, aspartate and taurine in the hippocampus of anaesthetised rats, using the microdialysis technique. SK & F 89976-A increased extracellular GABA levels under K(+)-depolarised conditions and did not affect extracellular glutamate, aspartate and taurine levels, indicating its selective effect on GABA uptake L-trans-PDC dose dependently increased basal and K(+)-evoked extracellular glutamate levels, and did not affect extracellular GABA levels, but increased basal aspartate and taurine levels. The K(+)-evoked release of GABA and glutamate, measured in the presence of both SK & F 89976-A and L-trans-PDC, was Ca(2+)-dependent for about 50% and 65%, respectively. In contrast, the release of the putative amino acid transmitters aspartate and taurine was not Ca(2+)-dependent. These results indicate that (1) in rat hippocampus uptake carriers actively regulate extracellular GABA and glutamate levels, (2) the GABA and glutamate released by K+ was derived from both Ca(2+)-dependent (presumably vesicular) and Ca(2+)-independent (presumably cytosolic) pools, whereas aspartate and taurine release was exclusively from Ca(2+)-independent pools.

Activity-dependent neurotransmitter release kinetics: correlation with changes in morphological distributions of small and large vesicles in central nerve terminals

In central nerve terminals transmitter release is tightly regulated and thought to occur in a number of steps. These steps include vesicle mobilization and docking prior to neurotransmitter release. Intrasynaptic changes in vesicle distribution were determined by electron microscopical analysis and neurotransmitter release was monitored by biochemical measurements. We correlated Ku200a+u200a‐induced changes in distribution of small and large vesicles with the release of their transmitters. For small synaptic vesicles, amino acid release as well as recruitment to and docking at the active zone were activated within 1u2003s of depolarization. In contrast, the disappearance of large dense‐cored vesicles and the release of the neuropeptide cholecystokinin were much slower, and no docking was observed. Studies with diverse Ca2u200a+u200a channel blockers indicated that mobilization and neurotransmitter release from both vesicle types were regulated by multiple Ca2u200a+u200a channels, although in different ways. Neurotransmitter release from small synaptic vesicles was predominantly regulated by P‐type Ca2u200a+u200a channels, whereas primarily Q‐type Ca2u200a+u200a channels regulated neurotransmitter release from large dense‐cored vesicles. The different Ca2u200a+u200a channnel types directly regulated mobilization of and neurotransmitter release from small synaptic vesicles whereas, by their cooperativity in raising the intracellular Ca2u200a+u200a concentration above release threshold, they more indirectly regulated large dense‐cored vesicle exocytosis.

Ba2+ replaces Ca2+/calmodulin in the activation of protein phosphatases and in exocytosis of all major transmitters

Exocytosis from nerve terminals is triggered by depolarization-evoked Ca2+ entry, which also activates calmodulin and stimulates protein phosphorylation. Ba2+ is believed to replace Ca2+ in triggering exocytosis without activation of calmodulin and can therefore be used to unravel aspects of presynaptic function. We have analysed the cellular actions of Ba2+ in relation to its effect on transmitter release from isolated nerve terminals. Barium evoked specific release of amino acid transmitters, catecholamines and neuropeptides (EC50 0.2-0.5 mM), similar to K-/Ca(2+)-evoked release both in extent and kinetics. Ba(2+)-and Ca(2+)-evoked release were not additive. In contrast to Ca2+, Ba2+ triggered release which was insensitive to trifluoperizine and hardly stimulated protein phosphorylation. These observations are in accordance with the ability of Ba2+ to replace Ca2+ in exocytosis without activating calmodulin. Nevertheless, calmodulin appears to be essential for regular (Ca(2+)-triggered) exocytosis, given its sensitivity to trifluoperizine. Both Ba(2+)-and Ca(2+)-evoked release were blocked by okadaic acid. Furthermore, anti-calcineurin antibodies decreased Ba(2+)-evoked release. In conclusion, Ba2+ replaces Ca2+/calmodulin in the release of the same transmitter pool. Calmodulin-dependent phosphorylation appears not to be essential for transmitter release. Instead, our data implicate both Ca(2+)-dependent and -independent dephosphorylation in the events prior to neurotransmitter exocytosis.

Anti-B-50 (GAP-43) antibodies decrease exocytosis of glutamate in permeated synaptosomes

The involvement of the protein kinase C substrate, B-50 (GAP-43), in the release of glutamate from small clear-cored vesicles in streptolysin-O-permeated synaptosomes was studied by using anti-B-50 antibodies. Glutamate release was induced from endogenous as well as 3H-labelled pools in a [Ca(2+)]-dependent manner. This Ca(2+)-induced release was partially ATP dependent and blocked by the light-chain fragment of tetanus toxin, demonstrating its vesicular nature. Comparison of the effects of anti-B-50 antibodies on glutamate and noradrenaline release from permeated synaptosomes revealed two major differences. Firstly, Ca(2+)-induced glutamate release was decreased only partially by anti-B-50 antibodies, whereas Ca(2+)-induced noradrenaline release was inhibited almost completely. Secondly, anti-B-50 antibodies significantly reduced basal glutamate release, but did not affect basal noradrenaline release. In view of the differences in exocytotic mechanisms of small clear-cored vesicles and large dense-cored vesicles, these data indicate that B-50 is important in the regulation of exocytosis of both types of neurotransmitters, probably at stages of vesicle recycling and/or vesicle recruitment, rather than in the Ca(2+)-induced fusion step.

Role of Calcineurin in Ca2+-Induced Release of Catecholamines and Neuropeptides

Abstract: Neurotransmission requires rapid docking, fusion, and recycling of neurotransmitter vesicles. Several of the proteins involved in this complex Ca2+‐regulated mechanism have been identified as substrates for protein kinases and phosphatases, e.g., the synapsins, synaptotagmin, rabphilin3A, synaptobrevin, munc18, MARCKS, dynamin I, and B‐50/GAP‐43. So far most attention has focused on the role of kinases in the release processes, but recent evidence indicates that phosphatases may be as important. Therefore, we investigated the role of the Ca2+/calmodulin‐dependent protein phosphatase calcineurin in exocytosis and subsequent vesicle recycling. Calcineurin‐neutralizing antibodies, which blocked dynamin I dephosphorylation by endogenous synaptosomal calcineurin activity, but had no effect on the activity of protein phosphatases 1 or 2A, were introduced into rat permeabilized nerve terminals and inhibited Ca2+‐induced release of [3H]noradrenaline and neuropeptide cholecystokinin‐8 in a specific and concentration‐dependent manner. Our data show that the Ca2+/calmodulin‐dependent phosphatase calcineurin plays an essential role in exocytosis and/or vesicle recycling of noradrenaline and cholecystokinin‐8, transmitters stored in large dense‐cored vesicles.