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We have studied the morphological changes induced by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment on pure cholinergic synaptosomes from Torpedo electric organ. These changes were studied by both ultrathin sections and freeze-fracture techniques. We found that after a treatment with TPA, a redistribution of synaptic vesicles inside the nerve endings and exocytotic images could be observed. Also, TPA, under conditions that induced the acetylcholine release, did not change the density of intramembrane particles at the synaptosomal protoplasmic hemimembrane leaflet. Similar results were found when calcium was not present in the extrasynaptosomal medium, and our results suggest that acetylcholine release induced by phorbol ester is probably mediated by exocytosis of synaptic vesicles.

We have studied the action of tetanus toxin on the release of acetylcholine from a subcellular fraction of cholinergic nerve terminals (synaptosomes) isolated from the Torpedo electric organ. We have also studied the morphological changes induced by chemical stimulation on the presynaptic plasma membrane of poisoned synaptosomes. These changes were studied by means of freeze-fracture techniques. We found that tetanus toxin blocks the release of acetylcholine from isolated nerve terminals in a dose-dependent manner. The maximal inhibition is achieved at a concentration of 12.5 nM in 10 min. This effect is prevented by tetanus toxin antiserum. Tetanus toxin also blocks the rearrangement of intramembrane particles at plasma membrane of poisoned synaptosomes, specifically the decrease of small (less than or equal to 9.5 nm diameter) intramembranous particles at the protoplasmic hemimembrane leaflet and the increase of large (greater than 9.5 nm diameter) intramembrane particles at the external hemimembrane leaflet induced by potassium stimulation. These results suggest that intramembrane particle rearrangement could be related to acetylcholine secretion.

Pure cholinergic synaptosomes isolated from the electric organ of Torpedo marmorata were stimulated by calcium ionophore A-23187. The effect of time course of stimulation on the changes in intramembrane particles (IMPs) on presynaptic membranes was studied by quick-freezing and aldehyde-fixation freeze-fracture. We showed that the decrease of small-particle density at the P-face and the increase of large-particle density at the E-face was maximum after 30 sec of A-23187 stimulation. Later, the density of synaptic vesicles decreased. We suggest that the redistribution of IMPs on the presynaptic membrane and acetylcholine (ACh) release from pure cholinergic synaptosomes have a similar time course when triggered by A-23187.

Botulinum neurotoxin, a strong blocker of acetylcholine release at peripheral cholinergic synapses, inhibits depolarization-stimulated protein phosphorylation in pure cholinergic synaptosomes isolated from the electric organ of Torpedo marmorata. Moreover, tetrodotoxin has the same effect on protein phosphorylation when cholinergic synaptosomes are depolarized by veratridine. Correlation between presynaptic protein phosphorylation and acetylcholine release is suggested by the fact that botulinum neurotoxin blocks specifically neurotransmitter release without affecting membrane depolarization and calcium fluxes in our synaptosomal preparation.

We have investigated the redistribution of filipin-cholesterol complexes at freeze-fractured presynaptic membrane of pure cholinergic synaptosomes isolated from Torpedo electric organ during acetylcholine release. After chemical depolarization, filipin-induced lesions increase at the presynaptic membrane. These changes do not take place when synaptosomes are stimulated in a calcium-free medium. Botulinum neurotoxin type A blocks both acetylcholine release and the rearrangement of filipin-induced lesions induced by depolarization. Since botulinum neurotoxin type A does not block either membrane depolarization or calcium entry into the nerve terminal, our results suggest that the redistribution of filipin-cholesterol complexes is linked to the acetylcholine release process.

Tetanus toxin (TeTx) is a neurotransmission impairing toxin that acts on several neurotransmitter systems. TeTx also inhibits the K+-induced release of acetylcholine (ACh) from synaptosomes isolated from the electric organ of Torpedo. Neither the membrane potential and depolarization, nor the depolarization-induced calcium uptake into cholinergic nerve terminals is modified after TeTx poisoning. On the other hand, it is known that, when cholinergic nerve terminals are stimulated, there is a release of ATP associated with the release of ACh. We have explored the action of TeTx on this co-release, and have found that there is no action of TeTx on the nucleotide release. Thus, TeTx blocks ACh release without modifying ATP release.

The action of botulinum neurotoxin on acetylcholine release, and on the structural changes at the presynaptic membrane associated with the transmitter release, was studied by using a subcellular fraction of cholinergic nerve terminals (synaptosomes) isolated from the Torpedo electric organ. Acetylcholine and ATP release were continuously monitored by chemiluminescent methods. To catch the membrane morphological changes, the quick-freezing method was applied. Our results show that botulinum neurotoxin inhibits the release of acetylcholine from these isolated nerve terminals in a dose-dependent manner, whereas ATP release is not affected. The maximal inhibition (70%) is achieved at neurotoxin concentrations as low as 125 pM with an incubation time of 6 min. This effect is not linked to an alteration of the integrity of the synaptosomes since, after poisoning by botulinum neurotoxin type A, they show a nonmodified occluded lactate dehydrogenase activity. Moreover, membrane potential is not altered by the toxin with respect to the control, either in resting condition or after potassium depolarization. In addition to acetylcholine release inhibition, botulinum neurotoxin blocks the rearrangement of the presynaptic intramembrane particles induced by potassium stimulation. The action of botulinum neurotoxin suggests that the intramembrane particle rearrangement is related to the acetylcholine secretion induced by potassium stimulation in synaptosomes isolated from the electric organ of Torpedo marmorata.