Glutamate and dynorphin release from a subcellular fraction enriched in hippocampal mossy fiber synaptosomes

A procedure is described for the isolation of intact hippocampal mossy fiber synaptosomes. Electron microscopic examination revealed numerous synaptosomal profiles which are clearly of mossy fiber origin, indicated by their large size (2-6 micron diameter) and characteristic morphology. Furthermore, this fraction is enriched in zinc and dynorphin B which appear to be concentrated in mossy fiber terminals in vivo. Synaptosomes isolated by this procedure accumulated 2-deoxyglucose and retained 88% of total lactate dehydrogenase activity after incubation at 30 degrees C for 60 minutes, indicating a high degree of membrane integrity. Oxygen consumption was stimulated 4-fold by veratridine (0.1 mM) and inhibited 90% by ouabain (1 mM), suggesting that synaptosomal metabolism remained tightly coupled to ouabain-sensitive ATPase activity. Potassium-stimulated (45 mM) release of dynorphin B was completely dependent upon the presence of extrasynaptosomal calcium, while only 30% of the evoked release of glutamate was calcium-dependent. D-aspartate, which exchanges glutamate out of the cytoplasmic pool, virtually eliminated the calcium-independent component of glutamate release. This synaptosomal preparation will be useful in identifying the factors that modulate the release of amino acid and opioid neurotransmitters from hippocampal nerve terminals and in the investigation of their presynaptic mechanisms of action.