Met5-enkephalin is localized within axon terminals in the subfornical organ: vascular contacts and interactions with neurons containing gamma-aminobutyric acid.

Met5-enkephalin inhibits sodium and water excretion and antagonizes the central actions of angiotensin II in subfornical organ of rat brain. We examined the ultrastructural basis for enkephalin modulation in this circumventricular region. Additionally, we examined the possibility that there might be cellular sites for functional interactions involving Met5-enkephalin and gamma-aminobutyric acid (GABA), a known inhibitory transmitter throughout the central nervous system. Met5-enkephalin and GABA were identified in single coronal sections through the subfornical organ using immunoperoxidase and silver-enhanced immunogold labeling methods, respectively. Enkephalin-like immunoreactivity was most prominently localized within axon terminals. These were distributed primarily in the central, highly vascular, regions of the subfornical organ. Enkephalin-labeled terminals were apposed to the basement membranes of fenestrated capillaries and also formed symmetric, inhibitory type synapses with neurons. In terminals associated with either blood vessels or neurons, the enkephalin immunoreactivity was enriched in large (80-150 nm) dense core vesicles. The immunoreactive vesicles were usually located within portions of the axon in close proximity to astrocytic processes. In contrast, smaller vesicles in the same terminals were more often aggregated near the basement membrane of the capillaries and the active zone of the synapse. The targets of enkephalin-immunoreactive terminals were either unlabeled or GABA-labeled dendrites of local neurons. Enkephalin was also co-localized with GABA in perikarya and in axon terminals. Terminals containing only GABA were far more abundant than those containing enkephalin or enkephalin and GABA. GABA-immunoreactive terminals formed symmetric synapses on unlabeled dendrites some of which also received convergent input from terminals containing enkephalin. Additionally, the enkephalin-immunoreactive terminals were closely apposed to GABA-labeled and unlabeled terminals. These results suggest sites for nonsynaptic release of Met5-enkephalin from dense core vesicles in contact with astrocytes near blood vessels and synaptic complexes in the rat subfornical organ. Moreover, the observed dual localization and pre- and postsynaptic associations between neurons containing Met5-enkephalin and GABA indicate that inhibitory effects of opioids in the subfornical organ may be mediated or potentiated by GABA.