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

Abstract

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.

Structure of the subfornical organ: A review

“…2). In the scant enkephalinimmunoreactive neuronal perikarya, a few large densecore vesicles are immunoreactive for enkephalin (Pickel and Chan, 1994); neither the sparse immunogold labeling for GABA (Pickel and Chan, 1994) nor the diffuse labeling for angiotensin II (Pickel et al, 1986) revealed any preferential cellular location. It should be noted that there are subjectively assessable quantitative differences in these attributes, even in animals that have not been subjected to any experimental procedure.…”

“…Other neurotransmitters and neurohormones that have been detected biochemically in the SFO (for citations, see Palkovits, 1987;Summy-Long, 1987) include acetylcholine, gamma-aminobutyric acid (GABA), noradrenaline, dopamine, histamine, serotonin, luteinizing hormone releasing hormone (LHRH), thyrotropin releasing hormone (TRH), somatostatin (SST), vasopressin (AVP), oxytocin, enkephalin, dynorphin, substance P, and motilin, but have so far escaped histochemical or immunohistochemical detection in neuronal perikarya. Enkephalin labeling has been reported in only a few neuronal perikarya (Wamsley et al, 1980) and may be colocalized with labeling for GABA (Pickel and Chan, 1994).…”

“…Pericapillary spaces also contain LHRH-and SST-immunoreactive axons (Krisch and Leonhardt, 1980). Enkephalin-immunoreactive axon terminals, of local origin or from other brain regions, are found apposed to the basal lamina of fenestrated capillaries (where they may regulate blood flow or glucose metabolism), in contact with glia, and to form symmetric (inhibitory, probably of AV3V origin) synapses with neuronal perikarya and unlabeled or GABA-immunoreactive dendrites (Pickel and Chan, 1994). GABA immunoreactivity is also present in symmetric synapses on unlabeled dendrites (Pickel and Chan, 1994).…”

Sensorineural hearing loss in combined modality treatment of nasopharyngeal carcinoma

“…The rabbit antiserum raised against methionine 5 ‐ENK has been previously characterized for cross‐reactivity against several members of the opioid family of peptides (Pickel and Chan,1994; Velley et al,1991). Immunodot blots showed that the antiserum principally recognized methionine 5 ‐ENK except at high concentrations (1:2,000), where there was cross‐reactivity with Leu 5 ‐ENK (Larsson1981; Velley et al,1991).…”

Dynorphin and stress‐related peptides in rat locus coeruleus: Contribution of amygdalar efferents

“…The rabbit antiserum raised against methionine 5 -ENK has been previously characterized for cross-reactivity against several members of the opioid family of peptides (Velley et al, 1991;Pickel and Chan, 1994). Immunodot blots showed that the antiserum principally recognized methionine 5 -ENK except at high concentrations (1:2,000) where there was crossreactivity with Leu 5 -ENK (Larsson 1981;Velley et al, 1991).…”