Opioid peptide enkephalin: immunohistochemical mapping in rat central nervous system.

Abstract

Using specific antisera to methionine-enkephalin and leucine-enkephalin, we have visualized apparent enkephalin-containing neuronal fibers and terminals throughout the central nervous system of the rat. Immunoreactive enkephalin displays sharply defined localizations. Regions of highest immunofluorescent density include the laminae I and II of the spinal cord, the substantia gelatinosa of the caudal nucleus of nerve V, the vagal nuclei of the medulla, the periventricular and periaqueductal areas of the upper medulla and midbrain, dorsomedial thalamic regions, specific hypothalamic nuclei, the basal ganglia, particularly the globus pallidus and the central nucleus of the amygdala, and the lateral septum. In certain regions enkephalin immunofluorescence corresponds closely with the distribution of autoradiographic opiate receptor grains.The opiate-like pentapeptides methionine-enkephalin (Metenk) and leucine-enkephalin (Leu-enk) (1, 2) appear to be endogenous ligands for the opiate receptor. The regional localization of enkephalin in mammalian brain, determined biochemically, resembles that of opiate receptor binding (3-6). In subcellular fractionation studies, enkephalin is localized to synaptosomal fractions that contain nerve terminals (7). Autoradiographic studies of the opiate receptor reveal sharply defined localizations to structures mediating functions affected by opiates, such as pain perception (8)(9)(10). If the enkephalins are neurotransmitters or neuromodulators associated with opiate receptors, one might expect enkephalin to be localized microscopically to neuronal systems impinging on opiate receptors. Preliminary immunohistochemical studies show immunoreactive enkephalin fluorescence in nerve fibers and terminals with highest densities in areas enriched in opiate receptors (11). We now report a detailed mapping of the rat central nervous system for immunoreactive enkephalin.MATERIALS AND METHODS Antisera Preparation. Met-enk or Leu-enk (20 mg) was coupled to keyhole limpet hemocyanin (10 mg) by incubation for 30 min in distilled water at room temperature with 150 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The material was dialyzed extensively against distilled water, lyophilized, suspended in 3 ml of distilled water, and stored. Guinea pigs were immunized with 1 mg of the hemocyanincoupled enkephalin diluted 1:10 in saline and mixed 1:1 with Freund's complete adjuvant. Rabbits were injected with 1.6 mg of this conjugate. The immunization was repeated three to four times at 3 to 4 week intervals using incomplete Freund's adjuvant. The guinea pigs and rabbits were bled 7-10 days after the third and subsequent immunizations and the sera were tested for enkephalin binding in radioimmunoassays (ref. 12; Abbreviations: Met-enk, methionine-enkephalin; Leu-enk, leucineenkephalin.* To whom reprint requests should be addressed.Simantov, Childers, and Snyder, unpublished data). Enkephalin binding by these antisera was not displaced by high concentrations of substance P, glucagon, insulin...

Hypothalamic regulation of the cardiovascular and respiratory systems: role of specific opiate receptors

“…First, opioid peptides and opiate receptors are found in brain nuclei which are known to control haemodynamic and respiratory functions (Simantov, Kuhar, Uhl & Snyder, 1977;Dupont, Lepine, Langelier, Merand, Rouleau, Vaudry, Gros & Barden, 1980). Second, central opioid systems have been implicated in the pathophysiology of shock due to various causes Janssen & Lutherer, 1980).…”

Enkephalin-like immunoreactivity of olivocochlear nerve fibers in cochlea of guinea pig and cat

“…Immunohistochemical mapping of enkephalin-like immunoreactivity in the central nervous system was done (3)(4)(5), and it was noted that in certain regions enkephalin-like immunoreactivity and opiate receptors had a similar distribution (3,5). A much entertained working hypothesis is that enkephalins may act as neurotransmitters or modulate the action of neurotransmitters (6).…”

Methionine enkephalinergic neuronal activity in cerebral nuclei of spontaneously hypertensive rats.

“…From each frozen brain, coronal sections (25 fim) were cut serially and processed as described previously. 6 ' l2 They were then mounted on glass cover slips, and brought to reaction for 30 min at 37°C with fivefold diluted anti-ME rabbits sera.…”

“…These areas are the nucleus (n.) tractus solitarii, n. arcuatus, n. interstitialis striae terminalis, and magnocellular nuclei. 6 "…”