Robert R. Goodman

Immunohistochemical mapping of enkephalin containing cell bodies, fibers and nerve terminals in the brain stem of the rat

Enkephalin immunoreactive perikarya, fibers and nerve terminals, visualized by the indirect immunohistofluorescent method in colchicine-pretreated animals, are localized in many discrete regions of the rat brain stem. These specific immunohistofluorescent patterns are similar after staining with selective primary antisera directed against either methionine-enkephalin or leucine-enkephalin. Cell bodies are found in the substantia gelatinosa and interpolaris zones of the trigeminal nuclear complex, the nucleus of the solitary tract, in the vicinity of the nucleus raphé magnus, in the dorsal cochlear, medial vestibular, and paraolivary nuclei and, dorsal to this last region, in the parabrachial nuclei and the dorsal tegmental nucleus of Gudden, in the periaqueductal gray matter and interpeduncular nucleus and along the borders of the lateral lemniscus and medial geniculate. In some areas, such as the parabrachial region, nucleus of the solitary tract and substantia gelatinosa of the trigeminal nucleus, these perikarya are associated with densities of fibers and terminals. Other regions, such as the dorsal cochlear nucleus and the vicinity of the nucleus raphé magnus, contain cell bodies associated with low densities of processes and terminals. In still other nuclei, such as the nucleus of the facial nerve and the locus coeruleus, fiber and terminal densities without associated cell bodies are evident. Many of these enkephalin localizations can be rationalized on the basis of known actions of opiate drugs and the brain stem distribution of opiate receptors.

An endogenous morphine-like factor in mammalian brain

Abstract An endogenous morphine-like substance (MLF) found in rat and calf brains has a regional distribution correlating with that of opiate receptors, with the highest levels in the caudate and negligible amounts in the cerebellum. In binding assays MLF behaves like an opiate agonist. Sodium ion and enzyme and reagent treatment of membranes decrease its potency and manganese ion enhances it. MLF is localized in synaptosomal fractions, stored in an osmotically labile compartment, and can be degraded by carboxypeptidase A and leucine amino peptidase, implying a peptide structure. Its molecular weight is about 1000 as determined by gel chromatography.

Neurotensin-containing cell bodies, fibers and nerve terminals in the brain stem of the rat: immunohistochemical mapping.

Neurotensin immunoreactive perikarya, fibers and nerve terminals, visualized by the indirect immunohistofluorescent method in colchicine-pretreated animals, are localized in many discrete regions of the rat brain stem. Cell body groups are found in the inner aspect of the substantia gelatinosa of the caudal trigeminal nuclear complex, the nucleus of the solitary tract, the parabrachial nuclei, the locus coeruleus, the dorsal raphé nucleus, the periaqueductal gray matter, and the ventral tegmental area of Tsai. These areas of cell body density are accompanied by concentrations of fibers and terminals, while the occasional positive perikaryon noted in the dorsal cochlear nucleus is accompanied by only sparse fluorescent fiber/terminal patterns. Other brain stem regions, such as the floor of the fourth ventricle and aspects of the caudal ventrolateral reticular formation, possess substantial numbers of fibers and terminals that are not accompanied by cell bodies. Many aspects of this distribution coincide with the brain stem distribution of the enkephalin pentapeptides, though significant differences in localization are also evident. Interactions of neurotensin with other neurotransmitter candidates are also suggested by its presence in areas enriched in norepinephrine, dopamine, serotonin, and substance P. Certain neurotensin localizations suggest an association of the peptide with functional brain systems preferentially involving these regions. In particular periaqueductal gray and substantia gelatinosa neurotensin synapses are plausible sites for the analgesia elicited after intercisternal injection of low doses of neurotensin.

Autoradiographic localization of kappa opiate receptors to deep layers of the cerebral cortex may explain unique sedative and analgesic effects

The pharmacologically defined kappa drug 3H-ethylketazocine (3H-EKC) and 3H-bremazocine bind to unique sites, but also to mu and delta receptors. By displacing mu and delta interactions with morphine and D-Ala2, D-Leu5-enkephalin (DADL) respectively we have visualized selective receptors for 3H-EKC and 3H-bremazocine. These two kappa ligands are localized to sites different from mu and delta receptors labeled with 3H-dihydromorphine (3H-DHM) and 3H-DADL. The highest density and most selective localization of putative kappa receptors occurs in layers V and VI of the cerebral cortex. Cells in these layers project to the thalamus, regulating sensory input to the cortex. These deep cortical kappa receptors may account for the unique sedative and analgesic actions of kappa opiates.

Regional Distribution of Opioid Receptors

The development of biochemical techniques to quantitatively determine the interaction of drugs or neurotransmitters with their receptor site(s) was understandably met with enthusiasm by the scientific community. Homogenate binding studies provided a wealth of biochemical information, but only a limited amount of neuroanatomical data. The major problem was the limited resolution associated with macroscopic dissections, which resulted in only very general localizations of drug and neurotransmitter receptors. The development of autoradiography with its microscopic resolution was a major advance. Technical issues as well as the identification of various subtypes of opioid receptors have greatly complicated the interpretation of autoradiographic studies over the past decade. Initially, most investigators assumed the presence of a single class of receptor, as suggested by standard homogenate binding assays. To facilitate their studies, investigators chose radioligands upon the basis of their potency and technical factors, such as the ratio of specific to nonspecific binding, not aware that some compounds nonselectively labeled a variety of opioid receptor subtypes, whereas others were relatively selective. It soon became apparent that the various opioid receptor subtypes mediated different actions, underscoring the importance of establishing the differential distributions of subtypes of opiate binding sites.

Histochemical localization of the enkephalins

The opiate-like pentapeptides methionine-enkephalin and leucine-enkephalin (Hughes, 1975; Hughes et al., 1975; Simantov and Snyder, 1976) are found in the mammalian brain. In biochemical studies, their regional localization resembles that of opiate receptor binding (Hughes, 1975; Simantov et al., 1976a; Pasternak et al., 1975) and in subcellular fractionation studies, they are localized to the synaptosomal fractions that contain nerve terminals (Simantov et al., 1976b). Electrophysiologic studies indicate that the enkephalins can have potent effects on neuronal firing rates (Zieglgansberger et al., 1976; Duggan et al., 1976; Frederickson and Norris, 1976; Bradley et al., 1976; Young et al., 1977; Nicoll et al., 1977). Taken together, these findings suggest that the enkephalins may be neurotransmitters or neuromodulators in the mammalian brain.