Localization Of Enkephalin Research Articles

Immunohistochemical localization of enkephalin- and ACTH-related substances in the pituitary of the lamprey.

The distributions of ACTH-, alpha MSH-, beta LPH-, and enkephalin-related substances were determined immunohistochemically in the pituitary of the brook lamprey, Lampetra lamotenii. an antiserum directed against the middle region of ACTH reacted chiefly with cells in the pro-adenohypophysis. An antiserum specific for alpha MSH reacted with all of the cells of the meta-adenohypophysis, but did not react with any of the middle ACTH-positive cells in the pro-adenohypophysis. Several antisera which crossreact with both beta LPH and beta-endorphin did not react with any region of the lamprey pituitary. However, an antiserum directed against gamma LPH did react with a small population of cells in the meso-adenohypophysis. This reactivity could be blocked following pre-absorption with mouse beta LPH but was not blocked by synthetic beta-endorphin (1-31). Antisera directed against either met-enkephalin or leuenkephalin reacted with fibers in the anterior neurohypophysis, cells in the pro-adenohypophysis, and all the cells of the meta-adenohypophysis. This crossreactivity could be blocked following pre-absorption with the appropriate enkephalin, but not by pre-absorption with synthetic beta-endorphin (1-31) or dynorphin (1-13). In addition, the enkephalin-like reactivity in the adenohypophysis of the lamprey was coincident with middle ACTH-like immunoreactivity in the pro-adenohypophysis and with alpha MSH-like immunoreactivity in the meta-adenohypophysis. The absence of beta LPH/beta-endorphin immunoreactivity coincident with ACTH immunoreactivity, and the presence of enkephalin-like material in the adenohypophysis are unique to the lamprey.

Enkephalins are associated with adrenergic granules in bovine adrenal medulla

The subcellular localization of enkephalins was studied in the bovine adrenal medulla. In the adrenal medulla enkephalins (Met-enkephalin, Leu-enkephalin, Met-enkephalin-Arg 6-Phe 7 and Met-enkephalin-Arg 6-Gly 7-Leu 8) are found free and in the form of cryptic peptides included in larger precursors. Total Met-enkephalin immunoreactivity, which includes free and cryptic peptides, was determined after a sequential enzymatic treatment with trypsin and carboxypeptidase B. Total Met-enkephalin immunoreactivity, dopamine beta-hydroxylase and catecholamines were found to have a parallel distribution in the various subcellular fractions. The bulk of the total Met-enkephalin immu noreactivity (42%) was recovered in the large granule fraction. The large granule fraction also contained 38% of the total dopamine beta-hydroxylase activity, and 42% of the total catecholamines. Enkephalins are thus concentrated in the chromaffin granules. Chromaffin granules were also separated according to the method of Terland & coworkers 17 into two fractions: one containing the dense noradrenergic vesicles and the other containing lighter adrenergic vesicles. Total Met-enkephalin immunoreactivity was restricted to the fractions containing the lighter adrenergic vesicles. In these fractions the molar ratio of adrenaline to total Met-enkephalin immu noreactivity was 97. This study is in accord with immunocytochemical observations which have indicated that enkephalins are located in adrenergic and not in the noradrenergic cells in the bovine adrenal medulla.

Comparative localization of enkephalin and cholecystokinin immunoreactivities and heavy metals in the hippocampus

The present study is concerned with the cellular origins and identities of the hippocampal enkephalin and CCK-immunoreactive fibers and terminals. In the hippocampus of the rat, the guinea pig and the European hedgehog a system of enkephalin immunoreactive nerves emerges in the hilus of area dentata and can be followed to the apical dendrites of the hippocampal regio inferior pyramidal cells. This pattern of immunoreactive nerves corresponds to the hippocampal mossy fiber system as visualized by the Timm staining. Cholecystokinin immunoreactive nerve fibers and terminals reveal the same distribution in the guinea pig and the European hedgehog whereas in the rat the mossy fiber zone contains little or no cholecystokinin immunoreactivity. In the guinea pig degeneration of the mossy fibers after stereotactic lesions of the mossy fibers causes a complete loss of both enkephalin and cholecystokinin immunoreactivity in the mossy fiber zone. Only a few enkephalin immunoreactive cell bodies were scattered throughout the granular cell layer of area dentata, but inhibition of the axoplasmic transport by colchicine dramatically increased the number of enkephalin immunoreactive granule cell bodies. Enkephalin immunoreactive cell bodies were also detected in the hilus, throughout the pyramidal cell layer as well as in the stratum radiatum and stratum moleculare. Cholecystokinin immunoreactive cell bodies were seen in the hilus of the area dentata and in the stratum oriens, stratum pyramidale and stratum radiatum and the cell-rich layer of subiculum. No cholecystokinin immunoreactive cell bodies were observed in the granular cell layer of area dentata. Even after colchicine treatment the granule cells were devoid of cholecystokinin immunoreactivity. In the rat a system of nerves displaying enkephalin immunoreactivity was also observed in the superficial one-third of the molecular layer of area dentata, a zone which corresponds to the termination of the lateral perforant path. Another observation was that in the rat, but not in the guinea pig and the hedgehog, the terminal zone of both the medial perforant path and the zone of commissural and associational fibers of area dentata contained cholecystokinin immunoreactive molecules. In summary, our data show: (1) that the hippocampal mossy fibers contain enkephalin immunoreactive molecules; (2) that the cholecystokinin immunoreactivity in the mossy fiber zone is most likely also localized in the mossy fibers per se, although the granule cells seem devoid of cholecystokinin immunoreactivity; (3) zinc, here visualized as a Timm-positive substance, is also localized in the mossy fiber terminals; further, (4) other intrinsic cell bodies than the granule cells may contribute to both the enkephalin and cholecystokinin immunoreactive terminals within the hippocampus; (5) in the rat the lateral perforant path may be enkephalinergic; and (6) both the terminal zone of the medial perforant path and the associational and commissural fibers of the rat contain cholecystokinin immunoreactivity.

Sympathoadrenal preganglionic neurons: their distribution and relationship to chemically-coded fibers in the kitten intermediolateral cell column

The location of those sympathetic preganglionic neurons in the spinal cord that project to the adrenal medulla — the sympathoadrenal preganglionic (SAP) neurons — was studied by the method of retrograde axonal transport of the fluorescent dye Fast Blue. The distribution of chemically-coded fibers and their relationship to the SAP neurons was also investigated using the indirect immunofluorescence technique. In kittens, 5 of a 1% solution of Fast Blue was injected into the medulla of the left adrenal gland. After a survival period of 5 days, the spinal cords from C8 to L5 were sectioned and processed for the localization of enkephalin-, neurophysin-, oxytocin-, serotonin-, substance P- and somatostatin-like immunoreactivity. Retrogradely labeled neurons were found in the ipsilateral intermediolateral cell column (IML) (89.8% of all retrogradely labeled neurons) from T1 to L4, and in the contralateral IML (10.2%) from T1 to L4. The enkephalin, serotonin and substance P immunoreactive fibers appeared to surround both the retrogradely labeled and unlabeled IML neurons. The somatostatin immunoreactive fibers were observed only in proximity to the retrogradely labeled neurons. Only a sparse population of neurophysin and oxytocin immunoreactive fibers were observed in IML, and were not seen to be in apposition to the retrogradely labeled neurons.

Immunocytochemical localization of enkephalin in the lateral septum of the guinea-pig brain. A light- and electron-microscopic study.

By the use of antisera to met-enkephalin and leu-enkephalin, enkephalin-containing structures were visualized in the lateral septum of the guinea-pig brain. The present results do not reveal immunoreactive perikarya in this area. The immunostaining is exclusively located in numerous nerve fibers and endings mostly encompassing neuronal perikarya, which accounts for the fact that at the light-microscopic level cellular somata appear to be immunostained. The immunoreactive terminals and fibers contain granules approximately 110 nm in diameter and synaptic vesicles. The origin and the functional role of these numerous enkephalin terminals remain to be established.

Co-localization of vasoactive intestinal peptide (VIP) and enkephalins in chromaffin cells of the adrenal gland of amphibia. Stimulation of corticosteroid production by VIP

Recent studies have shown that biologically active peptides and monoaminergic neurotransmitters coexist in certain neuronal cell populations. Using the immunofluorescence technique, we have examined the localization of enkephalins, vasoactive intestinal peptide (VIP) and tyrosine hydroxylase in the adrenal gland of the frog Rana ridibunda . Most chromaffin cells which stained for tyrosine hydroxylase contained VIP-like immunoreactivity, whereas methionine- (Met-) and leucine- (Leu-) enkephalin-like immunoreactivity was detected in about 40% of the cells revealed by the anti-tyrosine hydroxylase serum. No VIP- or enkephalin-like immunoreactive nerve fibres were observed. Since in the frog, the chromaffin cells are in close contact with the adrenocortical (interrenal) tissue, a possible action of VIP and opiates on corticosteroidogenesis has been investigated. At doses 10 −6 and 10 −5M, 20-min infusions of synthetic porcine or chicken VIP elicited a significant increase in corticosterone and aldosterone production by perifused frog adrenals, in a dose-dependent manner. As compared to ACTH, VIP was several orders of magnitude less effective in stimulating corticosteroid production. Morphine, Met- and Leu-enkephalins (10 −5M) had no effect on spontaneous secretion of corticosteroids. In addition, Met- and Leu-enkephalins (10 −5M) did not alter the production of corticosterone induced by ACTH. These results suggest that VIP contained in the chromaffin cells of the frog adrenal gland may exert a local action in stimulating corticosteroid production by the interrenal tissue.

The distribution of enkephalin immunoreactive fibers and terminals in the monkey central nervous system: An immunohistochemical study

Using immunohistochemical techniques, the distribution of met-enkephalin fibers and terminals was studied in the central nervous system of adult old-world monkeys. Areas which showed the greatest density of immunoreactivity included substantia gelatinosa, nucleus tractus solitarius, nucleus parabrachialis, substantia nigra, median eminence, globus pallidus (external segment), patches within the striatum and the region of nucleus accumbens and the olfactory area. Striking and discrete zones of enkephalin immunoreactive fibers and terminals which did not conform to known nuclear boundaries were observed in the latter areas. The distribution of enkephalin in the monkey is compared to what has been described in the rat central nervous system. In general, the two species are similar, however, differences were observed in some areas including the hypoglossal nucleus, substantia nigra and in the region of the nucleus accumbens and olfactory area. The results are discussed with regard to the possible functional significance of enkephalin localization in regions related to regulation of pain, mood, and autonomie function.

Ultrastructural immunocytochemical localization of enkephalin in the goldfish preoptic nucleus

This study describes the ultastructural localization of the opioid peptide enkephalin (ENK) in the preoptic nucleus of the goldfish. Using immunocytochemical techniques, ENK could be seen in neurosecretory granules and throughout the cytoplasm of magnocellular neurons with an agranular distribution. ENK was also associated with small clear vesicles and large dense-core vesicles within certain axon terminals in the preoptic nucleus. In this report, we discuss the cellular and synaptic relationships of ENK neurons in the preoptic nucleus of the teleost.

Immunocytochemical localization of enkephalin and substance P in retina and eyestalk neurones of lobster.

Enkephalin and substance P are two peptides found in vertebrate nervous systems that have been intensively studied with regard to cellular distribution and receptor mechanisms1–3. Invertebrate nervous systems, which are extremely useful in elucidating general principles of neural functions4–6, can also be used as models for the analysis of peptidergic mechanisms7,8. The accessibility, regularity, small number and relatively large size of the neural components of one such system, the crustacean compound eye, have facilitated the accumulation of detailed information about their anatomy9–11 and physiology12–14, and provided insight into basic principles of intercellular communication12–14. We report here that immunoreactivity of enkephalin and substance P can be localized to specific neural elements in the visual system of the spiny lobster, Panulirus interruptus. Our observations suggest that this highly organized, simple in vivo system may be a convenient model to evaluate the cellular mechanisms of these peptides. Although several neuropeptides have been localized in vertebrate retinal inter-neurones15, our results are the first to detect a known peptide in primary photoreceptors.

Comparative immunocytochemical localization of enkephalin and somatostatin in the median eminence, hypothalamus and adjacent areas of the guinea-pig brain

Because of the presence of an enkephalin-related substance in the somatostatin terminals of the median eminence 47, an investigation was carried out on the staining for enkephalin of the somtostatin-containing perikarya localized in the hypothalamic periventricular nucleus. Only an occasional and weak enkephalin immunoreactivity was detected in these perikarya, making the common synthesis of the two peptides unlikely. Moreover, our results demonstrate an enkephalin staining in other fibers of the median eminence clearly distinct from the somatostatin ones. Enkephalin was demonstrated in perikarya distributed in the septum, preoptic area, dorsal and ventromedial hypothalamus, all regions known to possibly project into the median eminence. In none of these sites was enkephalin found in association with somatostatin. The present morphological results support the hypothesis that enkephalins may e exert their GH-releasing effect at the hypothalamic level and suggest that enkephalins might directly modulate the somatostatin release into the blood of the portal plexus.

Immunohistochemical localization of enkephalin in the central nervous system and pituitary of the lizard, Anolis carolinensis.

The topographical distribution of enkephalin in the central nervous system of the lizard, Anolis carolinensis, has been studied by the immunoperoxidase technique with antiserum to leucine-enkephalin. Immunoreactive enkephalin perikarya, fibers and probably terminals are widely distributed throughout the central nervous system, which agrees well with the distribution of enkephalins in the mammalian brain. Enkephalin-containing perikarya are found in the subpallium (septum, nucleus accumbens, striatum, amydgala), preoptic and hypothalamic region, ventromedial nucleus and ventromedial area of thalamus, pretectal geniculate nucleus and posterodorsal nucleus of pretectum, nucleus of the lateral lemniscus, locus ceruleus, spinal trigeminal nucleus, nucleus of the solitary tract, medial parvocellular nucleus, and dorsal horn of the spinal cord. Enkephalinergic fibers and terminals are found in the above-mentioned areas as well as in the pallium (medial and dorsal cortex, dorsal ventricular ridge), dorsomedial and anterior dorsolateral nucleus of the thalamus, habenula, nucleus of the stria medullaris, torus semicircularis, mesencephalic tegmental area, interpeduncular nucleus, mesencephalic trigeminal nucleus, central gray, reticular formation, raphe nucleus, substantia nigra, isthmus region, and nucleus of the trapezoid body. Enkephalinergic pathways appear to exist between the septum and the medial cortex, nucleus accumbens and nucleus of the lateral olfactory tract, striatum and certain mesencephalic structures, hypothalamus and tegmentum, and between nucleus of the lateral lemniscus and torus semicircularis. In the pituitary, cells of the pars intermedia, and certain cells of the rostral pars distalis also show immunoreactivity to enkephalin antiserum. The distribution of enkephalin immunoreactivity throughout the hypothalamus and in the median eminence suggests involvement in neuroendocrine regulation. Presence of enkephalin in many extrahypothalamic brain areas indicates its important role in various sensory functions and in behavioral and autonomic integration.

The immunocytochemical localization of enkephalin in the central nervous system of the rat.

The immunocytochemical localization of enkephalin-like immunoreactivity (ELI) throughout the rat central nervous system (CNS) was investigated. The detection of ELI-containing structures was facilitated through the use of (1) brains from colchicine-treated rats, (2) the proteolytic pretreatment of sections with pronase and (3) the "double-bridge" staining technique. Our findings confirm the presence of ELI in perikarya, neuronal processes and terminals in many areas of the CNS. In addition, the localization of ELI-containing perikarya is reported for the first time in the following areas: the olfactory bulb, the olfactory tubercle, the lateral preoptic nucleus, several nuclei within the amygdaloid nuclear complex, the hippocampus, the neocortex, the cingulate cortex, the posterior mammillary nucleus, the medial nucleus of the optic tract, the brachium of the inferior colliculus, the ventral tegmental nucleus, the locus ceruleus, the sub-ceruleal region, the lateral trapezoid nucleus, the nucleus reticularis lateralis, and lamina VII of the cervical spinal cord. Our results demonstrate ELI in neurons which are heterogeneous in size, some probably functioning as interneurons and others as projection neurons in different areas of the CNS. The location of these neurons within the brain suggests that these pentapeptides serve diverse functions which include, in addition to nociception, the regulation of neuroendocrine, respiratory, auditory, vestibular, and olfactory functions.

Electron microscopic localization of enkephalin in the median eminence and the adenohypophysis of the guinea-pig

By using different immunocytochemical methods, it has been possible to define the ultrastructural localization of enkephalins in the median eminence and in the adenohypophysis of the guinea-pig. In the median eminence the staining was essentially confined to the granules of nerve terminals in the palissade zone. When the immunocytochemical procedure occurred before embedding the tissue, the granules displayed several electron-dense dots revealing the presence of enkephalin. In addition this technique made it possible to observe a peripheral staining of the small vesicles. In the adenohypophysis three cell types bound antisera to leucine or methionine-enkephalins: the gonadogrophs, the thyrotrophs and the corticotrophs. In all three cases the reaction product was obtained on the granules. It is proposed that enkephalins might act on the pituitary secretions not only at the hypothalamic level but also at the pituitary level.

Immunocytochemical localization of enkephalin and substance P in the dorsal tegmental nuclei in human fetal brain.

The peroxidase-antiperoxidase (PAP) technique is used to examine the localization of methionine [Met 5]-enkephalin and substance P in the dorsal tegmental nuclei of the human fetal brain. Specific antiserum to both peptides is immunocytochemically detectable in neuronal perikarya and varicose processes in the human dorsal tegmental nuclei at 17-21 weeks of gestation. However the two peptides show a differential distribution in the central versus peripheral portions of the nucleus. Enkephalin-like immunoreactivity is primarily localized in perikarya along the pripheral borders of the tegmental nuclei, whereas substance P is present primarily in varicose terminals and a few perikarya in the more central portions. These findings suggest that substance P-containing terminals in dorsal tegmental nuclei are probably derived from afferent axons and that enkephalin is present in intrinsic neurons.

A topographic localization of enkephalin on the dopamine neurons of the rat substantia nigra and ventral tegmental area demonstrated by combined histofluorescence-immunocytochemistry

A topographic localization of enkephalin on the dopamine neurons of the rat substantia nigra and ventral tegmental area demonstrated by combined histofluorescence-immunocytochemistry

Immunohistochemical localization of enkephalin in rat forebrain

Discrete localization of enkephalin immunoreactive sites in the rat forebrain was undertaken using animals with and without colchicine pretreatment. Both the immunofluorescence and unlabeled PAP antibody techniques were employed using antisera directed against methionine-enkephalin. We have found several areas of enkephalin fiber and cell localizations not previously described, as well as confirming the results of other researchers. Thus, wer found large amounts of perikaryal enkephalin immunoreactivity in the lateral septal nucleus, bed nucleus of the stria terminalis, the preoptic nuclei, the ventral premammillary nucleus, prelateral mammillary nucleus, the medial mammillary nucleus, paraventricular nucleus, the periventricular, ventromedial, dorsomedial and posterior nuclei of the hypothalamus, the arcuate nucleus, and the ventral nucleus of the lateral geniculate body. High concentrations of immunoreactive neuronal fibers not immediately associated with enkephalin perikarya were seen in the nucleus accumbens, medial fore-brain bundle, globus pallidus, mammillothalamic tract, the subthalamic nucleus, and the caudal portion of the zona incerta. Many of the areas presented in this study correspond with areas known to exert some regulation over neuroendocrine function as well as various motor and sensory functions. Nuclei with high immunoreactive fiber content also correspond in many instances with areas reported to have high concentrations of opiate receptors. Possible enkephalin-containing tracts and the relationships of enkephalin-containing cells in fiber bundles are also pointed out.

Relationship between enkephalinergic neurons and the vasopressin-oxytocin neuroendocrine system of the cat: an immunohistochemical study.

Localization of enkephalins and opiate binding sites in the central nervous system of rats has been reported by several authors. These studies did not reveal an extensive enkephalinergic system in the hypothalamo-hypophyseal axis of rats. The present paper reports on an extensive enkephalinergic system in the cat hypothalamo-hypophyseal system. Sections of paraformaldehyde fixed cat hypothalami were incubated with anti-methionine enkephalin serum, anti-vasopressin serum, and anti-oxytocin serum. Immunohistochemical localization of methionine enkephalin fibers and terminals in the median eminence, hypophyseal stalk, and pars nervosa was similar, but not identical to the distribution of vasopressin and oxytocin in these structures. Neuronal perikarya localized with the three antisera in the nucleus supraopticus and nucleus paraventricularis were of a similar size and morphology. In cats treated with colchicine prior to sacrifice, the anti-methionine enkephalin serum revealed a group of periventricular cell bodies. Cell bodies were not localized in this area with anti-vasopressin or anti-oxytocin sera. The functional significance of such an extensive enkephalinergic system in the cat hypothalamo-hypophyseal axis is discussed.

Immunocytochemical localization of enkephalin in the neostriatum of rat brain: A light and electron microscopic study

The peroxidase-antiperoxidase (PAP) immunocytochemical technique was used to determine the light and electron microscopic localization of antisera directed against either methionine [Met5]-or leucine [Leu5]-enkephalin in the neostriatum of brains from untreated rats. By light microscopy, neuronal perikarya and processes showing enkephalin-like immunoreactivity (ELI) were unevenly distributed throughout the neostriatum. The greatest accumulation of neuronal structures showing ELI was in the ventro- and caudo- lateral portions of the nucleus. The labeled perikarya measured 10--15 micrometer in diameter and constituted about 15--20% of the total neurons in the neostriatum. By electron microscopy, examination of three areas from horizontal and coronal sections revealed no regional differences in types of neurons showing ELI or in their synaptic organization. All labeled neurons showed a relatively low intensity reaction product which was diffusely distributed throughout the perikarya and dendrites. The cytoplasm contained relatively few organelles, which included mitochondria, endoplasmic reticulum and numerous "alveolate" vesicles. The dendrites had many spiny processes which formed asymmetric synapses with unlabeled axon terminals containing all small clear vesicles. In contrast to the perikarya and dendrites a dense accumulation of reaction product was present in a few myelinated and numerous unmyelinated axons and axonal varicosities. Approximately 75% of the labeled varicosities did not form a specialized synaptic junction in a single plane of section. The remaining 25% of the labeled terminals formed asymmetric junctions primarily with unlabeled dendrites and rarely with unlabeled perikarya or axons. The morphology and synaptic relations of the neurons showing ELI suggest that they may belong to the general group of medium-sized spiny cells characterized in Golgi studies by Kemp and Powell ('71a). At least some of the peptide-containing neurons may also have a myelinated efferent axons.

Immunohistochemical localization of enkephalins in median eminence and adenohypophysis

Immunohistochemical staining for methionine-enkephalin and leucine-enkephalin were used to reveal an enkephalins-like substance in the median eminence and adenohypophysis of guinea pig and rat. Nerve endings were stained for enkephalins in the external layer of the median eminence. By successive staining of two different antigens, somatostatin fibers in the guinea pig were also shown to be enkephalin-immunoreactive. Staining of the adenohypophysis varied with the species and the antisera used. Intermediate lobe and corticomelanotrophs were stained moderately. In both species, and with all the antisera, thyrotrophs were shown to contain an immunoreactive substance. In the guinea pig, gonadotrophs were stained particularly by the anti-leucine-enkephalin antiserum used. The locus of enkephalin action on pituitary functions can be discussed: enkephalins possibly have an inhibiting action on somatostatin fibers, which agrees with the stimulating action of enkephalins on GH already known. Furthermore, enkephalins could have a direct action on pituitary glycoprotein proceducing cells, which would explain their inhibiting action demonstrated previously on LH, FSH and TSH.

Localization of leu-enkephalin in dense core vesicles of axon terminals

The localization of enkephalins by immunofluorescence has already shown that these pentapeptides are largely distributed in the central nervous system. In order to clearly identify the structures and organelles containing enkephalins, an immunohistochemical localization of Leu-enkephalin was performed at the ultrastructural level. Leu-enkephalin was detected in dense core vesicles (70–100 nm in diameter) of many nerve fibers in the substantia gelatinosa of the spinal cord, the globus pallidus and the nucleus parabrachialis. These findings support the hypothesis that Leu-enkephalin could act as a neuromodulator.