Enkephalin-like Immunoreactivity Research Articles

Spinal cord grafts: An intraocular approach to enigmas of nerve growth regulation

The possible usefulness of intraocular transplantation in studies of spinal cord growth and regeneration has been evaluated. Defined segments of fetal rat spinal cord were grafted to the anterior chamber of the eye of adult rats. Such grafts become vascularized from the host iris, grow and develop neuron types, myelinated fiber bundles, astroglial populations (as shown by GFA-immunoreactivity), and electrical activity reminiscent of such features in normal spinal cord tissue. The intraocular technique permits studies of intrinsic circuitries as well as conditions for formation of afferent and efferent connections with the host iris and with other central or peripheral tissues which can be grafted into contact with the spinal cord grafts. One example of an intrinsic system preserved in the grafts is a rich network of nerve fibers with enkephalin-like immunoreactivity. When combined with cerebral cortex, the enkephalin-positive neurons of the spinal cord graft are able to form only very limited projections to the cortex graft. Special emphasis was given the possible formation of adrenergic afférents to spinal cord grafts. No appreciable ingrowth of peripheral sympathetic nerves occurred. Locus coeruleus grafts have many organotypical electrophysiological characteristics and were able to innervate adjacent spinal cord grafts provided that the sensory innervation of the host iris was removed. Experiments such as these suggest that “negative neurotropic” factors may be present in spinal cord and possibly relate to the unique relationship between spinal ganglia and spinal cord.

Enkephalin-mediated basal ganglia influences over the optic tectum: immunohistochemistry of the tectum and the lateral spiriform nucleus in pigeon.

By using immunohistochemical techniques with antisera directed against either leucine-enkephalin or methionine-enkephalin (generously supplied by K.-J. Chang), four distinct bands of fibers with enkephalinlike immunoreactivity were demonstrated in the pigeon tectum: (1) a thin band of thick fibers and tightly clustered bulbous swellings in layer 3, (2) a broader band of fibers with less tightly clustered bulbous swellings in layer 5, (3) a broad band of numerous obliquely and radially oriented fibers that spanned layers 8-13, and (4) a band of sinuous fibers in layer 15. In addition, numerous enkephalinergic cell bodies with radially ascending processes were seen in layers 8-10. Since the neurons of the avian lateral spiriform nucleus (SpL) of the pretectum are known to contain enkephalin (Davis et al., '80; De Lanerolle et al., '81) and project to the tectum (Brecha et al., '76; Reiner et al., '82), unilateral electrolytic lesions were made of SpL. In birds with unilateral lesions of SpL, layers 8-13 of the ipsilateral tectum were nearly devoid of enkephalinergic fibers, but no alterations were seen in layers 3, 5, and 15. Since no other neurons in the vicinity of SpL are enkephalinergic and project to the tectum, the loss of enkephalin-immunoreactive fibers in the ipsilateral tectal layers 8-13 is attributable to the destruction of SpL. Although the source of the enkephalinergic fibers in tectal layers 3, 5, and 15 is unclear, part of the enkephalin pattern in layers 3 and 5 may derive from the ascending processes of the enkephalinergic neurons of layer 8-10. The present results indicate that SpL has an enkephalinergic projection to layers 8-13 of the ipsilateral tectum. The avian SpL receives its major input from the ascending processes of the enkephalinergic neurons of layers 8-10, nuclei that themselves receive major basal ganglia inputs (Reiner et al., '82) and projects to the tectal layers 8-13 (Reiner et al., '82), the layers of origin of the major tectal efferent projections (Reiner and Karten, '82). The enkephalinergic fibers in layers 8-13 may, thus, have some influence upon the motor output functions of the avian tectum.

Evidence for the existence of an enkephalin-containing pathway from the area just ventrolateral to the anterior hypothalamic nucleus to the lateral septal area of the rat

The afferent source of enkephalin-like immunoreactive (ELI) fibers in the lateral septal area of the rat was elucidated experimentally by means of an indirect immunofluorescence technique. These ELI fibers had almost completely disappeared on the operated side after the destruction of the area just ventrolateral to the anterior hypothalamic nucleus, where a number of ELI cells was detected. This fact strongly suggests that these ELI cells project ipsilaterally to the lateral septal area.

Distribution of enkephalin-like immunoreactivity in the rat main olfactory bulb

Enkephalin-like immunoreactivity was localized within the main olfactory bulb of the rat using immunohistochemical techniques. These studies utilized well characterized antisera directed to either leu 5- or met 5-enkephalin. Specificity was established by absorption of the antisera with either 10 μM synthetic leu 5- or met 5-enkephalin. Specific enkephalin-like immunoreactivity was observed within several different cell populations including (1) periglomerular cells, (2) granule cells and their processes within the external plexiform layer and (3) occasional short-axon (horizontal) cells within the granule and external plaxiform layers. The granule cell layer contained the greatest number of immunoreactive cells. Only a limited number of immunoreactive cells were found in both the periglomerular and granule cell layers, suggesting the enkephalin-containing neurons represent a sub-population within each layer. The absence of immunoreactive processes in the periventribular white matter, as well as the morphologies of immunoreactive bulbar neurons, indicates that enkephalin is found exclusively within intrinsic olfactory bulb neurons.

Immunocytochemical localization of enkephalin-like immunoreactivity in the retina of the guinea pig.

The distribution of enkephalin-like immunoreactivity in the retina of the guinea pig was studied. Indirect immunofluorescence techniques were used on retinae with and without colchicine pretreatment. In retinae not receiving colchicine pretreatment, enkephalin-like immunoreactivity was seen in fibers in the inner plexiform layer, predominantly in laminae 1, 3, and 5. In colchicine-pretreated retinae, enkephalin immunofluorescent cell bodies were seen in the inner margin of the inner nuclear layer in addition to the immunoreactive fibers. These cells showed morphological characteristics of amacrine cells. No enkephalin-like immunofluorescence was seen in the optic nerve, ganglion cell layer, or outer nuclear or plexiform layers. These findings of enkephalin-like immunoreactive cells and fibers in a mammalian retina add to the findings in nonmammalian retinae and suggest that the enkephalins play a role in primary sensory systems of mammalian and nonmammalian species.

Enkephalin-like immunoreactivity is restricted to the adrenaline cells in the hamster adrenal medulla.

Enkephalin-like immunoreactivity was demonstrated using the peroxide-antiperoxidase method. Only the adrenaline-storing cells of the hamster adrenal medulla were labelled.

Altered methionine enkephalin immunoreactivity in cerebral nuclei of spontaneously hypertensive rats.

In young SHR, methionine enkephalin-like immunoreactivity (ME) levels were lower than WKY in the tractus spinalis n. trigemini, n. reticularis lateralis, n. arcuatus and n. corporis mamillaris lateralis, whereas higher in the n. dorsalis n. vagi, n. amygdaloideus medialis and n. striae terminalis. In adult SHR, the alteration in these nuclei disappeared. ME levels were higher than those of WKY in the n. reticularis medialis and n. accumbens septi, while lower in the area lateralis hypothalami. The alteration was not due to hypertension, since acute hypertension induced with angiotensin II produced no change in these nuclei. The decrease in ME neuronal activity in bulbar sensory and somatosensory afferents, and n. arcuatus, and ME neuronal activation in the limbic depressor neurons may be involved in the development of hypertension and hyper-reactivity in SHR.

Enkephalin-like immunoreactivity in the gustatory lobes and visceral nuclei in the brains of goldfish and catfish

The distribution of enkephalin-like immunoreactivity was examined in the medulla of two species of teleost fish, Carassius auratus and Ictalurus nebulosus. The primary visceral sensory nuclei of these species are clearly divided into three segments: facial gustatory lobe, vagal gustatory lobe and a general visceral nucleus. Dense ELI occurs in the general visceral nucleus and in certain layers or portions of the vagal gustatory lobe. The facial gustatory lobe contains a few enkephalin-like immunoreactive fibers, far less than either of the other two primary visceral sensory areas. Large numbers of such immunoreactive fibers are also associated with the facial nucleus, nucleus ambiguus and certain neurons of the dorsal motor nucleus of the vagus. The primary gustatory area of the medulla should not be considered to be a unitary structure but includes a vagal portion rich in ELI and a facial portion largely devoid of such immunoreactivity. This appears to be a feature common to fish and mammals if not all vertebrate classes.

Enkephalin-like immunoreactivity in vocal control regions of the zebra finch brain

Singing in passerine birds is an androgen-dependent behavior typical of males, and in many species is learned during an early critical period. Brain regions which control song form a rather discrete, interconnected series of nuclei which have been described in the canary and zebra finch. These regions include the caudal nucleus of the hyperstriatum ventrale (HVc), the robust nucleus of the archistriatum (RA), the magnocellular nucleus of the neostriatum (MAN), area X of the lobus parolfactorius, nucleus interface (NIF), intercollicular nucleus (ICo), and the tracheosyringeal portion of the hypoglossal motor nucleus (nXIIts). In the present report, we describe cell bodies and terminals in these brain regions which contain enkephalin-like immunoreactivity (ELI). This study is the third in a series investigating the histochemical characteristics of the vocal control system in zebra finches.

Enkephalin-like immunoreactivity in the mossy fiber pathway of the hippocampal formation of the tree shrew ( Tupaia glis)

The distribution of enkephalin-like immunoreactivity within the hippocampal formation of the tree shrew ( Tupaia glis) has been investigated. By far the most conspicuous immunoreactive structures within the hippocampal formation were components of the mossy fiber pathway. Immunoreactive granule cells within the dentate gyrus could often be seen sending dendrites into the molecular layer and axons into the hilus of the dentate gyrus. Within the regio inferior of Ammon's horn, massive immunoreactive terminal swellings associated with thin fibers were located in the zone just above the pyramidal cell layer. While the mossy fiber pathway was the most intensely labeled portion of the hippocampal formation, occasionally sparse terminals and lightly immunoreactive cell bodies were noted in regio superior. These results indicate that the mossy fiber system may be an important pathway for opiate modulation of hippocampal activity.

Enkephalin immunoreactivity in Golgi cells and mossy fibres of mammalian, avian, amphibian and teleost cerebellum

The cerebella of cats, rats, rabbits, pigeons, fish and frogs were examined immunohistochemically with antibodies raised against leucine-5-enkephalin. Enkephalin-like immunoreactivity was found in Golgi cells in all species examined except pigeon and frog, and enkephalin-like immunoreactive mossy fibres were seen in all species examined. In albino rat, rabbit and goldfish, enkephalin-like immunoreactive Golgi cells were inhomogeneously distributed in the cerebellum. This was not the case with a pigmented rat strain or in cat or catfish. Rabbits, which are known to have ‘displaced’ Golgi cells in the molecular layer, likewise showed enkephalin-like immunoreactive Golgi cells in the molecular layer. The variations in structure of the immunoreactive mossy fibres matched the inter-species variations of mossy fibre morphology which have been established with silver-impregnation techniques. We suggest that enkephalin may play a role in afferent and interneuronal cerebellar synaptic communication.

Factors governing release of methionine enkephalin-like immunoreactivity from mesencephalon and spinal cord of the cat in vivo.

Factors governing release of methionine enkephalin-like immunoreactivity from mesencephalon and spinal cord of the cat in vivo.

Differential response of methionine-enkephalin in basal hypothalamus and preoptic area following hypothalamic deafferentation

Following identification and characterization of naturally occurring opiate peptides from the brain and pituitary gland 4,5 several investigators have examined the precise distribution of fl-endorphin and enkephalin-like immunoreactivity in the neurons of the rat brain 1,9,11-13. Enkephalin-like immunoreactivity has been localized by radioimmunoassays and by immunocytochemical means in various nuclei of the diencephalon 11-13. Of particular significance are the demonstrations of enkephalin containing neurons in the preoptic-tuberal pathway 11-13, and the ability of methionine-enkephalin (Met-enkephalin) administration to influence LH and prolactin secretion in the rat 2,3,10. In this communication we report the effects of disruption by surgical means of the preoptic-tuberal pathway on the Met-enkephalin levels in the basal tuberal regions and the anterior hypothalamic-preoptic area (AHPOA). Adult Charles River (CD) rats were maintained in air-conditioned rooms (76 ° F) with controlled light/dark cycle (lights on between 0500 and 1900 h). The rats were ovaliectomized under pentabarbitol anesthesia (40 mg/kg, i.p.) and underwent hypothalamic deafferentation with the help of Halasz-type knife which was lowered 1.6 mm behind the bregma when the rats were held in a stereotaxic instrument with the nosebar 7 mm below the ear bar. Two types of deafferentations were performed 6,7; for anterior hypothalamic deafferentation (AHD, knife dimensions, width -2.8 mm and height --: 2.2 ram) the knife was lowered to the base of the brain and rotated 90 ° to either side to transect anterolateral inputs to the medial-basal hypothalamus (MBH) along the caudal border of optic-chiasm; for complete hypothalamic deafferentation (CHD) the MBH was isolated from the rest of the brain by rotating the knife (width 3 mm, height -2.5 mm) full 360 °. The cut extended anteriorly along the caudal border of the optic-chiasm and caudally through the mammillary bodies. In sham deafferentation controls the knife was lowered to the base of the brain and lifted up without

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.

Localization of enkephalin-like immunoreactivity to identified axonal and neuronal populations of the rat hippocampus.

The distribution of enkephalin-like immunoreactivity in the hippocampal formation of the rat was analyzed. Two specific projection systems are described. The first emerges from the hilus of the dentate gyrus and appears to terminate with notably large boutons on the proximal apical and, to a lesser extent, basal dendrites of hippocampal regio inferior pyramidal cells. This projection corresponds in source, position, and character to the hippocampal mossy fiber system. The second axonal population enters the temporal hippocampal formation from the medial wall of the subicular complex and follows the hippocampal fissure to occupy stratum lacunosum-moleculare of the hippocampus proper and the distal third of the dentate gyrus molecular layer; this pattern corresponds to the distribution of afferent input from the lateral entorhinal cortex and/or perirhinal area. Lesions of the hilus or retrohippocampal area caused a selective depletion of immunoreactivity in the mossy fiber fields and molecular layers of the dentate gyrus, respectively. Enkephalin-like immunoreactivity was found within the somata of three types of hippocampal neurons: 1) granule cells of the dentate gyrus, 2) occasional pyramidal shaped cells of field CA1 stratum pyramidale, and 3) varied scattered interneurons. Of this last group, two types of interneurons were consistently seen. The first occupy the border between stratum radiatum and stratum lacunosum-moleculare and extend processes at right angles to the long axis of the pyramidal cell dentrites, whereas the second lie within stratum radiatum of field CA1 and extend processes in alignment with the long axis of the pyramidal cell dendrites. Cells containing enkephalin-like immunoreactivity were also observed in the subiculum and retrohippocampal region, most notably including layers II and III of the lateral entorhinal cortex-perirhinal area--the probable source of extrinsic immunoreactive input to the hippocampal formation. Intraventricular colchicine treatment intensified the immunoreactive staining of some hippocampal neurons but did not reveal any cell types not seen to be labeled in untreated rats.

Anatomical distribution and physiological effects of enkephalin in rat inferior olive

The opioid peptide enkephalin was evaluated as a possible synaptic transmitter in the inferior olivary complex using anatomical and physiological techniques. With standard immunohistochemical procedures, fibres and terminals containing enkephalin-like immunoreactivity were found to be heterogenously distributed in the inferior olive of the rat. Enkephalin-like immunoreactivity was found in the medial and dorsal accessory nuclei, but was sparse in the principal nucleus. Iontophoresis or pressure microapplication of Leu 5-enkephalin, or a peptidase-resistant enkephalin analogue, depressed both spontaneous and amino acid-evoked firing of inferior olivary complex cells. The effect appeared to be postsynaptic, since the excitatory effects of iontophoretically-applied dl-homocysteic acid were suppressed. No selective effect on a calcium-mediated component of the action potential was seen. We conclude that enkephalin may act as a neurotransmitter or neuromodulator at afferent synapses in the inferior olive.

Serotonergic and peptidergic projections to the spinal cord demonstrated by a combined retrograde HRP histochemical and immunocytochemical staining method

A simple technique is described for simultaneously visualizing the cells of origin of descending brain stem-spinal systems and immunocytochemically defined neurotransmitters. The combination of horseradish peroxidase (HRP) histochemistry, enhanced by the use of CoCl 2, with the unlabeled antibody, peroxidase-antiperoxidase (PAP) immunocytochemical staining method, revealed double labeled cells containing black punctate HRP granules within a homogeneous brown cytoplasm stained positively for serotonin (5-HT), substance P or enkephalin-like immunoreactivity. The advantages of this method over other double labeling techniques are discussed.

Localization of [Met 5]- and [Leu 5]-enkephalin-like immunoreactivity in nerve terminals in human paravertebral sympathetic ganglia

Both [Leu 5]- and [Met 5]-enkephalin have been localized immunohistochemically in nerve fibres and in small, intensely fluorescent cells of adult human sympathetic ganglia. The nerve fibres showing enkephalin-like immunoreactivity formed a network varying in density around the sympathetic neurons, some being closely related to the perikarya. No labelled neuronal cell bodies were found. No structures within the ganglion were labelled after reaction with antibodies to vasoactive intestinal polypeptide, adrenocorticotrophin or substance P. No differences between the distributions of [Leu 5]-and [Met 5]-enkephalin-like immunoreactivities were found. The physiological roles of enkephalins are still unknown, but it is possible that they might act as neurotransmitters or neuromodulators in the human sympathetic nervous system.

Immunocytochemical localization of catecholamine synthesizing enzymes and neuropeptides in area postrema and medial nucleus tractus solitarius of rat brain.

The catecholaminergic and peptidergic neurons in the area postrema and adjacent portion of the medial nucleus tractus solitarii (mNTS) were characterized by the immunocytochemical localization of the catecholamine synthesizing enzymes tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT) and two neuropeptides, substance P and (Leu5)-enkephalin. The catecholamine synthesizing enzymes TH and DBH, found jointly only in noradrenergic and adrenergic neurons, were localized in cells having a similar morphology and topographical distribution. These cells were located throughout the rostrocaudal and dorsoventral extent of the area postrema, as well as in neurons within the mNTS. The processes showing TH and DBH immunoreactivity appear to form reciprocal connections between the area postrema and mNTS. Phenylethanolamine-N-methyltransferase, the enzymatic marker found only in adrenergic neurons, was detected immunocytochemically in terminals distributed throughout the area postrema and in neuronal perikarya and varicosities within the adjacent mNTS. Like the catecholamine synthesizing enzymes TH and DBH, enkephalin-like immunoreactivity was localized to perikarya, proximal processes and varicose axon terminals within the area postrema and the adjacent mNTS. However, in contrast to the widespread distribution of the enzymes, the enkephalin-like immunoreactivity was localized predominantly along the dorsal and ventrolateral margins of the area postrema. The distribution of substance P immunoreactivity, which was detected only in varicose processes, paralleled the distribution of enkephalin-like immunoreactivity, being predominantly located along the dorsal and ventrolateral margins of the area postrema. Within the mNTS adjacent to the area postrema, substance P immunoreactivity was localized to neuronal perikarya, proximal processes and varicose axon terminals. Based upon the presence of appropriate biosynthetic enzyme markers and neuropeptide localization, these findings suggest that neurons within the area postrema contain noradrenalin and enkephalin and that the afferent axons contain substance P, adrenalin and, probably, noradrenalin.

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

The distribution of enkephalin-like immunoreactivity in the cochlea of the guinea pig and cat was studied. Indirect immunofluorescence immunohistochemistry using antisera generated against a methionine enkephalin-bovine thyroglobulin conjugate was applied to surface preparations of the organ of Corti and cryostat sections of the whole of the cochlea. In the cochlear osseous spiral lamina, immunofluorescence was localized to unmyelinated fibers of the intraganglionic spiral bundle. In the organ of Corti, immunofluorescence was localized to a small number of fibers at inner hair cells, the inner spiral bundle, and tunnel spiral bundle, to tunnel crossing fibers at the level of the tunnel floor, to an occasional spiral outer fiber, and to the synaptic region of outer hair cells in the three rows of the basal turn of the cochlea. Less immunofluorescence was found in this region as one progressed towards the apex, with none seen at the apex. At the most apical region the inner spiral bundle became patchy and the tunnel spiral bundle developed arcades. There was no immunofluorescence found in spiral ganglion cells, in auditory nerve fibers, or in the hair cells of the organ of Corti. The findings were the same in cat as in guinea pig, the latter being studied in more detail. It was concluded that efferent, olivocochlear neurons of the cochlea, synapsing predominantly with primary auditory nerve fibers from the inner sensory cells or with the sensory cells, contain enkephalin-like immunoreactivity. Also, the findings indicate that endings of olivocochlear neurons that synapse predominantly with outer hair cells contain enkephalin-like immunoreactivity. It has previously been shown that olivocochlear neurons are likely to be cholinergic.