An immunohistochemical study of methionine-enkephalin-Arg6-Gly7-Leu8-like immunoreactivity-containing neurons in the parasympathetic preganglionic regions of the rat spinal cord

The localization of the methionine-enkephalin-Arg6-Gly7-Leu8 (Met-Enk-Arg-Gly-Leu)-like immunoreactivity-containing neurons in the rat lumbosacral spinal cord was immunohistochemically examined by an antiserum very specific to Met-Enk-Arg-Gly-Leu. The immunoreactive neurons occupied the positions corresponding to the parasympathetic preganglionic nuclei determined by the previous horseradish peroxidase (HRP)-tracing experiments. The present study suggests that the parasympathetic preganglionic neurons in the rat lumbosacral spinal cord produce preproenkephalin A and its related peptides.     

Comparative distribution of three opioid systems in the lower brainstem of the monkey (Macaca fuscata)

The regional distribution of the three opioid peptide neuronal systems--proopiomelanocortin (POMC), proenkephalin A, and proenkephalin B--was investigated in the lower brainstem of Japanese monkeys (Macaca fuscata) by immunocytochemical techniques. Antiserum to beta-endorphin/beta-lipotropin, [Met]-enkephalin-Arg6-Gly7-Leu8, and human leumorphin were used to identify the POMC and the proenkephalin A and B systems, respectively. POMC-related immunoreactive material was not found in the neuronal perikarya in the lower brainstem; reactive fibers and apparent terminals were distributed in the substantia nigra, lemniscus lateralis, midbrain central gray, the nucleus raphes, nucleus parabrachialis lateralis, ventral area of the spinal trigeminal nerve, nucleus tractus solitarii, and in the reticular formation throughout the lower brainstem. Proenkephalin A-related immunoreactive neuronal perikarya were detected in the central gray, reticular formation, nucleus raphes, trapezoid body, nucleus parabrachialis lateralis and medialis, nucleus spinalis nervi trigemini, nucleus dorsalis nervi vagi, and in the nucleus tractus solitarii. Densely packed immunoreactive fibers were widely distributed in the substantia nigra, nucleus interpeduncularis, nucleus raphes, superior colliculus, periaqueductal central gray, nucleus parabrachialis lateralis and medialis, locus coeruleus, trapezoid body, nuclei cochleares, nucleus spinalis nervi trigemini, tractus spinalis nervi trigemini, nucleus tractus solitarii, nucleus dorsalis nervi vagi, nucleus gracilis, nucleus cuneatus, nucleus cuneatus accessorius, and in the reticular formation throughout the lower brainstem. Neuronal perikarya containing immunoreactive material related to proenkephalin B were found in the periaqueductal central gray, nucleus parabrachialis lateralis and medialis, nucleus tractus solitarii, and nucleus spinalis nervi trigemini. In addition, immunoreactive fibers were detected in the ventral tegmental area, substantia nigra, nucleus parabrachialis lateralis and medialis, nucleus vestibularis lateralis and medialis, and in some areas of the reticular formation. These anatomical findings demonstrate that these three opioid peptide neuronal systems are widely but uniquely distributed in the lower brainstem of the monkey.     

Distribution and possible origin of beta-endorphin and ACTH in discrete brainstem nuclei of rats

Immunoreactive ACTH and beta-endorphin in the lower brainstem nuclei of intact and brainstem-hemisected rats were quantified by radioimmunoassay. The distribution of these peptides was almost identical throughout the lower brainstem. The concentrations of ACTH and beta-endorphin were essentially equal when expressed on a molar basis. Both peptides were distributed unevenly in the lower brainstem. High concentrations were found in the periaqueductal central gray matter and the dorsal raphe nucleus, and moderate levels were present in the locus coeruleus, the parabrachial nuclei, the nucleus raphe magnus and in the nucleus of the solitary tract. beta-Endorphin was measurable in all 43 brainstem nuclei investigated; ACTH was non-detectable in the red and lateral cuneate nuclei. Except in certain areas in the medulla oblongata (nucleus of the solitary tract, lateral reticular nucleus, reticular formation) ACTH and beta-endorphin declined in brainstem nuclei 10 days after midbrain hemisections. Retrograde accumulation of ACTH and beta-endorphin was found in the arcuate nucleus 3 days after midbrain hemisection, which was mainly ipsilateral to the lesion. Data from brainstem-hemisected rats also indicated that ACTH and beta-endorphin in the nucleus of the solitary tract are primarily of local origin, whereas the lateral reticular nucleus (A1 and A5 catecholaminergic cell groups) and medullary reticular formation may receive ACTH and beta-endorphin innervation from both hypothalamic and medullary neurons.     

Localization of Met-enkephalin-Arg6-Gly7-Leu8 immunoreactivity in rat duodenum

The distribution of Met-enkephalin-Arg6-Gly7-Leu8 (Met-ENK-Arg6-Gly7-Leu8) in the rat duodenum was determined using specific antibodies against Met-ENK-Arg6-Gly7-Leu8 and the immunofluorescence microscope technique. Met-ENK-Arg6-Gly7-Leu8 immunoreactive perikarya have been detected in the myenteric plexus. These neuronal cell bodies were large in diameter and round in shape. Met-ENK-Arg6-Gly7-Leu8 immunostained nerve fibres were noted in both the circular muscle layer and, more abundantly, in interconnecting myenteric plexus nerve fibre bundles. These findings might indicate that Met-ENK-Arg6-Gly7-Leu8 has important physiological roles as neurotransmitter and/or neuromodulator in the human and mammalian gastrointestinal tract.     

The opioid octapeptide Met5-enkephalin-Arg6-Gly7-Leu8: characterization and distribution in rat spinal cord

The regional quantitation, immunohistochemical localization and molecular heterogeneity of Met5-enkephalin-Arg6-Gly7-Leu8 were examined in rat spinal cord with a specific radioimmunoassay. A rostrocaudal gradient in Met5-enkephalin-Arg6-Gly7-Leu8 content was observed; the highest levels occurred in sacral cord. Dorsal cord content was higher than that of ventral cord at all spinal segments. Immunohistochemical staining supported and refined the latter observation: a dense network of perikarya and fibers was found in Laminae I and II of the dorsal horn. Cell bodies were frequently observed in lamina IV. Additional terminals were seen around the central canal and in the ventral gray matter, often outlining perikarya of motor neurons. Total Met5-enkephalin-Arg6-Gly7-Leu8 immunoreactivity could be fractionated into two main components using gel filtration chromatography. Nearly half of the total immuno-reactivity eluted as a high molecular weight peptide; the other half which co-eluted with Met5-enkephalin-Arg6-Gly7-Leu8 was further identified to be authentic Met5-enkephalin-Arg6-Gly7-Leu8 on reverse phase high pressure liquid chromatography. The present data, in conjunction with our previous study of Met5-enkephalin and Met5-enkephalin-Arg6-Phe7 indicates that all opioid peptides derived from preproenkephalin A are present in spinal cord and most likely are stored in the same neurons. Immunohistochemical localization of Met5-enkephalin-Arg6-Gly7-Leu8 in dorsal and ventral cord suggest a role for this peptide in both sensory and motor integration.     

Brainstem afferents to the magnocellular basal forebrain studied by axonal transport, immunohistochemistry, and electrophysiology in the rat

Brainstem afferents to the magnocellular basal forebrain were studied by using tract tracing, immunohistochemistry and extracellular recordings in the rat. WGA-HRP injections into the horizontal limb of the diagonal band (HDB) and the magnocellular preoptic area (MgPA) retrogradely labelled many neurons in the pedunculopontine and laterodorsal tegmental nuclei, dorsal raphe nucleus, and ventral tegmental area. Areas with moderate numbers of retrogradely labelled neurons included the median raphe nucleus, and area lateral to the medial longitudinal fasciculus in the pons, the locus ceruleus, and the medial parabrachial nucleus. A few labelled neurons were seen in the substantia nigra pars compacta, mesencephalic and pontine reticular formation, a midline area in the pontine central gray, lateral parabrachial nucleus, raphe magnus, prepositus hypoglossal nucleus, nucleus of the solitary tract, and ventrolateral medulla. A similar but not identical distribution of labelled neurons was seen following WGA-HRP injections into the nucleus basalis magnocellularis. The possible neurotransmitter content of some of these afferents to the HDB/MgPA was examined by combining retrograde Fluoro-Gold labelling and immunofluorescence. In the mesopontine tegmentum, many retrogradely labelled neurons were immunoreactive for choline acetyltransferase. In the dorsal raphe nucleus, some retrogradely labelled neurons were positive for serotonin and some for tyrosine hydroxylase (TH); however, the majority of retrogradely labelled neurons in this region were not immunoreactive for either marker. The ventral tegmental area, substantia nigra pars compacta, and locus ceruleus contained retrogradely labelled neurons which were also immunoreactive for TH. Of the retrogradely labelled neurons occasionally observed in the nucleus of the solitary tract, prepositus hypoglossal nucleus, and ventrolateral medulla, some were immunoreactive for either TH or phenylethanolamine-N-methyltransferase. To characterize functionally some of these brainstem afferents, extracellular recordings were made from antidromically identified cortically projecting neurons, mostly located in the HDB and MgPA. In agreement with most previous studies, about half (48%) of these neurons were spontaneously active. Electrical stimulation in the vicinity of the pedunculopontine tegmental and dorsal raphe nuclei elicited either excitatory or inhibitory responses in 21% (13/62) of the cortically projecting neurons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution of somatostatin-28 (1-12) in the cat brainstem: an immunocytochemical study.

We studied the distribution of somatostatin-28 (1-12)-immunoreactive fibers and cell bodies in the cat brainstem. A moderate density of cell bodies containing the peptide was observed in the ventral nucleus of the lateral lemniscus, accessory dorsal tegmental nucleus, retrofacial nucleus and in the lateral reticular nucleus, whereas a low density of such perikarya was found in the interpeduncular nucleus, nucleus incertus, nucleus sagulum, gigantocellular tegmental field, nucleus of the trapezoid body, nucleus praepositus hypoglosii, lateral and magnocellular tegmental fields, nucleus of the solitary tract, nucleus ambiguous and in the nucleus intercalatus. Moreover, a moderate density of somatostatin-28 (1-12)-immunoreactive processes was found in the dorsal nucleus of the raphe, dorsal tegmental nucleus, accessory dorsal tegmental nucleus, periaqueductal gray and in the marginal nucleus of the brachium conjunctivum. Finally, few immunoreactive fibers were visualized in the interpeduncular nucleus, cuneiform nucleus, locus coeruleus, nucleus incertus, superior and inferior central nuclei, nucleus sagulum, ventral nucleus of the lateral lemniscus, nucleus praepositus hypoglosii, medial vestibular nucleus, K?lliker-Fuse area, nucleus ambiguous, retrofacial nucleus, postpyramidal nucleus of the raphe, nucleus of the solitary tract, dorsal motor nucleus of the vagus, lateral reticular nucleus and laminar and alaminar spinal trigeminal nuclei.     

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.     

Distribution of proenkephalin-derived peptides in the brain of Rana esculenta

The immunocytochemical distribution of authentic proenkephalin-containing perikarya and nerve fibers in the brain of Rana esculenta was determined with antisera directed toward different epitopes of preproenkephalin. The pattern of proenkephalinlike immunoreactivity was similar with antisera directed toward [Met5]-enkephalin, [Met5]-enkephalin-Arg6, [Met5]-enkephalin-Arg6-Phe7, [Leu5]-enkephalin, and metorphamide; however, the intensity of the labelling varied depending on the target antigen. Proenkephalin-containing perikarya were located in all major subdivisions of the brain except the metencephalon. In the telencephalon, immunoreactive perikarya were detected in the dorsal, medial, and lateral pallium; the medial septal nucleus; the dorsal and ventral striatum; and the amygdala. In the diencephalon, immunoreactive perikarya were detected in the preoptic nucleus, in the dorsal and ventral caudal hypothalamus, and in an area that appeared to be homologous to the paraventricular nucleus. In the mesencephalon, numerous immunoreactive perikarya were detected in layer 6 of the optic tectum and a few scattered perikarya were detected in layer 4 of the optic tectum. Immunoreactive perikarya also occurred in the laminar nucleus of the torus semicircularis. In the rhombencephalon, immunoreactive perikarya were detected in the obex region and the nucleus of the solitary tract. Immunoreactive fibers of varying density were observed in all major subdivisions of the brain with the densest accumulations of fibers occurring in the dorsal pallium, the lateral and medial forebrain bundles, the amygdala, the periventricular hypothalamus, the superficial region of the caudolateral brainstem, and in a tract that appeared to be homologous to the tractus solitarius. The extensive system of proenkephalin-containing perikarya and nerve fibers in the brain of an amphibian showed many similarities to the distribution of proenkephalin-containing perikarya and nerve fibers previously described for the amniote brain.     

Localization of met-enkephalin-like immunoreactivity within pain-related nuclei of cervical spinal cord, brainstem and midbrain in the cat

Met-enkephalin immunoreactivity was investigated with an indirect immunoperoxidase technique in the cervical spinal cord, brainstem and midbrain of the cat, paying special attention to pain-related nuclei. Different technical conditions were used to reveal preferentially met-enkephalin-containing fibres and terminals or perikarya. Immunoreactive fibres and terminals were revealed optimally in sections from control animals incubated with detergent (Triton X-100). Immunoreactive perikarya were revealed in colchicine treated animals. Comparison between different routes of administration showed that local injections of colchicine are needed to reveal optimally immunoreactive perikarya in nuclei located far from the ventricles. Met-enkephalin-containing fibres and terminals are widely distributed in the posterior brain and spinal cord. The densest network of immunoreactive fibres are observed in the superficial layers of the cervical spinal cord and the caudal trigeminal nucleus, in the nucleus of the solitary tract, the nucleus of the facial nerve, the nucleus of the prepositus hypoglossi, the nucleus raphe pallidus, the medial vestibular nucleus, the interpedoncular nucleus and the substantia nigra. A moderate staining of fibres is observed in various nuclei including the ventral horn of the spinal cord and caudal trigeminal nucleus, the brainstem and midbrain reticular formation, the inferior olivary complex, the nucleus of the descending trigeminal tract and the periaqueductal grey. Met-enkephalin-containing perikarya are present in all the nuclei cited before, except in the inferior olivary complex. The densest aggregation of enkephalin-like perikarya is observed in the nucleus raphe magnus, nucleus raphe obscurus, nucleus raphe pallidus, nucleus reticularis gigantocellularis pars α and nucleus reticularis lateralis. The general distribution of enkephalin-containing structures in the cervical spinal cord, brainstem and midbrain of the cat appears very similar to that of the rat except in the substantia nigra where met-enkephalin cell bodies are found in the cat but not in the rat. In particular the pain-related nuclei present a similar distribution of the peptide in the two species; however, met-enkephalin-containing cell bodies are much more numerous in the cat than in the rat (notably in the reticular formation). Similar types of metenkephalin innervation occur in the dorsal and intermediate grey of the spinal cord and of the caudal trigeminal nucleus supporting further that the functional organizations of these regions are closely related.     

A comparative study of the immunohistochemical localization of a presumptive proctolin-like peptide, thyrotropin-releasing hormone and 5-hydroxytryptamine in the rat central nervous system

A proctolin (PROC)-like peptide was studied immunohistochemically in the hypothalamus, lower brainstem and spinal cord of the rat using an antiserum against PROC conjugated to thyroglobulin. Neuronal cell bodies containing PROC-like immunoreactivity (PROC-LI) were observed in the dorsomedial, paraventricular and supraoptic nuclei of the hypothalamus and in the nucleus raphe magnus, nucleus raphe pallidus, nucleus raphe obscurus and nucleus interfascicularis nervi hypoglossi in the medulla oblongata. Fibers containing PROC-LI were seen in the median eminence and in other hypothalamic nuclei, and in the lower brainstem in cranial motor nuclei including the dorsal motor nucleus of the vagus nerve, the motor trigeminal nucleus, the facial nucleus and nucleus ambiguous, and in lower numbers in the nucleus of the solitary tract and locus coeruleus. Fibers containing PROC-LI were also located in the spinal cord, in the intermediolateral cell column at thoracic levels and in the ventral horns at all levels of the spinal cord. After transection of the spinal cord, all PROC-immunoreactive fibers below the lesion disappeared. Following injection of Fast blue into the thoracic spinal cord, retrogradely labeled cells in the nuclei raphe pallidus, obscurus and magnus and nucleus interfasciculari nervi hypoglossi were seen to contain PROC-LI. PROC-LI had a similar distribution as thyrotropin-releasing hormone (TRH)-LI in the above-mentioned areas and coexistence of TRH-LI and PROC-LI was shown in cell bodies in the hypothalamus and medulla oblongata. PROC-LI could also be shown to coexist with 5-hydroxytryptamine (5-HT)-LI in neuronal cell bodies in the lower brainstem. The results demonstrate the occurrence of a PROC-like peptide in the mammalian nervous system, and these neurons seem to be at least largely identical to previously described TRH systems. A possible involvement of the PROC-like peptide in spinal motor control is discussed in relation to the well-established role of PROC in control of motor behavior in insects and invertebrates.     

Some anatomical observations on the projections from the hypothalamus to brainstem and spinal cord: an HRP and autoradiographic tracing study in the cat

The hypothalamus is closely involved in a wide variety of behavioral, autonomic, visceral, and endocrine functions. To find out which descending pathways are involved in these functions, we investigated them by horseradish peroxidase (HRP) and autoradiographic tracing techniques. HRP injections at various levels of the spinal cord resulted in a nearly uniform distribution of HRP-labeled neurons in most areas of the hypothalamus except for the anterior part. After HRP injections in the raphe magnus (NRM) and adjoining tegmentum the distribution of labeled neurons was again uniform, but many were found in the anterior hypothalamus as well. Injections of 3H-leucine in the hypothalamus demonstrated that: The anterior hypothalamic area sent many fibers through the medial forebrain bundle (MFB) to terminate in the ventral tegmental area of Tsai (VTA), the rostral raphe nuclei, the nucleus Edinger-Westphal, the dorsal part of the substantia nigra, the periaqueductal gray (PAG), and the interpeduncular nuclei. Further caudally a lateral fiber stream (mainly derived from the lateral parts of the anterior hypothalamic area) distributed fibers to the parabrachial nuclei, nucleus subcoeruleus, locus coeruleus, the micturition-coordinating region, the caudal brainstem lateral tegmentum, and the solitary and dorsal vagal nucleus. Furthermore, a medial fiber stream (mainly derived from the medial parts of the anterior hypothalamic area) distributed fibers to the superior central and dorsal raphe nucleus and to the NRM, nucleus raphe pallidus (NRP), and adjoining tegmentum. The medial and posterior hypothalamic area including the paraventricular hypothalamic nucleus (PVN) sent fibers to approximately the same mesencephalic structures as the anterior hypothalamic area. Further caudally two different fiber bundles were observed. A medial stream distributed labeled fibers to the NRM, rostral NRP, the upper thoracic intermediolateral cell group, and spinal lamina X. A second and well-defined fiber stream, probably derived from the PVN, distributed many fibers to specific parts of the lateral tegmental field, to the solitary and dorsal vagal nuclei, and, in the spinal cord, to lamina I and X, to the thoracolumbar and sacral intermediolateral cell column, and to the nucleus of Onuf. The lateral hypothalamic area sent many labeled fibers to the lateral part of the brainstem and many terminated in the caudal brainstem lateral tegmentum, including the parabrachial nuclei, locus coeruleus, nucleus subcoeruleus, and the solitary and dorsal vagal nuclei.(ABSTRACT TRUNCATED AT 400 WORDS)     

Afferents to the nucleus reticularis parvicellularis of the cat medulla oblongata: A tract-tracing study with cholera toxin B subunit

The aim of this study was to examine anatomical evidence in cats of whether the nucleus reticularis parvicellularis (Pc) is part of the circuit responsible for the inhibition of brainstem motoneurons during paradoxical sleep. For this purpose, we made iontophoretic injections of the retrograde and anterograde tracer cholera toxin B subunit (CTb) in the Pc. After CTb injections in the Pc, a large number of retrogradely labeled neurons were seen in the central nucleus of the amygdala, the lateral part of the bed nucleus of the stria terminalis, the posterior hypothalamic areas, the mesencephalic reticular formation, the nucleus locus subcoeruleus, the nucleus pontis caudalis, other portions of the Pc, the nucleus reticularis dorsalis, the trigeminal sensory complex, and the nucleus of the solitary tract. We further found that the Pc receives (1) serotoninergic afferents from the raphe dorsalis, magnus, and obscurus nuclei; (2) noradrenergic inputs from the dorsolateral pontine tegmentum; (3) cholinergic afferents from the lateral medullary reticular formation; (4) substance P-like afferents from the central nucleus of the amygdala, bed nucleus of the stria terminalis, periaqueductal gray, and nucleus of the solitary tract; and (5) methionine-enkephalin-like projections from the periaqueductal gray, the nucleus of the solitary tract, the lateral pontine and medullary reticular formation, and the spinal trigeminal nucleus. We further found that the Pc do not receive afferents from brainstem structures responsible for muscle atonia, such as the ventromedial medulla and the dorsomedial pontine tegmentum, and therefore may not be part of the circuit inhibiting the brainstem motoneurons during paradoxical sleep. © 1994 Wiley-Liss, Inc.     

Descending projections of the posterior nucleus of the hypothalamus: Phaseolus vulgaris leucoagglutinin analysis in the rat

No previous report in any species has systematically examined the descending projections of the posterior nucleus of the hypothalamus (PH). The present report describes the descending projections of the PH in the rat by using the anterograde anatomical tracer, Phaseolus vulgaris leucoagglutinin. PH fibers mainly descend to the brainstem through two routes: dorsally, within the central tegmental tract; and ventromedially, within the mammillo-tegmental tract and its caudal extension, ventral reticulo-tegmental tracts. PH fibers were found to distribute densely to several nuclei of the brainstem. They are (from rostral to caudal) 1) lateral/ventrolateral regions of the diencephalo-mesopontine periaqueductal gray (PAG); 2) the peripeduncular nucleus; 3) discrete nuclei of pontomesencephalic central gray (dorsal raphe nucleus, laterodorsal tegmental nucleus, and Barrington's nucleus); 4) the longitudinal extent of the central core of the mesencephalic through medullary reticular formation (RF); 5) the ventromedial medulla (nucleus gigantocellularis pars alpha, nucleus raphe magnus, and nucleus raphe pallidus); 6) the ventrolateral medulla (nucleus reticularis parvocellularis and the rostral ventrolateral medullary region); and 7) the inferior olivary nucleus. PH fibers originating from the caudal PH distribute much more heavily than those from the rostral PH to the lower brainstem. The PH has been linked to the control of several important functions, including respiration, cardiovascular activity, locomotion, antinociception, and arousal/wakefulness. It is likely that descending PH projections, particularly those to the PAG, the pontomesencephalic RF, Barrington's nucleus, and parts of the ventromedial and ventrolateral medulla, serve a role in a PH modulation of complex behaviors involving an integration of respiratory, visceromotor, and somatomotor activity. © 1996 Wiley-Liss, Inc.     

Immunocytochemical localization of the choline acetyltransferase containing neuron system in the rat lower brain stem

This distribution of choline acetyltransferase (CHAT) immunoreactivity (CHAT-I) in the rat lower brain stem was analyzed using a highly sensitive avidin-biotin immunocytochemical method and 3-amino-9-ethyl-carbazole visualization. A much wider and more abundant distribution of CHAT-I structures in the lower brain stem was demonstrated than in earlier studies. The following areas were newly identified as areas rich in CHAT-I fibers: the interpeduncular nucleus, medial geniculate body, central gray matter of pons, pontine nucleus, parabigeminal nucleus, dorsal tegmental nucleus of Gudden, lateral trapezoid nucleus, inferior colliculus, dorsal and ventral cochlear nuclei, medial and lateral vestibular nuclei, reticular formation of medulla oblongata, and gelatinosa of caudal trigeminal spinal tract nucleus. In addition to the areas in which they have been known to exist, CHAT-I perikarya were found in the caudal portion of substantia nigra pars reticulata, the area between trigeminal motor nucleus and superior olivary nucleus, the medial and spinal vestibular nucleus, prepositus hypoglossal nucleus, raphe magnus and obscurus, ventromedial portion of solitary tract nucleus and its just ventral reticular formation, and caudal trigeminal spinal tract nucleus.     

Differential colocalization of neuropeptide Y- and methionine-enkephalin-Arg6-Gly7-Leu8-like immunoreactivity in catecholaminergic neurons in the rat brain stem

The present study, using a combination of catecholamine (CA) histofluorescence and peptide immunocytochemistry in the same tissue sections, investigated the coexistence of neuropeptide Y (NPY) and methionine-enkephalin-Arg6-Gly7-Leu8 (MEAGL)-like immunoreactivity (LI) in catecholaminergic neurons of colchicine-treated rat brain stems. Of the total number of catecholaminergic neurons in the A1/C1, A2/C2, A3, A4, and A6 regions approximately 83, 28, 98, 76, and 36%, respectively, contained both NPY-LI and CA. Of the total number of catecholaminergic neurons in A1/C1, A2/C2, A3, and A5 regions, approximately 47, 4, 8, and 17%, respectively, contained both MEAGL-LI and CA. Moreover, about 24% of the catecholaminergic neurons in the A1/C1 region contained both NPY- and MEAGL-LI. Neither the noradrenergic neurons (A7) in the pons nor any of the dopaminergic neurons in the midbrain (A8, A9, A10) contained NPY- or MEAGL-LI. Neurons containing both NPY- and MEAGL-like immunoreactive peptides without CA were not found in the rat brain stem. These findings indicate that catecholaminergic neurons in the brain stem of the rat can be subdivided into distinct subgroups on the basis of the coexistence of specific peptides.     

Co-localization of substance P and Met-enkephalin-Arg6-Gly7-Leu8 in the intraspinal neurons of the rat, with special reference to the neurons in the substantia gelatinosa

A double-labeling immunofluorescence technique was employed to investigate the co-localization of the functionally antagonistic neuropeptides, substance P and enkephalins, within intraspinal neurons of the rat. Anti-Met-enkephalin-Arg6-Gly7-Leu8 (Enk-8) antiserum was used as a marker of the preproenkephalin A neuron system. The observations were focused on the lumbar spinal cord. Co-localization was most prominent within neurons in the substantia gelatinosa, in which more than 95% of substance P-like immunoreactivity neurons showed Enk-8-like immunoreactivity. These double-labeled cells corresponded to 45% of Enk-8-like immunoreactive neurons in the same area. This suggests that SP/Enk-8 interaction occurs at the axon terminals of the substantia gelatinosa neurons. In deeper layers of the dorsal horn (laminae III, IV), only 14% and 6% of SP-like immunoreactive and Enk-8-like immunoreactive neurons were double labeled, respectively. Co-localization was also observed in neurons located in the laminae I, V, VII and X, suggesting concomitant involvement of these peptides in a variety of spinal cord functions.     

Immunocytochemical analysis of proenkephalin-derived peptides in the amphibian hypothalamus and optic tectum

[Met5]-Enkephalin-, [Met5]-enkephalin-Arg6-, [Met5]-enkephalin-Arg6-Phe7-, metorphamide- and BAM 22P-like peptides could be localized in the amphibian brain by immunocytochemistry. However, a [Met5]-enkephalin-Arg6-Gly7-Leu8-like peptide could not be detected in the brain of any anuran species with an antiserum that was capable of detecting this octapeptide in mammalian brain. A synenkephalin-like peptide also could not be detected in the anuran brain with an antiserum that was capable of detecting the antigen in bovine and porcine brain. Although the intensity of proenkephalin-like immunoreactivity depended on the antiserum used, its distribution appeared to be identical with all of the effective antisera. Antisera directed against somatostatin and corticotropin-releasing factor stained perikarya, nerve fibers and terminals in the anuran brain with a distribution that was different from antisera directed against proenkephalin-derived peptides. The distribution of proenkephalin-containing perikarya and nerve fibers in the regions of the anuran brain selected for study showed many similarities to the distribution of proenkephalin-containing perikarya and nerve fibers in the same regions of the amniote brain.     

大鼠中脑导水管周围灰质和中缝核簇内P物质受体定位分布的免疫细胞化学研究

本研究用免疫细胞化学染色技术对大鼠中脑导水管周围灰质（ＰＡＧ）和中缝核簇内Ｐ物质受体的精确定位分布进行了系统地观察。Ｐ物质受体阳性的神经元胞体和纤维主要密集地分布于ＰＡＧ吻段的背侧部、中段的外侧部和尾段的腹侧部。在线形核、中缝背核、中缝正中核、中缝桥核、中缝大核和中缝隐核可见中等密度至高密度的阳性神经元胞体和纤维；在中央下核和中缝苍白核内仅见少量阳性纤维。     

Distribution of galanin-like immunoreactivity in the brain of Rana esculenta and Xenopus laevis.

The immunocytochemical distribution of galanin-containing perikarya and nerve terminals in the brain of Rana esculenta and Xenopus laevis was determined with antisera directed toward either porcine or rat galanin. The pattern of galanin-like immunoreactivity appeared to be identical with antisera directed toward either target antigen. The distribution of galanin-like immunoreactivity was similar in Rana esculenta and Xenopus laevis except for the absence of a distinct laminar distribution of immunoreactivity in the optic tectum of Xenopus laevis. Galanin-containing perikarya were located in all major subdivisions of the brain except the metencephalon. In the telencephalon, immunoreactive perikarya were detected in the pars medialis of the amygdala and the preoptic area. In the diencephalon, immunoreactive perikarya were detected in the caudal half of the suprachiasmatic nucleus, the nucleus of the periventricular organ, the ventral hypothalamus, and the median eminence. In the mesencephalon, immunoreactive perikarya were detected near the midline of the rostroventral tegmentum, in the torus semicircularis and, occasionally, in lamina A and layer 6 of the optic tectum. In the myelencephalon, labelled perikarya were detected only in the caudal half of the nucleus of the solitary tract. Immunoreactive nerve fibers of varying density were observed in all subdivisions of the brain with the densest accumulations of fibers occurring in the pars lateralis of the amygdala and the preoptic area. Dense accumulations of nerve fibers were also found in the lateral septum, the medial forebrain bundle, the periventricular region of the diencephalon, the ventral hypothalamus, the median eminence, the mesencephalic central gray, the laminar nucleus of the torus semicircularis, several laminae of the optic tectum, the interpeduncular nucleus, the isthmic nucleus, the central gray of the rhombencephalon, and the dorsolateral caudal medulla. The extensive system of galanin-containing perikarya and nerve fibers in the brain of representatives of two families of anurans showed many similarities to the distribution of galanin-containing perikarya and nerve fibers previously described for the mammalian brain.