Immunohistochemical evidence for coexistence of methionine-enkephalin and tyrosine hydroxylase in neurons of the locus coeruleus complex projecting to the spinal cord of the cat.

Previous studies have revealed the presence of pontospinal neurons with either methionine-enkephalin- or tyrosine hydroxylase-like immunoreactivity in the dorsolateral pontine tegmentum of the cat. Using a combined fast blue retrograde transport technique and simultaneous immunofluorescence histochemistry, the present study was designed to reveal the coexistence of enkephalin and tyrosine hydroxylase in cat coerulospinal neurons and to determine if and to what extent the coerulospinal pathway is heterogeneous. Fast blue-labelled neurons with tyrosine hydroxylase- and enkephalin-like immunoreactivities were found in the nucleus locus coeruleus, nucleus subcoeruleus, K?lliker-Fuse nucleus, and the medial and lateral parabrachial nuclei. Approximately 87% of tyrosine hydroxylase-like immunoreactive neurons had enkephalin-like immunoreactivity, whereas about 76% of the enkephalin-like immunoreactive neurons had tyrosine hydroxylase-like immunoreactivity. About 71% of all coerulospinal neurons exhibited both tyrosine hydroxylase- and enkephalin-like immunoreactivities. These findings indicate that coerulospinal activity may lead to spinal cord effects reflecting both norepinephrine and enkephalin activity in most cases but do not rule out each transmitter's isolated functions.     

The nuclei of origin of brainstem enkephalin and cholecystokinin projections to the spinal trigeminal nucleus of the rat

The sites of origin of brain stem enkephalin and cholecystokinin projections to the rodent spinal trigeminal nucleus were studied utilizing the combined retrograde transport-peroxidase antiperoxidase immunohistochemical technique. Several brain stem areas were found to contain enkephalin-like immunoreactive double-labeled neurons following injection of wheat germ agglutinin-horseradish peroxidase or horseradish peroxidase into the spinal trigeminal nucleus. The largest numbers of enkephalin double-labeled neurons were identified in the nucleus pontis oralis, nucleus raphe medianis, medial vestibular nucleus and the midbrain periaqueductal gray. Enkephalin projections to the spinal trigeminal nucleus were also found to originate from the nucleus solitarius, nucleus raphe pallidus, nucleus raphe magnus, nucleus raphe dorsalis, nucleus reticularis paragigantocellularis, nucleus reticularis gigantocellularis pars alpha and the deep mesencephalic nucleus. In contrast to the numerous sources of enkephalin input to the spinal trigeminal nucleus, cholecystokinin projections to this region were limited to four brain stem nuclei. These included the nucleus solitarius, raphe obscurus, nucleus paragigantocellularis and the ventral reticular nucleus of the medulla. The finding that only a small number of brain stem cholecystokinin-like immunoreactive neurons project to the spinal trigeminal nucleus supports the hypothesis that most of the cholecystokinin input to the spinal trigeminal nucleus arises from primary afferent trigeminal fibers. The spinal trigeminal nucleus is known to play a role in processing sensory information and in the transmission of orofacial nociception. The present study identifies several brain stem sites which provide enkephalin and/or cholecystokinin input to the spinal trigeminal nucleus. Several of these nuclei have been implicated as components of the endogenous pain control system and the present results raise the possibility that they may modulate incoming orofacial nociception by releasing the endogenous opioid, enkephalin. Cholecystokinin, on the other hand, has been demonstrated in other studies to attenuate the action of opiates and thus may play an opposing role in the spinal trigeminal nucleus.     

Expression of eight neuropeptides in intraocular spinal cord grafts: organotypical and disturbed patterns as evidenced by immunohistochemistry

The present study examines the distribution of several neuropeptides, as revealed by immunohistochemistry in the isolated cord. Fetal rat spinal cord was grafted to the anterior chamber of the adult Sprague-Dawley albino rats. After intraocular maturation for 2-3 months, the amount and distribution of somatostatin, neuropeptide Y, substance P, enkephalin, vasoactive intestinal peptide, peptide histidine-isoleucine, calcitonin gene-related peptide and cholecystokinin immunoreactive terminals and cell bodies were analysed using indirect fluorescence immunohistochemistry. The visualization of immunoreactive cell bodies in the grafts was enhanced using a novel intraocular colchicine treatment. In the graft a rich network of somatostatin-positive terminals was found with a high density in well-demarcated areas reminiscent of substantia gelatinosa of the dorsal horn of normal spinal cord. A large number of small- to medium-sized somatostatin neurons was found throughout the grafts without colchicine treatment. This is in contrast to normal spinal cord, where positive neurons were difficult to visualize without colchicine and were mainly confined to the dorsal horn. Neuropeptide Y had a distribution in the grafts similar to that of somatostatin and neuropeptide Y cells were found throughout the grafts without colchicine treatment. In normal spinal cord, neuropeptide Y-positive fibers were found mainly in substantia gelatinosa with a sparse network in the ventral horn. Enkephalin-positive fibers were found throughout the grafts. The distribution of fibers resembled that of somatostatin and neuropeptide Y with distinct zones of high fiber density in well-demarcated areas, whereas the density of nerve fibers in the rest of the graft neuropil was moderate to low. The distribution of substance P was similar to that of enkephalin. After colchicine treatment, both enkephalin- and substance P-positive cell bodies were visualized. In the intact spinal cord both peptides were seen in the entire gray matter with the highest concentrations in the superficial laminae of the dorsal horn. Antisera against calcitonin gene related-peptide, revealed a sparse terminal network and many large cells, which might represent motoneurons. A sparse network of varicose cholecystokinin-immunoreactive fibers was found evenly distributed in the grafts. In normal spinal cord a dense cholecystokinin-positive network of primary sensory afferent origin was found in the dorsal horn. In the grafts cholecystokinin cell bodies were seen after colchicine treatment.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cell suspension grafts of noradrenergic locus coeruleus neurons in rat hippocampus and spinal cord: reinnervation and transmitter turnover

Fetal noradrenergic neurons from the brain stem locus coeruleus region can be successfully grafted as a dissociated cell suspension provided that the dissociation is done in the absence of any trypsin digestion step. The survival, fiber outgrowth and biochemical function of locus coeruleus neurons, taken from 13- to 15-day-old rat embryos, have been studied after injection into the dorsal hippocampal formation and the thoracolumbar spinal cord in adult rats. All rats were treated with an i.v. injection of 6-hydroxydopamine prior to grafting to remove the intrinsic locus coeruleus projections to these areas, and they were taken for fluorescence histochemical or biochemical analyses 2-7 months after transplantation. Up to 330 surviving noradrenaline neurons were found at each implantation site (injected with 2-3 microliters of cell suspension) which represents an estimated survival rate of about 40%. In the most successful cases the entire dorsal hippocampal formation, and an approximately 4 cm long segment of the thoracolumbar spinal cord, was supplied with a new noradrenaline-containing terminal network, which reached normal densities in the regions closest to the grafts. In the hippocampal formation, in particular, the ingrowing axons re-established a laminar innervation pattern which resembled that of the normal locus coeruleus afferents. In the hippocampus, two 2-microliters injections of locus coeruleus cell suspension restored the total hippocampal noradrenaline content to an average of 55%, and the noradrenaline synthesis rate (as assessed by the rate of DOPA accumulation after synthesis inhibition) was found to be close to normal in the graft-reinnervated specimens. In the spinal cord, two 3-microliters injections restored the noradrenaline level in the thoracolumbar cord (a 4.5 cm long segment) to an average of 22% of normal, with the highest individual levels being close to normal. Determinations of the noradrenaline metabolite 3,4-dihydroxy-phenylethyleneglycol indicated that the rate of noradrenaline metabolism in the graft-reinnervated spinal cord was close to that of the normal intact spinal cord. The results demonstrate the potential of the suspension grafting technique for extensive noradrenergic reinnervation of the hippocampal formation or large portions of the spinal cord. Fetal locus coeruleus neurons implanted in this way can re-establish fairly normal terminal innervation patterns and reinstate noradrenaline turnover and metabolism in a previously denervated central target.     

Synaptic connections of enkephalin-immunoreactive nerve terminals in the neostriatum: a correlated light and electron microscopic study

Summary Two different antisera to leucine-enkephalin were used to study the localization of enkephalin-like immunoreactive material in the neostriatum and globus pallidus of the rat, by means of the unlabelled antibody-enzyme method. Thin immunoreactive varicose fibres are scattered throughout the neostriatum. In the ventral striatum, fibres come together and follow a relatively straight course for several micrometers, forming tube-like structures which can be traced to cell bodies; these cell bodies are completely surrounded by immunoreactive fibres. Occasional immunoreactive varicose fibres are also found close to another type of neuron throughout the whole neostriatum.Examination by electron microscopy of immunoreactive structures that had been identified first in the light microscope, showed that each of the nearly 200 varicosities examined was a vesicle-containing bouton that formed a synaptic contact. Rarely were asymmetrical synaptic contacts found between immunoreactive boutons and dendritic spines. All other synapses formed by enkephalin-immunoreactive boutons were symmetrical. Two types of postsynaptic neuron were identified; the first type was a medium-sized neuron with the ultrastructural features of a typical striatal spiny neuron. The second type had a larger perikaryon surrounded by numerous immunoreactive varicosities that were found to be boutons forming symmetrical synapses. The long dendrites of this second type of neuron likewise received a dense input of immunoreactive boutons forming symmetrical synapses; such ensheathed dendrites were found to be the tube-like structures seen in the light microscope. The ultrastructural features of these neurons, notably a highly indented nucleus, were those of a rare type of striatonigral neuron. In the globus pallidus, all the enkaphalin-immunoreactive boutons studied formed symmetrical synapses with ensheathed dendrites and perikarya that were similar to the latter type of postsynaptic neuron in the neostriatum. Axo-axonic synapses involving immunoreactive boutons were not seen in our material.The results are consistent with the view that enkephalin-like substances may be synaptic transmitters in the neostriatum and that they may have different actions according to the nature of the postsynaptic target. The finding that one type of neostriatal neuron, and a very similar neuron in the globus pallidus, receives multiple enkephalin-immunoreactive boutons all over its perikaryon and along its dendrites indicates a potentially important role of enkephalin in the convergence of information within the neostriatum and pallidum on to output neurons.     

Locus coeruleus terminals in intraocularly transplanted spinal cords as compared with catecholamine terminals in normal spinal cords: Their synaptic densities and functional considerations

Locus coeruleus terminals in intraocularly transplanted spinal cords and catecholamine terminals in defined areas of normal spinal cords were investigated qualitatively and quantitatively by immunoelectron microscopy. Results showed that the morphological features of synapses formed in the grafts closely resembled those of normal spinal cords. The incidences of synapses per varicosities, as observed in single sections, were 30.1, 40.2 and 22.8% for the ventral horn, dorsal horn and grafted spinal cord, respectively. In all three groups, most of the postsynaptic targets were small dendrites, although high frequencies of large dendrites were found in the ventral horn. Spines and axons in the grafts were also postsynaptic targets. Several characteristics of relative immaturity were observed in the grafts. It is suggested that the inhibition of spinal neurons by locus coeruleus terminals may be mediated not only by volume transmission through nonsynaptic contacts, but also by direct contacts with catecholamine terminals, and that the excitation of facilitation observed at those terminals may be explained by the suppression of inhibitory neurons by axoaxonic contacts.     

人胎蓝斑神经元的电镜观察

为了探讨人蓝斑神经元在胚胎发育过程中的超微结构和突触形成特征 ,为蓝斑 -脊髓移植选择适宜胎龄提供形态学资料。用透射电镜观察了 4～ 8个月人胎蓝斑神经元在胚胎发育过程中的变化。结果证明 :胎龄 4个月人胎蓝斑神经元显示不成熟细胞特征 ,胎龄 6个月为发育成熟过程中的细胞特征 ,胎龄 8个月为成熟细胞特征。提示进行人胎蓝斑 -脊髓移植时以 4个月胎龄蓝斑作移植供体较为适宜     

Locus coeruleus neurons in the rat containing neuropeptide Y, tyrosine hydroxylase or galanin and their efferent projections to the spinal cord, cerebral cortex and hypothalamus

The efferent projections of locus coeruleus neurons which contain neuropeptide Y-, tyrosine hydroxylase- or galanin-like immunoreactivity were investigated using the indirect immunofluorescence technique combined with the retrograde transport of the fluorescent substance Fast Blue. Four groups of rats received injections of Fast Blue: (1) bilaterally into the mid-thoracic spinal cord (T6-T7); (2) unilaterally into the low cervical spinal cord (C4-C5); (3) unilaterally into the paraventricular, periventricular and dorsomedial hypothalamic nuclei; and (4) unilaterally into five sites in the cerebral cortex (frontal, cingulate and striate cortex). Efferent projections to the spinal cord, hypothalamus and cerebral cortex from neuropeptide Y-, tyrosine hydroxylase- and galanin-containing locus coeruleus cells were observed. A higher percentage of the peptidergic locus coeruleus neurons projected to the hypothalamus than to the spinal cord or cerebral cortex. The distribution and morphology of the neuropeptide Y- and galanin-containing neurons in the locus coeruleus were also investigated. Neuropeptide Y-like immunoreactivity and galanin-like immunoreactivity were found in small, medium and large multipolar neurons, as well as in fusiform locus coeruleus cells. The neuropeptide Y- and galanin-immunoreactive neurons were found throughout the locus coeruleus. In the caudal locus coeruleus, they were primarily located in the dorsal portion. Neuropeptide Y-like immunoreactivity and galanin-like immunoreactivity were only seen in a few tyrosine hydroxylase-positive neurons of the subcoeruleus group. The data show that the peptide-containing locus coeruleus neurons have efferent projections to the spinal cord, hypothalamus and cerebral cortex. The locus coeruleus may be divided into functional subdivisions dependent on the region of the locus coeruleus, the neurotransmitter/neuropeptide(s) contained within the neurons and their efferent projections.     

Conditions for adrenergic hyperinnervation in hippocampus: I. Histochemical evidence from intraocular double grafts

Summary The developing hippocampal anlage of rats was homologously grafted to the anterior chamber of the eye of adult recipients. After intraocular maturation of the hippocampal graft and removal of the sympathetic innervation of the eye by ipsilateral superior cervical ganglionectomy, four types of monoamine neurons were transplanted to the eye chamber: Peripheral sympathetic neurons, central adrenergic neurons of locus coeruleus, central dopaminergic neurons of substantia nigra, and central 5-hydroxytryptamine neurons of the lower raphé nuclei. All four classes of monoamine neurons were able to reinnervate the hippocampal graft, but the fiber ingrowth differed markedly. Although peripheral sympathetic neurons produced a pattern of adrenergic innervation in the hippocampal graft which resembled innervation of the hippocampus by the locus coeruleus in the brain, locus coeruleus neurons themselves produced an extremely dense plexus of fibers within the graft. This hyperinnervation remained intact for up to 9 months, the longest period of time studied. The locus coeruleus graft itself received fibers from the hippocampus graft, as demonstrated by the retrograde transport of horseradish peroxidase. We conclude that the hippocampal graft exerts a much stronger growth stimulation on the locus coeruleus neurons than on the peripheral sympathetic neurons. The difference between innervation patterns suggest that both presynaptic and postsynaptic influences determine fiber ingrowth in the central nervous system.     

Fetal locus coeruleus transplanted into the transected spinal cord of the adult rat: some observations and implications

The 16-day-old fetal locus coeruleus can survive and grow, when transplanted into the spinal cavity created by transection of the adult spinal cord. The implants usually break up into smaller cell groups which become lodged in the host spinal cord, mainly in the caudal region. There was a marked proliferation of the damaged coerulospinal noradrenergic fibers in the rostral, ventral horn region of the cord. A neurotrophic substance produced by the locus coeruleus implants is probably responsible for this effect. Evidence for vigorous axonal growth of noradrenergic fibers derived from the implants in the caudal region of the cord was obtained. In 2/12 cases, the surviving locus coeruleus did not grow into the host tissue. Neosympathetic innervation of the caudal region of the cord occurred both in cases in which the implants survived, or did not survive. The survival rate of the transected, implanted animals is greater than 90%. The success rate of fusion of the implant with the host tissue is 40%. Based on these results, and other reports published recently, it is concluded: (1) that the purely technical difficulties of transplantation and survival of the fetal locus coeruleus in the transected spinal cord of the young adult rat on a long-term basis can be successfully solved. (2) For optimum reinnervation, the transplant should be placed in the lumbar intumescence, and not in the spinal cavity created by transection. (3) The spinal cavity created by transection must be bridged by some other method. The use of fetal spinal cord tissue on fetal mesencephalic tissue may prove to be useful for this purpose. (4) Transection of the cord must be done sub-pially, in order to minimize retraction and compression of the cord. The damaged pia must be resutured. (5) A second fetal locus coeruleus implant should be placed adjacent to the exposed surface of the rostral region of the cord. The results are discussed both in the context of recent efforts to try to restore functional activity to the damaged spinal cord, and in terms of efforts to try to understand the problems involved in fostering the growth of fetal brain tissue in the damaged spinal cord of an adult host animal.     

Locus coeruleus neurons projecting to the forebrain and the spinal cord in the cat

The intranuclear organization of the cat locus coeruleus neurons was investigated anatomo-physiologically. The locus coeruleus neurons project to the forebrain through the dorsal noradrenergic bundle and to the spinal cord. Horseradish peroxidase, a retrograde tracer, was pressure-injected into either the dorsal noradrenergic bundle or the ventrolateral funiculus of the high cervical cord (C1-C2). The cats (n = 12) were killed after a 2- or 3-day survival period. The frontal sections (100 micron) throughout the locus coeruleus were observed by light microscope after carrying out the diaminobenzidine reaction. The labeled locus coeruleus neurons were located predominantly in the rostral locus coeruleus proper and locus coeruleus alpha when horseradish peroxidase was injected into the dorsal noradrenergic bundle, whereas they were predominantly located in the caudal locus coeruleus alpha and subcoeruleus when horseradish peroxidase was injected into the spinal cord. In the electrophysiological experiments, cats (n = 30) were anesthetized with alpha-chloralose and two stimulating electrodes were placed stereotaxically in the dorsal noradrenergic bundle and the ipsilateral ventrolateral funiculus of the high cervical cord (C1-C2), respectively. Monophasic square-wave pulses (2.5 Hz, 100 microsecond duration, 800 microA) were delivered. A recording glass electrode, filled with 2 M NaCl saturated with Fast Green, was placed in the locus coeruleus. Neurons with different conduction velocities, which were evoked by the antidromic stimulation of the dorsal noradrenergic bundle and spinal cord, were verified in the locus coeruleus and the adjacent areas. The slow conductive neurons with a conduction velocity of less than 1 m/s had a slow firing rate (1.6 +/- 0.9/s). They were located predominantly in the rostral locus coeruleus proper and locus coeruleus alpha by the dorsal noradrenergic bundle stimulation. From the anatomical and electrophysiological experimental results, it was concluded that the conduction velocities of the horseradish peroxidase-labeled neurons observed in locus coeruleus proper and locus coeruleus alpha were mostly slow and less than 1 m/s. Most of the slow conductive neurons were considered to be noradrenergic. Neurons evoked antidromically by both the dorsal noradrenergic bundle and spinal cord stimulation were not observed.     

Arcuate nucleus projections to brainstem regions which modulate nociception

Anterograde tracing studies were conducted in order to identify efferents from the arcuate nucleus, which contains the hypothalamic opiocortin neuronal pool. Phaseolus vulgaris leucoagglutinin (PHA-L) was stereotaxically iontophoresed into the arcuate nucleus and the terminal fields emanating from the labelled perikarya were identified immunocytochemically. PHA-L-immunoreactive (-ir) fibers were identified in nucleus accumbens, lateral septal nucleus, bed nucleus of the stria terminalis, medial and lateral preoptic areas, anterior hypothalamus, amygdaloid complex, lateral hypothalamus, paraventricular nucleus, zona incerta, dorsal hypothalamus, periventricular gray, medial thalamus and medial habenula. In the brainstem, arcuate terminals were identified in the periaqueductal gray (PAG), dorsal raphe nucleus (DRN), nucleus raphe magnus (NRM), nucleus raphe pallidus, locus coeruleus, parabrachial nucleus, nucleus reticularis gigantocellularis pars alpha, nucleus tractus solitarius and dorsal motor nucleus of the vagus nerve. Dual immunostaining was used to identify the neurochemical content of neurons in arcuate terminal fields in the brainstem. Arcuate fiber terminals established putative contacts with serotonergic neurons in the ventrolateral PAG, DRN and NRM and with noradrenergic neurons in periventricular gray, PAG and locus coeruleus. In the PAG, arcuate fibers terminated in areas with neurons immunoreactive to substance P, neurotensin, enkephalin and cholecystokinin (CCK) and putative contacts were identified with CCK-ir cells. This study provides neuroanatomical evidence that putative opiocortin neurons in the arcuate nucleus influence a descending system which modulates nociception.     

Ultrastructural localization of enkephalin-like immunoreactivity in developing rat cerebellum

Methionine enkephalin, an endogenous opioid peptide, participates in the regulation of growth in the developing brain. In the present study, enkephalin-like immunoreactivity was localized in the cerebellum of developing and adult rats by immunoelectron microscopy. In 10-day-old animals, enkephalin-like immunoreactivity was found in the somata of proliferating, migrating and differentiating neural cells, and was associated with the plasma membrane, microtubules, filaments, mitochondria, endoplasmic reticulum and nuclear envelope. Both neurons and glia in the cerebellum of the preweaning rat displayed a similar profile of immunoreactivity. Reaction product was also detected in the dendrites and dendritic spines of Purkinje cells where it was concentrated in postsynaptic densities. The majority of internal granule neurons in 10-day-old animals were not immunoreactive, nor were axons, glial processes and postsynaptic elements (with the exception of mossy fiber terminals). At weaning (Day 21), enkephalin-like immunoreactivity was confined primarily to the somata of Purkinje, basket and stellate neurons, and to Purkinje cell dendrites and synaptic spines. Adult rats (day 75) exhibited no enkephalin-like immunoreactivity. These results establish that enkephalin or an enkephalin-like substance can be detected during the ontogeny of both neurons and glia in the cerebellar cortex, and appears to be associated with certain structural elements.     

Brainstem projections to spinal motoneurons: an update

1. The existence of direct projections to spinal motoneurons and interneurons from the raphe pallidus and obscurus, the adjoining ventral medial reticular formation and the locus coeruleus and subcoeruleus is now well substantiated by various anatomical techniques. 2. The spinal projections from the raphe nuclei and the adjoining medial reticular formation contain serotonergic and non-serotonergic fibres. These projections also contain various peptides, several of which are contained within the serotonergic fibres. Whether still other transmitter substances (e.g. acetylcholine) are present in the various descending brainstem projections to motoneurons remains to be determined. 3. The spinal projections from the locus coeruleus and subcoeruleus are mainly noradrenergic, but there also exists a non-noradrenergic spinal projection. 4. Pharmacological, physiological and behavioural studies indicate an overall facilitatory action of noradrenaline and serotonin (including several peptides) on motoneurons. This may lead to an enhanced susceptibility for excitatory inputs from other sources. 5. The brainstem areas in question receive an important projection from several components of the limbic system. This suggests that the emotional brain can exert a powerful influence on all regions of the spinal cord and may thus control both its sensory input and motor output.     

Distribution of nerve growth factor receptor-like immunoreactivity in the adult rat central nervous system. Effect of colchicine and correlation with the cholinergic system--II. Brainstem, cerebellum and spinal cord

Multiple nuclei and fiber tracts in the adult rat brainstem and spinal cord were found to contain nerve growth factor receptor-related protein, as recognized by the monoclonal antibody 192-IgG. Both cholinergic and non-cholinergic sensory and motor regions demonstrated immunoreactive cell bodies and fibers. Nerve growth factor receptor-immunoreactive cells were seen in the mesencephalic nucleus of trigeminal nerve, superior colliculus, parabrachial, prepositus hypoglossal, raphe, dorsal and ventral cochlear, interstitial nucleus of the vestibular nerve, ambiguus and reticular nuclei, cerebellum and ventral spinal cord. Immunoreactive cells resembling neuroglia were distributed subpially along the superior colliculus. Intracerebroventricular injection of colchicine resulted in significantly increased nerve growth factor receptor immunoreactivity in all previously positive neurons and especially in certain neurons of the cochlear and ambiguus nuclei. It also resulted in the visualization of receptor immunoreactivity in certain neurons which were normally non-immunoreactive including cerebellar Purkinje cells, neurons of the central gray, locus coeruleus, facial, dorsal motor vagal and hypoglossal nuclei. In normal animals, nerve growth factor receptor-immunoreactive fibers and varicosities occurred in the trigeminal nerve nuclei, pontine, vestibular, parabrachial, facial, hypoglossal, dorsal motor vagal, solitary, gracile and cuneate nuclei and spinal cord. Although most fiber-like immunoreactive structures were probably axons and nerve terminals, neuroglial or extracellular localizations could not be excluded in some areas. For example, the medial nucleus of the inferior olive and most cerebellar nuclei contained diffuse non-fibrillar receptor immunoreactivity. The presence of nerve growth factor receptor-like immunoreactivity in cell bodies and fibers of several sensory and motor areas of the adult rat brainstem, cerebellum and spinal cord suggests multifocal actions of nerve growth factor or a nerve growth factor-like substance. Although the degree of overlap between nerve growth factor receptor- and choline acetyltransferase-containing regions in the brainstem is not as great as in the forebrain, our findings suggest a potential influence of nerve growth factor or nerve growth factor-like substances on cholinergic systems outside the forebrain. Furthermore, the disparities which occur imply that non-cholinergic nerve growth factor receptor-containing neurons of the brainstem, cerebellum and spinal cord may be affected by such trophic substances.     

Non-dopaminergic catecholaminergic neurons of mesencephalic and medullary nuclei contain different levels of dopamine

The present study addresses the question whether metabolic dopamine can be immunocytochemically detected within nondopaminergic catecholaminergic axonal fibers. For this purpose, confocal microscopy was used to analyze sections treated for the double fluorescence immunostaining of dopamine and either noradrenaline or phenylethanolamine-N- methyltransferase (the enzyme in adrenergic neurons that converts noradrenaline into adrenaline). Our data demonstrate that throughout the brain and spinal cord, the majority of the axonal fibers that reacted with the anti-phenylethanolamine-N-methyltansferase antibodies also exhibited faint to intense dopamine immunoreactivity. Similarly noradrenaline and dopamine immunoreactivities were frequently colocalized within axonal fibers innervating brain and spinal cord regions that receive a dense innervation from medullary noradrenergic neurons. On the contrary, dopamine was rarely detected within noradrenaline-immunoreactive fibers in those regions where the nomdrenergic innervation essentially arises from noradrenergic neurons of the locus coeruleus. A similar differential dopamine immunostaining was observed in the corresponding neuronal perikarya of the medulla oblongata and the locus coeruleus. These data indicate that two types of non-dopaminergic catecholaminergic neurons can be distinguished according to their content in dopamine: (i) the noradrenergic and adrenergic neurons located in the medulla oblongata, whose cell bodies and axons contain high concentrations of metabolic dopamine and (ii) the noradrenergic neurons located in the mesencephalon, which contain low levels of metabolic dopamine.     

Correlation between met-enkephalin and substance P immunoreactivity in the primate globus pallidus

Using immunohistochemical techniques, the distribution of enkephalin and substance P immunoreactive fibers and terminals was studied in the globus pallidus of the non-human primate. In the external segment of the globus pallidus, enkephalin immunoreactivity was very dense while only sparse to moderate substance P staining was observed. Enkephalin immunoreactivity in the inner portion of the internal segment was moderate while such fibers were sparse in the outer portion of the internal segment. Substance P immunoreactivity was dense throughout the internal segment of the globus pallidus.The pattern of enkephalin and substance P immunoreactivity in the globus pallidus of the non-human primate as reported in the present study is of interest with regard to pallidal efferents. The pallidosubthalmic projection, arising from the external segment of the globus pallidus, is likely to be strongly influenced by the very dense network of enkephalin immunoreactive fibers and terminals in this region. Conversely, substance P immunoreactive elements are sparse in the external segment, and are, therefore, unlikely to influence significantly activity carried by the pallidosubthalmic projection. Since the inner portion of the internal segment contains moderate enkephalin immunoreactivity and dense substance P immunoreactivity, the information carried by the lenticular fasiculus may be modulated by both of these putative transmitters. On the other hand, based on the densities of immunoreactivity, the ansa lenticularis, which arises from the outer portion of the internal segment, is likely to be under greater influence of the dense substance P projection to this area.     

Enkephalin-like immunoreactivity within the telencephalon of the reptile Caiman crocodilus

Immunohistochemical methods were used to characterize the distribution of staining for leucine enkephalin-like and methionine enkephalin-like immunoreactivities in the telencephalon of Caiman crocodilus. Very similar distributions of both leucine enkephalin-like and methionine enkephalin-like immunoreactivity were observed. The greatest accumulations of enkephalin-like immunoreactive material were observed within the ventrolateral area of the telencephalon, a region considered comparable to the mammalian corpus striatum and avian paleostriatal complex (i.e. basal ganglia) on the basis of embryological, anatomical and histochemical criteria. Within the ventrolateral area, many small immunoreactive neuron cell bodies were observed, particularly within the rostromedial small-celled component of the ventrolateral telencephalic area. A rich plexus of fibers displaying enkephalin-like immunoreactivity invests the entire ventrolateral area including the large-celled subdivision. A system of thick, coarse, radially-directed immunoreactive fibers running between medial and dorsal portions of the ventrolateral area and more ventral portions was observed in this study. Other structures in the caiman telencephalon, containing large numbers of neural elements displaying enkephalin-like immunoreactivity, were the ventral paleostriatum (a region considered comparable to the ventral pallidum of mammals), the lateral septal nucleus and the nucleus accumbens. The corticoid areas contained far fewer elements displaying enkephalin-like immunoreactivity, although immunoreactive fibers and cell bodies were observed within the medial, dorsal and lateral corticoid areas, particularly at caudal levels. The dorsal ventricular ridge contains the lowest number of immunoreactive cells and fibers of any structure within the caiman telencephalon although occasional neurons displaying enkephalin-like immunoreactivity were encountered in the dorsal ventricular ridge. The results are compared to the distribution of enkephalin within the cerebral hemispheres of mammals, birds and other reptiles.     

Cholinergic neurons expressing neuromedin K receptor (NK3) in the basal forebrain of the rat: a double immunofluorescence study

By using a double immunofluorescence method we have examined the distribution of cholinergic neurons expressing neuromedin K receptor (NK3) in the rat brain and spinal cord. The distribution of neuromedin K receptor-like immunoreactive neurons completely overlapped with that of choline acetyltransferase-positive neurons in certain regions of the basal forebrain, e.g. the medial septal nucleus, nucleus of the diagonal band of Broca, magnocellular preoptic nucleus and substantia innominata. Partially overlapping distributions of neuromedin K receptor-like immunoreactive and choline acetyltransferase-positive neurons were found in the basal nucleus of Meynert, globus pallidus, ventral pallidum of the forebrain, tegmental nuclei of the pons and dorsal motor nucleus of the vagus. Neurons showing both neuromedin K receptor-like and choline acetyltransferase immunoreactivities, however, were found predominantly in the medial septal nucleus, nucleus of the diagonal band of Broca and magnocellular preoptic nucleus of the basal forebrain: 66-80% of these choline acetyltransferase-positive neurons displayed neuromedin K receptor-like immunoreactivity. Neurons showing both neuromedin K receptor-like and choline acetyltransferase immunoreactivities were hardly detected in other aforementioned regions of the forebrain, brainstem and spinal cord. The present study has provided morphological evidence for direct physiological modulation or regulation of cholinergic neurons by tachykinins through the neuromedin K receptor in the basal forebrain of rats.     

Distribution of enkephalin-immunoreactive nerve fibres and terminals in the region of the nucleus basalis magnocellularis of the rat: a light and electron microscopic study

Summary This investigation was carried out on the distribution of enkephalin-containing nerve fibres and terminals in the region of the nucleus basalis magnocellularis (NBM) of the rat. At the light microscope (LM) level, enkephalin-immunoreactive sites and endogenous choline acetyltransferase (ChAT) were demonstrated by employing the two-colour immunoperoxidase staining technique, using highly specific monoclonal antibodies against enkephalin and ChAT. A pharmacohistochemical procedure to reveal acetylcholinesterase (AChE)-synthesizing neurons combined with the peroxidase-antiperoxidase (PAP) immunocytochemical technique to detect endogenous enkephalins, provided ultrastructural data on the relationships of neuronal elements containing AChE and enkephalins in the region of the NBM.At the LM level, cholinergic neurons of the NBM were surrounded by a dense network of enkephalin-immunoreactive nerve fibres. Electron microscopic (EM) observations of histochemically characterized structures, that were first identified in the LM, revealed that intensely AChE-stained structures in the region of the NBM received sparse synaptic inputs from enkephalin immunoreactive terminals. Synaptic inputs of immunoreactive terminals onto intensely AChE-stained neuron cell bodies were not detected. Synaptic contacts onto proximal AChE-positive dendrites were sparse, but the density increased on more distal regions of the dendrites. All immunoreactive boutons studied established symmetrical synaptic contacts with AChE-positive post-synaptic structures. The pattern of the synaptic input to these cells differs strikingly from that onto typical globus pallidus neurons. The perikarya and dendrites of the latter neurons were characteristically ensheathed in immunoreactive synaptic boutons.Results are consistent with the view that enkephalin-like substances in the rat might be synaptic transmitters or neuromodulators in the area of the NBM and that cholinergic neurons of the NBM (Ch4) are integrated into the circuitry of the basal ganglia. Enkephalins may play an important role regulating the extrinsic cholinergic innervation of the neocortex.