Axonal transport in serotonin neurons of the midbrain raphe.

The projections of serotonin-containing neurons of the midbrain raphe nuclei (nucleus raphe dorsalis, nucleus centralis superior) are studied by analysis of axonal transport of labeled amino acids. These results are correlated with regional alterations of serotonin content following midbrain raphe lesions which produce significant serotonin depletion in nearly all regions of the central nervous system. Twenty-four hours following injection of 100 muCi [3H]proline, raphe neurons have taken up labeled material and transported it, presumably as protein, to telencephalon, diencephalon, brain stem, the cerebellum and the spinal cord. This transport appears to take place predominantly in serotonin neurons. After injection of 100 muCi [3H]5-HTP into nucleus raphe dorsalis or nucleus centralis superior, the pattern of regional distribution of transported material is very similar to that obtained with tritiated proline. Selective lesions of serotonin terminals with 5.6-DHT result in greatly diminished axonal transport of proteins to all telencephalic, diencephalic and mesencephalic areas as well as to cerebellum, pons-medulla and spinal cord. Unilateral destruction of the medial forebrain bundle results in significant reduction in axonal transport of labeled material to ipsilateral telencehalon and thalamus. These results provide further support for the view that serotonin neurons of the midbrain raphe nuclei project widely throughout the neuraxis to telencephalon, diencephalon, brain stem, cerebellum and spinal cord.     

Immunohistochemical demonstration of serotonin neuron system in the central nervous system of the Japanese dogfish, Scyliorhinus torazame (Chondrichthyes)

The distribution of serotonin immunoreactivity in the brain of the Japanese dogfish (Scyliorhinus torazame) was studied with the peroxidase-antiperoxidase immunohistochemical method with serotonin antiserum. The somata of the serotonin neurons were located in the raphe regions of the brain stem and the spinal cord. A small number of serotonergic neurons were also distributed as cerebrospinal-fluid contacting neurons in the organum vasculosum hypothalami (OVH) and the paraventricular organ (PVO). These neurons were classified into nine groups (S1-S9): spinal cord (S1), rhombencephalon (S2-S6), mesencephalon (S7), and prosencephalon (OVH, S8 and PVO, S9). Processes of the serotonin neurons were widely distributed in the central nervous system, forming dense networks in various regions. The highest concentrations of these fibers were in the dorsal area of the vagal lobe, the nucleus isthmi, the tuberculum posterium, the distal part of the PVO, and the dorsal regions of the olfactory tubercle.     

Raphe dorsalis-spinal cord cografts in oculo: electrophysiological evidence for an excitatory serotonergic innervation of transplanted spinal neurons

Intraocular replicas of descending serotonergic bulbospinal pathways were constructed by means of sequential intraocular grafting of nucleus raphe dorsalis and spinal cord. Using extracellular recordings we have studied the functional connections between such double grafts. Superfusion of single spinal cord grafts with serotonin causes an increase in spontaneous activity. This excitation is reversibly blocked by the specific 5-hydroxytryptamine (5-HT) antagonist metergoline. Stimulation of the raphe part of nucleus raphe dorsalis-spinal cord double grafts causes a long-lasting excitation of the spinal neurons similar to that seen in single spinal cord grafts given serotonin. The electrically induced excitation could also be reversibly blocked with metergoline. It is concluded that serotonin-containing nerves from grafts of nucleus raphe dorsalis are not only morphologically organotypic, but also form functional contacts with neurons in cografted spinal cord. The results further support an excitatory or modulatory role of the descending spinal serotonergic pathways and demonstrate that functional contacts can be established between isolated CNS grafts when 5-HT fibers invade immature or mature spinal cord tissue.     

The development of serotonergic raphespinal projections in Xenopus laevis

The development of serotonin-immunoreactive neurons in the central nervous system of Xenopus laevis larvae has been studied with special emphasis on the development of the raphe nuclei and raphespinal projections. The first serotonergic neurons were observed in the rostral part of the brain stem at stage 25, only 28 hr after fertilization. By stage 28 some 20 serotonin-immunoreactive neurons were found in the rostral part of the brain stem, bearing small protrusions on the ventromedial side of the soma. These initial axonal outgrowths reach the rostral part of the spinal cord at stage 32. By stage 35/36 the growth cones of the descending serotonergic axons in the spinal cord have reached the level of the anus (10th to 15th myotome). Up to stage 45 the majority of the descending serotonergic axons was found in the dorsolateral part of the marginal zone of the spinal cord. After stage 45 some serotonergic axons were also found scattered over other parts of the spinal marginal zone. Collateral branches were first observed in the caudal part of the brain stem at stage 35/36. Later they occurred also in the rostral (stage 43) and caudal (stage 50) spinal cord, usually on fibers in the ventral half of the spinal cord. The number of serotonergic neurons in the central nervous system (brain stem and hypothalamus) increased steadily throughout development until stage 45. After that the total number of serotonergic neurons in the central nervous system increased about two times faster than the number of serotonergic neurons in the raphe nuclei, due to a massive increase of serotonergic neurons in the hypothalamus. The present study shows that young, just differentiated raphe neurons already contain serotonin. The generation of these neurons appears to take place in the ventricular zone (matrix) of the brain stem between the caudal border of the mesencephalon and the entrance of the nervus octavus. From here these neurons seem to migrate to their final destination. The distribution of serotonin-immunoreactive neurons in the brain stem suggests that a superior (not described so far in Anura) and an inferior raphe nucleus can be distinguished in Xenopus. A rostrocaudal gradient seems to be present in the production of serotonergic neurons which project to the spinal cord. Spinal projections from the raphe nuclei are particularly extensive from the nucleus raphes inferior and gradually decrease rostralwards. In the rostral part of the nucleus raphes superior almost no neurons projecting to the spinal cord are found.     

Intraocular grafts of nucleus raphe dorsalis provide cografted spinal cord with a serotonergic innervation

Nucleus raphe dorsalis was sequentially cografted with spinal cord to the anterior chamber of the eye of adult rats. After maturation in oculo, the double grafts were examined histologically and immunohistologically utilizing an antiserum to serotonin (5HT). No 5HT-like immunoreactivity could be detected in grafts of single spinal cord or in irides of nongrafted eyes. In single raphe grafts, positive cell bodies and a dense terminal plexus were found. 5HT-positive fibers also penetrated into the irides forming dense networks in the walls of blood vessels and elsewhere in the irides. When spinal cord was cografted to maturated nucleus raphe dorsalis grafts, the raphe graft provided the spinal cord graft with a serotonergic innervation. Interestingly, when the grafting procedure was done in reversed order, i.e., the spinal cord was grafted first and left to mature before immature raphe grafts were added to the eye, the average density of 5HT-positive nerve terminals in the spinal cord part was clearly higher. It is concluded that immature as well as mature isolated spinal cord tissue can become innervated by adjacent central 5HT-neurons. Thus isolated replicas of descending serotonergic pathways to the spinal cord, suitable for electrophysiological analysis, are obtained in oculo.     

Evidence for coexistence between calcitonin gene-related peptide and serotonin in the bulbospinal pathway in the monkey

By the use of the indirect immunofluorescence and in situ hybridization techniques, the distribution of calcitonin gene-related peptide (CGRP)-like immunoreactivity (LI) and CGRP mRNA was studied in the spinal cord as well as in the midline raphe nuclei and the hypoglossal nucleus in the medulla oblongata of the monkey (Macaca fascicularis). In the spinal cord only a few large neurons in the motor nucleus contained CGRP-LI, while a majority of the neurons in the hypoglossal nucleus contained CGRP-LI. A relatively dense innervation by CGRP-immunoreactive (IR) fibers was also seen close to cell bodies and proximal dendrites of large neurons in the motor nucleus, especially in its ventral part. 5-Hydroxytryptamine (5-HT)-, substance P- and thyrotropin-releasing hormone (TRH)-IR varicosities were also observed in a similar position around large neurons in the motor nucleus. Double labeling disclosed that the majority of CGRP-IR axon terminals also contained 5-HT-LI. Expression of CGRP mRNA was found in neurons in the medullary midline raphe nuclei and in large neurons in the motor nucleus at the cervical spinal cord level. In adjacent sections of the medulla oblongata, CGRP-labeled neurons in the midline raphe nuclei also expressed preprotachykinin mRNA. The present results show that CGRP- and 5-HT-LI coexist in fibers within the motor nucleus of the monkey spinal cord and that this coexistence is probably due to the presence of CGRP in the descending bulbospinal, serotonergic pathway.     

Origins of serotonergic projections to the spinal cord in rat: An immunocytochemical-retrograde transport study

The origins of the serotonergic projections to the spinal cord in the rat were determined by employing the retrograde cell marker HRP coupled with the unlabeled antibody, peroxidase-antiperoxidase immunocytochemical method of Sternberger. Large numbers of stained neurons (> 70%) in the medullary raphe nuclear complex were found to contain both HRP retrogradely transported from the spinal cord and positive 5-HT staining. These serotonergic cell groups, including the nucleus raphe obscurus, raphe pallidus, raphe magnus, and the ventral parts of the reticular formation, project to all spinal cord levels. In addition, some neurons contained HRP granules, but were unstained for 5-HT, suggesting that they may contain other non-serotonergic neurotransmitters. More rostrally in the midbrain reticular formation, many 5-HT neurons were found to have projections exclusively to the cervical spinal cord. These findings indicate that the descending serotonin inputs to the spinal cord originate not only from the serotonergic neurons located in the medullary raphe complex, but also from other new sources located in the central gray and reticular formation of the midbrain.     

Tracing specific transmitter pathways in the rat CNS: combination of [H]serotonin retrograde labelling with immunocytochemical detection of endogenous transmitters

Selective retrograde labelling with [³H]serotonin ([³H]5-HT) can be used to identify serotonergic cell bodies after specific [³H]5-HT uptake by the corresponding nerve terminals. In the present study, we demonstrate that autoradiography of this [³H]5-HT radiolabelling can be combined with immunocytochemical detection of endogenous serotonin, GABA or substance P on the same tissue section. The midbrain raphe serotonergic projections to the olfactory bulb and the spinal projections of medullary serotonergic nuclei were investigated. The specificity of retrograde labelling with [³H]5-HT was confirmed by immunoreactivity of the radiolabelled cells for serotonin, using an antiserum specific for formaldehyde-fixed serotonin. After spinal injections of [³H]5-HT, many retrogradely labelled cells in the medullary raphe were immunopositive for substance P, and a few for GABA. These results are in agreement with the available information on the co-existence of putative transmitters in the spinal projections of caudal raphe neurons. Therefore, autoradiography of [³H]5-HT retrograde labelling combined with immunocytochemistry offers a possibility to test the specificity of transmitter-selective retrograde labelling, to identify transmitter-defined neuronal interactions and to investigate the projection fields of multitransmitter containing neurons.     

In vivo-synthesized radioactively labelled α-methyl serotonin as a selective tracer for visualization of brain serotonin neurons

To investigate the use of α-[³H]methyl tryptophan (α-[³H]MTrp) as a tracer for the in vivo study of brain serotonergic neurons, we examined whether α-[³H]MTrp and its metabolite α-[³H]methyl serotonin (α-[³H]M5-HT) selectively label serotonergic neurons and whether once accumulated in these neurons, the radioactive metabolite behaves like endogenous serotonin. Rats received a systemic injection of 1–5 mCi of α-[³H]MTrp and 24 h later their brains were immediately removed or fixed by perfusion before removal. Tissue sections in which serotonergic neurons had been immunostained for 5-HT or its synthesizing enzyme, tryptophan hydroxylase, were processed for radioautography at the light and electron microscopic level. In another group of rats, the release of radioactivity from different brain areas was studied both under basal and depolarizing conditions. In the dorsal raphe nucleus, the light microscopic examination revealed almost complete colocalization between serotonergic neurons and those that accumulated radioactivity, with a heterogeneity in the content of α-[³H]M5-HT among the various cells. At the ultrastructural level, immunoidentified serotonergic perikarya and dendritic processes in the dorsal raphe nucleus, as well as nerve terminals in the cerebral cortex were also found to contain α-[³H]M5-HT. Under basal conditions, radioactivity was released from the brainstem raphe region and from projection areas such as the striatum and hippocampus. The basal output of α-[³H]M5-HT increased approximately twofold after a depolarizing 50 mM KCl solution was added to the perfusion fluid. These findings suggest that newly synthesized α-[³H]M5-HT can be released both at somatodendritic and terminal sites. In sum, the present results demonstrate the selectivity of α-[³H]MTrp as a tracer for serotonergic cells, and further suggest that α-[³H]MTrp radiolabelling provides for a direct assessment of the in vivo dynamics of brain serotonergic neurons at the cellular level. © 1995 Wiley-Liss, Inc.     

Brainstem location of serotonin neurons projecting to the caudal neurosecretory complex

Serotonergic fibers in the caudal neurosecretory complex (CNc) of poeciliids originate from neurons within, and extrinsic to this spinal cord nucleus. In the present study, retrograde tracing and immunofluorescence techniques were combined to localize extrinsic serotonergic projection neurons. The entire spinal cord and brain were sectioned after Fast Blue (FB) or horseradish peroxidase (HRP) was implanted in the CNc. No HRP or FB filled neurons were found in the spinal cord. Retrogradely filled neurons were found bilaterally in dorsolateral and ventromedial reticular nuclei, and the dorsal midbrain tegmentum. Fusiform cells in the medullary fasciculus longitudinalis medialis filled with FB but not HRP. Serotonin immunopositive neurons were found surrounding the third ventricle, in the raphe and in medullary reticular nuclei. Double labelled neurons in the medial reticular nucleus were determined to be the source of serotonergic projections to the CNc. Reticular projection nuclei are strategically situated to receive visceral sensory input from rhombencephalic cranial nerves. These putative pathways may provide an anatomical substrate by which visceral sensory information is transmitted to the CNc.     

The distribution of 5-HT immunoreactive systems in the brain of a saurian, the chameleon

The distribution of serotonin immunoreactive cell bodies and fibers was studied in the chameleon brain by using the immunohistochemical technique with antisera against serotonin coupled to a carrier with glutaraldehyde. Serotonin perikarya were found in the caudal midbrain tegmentum, in the lateral part of the nucleus reticularis isthmi, the lateral part of the nucleus interpeduncularis and along the midline in the raphe superior. More caudally, the serotonin immunoreactive cell bodies were located along the nucleus raphe inferior and ventrolaterally in the vicinity of the olivary complex. No immunoreactive cell bodies were found in the spinal cord nor in the paraventricular organ (PVO) of the hypothalamus. Immunoreactive fibers were observed in the entire brain. Prominent concentrations were found in the dorsal cortex, lateral septum, lateral geniculate nucleus, median eminence, pretectal nucleus, nucleus interpeduncularis, vestibular nucleus and olivary complex. Descending serotonin immunoreactive fibers were found in particular in the ventral motoneuron area in the spinal cord. One of the most interesting findings in this study was the lack of immunoreactive CSF contacting neurons in the PVO and the observation of an extensive plexus of supraependymal fibers, a feature reported so far only in mammals.     

Origin of the serotonergic innervation to the rat dorsolateral hypothalamus: retrograde transport of cholera toxin and upregulation of tryptophan hydroxylase mRNA expression following selective nerve terminals lesion.

The regulation of serotonin synthesis was investigated in the serotonergic neurons, which provide afferents to the dorsolateral hypothalamus (DLH). The origin of the DLH projection neurons within the raphe nucleus was identified by retrograde transport of Cholera toxin (CTb) and their serotonergic nature confirmed by tryptophan hydroxylase (TPH) immunocytochemistry. Disruption of serotonin synthesis steady-state was induced unilaterally by a selective and local destruction of serotonergic nerve terminals with 5,7-dihydroxytryptamine (5,7-DHT), stereotaxically injected in the right DLH. The results show that most of the serotonergic dorsal raphe neurons projecting to the DLH have an ipsilateral localization within the lateral aspects of the nucleus. In rats with unilateral DLH lesion, a population of serotonergic cells within the raphe nucleus exhibited a clear increase in TPH mRNA. These cells were about five times more numerous in the ipsilateral as compared to the contralateral dorsal raphe nucleus and they had, for the most part, a lateral localization within the raphe nucleus. Sham-operated rats did not exhibit any upregulation of TPH mRNA. Together, the present results provide the first demonstration that a discreet and selective destruction of serotonergic terminals induces a circumscribed and striking increase in TPH mRNA expression in a subset of brainstem serotonergic neurons projecting to and/or passing through the DLH. On the basis of these results and previous in vivo measurements of TPH activity (e.g., 5-HT synthesis), we suggest that this upregulation in TPH mRNA expression results from the loss of pre-synaptic and/or post-synaptic regulation of serotonin synthesis. These new findings raise important issues related to the repercussions of a local disruption in serotonergic neurotransmission on brain areas remote from the site of injury.     

GABAergic innervation of serotonergic neurons in the dorsal raphe nucleus of the rat studied by electron microscopy double immunostaining.

A double immunocytochemical method combining the preembedding PAP technique and the postembedding immunogold technique was used to examine interactions between GABAergic and serotonergic neurons in the same tissue sections of the dorsal raphe nucleus of the rat. A large number of immunogold stained GABAergic axon terminals were found to be presynaptic to strongly PAP immunostained serotonergic perikarya and dendrites. The types of synapses were mostly symmetrical although a few asymmetrical ones were also found. No axo-axonic synapse between the GABAergic axon terminals and the serotonergic neuronal profiles was found. These results suggest that GABAergic neurons could modulate serotonergic neurons in the dorsal raphe nucleus through synaptic relations.     

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.     

Colocalization of substance P or enkephalin in serotonergic neuronal afferents to the hypoglossal nucleus in the rat

The serotonergic innervation of the hypoglossal nucleus originates from the caudal raphe nuclei. Non-serotonergic neurons in the caudal raphe nuclei also project to the hypoglossal nucleus. We employed a triple-fluorescence technique to determine whether the substance P- or the enkephalin-containing neurons in the caudal raphe nuclei that projected to the hypoglossal nucleus also contained serotonin. Rhodamine latex microspheres were injected into the hypoglossal nucleus, and then serotonin and peptide dual-immunofluorescence was performed to colocalize perikarya containing serotonin, substance P, and rhodamine microspheres; or perikarya containing serotonin, enkephalin, and rhodamine microspheres. Our results demonstrate that most substance P-containing neuronal afferents to the hypoglossal nucleus colocalize serotonin. In contrast, few enkephalin-containing neuronal afferents to the hypoglossal nucleus also contain serotonin. These data suggest that substance P projections to the hypoglossal nucleus are a subset of serotonergic projections and that limited overlap exists between the populations of enkephalinergic and serotonergic neuronal afferents to the hypoglossal nucleus. Either substance P- or enkephalin-containing somata account for a very small proportion of non-serotonergic caudal raphe projections to the hypoglossal nucleus. Finally, these data demonstrate the medial tegmental field origins of the substance P projections and the enkephalin projections to the hypoglossal nucleus. J. Comp. Neurol. 391:491–505, 1998. © 1998 Wiley-Liss, Inc.     

Serotonin-storing secretory granules from thyroid parafollicular cells

The subcellular storage of 5-HT was studied in sheep thyroid parafollicular cells. These cells are neural crest derivatives and were investigated as a serotonergic model system. Light and electron microscopic immunocytochemistry was used to examine the distributions of 5-HT, 45 and 56 kDa forms of 5-HT binding protein (SBP), and calcitonin. A single type of parafollicular cell was found that contained calcitonin, 5-HT, and 45 kDa SBP but not 56 kDa SBP. The secretory granules of parafollicular cells all displayed calcitonin immunoreactivity, and many were also immunoreactive for 5-HT and 45 kDa SBP. Granules were isolated, first by size and then by density, on successive metrizamide gradients. These provided a granular fraction that was enriched in calcitonin, endogenous 5-HT, and 45 kDa SBP. Immunoblots confirmed the presence of 45 kDa SBP in the isolated granules and in suspensions of parafollicular cells that were purified by an affinity chromatographic technique. Parafollicular cell granules did not appear to contain substantial stores of ATP. Granules isolated on Percoll gradients were morphologically homogeneous and took up 3H-5-HT. The specificity of this uptake was confirmed by quantitative electron microscopic radioautography. The granular uptake of 3H-5-HT was inhibited by reserpine (10 microM). It is concluded that parafollicular cell granules are different from other amine-storing vesicles that do contain ATP; nevertheless, since parafollicular cell granules store 5-HT and have the same 45 kDa SBP as is found in serotonergic axon terminals, parafollicular cell granules may be analogous to the synaptic vesicles of serotonergic neurons.     

Co-localization of N-acetyl-aspartyl-glutamate in central cholinergic, noradrenergic, and serotonergic neurons

An immunohistochemical technique for simultaneously visualizing two different antigens has been used to investigate the presence of the acidic dipeptide, N-acetyl-aspartyl-glutamate (NAAG), in cholinergic, noradrenergic-adrenergic, and serotonergic neurons within CNS. The brain slices were processed sequentially with purified antisera against NAAG and then monoclonal antibody against choline acetyltransferase (ChAT), a marker for cholinergic neurons, or antiserum against dopamine-beta-hydroxylase (DBH), a marker of noradrenergic-adrenergic neurons, or antiserum against serotonin (5HT). Both antigens were revealed by the peroxidase reaction but with different chromogens, which are easily distinguishable. An intense double staining of NAAG-like immunoreactivity (NAAG-LI) and ChAT was observed in the motoneurons of the spinal cord as well as in the several motor components of cranial nerve nuclei including facial, ambiguus, and trigeminal nuclei. A partial colocalization of NAAG-LI and ChAT was evident in the perikarya of the basal forebrain cholinergic system, whereas cholinergic neurons of the medial septum exhibited only sporadic staining for NAAG-LI. A complete coexistence of NAAG-LI and DBH was observed in the locus coeruleus. Most of the other noradrenergic and adrenergic cell groups of the medulla region exhibited substantial co-localization with the exception of the A2 cell group, which was virtually devoid of NAAG-LI. In the dorsal raphe, only a low percentage of serotonergic neurons stained for NAAG-LI. The co-existence of NAAG-LI and serotonin was more evident in the neurons of the median raphe, although the majority of cells failed to show double staining.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extent of colocalization of serotonin and GABA in the neurons of the rat raphe nuclei

Previous investigations of the distribution of neurons containing both serotonin and GABA in the brainstem raphe nuclei have yielded discrepant results amongst different authors. This study attempted to clarify the distribution as well as the proportions of raphe and other brainstem neurons that contain both neurotransmitters. All the nine serotonergic cell groups known to be present in the brainstem were examined with an indirect immunofluorescence method using antibodies against serotonin and glutamic acid decarboxylase in colchicine-treated rats. Sections were incubated either simultaneously or sequentially for the two immunolabels. Brainstem neurons that were labelled for both markers were generally infrequent. Of all the serotonin cell groups in the brainstem, the nucleus raphe magnus contained the most double-labelled cells (a mean of 3.6% of a total of 625–1155 serotonin-immunoreactive cells counted in this nucleus), followed by the nucleus raphe obscurus (1.5% of a total of 220–550 serotonin-immunoreactive neurons counted). The dorsal, median and pontine raphe nuclei as well as the supralemniscal nucleus (the B9 group) contained very few double-labelled cells, which comprised a mean of 0.1–0.7% of all serotonin-immunoreactive cells in each of these nuclei. No double labelled cells were present in the caudal linear raphe nucleus or the nucleus raphe pallidus, nor in the B4 group. These results suggest that only a very small percentage of serotonergic neurons in the medullary raphe nuclei (raphe magnus and raphe obscurus) also contain GABA, whereas such cells are virtually absent in the midbrain raphe nuclei or in the non-raphe serotonergic cell groups in the brainstem.     

Origins of serotonergic projections to the lumbar spinal cord in the monkey using a combined retrograde transport and immunocytochemical technique

A newly developed technique employing the retrograde transport of horseradish peroxidase combined with immunocytochemistry is used to identify the cells of origin of descending spinal pathways and their putative neurotransmitters. With this technique the brainstem origins of descending serotonergic (5HT) pathways to the lumbar spinal cord have been determined in the monkey. Numerous 5HT stained neurons are found in the nucleus raphe obscurus and raphe magnus and in the adjacent reticular formation projecting to the lumbar spinal cord. The nucleus raphe pallidus contains relatively fewer descending 5HT neurons. In addition to the spinally projecting neurons containing 5HT, large multipolar shaped neurons within the raphe nuclei were found to project to the spinal cord, but these do not stain for 5HT immunoreactivity. These findings indicate that the raphe nuclear complex provides both serotonergic and non-serotonergic inputs to the spinal cord. The advantages and uses of the present double labeling method for localizing other neurotransmitter substances in identified neuronal pathways are discussed.     

Thyrotropin-releasing hormone-immunoreactive neurons project from the ventral medulla to the intermediolateral cell column: partial coexistence with serotonin

Projections from medullary thyrotropin-releasing hormone (TRH) containing neurons to the intermediolateral cell column (IML) of the thoracic spinal cord were studied in the rat. Lesions of the ventral medullary reticular formation nuclei, nucleus paragigantocellularis lateralis and nucleus interfascicularis hypoglossi, decreased the thyrotropin-releasing hormone immunoreactivity in the IML. The ventral horn and dorsal horn contents of TRH were also reduced in rats with nucleus paragigantocellularis lateralis lesions. Coexistence of spinal cord TRH and serotonin was evaluated and quantified in 5,7-dihydroxytryptamine-treated rats. Treatment with the serotonin neurotoxin reduced the TRH content of the IML by 45% and of the ventral horn by 92%. These data show that TRH containing neurons project from the ventral medulla to IML and that approximately one-half of these TRH neurons are also serotonergic. Comparisons of the effects of the same lesions on the substance P and TRH content of the IML show that neither the origin of the SP and TRH neuronal projections to the IML, nor their coexistence with serotonin, are identical.