Serotonergic innervation of the isthmo-optic nucleus of the pigeon centrifugal visual system. An immunocytochemical electron microscopic study

The ultrastructural features of serotonergic fibers, terminals and synaptic contacts were studied with the pre-embedding immunocytochemical method in the isthmo-optic nucleus of the pigeon centrifugal visual system. The 5-HT immunoreactive (-ir) profiles were diffusely distributed and their density was low. The labeled axons were thin and unmyelinated (mean diameter=0.21+/-0.03 microm) though a few larger myelinated axons were observed (mean diameter=0.51+/-0.07 microm). The 5-HT-ir terminals or varicosities were small (diameter=0.71+/-0.54 microm) and contained small agranular synaptic vesicles (diameter=28.5+/-6.9 nm) and large granular vesicles (diameter=102.2+/-19.5 nm). The latter only constituted approximately 1% of the total profiles containing synaptic vesicles in the isthmo-optic nucleus. In single thin sections, only 5% of the 5-HT-ir varicosities exhibited an active asymmetrical zone synapsing upon dendritic profiles of centrifugal visual neurons. Calculations indicated that 17% of these 5-HT-ir varicosities were actually engaged in junctional synaptic relationships, whereas the remaining (83%) were nonjunctional. The data suggest that, within the isthmo-optic nucleus, 5-HT acts both at synaptic junctions (wiring transmission) and at a distance via the extracellular space (volume transmission). These 5-HT afferents could thus modulate the activity of the retinopetal neurons and visual information processing.     

Quantitative immunogold evidence that glutamate is a neurotransmitter in afferent synaptic terminals within the isthmo-optic nucleus of the pigeon centrifugal visual system

A quantitative electron microscopic analysis of glutamate (GLU) immunoreactivity using the post-embedding immunogold technique was carried out within the isthmo-optic nucleus (ION) of the pigeon centrifugal visual system (CVS). Measurements were performed in each of eight different categories of axon terminals, including those that were GABA-immunoreactive (-ir), considered representing control profiles and identified using a single or double-label immunocytochemical procedure. The results demonstrated that the glutamate immunogold particle densities for both mitochondrial and vesicular pools and for total surface area of bouton profiles were significantly higher in P1a, P1b and P2b terminals and not significantly different in P4 and P5 terminals compared to those recorded in control GABA-ir terminals (P2a, P2c, P3). Moreover, the values measured in GLU-ir positive profiles were all significantly higher than in either P4 or P5 terminals. The results suggest that tectal neurons, which provide the main input to the ION cells, are either inhibitory GABA-ir possibly associated with P2c and/or P3 terminals or excitatory GLU-ir via P1a, P1b and P2b terminals. Such differential effects of tectal afferents may be the basis for the modulation of centrifugal activity and consequently of end target retinal ganglion cell responses. The data are relevant to hypotheses implicating the avian CVS in mechanisms of selective enhancement of visual attention to either novel or meaningful stimuli within the visual field.     

Serotonin distribution in the brain of the plainfin midshipman: Substrates for vocal-acoustic modulation and a reevaluation of the serotonergic system in teleost fishes

Serotonin (5-HT) is a modulator of neural circuitry underlying motor patterning, homeostatic control, and social behavior. While previous studies have described 5-HT distribution in various teleosts, serotonergic raphe subgroups in fish are not well defined and therefore remain problematic for cross-species comparisons. Here we used the plainfin midshipman fish, Porichthys notatus, a well-studied model for investigating the neural and hormonal mechanisms of vertebrate vocal-acoustic communication, to redefine raphe subgroups based on both stringent neuroanatomical landmarks as well as quantitative cell measurements. In addition, we comprehensively characterized 5-HT-immunoreactive (-ir) innervation throughout the brain, including well-delineated vocal and auditory nuclei. We report neuroanatomical heterogeneity in populations of the serotonergic raphe nuclei of the brainstem reticular formation, with three discrete subregions in the superior raphe, an intermediate 5-HT-ir cell cluster, and an extensive inferior raphe population. 5-HT-ir neurons were also observed within the vocal motor nucleus (VMN), forming putative contacts on those cells. In addition, three major 5-HT-ir cell groups were identified in the hypothalamus and one group in the pretectum. Significant 5-HT-ir innervation was found in components of the vocal pattern generator and cranial motor nuclei. All vocal midbrain nuclei showed considerable 5-HT-ir innervation, as did thalamic and hindbrain auditory and lateral line areas and vocal-acoustic integration sites in the preoptic area and ventral telencephalon. This comprehensive atlas offers new insights into the organization of 5-HT nuclei in teleosts and provides neuroanatomical evidence for serotonin as a modulator of vocal-acoustic circuitry and behavior in midshipman fish, consistent with findings in vocal tetrapods.     

Simultaneous Demonstration of Serotonin-immunoreactive Terminals and GABAergic Neurons in the VPL Nucleus of the Cat Thalamus

Pre-embedding immunoperoxidase (for serotonin) and postembedding immunogold (for γ-aminobutyric acid; GABA) labelling were combined at light and electron microscopic levels to demonstrate the neuronal targets of serotonin (5-HT) afferents in the ventral posterior lateral nucleus (VPL) of the cat thalamus. 5-HT-immunoreactive fibres and terminal varicosities were found in close proximity to GABA-immunoreactive interneurons and non-GABAergic relay neurons. Ultrastructurally, the vast majority of 5-HT terminals made close membrane contacts without overt membrane specializations with GABAergic axon terminals, GABAergic presynaptic dendrites and GABAergic somata. A very small number of 5-HT terminals formed typical asymmetrical synapses with GABAergic presynaptic dendrites and with dendritic shafts of relay cells. Some 5-HT terminals participated with the presynaptic dendrites in triadic synaptic arrangements. These findings suggest a dual innervation pattern by 5-HT afferents in VPL and the release of 5-HT in large part at sites not associated with morphologically detectable synapses.     

Topography of serotonin neurons in the dorsal raphe nucleus that send axon collaterals to the rat prefrontal cortex and nucleus accumbens

Diverse physiological actions have been reported for 5-hydroxytryptamine (5-HT, serotonin) in the medial prefrontal cortex (MPFC) and the nucleus accumbens (Acb) suggesting that the 5-HT innervation of these forebrain areas may be derived from different populations of neurons. We examined this possibility by mapping the distribution of 5-HT-immunoreactive (ir) and non-5HT-ir neurons containing retrograde labeling following injections of different tracers into both these target regions. The analysis was focused in the dorsal raphe nucleus (DRN) of the midbrain, since 5-HT pathways to the MPFC and Acb primarily originate from this area. Volume microinjections of the fluorescent retrograde tracer, Fluoro-Gold (FG), were placed into the MPFC and microinjections of cholera toxin B subunit coupled to 15 nm gold particles (CT-Au) were placed into the Acb of the same animal. Sections through the DRN containing retrogradely labeled neurons were further processed for immunofluorescent localization of 5-HT using a rhodamine marker. Neurons retrogradely labeled from the Acb were greater in number overall than those projecting to the MPFC. In addition, Acb-projecting neurons extended into the lateral wings of the DRN, whereas MPFC-projecting neurons were more restricted to the midline. Both groups of retrogradely labeled neurons, however, were more numerous in the caudal aspect of the dorsal raphe nucleus and were scattered amongst 5-HT immunoreactive perikarya. Of783 ± 69 CT-Au labeled cells, 15% also contained the FG label and 11% contained FG and 5-HT immunoreactivity. Of613 ± 48 FG labeled cells, 24% also contained the CT-Au label and 21% were also immunoreactive to 5-HT. The results suggest a more prominent input to the Acb from both 5-HT-ir and non-5-HT-ir neurons in the caudal aspect of the DRN and further indicate that while most 5-HT-ir and non-5-HT-ir neurons project differentially to both forebrain regions, a few cells also show collateralization to the MPFC and Acb. Such collateralization of single serotonergic neurons to divergent targets mey integrate cognitive and motor activities in response to pharmacological manipulations of ascending serotonergic pathways.     

Serotonin-containing structures in the nucleus raphe dorsalis of the cat: an ultrastructural analysis of dendrites, presynaptic dendrites, and axon terminals

In the nucleus raphe dorsalis of the cat, an electron microscopic immunocytochemistry method was used to identify the fine structure of serotoninergic dendritic profiles and axon terminals analyzed in serial sections. Two classes of serotoninergic dendrites were distinguished in the nucleus. The first class was constituted by conventional serotonin (5-HT) dendrites that were contacted by unlabeled axon terminals containing differing populations of synaptic vesicles. The second class consisted of serotoninergic dendrites that contained vesicles in their dendritic shafts. Such 5-HT dendrites were further subdivided into two groups according to their synaptic contacts. In some 5-HT vesicle-containing dendrites, the vesicles were densely packed in small clusters and were associated with a well-defined synaptic specialization. These dendrites were classified as serotoninergic presynaptic dendrites and established synaptic contacts with unlabeled and labeled dendrites and were contacted by unlabeled axon terminals. In other 5-HT vesicle-containing dendrites, extensive serial section examination showed that the vesicles could be observed near the membrane but were never found to be associated with any synaptic membrane specialization. Serotoninergic axon terminals that were presumed to be recurrent collaterals of 5-HT neurons were present in the nucleus. Some of them were observed in synaptic contact with dendrites or dendritic protrusions whereas others did not exhibit synaptic specializations. The existence of serotoninergic dendrodendritic synaptic contacts and axon terminals suggests direct local interactions between serotoninergic neurons within the nucleus raphe dorsalis.     

Ontogeny of descending serotonergic innervation and evidence for intraspinal 5-HT neurons in the mouse spinal cord

Neuronal networks in the mouse spinal cord express serotonin (5-HT)-induced rhythmic motor activity at early developmental stages (embryonic day (E) 12.5). Later in development, by post-natal day (P) 10, the 5-HT-evoked rhythmic motor activity matures and acquires an adult locomotor-like pattern. With the view to establishing a relationship between the ontogeny of locomotor networks and the maturation of spinal 5-HT systems, we have traced 5-HT immunoreactivity in the mouse spinal cord from E12.5 to PN10. By E12.5, descending 5-HT immunoreactive (5-HT-ir) fibers that likely originate from raphe nuclei were detected in the ventral and lateral funiculi, at anterior cervical spinal levels, but not at more caudal levels. Descending 5-HT-ir axons reached thoracic levels at E14.5 and lumbar levels at E16.5. Some 5-HT-ir fibers could be detected in the ventral and intermediate gray matter by E16.5, whereas the dorsal gray matter was not invaded before PN0. At PN10, a dense serotonergic innervation was restricted to the gray matter with a high concentration of 5-HT-ir fibers in three areas: dorsal horn, ventral horn (where motoneurons are located) and intermediate area. Surprisingly, from E16.5 to PN10, 5-HT-ir intraspinal neurons were found, exclusively at sacral levels. Their somata lay in the gray matter around the central canal and preferentially in the ventro-median part of the ventral horn. The functional significance of these sacral 5-HT-ir neurons is discussed.     

Brainstem afferents to the tuberomammillary nucleus in the rat brain with special reference to monoaminergic innervation

Monoaminergic innervation of a histamine-producing cell group, the tuberomammillary nucleus in the posterior hypothalamus, was investigated in the rat by light and electron microscopic immunohistochemical techniques. Immunohistochemical staining of sections of the posterior hypothalamus was demonstrated afferent fibers immunoreactive to tyrosine hydroxylase in ventral and medial subgroups of the tuberomammillary nucleus afferent fibers immunoreactive to tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), phenyletanolamine-N-methyltransferase (PNMT), and serotonin (5-HT). TH- and DBH-immunoreactive fibers were similar and were evenly and densely distributed throughout the tuberomammillary nucleus. Fibers stained with 5-HT antibodies were also present throughout the tuberomammillary nucleus but exhibited the densest labeling in the dendritic layer adjacent to the glia limitans in the ventral subgroup. Innervation by PNMT-immunoreactive axons was sparse. Electron microscopic analysis of TH-, DBH-, and 5-HT-immunoreactive fibers in the tuberomammillary nucleus revealed vesicle-containing terminal boutons, which formed synapses with dendrites of varying size. Synaptic contacts with nerve cell bodies were not found. Retrograde transport of the fluorescent dye Fast Blue injected into the tuberomammillary nucleus, combined with immunofluorescent staining with anti-TH, anti-DBH, anti-PNMT, and anti-5-HT antibodies, showed that monoaminergic input to the tuberomammillary nucleus originated mainly from the adrenergic and noradrenergic cell groups C1-C3 and A1-A2, respectively, and from the serotoninergic cell groups B5-B9 as designated by Dahlstr?m and Fuxe ('65). Few double-labeled neurons were found in the nucleus locus coeruleus and the dopaminergic cell groups of the rostral brain stem. The present findings suggest that the activity of the histamine-producing neurons of the tuberomammillary nucleus is influenced by monoaminergic neurons in the ventrolateral and dorsomedial medulla oblongata and the raphe nuclei of the rostral brainstem.     

Serotonergic projections and serotonin receptor expression in the reticular nucleus of the thalamus in the rat

The reticular nucleus (RT) of the thalamus, a thin sheet of GABAergic neurons located between the external medullary lamina and the internal capsule of the thalamus, has functionally distinct afferent and efferent connections with thalamic nuclei, the neocortex, the basal forebrain and the brainstem. RT is critically positioned to rhythmically pace thalamocortical networks leading to the generation of spindle activity during the early phases of sleep and during absence (spike-wave) seizures. Serotonin, acting on 5-HT(1A) receptors on parvalbumin-containing cells of RT, has been implicated in this rhythmicity. However, the precise source(s) of 5-HT afferents to the RT remains to be determined. In the present study, we injected the retrograde tracer, Fluorogold, into dorsal and ventral regions of RT to determine the origins of raphe input to RT. We further characterized the distribution of 5-HT fibers to RT by using immunohistochemistry for 5-HT and for the 5HT transporter (SERT) detection. Finally, we described the presence of the two major postsynaptic 5-HT receptors in RT, 5-HT(1A) and 5-HT(2A) receptors. Our results show that the dorsal raphe nucleus and the supralemniscal nucleus (B9) of the midbrain are the principal sources of raphe projections to RT. In addition, serotonergic fibers (5-HT and SERT positive) were richly distributed throughout RT, and 5-HT(1A) and 5-HT(2A) receptors were highly expressed on RT neurons and dendrites. These findings suggest a significant 5-HT modulatory influence on GABAergic neurons of RT in the control of rhythmical (or spindle) activity in thalamocortical systems directly associated with sleep and possibly with absence seizures.     

Ultrastructural localization of 5-hydroxytryptamine1A receptors in the rat brain

5-Hydroxytryptamine_1A (5-HT_1A) receptors have been visualized at the electron microscopic level in selected areas (dorsal raphe nucleus, hippocampus, septum) of the rat brain using specific anti-peptide antibodies. 5-HT_1A receptor immunoreactivity was found almost exclusively in the somatodendritic compartment of neurons and was very rarely observed within processes possibly belonging to glial cells. The immunoenzymatic reaction product was associated exclusively with dendritic spines in the dorsal hippocampus, whereas in the dorsal raphe nucleus and the septal complex, immunoreactivity was found in both dendritic processes and somata. Although some immunolabeling was observed within the cytoplasm of cell bodies, 5-HT_1A receptor immunoreactivity was essentially confined to the plasma membrane where it was unevenly distributed. It was frequently associated with synapses (except in the dorsal raphe nucleus), but was also found extrasynaptically in both somata and dendrites. These data suggest that the action of serotonin via 5-HT_1A receptor could occur through junctional as well as nonjunctional transmission. © 1996 Wiley-Liss, Inc.     

Differential origin of brainstem serotoninergic projections to the midbrain periaqueductal gray and superior colliculus of the rat

Previous studies have shown that both the midbrain periaqueductal gray (PAG) and the superior colliculus receive a significant serotoninergic (5-HT) innervation. In the present study the origins of these 5-HT projections to the rodent PAG and superior colliculus were analyzed by using a combined immunohistochemical-retrograde transport technique. Thirteen brainstem regions were found to contain double-labelled 5-HT-like immunoreactive neurons following HRP injections into the PAG while only four brainstem nuclei contained double-labelled neurons following superior collicular injections. After HRP deposits into the ventral PAG, the largest percentage of double-labelled neurons was identified in nucleus raphe magnus, pars alpha of the nucleus gigantocellularis, and the paragigantocellular nucleus. The dorsal PAG, on the other hand, received the largest percentage of its 5-HT projections from nuclei raphe dorsalis, raphe obscurus, raphe pontis, and raphe medianis. The 5-HT input to the superior colliculus was found to arise exclusively from nuclei raphe dorsalis, raphe medianis, and raphe pontis and from the contralateral periaqueductal gray. Raphe nuclei were found to contribute serotoninergic projections to both the PAG and the superior colliculus while reticular nuclei contributed 5-HT projections only to the PAG. Injections of the fluorescent retrograde tracers true blue and nuclear yellow were then made into the PAG and superior colliculus to ascertain if neurons located in raphe nuclei that projected to both structures provided axon collaterals to both areas. Generally, less than 10% of raphe neurons projecting to the superior colliculus were identified as providing axon collaterals to the PAG. The present results demonstrate major quantitative and qualitative differences in the origin of 5-HT projections to the ventral PAG and superior colliculus. The origin of 5-HT input to the dorsal PAG, on the other hand, showed many similarities to the origin of 5-HT innervation of the superior colliculus. These data also indicate that approximately 35% of raphe neurons provide nonserotoninergic projections to the PAG and superior colliculus.     

Changes in serotonergic neurons in the brain of pyrithiamine-induced acute thiamine-deficient mice

We examined changes in 5-hydroxytriptamine (5-HT, serotonin) neurons in pyrithiamine-induced thiamine deficiency in mice immunohistochemically. Extensive decreases in the densities of 5-HT-immunoreactive fibers were detected in the lateral septal nucleus, the thalamus, the medial mammillary nucleus, the dorsal and the median raphe nuclei, the raphe obscurus nucleus, the tegmental area, the cerebellum and the vestibular nucleus, though only a small decrease was detected in the inferior colliculus. Most remarkably, degenerative winding fibers were detected between the deep mesencephalic nucleus and the ventral tegmental area. Increases in intensity of 5-HT immunoreactivity in the dorsal raphe nucleus and decreases in the number of 5-HT-immunoreactive cell bodies in the dorsal and the median raphe nuclei were detected. These results demonstrated the differential vulnerability of 5-HT neurons in thiamine-deficient mice. This is the first report to demonstrate changes in 5-HT neurons immunohistochemically throughout the brain of pyrithiamine-induced thiamine deficient mouse. Received: 15 January 1999 / Revised 22 March 1999 / Accepted: 24 March 1999     

Ontogeny of 5-HT neurons in the brainstem of the lamprey, Petromyzon marinus

This study examined the spatial and temporal distribution of serotonin-immunoreactive (5-HT-ir) neurons in the brainstem of Petromyzon marinus at three developmental stages, larval, postmetamorphic, and reproductive. Computer-assisted 3-D reconstructions were made of the three main 5-HT-ir neuron groups. The rostralmost brainstem group was located near the posterior commissure, the second group at the isthmus, and the third group in the bulbar area. For each of those groups, the distribution of the 5-HT-ir neurons was very similar in the three developmental stages examined, suggesting that the 5-HT system is relatively mature early in larval animals. The soma of 5-HT-ir neurons increased in size and their dendritic fields increased in complexity with development. Furthermore, the number of 5-HT-ir neurons in each group increased significantly from the larval to the reproductive stage. To determine whether this was due to the genesis of 5-HT neurons, bromodeoxyuridine (BrdU) was injected into larval, metamorphosing, and postmetamorphic lampreys. These experiments revealed a few neurons colocalizing BrdU and 5-HT in metamorphosing animals. Taken together, the present results suggest that 5-HT neurons increase in number during maturation and that neurogenesis could, at least partially, contribute to the appearance of new 5-HT cells at different developmental stages.     

Evidence for cholecystokinin-like immunoreactive neurons in the rat medulla oblongata which project to the spinal cord

The distribution of cholecystokinin-like immunoreactive (CCK-LI) neurons has been mapped in the rat medulla after local and intracerebroventricular colchicine injections. CCK-positive neurons were found in the nucleus raphe magnus, nucleus raphe pallidus, nucleus raphe obscurus, nucleus paragigantocellularis pars α, and a population of ventral medullary neurons. Combined retrograde tracing with the fluorescent dye True Blue and indirect immunofluorescence for visualizing CCK neurons suggested that there was a CCK-LI system originating in the medulla and projecting to the spinal cord. Additional double labelling experiments established that some of these CCK-LI containing neurons also contain 5-HT.     

Distribution of 125I-galanin binding sites, immunoreactive galanin, and its coexistence with 5-hydroxytryptamine in the cat spinal cord: biochemical, histochemical, and experimental studies at the light and electron microscopic level.

The distribution of galanin-like immunoreactivity (GAL-LI) in the spinal cord of the cat was studied by use of indirect histochemistry and the peroxidase-antiperoxidase (PAP) technique. In the ventral horn GAL-immunoreactive (IR) axonal fibers and terminals were most frequent in the ventral part of the motor nucleus. The GAL-IR axons also contained 5-hydroxytryptamine (5-HT)-LI, and they disappeared after spinal cord transection. It was concluded that these GAL-IR fibers belong to the serotoninergic bublospinal pathway. In the medulla oblongata from normal cats, scattered GAL-IR cell bodies were encountered within the nucleus raphe obscurus and nucleus raphe pallidus. Electron microscopic observations revealed that the fine structure of the GAL-IR axonal boutons in the motor nucleus was similar to that of 5-HT-IR boutons with a varying number of immunoreactive large dense core vesicles. The postsynaptic element in all cases studied was a dendrite. A dense GAL-IR axonal plexus was found in the superficial laminae I-II of the dorsal horn. Coexistence was found between the GAL- and substance P-LI in fibers within the dorsal horn plexus. Spinal cord transection did not alter the pattern of GAL-LI in the dorsal horn, while the vast majority of GAL-IR axonal swellings disappeared following dorsal root sectioning. Electron microscopic observations in lamina II (substantia gelatinosa) revealed that the GAL-IR axonal terminals could be divided into two main groups. One with small to medium-sized axonal boutons formed synaptic contacts with both dendritic and axonal profiles. The other formed the central axon terminals of glomeruli, suggesting that GAL-LI may be present in C-type primary afferents. Numerous small GAL-IR cell bodies were encountered in laminae II and III. GAL-IR cell bodies were also observed in lamina X. The dorsal root ganglia contained a low but consistent number of small to medium-sized GAL-IR cell bodies, which all contained immunoreactive calcitonin gene-related peptide (CGRP). Following peripheral sciatic nerve transection, the number and the labeling intensity of GAL-IR cell bodies in the corresponding dorsal root ganglia showed a moderate increase. Radioimmunoassay revealed that the concentration of GAL-LI increased along the rostrocaudal axis of the normal spinal cord, and was about three times higher in the dorsal than in the ventral regions. The concentration in the dorsal root ganglia was intermediate to those seen in the corresponding dorsal and ventral cord regions.(ABSTRACT TRUNCATED AT 400 WORDS)     

The centrifugal visual system of a palaeognathous bird,the Chilean Tinamou (Nothoprocta perdicaria)

The avian centrifugal visual system, which projects from the brain to the retina, has been intensively studied in several Neognathous birds that have a distinct isthmo‐optic nucleus (ION). However, birds of the order Palaeognathae seem to lack a proper ION in histologically stained brain sections. We had previously reported in the palaeognathous Chilean Tinamou (Nothoprocta perdicaria) that intraocular injections of Cholera Toxin B subunit retrogradely label a considerable number of neurons, which form a diffuse isthmo‐optic complex (IOC). In order to better understand how this IOC‐based centrifugal visual system is organized, we have studied its major components by means of in vivo and in vitro tracing experiments. Our results show that the IOC, though structurally less organized than an ION, possesses a dense core region consisting of multipolar neurons. It receives afferents from neurons in L10a of the optic tectum, which are distributed with a wider interneuronal spacing than in Neognathae. The tecto‐IOC terminals are delicate and divergent, unlike the prominent convergent tecto‐ION terminals in Neognathae. The centrifugal IOC terminals in the retina are exclusively divergent, resembling the terminals from “ectopic” centrifugal neurons in Neognathae. We conclude that the Tinamou's IOC participates in a comparable general IOC‐retina‐TeO‐IOC circuitry as the neognathous ION. However, the connections between the components are structurally different and their divergent character suggests a lower spatial resolution. Our findings call for further comparative studies in a broad range of species for advancing our understanding of the evolution, plasticity and functional roles of the avian centrifugal visual system.     

Serotonin-containing projections to the thalamus in the rat revealed by a horseradish peroxidase and peroxidase antiperoxidase double-staining technique

The retrograde transport of horseradish peroxidase (HRP) has been used in combination with peroxidase antiperoxidase (PAP) immunocytochemistry in order to investigate serotonin-containing projections to the thalamus of the rat. Sections were histochemically stained to reveal retrogradely transported HRP and then PAP immunostained using a monoclonal anti-serotonin (5-HT) antibody. Following HRP injections into the ventral thalamus, retrogradely labelled cells were observed in a number of sites in the brainstem and including areas known to be rich in 5-HT-containing neurons. At rostral levels of the dorsal raphe nucleus, retrogradely labelled cells were observed both on the midline and in a distinct lateral group extending diffusely into the periaqueductal gray (PAG). In both of these areas many 5-HT-immunoreactive HRP retrogradely labelled neurons were observed. However, except for the most rostral levels of the dorsal raphe nucleus, such double-labelled cells represented only a small proportion of the total population of 5-HT-immunoreactive neurons. In the lateral group, the retrograde labelling was mainly unilateral to the injection site but some contralateral labelling was also seen. At caudal levels of the dorsal raphe nucleus, retrogradely labelled cells were observed predominantly in the lateral group. At the level of the dorsolateral tegmental nucleus, few 5-HT of 5-HT/HRP labelled cells were observed in the lateral group, although HRP retrogradely labelled neurons were present. Double-stained cells were detected also in the medial raphe nucleus (corresponding to the B8 cell group according to the nomenclature of Dahlström and Fuxe¹³), among the fibres of the medial lemniscus (B9), and in nucleus raphe pontis (B5).     

Cytoarchitecture of the nucleus raphe dorsalis of the rat

The nucleus raphe dorsalis of the rat was investigated by means of the Golgi rapid impregnation technique: a) Type 1 neurons: polygonal neurons with somatic spines. The axons which course towards the ventral tegmental area, there by giving off a few collaterals. b( Type 2 neurons: fusiform neurons the axons of which course in a latero-dorsal direction, emitting a few collaterals, too. c) Type 3 neurons: small pyriform neurons. The axons of these cells do not show any favoured route within the nucleus. The type 1 and type 2 neurons are considered to be efferent neurons having different modes of projection and termination. The type 1 neurons are supposed to represent the 5-HT ergic raphe dorsalis neurons. The type 3 neurons, are in probability, raphe interneurons. The findings presented here are indicative of the nucleus raphe dorsalis of the rat to have a more intricate cytoarchitecture as has been thought previously.     

Serotoninergic axonal contacts on identified cat spinal dorsal horn neurons and their correlation with nucleus raphe magnus stimulation

This study examined the distribution of serotoninergic (5-HT) immunoreactive axonal contacts on spinal laminae I and II neurons by combining the intracellular horseradish peroxidase (HRP) method with immunocytochemistry. In addition, the 5-HT distribution was correlated with effects produced by electrical stimulation within the nucleus raphe magnus (NRM). Responses of lamina I neurons and lamina II stalked cells to noxious stimulation were markedly suppressed during NRM stimulation. In contrast, responses of nociceptive lamina IIa islet or non-nociceptive lamina IIb islet cells remained unchanged during nucleus raphe magnus stimulation. These inhibitory influences were positively correlated with the distribution of 5-HT immunoreactive contacts on these neurons. Nociceptive lamina I neurons and lamina II stalked cells received a significantly greater number of contacts (average of 74 and 63, respectively) than either nociceptive lamina IIa islet or non-nociceptive lamina IIb islet cells (average of 25 and eight contacts, respectively). Irrespective of cell type, most 5-HT contacts occurred on dendritic shafts rather than spines. These data reveal a differential distribution of 5-HT contacts on neurons in spinal laminae I and II, and indicate that at least a portion of the NRM modulation of dorsal horn neuronal activity is serotoninergic and concentrated on the dendritic shafts of nociceptive lamina I neurons and lamina II stalked cells.