Serotoninergic Axons Research Articles

Immunohistochemical evidence of serotoninergic regulation of vasoactive intestinal polypeptide (VIP) in the rat suprachiasmatic nucleus

By applying a double-immunolabeling technique to preembedded tissue preparations, we demonstrated the existence of serotoninergic innervation to neurons containing vasoactive intestinal polypeptide (VIP) in the rat suprachiasmatic nucleus (SCN). Immunoreactivity for serotonin and VIP was revealed by the presence of diaminobenzidine (DAB) reaction products and silver-intensified DAB reaction products, respectively; in a further stage, the silver grains were substituted with gold particles. DAB reaction products were precipitated on the surface of vesicular structures, while gold particles were scattered diffusely throughout the neuroplasma at various densities. Serotoninergic axons were numerous and closely packed together, occasionally forming synaptic junctions with gold-labeled VIP-containing neurons. At these synaptic junctions, small vesicular structures accumulated to form a coat under the presynaptic membrane, and the postsynaptic membrane was lined with a homogeneous accumulation of fine deposits. This postsynaptic apparatus varied in appearance; some parts were flat and thin, while others were of irregular thickness. Serotoninergic fibers also formed synaptic junctions with unidentified neurons, in which postsynaptic membrane specialization was also observable. As VIP-containing neurons are known to be synapsed by somatostatin (SRIH)-containing neurons, their regulation must involve both serotonin and SRIH at least.

The serotoninergic innervation of cerebral cortex: Different classes of axon terminals arise from dorsal and median raphe nuclei

The objective of the present study was to characterize the morphology of serotoninergic axons in cerebral cortex of the rat and to determine whether dissimilar axon terminals arise from the dorsal vs. the median raphe nuclei. The anterograde tracer PHA-L was administered by iontophoresis into the dorsal (DR) and median (MR) raphe nuclei, and the morphologic features of the respective axonal projections from raphe to forebrain were analyzed. We have observed consistent structural differences between the axons from these two nuclei. Anterogradely labeled axons which arise from cells in the MR are characterized by large, spherical varicosities (type M axons) and by variations in axonal diameter. In contrast, DR fibers are very fine and typically have small, pleomorphic varicosities that are granular or fusiform in shape (type D axons). Similar features of serotonin (5-HT) axon morphology are also evident in 5-HT immunocytochemical preparations. In addition to structural differences, there is a differential topographic distribution of MR vs. DR fibers, with MR axons concentrated in particular areas of limbic cortex such as dentate gyrus, posterior cingulate, and entorhinal areas as well as in parietal cortex. Immunofluorescence with dual labels shows that over two-thirds of the raphe-cortical axons are serotoninergic. The dissimilarities in axon morphology indicate that individual raphe nuclei may form different patterns of synaptic organization. Based on the evidence that the dorsal and median raphe nuclei give rise to morphologically different axon terminals, we conclude that 5-HT axons in cortex may be subdivided into two distinct projections. This proposal is in accord with other, recent data showing that the two axon types have different pharmacologic properties and are likely to be functionally different.

Synaptic connections between serotoninergic axon terminals and tyrosine hydroxylase-immunoreactive neurons in the arcuate nucleus of the rat hypothalamus. A combination of electron microscopic autoradiography and immunocytochemistry

A combination of electron microscopic autoradiography and immunocytochemistry was used to examine the connections between serotoninergic axons and dopaminergic neurons of the arcuate nucleus of the rat hypothalamus. The serotoninergic elements were identified after selective uptake of tritiated serotonin and the dopaminergic neurons with tyrosine hydroxylase cytochemistry. Synaptic junctions between labeled serotoninergic nerve endings and tyrosine hydroxylase-immunoreactive neurons were observed, providing a structural basis for a direct influence of serotoninergic elements on tuberoinfundibular dopaminergic neurons.

An immunohistochemical study of serotonin development in the opossum cerebellum.

In the present study we have used the indirect antibody peroxidase anti-peroxidase technique to analyze the development of serotonin in the cerebellum of pouch young opossums ranging in age from birth (postnatal day (PD) 1) - to PD 47. The pathways by which serotoninergic axons enter the cerebellum appear to change during development. Between PD 1 and PD 11 varicose serotoninergic fibers course though the continuity between the tectum and the dorsal medial aspect of the cerebellar plate and distribute primarily to the intermediate zone. By PD 11 serotoninergic fibers enter the cerebellum via a connecting band between the cerebellum and the dorsal lateral aspect of the rostral medulla. Fibers entering the cerebellum via this later route course around the lateral recess of the fourth ventricle and initially distribute to ventral and lateral areas of the cerebellum. At later developmental ages (PD 14-PD 47) serotoninergic fibers are present in: 1) the cellular zone of migration between the Purkinje cell layer and deep nuclei; 2) the Purkinje cell layer; 3) the internal granule cell layer; and 4) the deep cerebellar nuclei. The external granule cell and the molecular layers rarely contain serotoninergic fibers. The present study has shown that serotonin is present in the cerebellar anlage on PD 1 (within 13 days of conception). This is prior to the arrival of other major afferent systems (King et al. 1982; Bishop et al. 1983; Martin et al. 1983; Morgan et al. 1983). Further, serotoninergic axons reach the cerebellum via different routes at different stages of development. The presence of serotonin in the intermediate zone early in development supports the hypothesis that it may influence neuronal migration and differentiation (Lauder and Krebs 1976). Axons containing serotonin and arriving later in development may function in synaptic transmission, a role proposed for this indoleamine in the adult (Strahlendorf et al. 1979).

Demonstration of serotoninergic axons terminating on luteinizing hormone-releasing hormone neurons in the preoptic area of the rat using a combination of immunocytochemistry and high resolution autoradiography

The synaptic relationship between serotoninergic terminals and luteinizing hormone-releasing hormone-containing neurons was investigated in the medial preoptic area using a combined technique. Axon terminals selectively taking up 5-[ 3H]hydroxytryptamine were labelled autoradiographically and luteinizing hormone-releasing hormone-containing neuronal elements were identified by means of immunocytochemistry. Synaptic contacts were observed between tritiated 5-hydroxytryptamine-labelled boutons and luteinizing hormone-releasing hormone-immunoreactive dendrites. About 5% of the boutons which formed synapses with luteinizing hormone-releasing hormone-immunoreactive dendrites were found to be labelled by the tritiated indolamine. Luteinizing hormone-releasing hormone-immunoreactive axon terminals occurred as presynaptic elements in contact with unidentified dendritic spines, shafts or perikarya. These observations provide morphological basis for the idea that 5-hydroxytryptamine-containing neurons can act directly on luteinizing hormone-releasing hormone release. Further, they support the assumption that luteinizing hormone-releasing hormone is not only a neurohormone but may also function as a neurotransmitter or neuromodulator.

Perivascular serotoninergic neurons: Somatodendritic contacts and axonic innervation of blood vessels

As shown by histofluorescence studies, a number of brain stem serotoninergic neurons have their cell bodies and dendrites in intimate contact with parenchymal blood vessels, including arterioles (perivascular serotoninergic neurons). Another, apparently distinct, phenomenon which was evidenced is the existence of a diffuse innervation by long serotoninergic axons of intrinsic blood vessels. These axons seem to be continuous with an adventitial nerve fiber network surrounding extrinsic blood vessels. A novel significant finding was that some of the perivascular neurons themselves can project an axonic process into the wall of the same vessel they embrace with their cell body and dendrites (a receptive as well as a vasomotor function?).

Distribution of 5-hydroxytryptamine (serotonin) in the brain of the teleost Gasterosteus aculeatus L.

The distributions of serotoninergic neurons in the brain of the three-spined stickleback was demonstrated with the indirect peroxidase-antiperoxidase (PAP) immunohistochemical method with antibodies against serotonin. Serotoninergic perikarya were demonstrated in the brainstem reticular formation (nucleus raphe dorsalis, nucleus raphe medialis, and nucleus tegmenti dorsalis lateralis) and in the periventricular ventral thalamus and hypothalamus (nucleus ventromedialis thalami, nucleus posterioris periventricularis, nucleus recessus lateralis, and nucleus recessus posterioris). After pharmacological pretreatment of the animals with a monoamine oxidase inhibitor, serotoninergic perikarya were also visualized in area praetectalis and in the medial brainstem, caudal to nucleus raphe medialis. Whereas the cell groups of the brainstem give rise to both ascending and descending pathways, it was not possible to analyze the distribution of efferent projections from the diencephalic cell groups. Distribution of serotoninergic axons showed marked regional differences. Only scattered varicose fibers were demonstrated in the cerebellum, the facial lobes, and the lateral line lobes. In the mesencephalon, the dorsal periventricular tegmentum and the central gray receive only small numbers of serotoninergic axons, while torus semicircularis and the visual layers of tectum opticum are profusely innervated. In the diencephalon, the hypothalamus and ventral thalamus generally display the highest density of serotoninergic axons. Exceptions are found in nucleus glomerulosus and the ventromedial portion of lobus inferioris, where densities are low. In the telencephalon, the density of serotoninergic axons is very high in area dorsalis pars medialis and pars lateralis dorsalis, but low in area dorsalis pars dorsalis and pars lateralis ventralis, and intermediate in area ventralis.

Different roles of catecholaminergic and serotoninergic neurons of the medial forebrain bundle on male rat sexual behavior

The medial forebrain bundle (MFB) plays an important role in the central elaboration of male sexual behavior. However the role of its ascending monoaminergic systems is not well understood. By selective lesions of catecholaminergic and serotoninergic axons of the MFB, produced with 6-OHDA and 5-7-DHT respectively, its possible involvement in male sexual behavior was studied. The evaluation of the behavioral patterns was studied by means of a microcomputer which also permitted the quantification of sequences in the sexual behavior patterns. Lesions with 5–7 DHT increased the intromission-ejaculation sequences and the percentage of ejaculatory males, without modifying other behavioral tests. Lesions with 6-OHDA diminished the number of “active” sexual patterns and motor activity. In order to check the specificity of the chemical lesions, the in vitro uptake of labeled norepinephrine and serotonin was measured, 5–7 DHT lesioned rats show a reduction in the uptake of 5-HT-C 14 in the MPOA while 6-OHDA group show a reduction of NA-H 3 in the striatum.

Ultrastructure of chemically defined neuron systems in the dorsal horn of the monkey. III. Serotonin immunoreactivity

The ultrastructural organization of serotoninergic axons and terminals in the superficial dorsal horn of the monkey was examined by the PAP immunohistochemical method. Terminals with serotonin-like immunoreactivity (SLI) were identified in lamina I (marginal zone) and lamina II o (outer substantia gelatinosa). Labelled profiles contained many small, round, clear vesicles and usually a few granular vesicles (70 nm diameter). Most synaptic junctions were symmetrical with sparse pre- and post-synaptic densities. Most frequently, terminals formed axodendritic synapses on large and small dendrites; axosomatic and axospinous contacts were infrequent. In addition SLI terminals were found apposed to unlabelled LGV-type terminals (containing several large granular vesicles of 75–90 nm). The appositions commonly met some criteria of axo-axonic synapses and the SLI terminal was suspected to be presynaptic. The unlabelled LGV terminal was often presynaptic to a dendrite, and it had characteristics similar to those observed for some primary afferents, particularly those which may contain substance P, a proposed transmitter for nociceptive C-fibers. Most of these ‘triplet’ complexes (SLI terminal apposing and LGV terminal synapsing onto dendrite) were found in the apical region of lamina I. The axodendritic and axosomatic serotoninergic contacts onto dorsal horn neurons may be a basis for some of the reported post-synaptic effects on dorsal horn cells of either local serotonin iontophoresis or of stimulation of the brainstem raphe, the probable origin of the serotoninergic terminals. These effects include both depression and excitation of the responses of the dorsal horn cells to electrical or natural stimulation of primary afferents, particularly C-fibers and nociceptors. Likewise, the contacts of SLI terminals with LGV terminals may provide a morphological substrate for the presynaptic effects also observed for serotonin iontophoresis or raphe stimulation, including changes in the excitability of primary afferent C-fibers.

Deglutition.

Deglutition.

Serotoninergic axon terminals in the rat dorsal accessory olive: normal ultrastructure and light microscopic demonstration of regeneration after 5,6-dihydroxytryptamine lesioning.

The serotoninergic innervation of the lateral portion of the dorsal accessory nucleus of inferior olive (DAO) was studied using high resolution radioautography after intraventricular or intracisternal administration of tritiated serotonin ([3H]5-HT). In normal adult rats, the axonal varicosities labelled with [3H]5-HT were of round-elongate shape and averaged 0.6–1.0μm in diameter. They contained microcanaliculi (15–25 nm in diameter), tubular-vesicular elements (25–40 nm) and large granular vesicules (80 nm), as well as mitochondria and smooth endoplasmic reticulum. Among 750 thin-sectional profiles of labelled varicosities, only 5% showed a differentiated area of membrane specialization which suggests that not more than one out of 7–8 varicosities was engaged in a synaptic junction. This serotoninergic innervation was therefore categorized as non-junctional. There were nevertheless certain structural features suggestive of polarity even in the ‘non-synaptic’ serotoninergic varicosities of the lateral DAO: many labelled profiles exhibited aggregation of their microcanalicular and tubular-vesicular organelles against a part of their membrane apposed to a dendritic process, while the remainder of their periphery was usually surrounded by glia. These observations reinforced the supposition that non-junctional as well as junctional serotoninergic varicosities might release transmitter.

Mechanisms of regrowth in the bulbospinal serotonin system following 5,6-dihydroxytryptamine induced axotomy. II. Fluorescence histochemical observations

The degenerative and regenerative processes following 75 μg 5,6-dihydroxytryptamine (5,6-DHT) have been investigated in the bulbospinal serotoninergic system with a fluorescence histochemical technique. Acutely, 1–3 weeks after 5,6-DHT an extensive disappearance of serotoninergic terminals was observed in the spinal gray, inferior olive, and the facial and hypoglossal nuclei, while the spinal trigeminal nucleus and regions of the reticular formation were partially denervated. Concomitantly, large numbers of swollen, highly fluorescent profiles, interpreted as the proximal stumps of lesioned serotoninergic axons, appeared in the medulla oblongata and most rostral cervical spinal cord. The first signs of regenerative sprouting from the swollen proximal stumps appeared one week after 5,6-DHT, and was prominent during subsequent weeks. The rate of reinnervation of different target areas was dependent on the distance to the regrowing axons; it was most rapid in brain stem nuclei. About one month after 5,6-DHT a terminal plexus of normal density had reappeared in both inferior olive and the facial nucleus. The axonal proliferation continued, however, to produce increasingly abnormal hyperinnervations. Certain abnormalities in terminal fiber morphology occurred: the fibers were coarser and exhibited higher fluorescence than normal, and in the facial and motor trigeminal nuclei rather thick, smooth fibers with pericellular arrangement appeared after long survival times (7–19 months). The reinnervation of spinal cord followed a slower time course. A normal amount of innervation was only reformed in the rostral 3–4 mm of the cervical spinal cord, while thoracic and lumbar levels only recovered a small part of the original terminal density. The regenerative growth demonstrated a high degree of specificity, which indicates precise mechanisms of guidance and recognition. These are presumably similar, or identical, to those regulating ontogenetic development of the serotoninergic system. Limitations of these regulatory mechanisms were, however, demonstrated both by the abnormal proximo-distal distribution of regenerated terminals, and in the inferior olivary complex, where the regrowing fibers invaded areas normally devoid of serotoninergic innervation. It is suggested that the drive of axonal growth is derived from an inherent program of the serotoninergic neurons to establish a predetermined amount of axonal arborizations. The role of the target tissue may be a mere provision of cues for recognition. The regulatory neuronal program seems to lack a mechanism to terminate further proliferation when an innervation of normal density has been reformed.

The indolaminergic innervation of the inferior olive. 2. Relation to harmaline induced tremor

The possible involvement of serotoninergic mechanisms in the induction of harmaline generated tremor in the inferior olive has been investigated electrophysiologically in the cat and rat. Mass recordings of Purkinje cell activity in the cat showed that harmaline induces strong, synchronous and rhythmic activity in those parts of the climbing fibre system originating in the caudal part of the medial accessory olive and the caudolateral parts of the dorsal accessory nucleus. These are the areas of the cat olive shown to receive a dense serotoninergic innervation. In the rat, the selective removal of the serotoninergic innervation — produced by an intraventricular injection of 5,6-dihydroxytryptamine, or 5,7-dihydroxytryptamine in combination with desipramine — caused a significant attenuation of both the tremor and the climbing fibre activity induced by an intravenous harmaline injection. In the 5,6-dihydroxytryptamine-treated animals the reappearance of the harmaline tremor seemed to parallel the regrowth of new serotoninergic axon sprouts in the inferior olive. On the basis of the present results it is proposed that the serotoninergic afferents to the accessory olivary nuclei are of critical importance for the tremor induction of harmaline in the inferior olive. It is suggested that harmaline, rather than acting directly on the olivary neurones, exerts its effect through an interference with a serotoninergic (possibly inhibitory) innervation of these cells.

Stereology: applications to biomedicalresearch.

Stereology: applications to biomedicalresearch.