Serotonin-immunoreactive Varicosities Research Articles

Adolescent cocaine exposure and offensive aggression: involvement of serotonin neural signaling and innervation in male Syrian hamsters

Repeated low-dose cocaine treatment (0.5 mg/kg/day) during adolescence facilitates offensive aggression in male Syrian hamsters ( Mesocricetus auratus). The current study assessed whether adolescent cocaine-facilitated offensive aggression was inhibited by increased serotonin activity and if cocaine exposure during this developmental period influenced serotonin development in the primary aggression areas of hamster brain. In a first experiment, hamsters were treated with low doses of cocaine throughout adolescence and then scored for offensive aggression following the systemic administration of vehicle or fluoxetine, a selective serotonin reuptake inhibitor. Vehicle-treated hamsters showed high levels of offensive aggression, while treatment with fluoxetine inhibited the cocaine-facilitated aggressive response. Only one out of ten fluoxetine-treated animals both attacked and bit intruders, compared to nine out of ten saline-treated animals. In a second experiment, hamsters were administered low doses of cocaine or saline throughout adolescence, tested for offensive aggression, and then examined for differences in serotonin afferent innervation to regions of the hamster brain implicated in aggressive responding. Aggressive cocaine-treated hamsters showed significant reductions (35–50%) in the number of serotonin immunoreactive varicosities and fibers in several aggression areas, including the anterior hypothalamus, lateral septum, medial amygdala, and bed nucleus of the stria terminalis. Together, these results support a role for serotonin innervation and function in adolescent cocaine-facilitated offensive aggression.

Apposition of enkephalin- and neurotensin-immunoreactive neurons by serotonin-immunoreactive varicosities in the rat spinal cord

The descending serotonergic system provides a powerful inhibitory input to the dorsal horn of the spinal cord. Little is known about the chemical identity of the spinal neurons that the serotonergic system innervates, although spinal enkephalinergic neurons are likely candidates. This study investigated the apposition of serotonin-immunoreactive varicosities onto enkephalin- and neurotensin-immunoreactive neurons in the rat lumbosacral spinal cord. Using a double immunofluorescence technique, serotonin-immunoreactive varicosities were observed to abut the soma or proximal dendrites of [Met]enkephalin- and neurotensin-immunoreactive neurons. Nearly 75% of all [Met]enkephalin- and neurotensin-immunoreactive neurons were apposed by serotonin-immunoreactive varicosities in the marginal zone and dorsal gray commissure. In substantia gelatinosa, approximately half of the [Met]enkephalin- and neurotensin-immunoreactive neurons were juxtaposed by serotonin-immunoreactive varicosities. [Met]enkephalin-immunoreactive neurons also were bordered by serotonin-immunoreactive varicosities in the nucleus proprius (65%) and sacral parasympathetic nucleus (75%). The results of this study suggest that the descending serotonergic system mediates nociception via probable contacts with intrinsic enkephalin and neurotensin spinal systems. The mode of action of spinal serotonin on enkephalin and neurotensin neurons may be through “volume” transmission vs synaptic or “wiring” transmission.

Behavioral and Neurobiological Consequences of Social Subjugation during Puberty in Golden Hamsters

In golden hamsters, offensive aggression is facilitated by vasopressin and inhibited by serotonin. We tested whether these neurotransmitter systems respond to modifications resulting from the stress of threat and attack (i.e., social subjugation) during puberty. Male golden hamsters were weaned at postnatal day 25 (P25), exposed daily to aggressive adults from P28 to P42, and tested for offensive aggression as young adults (P45). The results showed a context-dependent alteration in aggressive behavior. Subjugated animals were more likely to attack younger and weaker intruders than nonsubjugated controls. Conversely, subjugated animals were less likely to attack animals of similar size and age. After testing, the animals were killed, and their brains were collected to determine whether these behavioral changes are underlined by changes in the vasopressin and serotonin systems. Social subjugation resulted in a 50% decrease in vasopressin levels within the anterior hypothalamus, a site involved in the regulation of aggression. Furthermore, whereas the density of vasopressin-immunoreactive fibers within the area was not significantly altered in subjugated animals, the number of serotonin-immunoreactive varicosities within the anterior hypothalamus and lateral septum was 20% higher in subjugated animals than in their controls. These results establish puberty as a developmental period sensitive to environmental stressors. Furthermore, the results show that changes in the vasopressin and serotonin systems can correlate with behavioral alterations, supporting the role of these two neurotransmitters in the regulation of aggression.

Quantitative and morphometric data indicate precise cellular interactions between serotonin terminals and postsynaptic targets in rat substantia nigra

We have quantified the density of serotonin axonal varicosities, their synaptic incidence and their distribution among potential targets in the pars reticulata and pars compacta of the rat substantia nigra. Serotonin axonal varicosities, counted at the light microscopic level following in vitro [3H]serotonin uptake and autoradiography, amounted to 9×106/mm3 in the pars reticulata and 6×106/mm3 in the pars compacta, among the densest serotonin innervations in brain. As determined at the electron microscopic level following immunolabelling for serotonin, virtually all serotonin varicosities in the pars reticulata and 50% of those in the pars compacta formed a synapse, essentially with dendrites. The combination of serotonin immunocytochemistry with tyrosine hydroxylase immunolabelling of dopamine neurons reveals that 20% of the serotonin synaptic contacts in the pars reticulata are on dopamine dendrites and 6% are on a type of unlabelled dendrite characterized by its peculiarly high cytoplasmic content of microtubules. The comparison of the diameter of the dendritic profiles that were in synaptic contact with serotonin-immunoreactive varicosities with the diameter of all other dendritic profiles of the same type suggests that serotoninergic varicosities innervate dopamine dendrites uniformly along their length, whereas they tend to contact microtubule-filled dendrites in more proximal regions and the other, unidentified dendrites in more distal regions. Furthermore, the size of the serotonin-immunoreactive varicosities and of their synaptic junctions is significantly smaller on dopamine dendrites and larger on microtubule-filled dendrites than on other, unidentified dendrites, indicating that the nature of the postsynaptic target is an important determinant of synaptic dimensions.These data should help to clarify the role of serotonin in the nigral control of motor functions. They indicate that this dense serotonin input to the substantia nigra is very precisely organized, acting through both “non-junctional” and “junctional” modes of neurotransmission in the pars compacta, which projects to the neostriatum and the limbic system, whereas the predominant mode of serotonin transmission appears to be of the “junctional” type in the pars reticulata, where serotonin can finely control the motor output of the basal ganglia by acting on the GABA projection neurons either directly or through the local release of dopamine by dopaminergic dendrites. The data also raise the possibility that the postsynaptic targets have trophic retrograde influences on serotoninergic terminals.

Serotoninergic innervation of nonprincipal cells in the cerebral cortex of the lizard Podarcis hispanica.

The mechanism of serotoninergic transmission in the neo- and archicortex of mammals is complex, including both synaptic and nonsynaptic components, direct actions on principal cells, and indirect effects mediated by GABAergic interneurons. Here we studied the termination pattern and synaptic organization of the serotoninergic afferents in the cerebral cortex of the lizard, Podarcis hispanica, which is considered to correspond in part to the mammalian hippocampal formation, with the aim of unraveling basic, phylogenetically preserved rules in the connectivity of this pathway. We demonstrate that serotoninergic afferents, visualized by immunostaining for serotonin itself, establish multiple synaptic contacts with different subpopulations of nonprincipal cells containing parvalbumin, neuropeptide Y, and opioid peptides. The former two subpopulations contain GABA, whereas the opioid-immunoreactive neurons are most likely GABA-negative cells. Evidence is provided at the electron microscopic level that serotonin-immunoreactive varicosities establish conventional asymmetric synaptic contacts with their nonprincipal targets, but nonsynaptic varicosities also exist. We conclude that, similarly to mammals, a selective synaptic innervation of nonprincipal, possibly inhibitory, neurons is among the mechanisms of serotoninergic modulation of cerebral cortical activity in the lizard.

Organization of the serotonergic innervation of spinal neurons in rats—III. Differential serotonergic innervation of somatic and parasympathetic preganglionic motoneurons as determined by patterns of co-existing peptides

The spinal cord is innervated by brainstem serotonergic neurons, some of which contain substance P and/or thyrotropin-releasing hormone in addition to serotonin. These neurons project at least three types of axons to the spinal cord: those containing both substance P and thyrotropin-releasing hormone, those containing thyrotropin-releasing hormone but not substance P, and those containing neither substance P nor thyrotropin-releasing hormone. However, the organization of the different types of serotonergic processes is unclear. In the present studies, the types of serotonergic axons projecting to two kinds of spinal neurons were examined. Somatic and parasympathetic preganglionic motoneurons were labeled retrogradely from the pelvic or sciatic nerve, respectively. Sections containing these neurons were stained either for serotonin and substance P, or for serotonin and thyrotropin-releasing hormone. Of a total of 428 profiles examined that were retrogradely labeled from the sciatic nerve, 425 (99%) were apposed by serotonin-immunoreactive varicosities; similarly, of a total of 382 profiles examined that were retrogradely labeled from pelvic nerve, 353 (92%) were apposed by serotonin-immunoreactive varicosities. However, differences appeared to exist between the types of serotonergic varicosities innervating these two groups of neurons. Among the profiles labeled from the sciatic nerve, it was estimated that over 97% were apposed by serotonin-immunoreactive varicosities in which serotonin co-existed with substance P and thyrotropin-releasing hormone. In contrast, among the profiles labeled from pelvic nerve that were apposed by serotonin-immunoreactive varicosities, it was estimated that less than 1% were apposed by serotonin-immunoreactive varicosities containing both thyrotropin-releasing hormone and substance P. We estimate that most of the remainder (about 80%) were apposed by serotonin-immunoreactive varicosities containing thyrotropin-releasing hormone but not substance P. We conclude that both the cell bodies of neurons retrogradely labeled from the pelvic nerve and those labeled from the sciatic nerve were apposed by serotonin varicosities. However, these two systems of neurons appear to be innervated largely by two different populations of serotonergic cells. This suggests that the raphe-spinal serotonergic system may independently modulate the activities of somatic motoneurons and parasympathetic preganglionic motoneurons.

Organization of the serotonergic innervation of spinal neurons in rats—I. Neuropeptide coexistence in varicosities innervating some spinothalamic tract neurons but not in those innervating postsynaptic dorsal column neurons

Previous studies have suggested that peptides such as substance P and thyrotropin-releasing hormone coexist with serotonin in the same varicosities in the ventral horn and intermediate gray of the spinal cord in rat. However, coexistence of these peptides with serotonin is rare in fibers in the superficial dorsal horn. Since it has been proposed that serotonergic fibers in the superficial dorsal horn act to modulate nociception, it was hypothesized that the serotonergic neurons that contain neither substance P nor thyrotropin-releasing hormone might constitute a specifically antinociceptive subset of serotonergic neurons. This being the case, it would be expected that different types of serotonergic neurons innervate nociceptive and non-nociceptive spinal neurons. In order to test this hypothesis, a group of cells that include nociceptive neurons (spinothalamic tract neurons) and a group of predominantly non-nociceptive neurons (postsynaptic dorsal column neurons) in the spinal cord of rat were retrogradely labeled. Sections of the spinal cord containing retrogradely labeled spinothalamic tract or postsynaptic dorsal column neurons were stained for serotonin and either substance P or thyrotropin-releasing hormone using two-color immunohistochemistry. A retrogradely labeled cell was classified as “apposed” if there was no discernible distance between an immunohistochemically labeled varicosity and the cell. Eighty per cent of spinothalamic tract and 83% of postsynaptic dorsal column profiles were apposed by serotonin-immunoreactive varicosities in the spinal cord. Thirty-one per cent of the spinothalamic tract profiles that were apposed by serotonergic varicosities were apposed by serotonergic varicosities that were also stained for thyrotropin-releasing hormone. The distribution of the latter spinothalamic neurons was similar to that reported for spinothalamic tract neurons responsive to joint movement. In addition, at least 63% of the spinothalamic tract profiles which were apposed by serotonergic varicosities were apposed by “serotonin-only” varicosities, including most spinothalamic tract neurons in the marginal zone, suggesting that at least some “serotonin-only” neurons are antinociceptive. However, contrary to the hypothesis, at least 94% of the postsynaptic dorsal column profiles apposed by serotonergic varicosities were apposed by “serotonin-only” varicosities. These findings suggest that there may be a relationship between the sensory modality to which a spinal neuron responds and the type of serotonergic innervation it receives. However, it appears that “serotonin-only” neurons may not constitute a specifically antinociceptive category of serotonergic neurons.

Enkephalin, substance P, and serotonin axonal input to c-fos-like immunoreactive neurons of the rat spinal cord

Mustard oil, which stimulates small diameter afferents, was used to evoke the expression of the oncogene c-fos in the lumbar spinal cord. C-fos-like immunoreactivity was concentrated in, but not limited to, neuronal nuclei of laminae I and II of the lumbar dorsal horn. Double-label immunocytochemistry was used to determine if neurons which expressed c-fos-like immunoreactivity received axonal input from enkephalin-, substance P- or serotonin-immunoreactive neurons. The analysis of vibratome and semithin plastic-embedded tissue sections demonstrated that the majority of c-fos-like immunoreactive neurons received input from enkephalin-, substance P- or serotonin-immunoreactive axonal varicosities.

Serotoninergic varicosities make synaptic contacts with pleural sensory neurons of Aplysia

Serotonin is a modulatory neurotransmitter that produces many of the cellular changes associated with sensitization of reflexes in Aplysia. These changes have been carefully documented in sensory neurons located in the abdominal ganglion that mediate the gill-siphon withdrawal reflex and in sensory neurons located in the pleural ganglion that mediate the tail-siphon withdrawal reflex. Although serotonin appears to be necessary for sensitization, there is no direct evidence that serotoninergic neurons make synaptic contacts with sensory neurons. In this study, the immunoperoxidase technique was used to label serotonin-immunoreactive neurites surrounding the cell bodies of sensory neurons in the pleural ganglion. Serotonin-immunoreactive neurites had varicosities whose mean short axis diameter was 1.1 +/- 0.6 microns (mean +/- S.D.). The shape of the size distribution was skewed toward larger sizes, however, suggesting that there were multiple subpopulations of varicosities. One subpopulation was that of varicosities located at branch points whose average short axis diameter was larger than normal (1.7 +/- 0.5 microns). Serotonin-immunoreactive varicosities were directly apposed to the sensory neurons without intervening glial cells. In most contacts, serotonin-immunoreactive neurites invaginated into the plasma membranes of the sensory neurons. There were also a few contacts onto spinelike processes, but these were flat rather than invaginated. Serotoninergic neurons whose activity produces changes in the electrophysiological properties of sensory neurons have been identified, but this study provides the first direct evidence for synaptic connections between serotoninergic neurons and sensory neurons in Aplysia.

Serotonin-immunoreactive varicosities in the cell body region and neural sheath of the snail, Helix pomatia, ganglia: An electron microscopic immunocytochemical study

The distribution and connections of serotonin-immunoreactive fibers in the cell body region and neural sheath of the central ganglia of the snail, Helix pomatia, have been examined. The cell body region of the ganglia is supplied by an extremely dense network of varicose serotonin-immunoreactive fibers which surround neuronal perikarya in the ganglia. Immunoreactive processes also run to the neural sheath of both the ganglia and the peripheral nerve roots, forming a dense network. Electron microscopy revealed five different connections of serotonin-immunoreactive varicosities, according to their target: (i) non-specialized contacts with neuronal perikarya; (ii) non-specialized contacts with axon processes on the surface of the peripheral nerve roots; (iii) non-specialized neuromuscular connections with smooth muscle fibers in the neural sheath; (iv) varicosities engulfed by glial processes in both the cell body region and neural sheath; (v) varicosities embedded in the connective tissue elements of the sheath either partly or completely free of glial processes. In all cases of appositions no membrane specializations could be observed on either site of the contacts. These observations provide morphological evidence for non-synaptic regulatory actions of serotonin-containing neurons in Helix central nervous system: (i) modulation of the activity of neuronal perikarya; (ii) involvement in neuromuscular regulation; (iii) neurohormonal modulation of peripheral processes by release through the neural sheath.

Quantitative morphometric analysis of two types of serotonin-immunoreactive nerve fibres differentially responding to p-chlorophenylalanine treatment in the rat brain

Two types of serotonin neurons differentially responding to treatment with p-chlorophenyl-alanine, a potent inhibitor of serotonin synthesis, have been demonstrated in the rat brain by immunohistochemistry using a serotonin monoclonal antibody in combination with a quantitative morphometric analysis. In control rats, the numerical density of serotonin-immunoreactive varicosities and terminals varied considerably from region to region. In p-chlorophenylalanine-treated rats, the numerical density was reduced in every region, but the degree of reduction in each region differed markedly. No correlation was seen in values of the numerical density between the control and p-chlorophenylalanine-treated rats, indicating that serotonin-positive axons in p-chlorophenylalaninetreated rats were present independent of the degrees of the axon density seen in the controls. It is postulated, therefore, that there are at least two types of positive axons; one highly sensitive to p-chlorophenylalanine treatment and another relatively resistant to it. Since the numerical density in any region was always smaller in p-chlorophenylalanine-treated rats than in the controls, p-chlorophenylalanine-sensitive axons appeared to be more or less distributed in every region where positive axons were detectable under normal conditions. However, a number of regions were innervated purely by p-chlorophenylalanine-sensitive axons. By contrast, p-chlorophenylalanine-insensitive axons were observed in several definite regions. These regions appeared to be related to the limbic system, or cranial motor and parasympathetic nuclei. In the latter nuclei, p-chlorophenylalanine-insensitive axons seemed to possess such common morphological features as having relatively large varicosities and being situated adjacent to neural somata, suggesting their inhibitory roles in neurotransmission. The present result indicates that the two types of serotonin axons may play important roles in the behavioural events observed in rats following p-chlorophenylalanine treatment.

Synaptic organization of serotonin-immunoreactive fibers in primary visual cortex of the macaque monkey.

The macaque neocortex is very densely innervated by serotonin-containing fibers. The highest density of these fibers is in primary sensory regions such as the primary visual cortex. By using an antibody against serotonin, we analyzed the distribution and morphology of serotonin-immunoreactive fibers and synapses in the primary visual cortex of the adult cynomolgus monkey. In addition, we quantified the laminar distribution of labeled varicosities and the distances between varicosities in single fibers. While serotonin-immunoreactive fibers are found in all cortical layers, at least three bands of heightened density of innervation were readily recognized that were coincident with 1) layer IIIB to IVC alpha, 2) layer VA, and 3) layer VIB. Layer IVC alpha of area 17 contained more varicosities per unit area than any other sublayer. There was a high degree of variability in the intervaricosity distances along single fibers; more than half were longer than 10 microns. At the electron microscopic level, synaptic contacts were also observed throughout the entire thickness of area 17, with the highest frequency in layer IV. The labeled varicosities were packed with electron-lucent synaptic vesicles and formed synaptic complexes with small, but conspicuous, post-synaptic densities. Dendritic shafts were the most common postsynaptic target of the labeled synapses. Among these characteristically slender post-synaptic shafts, profiles with structural features of both spiny and smooth dendrites were observed. The small diameter of most of the postsynaptic dendrites indicated that distal dendrites were preferentially contacted by serotonin-immunoreactive varicosities. Although direct identification of the postsynaptic neurons will be required for complete characterization of this circuitry, the distribution of serotonin-immunoreactive varicosities suggests that serotoninergic interactions in the primary visual cortex of the cynomolgus monkey are directed predominantly at the distal dendrites of granular and infragranular neurons rather than at targets in the supragranular layers.

Immunocytochemical evidence for substance P and serotonin input to medullary bulbospinal adrenergic neurons

Using two-color immunoperoxidase staining combined with the retrograde transport of horseradish peroxidase injected into the rostral thoracic spinal cord, substance P-immunoreactive (SPI) and serotonin-immunoreactive (5HTI) varicosities have been observed in contiguity with medullary bulbospinal phenylethanolamine N-methyl transferase-immunoreactive (PNMTI) neurons of the C1, C2, and C3 cell groups. Since PNMTI terminals in the spinal cord are concentrated among sympathetic preganglionic neurons (SPGN) in the intermediolateral cell column, the close anatomical associations shown in the present study indicate that substance P- and serotonin-containing pathways in the medulla likely affect activity of SPGN via adrenergic bulbospinal neurons.

The organization of serotonin fibers in the mammalian superior colliculus

The distribution of serotonin immunoreactivity in the superior colliculus (SC) of the rat, hamster, chipmunk, cat, and monkey was studied using a sensitive immunohistochemical method. In all of these animals, serotonin immunoreactivity formed a dense network of varicose fibers throughout the SC. These fibers had a characteristic arrangement corresponding to the laminar structures of the SC. Except in the chipmunk, serotonergic fibers were more dense in the stratum griseum superficiale than in the other layers. In the SC of the chipmunk, these fibers appeared evenly distributed. To explore the degree of serotonergic innervation in each layer, a semi-quantitative assay of serotonin immunoreactive varicosities was conducted in the rat, chipmunk, cat, and monkey. Peaks in varicose density were seen in the stratum griseum superficiale, the stratum griseum intermedium and the stratum griseum profundum. In the rat, cat, and monkey, the highest density of these varicosities was in the stratum griseum superficiale. On the other hand, the stratum griseum intermedium of the chipmunk SC received the greatest innervation of serotonergic varicose fibers.

The distribution and cells of origin of serotonergic inputs to the paraventricular and supraoptic nuclei of the rat

The distribution of serotonin-immunoreactive varicosities in the paraventricular (PVH) and supraoptic (SO) nuclei was charted in normal immunohistochemical material and the probable cells of origin of these projections were identified using a combined retrograde transport-immunohistochemical method. The density of serotonergic fibers in the PVH and the SO is quite low relative to that in the immediately surrounding neuropil, in striking contrast to noradrenergic inputs to the nuclei. Immunoreactive fibers are concentrated in specific parts of the parvocellular division of the PVH, whereas in the magnocellular division of the nucleus, and in the SO, they are found mostly in regions where oxytocinergic cells predominate. These projections appear to arise from 3 distinct serotonergic cell groups (B7, B8 and B9) in the midbrain.

The ultrastructural localization of serotonin immunoreactivity within the nucleus of the solitary tract of the cat

Using a modification of the peroxidase-antiperoxidase technique, serotonin immunoreactivity was localized at the ultrastructural level in the nucleus of the solitary tract of the cat. Structures containing serotonin immunoreactivity included unmyelinated axons, varicosities (0.5 to 2 micrometers in diameter), and synaptic terminals. The serotonin-containing synaptic terminals were found less frequently than axons or varicosities. Within unmyelinated axons and varicosities, the immunoreactivity was associated mainly with large granular vesicles (80 to 150 nm). While large granular vesicles were found in all immunoreactive structures, greater numbers were observed in axons and nonsynaptic varicosities. Serial sections of several nonsynaptic serotonin-immunoreactive varicosities indicated the lack of synaptic specializations associated with these structures. In a typical section, only one or two granular vesicles were in synaptic terminals which contained numerous small clear vesicles. Serotonin-immunoreactive terminals formed asymmetrical contacts with dendrites and spines. No synaptic contacts involving immunoreactive terminals were found on cell bodies or other axonal structures. Serotonin-containing neuronal perikarya within the nucleus of the solitary tract were never observed. The abundance of nonsynaptic varicosities containing large granular vesicles suggests a possible neurohumoral role for serotonin within the feline nucleus of the solitary tract. This is discussed in relation to previous reports concerning the paucity of genuine synaptic contacts involving serotonin in other regions of the central nervous system. The presence of serotonin-immunoreactive terminals in the nucleus of the solitary tract also suggests its function as a putative neurotransmitter.