Body sodium overload modulates the firing rate and fos immunoreactivity of serotonergic cells of dorsal raphe nucleus.

In order to determine whether serotonergic (5HT) dorsal raphe nucleus (DRN) cells are involved in body sodium status regulation, the effect of a s.c. infusion of either 2 M or 0.15 M NaCl on 5HT DRN neuron firing was studied using single unit extracellular recordings. In separate groups of 2 M and 0.15 M NaCl-infused rats, water intake, oxytocin (OT) plasma concentration, urine and plasma sodium and protein concentrations were also measured. Also, to determine the involvement of particular brain nuclei and neurochemical systems in body sodium overload (SO), animals from both groups were perfused for brain immunohistochemical detection of Fos, Fos-OT and Fos-5HT expression. SO produced a significant increase in serotonergic DRN neuron firing rate compared to baseline and 0.15 M NaCl-infused rats. As expected, 2 M NaCl s.c. infusion also induced a significant increase of water intake, diuresis and natriuresis, plasma sodium concentration and osmolality, even though plasma volume did not increase as indicated by changes in plasma protein concentration. The distribution of neurons along the forebrain and brainstem expressing Fos after SO showed the participation of the lamina terminalis, extended amygdala, supraoptic and paraventricular hypothalamic nuclei in the neural network that controls osmoregulatory responses. Both Fos-OT immunoreactive and plasma OT concentration increased after s.c. hypertonic sodium infusion. Finally, matching the "in vivo" electrophysiological study, SO doubled the number of Fos-5HT immunolabeled cells within the DRN. In summary, the results characterize the behavioral, renal and endocrine responses after body sodium overload without volume expansion and specify the cerebral nuclei that participate at different CNS levels in the control of these responses. The electrophysiological approach also allows us to determine in an "in vivo" model that DRN 5HT neurons increase their firing frequency during an increase in systemic sodium concentration and osmolality, possibly to modulate sodium and water intake/excretion and avoid extracellular volume expansion.

Spinal cord transection modifies neuronal nitric oxide synthase expression in medullar reticular nuclei and in the spinal cord and increases parvalbumin immunopositivity in motoneurons below the site of injury in experimental rabbits.

Using immunohistochemistry, we detected the expression of neuronal nitric oxide synthase (nNOS) in ventral medullary gigantocellular reticular nuclei and in the lumbosacral spinal cord 10 days after thoracic transection in experimental rabbits. We tried to determine whether neurons located below the site of injury are protected by the calcium binding protein parvalbumin (PV). Changes of nNOS immunoreactivity (IR) in spinal cord were correlated with the level of nNOS protein in dorsal and ventral horns. Ten days after transection, nNOS was upregulated predominantly in lateral gigantocellular nuclei. In the spinal cord, we revealed a significant increase of nNOS protein in the dorsal horn. This is consistent with a higher density of punctate and fiber-like immunostaining for nNOS in laminae III-IV and the up-regulation of nNOS-IR in neurons of the deep dorsal horn. After surgery, the perikarya of motoneurons remained nNOS immunonegative. Contrary to nNOS, the PV-IR was upregulated in α-motoneurons and small-sized neurons of the ventral horn. However, its expression was considerably reduced in neurons of the deep dorsal horn. The findings indicate that spinal transection affects nNOS and PV in different neuronal circuits.

Inflammatory stimuli reduce survival of serotonergic neurons and induce neuronal expression of indoleamine 2,3-dioxygenase in rat dorsal raphe nucleus organotypic brain slices.

Neuroinflammation results in dysregulation of serotonergic neurons in the dorsal raphe nucleus (doR) and is considered to play an important role in the pathophysiology of depression. The aim of the present study was to induce neuroinflammation in a simple doR brain slice model using lipopolysaccharide (LPS), interferon-gamma (IFNγ), beta-amyloid1₋42 or tumor necrosis factor-alpha and to explore the survival of serotonergic neurons and the expression of the tryptophan degrading enzyme indoleamine 2,3-dioxygenase (IDO). Administration of pro-inflammatory stimuli reduced survival of serotonergic neurons in doR slices and increased IDO expression. IFNγ most potently induced IDO expression, which co-localized with neurons, including serotonergic neurons, but not with microglia or astrocytes. IFNγ did not induce PI-positive staining in slices, but increased the average nuclei size of IDO-positive cells. The inflammation-induced decline did not return to control levels, when slices were withdrawn from inflammation, pointing to neurodegeneration. The growth factors BDNF or GDNF did not counteract the inflammation-induced decrease in serotonergic neurons, except for LPS-induced neuronal decline. The inflammation-induced effect was not blocked by the NMDA-receptor antagonist MK-801. Further LPS, but not IFNγ increased inflammatory markers and microglia activity. In conclusion, our data show that a range of inflammatory stimuli decline serotonergic neurons in doR slices and upregulate IDO expression. The data suggest that IDO does not contribute to serotonergic decline, but may serve as a marker of neurodegeneration. Neuroinflammation may contribute to the development of depression.

Early-life treatment of antiserotonin antibodies alters sensitivity to serotonin receptors, nociceptive stimulus and serotonin metabolism in adult rats.

A single systemic administration of serotonin (5-HT) antibodies on day-1 of the life of rat has been investigated for neurotransmitter contents in nucleus raphe, and several discrete brain regions, as well as for serotoninergic syndromes and nociceptive responses in adult animals. 5-HT antiserum raised in rabbits were purified and characterized prior to subcutaneous administration in neonatal rats. Control animals received normal rabbit serum. Antibodies tagged with radioactive iodine were traced in the brains of rat pups treated subcutaneously. These animals at adulthood, exhibited an increase in body weight, increased sensitivity to serotonin agonist 5-methoxy-N-N-dimethyl tryptamine, and to nociceptive stimulus to subcutaneously administered formalin. Animals neonatally treated with 5-HT antiserum once on day 1 of life, exhibited significant decrease in the contents of serotonin and its metabolite as compared to normal serum treated animals specifically in nucleus raphe dorsalis, but not in substantia gresia centralis, nucleus accumbens, nucleus caudatus putamen, substantia nigra or tuberculum olfactorium during the study period of seven days to four months. The contents of dopamine or norepinephrine were not consistently altered in any of the nuclei studied. Since 5-HT is known to act as a trophic factor for its own development and its target areas, exposure to 5-HT antibodies during birth might have adversely affected the development of the serotoninergic system and resulted in long-lasting changes in behavior and 5-HT levels in the brain. These results have strong implications for the treatment of childhood developmental disorders such as autism where hyperserotoninemia is associated with the disease syndromes.

[Presynaptic 5-HT1A receptors in immunomodulation].

It is shown that a selective agonist of 5-HT1A receptors 8-OH-DPAT in a low dose (0.1 mg/kg), which is known to affect mainly the presynaptic 5-HT1A receptors increased the immune response at the peak of reactions (the forth or fifth day after immunization with sheep red blood cells - SRBC) in CBA mice and Wistar rats. Treatment of the animals with the drug 15 min prior to antigen injection raised the number of plaque-forming cells (lgM-PFC) and rosette-forming cells (RFC) in the spleen. The preliminary blockade of 5-HT1A receptor with a selective antagonist of 5-HT1A receptors WAY-100635 (0.1 mg/kg) prevented the immunostimulating effect of 5-HT 1A receptors agonist 8-OH-DPAT, whereas WAY-100635 administration alone in the same dose didn't change the immune response. Activation of 5-HT1A receptors under conditions of electrical lesion of 5-HTergic neurons of the nucleus raphe was unable to enhance the immune reactions, as it did in sham-operated rats. The data obtained indicate that the somatodendric 5-HT1A autoreceptors are involved in immunomodulation.

Neuroimmunological activation of the afferent laryngeal neural circuit in experimentally induced laryngeal inflammation.

CONCLUSIONS: These results show that laryngeal inflammatory reactions may induce the expression of proinflammatory cytokines along the afferent laryngeal circuit and in nuclei associated with the HPA axis. Local laryngeal inflammation may induce functional and physiologic alterations in the laryngeal neural system via neuroimmunologic reactions. OBJECTIVE: Idiopathic laryngeal disorders associated with various neurologic conditions such as spasmodic dysphonia, idiopathic vocal fold paralysis and sudden infant death syndrome are causally related to upper respiratory tract infections, and it can be speculated that these disorders result in neurophysiologic alterations. The goal of this study was to identify the neurophysiologic effect on the central nervous system of local inflammatory alterations in the larynx. MATERIAL AND METHODS: The expression of c-fos and IL-1beta was identified after injecting saline solution, 10 microg of lipopolysaccharide or 100 microg of lipopolysaccharide into the larynx of 12 rats. RESULTS: The inflammatory cytokine IL-1beta was mainly expressed in the inferior olivary nucleus and raphe nucleus, which are associated with the hypothalamic-pituitary-adrenal (HPA) axis. IL-1beta expression was also found in the nuclei of afferent nervous pathways of the superior laryngeal nerve, such as the nucleus tractus solitarius, nucleus ambiguus, lateral reticular nucleus, magnocellular reticular nucleus and paragigantocellular reticular nucleus.

Electron microscopic examination of the orexin immunoreactivity in the dorsal raphe nucleus.

The ultrastructure and the synaptic relationships of the orexin-A-like immunoreactive fibers in the dorsal raphe nucleus were examined with an immunoelectron microscopic method. At the electron microscopic level, most of the immunoreactive fibers, a varicosity appearance at the light microscopic level, were found as axon terminals. The large dense-cored vesicles contained in the immunoreactive axon terminals were the most intensely immunostained organellae. These axon terminals were often found to make synapses. While the axo-dendritic synapses were usually asymmetric in appearance, the axo-somatic synapses were symmetric. Orexin-A-like immunoreactive processes with no synaptic vesicles were also found. These processes often received asymmetric synapses. With less frequency, the synapses were found between the orexin-like immunoreactive processes. The results suggest that the orexin peptides are stored in the large dense-cored vesicles; the orexin-containing fibers may have influences on the physiological activities of the dorsal raphe nucleus through direct synaptic relationships.

A novel monoclonal antibody recognizes a previously unknown subdivision of the habenulo-interpeduncular system in zebrafish.

The habenulo-interpeduncular system is an evolutionarily conserved structure found in the brain of almost all vertebrates. We prepared a monoclonal antibody (6G11) which very specifically recognizes only a part of this system. 6G11 is a monoclonal antibody prepared from a neuronal membrane protein in adult zebrafish brain. In western blot analysis of the adult zebrafish brain, the antibody recognized a 95 kDa protein, and the class of the antibody was determined to be IgM. The 6G11 antigen was not detected in zebrafish muscle, intestine, testis or ovary. A group of neurons stained by the 6G11 antibody was located in the caudomedial part of the zebrafish habenula. The 6G11-immunopositive neurons extended their axons into the fasciculus retroflexus (FR). One group of immunopositive neurons projected toward the interpeduncular nucleus (IPN), especially to the intermediate and the central subnucleus (type 1 neuron). The other group projected to the ventral midline at the level of the raphe nucleus; these axons passed ipsilaterally beside the IPN and converged in the ventral midline under the raphe nucleus (type 2 neuron). Both type 1 and type 2 fibers are relatively minor components of the FR. Little has previously been known about this topological pattern in any species. The 6G11 monoclonal antibody could be a useful tool for expanding knowledge of the habenulo-interpeduncular system.

Dopamine-degrading activity of monoamine oxidase in locus coeruleus and dorsal raphe nucleus neurons. A histochemical study in the rat.

Dopamine-degrading activity of monoamine oxidase (MAO) was detected in the rat using a new histochemical method, with dopamine as the substrate. Our new method, designed to minimise the non-enzymatic oxidation of dopamine, was applied in combination with tyrosine hydroxylase (TH) and serotonin immunohistochemistry. We showed that the distribution pattern of MAO neurons was similar to that of TH-immunoreactive neurons (i.e. noradrenergic neurons) in the locus coeruleus (LC) and to that of serotonergic neurons in the dorsal raphe nucleus (DR). Since LC neurons form dopamine during noradrenaline biosynthesis, and DR neurons produce dopamine from exogenously administered L-dopa, our results indicate that dopamine produced in LC and DR neurons may be degraded, at least in part, by MAO.

Target regulation of neuronal differentiation in a temperature-sensitive cell line derived from medullary raphe.

Following infection of dissociated embryonic day 13 rat medullary raphe cells with a retrovirus encoding the temperature-sensitive mutant of SV40 large T antigen, a clonal cell line, RN33B, was isolated by serial dilution. At 33 degrees C, RN33B cells divide with a doubling time of 48 h and show T antigen, vimentin, nestin, diffuse neuron-specific enolase, and low and medium molecular weight neurofilament immunoreactivities. RN33B cells are immortal, but not transformed, as they will not grow in soft agar. At non-permissive temperature (38.5 degrees C), T antigen expression is markedly decreased and RN33B cells cease mitotic activity and differentiate with phase bright cell bodies and 'neuritic-like' processes. Differentiated RN33B cells express enhanced neuronal-specific protein expression but do not synthesize astrocytic or oligodendrocytic-specific proteins. Moreover, differentiated RN33B cells returned to 33 degrees C re-express T antigen, but do not de-differentiate or begin dividing. Co-culture with embryonic hippocampus and cerebral cortex, but not medullary raphe or spinal cord, resulted in significantly greater survival, more complex neuronal morphology, and enhanced expression of neuronal-specific antigens. Immunohistochemical and Northern blot analysis revealed high levels of low affinity NGF receptor protein and mRNA in differentiated RN33B cells. PCR analysis demonstrated the presence of trkB, but not trkA or trkC, mRNA in both undifferentiated and differentiated RN33B cells. These data suggest that the observed target regulation of RN33B cell neuronal differentiation in co-culture may be mediated by neurotrophin(s).

Electroacupuncture accelerated the expression of c-fos protooncogene in serotonergic neurons of nucleus raphe dorsalis.

It has been proposed that the FOS protein encoded by c-fos protooncogene functions as a nuclear "third messenger" molecule that couples short-term extracellular signals to long-term alterations in cell function, by regulating the expression of specific target genes. In the present study, immunocytochemical double staining technique was used to investigate the effects of electroacupuncture on the expression of c-fos oncogene in the serotonergic neurons in the nucleus raphe dorsalis (NRD) that has been known to play an important role in the endogenous analgesic system of the brain. The number of FOS positive serotonergic cells in the NRD increased significantly after the electroacupuncture stimulation. These results indicate that electroacupuncture can activate central serotonergic neurons at gene expression level.

Relationship between acupuncture-induced immunity and the regulation of central neurotransmitters in the rabbit: VI. The influence of NDR stimulation on acupuncture regulation of immune function in rabbit.

T-lymphocyte transformation ability decreased and the function of the B-lymphocyte and mononuclear leucocyte/macrophage system increased when the activity of central serotonergic neurons was enhanced by the stimulation of nucleus dorsal raphe (NDR). These changes induced by NDR stimulation were not effected by electroacupuncture (EA) when EA was performed on the background of NDR stimulation. By comparing these results with the previous resultant that immunoreactions were affected by locus coeruleus (LC) stimulation and LC stimulation plus EA, some regularity had been discovered. So, a hypothesis was put forward by us that the catecholaminergic system plays a main role in EA regulation of cellular immunity while the serotonergic system plays such a role in EA regulation of humoral immunity.

The presence of serotonin-immunoreactive nerve fibers in the optic nerve of the mouse.

Serotonin immunoreactivity was investigated in the optic nerve of the mouse by use of mono- and polyclonal antisera and the indirect peroxidase-antiperoxidase technique. A number of serotonin-immunoreactive nerve fibers with beaded appearance were demonstrated mainly orientated along its longitudinal axis. Since several studies have failed to demonstrate perikarya immunoreactive to serotonin in the mammalian retina, the presence of serotoninergic fibers in the optic nerve further supports a retinopetal innervation.

Evidence for leucine-enkephalin immunoreactive neurons in the medulla which project to spinal cord in squirrel monkey.

Rhodamine-labeled latex microsphere injections were combined with horseradish peroxidase immunohistochemistry in squirrel monkeys to reveal neurons in the medullary raphe and medial reticular formation which projected to spinal cord and were positive for leucine-enkephalin-like immunoreactivity. Double-labeled cells were found primarily in nucleus raphe magnus, the reticular nucleus magnocellularis, and the lateral reticular nucleus. These results provide evidence for a descending projection from enkephalin-containing cells of the rostral ventral medulla, a region which has been strongly implicated in antinociception.

Somatostatin-like immunoreactivity in the midbrain of the cat.

Somatostatin (SS) immunoreactivity was localized in cat brain sections with an immunoperoxidase technique. Cell bodies in the midbrain containing SS immunoreactivity were found in the superficial and intermediate gray layers of the superior colliculus, the interpeduncular nucleus, the raphe, the inferior colliculus and nucleus of its brachium, the nucleus of the optic tract, and the lateral tegmental field. Additional positive neurons were seen in the parabigeminal nucleus and in the dorsal periaqueductal gray in kitten material. Immunoreactive fibers were observed in the periaqueductal gray and in the midbrain tegmentum, with particularly dense labeling just dorsal to the substantia nigra and in the parabrachial nuclei. This is the first report of the distribution of SS immunoreactivity in the midbrain of the cat. It is concluded that somatostatin has a distribution compatible with a role as a major neurotransmitter/neuromodulator within certain midbrain nuclei, especially the interpeduncular nucleus and the superior colliculus.

Distribution of enkephalin-immunoreactive cell bodies in relation to serotonin-containing neurons in the raphe nuclei of the cat: immunohistochemical evidence for the coexistence of enkephalins and serotonin in certain cells.

In this study we have examined the distribution of enkephalin-like immunoreactive (ELI) cell bodies in the cat raphe nuclei pallidus (NRP), obscurus (NRO), magnus (NRM), pontis and dorsalis (NRD) after intraventricular administration of colchicine. All the raphe nuclei examined were observed to contain ELI cell bodies along their whole caudorostral extent. By comparing consecutive sections treated separately with anti-5-HT and enkephalin-antiserum it was observed that certain 5-HT cells in each raphe nucleus contain ELI material. A quantitative estimation was attempted. In NRP and NRO approximately half of the total immunoreactive neuronal population appeared to be immunoreactive for both 5-HT and the enkephalins. In NRM the proportion would be one-third, whereas it seemed almost negligible in NRD. Among the 5-HT cells, approximately two-thirds might be ELI in NRP and NRO, and one-half in NRM.

[Changes in the level of serotonin in the brain and immunocompetent organs during the formation of the immune response]. / Izmenenie urovnia serotonina v mozge i immunokompetentnykh organakh pri formirovanii immunnogo otveta.

The serotonin content increased in adrenals within 20 min after immunization of rats with sheep red blood cells, and diminished in ventral part of the anterior hypothalamus, hippocampus and in thymus. The number of antigen--responsive cells was increased twofold in bone marrow after the 1000-fold increase of the antigen dose. On the 4-5th days, the dependence of antibody-forming cells and rosette-forming cells number on the antigen dose was still observed in the spleen. The correlation between the serotonin level in the above brain structures and immunocompetent organs and the intensity of immune responses is discussed.

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 fibers 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 alpha 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 met-enkephalin 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.