In vivo release of serotonin in two raphe nuclei (raphe dorsalis and magnus) of the cat

The release of ³H-serotonin (³H-5-HT) endogenously synthesized from ³H-tryptophan was estimated in both dorsalis and magnus (MRN) raphe nuclei of anaesthetized “encéphale isolé” cats, by using push-pull cannulae. Resting steady state in the release of ³H-5-HT was observed 30 min after the beginning of superfusion with L-³H-tryptophan. The amounts of ³H-5-HT released in the DRN and the MRN are much greater than those measured simultaneously in the caudate nucleus. A marked increase either in the 5-HT release was seen in the presence of fluoxetine, a potent reuptake blocker of 5-HT, or during local depolarization with potassium chloride. The spontaneous release was diminished by removing Ca⁺⁺ and by adding cobalt to the medium. Tetrodotoxin (TTX) decreased the 5-HT release in the DRN and, based on previously established, blocked the stimulating effect of batrachotoxin. According to the pharmacological characteristics of the monoamine dendritic release determined for dopamine in the substantia nigra [17], our results suggest that 5-HT release processes in the DRN correspond to a release from nerve endings, not from dendrites. The purpose of this study was to determine if the 5-HT released in the DRN is released from either axon terminals or dendrites. Morphological studies performed on the DRN do not consistently demonstrate the high density of serotoninergic varicosities in the DRN. In addition, two types of 5-HT axonal varicosities, characterized by their synaptic or non-synaptic junctions, are present in the brain. The concept that the quantities of 5-HT released could vary from one type compared to the other is discussed.     

Developmental analysis of raphe dorsalis unit activity in the rat

Unit activity of raphe dorsalis neurons was recorded in rats between 3 and 24 days of age. Both a regular discharge pattern and a slow firing rate were observed as early as day 3. The frequency of discharge was not statistically different at any age from the mean discharge rate in the adult rat. It was concluded that the electrophysiological activity of 5-HT neurons develops early in the process of maturation, prior to that of the biochemistry and anatomy of these neurons.     

Immunocytochemical evidence for the existence of GABAergic neurons in the nucleus raphe dorsalis. possible existence of neurons containing serotonin and GABA

It has been established that nerve cell bodies of the nucleus raphe dorsalis (NRD) belong to ascending 5-hydroxytryptamine systems. These neurons could be modulated by GABAergic interneurons or interposed GABA neurons. A high glutamate decar☐ylase (GAD) activity in the NRD and a specific high-affinity uptake mechanism for GABA suggest the presence of GABA synthesizing elements in the NRD. Anti-GAD antibodies were used by an immunocytochemical procedure to demonstrate the presence of GABAergic elements. Anti-GAD antibodies were previously tested in the cerebellum and substantia nigra. Large amounts of GAD-positive reaction product were observed in the cytoplasm of some neurons (fusiform, ovoid or multipolar) or appeared as punctate deposits apposed to dendrites, soma and dispersed in the neuropil of the NRD. At the electron microscopic level, GAD-positive reaction product was observed within the cytoplasm of numerous somata in sections from colchicine-treated rats. GAD-positive staining was observed in numerous fibers or axonal terminals and two types of morphologically different fibers could be distinguished. The first displays small clear vesicles and few large granular vesicles (LGV) (80–100 nm), the second displays only clear round vesicles (40–60 nm). After 5,7-dihydroxytryptamine treatment (a neurotoxic for 5-HT terminals), the immunocytochemical labeling is much decreased. Some reactive neurons are still dispersed in the nucleus but the fibers containing LGV are no longer observed. These results strongly suggest that some neuronal elements in the NRD are morphologically, pharmacologically and anatomically similar to 5-HT neurons described at this level. Such cell elements could possess a double GABA and 5-HT potentiality. If this is not the case, a population of GABA neurons could be sensitive to 5,7-DHT and so have the capacity to take up 5-HT. The other reactive elements, insensitive to 5,7-DHT, could represent the GABAergic interneurons postulated at this level. Numerous GAD positive fibers or axon terminals were observed in synaptic contact with dendrites, axons or soma of other neurons. The chemical nature of the neuronal postsynaptic elements remains unknown. These findings strongly support the hypothesis for GABA-mediated inhibition in the NRD.     

Genetic studies of daily variations of first-step enzymes of monoamines metabolism in the brain of inbred strains of mice and hybrids. I. Daily variations of tryptophan hydroxylase activity in the nuclei raphe dorsalis, raphe centralis and in the striatum

A daily variation of tryptophan hydroxylase (TrH) activity was observed in the raphe dorsalis (RD), raphe centralis (RC) and striatum (St) of 3 inbred strains of mice (BALB/c, C57BL/6, C57BR) and of the reciprocal hybrids obtained from Balb/c and C57BL6. Significant differences of the characteristics of these rhythms have been found in the same strain between different structures and for the same structure between different strains. In RD and RC hybridization led to less defined daily variations which, in the striatum, remained well synchronized and could be controlled by a dominant genetic mechanism part. These results help to discuss evidence for selective control mechanisms of regulation responsible for daily variation of TrH in the 5-HT cell bodies and terminals and their relative independence.     

The effects of protein deprivation on the nucleus raphe dorsalis: A morphometric golgi study in rats of three age groups

In a previous study we identified 3 cell types in the nucleus raphe dorsalis (NRD): fusiform, multipolar and ovoid. In the present study, we have investigated the effect of an 8% casein diet on these 3 cell types using quantitative techniques on rapid Golgi-impregnated neurons from rats of 3 different ages: 30, 90 and 220 days. Major and minor axes of the cell body and dendritic diameter were unaffected and primary dendritic linear extent was only slightly affected by the diet. All 3 cell types in control rats showed an increase in synaptic spines on both primary and secondary dendrites between 30 and 90 days followed by a decrease for all 3 of the cell types at 220 days. Protein-deprived rats failed to show these age-related changes. Other parameters of comparison showed clear differences between the 3 cell types. These differences could be readily seen when total synaptic spine input to the primary and secondary dendrites was calculated from the data on dendritic number, linear extent and spine density. When viewed in this way the fusiform and ovoid cells show either little change or a decreased synaptic input at all ages, while the presumed serotonergic multipolar cells showed an increase. This is in agreement with neurochemical studies in these rats showing increased levels of this biogenic amine in protein malnourished rats.     

Effects of acute administration of SCH 23390 on dopamine and serotonin turnover in major dopaminergic areas and mesencephalic raphe nuclei — Comparison with ritanserin

1. The effects of acute administration of SCH 23390 (0.05 and 0.25 mg/kg s.c.), a dopamirie D-1 receptor antagonist having also a moderate serotonin-S₂ (5-HT-2) receptor blocking activity, and ritanserin (0.5 mg/kg), a specific 5-HT-2 antagonist, on dopamine (DA) and serotonin (5-HT) turnover were investigated in dopaminergic (nucleus caudatus, nucleus accumbens, substantia nigra, A10 area) and serotonergic (nucleus raphe dorsalis and nucleus raphe medialis) rat brain nuclei.

2. Acute SCH 23390 (both doses) increased the metabolism of DA and tended to augment the rate of DA synthesis (accumulation of DOPA after inhibition of aromatic acid decarboxylase) in the nucleus accumbens, but not in the nucleus caudatus. In addition, SCH 23390 had a moderate effect on DA metabolism in substantia nigra. SCH 23390 did not alter the turnover of 5-HT in any of the nuclei studied.

3. Acute administration of ritanserin did not modify 5-HT or DA turnover in any of the nuclei studied.

4. In conclusion, these results suggest that acute SCH 23390 administration preferentially activates the mesolimbic DA system. The lack of effect of ritanserin on DA or 5-HT turnover in nigrostriatal and mesolimbic DAergic areas suggests that under basal conditions the blockade of 5-HT₂ receptors do not change monoamine metabolism in these areas. The role of 5-HT-2 blockade in the actions of SCH 23390 on DA turnover appears thus to be of a minor importance.     

Changes in serotonin-immunoreactivity in the dorsal and median raphe nuclei of rats exposed to 2,4-dichlorophenoxyacetic acid through lactation

Comparison of serotonin-immunoreactive (SER-IR) neurons in nucleus raphe dorsalis (NRD) and median raphe nucleus (MRN) of 25-d-old rat pups exposed to 70 mg/kg/d 2,4-dichloro-phenoxyacetic acid through mothers milk and control pups was made using an immunohistochemical analysis. Significant 2,4-D-treatment-related increase in size and density of SER-IR neuronal somata as well as in fiber length were observed. We postulate that exposure to 2,4-dichlorophe-noxyacetic acid on the first day of life would modify the synthesis of 5-HT or the maturation of the brain serotonergic system.     

Deficits in the mid-brain raphe nuclei and striatum of the AS/AGU rat, a protein kinase C-gamma mutant

The AS/AGU rat carries a recessive mutation (agu) in the gene coding for the gamma isoform of protein kinase C. The rat is characterized by disordered locomotion and progressive dysfunction of the nigrostriatal dopaminergic (DA) system. This dysfunction begins with a failure to release DA within the striatum and culminates in cell loss within the substantia nigra pars compacta. The present study examines another midbrain aminergic system with input to the basal ganglia, the serotonergic (5-HT) raphe-striatal system originating in the dorsal raphe nucleus. By 3 months after birth, there is a very substantial reduction in the extracellular levels of 5-HT in the dorsal caudate-putamen of the mutants compared with controls (c. 70%). This is accompanied by a proportional increase in the levels of the 5-HT metabolite 5-hydroxyindole acetic acid (5-HIAA). At a later age, there are reductions in whole tissue 5-HT (and increases in 5-HIAA) in both the striatum and the region containing the dorsal raphe nucleus, as well as numbers of 5-HT-immunoreactive cells in the dorsal raphe nucleus. The median raphe appears to be unaffected. The results are seen in terms of an initial dysfunction in transmitter release leading to cell death, perhaps through the formation of free radicals or neurotoxins.     

Ontogenesis of unit activity in the raphe dorsalis of the behaving kitten: Its relationship with the states of vigilance

The relationship between the spontaneous unit activity in the raphe dorsalis (RD), and the sleep-wakefulness cycles, was analyzed in the cat from birth to 40 days of age. Electrodes for polygraphic sleep monitoring were implanted under anesthesia, and unit recordings were obtained from bundles of microwires positioned in the RD area in kittens of different ages. Attention was paid only to units with slow firing in wakefulness (W) (1-6 spikes/s), and two types of discharge patterns during this state were obtained: a 'regular' type, whose discharge in W had the same characteristics of regularity as those described for the adult under the same conditions, was always found inside the RD. An 'irregular' type was always found in sites outside the RD. Injections of different doses of 5-methoxy-N,N-dimethyltryptamine (5-MeODMT i.m.) induced a transient decrease in the firing rate of the regular type of cells, and no change for the units of the irregular type, suggesting that the regular neurons were of serotoninergic nature. Whereas the cells of the irregular type exhibited an increase of discharge frequency in active or paradoxical sleep (AS-PS), those which fired in a clock-like manner during W exhibited a rate of discharge which progressively decreased in quiet or slow wave sleep (QS-SWS) and even more in AS-PS. Such a pattern was qualitatively close to the adult one at all ages, but the discharge rate in AS was significantly higher during the first and second weeks of life than later on. The observation that these serotoninergic neurons exhibited at birth an adult-like pattern of discharge during W, indicates that during ontogenesis there was no direct relationship between the RD activity and the behavioral output. It is proposed that the RD neuronal discharge would be largely under genetic influences, and that the maturation of sleep regulations at the brainstem and mesencephalic levels is achieved only after the second week of postnatal age.     

Effect of pyridoxine deficiency in young rats on high-affinity serotonin and dopamine receptors

The high-affinity bindings of [3H]-5-hydroxytryptamine to serotonin S-1 receptors, [3H]-ketanserin to serotonin S-2 receptors in the cerebral cortex, [3H]-fluphenazine to dopamine D-1 receptors, and [3H]-spiroperidol to dopamine D-2 receptors in the corpus striatum were studied in pyridoxine-deficient rats and compared to pyridoxine-supplemented controls. There was a significant increase in the maximal binding (Bmax) of serotonin S-1 and S-2 receptors with a significant decrease in their binding affinities (Kd). However, there were no significant changes either in the maximal binding or binding affinity of striatal dopamine D-1 and D-2 receptors. Receptor sensitivity seems to correlate negatively with the corresponding neurotransmitter concentrations in the pyridoxine-deficient rats.     

Asymmetric dopamine and serotonin metabolism in nigrostriatal and limbic structures of the trained circling rat

The trained circling rat model was used to investigate dopamine and serotonin metabolism in extrapyramidal and limbic structures during turning behavior. We have previously reported that dopamine turnover is increased during circling in the caudate contralateral to the circling direction in this behavioral model. We have now studied changes in dopamine and serotonin turnover in nucleus accumbens, substantia nigra and amygdala. As in the caudate, dopamine production in nucleus accumbens was selectively increased on the contralateral side after 20 min of circling. By contrast, dopamine turnover in substantia nigra exhibited a relative decline on the contralateral side. Dopamine synthesis in the amygdala was not affected by circling. Selective changes in serotonin metabolism were also seen in these brain regions. In caudate and accumbens, serotonin turnover was unaffected by circling. However, both substantia nigra and amygdala showed significant, progressive increases in serotonin metabolism in the contralateral side after 20 and 70 min of circling. These results show that extrapyramidal and limbic dopamine and serotonin metabolism are involved in turning behavior of normal animals. Multiple transmitters of the nigrostriatal pathway and the limbic system appear to interact to modulate voluntary circling behavior.     

Association between increased serotonin metabolism in rat brainstem nuclei and development of spontaneous hypertension

Spontaneously hypertensive rats have increased brain stem serotonin turnover localized to the nuclei raphe magnus, raphe pallidus, tractus solitarius and reticularis lateralis. It occurs only in young animals coinciding with the development of hypertension. In the locus coeruleus, increased serotonin metabolism is present during both the early and the established phase of hypertension. Our results provide evidence for serotonergic overactivity in specific brainstem nuclei during the development of spontaneous hypertension in the rat.     

Differential pulse voltammetry: Parallel peak 3 changes with vigilance states in raphe dorsalis and raphe magnus of chronic freely moving rats and evidence for a 5-HT contribution to these peaks after monoamine oxidase inhibitors

Nuclei raphe dorsalis (RDN) and magnus (RMN) were simultaneously studied using the differential pulse voltammetry (DPV) technique in chronic freely moving rats during their sleep-waking cycle. Parallel variations in peak 3 (due to 5-hydroxyindoles) were observed in both these areas: the peak 3 heights were maximum in both RDN and RMN during waking (W), decreased in slow wave sleep (SWS) and were minimum in paradoxical sleep (PS). Monoamine oxidase inhibitor (MAOI) treatments induce at first a decrease of peak 3 (−50% compared to the control values), but there was a subsequent increase (+100% compared to the control values.) This suggests that 5-hydroxytryptamine (5-HT) can contribute to peak 3 measured in vivo.     

Differential responses of serotonergic and non-serotonergic neurons in nucleus raphe magnus to systemic morphine in rats

Forty-eight raphe-spinal units in nucleus raphe magnus were identified by antidromic stimulation and further classified into serotonergic and non-serotonergic populations according to their conduction velocity and spontaneous discharge rate. Following morphine administration (5 mg/kg, i.p.) they showed different responses: excitatory, depressive or non-responsive. It was found that 6 of 7 nonresponsive, 3 of 20 depression-responsive and only 1 of 21 excitation-responsive units proved to be serotonergic neurons, indicating that the involvement of serotonergic neurons in morphine analgesia is probably insignificant. It is suggested that the raphe-spinal fiber systems, both excitatory and inhibitory are originated mainly from the non-serotonergic neurons.     

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 rat raphe nuclei pallidus (NRP), obscurus (NRO), magnus (NRM), pontis and dorsalis (NRD) after intratissular or 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.     

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

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

Influence of inhibitory and excitatory inputs on serotonin efflux differs in the dorsal and median raphe nuclei

The dorsal (DRN) and median raphe nuclei (MRN) are two major sources of serotonergic projections to forebrain that are involved in regulation of behavioral state and motor activity, and implicated in affective disorders such as depression and schizophrenia. To investigate afferent influences on serotonergic neurons, this study compared the role of endogenous GABA and glutamate in the DRN and MRN using microdialysis and measurement of locomotor activity in freely behaving rats. Local infusion of the GABA(A) receptor antagonist bicuculline increased serotonin (5-HT) efflux in the DRN but not the MRN. In contrast, infusion of glutamate receptor antagonists produced larger decreases in 5-HT efflux in the MRN compared with the DRN. Moreover, glutamate receptor antagonists attenuated the increase in 5-HT efflux produced by GABA receptor blockade in the DRN. Thus, the disinhibitory effect of GABA blockers could be ascribed in part to an enhanced influence of glutamate. Measurements of locomotor activity indicate that changes in 5-HT were not simply correlated with behavioral activity induced by drug infusion. In summary, the role of inhibitory and excitatory afferents was strikingly different in the DRN and MRN. GABA afferents were the predominant tonic influence on serotonergic neurons in the DRN. In contrast, glutamatergic but not GABAergic afferents had a strong tonic influence on serotonergic neurons in the MRN.     

Regulation of cerebral serotonin binding and metabolism in sleep-deprived rats

Two different binding sites are found for serotonin binding to crude membrane fractions isolated from rat brain. After 12 h sleep deprivation (SD) the dissociation constant of the high affinity binding KD1 = 1.4 mM, remains unchanged whereas the dissociation constant, KD2, of the low affinity binding increased from 26 to 42 nM. The number of binding sites are only moderately affected. The dissociation constant of the low affinity binding, KD2, is decreased towards control values if SD is followed by 1 or 4 h sleep. As indicated by serotonin and 5-hydroxyindoleacetic acid analyses, a statistically significant increase in serotonin metabolism is found only, if SD is followed by 1 h recovery sleep.     

Changes in mediobasal hypothalamic dopamine and indoleamine metabolism after superior cervical ganglionectomy of rats

Summary Eight days after bilateral superior cervical ganglionectomy (Gx) of rats, norepinephrine content of medial basal hypothalamus (MBH) decreased significantly by 44–50%. To obtain information on other possible neurochemical sequela of Gx in MBH, we examined the metabolism of dopamine and serotonin in MBH of Gx rats by employing a high pressure liquid chromatography procedure. Eight days after Gx, MBH dopamine levels augmented significantly. Assessment of dopamine metabolism by measuring dihydroxyphenylacetic acid (DOPAC)/dopamine and homovanillic acid (HVA)/ dopamine indexes indicated a significant decrease of MBH DOPAC/dopamine ratio after Gx. MBH serotonin levels increased, and 5-hydroxyindoleacetic acid (5-HIAA)/serotonin index decreased significantly in Gx rats. To examine the interaction Gx-induced changes on MBH dopamine and serotonin with the modified hormonal milieu produced by an ectopic pituitary transplant, adult male rats bearing an ectopic pituitary within the pectoral muscles from day 5 of life were submitted to Gx on day 60 of life and were studied 8 days later. MBH dopamine content increased significantly after pituitary grafting, an effect counteracted by a subsequent Gx, while Gx alone augmented MBH dopamine levels. DOPAC and HVA contents augmented in pituitary-grafted animals, an effect counteracted by Gx. Gx increased MBH serotonin content in control but not in pituitary-grafted rats. After pituitary grafting a decrease in MBH 5-HIAA levels was found, an effect reversed by Gx. Pituitary transplants brought about a significant increase of MBH DOPAC/dopamine index, and a significant decrease in 5-HIAA/serotonin index, both effects being counteracted by Gx. Gx of control rats resulted in a significant decrease of MBH 5-HIAA/serotonin index. Analyzed as a main effect in a factorial analysis of variance, Gx decreased MBH DOPAC/dopamine and HVA/dopamine indexes significantly. Plasma prolactin increased in pituitary-grafted rats, an effect further increased by a subsequent Gx. In pituitary-grafted, Gx rats plasma GH levels augmented significantly. The data suggest that superior cervical ganglion removal affects differentially dopamine and indoleamine metabolism in MBH of control and pituitary-grafted rats.     

Simultaneous determination of the formation rate of dopamine and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in various rat brain areas

Administration of the MAO-inhibitor pargyline resulted in an increase of dopamine (DA) and an exponential decrease of the metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in arious rat brain areas. From these curves the rate of formation of DA and the rate of removal of DOPAC were calculated, within one experiment, for the striatum, tuberculum olfactorium and frontal cortex. When the accumulation rate of 3-methoxytyramine was taken into account it appeared that the rate of DA formation was in good agreement with the rate of DOPAC removal in the brain areas studied. The contribution of the DA formation to the synthesis of noradrenaline in the frontal cortex appeared negligible. The earlier reported rapid rise of strittal DA levels after administration of aminotetralin-derived DA agonists suggests that the synthesis rate of the biogenic amine is higher than its rate of metabolism. It appeared from the present study that an increased rate of synthesis of DA induced by the DA agonists was responsible for this discrepancy.