In vitro H-serotonin (5-HT) synthesis and release in BALBc and C57BL mice. I. Terminal areas

"In vitro," 3H-5-HT and 3H-5-HIAA newly synthesized from 3H-TRP are measured in the caudate nucleus and the hippocampus of C57BL and BALBc mice. Higher synthesis, utilization and release are to be found in C57BL than in BALBc strain. In the hippocampus of C57BL this higher synthesis is due both to higher tryptophan hydroxylase activity and to higher tryptophan uptake ability. But in the caudate nucleus the initial accumulation of tryptophan is similar in both strains. Finally the two forms of monoamine oxidase (A and B) show also similar activities in both strain. These data will be compared to those obtained at the nerve cell body level in the paper (II).     

Distribution and quantification of 5-HT nerve cell bodies in the nucleus raphe dorsalis area of C57BL and BALBc mice. Relationship between anatomy and biochemistry

BALBc and C57BL mice have been shown to have a different 5-HT metabolism. The present study compares the number and the distribution of 5-HT cell bodies in the nucleus raphe dorsalis area (B7 + B6) of these strains. By using 5-HT immunohistochemistry, we found a higher number of 5-HT neurons in the most caudal part of NRD (B6) of BALBc mice compared to C57BL. This difference may be correlated with a higher level of endogenous 5-HT, a higher uptake capacity toward exogenous [³H]5-HT, and a lower release of the amine in this same area of BALBc mice compared to C57BL. It could also imply a significant participation of the nerve cell bodies in the regulation of 5-HT transmission inside 5-HT nuclei.     

Endogenous serotonin (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) levels in large regions and in discrete brain areas of C57BL and BALBc mice at three times of the day

The serotoninergic system has been analysed in the brainstem and the forebrain of adult C57BL and BALBc mice. Endogenous 5-HT and 5-HIAA levels have also been measured in the different raphe nuclei and in the caudate nucleus and the hippocampus of these strains, using the micropunch technique and the radioenzymatic method (for 5-HT) and a new radioimmunoassay (for 5-HIAA). This investigation was performed at three times of the day: 9:00 hr; 14:00 hr; 20:00 hr to underline the differences between the strains. Most of the time, higher 5-HT and 5-HIAA levels are found in the brainstem and the raphe nuclei of the BALBc strain compared to C57BL. In the forebrain the differences are reversed, except in the caudate nucleus and the hippocampus where the levels of 5-HT are identical in the two strains. Moreover the 5-HIAA/5-HT ratio shows a higher turn-over of the amine in the C57BL strain.     

Developmental variations of brain serotonin, tryptophan, 5-hydroxyindole acetic acid, and noradrenaline and dopamine content in two inbred strains of mice

Endogenous levels of tryptophan (TRP), serotonin (5-HT), 5-hydroxyindol acetic acid (5-HIAA), noradrenaline (NA) and dopamine (DA), were measured in the brain-stem and anterior regions of C57BL and BALBc mice whose ages ranged from 1 day postnatal to 12 weeks. In C57BL mice levels of NA were found to be higher in the brainstem from week 1 onwards (except at week 3), and in the anterior region from week 6 onwards than in BALBc mice. 5-HT levels were found to be higher in C57BL mice from day 1 to week 2 in the brain-stem, and in the anterior region at week 1 and from week 5 onwards, compared to BALBc. The evolution of DA levels in the anterior region shows opposite differences before and after week 3-4. Before week 3 lower levels of DA were found in C57BL mice and the reverse is observed from week 5 onwards. Moreover during the postnatal development, the variation of the different components of the serotoninergic metabolism (TRP, 5-HT, 5-HIAA) is similar to that observed in other species.     

Raphe projections to the locus coeruleus in the rat

Afferent projections to the locus coeruleus from the various raphe nuclei, particularly of the midbrain (nuclei raphe dorsalis and medianus) and pons (nuclei raphe pontis and magnus), have been studied in the rat by retrograde transport methods using horseradish peroxidase (HRP). The locus coeruleus, in both its dorsomedial and ventrolateral divisions, and in its various anterior-posterior components, were injected with 0.05 μl of horseradish peroxidase following which various structures of the brainstem, particularly the raphe nuclei, were examined for HRP reactive cells. It was found that injections in most components of the locus coeruleus were associated with HRP positive cells in varying degrees of density in the nuclei raphe dorsalis, medianus, pontis, and magnus, with considerably sparser labelling in the anterior aspects of the medullary raphe nuclei pallidus and obscurus. Labelled cells were also seen in the nuclei of the solitary tract, contralateral locus coeruleus, lateral reticular areas of the pons and midbrain, nuclei pontis oralis and caudalis, vestibular nuclei, mesencephalic nucleus of the trigeminal nerve, fastigial nuclei of cerebellum and medial parabrachial nuclei. These data, showing widespread innervation of the locus coeruleus from all raphe nuclei, as well as many other brainstem areas, in the rat support the general view of heavy innervation of the locus coeruleus from both extra-raphe and raphe nulcei. These latter raphe projections, probably serotonergic in nature, provide anatomical support for the various experiments indicating considerable regulation of locus coeruleus activities, such as phasic events of REM sleep, among others, by most of the raphe nuclei. Thus, various activities of the locus coeruleus could be modulated or regulated by widespread projections from most raphe nuclei as well as several other regions of the brainstem.     

Alpha 4 nicotinic acetylcholine receptor subunit links cholinergic to brainstem monoaminergic neurotransmission

Agonists of nicotinic receptors containing the alpha4-subunit produce antinociception accompanied by several adverse side effects. The purpose of this study was to determine the distribution of the alpha4-subunit of nicotinic acetylcholine receptors (nAChR) in brainstem monoaminergic nuclei that may contribute to these effects using dual labeling immunofluorescence methods. The alpha4-subunit immunoreactivity was enriched in serotonergic (nucleus raphe magnus, pallidus, obscurus, and dorsalis) and noradrenergic (A5, locus coeruleus (LC), A7) areas associated with antinociception, where it was commonly colocalized with serotonin (5-HT) or tyrosine hydroxylase (TH) immunoreactivity. However, it was also noted that alpha4 was present in all other brainstem monoaminergic nuclei examined (adrenergic C1-C3, noradrenergic A1-alpha4, dopamine A9 and A10, nucleus raphe medianus). To determine if alpha4 agonists could impact neural activity in brainstem, monoaminergic nuclei that are associated with antinociception, the expression of c-Fos in response to the systemic administration of epibatidine (2.5, 5, or 10 microg/kg) was examined. Epibatidine produced a robust (2-5-fold) increase in c-Fos expression, which was not dose dependent, in all of these areas examined except the nucleus raphe magnus. These results suggest that the alpha4 subunit is positioned to mediate the effects of acetylcholine widely across many, if not all, monoaminergic neurons in the brainstem. These observations emphasize the potential involvement of noradrenergic, as well as serotonergic mechanisms in epibatidine's analgesic effects, and they also suggest that even selective alpha4 ligand may have widespread effects on brain monoamine neurotransmission.     

Brainstem enkephalinergic projections to spinal autonomic nuclei.

The present studies in the rat employed a combined retrograde transport-immunocytochemical technique to determine the origin in the brainstem of enkephalin (Enk) projections to spinal sympathetic nuclei, including the intermediolateralis nucleus, pars principalis (ILp). We found that Enk projections to the ILp nucleus are found in such serotonergic-containing areas as the raphe obscurus; raphe pallidus; gigantocellular reticular nucleus, pars alpha; paragigantocellular lateral nucleus; raphe magnus; and the rostral extension of the raphe magnus nucleus. The adrenergic-containing rostroventrolateral reticular nucleus as well as the noradrenergic-containing areas A5, A7, ventral locus coeruleus, subcoeruleus, and fiber pathway linking the locus coeruleus and A5/A7 send Enk projections to ILp. In the pons, a large contralateral Enk projection to spinal sympathetic nuclei was found medial to the facial nerve and medial to the motor nucleus of the trigeminal nerve. These observations show the existence of a large number of Enk brainstem regions that can influence spinal autonomic centers via descending supraspinal projections.     

Serotonergic, noradrenergic and galaninergic projections to the nucleus parafascicularis.

Immunocytochemical staining for serotonin (5-HT), tyrosine hydroxylase (TH) and galanin (GAL) was combined with horseradish peroxidase (HRP) retrograde tract-tracing technique to analyze the localizations of 5-HT-, catecholamine (CA)- and GAL-containing neurons in the brainstem which project to the nucleus parafascicularis (PF) in rats. It is demonstrated that most of the retrogradely HRP-labeled neurons (70%) in bilateral periaqueductal gray (PAG) and raphe nuclei are positively immunostained by antiserum to 5-HT, and that most of the retrogradely HRP-labeled neurons (over 80%) in bilateral locus coeruleus (LC) are positively immunostained by antisera to both TH and GAL. The possible functions of these PF-petal serotonergic, catecholaminergic (actually noradrenergic) and galaninergic projections are discussed.     

Acute effects of moclobemide and deprenyl on 5-HT synthesis rates in the rat brain: An autoradiographic study

Serotonin (5-HT), norepinephrine (NE) and dopamine (DA) released from nerve terminals in the brain are primarily removed from the synaptic cleft by a reuptake mechanism. In part, the homeostasis is maintained by monoamine oxidase (MAO) deamination achieved primarily intracellularly. The present study's aim was to examine the effect of the acute administration of the MAO inhibitors, moclobemide (a MAO-A inhibitor) and deprenyl (a MAO-B inhibitor), on 5-HT synthesis rates, measured in discrete regions of the rat brain by an autoradiographic method, using alpha-[14C]methyl-l-tryptophan as a tracer. MAO inhibitors have different effects on 5-HT synthesis rates in the cell bodies and areas of the nerve terminals. Moclobemide (10 mg/kg, i.p. 30 min before the tracer injection) and deprenyl (3 mg/kg, i.p. 2 h before the tracer injection) decreased the 5-HT synthesis rates in the dorsal (-18% and -22%) and median (-22% and -33%) raphe, respectively. Moclobemide also significantly decreased 5-HT synthesis in the entire nerve terminal areas investigated. The reductions were between 23% (cingulate cortex) and 50% (locus coeruleus). Deprenyl did not significantly affect 5-HT synthesis in the nerve terminals. The present results suggest that MAO-A, and to a lesser extent, MAO-B, are involved in the regulation of 5-HT synthesis in the rat brain. The mechanism(s) of MAO inhibitors' action on 5-HT synthesis in the raphe nuclei are probably related to an increase in the extraneuronal 5-HT concentration and also to the interaction between the serotonergic and catecholaminergic neurons. The reduction of 5-HT synthesis in the raphe nuclei likely occurs by an action of extracellular 5-HT via the dendritic autoreceptors with a possible contribution from the action of extracellular DA and NE. In the terminal regions, the most likely mechanism is via the presynaptic autoreceptors through which elevated extraneuronal 5-HT acts on synthesis control. However, there is also a possibility that the elevation in intraneuronal 5-HT directly inhibits its synthesis, especially following deprenyl treatment. A great influence of moclobemide on 5-HT synthesis could be related to its antidepressant action.     

Genetic studies of daily variations of first-step enzymes of monoamines metabolism in the brain of inbred strains of mice and hybrids. II. Daily variations of tyrosine hydroxylase activity in the locus coeruleus

Daily variations of tyrosine hydroxylase (TH) activity in the locus coeruleus of 3 inbred strains of mice (BALB/c; C57BL6; C57Br) and of the F₁ hybrids obtained from BALB/c and C57BL/6 are discussed. Precise characteristics of the circadian rhythms were observed in each strain. They were found significantly different in two genetically pure parents (BALB/c and C57BL6). In their two F₁ hybrids the daily variation of TH activity was similar to that observed in one of the parents (C57BL6). This strongly suggests selective and genetically controlled mechanisms of regulation responsible for the daily variation of TH activity in the locus coeruleus of mice.     

Alterations in tyrosine hydroxylase activity elicited by raphe nuclei lesions in the rat locus coeruleus: evidence for the involvement of serotonin afferents

The time course of the variations in tyrosine hydroxylase (TH) activity was measured in the rat locus coeruleus (LC) after lesions of the nucleus raphe dorsalis (NRD), nucleus raphe centralis superior (NRCS) and nucleus raphe pontis (NRP). A certain number of lesions were performed in the raphe magnus (RM), the caudal and rostral NRP and the caudal and rostral NRCS, lateral to raphe nuclei and in adrenalectomized animals. The serotonin (5-HT) content in the LC was also determined after these lesions. Only raphe nuclei producing significant decreases in the 5-HT content in the LC are successful in provoking increases in the TH activity in the LC, thus these results suggest that the noradrenaline (NA) synthesis in the LC may be regulated by 5-HT afferents. Moreover, intraventricular injections of 5,6-dihydroxytryptamine (5,6-DHT) and administration of parachlorophenylalanine (PCPA) also produce significant increases in TH in the LC. After immunotitrations of TH in the LC it was shown that, with exception of a high dose of 5,6-DHT (75 micrograms), all these treatments provoke an increase in the concentration of the enzyme. It therefore seems that one of the functional roles of 5-HT in the LC could be the regulation of the concentration of TH.     

Origins of the glycinergic inputs to the rat locus coeruleus and dorsal raphe nuclei: a study combining retrograde tracing with glycine immunohistochemistry

The amino acid glycine is a major inhibitory neurotransmitter in the brainstem and is likely involved in the tonic inhibition of the monoaminergic neurons during all sleep-waking stages. In order to determine the neurons at the origin of the glycinergic innervation of the two principal monoaminergic nuclei, the locus coeruleus and the dorsal raphe of the rat, we applied a double-labelling technique, combining retrograde transport of cholera-toxin B subunit with glycine immunohistochemistry. Using this technique, we found that the locus coeruleus and dorsal raphe nuclei receive a common glycinergic innervation from the ventral and ventrolateral periaqueductal grey, including the adjacent deep mesencephalic reticular nucleus. Small additional glycinergic inputs to these nuclei originated from the lateral paragigantocellular nucleus and the rostral ventromedial medullary reticular formation. The potential role of these glycinergic inputs in the control of the excitability of the monoaminergic neurons of the locus coeruleus and dorsal raphe nuclei is discussed.     

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.     

A comparative study of the immunohistochemical localization of a presumptive proctolin-like peptide, thyrotropin-releasing hormone and 5-hydroxytryptamine in the rat central nervous system

A proctolin (PROC)-like peptide was studied immunohistochemically in the hypothalamus, lower brainstem and spinal cord of the rat using an antiserum against PROC conjugated to thyroglobulin. Neuronal cell bodies containing PROC-like immunoreactivity (PROC-LI) were observed in the dorsomedial, paraventricular and supraoptic nuclei of the hypothalamus and in the nucleus raphe magnus, nucleus raphe pallidus, nucleus raphe obscurus and nucleus interfascicularis nervi hypoglossi in the medulla oblongata. Fibers containing PROC-LI were seen in the median eminence and in other hypothalamic nuclei, and in the lower brainstem in cranial motor nuclei including the dorsal motor nucleus of the vagus nerve, the motor trigeminal nucleus, the facial nucleus and nucleus ambiguous, and in lower numbers in the nucleus of the solitary tract and locus coeruleus. Fibers containing PROC-LI were also located in the spinal cord, in the intermediolateral cell column at thoracic levels and in the ventral horns at all levels of the spinal cord. After transection of the spinal cord, all PROC-immunoreactive fibers below the lesion disappeared. Following injection of Fast blue into the thoracic spinal cord, retrogradely labeled cells in the nuclei raphe pallidus, obscurus and magnus and nucleus interfasciculari nervi hypoglossi were seen to contain PROC-LI. PROC-LI had a similar distribution as thyrotropin-releasing hormone (TRH)-LI in the above-mentioned areas and coexistence of TRH-LI and PROC-LI was shown in cell bodies in the hypothalamus and medulla oblongata. PROC-LI could also be shown to coexist with 5-hydroxytryptamine (5-HT)-LI in neuronal cell bodies in the lower brainstem. The results demonstrate the occurrence of a PROC-like peptide in the mammalian nervous system, and these neurons seem to be at least largely identical to previously described TRH systems. A possible involvement of the PROC-like peptide in spinal motor control is discussed in relation to the well-established role of PROC in control of motor behavior in insects and invertebrates.     

Distribution and cellular localization of mRNA coding for 5-HT1A receptor in the rat brain: correlation with receptor binding.

In order to localize the cells expressing 5-HT1A receptors in the rat brain, we used in situ hybridization histochemistry to visualize the distribution of the mRNA coding for 5-HT1A receptors. Oligonucleotides derived from different parts of the coding region of the rat 5-HT1A receptor gene were used as hybridization probes. 5-HT1A binding sites were visualized on consecutive sections by receptor autoradiography using 3H-8-hydroxy-2-(di-n-propylamino)tetralin as ligand. The highest levels of hybridization were observed in the dorsal raphe nucleus, septum, hippocampus, entorhinal cortex, and interpeduncular nucleus. Positive hybridization signals were also present in other areas, such as the olfactory bulb; cerebral cortex; some thalamic and hypothalamic nuclei; several nuclei of the brainstem, including all the remaining raphe nuclei, nucleus of the solitary tract, and nucleus of the spinal tract of the trigeminus; and the dorsal horn of the spinal cord. The distribution and abundance of 5-HT1A receptor mRNA in different rat brain areas generally correlate with those of the binding sites, suggesting that 5-HT1A receptors are predominantly somatodendritic receptors.     

Dopamine and serotonin release in dorsal striatum and nucleus accumbens is differentially modulated by morphine in DBA/2J and C57BL/6J mice

Numerous studies have demonstrated that genetic factors significantly influence opioid ability to induce behavioral modification in mice. This differential sensitivity has been extensively studied, particularly in the DBA/2J and C57BL/6J strains. In the present study, using the "in vivo" microdialysis technique in these strains, we investigated the effect of morphine administration on the extracellular levels of dopamine (DA), serotonin (5-HT), and their metabolites in the nucleus accumbens and dorsal striatum--areas thought to be involved in morphine-induced locomotor hyperactivity. In the nucleus accumbens, morphine (20 mg/kg) significantly increased extracellular levels of DA in both strains. However, in dorsal striatum the morphine-induced increase of extracellular DA was lower in DBA/2J mice than in C57BL/6J. Moreover, morphine significantly stimulated 5-HT and 5-hydroxyindolacetic acid (5-HIAA) release both in nucleus accumbens and dorsal striatum of C57BL/6J mice, whereas it decreased 5-HT release without modifying 5-HIAA levels in DBA/2J mice. These results suggest that the different behavioral and biochemical responses to acute morphine described in these two strains could be mediated by different sensitivity of both the dopaminergic and the serotonergic systems.     

Immunohistochemical localization of serotoninergic, enkephalinergic, and catecholaminergic cells in the brainstem and diencephalon of a cartilaginous fish, Hydrolagus colliei

We localized serotonin (5-HT), leu-enkephalin (LENK), and tyrosine hydroxylase (TH) immunoreactive cells in the brain of a holocephalian, Hydrolagus colliei, by use of antibodies made in rabbit and the peroxidase-antiperoxidase technique. Only three locations contained TH+ cells, the caudal myelencephalon, the locus coeruleus, and the diencephalon. Of these locations, the diencephalon contained the most cells and the locus coeruleus the least cells. The caudal TH+ myelencephalic cells formed a single large group that spanned both the dorsal and ventral portions of the brain (A1A2). The diencephalic TH+ cells were located in the posterior tuberculum, in the ventromedial and ventrolateral thalamic nuclei, and in the inferior lobe of the hypothalamus. Hydrolagus differed from mammals and the elasmobranchs, their sister group, in that no substantia nigra (A9), ventral tegmental area (A10), or A5 cell group was found. Distribution of LENK+ and 5-HT+ cells were similar to each other; the raphe nuclei contained most of the 5-HT+ and LENK+ cells. These 5-HT+ and LENK+ cells were found at all rostrocaudal levels of the myelencephalon. The nucleus reticularis magnocellularis, reticularis paragigantocellularis lateralis, the ventral met- and mesencephalon (B7 and B9 cell groups), the hypothalamus, and the pretectal area contained additional 5-HT+ and LENK+ cells. The solitary complex contained LENK+ cells but not but 5-HT+ cells. A dorsal raphe nucleus, which is the largest 5-HT+ cell group in mammals, was absent in Hydrolagus. A dorsal raphe nucleus is present in one galeomorph shark radiation but is absent in three radiations of batoids (rays, skates, and guitarfish). Thus even within cartilaginous fish, there are differences in the distribution of neurochemicals and possibly nuclei within their brains.     

Differential effects ofp-chlorophenylalanine on indoleamines in brainstem nuclei and spinal cord of rats. II. Identification of immunohistochemically stained structures using computer-assisted image enhancement techniques

Following intraperitoneal injection ofp-chlorophenylalanine (PCPA, 400 and 600 mg/kg) on 3 consecutive days, the brainstem and lumbar cord of rats were removed, frozen-sectioned and immunohistochemically stained (PAP method) for serotonin (5-HT). Using computer-assisted image analysis, the density of 5-HT staining in control, 400 and 600 mg/kg PCPA groups was determined. The mean number of pixels (representing 5-HT staining) was determined in 6 areas in the brainstem containing 5-HT cell bodies (nuclei raphe pallidus, raphe obscurus, rostral and caudal raphe magnus, raphe dorsalis and paragigantocellularis lateralis) and in the dorsal and ventral spinal cord. The resultssuggest a differential depletion of 5-HT within brainstem nuclei following PCPA treatment in that the most marked dose-related reductions were observed in nucleus raphe obscurus and caudal magnus. Furthermore, a computer program designed to isolate terminal structures in the spinal cord identified a differential depletion of 5-HT terminals in the dorsal horn versus the ventral horn. The present study describes 3 analytical approaches combining immunohistochemistry with the computer-assisted image analysis technique and allows comparison between groups of animals which received the same or different drug treatments.     

Developmental regulation of spinal motoneurons by monoaminergic nerve fibers

1. In rats, both diameter and area of the cell bodies of spinal MNs increase rapidly during the first few postnatal weeks and slowly thereafter. The total dendritic length, radial extent and arbor area of spinal MNs also increase significantly throughout the first few postnatal weeks. This development is coincident with motor development in rat, which progresses rapidly during the first two to four weeks of life. The dendritic length and radial extent of spinal MNs increase more significantly in the cervical cord than in the lumbar cord throughout the first three postnatal days, and are possibly related to the motor development, with a rostro-caudal gradient. 2. All monoaminergic neurons projecting their axons to the spinal cord are located in the brainstem. namely in the locus coeruleus, the subcoeruleus and the medulla raphe nuclei in rats. The NA neurons of the locus coeruleus begin to be detected at ED 10-13, slightly earlier than the 5HT neurons in the raphe nuclei, which are first detected at ED 13. At ED 16, the NA fibers are seen in the ventral funiculus only at the cervical level, and many NA fibers are seen in the ventral horns at all levels at ED 18. The 5HT fibers reach the caudalmost levels of spinal cord by ED 16-17, which is earlier than NA fibers; this occurs in spite of the earlier ontogeny of NA neurons in the locus coeruleus than that of 5HT neurons in the raphe nuclei. 3. The monoamine system is thought to exert a variety of modulatory effects on target neurons during both pre- and postnatal periods, and many reports support the idea that monoamine systems have a "neurotrophic effect." On the other hand, important roles of NA and 5HT in MN activity and/or motor behavior have also been reported. It is suggested, therefore, that monoaminergic systems play important roles in motor development through a two-step mechanism: during early developmental stage. monoaminergic systems mainly act as neurotrophic agents on spinal MNs, which are the final motor output neurons; thereafter, they mainly play neuromodulatory roles on MN activities.