Distribution and synaptic relations of NOS neurons in the dorsal raphe nucleus: A comparison to 5-HT neurons

Anti-nitric oxide synthase antibody was used to study the distribution, cytowchhecture, and synaptic relations of nitric oxide synthase-like immunoreactive neurons in the whole rostral-caudel length of the dorsal raphe nucleus of the rat and compared them with serotonergic neurons. Results showed that the distribution of the nitric oxide synthase in the dorsal raphe nucleus was similar to that of the serotonergic neurons at the rostral part of the dorsal raphe nucleus, including the mediodorsal and the medioventral cell groups, and changed at the middle and caudal parts of the dorsal raphe nucleus. The cytoarchitecture of the nitric oxide synthase-like immunoreactive neurons in the medioventrai cell group of the dorsal raphe nucleus was similar to that of the serotonergic neurons. Similar to the serotonergic neurons there, nitric oxide synthase-like immunoroactive neurons also received synapses from axon terminals that contained round, or flattened vesicles, or both kinds. Different to the serotonergic neurons, the few nitric oxide synthase-like immunoroactive axon terminals that were in this area formed synapses.     

Further studies on the activation of rat median raphe serotonergic neurons by inescapable sound stress

Previous studies, using a biochemical measure of serotonergic neuronal function, show that inescapable, randomly presented sound pulses activate serotonergic neurons in the rat median raphe but not dorsal raphe nucleus. The present study reveals that this activation also occurs in serotonin projection areas, in hippocampus, nucleus accumbens and cortex but not in caudate nucleus. The selectivity of this response is examined by comparing the response to sound stress with that produced by morphine, a treatment known to selectively activate dorsal raphe but not median raphe serotonergic neurons. Two approaches are used in Sprague-Dawley rat to measure the activation of serotonergic neurons: (1) determination ex vivo of accumulation of 5-hydroxytryptophan (5-HTP) in tissue from the dorsal and median raphe nuclei, hippocampus, cortex, caudate nucleus, and nucleus accumbens following in vivo inhibition of aromatic amino acid decarboxylase; and (2) measurement of extracellular serotonin levels in hippocampus, caudate nucleus, and nucleus accumbens. Sound stress increases 5-HTP accumulation in median raphe nucleus, hippocampus, cortex, and nucleus accumbens, but not dorsal raphe nucleus or caudate nucleus. Sound stress also enhances extracellular serotonin levels in hippocampus and nucleus accumbens, but not caudate nucleus. In contrast, the morphine treatment enhances 5-HTP accumulation in dorsal raphe nucleus, cortex and caudate nucleus, but not in median raphe nucleus, hippocampus or nucleus accumbens. Furthermore, it increases extracellular serotonin levels in only the caudate nucleus. The combined effects of sound stress and morphine on 5-HTP accumulation are identical to those obtained by each treatment individually. These findings provide further support for the presence of serotonergic neurons within the median raphe nucleus that have a unique response profile. These neurons may have an important role in responses or adaptations to stress.     

Origin of the serotonergic innervation to the rat dorsolateral hypothalamus: retrograde transport of cholera toxin and upregulation of tryptophan hydroxylase mRNA expression following selective nerve terminals lesion.

The regulation of serotonin synthesis was investigated in the serotonergic neurons, which provide afferents to the dorsolateral hypothalamus (DLH). The origin of the DLH projection neurons within the raphe nucleus was identified by retrograde transport of Cholera toxin (CTb) and their serotonergic nature confirmed by tryptophan hydroxylase (TPH) immunocytochemistry. Disruption of serotonin synthesis steady-state was induced unilaterally by a selective and local destruction of serotonergic nerve terminals with 5,7-dihydroxytryptamine (5,7-DHT), stereotaxically injected in the right DLH. The results show that most of the serotonergic dorsal raphe neurons projecting to the DLH have an ipsilateral localization within the lateral aspects of the nucleus. In rats with unilateral DLH lesion, a population of serotonergic cells within the raphe nucleus exhibited a clear increase in TPH mRNA. These cells were about five times more numerous in the ipsilateral as compared to the contralateral dorsal raphe nucleus and they had, for the most part, a lateral localization within the raphe nucleus. Sham-operated rats did not exhibit any upregulation of TPH mRNA. Together, the present results provide the first demonstration that a discreet and selective destruction of serotonergic terminals induces a circumscribed and striking increase in TPH mRNA expression in a subset of brainstem serotonergic neurons projecting to and/or passing through the DLH. On the basis of these results and previous in vivo measurements of TPH activity (e.g., 5-HT synthesis), we suggest that this upregulation in TPH mRNA expression results from the loss of pre-synaptic and/or post-synaptic regulation of serotonin synthesis. These new findings raise important issues related to the repercussions of a local disruption in serotonergic neurotransmission on brain areas remote from the site of injury.     

Evidence of the modulatory role of serotonin in acetylcholine release from striatal interneurons

The release of acetylcholine was studied in isolated striatal slices of the rat. The spontaneous and ouabain-stimulated release of acetylcholine was higher in those slices where serotonergic input was somehow impaired: raphe nuclei lesion orp-chlorophenylalanine pretreatment or 5,7-dihydroxytryptamine pretreatment resulted in a higher release.l-(m-chlorophenyl)-piperazine, a pure serotonin receptor stimulant andd-fenfluramine, a serotonin releaser significantly reduced the release of acetylcholine evoked by ouabain. Serotonin antagonists (cyproheptadine, mianserine and methysergide) prevented the effect of serotonin agonists. When the serotonergic neurons were destroyed either byp-chlorophenylalanine or by 5,7-dihydroxytryptamine pretreatmentd-fenfluramine had no inhibitory action; however, the effect ofl(m-chlorophenyl)-piperazine was not affected.It is suggested that there is a link between serotonergic and cholinergic neurons in the striatum: serotonin released from raphe-striatal neurons is able to inhibit the release of acetylcholine from striatal interneurons.     

Dorsal raphe neurons: self-inhibition by an amphetamine-induced release of endogenous serotonin

A direct infusion of amphetamine into the dorsal raphe nucleus of the rat inhibited the activity of serotonergic neurons in this site. An intravenous injection of 5-methoxy-N,N-dimethyltryptamine, a serotonin autoreceptor agonist, mimicked this effect. The amphetamine-induced depression of firing rate was blocked by a subsequent injection of methiothepin, a putative serotonin autoreceptor antagonist, but not by pretreatment with α-methyl-p-tyrosine which depletes brain catecholamines. Amphetamine infusions into the surrounding periaqueductal gray or brainstem reticular formation failed to change dorsal raphe activity. The results of these studies indicate that endogenous serotonin, which can be released by a direct infusion of amphetamine, suppresses neuronal activity in the dorsal raphe nucleus by a process of self-inhibition.     

Evidence that serotonergic neurons in the dorsal raphe nucleus exert a stimulatory effect on the secretion of renin but not of corticosterone

Data from previous experiments in rats indicate that release of serotonin in the central nervous system increases renin and corticosterone secretion. To determine which serotonergic neurons are involved, lesions of the dorsal or median raphe nuclei were produced by local injections of 5,7-dihydroxytryptamine (5,7-DHT) in rats, and 2 weeks later, the renin responses to parachloroamphetamine (PCA) were determined. Plasma corticosterone was also measured. PCA produced significant increases in plasma renin activity and plasma corticosterone in sham-lesioned animals and animals with median raphe lesions. The plasma corticosterone response to PCA was also normal in rats with dorsal raphe lesions but the renin response was significantly reduced. The data support the hypothesis that serotonergic neurons in the dorsal raphe nucleus are part of a neural pathway mediating increased renin secretion, and that the stimulatory effect of serotonin on corticosterone secretion is mediated by a different pathway.     

Melanin-concentrating hormone does not modulate serotonin release in primary cultures of fetal raphe nucleus neurons

Melanin-concentrating hormone (MCH) is a neuropeptide present in neurons located in the hypothalamus that densely innervate serotonergic cells in the dorsal raphe nucleus (DRN). MCH administration into the DRN induces a depressive-like effect through a serotonergic mechanism. To further understand the interaction between MCH and serotonin, we used primary cultured serotonergic neurons to evaluate the effect of MCH on serotonergic release and metabolism by HPLC-ED measurement of serotonin (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) levels. We confirmed the presence of serotonergic neurons in the E14 rat rhombencephalon by immunohistochemistry and showed for the first time evidence of MCHergic fibers reaching the area. Cultures obtained from rhombencephalic tissue presented 2.2 ± 0.7% of serotonergic and 48.9 ± 5.4% of GABAergic neurons. Despite the low concentration of serotonergic neurons, we were able to measure basal cellular and extracellular levels of 5-HT and 5-HIAA without the addition of any serotonergic-enhancer drug. As expected, 5-HT release was calcium-dependent and induced by depolarization. 5-HT extracellular levels were significantly increased by incubation with serotonin reuptake inhibitors (citalopram and nortriptyline) and a monoamine-oxidase inhibitor (clorgyline), and were not significantly modified by a 5-HT1A autoreceptor agonist (8-OHDPAT). Even though serotonergic cells responded as expected to these pharmacological treatments, MCH did not induce significant modifications of 5-HT and 5-HIAA extracellular levels in the cultures. Despite this unexpected result, we consider that assessment of 5-HT and 5-HIAA levels in primary serotonergic cultures may be an adequate approach to study the effect of other drugs and modulators on serotonin release, uptake and turnover.     

Corticotropin-releasing factor in the dorsal raphe nucleus increases medial prefrontal cortical serotonin via type 2 receptors and median raphe nucleus activity

Interactions between central corticotropin-releasing factor (CRF) and serotonergic systems are believed to be important for mediating fear and anxiety behaviors. Recently we demonstrated that infusions of CRF into the rat dorsal raphe nucleus result in a delayed increase in serotonin release within the medial prefrontal cortex that coincided with a reduction in fear behavior. The current studies were designed to study the CRF receptor mechanisms and pathways involved in this serotonergic response. Infusions of CRF (0.5 μg/0.5 μL) were made into the dorsal raphe nucleus of urethane-anesthetized rats following either inactivation of the median raphe nucleus by muscimol (25 ng/0.25 μL) or antagonism of CRF receptor type 1 or CRF receptor type 2 in the dorsal raphe nucleus with antalarmin (25–50 ng/0.5 μL) or antisauvagine-30 (2 μg/0.5 μL), respectively. Medial prefrontal cortex serotonin levels were measured using in-vivo microdialysis and high-performance liquid chromatography with electrochemical detection. Increased medial prefrontal cortex serotonin release elicited by CRF infusion into the dorsal raphe nucleus was abolished by inactivation of the median raphe nucleus. Furthermore, antagonism of CRF receptor type 2 but not CRF receptor type 1 in the dorsal raphe nucleus abolished CRF-induced increases in medial prefrontal cortex serotonin. Follow-up studies involved electrical stimulation of the central nucleus of the amygdala, a source of CRF afferents to the dorsal raphe nucleus. Activation of the central nucleus increased medial prefrontal cortex serotonin release. This response was blocked by CRF receptor type 2 antagonism in the dorsal raphe. Overall, these results highlight complex CRF modulation of medial prefrontal cortex serotonergic activity at the level of the raphe nuclei.     

Involvement of lateral habenula-dorsal raphe neurons in the differential regulation of striatal and nigral serotonergic transmission cats

The importance of the lateral habenula-dorsal raphe pathway in the control of in vivo [3H]serotonin release in the cat basal ganglia was examined using the push-pull cannula technique and an isotopic method for the estimation of [3H]serotonin continuously formed from [3H]tryptophan. [3H]Serotonin was measured in both caudate nuclei and substantiae nigra and, in some cases, in the dorsal raphe. Electrical stimulation of the lateral habenula decreased [3H]serotonin release in all structures studied. Blockade of the GABA inhibitory pathway to the lateral habenula by the local application of picrotoxin reduced [3H]serotonin release in both substantiae nigra and increased release of the 3H-amine in the dorsal raphe but was without effect on [3H]serotonin release in either caudate nucleus. This inhibition of nigral [3H]serotonin release was antagonized by simultaneous application of picrotoxin to the dorsal raphe. Substance P delivery to the dorsal raphe produced the same effects on [3H]serotonin release as described for picrotoxin application to the lateral habenula except that inhibition of nigral [3H]serotonin release was not prevented by local co-administration of picrotoxin. These results suggest that the lateral habenula can control serotonergic transmission in the basal ganglia and that this regulation may be different for those serotonergic neurons innervating the caudate nucleus versus those projecting to the substantia nigra.     

Selective effects of apomorphine on dorsal raphe neurons: A cytofluorimetric study

Using a quantitative cytofluorimetric method to detect changes in the intracellular levels of serotonin (5-HT) in individual neurons in rat brain, we have found that the dopaminergic agonist apomorphine increases 5-HT content of dorsal raphe cell bodies without affecting cells in the median raphe nucleus. Liquid chromatographic studies revealed that apomorphine also elevated the concentrations of both 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in striatum, the projection site for dorsal raphe neurons. Conversely, the dopaminergic antagonist haloperidol, at a dose of 0.8 mg/kg, decreased 5-HT levels in dorsal raphe cells. A lower dose of haloperidol (0.4 mg/kg), which had no significant effect alone, completely blocked the effect of apomorphine in the dorsal raphe. These results support the hypothesis that the effects of apomorphine on serotonergic neurons are secondary to dopamine receptor stimulation.     

Inescapable shock activates serotonergic neurons in all raphe nuclei of rat

Animal studies examining the effects of stress upon brain serotonergic neurons have not presented a clearcut and consistent picture. One stressor that has been shown to exert a consistently strong effect on serotonin release and c-fos activation in the dorsal raphe nucleus of rats is a series of inescapable electrical shocks. Using immunohistochemical double labeling for c-fos activation and serotonin, we examined the effects of delivering 100 inescapable tailshocks to rats on serotonergic neuronal activation throughout the brainstem raphe system. This stimulus exerted a consistent and strong activation of the entire midline brain stem system of serotonergic neurons. The implications of these findings for animal models of human psychopathology are discussed.     

Evidence for decreased transport of tryptophan hydroxylase in Alzheimer''s disease

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the synthesis of serotonin and a specific marker for serotonergic neurons. These neurons are affected in Alzheimer's disease (AD) in several ways: serotonin is decreased in axon terminals, serotonin neurons accumulate neurofibrillary protein, and these neurons are lost in AD brains. One subcellular mechanism which may underlie degeneration of neurons in AD is decreased axonal transport with accumulation of enzymes and their potentially toxic metabolites in the cell body. To determine whether there is a defect in axonal transport in serotonin neurons in AD we measured TPH activity, serotonin and its oxidative metabolite 5-hydroxyindoleacetic acid (5-HIAA) in dorsal raphe cell bodies from Alzheimer and control cases. TPH activity is increased 4.7-fold in raphe neuron cell bodies in Alzheimer brains. Serotonin and 5-HIAA are increased by 4.0- and 2.0-fold, respectively in Alzheimer compared to control raphe cell bodies. In contrast, in synaptic terminals of the amygdala 5-HT and 5-HIAA were decreased by 41% and 50%, respectively in the same AD cases. We propose that the accumulation of TPH and its products in the raphe perikarya in AD results from a diminished transport of TPH to axon terminals. The accumulation of oxidative metabolites of serotonin may contribute to the degeneration of serotonergic neurons in AD.     

Gabaergic interneurons in the dorsal raphe mediate the effects of apomorphine on serotonergic system

Apomorphine (APO) has been shown to elevate the concentrations of serotonin (5-HT) and its major metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the mesostriatal but not the mesolimbic serotonergic systems. We have previously demonstrated that the serotonergic actions of APO were secondary to dopamine (DA) autoreceptor stimulation in the substantia nigra. Using picrotoxin as a pharmacological tool, we have presently found that these effects of APO were also indirectly mediated through gamma-aminobutyric acid (GABA) neurons. In examination of the exact anatomical locus of GABA neurons responsible for the observed effects of APO, the results indicate that bilateral lateral habenular lesions did not block the effects of APO on 5-HT neurons, while direct picrotoxin infusion to the dorsal raphe, at a dose having no significant influence by itself, antagonized APO's actions. Together with the anatomical, biochemical and histofluorescent findings, it is suggested that APO influences dorsal raphe 5-HT by stimulation of DA autoreceptors in the substantia nigra; therefore, inhibition of DA neuron activity and the nigro-raphe pathway. Normally, DA probably exerts an excitatory influence on gabaergic interneurons in the dorsal raphe, and these inhibitory interneurons then synapse on 5-HT neurons in the same area. Activation of 5-HT neurons were explained by a disinhibitory effect as a result of reduced release of GABA due to feedback inhibition of DA neuron firing following APO activation of DA autoreceptors in the substantia nigra. The striatal presynaptic and postsynaptic DA receptors, however, do not appear to mediate the above effects of APO.     

Stimulation of the nucleus raphe obscurus produces marked serotonin release into the dorsal medulla of fed but not fasted rats — glutamatergic dependence

Serotonin interacts with TRH at the dorsal vagal complex (DVC) to augment gastric functional parameters. To ascertain physiologic relevance, patterns of stimulated release at the terminal field were characterized. Stimulation of the nucleus raphe obscurus (nRO) by kainic acid (423 pmol/10 nl) produced marked release of serotonin into dorsal medullary dialysates containing the DVC in freely fed, but not 24-h fasted rats. Probe infusion of kynurenic acid (1 mM), but not acute bilateral cervical vagotomy attenuated nRO-stimulated serotonin release in fed animals. The results suggest that the fed state facilitates serotonin release into the dorsal medulla by a mechanism mediated by activation of excitatory amino acid receptors in the dorsal medulla. Enhanced serotonergic neurotransmission at the DVC may comprise a heretofore unrecognized component of the integrated vago-vagal response to a meal.     

Superior colliculus stimulation enhances neocortical serotonin release and electrocorticographic activation in the urethane-anesthetized rat

Recent evidence indicates that the superior colliculus (SC), in addition to its functions in sensory detection, also participates in controlling the generalized activation state of the forebrain, as measured by the electroencephalogram (EEG) or electrocorticogram (ECoG). The mechanisms by which the SC modulates forebrain activation are not well understood. By using in vivo microdialysis, we examined the role of serotonin release as a mechanism by which the SC can control neocortical activity in the urethane-anesthetized rat. Electrical 100 Hz stimulation of the SC increased frontal cortex serotonin output to 116, 118, and 140% of baseline levels for stimulation intensities of 0.5, 0.75, and 1.0 mA, respectively. Further, 75% of extracellularly recorded single (putative serotonergic) dorsal raphe neurons increased their discharge rate in response to 100 Hz stimulation of the SC. Stimulation of the SC also suppressed frontal cortex low frequency (1-6 Hz) synchronized ECoG activity, replacing it with high-frequency desynchronization. This activation response was resistant to cholinergic-muscarinic receptor antagonists (atropine, 50 mg/kg; scopolamine, 2 mg/kg), but was reduced or abolished by systemic treatment with the serotonergic receptor antagonists ketanserin (10 mg/kg) or methiothepin (5 mg/kg). These data suggest that efferents from the SC, possibly by an excitatory action on serotonergic dorsal raphe cells, produce an enhanced release of serotonin and ECoG activation in the neocortex. The stimulation of cortical serotonin output may constitute a mechanism by which the SC acts on the forebrain to increase cortical excitability in response to sensory stimuli processed by SC neurons.     

Effects of MDMA ('ecstasy') on firing rates of serotonergic, dopaminergic, and noradrenergic neurons in the rat

3,4-Methylenedioxymethamphetamine (MDMA), a non-hallucinogenic drug of abuse, potently depressed firing rates of a subpopulation of serotonin neurons in the dorsal and median raphe. High neurotoxic doses depressed those serotonin neurons unresponsive to low doses. Noradrenaline neurons in the locus coeruleus were also depressed by moderate doses. Dopamine neurons were unaffected. It is concluded that MDMA's unique psychological effects are mediated through a subpopulation of serotonergic and noradrenergic neurons, presumably through effects on release mechanisms.     

In vivo-synthesized radioactively labelled α-methyl serotonin as a selective tracer for visualization of brain serotonin neurons

To investigate the use of α-[³H]methyl tryptophan (α-[³H]MTrp) as a tracer for the in vivo study of brain serotonergic neurons, we examined whether α-[³H]MTrp and its metabolite α-[³H]methyl serotonin (α-[³H]M5-HT) selectively label serotonergic neurons and whether once accumulated in these neurons, the radioactive metabolite behaves like endogenous serotonin. Rats received a systemic injection of 1–5 mCi of α-[³H]MTrp and 24 h later their brains were immediately removed or fixed by perfusion before removal. Tissue sections in which serotonergic neurons had been immunostained for 5-HT or its synthesizing enzyme, tryptophan hydroxylase, were processed for radioautography at the light and electron microscopic level. In another group of rats, the release of radioactivity from different brain areas was studied both under basal and depolarizing conditions. In the dorsal raphe nucleus, the light microscopic examination revealed almost complete colocalization between serotonergic neurons and those that accumulated radioactivity, with a heterogeneity in the content of α-[³H]M5-HT among the various cells. At the ultrastructural level, immunoidentified serotonergic perikarya and dendritic processes in the dorsal raphe nucleus, as well as nerve terminals in the cerebral cortex were also found to contain α-[³H]M5-HT. Under basal conditions, radioactivity was released from the brainstem raphe region and from projection areas such as the striatum and hippocampus. The basal output of α-[³H]M5-HT increased approximately twofold after a depolarizing 50 mM KCl solution was added to the perfusion fluid. These findings suggest that newly synthesized α-[³H]M5-HT can be released both at somatodendritic and terminal sites. In sum, the present results demonstrate the selectivity of α-[³H]MTrp as a tracer for serotonergic cells, and further suggest that α-[³H]MTrp radiolabelling provides for a direct assessment of the in vivo dynamics of brain serotonergic neurons at the cellular level. © 1995 Wiley-Liss, Inc.     

鱼藤酮对大鼠脑内5-羟色胺能神经元损伤的机制研究

目的研究鱼藤酮对大鼠脑内5-羟色胺能神经元的毒性作用及其机制。方法健康成年雄性Wistar大鼠背部皮下注射鱼藤酮制备帕金森病动物模型。采用免疫组化、蛋白印迹及分光吸光度法检测大鼠中脑中缝背核5-羟色胺能神经元的损伤及中缝背核氧化应激参数(丙二醛和还原型谷胱甘肽)的改变。结果和对照组相比,鱼藤酮组大鼠脑内中缝背核5-羟色胺(5-HT)免疫反应阳性神经元数明显少于对照组(P<0.01),Western blot结果显示中缝背核5-HT表达在鱼藤酮组明显降低(P<0.01);鱼藤酮组大鼠中缝背核区域丙二醛含量明显增高(P<0.01)、还原型谷胱甘肽(GSH)含量明显减少(P<0.01)。结论鱼藤酮在损伤大鼠脑内多巴胺能神经元的同时对5-HT能神经元也具有明显的毒性作用,氧化应激可能是导致5-HT神经元损伤的主要原因。     

Fibroblast Growth Factor-5 Promotes Differentiation of Cultured Rat Septal Cholinergic and Raphe Serotonergic Neurons: Comparison with the Effects of Neurotrophins

Fibroblast growth factor-5 (FGF-5) is a member of the fibroblast growth factor gene family, which has a signal sequence characteristic of secretory proteins. FGF-5 mRNA has previously been shown to be present in the adult mouse brain. Here we demonstrate that recombinant FGF-5 has neurotrophic activity on cultured rat septal cholinergic and raphe serotonergic neurons. The effect of FGF-5 on serotonin uptake was stronger than that evoked with either brain-derived neurotrophic factor or neurotrophin-3. FGF-5 also increased the choline acetyltransferase activity of cultured rat septal cholinergic neurons, the effect being additive to that of nerve growth factor. In situ hybridization experiments and immunohistochemistry using a specific anti-FGF-5 antibody demonstrated that FGF-5 is expressed in rat hippocampal neurons. Like nerve growth factor mRNA, the levels of FGF-5 mRNA in the rat hippocampus increased substantially during early postnatal development. In addition, injection of the muscarinic receptor agonist pilocarpine elevated FGF-5 mRNA. The presence of the secretory FGF-5 in the rat hippocampus, a target field of septal cholinergic and raphe serotonergic neurons, suggests that FGF-5 acts as a trophic factor for these neurons also in vivo.