The role of serotonergic neurons in dorsal raphe, median raphe and anterior hypothalamic pressor mechanisms

The role of serotonergic neurons in the dorsal raphe and median raphe in the pressor response to electrical stimulation of these areas, and the contribution of these neurons to the pressor response to serotonin (5-HT) in the anterior hypothalamus-preoptic area (AH/PO) have been studied by the use of local injections of 5,7-dihydroxytryptamine (5,7-DHT), a neurotoxin selective for 5-hydroxytryptamine (5-HT). When blood pressure was recorded in urethane-anesthetized rats, selective lesions of 5-HT-containing neurons in the dorsal raphe nucleus reduced by 60% the pressor response to electrical stimulation (50 Hz, 100-150 microA, 0.3 msec pulse duration) of this nucleus. On the other hand, selective lesion of 5-HT-containing neurons in the median raphe nucleus had no effect on the pressor response to electrical stimulation of this area. Injection of 5,7-dihydroxytryptamine into the anterior hypothalamus/preoptic area resulted in an increased pressor response to the injection of 5-HT (5 nmol) into the lesioned area 10 days later. Furthermore, the destruction of 5-HT-containing neurons in the dorsal raphe nucleus resulted in an enhanced pressor response to the injection of 5-HT (5 nmol) into the anterior hypothalamus/preoptic area, while the destruction of 5-HT-containing neurons in the median raphe nucleus had no effect on the pressor response to the injection of 5-HT (5 nmol) into the anterior hypothalamus/preoptic area. Therefore, it appears that 5-HT neurons in the dorsal raphe nucleus are important in the pressor response to electrical stimulation and are involved in a pressor mechanism in the anterior hypothalamus/preoptic area.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential mechanisms underlie the regulation of serotonergic transmission in the dorsal and median raphe nuclei by mirtazapine: a dual probe microdialysis study

Rationale

Blockade of α₂ adrenoceptors and histamine H₁ receptors plays important roles in the antidepressant and hypnotic effects of mirtazapine.

Objectives

However, it remains unclear how mirtazapine’s actions at these receptors interact to affect serotonergic transmission in the dorsal (DRN) and median (MRN) raphe nuclei.

Method

Using dual-probe microdialysis, we determined the roles of α₂ and H₁ receptors in the effects of mirtazapine on serotonergic transmission in the DRN and MRN and their respective projection regions, the frontal (FC) and entorhinal (EC) cortices.

Results

Mirtazapine (<30 μM) failed to alter extracellular serotonin levels when perfused alone into the raphe nuclei, but when co-perfused with a 5-HT_1A receptor antagonist, mirtazapine increased serotonin levels in the DRN, MRN, FC, and EC. Serotonin levels in the DRN and FC were decreased by blockade and increased by activation of H₁ receptors in the DRN. Serotonin levels in the MRN and EC were not affected by H₁ agonists/antagonists perfused in the MRN. The increase in serotonin levels in the DRN and FC induced by DRN H₁ receptor activation was attenuated by co-perfusion with mirtazapine. Furthermore, the increase in serotonin levels (DRN/FC) induced by DRN α₂ adrenoceptor blockade was attenuated by concurrent DRN H₁ blockade, whereas the increase in serotonin levels (MRN/EC) induced by MRN α₂ adrenoceptor inhibition was unaffected by concurrent MRN H₁ receptor blockade.

Conclusion

These results suggest that enhanced serotonergic transmission resulting from α₂ adrenoceptor blockade is offset by subsequent activation of 5-HT_1A receptors and, in the DRN but not MRN, H₁ receptor inhibition. These pharmacological actions of mirtazapine may explain its antidepressant and hypnotic actions.     

Differential clonidine effects on EEG following lesions of the dorsal and median raphe nuclei in rats

The effects of clonidine on EEG activity and gross behavior were studied in rats with electrolytic lesions of the median (MR) and dorsal (DR) raphe nuclei. Lesioned animals showed significant depletion in forebrain serotonin concentrations. Clonidine (0.1 mg/kg and 0.2 mg/kg IP) produced synchronization in cortical EEG pattern and markedly increased alpha and theta activities in unlesioned animals. Clonidine treatment resulted also in a sedative response. In MR lesioned rats clonidine effect upon EEG was significantly reduced and, additionally, sedative response was not seen. On the other hand clonidine effect on EEG was markedly increased in rats with lesioned DR. These results are discussed on the basis of possible interaction between serotonergic and noradrenergic neurons in the brain.     

Correction to: Differential mechanisms underlie the regulation of serotonergic transmission in the dorsal and median raphe nuclei by mirtazapine: a dual probe microdialysis study

Psychopharmacology - The authors have made unintentional errors in data analysis and consequently in four figures.     

Serotonin depletion in the dorsal and ventral hippocampus: effects on locomotor hyperactivity, prepulse inhibition and learning and memory

We present an overview of our studies on the differential role of serotonergic projections from the median raphe nucleus (MRN) and dorsal raphe nucleus (DRN) in behavioural animal models with relevance to schizophrenia. Stereotaxic microinjection of the serotonin neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) into the MRN or one of its main projections regions, the dorsal hippocampus, induced a marked enhancement of phencyclidine-induced locomotor hyperactivity and a disruption of prepulse inhibition (PPI) in rats. There was no enhancement of locomotor hyperactivity induced by amphetamine or MK-801 or after 5,7-DHT lesions of the DRN or ventral hippocampus. Rats with dorsal hippocampus lesions did not show significant changes in the Y-maze test for short-term spatial memory, the Morris water maze for long-term spatial memory, or in the T-maze delayed alternation test for working memory. These chronic lesion studies suggest a modulatory influence of serotonergic projections from the MRN to the dorsal hippocampus on phencyclidine effects and prepulse inhibition, but not on different forms of learning and memory. The results provide new insight into the role of serotonin in the dorsal hippocampus in aspects of schizophrenia.     

中缝背核投射到基底外侧杏仁核的5-羟色胺能纤维对睡眠的调节作用

目的　观察大鼠中缝背核 (DRN)到基底外侧杏仁核(BLA)的 5 HT能纤维投射在睡眠 -觉醒调节中的作用。方法　采用脑立体定位 ,核团微量注射和多导睡眠描记 (PSG)方法。结果　DRN内微量注射L Glu ,可使觉醒 (W )增加 ,慢波睡眠 (SWS)和异相睡眠 (PS)明显减少。在双侧BLA微量注射非选择性 5 HT受体阻断剂麦角新碱 (MS)可以逆转DRN内微量注射L Glu的效应 ,SWS增加 ,W减少 ,但PS没有变化 ;DRN内微量注射PCPA ,导致SWS增加 ,W减少 ,但当在DRN内微量注射PCPA后 ,双侧BLA内微量注射 5 HTP可以逆转PCPA所引起的睡眠增加效应 ,使SWS减少 ,W增加 ,但PS没有变化。结论　DRN对睡眠 -觉醒的调节作用部分通过DRN到BLA的 5 HT能纤维投射介导的     

Collateral inhibition of serotonergic neurones in the rat dorsal raphe nucleus: pharmacological evidence.

Previous studies have shown that serotonergic (5-HT) neurones in the dorsal raphe are inhibited following electrical stimulation of the major ascending 5-HT fibre pathway in the ventromedial tegmentum (VMT); this effect appears to be mediated directly through 5-HT axon collaterals. This possibility was tested further in the present study by pharmacological manipulations of the 5-HT system. When rats were pretreated with either para-chlorophenylalanine (P-CPA, which inhibits trypto phan hydroxylase, the rate-limiting enzyme for the 5-HT synthesis) or reserpine (which is believed to block storage of biogenic amines) or both, the inhibitory responses of antidromically identified 5-HT neurones to VMT stimulation were either totally prevented or markedly reduced in the great majority of cells tested. Furthermore, the P-CPA effects were reversed by the injection of 5-hydroxytryptophan (the immediate precursor of 5-HT which bypasses the synthesis step inhibited by P-CPA); low doses of l-dihydroxyphenylalanine (a precursor of catecholamines) were not effective. In some cells after the drug treatment, prolonged periods of suppression of firing of 5-HT cells still occurred following VMT stimulation. In such cells, continuous stimulation of the VMT (at 10 Hz for 3 min). which presumably depleted residual transmitter, diminished the VMT-induced effect. Of some possible 5-HT antagonists tested, only metergoline (1–2 mg/kg, i.v.) was effective in antagonizing the VMT induced depressant effect on 5-HT neurones; however, the development of the effect was extremely delayed. The present results show that the VMT-induced depression of firing of 5-HT neurones depends on the availability of 5-HT. This finding is consistent with the view that the inhibitory responses of 5-HT neurones to VMT stimulation are mediated directly through 5-HT axon collaterals (or dendo-dendritic junctions).     

Different behavioral responses of rats to kainate injections into the dorsal and median raphe nuclei

A local injection of kainic acid (KA) into the dorsal raphe nucleus (NRD) increased the motor activity and produced head shakes, hind limb abduction, forepaw treading and sniffing. This syndrome was antagonized partly by cyproheptadine and completely by naloxone. An injection of KA into the median raphe nucleus (NRM) produced sedation, catalepsy and analgesia, which were accompanied by a decrease in the beta-endorphin immunoreactivity in the mesencephalon. Naloxone completely reversed the behavioral inhibition after KA injections into the NRM, while a pretreatment with cyproheptadine augmented the catalepsy. KA injected into the NRD and NRM depressed the forebrain level of serotonin and slightly elevated that of 5-hydroxyindoleacetic acid. The results demonstrate that besides serotonin the opioid system is also involved in various effects induced by activation of the raphe nuclei.     

Modulatory effect of p-chlorophenylalanine microinjected into the dorsal and median raphe nuclei on cocaine-induced behaviour in the rat.

The present study examined whether a potentiation of cocaine-induced behaviour in rats following peripheral pretreatment with the 5-hydroxytryptamine (5-HT) biosynthesis inhibitor p-chlorophenylalanine may be due to depletion of 5-HT in the dorsal raphe nucleus and/or median raphe nucleus. Following peripheral pretreatment with p-chlorophenylalanine (100 mg/kg, i.p.) for 3 consecutive days, a potentiation of cocaine-induced locomotor activity and rears was observed. To investigate a possible involvement of serotonergic neurones arising in the midbrain raphe nuclei in the observed potentiation, p-chlorophenylalanine (0.5 microg) was microinjected in either the dorsal raphe nucleus or median raphe nucleus followed by behavioural testing 48 h later. Application of p-chlorophenylalanine in the dorsal raphe nucleus resulted in an enhancement of cocaine-induced locomotor activity and head bobs. In contrast, the stimulant effect of cocaine on behaviour was not altered by microinjection of p-chlorophenylalanine in the median raphe nucleus. Peripheral and central administration of p-chlorophenylalanine did not consistently alter the baseline behaviour of saline-treated animals. Biochemical results indicated only a moderate depletion of 5-HT in the midbrain raphe nuclei following peripheral p-chlorophenylalanine administration. Surprisingly, the central application of p-chlorophenylalanine in the dorsal raphe nucleus and median raphe nucleus did not alter the 5-HT levels in the midbrain raphe nucleus investigated. In addition, peripheral and central administration of p-chlorophenylalanine did not alter the 5-HT levels in the nucleus accumbens. In conclusion, the behavioural results suggest that the potentiation of cocaine-induced behaviour following peripheral p-chlorophenylalanine administration may be attributed to the dorsal raphe nucleus but not the median raphe nucleus suggesting that, serotonergic dorsal raphe nucleus neurones may normally mediate a tonic inhibitory effect on cocaine-induced behaviour. Furthermore, the biochemical data may indicate the existence of neurochemical resistance of the midbrain raphe nuclei to the 5-HT depleting effects of p-chlorophenylalanine.     

Modulation of cocaine-induced locomotor activity, rears and head bobs by application of WAY100635 into the dorsal and median raphe nuclei of the rat

The present study investigated the role of somatodendritic 5-HT1A autoreceptors located in the dorsal and median raphe nuclei on the stimulant effect of cocaine on locomotor activity, rears and head bobs in female Glaxo Wistar rats. Cocaine was administered at a submaximal dose of 15 mg/kg i.p. to enable either a potentiation or attenuation to be observed. The selective 5-HT1A antagonist WAY100635 (0.21 ng or 21 ng) or saline was microinjected in the dorsal or median raphe nuclei followed by the peripheral administration of cocaine 60 min later. WAY 100635 microinjected in the dorsal or median raphe nuclei did not consistently alter the locomotor activity and the number of rears of saline-treated animals. Microinjection of WAY100635 in the dorsal raphe nucleus potentiated cocaine-induced locomotor activity and the number of head bobs. The number of rears induced by cocaine was not significantly altered by WAY100635 microinjected in the dorsal raphe nucleus. In contrast, microinjection of WAY100635 in the median raphe nucleus did not alter the stimulant effect of cocaine on locomotor activity, rears or head bobs. It may be suggested from these results that stimulation of somatodendritic 5-HT1A autoreceptors located in the dorsal raphe nucleus mediates an inhibitory effect on cocaine-induced locomotor activity and head bobs, whereas somatodendritic 5-HT1A autoreceptors in the median raphe nucleus are not involved in the inhibitory role of 5-HT on the stimulant effect of cocaine on locomotor activity and head bobs. A differential involvement of the midbrain raphe nuclei may exist controlling the stimulant effect of cocaine on locomotor activity and head bobs.     

Neuronal tryptophan hydroxylase mRNA expression in the human dorsal and median raphe nuclei: major depression and suicide.

Major depressive disorder (MDD) and suicide are associated with deficient serotonergic neurotransmission. Tryptophan hydroxylase (TPH) is the rate-limiting biosynthetic enzyme for serotonin. Previously, we reported elevated levels of TPH protein in the dorsal raphe nucleus (DRN) of depressed suicides and now examine expression of neuronal TPH2 mRNA in a cohort of matched controls and depressed suicides (n = 11 pairs). DRN TPH2 mRNA was measured by densitometric analysis of autoradiograms from in situ hybridization histochemistry experiments. TPH2 mRNA is confirmed as the raphe-specific isoform of TPH in human brain, and is expressed in neurons throughout the anteroposterior extent of the DRN and median raphe nucleus (MRN). TPH2 mRNA expression correlates with TPH protein distribution in the DRN, and has a negative correlation with age. In drug-free suicides, TPH2 expression is 33% higher in the DRN and 17% higher in the MRN as compared to matched nonpsychiatric controls. Higher levels of TPH2 mRNA were found throughout the entire extent of the rostrocaudal axis of the DRN, and were not specific to any single subnucleus. Higher TPH2 mRNA expression may explain more TPH protein observed in depressed suicides and reflect a homeostatic response to deficient brain serotonergic transmission.     

Harmane inhibits serotonergic dorsal raphe neurons in the rat

Rationale Harmane and norharmane (two beta-carbolines) are tobacco components or products. The effects of harmane and norharmane on serotonergic raphe neurons remain unknown. Harmane and norharmane are inhibitors of the monoamine oxidases A (MAO-A) and B (MAO-B), respectively. Objectives To study the effects of harmane, norharmane, befloxatone (MAOI-A), and selegiline (MAOI-B) on the firing of serotonergic neurons. To compare the effects of these compounds to those of nicotine (whose inhibitory action on serotonergic neurons has been previously described). The effects of cotinine, a metabolite of nicotine known to interact with serotonergic systems, are also tested. Methods In vivo electrophysiological recordings of serotonergic dorsal raphe neurons in the anaesthetized rat. Results Nicotine, harmane, and befloxatone inhibited serotonergic dorsal raphe neurons. The other compounds had no effects. The inhibitory effect of harmane (rapid and long-lasting inhibition) differed from that of nicotine (short and rapidly reversed inhibition) and from that of befloxatone (slow, progressive, and long-lasting inhibition). The inhibitory effects of harmane and befloxatone were reversed by the 5-HT1A antagonist WAY 100 635. Pretreatment of animals with p-chlorophenylalanine abolished the inhibitory effect of befloxatone, but not that of harmane. Conclusions Nicotine, harmane, and befloxatone inhibit the activity of raphe serotonergic neurons. Therefore, at least two tobacco compounds, nicotine and harmane, inhibit the activity of serotonergic neurons. The mechanism by which harmane inhibits serotonergic dorsal raphe neurons is likely unrelated to a MAO-A inhibitory effect.     

Evidence that PGE2 in the dorsal and median raphe nuclei is involved in LPS-induced anorexia in rats

Anorexia is an element of the acute-phase immune response. Its mechanisms remain poorly understood. Activation of inducible cyclooxygenase-2 (COX-2) in blood-brain-barrier endothelial cells and subsequent release of prostaglandins (e.g., prostaglandin E2, PGE2) may be involved. Therefore, we sought to relate the effects of prostaglandins on the anorexia following gram-negative bacterial lipopolysaccharide treatment (LPS) to neural activity in the dorsal and median raphe nuclei (DRN and MnR) in rats. COX-2 antagonist (NS-398, 10 mg/kg; IP) administration prior to LPS (100 μg/kg; IP) prevented anorexia and reduced c-Fos expression the DRN, MnR, nucleus tractus solitarii and several related forebrain areas. These data indicate that COX-2-mediated prostaglandin synthesis is necessary for LPS anorexia and much of the initial LPS-induced neural activation. Injection of NS-398 into the DRN and MnR (1 ng/site) attenuated LPS-induced anorexia to nearly the same extent as IP NS-398, suggesting that prostaglandin signaling in these areas is necessary for LPS anorexia. Because the DRN and MnR are sources of major serotonergic projections to the forebrain, these data suggest that serotonergic neurons originating in the midbrain raphe play an important role in acute-phase response anorexia.     

Chemical lesions of both dorsal and median raphe nuclei and changes in social and aggressive behaviour in rats

Microinjections of 5,7-dihydroxytryptamine into both the dorsal and median raphe nuclei resulted in 90% depletions of striatal and hippocampal 5-HT concentrations. Compared with vehicle-injected controls the lesioned rats showed reduced active social interaction scores in all four of the test conditions and also reduced levels of locomotor activity. The lesioned rats did not differ from the controls in their latency to start drinking in a novel environment; or in their response to intruder rats placed in their homo cages, or in their behaviour as intruders when they were placed in the home-cages of unoperated rats. The difficulties of interpreting the behavioural effects of a lesion when the lesion produces hypoactivity, and the differences between the effects of these joint lesions of both dorsal and median raphe nuclei and the effects of separate lesions of each nucleus are discussed.     

Effects of corticotropin-releasing factor on neuronal activity in the serotonergic dorsal raphe nucleus.

The present study examined the regional localization of corticotropin-releasing factor (CRF)- and 5-hydroxytryptamine (5-HT)-immunoreactive (IR) fibers within the rat dorsal raphe nucleus (DRN) using immunohistochemistry. Additionally, the effects of CRF, administered intracerebroventricularly (0.1-3.0 micrograms) or intraraphe (0.3-30 ng), on discharge rates of putative 5-HT DRN neurons were quantified using in vivo single unit recording in halothane-anesthetized rats. CRF-IR fibers were present at all rostrocaudal levels of the DRN and exhibited a topographical distribution. CRF produced predominantly inhibitory effects on DRN discharge at lower doses and these effects diminished or became excitatory at higher doses. Inhibition of DRN discharge by CRF was attenuated by the nonselective CRF antagonist, DPheCRF12-41 and the CRF-R1-selective antagonist, antalarmin, implicating the CRF-R1 receptor subtype in these electrophysiological effects. The present findings provide anatomical and physiological evidence for an impact of CRF on the DRN-5HT system.     

An analysis of dorsal and median raphe self-stimulation: effects of parachlorophenylalanine.

The role of serotonergic systems in intracranial self-stimulation (ICSS) of the dorsal and median raphe nuclei of rats was investigated. Intragastric administration of 400 mg/kg of parachlorophenylalanine (PCPA) depressed ICSS rates in the group with dorsal raphe electrode placements over a similar time course to the depletion of brain serotonin which results from treatment with PCPA. An intrasessional analysis of these behavioral changes on the fourth day after PCPA revealed that dorsal raphe ICSS was depressed over both halves of the 2 hr test session, whereas a significant depression in median raphe ICSS occurred only in the last hr of the session. The data from these studies suggest that brain serotonin systems contribute to the phenomenon of brain-stimulation reward in the dorsal and median raphe nuclei. The involvement of multiple neurochemical substrates of brain stimulation reward is discussed.     

Effects of local application of 5-hydroxytryptamine into the dorsal or median raphe nuclei on haloperidol-induced catalepsy in the rat

The effects of local application of the endogenous brain neurotransmitter 5-hydroxytryptamine (5-HT; serotonin) into the dorsal (DR) or median (MR) raphe nuclei on haloperidol-induced catalepsy (CAT) in rats were studied. Local application of 5-HT (40 microg, -10 min) into the DR or MR, respectively, produced a significant reversal of haloperidol-induced CAT. Lower doses (5 or 25 microg) of 5-HT were ineffective. Compared to previous studies using the selective 5-HT1A receptor agonist 8-OH-DPAT, the non-selective endogenous serotonin receptor agonist 5-HT was significantly less potent in this paradigm. Furthermore, the observed anticataleptic effect of 5-HT was seen following injections into both DR or MR nuclei. The reversal of CAT by local application of 5-HT (40 microg) into the DR was significant also at 70 min after 5-HT administration, with the same tendency for 5-HT injections into the MR. At this time interval, other serotonergic behavioral symptoms like head twitches and wet-dog shakes also emerged. The early reversal of CAT by local 5-HT administration into the MR is in all probability mediated via stimulation of 5-HT1A autoreceptors on raphe serotonergic cell bodies. The reversal of CAT following 5-HT injections into the DR might alternatively be mediated via functional mechanisms other than stimulation of 5-HT1A autoreceptors. The anticataleptic effects observed at the later observation time could be due to stimulation of postsynaptic 5-HT2 receptors following diffusion of 5-HT into 5-HT2 receptor rich areas of the brain.     

Orexin A excites serotonergic neurons in the dorsal raphe nucleus of the rat

Orexin A (10-300 nM) strongly excited dorsal raphe serotonergic neurons maintained in vitro. The depolarization persisted in the presence of tetrodotoxin (TTX, 0.5 microM) and was associated with an increase in input resistance. These results have relevance in the context of food intake regulation and the disease, narcolepsy.     

Neurokinins activate local glutamatergic inputs to serotonergic neurons of the dorsal raphe nucleus.

It has been proposed that antidepressant effects of neurokinin NK(1) receptor blockade may result from an increase in serotonin (5-HT) transmission. However, the mechanism by which neurokinins influence 5-HT neurons is not known. In this study, local NK(1) and NK(3) receptor-mediated responses in 5-HT neurons of the dorsal raphe nucleus (DRN) were studied using intracellular recording in rat brain slices. Bath application of the NK(1) receptor agonist substance P (SP) or the NK(3) receptor agonists senktide and NKB induced a robust increase in "spontaneous" excitatory postsynaptic currents (EPSCs) in 5-HT neurons. The EPSCs were blocked by the AMPA/kainate glutamate receptor antagonist CNQX and the fast Na(+) channel blocker tetrodotoxin (TTX), indicating that the increase in EPSCs resulted from an increase in impulse flow in local glutamatergic neuronal afferents. The neurokinins agonists had no direct excitatory effects on 5-HT neurons and no NK(1) or NK(3) receptor immunolabeling was found in 5-HT-labeled neurones. However, neurokinins, by increasing excitatory postsynaptic potentials (EPSPs), did increase the spiking of 5-HT neurons. The SP- and NKB-induced EPSCs were preferentially blocked by NK(1) and NK(3) antagonists, and there was minimal cross-desensitization between agonists at the two receptors. We conclude that neurokinins, via distinct NK(1) and NK(3) receptors, could promote 5-HT transmission, at least in part, by exciting a local population of glutamatergic inputs to 5-HT neurons in the DRN. However, these local excitatory effects, viewed within the context of the global effects of neurokinins on 5-HT neurons, reveal important differences between the functional role of NK(1) and NK(3) receptors.