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Dendritic spines are the basic structural units of neuronal plasticity. Intracellular signaling cascades that promote spinogenesis have centered on RhoGTPases. We found that ovarian steroids increase gene expression of RhoGTPases [Ras homolog gene family member A (RhoA), cell division control protein 42 homolog (Cdc42), and ras-related C3 botulinum toxin substrate (Rac)] in laser-captured serotonin neurons. We sought to confirm that the increases observed in gene expression translate to the protein level. In addition, a preliminary study was conducted to determine whether an increase in spines occurs via detection of the spine marker protein, postsynaptic density-95 (PSD-95). Adult ovariectomized (Ovx) monkeys were treated with estradiol (E), progesterone (P), or E+P for 1 month. Sections through the dorsal raphe nucleus were immunostained for RhoA and Cdc42 (n=3-4/group). The number and positive pixel area of RhoA-positive cells and the positive pixel area of Cdc42-positive fibers were determined. On combining E- and E+P-treated groups, there was a significant increase in the average and total cell number and positive pixel area of RhoA-positive cells. E, P, and E+P treatments, individually or combined, also increased the average and total positive pixel area of Cdc42-positive fibers. With remaining sections from two animals in each group, we conducted a preliminary examination of the regulation of PSD-95 protein expression. PSD-95, a postsynaptic scaffold protein, was examined with immunogold silver staining (n=2/group), and the total number of PSD-95-positive puncta was determined with stereology across four levels of the dorsal raphe. E, P, and E+P treatment significantly increased the total number of PSD-95-positive puncta. Together, these findings indicate that ovarian steroids act to increase gene and protein expression of two pivotal RhoGTPases involved in spinogenesis and preliminarily indicate that an increased number of spines and/or synapses result from this action. Increased spinogenesis on serotonin dendrites would facilitate excitatory glutamatergic input and in turn, increase serotonin neuronal activity throughout the brain., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background and Purpose: To evaluate the hypothesis that activation of somatodendritic 5-HT(1A) autoreceptors in the dorsal raphe nucleus (DRN) produces the anti-emetic/anti-nausea effects of cannabidiol (CBD), a primary non-psychoactive cannabinoid found in cannabis., Experimental Approach: The potential of systemic and intra-DRN administration of 5-HT(1A) receptor antagonists, WAY100135 or WAY100635, to prevent the anti-emetic effect of CBD in shrews (Suncus murinus) and the anti-nausea-like effects of CBD (conditioned gaping) in rats were evaluated. Also, the ability of intra-DRN administration of CBD to produce anti-nausea-like effects (and reversal by systemic WAY100635) was assessed. In vitro studies evaluated the potential of CBD to directly target 5-HT(1A) receptors and to modify the ability of the 5-HT(1A) agonist, 8-OH-DPAT, to stimulate [(35) S]GTPγS binding in rat brainstem membranes., Key Results: CBD suppressed nicotine-, lithium chloride (LiCl)- and cisplatin (20 mg·kg(-1) , but not 40 mg·kg(-1) )-induced vomiting in the S. murinus and LiCl-induced conditioned gaping in rats. Anti-emetic and anti-nausea-like effects of CBD were suppressed by WAY100135 and the latter by WAY100635. When administered to the DRN: (i) WAY100635 reversed anti-nausea-like effects of systemic CBD, and (ii) CBD suppressed nausea-like effects, an effect that was reversed by systemic WAY100635. CBD also displayed significant potency (in a bell-shaped dose-response curve) at enhancing the ability of 8-OH-DPAT to stimulate [(35) S]GTPγS binding to rat brainstem membranes in vitro. Systemically administered CBD and 8-OH-DPAT synergistically suppressed LiCl-induced conditioned gaping., Conclusions and Implications: These results suggest that CBD produced its anti-emetic/anti-nausea effects by indirect activation of the somatodendritic 5-HT(1A) autoreceptors in the DRN., Linked Articles: This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Corticotropin-releasing factor (CRF) and serotonin are important transmitters of the pathophysiology of mood disorder. To clarify the mechanisms of action of lamotrigine (LTG) and carbamazepine (CBZ), we determined their effects on serotonin release associated with CRF in rat dorsal raphe nucleus (DRN) and median prefrontal cortex (mPFC) using dual-probe microdialysis. Neither perfusion with CRF1 nor CRF2 antagonists into DRN-affected serotonin release in DRN and mPFC. Perfusion of 10 μM CRF into DRN increased serotonin release in both regions, whereas 0.1 μM CRF decreased and had no effect on serotonin release in DRN and mPFC, respectively. Pre-perfusion with CRF1 antagonist into DRN inhibited 0.1 μM CRF-induced serotonin reduction, whereas pre-perfusion with CRF2 antagonist in DRN inhibited 10 μM CRF-induced serotonin elevation, without affecting 0.1 μM CRF-induced serotonin reduction. LTG perfusion concentration dependently decreased serotonin releases in DRN and mPFC. Therapeutic and supratherapeutic concentrations of CBZ increased and decreased serotonin releases in both regions, respectively. Pre-perfusion with sub-therapeutic concentration LTG inhibited CRF1-induced serotonin reduction without affecting CRF2-induced serotonin release, whereas pre-perfusion with therapeutic concentration of LTG inhibited both CRF1- and CRF2-actions. In contrast, both therapeutic and supratherapeutic concentrations of CBZ inhibited CRF2-induced serotonin release without affecting CRF1-induced serotonin reduction. Neither LTG nor CBZ affected the CRF-induced cAMP production in cells over-expressing CRF1 and CRF2 receptors. This study demonstrated that inhibition of CRF2-receptor-mediated serotonergic transmission is a mechanism shared by LTG and CBZ, two clinically related compounds, whereas LTG but not CBZ inhibits CRF1-receptor-mediated serotonergic transmission. Therefore, these mechanisms may contribute to the clinical actions of these agents.

Dopamine and serotonin (5-HT) in the nucleus accumbens (ACC) and ventral tegmental area of the mesoaccumbens reward pathways have been implicated in the mechanisms underlying development of alcohol dependence. We used a C57BL/6J mouse model with increased voluntary alcohol-drinking behavior by exposing the mice to alcohol vapor for 20 consecutive days. In the alcohol-exposed mice, the expression of 5-HT(2C) receptor mRNA increased in the ACC, caudate nucleus and putamen, dorsal raphe nucleus (DRN), hippocampus and lateral hypothalamus, while the protein level of 5-HT(2C) receptor significantly increased in the ACC. The expression of 5-HT(7) receptor mRNA increased in the ACC and DRN. Contents of 5-HT decreased in the ACC shell (ACC(S) ) and DRN of the alcohol-exposed mice. The basal extracellular releases of dopamine (DA) and 5-HT in the ACC(S) increased more in the alcohol-exposed mice than in alcohol-naïve mice. The magnitude of the alcohol-induced ACC(S) DA and 5-HT release in the alcohol-exposed mice was increased compared with the control mice. Intraperitoneal (i.p.) administration or local injection into ACC(S) of the 5-HT(2C) receptor antagonist, SB-242084, suppressed voluntary alcohol-drinking behavior in the alcohol-exposed mice. But the i.p. administration of the 5-HT(7) receptor antagonist, SB-258719, did not have significant effects on alcohol-drinking behavior in the alcohol-exposed mice. The effects of the 5-HT(2C) receptor antagonist were not observed in the air-exposed control mice. These results suggest that adaptations of the 5-HT system, especially the upregulation of 5-HT(2C) receptors in the ACC(S) , are involved in the development of enhanced voluntary alcohol-drinking behavior., (© 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Mimosa pudica (Linn.) (M. pudica L.) is a plant used in some countries to treat anxiety and depression. In the present study we investigated the effects of an aqueous extract of M. pudica L. on mouse anxiety-like behaviour using the elevated T maze, and on regulation of dorsal raphe nucleus (DRN) 5-hydroxytryptamine (5-HT) neuronal activity using an in-vitro mouse brain slice preparation. Acute treatment with M. pudica L. extract had an anxiolytic effect on behaviour in the elevated T maze, specifically on inhibitory avoidance behaviour. Acute application of the extract alone had no effect on the activity of DRN 5-HT neurones. However, when co-applied with the GABA(A) receptor agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol), the extract enhanced the inhibitory effect of the THIP on DRN 5-HT neurones. These observed effects of M. pudica L. on both behaviour and GABA modulation of 5-HT neuronal activity are similar to the effects of diazepam, the established anxiolytic and positive modulator of the GABA(A) receptor. This study suggests that the aqueous extract of M. pudica L. contains a positive modulator of GABA(A) receptor function and provides impetus for further investigation of the neuropharmacologically active constituents of the extract.

Obstructive sleep apnoea (OSA) is characterized by periods of upper airway collapse accompanied by repeated episodes of hypoxia. In experimental animals repeated bouts of hypoxia may evoke sustained augmentation of phrenic nerve activity, known as phrenic long-term facilitation (pLTF). This form of physiological compensation might contribute to stable breathing, minimizing the occurrence of apnoeas and/or hypopnoeas during sleep in patients with OSA. Serotonin (5-HT) has been shown to modulate respiratory neuronal activity, possibly via projections originating in the raphe nuclei. Our model focuses on the effects of 5-HT1A receptors blockade by selective antagonist WAY-100635 into the caudal raphe region on phrenic long-term facilitation after exposure to acute intermittent hypoxia (AIH) episodes. Adult, male, urethane-anaesthetized, vagotomized, paralyzed and mechanically ventilated Sprague-Dawley rats were exposed to AIH protocol. Experimental group received microinjection of WAY-100635 into the caudal raphe nucleus, whereas the control group received saline into the same site. Peak phrenic nerve activity and respiratory rhythm parameters were analysed during five hypoxic episodes, as well as at 15, 30 and 60 min after the end of hypoxias. In the control group, 1 h post-hypoxia pLTF was developed. Microinjections of selective 5-HT1A receptor antagonist WAY-100635 into the raphe nuclei prior to the AIH protocol prevented induction of pLTF. These results suggest that 5-HT1A receptor activation at supraspinal level is important for induction of pLTF, which is suggested to be an important respiratory neuroplasticity model in animal studies that possibly correlates with OSA in humans., (© 2011 European Sleep Research Society.)

Genetic variations in certain components of the glucocorticoid receptor (GR) chaperone complex have been associated with the development of stress-related affective disorders and individual variability in therapeutic responses to antidepressants. Mechanisms that link GR chaperoning and stress susceptibility are not well understood. Here, we show that the effects of glucocorticoid hormones on socioaffective behaviors are critically regulated via reversible acetylation of Hsp90, a key component of the GR chaperone complex. We provide pharmacological and genetic evidence indicating that the cytoplasmic lysine deacetylase HDAC6 controls Hsp90 acetylation in the brain, and thereby modulates Hsp90-GR protein-protein interactions, as well as hormone- and stress-induced GR translocation, with a critical impact on GR downstream signaling and behavior. Pet1-Cre-driven deletion of HDAC6 in serotonin neurons, the densest HDAC6-expressing cell group in the mouse brain, dramatically reduced acute anxiogenic effects of the glucocorticoid hormone corticosterone in the open-field, elevated plus maze, and social interaction tests. Serotonin-selective depletion of HDAC6 also blocked the expression of social avoidance in mice exposed to chronic social defeat and concurrently prevented the electrophysiological and morphological changes induced, in serotonin neurons, by this murine model of traumatic stress. Together, these results identify HDAC6 inhibition as a potential new strategy for proresilience and antidepressant interventions through regulation of the Hsp90-GR heterocomplex and focal prevention of GR signaling in serotonin pathways. Our data thus uncover an alternate mechanism by which pan-HDAC inhibitors may regulate stress-related behaviors independently of their action on histones.

Recovery of neurological function after traumatic injury of the adult mammalian central nervous system is limited by lack of axonal growth. Myelin-derived inhibitors contribute to axonal growth restriction, with ephrinB3 being a developmentally important axonal guidance cue whose expression in mature oligodendrocytes suggests a role in regeneration. Here we explored the in vivo regeneration role of ephrinB3 using mice lacking a functional ephrinB3 gene. We confirm that ephrinB3 accounts for a substantial portion of detergent-resistant myelin-derived inhibition in vitro. To assess in vivo regeneration, we crushed the optic nerve and examined retinal ganglion fibers extending past the crush site. Significantly increased axonal regeneration is detected in ephrinB3(-/-) mice. Studies of spinal cord injury in ephrinB3(-/-) mice must take into account altered spinal cord development and an abnormal hopping gait before injury. In a near-total thoracic transection model, ephrinB3(-/-) mice show greater spasticity than wild-type mice for 2 mo, with slightly greater hindlimb function at later time points, but no evidence for axonal regeneration. After a dorsal hemisection injury, increased corticospinal and raphespinal growth in the caudal spinal cord are detected by 6 wk. This increased axonal growth is accompanied by improved locomotor performance measured in the open field and by kinematic analysis. Thus, ephrinB3 contributes to myelin-derived axonal growth inhibition and limits recovery from adult CNS trauma.

Aim: It has been suggested that the medullary raphe (MR) plays a key role in the physiological responses to hypoxia. As opioid μ-receptors have been found in the MR, we studied the putative role of opioid μ-receptors in the rostral MR (rMR) region on ventilation in normal and 7% hypoxic conditions., Methods: We measured pulmonary ventilation (VE) and the body temperatures (Tb) of male Wistar rats before and after the selective opioid μ-receptor antagonist CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2, cyclic, 0.1 μg per 0.1 μL) was microinjected into the rMR during normoxia or after 60 min of hypoxia., Results: The animals treated with intra-rMR CTAP exhibited an attenuation of the ventilatory response to hypoxia (430 ± 86 mL kg(-1) min(-1)) compared with the control group (790 ± 82 mL kg(-1) min(-1) ) (P < 0.05). No differences in the Tb were observed between groups during hypoxia., Conclusion: These data suggest that opioids acting on μ-receptors in the rMR exert an excitatory modulation of hyperventilation induced by hypoxia., (© 2011 The Authors. Acta Physiologica © 2011 Scandinavian Physiological Society.)

Background: Serotonin (5-HT) has a role in respiratory function and dysfunction. Although 5-HT affects respiratory drive to both phrenic and cranial motoneurons, relatively little is known about the role of 5-HT receptor subtypes in the control of upper airway muscle (UAM) respiratory activity., Materials and Methods: Here, we performed central injections of 5-HT1A agonist (8-OHDPAT) or antagonist (WAY100635) in anesthetized rats and analyzed changes in the electromyographic activity of several UAM and other cardiorespiratory parameters. We also compared the pattern of Fos expression induced after central injection of a control solution or 8-OHDPAT., Results: Results showed that 8-OHDPAT induced a robust increase in UAM activity, associated with either tachypnea under volatile anesthesia or bradypnea under liquid anesthesia. Injection of WAY100635 switched off UAM respiratory activity and led to bradypnea, suggesting a tonic excitatory role of endogenous 5-HT1A receptor activation. Co-injection of the agonist and the antagonist blocked the effects produced by each drug alone. Besides drug-induced changes in respiratory frequency, only slight increases in surface of diaphragm bursts were observed. Significant increases in Fos expression after 5-HT1A receptor activation were seen in the nucleus tractus solitarius, nucleus raphe pallidus, parapyramidal region, retrotrapezoid nucleus, lateral parabrachial, and Kölliker-Fuse nuclei. This restricted pattern of Fos expression likely identified the neural substrate responsible for the enhancement of UAM respiratory activity observed after 8-OHDPAT injection., Conclusions: These findings suggest an important role for the 5-HT1A receptors in the neural control of upper airway patency and may be relevant to counteract pharyngeal atonia during obstructive sleep apneas.

The serotonergic system is one of the major systems targeted in the pharmacological treatment of mood disorders including depression. Fluoxetine, one of the selective serotonin reuptake inhibitors (SSRIs), has been reported to induce the expression of tryptophan hydroxylase (TPH), the rate-limiting enzyme in the biosynthesis of serotonin. The 14-3-3 protein family not only activates neuronal enzymes, including TPH, but also plays a role in a wide variety of cell signaling. The aim of the present study was to determine whether fluoxetine regulates both the interaction of TPH and 14-3-3 proteins as well as the increase of those proteins in the dorsal raphe nucleus and the hippocampus. Sprague-Dawley rats were administered fluoxetine or vehicle for 5 and 14 days and sacrificed at 5 and 14 days after initial treatment. The intensity of immunoreactivity for TPH and 14-3-3 proteins in the dorsal raphe nucleus of the midbrain and in the hippocampus was measured, and the colocalization of both proteins was observed with double-labeling immunofluorescence. At 5 days after initial treatment with fluoxetine, immunoreactivity of 14-3-3 protein increased in both the dorsal raphe nucleus and the hippocampus, while that of TPH did not change in either region. In addition, at 14 days after initial treatment with fluoxetine, immunoreactivity of 14-3-3 protein significantly increased in both the dorsal raphe nucleus and hippocampus, while that of TPH showed few changes in either region. Colocalization of TPH and 14-3-3 proteins was observed in the cell bodies of dorsal raphe nucleus, whereas it was not observed in the hippocampus. These results suggest that the time-dependent regulation of 14-3-3 protein may be one of the various factors associated with delayed pharmacological effects of SSRIs., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The ascending 5-HT system has been and continues to be the subject of much research. The majority of in vivo electrophysiological and neurochemical studies of 5-HT function in rodents have been conducted in animals under anaesthesia - usually chloral hydrate or urethane. However, the effects of anaesthetics, on 5-HT function have not been systematically investigated. Here we used in vitro electrophysiology in dorsal raphe slices, to determine the effects of anaesthetically relevant concentrations of chloral hydrate (100 μM and 1 mM), urethane (10 and 30 mM), pentobarbitone (10 and 100 μM) and ketamine (10, 100 and 300 μM) on regulators of 5-HT firing activity. We examined i) basal firing (driven by α(1) adrenoceptors), ii) the excitatory response to N-methyl-d-aspartate (NMDA), iii) the 5-HT(1A) autoreceptor-mediated inhibitory response to 5-HT and iv) the GABA(A) receptor-mediated inhibitory response to 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridinyl-3-ol (THIP, gaboxadol). Pentobarbitone selectively enhanced the response to THIP. Ketamine decreased basal firing, attenuated the response to NMDA, and enhanced responses to both 5-HT and THIP. Chloral hydrate had marginal effects on basal firing, slightly attenuated the NMDA response, and enhanced both the 5-HT and THIP responses. Urethane increased basal firing, decreased the NMDA response, increased the response to THIP, but had no effect on the 5-HT response. Our data indicate that all anaesthetics tested significantly affect the regulators of 5-HT neuronal function. These findings will aid in the interpretation of previous reports of in vivo studies of the 5-HT system and will allow researchers to make a rational selection of anaesthetic for future studies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

3,4-Methylenedioxymethamphetamine (MDMA, 'ecstasy') is a recreational drug widely used by adolescents and young adults. Although its rewarding effects are well established, there is controversy on its addictive potential. We aimed to compare the consequences of active and passive MDMA administration on gene expression in the mouse brain since all previous studies were based on passive MDMA administration. We used a yoked-control operant intravenous self-administration paradigm combined with microarray technology. Transcriptomic profiles of ventral striatum, frontal cortex, dorsal raphe nucleus and hippocampus were analysed in mice divided in contingent MDMA, yoked MDMA and yoked saline groups, and several changes were validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The comparison of contingent MDMA and yoked MDMA vs. yoked saline mice allowed the identification of differential expression in several genes, most of them with immunological and inflammatory functions, but others being involved in neuroadaptation. In the comparison of contingent MDMA vs. yoked MDMA administration, hippocampus and the dorsal raphe nucleus showed statistically significant changes. The altered expression of several genes involved in neuroadaptative changes and synapse function, which may be related to learning self-administration behaviour, could be validated in these two brain structures. In conclusion, our study shows a strong effect of MDMA administration on the expression of immunological and inflammatory genes in all the four brain regions studied. In addition, experiments on MDMA self-administration suggest that the dorsal raphe nucleus and hippocampus may be involved in active MDMA-seeking behaviour, and show specific alterations on gene expression that support the addictive potential of this drug., (© 2011 The Authors. Genes, Brain and Behavior © 2011 Blackwell Publishing Ltd and International Behavioural and Neural Genetics Society.)

This study investigated the participation of median raphe nucleus (MnR) α1-adrenergic receptors in the control of feeding behaviour. The α1-adrenergic agonist phenylephrine (PHE) and α2-adrenergic agonist clonidine (CLON) (at equimolar doses of 0, 6 and 20 nmol) were injected into the MnR of: a) rats submitted to overnight fasting (18 h); or b) rats maintained with 15 g of lab chow/day for 7 days. Immediately after the drug injections, the animals were placed in the feeding chamber and feeding and non-ingestive behaviours such as grooming, rearing, resting, sniffing and locomotion were recorded for 30 min. The results showed that both doses of PHE injected into the MnR of overnight fasted animals decreased food intake accompanied by an increase in the latency to start feeding. A reduction in feeding duration was observed only after treatment of the MnR with the 20 nmol dose of PHE. Both locomotion duration and sniffing frequency increased after injection with the highest dose PHE into the MnR. Feeding frequency and the other non-ingestive behaviours remained unchanged after PHE treatment in the MnR. Both doses of PHE injected into the MnR of food-restricted rats decreased food intake. This hypophagic response was accompanied by a decrease in feeding duration only after treatment of the MnR with the highest dose of PHE. The latency to start feeding and feeding frequency were not affected by injection of either dose of PHE into the MnR. While both doses of PHE increased sniffing duration, the highest dose of PHE increased resting duration and resting frequency. Treatment with CLON into the MnR did not affect feeding behaviour in either of the food deprivation conditions. The present results indicate the inhibitory functional role of α1-adrenergic receptors within the MnR on feeding behaviour., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In urethane/α-chloralose anesthetized rats, cold exposure increased brown adipose tissue sympathetic nerve activity (BAT SNA: +699 ± 104% control). Intravenous administration of 2-deoxy-D-glucose (2-DG; 200 mg·ml(-1)·kg(-1)) reversed the cold-evoked activation of BAT SNA (nadir: 139 ± 36% of control) and decreased BAT temperature (-1.1 ± 0.2°C), expired CO(2) (-0.4 ± 0.1%), and core temperature (-0.5 ± 0.0). Similarly, unilateral nanoinjection of the glucoprivic agent 5-thioglucose (5-TG; 12 μg/100 nl) in the ventrolateral medulla (VLM) completely reversed the cold-evoked increase in BAT SNA (nadir: 104 ± 7% of control), and decreased T(BAT) (-1.4 ± 0.3°C), expired CO(2) (-0.2 ± 0.0%), and heart rate (-35 ± 10 beats/min). The percentage of rostral raphé pallidus (RPa)-projecting neurons in the dorsal hypothalamic area/dorsomedial hypothalamus that expressed Fos in response to cold exposure (ambient temperature: 4-10°C) did not differ between saline (28 ± 6%) and 2-DG (30 ± 5%) pretreated rats, whereas the percentage of spinally projecting neurons in the RPa/raphé magnus that expressed Fos in response to cold exposure was lower in 2-DG- compared with saline-pretreated rats (22 ± 6% vs. 42 ± 5%, respectively). The increases in BAT SNA evoked by nanoinjection of bicuculline in the RPa or by transection of the neuraxis at the pontomedullary border were resistant to inhibition by glucoprivation. These results suggest that neurons within the VLM play a role in the glucoprivic inhibition of BAT SNA and metabolism, that this inhibition requires neural structures rostral to the pontomedullary border, and that this inhibition is mediated by a GABAergic input to the RPa.

Background: Serotonergic system participates in a wide range of physiological processes and behaviors, but its role is generally considered as modulatory and noncrucial, especially concerning life-sustaining functions. We recently created a transgenic mouse line in which a functional deficit in serotonin homeostasis due to excessive serotonin autoinhibition was produced by inducing serotonin 1A receptor (Htr1a) overexpression selectively in serotonergic neurons (Htr1a raphe-overexpressing or Htr1a(RO) mice). Htr1a(RO) mice exhibit episodes of autonomic dysregulation, cardiovascular crises and death, resembling those of sudden infant death syndrome (SIDS) and revealing a life-supporting role of serotonergic system in autonomic control. Since midbrain serotonergic neurons are chemosensitive and are implicated in arousal we hypothesized that their chemosensitivity might be impaired in Htr1a(RO) mice., Principal Findings: Loose-seal cell-attached recordings in brainstem slices revealed that serotonergic neurons in dorsal raphe nucleus of Htr1a(RO) mice have dramatically reduced responses to hypercapnic challenge as compared with control littermates. In control mice, application of 9% CO(2) produced an increase in firing rate of serotonergic neurons (0.260 ± 0.041 Hz, n=20, p=0.0001) and application of 3% CO(2) decreased their firing rate (-0.142 ± 0.025 Hz, n=17, p=0.0008). In contrast, in Htr1a(RO) mice, firing rate of serotonergic neurons was not significantly changed by 9% CO(2) (0.021 ± 0.034 Hz, n=16, p=0.49) and by 3% CO(2) (0.012 ± 0.046 Hz, n=12, p=0.97)., Conclusions: Our findings support the hypothesis that chemosensitivity of midbrain serotonergic neurons provides a physiological mechanism for arousal responses to life-threatening episodes of hypercapnia and that functional impairment, such as excessive autoinhibition, of midbrain serotonergic neuron responses to hypercapnia may contribute to sudden death.

Several lines of evidence support the involvement of serotonergic (5-HT) neurons of the median raphe nucleus (MRN) in anxiety-like behaviour. In this context, it is known that blockade of 5-HT(1A) somatodendritic autoreceptors in the midbrain raphe nuclei increases the firing rate of these neurons, disinhibiting 5-HT release in postsynaptic target areas such as amygdala, hippocampus and periaqueductal grey matter (PAG). However, while activation of 5-HT(1A) or 5-HT(2) receptors in forebrain targets such as the amygdala or hippocampus enhances anxiety-like behaviours in rodents, stimulation of both receptor subtypes in the midbrain PAG markedly reduces anxiety-like behaviour. In view of these findings, the present study investigated whether the anti-anxiety effects induced by pharmacological disinhibition of 5-HT neurons in the MRN are attenuated by the blockade of 5-HT(2) receptors within the PAG. Mice received combined intra-PAG injection with ketanserin (10 nmol/0.1 μl), a 5-HT(2) receptor antagonist, followed by intra-MRN injection of WAY-100635 (5.6 nmol/0.1 μl), a highly selective 5-HT(1A) receptor antagonist. They were then individually exposed to the elevated plus-maze (EPM), with the videotaped behavioural sessions subsequently scored for both conventional and ethological measures. The results confirmed that intra-MRN infusion of WAY100635 reduces behavioural indices of anxiety without significantly altering general activity measures, and further showed that this effect was completely blocked by intra-PAG pretreatment with an intrinsically-inactive dose of ketanserin. Together, these results suggest that 5HT(2) receptor populations located within the midbrain PAG play a significant role in the reduction of anxiety observed following disinhibition of 5-HT neurons in the MRN., (Copyright © 2011. Published by Elsevier B.V.)

Serotonin reuptake inhibitor (SRI) antidepressants such as fluoxetine (Prozac), promote hippocampal neurogenesis. They also increase the levels of the bcl-2 protein, whose overexpression in transgenic mice enhances adult hippocampal neurogenesis. However, the mechanisms underlying SRI-mediated neurogenesis are unclear. Recently, we identified the microRNA miR-16 as an important effector of SRI antidepressant action in serotonergic raphe and noradrenergic locus coeruleus (LC). We show here that miR-16 mediates adult neurogenesis in the mouse hippocampus. Fluoxetine, acting on serotonergic raphe neurons, decreases the amount of miR-16 in the hippocampus, which in turn increases the levels of the serotonin transporter (SERT), the target of SRI, and that of bcl-2 and the number of cells positive for Doublecortin, a marker of neuronal maturation. Neutralization of miR-16 in the hippocampus further exerts an antidepressant-like effect in behavioral tests. The fluoxetine-induced hippocampal response is relayed, in part, by the neurotrophic factor S100β, secreted by raphe and acting via the LC. Fluoxetine-exposed serotonergic neurons also secrete brain-derived neurotrophic factor, Wnt2 and 15-Deoxy-delta12,14-prostaglandin J2. These molecules are unable to mimic on their own the action of fluoxetine and we show that they act synergistically to regulate miR-16 at the hippocampus. Of note, these signaling molecules are increased in the cerebrospinal fluid of depressed patients upon fluoxetine treatment. Thus, our results demonstrate that miR-16 mediates the action of fluoxetine by acting as a micromanager of hippocampal neurogenesis. They further clarify the signals and the pathways involved in the hippocampal response to fluoxetine, which may help refine therapeutic strategies to alleviate depressive disorders.

Even though neuronal toxicity due to organomercury compounds is well known, thimerosal, an organomercury compound, is widely used in pediatric vaccine preservation. In the present study, we examined whether embryonic exposure to thimerosal affects early development of serotonergic neurons. Thimerosal (1mg Hg/kg) was intramuscularly administered to pregnant rats on gestational day 9 (susceptible time window for development of fetal serotonergic system), and fetal serotonergic neurons were assessed at embryonic day 15 using anti-serotonin antibodies. A dramatic increase in the number of serotonergic neurons localized to the lateral portion of the caudal raphe was observed in thimerosal group (1.9-fold increase, p<0.01 compared to control). These results indicate that embryonic exposure to thimerosal affects early development of serotonergic neurons., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Orexin (hypocretin) neurons, located exclusively in the PeF-LH, which includes the perifornical area (PeF), the lateral hypothalamus (LH), and lateral portions of the medial hypothalamus, have widespread projections and influence many physiological functions, including the autonomic regulation of body temperature and energy metabolism. Narcolepsy is characterized by the loss of orexin neurons and by disrupted sleep, but also by dysregulation of body temperature and by a strong tendency for obesity. Heat production (thermogenesis) in brown adipose tissue (BAT) contributes to the maintenance of body temperature and, through energy consumption, to body weight regulation. We identified a neural substrate for the influence of orexin neurons on BAT thermogenesis in rat. Nanoinjection of orexin-A (12 pmol) into the rostral raphe pallidus (rRPa), the site of BAT sympathetic premotor neurons, produced large, sustained increases in BAT sympathetic outflow and in BAT thermogenesis. Activation of neurons in the PeF-LH also enhanced BAT thermogenesis over a long time course. Combining viral retrograde tracing from BAT, or cholera toxin subunit b tracing from rRPa, with orexin immunohistochemistry revealed synaptic connections to BAT from orexin neurons in PeF-LH and from rRPa neurons with closely apposed, varicose orexin fibers, as well as a direct, orexinergic projection from PeF-LH to rRPa. These results indicate a potent modulation of BAT thermogenesis by orexin released from the terminals of orexin neurons in PeF-LH directly into the rRPa and provide a potential mechanism contributing to the disrupted regulation of body temperature and energy metabolism in the absence of orexin.

Addiction is a brain chronic relapsing disorder associated with emotional distress. The serotonergic system and especially the 5-HT(1A) receptor crucially regulate emotional behaviors both in humans and rodents. Using [(35)S]GTPγS autoradiography in mice, we show that 5-HT(1A) receptor function is enhanced by chronic morphine treatment in the medial prefrontal cortex, and decreased in dorsal raphe nucleus one week later, two regions involved in emotional processing. These molecular adaptations could contribute to the development of emotional disorders experienced by former opiate addicts., (Copyright © 2011 Elsevier B.V. and ECNP. All rights reserved.)

Uncontrollable stressors produce behavioral changes that do not occur if the organism can exercise behavioral control over the stressor. Previous studies suggest that the behavioral consequences of uncontrollable stress depend on hypersensitivity of serotonergic neurons in the dorsal raphe nucleus (DRN), but the mechanisms involved have not been determined. We used ex vivo single-unit recording in rats to test the hypothesis that the effects of uncontrollable stress are produced by desensitization of DRN 5-HT(1A) autoreceptors. These studies revealed that uncontrollable, but not controllable, tail shock impaired 5-HT(1A) receptor-mediated inhibition of DRN neuronal firing. Moreover, this effect was observed only at time points when the behavioral effects of uncontrollable stress are present. Furthermore, temporary inactivation of the medial prefrontal cortex with the GABA(A) receptor agonist muscimol, which eliminates the protective effects of control on behavior, led even controllable stress to now produce functional desensitization of DRN 5-HT(1A) receptors. Additionally, behavioral immunization, an experience with controllable stress before uncontrollable stress that prevents the behavioral outcomes of uncontrollable stress, also blocked functional desensitization of DRN 5-HT(1A) receptors by uncontrollable stress. Last, Western blot analysis revealed that uncontrollable stress leads to desensitization rather than downregulation of DRN 5-HT(1A) receptors. Thus, treatments that prevent controllable stress from being protective led to desensitization of 5-HT(1A) receptors, whereas treatments that block the behavioral effects of uncontrollable stress also blocked 5-HT(1A) receptor desensitization. These data suggest that uncontrollable stressors produce a desensitization of DRN 5-HT(1A) autoreceptors and that this desensitization is responsible for the behavioral consequences of uncontrollable stress.

The present report examined the effects of midbrain raphe nuclei injections of nitric oxide synthase inhibitors NG-nitro-L-arginine methyl ester (L-NAME) and 7-nitroindazole (7-NI) on eating behavior. L-NAME (5-500 pmol) and 7-NI (2-200 pmol) were administered into either the dorsal or median raphe nucleus. Both nitric oxide synthase inhibitors decreased food intake in adult male Sprague-Dawley rats when injected into either raphe site. Further, eating elicited by dorsal and median raphe injections of the 5-HT1A agonist 8-OH-DPAT (0.8 nmol) was attenuated by L-NAME or 7-NI pretreatment. Our findings indicate that nitric oxide acts within the raphe to alter food intake. The inhibitory effects of L-NAME and 7-NI on eating elicited by 8-OH-DPAT further suggest that nitric oxide and 5-HT systems interact in the control of food intake.

It has been reported that the sedative component of pentobarbital is mediated by GABA receptors in an endogenous sleep pathway and the ventrolateral preoptic area (VLPO)-tuberomammillary nucleus (TMN) or VLPO-dorsal raphe nucleus (DRN) neural circuit is important in the sedative response to pentobarbital. Our previous findings indicated that the VLPO-TMN neuronal circuit may play crucial part in the augmentative effect of diltiazem on pentobarbital sleep and the serotonergic system may be involved. This study was designed to investigate the role of DRN and the serotonergic receptors 5-HT(1A) and 5-HT(2A/2C) in the augmentative effect of diltiazem on pentobarbital-induced hypnosis in rats. The results showed that diltiazem (5mg/kg, i.g.) significantly reversed pentobarbital-induced (35 mg/kg, i.p.) reduction of c-Fos expression in 5-HT neurons of DRNV (at -7.5mm Bregma), DRND, DRNVL and MRN (at -8.0mm Bregma). However it did not influence this reducing effect of pentobarbital on non-5-HT neurons either in DRN or in MRN. Moreover, the effect of diltiazem (1 or 2mg/kg, i.g.) on pentobarbital-induced (35 mg/kg, i.p.) hypnosis was significantly inhibited by 5-HT(1A) agonist 8-OH-DPAT (0.5mg/kg, i.p.) and 5-HT(2A/2C) agonist DOI (0.5mg/kg, i.p.), and potentiated by 5-HT(1A) antagonist p-MPPI (2mg/kg, i.p.) and 5-HT(2A/2C) antagonist ritanserin (2mg/kg, i.p.), respectively. From these results, it should be presumed that the augmentative effect of diltiazem on pentobarbital-induced sleep may be related to 5-HT(1A) and 5-HT(2A/2C) receptors, and DRN may be involved. In addition, it also suggested that the DRN may play a multi-modulating role in sleep-wake regulation rather than being recognized simply as arousal nuclei., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The raphe pallidus (RPa) and Bötzinger complex (BötC) represent two important nuclei which project to spinal phrenic motor neurons. Stimulation of the RPa produces facilitative effects on respiratory activity, whereas stimulation of the BötC induces inhibitory effects on respiratory activity. In the present study, we examined the modulatory effects of serotonergic (5-hydroxytryptamine, 5-HT) RPa neurons on the inhibitory response of the phrenic nerve activity elicited from the BötC in rats. Experiments were performed on spontaneously breathing, urethane-anesthetized adult rats. Either high-frequency stimulation or glutamatergic chemical activation of the RPa region significantly attenuated the BötC-induced inhibition of the phrenic nerve. This attenuation showed a post-stimulation time and intensity dependency. Pharmacological experiments showed that intravenous injection of methysergide, a broad-spectrum antagonist of 5-HT receptors, markedly reduced the respiratory facilitation induced by electrical stimulation of the RPa. Furthermore, microinjections of methysergide into the cerebrospinal fluid around the phrenic motor nucleus (PMN) region at spinal cord segments C4 and C5 significantly decreased the RPa-related attenuation effects on BötC-evoked inhibition of phrenic nerve discharge. These results suggest that RPa serotonergic neurons could modulate the inhibition of phrenic nerve activity induced by BötC. Moreover, as the relevant 5-HT receptors for RPa's modulatory effects are located in the cervical spinal cord, 5-HT may, in part, function as a modulator to suppress the BötC neuronal activity via direct RPa-PMN and BötC-PMN convergent projection pathways to phrenic motoneurons., (© 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Serotonergic neurons in the median raphe nucleus (MnR) are stimulated by α(1)-adrenergic agonists and inhibited by α(2)-agonists. This study investigated the effect of the blockade of the MnR α(1)-adrenergic receptors of free feeding rats as an attempt to elucidate the functional role of these receptors in the control of feeding behavior. In addition, an α(2)-receptor antagonist was also administered in the MnR in order to strengthen the previous suggestion that α(2)-adrenergic receptors participate in the control of feeding behavior, probably decreasing the facilitatory influence on MnR serotonergic neurons. The α(1)-adrenergic antagonist prazosin (PRA, 40 nmol) or vehicle was injected into the MnR 15 min before treatment with phenylephrine (PHE, 0.2 nmol). The α(2)-adrenergic antagonist yohimbine (YOH, 40 nmol) was administered 15 min before clonidine (CLO, 20 nmol) or vehicle in free-feeding rats. After the injections, the animals were placed in the feeding chamber for 30 min to evaluate the ingestive and non-ingestive behaviors. At the end of the experiment the quantity of food and water consumed were measured. While treatment with PRA in the MnR followed by PHE did not change the feeding behavior, PRA injection alone into the MnR caused hyperphagia accompanied by a reduction in the latency to start eating, an increase in feeding frequency and an increase in the feeding duration. Pretreatment with YOH in the MnR blocked the hyperphagic effect induced by CLO. The present data reinforce our previous suggestion that the MnR α(2)-adrenergic receptors participate in the control of feeding behavior, probably decreasing the facilitatory influence on MnR serotonergic neurons of free-feeding animals. Furthermore, these results indicate that this influence is tonically mediated by α(1)-adrenergic receptors upon MnR neurons, which inhibit food intake., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The role of the acetylcholine-mediated system in the organization of postictal antinociception was investigated. For this purpose, nicotinic and muscarinic cholinergic receptor antagonists were microinjected into the nucleus raphe magnus (NRM), a key structure of the endogenous pain inhibitory system. After the tail-flick test baseline recording, male Wistar rats (N=8 per group) were submitted to stereotaxic surgery for the introduction of a guide cannula aiming at the NRM. Five days after surgery, atropine or mecamylamine (1 µg/0.2 µL, 3 µg/0.2 µL, or 5 µg/0.2 µL) was microinjected into the NRM. The tail-flick withdrawal latency was recorded immediately after peripheral treatment with pentylenetetrazole (PTZ) (64 mg/kg), in two different interictal time windows, and for 130 minutes after the last seizure evoked by intraperitoneal injection of PTZ. The blockade of GABA-mediated Cl(-) influx caused tonic-clonic convulsions in all animals followed by sustained postictal antinociception lasting 110 minutes after seizures; the nociceptive threshold was also found to be high in interictal periods. Pretreatment of the NRM with either atropine or mecamylamine antagonized both interictal and postictal antinociception, suggesting the involvement of cholinergic mechanisms recruiting muscarinic and nicotinic cholinergic receptors of the NRM in the organization of tonic-clonic seizure-induced antinociception., (Copyright © 2011 Elsevier Inc. All rights reserved.)

This study of the participation of the serotonergic system in the inhibitory effect of estrogen on induced sodium appetite in female rats explores sodium appetite induced by Furosemide and low sodium diet treatment (DEP) in normally cycling rats and in ovariectomized rats with and without estradiol replacement (OVX, OVX+E(2)). We also analyzed the neural activity of serotonergic neurons of the dorsal raphe nucleus (DRN) as well as the activity of other brain nuclei previously found to be involved in sodium and water balance in sodium depleted animals without access to the intake test. For this purpose, we examined the brain Fos, Fos-serotonin and Fos-vasopressin immunoreactivity patterns in diestrus (D), estrus (E), OVX and OVX+E(2) rats subjected to DEP. Female rats in E and OVX+E(2) exhibited a significant decrease in induced sodium intake compared with females in D and OVX. This estrogen-dependent inhibition on induced sodium appetite (approximately 50% reduction) can be correlated with changes in Fos activation observed in the organum vasculosum of the lamina terminalis (OVLT) and DRN, in response to sodium depletion. Given our previous observations in males, the expected sodium depletion-induced activity of the OVLT was found to be absent in OVX+E(2) females, while the usual inhibitory tonic activity of serotonergic neurons of the DRN, instead of decreasing after sodium depletion, increases or remains unchanged in OVX+E(2)-DEP and E-DEP females, respectively. Regarding urinary water and sodium excretion 3h after furosemide treatment, E-DEP and OVX+E(2)-DEP animals excreted smaller volumes of more highly concentrated urine than depleted D and OVX rats. Twenty hours after sodium depletion, the same groups of animals also showed a significant increase in the number of Fos-AVP immunoreactive neurons within the supraoptic nucleus, compared with D-DEP. In summary, our results demonstrate an estrogen-dependent inhibition of induced sodium appetite in normally cycling rats and ovariectomized animals with estradiol replacement, which may involve an interaction between excitatory neurons of the OVLT and inhibitory serotonergic cells of the DRN. The main finding is thus serotonergic system involvement as a possible mechanism in the inhibitory action of estrogen on induced sodium appetite., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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, 10mg/kg; IP) administration prior to LPS (100μg/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 (1ng/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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Selective serotonin (5-HT) re-uptake inhibitors (SSRIs) are commonly used in the treatment of generalized anxiety disorder in Humans. However, because only few animal models display overt anxious-like behavior, detailed preclinical studies of the anxiolytic properties of antidepressants are still lacking. Here, we studied the neurochemical and behavioral effects of a double 5-HT(1A/1B) receptor knockout in mice (5-HT(1A/1B)-/-) as compared to their wild-type littermates (5-HT(1A/1B)+/+). It is known that single deletion of either 5-HT(1A) or 5-HT(1B) receptor induces behavioral changes that are not correlated with differences in brain serotonergic tone. Deletion of both receptors resulted in (i) higher emotionality of animals, as observed in three unconditioned paradigms of anxiety (open field, elevated plus maze and novelty suppressed feeding tests); (ii) a ≈200% increase in the mean spontaneous firing rate of 5-HT neurons in the dorsal raphe nucleus (DRN) compared to 5-HT(1A/1B)+/+ mice; (iii) elevated basal dialysate levels of 5-HT in the DRN and frontal cortex; (iv) an exaggerated response to acute paroxetine administration in microdialysis experiments, and (v) increased basal core body temperature. These findings suggest that the deletion of both autoreceptors induces a strong anxious-like behavioral state associated with increased 5-HT neurotransmission. Interestingly, 5-HT(1A/1B)-/- mice are still sensitive to the acute administration of diazepam. Moreover, while deletion of both receptors impacted on the response to acute SSRI treatment in the forced swim test, anxiolytic-like effects of a chronic SSRI treatment were still observed in 5-HT(1A/1B)-/- mice. Thus, the 5-HT(1A/1B)-/- mouse model could be of great interest to unveil the mechanisms of action of the anxiolytic effects of SSRIs., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Previous studies have shown that microinjections of the GABA-A agonist muscimol into the median raphe nucleus (MR) result in large increases in the intake of solid foods. In the current study, we used microstructural techniques to characterize the effects of intra-MR muscimol injections on the consumption of either a 0.05 M or a 0.29 M sucrose solution. After injections of either saline or muscimol, animals consumed more of the 0.29 M than the 0.05 M solution, an effect which resulted primarily from increases in the initial rate of consumption with no change in the rate at which licking decayed across the test session. In contrast, intra-MR muscimol injections had little effect on the initial licking rate, but greatly increased meal duration, indicating that this treatment affected ingestion in a different way than did altering the sucrose concentration. Muscimol injections produced a significantly larger increase in the intake of the 0.29 M than of the 0.05 M solution. Intra-MR muscimol injections did not alter the within burst rate of licking, suggesting that they did not affect the functioning of the licking pattern generator. In contrast, these injections did increase the number of licks contained within "clusters," that is groups of licks separated from each other by intervals of more than 0.5 sec. These findings show that inactivation of the MR produces a powerful effect on the intake of liquid diets, and that the nature of this effect is different from that produced here by changes in sucrose concentration and from those reported after pharmacological manipulations of a number of other brain systems. We additionally discuss several theoretical issues arising in the interpretation of microstructural data. (PsycINFO Database Record (c) 2011 APA, all rights reserved).

Ghrelin promotes growth hormone (GH) secretion and feeding. Recent studies further showed that ghrelin displayed a defending effect against the depressive-like symptoms and affected sleep in animals and humans. Serotonergic system is considered to be implicated in feeding, depression and other mood disorders, and sleep. The dorsal raphe nucleus (DRN) utilizes serotonin (5-HT) as its major neurotransmitter and expresses GH secretagogue receptors (GHS-Rs). Therefore, the present study was carried out to examine the electrophysiological effect of ghrelin on rat DRN neurons in vitro and determine the ionic mechanism involved. Whole-cell recording revealed that ghrelin depolarized DRN neurons dose-dependently in tetrodotoxin-containing artificial cerebrospinal fluid (TTX ACSF). Pretreatment with [D-Lys(3)]-GHRP-6, a selective antagonist for GHS-Rs, antagonized the ghrelin-induced depolarization. The depolarization was significantly reduced in a low-Na(+) TTX ACSF and in a high-K(+) TTX ACSF and was abolished in the combination of both ACSFs, suggesting that the ghrelin-induced depolarization is mediated by a dual ionic mechanism including an increase in nonselective cationic conductance and a decrease in K(+) conductance. The experiments on the reversal potential also supported an involvement of the dual ionic mechanism in the ghrelin-induced depolarization. On the basis of their electrophysiological and pharmacological properties, approximately 80% of DRN neurons were classified as putative 5-HT-containing neurons and ghrelin depolarized 75% of them. These results suggest that DRN neurons, especially 5-HT-containing neurons, might be involved in the neural mechanisms through which ghrelin participates in the development and/or regulation of feeding behavior, sleep-wake states and depressive-like symptoms., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Tobacco use is a major public health problem, and although many smokers report that they want to quit, only a small percentage succeed. Side effects associated with nicotine withdrawal, including depression, anxiety, and restlessness, certainly contribute to the low success rate. The dorsal raphe nucleus (DRN) is a serotonergic center with many functions, including control of mood and emotional state. We investigated the effect of nicotine on DRN neurons that project to the nucleus accumbens (NAc), an area involved in reward-related behaviors. Using a retrograde labeling method, we found that 75% of DRN-NAc projection neurons are serotonergic. In coronal slices that include the DRN, whole cell recordings were conducted on neurons identified by fluorescent backlabeling from NAc or randomly selected within the nucleus. Nicotine increased action potential firing rates in a subset of DRN neurons. Voltage-clamp recording revealed nicotinic acetylcholine receptor (nAChR)-mediated inward currents that contribute to the nicotine-induced excitation. Nicotinic receptors also indirectly affect excitability by modulating synaptic inputs to these neurons. Nicotine enhanced excitatory glutamatergic inputs to a subset of DRN-NAc projection neurons, while inhibitory γ-aminobutyric acid (GABA)ergic inputs were modulated either positively or negatively in a subset of these neurons. The net effect of nAChR activation is enhancement of serotonergic output from DRN to the NAc, which may contribute to the effects of nicotine on mood and affect.

Our previous studies indicated that L-type calcium channel blocker diltiazem could potentiate pentobarbital-induced hypnosis through serotonergic system. In view of the important role of dorsal raphe nucleus (DRN) on the sleep regulation and the pharmacological actions of calcium channel blocker, we presumed that Ca(2+) in the DRN may play an important role in sleep regulation in pentobarbital treated rats. Therefore, we investigated whether the Ca(2+) modulation in DRN by the microinjection of L-type Ca(2+) channel antagonist diltiazem, agonist BAY-K-8644, Ca(2+) chelator EGTA and CaCl(2) would alter the sleep parameters in pentobarbital treated rats. Results showed that perfusion of the agents attenuating Ca(2+) function, such as diltiazem (5 or 20 nmol) or EGTA (3 or 6 pmol) into DRN significantly increased pentobarbital (35 mg/kg, i.p.)-induced total sleep (TS), non-rapid eye movement (NREM) sleep and the slow wave sleep (SWS) ratio in NREM sleep. On the contrary, the DRN injection of the agents improving Ca(2+) function, such as BAY-K-8644 (10 nmol) or CaCl(2) (50 or 100 nmol) significantly reduced pentobarbital (35 mg/kg, i.p.)-induced TS, NREM sleep, rapid eye movement (REM) sleep and REM sleep ratio in TS without influence on SWS. These results suggested that the suppression of Ca(2+) function in DRN could increase NREM sleep including SWS, and the elevation of Ca(2+) function could reduce both NREM and REM sleep in pentobarbital treated rats., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Shivering is a remarkable somatomotor thermogenic response that is controlled by brain mechanisms. We recorded EMGs in anaesthetized rats to elucidate the central neural circuitry for shivering and identified several brain regions whose thermoregulatory neurons comprise the efferent pathway driving shivering responses to skin cooling and pyrogenic stimulation. We simultaneously monitored parameters from sympathetic effectors: brown adipose tissue (BAT) temperature for non-shivering thermogenesis and arterial pressure and heart rate for cardiovascular responses. Acute skin cooling consistently increased EMG, BAT temperature and heart rate and these responses were eliminated by inhibition of neurons in the median preoptic nucleus (MnPO) with nanoinjection of muscimol. Stimulation of the MnPO evoked shivering, BAT thermogenesis and tachycardia, which were all reversed by antagonizing GABA(A) receptors in the medial preoptic area (MPO). Inhibition of neurons in the dorsomedial hypothalamus (DMH) or rostral raphe pallidus nucleus (rRPa) with muscimol or activation of 5-HT1A receptors in the rRPa with 8-OH-DPAT eliminated the shivering, BAT thermogenic, tachycardic and pressor responses evoked by skin cooling or by nanoinjection of prostaglandin (PG) E2, a pyrogenic mediator, into the MPO. These data are summarized with a schematic model in which the shivering as well as the sympathetic responses for cold defence and fever are driven by descending excitatory signalling through the DMH and the rRPa, which is under a tonic inhibitory control from a local circuit in the preoptic area. These results provide the interesting notion that, under the demand for increasing levels of heat production, parallel central efferent pathways control the somatic and sympathetic motor systems to drive thermogenesis.

The effects of the selective 5-HT(3) receptor agonist m-chlorophenylbiguanide (m-CPBG), and of the NMDA (N-methyl-D-aspartate) and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionate)/kainate antagonists AP-5 [(±)-2-amino-5-phosphono-pentanoic acid] and CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), respectively, were studied in adult male Wistar rats implanted for chronic sleep recordings. The compounds were microinjected directly into the dorsal raphe nucleus (DRN) during the light period of the 12-h light/12-h dark cycle. Infusion of m-CPBG (2 and 4mM) into the DRN induced a significant reduction of rapid-eye-movement sleep (REMS) and of the number of REM periods. Local infusion of AP-5 (0.5-1 mM) and CNQX (2 mM) significantly increased slow wave sleep (SWS). Pretreatment with AP-5 (0.5 mM) or CNQX (0.5 mM) antagonized the m-CPBG-induced suppression of REMS. It is proposed that the reduction of REMS after microinjection of m-CPBG into de DRN is related to the activation of glutamatergic interneurons that express the 5-HT(3) receptor and make synaptic contacts with serotonergic cells. The resultant increase of serotonin release at postsynaptic sites involved in the induction of REMS would provoke the suppression of the behavioral state. Our findings provide, in addition, new details concerning the pharmacology of DRN serotonergic neurons in the rat that may become relevant to the development of drugs for enhancing cortical and subcortical serotonergic neurotransmission., (Copyright © 2011 Elsevier Inc. All rights reserved.)

This chapter reviews the neurobiological effects of stress sensitivity and s-citalpram (CIT) treatment observed in our nonhuman primate model of functional hypothalamic amenorrhea (FHA). This type of infertility, also known as stress-induced amenorrhea, is exhibited by cynomolgus macaques. In small populations, some individuals are stress-sensitive (SS) and others are highly stress-resilient (HSR). The SS macaques have suboptimal secretion of estrogen and progesterone during normal menstrual cycles. SS monkeys also have decreased serotonin gene expression and increased CRF expression compared to HSR monkeys. Recently, we found that CIT treatment improved ovarian steroid secretion in SS monkeys, but had no effect in HSR monkeys. Examination of the serotonin system revealed that SS monkeys had significantly lower Fev (fifth Ewing variant, rodent Pet1), TPH2 (tryptophan hydroxylase 2), 5HT1A autoreceptor and SERT (serotonin reuptake transporter) expression in the dorsal raphe than SR monkeys. However, CIT did not alter the expression of either Fev, TPH2, SERT or 5HT1A mRNAs. In contrast, SS monkeys tended to have a higher density of CRF fiber innervation of the dorsal raphe than HSR monkeys, and CIT significantly decreased the CRF fiber density in SS animals. In addition, CIT increased CRF-R2 gene expression in the dorsal raphe. We speculate that in a 15-week time frame, the therapeutic effect of S-citalopram may be achieved through a mechanism involving extracellular serotonin inhibition of CRF and stimulation of CRF-R2, rather than alteration of serotonin-related gene expression., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The aim of this study was to evaluate by quantitative receptor autoradiography the interactions between Neuropeptide Y Y1 (NPY Y1) and Galanin (GAL) receptors in the dorsal raphe nucleus (DRN) where both GAL receptors and NPY Y1 receptors exist. The ability of the GAL receptor antagonist M35 to block the GAL action was also evaluated. Double immunocytochemical staining of 5-hydroxytryptmine and c-Fos and stereology techniques were used to study the specific cell activation in the DRN after the intracerebroventricular coinjections of GAL and the NPY Y1/Y5 agonist [(125)I] Leu(31),Pro(34)PYY. GAL (0.3 nM) decreases [(125)I] Leu(31),Pro(34)PYY binding in the DRN by 48% (p < 0.01) as shown by quantitative receptor autoradiography. This effect was reversed with the GAL receptor antagonist M35. Intracerebroventricular coinjections of NPY Y1/Y5 agonist and GAL reduced the c-Fos expression in the serotoninergic cells induced by the NPY Y1/Y5 agonist in DRN. These results indicate the existence of antagonistic interactions between GAL receptors and NPY Y1 receptors in the DRN that may be of relevance in mood disorders., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Neuroinflammation results in dysregulation of serotonergic neurons in the dorsal raphe nucleus (doR) and is considered to play an important role in the pathophysiology of depression. The aim of the present study was to induce neuroinflammation in a simple doR brain slice model using lipopolysaccharide (LPS), interferon-gamma (IFNγ), beta-amyloid₁₋₄₂ or tumor necrosis factor-alpha and to explore the survival of serotonergic neurons and the expression of the tryptophan degrading enzyme indoleamine 2,3-dioxygenase (IDO). Administration of pro-inflammatory stimuli reduced survival of serotonergic neurons in doR slices and increased IDO expression. IFNγ most potently induced IDO expression, which co-localized with neurons, including serotonergic neurons, but not with microglia or astrocytes. IFNγ did not induce PI-positive staining in slices, but increased the average nuclei size of IDO-positive cells. The inflammation-induced decline did not return to control levels, when slices were withdrawn from inflammation, pointing to neurodegeneration. The growth factors BDNF or GDNF did not counteract the inflammation-induced decrease in serotonergic neurons, except for LPS-induced neuronal decline. The inflammation-induced effect was not blocked by the NMDA-receptor antagonist MK-801. Further LPS, but not IFNγ increased inflammatory markers and microglia activity. In conclusion, our data show that a range of inflammatory stimuli decline serotonergic neurons in doR slices and upregulate IDO expression. The data suggest that IDO does not contribute to serotonergic decline, but may serve as a marker of neurodegeneration. Neuroinflammation may contribute to the development of depression., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Midbrain raphe nuclei provide strong serotonergic projections to the hippocampus, in which serotonin (5-HT) exerts differential effects mediated by multiple 5-HT receptor subtypes. The functional relevance of this diversity of information processing is poorly understood. Here we show that serotonin via 5-HT(1B) heteroreceptors substantially reduces synaptic excitation of cholecystokinin-expressing interneurons in area CA1 of the rat hippocampus, in contrast to parvalbumin-expressing basket cells. The reduction is input specific, affecting only glutamatergic synaptic transmission originating from CA1 pyramidal cells. As a result, serotonin selectively decreases feedback inhibition via 5-HT(1B) receptor activation and subsequently increases the integration time window for spike generation in CA1 pyramidal cells. Our data imply an important role for serotonergic modulation of GABAergic action in subcortical control of hippocampal output.

The anxiolytic effect of fluoxetine (Flx) was often ineffective in postmenopausal and estrogen-deficient patients, but such effect had not been experimentally demonstrated, particularly in the female rat model of estrogen deficiency. Here we determined the anxiety-like behaviors in ovariectomized (Ovx) rats treated for 4weeks with 10μg/kg 17β-estradiol s.c. (Ovx+E2), 10mg/kg Flx p.o. (Ovx+Flx) or a combination of both (Ovx+E2+Flx). Since Flx is known to induce anxiolysis in males, we first evaluated the Flx regimen in male rats. The results showed that anxiety-like behaviors were reduced in Flx-treated male rats. In contrast, Ovx+Flx rats still exhibited the same anxiety-like behaviors as in Ovx rats. Both Ovx+E2 and Ovx+E2+Flx rats, however, showed comparable reductions in anxiety-like behaviors, suggesting that Flx had no anxiolytic-like effect. Furthermore, E2 and E2+Flx similarly upregulated the mRNA expression of serotonin reuptake transporter (SERT) and tryptophan hydroxylase-2 in the dorsal raphé of Ovx rats, while having no effect on SERT expression in the frontal cortex, hippocampus, septum, amygdala and periaqueductal gray. In conclusion, Flx induced anxiolytic-like action in male rats. In Ovx rats, it was E2 and not Flx that exerted the anxiolytic-like action, which was mediated, in part, by altering serotonin metabolism in the dorsal raphé., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Estradiol (E2) exerts an inhibitory effect on food intake in a variety of species. While compelling evidence indicates that central, rather than peripheral, estrogen receptors (ERs) mediate this effect, the exact brain regions involved have yet to be conclusively identified. In order to identify brain regions that are sufficient for E2's anorectic effect, food intake was monitored for 48 h following acute, unilateral, microinfusions of vehicle and two doses (0.25 and 2.5 μg) of a water-soluble form of E2 in multiple brain regions within the hypothalamus and midbrain of ovariectomized rats. Dose-related decreases in 24-h food intake were observed following E2 administration in the medial preoptic area (MPOA), arcuate nucleus (ARC), and dorsal raphe nucleus (DRN). Within the former two brain areas, the larger dose of E2 also decreased 4-h food intake. Food intake was not influenced, however, by similar E2 administration in the paraventricular nucleus, lateral hypothalamus, or ventromedial nucleus. These data suggest that E2-responsive neurons within the MPOA, ARC, and DRN participate in the estrogenic control of food intake and provide specific brain areas for future investigations of the cellular mechanism underlying estradiol's anorexigenic effect., (Copyright © 2011 Elsevier Inc. All rights reserved.)

To control the breathing rhythm the medullary respiratory network generates periodic salvo activities for inspiration, post-inspiration and expiration. These are under permanent modulatory control by serotonergic neurons of the raphe which governs the degree of phosphorylation of the inhibitory glycine receptor α3. The specific activation of serotonin receptor type 1A (5-HTR(1A)), which is strongly expressed in the respiratory neurons, functions via inhibition of adenylate cyclase and the resulting reduction of the intracellular cAMP level and a gradual dephosphorylation of the glycine receptor type α3 (GlyRα3). This 5-HTR(1A)-GlyRα3 signal pathway is independent of the µ-opioidergic transduction pathway and via a synaptic inhibition caused by an increase in GlyRα3 stimulates a disinhibition of some target neurons not only from excitatory but also from inhibitory neurons. Our physiological investigations show that this 5-HTR(1A)-GlyRα3 modulation allows treatment of respiratory depression due to opioids without affecting the desired analgesic effects of opioids. The molecular mechanism presented here opens new pharmacological possibilities to treat opioid-induced respiratory depression and respiratory disorders due to disturbed inhibitory synaptic transmission, such as hyperekplexia.

Post-weaning social isolation of rats is utilized as a model of early life stress. We have previously demonstrated that rats exposed to post-weaning social isolation exhibit greater anxiety-like behaviors as adults. Furthermore, these rats exhibit greater density of corticotropin-releasing factor (CRF) type 2 receptors in the dorsal raphe nucleus. Therefore, we examined whether antagonism of CRF(2) receptors in the dorsal raphe nucleus reverses the effects of post-weaning social isolation on anxiety states. Male rats were reared in isolation or in groups from day of weaning (postnatal day [PND] 21) to mid-adolescence (PND42) and then allowed to develop to early adulthood housed in groups. At PND62, rats were either infused with vehicle, the CRF(1) receptor antagonist antalarmin (0.25-0.5 μg) or the CRF(2) receptor antagonist antisauvagine-30 (2 μg) into the dorsal raphe nucleus, 20 min prior to being introduced to the elevated plus maze. Isolation-reared rats showed reduced open arm behavior compared to group-reared rats, confirming the anxiogenic effects of post-weaning social isolation. Infusion of the CRF(2) receptor antagonist, but not the CRF(1) receptor antagonist, into the dorsal raphe nucleus of isolation-reared rats increased open arm behavior when compared to that of group-reared rats. Overall, the findings suggest that CRF(2) receptors within the dorsal raphe nucleus mediate anxiety-like states following post-weaning social isolation, and CRF(2) receptors may represent an important target for the treatment of anxiety disorders following early life stressors., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Neuroinflammation is a key mechanism contributing to long-term neuropathology observed after neonatal hypoxia-ischemia (HI). Minocycline, a second-generation tetracycline, is a potent inhibitor of neuroinflammatory mediators and is successful for at least short-term amelioration of neuronal injury after neonatal HI. However the long-term efficacy of minocycline to prevent injury to a specific neuronal network, such as the serotonergic (5-hydroxytryptamine, 5-HT) system, is not known. In a post-natal day 3 (P3) rat model of preterm HI we found significant reductions in 5-HT levels, 5-HT transporter expression and numbers of 5-HT-positive dorsal raphé neurons 6 weeks after insult compared to control animals. Numbers of activated microglia were significantly elevated in the thalamus and dorsal raphé although the greatest numbers were observed in the thalamus. Brain levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were also significantly elevated on P45 in the thalamus and frontal cortex. Post-insult administration of minocycline for 1 week (P3-P9) attenuated the P3 HI-induced increases in numbers of activated microglia and levels of TNF-α and IL-1β on P45 with concurrent changes in serotonergic outcomes. The parallel prevention of P3 HI-induced serotonergic changes suggests that inhibition of neuroinflammation within the first week after P3 HI injury was sufficient to prevent long-term neuroinflammation as well as serotonergic system damage still evident at 6 weeks. Thus early, post-insult administration of minocycline may target secondary neuroinflammation and represent a long-term therapy to preserve the integrity of the central serotonergic network in the preterm neonate., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

We present a new hypothesis for the efficacy of selective serotonin reuptake inhibitors (SSRIs). We propose that SSRIs bring the response to the phasic firing of raphe nucleus cells back to normal, even though the average extracellular 5HT concentration remains low. We discuss burst firing in the raphe nuclei and use mathematical models to argue that tonic firing and phasic firing may be decoupled and may come from different mechanisms. We use a mathematical model for serotonin synthesis, release, and reuptake in terminals to illustrate the responses in terminal regions to bursts in a normal individual and in an individual with low vesicular serotonin. We then show that acute doses of SSRIs do not bring the response to bursts back to normal, but that chronic doses do return the response to normal. These model results need to be confirmed by new electrophysiological and pharmacological experiments., (© Georg Thieme Verlag KG Stuttgart · New York.)

Antagonism of the melanin-concentrating hormone (MCH) receptor 1 (MCH-R1) has been recently shown to have antidepressant-like profile in rats. However, the mechanisms by which the MCHergic system participates in the modulation of emotional states are still to be determined. In the present study we confirmed the presence of MCHergic fibers within the dorsal raphe nucleus (DRN), a serotonergic nucleus involved in the physiopathology of major depression. We also assessed the effects of the administration of MCH and anti-MCH antibody (immunoneutralization) into the DRN using the forced swim test in rats, an animal model to screen antidepressant drugs. We found that a low dose of MCH (50 ng) evoked a depressive-like behavior indicated by a significant increase in the immobility time as well as a decrease in climbing behavior. Furthermore, the depressive-like response was prevented by pretreatment with fluoxetine. Consistent with these results, the immunoneutralization of MCH produced an antidepressant-like effect. By means of the open field test we discarded that these effects were related to unspecific changes in motor activity. Our results suggest that the MCHergic neurons are involved in the regulation of emotional behaviors through the modulation of the serotonergic neuronal activity within the DRN. In addition, the present results are in agreement with previous reports showing that antagonism of the MCHergic system may be a novel therapeutic strategy for the treatment of depressive disorders., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: Serotonin 1B (5-HT(1B)) autoreceptors regulate release of serotonin from terminals of dorsal raphe nucleus (DRN) projections. Expression of 5-HT(1B) in the DRN inversely correlates with behavioral measures of emotion, and viral-mediated overexpression of 5-HT(1B) receptors in the middle DRN inversely reduces measures of fear and anxiety in unstressed rats. Because the caudal subregion of the DRN is important in translating stress into emotional dysregulation, we explored behavioral functions of 5-HT(1B) autoreceptors in the caudal DRN., Methods: We manipulated 5-HT(1B) autoreceptor function in rats using either viral-mediated gene transfer into the caudal DRN or systemic injections of the 5-HT(1B) agonist 3-(1,2,5,6-tetrahydro-4-pyridyl)-5-propoxypyrrolo[3,2-b]pyridine (CP-94253). Rats were tested in forced swim test, open field test, and contextual fear conditioning., Results: Overexpression of 5-HT(1B) in the caudal DRN increased swimming in the forced swim test. It did not alter locomotion or thigmotaxis in the open field test but did reduce conditioned freezing. Freezing was reduced when 5-HT(1B) overexpression was present only during testing but not training. The CP-94253 exerted an inverted U-shaped dose response curve on conditioned freezing, with most pronounced effects seen at 1 mg/kg. At this dose, CP-94253 administered before a fear retention test reduced freezing both during that session and in subsequent drug-free testing, but only when drug was paired with re-exposure to the fear context., Conclusions: The 5-HT(1B) autoreceptors originating in the caudal DRN regulate behavioral expression of helplessness and fear. Because systemic pharmacologic treatment with a 5-HT(1B) agonist facilitates reductions in fear, 5-HT(1B) receptors may be a target for the treatment of certain anxiety disorders., (Copyright © 2011 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)