Intracerebroventricular administration of corticotropin-releasing factor antagonist attenuates arousal response accompanied by yawning behavior in rats

We have reported that an arousal response accompanied by yawning behavior can be evoked by electrical and chemical stimulation of the hypothalamic paraventricular nucleus (PVN) in rats, although the mechanism responsible for the arousal response accompanied by yawning evoked by PVN stimulation is still unknown. In the present study, we examined the involvement of corticotropin-releasing factor (CRF) in the arousal response during yawning induced by electrical stimulation of the PVN in anesthetized, spontaneous breathing rats using intracerebroventricular (icv) injection of alpha-helical CRF, a CRF antagonist (4.2 microg, lateral ventricle). The electrocorticogram (ECoG) was recorded to evaluate arousal responses during yawning. Fast Fourier transform was used to obtain the power spectrum in delta (0.5-4 Hz), theta (4-8 Hz), alpha (8-13 Hz), and beta (13-20 Hz) bands. We also recorded the intercostal electromyogram as an index of inspiratory activity and blood pressure (BP) as an index of autonomic function to evaluate yawning response. PVN stimulation induced significant increases in relative powers of theta, alpha, and beta bands, but not delta band, concurrent with yawning events regardless of icv injection, though the relative powers after icv injection of alpha-helical CRF were significantly lower than those after saline injection. These findings suggest that CRF neurons in the PVN are primarily responsible for the arousal response accompanied by yawning behavior.     

Microinjection of urocortin 2 into the dorsal raphe nucleus activates serotonergic neurons and increases extracellular serotonin in the basolateral amygdala

The intra dorsal raphe nucleus (DRN) administration of corticotropin releasing hormone (CRF) inhibits serotonergic (5-HT) activity in this structure, an effect blocked by antagonists selective for the type 1 CRF receptor (CRF1). The DRN has a high density of the type 2 receptor (CRF2), and so the present experiments explored the impact of CRF2 activation within the DRN on 5-HT function. The intra-DRN administration of the selective CRF2 agonist urocortin 2 (Ucn 2) dose dependently increased 5-HT efflux in the basolateral amygdala, a projection region of the DRN. Intra-DRN Ucn 2 also increased c-fos expression in labeled 5-HT neurons. Both of these effects of Ucn 2 were completely blocked by intra-DRN antisauvagine-30 (ASV-30), a relatively selective CRF2 antagonist. These data suggest that CRF1 and CRF2 activation within the DRN affect 5-HT neurons in opponent fashion. Implications of these results for understanding the behavioral effects of CRF and other CRF-like ligands are discussed.     

Use of peripheral blood transcriptome biomarkers for epilepsy prediction

Fenfluramine reduces hunger and promotes body weight loss by increasing central serotonin (5-HT) signaling. More recently, neuropeptides have been linked to the regulation of feeding behavior, metabolism and body weight. To examine possible interactions between 5-HT and neuropeptides in appetite control, fenfluramine (200 nmol/0.5 μl/side) was administered directly into the hypothalamic paraventricular nuclei (PVN) of male rats. Bilateral fenfluramine produced significant hypophagia and increased expression of PVN corticotropin releasing factor (CRF) mRNA and neuropeptide Y (NPY) mRNA in the arcuate nucleus within the first hour after drug administration. Fenfluramine's effects on feeding behavior and mRNA expression were blocked by PVN injections of a 5-HT(1-2) receptor antagonist, metergoline (15 nmol/0.5 μl/side). These data suggest that 5-HT neurons targeting hypothalamic paraventricular CRF neurons may participate in an appetite control circuit for reducing food intake.     

Stress potentiation of morphine-induced dopamine efflux in the nucleus accumbens shell is dependent upon stressor uncontrollability and is mediated by the dorsal raphe nucleus

A single session of uncontrollable (inescapable tailshock, IS), but not controllable (escapable tailshock, ES), stress is known to selectively potentiate subsequent morphine-conditioned place preference in a dorsal raphe nucleus (DRN) serotonin (5-HT) dependent manner. Here, in vivo microdialysis is used to test the hypothesis that prior IS, but not ES, will potentiate morphine-induced dopamine (DA) efflux in the nucleus accumbens (NAc) shell and that this will occur by a pathway involving DRN 5-HT neurons. Male Sprague-Dawley rats were exposed to yoked IS, ES, or no stress. Twenty-four hours later, morphine (3 mg/kg s.c.) or saline was administered during microdialysis. As predicted, prior IS selectively potentiated morphine-induced DA, but not 5-HT, efflux in the NAc. This potentiation was due to morphine's action in the DRN because it was blocked by intra-DRN microinjection of the opioid antagonist naltrexone (10 microg). IS potentiation of morphine-induced DA efflux in the NAc was also dependent upon activation of 5-HT neurons in the DRN because it was blocked by intra-DRN microinjection of the 5-HT1A autoreceptor agonist 8-hydroxy-2-di-n-(propylamino)-tetralin (1 microg). No effect of IS was found on morphine-induced 5-HT or DA efflux in the ventral tegmental area. These results suggest a neural substrate for stress potentiation of morphine reward involving 5-HT neurotransmission in the DRN.     

Nociceptin/orphanin FQ decreases serotonin efflux in the rat brain but in contrast to a kappa-opioid has no antagonistic effect on mu-opioid-induced increases in serotonin efflux

Similar to kappa-opioids, nociceptin/orphanin FQ (OFQ) exerts anti-mu-opioid actions. This may involve interactions within the circuitry controlling 5-HT neurons in the dorsal raphe nucleus (DRN) that project to the nucleus accumbens (NAcc). To test this hypothesis, we compared the effects of OFQ and kappa-opioids on 5-HT efflux in the CNS of freely behaving rats. First, OFQ (30-300 microM) infused into the DRN for 120 min dose-dependently decreased 5-HT efflux in the DRN. The opioid receptor-like 1 (ORL-1) antagonist [Nphe(1)]nociceptin(1-13)NH(2) blocked this effect. Using dual-probe microdialysis we observed that OFQ (300 microM) infused into the DRN for 120 min produced parallel decreases in 5-HT efflux in the DRN and NAcc, suggesting that ORL-1 receptors in the DRN inhibit serotonergic neurons projecting to the NAcc. Also, 5-HT efflux in the NAcc was dose-dependently decreased during OFQ (30-300 microM) infusion into the NAcc. This suggests that OFQ can reduce 5-HT efflux in the NAcc both by inhibiting serotonergic neurons in the DRN and by stimulating ORL-1 receptors in the NAcc. Similar to OFQ, the kappa-opioids U-50,488 (300 microM) and dynorphin A(1-13) (300 microM) infused into the DRN for 120 min decreased 5-HT efflux in the DRN. This effect was blocked only by the kappa-opioid receptor antagonist nor-BNI. Lastly, we compared the ability of OFQ and U-50,488 to block mu-opioid-induced increases in 5-HT. The kappa-opioid U-50,488 (1000 microM) attenuated the increase in 5-HT induced by the mu-opioid agonist endomorphin-1 (300 microM) in the DRN. In contrast, OFQ (300-1000 microM) did not alter mu-opioid-induced increases in 5-HT efflux. In summary, kappa-opioids and OFQ both decreased 5-HT efflux in the CNS. However, in contrast to kappa-opioids, which reversed mu-opioid-induced increases in 5-HT efflux, the anti-mu-opioid effects of OFQ apparently do not involve changes in 5-HT transmission under our experimental conditions.     

Chronic low dose ovine corticotropin releasing factor or urocortin II into the rostral dorsal raphe alters exploratory behavior and serotonergic gene expression in specific subregions of the dorsal raphe

Corticotropin releasing factor (CRF) family peptides play key roles in integrating neural responses to stress. Both major CRF receptors have been pharmacologically identified in the dorsal raphe nucleus (DRN), a stress sensitive and internally heterogeneous nucleus supplying many forebrain regions with serotonergic input. Despite the involvement of chronic stress and serotonergic dysfunction in human mood and anxiety disorders, little is known about the effects of chronic CRF receptor activation on the DRN. We infused ovine CRF (1 ng/h), urocortin II (UCNII, 1 ng/h), or vehicle alone into rat DRN over 6 days. During infusion, animals were allowed to freely explore an open field for 15 min on each of 2 days, with the addition of a novel object on the second day. Following behavioral testing, 5-HT1A, 5-HT1B, 5-HT transporter (SERT), and tryptophan hydroxylase-2 (Tph2) expression was examined through the DRN by in situ hybridization. Ovine CRF infusion resulted in significantly decreased novel object touches, climbs, as well as increased latency to first novel object contact. UCNII had a similar but less dramatic effect, decreasing only climbing behavior. Both ovine CRF and UCNII blunted the decrease in corner time expected on re-exposure to the open field. Both peptides also produced regionally specific changes in gene expression: 5-HT1A expression was increased 30% in the mid-rostral ventromedial DRN, while SERT was decreased by 30% in the mid-caudal shell dorsomedial DRN. There also appeared to be a shift in the relative level of Tph2 expression between the ventromedial and core dorsomedial DRN at the mid-rostral level. Changes in 5-HT1A, SERT, and relative Tph2 mRNA abundance were correlated with novel object exploration. These findings suggest chronic intra-DRN administration of CRF agonists decreases exploratory behavior, while producing subregionally limited changes in serotonergic gene expression. These studies may be relevant to mechanisms underlying behavioral changes after chronic stress.     

Aggressive encounters alter the activation of serotonergic neurons and the expression of 5-HT1A mRNA in the hamster dorsal raphe nucleus

Serotonergic (5-HT) neurons in the dorsal raphe nucleus (DRN) have been implicated in stress-induced changes in behavior. Previous research indicates that stressful stimuli activate 5-HT neurons in select subregions of the DRN. Uncontrollable stress is thought to sensitize 5-HT neurons in the DRN and allow for an exaggerated 5-HT response to future stimuli. In the current study, we tested the hypothesis that following aggressive encounters, losing male Syrian hamsters would exhibit increased c-Fos immunoreactivity in 5-HT DRN neurons compared to winners or controls. In addition, we tested the hypothesis that losers would have decreased 5-HT1A mRNA levels in the DRN compared to winners or controls. We found that a single 15-min aggressive encounter increased c-Fos expression in 5-HT and non-5-HT neurons in losers compared to winners and controls. The increased c-Fos expression in losers was restricted to ventral regions of the rostral DRN. We also found that four 5-min aggressive encounters reduced total 5-HT1A mRNA levels in the DRN in losers compared to winners and controls, and that differences in mRNA levels were not restricted to specific DRN subregions. These results suggest that social defeat activates neurons in select subregions of the DRN and reduces message for DRN 5-HT1A autoreceptors. Our results support the hypothesis that social stress can activate 5-HT neurons in the DRN, reduce 5-HT1A autoreceptor-mediated inhibition, and lead to hyperactivity of 5-HT neurons.     

An activation of parvocellular oxytocinergic neurons in the paraventricular nucleus in oxytocin-induced yawning and penile erection

Intracerebroventricular (ICV) or PVN local injections of oxytocin induce yawning and penile erection, for which a positive feedback mechanism for the PVN oxytocinergic activation is suggested, but this had not been directly substantiated in vivo. We have assessed the behavioral effects and activity of oxytocinergic neurons with double-staining for c-Fos and oxytocin in the PVN after ICV administration of oxytocin in adult male rats. ICV oxytocin injections (50 and 200 ng) dose-dependently induced yawning and penile erection and significantly increased the percentage of c-Fos positive oxytocin neurons in the medial, dorsal and lateral parvocellular subdivision of the PVN. However, increases in the magnocellular portion were not significant. We also found that lithium chloride (LiCl, 0.5 and l.0 mEq), a compound known to activate oxytocinergic neurons, also significantly increased the percentage of c-Fos positive oxytocin neurons in all PVN portions. However, LiCl did not induce yawning and penile erection, but counteracted the oxytocin-induced yawning and penile erection. These results suggest that if the activation of oxytocinergic neurons in the PVN is important for mediating oxytocin-induced yawning and penile erection, a selective activation of parvocellular oxytocinergic neurons in the PVN is likely to be involved.     

Coordinate regulation of noradrenergic and serotonergic brain regions by amygdalar neurons

Based on the importance of the locus coeruleus-norepinephrine (LC-NE) system and the dorsal raphe nucleus-serotonergic (DRN-5-HT) system in stress-related pathologies, additional understanding of brain regions coordinating their activity is of particular interest. One such candidate is the amygdalar complex, and specifically, the central nucleus (CeA), which has been implicated in emotional arousal and is known to send monosynaptic afferent projections to both these regions. Our present data using dual retrograde tract tracing is the first to demonstrate a population of amygdalar neurons that project in a collateralized manner to the LC and DRN, indicating that amygdalar neurons are positioned to coordinately regulate the LC and DRN, and links these brain regions by virtue of a common set of afferents. Further, we have also characterized the phenotype of a population of these collaterally projecting neurons from the amygdala as containing corticotropin releasing factor or dynorphin, two peptides heavily implicated in the stress response. Understanding the co-regulatory influences of this limbic region on 5HT and NE regions may help fill a gap in our knowledge regarding neural circuits impacting these systems and their adaptations in stress.     

Unilateral lesion of the nigrostriatal pathway induces an increase of neuronal firing of the midbrain raphe nuclei 5-HT neurons and a decrease of their response to 5-HT1A receptor stimulation in the rat

Several studies have shown that the 5-hydroxytryptamine (serotonin, 5-HT) system is severely affected after degeneration of nigrostriatal dopaminergic neurons. In the present study, we examined the changes in the firing rate and firing pattern of the dorsal and median raphe nuclei (DRN and MRN) 5-HT neurons, and the effect of the selective 5-HT_1A receptor agonist (R)-(+)-8-hydroxy-2-(dipropylamino)tetralin hydrobromide (8-OH-DPAT) and antagonist (N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-2-pyridylcyclohexane carboxamide maleate salt (WAY-100635) on the neuronal firing in rats with 6-hydroxydopamine (6-OHDA) lesions of the substantia nigra pars compacta by using extracellular recording. The unilateral lesion of the nigrostriatal pathway significantly increased the mean firing rate of DRN and MRN 5-HT neurons compared with normal rats, and the firing pattern of these neurons also changed significantly towards a more bursty one. The lower dose of 8-OH-DPAT, 4 μg/kg (cumulative doses, i.v.), completely inhibited the firing activity of all DRN and MRN 5-HT neurons examined in normal and sham rats. In contrast to normal and sham rats, only the higher doses of 8-OH-DPAT, 128 and 64 μg/kg, completely inhibited the firing rate of DRN and MRN 5-HT neurons in 6-OHDA-lesioned rats, respectively. Furthermore, the local application of 8-OH-DPAT, 1.5 μg, in the DRN completely inhibited the firing rate of 5-HT neurons in normal and sham rats, while having no effect on firing rate in the lesioned rats. Altogether, these results indicate that lesion of the nigrostriatal pathway leads to hyperactivity of DRN and MRN 5-HT neurons, suggesting the implication of the DRN and MRN in the pathophysiology of Parkinson's disease, and the decreased response of these 5-HT neurons to 5-HT_1A receptor stimulation, reflecting 5-HT_1A receptor dysfunction in 6-OHDA-lesioned rats.     

In vivo assessment of the midbrain raphe nuclear regulation of serotonin release in the hamster suprachiasmatic nucleus

Serotonin (5-HT) plays important regulatory roles in mammalian circadian timekeeping; however, little is known concerning the regulation of serotonergic activity in the circadian clock located in the suprachiasmatic nuclei (SCN). By using in vivo microdialysis to measure 5-HT release we demonstrated that electrical or pharmacological stimulations of the dorsal or median raphe nuclei (DRN and MRN, respectively) can alter basal release of 5-HT in the hamster SCN. There were similar increases in SCN 5-HT release after electrical stimulation of either the MRN or DRN, indicating that both could contribute to the serotonergic activity in the SCN. Systemic pretreatment with the 5-HT antagonist metergoline abolished DRN-induced SCN 5-HT release but had little effect on MRN-induced SCN 5-HT release, suggesting different pathways for these nuclei in regulating 5-HT output in the SCN. Microinjections of the 5-HT1A autoreceptor agonist 8-OH-DPAT or antagonist WAY 100635 into the MRN caused significant inhibition and stimulation of SCN 5-HT release, respectively. Both drugs had substantially less effect in the DRN. These differential drug actions indicate that somatodendritic 5-HT1A autoreceptors on MRN neurons provide the prominent raphe autoregulation of 5-HT output in the SCN. Collectively the current results are evidence that DRN as well as MRN neurons can contribute to the regulation of 5-HT release in the hamster SCN. On the basis of the current observations and those from recent anatomic tracing studies of serotonergic projections to SCN it is hypothesized that DRN input to the SCN could be mediated by a DRN --> MRN --> SCN pathway involving a 5-HT-sensitive multisynaptic interaction between the DRN and MRN neurons.     

高频电刺激大鼠丘脑底核抑制背侧中缝核5-羟色胺表达

本研究探讨高频电刺激丘脑底核对大鼠背侧中缝核5-羟色胺(5-HT)表达的影响。实验动物分两组,刺激组给予高频电流(130Hz,100μA,60μs)刺激大鼠右侧丘脑底核,对照组大鼠右侧丘脑底核植入电极,但无电流输出。刺激结束后,用免疫组织化学方法染色背侧中缝核5-HT能神经元,检测背侧中缝核5-HT能神经元的数量和平均灰度值。结果显示电刺激组背侧中缝核5-HT阳性神经元数目与对照组比明显减少(t(13)=3.786,P=0.002),并且神经递质5-HT表达量减少,平均灰度值显著增高(t(13)=7.917,P<0.001)。本实验结果表明高频电刺激丘脑底核对背侧中缝核5-HT能神经元有抑制作用,在应用高频电刺激丘脑底核治疗Parkison病运动障碍时出现的情绪障碍可能与其有关。     

Somatosensory regulation of serotonin release in the central nucleus of the amygdala is mediated via corticotropin releasing factor and gamma-aminobutyric acid in the dorsal raphe nucleus

Noxious cutaneous stimulation increases, whereas innocuous cutaneous stimulation decreases serotonin (5-HT) release in the central nucleus of the amygdala (CeA) in anesthetized rats. In the present study, we investigated the contribution of corticotropin releasing factor (CRF) receptors and gamma-aminobutyric acid (GABA) receptors in the dorsal raphe nucleus (DRN) to those responses. Release of 5-HT in the CeA was monitored by microdialysis before and after 10-min stimulation by pinching or stroking. Increased 5-HT release in the CeA in response to pinching was abolished by CRF₂ receptor antagonism in the DRN. Decreased 5-HT release in the CeA in response to stroking was abolished by either CRF₁ receptor antagonism or GABA_A receptor antagonism in the DRN. These results suggest that opposite responses of 5-HT release in the CeA to noxious versus innocuous stimulation of the skin are due to separate contributions of CRF₂, CRF₁ and GABA_A receptors in the DRN.     

Opioidergic, GABAergic and serotonergic neurotransmission in the dorsal raphe nucleus modulates tonic immobility in guinea pigs

Tonic immobility (TI) is an innate defensive behavior that can be elicited by physical restriction and postural inversion and is characterized by a profound and temporary state of akinesis. Our previous studies demonstrated that the stimulation of serotonin receptors in the dorsal raphe nucleus (DRN) appears to be biphasic during TI responses in guinea pigs (Cavia porcellus). Serotonin released by the DRN modulates behavioral responses and its release can occur through the action of different neurotransmitter systems, including the opioidergic and GABAergic systems. This study examines the role of opioidergic, GABAergic and serotonergic signaling in the DRN in TI defensive behavioral responses in guinea pigs. Microinjection of morphine (1.1 nmol) or bicuculline (0.5 nmol) into the DRN increased the duration of TI. The effect of morphine (1.1 nmol) was antagonized by pretreatment with naloxone (0.7 nmol), suggesting that the activation of μ opioid receptors in the DRN facilitates the TI response. By contrast, microinjection of muscimol (0.5 nmol) into the DRN decreased the duration of TI. However, a dose of muscimol (0.26 nmol) that alone did not affect TI, was sufficient to inhibit the effect of morphine (1.1 nmol) on TI, indicating that GABAergic and enkephalinergic neurons interact in the DRN. Microinjection of alpha-methyl-5-HT (1.6 nmol), a 5-HT(2) agonist, into the DRN also increased TI. This effect was inhibited by the prior administration of naloxone (0.7 nmol). Microinjection of 8-OH-DPAT (1.3 nmol) also blocked the increase of TI promoted by morphine (1.1 nmol). Our results indicate that the opioidergic, GABAergic and serotonergic systems in the DRN are important for modulation of defensive behavioral responses of TI. Therefore, we suggest that opioid inhibition of GABAergic neurons results in disinhibition of serotonergic neurons and this is the mechanism by which opioids could enhance TI. Conversely, a decrease in TI could occur through the activation of GABAergic interneurons.     

Serotonergic and nonserotonergic dorsal raphe neurons are pharmacologically and electrophysiologically heterogeneous

The dorsal raphe nucleus (DRN) projects serotonergic axons throughout the brain and is involved in a variety of physiological functions. However, it also includes a large population of cells that contain other neurotransmitters. To clarify the physiological and pharmacological differences between the serotonergic and nonserotonergic neurons of the DRN, their postsynaptic responses to 5-hydroxytryptamine (5-HT, serotonin) and to selective activation of 5-HT1A or 5-HT2A/C receptors and their action potential characteristics were determined using in vitro patch-clamp recordings. The slices containing these neurons were then immunostained for tryptophan hydroxylase (TPH), a marker of serotonergic neurons. It was found that subpopulations of both serotonergic and nonserotonergic neurons responded to 5-HT with outward (i.e., inhibitory) and inward (i.e., excitatory) currents, responded to both 5-HT1A and 5-HT2A/C receptor activation with outward and inward currents, respectively, and displayed overlapping action potential characteristics. These findings suggest that serotonergic and nonserotonergic neurons in the DRN are both heterogeneous with respect to their individual pharmacological and electrophysiological characteristics. The findings also suggest that the activity of the different populations of DRN neurons will display heterogeneous changes when the serotonergic tone in the DRN is altered by neurological disorders or by drug treatment.     

Studies on the neuroendocrine role of serotonin

The aim of the thesis was to investigate in male Wistar rats, the involvement of serotonin (5-HT) and 5-HT receptors in the regulation of the gene expression of hypothalamic hormones and in the secretion of the pituitary gland hormones prolactin (PRL), adrenocorticotropic hormone (ACTH), vasopressin (AVP) and oxytocin in basal and stress conditions. Furthermore, to study the significance of some distinctive central nuclei in these processes, and the metabolism of 5-HT in the hypothalamus and the dorsal raphe nucleus (DRN). The experiments were focused on (1) determination of involved neurons and nuclei (2) the hypothalamic level and (3) the pituitary gland level of regulation. The studies were typically performed in vivo but some studies were performed in vitro. Stereotactically neurotoxic lesion with 5,7-dihydroxy-5-HT in the dorsal raphe nucleus (DRN) or the hypothalamic paraventricular nucleus (PVN) reduced the ACTH and AVP response to stress, indicating an importance of these structures for this response. In situ hybridization on rat brain slices with oligopeptides showed an increase of corticotropin releasing hormone (CRH) mRNA in the PVN and proopiomelanocortin in the anterior pituitary lobe upon stimulation of the 5-HT1A, 5-HT1B, 5-HT2A and 5-HT2C receptors. Stimulation of 5-HT2A+2C receptors increased AVP mRNA in the PVN but not in the supraoptic nucleus (SON), whereas the level of oxytocin (OT) mRNA was increased both in the SON and the PVN and this effect was in addition mediated via 5-HT1A+1B receptors. Serotonin infused directly into the PVN by microdialysis stimulated local release of AVP. CRH was found to have a major role but not a complete responsibility in the 5-HT-induced release of ACTH, since immunoneutralisation of CRH inhibited the POMC gene expression and the ACTH response and since 5-HT and 5-HT antagonists were able to modulate the ACTH release from anterior pituitary gland cells in vitro. Through the years of investigation, the classification of the 7 main groups of 5-HT receptors (5-HT1 - 5-HT7) has changed due to molecular biological characterisation of the receptors and new receptors have been identified. With a battery of 5-HT agonists and antagonists several pharmacological experiments were performed with systemically or central administration of compounds and radioimmuno assay of plasma for pituitary gland hormone levels. Specific substances were not available for all 5-HT receptors and subreceptors thus some conclusions are a based on combination of experiments. The 5-HT induced PRL response is mediated via 5-HT1A, 5-HT2A, 5-HT2C and 5-HT3 receptors. In addition an involvement of 5-HT1B, 5-HT5 or 5-HT7 receptors seem possible. The ACTH response to 5-HT is mediated via 5-HT1A, 5-HT1B, 5-HT2A and 5-HT2C receptors and an involvement of the 5-HT4, 5-HT5 and 5-HT7 receptors is proposed. Peripheral secretion of AVP upon stimulation with 5-HT is mediated via 5-HT2C, 5-HT4 and 5-HT7 receptors but not 5-HT1A receptors. The secretion of OT is primarily mediated via 5-HT1A, 5-HT2C and 5-HT4 receptors and probably also 5-HT1B, 5-HT2A, 5-HT5A and 5-HT7 receptors. Physical and psychological stress activates hippocampal and hypothalamic 5-HT neurons. In contrast to other stress factors, restraint stress increases the content of 5-HT in the DRN but do not increase the metabolism of 5-HT and does not induce changes in hypothalamic levels of 5-HT. Large variations are found in the literature with different kinds of stress, different measurements and different time schedules. Restraint or ether stress induced secretion of PRL involves 5-HT2 and 5-HT3 receptors, whereas the ACTH secretion is mediated via 5-HT1A, 5-HT2A and 5-HT2C receptors. In the present study restraint stress increased AVP secretion, but opposite findings has reported possibly due to differences in the stress procedure. The 5-HT2, 5-HT3 and 5-HT4 receptor is involved in the AVP response to restraint whereas the OT response involves the 5-HT1A and the 5-HT2 receptor. The 5-HT2 receptor is involved in the OT response to dehydration or haemorrhage, whereas the AVP responses to these stressors probably do not involve 5-HT. It can be concluded that 5-HT is involved in basal and stress-induced regulation of PRL, ACTH, AVP and oxytocin mainly via the 5-HT2A+2C receptors but other receptors are also important but differs from hormone to hormone. Serotonin affect the secretion of CRH and ACTH both at the hypothalamic, pituitary portal and pituitary gland level, and possibly also at the adrenal level.     

Circadian clock resetting by behavioral arousal: neural correlates in the midbrain raphe nuclei and locus coeruleus

Some procedures for stimulating arousal in the usual daily rest period (e.g., gentle handling, novel wheel-induced running) can phase shift circadian rhythms in Syrian hamsters, while other arousal procedures are ineffective (inescapable stress, caffeine, modafinil). The dorsal and median raphe nuclei (DRN, MnR) have been implicated in clock resetting by arousal and, in rats and mice, exhibit strong regionally specific responses to inescapable stress and anxiogenic drugs. To examine a possible role for the midbrain raphe nuclei in the differential effects of arousal procedures on circadian rhythms, hamsters were aroused for 3 h in the mid-rest period by confinement to a novel running wheel, gentle handling (with minimal activity) or physical restraint (with intermittent, loud compressed air stimulation) and sacrificed immediately thereafter. Regional expression of c-fos and tryptophan hydroxylase (TrpOH) were quantified immunocytochemically in the DRN, MnR and locus coeruleus (LC). Neither gentle handling nor wheel running had a large impact on c-fos expression in these areas, although the manipulations were associated with a small increase in c-Fos in TrpOH-like and TrpOH-negative cells, respectively, in the caudal interfascicular DRN region. By contrast, restraint stress significantly increased c-Fos in both TrpOH-like and TrpOH-negative cells in the rostral DRN and LC. c-Fos-positive cells in the DRN did not express tyrosine hydroxylase. These results reveal regionally specific monoaminergic correlates of arousal-induced circadian clock resetting, and suggest a hypothesis that strong activation of some DRN and LC neurons by inescapable stress may oppose clock resetting in response to arousal during the daily sleep period. More generally, these results complement evidence from other rodent species for functional topographic organization of the DRN.     

Electrophysiological and pharmacological properties of GABAergic cells in the dorsal raphe nucleus

The dorsal raphe nucleus (DRN) is the origin of the central serotonin [5-hydroxytryptamine (5-HT)] system and plays an important role in the regulation of many physiological functions such as sleep/arousal, food intake and mood. In order to understand the regulatory mechanisms of 5-HT system, characterization of the types of neurons is necessary. We performed electrophysiological recordings in acute slices of glutamate decarboxylase 67–green fluorescent protein knock-in mice. We utilized this mouse to identify visually GABAergic cells. Especially, we examined postsynaptic responses mediated by 5-HT receptors between GABAergic and serotonergic cells in the DRN. Various current responses were elicited by 5-HT and 5-HT_1A or 5-HT_2A/2C receptor agonists in GABAergic cells. These results suggested that multiple 5-HT receptor subtypes overlap on GABAergic cells, and their combination might control 5-HT cells. Understanding the postsynaptic 5-HT feedback mechanisms may help to elucidate the 5-HT neurotransmitter system and develop novel therapeutic approaches.     

Stress induces CRF release in the paraventricular nucleus, and both CRF and GABA release in the amygdala

In the hypothalamus, corticotropin-releasing factor (CRF) initiates the hypothalamic-pituitary-adrenal (HPA) axis response to stress, resulting in the release of glucocorticoids, including cortisol. Extrahypothalamic CRF, particularly in the limbic system, also appears to play a role in the stress response. To further define brain CRF response to stress, immunosensor-based microdialysis probes were used to measure the extracellular levels of CRF in the paraventricular nucleus of the hypothalamus (PVN) and in the amygdala of sheep during a predator (dog) exposure stress. In addition, gamma amino butyric acid (GABA) was measured in the amygdala and cortisol was measured in venous blood. Exposure to the predator stress increased CRF in the PVN and both CRF and GABA in the amygdala. These were followed in time by a rise in venous cortisol. Application of a CRF antagonist to the amygdala, immediately prior to stress, had a small effect on the subsequent observed stress responses. This treatment, however, significantly reduced the responses to a repeat stress administered 2 days later, compared to nontreated animals. Application of a GABA antagonist to the amygdala prior to stress had no effect on the subsequent observed stress response but increased the response to the stress repeated 2 days later. Perfusion with 4-aminopyridine, a neuronal depolarising agent, into the PVN induced a release of CRF accompanied shortly thereafter by a small increase in CRF in the amygdala, and 5-10 min later by an increase in venous cortisol. Perfusion into the amygdala increased the levels of both CRF and GABA but had no effect on either PVN CRF or venous cortisol. These data support roles for both the PVN and amygdala in stress responsiveness. It suggests further that actions at the amygdala can strongly influence subsequent responsiveness to a further stress, mediated in part by both CRF and GABA actions.     

Noradrenergic inputs to the paraventricular hypothalamus contribute to hypothalamic-pituitary-adrenal axis and central Fos activation in rats after acute systemic endotoxin exposure

Noradrenergic (NA) neurons within the nucleus of the solitary tract (NST) and caudal ventrolateral medulla (VLM) innervate the hypothalamic paraventricular nucleus (PVN) to initiate and modulate hypothalamic-pituitary-adrenal (HPA) axis responses to interoceptive stress. Systemic endotoxin (i.e. bacterial lipopolysaccharide, LPS) activates NA neurons within the NST and VLM that project to the PVN and other brain regions that receive interoceptive signals. The present study examined whether NA neurons with axonal inputs to the PVN are necessary for LPS to activate Fos expression within the PVN and other interoceptive-related brain regions, and to increase plasma corticosterone. Male Sprague-Dawley rats received bilateral stereotaxic microinjections of DSAP (saporin toxin conjugated to an antibody against dopamine-beta-hydroxylase, DbH) into the PVN to destroy NA inputs. Control rats were microinjected with vehicle into the PVN or received no PVN injections. Two weeks later, DSAP and control rats were injected i.p. with LPS (200 microg/kg BW) or saline vehicle, and perfused with fixative 2.5-3 h later. Brain tissue sections were processed to reveal nuclear Fos protein and cytoplasmic DbH immunolabeling. DSAP lesions depleted NA terminals in the PVN and bed nucleus of the stria terminalis, reduced the number of NA cell bodies in the NST and VLM, attenuated PVN Fos activation after LPS, and attenuated LPS-induced increases in plasma corticosterone. These findings support the view that NA projections from hindbrain to hypothalamus are necessary for a full HPA axis response to systemic immune challenge.