Gastrointestinal effects of intracerebroventricularly injected nociceptin/orphaninFQ in rats

Nociceptin/orphanin FQ/(N/OFQ), a novel heptadecapeptide recently isolated from porcine and rat brain, is the endogenous ligand of the N/OFQ peptide receptor (NOP, previously known as ORL-1). In this study we examined the effects of intracerebroventricularly (icv) injected N/OFQ on gastric emptying, gastrointestinal transit, colonic propulsion and gastric acid secretion in rats. N/OFQ (0.01-10 nmol/rat) significantly delayed gastric emptying of a phenol red meal, inhibited transit of a non-absorbable charcoal marker through the small intestine and increased the mean colonic bead expulsion time. These N/OFQ-motor effects were abolished by the NOP receptor selective antagonist [NPhe(1)]N/OFQ(1-13)-NH(2) (50 nmol/rat), but were unaltered by the classical opioid receptor antagonist, naloxone (9.2 micromol/kg). Icv injected N/OFQ (10 nmol/rat) decreased gastric acid secretion in 2-h pylorus ligated rats in a naloxone sensitive manner. [NPhe(1)]N/OFQ(1-13)-NH(2) (100 nmol/rat) icv administered alone stimulated gastric acid secretion. These results indicate that N/OFQ activates via NOP receptor stimulation a central inhibitory pathway modulating gastrointestinal propulsive activity and gastric acid secretion in rats.     

Role of dorsal raphe nucleus serotonin 5-HT1A receptor in the regulation of REM sleep

Cholinergic neurons in the laterodorsal (LDT) and the pedunculopontine (PPT) tegmental nuclei act to promote REM sleep (REMS). The predominantly glutamatergic neurons of the REMS-induction region of the medial pontine reticular formation are in turn activated by cholinergic cells, which results in the occurrence of tonic and phasic components of REMS. All these neurons are inhibited by serotonergic (5-HT), noradrenergic, and presumably histaminergic (H2 receptor) and dopaminergic (D2 and D3 receptor) cells. 5-Hydroxytryptamine-containing neurons in the dorsal raphe nucleus (DRN) virtually cease firing when an animal starts REMS, consequently decreasing the release of 5-HT during this state. The activation of GABA(A) receptors is apparently responsible for this phenomenon. Systemic administration of the selective 5-HT1A receptor agonist 8-OHDPAT induces dose-dependent effects; i.e. low doses increase slow wave sleep and reduce waking, whereas large doses increase waking and reduce slow wave sleep and REM sleep. Direct injection of 8-OHDPAT or flesinoxan, another 5-HT1A agonist into the DRN, or microdialysis perfusion of 8-OHDPAT into the DRN significantly increases REMS. On the other hand, infusion of 8-OHDPAT into the LDT selectively inhibits REMS, as does direct administration into the DRN of the 5-HT1A receptor antagonists pindolol or WAY 100635. Thus, presently available evidence indicates that selective activation of the somatodendritic 5-HT1A receptor in the DRN induces an increase of REMS. On the other hand, activation of the postsynaptic 5-HT1A receptor at the level of the PPT/LDT nuclei decreases REMS occurrence.     

Lack of effect of corticotropin releasing factor on hypothalamic dopamine and serotonin synthesis turnover rates in rats

Catecholamine and serotonin neurons in the hypothalamus regulate the secretion of corticotropin releasing factor (CRF). We considered the possibility that CRF might in turn affect the activity of these aminergic neurons. We examined the effect of intracisternal administration of synthetic CRF on the synthesis turnover rates of dopamine and serotonin in the hypothalamus of adult male rats using two different methods to assess turnover. In one study, we measured the accumulation of L-dihydroxyphenylalanine (L-DOPA) or 5-hydroxytryptophan (5-HTP) in mediobasal hypothalamus after L-aromatic amino acid decarboxylase inhibition with m-hydroxybenzylhydrazine 20 min before sacrifice, and in the second study we measured the accumulation of dopamine, norepinephrine, epinephrine and serotonin after monoamine oxidase inhibition with pargyline 20 min before sacrifice. The commercial CRF which we administered intraarterially increased plasma ACTH and corticosterone concentrations. Intracerebral CRF 5 to 20 micrograms 20 min before sacrifice or 20 micrograms 110 min before sacrifice did not alter the m-hydroxybenzylhydrazine-induced accumulation of L-DOPA or 5-HTP when compared with saline vehicle-injected controls. CRF 20 micrograms did not alter basal concentration or pargyline-induced accumulation of the catecholamines or serotonin in whole hypothalamus when compared with saline vehicle-injected controls. Thus, intracisternal administration of CRF did not alter hypothalamic dopamine or serotonin synthesis rates as assessed by two nonsteady state turnover methods. The data suggest that the release of CRF from neurons in hypothalamus does not alter the activity of catecholamine or serotonin neurons in the hypothalamus of normal adult male rats.     

Altered serotonin and norepinephrine metabolism in rat dorsal raphe nucleus after drug-induced hypertension

The effect of drug-induced hypertension on neurotransmitter release from dorsal raphe nucleus was studied by in vivo electrochemical electrodes in urethane anesthetized male Sprague-Dawley rats. Carbon paste electrodes were stereotaxically placed into dorsal raphe nucleus and neurotransmitter release was estimated electrochemically. Blood pressure was recorded from a femoral arterial catheter. Voltammograms taken from dorsal raphe nucleus showed two distinct peaks corresponding to norepinephrine and 5-hydroxyindole acetic acid (5-HIAA). After basal blood pressure and neurotransmitter release were monitored for 30 min, blood pressure was raised 50 mmHg by continuous intravenous infusion of L-phenylephrine hydrochloride. Drug infusion was discontinued after 50 min, but blood pressure and neurotransmitter release were measured for an additional 2 hr. Results showed that the 5-HIAA response increased immediately after the initiation of hypertension and remained elevated. By contrast, norepinephrine release initially decreased, then returned to the basal level and then rose in parallel with 5-HIAA to a level above baseline as drug-induced hypertension was discontinued. The same experimental protocol was used to study the electrochemical response to drug-induced hypotension. Blood pressure was lowered 20 mmHg by intravenous infusion of sodium nitroprusside dihydrate. During hypotension, no changes were seen in either transmitter response. However, as reflex hypertension appeared following discontinuation of the sodium nitroprusside infusion, the 5-HIAA response increased and the norepinephrine response decreased. These results show that drug-induced and reflex hypertension reduce norepinephrine release and increase serotonin turnover in dorsal raphe nucleus in anesthetized normotensive rats. These reciprocal changes appear to be a part of the neural response to hypertension.     

Disruption of model-based decision making by silencing of serotonin neurons in the dorsal raphe nucleus

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Role of corticotropin releasing factor in anxiety disorders: a translational research perspective

Anxiety disorders are a group of mental disorders that include generalized anxiety disorder (GAD), panic disorder, phobic disorders (e.g., specific phobias, agoraphobia, social phobia) and posttraumatic stress disorder (PTSD). Anxiety disorders are among the most common of all mental disorders and, when coupled with an awareness of the disability and reduced quality of life they convey, they must be recognized as a serious public health problem. Over 20 years of preclinical studies point to a role for the CRF system in anxiety and stress responses. Clinical studies have supported a model of CRF dysfunction in depression and more recently a potential contribution to specific anxiety disorders (i.e., panic disorder and PTSD). Much work remains in both the clinical and preclinical fields to inform models of CRF function and its contribution to anxiety. First, we will review the current findings of CRF and HPA axis abnormalities in anxiety disorders. Second, we will discuss startle reflex measures as a tool for translational research to determine the role of the CRF system in development and maintenance of clinical anxiety.     

Role of cyclic AMP in corticotropin releasing factor mediated ACTH release

To elucidate the role of cAMP in the secretion of ACTH, the effect of (1) three phosphodiesterase inhibitors, (2) forskolin, and (3) 8Bromo-cAMP, on CRF mediated ACTH release was studied in rat pituitary cell culture. The action of glucocorticoids on CRF induced cAMP accumulation and ACTH release was investigated. Isobutyl-methylxanthine (IBMX), caffeine, and forskolin augmented the release of ACTH induced from CRF 1.0 nM by 17%, 39%, and 20%, respectively. Also IBMX and caffeine potentiated CRF 10 nM stimulated ACTH release by 32% and 20%. Doses of forskolin and 8Bromo-cAMP, which alone stimulate large amounts of ACTH release, did not increase the amount of ACTH released from CRF 100 nM stimulated cells. Cortisol (500 nM) and corticosterone (500 nM) inhibited CRF induced intracellular cAMP by 39% and 26% while inhibiting pituitary ACTH release by 40% and 52%. In conclusion, cAMP plays an important role in the mechanism of ACTH secretion and it appears the final intracellular mechanism of CRF stimulated ACTH is via cAMP. Also, glucocorticoids exert their inhibitory influence prior to cAMP generation.     

Effects of corticotropin releasing factor on locomotor activity in hypophysectomized rats

Corticotropin releasing factor (CRF) injected intracerebroventricularly to hypophysectomized and sham hypophysectomized rats produced a dose dependent increase in locomotor activity, but in untreated hypophysectomized rats 10× more CRF was needed to produce a significant increase in activity. Concomitant daily supplements of rat growth hormone, thyroxine, and corticosterone to the hypophysectomized rats eliminated locomotor activity differences between the two groups. There was no statistically significant difference in locomotor response to either saline, 0.1 μg CRF, 1.0 μg CRF or 10.0 μg CRF in the group of animals receiving hormonal supplements. These results demonstrate that CRF can produce behavioral activation in rats independently of its effects on releasing hormones from the pituitary gland.     

The median raphe nucleus participates in the depressive-like behavior induced by MCH: Differences with the dorsal raphe nucleus

An emerging body of evidence involves the hypothalamic neuropeptide melanin-concentrating hormone (MCH) in the regulation of emotional states. We have reported a pro-depressive effect induced by MCH after its microinjection into the dorsal raphe nucleus (DR) evaluated in the forced swimming test (FST) in rats. Here we extended this study to the median raphe nucleus (MnR). Firstly, the presence of MCH-containing fibers in the rat MnR was analyzed by means of immunohistochemistry. Secondly, the behavioral effect induced by the microinjection of MCH into the MnR was assessed using the FST. Morphological results showed a large density of MCHergic fibers within the MnR. Behavioral results indicated that 100 ng of MCH (but not 50 ng) significantly increased the immobility time and decreased the swimming time, demonstrating a depressive-like effect. In contrast, climbing behavior was not significantly affected. Present findings revealed that the MnR neurons participate in the MCHergic control of affective-related behavioral responses. However, the behavioral patterns induced by MCH in the MnR and DR were different. This could be explained by anatomical and physiological differences between both nuclei.     

Single-cell activity and network properties of dorsal raphe nucleus serotonin neurons during emotionally salient behaviors

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Expression of serotonin transporter in the dorsal raphe nucleus during the early postnatal period in the normal state and under prenatal deficiency of the serotonergic system in rats

Expression of the serotonin transporter protein (5-NTT) in the dorsal raphe nucleus (DNR) during the early postnatal period was investigated in laboratory Wistar rats. Immunocytochemical labeling showed that during the first 3 postnatal weeks the intensity of 5-NTT expression in DNR of control animals changes. During the earliest postnatal stages, most of DNR subnuclear neurons (dorsal, DNR-d; ventral, DNR-v; lateral, DNR-lat) were found to intensely express 5-NTT. 5-NTT localization sites were revealed on the membrane surface of neuronal cell bodies and their processes in neuropil. On P10, the number of 5-NTT expressing neurons and 5-NTT binding sites decreases. At this time, the 5-NTT binding sites were shown to undergo redistribution becoming very few on neuronal cell bodies and dendrites, but rather densely packed in the axonal membrane. The number of 5-NTT expressing neurons and density of 5-NTT localization sites in neuropil gradually increases with age. The reduction in the serotonin level in all DNR regions during prenatal development leads to the reduction both in the number of 5-NTT expressing neurons and 5-NTT localization sites during the early postnatal period. This tendency was shown to persist with age.     

Regulation of nociceptin/orphaninFQ secretion by immune cells and functional modulation of interleukin-2

Previous research has demonstrated that numerous populations of immune cell, including lymphocytes, synthesize nociceptin (N/OFQ) precursor mRNA although little is known regarding the immunological role of N/OFQ. In the present study we have demonstrated significant effects of mitogens, pro-inflammatory cytokines, cyclic AMP analogues, glucocorticoids and CRF on N/OFQ secretion by rat splenocytes in vitro. N/OFQ (10(-14) to 10(-10)M) was also shown to inhibit proliferation of Con A-activated splenocytes and production of IL-2 in vitro. In summary we have shown how a variety of stimuli relevant to inflammation can regulate endogenous N/OFQ secretion by splenocytes in vitro. We also suggest that N/OFQ may promote anti-inflammatory actions via suppression of IL-2 in vivo.     

Regulation of nucleus accumbens dopamine release by the dorsal raphe nucleus in the rat

The effects of microinfusingl-glutamate, serotonin (5-HT), (±)-8-hydroxy-2-(di-N-propylamino) tetralin (8-OH DPAT; a 5-HT_1A agonist), and muscimol (a GABA_A agonist) into the dorsal raphe nucleus on the extracellular levels of 5-HT, dopamine (DA) and their metabolites in the nucleus accumbens were studied in unanesthetized, freely moving, adult male Wistar rats, using the technique of microdialysis coupled with small-bore HPLC. Administration of 0.75 gl-glutamate produced a 25–50% increase (P<0.05) in the extracellular levels of both 5-HT and DA. On the other hand, infusion of 8-OH DPAT and, to a lesser extent, 5-HT produced a significant (P<0.05) decrease in the extracellular levels of both 5-HT and DA. Muscimol (0.25 or 0.50 g) had little effect on the extracellular concentrations of 5-HT or DA following its administration. In general, the extracellular levels of the major metabolites of 5-HT and DA in the nucleus accumbens were not altered by microinfusion of any of the agents. The data indicate that (a) the 5-HT neurons projecting to the nucleus accumbens from the dorsal raphe nucleus can be activated by excitatory amino acid receptors and inhibited by stimulation of 5-HT_1A autoreceptors, and (b) the dorsal raphe nucleus 5-HT neuronal system may regulate the ventral tegmental area DA projection to the nucleus accumbens.Special issue dedicated to Dr. Morris H. Aprison     

PTSD促进大鼠中缝背核细胞色素c表达

目的研究创伤后应激障碍(PTSD)大鼠中缝背核神经元细胞色素C(Cyt-c)的表达变化。方法应用无连续单一刺激(SPS)方法建立PTSD大鼠模型,随机分为SPS刺激后1d、4d、7d和对照组,应用酶组织化学法和RT-PCR方法观察中缝背核神经元Cyt-c的表达变化。结果光镜酶细胞化学法和RT-PCR法显示中缝背核神经元Cyt-c染色阳性细胞于SPS刺激后1d明显高于对照组,4d逐渐增高,并于7d达到高峰。电镜下显示Cyt-c阳性反应产物主要分布在中缝背核神经元线粒体膜,SPS刺激后可见Cyt-c释放到胞浆中。结论 SPS刺激引起Cyt-c在PTSD大鼠中缝背核神经元呈过表达。     

3,4-methylenedioxymethamphetamine (MDMA)-induced release of endogenous serotonin from the rat dorsal raphe nucleus in vitro: Effects of fluoxetine and tryptophan

The serotonin releasing action of 3,4-methylenedioxymethamphetamine on slices of dorsal raphe nucleus from rat was investigated. The slices were maintained in a gas-liquid interface perfusion chamber used for electrophysiological recording. Microdialysis probes designed for use on the slice surface were employed to measure the release of endogenous serotonin which was determined using liquid chromatography with electrochemical detection. Three minute duration exposure of the slices to 100 micromolar 3,4-methylenedioxymethamphetamine caused a long lasting release of endogenous serotonin. Fluoxetine, a serotonin transport inhibitor, reduced the amount of serotonin release. Tryptophan added to the perfusion solution increased both the duration and amount of serotonin released. These results further support earlier work on the mechanism of 3,4-methylenedioxymethamphetamine induced inhibition of serotonin neuronal firing.     

Activation of corticotropin releasing factor receptor type 2 in the heart by corticotropin releasing factor offers cytoprotection against ischemic injury via PKA and PKC dependent signaling

Corticotrophin-releasing factor receptor 2β (CRFR2β) is expressed in the myocardium. In the present study we explore whether acute treatment with the neuropeptide corticotrophin-releasing factor (CRF) could induce cytoprotection against a lethal ischemic insult in the heart (isolated murine neonatal cardiac myocytes and the isolated Langendorff perfused rat heart) by activating CRFR2. In vitro, CRF offered cytoprotection when added prior to lethal simulated ischemic stress by reducing apoptotic and necrotic cell death. Ex vivo, CRF significantly reduced infarct size from 52.1±3.1% in control hearts to 35.3±3.1% (P<0.001) when administered prior to a lethal ischemic insult. The CRF peptide did not confer cytoprotection when administered at the point of hypoxic reoxygenation or ischemic reperfusion. The acute effects of CRF treatment are mediated by CRF receptor type 2 (CRFR2) since the cardioprotection ex vivo was inhibited by the CRFR2 antagonist astressin-2B. Inhibition of the mitogen activated protein kinase-ERK1/2 by PD98059 failed to inhibit the effect of CRF. However, both protein kinase A and protein kinase C inhibitors abrogated CRF-mediated protection both ex vivo and in vitro. These data suggest that the CRF peptide reduces both apoptotic and necrotic cell death in cardiac myocytes subjected to lethal ischemic induced stress through activation of PKA and PKC dependent signaling pathways downstream of CRFR2.     

In vivo efflux of serotonin in the dorsal raphe nucleus of 5-HT1A receptor knockout mice

In the dorsal raphe nucleus (DR), extracellular serotonin (5-HT) regulates serotonergic transmission through 5-HT1A autoreceptors. In this work we used in vivo microdialysis to examine the effects of stressful and pharmacological challenges on DR 5-HT efflux in 5-HT1A receptor knockout (5-HT1A-/-) mice and their wild-type counterparts (5-HT1A+/+). Baseline 5-HT concentrations did not differ between both lines of mice, which is consistent with a lack of tonic control of 5-HT1A autoreceptors on DR 5-HT release. (R)-(+)-8-Hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT, 0.5 mg/kg) reduced 5-HT levels to 30% of basal values in 5-HT1A+/+ mice, but not in 5-HT1A-/- mice. The selective 5-HT1B receptor agonist 1,4-dihydro-3-(1,2,3,6-tetrahydro-4-pyridinyl)-5H-pyrrolo[3,2-b]pyridin-5-one dihydrochloride (CP 93129, 300 micro m) reduced dialysate 5-HT to the same extent (30-40% of baseline) in the two genotypes, which suggests a lack of compensatory changes in 5-HT1B receptors in the DR of such mutant mice. Both a saline injection and handling for 3 min increased DR dialysate 5-HT in mutants, but not in 5-HT1A+/+ mice. Fluoxetine (5 and 20 mg/kg) elevated 5-HT in a dose-dependent manner in both genotypes. However, this effect was markedly more pronounced in the 5-HT1A-/- mice. The increased responsiveness of the extracellular 5-HT in the DR of 5-HT1A receptor knockout mice reflects a lack of the autoinhibitory control exerted by 5-HT1A autoreceptors.