Serotonin depletion reduces both acquisition and expression of context-conditioned fear.

OBJECTIVE: Whereas numerous experimental and clinical studies suggest a complex involvement of serotonin in the regulation of anxiety, it remains to be clarified if the dominating impact of this transmitter is best described as anxiety-reducing or anxiety-promoting. The aim of this study was to assess the impact of serotonin depletion on acquisition, consolidation, and expression of conditioned fear. METHODS: Male Sprague-Dawley rats were exposed to foot shocks as unconditioned stimulus and assessed with respect to freezing behaviour when re-subjected to context. Serotonin depletion was achieved by administration of a serotonin synthesis inhibitor, para-chlorophenylalanine (PCPA) (300 mg/kg daily × 3), (i) throughout the period from (and including) acquisition to (and including) expression, (ii) during acquisition but not expression, (iii) after acquisition only, and (iv) during expression only. RESULTS: The time spent freezing was significantly reduced in animals that were serotonin-depleted during the entire period from (and including) acquisition to (and including) expression, as well as in those being serotonin-depleted during either acquisition only or expression only. In contrast, PCPA administrated immediately after acquisition, that is during memory consolidation, did not impact the expression of conditioned fear. CONCLUSION: Intact serotonergic neurotransmission is important for both acquisition and expression of context-conditioned fear.

Role of serotonin on the intestinal mucosal immune response to stress-induced diarrhea in weaning mice.

BACKGROUND: During weaning, babies and young animal often experience diarrhea from food intolerance and/or decreasing levels of maternal antibodies, and diarrhea tends to be particularly severe during the early-weaned period, which often exhibits an underdeveloped immune system, a disturbed gut environment and results in nutrient malabsorption and dehydration. It was deduced that neuroendocrine might have close relation with diarrhea, especially 5-HT. METHODS: To explore the role of serotonin (5-HT) in weaning mice subjected to stress-induced diarrhea, 21-day-old weaned mice were divided into the following groups: control group, stress-induced diarrhea group (restrained by binding the hind limbs and intragastric administration of folium sennae with 0.4 g/mL, 15 mL/kg body weight) and para-chlorophenylalanine (PCPA) + stress-induced diarrhea group (30 mg/mL, 300 mg/kg body weight PCPA intraperitoneal injection before stress-induced diarrhea treatment). RESULTS: Based on results from enzyme-linked immunosorbent assays, histological staining, lymphocyte proliferation assays and flow cytometry analysis, we found that the mice experienced increases in several stress markers, which coincided with severe diarrhea and an increase in 5-HT levels. However, pre-treatment with PCPA resulted in a decrease in the stress indicators and the severity of diarrhea, which correlated with decreased 5-HT levels. Interestingly, stress-induced diarrhea caused changes in various aspects of the immune system, including the amount of intraepithelium lymphocytes, CD4+/CD8+ T lymphocyte populations, B and T lymphocyte proliferation, and the secretion of sIgA and cytokines in the small intestine and ileum. However, these immune system changes could be reversed upon treatment with PCPA. CONCLUSIONS: We observed a distinct correlation between 5-HT levels and the occurrence of stress-induced diarrhea in weaning mice, which may result in the deregulation of the mucosal immune system.

Modulatory Action by the Serotonergic System: Behavior and Neurophysiology in Drosophila melanogaster.

Serotonin modulates various physiological processes and behaviors. This study investigates the role of 5-HT in locomotion and feeding behaviors as well as in modulation of sensory-motor circuits. The 5-HT biosynthesis was dysregulated by feeding Drosophila larvae 5-HT, a 5-HT precursor, or an inhibitor of tryptophan hydroxylase during early stages of development. The effects of feeding fluoxetine, a selective serotonin reuptake inhibitor, during early second instars were also examined. 5-HT receptor subtypes were manipulated using RNA interference mediated knockdown and 5-HT receptor insertional mutations. Moreover, synaptic transmission at 5-HT neurons was blocked or enhanced in both larvae and adult flies. The results demonstrate that disruption of components within the 5-HT system significantly impairs locomotion and feeding behaviors in larvae. Acute activation of 5-HT neurons disrupts normal locomotion activity in adult flies. To determine which 5-HT receptor subtype modulates the evoked sensory-motor activity, pharmacological agents were used. In addition, the activity of 5-HT neurons was enhanced by expressing and activating TrpA1 channels or channelrhodopsin-2 while recording the evoked excitatory postsynaptic potentials (EPSPs) in muscle fibers. 5-HT2 receptor activation mediates a modulatory role in a sensory-motor circuit, and the activation of 5-HT neurons can suppress the neural circuit activity, while fluoxetine can significantly decrease the sensory-motor activity.

Serum serotonin reduced the expression of hepatic transporter Mrp2 and P-gp via regulating nuclear receptor CAR in PI-IBS rats.

Hepatic transporters and drug metabolizing enzymes (DMEs) play important roles in the pharmacological effects and (or) side-effects of many drugs, and are regulated by several mediators, including neurotransmitters. This work aimed to investigate whether serum levels of 5-hydroxytryptamine (5-HT) affected the expression of hepatic transporters or DMEs. The expression of hepatic transporters was assessed using the Western-blot technique in a 2,4,6-trinitrobenzenesulfonic-acid-induced rat model of post-infectious irritable bowel syndrome (PI-IBS), in which serum levels of 5-HT were significantly elevated. To further clarify the underlying mechanism, the 5-HT precursor 5-hydroxytryptophan (5-HTP) and the 5-HT depleting agent parachlorophenylalanine (pCPA) were applied to adjust serum levels of 5-HT. Serum levels of 5-HT were measured using LC-MS/MS; the expression of hepatic transporters, DMEs, and nuclear receptors were examined by Western-blot technique. Our results showed that in PI-IBS rats the expression of multidrug resistance protein 2 (Mrp2) was significantly decreased, while colonic enterochromaffin cell density and serum levels of 5-HT were all significantly increased. Moreover, 5-HTP treatment significantly increased serum levels of 5-HT and decreased the expression of Mrp2 and glycoprotein P (P-gp), whereas treatment with pCPA markedly decreased serum levels of 5-HT and increased the expression of Mrp2 and P-gp. Our results indicated that serum 5-HT regulates the expression of Mrp2 and P-gp, and the underlying mechanism may be related to the altered expression of the nuclear receptor constitutive androstane receptor (CAR).

[Participation of serotonin in the mechanisms of the formation of trigeminal motor nucleus].

The formation of trigeminal motor nucleus (TMN) was studied in the early postnatal period in 21 female Wistar rats which received the serotonin biosynthesis inhibitor para-chloro-phenylalanine at prenatal Day 16 (the period of serotoninergic system formation). It was shown that the serotonin deficit during the prenatal period in rats resulted in the changes of TMN structural organization. In the early postnatal period, the delay of neuropil development, the reduction of cell body size with the partial loss of Nissl substance in some of the neurons, the presence of degenerating neurons with the signs of hyperchromatosis in all the parts of the nucleus, especially in TMN ventromedial part, were detected. At later stages, the destruction of motoneurons became slower, though some of them had morphological abnormalities. With the increase of the postnatal age (by Day 20) the number of motor neurons decreased, apparently, as a result of the gradual intensification of cell death. Simultaneously with the motor neuron degeneration in TMN parts studied, the astrocytic gliosis was observed.

Diet and companionship modulate pain via a serotonergic mechanism.

Treatment of severe chronic and acute pain in sickle cell disease (SCD) remains challenging due to the interdependence of pain and psychosocial modulation. We examined whether modulation of the descending pain pathway through an enriched diet and companionship could alleviate pain in transgenic sickle mice. Mechanical and thermal hyperalgesia were reduced significantly with enriched diet and/or companionship. Upon withdrawal of both conditions, analgesic effects observed prior to withdrawal were diminished. Serotonin (5-hydroxytryptamine, 5-HT) was found to be increased in the spinal cords of mice provided both treatments. Additionally, 5-HT production improved at the rostral ventromedial medulla and 5-HT accumulated at the dorsal horn of the spinal cord of sickle mice, suggesting the involvement of the descending pain pathway in the analgesic response. Modulation of 5-HT and its effect on hyperalgesia was also investigated through pharmaceutical approaches. Duloxetine, a serotonin-norepinephrine reuptake inhibitor, showed a similar anti-nociceptive effect as the combination of diet and companionship. Depletion of 5-HT through p-chlorophenylalanine attenuated the anti-hyperalgesic effect of enriched diet and companionship. More significantly, improved diet and companionship enhanced the efficacy of a sub-optimal dose of morphine for analgesia in sickle mice. These findings offer the potential to reduce opioid use without pharmacological interventions to develop effective pain management strategies.

iTRAQ-based proteomics analysis on insomnia rats treated with Mongolian medical warm acupuncture.

OBJECTIVE: To explore the proteomic changes in the hypothalamus of rats treated with Mongolian medical warm acupuncture for insomnia therapy based proteomics. METHOD: We used an iTRAQ-based quantitative proteomic approach to identify proteins that potential molecular mechanisms involved in the treatment of insomnia by Mongolian medical warm acupuncture. RESULT: In total, 7477 proteins were identified, of which 36 proteins showed increased levels and 45 proteins showed decreased levels in insomnia model group (M) compared with healthy control group (C), 72 proteins showed increased levels and 44 proteins showed decreased levels from the warm acupuncture treated insomnia group (W) compared with healthy controls (C), 28 proteins showed increased levels and 17 proteins showed decreased levels from the warm acupuncture-treated insomnia group (W) compared with insomnia model group (M). Compared with healthy control groups, warm acupuncture-treated insomnia group showed obvious recovered. Bioinformatics analysis indicated that up-regulation of neuroactive ligand-receptor interaction and oxytocin signaling was the most significantly elevated regulate process of Mongolian medical warm acupuncture treatment for insomnia. Proteins showed that increased/decreased expression in the warm acupuncture-treated insomnia group included Prolargin (PRELP), NMDA receptor synaptonuclear-signaling and neuronal migration factor (NSMF), Transmembrane protein 41B (TMEM41B) and Microtubule-associated protein 1B (MAP1B) to adjust insomnia. CONCLUSION: A combination of findings in the present study suggest that warm acupuncture treatment is efficacious in improving sleep by regulating the protein expression process in an experimental rat model and may be of potential benefit in treating insomnia patients with the added advantage with no adverse effects.

Effects of dopamine and serotonin synthesis inhibitors on the ketamine-, d-amphetamine-, and cocaine-induced locomotor activity of preweanling and adolescent rats: sex differences.

The pattern of ketamine-induced locomotor activity varies substantially across ontogeny and according to sex. Although ketamine is classified as an NMDA channel blocker, it appears to stimulate the locomotor activity of both male and female rats via a monoaminergic mechanism. To more precisely determine the neural mechanisms underlying ketamine's actions, male and female preweanling and adolescent rats were pretreated with vehicle, the dopamine (DA) synthesis inhibitor ∝-methyl-DL-p-tyrosine (AMPT), or the serotonin (5-HT) synthesis inhibitor 4-chloro-DL-phenylalanine methyl ester hydrochloride (PCPA). After completion of the pretreatment regimen, the locomotor activating effects of saline, ketamine, d-amphetamine, and cocaine were assessed during a 2 h test session. In addition, the ability of AMPT and PCPA to reduce dorsal striatal DA and 5-HT content was measured in male and female preweanling, adolescent, and adult rats. Results showed that AMPT and PCPA reduced, but did not fully attenuate, the ketamine-induced locomotor activity of preweanling rats and female adolescent rats. Ketamine (20 and 40 mg/kg) caused a minimal amount of locomotor activity in male adolescent rats, and this effect was not significantly modified by AMPT or PCPA pretreatment. When compared to ketamine, d-amphetamine and cocaine produced different patterns of locomotor activity across ontogeny; moreover, AMPT and PCPA pretreatment affected psychostimulant- and ketamine-induced locomotion differently. When these results are considered together, it appears that both dopaminergic and serotonergic mechanisms mediate the ketamine-induced locomotor activity of preweanling and female adolescent rats. The dichotomous actions of ketamine relative to the psychostimulants in vehicle-, AMPT-, and PCPA-treated rats, suggests that ketamine modulates DA and 5-HT neurotransmission through an indirect mechanism.

Conditional mouse mutants highlight mechanisms of corticotropin-releasing hormone effects on stress-coping behavior.

Hypersecretion of central corticotropin-releasing hormone (CRH) has been implicated in the pathophysiology of affective disorders. Both, basic and clinical studies suggested that disrupting CRH signaling through CRH type 1 receptors (CRH-R1) can ameliorate stress-related clinical conditions. To study the effects of CRH-R1 blockade upon CRH-elicited behavioral and neurochemical changes we created different mouse lines overexpressing CRH in distinct spatially restricted patterns. CRH overexpression in the entire central nervous system, but not when overexpressed in specific forebrain regions, resulted in stress-induced hypersecretion of stress hormones and increased active stress-coping behavior reflected by reduced immobility in the forced swim test and tail suspension test. These changes were related to acute effects of overexpressed CRH as they were normalized by CRH-R1 antagonist treatment and recapitulated the effect of stress-induced activation of the endogenous CRH system. Moreover, we identified enhanced noradrenergic activity as potential molecular mechanism underlying increased active stress-coping behavior observed in these animals. Thus, these transgenic mouse lines may serve as animal models for stress-elicited pathologies and treatments that target the central CRH system.

Serotonergic mediation of the antidepressant-like effect of the green leaves odor in mice.

The green odor (GO) that emanates from green leaves has been observed to have many physiological actions in mammals and may be associated with a healing effect in humans. This study examined the effect of GO (we used a mixture of cis-3-hexenol and trans-2-hexenal) on behavior in the forced swim test (FST) of depression in mice. Exposure of GO showed the antidepressant-like effect in the FST, i.e., a significant decrease in immobility time and increase in swimming time, but no change in climbing time. The behavioral responses of GO-exposed animals to FST were similar to those observed for animals given citalopram, which is a selective serotonin reuptake inhibitor. In contrast, desipramine, which is a selective noradrenaline reuptake inhibitor, decreased immobility time and increased climbing time without affecting swimming time. To examine the involvement of the serotonergic system in mediating the antidepressant-like action of GO, we performed further FST examinations in which GO-exposed mice were treated with p-chlorophenylalanine (PCPA). Prior PCPA administration induced depletion of central 5-HT in the brain and completely diminished the GO effect on the behavioral responses seen during the FST. No changes in locomotor activity after GO inhalation were observed. These results indicate that acute exposure to GO has an antidepressant-like effect that may involve the serotonergic system.

Antidepressant-like activity of n-hexane extract of nutmeg (Myristica fragrans) seeds in mice.

The present study was undertaken to investigate the effect of an n-hexane extract of Myristica fragrans seeds on depression in mice by using the forced swim test (FST) and the tail suspension test (TST). M. fragrans extract (5, 10, and 20 mg/kg) was administered orally for 3 successive days to different groups of Swiss male young albino mice. M. fragrans extract significantly decreased immobility periods of mice in both the FST and the TST. The 10 mg/kg dose was found to be most potent, as indicated by the greatest decrease in the immobility period compared with the control. Furthermore, this dose of the extract was found to have comparable potency to imipramine (15 mg/kg i.p.) and fluoxetine (20 mg/kg i.p.). The extract did not have a significant effect on locomotor activity of mice. Prazosin (62.5 microg/kg i.p.; an alpha (1)-adrenoceptor antagonist), sulpiride (50 mg/kg i.p.; a selective D(2) receptor antagonist), and p-chlorophenylalanine (100 mg/kg i.p.; an inhibitor of serotonin synthesis) significantly attenuated the M. fragrans extract-induced antidepressant-like effect in the TST. Thus, extract of M. fragrans elicited a significant antidepressant-like effect in mice, when assessed in both the TST and the FST. The antidepressant-like effect of the extract seems to be mediated by interaction with the adrenergic, dopaminergic, and serotonergic systems.

The Antidepressant Effects of an mGlu2/3 Receptor Antagonist and Ketamine Require AMPA Receptor Stimulation in the mPFC and Subsequent Activation of the 5-HT Neurons in the DRN.

We have reported the antidepressant effects of both metabotropic glutamate 2/3 (mGlu2/3) receptor antagonists and ketamine in several animal models, and proposed that serotonergic (5-HTergic) transmission is involved in these actions. Given that the projections from the medial prefrontal cortex (mPFC) to the dorsal raphe nucleus (DRN), where the majority of serotonin (5-HT) neurons exist, are reportedly involved in the antidepressant effects, in this study, we investigated using the forced swimming test (FST) of C57BL/6J male mice, the role of 5-HT neurons in the DRN regulated by the mPFC-DRN projections in the antidepressant effects of an mGlu2/3 receptor antagonist, LY341495, and ketamine. Following systemic administration/microinjection into the mPFC, both LY341495 and ketamine were found to exert antidepressant effects in the FST, and the effects were attenuated by depletion of 5-HT by treatment with an inhibitor of 5-HT synthesis, PCPA. The antidepressant effects of LY341495 and ketamine were also blocked by systemic administration/microinjection into the mPFC of an AMPA receptor antagonist, NBQX. Moreover, systemic administration/microinjection into the mPFC of LY341495 and ketamine significantly increased the c-Fos expression in the 5-HT neurons in the DRN, and the effect of systemic administration of these drugs on the neuronal c-Fos expression was attenuated by microinjection of NBQX into the mPFC. Our findings suggest that activation of 5-HT neurons in the DRN regulated by stimulation of the AMPA receptor in the mPFC may be involved in the antidepressant effects of an mGlu2/3 receptor antagonist and ketamine.

Loss of flight and associated neuronal rhythmicity in inositol 1,4,5-trisphosphate receptor mutants of Drosophila.

Coordinated flight in winged insects requires rhythmic activity of the underlying neural circuit. Here, we show that Drosophila mutants for the inositol 1,4,5-trisphosphate (InsP(3)) receptor gene (itpr) are flightless. Electrophysiological recordings from thoracic indirect flight muscles show increased spontaneous firing accompanied by a loss of rhythmic flight activity patterns normally generated in response to a gentle puff of air. In contrast, climbing speed, the jump response, and electrical properties of the giant fiber pathway are normal, indicating that general motor coordination and neuronal excitability are much less sensitive to itpr mutations. All mutant phenotypes are rescued by expression of an itpr(+) transgene in serotonin and dopamine neurons. Pharmacological and immunohistochemical experiments support the idea that the InsP(3) receptor functions to modulate flight specifically through serotonergic interneurons. InsP(3) receptor action appears to be important for normal development of the flight circuit and its central pattern generator.

In vivo resetting of the hamster circadian clock by 5-HT7 receptors in the suprachiasmatic nucleus.

Serotonin (5-HT) has been strongly implicated in the regulation of the mammalian circadian clock located in the suprachiasmatic nuclei (SCN); however, its role in behavioral (nonphotic) circadian phase resetting remains elusive. Central to this issue are divergent lines of evidence that the SCN may, or may not, be a target for the phase-resetting effects of 5-HT. We have addressed this question using a novel reverse-microdialysis approach for timed perfusions of serotonergic and other agents to the Syrian hamster SCN with durations equivalent to the increases in in vivo 5-HT release during phase-resetting behavioral manipulations. We found that 3 hr perfusions of the SCN with either 5-HT or the 5-HT(1A,7) receptor agonist 2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydro-naphthalene (8-OH-DPAT) at midday advanced the phase of the free-running circadian rhythm of wheel-running assessed using an Aschoff type II procedure. Phase shifts induced by 8-OH-DPAT were enhanced more than threefold by pretreatment with the 5-HT synthesis inhibitor para-chlorophenylalanine. Phase advances induced by SCN 8-OH-DPAT perfusion were significantly inhibited by the 5-HT(2,7) receptor antagonist ritanserin and by the more selective 5-HT(7) receptor antagonist DR4004, implicating the 5-HT(7) receptor in mediating this phase resetting. Concurrent exposure to light during the 8-OH-DPAT perfusion abolished the phase advances. Furthermore, coperfusion of the SCN with TTX, which blocked in vivo 5-HT release, did not suppress intra-SCN 8-OH-DPAT-induced phase advances. These results indicate that 5-HT(7) receptor-mediated phase resetting in the SCN is markedly influenced by the degree of postsynaptic responsiveness to 5-HT and by photic stimulation. Finally, 5-HT may act directly on SCN clock cells to induce in vivo nonphotic phase resetting.

Role of serotonin (5-HT) in the antidepressant-like properties of neuropeptide Y (NPY) in the mouse forced swim test.

Neuropeptide Y (NPY) is thought to be implicated in depressive disorders. The mouse forced swim test (FST) is an animal model widely used as a predictor of the efficacy of antidepressant drugs. The present study was undertaken to explore the possible contribution of endogenous serotonin (5-HT) systems in the behavioral effects elicited by NPY in this model. The selective serotonin re-uptake inhibitor (SSRI), fluoxetine, was also tested for comparison. 5-HT was depleted prior to testing by the administration of the tryptophan hydroxylase inhibitor p-chlorophenylalanine (PCPA; 300 mg/kg, i.p., each day for 3 days; control mice received saline-vehicle over the same period). On the fourth day, mice received NPY (3 nmol, I.C.V.), fluoxetine (16 mg/kg, i.p.) or saline injections before testing in the FST. Both NPY and fluoxetine significantly reduced immobility time in saline-treated control animals. Pre-treatment with PCPA significantly blocked the effects of fluoxetine in the FST, confirming the role of endogenous 5-HT. Similarly, pre-treatment with PCPA also significantly attenuated the anti-immobility effects of NPY, thus suggesting a role for 5-HT in the effects of NPY in the FST. Quantitative receptor autoradiography revealed increases in specific [125I][Leu31, Pro34]PYY sites that were sensitive to BIBP3226 (Y1-like sites) in various brain regions. Specific [125I]GR231118 and [125I]PYY(3-36) binding levels were not changed following PCPA treatment, suggesting that depletion of endogenous 5-HT resulted in an apparent increase in the level of Y1 sites in their high-affinity state. Taken together, these results suggest a role for 5-HT-related systems in the antidepressant-like properties of NPY.

Effects of central and peripheral depletion of serotonergic system on carrageenan-induced paw oedema.

The role of serotonergic system was investigated on peripheral inflammation induced by intraplantary injection of carrageenan. Para-chlorophenylalanine (PCPA) was administered intracerebroventriculary (50, 100 microg/rat) or intraperitoneally (150 mg/kg, 3 days) and 2 or 24 h later, respectively, inflammation was induced by injection of carrageenan. Paw oedema was decreased significantly in PCPA-treated (100 microg/rat, i.c.v.) rats compared to control groups. Injection of exogenous serotonin (i.c.v.) by dose of 0.70 nmol/10 microl/rat, but not the dose of 0.35 nmol/10 microl/rat, 15 min after induction of inflammation completely reversed the anti-inflammatory effects of PCPA. Myeloperoxidase activity in inflamed paws was reduced significantly in groups received PCPA (either i.c.v. or i.p.) compared to controls. Exogenous serotonin (0.70 nmol/10 microl/rat) reduced inflammatory response when injected (i.c.v.) 30 min before or 30 min after the induction of inflammation. Injection of serotonin at the time of induction of inflammation had no inflammatory/anti-inflammatory effect. These results suggest that serotonin, as a neurotransmitter in central nervous system, may be involved in modulating peripheral inflammation.

Parachlorophenylalanine exacerbates oxidative stress induced by gentamicin in rats.

The present work studies the effect of parachlorophenylalanine (PCPA, 200 mg/kg intraperitoneally/48 hr for 7 days) on the oxidative stress and nephropathy induced by gentamicin (80 mg/kg intraperitoneally/daily for 7 days) in Wistar rats. The effect of PCPA on lipid peroxidation products and reduced glutathione content in renal and brain tissue, as well as on 5HT content in brain was assessed. Catalase and superoxide dismutase activities were determined in brain tissue. Blood urea nitrogen and creatinine in plasma and total protein content in urine were also measured. Gentamicin caused significant increases in proteinuria, non-protein nitrogen compounds and lipid peroxidation markers, together with decreases in both reduced glutathione content in renal and brain tissue and enzymatic activities in brain homogenates. PCPA harnessed the effect of gentamicin in the brain and the kidney, while PCPA alone induced brain oxidative stress. These results support the prooxidant action of PCPA in brain tissue and its capacity to exacerbate the oxidative stress and renal dysfunction induced by gentamicin, as well as the possible antioxidant property of serotonin.

Role of Serotonin in MODS: Deficiency of Serotonin Protects Against Zymosan-Induced Multiple Organ Failure in Mice.

Zymosan-induced multiple organ dysfunction syndrome (MODS) is a multifactorial pathology that involves the deterioration of function of several organs. 5-Hydroxytryptamine (5-HT) is a small monoamine molecule that is primarily known for its role as a neurotransmitter. Previous studies have shown that 5-HT could serve as an important inflammatory mediator in the peripheral immune system. In the present study, we investigated the effect of 5-HT on the development of non-septic shock caused by zymosan in mice. Tryptophan hydroxylase 1-knockout mice (TPH1, leading to the absence of 5-HT), TPH1 + 5-hydroxytryptophan (precursor of 5-HT) treatment mice, wild-type (TPH1) mice, and wild-type plus p-chlorophenylalanine (PCPA, TPH1 inhibitor) treatment mice received zymosan intraperitoneally at a dose of 500 mg/kg. Organ failure and systemic inflammation in the mice were assessed 18 h after the administration of zymosan. Deficiency of 5-HT caused a significant reduction of the 1) peritoneal exudate formation, 2) neutrophil infiltration, 3) MODS, 4) nitrosative stress, and 5) cytokine formation. In addition, at the end of the observation period (7 days), deficiency of 5-HT in the mice was shown to be able to alleviate the severe illness characterized as systemic toxicity, significant loss of body weight, and high mortality caused by zymosan. In conclusion, the lack of 5-HT by genetic knockout or by pharmacologic inhibition of the TPH1 enzyme significantly attenuated zymosan-induced MODS.

Insomnia Caused by Serotonin Depletion is Due to Hypothermia.

STUDY OBJECTIVE: Serotonin (5-hydroxytryptamine, 5-HT) neurons are now thought to promote wakefulness. Early experiments using the tryptophan hydroxylase inhibitor para-chlorophenylalanine (PCPA) had led to the opposite conclusion, that 5-HT causes sleep, but those studies were subsequently contradicted by electrophysiological and behavioral data. Here we tested the hypothesis that the difference in conclusions was due to failure of early PCPA experiments to control for the recently recognized role of 5-HT in thermoregulation. DESIGN: Adult male C57BL/6N mice were treated with PCPA (800 mg/kg intraperitoneally for 5 d; n = 15) or saline (n = 15), and housed at 20 °C (normal room temperature) or at 33 °C (thermoneutral for mice) for 24 h. In a separate set of experiments, mice were exposed to 4 °C for 4 h to characterize their ability to thermoregulate. MEASUREMENTS AND RESULTS: PCPA treatment reduced brain 5-HT to less than 12% of that of controls. PCPA-treated mice housed at 20 °C spent significantly more time awake than controls. However, core body temperature decreased from 36.5 °C to 35.1 °C. When housed at 33 °C, body temperature remained normal, and total sleep duration, sleep architecture, and time in each vigilance state were the same as controls. When challenged with 4 °C, PCPA-treated mice experienced a precipitous drop in body temperature, whereas control mice maintained a normal body temperature. CONCLUSIONS: These results indicate that early experiments using para-chlorophenylalanine that led to the conclusion that 5-hydroxytryptamine (5-HT) causes sleep were likely confounded by hypothermia. Temperature controls should be considered in experiments using 5-HT depletion.

Evidence for postsynaptic mediation of the hypothermic effect of 5-HT1A receptor activation.

1. The 5-HT1A ligand BMY 7378 (8-[2[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]8-azaspirol [4,5]-decane-7,9-dione dihydrochloride, 0.032-2 mg kg-1, s.c.) caused hyperphagia, a response to the activation of presynaptic 5-HT1A receptors. 2. BMY 7378 (8 mg kg-1, s.c.) and the 5-HT1A agonist (8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), 0.10 and 0.25 mg kg-1 s.c.) also caused hypothermia. This was inhibited by (-)-pindolol (1-mg kg-1, i.p.) and not prevented by pretreatments with p-chlorophenylalanine which grossly depleted 5-hydroxytryptamine (5-HT) from terminal regions. The hypothermic effects are explicable by activation of postsynaptic 5-HT1A receptors. Infusion of BMY 7378 (8-64 micrograms) into the dorsal raphe was without convincing hypothermic effect. 3. BMY 7378 (8 mg kg-1, s.c.) inhibited another effect of activation of postsynaptic 5-HT1A receptors, i.e., the induction of components of the 5-HT syndrome by 8-OH-DPAT (0.5, 1.0 mg kg-1, s.c.) which suggests that BMY 7378 has antagonistic as well as agonistic effects at these sites. 4. Partial agonist properties of BMY 7378 at postsynaptic sites were also indicated by doses for hypothermia being much greater than those for hyperphagia i.e., ED50 (hypothermia) greater than 2 mg kg-1, ED50 (hyperphagia) = 0.010 mg kg-1. This contrasts with the similar ED50 values for both the hypothermic (ED50 = 0.08-0.10 mg kg-1) and hyperphagic (ED50 = 0.06-0.10 mg kg-1) effects of 8-OH-DPAT.5. The evidence obtained for mediation of the hypothermic response to 5-HTIA agonists by postsynaptic sites is relevant to the interpretation of the effects on it of antidepressant treatments and depressive illness.