Increased anxiety of mice lacking the serotonin1A receptor

Brain serotonin (5-HT) has been implicated in a number of physiological processes and pathological conditions. These effects are mediated by at least 14 different 5-HT receptors. We have inactivated the gene encoding the 5-HT_1A receptor in mice and found that receptor-deficient animals have an increased tendency to avoid a novel and fearful environment and to escape a stressful situation, behaviors consistent with an increased anxiety and stress response. Based on the role of the 5-HT_1A receptor in the feedback regulation of the 5-HT system, we hypothesize that an increased serotonergic neurotransmission is responsible for the anxiety-like behavior of receptor-deficient animals. This view is consistent with earlier studies showing that pharmacological activation of the 5-HT system is anxiogenic in animal models and also in humans.     

Structural requirements for 5-HT2A and 5-HT1A serotonin receptor potentiation by the biologically active lipid oleamide

Oleamide is an endogenous fatty acid primary amide that possesses sleep-inducing properties in animals and that has been shown to effect serotonergic receptor responses and block gap junction communication. Herein, the potentiation of the 5-HT_1A receptor response is disclosed, and a study of the structural features of oleamide required for potentiation of the 5-HT_2A and 5-HT_1A response to serotonin (5-HT) is described. Of the naturally occurring fatty acids, the primary amide of oleic acid (oleamide) is the most effective at potentiating the 5-HT_2A receptor response. The structural features required for activity were found to be highly selective. The presence, position, and stereochemistry of the Δ⁹-cis double bond is required, and even subtle structural variations reduce or eliminate activity. Secondary or tertiary amides may replace the primary amide but follow a well defined relationship requiring small amide substituents, suggesting that the carboxamide serves as a hydrogen bond acceptor but not donor. Alternative modifications at the carboxamide as well as modifications of the methyl terminus or the hydrocarbon region spanning the carboxamide and double bond typically eliminate activity. A less extensive study of the 5-HT_1A potentiation revealed that it is more tolerant and accommodates a wider range of structural modifications. An interesting set of analogs was identified that inhibit rather than potentiate the 5-HT_2A, but not the 5-HT_1A, receptor response, further suggesting that such analogs may permit the selective modulation of serotonin receptor subtypes and even have opposing effects on the different subtypes.     

Rapid modulation of spine morphology by the 5-HT2A serotonin receptor through kalirin-7 signaling

The 5-HT_2A serotonin receptor is the most abundant serotonin receptor subtype in the cortex and is predominantly expressed in pyramidal neurons. The 5-HT_2A receptor is a target of several hallucinogens, antipsychotics, anxiolytics, and antidepressants, and it has been associated with several psychiatric disorders, conditions that are also associated with aberrations in dendritic spine morphogenesis. However, the role of 5-HT_2A receptors in regulating dendritic spine morphogenesis in cortical neurons is unknown. Here we show that the 5-HT_2A receptor is present in a subset of spines, in addition to dendritic shafts. It colocalizes with PSD-95 and with multiple PDZ protein-1 (MUPP1) in a subset of dendritic spines of rat cortical pyramidal neurons. MUPP1 is enriched in postsynaptic density (PSD) fractions, is targeted to spines in pyramidal neurons, and enhances the localization of 5-HT_2A receptors to the cell periphery. 5-HT_2A receptor activation by the 5-HT₂ receptor agonist DOI induced a transient increase in dendritic spine size, as well as phosphorylation of p21-activated kinase (PAK) in cultured cortical neurons. PAK is a downstream target of the neuronal Rac guanine nucleotide exchange factor (RacGEF) kalirin-7 that is important for spine remodeling. Kalirin-7 regulates dendritic spine morphogenesis in neurons but its role in neuromodulator signaling has not been investigated. We show that peptide interference that prevents the localization of kalirin-7 to the postsynaptic density disrupts DOI-induced PAK phosphorylation and spine morphogenesis. These results suggest a potential role for serotonin signaling in modulating spine morphology and kalirin-7''s function at cortical synapses.     

Perturbed dentate gyrus function in serotonin 5-HT2C receptor mutant mice

Serotonin systems have been implicated in the regulation of hippocampal function. Serotonin 5-HT_2C receptors are widely expressed throughout the hippocampal formation, and these receptors have been proposed to modulate synaptic plasticity in the visual cortex. To assess the contribution of 5-HT_2C receptors to the serotonergic regulation of hippocampal function, mice with a targeted 5-HT_2C-receptor gene mutation were examined. An examination of long-term potentiation at each of four principal regions of the hippocampal formation revealed a selective impairment restricted to medial perforant path–dentate gyrus synapses of mutant mice. This deficit was accompanied by abnormal performance in behavioral assays associated with dentate gyrus function. 5-HT_2C receptor mutants exhibited abnormal performance in the Morris water maze assay of spatial learning and reduced aversion to a novel environment. These deficits were selective and were not associated with a generalized learning deficit or with an impairment in the discrimination of spatial context. These results indicate that a genetic perturbation of serotonin receptor function can modulate dentate gyrus plasticity and that plasticity in this structure may contribute to neural mechanisms underlying hippocampus-dependent behaviors.     

Serotonin Agonists and Antagonists in Obstructive Sleep Apnea

Obstructive sleep apnea hypopnea syndrome (OSAHS) is a prevalent disorder associated with substantial cardiovascular and neurobehavioral morbidity. Yet this is a disorder for which there are no widely effective pharmacotherapies. The pathophysiology of obstructive sleep apnea namely, normal respiration in waking with disordered breathing only in sleep, suggests that this disorder should be readily amenable to drug therapy. Over the past 10 years, we have gained tremendous insight into the neurochemical mechanisms involved in state-dependent control of respiration. It is apparent from this work that there are many potential avenues for pharmacotherapies, including several seemingly conflicting directions for serotonergic therapies.Serotonin delivery is reduced to upper airway dilator motor neurons in sleep, and this contributes, at least in part, to sleep-related reductions in dilator muscle activity and upper airway obstruction. The dilator motor neuron post-synaptic serotonin receptors are 5-HT_2A and 5-HT_2C subtypes, and in adults the presynaptic 5-HT receptor in motor nuclei is 5-HT_1B, an inhibitory receptor. Serotonin receptors are also found within central respiratory neuronal groups, and these receptor subtypes include 5-HT_1A (inhibitory) and 5-HT₂ receptors. Peripherally, stimulation of 5-HT_2A, 5-HT_2C and 5-HT₃ receptor subtypes have an inhibitory effect on respiration via action at the nodose ganglion. Many of these receptor subtypes and their signal transduction pathways may be affected by oxidative stress in obstructive sleep apnea. These alterations will make finding drug therapies for sleep apnea more challenging, but not insurmountable. Future directions are suggested for elucidating safe, well-tolerated serotonergic drugs for this disorder.Tryptophan was one of the first serotonergic drugs tested for OSAHS. This drug was withdrawn from the market as a result of reports linking tryptophan use with eosinophilic myalgia syndrome and life-threatening pulmonary hypertension. Newer drugs with serotonergic activity tested in persons with sleep-disordered breathing include buspirone, fluoxetine and paroxetine. Trials are presently being conducted to evaluate the effects of 5-HT_2A and 5-HT₃ antagonists on OSAHS. Many of the drugs tested have not shown significant improvement in sleep apnea. However, with continued effort to elucidate the pharmacology of neurochemical control of state-dependent changes in respiratory control, the availability of pharmacological therapy for this disorder is not too far away.     

Genetic deletion of MAO-A promotes serotonin-dependent ventricular hypertrophy by pressure overload

The potential role of serotonin (5-HT) in cardiac function has generated much interest in recent years. In particular, the need for a tight regulation of 5-HT to maintain normal cardiovascular activity has been demonstrated in different experimental models. However, it remains unclear how increased levels of 5-HT could contribute to the development of cardiac hypertrophy. Availability of 5-HT depends on the mitochondrial enzyme monoamine oxidase A (MAO-A). Therefore, we investigated the consequences of MAO-A deletion on ventricular remodeling in the model of aortic banding in mice. At baseline, MAO-A deletion was associated with an increase in whole blood 5-HT (39.4 ± 1.9 μM vs. 24.0 ± 0.9 μM in KO and WT mice, respectively). Cardiac 5-HT_2A, but not 5-HT_2B receptors were overexpressed in MAO-A KO mice, as demonstrated by real-time PCR and Western-blot experiments. After aortic banding, MAO-A KO mice demonstrated greater increase in heart wall thickness, heart to body weight ratios, cardiomyocyte cross-section areas, and myocardial fibrosis compared to WT. Exacerbation of hypertrophy in KO mice was associated with increased amounts of 5-HT in the heart. In order to determine the role of 5-HT and 5-HT_2A receptors in ventricular remodeling in MAO-A KO mice, we administered the 5-HT_2A receptor antagonists ketanserin (1 mg/kg/day) or M100907 (0.1 mg/kg/day) during 4 weeks of aortic banding. Chronic administration of these antagonists strongly prevented exacerbation of ventricular hypertrophy in MAO-A KO mice. These results show for the first time that regulation of peripheral 5-HT by MAO-A plays a role in ventricular remodeling via activation of 5-HT_2A receptors.     

5-HT4 and 5-HT2 receptors antagonistically influence gap junctional coupling between rat auricular myocytes

5-hydroxytryptamine-4 (5-HT₄) receptors have been proposed to contribute to the generation of atrial fibrillation in human atrial myocytes, but it is unclear if these receptors are present in the hearts of small laboratory animals (e.g. rat). In this study, we examined presence and functionality of 5-HT₄ receptors in auricular myocytes of newborn rats and their possible involvement in regulation of gap junctional intercellular communication (GJIC, responsible for the cell-to-cell propagation of the cardiac excitation). Western-blotting assays showed that 5-HT₄ receptors were present and real-time RT-PCR analysis revealed that 5-HT_4b was the predominant isoform. Serotonin (1 μM) significantly reduced cAMP concentration unless a selective 5-HT₄ inhibitor (GR113808 or ML10375, both 1 μM) was present. Serotonin also reduced the amplitude of L-type calcium currents and influenced the strength of GJIC without modifying the phosphorylation profiles of the different channel-forming proteins or connexins (Cxs), namely Cx40, Cx43 and Cx45. GJIC was markedly increased when serotonin exposure occurred in presence of a 5-HT₄ inhibitor but strongly reduced when 5-HT_2A and 5-HT_2B receptors were inhibited, showing that activation of these receptors antagonistically regulated GJIC. The serotoninergic response was completely abolished when 5-HT₄, 5-HT_2A and 5-HT_2B were simultaneously inhibited. A 24 h serotonin exposure strongly reduced Cx40 expression whereas Cx45 was less affected and Cx43 still less. In conclusion, this study revealed that 5-HT₄ (mainly 5-HT_4b), 5-HT_2A and 5-HT_2B receptors coexisted in auricular myocytes of newborn rat, that 5-HT₄ activation reduced cAMP concentration, I_CaL and intercellular coupling whereas 5-HT_2A or 5-HT_2B activation conversely enhanced GJIC.     

Serotonin responsiveness through 5-HT2A and 5-HT4 receptors is differentially regulated in hypertrophic and failing rat cardiac ventricle

Cardiac ventricular responsiveness to serotonin appears in rat postinfarction congestive heart failure (CHF), mainly mediated by 5-HT₄ receptors in chronic dilated CHF and 5-HT_2A receptors in acute CHF. To differentiate between the effects of left ventricular (LV) hypertrophy and failure on 5-HT_2A- and 5-HT₄-mediated inotropic serotonin response, male Wistar rats with increasing LV hypertrophy (AB1-3) and failure (ABHF) 6 weeks after banding of the ascending aorta were screened for contractile function in vivo (echocardiography) and ex vivo in LV papillary muscles, and mRNA expression level determined by RT-PCR. Both AB1-3 and ABHF displayed LV hypertrophy and remodelling. In ABHF, systolic LV and left atrial diameter increased and cardiac output decreased compared to AB3. Serotonin induced a positive inotropic response (PIR) in papillary muscles correlated with the degree of hypertrophy reaching a maximum in ABHF. Both 5-HT_2A and 5-HT₄ receptors contributed to the PIR. The 5-HT_2A contribution increased with increasing hypertrophy, and the 5-HT₄ contribution increased upon transition to heart failure. No 5-HT_2B-mediated PIR was observed, consistent with increased 5-HT_2B mRNA only in non-cardiomyocytes. The 5-HT_2A, 5-HT_2B and 5-HT₄ mRNA levels increased in AB1-3 and increased further in ABHF compared to AB3, but did not correlate with degree of hypertrophy. 5-HT_2A mRNA was also increased in LV of terminally failing human hearts. In conclusion, functional 5-HT_2A and 5-HT₄ receptors are differentially induced in LV hypertrophy and failure. While the 5-HT_2A-mediated PIR is linearly correlated with the degree of hypertrophy, the 5-HT₄-mediated PIR seems to increase with LV dilatation, as also seen in postinfarction CHF.     

Effects of Maternal Ethanol Consumption and Buspirone Treatment on 5-HT1A and 5-HT2A Receptors in Offspring

In utero ethanol exposure results in a decreased concentration of serotonin (5-HT) in brain regions containing the cell bodies of 5-HT neurons and their cortical projections. The concentration of 5-HT reuptake sites is also reduced in several brain areas. The present study extended prior work by evaluating the effects of chronic maternal ethanol consumption and maternal buspirone treatment on 5-HT_1A and 5-HT_2A receptors in multiple brain areas of offspring. Receptors were quantitated early in postnatal development and at an age when the 5-HT networks are normally well-established. Because fetal 5-HT functions as an essential neurotrophic factor, these studies also determined whether treatment of pregnant rats with buspirone, a 5-HT_1A agonist, could overcome the effects of the fetal 5-HT deficit and prevent ethanol-associated receptor abnormalities. The results demonstrated that in utero ethanol exposure significantly alters the binding of 0.1 nM [³H]-8-hydroxy-dipropylaminotetralin to 5-HT_1A receptors in developing animals. Ethanol impaired the development of 5-HT_1A receptors in the frontal cortex, parietal cortex, and lateral septum; these receptors did not undergo the normal developmental increase between postnatal days 19 and 35. The dentate gyms was also sensitive to the effects of in utero ethanol exposure. 5-HT_1A receptors were increased in this region at 19 days. Maternal buspirone treatment prevented the ethanol-associated abnormalities in 5-HT_1A receptors in the dentate gyms, frontal cortex, and lateral septum. Neither maternal ethanol consumption nor buspirone treatment altered the binding of 2 nM [³H]ketanserin to 5-HT_2A receptors in the ventral dentate gyrus, dorsal raphe, parietal and frontal cortexes, striatum, substantia nigra, or nucleus accumbens.     

Traxoprodil,a selective antagonist of the NR2B subunit of the NMDA receptor,potentiates the antidepressant-like effects of certain antidepressant drugs in the forced swim test in mice

One of the newest substances, whose antidepressant activity was shown is traxoprodil, which is a selective antagonist of the NR2B subunit of the NMDA receptor. The main goal of the present study was to evaluate the effect of traxoprodil on animals’ behavior using the forced swim test (FST), as well as the effect of traxoprodil (10 mg/kg) on the activity of antidepressants, such as imipramine (15 mg/kg), fluoxetine (5 mg/kg), escitalopram (2 mg/kg) and reboxetine (2.5 mg/kg). Serotonergic lesion and experiment using the selective agonists of serotonin receptors 5-HT_1A and 5-HT₂ was conducted to evaluate the role of the serotonergic system in the antidepressant action of traxoprodil. Brain concentrations of tested agents were determined using HPLC. The results showed that traxoprodil at a dose of 20 and 40 mg/kg exhibited antidepressant activity in the FST and it was not related to changes in animals’ locomotor activity. Co-administration of traxoprodil with imipramine, fluoxetine or escitalopram, each in subtherapeutic doses, significantly affected the animals’ behavior in the FST and, what is important, these changes were not due to the severity of locomotor activity. The observed effect of traxoprodil is only partially associated with serotonergic system and is independent of the effect on the 5-HT_1A and 5-HT₂ serotonin receptors. The results of an attempt to assess the nature of the interaction between traxoprodil and the tested drugs show that in the case of joint administration of traxoprodil and fluoxetine, imipramine or escitalopram, there were interactions in the pharmacokinetic phase.     

Serotonin receptor subtypes influence prolactin secretion in the turkey

Serotonin (5-HT) stimulation of prolactin (PRL) secretion is mediated through the dopaminergic (DAergic) system, with 5-HT ligands having no direct effect on pituitary PRL release. Infusion of 5-HT into the third ventricle (ICV) or electrical stimulation (ES) of the medial preoptic area (POM) or the ventromedial nucleus (VMN) induces an increase in circulating PRL in the turkey. These increases in PRL do not occur when a selective antagonist blocks the D₁ dopamine (DA) receptors in the infundibular area (INF). In this study, the ICV infusion of (R)(−)-DOI hydrochloride (DOI), a selective 5-HT_2A eceptor agonist, caused PRL to increase. Pretreatment with Ketanserin tartrate salt (KETAN), a selective 5-HT_2A receptor antagonist, blocked DOI-induced PRL secretion, attesting to the specificity of the response. DOI-induced PRL secretion was prevented when the D₁ DA receptors in the INF were blocked by the D₁ DA receptor antagonist, R(+)-SCH-23390 hydrochloride microinjection, suggesting that the DAergic activation of the vasoactive intestinal peptide (VIP)/PRL system is mediated by a stimulatory 5-HT_2A receptor subtype. The DOI-induced PRL increase did not occur when (±)-8-OH-DPAT (DPAT) was concurrently infused. DPAT is a 5-T_1A receptor agonist which appears to mediate the inhibitory influence of 5-HT on PRL secretion. When DPAT was microinjected directly into the VMN, it blocked the PRL release affected by ES in the POM. These data suggested that when 5-HT_2A receptors are activated, they influence the release of DA to the INF. When 5-HT_1A receptors are stimulated, they somehow inhibit the PRL-releasing actions of 5-HT_2A receptors. This inhibition could take place centrally, or it could occur postsynaptically at the pituitary level. It is known that D₂ DA receptors in the pituitary antagonize PRL-releasing effect of VIP. A release of DA to the pituitary, initiated by 5-HT_1A receptors, could effectively inhibit PRL secretion.     

Mast Cell Degranulation Induces Delayed Rectal Allodynia in Rats: Role of Histamine and 5-HT

Visceral hypersensitivity is a common feature offunctional bowel disorders, where an increased number ofmast cells have often been described. Thus, weinvestigated the effect of an experimental mast cell degranulation induced by BrX-537A on somatic(tail heating) and visceral (rectal distension)sensitivity in rats and the involvement of histamineand/or serotonin on this last response. After BrX-537Aadministration, the latency of tail withdrawal reflex wasshortened within the 2- to 8-hr period. Moreover,BrX-537A reduced the distension volume threshold from0.8 ml to 0.4 ml inducing allodynia, from 6 to 12 hrafter its administration. This effect was suppressedby doxantrazole (mast cell stabilizing agent) and WAY100635 (5-HT_1A receptor antagonist), andreproduced by 5-HTP (5-HT precursor) and 8-OH-DPAT(5-HT_1A receptor agonist). However, neither granisetron(5-HT3 receptor antagonist) nor H1, H2, or H3 histaminereceptor antagonists modified the BrX-537A-inducedallodynia. Consequently, mast cell degranulation initiates a delayed somatic and visceralallodynia, with the participation of serotonin, through5-HT_1A receptor activation, on the visceralresponse.     

Decrease in Gastric Sensitivity to Distension by 5-HT1A Receptor Agonists in Rats

Using an in vivo model for evaluation of gastricsensitivity in awake rats, we aimed to determine whether5-hydroxytryptamine 1A (5-HT_1A) agonistsmodify pain threshold and gastric compliancespecifically through 5-HT_1A receptors. Isobaricgastric distensions were performed with a barostat usingsteps of 5 mm Hg in male rats equipped with a gastricballoon and electrodes implanted in the neck muscles.Gastric distension at 15 or 20 mm Hg induced a typicalposture associated with contractions of the neckmuscles. Rats received drugs 30 min before gastricdistension. The 5-HT receptor agonist8-hydroxy-2-(di-n-propylamino)tetra1A lin (8-OH-DPAT),administered intraperitoneally (0.5 mg/kg) increasedgastric pain threshold and gastric tone. These effectswere reproduced when administered centrally (0.05 mg/kg) and blocked by intracerebroventricularadministration of the 5-HT_1A antagonist WAY100635. Flesinoxan (4 mg/kg, intraperitoneally), another5-HT_1A agonist reproduced the effects of8-OH-DPAT on pain threshold and gastric tone and the alpha-receptorantagonist yohimbine did not modify the action of8-OH-DPAT. Our results indicate that activation of5-HT_1A receptors at the level of the centralnervous system increases gastric tone and decreases gastric sensitivityto distension.     

Treatment of Pregnant Alcohol-Consuming Rats with Buspirone: Effects on Serotonin and 5-Hydroxyindoleacetic Acid Content in Offspring

This laboratory previously demonstrated that in utero ethanol exposure markedly impairs the development of the serotonergic system in rat brain. Developmental abnormalities could be detected as early as G15 in the brainstem and G19 in the cortex. Because of the importance of fetal serotonin (5-HT) and 5-HT_1A receptors for the normal development of 5-HT containing neurons, we initiated studies to determine whether administration of a 5-HT_1A agonist, buspirone, to pregnant rats could overcome the adverse effects of in utero ethanol exposure on the developing serotonergic system in offspring. Female, Sprague-Dawley rats were given daily subcutaneous injections of buspirone (4.5 mg/kg) from gestational day 13 (G13) to G20. 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) content were determined in the cortex and cortical regions. These experiments demonstrated that the ethanol-associated abnormalities in the development of the serotonergic system can be partially overcome by in utero exposure to buspirone. Specifically, whereas untreated ethanol rats had a deficiency of 5-HT and/or 5-HIAA in whole cortex on PN5, and in the motor cortex on PN19 and 35, no significant differences were detected in these regions of the age-matched offspring of buspirone-treated, ethanol-fed rats. In contrast, the 5-HT and 5-HIAA deficiency in the somatosensory cortex of 19-dayold offspring of ethanol-fed rats was not corrected by in utero buspirone treatment. These results suggest that the abnormal development of cortical projections of serotonergic neurons may be due in part to the low fetal 5-HT content in ethanol-exposed rats and may potentially be overcome by in utero treatment with a 5-HT_1A agonist. However, additional studies are needed to elucidate the importance of dose and timing of buspirone administration, and the effects of in utero buspirone exposure on other components of serotonergic and nonserotonergic neurons.     

Gq/5-HT2c receptor signals activate a local GABAergic inhibitory feedback circuit to modulate serotonergic firing and anxiety in mice

Serotonin 2c receptors (5-HT_2c-Rs) are drug targets for certain mental disorders, including schizophrenia, depression, and anxiety. 5-HT_2c-Rs are expressed throughout the brain, making it difficult to link behavioral changes to circuit specific receptor expression. Various 5-HT-Rs, including 5-HT_2c-Rs, are found in the dorsal raphe nucleus (DRN); however, the function of 5-HT_2c-Rs and their influence on the serotonergic signals mediating mood disorders remain unclear. To investigate the role of 5-HT_2c-Rs in the DRN in mice, we developed a melanopsin-based optogenetic probe for activation of Gq signals in cellular domains, where 5-HT_2c-Rs are localized. Our results demonstrate that precise temporal control of Gq signals in 5-HT_2c-R domains in GABAergic neurons upstream of 5-HT neurons provides negative feedback regulation of serotonergic firing to modulate anxiety-like behavior in mice.Serotonin (5-hydroxytryptamine, or 5-HT) is an important modulator of anxiety circuits (1). The diverse effects of serotonin are mediated through various 5-HT receptors (5-HT-Rs), including 5-HT_1–7-Rs (2). Recent pharmacologic and genetic studies have highlighted an important role of 5-HT_2c-Rs in anxiety disorders; however, the interpretation of physiological and behavioral data remains difficult owing to a lack of selective pharmacologic ligands (3).5-HT_2c-Rs are expressed in various cell types and brain regions of the anxiety circuit, including the amygdala and the dorsal raphe nucleus (DRN), a midbrain region containing high concentrations of 5-HT neurons. It has been suggested that 5-HT_2c-Rs are expressed in GABAergic neurons, and that 5-HT_2c-R activation may contribute to an inhibitory feedback control of 5-HT cell firing (4). The functional and behavioral consequences of such a possible inhibitory feedback mechanism for 5-HT firing have not yet been investigated, however.Unfortunately, current techniques for identifying the functions of 5-HT_2c-Rs in vertebrate brains are of limited value. For example, agonists and antagonists of 5-HT_2c-Rs are often unspecific, and their action is not restricted to a specific cell type. Complete and conditional knockouts of the receptor gene have limited control of developmental and compensation effects by other G-protein–coupled receptors (GPCRs), and none of the current techniques allows for the physiological control of the 5-HT_2c-R activation on a millisecond to second time scale.To overcome the limitations of pharmacologic and genetic approaches, we have developed a new light-activated GPCR based on vertebrate melanopsin (vMo). Both 5-HT_2c-Rs and vMo couple to the Gq signaling pathway (5, 6). To investigate the functional consequence of Gq signal activation in the cell types and cellular structures where 5-HT_2c-Rs are located, we virally expressed vMo carrying the C terminus (CT) of the 5-HT_2c-R in GABAergic neurons in the DRN. We found that light activation of vMo-CT_5-HT2c decreases the firing of 5-HT neurons and modulates anxiety behaviors in mice. Our results demonstrate a previously unidentified, autoregulatory negative feedback mechanism for the firing of serotonergic neurons to control anxiety in mice.     

Drugs affecting serotonin receptors

A greater understanding of the various serotonin receptor subtypes has led to a clearer appreciation of the role of serotonin in gastrointestinal motility, sensation and secretion. Serotonin is definitely involved in the aetiopathogenesis of cisplatin-induced emesis and carcinoid diarrhoea. The application of serotonergic drugs in clinical therapeutics for gut disturbances is presently dominated by the use of 5-HT₃ antagonists for acute chemotherapy-induced nausea and vomiting, and the use of substituted benzamides which are 5-HT₄ agonists stimulating gut motor function through 5-HT₄ neuronal receptors. The best-studied 5-HT₄ agonist is cisapride, which has been shown to stimulate motility at several levels of the gut. Cisapride is approved for healing and maintenance treatment of reflux oesophagitis and is used in several countries for the alleviation of symptoms consistent with regional stasis, from dyspepsia to constipation.Carcinoid diarrhoea is a prototypic disease associated with deranged serotonin metabolism, and a rationale for using 5-HT₃ or 5-HT₄ antagonists is based on the recent appreciation of the important role of impaired gut motor function in carcinoid diarrhoea. In the future, greater understanding of the serotonin receptor subtypes and their role in gut disorders may lead to novel approaches to alleviate increased visceral perception of functional gastrointestinal disorders, to correct changes in colonic capacitance, or to alter gastrointestinal motility that contributes to diarrhoea or constipation. However, at the present time, it must be stressed that these uses are still at an experimental stage and that careful validation and proper controlled studies are still required.     

Presynaptic serotonin 2A receptors modulate thalamocortical plasticity and associative learning

Higher-level cognitive processes strongly depend on a complex interplay between mediodorsal thalamus nuclei and the prefrontal cortex (PFC). Alteration of thalamofrontal connectivity has been involved in cognitive deficits of schizophrenia. Prefrontal serotonin (5-HT)_2A receptors play an essential role in cortical network activity, but the mechanism underlying their modulation of glutamatergic transmission and plasticity at thalamocortical synapses remains largely unexplored. Here, we show that 5-HT_2A receptor activation enhances NMDA transmission and gates the induction of temporal-dependent plasticity mediated by NMDA receptors at thalamocortical synapses in acute PFC slices. Expressing 5-HT_2A receptors in the mediodorsal thalamus (presynaptic site) of 5-HT_2A receptor-deficient mice, but not in the PFC (postsynaptic site), using a viral gene-delivery approach, rescued the otherwise absent potentiation of NMDA transmission, induction of temporal plasticity, and deficit in associative memory. These results provide, to our knowledge, the first physiological evidence of a role of presynaptic 5-HT_2A receptors located at thalamocortical synapses in the control of thalamofrontal connectivity and the associated cognitive functions.The prefrontal cortex (PFC) is a brain region critical for many high-level cognitive processes, such as executive functions, attention, and working and contextual memories (1). Pyramidal neurons located in layer V of the PFC integrate excitatory glutamatergic inputs originating from both cortical and subcortical areas. The latter include the mediodorsal thalamus (MD) nuclei, which project densely to the medial PFC (mPFC) and are part of the neuronal network underlying executive control and working memory (2–4). Disruption of this network has been involved in cognitive symptoms of psychiatric disorders, such as schizophrenia (3, 5). These symptoms severely compromise the quality of life of patients and remain poorly controlled by currently available antipsychotics (3, 6).The PFC is densely innervated by serotonin (5-hydroxytryptamine, 5-HT) neurons originating from the dorsal and median raphe nuclei and numerous lines of evidence indicate a critical role of 5-HT in the control of emotional and cognitive functions depending on PFC activity (7, 8). The modulatory action of 5-HT reflects its complex pattern of effects on cortical network activity, depending on the 5-HT receptor subtypes involved, and on receptor localization in pyramidal neurons, GABAergic interneurons or nerve terminals of afferent neurons.Among the 14 5-HT receptor subtypes, the 5-HT_2A receptor is a Gq protein-coupled receptor (9, 10) particularly enriched in the mPFC, with a predominant expression in apical dendrites of layer V pyramidal neurons (11–14). Moreover, a low proportion of 5-HT_2A receptors was detected presynaptically on thalamocortical fibers (12, 15–17).Activation of 5-HT_2A receptors exerts complex effects upon the activity of the PFC network (18). The most prominent one is an increase in pyramidal neuron excitability, which likely results from the inhibition of slow calcium-activated after hyperpolarization current (19). 5-HT_2A receptor stimulation also increases the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) in pyramidal neurons (19–22). The prevailing view is that postsynaptic 5-HT_2A receptors expressed on pyramidal neurons located in layer V are key modulators of glutamatergic PFC network activity (14, 21–24). However, the role of presynaptic 5-HT_2A receptors located on thalamic afferents in the modulation of glutamatergic transmission at thalamocortical synapses remains unexplored.Here, we addressed this issue by combining electrophysiological recordings in acute PFC slices with viral infections to specifically rescue the expression of 5-HT_2A receptors at the presynaptic site (MD) or postsynaptic site (PFC) in 5-HT_2A receptor-deficient (5-HT_2A^−/−) mice (25). We focused our study on NMDA transmission in line with previous findings indicating that many symptoms of schizophrenia might arise from modifications in PFC connectivity involving glutamatergic transmission at NMDA receptors (26, 27). To our knowledge, we provide the first direct evidence that stimulation of presynaptic 5-HT_2A receptors at thalamocortical synapses gates the induction of spike timing-dependent long-term depression (t-LTD) by facilitating the activation of presynaptic NMDA receptors at these synapses. In line with the role of t-LTD in associative learning (28), these studies were extended by behavioral experiments to explore the role of presynaptic 5-HT_2A receptors at thalamocortical synapses in several paradigms of episodic-like memory.     

Increased Expression of 5-HT3 and NK1 Receptors in 5-Fluorouracil-Induced Mucositis in Mouse Jejunum

Background and Objective

Although 5-fluorouracil (5-FU) is a widely used as chemotherapy agent, severe mucositis develops in approximately 80 % of patients. 5-FU-induced small intestinal mucositis can cause nausea and vomiting. The current study was designed to investigate peripheral alterations due to the 5-FU-induced mucositis of neuronal and non-neuronal 5-HT₃ and NK₁ receptor expression by immunohistochemical analysis.

Methods

5-FU was administered by i.p. injection to C57BL/6 mice. After 4 days, segments of the jejunum were removed. The specimens were analyzed by immunohistochemistry, real-time PCR, and enzyme immunoassay.

Results

The numbers of 5-HT₃ receptor immunopositive cells and nerve fibers in mucosa were increased by 5-FU treatment. The 5-HT₃ receptor immunopositive cell bodies were found only in jejunal submucosa and myenteric plexus in the 5-FU-treated mice. The numbers of NK₁ receptor cells in mucosa and immunopositive expression of NK₁ receptors in deep muscular plexus were dramatically increased in 5-FU-treated mice. Real-time PCR demonstrated that 5-FU treatment significantly increased mRNA levels of 5-HT_3A, 5-HT_3B, and NK₁ receptors. The amounts of 5-HT and substance P increased after 5-FU treatment. The 5-HT₃ or NK₁ receptor immunopositive cells colocalized with both 5-HT and substance P. Furthermore, 5-HT₃ and NK₁ receptors colocalized with CD11b.

Conclusions

The 5-HT₃ and NK₁ immunopositive macrophages and mucosal mast cells in lamina propria release 5-HT and substance P, which in turn activate their corresponding receptors on mucosal cells in autocrine and paracrine manners. It is assumed to result in the release of 5-HT and substance P in mucosa.