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MR33317 was synthesized as an acetylcholinesterase-inhibitor and an agonist at brain 5-HT 4 -receptors. MR33317 might be used to treat Morbus Alzheimer. This therapeutic action of MR33317 might be based on MR33317´s dual synergistic activity. We tested the hypothesis that MR33317 also stimulates 5-HT 4 -receptors in the heart. MR33317 (starting at 10 nM) increased force of contraction and beating rate in isolated atrial preparations from mice with cardiac confined overexpression of the human 5-HT 4 -serotonin receptor (5-HT 4 -TG) but was inactive in wild type mouse hearts (WT). Only in the presence of the phosphodiesterase III-inhibitor cilostamide, MR33317 raised force of contraction under isometric conditions in isolated paced (1 Hz) human right atrial preparations (HAP). This increase in force of contraction in human atrium by MR33317 was attenuated by 10 µM tropisetron or GR125487. These data suggest that MR33317 is an agonist at human 5-HT 4 -serotonin receptors in the human atrium. Clinically, one would predict that MR33317 may lead to atrial fibrillation., (© 2024. The Author(s).)

Tegaserod (1-{[(5-methoxy-1H-indol-3-yl)methyliden]amino}-3-pentylguanidine) is a potent agonist at human recombinant 5-HT 4 serotonin receptors. Consequently, tegaserod is utilized in the treatment of bowel diseases. The objective of this study was to test the hypothesis that tegaserod stimulates human cardiac atrial 5-HT 4 -receptors via cyclic adenosine monophosphate (cAMP)-dependent pathways. Tegaserod exerted positive inotropic effects (PIEs) and positive chronotropic effects (PCEs) in isolated left and right atrial preparations, respectively, from mice with cardiac-specific overexpression of the human 5-HT 4 serotonin receptor (5-HT 4 -TG) in a concentration- and time-dependent manner. However, no effect was observed in the hearts of littermates of wild-type mice (WT). Western blot analysis revealed that the expression of 5-HT 4 receptors was significantly higher in 5-HT 4 -TG mice compared to WT mice. The specificity of the signal for the 5-HT 4 receptor was confirmed by the absence of the signal in the hearts of 5-HT 4 receptor knockout mice. Furthermore, tegaserod increased the force of contraction (at concentrations as low as 10 nM), reduced the time of tension relaxation, and increased the rate of tension development in isolated electrically stimulated (at a rate of 60 beats per minute) human right atrial preparations (HAPs, obtained during open-heart surgery) when administered alone. The potency and efficacy of tegaserod to raise the force of contraction were enhanced in the presence of cilostamide, a phosphodiesterase III inhibitor. The positive inotropic effect of tegaserod in HAPs was found to be attenuated by the 5-HT 4 serotonin receptor antagonist GR 125487 (0.1 µM). The efficacy of tegaserod (10 µM) in raising the force of contraction in HAPs was less pronounced than that of serotonin (10 µM) or isoprenaline (1 µM). Tegaserod shifted the concentration-response curve of the force of contraction to serotonin to the right in HAPs, indicating that it is a partial agonist at 5-HT 4 serotonin receptors in this model. We propose that the mechanism of action of tegaserod in HAPs involves cAMP-dependent phosphorylation of cardiac regulatory proteins.

Zacopride (4-amino-5-chloro-2-methoxy-N-(quinuclidin-3-yl)-benzamide) is a potent agonist in human 5-HT 4 serotonin receptors in vitro and in the gastrointestinal tract. Zacopride was studied as an antiemetic drug and was intended to treat gastric diseases. Zacopride has been speculated to be useful as an antiarrhythmic agent in the human ventricle by inhibiting cardiac potassium channels. It is unknown whether zacopride is an agonist in human cardiac 5-HT 4 serotonin receptors. We tested the hypothesis that zacopride stimulates human cardiac atrial 5-HT 4 serotonin receptors. Zacopride increased the force of contraction and beating rate in isolated atrial preparations from mice with cardiac-specific overexpression of human 5-HT 4 serotonin receptors (5-HT 4 -TG). However, it was inactive in wild-type mouse hearts (WT). Zacopride was as effective as serotonin in raising the force of contraction and beating rate in atrial preparations of 5-HT 4 -TG. Zacopride raised the force of contraction in human right atrial preparations (HAP) in the absence and presence of the phosphodiesterase III inhibitor cilostamide (1 µM). The positive inotropic effect of zacopride in HAP was attenuated by either 10 µM tropisetron or 1 µM GR125487, both of which are antagonists at 5-HT 4 serotonin receptors. These data suggest that zacopride is also an agonist at 5-HT 4 serotonin receptors in the human atrium., (© 2024. The Author(s).)

Mosapride (4-amino-5-chloro-2-ethoxy-N-[[4-[(4-fluorophenyl) methyl]-2-morpholinyl]-methyl] benzamide) is a potent agonist at gastrointestinal 5-HT 4 receptors. Mosapride is an approved drug to treat several gastric diseases. We tested the hypothesis that mosapride also stimulates 5-HT 4 receptors in the heart. Mosapride increased the force of contraction and beating rate in isolated atrial preparations from mice with cardiac overexpression of human 5-HT 4 -serotonin receptors (5-HT 4 -TG). However, it is inactive in wild-type mouse hearts (WT). Mosapride was less effective and potent than serotonin in raising the force of contraction or the beating rate in 5-HT 4 -TG. Only in the presence of cilostamide (1 μM), a phosphodiesterase III inhibitor, mosapride, and its primary metabolite time dependently raised the force of contraction under isometric conditions in isolated paced human right atrial preparations (HAP, obtained during open heart surgery). In HAP, mosapride (10 μM) reduced serotonin-induced increases in the force of contraction. Mosapride (10 µM) shifted the concentration-response curves to serotonin in HAP to the right. These data suggest that mosapride is a partial agonist at 5-HT 4 -serotonin receptors in HAP., (© 2024. The Author(s).)

Torreya grandis (T. grandis) oil has been reported to alleviate symptoms of slow transit constipation (STC). However, the impact of sciadonic acid (SA), a distinctive fatty acid found in T. grandis oil, on the pathological progression of STC remains unclear. This study aimed to evaluate the effect of SA on STC and uncover the underlying mechanisms. The STC model was established by feeding Balb/c mice with loperamide. After 2 weeks of intervention, SA significantly improved weight loss and intestinal motility decline induced by STC, along with enhancing plasma indices and reducing colon pathological damage. SA effectively reversed the STC-induced decrease in the 5-HT4/cAMP/PKA/AQP4 signaling pathway genes and expression. Furthermore, 16S rRNA analysis demonstrated that SA mitigated the imbalance of the intestinal microbiota induced by STC, by reducing the ratio of Firmicutes to Bacteroidetes (F/B) and increasing the abundance of beneficial bacteria such as Akkermansia. In conclusion, SA intervention alleviated colonic dysfunction in STC mice. The activation of the SA-mediated 5-HT4/cAMP/PKA/AQP4 signaling pathway may serve as a potential target for STC treatment. These findings suggest that SA holds promise as a treatment option for STC and could potentially be extended to other related gut diseases for further investigation., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Postprandial dyslipidemia is commonly present in people with type 2 diabetes and obesity and is characterized by overproduction of apolipoprotein B48-containing chylomicron particles from the intestine. Peripheral serotonin is emerging as a regulator of energy homeostasis with profound implications for obesity; however, its role in dietary fat absorption and chylomicron production is unknown. Chylomicron production was assessed in Syrian golden hamsters by administering an olive oil gavage and IP poloxamer to inhibit lipoprotein clearance. Administration of serotonin or selective serotonin reuptake inhibitor, fluoxetine, increased postprandial plasma triglyceride (TG) and TG-rich lipoproteins. Conversely, inhibiting serotonin synthesis pharmacologically by p-chlorophenylalanine (PCPA) led to a reduction in both the size and number of TG-rich lipoprotein particles, resulting in lower plasma TG and apolipoprotein B48 levels. The effects of PCPA occurred independently of gastric emptying and vagal afferent signaling. Inhibiting serotonin synthesis by PCPA led to increased TG within the intestinal lumen and elevated levels of TG and cholesterol in the stool when exposed to a high-fat/high-cholesterol diet. These findings imply compromised fat absorption, as evidenced by reduced lipase activity in the duodenum and lower levels of serum bile acids, which are indicative of intestinal bile acids. During the postprandial state, mRNA levels for serotonin receptors (5-HTRs) were upregulated in the proximal intestine. Administration of cisapride, a 5-HT4 receptor agonist, alleviated reductions in postprandial lipemia caused by serotonin synthesis inhibition, indicating that serotonin controls dietary fat absorption and chylomicron secretion via 5-HT4 receptor., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com. See the journal About page for additional terms.)

Temporal discounting, in which the recipient of a reward perceives the value of that reward to decrease with delay in its receipt, is associated with impulsivity and psychiatric disorders such as depression. Here, we investigate the role of the serotonin 5-HT 4 receptor (5-HT 4 R) in modulating temporal discounting in the macaque dorsal caudate nucleus (dCDh), the neurons of which have been shown to represent temporally discounted value. We first mapped the 5-HT 4 R distribution in macaque brains using positron emission tomography (PET) imaging and confirmed dense expression of 5-HT 4 R in the dCDh. We then examined the effects of a specific 5-HT 4 R antagonist infused into the dCDh. Blockade of 5-HT 4 R significantly increased error rates in a goal-directed delayed reward task, indicating an increase in the rate of temporal discounting. This increase was specific to the 5-HT 4 R blockade because saline controls showed no such effect. The results demonstrate that 5-HT 4 Rs in the dCDh are involved in reward-evaluation processes, particularly in the context of delay discounting, and suggest that serotonergic transmission via 5-HT 4 R may be a key component in the neural mechanisms underlying impulsive decisions, potentially contributing to depressive symptoms., (© 2024. The Author(s).)

Parkinson's disease is caused by a selective vulnerability and cell loss of dopaminergic neurons of the Substantia Nigra pars compacta and, consequently, striatal dopamine depletion. In Parkinson's disease therapy, dopamine loss is counteracted by the administration of L-DOPA, which is initially effective in ameliorating motor symptoms, but over time leads to a burdening side effect of uncontrollable jerky movements, termed L-DOPA-induced dyskinesia. To date, no efficient treatment for dyskinesia exists. The dopaminergic and serotonergic systems are intrinsically linked, and in recent years, a role has been established for pre-synaptic 5-HT1a/b receptors in L-DOPA-induced dyskinesia. We hypothesized that post-synaptic serotonin receptors may have a role and investigated the effect of modulation of 5-HT4 receptor on motor symptoms and L-DOPA-induced dyskinesia in the unilateral 6-OHDA mouse model of Parkinson's disease. Administration of RS 67333, a 5-HT4 receptor partial agonist, reduces L-DOPA-induced dyskinesia without altering L-DOPA's pro-kinetic effect. In the dorsolateral striatum, we find 5-HT4 receptor to be predominantly expressed in D2R-containing medium spiny neurons, and its expression is altered by dopamine depletion and L-DOPA treatment. We further show that 5-HT4 receptor agonism not only reduces L-DOPA-induced dyskinesia, but also enhances the activation of the cAMP-PKA pathway in striatopallidal medium spiny neurons. Taken together, our findings suggest that agonism of the post-synaptic serotonin receptor 5-HT4 may be a novel therapeutic approach to reduce L-DOPA-induced dyskinesia., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

To better understand mechanisms of serotonin- (5-HT) mediated vasorelaxation, isolated lateral saphenous veins from cattle were assessed for vasoactivity using myography in response to increasing concentrations of 5-HT or selective 5-HT receptor agonists. Vessels were pre-contracted with 1 × 10 -4  M phenylephrine and exposed to increasing concentrations of 5-HT or 5-HT receptor agonists that were selective for 5-HT 1B , 5-HT 2B , 5-HT 4 , and 5-HT 7 . Vasoactive response data were normalized as a percentage of the maximum contractile response induced by the phenylephrine pre-contraction. At 1 × 10 -7  M 5-HT, a relaxation was observed with an 88.7% decrease (p < 0.01) from the phenylephrine maximum. At 1 × 10 -4  M 5-HT, a contraction was observed with a 165% increase (p < 0.01) from the phenylephrine maximum. Increasing concentrations of agonists selective for 5-HT 2B , 5-HT 4 , or 5-HT 7 resulted in a 27%, 92%, or 44% (p < 0.01) decrease from the phenylephrine maximum, respectively. Of these 5-HT receptor agonists, the selective 5-HT 4 receptor agonist resulted in the greatest potency (-log EC 50 ) value (6.30) compared with 5-HT 2B and 5-HT 7 receptor agonists (4.21 and 4.66, respectively). To confirm the involvement of 5-HT 4 in 5-HT-mediated vasorelaxation, blood vessels were exposed to either DMSO (solvent control) or a selective 5-HT 4 antagonist (1 × 10 -5  M) for 5-min prior to the phenylephrine pre-contraction and 5-HT additions. Antagonism of the 5-HT 4 receptor attenuated the vasorelaxation caused by 5-HT. Approximately 94% of the vasorelaxation occurring in response to 5-HT could be accounted for through 5-HT 4 , providing strong evidence that 5-HT-mediated vasorelaxation occurs through 5-HT 4 activation in bovine peripheral vasculature., (© 2024 The Author(s). Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Several fungi belonging to the genus Psilocybe, also called "magic mushrooms", contain the hallucinogenic drugs psilocybin and psilocin. They are chemically related to serotonin (5-HT). In addition to being abused as drugs, they are now also being discussed or used as a treatment option for depression. Here, we hypothesized that psilocybin and psilocin may act also on cardiac serotonin receptors and studied them in vitro in atrial preparations of our transgenic mouse model with cardiac myocytes-specific overexpression of the human 5-HT 4 receptor (5-HT 4 -TG) as well as in human atrial preparations. Both psilocybin and psilocin enhanced the force of contraction in isolated left atrial preparations from 5-HT 4 -TG, increased the beating rate in isolated spontaneously beating right atrial preparations from 5-HT 4 -TG and augmented the force of contraction in the human atrial preparations. The inotropic and chronotropic effects of psilocybin and psilocin at 10 µM were smaller than that of 1 µM 5-HT on the left and right atria from 5-HT 4 -TG, respectively. Psilocybin and psilocin were inactive in WT. In the human atrial preparations, inhibition of the phosphodiesterase III by cilostamide was necessary to unmask the positive inotropic effects of psilocybin or psilocin. The effects of 10 µM psilocybin and psilocin were abrogated by 10 µM tropisetron or by 1 µM GR125487, a more selective 5-HT 4 receptor antagonist. In summary, we demonstrated that psilocin and psilocybin act as agonists on cardiac 5-HT 4 receptors., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Methylation of serotonin-related genes has been proposed as a plausible gene-by-environment link which may mediate environmental stress, depressive and anxiety symptoms. DNA methylation is often measured in blood cells, but little is known about the association between this peripheral epigenetic modification and brain serotonergic architecture. Here, we evaluated the association between whole-blood-derived methylation of four CpG sites in the serotonin transporter (SLC6A4) and six CpG sites of the tryptophan hydroxylase 2 (TPH2) gene and in-vivo brain levels of serotonin transporter (5-HTT) and serotonin 4 receptor (5-HT 4 ) in a cohort of healthy individuals (N = 254) and, for 5-HT 4, in a cohort of unmedicated patients with depression (N = 90). To do so, we quantified SLC6A4/TPH2 methylation using bisulfite pyrosequencing and estimated brain 5-HT 4 and 5-HTT levels using positron emission tomography. In addition, we explored the association between SLC6A4 and TPH2 methylation and measures of early life and recent stress, depressive and anxiety symptoms on 297 healthy individuals., Results: We found no statistically significant association between peripheral DNA methylation and brain markers of serotonergic neurotransmission in patients with depression or in healthy individuals. In addition, although SLC6A4 CpG2 (chr17:30,236,083) methylation was marginally associated with the parental bonding inventory overprotection score in the healthy cohort, statistical significance did not remain after accounting for blood cell heterogeneity., Conclusions: We suggest that findings on peripheral DNA methylation in the context of brain serotonin-related features should be interpreted with caution. More studies are needed to rule out a role of SLC6A4 and TPH2 methylation as biomarkers for environmental stress, depressive or anxiety symptoms., (© 2024. The Author(s).)

In recent years, particular attention has been paid to the serotonin 4 receptor, which is well expressed in the brain, but also peripherally in various organs. The cerebral distribution of this receptor is well conserved across species, with high densities in the basal ganglia, where they are expressed by GABAergic neurons. The 5-HT 4 receptor is also present in the cerebral cortex, hippocampus, and amygdala, where they are carried by glutamatergic or cholinergic neurons. Outside the central nervous system, the 5-HT 4 receptor is notably expressed in the gastrointestinal tract. The wide distribution of the 5-HT 4 receptor undoubtedly contributes to its involvement in a plethora of functions. In addition, the modulation of this receptor influences the release of serotonin, but also the release of other neurotransmitters such as acetylcholine and dopamine. This is a considerable asset, as the modulation of the 5-HT 4 receptor can therefore play a direct or indirect beneficial role in various disorders. One of the main advantages of this receptor is that it mediates a much faster antidepressant and anxiolytic action than classical selective serotonin reuptake inhibitors. Another major benefit of the 5-HT 4 receptor is that its activation enhances cognitive performance, probably via the release of acetylcholine. The expression of the 5-HT 4 receptor is also altered in various eating disorders, and its activation by the 5-HT 4 agonist negatively regulates food intake. Additionally, although the cerebral expression of this receptor is modified in certain movement-related disorders, it is still yet to be determined whether this receptor plays a key role in their pathophysiology. Finally, there is no longer any need to demonstrate the value of 5-HT 4 receptor agonists in the pharmacological management of gastrointestinal disorders.

Hyperglycemia drives dysfunction of the intestinal barrier. 5-Hydroxytryptaine 4 receptor (5-HT 4 R) agonists have been considered therapeutics for constipation in clnic. However, the roles of 5-HT 4 R activation in mucosa should be fully realized. Here, we investigate the effects of 5-HT 4 R activation on diabetes-induced disruption of the tight junction (TJ) barrier in the colon. Not surprisingly, the TJ barrier in diabetic mice with or without 5-HT 4 R is tremendously destroyed, as indicated by increased serum fluorescein isothiocyanate (FITC)-dextran and decreased transepithelial electrical resistance (TER). Simultaneously, decreased expressions of TJ proteins are shown in both wild-type (WT) and 5-HT 4 R knockout (KO) mice with diabetes. Notably, chronic treatment with intraperitoneal injection of a 5-HT 4 R agonist in WT mice with diabetes repairs the TJ barrier and promotes TJ protein expressions, including occludin, claudin-1 and ZO-1, in the colon, whereas a 5-HT 4 R agonist does not improve TJ barrier function or TJ protein expressions in 5-HT 4 R KO mice with diabetes. Furthermore, stimulation of 5-HT 4 R inhibits diabetes-induced upregulation of myosin light chain kinase (MLCK), Rho-associated coiled coil protein kinase 1 (ROCK1), and phosphorylated myosin light chain (p-MLC), which are key molecules that regulate TJ integrity, in the colonic mucosa of WT mice. However, such action induced by a 5-HT 4 R agonist is not observed in 5-HT 4 R KO mice with diabetes. These findings indicate that 5-HT 4 R activation may restore TJ integrity by inhibiting the expressions of MLCK, ROCK1 and p-MLC, improving epithelial barrier function in diabetes.

Background: A prominent finding in major depressive disorder (MDD) is distorted stress hormone dynamics, which is regulated by serotonergic brain signaling. An interesting feature of the cerebral serotonin system is the serotonin 4 receptor (5-HT4R), which is lower in depressed relative to healthy individuals and also has been highlighted as a promising novel antidepressant target. Here, we test the novel hypothesis that brain 5-HT4R availability in untreated patients with MDD is correlated with cortisol dynamics, indexed by the cortisol awakening response (CAR). Further, we evaluate if CAR changes with antidepressant treatment, including a selective serotonin reuptake inhibitor, and if pretreatment CAR can predict treatment outcome., Methods: Sixty-six patients (76% women) with a moderate to severe depressive episode underwent positron emission tomography imaging with [11C]SB207145 for quantification of brain 5-HT4R binding using BPND as outcome. Serial home sampling of saliva in the first hour from awakening was performed to assess CAR before and after 8 weeks of antidepressant treatment. Treatment outcome was measured by change in Hamilton Depression Rating Scale 6 items., Results: In the unmedicated depressed state, prefrontal and anterior cingulate cortices 5-HT4R binding was positively associated with CAR. CAR remained unaltered after 8 weeks of antidepressant treatment, and pretreatment CAR did not significantly predict treatment outcome., Conclusions: Our findings highlight a link between serotonergic disturbances in MDD and cortisol dynamics, which likely is involved in disease and treatment mechanisms. Further, our data support 5-HT4R agonism as a promising precision target in patients with MDD and disturbed stress hormone dynamics., (© The Author(s) 2023. Published by Oxford University Press on behalf of CINP.)

Sexual dysfunction is prominent in Major Depressive Disorder (MDD) and affects women with depression more than men. Patients with MDD relative to healthy controls have lower brain levels of the serotonin 4 receptor (5-HT 4 R), which is expressed with high density in the striatum, i.e. a key hub of the reward system. Reduced sexual desire is putatively related to disturbed reward processing and may index anhedonia in MDD. Here, we aim to illuminate plausible underlying neurobiology of sexual dysfunction in unmedicated patients with MDD. We map associations between 5-HT 4 R binding, as imaged with [ 11 C]SB207145 PET, in the striatum, and self-reported sexual function. We also evaluate if pre-treatment sexual desire score predicts 8-week treatment outcome in women. From the NeuroPharm study, we include 85 untreated MDD patients (71% women) who underwent eight weeks of antidepressant drug treatment. In the mixed sex group, we find no difference in 5-HT 4 R binding between patients with sexual dysfunction vs normal sexual function. However, in women we find lower 5-HT 4 R binding in the sexual dysfunctional group compared to women with normal sexual function (β = -0.36, 95%CI[-0.62:-0.09], p = 0.009) as well as a positive association between sexual desire and 5-HT 4 R binding (β = 0.07, 95%CI [0.02:0.13], p = 0.012). Sexual desire at baseline do not predict treatment outcome (ROC curve AUC = 52%[36%:67%]) in women. Taken together, we find evidence for a positive association between sexual desire and striatal 5-HT 4 R availability in women with depression. Interestingly, this raises the question if direct 5-HT 4 R agonism can target reduced sexual desire or anhedonia in MDD., (© 2023. The Author(s).)

Brain serotonergic (5-HT) signaling is posited to modulate neural responses to emotional stimuli. Dysfunction in 5-HT signaling is implicated in major depressive disorder (MDD), a disorder associated with significant disturbances in emotion processing. In MDD, recent evidence points to altered 5-HT 4 receptor (5-HT 4 R) levels, a promising target for antidepressant treatment. However, how these alterations influence neural processing of emotions in MDD remains poorly understood. This is the first study to examine the association between 5-HT 4 R binding and neural responses to emotions in patients with MDD and healthy controls. The study included one hundred and thirty-eight participants, comprising 88 outpatients with MDD from the NeuroPharm clinical trial (ClinicalTrials.gov identifier: NCT02869035) and 50 healthy controls. Participants underwent an [ 11 C]SB207145 positron emission tomography (PET) scan to quantify 5-HT 4 R binding (BP ND ) and a functional magnetic resonance imaging (fMRI) scan during which they performed an emotional face matching task. We examined the association between regional 5-HT 4 R binding and corticolimbic responses to emotional faces using a linear latent variable model, including whether this association was moderated by depression status. We observed a positive correlation between 5-HT 4 R BP ND and the corticolimbic response to emotional faces across participants (r = 0.20, p = 0.03). This association did not differ between groups (parameter estimate difference = 0.002, 95% CI = -0.008: 0.013, p = 0.72). Thus, in the largest PET/fMRI study of associations between serotonergic signaling and brain function, we found a positive association between 5-HT 4 R binding and neural responses to emotions that appear unaltered in MDD. Future clinical trials with novel pharmacological agents targeting 5-HT 4 R are needed to confirm whether they ameliorate emotion processing biases in MDD., (© 2023. The Author(s).)

Importance: The cerebral serotonin 4 (5-HT4) receptor is a promising novel target for treatment of major depressive disorder (MDD), and pharmacological stimulation of the 5-HT4 receptor has been associated with improved learning and memory in healthy individuals., Objective: To map the neurobiological signatures of patients with untreated MDD compared with healthy controls and to examine the association between cerebral 5-HT4 receptor binding and cognitive functions in the depressed state., Design, Setting, and Participants: This case-control study used baseline data from the NeuroPharm clinical depression trial in Denmark. Adult participants included antidepressant-free outpatients with a current moderate to severe depressive episode and healthy controls. All participants completed positron emission tomography (PET) scanning with [11C]SB207145 for quantification of brain 5-HT4 receptor binding, but only the patients underwent cognitive testing. Data analyses were performed from January 21, 2020, to April 22, 2022., Main Outcomes and Measures: The main study outcome was the group difference in cerebral 5-HT4 receptor binding between patients with MDD and healthy controls. In addition, the association between 5-HT4 receptor binding and verbal memory performance in the patient group was tested. Other cognitive domains (working memory, reaction time, emotion recognition bias, and negative social emotions) were assessed as secondary outcomes., Results: A total of 90 patients with untreated MDD (mean [SD] age, 27.1 [8.2] years; 64 women [71.1%]) and 91 healthy controls (mean [SD] age, 27.1 [8.0] years; 55 women [60.4%]) were included in the analysis. Patients with current MDD had significantly lower cerebral 5-HT4 receptor binding than healthy controls (-7.0%; 95% CI, -11.2 to -2.7; P = .002). In patients with MDD, there was a correlation between cerebral 5-HT4 receptor binding and verbal memory (r = 0.29; P = .02)., Conclusions and Relevance: Results of this study show that cerebral 5-HT4 receptor binding was lower in patients with MDD than in healthy controls and that the memory dysfunction in patients with MDD was associated with lower cerebral 5-HT4 receptor binding. The cerebral 5-HT4 receptor is a promising treatment target for memory dysfunction in patients with MDD.

Serotonin acts solely via 5-HT 4 -receptors to control human cardiac contractile function. The effects of serotonin via 5-HT 4 -receptors lead to positive inotropic and chronotropic effects, as well as arrhythmias, in the human heart. In addition, 5-HT 4 -receptors may play a role in sepsis, ischaemia, and reperfusion. These presumptive effects of 5-HT 4 -receptors are the focus of the present review. We also discuss the formation and inactivation of serotonin in the body, namely, in the heart. We identify cardiovascular diseases where serotonin might play a causative or additional role. We address the mechanisms which 5-HT 4 -receptors can use for cardiac signal transduction and their possible roles in cardiac diseases. We define areas where further research in this regard should be directed in the future, and identify animal models that might be generated to this end. Finally, we discuss in what regard 5-HT 4 -receptor agonists or antagonists might be useful drugs that could enter clinical practice. Serotonin has been the target of many studies for decades; thus, we found it timely to summarise our current knowledge here.

Ergotamine (2'-methyl-5'α-benzyl-12'-hydroxy-3',6',18-trioxoergotaman) is a tryptamine-related alkaloid from the fungus Claviceps purpurea . Ergotamine is used to treat migraine. Ergotamine can bind to and activate several types of 5-HT 1 -serotonin receptors. Based on the structural formula of ergotamine, we hypothesized that ergotamine might stimulate 5-HT 4 -serotonin receptors or H 2 -histamine receptors in the human heart. We observed that ergotamine exerted concentration- and time-dependent positive inotropic effects in isolated left atrial preparations in H 2 -TG (mouse which exhibits cardiac-specific overexpression of the human H 2 -histamine receptor). Similarly, ergotamine increased force of contraction in left atrial preparations from 5-HT 4 -TG (mouse which exhibits cardiac-specific overexpression of the human 5-HT 4 -serotonin receptor). An amount of 10 µM ergotamine increased the left ventricular force of contraction in isolated retrogradely perfused spontaneously beating heart preparations of both 5-HT 4 -TG and H 2 -TG. In the presence of the phosphodiesterase inhibitor cilostamide (1 µM), ergotamine 10 µM exerted positive inotropic effects in isolated electrically stimulated human right atrial preparations, obtained during cardiac surgery, that were attenuated by 10 µM of the H 2 -histamine receptor antagonist cimetidine, but not by 10 µM of the 5-HT 4 -serotonin receptor antagonist tropisetron. These data suggest that ergotamine is in principle an agonist at human 5-HT 4 -serotonin receptors as well at human H 2 -histamine receptors. Ergotamine acts as an agonist on H 2 -histamine receptors in the human atrium.

Concurrent anxiety is frequent in major depressive disorder and a shared pathophysiological mechanism between anxiety and other depressive symptoms is plausible. The serotonin 4 receptor (5-HT 4 R) has been implicated in both depression and anxiety. This is the first study to investigate the association between the cerebral 5-HT 4 R binding and anxiety in patients with depression before and after antidepressant treatment and the association to treatment response. Ninety-one drug-free patients with depression were positron emission tomography scanned with the 5-HT 4 R ligand [ 11 C]-SB207145. Depression severity and concurrent anxiety was measured at baseline and throughout 8 weeks of antidepressant treatment. Anxiety measures included four domains: anxiety/somatization factor score; Generalized Anxiety Disorder 10-items (GAD-10) score; anxiety/somatization factor score ≥7 (anxious depression) and syndromal anxious depression. Forty patients were rescanned at week 8. At baseline, we found a negative association between global 5-HT 4 R binding and both GAD-10 score (p < 0.01) and anxiety/somatization factor score (p = 0.06). Further, remitters had a higher baseline anxiety/somatization factor score compared with non-responders (p = 0.04). At rescan, patients with syndromal anxious depression had a greater change in binding relative to patients with non-syndromal depression (p = 0.04). Concurrent anxiety in patients with depression measured by GAD-10 score and anxiety/somatization factor score is negatively associated with cerebral 5-HT 4 R binding. A lower binding may represent a subtype with reduced natural resilience against anxiety in a depressed state, and concurrent anxiety may influence the effect on the 5-HT 4 R from serotonergic antidepressants. The 5-HT 4 R is a promising neuroreceptor for further understanding the underpinnings of concurrent anxiety in patients with depression., (© 2022. The Author(s).)

Diabetes leads to intestinal barrier dysfunction. 5-Hydroxytryptamine 4 receptor (5-HT 4 R) is distributed in the colonic mucosa, but little is known about the role of its activation in diabetes-evoked colonic barrier dysfunction. This study investigates whether activation of 5-HT 4 Rs on goblet cells (GCs) protects the colon from commensal bacterial translocation in diabetic mice. Expression of 5-HT 4 R detected inside the colonic epithelium by RNAscope in situ hybridization was further observed within the mucin 2 (MUC2)-immunoreactive GCs. In diabetic mice, neither 5-HT 4 R transcription nor protein levels were altered compared with those in nondiabetic mice. Bacterial translocation was characterized by 16S rRNA RNAscope in situ hybridization and manifested in both crypts and lamina propria of the colon in diabetic mice. Mucin production and MUC2 expression were significantly decreased in diabetic mice. Furthermore, the loss of mitochondrial cristae of GCs and the down-regulation of mitofilin, the core protein maintaining mitochondrial homeostasis, were observed in diabetic mice. Long-term treatment with 5-HT 4 R agonist in diabetic mice not only prevented bacterial penetration of the whole colonic mucosa but also promoted mucin production and MUC2 expression. Markedly, 5-HT 4 R agonist also restored the mitochondrial cristae of GCs and up-regulated mitofilin. However, co-administration of 5-HT 4 R antagonist abolished the effects of 5-HT 4 R agonist on diabetic mice. These findings indicate that 5-HT 4 R in colonic mucosa is an effective target for the treatment of diabetes-induced colonic mucous barrier dysfunction., (Copyright © 2022 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Because of their importance in the regulation of gut functions, several therapeutic targets involving serotonin-related proteins have been developed or repurposed to treat motility disorders, including serotonin transporter inhibitors, tryptophan hydroxylase blockers, 5-HT3 antagonists, and 5-HT4 agonists. This chapter focuses on our discovery of 5-HT4 receptors in the epithelial cells of the colon and our efforts to evaluate the effects of stimulating these receptors. 5-HT4 receptors appear to be expressed by all epithelial cells in the mouse colon, based on expression of a reporter gene driven by the 5-HT4 receptor promoter. Application of 5-HT4 agonists to the mucosal surface causes serotonin release from enterochromaffin cells, mucus secretion from goblet cells, and chloride secretion from enterocytes. Luminal administration of 5-HT4 agonists speeds up colonic motility and suppresses distention-induced nociceptive responses. Luminal administration of 5-HT4 agonists also decreases the development of, and improves recovery from, experimental colitis. Recent studies determined that the prokinetic actions of minimally absorbable 5-HT4 agonists are just as effective as absorbable compounds. Collectively, these findings indicate that targeting epithelial receptors with non-absorbable 5-HT4 agonists could offer a safe and effective strategy for treating constipation and colitis., (© 2022. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Alzheimer's disease (AD) is a neurodegenerative disease characterized by memory decline and impaired hippocampal synaptic plasticity. The serotonin 5-HT4 receptor is involved in learning and memory processes. This study explored the effects of chronic stimulation of 5-HT4R on cognition, memory, long-term potentiation (LTP), paired-pulse ratio (PPR), and neuronal apoptosis in a rat model of amyloid-beta (Aβ)-induced AD. Thirty-five male Wistar rats were randomly divided into three groups as follows: the sham, Aβ, and Aβ + BIMU8 groups. Aβ (6 µg/µl) was administrated by intracerebroventricular (icv) injection. The animals were treated with BIMU8 (1 μg/μL, ICV) as a 5-HT4R agonist for 30 days. Memory and behavioral changes were assessed by the passive avoidance learning, novel object recognition, open field, and elevated plus maze tests. Hippocampal synaptic plasticity was evaluated in the dentate gyrus (DG) in response to the stimulation applied to the perforant pathway. Furthermore, neuronal apoptosis was measured in the hippocampus. Data were analyzed by SPSS version 19 using one-way ANOVA, followed by Tukey's post hoc test. Aβ induced memory deficits and neuronal loss and inhibited LTP induction. Aβ also increased the normalized PPR. BIMU8 enhanced the slope of the field excitatory postsynaptic potential in LTP and improved cognition behavior. Paired-pulse inhibition or facilitation was not affected by LTP induction in Aβ animals receiving the BIMU8. It can be concluded that the stimulation of the 5-HT 4 receptor modulated the Aβ-induced cognition and memory deficits, probably via a decrease in the hippocampal apoptotic neurons and an improvement in the hippocampal synaptic functions without involving its inhibitory interneurons., (Copyright © 2021. Published by Elsevier B.V.)

In the past, we generated transgenic mice that overexpress the human histamine 2 (H 2 )-receptor (H 2 -TG) or that overexpress the human serotonin 4 (5-HT 4 )-receptor (5-HT 4 -TG) in the heart. Here, we crossbred these lines of mice to generate double transgenic mice that overexpress both receptors (DT). This was done to study a conceivable interaction between these receptors in the mouse heart as a model for the human heart. When in left atria, initially, force of contraction was elevated maximally with 1 µM serotonin, and subsequently, histamine was cumulatively applied; a biphasic effect of histamine was noted: the force of contraction initially decreased, maximally at 10 nM histamine, and thereafter, the force of contraction increased again at 1 µM histamine. Notably, functional interaction between 5-HT and histamine was also identified in isolated electrically stimulated trabeculae carneae from human right atrium (obtained during cardiac surgery). These functional and biochemical data together are consistent with a joint overexpression of inotropically active H 2 -receptors and 5-HT 4 -receptors in the same mouse heart. We also describe an antagonistic interaction on the force of contraction of both receptors in the mouse atrium (DT) and in the human atrial muscle strips. We speculate that via this interaction, histamine might act as a "brake" on the cardiac actions of 5-HT via inhibitory GTP-binding proteins acting on the activity of adenylyl cyclase., (© 2021. The Author(s).)

The hippocampus has long been considered as a key structure for memory processes. Multilevel alterations of hippocampal function have been identified as a common denominator of memory impairments in a number of psychiatric and neurodegenerative diseases. For many years, the glutamatergic and cholinergic systems have been the main targets of therapeutic treatments against these symptoms. However, the high rate of drug development failures has left memory impairments on the sideline of current therapeutic strategies. This underscores the urgent need to focus on new therapeutic targets for memory disorders, such as type 4 serotonin receptors (5-HT 4 Rs). Ever since the discovery of their expression in the hippocampus, 5-HT 4 Rs have gained growing interest for potential use in the treatment of learning and memory impairments. To date, much of the researched information gathered by scientists from both animal models and humans converge on pro-mnesic and anti-amnesic properties of 5-HT 4 Rs activation, although the mechanisms at work require more work to be fully understood. This review addresses a fundamental, yet poorly understood set of evidence of the potential of 5-HT 4 Rs to re-establish or limit hippocampal alterations related to neurological diseases. Most importantly, the potential of 5-HT 4 Rs is translated by refining hypotheses regarding the benefits of their activation in memory disorders at the hippocampal level.

Cognitive deficits commonly accompany psychiatric disorders but are often underrecognised, and difficult to treat. The 5-HT 4 receptor is a promising potential treatment target for cognitive impairment because in animal studies 5-HT 4 receptor agonists enhance hippocampal-dependent memory processes. To date, there has been little work translating these effects to humans. We tested whether short-term administration of the 5-HT 4 partial agonist, prucalopride, modified behavioural and neural (fMRI) memory processing in 44 healthy human volunteers using an experimental medicine model. We found that participants who had received six days of prucalopride treatment were significantly better at recalling previously seen neutral images and distinguishing them from new images. At a neural level, prucalopride bilaterally increased hippocampal activity and activity in the right angular gyrus compared with placebo. Taken together, these findings demonstrate the potential of 5-HT 4 -receptor activation for cognitive enhancement in humans, and support the potential of this receptor as a treatment target for cognitive impairment., (© 2021. The Author(s).)

We investigated whether hypothermia would be arrhythmogenic in mice that overexpress the human 5-HT 4 receptor only in their cardiac myocytes (5-HT 4 -TG). Contractile studies were performed in isolated, electrically driven (1 Hz) left and spontaneously beating right atrial preparations of 5-HT 4 -TG and littermate wild-type control mice (WT). Hypothermia (23 °C) decreased the force of contraction in the mouse right and left atrial preparations. Moreover, the concentration-dependent positive inotropic effects of 5-HT were blunted but still shifted to lower 5-HT concentrations in the left 5-HT 4 -TG atria in hypothermia compared to normothermia (37 °C). Furthermore, hypothermia increased the incidence of right atrial arrhythmias in 5-HT 4 -TG more than in WT mice. In contrast, at 37 °C, lowering the potassium concentration from 5.2 to 2.0 mM also induced arrhythmias in the right atrium, but with a similar incidence in WT and 5-HT 4 -TG mice. In contrast, 10 μM d,l-sotalol and 300 μM erythromycin did not induce arrhythmias. Hypothermia was accompanied by the increased expression of heat shock protein 70 (HSP70) in WT but not in 5-HT 4 -TG mice. We concluded that without the stimulation of 5-HT 4 -receptors by exogenous agonists, a simple temperature reduction can increase arrhythmias in 5-HT 4 -TG mice. It is tempting to speculate that in human patients, 5-HT 4 receptors might contribute to potentially deadly hypothermia-induced arrhythmias., (Copyright © 2021 Elsevier B.V. All rights reserved.)

A series of oxadiazole derivatives were synthesized and evaluated as 5-hydroxytryptamine-4 receptor (5-HT 4 R) partial agonists for the treatment of cognitive deficits associated with Alzheimer's disease. Starting from a reported 5-HT 4 R antagonist, a systematic structure-activity relationship was conducted, which led to the discovery of potent and selective 5-HT 4 R partial agonist 1-isopropyl-3-{5-[1-(3-methoxypropyl) piperidin-4-yl]-[1,3,4]oxadiazol-2-yl}-1H-indazole oxalate (Usmarapride, 12l ). It showed balanced physicochemical-pharmacokinetic properties with robust nonclinical efficacy in cognition models. It also showed disease-modifying potential, as it increased neuroprotective soluble amyloid precursor protein alpha levels, and dose-dependent target engagement and correlation of efficacy with oral exposures. Phase 1 clinical studies have been completed and projected efficacious concentration was achieved without any major safety concerns. Phase 2 enabling long-term safety studies have been completed with no concerns for further development.

Issues with inflated false positive rates (FPRs) in brain imaging have recently received significant attention. However, to what extent FPRs present a problem for voxelwise analyses of Positron Emission Tomography (PET) data remains unknown. In this work, we evaluate the FPR using real PET data under group assignments that should yield no significant results after correcting for multiple comparisons. We used data from 159 healthy participants, imaged with the serotonin transporter ([ 11 C]DASB; N = 100) or the 5-HT 4 receptor ([ 11 C]SB207145; N = 59). Using this null data, we estimated the FPR by performing 1,000 group analyses with randomly assigned groups of either 10 or 20, for each tracer, and corrected for multiple comparisons using parametric Monte Carlo simulations (MCZ) or non-parametric permutation testing. Our analyses show that for group sizes of 10 or 20, the FPR for both tracers was 5-99% using MCZ, much higher than the expected 5%. This was caused by a heavier-than-Gaussian spatial autocorrelation, violating the parametric assumptions. Permutation correctly controlled the FPR in all cases. In conclusion, either a conservative cluster forming threshold and high smoothing levels, or a non-parametric correction for multiple comparisons should be performed in voxelwise analyses of brain PET data.

Serotonin receptor 4 (5-HT 4 R) plays an important role in regulating mood, anxiety, and cognition, and drugs that activate this receptor have fast-acting antidepressant (AD)-like effects in preclinical models. However, 5-HT 4 R is widely expressed throughout the central nervous system (CNS) and periphery, making it difficult to pinpoint the cell types and circuits underlying its effects. Therefore, we generated a Cre-dependent 5-HT 4 R knockout mouse line to dissect the function of 5-HT 4 R in specific brain regions and cell types. We show that the loss of functional 5-HT 4 R specifically from excitatory neurons of hippocampus led to robust AD-like behavioral responses and an elevation in baseline anxiety. 5-HT 4 R was necessary to maintain the proper excitability of dentate gyrus (DG) granule cells and cell type-specific molecular profiling revealed a dysregulation of genes necessary for normal neural function and plasticity in cells lacking 5-HT 4 R. These adaptations were accompanied by an increase in the number of immature neurons in ventral, but not dorsal, dentate gyrus, indicating a broad impact of 5-HT 4 R loss on the local cellular environment. This study is the first to use conditional genetic targeting to demonstrate a direct role for hippocampal 5-HT 4 R signaling in modulating mood and anxiety. Our findings also underscore the need for cell type-based approaches to elucidate the complex action of neuromodulatory systems on distinct neural circuits., (© 2021. The Author(s).)

Animal experimental studies suggest that 5-HT4 receptor activation holds promise as a novel target for the treatment of depression and cognitive impairment. 5-HT4 receptors are post-synaptic receptors that are located in striatal and limbic areas known to be involved in cognition and mood. Consistent with this, 5-HT4 receptor agonists produce rapid antidepressant effects in a number of animal models of depression, and pro-cognitive effects in tasks of learning and memory. These effects are accompanied by molecular changes, such as the increased expression of neuroplasticity-related proteins that are typical of clinically useful antidepressant drugs. Intriguingly, these antidepressant-like effects have a fast onset of their action, raising the possibility that 5-HT4 receptor agonists may be a particularly useful augmentation strategy in the early stages of SSRI treatment. Until recently, the translation of these effects to humans has been challenging. Here, we review the evidence from animal studies that the 5-HT4 receptor is a promising target for the treatment of depression and cognitive disorders, and outline a potential pathway for the efficient and cost-effective translation of these effects into humans and, ultimately, to the clinic.

5-HT 4 receptors are G protein-coupled receptors that link to the stimulatory protein Gs which activates adenylate cyclase to increase intracellular cyclic AMP which then activates protein kinase A (PKA). 5-HT 4 receptors are expressed by neurons in the central and peripheral nervous systems especially the enteric nervous system (ENS). In general, 5-HT 4 receptors are stimulatory and their activation in the ENS enhances neurotransmitter release and propulsive motility patterns. 5-HT 4 receptors are expressed by enterochromaffin (EC) cells, Goblet cells, and most enteric neurons. The study by Konen and colleagues in this issue of Neurogastroenterology and Motility features two novel 5-HT 4 receptor agonists (5-HT 4 -LA1 and 5-HT 4 -LA-2) that are not absorbed from the gastrointestinal tract of mice and act locally in the colonic mucosa to stimulate propulsive motility. The authors show that 5-HT 4 -LA1 and 5-HT 4 -LA2 were not absorbed from the colon and that both drugs stimulated colonic transit when administered by gavage. Both agonists stimulated colonic glass bead expulsion, and 5-HT 4 LA1 activation stimulated fecal output and increased fecal water content. These effects were detected in young and aged mice. 5-HT 4 receptors were also localized to the epithelium of the human duodenum, ileum, and colon. These studies highlight novel 5-HT 4 receptor agonists that have prokinetic actions on the GI tract. These drugs are not absorbed and act locally in the gut mucosa to stimulate propulsive motility while minimizing access to systemic 5-HT 4 receptors and avoiding potential unwanted side effects., (© 2021 John Wiley & Sons Ltd.)

Alzheimer's disease (AD) is a neurodegenerative disorder whose prevalence has an incidence in senior citizens. Unfortunately, current pharmacotherapy only offers symptom relief for patients with side effects such as bradycardia, nausea, and vomiting. Therefore, there is a present need to provide other therapeutic alternatives for treatments for these disorders. The 5-HT 4 receptor is an attractive therapeutic target since it has a potential role in central and peripheral nervous system disorders such as AD, irritable bowel syndrome, and gastroparesis. Quantitative structure-activity relationship analysis of a series of 62 active compounds in the 5-HT 4 receptor was carried out in the present work. The structure-activity relationship was estimated using three-dimensional quantitative structure-activity relationship (3D-QSAR) techniques based on these structures' field molecular (force and Gaussian field). The best force-field QSAR models achieve a value for the coefficient of determination of the training set of R 2 training = 0.821, and for the test set R 2 test = 0.667, while for Gaussian-field QSAR the training and the test were R 2 training = 0.898 and R 2 test = 0.695, respectively. The obtained results were validated using a coefficient of correlation of the leave-one-out cross-validation of Q 2 LOO = 0.804 and Q 2 LOO = 0.886 for force- and Gaussian-field QSAR, respectively. Based on these results, novel 5-HT 4 partial agonists with potential biological activity (pEC 50 8.209-9.417 for force-field QSAR and 9.111-9.856 for Gaussian-field QSAR) were designed. In addition, for the new analogues, their absorption, distribution, metabolism, excretion, and toxicity properties were also analyzed. The results show that these new derivatives also have reasonable pharmacokinetics and drug-like properties. Our findings suggest novel routes for the design and development of new 5-HT 4 partial agonists.

Ongoing activity in nociceptors, a driver of spontaneous pain, can be generated in dorsal root ganglion neurons in the absence of sensory generator potentials if one or more of three neurophysiological alterations occur - prolonged depolarization of resting membrane potential (RMP), hyperpolarization of action potential (AP) threshold, and/or increased amplitude of depolarizing spontaneous fluctuations of membrane potential (DSFs) to bridge the gap between RMP and AP threshold. Previous work showed that acute, sustained exposure to serotonin (5-HT) hyperpolarized AP threshold and potentiated DSFs, leading to ongoing activity if a separate source of maintained depolarization was present. Cellular signaling pathways that increase DSF amplitude and promote ongoing activity acutely in nociceptors are not known for any neuromodulator. Here, isolated DRG neurons from male rats were used to define the pathway by which low concentrations of 5-HT enhance DSFs, hyperpolarize AP threshold, and promote ongoing activity. A selective 5-HT 4 receptor antagonist blocked these 5-HT-induced hyperexcitable effects, while a selective 5-HT 4 agonist mimicked the effects of 5-HT. Inhibition of cAMP effectors, protein kinase A (PKA) and exchange protein activated by cAMP (EPAC), attenuated 5-HT's hyperexcitable effects, but a blocker of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels had no significant effect. 5-HT 4 -dependent PKA activation was specific to DRG neurons that bind isolectin B4 (a nonpeptidergic nociceptor marker). 5-HT's effects on AP threshold, DSFs, and ongoing activity were mimicked by a cAMP analog. Sustained exposure to 5-HT promotes ongoing activity in nonpeptidergic nociceptors through the G s -coupled 5-HT 4 receptor and downstream cAMP signaling involving both PKA and EPAC., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Atrial fibrillation (AF)-associated remodeling includes contractile dysfunction whose reasons are only partially resolved. Serotonin (5-HT) increases contractile force and causes arrhythmias in atrial trabeculae from patients in sinus rhythm (SR). In persistent atrial fibrillation (peAF), the force responses to 5-HT are blunted and arrhythmic effects are abolished. Since force but not arrhythmic responses to 5-HT in peAF could be restored by PDE3 + PDE4 inhibition, we sought to perform real-time measurements of cAMP to understand whether peAF alters PDE3 + PDE4-mediated compartmentation of 5-HT 4 receptor-cAMP responses. Isolated human atrial myocytes from patients in SR, with paroxysmal AF (paAF) or peAF, were adenovirally transduced to express the FRET-based cAMP sensor Epac1-camps. Forty-eight hours later, cAMP responses to 5-HT (100 μM) were measured in the absence or concomitant presence of the PDE3 inhibitor cilostamide (0.3 μM) and the PDE4 inhibitor rolipram (1 μM). We successfully established real-time cAMP imaging in AF myocytes. 5-HT increased cAMP in SR, paAF, and peAF, but in line with previous findings on contractility, this increase was considerably smaller in peAF than in SR or paAF. The maximal cAMP response to forskolin (10 μM) was preserved in all groups. The diminished cAMP response to 5-HT in peAF was recovered by preincubation with cilostamide + rolipram. We uncovered a significantly diminished cAMP response to 5-HT 4 receptor stimulation which may explain the blunted 5-HT inotropic responses observed in peAF. Since both cAMP and force responses but not arrhythmic responses were recovered after concomitant inhibition of PDE3 + PDE4, they might be regulated in different subcellular microdomains.

The type 4 serotonin receptor (5-HT4R) is highly involved in cognitive processes such as learning and memory. Behavioral studies have shown a beneficial effect of its activation and conversely reported memory impairments by its blockade. However, how modulation of 5HT4R enables modifications of hippocampal synaptic plasticity remains elusive. To shed light on the mechanisms at work, we investigated the effects of the 5-HT4R agonist RS67333 on long-term potentiation (LTP) within the hippocampal CA1 area. Although high-frequency stimulation-induced LTP remained unaffected by RS67333, the magnitude of LTP induced by theta-burst stimulation was significantly decreased. This effect was blocked by the selective 5-HT4R antagonist RS39604. Further, 5-HT4R-induced decrease in LTP magnitude was fully abolished in the presence of bicuculline, a GABAAR antagonist; hence, demonstrating involvement of GABA neurotransmission. In addition, we showed that the application of a GABABR antagonist, CGP55845, mimicked the effect of 5-HT4R activation, whereas concurrent application of CGP55845 and RS67333 did not elicit an additive inhibition effect on LTP. To conclude, through investigation of theta burst induced functional plasticity, we demonstrated an interplay between 5-HT4R activation and GABAergic neurotransmission within the hippocampal CA1 area., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

More than 30% of all pharmaceuticals target G-protein-coupled receptors (GPCRs). Here, we present a GPCR-based screen in yeast to identify ligands for human serotonin receptor 4 (5-HTR 4 ). Serotonin receptor 4 agonists are used for the treatment of irritable bowel syndrome with constipation. Specifically, the HTR 4 -based screen couples activation of 5-HTR 4 on the yeast cell surface to luciferase reporter expression. The HTR 4 -based screen has a throughput of one compound per second allowing the screening of more than a thousand compounds per day.

Objective: Sex steroid hormones potently shape brain functions, including those critical to maintain mental health such as serotonin signaling. Use of oral contraceptives (OCs) profoundly changes endogenous sex steroid hormone levels and dynamics. Recent register-based studies show that starting an OC is associated with increased risk of developing depression. Here, we investigate whether use of OCs in healthy women is associated with a marker of the serotonin system in terms of serotonin 4 receptor (5-HT4R) brain imaging., Methods: [ 11 C]SB207145-PET imaging data on 53 healthy women, of whom 16 used OCs, were available from the Cimbi database. We evaluated global effects of OC use on 5-HT4R binding in a latent variable model based on 5-HT4R binding across cortical and subcortical regions., Results: We demonstrate that OC users have 9-12% lower global brain 5-HT4R binding potential compared to non-users. Univariate region-based analyses (pallidostriatum, caudate, hippocampus, amygdala, anterior cingulate cortex, and neocortex) supported the global effect of OC use with the largest difference present in the hippocampus (-12.8% (95% CI [-21.0; -3.9], P corrected  = 0.03)., Conclusion: We show that women who use OCs have markedly lower brain 5-HT4R binding relative to non-users, which constitutes a plausible molecular link between OC use and increased risk of depressive episodes. We propose that this reflects a reduced 5-HT4R gene expression, possibly related to a blunted ovarian hormone state among OC users., (© 2020 The Authors. Acta Psychiatrica Scandinavica published by John Wiley & Sons Ltd.)

Serotonin (5-hydroxytryptamine [5-HT]) synthesized and released in enterochromaffin (EC) cells participates in various functions in the gastrointestinal tract by acting on a diverse range of 5-HT receptors (HTRs) expressed on smooth muscle, enteric neurons, and epithelial cells. We previously observed that genes encoding HTR2A, HTR2B, and HTR4 are expressed in murine intestinal organoids, suggesting the expression of these HTRs in intestinal epithelial cells. The present study investigated the localization of these HTRs in the murine intestine by immunofluorescence staining. HTR2A, HTR2B, and HTR4 localized in individual solitary cells in the epithelium, while HTR2C was observed in the lamina propria. In the epithelium, HTR2A, HTR2B, and HTR4 colocalized with 5-HT, and HTR4 colocalized with glucagon-like peptide 1 (GLP-1) and peptide YY (PYY). Murine intestinal organoids show a colocalization pattern that is similar to in vivo HTR2A and HTR4 with 5-HT, GLP-1, and PYY. Intraperitoneal and intragastric administration of tegaserod, an HTR4 agonist, failed to alter plasma GLP-1 levels in fasted mice. However, intragastric but not intraperitoneal administration of tegaserod reduced dietary lipid-induced increases of plasma GLP-1 levels. This action of tegaserod was inhibited by co-administration of RS39604, an HTR4 antagonist. These results suggest that murine ileal GLP-1/PYY-producing enteroendocrine (EE) cells express HTR4, while 5-HT-producing EC cells express HTR2A, HTR2B, and HTR4. In addition, the observations regarding in vivo GLP-1 secretion suggest that HTR4 signaling in ileal EE cells suppresses dietary lipid-induced GLP-1 secretion. We thus propose that EC and EE cells may interact with each other through paracrine signaling mechanisms.

Spinal cord injury (SCI) often leads to defecation dysfunction. Sacral nerve electrical stimulation (SNS) therapy could improve defecation function. The present study aimed to assess SNS therapy, with regard to the levels of serotonin (5‑HT) and its receptors (5‑HT3AR and 5‑HT4R) in the colon and sacral cord, a rat model of acute severe SCI was used. This rat model was made using the New York University Impactor device. Model rats were randomized to the SCI and SNS (electrical stimulation on the S3 nerve) groups. After 14 days of treatment, enteric transmission function was assessed. 5‑HT and 5‑HT3AR/5‑HT4R were measured by ELISA, quantitative PCR, immunohistochemistry and western blotting. In SCI rats, SNS significantly increased the quantity of feces, shortened the time to the first fecal passage, and improved fecal texture and colon histology. SNS elevated 5‑HT contents in the colon and spinal cord, and enhanced 5‑HT3AR/5‑HT4R protein expression and distribution in the colonic myenteric plexus and mucosa, sacral intermediolateral nucleus and dorsal horn. SNS upregulated the relative expression levels of 5‑HT3AR/5‑HT4R mRNA and protein in the colon and spinal cord. SNS can improve defecation and accelerate the recovery of colonic transmission functions in rat models of acute SCI. These effects involved upregulation of the 5‑HT/5‑HT3AR/5‑HT4R axes.

Introduction: Numerous chemotypes have been described over time in order to generate potent and selective 5-HT 4 R ligands. Both agonists and antagonists have demonstrated their interest in several disease models. This culminates with the FDA approval of tegaserod and prucalopride in the recent years., Areas Covered: This review summarizes the patent applications from 2014 to present, dedicated to the use or the description of novel 5-HT 4 R modulators. Several novel ligands and scaffolds have been industrially protected mainly in the field of central nervous system (CNS) pathologies as well as gastrointestinal disorders, including the combination with other drugs or for veterinary uses., Expert Opinion: The therapeutic potential of 5-HT 4 R modulators has been explored for several years in animal models, but also linked to potential safety issues with initial ligands. The current use of prucalopride in humans demonstrates that its toxicity is not linked to the target and that 5-HT 4 R modulators are safe in humans. Therefore, an important number of studies and patents has continued in the recent years to expand the use of 5-HT 4 R modulators, not only to treat gastrointestinal disorders, but also for CNS pathologies. This article details current efforts in this development.

The latest estimates from world health organization suggest that more than 450 million people are suffering from depression and other psychiatric conditions. Of these, 50-60% have been reported to have progression of gut diseases. In the last two decades, researchers introduced incipient physiological roles for serotonin (5-HT) receptors (5-HTRs), suggesting their importance as a potential pharmacological target in various psychiatric and gut diseases. A growing body of evidence suggests that 5-HT systems affect the brain-gut axis in depressive patients, which leads to gut comorbidity. Recently, preclinical trials of 5-HT4R agonists and antagonists were promising as antipsychotic and prokinetic agents. In the current review, we address the possible pharmacological role and contribution of 5-HT4R in the pathophysiology of chronic depression and associated gut abnormalities. Physiologically, during depression episodes, centers of the sympathetic and parasympathetic nervous system couple together with neuroendocrine systems to alter the function of hypothalamic-pituitary-adrenal (HPA) axis and enteric nervous system (ENS), which in turn leads to onset of gastrointestinal tract (GIT) disorders. Consecutively, the ENS governs a broad spectrum of physiological activities of gut, such as visceral pain and motility. During the stages of emotional stress, hyperactivity of the HPA axis alters the ENS response to physiological and noxious stimuli. Consecutively, stress-induced flare, swelling, hyperalgesia and altered reflexes in gut eventually lead to GIT disorders. In summary, the current review provides prospective information about the role and mechanism of 5-HT4R-based therapeutics for the treatment of depressive disorder and possible consequences for the gut via brain-gut axis interactions. This article is part of the special issue entitled 'Serotonin Research: Crossing Scales and Boundaries'., Competing Interests: Declaration of competing interest Authors declare no competing interest associated with manuscript. All the authors read and approved the final manuscript., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Prucalopride, a high affinity, selective serotonin type 4 (5-HT 4 ) receptor agonist, was associated with increased neoplasia incidence (in endocrine tissues and liver) in 2-year rodent bioassays, without evidence of a genotoxic mechanism of action. Proposed mechanisms of action involve prolactin and the constitutive androstane receptor (CAR). Epigenetic mechanisms and their relevance to humans are discussed. Data from in vitro and in vivo rodent studies demonstrated that prucalopride-related stimulation of prolactin secretion (via dopamine receptor D2 antagonism at high doses) is a rodent-specific, non-genotoxic mechanism for inducing hyperplasia and neoplasia in prolactin receptor-expressing endocrine tissues. Additional data demonstrated that CAR-mediated liver enzyme induction underlies the observed hepatocellular adenomas and thyroid follicular adenomas in rodents. A 12-month neonatal mouse carcinogenicity study confirmed the lack of a genotoxic mechanism of action. Furthermore, tumors were observed only at very high exposures (200 and 63 fold higher in mice and rats, respectively, than human exposure after a daily therapeutic dose of 2 mg). The studies indicate that non-genotoxic, rodent-specific, epigenetic mechanisms that are considered clinically irrelevant are responsible for the increased incidence of neoplasias associated with very high exposure to prucalopride in rodents, and that prucalopride does not pose a carcinogenic safety risk to humans., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Hong Wang was an employee of Shire, a member of the Takeda group of companies, at the time of this analysis and is now an employee of Pfizer. Zhen Lou was an employee of Shire, a member of the Takeda group of companies, at the time of this analysis and is now an employee of H3 Biomedicine. Kate Lane was an employee of Shire, a member of the Takeda group of companies, at the time of this analysis and is now an employee of Translate Bio. Suezanne Parker was an employee of Shire, a member of the Takeda group of companies, at the time of this analysis and is now an employee of Arcturus Therapeutics. Michael Placke was an employee of Shire, a member of the Takeda group of companies, at the time of this analysis. These studies were funded by Johnson & Johnson. Writing support was provided by Anja Becher, PhD, of PharmaGenesis London, London, UK, and funded by Shire, a member of the Takeda group of companies., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

A rigidification strategy was applied to the preclinical candidate donecopride, an acetylcholinesterase inhibitor possessing 5-HT 4 R agonist activity. Inspired by promising bioactive benzisoxazole compounds, we have conducted a pharmacomodulation study to generate a novel series of multitarget directed ligands. The chemical synthesis of the ligand was optimized and compounds were evaluated in vitro against each target and in cellulo. Structure-activity relationship was supported by docking analysis in human acetylcholinesterase binding site. Among the synthesized compounds, we have identified a novel hybrid 32a (3-[2-[1-(cyclohexylmethyl)-4-piperidyl]ethyl]-4-methoxy-1,2-benzoxazole) able to display nanomolar acetylcholinesterase inhibitory effects and nanomolar Ki for 5-HT 4 R.

The serotonin receptor 4b (5-HTR 4b ) is expressed throughout the gastrointestinal tract, and its agonists are used in the treatment of irritable bowel syndrome with constipation (IBS-C). Today, there are no rapid assays for the identification of 5-HTR 4b agonists. Here, we developed a luciferase-based 5-HTR 4b assay capable of assessing one compound per second with a 38-fold dynamic range and nM limit of detection for serotonin. We used the assay to screen more than 1000 natural products and anti-infection agents and identified five new 5-HTR 4b ligands: hordenine, halofuginone, proflavine, ethacridine, and revaprazan. We demonstrate that hordenine (antibiofilm), halofuginone (antiparasitic), and revaprazan (gastric acid reducer) activate 5-HTR 4b in human colon epithelial cells, leading to increased cell motility or wound healing. The 5-HTR 4b assay can be used to screen larger pharmaceutical libraries to identify novel treatments for IBS-C. This work shows that antimicrobials interact not only with the gut microbiota, but also with the human host.

Introduction : Chronic idiopathic constipation (CIC) is a kind of constipation in which the patient experiences constipation more than 3 months without any identifiable cause. Prucalopride is one such treatment considered for relieving symptoms of CIC regarding due to its selectivity for the 5HT4 receptor. Areas covered : This article is based on a PubMed and clinicaltrials.gov search for studies undertaken over the past 19 years (2000-2019) using the following keywords either alone or in combination: Prucalopride, chronic idiopathic constipation, chronic constipation, 5HT4 receptor, Resolor and Motegrity. Expert opinion : Prucalopride should be considered as one of the safe options for the treatment of CIC especially when previous treatments have failed. It can be helpful in the treatment of constipation caused by irritable bowel syndrome or spinal cord injury, opioid-induced constipation, post-operative ileus, and intestinal/colonic pseudo-obstruction. The major drawback of prucalopride is its high cost, which makes it less accessible to all patients.

Facing the complexity of Alzheimer's disease (AD), it is now currently admitted that a therapeutic pleiotropic intervention is needed to alter its progression. Among the major hallmarks of the disease, the amyloid pathology and the oxidative stress are closely related. We propose in this study to develop original Multi-Target Directed Ligands (MTDL) able to impact at the same time Aβ protein accumulation and toxicity of Reactive Oxygen Species (ROS) in neuronal cells. Such MTDL were obtained by linking on a central piperidine two scaffolds of interest: a typical aminochlorobenzophenone present in numerous 5-HT 4 R agonists, and diverse antioxidant chemotypes. Interestingly, the most active compound 9g possesses a Ki of 12.7 nM towards 5-HT 4 R and an antioxidant activity in vitro and in cellulo., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Serotonin (5-HT) homeostasis is critical for the brain development which influences neurogenesis, neuronal migration, and circuit formation. Distinctive distribution patterns of serotonin receptors (5-HTRs) in the brain govern various physiological activities. Amongst the 5-HTRs, serotonin 4 receptor (5-HT4R) is widely expressed in embryonic forebrain and affects neuronal development, synaptogenesis, and behavior, but its specific role in brain development is still not completely understood. Therefore, in the present study, we addressed the roles of 5-HT4R in the growth of hippocampal neurons during the development of mice brain. We cultured hippocampal neurons of the mouse at embryonic day 18 and then treatment of 5-HT4R agonist RS67333 was employed. We found RS67333 significantly increased the axonal length, diameter and branching along with total dendritic length, number of primary dendrites and their branching. In addition, these effects were neutralized by the concomitant treatment of 5-HT4R antagonist GR125487, which confirmed the specific role of the 5-HT4R in the growth of axon and dendrites. Further, the treatment of RS67333 upregulated the mRNA expression of collapsin response mediator protein-2 (CRMP2) and non-phosphorylated CRMP2 (npCRMP2) together with neurotrophic factors (BDNF, NT-3, NGF) and TRK-A. Additionally, the current research findings reveal that the knockdown of CRMP2 inhibited RS67333-induced growth of the axons and dendrites, which indicates that CRMP2 is required for the 5-HT4R-mediated growth of the axons and dendrites. Overall, the findings of the present in vitro study enrich the understanding and provide insight roles of 5-HT4R in embryonic brain development by promoting the growth of hippocampal neurons., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Previously, we found that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease (PD) model mice (PD mice) showed facilitation of hippocampal memory extinction via reduced cyclic adenosine monophosphate (cAMP)/cAMP-dependent response element-binding protein (CREB) signaling, which may cause cognitive impairment in PD. Serotonergic neurons in the median raphe nucleus (MnRN) project to the hippocampus, and functional abnormalities have been reported. In the present study, we investigated the effects of the serotonin 5-HT 4 receptor (5-HT 4 R) agonists prucalopride and velusetrag on the facilitation of memory extinction observed in PD mice. Both 5-HT 4 R agonists restored facilitation of contextual fear extinction in PD mice by stimulating the cAMP/CREB pathway in the dentate gyrus of the hippocampus. A retrograde fluorogold-tracer study showed that γ-aminobutyric acid-ergic (GABAergic) neurons in the reticular part of the substantia nigra (SNr), but not dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNpc), projected to serotonergic neurons in the MnRN, which are known to project their nerve terminals to the hippocampus. It is possible that the degeneration of the SNpc DAergic neurons in PD mice affects the SNr GABAergic neurons, and thereafter, the serotonergic neurons in the MnRN, resulting in hippocampal dysfunction. These findings suggest that 5HT4R agonists could be potentially useful as therapeutic drugs for treating cognitive deficits in PD.

Background: Activating luminal 5-HT 4 receptors results in the release of 5-HT from enterochromaffin cells into the lamina propria to modulate colonic motility. Our aim was to evaluate characteristics of colonic motor patterns involved in the prokinetic effects of intraluminal prucalopride in the rabbit colon., Methods: Colonic motor patterns were studied ex vivo using simultaneous spatiotemporal diameter mapping and pressure sensing., Key Results: Intraluminal prucalopride and intraluminal exogenous 5-HT strongly evoked or enhanced the colonic motor complex at all levels of excitation beginning with generation of clusters of fast propagating contractions (FPCs), then development of long-distance contractions (LDCs) within the clusters, and finally forceful LDCs as the highest level of excitation. Intraluminal prucalopride and intraluminal exogenous 5-HT stimulated propulsive motor activity in a dose-dependent and antagonist-sensitive manner by increasing the contraction amplitude, intraluminal pressure, frequency, velocity, and degree of propagation of the colonic motor complex., Conclusions and Inferences: Activating mucosal 5-HT 4 receptors via intraluminal prucalopride or 5-HT increases propulsive motor activity in a graded manner; that is, depending on starting conditions, amplitudes or frequencies of an activity may increase or a new pattern may be initiated. Our data support further studies into delivering 5-HT 4 receptor agonists via colon-targeted drug delivery systems and studies into the role of luminal 5-HT as an essential requirement for normal colon motor pattern generation., (© 2019 John Wiley & Sons Ltd.)