Synthesis and Structure-Activity Relationships of 2,5-Dimethoxy-4-Substituted Phenethylamines and the Discovery of CYB210010: A Potent, Orally Bioavailable and Long-Acting Serotonin 5-HT2 Receptor Agonist.

Structure-activity studies of 4-substituted-2,5-dimethoxyphenethylamines led to the discovery of 2,5-dimethoxy-4-thiotrifluoromethylphenethylamines, including CYB210010, a potent and long-acting serotonin 5-HT2 receptor agonist. CYB210010 exhibited high agonist potency at 5-HT2A and 5-HT2C receptors, modest selectivity over 5-HT2B, 5-HT1A, 5-HT6, and adrenergic α2A receptors, and lacked activity at monoamine transporters and over 70 other proteins. CYB210010 (0.1-3 mg/kg) elicited a head-twitch response (HTR) and could be administered subchronically at threshold doses without behavioral tolerance. CYB210010 was orally bioavailable in three species, readily and preferentially crossed into the CNS, engaged frontal cortex 5-HT2A receptors, and increased the expression of genes involved in neuroplasticity in the frontal cortex. CYB210010 represents a new tool molecule for investigating the therapeutic potential of 5-HT2 receptor activation. In addition, several other compounds with high 5-HT2A receptor potency, yet with little or no HTR activity, were discovered, providing the groundwork for the development of nonpsychedelic 5-HT2A receptor ligands.

Functional interaction of H2-receptors and 5HT4-receptors in atrial tissues isolated from double transgenic mice and from human patients.

In the past, we generated transgenic mice that overexpress the human histamine 2 (H2)-receptor (H2-TG) or that overexpress the human serotonin 4 (5-HT4)-receptor (5-HT4-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 H2-receptors and 5-HT4-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.

New insights into methoxetamine mechanisms of action: Focus on serotonergic 5-HT2 receptors in pharmacological and behavioral effects in the rat.

Methoxetamine (MXE) is a dissociative substance of the arylcyclohexylamine class that has been present on the designer drug market as a ketamine-substitute since 2010. We have previously shown that MXE (i) possesses ketamine-like discriminative and positive rewarding effects in rats, (ii) affects brain processing involved in cognition and emotional responses, (iii) causes long-lasting behavioral abnormalities and neurotoxicity in rats and (iv) induces neurological, sensorimotor and cardiorespiratory alterations in mice. To shed light on the mechanisms through which MXE exerts its effects, we conducted a multidisciplinary study to evaluate the various neurotransmitter systems presumably involved in its actions on the brain. In vivo microdialysis study first showed that a single administration of MXE (0.25 and 0.5 mg/kg, i.v.) is able to significantly alter serotonin levels in the rat medial prefrontal cortex (mPFC) and nucleus accumbens. Then, we observed that blockade of the serotonin 5-HT2 receptors through two selective antagonists, ketanserin (0.1 mg/kg, i.p.) and MDL 100907 (0.03 mg/kg, i.p.), at doses not affecting animals behavior per se, attenuated the facilitatory motor effect and the inhibition on visual sensory responses induced by MXE (3 mg/kg, i.p.) and ketamine (3 mg/kg, i.p.), and prevented MXE-induced reduction of the prepulse inhibition in rats, pointing to the 5-HT2 receptors as a key target for the recently described MXE-induced sensorimotor effects. Finally, in-vitro electrophysiological studies revealed that the GABAergic and glutamatergic systems are also likely involved in the mechanisms through which MXE exerts its central effects since MXE inhibits, in a concentration-dependent manner, NMDA-mediated field postsynaptic potentials and GABA-mediated spontaneous currents. Conversely, MXE failed to alter both the AMPA component of field potentials and presynaptic glutamate release, and seems not to interfere with the endocannabinoid-mediated effects on mPFC GABAergic synapses. Altogether, our results support the notion of MXE as a NMDA receptor antagonist and shed further lights into the central mechanisms of action of this ketamine-substitute by pointing to serotonin 5-HT2 receptors as crucial players in the expression of its sensorimotor altering effects and to the NMDA and GABA receptors as potential further important targets of action.

Acute intrathecal administration of quipazine elicits air-stepping behavior.

Serotonin plays a pivotal role in the initiation and modulation of locomotor behavior in the intact animal, as well as following spinal cord injury. Quipazine, a serotonin 2 receptor agonist, has been used successfully to initiate and restore motor behavior in rodents. Although evidence suggests that the effects of quipazine are spinally mediated, it is unclear whether intrathecal (IT) quipazine administration alone is enough to activate locomotor-like activity or whether additional stimulation is needed. Thus, the current study examined the effects of IT administration of quipazine in postnatal day 1 rats in two separate experiments. In experiment 1, quipazine (0.1, 0.3, or 1.0 mg/kg) was dissolved in saline and administered via IT injection to the thoracolumbar cord. There was no significant effect of drug on hindlimb alternating stepping. In experiment 2, quipazine (0.3 or 1.0 mg/kg) was dissolved in a polysorbate 80-saline solution (Tween 80) and administered via IT injection. Polysorbate 80 was used to disrupt the blood-brain barrier to facilitate absorption of quipazine. The injection was followed by tail pinch 5 minutes post-injection. A significant increase in the percentage of hindlimb alternating steps was found in subjects treated with 0.3 mg/kg quipazine, suggesting that IT quipazine when combined with sensory stimulation to the spinal cord, facilitates locomotor-like behavior. These findings indicate that dissolving the drug in polysorbate 80 rather than saline may heighten the effects of IT quipazine. Collectively, this study provides clarification on the role of quipazine in evoking spinally-mediated locomotor behavior.

The Association Between Antidepressant Effect of SSRIs and Astrocytes: Conceptual Overview and Meta-analysis of the Literature.

Major depressive disorders (MDD) a worldwide psychiatric disease, is yet to be adequately controlled by therapies; while the mechanisms of action of antidepressants are yet to be fully characterised. In the last two decades, an increasing number of studies have demonstrated the role of astrocytes in the pathophysiology and therapy of MDD. Selective serotonin reuptake inhibitors (SSRIs) are the most widely used antidepressants. It is generally acknowledged that SSRIs increase serotonin levels in the central nervous system by inhibiting serotonin transporters, although the SSRIs action is not ideal. The SSRIs antidepressant effect develops with considerable delay; their efficacy is low and frequent relapses are common. Neither cellular nor molecular pharmacological mechanisms of SSRIs are fully characterised; in particular their action on astrocytes remain underappreciated. In this paper we overview potential therapeutic mechanisms of SSRIs associated with astroglia and report the results of meta-analysis of studies dedicated to MDD, SSRIs and astrocytes. In particular, we argue that fluoxetine, the representative SSRI, improves depressive-like behaviours in animals treated with chronic mild stress and reverses depression-associated decrease in astrocytic glial fibrillary acidic protein (GFAP) expression. In addition, fluoxetine upregulates astrocytic mRNA expression of 5-hydroxytriptamin/serotonin2B receptors (5-HT2BR). In summary, we infer that SSRIs exert their anti-depressant effect by regulating several molecular and signalling pathways in astrocytes.

Brain serotonin 2A receptor binding predicts subjective temporal and mystical effects of psilocybin in healthy humans.

BACKGROUND: Psilocybin is a serotonergic psychedelic with psychoactive effects mediated by serotonin 2A receptor (5-HT2AR) activation. It produces an acute psychedelic altered state of consciousness with a unique phenomenology that can be temporally characterized by three intensity phases: onset of psychoactive effect, a peak plateau and return to normal consciousness. AIMS: We evaluated whether pre-drug brain 5-HT2AR binding predicted the three phases of psilocybin subjective drug intensity (SDI) and retrospective self-report of mystical type experiences in healthy individuals. METHOD: Sixteen participants completed a pre-drug [11C]Cimbi-36 positron emission tomography scan to assess 5-HT2AR binding. On a separate day, participants completed a single psilocybin session (oral dose range 0.2-0.3 mg/kg), during which SDI was assessed every 20 min. The Mystical Experience Questionnaire (MEQ) was completed at the end of the session. The three SDI phases were modelled using segmented linear regressions. We evaluated the associations between neocortex 5-HT2AR binding and SDI/MEQ outcomes using linear regression models. RESULTS: Neocortex 5-HT2AR was statistically significantly negatively associated with peak plateau duration and positively with time to return to normal waking consciousness. It was also statistically significantly negatively associated with MEQ total score. CONCLUSION: This is the first study to investigate how individual brain 5-HT2AR binding predicts subjective effects of a single dose of psilocybin. Our findings reinforce the role of cerebral 5-HT2AR in shaping the temporal and mystical features of the psychedelic experience. Future studies should examine whether individual brain levels of 5-HT2AR have an impact on therapeutic outcomes in clinical studies.

Evaluation of the hippocampal immunoreactivity of the serotonin 5-HT1A, 5-HT2 and 5-HT7 receptors in a pilocarpine temporal lobe epilepsy rat model with fast ripples.

Fast ripples (FRs) are found in the hippocampus of epileptic brains, and this fast electrical activity has been described as a biomarker of the epileptogenic process itself. Results from our laboratory, such as the observation of decreased seizure rates and FR incidence at a specific citalopram dose, have suggested that serotonin (5-HT) may play a key role in the FR generation process. Therefore, to gather more details about the state of the serotoninergic system in the hippocampus under an epileptogenic process, we studied the immunoreactivity of three 5-HT receptors (5-HT1A, 5-HT2 and 5-HT7) as well as the extracellular levels of 5-HT in the hippocampal tissue of epileptic rats with FR. Wistar rats (210-300 g) were injected with a single dose of pilocarpine hydrochloride (2.4 mg/2 µl) in the right lateral ventricle and video-monitored 24 h/d to detect spontaneous and recurrent seizures; microelectrodes were implanted in the dentate gyrus (DG) and CA3 and CA1 regions of these rats ipsilateral to the pilocarpine injection site 1 day after the first spontaneous seizure was observed, and only rats who suffered FR events were used in this work. Thirty-three days after the first spontaneous seizure, an immunostaining procedure and high performance liquid chromatography were performed to measure the 5-HT levels. A general depletion of the 5-HT and 5-HIIA levels in hippocampal tissue from epileptic animals compared with those in controls was observed; in addition, a general decrease in immunoreactivity for the three receptors was found, especially in the DG, which may support the establishment of an excitatory/inhibitory imbalance in the trisynaptic circuit that underlies the FR generation process.

Low Basicity as a Characteristic for Atypical Ligands of Serotonin Receptor 5-HT2.

Serotonin receptors are extensively examined by academic and industrial researchers, due to their vital roles, which they play in the organism and constituting therefore important drug targets. Up to very recently, it was assumed that the basic nitrogen in compound structure is a necessary component to make it active within this receptor system. Such nitrogen interacts in its protonated form with the aspartic acid from the third transmembrane helix (D3x32) forming a hydrogen bond tightly fitting the ligand in the protein binding site. However, there are several recent studies that report strong serotonin receptor affinity also for compounds without a basic moiety in their structures. In the study, we carried out a comprehensive in silico analysis of the low-basicity phenomenon of the selected serotonin receptor ligands. We focused on the crystallized representatives of the proteins of 5-HT1B, 5-HT2A, 5-HT2B, and 5-HT2C receptors, and examined the problem both from the ligand- and structure-based perspectives. The study was performed for the native proteins, and for D3x32A mutants. The investigation resulted in the determination of nonstandard structural requirements for activity towards serotonin receptors, which can be used in the design of new nonbasic ligands.

Involvement of 5-HT2 serotonin receptors in cognitive defects induced by aristolochic acid I in mice.

Aristolochic acids (AAs) are a natural bioactive substance found in Chinese herbs, which are widely used for treating diseases. Many studies have demonstrated that AAs have various pharmacological function, while increasing reports indicated its toxicity. However, the role AAs in cognition remains poorly understood. This study explored the neurotoxic effect of aristolochic acid I (AAI), the most toxic component of the AAs family, on hippocampal synaptic plasticity and spatial cognition in mice. C57BL/6 mice were exposed to 5 mg/kg AAI for 4 weeks. After chronic treatment, AAI considerably increased the level of anxiety and the degree of behavioral despair in mice. Working and reference error rates were higher in the AAI exposed mice than in the control. This was further validated by the molecular docking studies, which AAI might interact with 5-HT2 serotonin receptor (5-HT2AR). Mechanism investigation indicated that AAI triggered inflammation in the hippocampus of mice through increasing the activity of Tnf-α-NF-κB-IL-6 signaling pathway. Conclusively, chronic AAI administration causes inflammation, and it possibly also serves as a potential antagonist of 5-HT2AR to influence the cognition function in C57BL/6 mice.

Discovery and SAR studies of 2-alkyl-3-phenyl-2,4,5,6,7,8-hexahydropyrazolo[3,4-d]azepines as 5-HT7/2 inhibitors leading to the identification of a clinical candidate.

We report here the synthesis and characterization of a dual 5-HT7 / 5-HT2 receptor antagonist 3-(4-Fluoro-phenyl)-2-isopropyl-2,4,5,6,7,8-hexahydro-1,2,6-triaza-azulene (4j). 4j is a high affinity 5-HT7 and 5-HT2A receptor ligand having a pKi = 8.1 at both receptors. It behaves as an antagonist in an in vitro functional assay for 5-HT2A and as an inverse agonist in an in vitro functional assay for 5-HT7. In a validated in vivo model for central 5-HT7 activity in rats, blockade of 5-carboxamidotryptamine (5-CT) induced hypothermia, 4j shows efficacy at low doses (ED50 = 0.05 mg/kg, p.o., 1 h) and maximal efficacy was observed at 0.3 mg/kg p.o. with a corresponding plasma concentration of ~27 ng/ml. In a validated in vivo model for central 5-HT2A activity, blockade of 2,5-dimethoxy-4-iodoamphetamine (DOI) induced head-twitches in mice, 4j shows efficacy at low doses with an ED50 = 0.3 mg/kg p.o. Ex vivo receptor binding studies demonstrate that 4j occupied 5-HT2A receptor binding sites in the frontal cortex of the rat brain with an ED50 in good agreement with the ED50 value for central functional effect mediated by 5-HT2A receptor (ED50 = 0.8 mg/kg, p.o., 1 h).

Blocking properties of terguride at the 5-HT2 receptor subtypes mediating cardiovascular responses in the rat.

In vitro studies have suggested that terguride blocks the contractile and relaxant responses produced by 5-hydroxytryptamine (5-HT) via 5-HT2A/2B receptors. This study has now investigated terguride's blocking properties on central/peripheral 5-HT2 receptors in anaesthetized or pithed rats. Male Wistar anaesthetized/pithed rats were cannulated for recording blood pressure and heart rate and for i.v. administration of several compounds. In both groups of rats, i.v. bolus injections of 5-HT or (±)-DOI (a 5-HT2 receptor agonist; 1-1000 µg/kg) produced dose-dependent increases in diastolic blood pressure and heart rate. These responses were dose-dependently antagonized by terguride (10-3000 µg/kg). In anaesthetized rats, i.v. bolus injections of BW723C86 (a 5-HT2B receptor agonist; 1-1000 µg/kg) produced dose-dependent increases in diastolic blood pressure and not dose-dependent increases in heart rate, while in pithed rats, these responses were attenuated. The vasopressor responses elicited by BW723C86 in anaesthetized rats were dose-dependently blocked by terguride (10-300 µg/kg), whereas its the tachycardic responses were dose-independently blocked. These results, taken together, suggest that terguride behaved as an antagonist at the 5-HT2 receptors located in the central nervous system and (or) the systemic vasculature. This is the first evidence demonstrating that terguride can block central/peripheral 5-HT2 receptors mediating cardiovascular responses in anaesthetized or pithed rats.

Antidepressant-like effects of kynurenic acid in a modified forced swim test.

BACKGROUND: Kynurenic acid (KYNA) is an L-tryptophan metabolite with neuromodulatory activities, regulating the release of neurotransmitters such as glutamate, dopamine (DA), and acetylcholine (Ach). Dysregulation of the kynurenine pathway has been associated with neurodegenerative, neurological, and psychological disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease, major depressive disorder, and schizophrenia. METHODS: The antidepressant-like effects of KYNA were studied with a modified mouse forced swimming test (FST), and the potential involvement of the serotonin (SER), norepinephrine, DA, Ach, N-methyl-D-aspartate, or gamma-aminobutyric acid subunit A (GABAA) receptors in its antidepressant-like effect was assayed by modified combination mouse FST. In combination studies, the mice were pretreated with the respective receptor antagonist, cyproheptadine (CPH), phenoxybenzamine, yohimbine, propranolol, haloperidol (HPD), atropine, MK-801, or bicuculline (BCL). RESULTS: The FST revealed that KYNA reversed immobility, climbing, and swimming times, suggesting the antidepressant-like effects of KYNA. Furthermore, the combination studies showed that CPH prevented the antidepressant-like effects of KYNA on immobility, climbing, and swimming times, whereas HPD reduced climbing time and BCL influenced immobility and climbing times and prevented the effects of KYNA on swimming time. CONCLUSIONS: The results demonstrated, for the first time, the presence of antidepressant-like effects of KYNA in a modified mouse FST. Furthermore, modified combination FST showed that the antidepressant-like actions of KYNA strongly interacted with 5-hydroxytryptamine type 2 SER-ergic receptors, weakly interacted with D2, D3, D4 DA-ergic receptors, and interacted moderately with GABAA receptors.

Involvement of monoaminergic targets in the antidepressant- and anxiolytic-like effects of the synthetic alkamide riparin IV: Elucidation of further mechanisms through pharmacological, neurochemistry and computational approaches.

Despite recent advances, current antidepressants have considerable limitations: late onset of action and the high profile of refractoriness. Biomedical research with natural products has gained growing interest in the last years, and had provide useful candidates for new antidepressants. Riparins are a group of natural alkamides obtained from Aniba riparia, which had marked neuroactive effects, mainly as antidepressant and antinociceptive agents. We made modifications of the basic structure of riparins, originating a synthetic alkamide, also known as riparin IV (RipIV). RipIV demonstrated a superior analgesic effect than its congeners and a marked antidepressant-like effect. However, the basic mechanism for the central effects of RipIV remains unknown. Here, we aimed to investigate the participation of monoaminergic neurotransmission targets in the antidepressant-like effects of RipIV. To do this, we applied a combined approach of experimental (classical pharmacology and neurochemistry) and computer-aided techniques. Our results demonstrated that RipIV presented antidepressant- and anxiolytic-like effects without modifying locomotion and motor coordination of mice. Also, RipIV increased brain monoamines and their metabolite levels. At the higher dose (100 mg/kg), RipIV increased serotonin concentrations in all studied brain areas, while at the lower one (50 mg/kg), it increased mainly dopamine and noradrenaline levels. When tested with selective receptor antagonists, RipIV antidepressant effect showed dependence of the activation of multiple targets, including D1 and D2 dopamine receptors, 5-HT2A/2, 5-HT3 receptors and α2 adrenergic receptors. Molecular docking demonstrated favorable binding conformation and affinity of RipIV to monoamine oxidase B (MAO-B), serotonin transporter (SERT), α1 receptor, D2 receptor, dopamine transporter (DAT) and at some extent GABA-A receptor. RipIV also presented a computationally predicted favorable pharmacokinetic profile. Therefore, this study demonstrated the involvement of monoaminergic targets in the mechanism of RipIV antidepressant-like action, and provide evidence of it as a promising new antidepressant.

Radiosynthesis and preclinical evaluation of [11 C]Cimbi-701 - Towards the imaging of cerebral 5-HT7 receptors.

The serotonin 7 (5-HT7 ) receptor is suggested to be involved in a broad variety of CNS disorders, but very few in vivo tools exist to study this important target. Molecular imaging with positron emission tomography (PET) would enable an in vivo characterization of the 5-HT7 receptor. However, no clinical PET radiotracer exists for this receptor, and thus we aimed to develop such a tracer. In this study, we present the preclinical evaluation of [11 C]Cimbi-701. Cimbi-701 was synthesized in a one-step procedure starting from SB-269970. Its selectivity profile was determined using an academic screening platform (NIMH Psychoactive Drug Screening Program). Successful radiolabeling of [11 C]Cimbi-701 and subsequent in vivo evaluation was conducted in rats, pigs and baboon. In vivo specificity was investigated by 5-HT7 and σ receptor blocking studies. P-gp efflux transporter dependency was investigated using elacridar. [11 C]Cimbi-701 could successfully be synthesized. Selectivity profiling revealed high affinity for the 5-HT7 (Ki = 18 nM), σ-1 (Ki = 9.2 nM) and σ-2 (Ki = 1.6 nM) receptors. In rats, [11 C]Cimbi-701 acted as a strong P-gp substrate. After P-gp inhibition, rat brain uptake could specifically be blocked by 5-HT7 and σ receptor ligands. In pig, high brain uptake and specific 5-HT7 and σ-receptor binding was found for [11 C]Cimbi-701 without P-gp inhibition. Finally, low brain uptake was found in baboons. Both the specific σ-receptor binding and the low brain uptake of [11 C]Cimbi-701 displayed in baboon discouraged further translation to humans. Instead, we suggest exploration of this structural class as results indicate that selective 5-HT7 receptor imaging might be possible when more selective non-P-gp substrates are identified.

G protein-coupled receptor binding and pharmacological evaluation of indole-derived thiourea compounds.

Four 2-(1H-indol-3-yl)ethylthiourea derivatives were prepared by condensation of 2-(1H-indol-3-yl)ethanamine with the corresponding aryl/alkylisothiocyanates in a medium-polarity solvent. Their structures were confirmed by spectral techniques, and the molecular structure of 3 was determined by X-ray crystal analysis. For all derivatives, the binding affinities at the 5-HT2A and 5-HT2C receptors, as well as their functional activities at the 5-HT1A and D2 receptors, were determined. The arylthioureas 1 and 4 were the most active at the 5-HT1A receptor, showing, at the same time, significant selectivity over the studied 5-HT2 and D2 receptor subtypes. The compounds were tested for their pharmacological activities within the central nervous system in relevant mouse models. The involvement of the serotonergic system in the activity of 1 and 4 was indicated. The antinociceptive action of 4 was linked to its anti-inflammatory activity.

5-HT2 receptor activation alleviates airway inflammation and structural remodeling in a chronic mouse asthma model.

AIMS: Although the bulk of research into the biology of serotonin 5-HT2A receptors has focused on its role in the CNS, selective activation of these receptors in peripheral tissues can produce profound anti-inflammatory effects. We previously demonstrated that the small molecule 5-HT2 receptor agonist (R)-2,5-dimethoxy-4-iodoamphetamine [(R)-DOI] inhibits TNF-α-mediated proinflammatory signaling cascades and inflammation via 5-HT2A receptor activation and prevents the development of, and inflammation associated with, acute allergic asthma in a mouse ovalbumin (OVA) model. Here, we investigated the ability of (R)-DOI to reverse inflammation and symptoms associated with established asthma in a newly developed model of chronic asthma. METHODS: An 18-week ovalbumin challenge period was performed to generate persistent, chronic asthma in BALB/c mice. Four once daily intranasal treatments of (R)-DOI were administered one week after allergen cessation, with respiratory parameters being measured by whole-body plethysmography (WBP). Cytokine and chemokine levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR) in homogenized lung tissue, bronchoalveolar (BALF) fluid was analyzed for chemokine modulation by multiplex assays, and Periodic Acid-Schiff and Masson's Trichrome staining was performed to determine goblet cell infiltration and overall changes to lung morphology. KEY FINDINGS: 5-HT2 activation via (R)-DOI attenuates elevated airway hyperresponsiveness to methacholine, reduces pulmonary inflammation and mucus production, and reduces airway structural remodeling and collagen deposition by nearly 70%. SIGNIFICANCE: Overall, these data provide support for the therapeutic potential of (R)-DOI and 5-HT2 receptor activation for the treatment of asthma, and identifies (R)-DOI as a novel therapeutic compound against pulmonary fibrosis.

Adaptation in 5-HT2 receptors-CaMKII signaling in lateral habenula underlies increased nociceptive-sensitivity in ethanol-withdrawn rats.

Alcoholics often experience hyperalgesia, especially during abstinence, yet the underlying cellular and molecular bases are unclear. Recent evidence suggests that 5-HT type 2 receptors (5-HT2Rs) at glutamatergic synapses on lateral habenula (LHb) neurons may play a critical role. We, therefore, measured paw withdrawal responses to thermal and mechanical stimuli, and alcohol intake in a rat model of intermittent drinking paradigm, as well as spontaneous glutamatergic transmission (sEPSCs), and firing of LHb neurons in brain slices. Here, we report that nociceptive sensitivity was higher in rats at 24 h withdrawal from chronic alcohol consumption than that of alcohol-naive counterparts. The basal frequency of sEPSCs and firings was higher in slices of withdrawn rats than that of Naïve rats, and 5-HT2R antagonists attenuated the enhancement. Also, an acute ethanol-induced increase of sEPSCs and firings was smaller in withdrawal than in Naïve rats; it was attenuated by 5-HT2R antagonists but mimicked by 5-HT2R agonists. Importantly, intra-LHb infusion of 5-HT2R agonists increased nociceptive sensitivity in Naïve rats, while antagonists or 5-HT reuptake blocker decreased nociceptive sensitivity and alcohol intake in withdrawn rats. Additionally, KN-62, a CaMKII inhibitor, attenuated the enhancement of EPSCs and firing induced by acute alcohol and by 5-HT2R agonist. Furthermore, intra-LHb KN-62 reduced nociceptive sensitivity and alcohol intake. Quantitative real-time PCR assay detected mRNA of 5-HT2A and 2C in the LHb. Thus adaptation in 5-HT2R-CaMKII signaling pathway contributes to the hyper-glutamatergic state, the hyperactivity of LHb neurons as well as the higher nociceptive sensitivity in rats withdrawn from chronic alcohol consumption.

Differences between young and aged rats in voiding frequency and detrusor muscle serotonergic contraction.

INTRODUCTION: The involvement of serotonin (5-HT) in increased lower urinary tract symptoms in aging is unclear. We sought to compare voiding function and 5-HT induced detrusor contraction between young and aged rats. METHODS: This study used young (2- to 3-month-old) and aged (26- to 30-month-old) male Fischer 344 rats. 1. Rats were housed in individual metabolic cages, and then the total volume of urination, volume per micturition, voiding frequency, and voiding interval were analyzed. 2. Using urinary bladder body strips, developed tension was recorded after cumulative addition of 5-HT (1-100 nM) in the absence or presence of tetrodotoxin (1 µM), and in the presence of tetrodotoxin with ketanserin (0.3-3 µM) or naftopidil (1 and 3 µM). We examined the effects of atropine, ketanserin, and naftopidil on electrical field stimulation (EFS)-induced contraction. RESULTS: 1. Compared to young rats, aged rats exhibited decreased voiding frequency and increased volume per micturition, but total volume of urination (normalized to body weight) did not differ. Moreover, voiding interval was significantly prolonged in aged rats during the active period. 2. In the presence of tetrodotoxin, pEC50 of 5-HT were significantly lower in aged rats than in young rats (P < 0.01), but the maximal response to 5-HT was not altered in the aged bladder. Ketanserin inhibited 5-HT-induced contraction in both groups, while suppression by naftopidil was relatively limited, especially in aged rats. EFS induced neurogenic contraction in a frequency-dependent manner. Atropine-resistant contraction was not inhibited by naftopidil, but was potentiated by ketanserin. CONCLUSIONS: Urination intervals were extended in aged rats, indicating that urination rhythm changed. In the senescent rat bladder, 5-HT induced detrusor contraction, but the effect of 5-HT and the naftopidil-sensitive contractile force were weaker than those in young rats. Additionally, 5-HT did not contribute to the increase in atropine-resistant EFS-induced contractions in aged rats.

Differential serotonin transporter (5-HTT) and 5-HT2 receptor density in limbic and neocortical areas of adults and children with autism spectrum disorders: implications for selective serotonin reuptake inhibitor efficacy.

As selective serotonin reuptake inhibitors (SSRIs) are among the most commonly prescribed medications in autism, we aimed to determine whether targets for SSRIs are differentially affected in three cortical areas in children and adults with autism compared to neurotypical individuals. Utilizing a large cohort of postmortem brain tissue (n = 14-19 per group), saturation ligand binding assays were conducted on sections from the anterior cingulate cortex (ACC), posterior cingulate cortex, and fusiform gyrus (FG). Specific binding to the 5-HT transporter (5-HTT) as well as to 5-HT2 and 1A receptors (5-HT2, 5-HT1A ) was quantified in superficial and deep layers of each region using the ligands [3 H]-citalopram (5-HTT), [3 H]-ketanserin (5-HT2 ), and [3 H]-8-OH-DPAT (5-HT1A ). A Welch's t-test was utilized to compare receptor densities (Bmax ), revealing a statistically significant decrease in 5-HTT within the ACC of the entire autism cohort. There was also a decrease in 5-HT2 receptor density in the ACC in the adult cohort, but not in child postmortem autism cases as compared to controls. Comparing linear regression lines of Bmax values plotted against age, shows a significantly lower intercept for 5-HTT in autism (p = 0.025). 5-HT2 density increases with age in control cases, whereas in autism there is a decrease with age and significantly different slopes between regression lines (p = 0.032). This suggests a deficit in 5-HTT within the ACC in individuals with autism, while decreases in 5-HT2 density are age-dependent. There were no differences in receptor densities in the posterior cingulate cortex or FG in autism and no differences in ligand affinity (KD ) across all regions and ligands examined.

Genetic and Functional Dissection of the Role of Individual 5-HT2 Receptors as Entry Receptors for JC Polyomavirus.

JC polyomavirus (JCPyV) is a ubiquitous human pathogen that causes progressive multifocal leukoencephalopathy (PML). The entry receptors for JCPyV belong to the 5-hydroxytryptamine 2 receptor (5-HT2R) family, but how individual members of the family function to facilitate infection is not known. We used proximity ligation assay (PLA) to determine that JCPyV interacts with each of the 5-HT2 receptors (5-HT2Rs) in a narrow window of time during entry. We used CRISPR-Cas9 to randomly introduce stop codons in the gene for each receptor and discovered that the second intracellular loop of each was necessary for infection. This loop contains a motif possibly involved in receptor internalization by ß-arrestin. Mutation of this motif and small interfering RNA (siRNA) knockdown of ß-arrestin recapitulated the results of our CRISPR-Cas9 screen, showing that this motif is critical. Our results have implications for the role these receptors play in virus infection and for their normal functioning as receptors for serotonin.