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.

Antagonism of 5-HT2 receptors attenuates self-sustained firing of human motor units.

5-HT2 receptors on motoneurones play a critical role in facilitating persistent inward currents (PICs). Although facilitation of PICs can enhance self-sustained firing after periods of excitation, the relationship between 5-HT2 receptor activity and self-sustained firing in human motor units (MUs) has not been resolved. MU activity was assessed from the tibialis anterior of 10 healthy adults (24.9 ± 2.8 years) during two contraction protocols. Both protocols featured steady-state isometric contractions with constant descending drive to the motoneurone pool. However, one protocol also included an additional phase of superimposed descending drive. Adding and then removing descending drive in the middle of steady-state contractions altered MU firing behaviour across the motor pool, where newly recruited units in the superimposed phase were unable to switch off (P = 0.0002), and units recruited prior to additional descending drive reduced their discharge rates (P < 0.0001, difference in estimated marginal means (∆) = 2.24 pulses/s). The 5-HT2 receptor antagonist, cyproheptadine, was then administered to determine whether changes in MU firing were mediated by serotonergic mechanisms. 5-HT2 receptor antagonism caused reductions in MU discharge rate (P < 0.001, ∆ = 1.65 pulses/s), recruitment threshold (P = 0.00112, ∆ = 1.09% maximal voluntary contraction) and self-sustained firing duration (P < 0.0001, ∆ = 1.77s) after the additional descending drive was removed in the middle of the steady-state contraction. These findings indicate that serotonergic neuromodulation plays a key role in facilitating discharge and self-sustained firing of human motoneurones, where adaptive changes in MU recruitment must occur to meet the demands of the contraction. KEY POINTS: Animal and cellular preparations indicate that somato-dendritic 5-HT2 receptors regulate the intrinsic excitability of motoneurones. 5-HT2 receptor antagonism reduces estimates of persistent inward currents in motoneurones, which contribute to self-sustained firing when synaptic inputs are reduced or removed. This human study employed a contraction task that slowly increased (and then removed) the additional descending drive in the middle of a steady-state contraction where marked self-sustained firing occurred when the descending drive was removed. 5-HT2 receptor antagonism caused widespread reductions in motor unit (MU) discharge rates during contractions, which was accompanied by reduced recruitment threshold and attenuation of self-sustained firing duration after the removal of the additional descending drive to motoneurones. These findings support the role that serotonergic neuromodulation is a key facilitator of MU discharge and self-sustained firing of human motoneurones, where adaptative changes in MU recruitment must occur to meet the demands of the contraction.

Excitatory drive to spinal motoneurones is necessary for serotonin to modulate motoneurone excitability via 5-HT2 receptors in humans.

Serotonin modulates corticospinal excitability, motoneurone firing rates and contractile strength via 5-HT2 receptors. However, the effects of these receptors on cortical and motoneurone excitability during voluntary contractions have not been explored in humans. Therefore, the purpose of this study was to investigate how 5-HT2 antagonism affects corticospinal and motoneuronal excitability with and without descending drive to motoneurones. Twelve individuals (aged 24 ± 4 years) participated in a double-blind, placebo-controlled, crossover study, whereby the 5-HT2 antagonist cyproheptadine was administered. Transcranial magnetic stimulation (TMS) was delivered to the motor cortex to produce motor evoked potentials (MEPs), and electrical stimulation at the cervicomedullary junction was used to generate cervicomedullary motor evoked potentials (CMEPs) in the biceps brachii at rest and during a range of submaximal elbow flexions. Evoked potentials were also obtained after a conditioning TMS pulse to produce conditioned MEPs and CMEPs (100 ms inter-stimulus interval). 5-HT2 antagonism reduced maximal torque (p < 0.001), and compared to placebo, reduced unconditioned MEP amplitude at rest (p = 0.003), conditioned MEP amplitude at rest (p = 0.033) and conditioned MEP amplitude during contractions (p = 0.020). 5-HT2 antagonism also increased unconditioned CMEP amplitude during voluntary contractions (p = 0.041) but not at rest. Although 5-HT2 antagonism increased long-interval intracortical inhibition, net corticospinal excitability was unaffected during voluntary contractions. Given that spinal motoneurone excitability was only affected when descending drive to motoneurones was present, the current study indicates that excitatory drive is necessary for 5-HT2 receptors to regulate motoneurone excitability but not intracortical circuits.

Blockade of 5-HT2 receptors suppresses motor unit firing and estimates of persistent inward currents during voluntary muscle contraction in humans.

Serotonergic neuromodulation contributes to enhanced voluntary muscle activation. However, it is not known how the likely motoneurone receptor candidate (5-HT2 ) influences the firing rate and activation threshold of motor units (MUs) in humans. The purpose of this study was to determine whether 5-HT2 receptor activity contributes to human MU behaviour during voluntary ramped contractions of differing intensity. High-density surface EMG (HDsEMG) of the tibialis anterior was assessed during ramped isometric dorsiflexions at 10, 30, 50 and 70% of maximal voluntary contraction (MVC). MU characteristics were successfully extracted from HDsEMG of 11 young adults (four female) pre- and post-ingestion of 8 mg cyproheptadine or a placebo. Antagonism of 5-HT2 receptors caused a reduction in MU discharge rate during steady-state muscle activation that was independent of the level of contraction intensity [P < 0.001; estimated mean difference (∆) = 1.06 pulses/s], in addition to an increase in MU derecruitment threshold (P < 0.013, ∆ = 1.23% MVC), without a change in force during MVC (P = 0.652). A reduction in estimates of persistent inward current amplitude was observed at 10% MVC (P < 0.001, ∆ = 0.99 Hz) and 30% MVC (P = 0.003, ∆ = 0.75 Hz) that aligned with 5-HT changes in MU firing behaviour attributable to 5-HT2 antagonism. Overall, these findings indicate that 5-HT2 receptor activity has a role in regulating the discharge rate in populations of spinal motoneurones when performing voluntary contractions. This study provides evidence of a direct link between MU discharge properties, persistent inward current activity and 5-HT2 receptor activity in humans. KEY POINTS: Activation of 5-HT receptors on the soma and dendrites of motoneurones regulates their excitability. Previous work using chlorpromazine and cyproheptadine has demonstrated that the 5-HT2 receptor regulates motoneurone activity in humans with chronic spinal cord injury and non-injured control subjects. It is not known how the 5-HT2 receptor directly influences motor unit (MU) discharge and MU recruitment in larger populations of human motoneurones during voluntary contractions of differing intensity. Despite the absence of change in force during maximal voluntary dorsiflexions, 5-HT2 receptor antagonism caused a reduction in MU discharge rate during submaximal steady-state muscle contraction, in addition to an increase in MU derecruitment threshold, irrespective of the submaximal contraction intensity. Reductions in estimates of persistent inward currents after 5-HT2 receptor antagonism support the viewpoint that the 5-HT2 receptor plays a crucial role in regulating motor activity, whereby a persistent inward current-based mechanism is involved in regulating the excitability of human motoneurones.

Role of 5-HT2 receptors family in the allergy-induced increased aorta contractile responses to 5-HT.

Asthma poses an increased risk for cardiovascular disorders, suggesting that allergy, which is an underlying process in asthma, causes atypical functioning of organs other than lungs. In a previous study in a guinea pig asthma model, we concluded that allergic sensitization increased aorta contractile responses to 5-HT. To further characterize these responses, here we explored the role of the 5-HT2 receptors family. We found that TCB-2 (5-HT2A agonist) and WAY161503 (5-HT2C agonist) induced aorta contractions resembling those elicited by 5-HT but less intense (~43 % and ~25 %, respectively). In these experiments, aortas from sensitized guinea pigs showed increased contractions to TCB-2, but not to WAY161503. In turn, MDL 100907 (5-HT2A antagonist) and RS-102221 (5-HT2C antagonist) caused a notably and a mild reduction of the 5-HT-induced contractions, respectively, with no differences seen between sensitized and non-sensitized tissues. BW723C86 (5-HT2B agonist) did not induce contractile responses and RS-127445 (5-HT2B antagonist) did not modify the contractile responses to 5-HT. In non-sensitized aortas, the pattern of protein expression of receptors was 5HT2B>5-HT2A=5-HT2C, which did not change in sensitized animals. In conclusion, we found that allergic sensitization increased the aorta contractile responses to 5-HT, partly mediated by enhanced responses of 5-HT2A receptors, which was unrelated to changes in the expression of these receptors.

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.

Transcriptomics-informed large-scale cortical model captures topography of pharmacological neuroimaging effects of LSD.

Psychoactive drugs can transiently perturb brain physiology while preserving brain structure. The role of physiological state in shaping neural function can therefore be investigated through neuroimaging of pharmacologically induced effects. Previously, using pharmacological neuroimaging, we found that neural and experiential effects of lysergic acid diethylamide (LSD) are attributable to agonism of the serotonin-2A receptor (Preller et al., 2018). Here, we integrate brain-wide transcriptomics with biophysically based circuit modeling to simulate acute neuromodulatory effects of LSD on human cortical large-scale spatiotemporal dynamics. Our model captures the inter-areal topography of LSD-induced changes in cortical blood oxygen level-dependent (BOLD) functional connectivity. These findings suggest that serotonin-2A-mediated modulation of pyramidal-neuronal gain is a circuit mechanism through which LSD alters cortical functional topography. Individual-subject model fitting captures patterns of individual neural differences in pharmacological response related to altered states of consciousness. This work establishes a framework for linking molecular-level manipulations to systems-level functional alterations, with implications for precision medicine.

Harnessing psilocybin: antidepressant-like behavioral and synaptic actions of psilocybin are independent of 5-HT2R activation in mice.

Depression is a widespread and devastating mental illness and the search for rapid-acting antidepressants remains critical. There is now exciting evidence that the psychedelic compound psilocybin produces not only powerful alterations of consciousness, but also rapid and persistent antidepressant effects. How psilocybin exerts its therapeutic actions is not known, but it is widely presumed that these actions require altered consciousness, which is known to be dependent on serotonin 2A receptor (5-HT2AR) activation. This hypothesis has never been tested, however. We therefore asked whether psilocybin would exert antidepressant-like responses in mice and, if so, whether these responses required 5-HT2AR activation. Using chronically stressed male mice, we observed that a single injection of psilocybin reversed anhedonic responses assessed with the sucrose preference and female urine preference tests. The antianhedonic response to psilocybin was accompanied by a strengthening of excitatory synapses in the hippocampus-a characteristic of traditional and fast-acting antidepressants. Neither behavioral nor electrophysiological responses to psilocybin were prevented by pretreatment with the 5-HT2A/2C antagonist ketanserin, despite positive evidence of ketanserin's efficacy. We conclude that psilocybin's mechanism of antidepressant action can be studied in animal models and suggest that altered perception may not be required for its antidepressant effects. We further suggest that a 5-HT2AR-independent restoration of synaptic strength in cortico-mesolimbic reward circuits may contribute to its antidepressant action. The possibility of combining psychedelic compounds and a 5-HT2AR antagonist offers a potential means to increase their acceptance and clinical utility and should be studied in human depression.

Serotonin 2 Receptors, Agomelatine, and Behavioral and Psychological Symptoms of Dementia in Alzheimer's Disease.

There are nearly 50 million Alzheimer's disease (AD) patients worldwide, 90% of whom develop behavioral and psychological symptoms of dementia (BPSD), which increase the mortality rate of patients, and impose an economic and care burden on families and society. As a neurotransmitter and neuromodulator, serotonin is involved in the regulation of psychoemotional, sleep, and feeding functions. Accumulating data support the importance of serotonin in the occurrence and development of BPSD. Studies have shown that reduction of serotonin receptors can increase depression and mental symptoms in AD patients. At present, there is no drug treatment for AD approved by the US Food and Drug Administration. Among them, agomelatine, as a new type of antidepressant, can act on serotonin 2 receptors to improve symptoms such as depression and anxiety. At present, research on BPSD is still in the preliminary exploratory stage, and there are still a lot of unknowns. This review summarizes the relationship between serotonin 2 receptors, agomelatine, and BPSD. It provides a new idea for the study of the pathogenesis and treatment of BPSD.

Receptor-mediated biological effects of extracts obtained from three Asplenium species.

This study reports the effects of aqueous extracts obtained from three fern species of Bulgarian origin: Asplenium ceterach L., Asplenium scolopendrium L., and Asplenium trichomanes L. on the contractility and bioelectrogenesis of rat gastric smooth muscle tissues. In the concentration range 0.015-0.150 mg/mL the three extracts contracted smooth muscle tissues in a concentration-dependent manner. The contractions caused by A. ceterach L. and A. scolopendrium L. extracts (0.150 mg/mL) were reduced by ketanserin (5 × 10-7 and 5 × 10-6 mol/L), an antagonist of serotonin 5-HT2 receptor. The contraction evoked by A. trichomanes L. (0.150 mg/mL) was significantly reduced by 1 × 10-6 mol/L atropine, an antagonist of muscarinic receptors, and turned into relaxation against the background of 3 × 10-7 mol/L galantamine. After combined pretreatment with galantamine and l-arginine (5 × 10-4 mol/L), this relaxation become more pronounced. The study demonstrates that constituents of A. ceterach L. and A. scolopendrium L. extracts act as agonists of 5-HT2 receptors and cause contraction by activating serotonergic signaling system. A. trichomanes L.-induced reaction is an additive result of two opposite-in-character effects. The dominant contraction is initiated by inhibition of acetylcholinesterase activity. The relaxation develops with pre-inhibited acetylcholinesterase, it is significantly potentiated by l-arginine, and therefore associated with nitrergic signaling pathway.

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.

Serotonin functions as a bidirectional guidance molecule regulating growth cone motility.

The neurotransmitter serotonin has been implicated in a range of complex neurological disorders linked to alterations of neuronal circuitry. Serotonin is synthesized in the developing brain before most neuronal circuits become fully functional, suggesting that serotonin might play a distinct regulatory role in shaping circuits prior to its function as a classical neurotransmitter. In this study, we asked if serotonin acts as a guidance cue by examining how serotonin alters growth cone motility of rodent sensory neurons in vitro. Using a growth cone motility assay, we found that serotonin acted as both an attractive and repulsive guidance cue through a narrow concentration range. Extracellular gradients of 50 µM serotonin elicited attraction, mediated by the serotonin 5-HT2a receptor while 100 µM serotonin elicited repulsion mediated by the 5-HT1b receptor. Importantly, high resolution imaging of growth cones indicated that these receptors signalled through their canonical pathways of endoplasmic reticulum-mediated calcium release and cAMP depletion, respectively. This novel characterisation of growth cone motility in response to serotonin gradients provides compelling evidence that secreted serotonin acts at the molecular level as an axon guidance cue to shape neuronal circuit formation during development.

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).

Serotonergic modulation of visual neurons in Drosophila melanogaster.

Sensory systems rely on neuromodulators, such as serotonin, to provide flexibility for information processing as stimuli vary, such as light intensity throughout the day. Serotonergic neurons broadly innervate the optic ganglia of Drosophila melanogaster, a widely used model for studying vision. It remains unclear whether serotonin modulates the physiology of interneurons in the optic ganglia. To address this question, we first mapped the expression patterns of serotonin receptors in the visual system, focusing on a subset of cells with processes in the first optic ganglion, the lamina. Serotonin receptor expression was found in several types of columnar cells in the lamina including 5-HT2B in lamina monopolar cell L2, required for spatiotemporal luminance contrast, and both 5-HT1A and 5-HT1B in T1 cells, whose function is unknown. Subcellular mapping with GFP-tagged 5-HT2B and 5-HT1A constructs indicated that these receptors localize to layer M2 of the medulla, proximal to serotonergic boutons, suggesting that the medulla neuropil is the primary site of serotonergic regulation for these neurons. Exogenous serotonin increased basal intracellular calcium in L2 terminals in layer M2 and modestly decreased the duration of visually induced calcium transients in L2 neurons following repeated dark flashes, but otherwise did not alter the calcium transients. Flies without functional 5-HT2B failed to show an increase in basal calcium in response to serotonin. 5-HT2B mutants also failed to show a change in amplitude in their response to repeated light flashes but other calcium transient parameters were relatively unaffected. While we did not detect serotonin receptor expression in L1 neurons, they, like L2, underwent serotonin-induced changes in basal calcium, presumably via interactions with other cells. These data demonstrate that serotonin modulates the physiology of interneurons involved in early visual processing in Drosophila.