Procognitive 5-HT6 antagonists in the rat forced swimming test: Potential therapeutic utility in mood disorders associated with Alzheimer's disease

Aims5-HT₆ receptor subtype is predominantly expressed in the brain, and preclinical evidence suggests its potential role in the cognitive function. Brain microdialysis studies demonstrated that 5-HT₆ antagonists enhance not only cholinergic but also monoaminergic neurotransmission, a property that may differentiate from acetylcholine esterase (AChE) inhibitors such as donepezil. In this study we compared the antidepressant-like effects of 5-HT₆ antagonists with donepezil to determine whether their different effects on monoamines are behaviorally relevant.Main methodsSelective 5-HT₆ antagonists (SB-399885 and SB-271046) and donepezil were evaluated in the rat forced swimming test since this is known to identify drugs such as antidepressants which can increase brain monoamine levels. Binding assay was undertaken by using [¹²⁵I]SB-258585 to measure brain 5-HT₆ receptor occupancy.Key findingsSystemic administration of SB-399885 (3 and 10 mg/kg, i.p.) and SB-271046 (10 and 30 mg/kg, i.p.) produced a significant reduction of immobility time in the rat forced swimming test with a similar profile in terms of 5-HT₆ receptor occupancy (62 and 96% for 3 and 10 mg/kg SB-399885 respectively; 56 and 84% for 10 and 30 mg/kg SB-271046 respectively). In contrast, donepezil (0.5 and 1 mg/kg i.p.) did not show any effects in this model.SignificanceThese data suggest that 5-HT₆ antagonists, at doses corresponding to those occupy central 5-HT₆ receptors, could have an antidepressive effect in humans. This may differentiate 5-HT₆ antagonists from AChE inhibitors with respect to the mood control in the symptomatic treatment of Alzheimer's disease.     

Characteristics of [14C]Guanidinium Accumulation in NG 108-15 Cell Exposed to Serotonin 5-HT3 Receptor Ligands and Substance P

Abstract: In the presence of substance P (SP; 10 μM), serotonin (5-HT; 1 μM) triggered a cation permeability in cells of the hybridoma (mouse neuroblastoma X rat glioma) clone NG 108-15 that could be assessed by measuring the cell capacity to accumulate [¹⁴C]guanidinium for 10-15 min at 37°C. In addition to 5-HT (EC₅₀, 0.33 μM), the potent 5-HT₃ receptor agonists 2-methyl-serotonin, phenylbiguanide, and m-chlorophenylbiguanide, and quipazine, markedly increased [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 10 μM SP. In contrast, 5-HT₃ receptor antagonists prevented the effect of 5-HT. The correlation (r= 0.97) between the potencies of 16 different ligands to mimic or prevent the effects of 5-HT on [¹⁴C]guanidinium uptake, on the one hand, and to displace [³H]zacopride specifically bound to 5-HT₃ receptors on NG 108-15 cells, on the other hand, clearly demonstrated that [¹⁴C]guanidinium uptake was directly controlled by 5-HT₃ receptors. Various compounds such as inorganic cations (La³⁺, Mn²⁺, Ba²⁺, Ni²⁺, and Zn²⁺), D-tubocurarine, and memantine inhibited [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 5-HT and SP, as expected from their noncompetitive antagonistic properties at 5-HT₃ receptors. However, ethanol (100 mM), which has been reported to potentiate the electrophysiological response to 5-HT₃ receptor stimulation, prevented the effects of 5-HT plus SP on [¹⁴C]guanidinium uptake. The cooperative effect of SP on this 5-HT₃-evoked response resulted neither from an interaction of the peptide with the 5-HT₃ receptor binding site nor from a possible direct activation of G proteins in NG 108-15 cells. Among SP derivatives, [D-Pro⁹]SP, a compound inactive at the various neurokinin receptor classes, was the most potent to mimic the stimulatory effect of SP on [¹⁴C]guanidinium uptake in NG 108-15 cells exposed to 5-HT. Although the cellular mechanisms involved deserve further investigations, the 5-HT-evoked [¹⁴C]guanidinium uptake appears to be a rapid and reliable response for assessing the functional state of 5-HT₃ receptors in NG 108-15 cells.     

Hippocampal Serotonin 5-HT1A Receptor Enhances Acetylcholine Release in Conscious Rats

Abstract: We investigated changes in the extracellular levels of acetylcholine (ACh) following local application of serotonergic agents to the dorsal hippocampus of freely moving rats by means of perfusion using a microdialysis technique. Perfusion of serotonin (5-HT; 10 μM, for 30 min at a rate of 3 μl/min), dissolved in Ringer's solution containing 10 μM eserine, showed no marked effect on the extracellular levels of ACh. 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 20 μM), a 5-HT_1A agonist, increased ACh levels, whereas 7-trifluoromethyl-4-(4-methyl-1 -piperazinyl)-pymoto[1,2-a]quinoxaline (CGS-12066B; 100 μM), a 5-HT_1B agonist, decreased it. Clomipramine (2 μM), an uptake inhibitor of 5-HT, had no effect on ACh levels. Following perfusion of 1-(2-methoxyphenyl)-4-[4- (2-phthalimido)butyl]piperazine (NAN-190; 10 μM), which is a selective 5-HT_1A antagonist, the effect of 8-OH-DPAT was totally abolished, whereas CGS-12066B decreased extracellular ACh levels. 5-HT, as well as Clomipramine, had a decreasing effect on ACh levels after pretreatment with NAN-190. These results indicate that the 5-HT_1A receptor, which exists in the dorsal hippocampus, enhances the spontaneous ACh release, and that the mechanism of serotonergic modulation of ACh release partly depends on both the stimulatory control via the 5-HT_1A receptor and the suppressive one via the 5-HT_1B receptor in the dorsal hippocampus of rats.     

The Serotonin 5-HT2C Receptor Is a Prominent Serotonin Receptor in Basal Ganglia: Evidence from Functional Studies on Serotonin-Mediated Phosphoinositide Hydrolysis

Abstract: Serotonin (5-hydroxytryptamine; 5-HT) 5-HT_2A and 5-HT_2C receptors belong to the class of phosphoinositide-specific phospholipase C (PLC)-linked receptors. Conditions were established for measuring 5-HT_2A-linked and 5-HT_2C-linked PLC activity in membranes prepared from previously frozen rat frontal cortex and caudate. In the presence of Ca²⁺ (300 nM) and GTPγS (1 µM), 5-HT increased PLC activity in caudate membranes. Pharmacological analysis using the selective 5-HT_2A antagonist, spiperone, and the nonselective 5-HT_2A/2C antagonist, mianserin, demonstrated that over half of the 5-HT-stimulated PLC activity was due to stimulation of 5-HT_2C receptors as opposed to 5-HT_2A receptors. Radioligand binding assays with [³H]RP 62203 and [³H]-mesulergine were used to quantify 5-HT_2A and 5-HT_2C sites, respectively, in caudate. From these data, the B_max for caudate 5-HT_2A sites and 5-HT_2C sites was 165.4 ± 9.7 fmol/mg of protein and 49.7 ± 3.3 fmol/mg of protein, respectively. In contrast to that in caudate, PLC activity in frontal cortex was stimulated by 5-HT in a manner that was inhibited by the 5-HT_2A-selective antagonists, spiperone and ketanserin. Taken together, the results indicate that 5-HT_2A- and 5-HT_2C-linked PLC activity can be discerned in brain regions possessing both receptor subtypes using membranes prepared from previously frozen tissue. More importantly, significant 5-HT_2C-mediated phosphoinositide hydrolysis was observed in caudate, despite the relatively low density of 5-HT_2C sites. The significance of these observations with respect to the physiological function of 5-HT_2C receptors is discussed.     

Alterations in hippocampal serotonergic and INSR function in streptozotocin induced diabetic rats exposed to stress: neuroprotective role of pyridoxine and <Emphasis Type="Italic">Aegle marmelose</Emphasis>

Diabetes and stress stimulate hippocampal 5-HT synthesis, metabolism and release. The present study was carried out to find the effects of insulin, Aegle marmelose alone and in combination with pyridoxine on the hippocampal 5-HT, 5-HT_2A receptor subtype, gene expression studies on 5-HT_2A, 5-HTT, INSR, immunohistochemical studies and elevated plus maze in streptozotocin induced diabetic rats. 5-HT content showed a significant decrease (p < 0.001) and a significant increase (p < 0.001) in 5-HIAA in hippocampus of diabetic rats compared to control. 5-HT receptor binding parameters B_max and K_d showed a significant decrease (p < 0.001) whereas 5-HT_2A receptor binding parameters B_max showed a significant decrease (p < 0.001) with a significant increase (p < 0.05) in K_d in hippocampus of diabetic rats compared to control. Gene expression studies of 5-HT_2A, 5-HTT and INSR in hippocampus showed a significant down regulation (p < 0.001) in diabetic rats compared to control. Pyridoxine treated in combination with insulin and A. marmelose to diabetic rats reversed the 5-HT content, B_max , K_d of 5-HT, 5-HT_2A and gene expression of 5-HT_2A, 5-HTT and INSR in hippocampus to near control. The gene expression of 5-HT_2A and 5-HTT were confirmed by immunohistochemical studies. Behavioural studies using elevated plus maze showed that serotonin through its transporter significantly increased (p < 0.001) anxiety-related traits in diabetic rats which were corrected by combination therapy. Our results suggest that pyridoxine treated in combination with insulin and A. marmelose has a role in the regulation of insulin synthesis and release, normalising diabetic related stress and anxiety through hippocampal serotonergic function. This has clinical significance in the management of diabetes.     

Mediation of serotonin-induced hyperventilation via 5-HT3-receptor in European eel Anguilla anguilla

The effects of serotonin (5-hydroxytryptamine) on ventilation were investigated by continuous measurements of intrabuccal pressure in unrestrained eel. Intravenous administration of 5-hydroxytryptamine (30 g·kg^-1) caused a large increase in ventilatory frequency (+100%) and amplitude (+140%). The 5-hydroxytryptamine-induced hyperventilation was blocked by the 5-HT₃-receptor antagonists metoclopramide (1.0 mg·kg^-1) or MDL72222 (1.0 mg·kg^-1), and was insensitive to the 5-HT_1/2-receptor antagonist methysergide (3.0 mg·kg^-1) and to the 5-HT₄-receptor antagonist DAU 6285 CL (3.0 mg·kg^-1). The hyperventilatory response to 5-hydroxytryptamine could be mimicked by the 5-HT₃ receptor agonist 1-phenylbiguanide (300 g·kg^-1). These results strongly implicate the 5-HT₃-receptor as the mediator of the 5-hydroxytryptamine-induced hyperventilation in eel.Abbreviations a.u. arbitrary units - 5-HT 5-hydroxytryptamine - SEM standard error of mean - VA ventilatory amplitude - VF ventilatory frequency - RBI 1-phenylbiguanide     

Polymorphism of the Serotonin System Genes in Some Finno-Ugric Populations

Polymorphic sites in the genes encoding monoamine oxidase A (MAO-A), serotonin transporter (hSERT) and 5-HT_2A receptor were typed in Khant and Komi ethnic groups with the purpose of revealing possible inerpopulation differences in genotype and allele frequencies. No statistically significant differences in the hSERT and 5-HT_2A gene frequencies were detected. At the same time, the populations examined had statistically significantly different MAO-A genotype and allele frequencies. These results obtained indicate the prevalence of the site gain alleles of theEcoRV and Fnu4HI RFLP loci at the MAO-A gene in Komis and the of the corresponding site loss alleles in Khants.     

Hydrogen‐rich water regulates effects of ROS balance on morphology,growth and secondary metabolism via glutathione peroxidase in Ganoderma lucidum

Ganoderma lucidum is one of the most important medicinal fungi, but the lack of basic study on the fungus has hindered the further development of its value. To investigate the roles of the redox system in G. lucidum, acetic acid (HAc) was applied as a reactive oxygen species (ROS) stress inducer, and hydrogen‐rich water (HRW) was used to relieve the ROS stress in this study. Our results demonstrate that the treatment of 5% HRW significantly decreased the ROS content, maintained biomass and polar growth morphology of mycelium, and decreased secondary metabolism under HAc‐induced oxidative stress. Furthermore, the roles of HRW were largely dependent on restoring the glutathione system under HAc stress in G. lucidum. To provide further evidence, we used two glutathione peroxidase (GPX)‐defective strains, the gpxi strain, the mercaptosuccinic acid (MS, a GPX inhibitor)‐treated wide‐type (WT) strain, and gpx overexpression strains for further research. The results show that HRW was unable to relieve the HAc‐induced ROS overproduction, decreased biomass, mycelium morphology change and increased secondary metabolism biosynthesis in the absence of GPX function. The gpx overexpression strains exhibited resistance to HAc‐induced oxidative stress. Thus, we propose that HRW regulates morphology, growth and secondary metabolism via glutathione peroxidase under HAc stress in the fungus G. lucidum. Furthermore, our research also provides a method to study the ROS system in other fungi.     

Synthesis and computer-aided analysis of the role of linker for novel ligands of the 5-HT6 serotonin receptor among substituted 1,3,5-triazinylpiperazines

A series of 2-amino-4-(4-methylpiperazin-1-yl)-1,3,5-triazines was designed based on previously published 2-amino-4-benzyl-(4-methylpiperazin-1-yl)-1,3,5-triazines in order to evaluate the role of a linker between the triazine moiety and an aromatic substituent for the human serotonin 5-HT₆ receptor affinity. As new linkers two carbon atoms (ethyl or ethenyl) or an oxyalkyl chain (methoxy, 2-ethoxy, 2-propoxy) were introduced. Affinities of the compounds for the 5-HT₆R as the main target, and for the 5-HT_1AR, 5-HT₇R and D₂R as competitive ones, were determined in the radioligand binding assays. Docking to the 5-HT₆R homology model was performed to support SAR analysis. Results showed that the branching of the methoxyl linker increased affinity for the human 5-HT₆R whereas an unsaturated bond within the linker dramatically reduced desirable activity. Both experimental and theoretical studies confirmed the previously postulated beneficial role of the aromatic size for interaction with the 5-HT₆R. Thus, the largest naphthyl moiety yielded the highest activity. In particular, 4-(4-methylpiperazin-1-yl)-6-(1-(naphthalen-1-yloxy)ethyl)-1,3,5-triazin-2-amine (24), the most potent 5-HT₆R agent found (K_i = 23 nM), can be a new lead structure for further search and development.     

Regulation of ion and water transport across the eel intestine: effects of acetylcholine and serotonin

Summary Both acetylcholine (ACh) and serotonin (5-HT) lowered the serosa-negative transepithelial potential difference (PD) and the short-circuit current (I_sc), accompanied by a decrease in NaCl and water absorption across the eel intestine. These inhibitory effects of ACh and 5-HT were blocked by atropine, a muscarinic receptor antagonist, and ICS-205930, a 5-HT₃ receptor antagonist, respectively. Even after blocking the ACh receptor with atropine, 5-HT inhibited the PD and I_sc, and ACh lowered them after blocking the 5-HT receptor with ICS-205930, indicating that ACh and 5-HT act independently. Similar inhibition in the PD and the I_sc was observed after electrical field stimulation (EFS) which is expected to release endogenous regulators. These effects of EFS were reduced by 70% after simultaneous addition of atropine and ICS-205930. Since atropine and ICS-205930 block ACh and 5-HT receptors, respectively, these results suggest that endogenous ACh and 5-HT are released by EFS.Abbreviations ACh acetylcholine - EFS electrical field stimulation - 5-HT serotonin - I _sc short-circuit current - PD transepithelial potential difference - R _t tissue resistance - TTX tetrodotoxin     

The Peptides Mimicking the Third Intracellular Loop of 5-Hydroxytryptamine Receptors of the Types 1B and 6 Selectively Activate G Proteins and Receptor-Specifically Inhibit Serotonin Signaling via the Adenylyl Cyclase System

The third intracellular loop (ICL3) of G protein-coupled receptors has, as a rule, a key role in their interaction with heterotrimeric G proteins. We synthesized peptides corresponding to the C-terminal region of the ICL3 (C-ICL3) of 5-hydroxytryptamine receptors of the type 1B (5-HT_1BR) and 6 (5-HT₆R) and studied their influence on the functional activity of adenylyl cyclase signaling system (ACSS) in synaptosomal membranes isolated from the rat brain. The 5-HT_1BR-peptide ARERKATKTL^307–316K-amide mimicking agonist-activated 5-HT_1BR reduced forskolin-stimulated adenylyl cyclase (AC) activity and activated pertussis toxin-sensitive G proteins. It lowered inhibitory effects of serotonin and 5-HT_1BR-agonists on forskolin-stimulated AC activity and their stimulating effects on GTP binding. This was not the case in the presence of 5-HT_1BR-antagonists. The 5-HT₆R-peptides mimicking 5-HT₆R activated both the basal AC activity and GTP binding of cholera toxin-sensitive G proteins. They lowered the stimulating effect of serotonin and 5-HT₆R-agonists on AC and G_s proteins, but in the presence of 5-HT₆R-antagonists their action was blocked. Of all the 5-HT₆R-peptides with linear and dimeric structure we studied the palmitoylated peptide KHSRKALKASL^258–268K(Pal)A-amide had a most pronounced effect both on the basal and 5-HT₆R-agonist-stimulated ACSS. The data was obtained indicating that the peptides corresponding to C-ICL3 of 5-HT_1BR and 5-HT₆R selectively activate G_i and G_s proteins, respectively, and in a receptor-specific manner reduce signal transduction via serotonin-sensitive ACSS in the rat brain. The results of the study give strong evidence in favor of active participation of C-ICL3 of these 5-HTRs in their coupling with the G proteins.     

Synthesis and structure-activity relationship of 1H-indole-3-carboxylic acid pyridine-3-ylamides: a novel series of 5-HT2C receptor antagonists

A novel series of 1H-indole-3-carboxylic acid pyridine-3-ylamides were synthesized and identified to show high affinity and selectivity for 5-HT_2C receptor. Among them, 1H-indole-3-carboxylic acid[6-(2-chloro-pyridin-3-yloxy)-pyridin-3-yl]-amide (15k) exhibits the highest affinity (IC₅₀ = 0.5 nM) with an excellent selectivity (>2000 times) over other serotonin (5-HT_1A, 5-HT_2A, and 5-HT₆) and dopamine (D₂–D₄) receptors.     

Divergent actions of serotonin receptor activation during fictive swimming in frog embryos

We have investigated the pharmacology underlying locomotor system responses to serotonin (5-HT) in embryos of the frog, Rana temporaria, to provide a comparison to studies in embryos of its close relative, Xenopus laevis. Our findings suggest that two divergent mechanisms underlie the modulation of locomotion by 5-HT in Rana. Bath-applied 5-HT or 5-carboxamidotyptamine, a 5-HT_1,5A,7 receptor agonist, can modulate fictive swimming in a dose-dependent manner, increasing burst durations and cycle periods. However, activation of 5-HT_1,7 receptors with R8-OHDPAT or 8-OHDPAT fails to mimic 5-HT, and in some cases exerts exactly the opposite response; decreasing burst durations and cycle periods. Elevating endogenous 5-HT levels by blocking re-uptake with clomipramine transiently increases burst durations. The receptors involved in this endogenous response include 5-HT_1A receptors, as in Xenopus, but also 5-HT₇ receptors. However, like the 8-OHDPAT enantiomers, prolonged re-uptake inhibition can result in a motor response in the opposite direction to exogenous 5-HT. This effect is not reversed by 5-HT_1A and/or 5-HT₇ receptor antagonism, implicating 5-HT_1B/1D receptors. Remarkably, antagonism of these receptors using methiothepin unmasks a dose-dependent response to clomipramine, reminiscent of exogenous 5-HT. Our data suggest that 5-HT_1A,7 and 5-HT_1B/1D receptors act as gain-setters of burst durations, whilst 5-HT_5A receptors are involved in the effects of bath-applied 5-HT on locomotion.     

Pharmacological Characterization of 5-Hydroxytryptamine3Receptors in Murine Brain and Ileum Using the Novel Radioligand [3H]RS-42358–197: Evidence for Receptor Heterogeneity

Abstract: Previous studies have demonstrated species-specific differences in 5-hydroxytryptamine₃ (5-HT₃) receptors, but unequivocal evidence of 5-HT₃ receptor subtypes, within a species, has not yet been obtained. The purpose of the current study was to test for heterogeneity in 5-HT₃ receptors in murine tissues. 5-HT₃ receptors in membranes derived from brain cerebral cortex of CD-1, C57BI/6, and Swiss Webster mice and ileum of CD-1 mice were labeled with the 5-HT₃ receptor antagonist [³H]RS-42358–197. Structurally diverse competing ligands were then used to characterize the binding site. [³H]RS-42358-197 bound with similar affinity in each of the cortical tissues (mean K_D= 0.14 nM; range, 0.06–0.32 nM) but bound with lower affinity in ileal tissue (2.5 nM). The density of sites labeled with [³H]RS-42358–197 ranged from 10.4 fmol/mg of protein in Swiss Webster mouse cortex to 44.2 fmol/mg of protein in Sprague-Dawley rat cortex. Displacing ligands produced a pharmacologic profile of the [³H]RS-42358–197 binding site consistent with it being a 5-HT₃ receptor: (R)-YM060 > (S)-zacopride > (R)-zaco-pride > MDL 72222 > 2-methyl-5-HT. However, 10-fold differences in the affinity of certain ligands were found when comparing 5-HT₃ binding sites in membranes from cerebral cortex of the different strains of mice and when comparing 5-HT₃ binding sites in brain and ileal membranes prepared from the CD-1 mouse strain. Ligands demonstrating selectivity included RS-42358–197, (R)-za-copride, 1-(m-chlorophenyl) biguanide, (R)-YM060, and MDL 72222. These studies demonstrate tissue-and strain-dependent differences in murine 5-HT₃ binding sites. This suggests that 5-HT₃ receptors exist as multiple subtypes within species and that subtype-selective 5-HT₃ ligands may be identified.     

Synthesis and structure–activity relationship of novel conformationally restricted analogues of serotonin as 5-HT6 receptor ligands

5-Hydroxytryptamine 6 receptors (5-HT₆R) are being perceived as the possible target for treatment of cognitive disorders as well as obesity. The present article deals with the design, synthesis, in vitro binding and structure–activity relationship of a novel series of tetracyclic tryptamines with the rigidized N-arylsulphonyl, N-arylcarbonyl and N-benzyl substituents as 5-HT₆ receptor ligands. The chiral sulphonyl derivatives 15a and 17a showed high affinity at 5-HT₆R with the K_i of 23.4 and 20.5?nM, respectively. The lead compound from the series 15a has acceptable ADME properties, adequate brain penetration and is active in animal models of cognition like Novel Object Recognition Task (NORT) and water maze.     

Genotype‐dependent consequences of traumatic stress in four inbred mouse strains

Post‐traumatic stress disorder (PTSD) is an anxiety disorder that develops in predisposed individuals following a terrifying event. Studies on isogenic animal populations might explain susceptibility to PTSD by revealing associations between the molecular and behavioural consequences of traumatic stress. Our study employed four inbred mouse strains to search for differences in post‐stress response to a 1.5‐mA electric foot shock. One day to 6 weeks after the foot shock anxiety, depression and addiction‐like phenotypes were assessed. In addition, expression levels of selected stress‐related genes were analysed in hippocampus and amygdala. C57BL/6J mice exhibited up‐regulation in the expression of Tsc22d3, Nfkbia, Plat and Crhr1 genes in both brain regions. These alterations were associated with an increase of sensitized fear and depressive‐like behaviour over time. Traumatic stress induced expression of Tsc22d3, Nfkbia, Plat and Fkbp5 genes and developed social withdrawal in DBA/2J mice. In 129P3/J strain, exposure to stress produced the up‐regulation of Tsc22d3 and Nfkbia genes and enhanced sensitivity to the rewarding properties of morphine. Whereas, SWR/J mice displayed increase only in Pdyn expression in the amygdala and had the lowest conditioned fear. Our results reveal a complex genetic background of phenotypic variation in response to stress and indicate the SWR/J strain as a valuable model of stress resistance. We found potential links between the alterations in expression of Tsc22d3, Nfkbia and Pdyn, and different aspects of susceptibility to stress.