3D QSAR based design of novel oxindole derivative as 5HT7 inhibitors.

To understand the structural requirements of 5-hydroxytryptamine (5HT7) receptor inhibitors and to design new ligands against 5HT7 receptor with enhanced inhibitory potency, a three-dimensional quantitative structure-activity relationship study with comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) for a data set of 56 molecules consisting of oxindole, tetrahydronaphthalene, aryl ketone substituted arylpiperazinealkylamide derivatives was performed. Derived model showed good statistical reliability in terms of predicting 5HT7 inhibitory activity of the molecules, based on molecular property fields like steric, electrostatic, hydrophobic, hydrogen bond donor and hydrogen bond acceptor fields. This is evident from statistical parameters like conventional r2 and a cross validated (q2) values of 0.985, 0.743 for CoMFA and 0.970, 0.608 for CoMSIA, respectively. Predictive ability of the models to determine 5HT7 antagonistic activity is validated using a test set of 16 molecules that were not included in the training set. Predictive r2 obtained for the test set was 0.560 and 0.619 for CoMFA and CoMSIA, respectively. Steric, electrostatic fields majorly contributed toward activity which forms the basis for design of new molecules. Absorption, distribution, metabolism and elimination (ADME) calculation using QikProp 2.5 (Schrodinger 2010, Portland, OR) reveals that the molecules confer to Lipinski's rule of five in majority of the cases.

Autoradiographic distribution of 5-HT7 receptors in the human brain using [3H]mesulergine: comparison to other mammalian species.

1. The main aim of this investigation was to delineate the distribution of the 5-HT(7) receptor in human brain. Autoradiographic studies in guinea-pig and rat brain were also carried out in order to revisit and compare the anatomical distribution of 5-HT(7) receptors in different mammalian species. 2. Binding studies were performed in rat frontal cortex membranes using 10 nm [(3)H]mesulergine in the presence of raclopride (10 microm) and DOI (0.8 microm). Under these conditions, a binding site with pharmacological characteristics consistent with those of the 5-HT(7) receptors was identified (rank order of binding affinity values: 5-CT>5-HT>5-MeOT>mesulergine approximately methiothepin>8-OH-DPAT=spiperone approximately (+)-butaclamol>>imipramine approximately (+/-)-pindolol>>ondansetron approximately clonidine approximately prazosin). 3. The autoradiographic studies revealed that the anatomical distribution of 5-HT(7) receptors throughout the human brain was heterogenous. High densities were found over the caudate and putamen nuclei, the pyramidal layer of the CA2 field of the hippocampus, the centromedial thalamic nucleus, and the dorsal raphe nucleus. The inner layer of the frontal cortex, the dentate gyrus of the hippocampus, the subthalamic nucleus and superior colliculus, among others, presented intermediate concentrations of 5-HT(7) receptors. A similar brain anatomical distribution of 5-HT(7) receptors was observed in all three mammalian species studied. 4. By using [(3)H]mesulergine, we have mapped for the first time the anatomical distribution of 5-HT(7) receptors in the human brain, overcoming the limitations previously found in radiometric studies with other radioligands, and also revisiting the distribution in guinea-pig and rat brain.

5-Hydroxytryptamine7 (5-HT7) receptor immunoreactivity-positive 'stigmoid body'-like structure in developing rat brains.

We examined the expression of 5-hydroxytryptamine(7) (5-HT(7)) receptor protein in developing and adult rats with immunohistochemical technique. In adult male rats, 5-HT(7) receptor immunoreactivity was detected in the septum, striatum, indusium griseum, tenia tecta, thalamus, hippocampus and hypothalamus in the forebrain as well as the pons and cerebellum. In brains of 1, 7, 15 and 21 days old male rats but not of adult ones, 5-HT(7) receptor immunoreactivity-positive dot-like structures were detected. The dot-like structures were visualized in hypothalamus, hippocampus, frontal cortex, brainstem and cerebellum at 1 day old male rats. In 7 days old male rats, the dot-like structures were found in the hypothalamus, medial preoptic area (MPA), bed nucleus of the stria terminalis (BST), amygdaloid nucleus and brainstem reticular formation. In 15 and 21 days old male and female rats, 5-HT(7) receptor immunoreactive dots were most clearly detected in MPA, hypothalamus, raphe pallidus, raphe magnus and brainstem reticular formation. The 5-HT(7) receptor immunoreactivity-positive dot-like structures were shown in the cytoplasm and they were less than 1 microm in diameter in 1 and 7 days old rats and became larger to 1-3 microm in 15 and 21 days old rats. From the distribution and morphologic features, the 5-HT(7) receptor immunoreactivity-positive dot-like structure found in developing rat brains is considered to be identical to a cytoplasmic inclusion named 'stigmoid body'.

Differential distribution of 5HT2A and NMDA receptors in single cells within the rat medial nucleus of the solitary tract.

Activation of serotonin (5-hydroxytryptamine; 5HT) receptors of the 2A subtype (5HT2A) in the intermediate portion of the medial nucleus tractus solitarius (mNTS) produces marked hypotension and bradycardia. This portion of the mNTS receives major input from glutamatergic baroreceptor afferents. Thus, the cardiorespiratory effects of 5HT2A agonists may be attributed, in part, to interactions involving the glutamatergic target neurons, some of which express N-methyl-D-aspartate (NMDA) glutamate receptors. To determine the functional sites for activation of 5HT2A receptors and their relationship to NMDA receptors in this region, we used electron microscopic immunocytochemistry for the localization of antipeptide antisera selectively recognizing each receptor protein in the intermediate mNTS in rat brain. Of 1,052 5HT2A-labeled profiles, 38% were dendrites and dendritic spines, 27% were unmyelinated axons, 14% were axon terminals, and 11% were glial processes. These 5HT2A-labeled profiles frequently contained NR1 gold particles with dendrites comprising 68% of the total dual-labeled profiles. In dendrites, the 5HT2A immunoreactivity was localized to cytoplasmic organelles or discretely distributed on synaptic or extrasynaptic segments of the plasma membrane. In contrast, NR1 immunoreactivity was prominently localized to postsynaptic junctions and these were distinct from the 5HT2A receptor labeling when coexpressed in the same dendrites. Dendrites containing both receptors composed 56% (224/399) of the total 5HT2A-labeled dendrites and 34% (224/659) of the total NR1-labeled dendrites. Our results provide the first ultrastructural evidence that in the intermediate mNTS, 5HT2A receptor agonists may affect the postsynaptic excitability of many of the same neurons that show NMDA-evoked responses to glutamate.

Evidence for regional heterogeneity in corticotropin-releasing factor interactions in the dorsal raphe nucleus.

The dorsal raphe nucleus (DR) is innervated by fibers containing the stress-related neurohormone corticotropin-releasing factor (CRF), which alters DR neuronal activity and serotonin release in rats. This study examined the relative distribution of CRF-immunoreactive fibers in the rat DR by using light level densitometry. Additionally, CRF-immunoreactive processes within specific subregions of the DR were examined at the ultrastructural level by using electron microscopy. CRF-immunoreactive fibers were organized within the DR along a caudal-rostral gradient, such that proceeding rostrally, innervation shifted from dorsolateral to ventromedial. Numerous CRF-immunoreactive axon terminals containing dense-core vesicles were found in both the caudal dorsolateral region and the rostral ventromedial/interfascicular region. These formed synaptic specializations with unlabeled dendrites and frequently contacted nonlabeled axon terminals. Semiquantitative analysis revealed certain differences between the two regions with respect to the types of associations made by CRF-immunoreactive terminals. Associations with dendrites were more frequent in the dorsolateral vs. ventromedial region (65% of 171 terminals vs. 39% of 233 terminals, respectively), whereas associations with axon terminals were more frequent in the ventromedial/interfascicular vs. the dorsolateral region (72% of 233 terminals vs. 57% of 171 terminals, respectively). Additionally, synaptic specializations between CRF-immunoreactive terminals and dendrites were more frequently asymmetric in the dorsolateral region (60%) and symmetric (49%) in the ventromedial/interfascicular region. Regional differences in CRF terminal interactions in the DR could account for the reported heterogeneous effects of CRF on DR neuronal activity and forebrain serotonin release. Importantly, the present results provide anatomical substrates for regulation of the DR by endogenous CRF.

Mammal-like striatal functions in Anolis. I. Distribution of serotonin receptor subtypes, and absence of striosome and matrix organization.

Serotonin (5-HT) 5-HT(2A) and 5-HT(2C) receptors are thought to play important roles in the mammalian striatum. As basal ganglia functions in general are thought highly conserved among amniotes, we decided to use in situ autoradiographic methods to determine the occurrence and distribution of pharmacologically mammal-like 5-HT(2A) and 5-HT(2C) receptors in the lizard, Anolis carolinensis, with particular attention to the striatum. We also determined the distributions of 5-HT(1A), 5-HT(1B/D), 5 HT(3), and 5-HT(uptake) receptors for comparison. All 5-HT receptors examined showed pharmacological binding specificity, and forebrain binding density distributions that resembled those reported for mammals. Anolis 5 HT(2A/C) and 5-HT(1A) site distributions were similar in both in vivo and ex vivo binding experiments. 5-HT(2A & C) receptors occur in both high and low affinity states, the former having preferential affinity for (125)I-(+/-)-2,5-dimethoxy-4-iodo-amphetamine hydrochloride ((125)I-DOI). In mammals (125)I-DOI binding shows a patchy density distribution in the striatum, being more dense in striosomes than in surrounding matrix. There was no evidence of any such patchy density of (125)I-DOI binding in the anole striatum, however. As a further indication that anoles do not possess a striosome and matrix striatal organization, neither (3)H-naloxone binding nor histochemical staining for acetylcholinesterase activity (AChE) were patchy. AChE did show a band-like striatal distribution, however, similar to that seen in birds.

Synaptic plasticity of 5-hydroxytryptamine-immunoreactive terminals in the phrenic nucleus following spinal cord injury: a quantitative electron microscopic analysis.

The present study was conducted to examine the plasticity of 5-hydroxytryptamine (5-HT)-immunoreactive terminals in the rat phrenic nucleus following an ipsilateral C2 spinal cord hemisection and 30-day survival period. A retrograde horseradish peroxidase (HRP) labeling technique was used to identify the phrenic motoneurons at the electron microscopic (EM) level. After employing a pre-embedding immunocytochemical technique, the ultrastructural characteristics of 5-HT-immunoreactive terminals were qualitatively and then quantitatively analyzed with a computerized morphometric system before and after injury in separate groups of rats. The results indicated that the majority of the 5-HT-labeled terminals formed axodendritic contacts, but some 5-HT-labeled terminals made axosomatic contacts. 5-HT terminals were associated with either asymmetrical or symmetrical synapses, and some displayed postsynaptic dense bodies. Approximately 2% of the 5-HT terminals had dense-core vesicles. Although the total number of labeled and unlabeled terminals in the phrenic nucleus was reduced after hemisection, the number of 5-HT terminals in the hemisected group was greater than that of the control group. Moreover, the total number and length of asymmetrical and symmetrical synaptic active zones per 5-HT terminal were significantly greater after injury. Finally, the total number of 5-HT terminals with multiple synapses was significantly greater in the hemisected group as compared to controls. It is possible that 5-HT synaptic plasticity may be part of the morphological substrate for the unmasking of the latent crossed phrenic pathway which mediates recovery of the ipsilateral hemidiaphragm paralyzed by C2 spinal cord hemisection.

Light and electron microscopic immunocytochemical visualization of 5-HT1B receptors in the rat brain.

Specific antipeptide antibodies were used for the immunohistochemical visualization of 5-HT1B receptors in the rat brain. A dense, specific 5-HT1B receptor-like immunoreactivity was found in the globus pallidus, the dorsal subiculum and the substantia nigra. At the light microscope level, immunostaining was diffuse within the neuropil but absent from cell bodies. Observations at the electron microscope level in the substantia nigra showed immunoperoxidase staining in fine unmyelinated axons and nerve terminals.

Contributions of raphe-cortical and thalamocortical axons to the transient somatotopic pattern of serotonin immunoreactivity in rat cortex.

Two experiments were carried out to evaluate the relative contributions of thalamocortical and raphe-cortical fibers to the transient somatotopically organized pattern of serotonin (5-HT) immunoreactivity that appears in the primary somatosensory cortex (SI) of rats during the first 2 weeks of life. In the first experiment, the specific 5-HT uptake inhibitors, fluoxetine and paroxetine, were administered systemically, animals were killed 3, 6, or 12 h later, and cortices evaluated for 5-HT immunoreactivity. Fluoxetine treatment had no appreciable effect on the density of 5-HT immunoreactivity in the cortex. Paroxetine treatment caused a reduction in 5-HT immunoreactivity which was maximal 6 h after administration. Examination of the cortices of these animals revealed a loss of very fine dust-like 5-HT immunoreactivity, but a vibrissae-related pattern remained visible in thicker fibers. In a second experiment, raphe-cortical fibers were destroyed by systemic administration of 5,7-dihydroxytryptamine on the day of birth. Six days after this manipulation, 5-HT was applied directly to the cortex in vivo and the animals were then killed and cortices processed to demonstrate 5-HT immunoreactivity. The cortices of these rats revealed a fine dust-like immunoreactivity organized in a somatotopic pattern, but only very few 5-HT-positive axons. The results of these experiments suggest that both raphe-cortical axons and thalamocortical fibers contribute to the patterned 5-HT immunoreactivity observed in SI of perinatal rats.

[The participation of mast cells and serotonin-immunoreactive fibers in the innervation of the cerebral vessels]. / Uchastie tuchnykh kletok i serotonin-immunoreaktivnykh volokon v innervatsii sosudov mozga.

Interrelations between adrenergic and serotoninergic nerve fibres and mast cells located near the vascular wall were studied on total preparations of rat brain pia mater. Histochemical method with glyoxylic acid revealed the dense network of adrenergic fibres and their terminal regions with variceal endings with mast cells located next to them. Immunohistochemical methods (fluorescent and method with use of peroxidase antiperoxidase complex) with the aid of antiserum serotonin showed the content of serotonin in these cells. Distribution of serotoninergic nerve fibres resemble that of adrenergic fibres with density being significantly lower. The fibres have frequent contacts with mast cells processes. These data indicate the presence of functional connection between serotonin positive nerve fibres and the possibility of their drawings in control of blood circulation of brain.

[The neuronal reaction of the rat neocortex to damage to the central serotoninergic system]. / Reaktsiia neironov neokorteksa krys na povrezhdenie tsentral'noi serotoninergicheskoi sistemy.

Pyramidal neurons of the motor cortex II-III layers developed degenerative alterations in response to a selective damage of the rat brain serotoninergic system, whereas those of the V-VI layers manifested adaptive changes. The finding could be due to a selective sensitivity of the target cells in respect to serotonin deficiency.

Embryonic expression of the 5-HT3 receptor subunit, 5-HT3R-A, in the rat: an in situ hybridization study.

The role of serotonin as a neurotransmitter in the vertebrate central and peripheral nervous systems has been extensively studied. In addition to its well-defined role in neurotransmission, serotonin has also been implicated in development. We have used in situ hybridization to localize 5-HT3 receptor mRNA in embryonic rat sections from Embryonic Day 10 (E10) to E18. Expression was first detected in the cranial sensory ganglia starting on E10. Expression was later detected in many regions within the developing CNS. In addition to the cranial sensory ganglia, expression was detected in many regions of the peripheral nervous system including dorsal root, sympathetic, and parasympathetic ganglia, and in the myenteric plexus of the enteric nervous system. Expression was also detected in many nonneuronal cell populations including the choroid plexus, cochlear duct, and olfactory epithelium. Expression in regions of active epithelial-mesenchymal interaction such as the tooth bud, lung, and submandibular gland may indicate a role for 5-HT3 receptors in the process of secondary induction.

Ultrastructure of the 5-hydroxytryptamine3 receptor.

We have determined the ultrastructure of 5-hydroxytryptamine3 (5-HT3) serotonin receptors purified from NG108-15 mouse neuroblastoma x rat glioma cells by electron microscopic examination of receptor particles embedded in uranyl acetate stain and metal replicas of rapidly frozen receptors. The 5-HT3 receptor can be modelled as a cylinder 11 nm in length and 8 nm in diameter with a closed end and a central cavity 3 nm in diameter. Analysis of the rotational symmetry of single receptor particles indicates that the 5-HT3 receptor is composed of five subunits arranged symmetrically around a central cavity. Together with evidence obtained for related proteins in other studies using ultrastructural, biochemical, or electrophysiological methods, our observations suggest that all members of the ligand-gated ion channel superfamily may possess a pentameric quaternary structure.

[The target cells of serotoninergic innervation in the locus coeruleus]. / Kletki-misheni serotoninergicheskoi innervatsii v golubovatom meste.

Method of selective chemical injury of locus ceruleus (LC) has revealed the following serotonergic structures: axonal varicosities, terminals and synapses. Serotonergic structures in LC are numerous. Small neurons with peculiar structures are described which are the targets of serotonergic innervation in LC and are responsible for interrelation with the proper noradrenergic cells and for intermediate interactions.

[Serotoninergic nerve fibers in the area of the substantia grisea and the ependyma of the cerebral aqueduct]. / Serotoninergicheskie nervnye volokna v zone serogo veshchestva i épendimy vodoprovoda mozga.

An electron microscopic study of the midbrain gravy with the help of degeneration caused by serotonin-like neurotoxin 5,7-dihydroxytryptamine has shown that serotonergic terminals are responsible for the innervation of dendrites, neuron bodies and ependyma cells of the cerebral aqueduct. The detection of light, dark with prevailing fine transparent or large granular synaptic vesicles as well as vacuole degeneration evidences of the existence of several sources of serotonergic innervation of the central gray substance. Continuous damage of perivascular astrocytes of the central gray by neurotoxin allows a suggestion to be made that they are also the object of serotonergic innervation. Immunomorphological data on the presence of serotoninergic neurons in the cerebral gray are confirmed.

Three-dimensional steric molecular modeling of the 5-hydroxytryptamine3 receptor pharmacophore.

A computer-based three-dimensional steric molecular model of the 5-hydroxytryptamine3 (5-HT3) receptor pharmacophore was defined on the basis of radioligand binding data. Analysis of published data led to the identification of 19 different chemical structures that share only a single known pharmacological property, i.e., less than 10 nM affinity for the 5-HT3 receptor. These 19 compounds were then categorized into seven chemical families, which derive from six main steric "core" structures. From the composite analysis of all 19 potent agents, nine steric chemical criteria were derived, which can be used to describe the 5-HT3 receptor pharmacophore. This information was then used to explain the 5-HT3 receptor inactivity of atrophine, a compound that differs structurally from ICS 205-930 in the steric properties of only a single key atom. The steric chemical information was also used to predict the activity of serotonergic compounds that had never been analyzed at 5-HT3 receptor binding sites. Two serotonergic drugs that meet all nine steric criteria were found to be active at the 5-HT3 receptor binding site (i.e., pizotifen, KI = 42 +/- 10 nM, and clozapine, KI = 52 +/- 8 nM). By contrast, two serotonergic agents that do not meet the criteria were found to be inactive at the 5-HT3 receptor binding site (i.e., ipsapirone and pirenperone, KI values greater than 1000 nM). This computer-based steric molecular modeling approach allows for the analysis and identification of 5-HT3 receptor-active agents with minimal dependence upon animals and radioactive compounds.