Learning is highly regulated by the pattern of training. In Aplysia, an important organism for the development of cellular and molecular models of learning, spaced versus massed application of the same stimulus leads to different forms of memory. A critical molecular step underlying memory is the serotonin (<em>5HT</em>)-mediated activation of the novel PKC Apl II. Here, we demonstrate that activation of PKC Apl II is highly sensitive to the pattern of <em>5HT</em> application. Spaced applications downregulate PKC translocation through PKA signaling, whereas massed applications lead to persistent translocation of PKC. Differential regulation of PKC translocation is mediated by competing feedback mechanisms that act through protein synthesis. These studies elucidate a fundamental molecular difference between spaced and massed training protocols.

1. Serotonin is a neurotransmitter in the central nervous system which has been implicated in the aetiology and pathogenesis of affective disorders. The serononergic system also plays several roles in the immune system through the expression of a number of its receptor subtypes in the immune cells. 2. Following release serotonin is inactivated by reuptake into neurons and other cells by a specific serotonin sodium and chloride-dependent transporter molecule, whose structure has been elucidated. 3. Measurement [3H]paroxetine binding showed that human lymphocytes contain a high-affinity serotonin transporter. 4. To assess the serotonin function in major depression, we investigated serotonin transporter density in blood lymphocytes from patients with this disorder and selected according to the interview of the American Psychiatric Association. 5. Patients were divided into two groups and treated with two different antidepressant drugs, one group receiving fluoxetine, a selective serotonin reuptake inhibitor, and another mirtazapine, an antagonist of alpha2-adrenergic auto and heteroreceptors, for a period of 6 weeks. 6. Blood samples were obtained before and after the treatment, lymphocytes were isolated by Ficoll/Hypaque gradient, subjected to differential adhesion to plastic, and cell membranes were prepared for binding assay of [3H]paroxetine. 7. Lymphocytes serotonin transporter number was significantly reduced, while the affinity was unchanged, in patients with major depression disorder as compare to controls. 8. In addition, there was a partial recovery in lymphocytes serotonin (<em>5HT</em>) transporter number in the period posterior to the antidepressants administration, accompanied with clinical and depression rating scales improvement. Serotonin was determined in platelet-poor plasma and in lymphocytes before and after drugs administration, showing a significant decrease in the patients treated compared to untreated and controls. 9. These results are evidence of the potential interaction between the nervous and immune systems. The mechanisms underlying this interaction are under study, and might be related to modifications in the expression or function of the serotonin transporters in lymphocytes of depressed patients.

Research has shown that spinal circuits have the capacity to adapt in response to training, nociceptive stimulation and peripheral inflammation. These changes in neural function are mediated by physiological and neurochemical systems analogous to those that support plasticity within the hippocampus (e.g., long-term potentiation and the NMDA receptor). As observed in the hippocampus, engaging spinal circuits can have a lasting impact on plastic potential, enabling or inhibiting the capacity to learn. These effects are related to the concept of metaplasticity. Behavioral paradigms are described that induce metaplastic effects within the spinal cord. Uncontrollable/unpredictable stimulation, and peripheral inflammation, induce a form of maladaptive plasticity that inhibits spinal learning. Conversely, exposure to controllable or predictable stimulation engages a form of adaptive plasticity that counters these maladaptive effects and enables learning. Adaptive plasticity is tied to an up-regulation of brain derived neurotrophic factor (BDNF). Maladaptive plasticity is linked to processes that involve kappa opioids, the metabotropic glutamate (mGlu) receptor, glia, and the cytokine tumor necrosis factor (TNF). Uncontrollable nociceptive stimulation also impairs recovery after a spinal contusion injury and fosters the development of pain (allodynia). These adverse effects are related to an up-regulation of TNF and a down-regulation of BDNF and its receptor (TrkB). In the absence of injury, brain systems quell the sensitization of spinal circuits through descending serotonergic fibers and the serotonin 1A (<em>5HT</em> 1A) receptor. This protective effect is blocked by surgical anesthesia. Disconnected from the brain, intracellular Cl(-) concentrations increase (due to a down-regulation of the cotransporter KCC2), which causes GABA to have an excitatory effect. It is suggested that BDNF has a restorative effect because it up-regulates KCC2 and re-establishes GABA-mediated inhibition.

Effect of 4-hydroxynonenal (HNE), a long-chain alpha, beta unsaturated aldehyde product, generated by the oxidation of omega-6 polyunsaturated fatty acids on the sensitivity of selected neurotransmitter receptors was studied in PC-12 cells. Cytotoxicity profiling was carried out at varying concentrations of HNE (0.1-50microM) for 30min to 24h. Trypan blue dye exclusion, MTT, LDH release and neutral red uptake (NRU) assays were carried out to assess the cytotoxicity of HNE. Cytotoxic response was found to be significant at 2h of exposure. Cytotoxicity of HNE at 50microM was exerted even at 90min. HNE 10-50microM was found to be cytotoxic, whereas, 2-5microM causes physiological stress only and 1-0.1microM non-cytotoxic. Effect on dopamine, cholinergic, serotonin and benzodiazepine receptors was studied at varying concentrations of HNE (1, 10, 25 and 50microM for 1-8h). A significant decrease in binding of 3H-QNB, 3H-Fluinitrazepam and 3H-Ketanserin, known to label cholinergic (muscarinic), benzodiazepine and serotonin (<em>5HT</em>(2A)) receptors respectively was observed at 1h exposure of PC-12 cells to HNE at 25 and 50microM concentrations. The decrease in the binding of (3)H-Spiperone, known to label dopamine (DA-D2) receptors was evident at 4h of exposure of PC-12 cells to HNE. The decrease in the binding with DA-D2 receptors continued till 8h. Effect on the binding of (3)H-Fluinitrazepam and 3H-Ketanserin appeared to be maximum at 25 and 50microM concentrations of HNE for 4h and 8h. The PC-12 cells appear to be vulnerable to cytotoxic concentrations of HNE. Experimental HNE exposure provides an intriguing model of toxicant-cell interactions involving neurotransmitter receptors in HNE neurotoxicity.

Rats were immobilised for 2 h/day. Twenty-four hours after the 1, 3 or 7 immobilisation periods they were injected with the <em>5HT</em> agonist 5-methoxy-N,N-dimethyltryptamine (5MeODMT; 5 mg/kg i.p.) and behavioural responses (i.e. hind limb abduction, forepaw treading, head weaving, tremor, Straub tail) compared with those of a control group. As we have previously observed after 7 (but not after 1 or 3 immobilisations) forepaw treading and tremor were enhanced and the other responses unaffected. Pretreatment with metyrapone (a corticosterone synthesis inhibitor 150 mg/kg i.p., 3 h before each immobilisation) did not affect the above responses to 1 immobilisation, increased tremor after 3 immobilisations and also increased forepaw treading, hind limb abduction and Straub tail after 7 immobilisations but decreased head weaving under the latter conditions. Metyrapone without immobilisation had no effect on responses to 5MeODMT. Twenty four hours after 1 or 3 (but not 7) immobilisation periods, rats placed for the first time in an open field showed less locomotion and rearing and more defaecation than control animals. Rats also given metyrapone exhibited normal open field behaviour after only 3 immobilisations. The drug also accelerated the return to normal on repeated immobilisation of the impairment of food intake and growth rate which occurred after a single immobilisation. The results as a whole suggest that metyrapone promotes behavioural adaptation to repeated immobilisation and that this is associated with enhanced postsynaptic responses to <em>5HT</em>. These findings suggest that immobilisation stress-induced changes might be relevant as an animal model for depression which incorporates reported biochemical abnormalities in the illness and is of relevance to proposals concerning its precipitation by stress.

Hyperactivity of the hypothalamic-pituitary-adrenal axis is a characteristic feature of depressive illness. The centrally located corticosteroid receptors, the glucocorticoid and mineralocorticoid receptors, are thought to be important modulators of this axis and changes in the levels of these receptors, particularly in the hippocampus, may underlie the hyperactivity observed. Various antidepressant drugs increase hippocampal mineralocorticoid and glucocorticoid receptor levels in vivo. These effects are thought to be mediated via alterations in monoaminergic neurotransmission. We examined whether serotonin (<em>5HT</em>) and noradrenaline (NA) have direct effects on glucocorticoid receptor and mineralocorticoid receptor expression in primary hippocampal neurones, and whether antidepressants also exert direct effects on target neurones. Exposure of hippocampal cells to <em>5HT</em> for 4 days increased both glucocorticoid and mineralocorticoid receptor mRNA and protein expression. The induction of mineralocorticoid receptor mRNA was completely blocked by the <em>5HT</em>(7) receptor antagonist SB 269970. In contrast glucocorticoid receptor induction was insensitive to the <em>5HT</em>(7) receptor, whilst studies with the <em>5HT</em>(1A) receptor agonist 8-hydroxy-2-(di-n-proplamino) tetralin hydrochloride and the <em>5HT</em>(1A) receptor antagonist N-[2-[4-2-[O-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide trihydrochloride (WAY 100635) suggest a partial role for <em>5HT</em>(1A) receptors in hippocampal glucocorticoid receptor regulation. Treatment with NA for 4 days also increased glucocorticoid receptor expression but had no effect on mineralocorticoid receptor expression. This was blocked by propanolol suggesting action via beta-adrenergic receptors. Similarly to NA, fluoxetine and amitriptyline also selectively increased glucocorticoid receptor mRNA and protein levels over this time course. However, glucocorticoid receptor induction by fluoxetine or amitriptyline was not blocked by WAY 100635 or propanolol. These results show that <em>5HT</em>, NA and antidepressants act directly but via distinct mechanisms on hippocampal neurones to regulate mineralocorticoid and glucocorticoid receptor expression. Thusly, manipulation of neurotransmitter or antidepressant levels in the brain may aid in reversing hypothalamic-pituitary-adrenal axis hyperactivity by restoring hippocampal corticosteroid receptor balance.

BACKGROUND

Enzyme-treated rice fiber (ERF) is a recently developed prebiotic product made from rice bran by heat-resistant amylase, protease and hemicellulase treatment. Although the detailed mechanism of inflammatory bowel disease (IBD) is still unclear, the role of the resident luminal bacteria and its interaction on the mucosal barrier seem to be an important factor in the development of IBD and its chronicity. With the objective of manipulating the intestinal microbiota in IBD, this study was carried out to evaluate the effects of ERF on IBD with using experimental colitis models.

METHODS

Three colitis models were used and they were induced by the oral administration of dextran sodium sulfate in male Sprague-Dawley rats or BALB/c mice and transferring CD4+ CD45RB(high) T cells to female SCID mice, sequentially their CD4+ T cells were retransferred to new SCID mice. The evaluation included the measurement of body weight, spleen weight, colon length, histological examination, serum and mucosal cytokine (tumor necrosis factor-alpha (TNF-α), an interferon-gamma (IFN-γ), interleukin-12 p70 (IL-12p70), IL-1β, IL-6, IL-4) analysis, mucosal serotonin (<em>5HT</em>), and organic acid production and a microbiota analysis of the cecal contents. The characteristics of T cell surface markers including CD4, CD69, CD45RB of spleen and mesenteric lymph nodes (MLN) were also analyzed. In addition, the effects of ERF on the change in the induction of dendritic cells (DCs) were evaluated.

RESULTS

The preventive effect of ERF on colitis was significantly superior to that of raw material rice bran or control group. An overexpression of inflammatory cytokine production was attenuated by ERF treatment, which was accompanied with a decrease in both the colonic mucosal damage and <em>5HT</em> production. Furthermore, ERF significantly attenuated the T cell activation (CD4+CD69+) of spleen and MLN, and this characteristic was inherited by the retransferred mice. ERF significantly suppressed the growth of Clostiridium, and increased short-chain fatty acids (acetate, propionate and butyrate) content in colitis. The relatively hydrophilic fraction of ERF (ethanol-methanol soluble fraction) is therefore considered to have a potent ability to attenuate the induction of DCs.

CONCLUSIONS

A new prebiotic, ERF, reduced inflammation by modulating the colonic environment and regulating immune cell differentiation. Although a more detailed study is required, this study showed the promising anti-inflammatory effects of an adjunctive prebiotic treatment for IBD.

In the mouse forced swimming test (FST) pretreatment with a subactive dose of lithium (1 mEq/kg), given IP 45 min before the test, facilitated the antidepressant activity of iprindole, fluoxetine, and moclobemide (given IP 30 min before the test). These antidepressants (ADS) were not active alone in the FST in this study. Moreover, when subactive lithium was combined with a wide range of ADS, each given at subactive doses, those ADS with serotoninergic properties (e.g. imipramine, citalopram, paroxetine, fluoxetine, trazodone, mianserin, and moclobemide) significantly reduced immobility times. ADS acting primarily on noradrenaline (NA) or dopamine (DA) systems (desipramine, maprotiline, viloxazine, and bupropion) did not significantly decrease immobility when given in combination with lithium. This was also the case for RO 16 6491 [a reversible, B specific monoamine oxidase inhibitor (MAOI)], nialamide, and pargyline (both irreversible, mixed MAOIs). The anti-immobility effect of iprindole in combination with lithium suggests either a direct or indirect action on the serotonin (<em>5HT</em>) system by this ADS whose mechanism of action remains obscure. These results, using an animal behavioral model of depression and combining our present knowledge of the acute action of various ADS, support the hypothesis that the potentiation by lithium of ADS is via direct <em>5HT</em> mechanisms, indirectly via a NA/<em>5HT</em> link, and/or by second messenger systems. Lithium may also facilitate the expression of antidepressant activity of ADS not active by themselves in the FST.

BACKGROUND

Selective attention deficit, characterised by the inability to differentiate relevant from irrelevant information, is considered to underlie many cognitive deficits of schizophrenia, and appears to be only marginally responsive to treatment with current antipsychotics.

OBJECTIVE

We compared the activity of the putative atypical antipsychotic SSR181507 (a dopamine D(2) receptor antagonist and <em>5HT</em>(1A) receptor agonist) with reference compounds, on disturbances of novelty discrimination in a social context in rats, a behavioural paradigm that putatively models selective attention deficit.

METHODS

A first (familiar) juvenile rat was presented to an adult rat for a period (P1) of 30 min. A second (novel) juvenile was then introduced at the end of P1 for a period (P2) of 5 min. The ability of the adult rat to discriminate between the two juveniles, presented at the same time, was evaluated by measuring the ratio of the time spent in interaction with the novel vs the familiar juvenile during P2.

RESULTS

Adult rats spent more time exploring the novel than the familiar juvenile. This novelty discrimination capacity was disrupted by: (1) parametric modification of the procedure (reduction of time spent in contact with the familiar juvenile during P1); (2) acute injection of psychotomimetics that are known to induce schizophrenia-like symptoms in humans, such as phencyclidine (PCP; 3 mg/kg, i.p.) and d-amphetamine (1 mg/kg, i.p.) and (3) neonatal treatment with PCP (three injections of 10 mg/kg, s.c.), a model based on the neurodevelopmental hypothesis of schizophrenia. The potential atypical antipsychotic SSR181507 (0.03-3 mg/kg, i.p.) and the atypical antipsychotics clozapine (0.1-1 mg/kg, i.p.) and amisulpride (1-3 mg/kg, i.p.) attenuated deficits in novelty discrimination produced by parametric manipulation and by acute or neonatal treatment with PCP. The typical antipsychotic haloperidol (up to 0.3 mg/kg, i.p.) attenuated only deficits in novelty discrimination produced by parametric modification.

CONCLUSIONS

Collectively, these results suggest that SSR181507 can alleviate disturbances of novelty discrimination in a social context in rats, and that this paradigm may represent a suitable animal model of selective attention deficits observed in schizophrenia.

Recent studies have shown that G-protein-coupled receptors (GPCRs) can assemble as high molecular weight homo- and hetero-oligomeric complexes. This can result in altered receptor-ligand binding, signaling, or intracellular trafficking. We have co-transfected HEK-293 cells with differentially epitope-tagged GPCRs from different subfamilies and determined whether oligomeric complexes were formed by co-immunoprecipitation and immunoblot analysis. This gave the surprising result that the <em>5HT</em>(1A) receptor was capable of forming hetero-oligomers with all GPCRs tested including the <em>5HT</em>(1B), <em>5HT</em>(1D), EDG(1), EDG(3), GPR(26), and GABA(B2) receptors. The testing of other GPCR combinations showed similar results with hetero-oligomer formation occurring for the <em>5HT</em>(1D) with the <em>5HT</em>(1B) and EDG(1) receptor. Control studies showed that these complexes were present in co-transfected cells before the time of lysis and that the hetero-oligomers were comprised of GPCRs at discrete stoichiometries. These findings suggest that GPCRs have a natural tendency to form oligomers when co-transfected into cells. Future studies should therefore investigate the presence and physiological role of GPCR hetero-oligomers in cells in which they are endogenously expressed.

In emission tomography, quantification of brain tracer uptake, metabolism or binding requires knowledge of the cerebral input function. Traditionally, this is achieved with arterial blood sampling. We propose a noninvasive alternative via the use of a blood vessel time-activity curve (TAC) extracted directly from dynamic positron emission tomography (PET) scans by cluster analysis. Five healthy subjects were injected with the <em>5HT</em>(2A)-receptor ligand [(18)F]-altanserin and blood samples were subsequently taken from the radial artery and cubital vein. Eight regions-of-interest (ROI) TACs were extracted from the PET data set. Hierarchical K-means cluster analysis was performed on the PET time series to extract a cerebral vasculature ROI. The number of clusters was varied from K = 1 to 10 for the second of the two-stage method. Determination of the correct number of clusters was performed by the 'within-variance' measure and by 3D visual inspection of the homogeneity of the determined clusters. The cluster-determined input curve was then used in Logan plot analysis and compared with the arterial and venous blood samples, and additionally with one of the currently used alternatives to arterial blood sampling, the Simplified Reference Tissue Model (SRTM) and Logan analysis with cerebellar TAC as an input. There was a good agreement (P < 0.05) between the values of Distribution Volume (DV) obtained from the K-means-clustered input function and those from the arterial blood samples. This work acts as a proof-of-principle that the use of cluster analysis on a PET data set could obviate the requirement for arterial cannulation when determining the input function for kinetic modelling of ligand binding, and that this may be a superior approach as compared to the other noninvasive alternatives.

To examine whether the hypothalamic corticotropin-releasing hormone (CRH) neuron is regulated by CRH, by products of the proopiomelanocortin (POMC) gene, and/or by glucocorticoids, we used a rat hypothalamic organ culture system in which rat CRH secretion from single explanted hypothalami was evaluated by an RIA (iCRH) specific for rat CRH. The effects of graded concentrations of ovine CRH (oCRH), adrenocorticotropin hormone (ACTH), beta-endorphin (beta-EP), alpha-melanocyte-stimulating hormone (alpha-MSH), corticotropin-like intermediate lobe peptide (CLIP), ovine beta-lipotropin (ovine beta-LPH), and dexamethasone (DEX) upon unstimulated and serotonin- (<em>5HT</em>), acetylcholine- (ACh), and norepinephrine-(NE) stimulated CRH secretion were determined. oCRH and DEX inhibited unstimulated iCRH secretion with ID50 at the 10(-8) M range. ACTH had no detectable suppressive effect at 10(-8) M. oCRH, ACTH, and DEX inhibited <em>5HT</em>-, ACh-, and NE-stimulated iCRH secretion in a dose-dependent fashion. beta-EP, alpha-MSH, and CLIP also inhibited <em>5HT</em>-induced iCRH secretion. Of the latter peptides, the strongest inhibitor was beta-EP and the weakest was CLIP. Ovine beta-LPH had only a weak inhibitory effect on <em>5HT</em>-induced iCRH secretion. Generally, the concentrations required for 50% suppression of neurotransmitter-stimulated iCRH secretion were significantly lower than those required for a similar suppression of unstimulated iCRH secretion. In conclusion, these data suggest the presence of multiple negative feedback loops involved in the regulation of the hypothalamic CRH neuron: an ultrashort CRH-mediated loop, a short, hypothalamic POMC-derived peptide loop, and a long, glucocorticoid-mediated negative feedback loop. The potency of these negative feedback loops may be determined by the state of activation of the CRH neuron.

The purpose of this study was to attempt to reproduce previous findings regarding the antagonist specificity of the <em>5HT</em> autoreceptor and to find additional antagonists of this receptor. Crude synaptosomal preparations of the rat hypothalamus were loaded with [3H]<em>5HT</em>, placed on glass microfiber filters and superfused with modified Krebs--Henseleit buffer at 37 degrees C. The release of [3H]<em>5HT</em> was stimulated by raising the buffer K+ concentration and was Ca2+-dependent. In the presence of 100 nM fluoxetine (a selective <em>5HT</em> uptake inhibitor), exogenous <em>5HT</em> inhibited the K+-induced release of [3H]<em>5HT</em> but did not affected basal [3H]<em>5HT</em> release. The K+-induced [3H]<em>5HT</em> release was maximally inhibited by 30 nM <em>5HT</em> to a level of 66.4 +/- 4.0% of control. The concentration of <em>5HT</em> required to inhibit half-maximally K+-induced [3H]<em>5HT</em> release was approx. 7 nM. Methiothepin and quipazine were found to block the inhibition of K+-induced [3H]<em>5HT</em> release by exogenous <em>5HT</em> (30 nM). The IC50S for blockade of the effects of <em>5HT</em> were approx. 3.8 and 670 nM for methiothepin and quipazine, respectively. Several other putative <em>5HT</em> antagonists, the dopamine receptor antagonist, spiperone and the alpha receptor antagonist, phentolamine, were without effect. Thus, the <em>5HT</em> autoreceptor appears to have a unique specificity for certain <em>5HT</em> antagonists. In addition, blockade of <em>5HT</em> autoreceptors may be one mechanism by which quipazine produces behavioral effects characteristic of a <em>5HT</em> receptor agonist.

Nineteen rats received injections of 5,7-dihydroxytryptamine into the dorsal and median raphe nuclei; 16 rats received sham injections. The rats underwent 50 daily training sessions under an interresponse-time-greater-than-15-seconds (IRT greater than 15 s) schedule of sucrose reinforcement. The lesioned group showed impaired acquisition of temporal differentiation, in that their response rates remained significantly higher and their obtained reinforcement frequencies significantly lower than those of the control (sham-lesioned) group. Comparison of the IRT frequency distributions obtained from the two groups during the last 5 days of training showed that the lesioned group produced a significantly higher proportion of very short IRTs (less than 3 s) than the control group; when these short IRTs were disregarded, the lesioned group displayed a significantly lower mean IRT and a significantly higher coefficient of variation than the control group. The levels of 5-hydroxytryptamine (<em>5HT</em>) and 5-hydroxyindoleacetic acid in the parietal cortex, hippocampus, amygdala, nucleus accumbens and hypothalamus were markedly reduced in the lesioned group, but the levels of noradrenaline and dopamine were not significantly affected by the lesion. The results suggest that destruction of the ascending <em>5HT</em>ergic pathways may reduce animals' capacity to inhibit positively reinforced operant behaviour, and may impair temporal discrimination.

The dorsal raphe (DR) is invested with nitric oxide synthase (NOS)-expressing profiles. To characterize the connections of NO-containing cells and further assess neurochemical relationships maintained by DR, the transmitter identity of the raphe projection to the trigeminal somatosensory system was examined. Rats were injected with retrograde tracer into vibrissae-related target areas or with anterograde tracer into DR. NADPH-diaphorase (NADPHd) histochemistry or NOS-immunostaining was combined with serotonin (<em>5HT</em>)- or serotonin transporter (SERT)-immunolabeling to examine: 1) the presence of NO in <em>5HT</em>-containing axons from DR; 2) the distribution of NO-containing fibers with respect to other nitrergic profiles in the somatosensory system; and 3) the propensity for individual projection neurons in specific subdivisions of DR to colocalize <em>5HT</em> and NO. Results confirm that "barrel-like" patches can be identified in several adult trigeminal relay nuclei by NADPHd histochemistry and demonstrate that fibers from DR contain <em>5HT</em> and NO. Observations include a high percentage of cortical midline projection neurons which contained NADPHd (70-80%) and coexpressed <em>5HT</em>. In contrast, approximately 40% of retrogradely labeled DR-thalamus cells in the lateral wing demonstrated NADPHd or <em>5HT</em> expression, but not both in the same neuron. Colocalization of NADPHd and <em>5HT</em> within individual DR projection neurons indicates that: i) DR is a source of nitrergic input to trigeminal structures, and ii) NO and <em>5HT</em> may be simultaneously released to influence information-processing within somatosensory targets. Disparities in NADPHd expression between retrogradely labeled DR neuronal subpopulations further suggest functional differences in the impact of NO on cortical and subcortical targets.

Site-directed mutagenesis and molecular modeling were used to investigate the molecular interactions involved in ligand binding to, and activation of, the rat 5-hydroxytryptamine(2A) (5-HT(2A)) serotonin (5-HT) receptor. Based on previous modeling studies utilizing molecular mechanics energy calculations and molecular dynamics simulations, four sites (S239[5.43], F240[5.44], F243[5.47], and F244[5.48]) in transmembrane region V were selected, each predicted to contribute to agonist and/or antagonist binding. The F243A mutation increased the affinity of (+/-)4-iodo-2, 5-dimethoxyphenylisopropylamine, decreased the binding of alpha-methyl-<em>5HT</em>, N-omega-methyl-<em>5HT</em>, ketanserin, ritanserin, and spiperone and had no effect on the binding of 5-HT and 5-methyl-N, N-dimethyltryptamine. The F240A mutant had no effect on the binding of any of the ligands tested, whereas F244A caused an agonist-specific decrease in binding affinity (3- to 10-fold). S239A caused a 6- to 13-fold decrease in tryptamine-binding affinity and a 5-fold increase in affinity of 4-iodo-2, 5-dimethoxyphenylisopropylamine. A subset of the agonists used in binding studies were used to determine the efficacies and potencies of these mutants to activate phosphoinositide hydrolysis. The F243A and F244A mutations reduced agonist stimulated phosphoinositide hydrolysis, whereas the S239A and F240A mutations had no effect. There was little correlation between agonist binding and second messenger production. Furthermore, molecular dynamics simulations, considering these data, produced ligand-bound structures utilizing substantially different bonding interactions even among structurally similar ligands (differing by as little as one methyl group). Taken together, these results suggest that relatively minor changes in either receptor or ligand structure can produce drastic and unpredictable changes in both binding interactions and 5-HT(2A) receptor activation. Thus, our finding may have major implications for the future and feasibility of receptor structure-based drug design.

Developmental exposure to unrelated neurotoxicants can nevertheless converge on common final targets so as to exacerbate damage or functional deficits. We examined the effects of developmental exposure to terbutaline, a beta2-adrenergic receptor agonist used to arrest preterm labor, and chlorpyrifos, a widely used organophosphate pesticide, on serotonin (<em>5HT</em>) systems. Treatments were chosen to parallel periods typical of human developmental exposures, terbutaline (10 mg/kg) on postnatal days (PN) 2-5 and chlorpyrifos (5 mg/kg) on PN11-14, with assessments conducted in juvenile and adolescent stages (PN21, PN30 and PN45), comparing each agent alone as well as sequential administration of both. By itself, terbutaline produced persistent <em>5HT</em> presynaptic hyperactivity as evidenced by increased <em>5HT</em> turnover in brain regions containing <em>5HT</em> terminal zones; this effect was similar to that seen in earlier studies with chlorpyrifos administration during the same early postnatal period. Later administration of chlorpyrifos (PN11-14) produced a transient increase in <em>5HT</em> turnover during the juvenile stage, and the sequential exposure paradigm, terbutaline followed by chlorpyrifos, showed a corresponding increase in effect over either agent alone. In combination with our earlier work on <em>5HT</em> receptors, these results indicate that terbutaline is a developmental neurotoxicant that targets the <em>5HT</em> system, findings that lend a mechanistic underpinning to clinical indications of elevated childhood psychiatric disorders in the offspring of women treated with beta-agonist tocolytics. Equally importantly, the interaction between terbutaline and chlorpyrifos suggests that tocolytic therapy may alter the subsequent susceptibility to common environmental toxicants.

Urapidil is a novel antihypertensive agent, chemically related to uracil. Its cardiovascular profile was evaluated in a variety of pharmacological models. Urapidil caused a significant decrease of blood pressure in intact rats, both hypertensive (SHR) and normotensive (WKY), as well as in alpha-glucochloralose-anaesthetized cats. Reflex tachycardia was not observed. An analysis in pithed rats showed that urapidil is an alpha-adrenoceptor blocking drug with an obvious selectivity for postsynaptic alpha 1- over alpha 2-adrenoceptors. The alpha 1-adrenoceptor blocking potency proved quantitatively less than that of prazosin. Experiments on isolated aorta preparations and radioligand binding studies confirmed the selectivity of urapidil for alpha 1- over alpha 2-adrenoceptors. The involvement of urapidil with presynaptic alpha 2-adrenoceptors proved negligible. Urapidil proved to possess modest but significant beta 1-adrenoceptor blocking activity, accompanied by a certain degree of intrinsic sympathomimetic activity (TSA) at the level of the cardiac beta 1-adrenoceptors. No significant interaction with vascular beta 2-adrenoceptors was observed. High doses of urapidil caused pressor effects of a probably unspecific nature; neither alpha-adrenoceptors nor <em>5HT</em>-receptors were involved. When injected into the vertebral artery of the cat, urapidil caused a significant central hypotensive effect which was different from that of clonidine and related drugs, since it could not be blocked by yohimbine (alpha 2-receptor antagonist). Similarly, the modest sedation produced by urapidil in mice remained uninfluenced by yohimbine. The urapidil molecule does not contain any stereoisomers. Accordingly, one and the same molecule possesses the following pharmacodynamic properties: postsynaptic alpha 1-adrenoceptor blockade; weak postsynaptic alpha 2-adrenoceptor blockade; modest but selective beta 1-adrenoceptor blockade with ISA; central hypotensive activity not mediated by central alpha 2-adrenoceptors.

OBJECTIVE

Alzheimer's disease (AD) patients suffer from behavioural and psychological symptoms of dementia (BPSD). A genetic component to BPSD development in AD has been demonstrated. Polymorphisms within serotonin receptors <em>5HT</em>(2A) and <em>5HT</em>(2C) have been previously investigated in a few interesting studies reviewed here, however, their role remains unclear.

METHODS

Our large cohort of 394 patients had longitudinal information on the BPSD (Neuropsychiatric Inventory), which was used to dichotomise patients into whether they had ever suffered from a given symptom within the study period and give each patient a severity score. These measures were related to the <em>5HT</em>(2A) T102C and <em>5HT</em>(2C) cys23ser genotype and allele frequencies.

RESULTS

Our data supports previous reports of an increased frequency of the C allele and CC genotype of the T102C variant of <em>5HT</em>(2A) with hallucinations, delusions, psychosis and aberrant motor behaviour, however, we dispute previous associations with depression and aggression. We describe for the first time an increase in the C allele and CC genotype frequencies of the cys23ser variant of <em>5HT</em>(2C) with anxiety and support previous associations with appetite disturbances in females.

CONCLUSIONS

This review and extension of previous data presents support for the role of <em>5HT</em>(2A) and <em>5HT</em>(2C) in the development of certain symptoms, although the effect size may be small.

GAP-43 has been implicated in axonal pathfinding and sprouting, synaptic plasticity, and neurotransmitter release. However, its effect on cortical development in vivo is poorly understood. We have previously shown that GAP-43 knockout (-/-) mice fail to develop whisker-related barrels or an ordered whisker map in the cortex. Here we used cytochrome oxidase (CO) histochemistry to demonstrate that GAP-43 heterozygous (+/-) mice develop larger than normal barrels at postnatal day 7 (P7), despite normal body and brain weight. Using serotonin transporter (<em>5HT</em>-T) histochemistry to label thalamocortical afferents (TCAs), we found no obvious abnormalities in other somatosensory areas or primary visual cortex of GAP-43 (+/-) mice. However, TCA projections to (+/-) primary auditory cortex were not as clearly defined. To clarify the mechanism underlying the large-barrel phenotype, we used lipophilic (DiI) axon labeling. We found evidence for multiple pathfinding abnormalities among GAP-43 (+/-) TCAs. These axons show increased fasciculation within the internal capsule, as well as abnormal turning and branching in the subcortical white matter. These pathfinding errors most likely reflect failures of signal recognition and/or transduction by ingrowing TCAs. In addition, many DiI-labeled (+/-) TCAs exhibit widespread, sparsely branched terminal arbors in layer IV, reflecting the large-barrel phenotype. They also resemble those found in rat barrel cortex deprived of whisker inputs from birth, suggesting a failure of activity-dependent synaptogenesis and/or synaptic stabilization in (+/-) cortex. Our findings suggest that reduced GAP-43 expression can alter the fine-tuning of a cortical map through a combination of pathfinding and synaptic plasticity mechanisms.

Multiple administrations of methamphetamine (METH) rapidly decreased serotonin (<em>5HT</em>) transporter (SERT) function in rat striatum and hippocampus. The purpose of this study was to identify the mechanisms/ factors contributing to this METH-induced decrease in SERT function. Multiple high-dose METH injections rapidly decreased <em>5HT</em> uptake without altering binding of the <em>5HT</em> transporter ligand paroxetine. Hyperthermia contributed to this deficit in transporter function in striatum and hippocampus, as prevention of METH-induced hyperthermia attenuated this decrease. A role for dopamine (DA) was suggested by findings that pretreatment with the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine, the D1 antagonist SCH-23390, or the D2 antagonist eticlopride attenuated the METH-induced decrease in striatal, but not hippocampal, SERT activity. These effects were independent of the ability of these DA-antagonizing drugs to prevent METH-induced hyperthermia. These results suggest that DA contributes to the decrease in SERT function caused by multiple METH injections in the striatum, but not hippocampus, and that hyperthermia facilitates these deficits in SERT function in both brain regions. In contrast, the response of SERT to a single administration of METH was DA and hyperthermia independent. These findings suggest that the mechanisms/ factors involved in decreasing SERT activity after a single administration of METH are distinct from that caused by multiple administrations.

Sexual dysfunction (SD) is a common and underestimated effect of antidepressants. Healthy volunteers are the most adequate group to study this adverse event avoiding influence of depression itself. Sexual acceptability of agomelatine (a melatonergic agonist and <em>5HT</em>(2C) antagonist) paroxetine and placebo by using the Psychotropic-Related Sexual Dysfunction Salamanca Sex Questionnaire (PRSEXDQ-SALSEX) was explored. A total of 92 healthy male volunteers were randomised to agomelatine (25 or 50 mg), paroxetine 20 mg or placebo for 8 weeks. SD, defined as at least one sexual impairment in one of the following PRSEXDQ-SALSEX items (decreased libido, delayed orgasm/ejaculation, anorgasmia/no ejaculation and erectile dysfunction), was evaluated at baseline and after 2, 4 and 8 weeks. At the last post-baseline assessment, SD was significantly lower in each agomelatine group (22.7% on 25 mg and 4.8% on 50 mg) than in the paroxetine group (85.7%; p < 0.0001). In the placebo group, 8.7% of volunteers reported a SD. The percentages of volunteers with moderate or severe SD were 4.5% for agomelatine 25 mg, 4.8% for agomelatine 50 mg, 61.9% for paroxetine 20 mg and 0% in the placebo group (p < or = 0.0001 agomelatine versus paroxetine). There is a much lower risk of having SD with agomelatine than paroxetine in healthy male volunteers, which confirms the better sexual acceptability profile of agomelatine compared with the SSRIs.

Monoamine serotonin (<em>5HT</em>) has been linked to aggression for many years across species. However, elaboration of the neurochemical pathways that govern aggression has proven difficult because monoaminergic neurons also regulate other behaviors. There are approximately 100 serotonergic neurons in the Drosophila nervous system, and they influence sleep, circadian rhythms, memory, and courtship. In the Drosophila model of aggression, the acute shut down of the entire serotonergic system yields flies that fight less, whereas induced activation of <em>5HT</em> neurons promotes aggression. Using intersectional genetics, we restricted the population of <em>5HT</em> neurons that can be reproducibly manipulated to identify those that modulate aggression. Although similar approaches were used recently to find aggression-modulating dopaminergic and Fru(M)-positive peptidergic neurons, the downstream anatomical targets of the neurons that make up aggression-controlling circuits remain poorly understood. Here, we identified a symmetrical pair of serotonergic PLP neurons that are necessary for the proper escalation of aggression. Silencing these neurons reduced aggression in male flies, and activating them increased aggression in male flies. GFP reconstitution across synaptic partners (GRASP) analyses suggest that <em>5HT</em>-PLP neurons form contacts with <em>5HT</em>1A receptor-expressing neurons in two distinct anatomical regions of the brain. Activation of these <em>5HT</em>1A receptor-expressing neurons, in turn, caused reductions in aggression. Our studies, therefore, suggest that aggression may be held in check, at least in part, by inhibitory input from <em>5HT</em>1A receptor-bearing neurons, which can be released by activation of the <em>5HT</em>-PLP neurons.