Behavioral response to emotional stress in rabbits: role of serotonin and serotonin2A receptors

Exposure to a novel environment is a stressor which modulates behavior, increases stress hormones and enhances the release of several neurotransmitters including serotonin (5-HT). Exposing rabbits to a novel environment significantly increases head-bob behavior but fails to alter either grooming or wet dog shakes compared with those observed in the home-cage. The goal of this study was to determine the role of 5-HT and its receptors in mediating novelty-elicited head-bob behavior. Reduction of central 5-HT levels after treatment with the serotonergic neurotoxin 5,7-DHT significantly decreased novelty-elicited head bobs by 40% compared with those in sham-lesioned rabbits, indicating that 5-HT mediates, in part, this behavior. Additionally, pretreatment with the 5-HT1A partial agonist and clinically used anxiolytic buspirone also significantly attenuated novelty-elicited head bobs. Pretreatment with the selective 5-HT2A antagonist M 100,907 significantly reduced novel environment-elicited head bobs by 40%. Furthermore, agonist-induced reduction of cortical 5-HT2A receptor density resulted in a significant 40% reduction in the number of head bobs elicited by the novel environment. These data demonstrate that rabbit head-bob behavior, an index of the response to novelty stress, is mediated, in part, by 5-HT activation of 5-HT2A receptors.     

Interactions of selective serotonin reuptake inhibitors with the serotonin 5-HT2C receptor

Interactions of the selective serotonin reuptake inhibitors (SSRIs) citalopram, fluoxetine and its main metabolite norfluoxetine, and the tricyclic anti-depressant (TCA) imipramine with the rat serotonin 5-HT_2C receptor in a clonal cell line and in the rat choroid plexus were investigated by radioligand binding and phosphoinositide (PI) hydrolysis assays. For comparison, the affinities of a variety of other antidepressants of different chemical classes for the cloned rat 5-HT_2C and 5-HT_2A receptors were also determined by radioligand binding assays. Fluoxetine displayed relatively high affinity for the 5-HT_2C receptor in the choroid plexus, with a K_i value for inhibition of [³H]mesulergine binding of 55.4 nM. The K_i values for imipramine, norfluoxetine and citalopram were 136 nM, 203 nM, and 298 nM, respectively. Similar rank order of potency was detected in PI hydrolysis assays, which showed that these drugs are antagonists at the 5-HT_2C receptor without exhibiting inverse agonist activity. [³H]Ketanserin (5-HT_2A) binding assays revealed that the SSRIs fluoxetine, norfluoxetine and citalopram show 10- to 23-fold selectivity for the 5-HT_2C receptor in vitro, whereas the TCA imipramine does not. Many other TCAs also had high to intermediate affinity for both 5-HT_2A and 5-HT_2C receptors. The present data provide evidence that fluoxetine, norfluoxetine and citalopram, along with many other antidepressant compounds, interact directly with the 5-HT_2C receptor.     

Cannabinoid 2 receptor- and beta Arrestin 2-dependent upregulation of serotonin 2A receptors

Recent evidence suggests that cannabinoid receptor agonists may regulate serotonin 2A (5-HT_2A) receptor neurotransmission in the brain, although no molecular mechanism has been identified. Here, we present experimental evidence that sustained treatment with a non-selective cannabinoid agonist (CP55,940) or selective CB2 receptor agonists (JWH133 or GP1a) upregulate 5-HT_2A receptors in a neuronal cell line. Furthermore, this cannabinoid receptor agonist-induced upregulation of 5-HT_2A receptors was prevented in cells stably transfected with either CB2 or β-Arrestin 2 shRNA lentiviral particles. Additionally, inhibition of clathrin-mediated endocytosis also prevented the cannabinoid receptor-induced upregulation of 5-HT_2A receptors. Our results indicate that cannabinoid agonists might upregulate 5-HT_2A receptors by a mechanism that requires CB2 receptors and β-Arrestin 2 in cells that express both CB2 and 5-HT_2A receptors. 5-HT_2A receptors have been associated with several physiological functions and neuropsychiatric disorders such as stress response, anxiety and depression, and schizophrenia. Therefore, these results might provide a molecular mechanism by which activation of cannabinoid receptors might be relevant to some cognitive and mood disorders in humans.     

Fe2+-mediated binding of serotonin and dopamine to skeletal muscle actin: resemblance to serotonin binding proteins

Fe²⁺ stimulates the binding of [³H]serotonin and [³H]dopamine to rabbit skeletal muscle actin. This binding is inhibited by reducing agents (sodium ascorbate, vitamin E), by superoxide dismutase and by sulfhydryl group-modifying reagents (N-ethylmaleimide, 2,2′-dinitro-5,5′-dithiobenzoic acid). The effect of Fe²⁺ is mimicked by oxidants (sodium periodate, potassium nitroso-disulfonate) and by superoxide radicals. Once formed, the binding cannot be decreased by a large excess of monoamine. It is proposed that Fe²⁺ catalyses the autoxidation of the monoamines by generating oxygen free radicals, and the oxidation products are likely to bind covalently to exposed cysteine residues of actin. Digestion of [³H]dopamine-labelled actin by cyanogen bromide and then by V8 protease(EC3.4.21.19) yields two labelled peptides whose apparent molecular weights (4.1 and 1.2 kDa) are compatible with the labelling of cysteine-10 and -374.Fe²⁺ also inactivates some of the binding sites on actin. This inactivation, and the covalent nature of the binding precludes the interpretation of monoamine saturation and competition binding data in terms of reversible bimolecular interactions.     

The ratios of serotonin2 and dopamine2 affinities differentiate atypical and typical antipsychotic drugs

Atypical antipsychotic drugs such as clozapine, fluperlapine, and melperone produce weak catalepsy in rodents, and minimal extrapyramidal symptoms and serum prolactin elevations in humans, compared to typical antipsychotic drugs such as haloperidol. The biological basis for these differences has been attributed to relatively weak blockade at D2 dopamine (DA) receptors, various effects at D1 receptors, or potent serotonin2 (5-HT2) antagonism, although other possibilities exist. To clarify the relative importance of actions at D1, D2, and 5-HT2 receptors for identification of candidate typical and atypical drugs, we determined the negative log of the Ki (pKi) value of 21 typical and 17 atypical antipsychotic drugs for the striatal D1 and D2 and frontal cortex 5-HT2 receptors of rodent brain. Cluster analysis identified that the 5-HT2/D2 ratio was the most successful means of utilizing this data to classify typical and atypical antipsychotic drugs correctly. A 92 percent accuracy was achieved.     

Functional crosstalk and heteromerization of serotonin 5-HT2A and dopamine D2 receptors

The serotonin 5-HT_2A receptor (5-HT_2AR) and dopamine D₂ receptor (D₂R) are high-affinity G protein-coupled receptor targets for two different classes of antipsychotic drugs used to treat schizophrenia. Interestingly, the antipsychotic effects are not based on the regulation of same signaling mediators since activation of the 5-HT_2AR and of the D₂R regulate G_q/11 protein and G_i/o protein, respectively. Here we use radioligand binding and second messenger production assays to provide evidence for a functional crosstalk between 5-HT_2AR and D₂R in brain and in HEK293 cells. D₂R activation increases the hallucinogenic agonist affinity for 5-HT_2AR and decreases the 5-HT_2AR induced inositol phosphate production. In vivo, 5-HT_2AR expression is necessary for the full effects of D₂R antagonist on MK-801-induced locomotor activity. Co-immunoprecipitation studies show that the two receptors can physically interact in HEK293 cells and raise the possibility that a receptor heterocomplex mediates the crosstalk observed. The existence of this 5-HT_2AR-D₂R heteromer and crosstalk may have implications for diseases involving alterations of serotonin and dopamine systems and for the development of new classes of therapeutic drugs.     

RNA editing of serotonin 2C receptor and major mental disorders

A-to-I RNA editing has mainly been found in various receptors and ion channels in the central nervous system, including the serotonin 2C receptor, glutamate receptor, GABA receptor, and potassium channel. Interestingly, most of them are suggested to be involved in the pathophysiology of major mental disorders such as schizophrenia, bipolar disorder, and major depression. Here we review studies examining the relationship between the serotonin 2C receptor and major mental disorders.     

Down-regulation of serotonin2, but not of beta-adrenergic receptors during chronic treatment with amitriptyline is independent of stimulation of serotonin2 and beta-adrenergic receptors

Antidepressant drugs down-regulate beta-adrenergic, alpha₂-adrenergic and serotonergic 5-HT₂ receptors with a time course that parallels their clinical efficacy, i.e. chronic administration is required (Crews and Smith, 1978; Svensson and Usdin, 1978; Banerjee, Kung, Riggi and Chanda, 1979; Bergstrom and Keller, 1979; Peroutka and Snyder, 1980). In the present study, it was found that the 5-HT₂ receptor antagonist, nefazadone (50 mg/kg per day) did not prevent the downregulation of 5-HT₂ receptors in the cerebral cortex produced by amitriptyline (10 mg/kg per day), when administered for 3 weeks. Moreover, treatment with nefazadone (50 mg/kg per day) alone for 3 weeks decreased binding to 5-HT₂ receptors in cerebral cortex. In contrast, administration of propranolol, the beta receptor antagonist, (10 mg/kg per day) with amitriptyline (10 mg/kg per day) for 3 weeks prevented the down-regulation of beta receptors, but did not alter the decrease in binding to 5-HT₂ receptors. In addition, the depletion of central stores of norepinephrine and serotonin by a 4-day treatment with reserpine (5 mg/kg per day) increased binding to beta receptors in the cerebral cortex and hippocampus, but did not affect binding to 5-HT₂ receptors in either region. These results suggest that the 5-HT₂ receptor is not down-regulated by direct stimulation by serotonin agonists and that the down-regulation of 5-HT₂ receptors by amitriptyline is independent of down-regulation of beta-adrenergic receptors.     

The neurotoxin 2'-NH2-MPTP degenerates serotonin axons and evokes increases in hippocampal BDNF

1-Methyl-4-(2'-aminophenyl)-1,2,3,6-tetrahydropyridine (2'-NH2-MPTP) causes long-term depletions in cortical and hippocampal serotonin (5-HT) and norepinephrine (NE) that are accompanied by acute elevations in glial fibrillary acidic protein (GFAP) and argyrophilia. To further investigate the hypothesis that these changes are reflective of serotonergic and noradrenergic axonal degeneration, 2'-NH2-MPTP was administered to mice and innervation densities were determined immunocytochemically. Regional responses of the neurotrophin, brain-derived neurotrophic factor (BDNF), to putative damage were also assessed. Three days after 2'-NH2-MPTP, 5-HT axons exhibited a beaded, tortuous appearance indicative of ongoing degeneration. At 21 days, numbers of serotonin axons were significantly decreased, with the greatest axonal losses occurring in cortex and hippocampus. Serotonin axons in the amygdala were contrastingly spared long-term damage, as were 5-HT and NE cell bodies in the brain stem. BDNF protein levels were selectively increased in the hippocampus 3 days post-dose and returned to normal 21 days later. These results, in conjunction with previous findings, demonstrate that 2'-NH2-MPTP causes degeneration of serotonergic axons innervating the cortex and hippocampus on par with depletions in neurotransmitter levels. Moreover, damage to the hippocampus, a brain region important for learning and memory, and the modulation of anxiety and stress responsiveness, results in a transitory increase in BDNF.     

Binding and function of serotonin 2 receptors following chronic administration of imipramine

Experiments were undertaken to determine whether alterations in the binding of 5-HT2 receptors in the cerebral cortex of rat brain, induced by antidepressant drugs, are related to behavioral changes observed in response to the administration of a precursor of serotonin. Chronic (5 days), but not acute, administration of an imipramine-yohimbine combination resulted in a significant decrease in the binding of 5-HT2 receptors in the frontal cortex and wet-dog shakes induced by 5-hydroxytryptophan (5-HTP). The decrease in the binding to 5-HT2 receptors was due entirely to a change in the concentration of binding sites with no modification in receptor affinity. The behavioral alteration resulted from a decrease in the maximal response to 5-HTP. Further experiments revealed that chronic (5 days) administration of imipramine alone altered the behavioral response to 5-HTP without changing the binding of 5-HT2 receptor binding and that changes in 5-HT2 binding in the cortex were not always accompanied by an alteration in the response to 5-HTP. These results suggest a lack of correlation between the drug-induced change in 5-HT2 binding in frontal cortex and the function of serotonin 2 receptors.     

Anaphylactic bronchoconstriction in BP2 mice: interactions between serotonin and acetylcholine

1. Immunized BP2 mice developed an acute bronchoconstriction in vivo and airway muscle contraction in vitro in response to ovalbumin (OA) and these contractions were dose dependent. 2. Methysergide or atropine inhibited OA-induced bronchoconstriction in vivo and airway muscle contraction in vitro. 3. Neostigmine potentiated the OA-induced bronchoconstriction in vivo and airway muscle contraction in vitro of BP2 mice. This potentiation was markedly reduced by the administration of methysergide or atropine and when the two antagonists were administered together, the responses were completely inhibited. 4. Neostigmine also potentiated the serotonin (5-HT)- and acetylcholine (ACh)-induced bronchoconstriction and this potentiation was significantly reversed by atropine. 5. These results indicate that OA provokes a bronchoconstriction in immunized BP2 mice by stimulating the release of 5-HT, which in turn acts via the cholinergic mediator, ACh.     

The serotonin 5-HT2A receptor agonist TCB-2: a behavioral and neurophysiological analysis

Rationale  

There are few reports on the high-affinity 5-HT_2A agonist (4-Bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide (TCB-2).     

Selective agents for serotonin2C (5-HT2C) receptor

The serotonin2C (5-HT2C) receptor has attracted a lot of attention owing to its role in appetite regulation, depression, obsessive-compulsive disorder (OCD), panic disorders, and substance abuse. This review summarizes non-patent and patent literature up to November 2005 that deals with the synthesis and characterization of selective 5-HT2C receptor agonists and antagonists. Highlights on structure-activity relationships have been included, when possible.     

Effects of selective serotonin and serotonin/noradrenaline reuptake inhibitors on extracellular serotonin in rat diencephalon and frontal cortex

Some clinical reports suggest that tricyclic antidepressants which block both noradrenaline and serotonin (5-HT) reuptake (SNRIs) are more effective than selective 5-HT reuptake inhibitors (SSRIs) in treating severe depression. Moreover, one neurochemical study reported larger increases in extracellular 5-HT in rat frontal cortex in response to the tricyclic antidepressant imipramine compared to the SSRI fluoxetine. However, imipramine, which blocks both 5-HT and noradrenaline reuptake, also binds with relatively high affinity to receptors for noradrenaline, histamine and acetylcholine. Thus, to test the hypothesis that compounds that inhibit both 5-HT and noradrenaline reuptake produce larger increases in 5-HT efflux, we compared the effects of acute systemic administration of several SNRIs and SSRIs. Extracellular 5-HT was measured using microdialysis probes implanted in the diencephalon and frontal cortex of unanesthetized rats. We tested the SSRIs paroxetine (0.3-10 mg/kg), citalopram (10-20 mg/kg) and fluoxetine (10 mg/kg), the nonselective tricyclic antidepressant imipramine (20 mg/kg) and the more selective SNRIs duloxetine (3-30 mg/kg) and venlafaxine (30-50 mg/kg). During the lights-off period, paroxetine and duloxetine increased 5-HT in the diencephalon approximately 300 and approximately 200%, respectively. During the lights-on period, paroxetine and duloxetine each increased 5-HT approximately 400% in the diencephalon. In the frontal cortex, both paroxetine and duloxetine increased 5-HT approximately 200%. Citalopram and venlafaxine each increased 5-HT in the diencephalon approximately 300%. Fluoxetine and imipramine increased 5-HT in the diencephalon by approximately 125 and approximately 80%, respectively. Thus, these results do not support the hypothesis that compared to SSRIs, compounds which inhibit both 5-HT and noradrenaline reuptake have a larger acute effect on extracellular 5-HT.     

2-Iodoimipramine, a novel ligand for the serotonin transporter

Iodoimipramine was synthesized by iodinating imipramine with ICI. Iodoimipramine competitively inhibits [3H]imipramine binding with a KI of 0.52 nM and also inhibits [3H]serotonin transport competitively, suggesting that serotonin, imipramine, and iodoimipramine all bind to the same site on the serotonin transporter. Association of [125I]iodoimipramine to platelet membranes in Na+ requires 20 min to reach equilibrium at 25 degrees and 1.5 hr at 0 degrees. [125I] Iodoimipramine binding at equilibrium is saturable and Na+ dependent, with a KD of 0.58 nM and a Bmax of 1.3 pmol/mg at 25 degrees. Serotonin competitively inhibits [125I]iodoimipramine binding, with a KI of 1.3 microM. [125I]Iodoimipramine bound at 0 degrees in the presence of Na+ does not dissociate unless the temperature is raised or Na+ is removed from the medium. At 25 degrees, dissociation of [125I] iodoimipramine from platelet membranes in the presence of Na+ is only partial, with 40% of the ligand remaining persistently bound over 5 hr after a 50-fold dilution.