Serotonin 5-HT2A receptor expression and functionality in postmortem frontal cortex of subjects with schizophrenia: Selective biased agonism via Gαi1-proteins.

Serotonin 5-HT2A receptors (5-HT2ARs) have been implicated in schizophrenia. However, postmortem studies on 5-HT2ARs expression and functionality in schizophrenia are scarce. The 5-HT2AR mRNA and immunoreactive protein expression were evaluated in postmortem tissue from dorsolateral prefrontal cortex (DLPFC) of antipsychotic-free (n = 18) and antipsychotic-treated (n = 9) subjects with schizophrenia, and matched controls (n = 27). Functional coupling of 5-HT2AR to G-proteins was tested by measuring the activation induced by the agonist (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride ((±)DOI) in antibody-capture [35S]GTPγS scintillation proximity assays (SPA). In antipsychotic-free schizophrenia subjects, 5-HT2AR mRNA expression and protein immunoreactivity in total homogenates was similar to controls. In contrast, in antipsychotic-treated schizophrenia subjects, lower mRNA expression (60±9% vs controls) and a trend to reduced protein immunoreactivity (86±5% vs antipsychotic-free subjects) just in membrane-enriched fractions was observed. [35S]GTPγS SPA revealed a significant ~6% higher stimulation of Gαi1-protein by (±)DOI in schizophrenia, whereas activation of the canonical Gαq/11-protein pathway by (±)DOI remained unchanged. Expression of Gαi1- and Gαq/11-proteins did not differ between groups. Accordingly, in rats chronically treated with clozapine, but not with haloperidol, a 30-40% reduction was observed in 5-HT2AR mRNA expression, 5-HT2AR protein immunoreactivity and [3H]ketanserin binding in brain cortical membranes. Overall, the data suggest a supersensitive 5-HT2AR signaling through inhibitory Gαi1-proteins in schizophrenia. Together with previous results, a dysfunctional pro-hallucinogenic agonist-sensitive 5-HT2AR conformation in postmortem DLPFC of subjects with schizophrenia is proposed. Atypical antipsychotic treatment would contribute to counterbalance this 5-HT2AR supersensitivity by reducing receptor expression.

Effect of photoperiodic alterations on depression-like behavior and the brain serotonin system in mice genetically different in tryptophan hydroxylase 2 activity.

Reduction of natural illumination in fall/winter months causes seasonal affective disorders (SAD) in vulnerable individuals. Neurotransmitter serotonin (5-HT) is involved in the mechanism of SAD. Tryptophan hydroxylase-2 (TPH2) is the key enzyme of 5-HT synthesis in the brain. C1473 G polymorphism in the Tph2 gene is a key factor defining the enzyme activity in the mouse brain. The main aims of the study were to investigate the effects of C1473 G polymorphism on behavior and brain 5-HT system responses to photoperiod alterations. The experiment was carried out on adult mouse males of B6-1473C and B6-1473 G congenic lines with normal and low TPH2 activities, respectively. B6-1473C and B6-1473 G mice were divided into four groups of 8 each and exposed for 28 days to standard-day (14 h light and 10 h darkness) or short-day (4 h light and 20 h darkness) conditions. No effect of photoperiod on locomotor, exploratory activities and anxiety in the open field test was observed. At the same time, photoperiod alterations affected depressive-like immobility in the forced swim test, the 5-HT, 5-hydroxyindoleacetic acid (5-HIAA) levels, 5-HIAA/5-HT ratio and the Htr2a mRNA level in hippocampus and midbrain. The effect of the interaction between C1473 G polymorphism and photoperiod on 5-HT level and 5-HIAA/5-HT ratio in hippocampus was revealed. Short-day conditions reduced the level and increased 5-HIAA/5-HT ratio in this structure only in B6-1473 G mice. At the same time, C1473 G polymorphism does not alter effects of short-day conditions on immobility time in the forced swim test and the Htr2a mRNA level in the brain.

The Effects of Hallucinogens on Gene Expression.

The classic serotonergic hallucinogens, or psychedelics, have the ability to profoundly alter perception and behavior. These can include visual distortions, hallucinations, detachment from reality, and mystical experiences. Some psychedelics, like LSD, are able to produce these effects with remarkably low doses of drug. Others, like psilocybin, have recently been demonstrated to have significant clinical efficacy in the treatment of depression, anxiety, and addiction that persist for at least several months after only a single therapeutic session. How does this occur? Much work has recently been published from imaging studies showing that psychedelics alter brain network connectivity. They facilitate a disintegration of the default mode network, producing a hyperconnectivity between brain regions that allow centers that do not normally communicate with each other to do so. The immediate and acute effects on both behaviors and network connectivity are likely mediated by effector pathways downstream of serotonin 5-HT2A receptor activation. These acute molecular processes also influence gene expression changes, which likely influence synaptic plasticity and facilitate more long-term changes in brain neurochemistry ultimately underlying the therapeutic efficacy of a single administration to achieve long-lasting effects. In this review, we summarize what is currently known about the molecular genetic responses to psychedelics within the brain and discuss how gene expression changes may contribute to altered cellular physiology and behaviors.

Differences in 5-HT2A and mGlu2 Receptor Expression Levels and Repressive Epigenetic Modifications at the 5-HT2A Promoter Region in the Roman Low- (RLA-I) and High- (RHA-I) Avoidance Rat Strains.

The serotonin 2A (5-HT2A) and metabotropic glutamate 2 (mGlu2) receptors regulate each other and are associated with schizophrenia. The Roman high- (RHA-I) and the Roman low- (RLA-I) avoidance rat strains present well-differentiated behavioral profiles, with the RHA-I strain emerging as a putative genetic rat model of schizophrenia-related features. The RHA-I strain shows increased 5-HT2A and decreased mGlu2 receptor binding levels in prefrontal cortex (PFC). Here, we looked for differences in gene expression and transcriptional regulation of these receptors. The striatum (STR) was included in the analysis. 5-HT2A, 5-HT1A, and mGlu2 mRNA and [3H]ketanserin binding levels were measured in brain homogenates. As expected, 5-HT2A binding was significantly increased in PFC in the RHA-I rats, while no difference in binding was observed in STR. Surprisingly, 5-HT2A gene expression was unchanged in PFC but significantly decreased in STR. mGlu2 receptor gene expression was significantly decreased in both PFC and STR. No differences were observed for the 5-HT1A receptor. Chromatin immunoprecipitation assay revealed increased trimethylation of histone 3 at lysine 27 (H3K27me3) at the promoter region of the HTR2A gene in the STR. We further looked at the Akt/GSK3 signaling pathway, a downstream point of convergence of the serotonin and glutamate system, and found increased phosphorylation levels of GSK3ß at tyrosine 216 and increased ß-catenin levels in the PFC of the RHA-I rats. These results reveal region-specific regulation of the 5-HT2A receptor in the RHA-I rats probably due to absence of mGlu2 receptor that may result in differential regulation of downstream pathways.

Monoaminergic descending pathways contribute to modulation of neuropathic pain by increasing-intensity treadmill exercise after peripheral nerve injury.

This study characterizes the impact of increasing-intensity treadmill exercise (iTR) on noradrenergic (NE) and serotonergic (5HT) modulation of neuropathic pain. Following sciatic nerve transection and repair (SNTR) rats developed significant mechanical and thermal hyperalgesia that was partially prevented by iTR performed during the first 2weeks after injury. Marked decrease in the expression of 5HT2A and α1A and ß-, but not α2A adrenergic receptors in the spinal cord dorsal horn was associated to SNTR and recovered by iTR, particularly in lamina II. iTR significantly increased 5HT2A in periaqueductal grey (PAG), raphe magnus (RM) and dorsal raphe nucleus (DRN), with a pattern suggesting reorganization of serotonergic excitatory interconnections between PAG and DRN. iTR also increased the expression of α1A in locus coeruleus (LC) and DRN, and ß2 in LC, indicating that exercise enhanced activity of NE neurons, likely by activating autologous projections from DRN and PAG. iTR hypoalgesia was antagonized by blockade of ß2 and 5HT2A receptors with administration of butoxamine and ketanserin. The neurotoxin DSP4 was injected to induce depletion of NE projections from LC before starting iTR. DSP4 treatment worsened mechanical hyperalgesia, but iTR hypoalgesia was similarly produced. Moreover, 5HT2A expression in LC further increased after DSP4 injection, all these results suggesting an intrinsic regulation of 5HT and NE activity between PAG, DRN and LC neurons activated by iTR. Finally, iTR significantly reduced microglial reactivity in LC and increased non-microglial BDNF expression, an effect that was reverted by butoxamine, implicating BDNF regulation in central 5HT/NE actions on neuropathic pain.

Characterization of the adrenocorticotrophic hormone - induced mouse model of resistance to antidepressant drug treatment.

Approximately 30-60% of patients treated with existing antidepressants fail to achieve remission of depressive symptoms leading to Treatment Resistant Depression (TRD). There is an urgent need to develop novel medications, which is highly limited by the non-availability of relevant animal models with good predictive validity. ACTH administration has been shown to result in the resistance to acute and chronic effects of imipramine. However, the pharmacology of the model and the mechanisms contributing to the resistance are not completely understood. Furthermore, it is not known whether the ACTH administered animals show signs of depression-like behavior. Accordingly, we characterized the behavioral profile and sensitivity to antidepressants in BALB/c mice treated with ACTH and to evaluate some of the mechanisms responsible for the behavioral effects. Daily treatment with ACTH for 14, 21 or 28days failed to produce a depression-like phenotype in the sucrose preference test, voluntary wheel running or FST. In contrast, the acute antidepressant response in the FST was no longer observed in ACTH mice treated with fluoxetine, imipramine, duloxetine or bupropion. Interestingly, the combination of fluoxetine and a low dose of olanzapine, or the combination of fluoxetine and bupropion was efficacious in ACTH treated mice. Further, the sensitivity to a GluN2B receptor antagonist, radiprodil was retained in the ACTH model. To understand the mechanism responsible for the diminished response in these mice, we evaluated p11 (S100A10) mRNA expression and 5-HT2A protein expression. p11 expression was decreased and 5-HT2A protein content increased in ACTH treated mice. In summary, this model may have utility for the identification of novel treatments for TRD.

Effects of gravity changes on gene expression of BDNF and serotonin receptors in the mouse brain.

Spaceflight entails various stressful environmental factors including microgravity. The effects of gravity changes have been studied extensively on skeletal, muscular, cardiovascular, immune and vestibular systems, but those on the nervous system are not well studied. The alteration of gravity in ground-based animal experiments is one of the approaches taken to address this issue. Here we investigated the effects of centrifugation-induced gravity changes on gene expression of brain-derived neurotrophic factor (BDNF) and serotonin receptors (5-HTRs) in the mouse brain. Exposure to 2g hypergravity for 14 days showed differential modulation of gene expression depending on regions of the brain. BDNF expression was decreased in the ventral hippocampus and hypothalamus, whereas increased in the cerebellum. 5-HT1BR expression was decreased in the cerebellum, whereas increased in the ventral hippocampus and caudate putamen. In contrast, hypergravity did not affect gene expression of 5-HT1AR, 5-HT2AR, 5-HT2CR, 5-HT4R and 5-HT7R. In addition to hypergravity, decelerating gravity change from 2g hypergravity to 1g normal gravity affected gene expression of BDNF, 5-HT1AR, 5-HT1BR, and 5-HT2AR in various regions of the brain. We also examined involvement of the vestibular organ in the effects of hypergravity. Surgical lesions of the inner ear's vestibular organ removed the effects induced by hypergravity on gene expression, which suggests that the effects of hypergravity are mediated through the vestibular organ. In summary, we showed that gravity changes induced differential modulation of gene expression of BDNF and 5-HTRs (5-HT1AR, 5-HT1BR and 5-HT2AR) in some brain regions. The modulation of gene expression may constitute molecular bases that underlie behavioral alteration induced by gravity changes.

Psychedelics as Medicines: An Emerging New Paradigm.

Scientific interest in serotonergic psychedelics (e.g., psilocybin and LSD; 5-HT2A receptor agonists) has dramatically increased within the last decade. Clinical studies administering psychedelics with psychotherapy have shown preliminary evidence of robust efficacy in treating anxiety and depression, as well as addiction to tobacco and alcohol. Moreover, recent research has suggested that these compounds have potential efficacy against inflammatory diseases through novel mechanisms, with potential advantages over existing antiinflammatory agents. We propose that psychedelics exert therapeutic effects for psychiatric disorders by acutely destabilizing local brain network hubs and global network connectivity via amplification of neuronal avalanches, providing the occasion for brain network "resetting" after the acute effects have resolved. Antiinflammatory effects may hold promise for efficacy in treatment of inflammation-related nonpsychiatric as well as potentially for psychiatric disorders. Serotonergic psychedelics operate through unique mechanisms that show promising effects for a variety of intractable, debilitating, and lethal disorders, and should be rigorously researched.

Risperidone induces long-lasting changes in the conditioned avoidance response and accumbal gene expression selectively in animals treated as adolescents.

Adolescence is a period of dynamic remodeling and maturation in the brain. Exposure to psychotropic drugs during adolescence can potentially alter neural maturation in the adolescent brain subsequently altering neural function at maturity. In this regard, antipsychotic drugs (APDs) are important given a notable global increase in prescription of these APDs to adolescents for a variety of behavioural symptoms and conditions over the past twenty years. However, there is a paucity of data on the long-term consequences of APDs on the adolescent brain. In this preclinical study, we have examined whether the adolescent brain is more susceptible than the adult brain to long-term neural changes induced by risperidone, which is the APD most frequently prescribed to adolescents. Rats were chronically treated (21 days) with 1.3 mg/kg/day risperidone or vehicle either as adolescents (postnatal day (PND) 36-56)) or adults (PND80-100). Behaviour was assessed using the well-described suppression of the conditioned avoidance response (CAR) by APDs. We examined CAR after all animals had reached maturity (PND127). We show that mature rats treated with risperidone as adolescents had increased CAR suppression compared to adults when rechallenged with this same drug. In the nucleus accumbens, significant downregulation of serotonergic 5HT2A receptors and catechol-o-methyl transferase mRNA levels was observed only in the adolescent treated animals. Impaired 5HT2A receptor signaling may explain the increased CAR suppression observed in rats treated with risperidone as adolescents. Magnetic resonance imaging (MRI), however, did not detect any risperidone-induced long-term brain structural change at maturity. These findings confirm that APD administration during adolescence may produce long-term behavioural and neurochemical alterations.

Altered serotonin (5-HT) 1D and 2A receptor expression may contribute to defective insulin and glucagon secretion in human type 2 diabetes.

Islet produced 5-hydroxy tryptamine (5-HT) is suggested to regulate islet hormone secretion in a paracrine and autocrine manner in rodents. Hitherto, no studies demonstrate a role for this amine in human islet function, nor is it known if 5-HT signaling is involved in the development of beta cell dysfunction in type 2 diabetes (T2D). To clarify this, we performed a complete transcriptional mapping of 5-HT receptors and processing enzymes in human islets and investigated differential expression of these genes in non-diabetic and T2D human islet donors. We show the expression of fourteen 5-HT receptors as well as processing enzymes involved in the biosynthesis of 5-HT at the mRNA level in human islets. Two 5-HT receptors (HTR1D and HTR2A) were over-expressed in T2D islet donors. Both receptors (5-HT1d and 5-HT2a) were localized to human alpha, beta and delta cells. 5-HT inhibited both insulin and glucagon secretion in non-diabetic islet donors. In islets isolated from T2D donors the amine significantly increased release of insulin in response to glucose. Our results suggest that 5-HT signaling participates in regulation of overall islet hormone secretion in non- diabetic individuals and over-expression of HTR1D and HTR2A may either contribute to islet dysfunction in T2D or arise as a consequence of an already dysfunctional islet.

The effect of a 5-HT2A receptor antagonist on pain-related behavior, endogenous 5-hydroxytryptamine production, and the expression 5-HT2A receptors in dorsal root ganglia in a rat lumbar disc herniation model.

STUDY DESIGN: Controlled, interventional, animal study. OBJECTIVE: To evaluate the effect of a 5-HT2A receptor antagonist on pain-related behavior, endogenous 5-hydroxytryptamine (5-HT) plasma levels, and expression of 5-HT2A receptors in dorsal root ganglia (DRGs) in a rat lumbar disc herniation model. SUMMARY OF BACKGROUND DATA: Application of nucleus pulposus on the nerve root induces immediate peripheral 5-HT production and the expression of 5-HT2A receptors in the adjacent DRG. However, the efficacy of a 5-HT2A receptor antagonist for pain relief in this situation and the mechanism remain unknown. METHODS: Autologous nucleus pulposus was applied to the left L5 nerve root of 91 adult female Sprague-Dawley rats. The selective 5-HT2A receptor antagonist sarpogrelate hydrochloride (SPG; 1 mg/kg or 10 mg/kg) or vehicle was administered orally once a day from 1 to 21 days postoperatively. Von Frey tests were used to test pain behavior before and after surgery. To assess the effect of SPG on endogenous 5-HT release surrounding the inflamed nerve root, we measured levels of 5-hydroxyindole acetic acid, a 5-HT metabolite, in plasma. Expression of 5-HT2A receptors in the left L5 DRG was examined with immunoblotting. RESULTS: The higher dose (10 mg/kg) of SPG significantly improved the mechanical withdrawal thresholds from 5 to 21 days after surgery compared with vehicle treatment. 5-hydroxyindole acetic acid in plasma was not significantly different among any groups at any time points. Both doses of SPG inhibited the expression of 5-HT2A receptors after surgery compared with vehicle treatment. CONCLUSION: A selective 5-HT2A receptor antagonist attenuated pain-related behavior and suppressed 5-HT2A receptor expression in the DRG, but did not affect peripheral 5-HT production. Selective 5-HT2A receptor antagonists may attenuate sciatica by blocking and downregulating 5-HT2A receptors in DRGs in lumbar disc herniation. LEVEL OF EVIDENCE: NA.

Serotonin activates cell survival and apoptotic death responses in cultured epithelial thyroid cells.

Anatomic and physiological interactions between central serotonergic system and thyroid gland are well established. However, the effects of locally available serotonin on the thyroid functions are poorly known. Here, we first demonstrate the expression of serotonin transporter SERT and 5-HT2A receptor subtype in rat thyroid epithelial cell line FRT both at mRNA and protein levels. In order to investigate the molecular mechanisms of serotonin action, FRT cells were exposed to increasing concentrations of the amine. Low concentrations of serotonin (up to 5 µM) enhanced FRT cell growth, and ERK1/2 and SMAD2/3 phosphorylation. Cell exposure to the selective 5-HT2A receptor agonist DOI recapitulated the effects of 5-HT on ERK1/2 phosphorylation. By contrast, administration of M100907, a specific 5-HT2A receptor inhibitor, prevented 5-HT induced ERK1/2 activation. On the other hand, high doses of serotonin (50 µM up to 1 mM) activated a caspase-3 mediated apoptosis of cells. Overall, our findings demonstrate that low levels of serotonin, interacting with 5-HT2A receptor, are able to activate proliferative signals in the thyroid epithelial cells, while high levels of serotonin cause pro-apoptotic responses, thus suggesting an active role of the amine in the thyroid functions and disorders.

Upregulation of serotonin-receptor-2a and serotonin transporter expression in the pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia.

PURPOSE: Congenital diaphragmatic hernia (CDH) is attributed to severe pulmonary hypoplasia and pulmonary hypertension (PH). PH is characterized by structural changes resulting in vascular remodeling. Serotonin, a potent vasoconstrictor, plays a central role in the development of PH. It exerts its constricting effects on the vessels via Serotonin receptor 2A (5-HT2A) and induces pulmonary smooth muscle cell proliferation via the serotonin transporter (5-HTT). This study was designed to investigate expressions of 5-HT2A and 5-HTT in the pulmonary vasculature of rats with nitrofen-induced CDH. METHODS: Rats were exposed to nitrofen or vehicle on D9. Fetuses were sacrificed on D21 and divided into nitrofen and control group (n=32). Pulmonary RNA was extracted and mRNA level of 5HT2A was determined by qRT-PCR. Protein expression of 5HT2A and 5-HTT was investigated by western blotting. Confocal immunofluorescence double-staining for 5-HT2A, 5-HTT, and alpha smooth muscle actin were performed. RESULTS: Pulmonary 5-HT2A gene expression levels were significantly increased in nitrofen-induced CDH compared to controls. Western blotting and confocal microscopy confirmed increased pulmonary protein expression in CDH lungs compared to controls. CONCLUSION: Increased gene and protein expression of 5HT2A and 5-HTT in the pulmonary vasculature of nitrofen-induced CDH lungs suggest that 5HT2A and 5-HTT are important mediators of PH in nitrofen-induced CDH.

Effects of developmental hyperserotonemia on juvenile play behavior, oxytocin and serotonin receptor expression in the hypothalamus are age and sex dependent.

There is a striking sex difference in the diagnosis of Autism Spectrum Disorder (ASD), such that males are diagnosed more often than females, usually in early childhood. Given that recent research has implicated elevated blood serotonin (hyperserotonemia) in perinatal development as a potential factor in the pathogenesis of ASD, we sought to evaluate the effects of developmental hyperserotonemia on social behavior and relevant brain morphology in juvenile males and females. Administration of 5-methoxytryptamine (5-MT) both pre- and postnatally was found to disrupt normal social play behavior in juveniles. In addition, alterations in the number of oxytocinergic cells in the lateral and medial paraventricular nucleus (PVN) were evident on postnatal day 18 (PND18) in 5-MT treated females, but not treated males. 5-MT treatment also changed the relative expression of 5-HT(1A) and 5-HT(2A) receptors in the PVN, in males at PND10 and in females at PND18. These data suggest that serotonin plays an organizing role in the development of the PVN in a sexually dimorphic fashion, and that elevated serotonin levels during perinatal development may disrupt normal organization, leading to neurochemical and behavioral changes. Importantly, these data also suggest that the inclusion of both juvenile males and females in studies will be necessary to fully understand the role of serotonin in development, especially in relation to ASD.

Stress and withdrawal from d-amphetamine alter 5-HT2A receptor mRNA expression in the prefrontal cortex.

Psychostimulant withdrawal results in emotional, behavioral, and cognitive impairments, which may be exacerbated by stress. However, little is known about the neurochemical changes that occur when these two conditions are experienced concomitantly. 5-HT2A receptor (5-HT2AR) mRNA expression in the prefrontal cortex (PFC) is diminished following withdrawal from d-amphetamine (AMPH) and may underlie the emotional and cognitive impairments observed in psychostimulant withdrawal, but whether stress affects 5-HT2AR mRNA expression during psychostimulant withdrawal is unknown. The goal of this study was to examine the impact of forced swim test (FST) exposure during AMPH withdrawal on 5-HT2AR mRNA expression in PFC. Animals were treated 3 times a day for 4 days with escalating doses of AMPH (1-10mg/kg) and 24h or 4 days after the final injection, animals were subjected to FST. At 24h of withdrawal, AMPH-treated animals showed greater immobility in FST and at 4 days of withdrawal, AMPH-treated animals did not show immobility. At 24h of withdrawal, animals showed lower 5-HT2AR mRNA expression in the PFC relative to saline-treated animals, and exposure to FST did not further decrease expression in these animals. At 4 days of withdrawal, AMPH-treated animals showed greater 5-HT2AR mRNA expression relative to saline-treated animals in the PFC, an effect that was diminished by exposure to FST. These data indicate that stress and short-term AMPH withdrawal affect prefrontal 5-HT2AR mRNA expression to a similar degree, and stress experienced during long-term AMPH withdrawal can diminish the recovery of 5-HT2AR mRNA expression. Together, these data suggest that exposure to stress during extended AMPH withdrawal could prolong withdrawal-induced, 5-HT2AR mRNA expression which could be related to 5-HT2AR mediated deficits.

The expression and role of serotonin receptor 5HTR2A in canine osteoblasts and an osteosarcoma cell line.

BACKGROUND: The significance of the serotonergic system in bone physiology and, more specifically, the importance of the five hydroxytryptamine receptor 2A (5HTR2A) in normal osteoblast proliferation have been previously described; however the role of serotonin in osteosarcoma remains unclear. Particularly, the expression and function of 5HTR2A in canine osteosarcoma has not yet been studied, thus we sought to determine if this indoleamine modulates cellular proliferation in vitro. Using real time quantitative reverse transcription PCR and immunoblot analyses, we explored receptor expression and signaling differences between non-neoplastic canine osteoblasts (CnOb) and an osteosarcoma cell line (COS). To elucidate specific serotonergic signaling pathways triggered by 5HTR2A, we performed immunoblots for ERK and CREB. Finally, we compared cell viability and the induction of apoptosis in the presence 5HTR2A agonists and antagonists. RESULTS: 5HTR2A was overexpressed in the malignant cell line in comparison to normal cells. In CnOb cells, ERK phosphorylation (ERK-P) decreased in response to both serotonin and a specific 5HTR2A antagonist, ritanserin. In contrast, ERK-P abundance increased in COS cells following either treatment. While endogenous CREB was undetectable in CnOb, CREB was observed constitutively in COS, with expression and exhibited increased CREB phosphorylation following escalating concentrations of ritanserin. To determine the influence of 5HTR2A signaling on cell viability we challenged cells with ritanserin and serotonin. Our findings confirmed that serotonin treatment promoted cell viability in malignant cells but not in normal osteoblasts. Conversely, ritanserin reduced cell viability in both the normal and osteosarcoma cells. Further, ritanserin induced apoptosis in COS at the same concentrations associated with decreased cell viability. CONCLUSIONS: These findings confirm the existence of a functional 5HTR2A in a canine osteosarcoma cell line. Results indicate that intracellular second messenger signal coupling of 5HTR2A is different between normal and malignant cells, warranting further research to investigate its potential as a novel therapeutic target for canine osteosarcoma.

Systems pharmacology identifies drug targets for Stargardt disease-associated retinal degeneration.

A systems pharmacological approach that capitalizes on the characterization of intracellular signaling networks can transform our understanding of human diseases and lead to therapy development. Here, we applied this strategy to identify pharmacological targets for the treatment of Stargardt disease, a severe juvenile form of macular degeneration. Diverse GPCRs have previously been implicated in neuronal cell survival, and crosstalk between GPCR signaling pathways represents an unexplored avenue for pharmacological intervention. We focused on this receptor family for potential therapeutic interventions in macular disease. Complete transcriptomes of mouse and human samples were analyzed to assess the expression of GPCRs in the retina. Focusing on adrenergic (AR) and serotonin (5-HT) receptors, we found that adrenoceptor α 2C (Adra2c) and serotonin receptor 2a (Htr2a) were the most highly expressed. Using a mouse model of Stargardt disease, we found that pharmacological interventions that targeted both GPCR signaling pathways and adenylate cyclases (ACs) improved photoreceptor cell survival, preserved photoreceptor function, and attenuated the accumulation of pathological fluorescent deposits in the retina. These findings demonstrate a strategy for the identification of new drug candidates and FDA-approved drugs for the treatment of monogenic and complex diseases.

The response of juxtacellular labeled GABA interneurons in the basolateral amygdaloid nucleus anterior part to 5-HT2A/2C receptor activation is decreased in rats with 6-hydroxydopamine lesions.

Here we report that juxtacellular labeled GABA interneurons in the basolateral amygdaloid nucleus anterior part (BLA) of rats with 6-hydroxydopamine lesions of the substantia nigra pars compacta (SNc) showed a more burst-firing pattern, while having no change in the firing rate. In sham-operated and the lesioned rats, systemic administration of 5-HT(2A/2C) receptor agonist DOI produced excitation, inhibition and unchanged in the firing rate of the interneurons, and the mean response of DOI was excitatory. However, cumulative dose producing excitation in the lesioned rats was higher than that of sham-operated rats. The local administration of DOI in the BLA also produced three types of responses in two groups of rats. Furthermore, the local administration of DOI excited the interneurons in sham-operated rats, whereas the mean firing rate of the interneurons in the lesioned rats was not affected at the same dose. The excitatory effect of the majority of the interneurons after systemic and local administration of DOI was not reversed by the selective 5-HT(2C) receptor antagonist SB242084, and the inhibitory effect of DOI in all the interneurons examined was reversed by GABA(A) receptor antagonist picrotoxinin. The SNc lesion in rats did not change the density of GAD67/5-HT(2A) receptor co-expressing neurons in the BLA. These results indicate that the SNc lesion changes the firing activity of BLA GABA interneurons. Moreover, DOI regulated the firing activity of the interneurons mainly through activation of 5-HT(2A) receptor, and the lesion led to a decreased response of the interneurons to DOI, which attributes to dysfunction of 5-HT(2A) receptor on these interneurons.

G-protein receptor kinase 5 regulates the cannabinoid receptor 2-induced up-regulation of serotonin 2A receptors.

We have recently reported that cannabinoid agonists can up-regulate and enhance the activity of serotonin 2A (5-HT2A) receptors in the prefrontal cortex (PFCx). Increased expression and activity of cortical 5-HT2A receptors has been associated with neuropsychiatric disorders, such as anxiety and schizophrenia. Here we report that repeated CP55940 exposure selectively up-regulates GRK5 proteins in rat PFCx and in a neuronal cell culture model. We sought to examine the mechanism underlying the regulation of GRK5 and to identify the role of GRK5 in the cannabinoid agonist-induced up-regulation and enhanced activity of 5-HT2A receptors. Interestingly, we found that cannabinoid agonist-induced up-regulation of GRK5 involves CB2 receptors, ß-arrestin 2, and ERK1/2 signaling because treatment with CB2 shRNA lentiviral particles, ß-arrestin 2 shRNA lentiviral particles, or ERK1/2 inhibitor prevented the cannabinoid agonist-induced up-regulation of GRK5. Most importantly, we found that GRK5 shRNA lentiviral particle treatment prevented the cannabinoid agonist-induced up-regulation and enhanced 5-HT2A receptor-mediated calcium release. Repeated cannabinoid exposure was also associated with enhanced phosphorylation of CB2 receptors and increased interaction between ß-arrestin 2 and ERK1/2. These latter phenomena were also significantly inhibited by GRK5 shRNA lentiviral treatment. Our results suggest that sustained activation of CB2 receptors, which up-regulates 5-HT2A receptor signaling, enhances GRK5 expression; the phosphorylation of CB2 receptors; and the ß-arrestin 2/ERK interactions. These data could provide a rationale for some of the adverse effects associated with repeated cannabinoid agonist exposure.

The effect of stress on gill basolateral membrane binding kinetics of 5-ht2 receptor ligands: potential implications for urea excretion mechanisms.

The goal of this study was to determine the relationship between cortisol and the toadfish serotonin 2A (5-HT2A ) receptor, which is believed to be responsible for the activation of the toadfish urea transporter, tUT. We hypothesize that elevations in cortisol would play a role in the regulation of the 5-HT2A receptor at the level of mRNA expression, ligand binding, and/or function. To test this idea, cortisol levels were manipulated by either crowding or through treatment with the cortisol synthesis blocker, metyrapone. Crowded fish had significantly higher circulating cortisol levels compared to uncrowded fish and cortisol levels in metyrapone-treated fish were significantly lower than saline-treated controls. No significant difference was measured in gill 5-HT2A mRNA expression levels between uncrowded and crowded, control- or metyrapone-treated fish. Furthermore, no significant difference was measured in [(3) H]-5-HT binding kinetics or in the competitive binding of the 5-HT2 agonist, α-methyl 5-HT, to isolated gill basolateral membranes of uncrowded or crowded toadfish. However, the binding maximum (Bmax ) of the 5-HT2A receptor antagonist, [(3) H]-ketanserin, was significantly different between all four groups of fish (metyrapone > control > crowded > uncrowded). Furthermore, metyrapone-treated fish excreted approximately twofold more urea compared to controls when injected with α-methyl 5-HT, a 5-HT2 receptor agonist shown to stimulate urea excretion. Our results suggest that cortisol may have differential effects on 5-HT receptor binding, which could have potential implications on the control of pulsatile urea excretion in toadfish.