Expression Of Serotonin Receptors Research Articles

Abstract 2534: The role of human 5-HTR2A in miRNA expression in the triple negative breast cancer cell line

Abstract In the United States, African American women are twice as likely to be diagnosed with triple-negative breast cancer (TNBC) than Caucasian women. African American women experience the highest death rates when diagnosed with breast cancer (Bca). TNBC is the most aggressive subtype of BCa and difficult to treat. Studies have demonstrated the expression of serotonin receptors (5-HTRs) on BCa cells and reported their role in tumor progression. The dysregulation of miRNAs has been associated with oncogenesis. miRNAs play a critical role in biological processes such as cell proliferation and death not only through targeting cell-cycle components but also by extensively regulating multiple signaling pathways. In the context of cancer, miRNAs have been observed to function as either oncogenes or tumor suppressors in cancer development. 5-Hydroxytryptamine receptor 2A (5-HTR2A), a G coupled-protein serotonin receptor, was implicated in cancer progression. In this study, we determined the miRNA expression profile related to 5-HTR2A biological activity in a TNBC cell line derived from an African American woman. The MDA-MB-468 cell line was cultured and an MTS assay performed to identify the effective concentration of (±)-DOI hydrochloride (5-HTR2A agonist) for treatment. Following treatment with 5 µM DOI, total RNA was isolated using Qiagen's AllPrep DNA/RNA Mini Kit following modified protocol for including miRNA. Total RNA was subjected to the NanoString human v3 miRNA kit targeting 800 endogenous miRNAs using color-coded probe sets. nSolver was used for differential miRNA expression and statistical analysis. For differential miRNA analysis DOI treated cells were compared with untreated MDA-MB 468 cells. The miRNA counts for each triplicate set were averaged. nSolver Analysis Software created by NanoString was used to determine fold change and statistical significance. Quality-control measures and normalization of data were also performed using the nSolver analysis package. After identifying significant (p<0.05) miRNAs and selecting those with a fold change greater than three, fourteen miRNAs were differentially expressed between the DOI treated and untreated BCa cell line (MDA-MB 468). The greatest differential expression observed in has-mir-205-5p. This oncogenic miRNA has been associated with cellular differentiation, migration, and proliferation. When comparing these fourteen miRNAs to the normal breast cell line (MCF-10a), only one miRNA (hsa-miR-221-3p), was found to be significant (p<0.05). In DOI treated TNBC (MDA-MB 468), hsa-miR-221-3p was downregulated by 3.44 and 1.81 compared to untreated TNBC (MDA-MB 468) and normal (MCF-10a), respectively. Reportedly, hsa-miR-221-3p (an oncogenic miRNA) is associated with TNBC and acts downstream of the oncogenic RAS-RAF-MAPK pathway. This study elucidates miRNAs contributing to TNBC development and progression in correlation with 5-HTR2A activity in vitro. Specifically, two oncogenic miRNAs were identified, has-mir-205-5p and hsa-miR-221-3p. Although their involvement in breast cancer progression has been studied, has-mir-205-5p and hsa-miR-221-3p should be validated as a consequence of 5-HTR2A biological activity. These miRNAS may be used as prognostic biomarkers in breast cancer management. Citation Format: Nicole L. Retland Moreland, Abrar Aloufi, Areej Alyahyawi, Afnan Shakoori, Joseph Aubee, Karl Thompson, Muneer Abbas. The role of human 5-HTR2A in miRNA expression in the triple negative breast cancer cell line [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2534.

Electroacupuncture: A New Approach for Improved Postoperative Sleep Quality After General Anesthesia.

General anesthesia produces a state of drug-induced unconsciousness that is controlled by the extent and duration of administered agents. Whether inhalation or intravenous in formulation, such agents may interfere with normal sleep–wake cycles, impairing postoperative sleep quality and creating complications. Electroacupuncture is a new approach widely applied in clinical practice during recent years. This particular technology helps regulate neurotransmitter concentrations in the brain, lowering norepinephrine and dopamine levels to improve sleep quality. It also alleviates surgical pain that degrades postoperative sleep quality after general anesthesia by downregulating immune activity (SP, NK-1, and COX-1) and upregulating serotonin receptor (5-HT1AR, 5-HT2AR) and endocannabinoid expression levels. However, large-scale, multicenter studies are still needed to determine the optimal duration, frequency, and timing of electroacupuncture for such use.

Peripheral serotonin regulates glucose and insulin metabolism in Holstein dairy calves

Peripheral serotonin regulates energy metabolism in several mammalian species, however, the potential contribution of serotonergic mechanisms as metabolic and endocrine regulators in growing dairy calves remain unexplored. Objectives were to characterize the role of serotonin in glucose and insulin metabolism in dairy calves with increased serotonin bioavailability. Milk replacer was supplemented with saline, 5-hydroxytryptophan (90 mg/d), or fluoxetine (40 mg/d) for 10-d (n = 8/treatment). Blood was collected daily during supplementation and on days 2, 7, and 14 during withdrawal. Calves were euthanized after 10-d supplementation or 14-d withdrawal periods to harvest liver and pancreas tissue. 5-hydroxytryptophan increased circulating insulin concentrations during the supplementation period, whereas both treatments increased circulating glucose concentration during the withdrawal period. The liver and pancreas of preweaned calves express serotonin factors (ie, TPH1, SERT, and cell surface receptors), indicating their ability to synthesize, uptake, and respond to serotonin. Supplementation of 5-hydroxytryptophan increased hepatic and pancreatic serotonin concentrations. After the withdrawal period, fluoxetine cleared from the pancreas but not liver tissue. Supplementation of 5-hydroxytryptophan upregulated hepatic mRNA expression of serotonin receptors (ie, 5-HTR1B, -1D, -2A, and -2B), and downregulated pancreatic 5-HTR1F mRNA and insulin-related proteins (ie, Akt and pAkt). Fluoxetine-supplemented calves had fewer pancreatic islets per microscopic field with reduced insulin intensity, whereas 5-hydroxytryptophan supplemented calves had increased islet number and area with greater insulin and serotonin and less glucagon intensities. After the 14-d withdrawal of 5-hydroxytryptophan, hepatic mRNA expression of glycolytic and gluconeogenic enzymes were simultaneously downregulated. Improving serotonin bioavailability could serve as a potent regulator of endocrine and metabolic processes in dairy calves.

Increasing serotonin bioavailability alters gene expression in peripheral leukocytes and lymphoid tissues of dairy calves

Dairy calves are born with a naïve immune system, making the pre-weaning phase a critical window for immune development. In the U.S., 40–60% of dairy farms feed milk replacer to pre-weaned calves, which are devoid of bioactive factors with immunological roles. Serotonin is a bioactive factor with immunoregulatory properties naturally produced by the calf and present in milk. Human and rodent immune cells express the serotonin machinery, but little is known about the role of serotonin in the bovine immune system. Supplementing milk replacer with 5-hydroxytryptophan (serotonin precursor) or fluoxetine (reuptake inhibitor) increases serotonin bioavailability. We hypothesized that increased serotonin bioavailability promotes serotonergic signaling and modulates the expression of immune related genes in peripheral leukocytes and immune-related tissues of dairy calves. The present experiment targeted candidate genes involved in serotonin production, metabolism, transport, signaling and immune regulation. We established that bovine peripheral leukocytes express all known serotonin receptors, and can synthesize, uptake and degrade serotonin due to the expression of serotonin metabolism-related genes. Indeed, we showed that increasing serotonin bioavailability alters gene expression of serotonin receptors and immune-related genes. Further research will determine whether manipulation of the serotonin pathway could be a feasible approach to bolster dairy calves’ immune system.

Serotonin (5-HT) Shapes the Macrophage Gene Profile through the 5-HT2B-Dependent Activation of the Aryl Hydrocarbon Receptor.

Macrophages can either promote or resolve inflammatory responses, and their polarization state is modulated by peripheral serotonin (5-hydroxytryptamine [5-HT]). In fact, pro- and anti-inflammatory macrophages differ in the expression of serotonin receptors, with 5-HT2B and 5-HT7 expression restricted to M-CSF-primed monocyte-derived macrophages (M-MØ). 5-HT7 drives the acquisition of profibrotic and anti-inflammatory functions in M-MØ, whereas 5-HT2B prevents the degeneration of spinal cord mononuclear phagocytes and modulates motility of murine microglial processes. Because 5-HT2B mediates clinically relevant 5-HT-related pathologies (valvular heart disease, pulmonary arterial hypertension) and is an off target of anesthetics, antiparkinsonian drugs, and selective serotonin reuptake inhibitors, we sought to determine the transcriptional consequences of 5-HT2B engagement in human macrophages, for which 5-HT2B signaling remains unknown. Assessment of the effects of specific agonists and antagonist revealed that 5-HT2B engagement modifies the cytokine and gene signature of anti-inflammatory M-MØ, upregulates the expression of aryl hydrocarbon receptor (AhR) target genes, and stimulates the transcriptional activation of AhR. Moreover, we found that 5-HT dose dependently upregulates the expression of AhR target genes in M-MØ and that the 5-HT-mediated activation of AhR is 5-HT2B dependent because it is abrogated by the 5-HT2B-specific antagonist SB204741. Altogether, our results demonstrate the existence of a functional 5-HT/5-HT2B/AhR axis in human macrophages and indicate that 5-HT potentiates the activity of a transcription factor (AhR) that regulates immune responses and the biological responses to xenobiotics.

From the gut to the heart: L. plantarum and inulin administration as a novel approach to control cardiac apoptosis via 5-HT2B and TrkB receptors in diabetes.

Type 2 diabetes mellitus, as a metabolic disorder, can lead to diabetic cardiomyopathy, identified by cardiomyocyte apoptosis and myocardial fibrosis. Brain-derived neurotrophic factor (BDNF) and serotonin are two neurotransmitters that can control cardiomyocyte apoptosis and myocardial fibrosis through their cardiac receptors. In the present study, we investigated the impacts of L. plantarum and inulin supplementation on the inhibition of cardiac apoptosis and fibrosis by modulating intestinal, serum, and cardiac levels of serotonin and BDNF as well as their cardiac receptors. Diabetes was induced by a high-fat diet and streptozotocin in male Wistar rats. Rats were divided into six groups and were supplemented with L. plantarum, inulin or their combination for 8 weeks. Finally, the rats were killed and levels of intestinal, serum, and cardiac parameters were evaluated. Concurrent administration of L. plantarum and inulin caused a significant rise in the expression of cardiac serotonin and BDNF receptors (P<0.001) as well as a significant fall in cardiac interstitial and perivascular fibrosis (P<0.001, both) and apoptosis (P=0.01). Moreover, there was a strong correlation of cardiac 5-Hydroxytryptamine 2B (5-HT2B) and tropomyosin receptor kinase B (TrkB) receptors with interstitial/perivascular fibrosis and apoptosis (P<0.001, both). Results revealed beneficial effects of L. plantarum, inulin or their combination on intestinal, serum, and cardiac serotonin and BDNF accompanied by higher expression of their cardiac receptors and lower levels of cardiac apoptotic and fibrotic markers. It seems that L. plantarum and inulin supplementation could be considered as a novel adjunct therapy to reduce cardiac complications of type 2 diabetes mellitus.

Ozone increases plasma kynurenine-tryptophan ratio and impacts hippocampal serotonin receptor and neurotrophic factor expression: Role of stress hormones

Air pollution is associated with adverse impacts on the brain, including cognitive decline and increased incidence of dementia, depression and anxiety; however, underlying mechanisms remain unclear. We have shown that both ozone and particulate matter activate the hypothalamic-pituitary-adrenal (HPA) axis, increasing plasma glucocorticoids and altering mRNA profiles in multiple tissues including the brain. HPA axis dysregulation has been associated with central nervous system impacts, including key effects in the hippocampus; accordingly, we hypothesized that pollutant-dependent increases in glucocorticoid levels impact biological pathways relevant to brain health. Fischer-344 rats were treated with metyrapone (0 or 50 mg/kg), a glucocorticoid synthesis inhibitor, and exposed to ozone (0 or 0.8 ppm) for 4 h (n = 5/group) to investigate the role of glucocorticoids in ozone-dependent effects on tryptophan metabolism and expression of serotonin receptors and neurotrophic factors. Ozone increased plasma levels of the tryptophan metabolite kynurenine (~2-fold) and decreased tryptophan levels (~1.2 fold). Hippocampal expression of serotonin receptors exhibited differential regulation following exposure, and expression of key neurotrophic factors (brain-derived neurotrophic factor, vascular endothelial growth factor A, insulin-like growth factor-1, tyrosine kinase receptor B, b-cell lymphoma 2) was decreased. Some, but not all effects were abrogated by metyrapone treatment, suggesting both glucocorticoid-dependent and -independent regulation. Exposure to exogenous corticosterone (10 mg/kg) followed by clean air reproduced the ozone effects that were blocked with metyrapone, confirming the specificity of effects to glucocorticoids. These results indicate that ozone can modify pathways relevant to brain health and establish a role for the HPA axis in mediating these effects.

The Role of the Gut‐Brain Axis in the Capacity for Learning in Zebrafish

Starved (low calorie) and overfed (high calorie) diets are common in food insecure areas and can shift gut microbiota diversity. While there is increasing evidence linking the gut microbiota composition to psychiatric disorders including anxiety and depression, little is known regarding the impact gut microbes have on learning behavior and capacity. Current literature supports that alterations in intestinal microbiota diversity and richness can influence serotonergic and dopaminergic neurotransmission. The aim of this study is to examine the influence of gut‐brain axis on learning capacity. We hypothesize that a decrease in microbial diversity and abundance will negatively impact learning in zebrafish. Fish were grouped and fed a control diet, a starved low caloric diet and an overfed high caloric diet. Afterwards microbial DNA and RNA from zebrafish brain tissue was isolated. Bacterial abundance was quantified with primers designed against 16s ribosomal RNA. Dopamine and serotonin receptor expression was examined using TaqMan primer probes. Overfed fish exhibited a significant increase weight relative to starved fish, in addition starved fish displayed a 7.0 mg decrease in weight (p&lt;.001, n=5). Both overfed and starved fish exhibited a decrease in intestinal microbial abundance. Associative learning was assessed using a plus‐maze protocol. Interestingly we observed modest, though not significant increases in learning in the starved fish relative to overfed and control fish. Coinciding with this data, overfed fish showed no significant changes in serotonin or dopamine receptor gene expression compared to control diets, however starved diets illustrated an increasing trend in serotonin receptor expression and a significant increase in dopamine D1 receptor expression (p&lt;.04, n=11). These results indicate that starved and overfed diets decrease gut microbial abundance and may influence learning capacity through the gut‐brain axis in starved fish.Support or Funding InformationElon University

Blockade of multiple monoamines receptors reduce insulin secretion from pancreatic \u03b2-cells

Clinical use of olanzapine frequently causes severe hyperglycemia as an adverse effect. In this study, we elucidated mechanisms by which olanzapine reduced insulin secretion using the hamster pancreatic β-cell line HIT-T15. Reverse transcriptional-PCR analysis revealed expression of dopamine (D2, D3 and D4), serotonin (5-HT2A, 5-HT2B, 5-HT2C, and 5-HT6), and histamine (H1 and H2) receptors in HIT-T15 cells. Olanzapine decreased insulin secretion from HIT-T15 cells at clinically relevant concentrations (64–160 nM). A dopamine D2 agonist, D3 antagonist, and D4 antagonist suppressed insulin secretion, whereas a D2 antagonist and D3 agonist increased it. A serotonin 5-HT2B agonist slightly increased insulin secretion, while a 5-HT2C antagonist slightly decreased it. Other agonists and antagonists for serotonin receptors did not affect insulin secretion. A histamine H1 agonist increased insulin secretion, whereas an H1 antagonist and H2 agonist suppressed it. Our results suggest that dopamine (D2, D3 and D4), serotonin (5-HT2B and 5-HT2C), and histamine (H1 and H2) receptors, which are expressed on pancreatic β-cells, directly modulate insulin secretion from pancreatic β-cells. Thus, olanzapine may induce hyperglycemia in clinical settings by suppressing insulin secretion from pancreatic β-cells through inhibition of dopamine D3, serotonin 5-HT2B and 5-HT2C, and histamine H1 receptors.

Expression of serotonin 2A, 2C, 6 and 7 receptor and IL-6 mRNA in experimental toxoplasmic encephalitis in mice

The neurotropic pathogen Toxoplasma gondii infects about one-third of the human population. Both acute and chronic (latent or life-long) forms of toxoplasmosis are associated with specific neurologic and neuropsychiatric symptoms. In the present study, swiss albino mice were inoculated intraperitoneally with 15–20 tissue cysts of the ME-49 strain of Toxoplasma gondii. The brain samples were collected on the days 10, 20, and 30 for determining the histopathological scores and the number of cysts. Furthermore, a real-time quantitative polymerase chain reaction (RT-PCR) was conducted to find out the gene expression levels of the serotonin 2A receptor (5-HTR2A), serotonin 2C receptor (5-HTR2C), serotonin 6 receptor (5-HTR6), serotonin 7 receptor (5-HTR7), and interleukin-6. The results were compared to the histopathological findings of encephalitic toxoplasmosis. The expression levels were observed to increase for all receptors; however at different time points of infection. This experimental model demonstrates that the expression of serotonin receptors was induced in Toxoplasma gondii infections, displaying unique findings for each of the individual receptors.

Serotonin and Dopamine Receptor Expression in Solid Tumours Including Rare Cancers

In preclinical studies serotonin stimulates and dopamine inhibits tumour growth and angiogenesis. Information regarding serotonin and dopamine receptor (5-HTR and DRD) expression in human cancers is limited. Therefore, we screened a large tumour set for receptor mRNA overexpression using functional genomic mRNA (FGmRNA) profiling, and we analysed protein expression and location of 5-HTR1B, 5-HTR2B, DRD1, and DRD2 with immunohistochemistry in different tumour types. With FGmRNA profiling 11,756 samples representing 43 tumour types were compared to 3,520 normal tissue samples to analyse receptor overexpression. 5-HTR2B overexpression was present in many tumour types, most frequently in uveal melanomas (56%). Receptor overexpression in rare cancers included 5-HTR1B in nasopharyngeal carcinoma (17%), DRD1 in ependymoma (30%) and synovial sarcoma (21%), and DRD2 in astrocytoma (13%). Immunohistochemistry demonstrated high 5-HTR2B protein expression on melanoma and gastro-intestinal stromal tumour cells and endothelial cells of colon, ovarian, breast, renal and pancreatic tumours. 5-HTR1B expression was predominantly low. High DRD2 protein expression on tumour cells was observed in 48% of pheochromocytomas, and DRD1 expression ranged from 14% in melanoma to 57% in renal cell carcinoma. In conclusion, 5-HTR1B, 5-HTR2B, DRD1, and DRD2 show mRNA overexpression in a broad spectrum of common and rare cancers. 5-HTR2B protein is frequently highly expressed in human cancers, especially on endothelial cells. These findings support further investigation of especially 5HTR2B as a potential treatment target.

Selective Regulation of 5-HT1B Serotonin Receptor Expression in the Striatum by Dopamine Depletion and Repeated L-DOPA Treatment: Relationship to L-DOPA-Induced Dyskinesias.

Dopamine and serotonin in the basal ganglia interact in a bidirectional manner. On the one hand, serotonin (5-HT) receptors regulate the effects of dopamine agonists on several levels, ranging from molecular signaling to behavior. These interactions include 5-HT receptor-mediated facilitation of dopamine receptor-induced gene regulation in striatal output pathways, which involves the 5-HT1B receptor and others. Conversely, there is evidence that dopamine action by psychostimulants regulates 5-HT1B receptor expression in the striatum. To further investigate the effects of dopamine and agonists on 5-HT receptors, we assessed the expression of 5-HT1B and other serotonin receptor subtypes in the striatum after unilateral dopamine depletion by 6-OHDA and subsequent treatment with L-DOPA (5mg/kg; 4weeks). Neither dopamine depletion nor L-DOPA treatment produced significant changes in 5-HT2C, 5-HT4, or 5-HT6 receptor expression in the striatum. In contrast, the 6-OHDA lesion caused a (modest) increase in 5-HT1B mRNA levels throughout the striatum. Moreover, repeated L-DOPA treatment markedly further elevated 5-HT1B expression in the dopamine-depleted striatum, an effect that was most robust in the sensorimotor striatum. A minor L-DOPA-induced increase in 5-HT1B expression was also seen in the intact striatum. These changes in 5-HT1B expression mimicked changes in the expression of neuropeptide markers (dynorphin, enkephalin mRNA) in striatal projection neurons. After repeated L-DOPA treatment, the severity of L-DOPA-induced dyskinesias and turning behavior was positively correlated with the increase in 5-HT1B expression in the associative, but not sensorimotor, striatum ipsilateral to the lesion, suggesting that associative striatal 5-HT1B receptors may play a role in L-DOPA-induced behavioral abnormalities.

Serotonin (5HTR2A and 5HTR3A) and GABA (GABAB) Receptor Genes Overexpression are Correlated with Non-small Cell Lung Cancer (NSCLC)

Background: Lung cancer is one of the main causes of cancer mortality in both men and women. Up to 80% of lung cancers are Non-small-cell lung cancer (NSCLC). With regards to the role of serotonin, as an autocrine growth factor for small-cell lung cancer cells and gammaaminobutyric acid (GABA) and its receptors as a regulator in many types of cancers; the current study was conducted to investigate the expression of serotonin and GABA gene receptors in lung cancer patients. Methods: Relative gene expression of two 5-hydroxytryptamine subtypes (5HTR2A and 5-HTR3A) and GABAB receptor was measured by quantitative polymerase chain reaction in peripheral blood mononuclear cell (PBMC) from 30 NSCLC patients visited in Imam Khomeini hospital, Tehran and 30 healthy controls. Results: Our results demonstrated that the expression of 5HTR3A, 5HTR2A and GABAB R genes was significantly higher in patients compared to the healthy individuals. Conclusion: According to our findings, 5-HT and GABA may be involved in the regulation of tumorigenesis via their receptors, thus playing an important role in lung cancer.

The microRNA-29a Modulates Serotonin 5-HT7 Receptor Expression and Its Effects on Hippocampal Neuronal Morphology.

miRNAs are master regulators of gene expression in diverse biological processes, including the modulation of neuronal cytoarchitecture. The identification of their physiological target genes remains one of the outstanding challenges. Recently, it has been demonstrated that the activation of serotonin receptor 7 (5-HT7R) plays a key role in regulating the neuronal structure, synaptogenesis, and synaptic plasticity during embryonic and early postnatal development of the central nervous system (CNS). In order to identify putative miRNAs targeting the 3'UTR of 5-HT7R mouse transcript, we used a computational prediction tool and detected the miR-29 family members as the only candidates. Thus, since miR-29a is more expressed than other members in the brain, we investigated its possible involvement in the regulation of neuronal morphology mediated by 5-HT7R. By luciferase assay, we show that miR-29a can act as a post-transcriptional regulator of 5-HT7R mRNA. Indeed, it downregulates 5-HT7R gene expression in cultured hippocampal neurons, while the expression of other serotonin receptors is not affected. From a functional point of view, miR-29a overexpression in hippocampal primary cultures impairs the 5HT7R-dependent neurite elongation and remodeling through the inhibition of the ERK intracellular signaling pathway. In vivo, the upregulation of miR-29a in the developing hippocampus parallels with the downregulation of 5-HT7R expression, supporting the hypothesis that this miRNA is a physiological modulator of 5-HT7R expression in the CNS.

Lead exposure affects 5HT2A serotonin receptor and transporter expression in pediatric age group: An explorative study in North-Western India

Lead exposure affects 5HT2A serotonin receptor and transporter expression in pediatric age group: An explorative study in North-Western India

Neurochemical Plasticity of Serotonin Receptors on Phrenic Motor Neurons: Effects of Cervical Spinal Injury and Intermittent Hypoxia

Serotonin is critical for multiple forms of spinal respiratory motor plasticity, including acute intermittent hypoxia (AIH) induced phrenic long‐term facilitation. Relevant serotonin receptor subtypes include 5HT2A, 5HT2B and 5HT7 receptors. Cervical spinal cord injury (cSCI) disrupts serotonergic projections to spinal respiratory motor neurons, transiently abolishing serotonin‐dependent, AIH‐induced phrenic motor plasticity. However, serotonergic innervation recovers with time post‐injury (&gt;8 wks), restoring the capacity for AIH‐induced phrenic motor plasticity. Repetitive “low dose” AIH exposure is emerging as a promising strategy to restore breathing ability following cSCI; conversely, “high dose” chronic intermittent hypoxia (CIH) simulating that experienced during sleep apnea undermines these functional benefits and triggers pathology. Despite the powerful influences of cSCI, repetitive AIH and CIH on breathing ability, we know little concerning the potential of these conditions to change the expression of serotonin receptors (neurochemical plasticity) in phrenic motor neurons. Thus, we tested the hypothesis that cSCI, repetitive AIH and CIH exert differential effects on serotonin receptor expression in phrenic motor neurons. 5HT2A, 5HT2B and 5HT7 receptor expression were determined in male Sprague Dawley rats with and without C2 spinal hemisection (C2Hx; 12 wks post‐injury) that were exposed to 28 days of: 1) normoxia; 2) daily AIH (10, 5‐min 10.5% O2 episodes with 5‐min intervals per day; 1.5 hrs/day); 3) moderate CIH (CIH 5/5: 5‐min 10.5% O2 episodes, 5‐min intervals; 8 hrs/day); and 4) high dose CIH (CIH 2/2: 2‐min 10.5% O2 episodes, 2 min intervals; 8 hrs/day). Rats were then perfused, and cervical spinal cords sectioned (40mm). C3 to C5 sections were processed for 5HT7 receptors and Cholera toxin B subunit (CtB, injected 14 days before C2Hx). Using a custom MATLAB algorithm, optical density in CtB‐positive phrenic motor neurons was quantified. Statistical analysis was performed using MANOVA (SAS JMP). C2Hx had marginal effects on 5HT7 receptor expression (p=0.07). Intermittent hypoxia exerted protocol‐specific effects on 5HT7 receptor expression (CIH 2/2 &gt; CIH 5/5 &gt; daily AIH; p&lt;0.001), and these effects were most prominent after C2Hx (injury/treatment interaction; p&lt;0.011). 5HT2A and 5HT2B quantification are ongoing. The injury‐specific effects of intermittent hypoxia on 5HT7 receptor expression are consistent with the notion that the injured spinal cord has unique properties. Phrenic motor neuron serotonin receptor plasticity may be significant as we work to develop new therapeutic strategies to improve breathing after spinal cord injury.Support or Funding InformationSupported by: NIH T32 HD043730 (LLA) OT2OD023854 (SPARC), NIH K12 HD055929 (EGR), McKnight Brain Institute.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Assessment of Brain Serotonin Receptors in an Fmr1 Knockout Mouse Model of Fragile X Syndrome

Fragile X syndrome (FXS) is the most common form of inherited mental retardation and autism. It is caused by a mutation of FMR1 that prevents transcription of mRNA coding for the protein, FMRP. FMRP regulates the translation of nearly 1000 proteins, including many involved in cell signaling, neurotransmission, synaptic development and plasticity. There are currently no approved medications to treat core symptoms of FXS. Several FMRP targets are functionally linked with the central serotonin system, and certain serotonin receptors can modulate neuropsychological states that are disrupted in FXS, suggesting distinct serotonin receptors may be viable targets for FXS pharmacotherapy. However, studies investigating the expression and function of serotonin receptors and the serotonin transporter (SERT) in the brains of Fmr1 knockout mice—a preclinical, genetic model of FXS—have not been conducted. Using radioligand autoradiography and saturation binding assays, we are assessing the expression of key serotonin proteins—5‐HT1, 5‐HT2A, 5‐HT2C, and 5‐HT7 receptors as well as SERT—in brains of male and female wild‐type (WT) compared to Fmr1 knockout (KO) mice. We are also assessing GABAA receptors (as a positive control), as alterations in brain GABAA receptor expression have been reported in Fmr1 KO mice. For autoradiography experiments, brains from WT and Fmr1 KO mice were cryosectioned at 20 μm and thaw‐mounted on glass slides. Slides were immersed in assay buffer with radioligands at ~Kd concentrations to target receptors of interest: 5‐HT1 (2 nM [3H]5‐CT with 1 μM SB269970); 5‐HT2A (2 nM [3H]Ketanserin and 2 nM [3H]Ketanserin with 1 μM M100907 to distinguish 5‐HT2A labeling from off‐targets); 5‐HT2C, (3 nM [3H]Mesulergine); 5‐HT7 (2 nM [3H]5‐CT with 500 nM cyanopindolol); SERT (3 nM [3H]Citalopram); and GABAA (3 nM [3H]Flumazenil). Slides were dried and exposed to film for 8–12 weeks prior to developing. We did not observe obvious differences between WT and Fmr1 KO mice in expression, across several neural systems (e.g. dorsal hippocampus, cingulate cortex), of any receptor assessed by autoradiography (N=3–6 mice/genotype), however, this may be due to the low resolution of the technique. Saturation binding experiments using homogenized whole brain tissue are in progress. Preliminary results suggest an increase in GABAA receptor expression in Fmr1 KO mice, whereas preliminary results suggest no genotypic differences in SERT expression. In vivo tests of 5‐HT2A receptor function, using the DOI‐elicited head‐twitch response (HTR) assay, revealed no significant differences between WT and Fmr1 KO mice in the number of HTRs elicited by 1 mg/kg (±)‐DOI (N=9 WT, N=9 Fmr1 KO, P=0.10). Locomotion and rearing, however, were significantly higher in Fmr1 KO compared to WT mice treated with (±)‐DOI (P&lt;0.02 for each). In vivo pharmacology tests of 5‐HT1, 5‐HT2C, and SERT function are in progress. Although clear conclusions cannot yet be made, our data currently suggest unaltered expression and function of key 5‐HT proteins in Fmr1 KO mice.Support or Funding InformationFRAXA Research Foundation, DOD W81XWH‐17‐1‐0329This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Autocrine production of reproductive axis neuropeptides affects proliferation of canine osteosarcoma in vitro

BackgroundOsteosarcoma strikes hundreds of people each year, of both advanced and younger ages, and is often terminal. Like many tumor types, these bone tumors will frequently undergo a neuroendocrine transition, utilizing autocrine and/or paracrine hormones as growth factors and/or promoters of angiogenesis to facilitate progression and metastasis. While many of these factors and their actions on tumor growth are characterized, some tumor-derived neuropeptides remain unexplored.MethodsUsing validated canine osteosarcoma cell lines in vitro, as well as cells derived from spontaneous tumors in dogs, we explored the autocrine production of two neuropeptides typically found in the hypothalamus, and most closely associated with reproduction: gonadotropin-releasing hormone (GnRH) and kisspeptin (Kiss-1). We evaluated gene expression and protein secretion of these hormones using quantitative RT-PCR and a sensitive radioimmunoassay, and explored changes in cell proliferation determined by MTS cell viability assays.ResultsOur current studies reveal that several canine osteosarcoma cell lines (COS, POS, HMPOS, D17, C4) synthesize and secrete GnRH and express the GnRH receptor, while COS and POS also express kiss1 and its cognate receptor. We have further found that GnRH and kisspeptin, exogenously applied to these tumor cells, exert significant effects on both gene expression and proliferation. Of particular interest, kisspeptin exposure stimulated GnRH secretion from COS, similarly to the functional relationship observed within the neuroendocrine reproductive axis. Additionally, GnRH and kisspeptin treatment both increased COS proliferation, which additionally manifested in increased expression of the bone remodeling ligand rankl within these cells. These effects were blocked by treatment with a specific GnRH receptor inhibitor. Both neuropeptides were found to increase expression of the specific serotonin (5HT) receptor htr2a, the activation of which has previously been associated with cellular proliferation, suggesting that production of these factors by osteosarcoma cells may act to sensitize tumors to circulating 5HT of local and/or enteric origin.ConclusionsHere we report that kisspeptin and GnRH act as autocrine growth factors in canine osteosarcoma cells in vitro, modulating RANKL and serotonin receptor expression in a manner consistent with pro-proliferative effects. Pharmacological targeting of these hormones may represent new avenues of osteosarcoma treatment.

5-HT3A serotonin receptor in the gastrointestinal tract: the link between immune system and enteric nervous system in the digestive form of Chagas disease.

Chagas disease is caused by Trypanosoma cruzi and remains one of the most neglected diseases in Latin America. One of its clinical forms is Chagas megacolon. Despite being known for more than half a century, detailed causes are still obscure. Recent evidence indicates a close relationship between the immune system and the enteric nervous system in the etiology of chagasic megacolon pathology. It is believed that low expression of the 5-HT3A serotonin receptor on lymphocytes could be linked to megacolon development. To test this hypothesis, this work investigated the distribution of CD4, CD8, and CD20 lymphocytes and their 5-HT3A receptor expression. The results demonstrated that Chagas patients without megacolon present a higher expression of the 5-HT3A receptor in all analyzed lymphocytes compared with Chagas patients with megacolon. These data suggest that the high expression of this receptor may lead to immunomodulation and prevent the development of Chagas megacolon.

Chronic exposure to dietary selenomethionine dysregulates the genes involved in serotonergic neurotransmission and alters social and antipredator behaviours in zebrafish (Danio rerio)

Selenium (Se) is a metalloid of potential interest from both a toxicological and nutritional perspective, having a range of safe intake. The adverse neuro-behavioural effects of Se have been investigated in both humans and fishes, but little is known about its effects on social behaviours or the serotonergic signaling pathway in the brain. In the present study, we investigated the effects of chorionic dietary exposure to Se (as selenomethionine) at different concentrations (control, 2.1, 11.6 or 31.5 μg/g dry wt.) on antipredator avoidance, shoaling behaviour, and social group preferences in adult zebrafish (Danio rerio). In addition, we also measured the expression of important genes in the serotonergic pathway that influence social behaviours. After 60 days of exposure, the highest dose (31.5 μg/g dry wt.) caused the highest level of baseline fear behaviour, with fish swimming lower in the water column and in tighter shoals compared to fish in the other treatments. With high levels of baseline fear, these fish did not significantly intensify fear behaviours in response to predation risk in the form of exposure to chemical alarm cues. When individual fish were given an opportunity to shoal with groups of differing sizes (3 vs. 4 individuals), fish exposed to the high dose spent less time with groups in general, and only control fish showed a significant preference for the larger group. In the zebrafish brain, we found significant upregulation in the mRNA expression of serotonin receptors (htr1aa and htr1b), a transporter (slc6a4a), and tryptophan hydroxylase-2 (tph2), whereas there was a downregulation of the monoamine oxidase (mao) gene. The results of this study suggest that disruption of serotonergic neurotransmission might have been responsible for Se-induced impairment of antipredator and social behaviour in zebrafish.