Serotonin-related Gene Expression Research Articles

Exposure to the pharmaceutical buspirone alters locomotor activity, anxiety-related behaviors, and transcripts related to serotonin signaling in larval zebrafish (Danio rerio)

Buspirone is a pharmaceutical used to treat general anxiety disorder by acting on the dopaminergic and serotoninergic system. Buspirone, like many human pharmaceuticals, has been detected in municipal wastewater; however, the environmental exposure risks are unknown for this psychoactive compound. We studied the effects of buspirone on the behavior of zebrafish, focusing on locomotor and anxiolytic behavior. We also measured transcripts associated with oxidative stress, neurotoxicity, and serotonin signaling to identify potential mechanisms underlying the behavioral changes. Concentrations ranged from environmentally relevant (nM) to physiologically active concentrations typical of human pharmaceuticals (μM). Buspirone treatment did not impact survival, nor did it induce deformities in zebrafish treated for 7 days up to 10 μM. There was a positive relationship between locomotor activity and buspirone concentration in dark periods of the visual motor response test. In the light-dark preference test, both the average time per visit to the dark zone and the percent cumulative duration in the dark zone were increased by 1 μM buspirone. Transcript levels of ache, manf, and mbp were decreased in larvae, while the expression of gap43 was increased following exposure to buspirone, indicating potential neurotoxic effects. There was also reduced expression of serotonin-related genes encoding receptors, transporters, and biosynthesis enzymes (i.e., 5ht1aa, sertb, and tph1a). These data increase understanding of the behavioral and molecular responses in zebrafish following waterborne exposure to neuroactive pharmaceuticals like buspirone.

Hazardous potential evaluation of biochar exposure on mice through analyses of gut-microbiome and fatty acids in brain

In an effort to enhance crop yield and soil fertility, the use of biochar has been widely practiced in a sector of agricultural industry. The use of biochar has been also deemed a strategic measure to abate catastrophic consequences (triggered by global warming) considering its intrinsic carbon negativity. Also, biochar has been fed into livestock as supplement due to its beneficial biological effects (such as the fast growth rate). Nonetheless, biosafety in line with the use of biochar has not been discussed with a fully transparent manner. As such, it is of great importance to scrutinize a hazardous potential when biochar is fed into livestock. To this end, we fed mice with control chow, 0.6 wt%, and 1.2 wt% biochar mixed ones. In the controlled group, anxiety symptoms were observed in the elevated plus maze test and light and dark box test, and the despair-like behavior was identified in forced swim test. The behavioral changes caused by biochar were higher than ≥ 3 times. Also, the trends in the main composition of fatty acids in mouse brain were changed. Decrease in serotonin-related gene expression was resulted from the controlled group. TPH and SERT (that are serotonin-related genes) also decreased ≤ 55% only from the biochar group. Interestingly, the diversity of microorganisms (microbiome) of mouse gut was enhanced. It was concluded that biochar likely affects mouse mood-related behavior and the gut microbiome population. Therefore, this study revealed that the exposure of biochar into the living organisms could have a negative effect, thereby leading to the adverse effects on the ecosystem. Also, the comprehensive/systematic investigations on a hazardous potential of biochar should be administrated with the top priority in the near future.

Riociguat normalizes altered lung expression of serotonin-related genes and renal damage markers in experimental pulmonary hypertension

Abstract Introduction Pulmonary arterial hypertension (PAH) leads to a progressive increase in pulmonary vascular resistance, right heart failure, adverse cardiorenal interactions and mortality. Preclinical studies have shown an association between elevated serotonin (5-HT) levels and disease progression, however, the local pathogenic roles of individual components of serotoninergic system in the lung in PAH remain less well defined. Purpose To test the hypothesis that experimental PAH is associated with alterations in the expression of serotoninergic genes in the lung and right ventricle and that short treatment of experimental PAH with riociguat could have an impact on the serotoninergic system. In addition, we examined whether riociguat could alleviate renal damage associated with cardiorenal interactions in PAH. Methods We used rat monocrotaline model of PAH (60 mg/kg, s.c.). 2 weeks after MCT, we treated two groups with riociguat (10 mg/kg/d, p.o.) until week 4 when rats were sacrificed. We divided rats into 4 groups. CON-control group, MCT-rats with established PAH, RIO-healthy rats with administered riociguat, and MCT+RIO-rats with established PAH treated with riociguat. We used RT-qPCR to monitor mRNA changes of brain natriuretic peptide (Nppb), components of serotoninergic system in the lungs and right ventricle, biomarkers of renal tubular damage Kim1 and podocyte damage Nphs1, an indicator of neurohumoral activation renin (Ren) and marker of renal hypoxia erythropoietin (Epo). In addition, we measured plasma NT-proBNP, serotonin, cGMP and a heart failure marker sST2 using ELISA. Results As expected, MCT caused RV hypertrophy and increased lung weight (p<0.05 vs CON). We observed a 10-fold increase of Nppb in the RV (p<0.05 vs CON), however, plasma levels of NT-proBNP and sST2 did not change. Short-term RIO did not revert these changes. MCT increased cGMP plasma concentration by more than 80% (p<0.05 vs CON), RIO did not influence plasma cGMP levels. However, in the lungs, MCT increased the expression Tph1, Htr1b and Htr2b genes and decreased that of Sert. Riociguat treatment in MCT+RIO normalized these changes (p<0.05 vs MCT) with the exception of decreased Sert expression. In the kidney, we observed a 5-fold increase in expression of Kim1 and a 50% increase in Nphs1 mRNA by MCT (p<0.05 vs CON), expression of both genes were normalized in MCT+RIO (p<0.05 vs MCT). MCT-related increases in Ren and Epo expressions were unaffected by RIO. Conclusion Serotonin-related genes alterations in the lungs of rats with experimental PAH are indicative of increased serotonin signalling and they are partly normalized after of short-term treatment with riociguat. At the same time, riociguat had beneficial effects on renal damage markers in PAH without affecting neurohumoral activation associated with PAH. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Research and Development Support AgencyScientific Grant Agency of the Ministry of Education, Science, Research and Sports of the Slovak Republic

The Effects of Mild Traumatic Brain Injury (mTBI) on Serotonergic Gene Expression

Traumatic brain injury (TBI) is a risk factor for suicide. Approximately 1.7 million TBIs occur each year and new TBI cases in the U.S. military have more than doubled in the last 5 years. The majority of military TBIs are mild in severity and can lead to chronic symptoms including, impulsivity, emotional instability, and impaired inhibition. Impulsivity is one of the most frequent behavioral changes seen following mTBI and involves poor response inhibition and/or poor decision making. Also, impulsivity is a key risk factor for suicide. The serotonergic system is an important modulator of impulsivity and, thus, a molecular target for understanding the behavioral changes in impulsivity that occur following mTBI. One major source of serotonin in the brain is the raphe nuclei in the pons. The serotonergic neurons in the raphe nuclei project to the prefrontal cortex, frontal cortex and hippocampus and are involved in many cognitive processes related to impulsivity and suicidality. In addition, both animal and human research indicates that the brainstem is affected in mTBI, particularly, in the lateral fluid percussion model utilized in this study. This provides additional support for the notion that mTBI may lead to dysfunction of the serotonin producing raphe nuclei located in the brain stem. In this study, changes in serotonergic gene expression in the raphe nuclei and target regions including the prefrontal cortex, frontal cortex and hippocampus were assessed using qRT‐PCR. Rats sustained a single mTBI induced using the lateral fluid percussion model and were sacrificed three months following injury. Brains were extracted, flash frozen, sectioned at 300um and tissue for the raphe nuclei and target regions was collected using a 1.5 mm tissue punch. RNA was extracted from the tissue punches, reverse transcribed into cDNA and qRT‐PCR was performed. The hypothesis states that mTBI leads to altered expression of serotonin related genes in the raphe nuclei and/or raphe nuclei target regions including the prefrontal cortex, frontal cortex, and hippocampus. Results show altered expression levels of tryptophan hydroxylase and serotonin transporter genes in both the medial raphe and dorsal raphe nuclei in mTBI rats compared to SHAM control rats. Changes in expression levels of the 5HT1A and 5HT2A receptor genes were also seen in the frontal cortex, prefrontal cortex, and ventral hippocampus. The current study serves to provide evidence for a link between TBI and altered serotonin levels to better understand the molecular mechanisms underlying impulsivity and suicidality in humans following TBI.

Oral Microbiota Changes Contribute to Autism Spectrum Disorder in Mice

The oral microbiota has been implicated in various neurologic conditions, including autism spectrum disorder (ASD), a category of neurodevelopmental disorders defined by core behavioral impairments. Recent data propose the etiopathogenetic role of intestinal microbiota in ASD. The aim of the present study was to elucidate whether the oral microbiota contributes to the pathogenesis of ASD. On the basis of microbial changes detected in the oral cavity of children with ASD, we transferred oral microbiota from donors with ASD and typical development (TD) into an antibiotic-mediated microbiota-depleted mouse model and found that the ASD microbiota is sufficient to induce ASD-like behaviors, such as impaired social behavior. Mice receiving oral microbiota from the ASD donor showed significantly different microbiota structures in their oral cavity and intestinal tract as compared with those receiving TD microbiota and those not receiving any bacterium. The prefrontal cortex of ASD microbiota recipient mice displayed an alternative transcriptional profile with significant upregulation of serotonin-related gene expression, neuroactive ligand-receptor interaction, and TGF-β signaling pathway relative to that in TD microbiota recipient mice. The expression of serotonin-related genes was significantly increased in ASD microbiota recipient mice and was associated with selective autistic behaviors and changes in abundance of specific oral microbiota, including species of Bacteroidetes [G-7], Porphyromonas, and Tannerella. Machine learning based on the causal inference method confirmed a contributing role of Porphyromonas sp. HMT 930 in ASD. Taken together, the oral microbiota of children with ASD can lead to ASD-like behaviors, differences in microbial community structures, and altered neurosignaling activities in recipient mice; this highlights the mouth-microbial-brain connections in the development of neuropathology and provides a novel strategy to fully understand the etiologic mechanism of ASD.

Moderate intensity of static magnetic fields can alter the avoidance behavior and fat storage of Caenorhabditis elegans via serotonin.

Man-made static magnetic fields (SMFs) widely exist in human life as a physical environmental factor.However, the biological responses to moderate SMFs exposure and their underlying mechanisms are largely unknown. The present study was focused on exploring the nervous responses to moderate-intensity SMFs at 0.5T and 1T in Caenorhabditis elegans (C. elegans). We found that SMFs at either 0.5T or 1T had no statistically significant effects on the locomotor behaviors, while the 1T magnetic field increased pharyngeal pumping. The avoidance behavior of the pathogenic Pseudomonas aeruginosa was greatly decreased in either 0.5T or 1T SMFs exposed nematodes, and the learning index was reducede from 0.52 ± 0.11 to 0.23 ± 0.17 and 0.16 ± 0.11, respectively. The total serotonin level was increased by 17.08% and 16.45% with the treatment of 0.5T and 1T SMF, compared to the control group; however, there were minimal effects of SMFs on other three neurotransmitters including choline, γ-aminobutyric acid (GABA), dopamine. RT-qPCR was used to further investigate the expression of serotonin-related genes, including rate-limiting enzymes, transcription factors and transport receptors. The expression levels of tph-1 and unc-86 genes were increased by SMF exposure, while those of ocr-2, osm-9, ser-1 and mod-1 genes were decreased. With the staining of lipid in either wild-type N2 or tph-1 mutants, we found that 0.5T and 1T SMFs decreased fat storage in C. elegans via serotonin pathway. Our study demonstrated that moderate-intensity SMFs induced neurobehavioral disorder and the reduction of fat storage by disturbing the secretion of serotonin in C. elegans, which provided new insights into elucidating nervous responses of C. elegans to moderate-intensity SMFs.

Trait Anxiety Mediated by Amygdala Serotonin Transporter in the Common Marmoset.

High trait anxiety is associated with altered activity across emotion regulation circuitry and a higher risk of developing anxiety disorders and depression. This circuitry is extensively modulated by serotonin. Here, to understand why some people may be more vulnerable to developing affective disorders, we investigated whether serotonin-related gene expression across the brain's emotion regulation circuitry may underlie individual differences in trait anxiety using the common marmoset (Callithrix jacchus, mixed sexes) as a model. First, we assessed the association of region-specific expression of the serotonin transporter (SLC6A4) and serotonin receptor (HTR1A, HTR2A, HTR2C) genes with anxiety-like behavior; and second, we investigated their causal role in two key features of the high trait anxious phenotype: high responsivity to anxiety-provoking stimuli and an exaggerated conditioned threat response. While the expression of the serotonin receptors did not show a significant relationship with anxiety-like behavior in any of the targeted brain regions, serotonin transporter expression, specifically within the right ventrolateral prefrontal cortex (vlPFC) and most strongly in the right amygdala, was associated positively with anxiety-like behavior. The causal relationship between amygdala serotonin levels and an animal's sensitivity to threat was confirmed via direct amygdala infusions of a selective serotonin reuptake inhibitor (SSRI), citalopram. Both anxiety-like behaviors, and conditioned threat-induced responses were reduced by the blockade of serotonin reuptake in the amygdala. Together, these findings provide evidence that high amygdala serotonin transporter expression contributes to the high trait anxious phenotype and suggest that reduction of threat reactivity by SSRIs may be mediated by their actions in the amygdala.SIGNIFICANCE STATEMENT Findings here contribute to our understanding of how the serotonin system underlies an individual's expression of threat-elicited negative emotions such as anxiety and fear within nonhuman primates. Exploration of serotonergic gene expression across brain regions implicated in emotion regulation revealed that serotonin transporter gene expression in the ventrolateral prefrontal cortex (vlPFC) and most strongly in the amygdala, but none of the serotonin receptor genes, were predictive of interindividual differences in anxiety-like behavior. Targeting of amygdala serotonin reuptake with selective serotonin reuptake inhibitors (SSRIs) confirmed the causal relationship between amygdala serotonin transporter and an animal's sensitivity to threat by reversing expression of two key features of the high trait-like anxiety phenotype: high responsivity to anxiety-provoking uncertain threat and responsivity to certain conditioned threat.

Expression of 22 serotonin-related genes in rat brain after sub-acute serotonin depletion or reuptake inhibition.

Although the assessment of expression of serotonin-related genes in experimental animals has become a common strategy to shed light on variations in brain serotonergic function, it remains largely unknown to what extent the manipulation of serotonin levels causes detectable changes in gene expression. We therefore chose to investigate how sub-acute depletion or elevation of brain serotonin influences the expression of a number of serotonin-related genes in six brain areas. Male Wistar rats were administered a serotonin synthesis inhibitor, para-chlorophenylalanine (p-CPA), or a serotonin reuptake inhibitor, paroxetine, for 3 days and then sacrificed. The expression of a number of serotonin-related genes in the raphe nuclei, hypothalamus, amygdala, striatum, hippocampus and prefrontal cortex was investigated using real-time quantitative PCR (rt-qPCR). While most of the studied genes were uninfluenced by paroxetine treatment, we could observe a robust downregulation of tryptophan hydroxylase-2 in the brain region where the serotonergic cell bodies reside, that is, the raphe nuclei. p-CPA induced a significant increase in the expression of Htr1b and Htr2a in amygdala and of Htr2c in the striatum and a marked reduction in the expression of Htr6 in prefrontal cortex; it also enhanced the expression of the brain-derived neurotrophic factor (Bdnf) in raphe and hippocampus. With some notable exceptions, the expression of most of the studied genes is left unchanged by short-term modulation of extracellular levels of serotonin.

Effects of maternal separation on serotonergic systems in the dorsal and median raphe nuclei of adult male Tph2-deficient mice

Previous studies have highlighted interactions between serotonergic systems and adverse early life experience as important gene x environment determinants of risk of stress-related psychiatric disorders. Evidence suggests that mice deficient in Tph2, the rate-limiting enzyme for brain serotonin synthesis, display disruptions in behavioral phenotypes relevant to stress-related psychiatric disorders. The aim of this study was to determine how maternal separation in wild-type, heterozygous, and Tph2 knockout mice affects mRNA expression of serotonin-related genes. Serotonergic genes studied included Tph2, the high-affinity, low-capacity, sodium-dependent serotonin transporter (Slc6a4), the serotonin type 1a receptor (Htr1a), and the corticosterone-sensitive, low-affinity, high-capacity sodium-independent serotonin transporter, organic cation transporter 3 (Slc22a3). Furthermore, we studied corticotropin-releasing hormone receptors 1 (Crhr1) and 2 (Crhr2), which play important roles in controlling serotonergic neuronal activity. For this study, offspring of Tph2 heterozygous dams were exposed to daily maternal separation for the first two weeks of life. Adult, male wild-type, heterozygous, and homozygous offspring were subsequently used for molecular analysis. Maternal separation differentially altered serotonergic gene expression in a genotype- and topographically-specific manner. For example, maternal separation increased Slc6a4 mRNA expression in the dorsal part of the dorsal raphe nucleus in Tph2 heterozygous mice, but not in wild-type or knockout mice. Overall, these data are consistent with the hypothesis that gene x environment interactions, including serotonergic genes and adverse early life experience, play an important role in vulnerability to stress-related psychiatric disorders.

P.3.16 DNA methylation at the myelin basic protein gene - susceptibility to prenatal stress in mouse and man

Previous studies have highlighted interactions between serotonergic systems and adverse early life experience as important gene x environment determinants of risk of stress-related psychiatric disorders. Evidence suggests that mice deficient in Tph2, the rate-limiting enzyme for brain serotonin synthesis, display disruptions in behavioral phenotypes relevant to stress-related psychiatric disorders. The aim of this study was to determine how maternal separation in wild-type, heterozygous, and Tph2 knockout mice affects mRNA expression of serotonin-related genes. Serotonergic genes studied included Tph2, the high-affinity, low-capacity, sodium-dependent serotonin transporter (Slc6a4), the serotonin type 1a receptor (Htr1a), and the corticosterone-sensitive, low-affinity, high-capacity sodium-independent serotonin transporter, organic cation transporter 3 (Slc22a3). Furthermore, we studied corticotropin-releasing hormone receptors 1 (Crhr1) and 2 (Crhr2), which play important roles in controlling serotonergic neuronal activity. For this study, offspring of Tph2 heterozygous dams were exposed to daily maternal separation for the first two weeks of life. Adult, male wild-type, heterozygous, and homozygous offspring were subsequently used for molecular analysis. Maternal separation differentially altered serotonergic gene expression in a genotype- and topographically-specific manner. For example, maternal separation increased Slc6a4 mRNA expression in the dorsal part of the dorsal raphe nucleus in Tph2 heterozygous mice, but not in wild-type or knockout mice. Overall, these data are consistent with the hypothesis that gene x environment interactions, including serotonergic genes and adverse early life experience, play an important role in vulnerability to stress-related psychiatric disorders.

Neuroinflammation and aberrant hippocampal plasticity in a mouse model of emotional stress evoked by exposure to ultrasound of alternating frequencies

Emotional stress is a form of stress evoked by processing negative mental experience rather than an organic or physical disturbance and is a frequent cause of neuropsychiatric pathologies, including depression. Susceptibility to emotional stress is commonly regarded as a human-specific trait that is challenging to model in other species. Recently, we showed that a 3-week-long exposure to ultrasound of unpredictable alternating frequencies within the ranges of 20-25 kHz and 25-45 kHz can induce depression-like characteristics in laboratory mice and rats. In an anti-depressant sensitive manner, exposure decreases sucrose preference, elevates behavioural despair, increases aggression, and alters serotonin-related gene expression. To further investigate this paradigm, we studied depression/distress-associated markers of neuroinflammation, neuroplasticity, oxidative stress and the activity of glycogen synthase kinase-3 (GSK-3) isoforms in the hippocampus of male mice. Stressed mice exhibited a decreased density of Ki67-positive and DCX-positive cells in the subgranular zone of hippocampus, and altered expression of brain-derived neurotrophic factor (BDNF), its receptor TrkB, and anti-apoptotic protein kinase B phosphorylated at serine 473 (AktpSer473). The mice also exhibited increased densities of Iba-1-positive cells, increased oxidative stress, increased levels of interleukin-1β (IL-1β), interleukin-6 (IL-6) in the hippocampus and plasma, and elevated activity of GSK-3 isoforms. Together, the results of our investigation have revealed that unpredictable alternating ultrasound evokes behavioural and molecular changes that are characteristic of the depressive syndrome and validates this new and simple method of modeling emotional stress in rodents.

Comparing the Expression of Genes Related to Serotonin (5-HT) in C57BL/6J Mice and Humans Based on Data Available at the Allen Mouse Brain Atlas and Allen Human Brain Atlas.

Brain atlases are tools based on comprehensive studies used to locate biological characteristics (structures, connections, proteins, and gene expression) in different regions of the brain. These atlases have been disseminated to the point where tools have been created to store, manage, and share the information they contain. This study used the data published by the Allen Mouse Brain Atlas (2004) for mice (C57BL/6J) and Allen Human Brain Atlas (2010) for humans (6 donors) to compare the expression of serotonin-related genes. Genes of interest were searched for manually in each case (in situ hybridization for mice and microarrays for humans), normalized expression data (z-scores) were extracted, and the results were graphed. Despite the differences in methodology, quantification, and subjects used in the process, a high degree of similarity was found between expression data. Here we compare expression in a way that allows the use of translational research methods to infer and validate knowledge. This type of study allows part of the relationship between structures and functions to be identified, by examining expression patterns and comparing levels of expression in different states, anatomical correlations, and phenotypes between different species. The study concludes by discussing the importance of knowing, managing, and disseminating comprehensive, open-access studies in neuroscience.

Reproductive steroid receptors and actions in the locus coeruleus of male macaques: Part of an aggression circuit?

This study was initiated to determine whether the noradrenergic (NE) neurons of the locus coeruleus (LC) could mediate the stimulatory action of androgens on serotonin-related gene expression in male macaques. These experiments follow our observations that serotonin neurons lack androgen receptors (ARs), and yet respond to androgens. Male Japanese macaques (Macaca fuscata) were castrated for 5–7months and then treated for 3months with [1] placebo, [2] T (testosterone), [3] DHT (dihydrotestosterone; non-aromatizable androgen) plus ATD (steroidal aromatase inhibitor), or [4] FLUT (Flutamide; androgen antagonist) plus ATD (n=5/group). The noradrenergic (NE) innervation of the raphe was determined with immunolabeling of axons with an antibody to dopamine-β-hydroxylase (DBH). Immunolabeling of tyrosine hydroxylase (TH) dendrites and corticotropin releasing hormone (CRH) axons innervating the LC was also determined. Due to the longer treatment period employed, the expression of the cognate nuclear receptors was sought. Androgen receptor (AR), estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) immunostaining was accomplished. Quantitative image analysis was applied and immunopositive neurons or axons with boutons were measured. Double-label of NE neurons for each receptor plus TH determined whether the receptors were localized in NE neurons. Androgens with or without aromatase activity significantly stimulated DBH axon density in the raphe (ANOVA, p=0.006), and LC dendritic TH (ANOVA, p<0.0001), similar to serotonin-related mRNA expression in the raphe. There were significantly more AR-positive neurons in T- and DHT+ATD-treated groups compared to placebo or FLUT+ATD-treated groups (ANOVA, p=0.0014). There was no difference in the number of positive-neurons stained for ERα or ERβ. The CRH axon density in the LC was significantly reduced with aromatase inhibition, suggesting that CRH depends on estrogen, not androgens (ANOVA, p=0.0023). Double-immunohistochemistry revealed that NE neurons did not contain AR. Rather, AR-positive nuclei were found in neighboring cells that are likely neurons. However, >80% of LC NE neurons contained ERα or ERβ. In conclusion, the LC NE neurons may transduce the stimulatory effect of androgens on serotonin-related gene expression. Since LC NE neurons lack AR, the androgenic stimulation of dendritic TH and axonal DBH may be indirectly mediated by other neurons. Estrogen, either from metabolism of T or from de novo synthesis, appears necessary for robust CRH innervation of the LC, which differs from female macaques.

High fat diet decreases beneficial effects of estrogen on serotonin-related gene expression in marmosets

The administration of estradiol-17β (E) to animal models after loss of ovarian steroid production has many beneficial effects on neural functions, particularly in the serotonin system in nonhuman primates (NHPs). E also has anorexic effects, although the mechanism of action is not well defined. In the US, obesity has reached epidemic proportions, and blame is partially directed at the Western style diet, which is high in fat and sugar. This study examined the interaction of E and diet in surgically menopausal nonhuman primates with a 2×2 block design. Marmosets (Callithrix jacchus; n=4/group) were placed on control-low fat diet (LFD; 14%kcal from fat) or high fat diet (HFD; 28%kcal from fat) 1month prior to ovariectomy (Ovx). Empty (placebo) or E-filled Silastic capsules were implanted immediately following Ovx surgery. Treatments extended 6months. The established groups were: placebo+LFD, E+LFD, placebo+HFD, or E+HFD. At necropsy, the brain was flushed with saline and harvested. The midbrain was dissected and a small block containing the dorsal raphe nucleus was processed for qRT-PCR using Evagreen (Biotinum). Genes previously found to impact serotonin neural functions were examined. Results were compared with 2-way ANOVA followed by Bonferroni post-hoc tests or Cohen's D analysis. There was a significant effect of treatment on tryptophan hydroxylase 2 (TPH2) across the groups (p=0.019). E stimulated TPH2 expression and HFD prevented E-stimulated TPH2 expression (p<0.01). Treatment differentially affected monoamine oxidase B (MAO-B) across the groups (p=0.05). E increased MAO-B with LFD, and this stimulatory effect was prevented by HFD (p<0.05). There was a significant difference between treatments in corticotrophin releasing factor-receptor 2 (CRF-R2) expression (p=0.012). E increased CRF-R2 and this stimulatory effect was blocked by HFD (p<0.01). Regardless of diet, E increased Fev mRNA (p=0.028) and decreased CRF-receptor 1 (CRF-R1) mRNA (p=0.04). HFD suppressed urocortin 1 (UCN1; stresscopin) expression (p=0.045) but E treatment had no effect. Monoamine oxidase A (MAO-A) was different due to treatment across the groups (p=0.028). MAO-A was increased in the E+HFD group (p<0.01) whereas previous studies showed E suppressed MAO-A in macaques. The serotonin reuptake transporter (SERT), the serotonin 1A receptor (5HT1A), estrogen receptor beta (ERβ) and progestin receptor (PR) expressions were not different between groups. Estrogen receptor alpha (ERα) was undetectable. In summary, the data indicate that important actions of hormone therapy in the serotonin system may be lost in the context of a HFD.

Relationships between androgens, serotonin gene expression and innervation in male macaques

Androgen administration to castrated individuals was purported to decrease activity in the serotonin system. However, we found that androgen administration to castrated male macaques increased fenfluramine-induced serotonin release as reflected by increased prolactin secretion. In this study, we sought to define the effects of androgens and aromatase inhibition on serotonin-related gene expression in the dorsal raphe, as well as serotonergic innervation of the LC. Male Japanese macaques (Macaca fuscata) were castrated for 5–7months and then treated for 3months with (1) placebo, (2) testosterone (T), (3) dihydrotestosterone (DHT; non-aromatizable androgen) and ATD (steroidal aromatase inhibitor), or (4) Flutamide (FLUT; androgen antagonist) and ATD (n=5/group). This study reports the expression of serotonin-related genes: tryptophan hydroxylase 2 (TPH2), serotonin reuptake transporter (SERT) and the serotonin 1A autoreceptor (5HT1A) using digoxigenin-ISH and image analysis. To examine the production of serotonin and the serotonergic innervation of a target area underlying arousal and vigilance, we measured the serotonin axon density entering the LC with ICC and image analysis. TPH2 and SERT expression were significantly elevated in T- and DHT+ATD-treated groups over placebo- and FLUT+ATD-treated groups in the dorsal raphe (p<0.007). There was no difference in 5HT1A expression between the groups. There was a significant decrease in the pixel area of serotonin axons and in the number of varicosities in the LC across the treatment groups with T>placebo>DHT+ATD=FLUT+ATD treatments. Comparatively, T- and DHT+ATD-treated groups had elevated TPH2 and SERT gene expression, but the DHT+ATD group had markedly suppressed serotonin axon density relative to the T-treated group. Further comparison with previously published data indicated that TPH2 and SERT expression reflected yawning and basal prolactin secretion. The serotonin axon density in the LC agreed with the area under the fenfluramine-stimulated prolactin curve, providing a morphological basis for the pharmacological results. This suggested that androgen activity increased TPH2 and SERT gene expression but, aromatase activity, and neural production of estradiol (E), may subserve axonal serotonin and determination of the compartment acted upon by fenfluramine. In summary, androgens stimulated serotonin-related gene expression, but aromatase inhibition dissociated gene expression from the serotonin innervation of the LC terminal field and fenfluramine-stimulated prolactin secretion.

P.2.a.017 Effects of hypergravity on serotonin-related gene expression in the mouse brain

P.2.a.017 Effects of hypergravity on serotonin-related gene expression in the mouse brain

The effect of short-term stress on serotonin gene expression in high and low resilient macaques.

Female cynomolgus monkeys exhibit different degrees of reproductive dysfunction with moderate metabolic and psychosocial stress. When stressed with a paradigm of relocation and diet for 60 days, or 2 menstrual cycles, highly stress resilient monkeys continue to ovulate during both stress cycles (HSR); medium stress resilient monkeys ovulate once (MSR) and stress sensitive monkeys do not ovulate for the entire 60 days (SS). This study examines serotonin-related gene expression in monkeys with different sensitivity to stress and exposed to 5 days of moderate stress. Monkeys were first characterized as HSR, MSR or SS. After resumption of menstrual cycles, each monkey was re-stressed for 5 days in the early follicular phase. The expression of 3 genes pivotal to serotonin neural function was assessed in the 3 groups of monkeys (n=4-5/group). Tryptophan hydroxylase 2 (TPH2), the serotonin reuptake transporter (SERT), and the 5HT1A autoreceptor mRNAs expression were determined at 4 morphological levels of the dorsal raphe nucleus with in situ hybridization (ISH) using digoxigenin-incorporated riboprobes. In addition, cFos was examined with immunohistochemistry. Positive pixel area and/or cell number were measured. All data were analyzed with ANOVA (3 groups) and with a t-test (2 groups). After 5 days of stress, TPH2, SERT, 5HT1A and cFos were significantly lower in the SS group than the HSR group (p<0.05, all). This pattern of expression was the same as the pattern observed in the absence of stress in previous studies. Therefore, the ratio of the HSR/SS expression of each serotonergic gene was calculated in the presence and absence of stress. There was little or no difference in the ratio of HSR/SS gene expression in the presence or absence of stress. Moreover, cFos expression indicates that overall, cell activation in the dorsal raphe nucleus and periaquaductal gray is lower in SS than HSR animals. These data suggest that the serotonin system may set the sensitivity or resilience of the individual, but serotonin-related gene expression may not rapidly respond to moderate stress in nonhuman primates.

Effect of a new potential psychotropic drug, 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-amine hydrochloride, on the expression of serotonin-related genes in the mouse brain

Study of molecular mechanisms of psychotropic drug action is the main aim of molecular psychopharmacology. New synthetic analog of variacin 8-(Trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-amine (TX-2153) was shown to produce anxiolytic and anticonvulsant effects on mice. Here the effect of chronic administration of TX-2153 on expression of some serotonin-related genes in mouse brain was investigated. The drug (10 mg/kg, per os, 16 days) was administered to adult males of ASC (Antidepressant Sensitive Catalepsy) mouse strain characterizing by alterations in behavior and brain serotonin system. The expression of genes encoding 1) the key enzyme of serotonin synthesis, tryptophan hydroxylase 2 (TPH2), 2) main enzyme of serotonin degradation, monoamine oxydase A (MAOA), 3) 5-HT transporter (SERT) and 4) 5-HT(1A) receptor was studied using quantitative RT-PCR. TX-2153 significantly reduced m-RNA level of 5-HT(1A) receptor and MAOA genes in the midbrain without any effect on expression of these genes in the frontal cortex and hippocampus. The drug failed to affect expression of TPH2 and SERT genes in the midbrain. The result indicates involvement of the brain 5-HT system in the molecular mechanism underlying the effect of TX-2153.

Interactions of Corticotropin-Releasing Factor, Urocortin and Citalopram in a Primate Model of Stress-Induced Amenorrhea

Background/Aims: We established a cynomolgus macaque model of stress-induced amenorrhea in which the application of combined metabolic and psychosocial stress suppressed ovulation in stress-sensitive (SS) individuals, but not in highly stress-resilient (HSR) individuals. We previously reported that SS monkeys have deficits in global serotonin release and serotonin-related gene expression in the raphe nucleus, and that administration of the selective serotonin reuptake inhibitor S-citalopram increased estrogen and progesterone production in SS monkeys. In this study, we questioned whether there was a difference in corticotropin-releasing factor (CRF) or urocortin (UCN) stress-related peptide systems in the midbrain raphe region when HSR and SS monkeys treated with placebo or S-citalopram are compared. Methods: Monkeys characterized as HSR or SS were administered placebo or S-citalopram for 15 weeks. CRF fibers in the dorsal raphe were detected with an antibody against human CRF. UCN1 fibers were immunostained in an area rostral to the dorsal raphe. The fibers were quantified by stereology and analyzed by two-way ANOVA. UCN1 cell bodies were counted in the supraoculomotor area near the Edinger-Westphal nucleus. Results: S-citalopram significantly decreased the CRF fiber density in SS animals, but not in HSR animals. SS monkeys had a significantly lower UCN1 fiber density compared to HSR monkeys, but S-citalopram treatment did not alter the UCN1 fiber density. SS animals treated with S-citalopram tended to have a higher number of UCN1-positive cell bodies than the other groups. Conclusion: S-citalopram decreased CRF fiber density and appears to increase the production of UCN1 in SS individuals, indicating the likelihood that serotonin is involved in regulating CRF and UCN1 in individuals who are sensitive to the effects of serotonin.

Stress sensitive female macaques have decreased fifth Ewing variant (Fev) and serotonin-related gene expression that is not reversed by citalopram

Female cynomolgus monkeys exhibit different degrees of reproductive dysfunction with moderate metabolic and psychosocial stress. When stressed with a paradigm of relocation and diet for 60 days or two menstrual cycles, highly stress resilient monkeys (HSR) continued to ovulate during the stress cycles whereas stress sensitive monkeys (SS) did not. After cessation of stress, monkeys characterized as HSR or SS were administered placebo (PL) or S-citalopram (CIT) for 15 weeks at doses that normalized ovarian steroid secretion in the SS animals and that maintained blood CIT levels in a therapeutic range. After euthanasia, the brain was perfused with 4% paraformaldehyde. The pontine midbrain was blocked and sectioned at 25 μm. The expression of four genes pivotal to serotonin neural function was assessed in the four groups of monkeys ( n=4/group). Fev (fifth Ewing variant) ETS transcription factor, tryptophan hydroxylase 2 (TPH2), the serotonin reuptake transporter (SERT), and the 5HT1A autoreceptor were determined at 7–8 levels of the dorsal raphe nucleus with in situ hybridization (ISH) using radiolabeled- and digoxygenin-incorporated riboprobes. Positive pixel area and cell number were measured with Slidebook 4.2 in the digoxigenin assay for Fev. Optical density (OD) and positive pixel area were measured with NIH Image software in the radiolabeled assays for TPH2, SERT and 5HT1A. All data were analyzed with two-way ANOVA. SS monkeys had significantly fewer Fev-positive cells and lower Fev-positive pixel area in the dorsal raphe than HSR monkeys. SS monkeys also had significantly lower levels of TPH2, SERT and 5HT1A mRNAs in the dorsal raphe nucleus than HSR monkeys. However, CIT did not alter the expression of either Fev, TPH2, SERT or 5HT1A mRNAs. These data suggest that SS monkeys have fewer serotonin (5-HT) neurons than HSR monkeys, and that they have deficient Fev expression, which in turn, leads to deficient TPH2, SERT and 5HT1A expression. In addition, the therapeutic effect of CIT is probably achieved through mechanisms other than alteration of 5-HT-related gene expression.