Effects of the Cc2d1a/Freud-1 Knockdown in the Hippocampus of BTBR Mice on the Autistic-Like Behavior, Expression of Serotonin 5-HT1A and D2 Dopamine Receptors, and CREB and NF-kB Intracellular Signaling.

The mechanisms of autism are of extreme interest due to the high prevalence of this disorder in the human population. In this regard, special attention is given to the transcription factor Freud-1 (encoded by the Cc2d1a gene), which regulates numerous intracellular signaling pathways and acts as a silencer for 5-HT1A serotonin and D2 dopamine receptors. Disruption of the Freud-1 functions leads to the development of various psychopathologies. In this study, we found an increase in the expression of the Cc2d1a/Freud-1 gene in the hippocampus of BTBR mice (model of autistic-like behavior) in comparison with C57Bl/6J mice and examined how restoration of the Cc2d1a/Freud-1 expression in the hippocampus of BTBR mice affects their behavior, expression of 5-HT1A serotonin and D2 dopamine receptors, and CREB and NF-κB intracellular signaling pathways in these animals. Five weeks after administration of the adeno-associated viral vector (AAV) carrying the pAAV_H1-2_shRNA-Freud-1_Syn_EGFP plasmid encoding a small hairpin RNA (shRNA) that suppressed expression of the Cc2d1a/Freud-1 gene, we observed an elevation in the anxiety levels, as well as the increase in the escape latency and path length to the platform in the Morris water maze test, which was probably associated with a strengthening of the active stress avoidance strategy. However, the Cc2d1a/Freud-1 knockdown did not affect the spatial memory and phosphorylation of the CREB transcription factor, although such effect was found in C57Bl/6J mice in our previous study. These results suggest the impairments in the CREB-dependent effector pathway in BTBR mice, which may play an important role in the development of the autistic-like phenotype. The knockdown of Cc2d1a/Freud-1 in the hippocampus of BTBR mice did not affect expression of the 5-HT1A serotonin and D2 dopamine receptors and key NF-κB signaling genes (Nfkb1 and Rela). Our data suggest that the transcription factor Freud-1 plays a significant role in the pathogenesis of anxiety and active stress avoidance in autism.

Fluoxetine regulates the neuronal differentiation of neural stem cells transplanted into rat brains after stroke by increasing the 5HT level.

Fluoxetine, a 5-HT uptake inhibitor, has been adopted for the treatment of post-stroke depression in recent years. It has been confirmed to induce neuronal regeneration in vivo, but its effect on inducing stem cell differentiation after transplantation has not yet been verified. To evaluate its regulatory effect on stem cell differentiation, fluoxetine was used in this study to treat rats with cerebral ischemia after neural stem cell (NSC) transplantation. The results showed that the proportion of NSCs differentiating into neurons significantly increased after fluoxetine treatment. In NSC adherent culture, the addition of 5-HT but not of fluoxetine significantly increased the neuronal differentiation ratio of NSCs. Moreover, the addition of 5-HT2A or 5-HT3A antagonists inhibited this effect. In addition, Western blotting revealed that the increase in 5-HT inhibited ERK2 phosphorylation and upregulated neurogenin1 expression. In conclusion, fluoxetine increased the 5-HT level and promoted neuronal differentiation, thereby upregulating neurogenin1 expression and downregulating ERK2 phosphorylation.

Protective effect of the combination of essential oil from patchouli and tangerine peel against gastric ulcer in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Essential oil (EO) is the main extract of patchouli and tangerine peel with antiinflammatory, antiulcer, and other functions. However, the efficacy and mechanism of the combination of EO from patchouli and tangerine peel against gastric ulcer (GU) are unclear. AIM OF THE STUDY: This study aims to reveal the protective effect of the combination of EO from patchouli and tangerine peel against GU in rats, as well as explore the optimal ratio and possible mechanism of EO in GU treatment. MATERIALS AND METHODS: The GU model is executed via water immersion and restraint stress. The repair effect of EO in different proportions on gastric mucosa injury and the effects on serum gastrin (GAS), pepsinogen C (PGC), prostaglandin E2 (PGE2), and 5-hydroxytryptamine in GU rats were observed. The optimal ratio obtained was used in the second part to set different dose groups for further experiment. The effects of the different EO doses on gastric mucosal ulcer formation and gastric acid secretion were evaluated. The morphology of chief and parietal cells were observed via transmission electron microscopy. The contents of GAS, PGC, substance P (SP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), cholecystokinin (CCK), PGE2, and motilin (MTL) in serum in different groups were detected via enzyme-linked immunosorbent assay. Expressions of epidermal growth factor (EGF) and trefoil factor 2 (TFF2) protein in gastric tissues were detected via immunohistochemistry, and expressions of c-Jun N-terminal kinase (JNK), P53, Bcl-2-associated X protein (Bax), and Caspase-3 protein in gastric tissues were detected via western blotting. RESULTS: The EO from patchouli and tangerine peel at 1:2 ratio of compatibility significantly improved gastric mucosal injury, decreased serum GAS and PGC contents, and increased the PGE2 level in serum (p < 0.05). The mixture of EO from patchouli and tangerine peel (Mix-EO) can reduce the formation of gastric mucosal ulcers, reduce gastric mucosal injury, improve the expansion of the endoplasmic reticulum of the chief cells, repair mitochondrial damage, and inhibit the secretion of gastric acid by parietal cells. Mix-EO at 300 mg/kg can reduce the expression of serum GAS, PGC, SP, CCK, and cAMP/cGMP (p < 0.05 or 0.01); increase the expression of EGF and TFF2 protein in gastric tissues (p < 0.01); and inhibit the expression of JNK, p53, Bax, and Caspase-3 proteins (p < 0.01). CONCLUSION: The combination of EO from patchouli and tangerine peel can repair the gastric mucosal damage in GU rats and prevent the occurrence of ulcers by inhibiting the secretion of gastric acid, enhancing the defensive ability of gastric mucosa, and suppressing the apoptosis of gastric epithelial cells. Moreover, the optimal compatible ratio of patchouli and tangerine peel is 1:2.

Effects of a Cc2d1a/Freud-1 Knockdown in the Hippocampus on Behavior, the Serotonin System, and BDNF.

The serotonin 5-HT1A receptor is one of the most abundant and widely distributed brain serotonin (5-HT) receptors that play a major role in the modulation of emotions and behavior. The 5-HT1A receptor gene (Htr1a) is under the control of transcription factor Freud-1 (also known as Cc2d1a/Freud-1). Here, using adeno-associated virus (AAV) constructs in vivo, we investigated effects of a Cc2d1a/Freud-1 knockdown in the hippocampus of C57BL/6J mice on behavior, the brain 5-HT system, and brain-derived neurotrophic factor (BDNF). AAV particles carrying the pAAV_H1-2_shRNA-Freud-1_Syn_EGFP plasmid encoding a short-hairpin RNA targeting mouse Cc2d1a/Freud-1 mRNA had an antidepressant effect in the forced swim test 5 weeks after virus injection. The knockdown impaired spatiotemporal memory as assessed in the Morris water maze. pAAV_H1-2_shRNA-Freud-1_Syn_EGFP decreased Cc2d1a/Freud-1 mRNA and protein levels. Furthermore, the Cc2d1a/Freud-1 knockdown upregulated 5-HT and its metabolite 5-hydroxyindoleacetic acid but not their ratio. The Cc2d1a/Freud-1 knockdown failed to increase mRNA and protein levels of Htr1a but diminished a 5-HT1A receptor functional response. Meanwhile, the Cc2d1a/Freud-1 knockdown reduced Creb mRNA expression and CREB phosphorylation and upregulated cFos mRNA. The knockdown enhanced the expression of a BDNF precursor (proBDNF protein), which is known to play a crucial part in neuroplasticity. Our data indicate that transcription factor Cc2d1a/Freud-1 is implicated in the pathogenesis of depressive disorders not only via the 5-HT1A receptor and transcription factor CREB but also through an influence on BDNF.

Serotonergic Neurotransmission System Modulator, Vortioxetine, and Dopaminergic D2/D3 Receptor Agonist, Ropinirole, Attenuate Fibromyalgia-Like Symptoms in Mice.

Fibromyalgia is a disease characterized by lowered pain threshold, mood disorders, and decreased muscular strength. It results from a complex dysfunction of the nervous system and due to unknown etiology, its diagnosis, treatment, and prevention are a serious challenge for contemporary medicine. Impaired serotonergic and dopaminergic neurotransmission are regarded as key factors contributing to fibromyalgia. The present research assessed the effect of serotonergic and dopaminergic system modulators (vortioxetine and ropinirole, respectively) on the pain threshold, depressive-like behavior, anxiety, and motor functions of mice with fibromyalgia-like symptoms induced by subcutaneous reserpine (0.25 mg/kg). By depleting serotonin and dopamine in the mouse brain, reserpine induced symptoms of human fibromyalgia. Intraperitoneal administration of vortioxetine and ropinirole at the dose of 10 mg/kg alleviated tactile allodynia. At 5 and 10 mg/kg ropinirole showed antidepressant-like properties, while vortioxetine had anxiolytic-like properties. None of these drugs influenced muscle strength but reserpine reduced locomotor activity of mice. Concluding, in the mouse model of fibromyalgia vortioxetine and ropinirole markedly reduced pain. These drugs affected emotional processes of mice in a distinct manner. Hence, these two repurposed drugs should be considered as potential drug candidates for fibromyalgia. The selection of a specific drug should depend on patient's key symptoms.

Effects of miR-143 and its target receptor 5-HT2B on agonistic behavior in the Chinese mitten crab (Eriocheir sinensis).

Chinese mitten crab (Eriocheir sinensis) as a commercially important species is widely cultured in China. However, E. sinensis is prone to agonistic behavior, which causes physical damage and wastes energy resources, negatively impacting their growth and survival. Therefore, understanding the regulatory mechanisms that underlie the switching of such behavior is essential for ensuring the efficient and cost-effective aquaculture of E. sinensis. The 5-HT2B receptor is a key downstream target of serotonin (5-HT), which is involved in regulating animal behavior. In this study, the full-length sequence of 5-HT2B gene was cloned. The total length of the 5-HT2B gene was found to be 3127 bp with a 236 bp 5'-UTR (untranslated region), a 779 bp 3'-UTR, and a 2112 bp open reading frame encoding 703 amino acids. Phylogenetic tree analysis revealed that the 5-HT2B amino acid sequence of E. sinensis is highly conserved with that of Cancer borealis. Using in vitro co-culture and luciferase assays, the miR-143 targets the 5-HT2B 3'-UTR and inhibits 5-HT2B expression was confirmed. Furthermore, RT-qPCR and Western blotting analyses revealed that the miR-143 mimic significantly inhibits 5-HT2B mRNA and protein expression. However, injection of miR-143 did not decrease agonistic behavior, indicating that 5-HT2B is not involved in the regulation of such behavior in E. sinensis.

First Evidence of Kv3.1b Potassium Channel Subtype Expression during Neuronal Serotonergic 1C11 Cell Line Development.

Kv3.1 channel is abundantly expressed in neurons and its dysfunction causes sleep loss, neurodegenerative diseases and depression. Fluoxetine, a serotonin selective reuptake inhibitor commonly used to treat depression, acts also on Kv3.1. To define the relationship between Kv3.1 and serotonin receptors (SR) pharmacological modulation, we showed that 1C11, a serotonergic cell line, expresses different voltage gated potassium (VGK) channels subtypes in the presence (differentiated cells (1C11D)) or absence (not differentiated cells (1C11ND)) of induction. Only Kv1.2 and Kv3.1 transcripts increase even if the level of Kv3.1b transcripts is highest in 1C11D and, after fluoxetine, in 1C11ND but decreases in 1C11D. The Kv3.1 channel protein is expressed in 1C11ND and 1C11D but is enhanced by fluoxetine only in 1C11D. Whole cell measurements confirm that 1C11 cells express (VGK) currents, increasing sequentially as a function of cell development. Moreover, SR 5HT1b is highly expressed in 1C11D but fluoxetine increases the level of transcript in 1C11ND and significantly decreases it in 1C11D. Serotonin dosage shows that fluoxetine at 10 nM blocks serotonin reuptake in 1C11ND but slows down its release when cells are differentiated through a decrease of 5HT1b receptors density. We provide the first experimental evidence that 1C11 expresses Kv3.1b, which confirms its major role during differentiation. Cells respond to the fluoxetine effect by upregulating Kv3.1b expression. On the other hand, the possible relationship between the fluoxetine effect on the kinetics of 5HT1b differentiation and Kv3.1bexpression, would suggest the Kv3.1b channel as a target of an antidepressant drug as well as it was suggested for 5HT1b.

N-3 PUFA improved pup separation-induced postpartum depression via serotonergic pathway regulated by miRNA.

Stress and ovarian hormone fluctuation are risk factors for postpartum depression (PPD). Previous studies suggested antidepressant-like effects of n-3 polyunsaturated fatty acids (PUFA), but their effect on dam animal with additional stress were not clear. The purpose of the present study was to investigate the hypothesis that n-3 PUFA improved PPD through the serotonergic and glutamatergic pathways by modulating miRNA. Rats were fed n-3 PUFA or control diet from gestation, with pup separation (PS) on postpartum days 2-14 and non-PS controls. N-3 PUFA reversed PS-induced depressive behaviors, including increased immobility, latencies to contact first pup and retrieve all pups, and decreased sucrose preference. N-3 PUFA also modulated the hypothalamic-pituitary-adrenal (HPA) axis by decreasing circulating levels of adrenocorticotropic hormone and corticosterone and expression of hypothalamic corticotrophin releasing factor and hippocampal miRNA-218 but increasing the hippocampal expression of glucocorticoid receptor. N-3 PUFA inhibited neuroinflammation by decreasing circulating levels of prostaglandin E2 and hippocampal expression of tumor necrosis factor-α, interleukin-6, and miRNA-155. In addition, n-3 PUFA up-regulated the serotonergic pathway by increasing circulating levels of serotonin and hippocampal expression of serotonin-1A receptor, cAMP response element binding protein (CREB), pCREB, brain-derived neurotrophic factor, and miRNA-182 but did not affect the glutamatergic pathway according to the hippocampal expression of N-methyl-D-aspartate receptor-2B. The present study suggested that n-3 PUFA improved PPD through the serotonergic pathway by modifying the HPA axis, neuroinflammation, and related miRNAs.

Induction of tryptophan hydroxylase in the liver of s.c. tumor model of prostate cancer.

Enhanced degradation of tryptophan (Trp) and thus decreased plasma Trp levels are common in several types of cancers. Although it is well known that Trp catabolism is induced in the tumor microenvironment by the enzymes expressed in cancer cells, immune cells, or both, few studies have examined systemic Trp catabolism in cancer pathophysiology. The present study aimed to evaluate Trp catabolism in both tumor and peripheral tissues using tumor-engrafted Copenhagen rats that were s.c. inoculated with AT-2 rat prostate cancer cells negative for expression of Trp catabolic enzymes. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomics showed significantly decreased plasma Trp levels in AT-2 engrafted rats, accompanied by increased kynurenine/Trp ratios in spleen and thymus and serotonin levels in liver and thymus. Quantitative PCR and enzymatic activity assays showed indoleamine-2, 3-dioxygenase, an inducible enzyme that catalyzes Trp to kynurenine, was increased in tumor tissues, whereas tryptophan-2,3-dioxygenase, a major Trp catabolic enzyme that regulates systemic level of Trp, tended to be increased in the liver of AT-2 engrafted rats. Furthermore, tryptophan hydroxylase-1 (TPH1), an enzyme that catalyzes the reaction of Trp to serotonin, was significantly increased in liver and spleen of AT-2 engrafted rats. Further histochemical analysis revealed that the induction of TPH1 in the liver could be attributed to infiltration of mast cells. A similar phenomenon was observed with nonneoplastic liver samples from colorectal cancer patients. These results suggested that Trp catabolism toward serotonin synthesis might be induced in peripheral remote tissues in cancer, which could have a pathophysiological effect on cancer.

The Role of Serotonin in Concanavalin A-Induced Liver Injury in Mice.

Serotonin is involved in the pathological processes of several liver diseases via the regulation of inflammatory response and oxidative stress. We aimed to investigate the role of serotonin in Concanavalin A- (Con A-) induced acute liver injury (ALI). ALI was induced in C57B/6 wild-type (WT) mice and tryptophan hydroxylase 1 (TPH1) knockout mice through tail vein injection of Con A (15 mg/kg body weight). Another group of TPH1 knockout ALI mice was supplied with 5-hydroxytryptophan (5-HTP) in advance to recover serotonin. The blood and liver tissues of mice were collected in all groups. Markedly increased serum levels of serotonin were identified after the injection of Con A. Increased serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and stronger hepatic tissue pathology were detected, suggesting that serotonin could mediate Con A-induced liver damage. Serotonin significantly facilitated the release of serum and intrahepatic inflammatory cytokines, including interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-17A (IL-17A), interferon-gamma (IFN-γ), and tumor necrosis-alpha (TNF-α), after the administration of Con A. In addition, serotonin significantly increased the intrahepatic levels of oxidative stress markers malonaldehyde (MDA), myeloperoxidase (MPO), and nitric oxide (NO) and decreased antioxidant stress indicator glutathione (GSH) in Con A-treated mice. Additionally, serotonin promoted hepatocyte apoptosis and autophagy based on B-cell lymphoma-2 (Bcl-2), Bcl-2-asociated X protein (Bax), and Beclin-1 levels and TUNEL staining. More importantly, serotonin activated nuclear factor kappa B (NF-κB) and upregulated the hepatic expressions of high mobility group protein B1 (HMGB1), toll-like receptor-4 (TLR4), and downstream molecules in Con A-mediated liver injury. Serotonin 2A receptor was upregulated in liver tissue after Con A injection, and serotonin 2A receptor antagonist Ketanserin protected against Con A-induced hepatitis. These results indicated that serotonin has the potential to aggravate Con A-induced ALI via the promotion of inflammatory response, oxidative stress injury, and hepatocyte apoptosis and the activation of hepatic HMGB1-TLR signaling pathway and serotonin 2A receptor.

Aggression based genome-wide, glutamatergic, dopaminergic and neuroendocrine polygenic risk scores predict callous-unemotional traits.

Aggression and callous, uncaring, and unemotional (CU) traits are clinically related behavioral constructs caused by genetic and environmental factors. We performed polygenic risk score (PRS) analyses to investigate shared genetic etiology between aggression and these three CU-traits. Furthermore, we studied interactions of PRS with smoking during pregnancy and childhood life events in relation to CU-traits. Summary statistics for the base phenotype were derived from the EAGLE-consortium genome-wide association study of children's aggressive behavior and were used to calculate individual-level genome-wide and gene-set PRS in the NeuroIMAGE target-sample. Target phenotypes were 'callousness', 'uncaring', and 'unemotional' sumscores of the Inventory of Callous-Unemotional traits. A total of 779 subjects and 1,192,414 single-nucleotide polymorphisms were available for PRS-analyses. Gene-sets comprised serotonergic, dopaminergic, glutamatergic, and neuroendocrine signaling pathways. Genome-wide PRS showed evidence of association with uncaring scores (explaining up to 1.59% of variance; self-contained Q = 0.0306, competitive-P = 0.0015). Dopaminergic, glutamatergic, and neuroendocrine PRS showed evidence of association with unemotional scores (explaining up to 1.33, 2.00, and 1.20% of variance respectively; self-contained Q-values 0.037, 0.0115, and 0.0473 respectively, competitive-P-values 0.0029, 0.0002, and 0.0045 respectively). Smoking during pregnancy related to callousness scores while childhood life events related to both callousness and unemotionality. Moreover, dopaminergic PRS appeared to interact with childhood life events in relation to unemotional scores. Our study provides evidence suggesting shared genetic etiology between aggressive behavior and uncaring, and unemotional CU-traits in children. Gene-set PRS confirmed involvement of shared glutamatergic, dopaminergic, and neuroendocrine genetic variation in aggression and CU-traits. Replication of current findings is needed.

Specific neural phase relation of serotonin and dopamine modulate the testicular activity in Japanese quail.

Specific phase relation of serotonin and dopamine modulate the hypothalamo-hypophyseal-gonadal axis as well as photosexual responses in Japanese quail, but the effect of these specific phase relations on testicular activity and steroidogenesis is not yet been investigated. We hypothesized that temporal phase relation induced alteration in local testicular gonadotropin-releasing hormone (GnRH)-Gonadotropin-inhibitory hormone (GnIH) and their receptor system may modulate the testicular activity and steroidogenesis through local (paracrine and autocrine) action. To validate this hypothesis, we have checked the alterations in the expression of gonadotropin-releasing hormone receptor (GnRH-R), gonadotropin-inhibitory hormone receptor (GnIH-R) messenger RNA (mRNA), growth hormone receptor (GH-R), proliferating cell nuclear antigen (PCNA), cell communication and gap junctional proteins (14-3-3 and connexin-43 [Cnx-43]), steroidogenic factor-1 (SF-1), steroidogenic acute regulatory (StAR) protein, steroidogenic enzyme (3ß-hydroxysteroid dehydrogenase [3ß-HSD]) in testis as well as androgen receptor (AR) in testis and epididymis of control, 8-, and 12-hr quail. Experimental findings clearly indicate the increased expression of GnIH-R mRNA and suppression of GnRH-R, GH-R, PCNA, 14-3-3, Cnx-43, SF-1, StAR, 3ß-HSD in testis as well as AR in testis and epididymis in 8-hr quail, while 12-hr quail exhibited the opposite results that is significantly decreased expression of GnIH-R mRNA and increased expression of GnRH-R, GH-R, PCNA, 14-3-3, Cnx-43, SF-1, StAR, 3ß-HSD in testis as well as AR in testis and epididymis. The significantly increased intratesticular testosterone has been observed in the 12-hr quail while, 8-hr quail showed opposite result. Hence, it can be concluded that 12-hr quail showed significantly increased testicular activity and steroidogenesis while opposite pattern was observed in 8-hr quail.

Associations Between Catecholaminergic and Serotonergic Genes and Persistent Breast Pain Phenotypes After Breast Cancer Surgery.

Genetic variations in the catecholaminergic and serotonergic pathways may contribute to the development and severity of persistent breast pain. However, investigations of these associations are limited. The purpose of this study was to evaluate for associations between breast pain phenotypes and single nucleotide polymorphisms among 15 genes involved in catecholaminergic and serotonergic neurotransmission. Women rated the presence and intensity of breast pain monthly for 6 months after breast cancer surgery. Distinct latent classes of patients were identified using growth mixture modeling. Logistic regression analyses identified significant differences between genotype or haplotype frequencies and the breast pain classes (ie, no breast pain [n = 96] vs mild breast pain [n = 141], moderate breast pain [n = 46], and severe breast pain [n = 27]). Polymorphisms in 5 genes were associated with membership in the mild pain class: ** beta-2-adrenergic receptor (ADRB2) rs2400707, beta adrenergic receptor kinase 2 (ADRBK2) HapA04, 5-hydroxytryptamine receptor 3A (HTR3A) rs10160548, solute-like carrier (SLC) family 6 member 2-noradrenaline transporter (SLC6A2) rs1566652, and tryptophan hydroxylase 2 (TPH2) rs11179000. Polymorphisms in 3 genes were associated with membership in the moderate pain class: 5-hydroxytryptamine receptor 2A (HTR2A) rs2296972, SLC6A2 rs17841327, and SLC6A3 rs403636. Polymorphisms in 3 genes were associated with membership in the severe pain class: COMT HPS haplotype, SLC family 6 member 2-noradrenaline transporter (SLC6A2) HapD01, and SLC family 6 member 3-noradrenaline transporter (SLC6A3) rs464049. The identification of these associations suggest possible underlying mechanisms that play a role in the development and severity of persistent breast pain. PERSPECTIVE: Findings from this study of women with breast cancer suggest that a number of catecholaminergic and serotonergic mechanisms may play a role in the development and severity of persistent breast pain phenotypes after surgery.

Effects of obesogenic diet and estradiol on dorsal raphe gene expression in old female macaques.

The beneficial effects of bioidentical ovarian steroid hormone therapy (HT) during the perimenopause are gaining recognition. However, the positive effects of estrogen (E) plus or minus progesterone (P) administration to ovariectomized (Ovx) lab animals were recognized in multiple systems for years before clinical trials could adequately duplicate the results. Moreover, very large numbers of women are often needed to find statistically significant results in clinical trials of HT; and there are still opposing results being published, especially in neural and cardiovascular systems. One of the obvious differences between human and animal studies is diet. Laboratory animals are fed a diet that is low in fat and refined sugar, but high in micronutrients. In the US, a large portion of the population eats what is known as a "western style diet" or WSD that provides calories from 36% fat, 44% carbohydrates (includes 18.5% sugars) and 18% protein. Unfortunately, obesity and diabetes have reached epidemic proportions and the percentage of obese women in clinical trials may be overlooked. We questioned whether WSD and obesity could decrease the positive neural effects of estradiol (E) in the serotonin system of old macaques that were surgically menopausal. Old ovo-hysterectomized female monkeys were fed WSD for 2.5 years, and treated with placebo, Immediate E (ImE) or Delayed E (DE). Compared to old Ovx macaques on primate chow and treated with placebo or E, the WSD-fed monkeys exhibited greater individual variance and blunted responses to E-treatment in the expression of genes related to serotonin neurotransmission, CRH components in the midbrain, synapse assembly, DNA repair, protein folding, ubiquitylation, transport and neurodegeneration. For many of the genes examined, transcript abundance was lower in WSD-fed than chow-fed monkeys. In summary, an obesogenic diet for 2.5 years in old surgically menopausal macaques blunted or increased variability in E-induced gene expression in the dorsal raphe. These results suggest that with regard to function and viability in the dorsal raphe, HT may not be as beneficial for obese women as normal weight women.

Activation of the ileal neuroendocrine tumor cell line P-STS by acetylcholine is amplified by histamine: role of H3R and H4R.

Neuroendocrine tumors may present with pseudoallergic reactions like diarrhea and idiopathic anaphylaxis. Here we present the P-STS human ileal neuroendocrine cell line as a model cell line for these tumors. Neuroendocrine markers and changes in cytoplasmic calcium concentration ([Ca2+]i) in response to several possible activators of 5-hydroxytryptamine (5-HT) release were analyzed. P-STS cells still expressed chromogranin A and synaptophysin after 2 years of culture. Tryptophan hydroxylase 1 mRNA and a low amount of 5-HT were also detected. Acetylcholine (ACh) caused a rise in [Ca2+]i. Somatostatin inhibited, whereas histamine (HA) but not the HA receptor ligand betahistine enhanced activation by ACh. The [Ca2+]i response to ACh/HA was inhibited by the HA receptor H3 (H3R) agonist methimepip and by the antidepressant imipramine. Further [Ca2+]i response studies indicated the presence of H4Rs and of a functional calcium sensing receptor. High or low affinity IgE receptor protein or mRNA were not detected. Taken together, neuroendocrine markers and response to intestinal neurotransmitters approve the P-STS cell line as a valuable model for enterochromaffin cells. Enhancement of their ACh-induced pro-secretory response by HA, with a role for H3R and H4R, suggests an amplifying role of neuroendocrine cells in allergen-induced diarrhea or anaphylaxis.

Prolonged Activation of the Htr2b Serotonin Receptor Impairs Glucose Stimulated Insulin Secretion and Mitochondrial Function in MIN6 Cells.

AIMS: Pancreatic ß-cells synthesize and release serotonin (5 hydroxytryptamine, 5HT); however, the role of 5HT receptors on glucose stimulated insulin secretion (GSIS) and the mechanisms mediating this function is not fully understood. The aims of this study were to determine the expression profile of 5HT receptors in murine MIN6 ß-cells and to examine the effects of pharmacological activation of 5HT receptor Htr2b on GSIS and mitochondrial function. MATERIALS AND METHODS: mRNA levels of 5HT receptors in MIN6 cells were quantified by RT qPCR. GSIS was assessed in MIN6 cells in response to global serotonergic activation with 5HT and pharmacological Htr2b activation or inhibition with BW723C86 or SB204741, respectively. In response to Htr2b activation also was evaluated the mRNA and protein levels of PGC1α and PPARy by RT-qPCR and western blotting and mitochondrial function by oxygen consumption rate (OCR) and ATP cellular content. RESULTS: We found that mRNA levels of most 5HT receptors were either very low or undetectable in MIN6 cells. By contrast, Htr2b mRNA was present at moderate levels in these cells. Preincubation (6 h) of MIN6 cells with 5HT or BW723C86 reduced GSIS and the effect of 5HT was prevented by SB204741. Preincubation with BW723C86 increased PGC1α and PPARy mRNA and protein levels and decreased mitochondrial respiration and ATP content in MIN6 cells. CONCLUSIONS: Our results indicate that prolonged Htr2b activation in murine ß-cells decreases glucose-stimulated insulin secretion and mitochondrial activity by mechanisms likely dependent on enhanced PGC1α/PPARy expression.

Raphe gene expression changes implicate immune-related functions in ventilatory plasticity following carotid body denervation in rats.

The regulation of blood gases in mammals requires precise feedback mechanisms including chemoreceptor feedback from the carotid bodies. Carotid body denervation (CBD) leads to immediate hypoventilation (increased PaCO2) in adult rats, but over a period of days and weeks ventilation normalizes due in part to central (brain) mechanisms. Here, we tested the hypothesis that functional ventilatory recovery following CBD correlated with significant shifts in medullary raphe gene expression of molecules/pathways associated with known or novel forms of neuroplasticity. Tissue punches were obtained from snap frozen brainstems collected from rats 1-2days or 14-15days post-sham or post-bilateral CBD surgery (verified by physiologic measurements), and subjected to mRNA sequencing to identify, quantify, and statistically compare gene expression level differences among these groups of rats. We found the greatest number of gene expression changes acutely after CBD (154 genes), with fewer changes in the weeks after CBD (69-80 genes) and the fewest changes in expression among the time control groups (39 genes). Little or no changes were observed for multiple genes associated with serotonin- or glutamate receptor-dependent forms of neuroplasticity. However, an unbiased assessment of gene expression changes using a bioinformatics pathway analysis highlighted multiple changes in gene expression in signaling pathways associated with immune function. These included several growth factors and cytokines associated with peripheral and innate immune systems. Thus, these medullary raphe gene expression data support a role for immune-related signaling pathways in the functional restoration of blood gas control after CBD, but little or no role for serotonin- or glutamate receptor-mediated plasticity.

Lentiviral vector-driven inhibition of 5-HT synthesis in B3 bulbo-spinal serotonergic projections - Consequences on nociception, inflammatory and neuropathic pain in rats.

Although it is well established that bulbo-spinal serotonergic projections contribute to pain control mechanisms, whether they exert anti- or pro-nociceptive modulations is still a matter of debate. In order to reappraise the role of 5-HT in descending controls, we used RNA interference to selectively inhibit 5-HT synthesis in B3 neurons and assess resulting changes in nociception. Rats were injected into the bulbar B3 group with a recombinant lentiviral vector, LV-shTPH2, encoding RNA interfering with tryptophan hydroxylase 2 expression. Together with the long term disappearance of this enzyme in the whole rostro-caudal extent of B3 group, 5-HT was markedly depleted selectively in the dorsal horn at all levels of the spinal cord. In contrast, immunolabeling of the 5-HT transporter was unaffected by LV-shTPH2 injection, indicating the preservation of serotonergic fibers integrity. Whereas mechanical and thermal nociceptive thresholds were unchanged by 5-HT depletion, marked reductions in intraplantar formalin (but not carrageenin)-evoked nocifensive responses, and, in contrast, significant increases in mechanical and thermal hyperalgesia evoked by sciatic nerve ligation were noted in LV-shTPH2-injected rats versus controls. Parallel changes in c-Fos immunolabeling within the dorsal horn confirmed that bulbo-spinal serotonergic projections modulate pain signaling under these various conditions. These results suggest that serotonergic neurons of the B3 group are only moderately concerned, if any, by acute nociception but exert modulatory influences under pain sensitizing conditions. The opposite changes in formalin injected- versus sciatic nerve ligated rats might be related to the implication of different receptors in 5-HT-mediated modulation of inflammatory versus neuropathic pain.

Association of SNPs in dopamine and serotonin pathway genes and their interacting genes with temperament traits in Charolais cows.

Cattle temperament is a complex trait, and molecular studies aimed at defining this trait are scarce. We used an interaction networks approach to identify new genes (interacting genes) and to estimate their effects and those of 19 dopamine- and serotonin-related genes on the temperament traits of Charolais cattle. The genes proopiomelanocortin (POMC), neuropeptide Y (NPY), solute carrier family 18, member 2 (SLC18A2) and FBJ murine osteosarcoma viral oncogene homologue (FOSFBJ) were identified as new candidates. Their potential to be associated with temperament was estimated according to their reported biological activities, which included interactions with neural activity, receptor function, targeting or synthesis of neurotransmitters and association with behaviour. Pen score (PS) and exit velocity (EV) measures were determined from 412 Charolais cows to calculate their temperament score (TS). Based on the TS, calm (n = 55; TS, 1.09 ± 0.33) and temperamental (n = 58; TS, 2.27 ± 0.639) cows were selected and genotyped using a 248 single-nucleotide variation (SNV) panel. Of the 248 variations in the panel, only 151 were confirmed to be polymorphic (single-nucleotide polymorphisms; SNPs) in the tested population. Single-marker association analyses between genotypes and temperament measures (EV, PS and/or TS) indicated significant associations of six SNPs from four candidate genes. The markers rs109576799 and rs43696138, located in the DRD3 and HTR2A genes, respectively, were significantly associated with both EV and TS traits. Four markers, rs110365063 and rs137756569 from the POMC gene and rs110365063 and rs135155082 located in SLC18A2 and DRD2, respectively, were associated with PS. The variant rs110365063 located in bovine SLC18A2 causes a change in the amino acid sequence from Ala to Thr. Further studies are needed to confirm the association of genetic profile with cattle temperament; however, our study represents important progress in understanding the regulation of cattle temperament by different genes with divergent functions.

Tetratricopeptide repeat domain 9A modulates anxiety-like behavior in female mice.

Tetratricopeptide repeat domain 9A (TTC9A) expression is abundantly expressed in the brain. Previous studies in TTC9A knockout (TTC9A-/-) mice have indicated that TTC9A negatively regulates the action of estrogen. In this study we investigated the role of TTC9A on anxiety-like behavior through its functional interaction with estrogen using the TTC9A-/- mice model. A battery of tests on anxiety-related behaviors was conducted. Our results demonstrated that TTC9A-/- mice exhibited an increase in anxiety-like behaviors compared to the wild type TTC9A+/+ mice. This difference was abolished after ovariectomy, and administration of 17-ß-estradiol benzoate (EB) restored this escalated anxiety-like behavior in TTC9A-/- mice. Since serotonin is well-known to be the key neuromodulator involved in anxiety behaviors, the mRNA levels of tryptophan hydroxylase (TPH) 1, TPH2 (both are involved in serotonin synthesis), and serotonin transporter (5-HTT) were measured in the ventromedial prefrontal cortex (vmPFC) and dorsal raphe nucleus (DRN). Interestingly, the heightened anxiety in TTC9A-/- mice under EB influence is consistent with a greater induction of TPH 2, and 5-HTT by EB in DRN that play key roles in emotion regulation. In conclusion, our data indicate that TTC9A modulates the anxiety-related behaviors through modulation of estrogen action on the serotonergic system in the DRN.