5HT Synthesis Research Articles

Differential effects on urethral function, epithelial morphology, and molecular profile in two different rat models of stress urinary incontinence by simulated birth trauma or estrogen deficiency

ABSTRACT Introduction Stress urinary incontinence (SUI) is the common type of urinary incontinence with a significantly negative impact on quality of life, including sexual dysfunction, in women. Although the etiology of SUI in women seems to be multifactorial, multiple vaginal parities and estrogen deficiency (ED) are considered to be major risk factors of SUI in elderly women. Thus, animal models of simulated birth trauma induced by multiple vaginal distention (VD) and ED induced by bilateral ovariectomy (OVX) have been used to study the SUI pathophysiology in rats. In previous studies, bilateral ovariectomy (OVX) reportedly induces the morphological changes in the rat urethral epithelium. It has also been reported that serotonergic (5HT+) paraneurons uniquely located in the urethra modulate urethral sensory function and that their numbers decrease in aged or diabetes rats. However, the difference of changes in the urethra such as those in epithelial morphology and expression of 5HT+ paraneurons in the urethra, in these two models has not been well characterized. Objective We therefore evaluated the histological changes of the urethral epithelium and 5HT+ cell expression, and urethral continence function in rats with OVX-induced ED or VD-induced simulated birth trauma. Methods Nulliparous SD rats were divided into: (1) sham group; (2) OVX 3-weeks group; (3) OVX 6-weeks group; (4) VD-1 group (1 time VD 2 weeks before assays); (5) VD-3 group (3 times VDs every 2 weeks). VD was induced by balloon catheter inflation for 4 hours in the vagina under isoflurane anesthesia. Urethral function was evaluated by the amplitude of urethral pressure responses during sneezing (A-URS) and urethral baseline pressure (UBP), which was measured by a microtransducer-tipped catheter inserted into the mid-urethra (n=6, each group). Following the urethral function analysis, the mid urethra was harvested, and the tissue sections (10 μm) were stained with Hematoxylin-eosin (H&E) and anti-5HT antibodies for immunofluorescent detection of 5HT+ paraneurons. In addition, in separate groups of rats, changes in urethral mRNA levels of tryptophan hydroxylase-1 (TPH1), a key enzyme of 5HT synthesis, were quantified by RT-PCR (n=6, each group). All data are represented as means ± SE. P < 0.05 was considered significant. Results OVX 6-weeks and VD-3 rats exhibited significant decreases in UBP and A-URS vs. sham (Fig. 1A). The mean epithelial thickness was significantly decreased in OVX 3, 6-weeks, VD-1 and 3 rats (Fig 1B,C) vs. sham. The number of urethral 5HT+ paraneurons was significantly decreased in OVX 3, 6 weeks, VD-1 and 3 rats (Fig. 2A,B) vs. sham. TPH-1 mRNA levels were significantly decreased in OVX 3, 6-weeks and VD-3 rats (Fig. 2C) vs. sham. Conclusions These results indicate that urethral epithelial atrophy and loss of 5HT+ paraneurons occur prior to SUI- inducing urethral dysfunction in both models, with the changes being rapid and greater in OVX compared to VD. These histological changes in the urethra may contribute to the establishment of SUI in ED and multiple birth trauma. Disclosure Work supported by industry: no.

High-fat diet induces neuroinflammation and reduces the serotonergic response to escitalopram in the hippocampus of obese rats

Clinical studies indicate that obese individuals have an increased risk of developing co-morbid depressive illness and that these patients have reduced responses to antidepressant therapy, including selective serotonin reuptake inhibitors (SSRIs). Obesity, a condition of chronic mild inflammation including obesity-induced neuroinflammation, is proposed to contribute to decreases in synaptic concentrations of neurotransmitters like serotonin (5HT) by decreasing 5HT synthesis in the dorsal raphe nucleus (DRN) and/or affecting 5HT reuptake in DRN target regions like the hippocampus. In view of these observations, the goal of the current study was to examine inflammatory markers and serotonergic dynamics in co-morbid obesity and depression. Biochemical and behavioral assays revealed that high-fat diet produced an obesity and depressive-like phenotype in one cohort of rats and that these changes were marked by increases in key pro-inflammatory cytokines in the hippocampus. In real time using fast scan cyclic voltammetry (FSCV), we observed no changes in basal levels of hippocampal 5HT; however responses to escitalopram were significantly impaired in the hippocampus of obese rats compared to diet resistant rats and control rats. Further studies revealed that these neurochemical observations could be explained by increases in serotonin transporter (SERT) expression in the hippocampus driven by elevated neuroinflammation. Collectively, these results demonstrate that obesity-induced increases in neuroinflammation adversely affect SERT expression in the hippocampus of obese rats, thereby providing a potential synaptic mechanism for reduced SSRI responsiveness in obese subjects with co-morbid depressive illness.

Design, Synthesis, and Biological Evaluation of New Peripheral 5HT2A Antagonists for Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is increasingly prevalent worldwide, causing serious liver complications, including nonalcoholic steatohepatitis. Recent findings suggest that peripheral serotonin (5-hydroxytryptamine, 5HT) regulates energy homeostasis, including hepatic lipid metabolism. More specifically, liver-specific 5HT2A knockout mice exhibit alleviated hepatic lipid accumulation and hepatic steatosis. Here, structural modifications of pimavanserin (CNS drug), a 5HT2A antagonist approved for Parkinson's disease, led us to synthesize new peripherally acting 5HT2A antagonists. Among the synthesized compounds, compound 14a showed good in vitro activity, good liver microsomal stability, 5HT subtype selectivity, and no significant inhibition of CYP and hERG. The in vitro and in vivo blood-brain barrier permeability study proved that 14a acts peripherally. Compound 14a decreased the liver weight and hepatic lipid accumulation in high-fat-diet-induced obesity mice. Our study suggests new therapeutic possibilities for peripheral 5HT2A antagonists in NAFLD.

Modified Liu-Jun-Zi decoction alleviates visceral hypersensitivity in functional dyspepsia by regulating EC cell-5HT3r signaling in duodenum.

Modified Liu-Jun-Zi (MLJZ) is derived from one of the most famous traditional Chinese prescription Liu-Jun-Zi. It exhibits therapeutic effects in functional dyspepsia (FD), but the underlying mechanisms remain not well understood. Enterochromaffin (EC) cells contribute to the pathogeneses of visceral hypersensitivity in functional gastrointestinal disorders. But whether and how EC cells in duodenum participate in the mechanism of FD remain unsettled. To detect the crucial factors related to EC cells, and to evaluate the therapeutic effect of MLJZ and to determine whether MLJZ relieves visceral hypersensitivity in FD by regulating EC cell-5-hydroxytryptamine 3 receptor (5HT3r) signaling. FD rats were established by iodoacetamide gavage combined with tail clamping method. The verification of FD model and the evaluation of the therapeutic effect of MLJZ was taken place by hematoxylin-eosin (HE) staining and visceral sensitivity measurement. The expression of EC cells and 5-hydroxytryptamine (5HT) in duodenum was detected by Immunohistochemistry (IHC) staining and enzyme-linked immunosorbent assay (ELISA). IHC staining and quantitative polymerase chain reaction (qPCR) were applied to measure the expression of tryptophan hydroxylase-1 (TPH1), paired box gene 4 (PAX4), transient receptor potential A1 (TRPA1), transient receptor potential C4 (TRPC4) and 5HT3r. Duodenum sections were stained by double immunofluorescence (IF) to study the synthesis of 5HT in EC cells. The gastric sensitivity increased in FD rats while MLJZ decoction significantly attenuated visceral hypersensitivity. The duodenum of FD rats displayed increased expressions of EC cells, 5HT, TPH1, PAX4 and 5HT3r. And the overexpression was reduced in response to MLJZ decoction treatment. EC cell-5HT3r signaling pathway is abnormally active in FD with visceral hypersensitivity. And MLJZ decoction can alleviates visceral hypersensitivity in FD by regulating EC cell-5HT3r signaling in duodenum.

Modulation of the Tryptophan Hydroxylase 1/Monoamine Oxidase-A/5-Hydroxytryptamine/5-Hydroxytryptamine Receptor 2A/2B/2C Axis Regulates Biliary Proliferation and Liver Fibrosis During Cholestasis.

Serotonin (5HT) is a neuroendocrine hormone synthetized in the central nervous system (CNS) as well as enterochromaffin cells of the gastrointestinal tract. Tryptophan hydroxylase (TPH1) and monoamine oxidase (MAO-A) are the key enzymes for the synthesis and catabolism of 5HT, respectively. Previous studies demonstrated that 5-hydroxytryptamine receptor (5HTR)1A/1B receptor agonists inhibit biliary hyperplasia in bile-duct ligated (BDL) rats, whereas 5HTR2B receptor antagonists attenuate liver fibrosis (LF) in mice. Our aim was to evaluate the role of 5HTR2A/2B/2C agonists/antagonists in cholestatic models. While in vivo studies were performed in BDL rats and the multidrug resistance gene 2 knockout (Mdr2-/- ) mouse model of PSC, in vitro studies were performed in cell lines of cholangiocytes and hepatic stellate cells (HSCs). 5HTR2A/2B/2C and MAO-A/TPH1 are expressed in cholangiocytes and HSCs from BDL rats and Mdr2-/- - mice. Ductular reaction, LF, as well as the mRNA expression of proinflammatory genes increased in normal, BDL rats, and Mdr2-/- - mice following treatment 5HTR2A/2B/2C agonists, but decreased when BDL rats and Mdr2-/- mice were treated with 5HTR2A/2B/2C antagonists compared to BDL rats and Mdr2-/- mice, respectively. 5HT levels increase in Mdr2-/- mice and in PSC human patients compared to their controls and decrease in serum of Mdr2-/- mice treated with 5HTR2A/2B/2C antagonists compared to untreated Mdr2-/- mice. In vitro, cell lines of murine cholangiocytes and human HSCs express 5HTR2A/2B/2C and MAO-A/TPH1; treatment of these cell lines with 5HTR2A/2B/2C antagonists or TPH1 inhibitor decreased 5HT levels as well as expression of fibrosis and inflammation genes compared to controls. Modulation of the TPH1/MAO-A/5HT/5HTR2A/2B/2C axis may represent a therapeutic approach for management of cholangiopathies, including PSC.

The Role of Integrin β3 in Nucleus Accumbens Function

Stress disorders, depression, anxiety, and substance use disorder, are regulated by serotonin (5HT). The dorsal raphe nucleus (DRN), the major site for neuronal 5HT synthesis, sends projections to the nucleus accumbens (NAc). NAc glutamatergic synapse adaptations are consistent with changes in mood and reward, key components of stress disorders. A subpopulation of DRN projection neurons express the β3 subunit of the integrin αvβ3 receptor. The integrin αvβ3 receptor regulates extracellular 5HT concentration by modulation of 5HT transporters. Previous work has implicated this cell adhesion receptor in modulating stress, as mice lacking the β3 subunit have increased vulnerability to chronic stress. However, how NAc function is regulated by the DRN β3 subunit and contributes to maladaptive reward behaviors as seen in stress disorders is unknown.Because the β3 subunit of the integrin avβ3 receptor is only expressed in a subpopulation of 5HT neurons, this suggests that it confers a specific function to this neuronal population. Using a mouse line with selective deletion of the β3 subunit within 5HT neurons, we are able to characterize its role in 5HT synaptic transmission. Using acute brain slice whole‐cell patch‐clamp electrophysiology, we characterize how the β3 subunit of 5HT inputs contribute to glutamatergic NAc synaptic transmission. Our data suggests that selective deletion of the β3 subunit in 5HT neurons blunts excitatory synaptic strength in the NAc. This suggests that the β3 subunit in 5HT neurons modulates excitatory input to the NAc, which may relate to stress response.Support or Funding InformationNIH T32 5T32MH064913‐15This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A novel serotonin-containing tuft cell subpopulation in mouse intestine

In this study, a novel subset of doublecortin-like kinase 1 (DCLK1)-immunoreactive (IR) tuft cells that also contain serotonin (5-hydroxytryptamine, 5HT) is described, in terms of their number, regional distribution, possible synthesis or reuptake of 5HT and proximity to 5-HT-containing enterochromaffin (EC) cells. The small intestine from C57BL/6J mice was divided into five segments while the large intestine was kept undivided. Double immunostaining was used to estimate numbers and topographic distribution of 5HT-IR (DCLK1/5HT) tuft cells and their possible expression of tryptophan hydroxylase (TPH) and serotonin transporter (SERT). Also, possible contacts between tuft cells and 5HT-IR EC cells were studied. In the small intestine, up to 80% of all tuft cells were identified as DCLK1/5HT-IR; in the large intestine, such cells were rare. The highest number of DCLK1/5HT-IR cells was found in the upper small intestine. The numbers of DCLK1/5HT-IR cells gradually decreased distally. DCLK1-IR tuft cells were not found to contain TPH, the rate-limiting enzyme in 5HT synthesis. SERT, the selective transporter for 5HT reuptake, could not convincingly be demonstrated in tuft cells. In villi and crypts, 3% and 10%, respectively, of all DCLK1-IR cells were in close proximity to EC cells. EC cells in close proximity to DCLK1-IR cells were, in villi and crypts, 3 and 8%, respectively. We conclude that DCLK1/5HT-IR cells constitute a novel subset of tuft cells that may have unique roles in the GI tract.

Serotonin Mediates Depression of Aggression After Acute and Chronic Social Defeat Stress in a Model Insect.

In all animals, losers of a conflict against a conspecific exhibit reduced aggressiveness, often coupled with depression-like symptoms, particularly after multiple defeats. While serotonin (5HT) is involved, discovering its natural role in aggression and depression has proven elusive. We show how 5HT influences aggression in male crickets, before, and after single and multiple defeats using serotonergic drugs, at dosages that had no obvious deleterious effect on general motility: the 5HT synthesis inhibitor alpha-methyltryptophan (AMTP), the 5HT2 receptor blocker ketanserin, methiothepin which blocks 5HT receptor subtypes other than 5HT2, 5HT's precursor 5-hydroxytryptophan (5HTP) and re-uptake inhibitor fluoxetine. Contrasting reports for other invertebrates, none of the drugs influenced aggression at the first encounter. However, the recovery of aggression after single defeat, which normally requires 3 h in crickets, was severely affected. Losers that received ketanserin or AMTP regained their aggressiveness sooner, whereas those that received fluoxetine, 5HTP, or methiothepin failed to recover within 3 h. Furthermore, compared to controls, which show long term aggressive depression 24 h after 6 defeats at 1 h intervals, crickets that received AMTP or ketanserin regained their full aggressiveness and were thus more resilient to chronic defeat stress. In contrast, 5HTP and fluoxetine treated crickets showed long term aggressive depression 24 h after only 2 defeats, and were thus more susceptible to defeat stress. We conclude that 5HT acts after social defeat via a 5HT2 like receptor to maintain depressed aggressiveness after defeat, and to promote the susceptibility to and establishment of long-term depression after chronic social defeat. It is known that the decision to flee and establishment of loser depression in crickets is controlled by nitric oxide (NO), whereas dopamine (DA), but not octopamine (OA) is necessary for recovery after defeat. Here we show that blocking NO synthesis, just like ketanserin, affords resilience to multiple defeat stress, whereas blocking DA receptors, but not OA receptors, increases susceptibility, just like fluoxetine. We discuss the possible interplay between 5HT, NO, DA, and OA in controlling aggression after defeat, as well as similarities and differences to findings in mammals and other invertebrate model systems.

29: Exposure to a high fat diet is associated with persistent alterations in behavior and the gut microbiome in juvenile offspring primates

Prior work from our consortium shows that maternal high-fat diet (HFD) consumption is associated with modifications in behavior and the central serotonergic system in juveniles at 1 year of age. Additionally, we have described dysbiosis of the gut microbiome occurring at this age with exposure to maternal HFD. Intriguingly, ∼90% of serotonin (5HT) is made in the gut, and microbes and their metabolites function as important regulators of 5HT production. Here, we aimed to determine the importance of exposure to a HFD as a persistent behavioral mediator in juvenile offspring by examining the associations between diet, behavior, and the gut microbiome. Using our Japanese macaque model of maternal HFD exposure, we performed 16S or whole genome shotgun (WGS) sequencing on stool samples (n=15) collected at 1 & 3 years of age. WGS was performed on the Illumina HiSeq platform and resultant sequences were trimmed/quality filtered using KneadData. Taxonomic classification and functional annotation of species-specific (MetaPhlAn2) and community level metabolic pathways (HUMAnN2) was performed. Stool 5HT levels were assayed by ELISA. Behavioral testing (i.e. novel objects, repetitive behavior) occurred to associate with 5HT and microbiome data. Exposure to HFD in gestation is associated with persistent behavioral modifications (increased anxiety) in offspring at 3 years of age (Fig A). Additionally, we found alterations in metabolic pathways by metagenomic analysis (0.9 billion reads, average 62 million reads/sample) that have been previously shown to modulate 5HT; this includes L-tryptophan biosynthesis, of which Phascolarctobacterium succinatutens was identified as a primary contributor (Fig B). Furthermore, we detected significant inverse correlations with specific taxa, including Phascolarctobacterium and 5HT at 3 years of age (Fig C). Altogether, our data suggest HFD exposure leads to persistent gut microbial dysbiosis associated with reductions in serotonergic signaling mediators and 5HT synthesis that are detectable at 3 years. The interplay between the gut microbiome and serotonergic signaling potentially contribute to behavioral modifications observed with HFD exposure during fetal life, even with consumption of a postnatal control diet.

Constitutively Elevated Blood Serotonin Is Associated with Bone Loss and Type 2 Diabetes in Rats.

Reduced peripheral serotonin (5HT) in mice lacking tryptophan hydroxylase (TPH1), the rate limiting enzyme for 5HT synthesis, was reported to be anabolic to the skeleton. However, in other studies TPH1 deletion either had no bone effect or an age dependent inhibition of osteoclastic bone resorption. The role of 5HT in bone therefore remains poorly understood. To address this issue, we used selective breeding to create rat sublines with constitutively high (high-5HT) and low (low-5HT) platelet 5HT level (PSL) and platelet 5HT uptake (PSU). High-5HT rats had decreased bone volume due to increased bone turnover characterized by increased bone formation and mineral apposition rate, increased osteoclast number and serum C-telopeptide level. Daily oral administration of the TPH1 inhibitor (LX1032) for 6 weeks reduced PSL and increased the trabecular bone volume and trabecular number of the spine and femur in high-5HT rats. High-5HT animals also developed a type 2 diabetes (T2D) phenotype with increased: plasma insulin, glucose, hemoglobin A1c, body weight, visceral fat, β-cell pancreatic islets size, serum cholesterol, and decreased muscle strength. Serum calcium accretion mediated by parathyroid hormone slightly increased, whereas treatment with 1,25(OH)2D3 decreased PSL. Insulin reduction was paralleled by a drop in PSL in high-5HT rats. In vitro, insulin and 5HT synergistically up-regulated osteoblast differentiation isolated from high-5HT rats, whereas TPH1 inhibition decreased the number of bone marrow-derived osteoclasts. These results suggest that constitutively elevated PSL is associated with bone loss and T2D via a homeostatic interplay between the peripheral 5HT, bone and insulin.

Role of peripheral serotonin in the insulin secretion and glucose homeostasis

The most studied roles of serotonin (5-hydroxytryptamine, 5HT) have been related to its action in the Central Nervous System (CNS). However, most of 5HT is produced outside the CNS, mainly in the enterochromaffin cells of the gut. Additionally, other tissues such as the endocrine pancreas, particularly β-cells, have its own serotonin system able to synthesize, secrete and respond to extracellular 5HT through cell surface receptors subtypes that have been grouped in 7 families (HTR1-7). Interestingly, 5HT is stored in granules and released together with insulin from β-cells and its biological significance is likely a combination of intra and extracellular actions. The expression of enzymes involved in 5HT synthesis and their receptors varied markedly in β-pancreatic cells during pregnancy, in parallel with an increase in their insulin secretion potential (probably through the action of Htr3a) and an increase in β-cell mass (through the action of Htr2b and Htr1d). In addition, it has been suggested that gut-derived 5HT may promote hepatic gluconeogenesis during prolonged fasting through Htr2b receptor. Taken together, these findings suggest that peripheral 5HT plays an important role in the regulation of glucose homeostasis through the differential expression and activation of 5-HT membrane receptors on the surface of hepatocytes, adipocytes and pancreatic β-cells.

Abstract 572: Serotonin and Selective Serotonin Reuptake Inhibitors in Platelet Function and RhoA Activation

Platelet hyperactivity leads to thrombotic disease, underlying cardiovascular (CV) pathologies including stroke and myocardial infarction. Clinical data suggest that selective serotonin reuptake inhibitors (SSRIs) have hemostatic effects, signifying a potentially novel mechanism to treat CV disease. SSRIs target the serotonin transporter (SERT), which maintains dense-granule serotonin (5HT) in platelets. Serotonin is also a platelet agonist, acting through 5HT2A receptors, yet the full role of 5HT in platelet physiology is not well understood. We hypothesized that SERT KO mice, mimicking chronic SSRI treatment, would exhibit a bleeding phenotype due to reduced platelet function. Consistent with previous reports, we demonstrated that SERT KO mice have decreased whole blood 5HT. There were comparable concentrations of the 5HT metabolite 5HIAA, suggesting that overall synthesis and metabolism of 5HT is unchanged. We next demonstrate that there is a bleeding phenotype in the SERT KO mice through a significantly increased tail-bleed time and increased thrombin time (TT) in whole blood. Additionally, SERT KO mice have reduced platelet retraction, a platelet function attributed to decreased RhoA activation. To determine if sub-chronic SSRI treatment alters platelet function, we treated both wild type and a hyperactive platelet (KI) mice with citalopram in drinking water for 6 days, one day exceeding the turnover rate of platelet production (15mg/kg/day). Activation of RhoA was examined to determine if reduced serotonylation leads to reduced platelet function. Collectively, these data suggest that genetic or pharmacological inhibition of SERT activity could lead to reduced platelet function. These findings may be due to a decrease in intracellular 5HT, as 5HT is still present for 5HT2A activation as determined by the presence of the metabolite. Future experiments will examine the mechanism of SSRI-mediated platelet activity attenuation, which may include reduced serotonylation of important signaling molecules.

Nanomolar Oxytocin Synergizes with Weak Electrical Afferent Stimulation to Activate the Locomotor CPG of the Rat Spinal Cord In Vitro

Synergizing the effect of afferent fibre stimulation with pharmacological interventions is a desirable goal to trigger spinal locomotor activity, especially after injury. Thus, to better understand the mechanisms to optimize this process, we studied the role of the neuropeptide oxytocin (previously shown to stimulate locomotor networks) on network and motoneuron properties using the isolated neonatal rat spinal cord. On motoneurons oxytocin (1 nM–1 μM) generated sporadic bursts with superimposed firing and dose-dependent depolarization. No desensitization was observed despite repeated applications. Tetrodotoxin completely blocked the effects of oxytocin, demonstrating the network origin of the responses. Recording motoneuron pool activity from lumbar ventral roots showed oxytocin mediated depolarization with synchronous bursts, and depression of reflex responses in a stimulus and peptide-concentration dependent fashion. Disinhibited bursting caused by strychnine and bicuculline was accelerated by oxytocin whose action was blocked by the oxytocin antagonist atosiban. Fictive locomotion appeared when subthreshold concentrations of NMDA plus 5HT were coapplied with oxytocin, an effect prevented after 24 h incubation with the inhibitor of 5HT synthesis, PCPA. When fictive locomotion was fully manifested, oxytocin did not change periodicity, although cycle amplitude became smaller. A novel protocol of electrical stimulation based on noisy waveforms and applied to one dorsal root evoked stereotypic fictive locomotion. Whenever the stimulus intensity was subthreshold, low doses of oxytocin triggered fictive locomotion although oxytocin per se did not affect primary afferent depolarization evoked by dorsal root pulses. Among the several functional targets for the action of oxytocin at lumbar spinal cord level, the present results highlight how small concentrations of this peptide could bring spinal networks to threshold for fictive locomotion in combination with other protocols, and delineate the use of oxytocin to strengthen the efficiency of electrical stimulation to activate locomotor circuits.

Consequences of a Maternal Vitamin B6 Deficiency on Hypercapnic Response in Rat Pups

Recently, maternal dietary deficiencies have been linked to brainstem neurotransmitter and homeostatic reflex dysfunctions and infant vulnerability to the Sudden Infant Death Syndrome (SIDS). In particular, a gestational and postnatal tryptophan (critical for serotonin, 5HT synthesis) deficiency was shown to induce a serotonergic defect in rat pups, and an attenuated ventilatory response to CO2. We study combined influences of 5HT and the γ‐aminobutyric acid (GABA) mediated chemosensitivity. Vitamin B6 is a coenzyme critical in both 5HT and GABA synthesis. We test the hypothesis that a maternal dietary B6 deficiency (B6d) will alter homeostasis in rat pups. We show multisystem failures with B6d beginning at parturition (P0), reflex disruption when B6d is started at P5, and little effect of B6d started at P10. Data suggest a critical period for B6d sensitivity and the potential that B6d‐induced 5HT and GABAergic deficiencies to produce homeostatic dysfunction that could also influence vulnerability to SIDS. Supported by NIH 2U54NS041069–06A1 and 5P20RR016466.

Serotonergic Signaling by Preimplantation Embryos Impacts Subsequent Trophoblast Differentiation and Pregnancy Failure.

Serotonin (5-hydroxytryptamine, 5HT) is a monoamine neurotransmitter that is also abundant outside the central nervous system where it circulates in blood platelets and mediates a broad variety of neuronal and non-neuronal biological functions. 5HT is produced in the mammalian reproductive tract, oocyte and preimplantation embryo where it may participate in the regulation of fertilization and early embryonic development. Preimplantation mouse embryos express several G protein coupled 5HT receptors, as well as the 5HT reuptake transporter, and are potentially capable of serotonergic signaling. 5HT is generated from tryptophan by tryptophan 5-hydroxylase (EC 1.14.16.4), an enzyme encoded in mice by two genes, Tph1 and Tph2, which are restricted in their expression to tissues either outside or within the central nervous system, respectively. Interestingly, Tph2, but not Tph1, is expressed in oocytes and preimplantation embryos. Transgenic mice homozygous for the deletion of Tph2 had a significant (14-fold) increase in pregnancy failure that was also evident in periimplantation development between embryonic day 4.5 (E4.5) and E7.5. However, morula-stage embryo (E2.5) production was not significantly affected by a lack of embryonic TPH2. TPH2 and 5HT were detected by immunofluorescence in wild type embryos from the 1-cell to morula stage, but decreased abruptly at the blastocyst stage. Developmental rates in vitro were similar for wild type and Tph2 null morulae to the hatched blastocyst stage, but trophoblast differentiation into migratory cells was delayed by about 24 hours (p<0.05) in knockout embryos cultured to form blastocyst outgrowths. Tph2 null mothers are able to produce 5HT in the reproductive tract using TPH1, but this was insufficient to support normal development of Tph2 null embryos. However, pregnancy failure rates were intermediate in Tph2 null mothers crossed with heterozygous males or in heterozygous mothers crossed with Tph2 null males, suggesting at least a partial rescue in vivo by 5HT-producing sibling embryos. We conclude that in the absence of endogenous 5HT synthesis, fertilization and preimplantation embryo production is normal, but implantation and pregnancy success rates are both reduced. These findings suggest that autocrine or local paracrine serotonergic signaling during preimplantation development, prior to blastocyst formation, ensures on time differentiation of the trophoblast for implantation. Activation of embryonic 5HT receptors could mediate interactions among blastomeres or induce gene expression that is required for trophoblast functions that take place during or after blastocyst implantation to maintain a viable fetus. The Tph2 null mouse is a unique model of implantation failure arising from trophoblast dysfunction that might also provide insight into placental insufficiency and associated perinatal disorders. Supported by the Intramural Research Program of the NICHD, NIH, and the Department of Veterans Affairs.

Analysis of extracellular levels of serotonin in rat amygdala after oral administration of large amounts of tryptophan

Serotonin (5HT) is an important neurotransmitter known to affect many behaviors. The synthesis of 5HT in the brain is dependent on the availability of tryptophan (Trp) from plasma. Therefore, oral administration of Trp affects 5HT synthesis in the brain. We focused on the amygdala, a brain structure central in the generation of both normal and pathological emotional behavior. The aim of this work was to investigate the effects of oral administration of large amounts of Trp on the extracellular levels of 5HT and its metabolite 5‐hydroxyindoleaceticacid (5HIAA) in the amygdala. The overnight fasted rats were administered Trp at concentrations ranging from 18 to 144 mg/100g BW by oral gavage. The lowest concentration of Trp employed is equivalent to that consumed in a 24‐h period by rats. Extracellular levels of 5HT and 5HIAA in rat amygdala were measured with in vivo microdialysis technique. During the observation period, no changes were observed in the levels of 5HT in all treatment groups. In contrast, the levels of 5HIAA started to increase from the basal level at one hour after oral administration of Trp, irrespective of the doses of Trp. The maximum levels of 5HIAA were almost the same among all Trp administered groups, but the higher doses of Trp had a more prolonged effect on the levels of 5HIAA. These results suggested that oral administration of large amounts of Trp induced an increase in neuronal activity in amygdala.

Anxiety-like behavior and cognitive flexibility in adult rats perinatally exposed to increased serotonin concentrations

Serotonin (5-hydroxytryptamine, 5HT) is a biologically active amine that regulates the development of 5HT neurons and target tissues during neurogenesis, while later it assumes the function of a neurotransmitter. Serotonin mediates many essential behaviors common to all mammals, and is held responsible for anxiety-like behavior and cognitive rigidity. Proper serotonin levels, controlled through 5HT synthesis and metabolism, are crucial for normal brain development. In this study we investigated anxiety-like behavior and cognitive flexibility in adult animals after exposing their developing brains to increased 5HT concentrations. Wistar rats were treated subcutaneously from gestational day 12 to post-natal day 21 with the immediate 5HT precursor 5-hydroxytryptophan (5HTP, 25mg/kg), a non-selective MAO inhibitor tranylcypromine (TCP, 2mg/kg), or saline. After reaching adulthood, animals were tested for anxiety-like behavior (exploratory behavior, thigmotactic behavior, social contact, and reaction to stressful stimulus) and cognitive flexibility (ability for reversal learning). Results of the behavioral studies corresponded with our previous neurochemical findings. Treatment with 5HTP, which has induced mild reduction in cortical 5HT concentrations, caused reduction in only one aspect of anxiety-like behavior (increased exploratory activity). Treatment with TCP, which lead to drastic reduction in 5HT concentration/function, resulted in a highly anxiolytic phenotype (reduced thigmotaxis, reaction to stress, and social anxiety) with improved cognitive flexibility. Although further neurochemical, anatomical and gene-expression studies are needed to elucidate the mechanisms underlying the observed behavior, we hope that our results will contribute to the understanding of the role of serotonin in anxiety-like behavior and cognitive rigidity.

Serotonin–mushroom body circuit modulating the formation of anesthesia-resistant memory in Drosophila

Pavlovian olfactory learning in Drosophila produces two genetically distinct forms of intermediate-term memories: anesthesia-sensitive memory, which requires the amnesiac gene, and anesthesia-resistant memory (ARM), which requires the radish gene. Here, we report that ARM is specifically enhanced or inhibited in flies with elevated or reduced serotonin (5HT) levels, respectively. The requirement for 5HT was additive with the memory defect of the amnesiac mutation but was occluded by the radish mutation. This result suggests that 5HT and Radish protein act on the same pathway for ARM formation. Three supporting lines of evidence indicate that ARM formation requires 5HT released from only two dorsal paired medial (DPM) neurons onto the mushroom bodies (MBs), the olfactory learning and memory center in Drosophila: (i) DPM neurons were 5HT-antibody immunopositive; (ii) temporal inhibition of 5HT synthesis or release from DPM neurons, but not from other serotonergic neurons, impaired ARM formation; (iii) knocking down the expression of d5HT1A serotonin receptors in α/β MB neurons, which are innervated by DPM neurons, inhibited ARM formation. Thus, in addition to the Amnesiac peptide required for anesthesia-sensitive memory formation, the two DPM neurons also release 5HT acting on MB neurons for ARM formation.

The effect in humans of ingesting different dietary proteins on plasma tryptophan, tyrosine, and the amino acid ratios that predict their brain uptake

Overnight‐fasted healthy males (n=6) with venous catheters ingested on four mornings 12oz V‐8 Juice containing 40 g of α‐lactalbumin (LAC), wheat gluten (GLUT), zein or starch. Blood was taken before ingestion and at 30 min intervals for 4 hr. Tryptophan (TRP), tyrosine (TYR) and other large neutral amino acids (LNAA) were assayed by HPLC. Predictors of brain TRP and TYR uptake (plasma TRP/∑LNAA, TYR/∑LNAA) were calculated. 0‐Time plasma TRP was ≈50 nmol/ml; TRP/∑LNAA was ≈0.010. LAC increased plasma TRP (3‐fold) and TRP/∑LNAA (50%) within 2 hr; starch did not change either TRP variable, while GLUT caused modest (25%) and zein larger reductions (50%) in both variables. The peak difference in plasma TRP/∑LNAA between LAC and zein was ≈3‐fold. Changes in TYR/∑LNAA were minimal. Plasma TRP/∑LNAA predicts brain TRP uptake and serotonin (5HT) synthesis from TRP in rats. The results thus suggest that in humans, LAC ingestion may raise and zein ingestion lower 5HT synthesis in/release by brain neurons, and perhaps produce 5HT‐mediated changes in mood and behavior.Supported by the Peterson Foundation and the UPMC Weight Management Center.

The Chronic Ingestion of Diets Containing Different Proteins Produces Marked Variations in Brain Tryptophan Levels and Serotonin Synthesis in the Rat

Serotonin (5HT) synthesis in brain is influenced by precursor (tryptophan (TRP)) concentrations, which are modified by food ingestion. Hence, in rats, a carbohydrate meal raises brain TRP and 5HT; a protein-containing meal does not, but little attention has focused on differences among dietary proteins. Recently, single meals containing different proteins have been shown to produce marked changes in TRP and 5HT. The present studies evaluate if such differences persist when rats ingest such diets chronically. Male rats were studied that ingested diets for 9 days containing zein, wheat gluten, soy protein, casein, or α-lactalbumin (17% dry weight). Brain TRP varied up to eightfold, and 5HT synthesis fivefold among the different protein groups. TYR and LEU concentrations, and catecholamine synthesis rate in brain varied much less. The effects of dietary protein on brain TRP and 5HT previously noted after single meals thus continue undiminished when such diets are consumed chronically.