Thermogenic brown and beige adipocytes convert chemical energy to heat by metabolizing glucose and lipids via uncoupling protein 1 (Ucp1), a process known as non-shivering thermogenesis. Serotonin (5-HT) neurons in the ventral medulla are known to regulate sympathetic efferent neurons in the intermediolateral nucleus (IML) necessary to maintain brown adipose tissue (BAT) activity. Previous studies show that mice lacking central 5-HT neurons are incapable of maintaining body temperature in cold, ambient conditions. Due to this direct linkage between 5-HT and thermoregulation, we hypothesized that central 5-HT neurons may be vital to the regulation of brown and beige adipocyte activity. Given that BAT consumes large amounts of substrate when active, we also hypothesized that inactivation of BAT due to deletion of the regulatory neural circuitry (5-HT neurons) would cause metabolic dysregulation. To test this, we generated mice in which the human diphtheria toxin (DT) receptor was selectively expressed in central 5-HT neurons under control of a Pet-1 promoter. Pet-1 is a transcription factor selectively located in mature, central 5-HT neurons. Coincidentally, some cells within pancreatic islets also express Pet-1, and contain adequate machinery to produce, release, and uptake 5-HT. Systemic treatment with DT eliminated 5-HT neurons and caused loss of thermoregulation, BAT steatosis, and a >50% decrease in Ucp1 expression in BAT and beige fat, indicative of reduced thermal production. In parallel, blood glucose increased 3.5-fold, free fatty acids 13.4-fold and triglycerides 6.5-fold. Intracerebroventricular (ICV) treatment with 1/30th the systemic dose of DT induced an even greater thermoregulatory impairment. The metabolic deficits following systemic DT treatment indicate that central 5-HT neurons are essential for proper metabolic regulation. However, such high levels of glucose and lipids also indicate failure of the pancreatic endocrine program following systemic treatment, likely due to moderate destruction of β-cells expressing Pet-1 and the DT receptor. Because ICV treatment caused even greater thermoregulatory and metabolic deficits, where little, if any, of the toxin would spread systemically, central 5-HT neurons are clearly essential for normal central regulation of metabolism. Interestingly, similar BAT and beige fat defects occurred in Lmx1bf/f/p mice, in which 5-HT neurons fail to develop in utero. Assessment of systemically treated animals using a euglycemic/hyperinsulinemic clamp showed extensive fasting hyperglycemia and systemic insulin resistance, coinciding with reduced glucose uptake in skeletal muscle and BAT. The hyperinsulinemic clamp failed to suppress hepatic glucose and fatty acid production, leading to the conclusion that loss of central 5-HT neurons disrupts central hepatic regulation. In attempts to induce BAT thermogenesis and metabolism, we optogenetically stimulated 5-HT neurons in the rostral raphe pallidus and measured BAT and body temperature along with blood glucose. Unfortunately, these stimulations were incapable of increasing BAT temperature and lowering blood glucose, perhaps limiting therapeutic potential of these 5-HT neurons. We conclude that 5-HT neurons are major players in central regulation of metabolic homeostasis, in part through recruitment and activation of brown and beige adipocytes and hepatic substrate production. Data also suggest that 5-HT neurons regulate glucose homeostasis via undefined neural mechanisms independently of BAT activity and pancreatic insulin secretion. Cumulative data on central 5-HT neurons indicate they are master regulators of whole-body metabolism.

In zebrafish, the ventilatory response to hypoxia first develops at 3 days post-fertilization (d.p.f.) before O2-chemoreceptive neuroepithelial cells (NECs) of the gill appear at 7 d.p.f. This indicates the presence of extrabranchial chemoreceptors in embryos and a developmental transition to primarily gill O2 sensing. This thesis examined the skin NECs, which reach peak density in embryos but decline as gill NECs appear. Exposure of embryos and larvae to chronic hypoxia prevented the loss of skin NECs, shifted peak basal ventilation to a later developmental stage, and induced a hypoventilatory response to acute hypoxia. Chronic exposure to hyperoxia rapidly diminished skin NECs, shifted peak ventilation to earlier stages and eliminated the response to acute hypoxia. Administration of the neurotoxin 6-hydroxydopamine degraded nerve terminals that contact skin NECs and reduced both basal ventilation frequency and the hypoxic ventilatory response. Thus, skin NECs are candidates for extrabranchial O2 chemoreceptors in developing zebrafish.

In zebrafish, the ventilatory response to hypoxia first develops at 3 days post-fertilization (d.p.f.) before O2-chemoreceptive neuroepithelial cells (NECs) of the gill appear at 7 d.p.f. This indicates the presence of extrabranchial chemoreceptors in embryos and a developmental transition to primarily gill O2 sensing. This thesis examined the skin NECs, which reach peak density in embryos but decline as gill NECs appear. Exposure of embryos and larvae to chronic hypoxia prevented the loss of skin NECs, shifted peak basal ventilation to a later developmental stage, and induced a hypoventilatory response to acute hypoxia. Chronic exposure to hyperoxia rapidly diminished skin NECs, shifted peak ventilation to earlier stages and eliminated the response to acute hypoxia. Administration of the neurotoxin 6-hydroxydopamine degraded nerve terminals that contact skin NECs and reduced both basal ventilation frequency and the hypoxic ventilatory response. Thus, skin NECs are candidates for extrabranchial O2 chemoreceptors in developing zebrafish.

In the absence of ligand, certain growth factor receptors can be activated via G protein-coupled receptor (GPCR) activation in a process termed transactivation. Serotonin (5-HT) receptors can transactivate the receptor tyrosine kinase (RTK) platelet-derived growth factor (PDGF) ?? receptors in smooth muscle cells, but it is not known if similar pathways occur in neuronal cells. Here, it is shown that 5-HT can transiently increase the phosphorylation of PDGF?? receptors in a time- and concentration-dependent manner in SH-SY5Y neuroblastoma cells. This transactivation pathway was pertussis-toxin sensitive, and was dependent on phospholipase C activity, intracellular calcium signaling and subsequent protein kinase C activation. Exogenous application of non-lethal concentrations of H2O2 induced the phosphorylation of PDGF?? receptors in a concentration-dependent fashion, similar to that observed with 5-HT. Further investigation revealed reactive oxygen species (ROS) production as a necessary component in the transactivation pathway, as scavenging ROS eliminated PDGF?? receptor phosphorylation. NADPH oxidase was determined to be the likely source of ROS given that the NADPH oxidase inhibitors diphenyleneiodonium chloride and apocynin abrogated PDGF?? receptor transactivation. The role of Src tyrosine kinase was also investigated, and its location in this signaling cascade was determined to be downstream of calcium signaling, but upstream of NADPH oxidase activity. In addition, the activation of ERK1/2 in this system was elucidated to be independent of PDGF?? receptor transactivation. Interestingly, 5-HT also transactivated TrkB receptors, another RTK whose function is implicated in clinical depression. Expectedly, the enzymes in this mechanism were consistent with those revealed in 5-HT-to-PDGF?? receptor signaling. This cross-talk between 5-HT and RTKs such as TrkB and PDGF?? receptors identifies a potentially important signaling link between the serotonergic system and neurotrophic factor signaling in neurons that could have implications in mental health disorders including depression. Furthermore, although transactivation pathways are commonly initiated by a GPCR, recent reports have demonstrated that selective serotonin reuptake inhibitors (SSRIs) were able to block 5-HT-induced transactivation of PDGF?? receptors, suggesting that in addition to GPCRs, monoamine transporters may also be involved in RTK transactivation. SH-SY5Y cells pretreated with the SSRI fluoxetine blocked 5-HT-induced transactivation of the PDGF?? receptors, but not PDGF-induced PDGF?? receptor activation. Upon further examination, it was discovered that during the pretreatment period, fluoxetine itself was transiently transactivating the PDGF?? receptor via 5-HT2 receptors. By the end of the pretreatment period, the effects of fluoxetine on PDGF?? receptor phosphorylation had returned to baseline, and a subsequent transactivating stimulus (5-HT) failed to ???re-transactivate??? the PDGF?? receptor. Additional investigations demonstrated that 5-HT pretreatment can block dopamine-induced PDGF?? receptor transactivation, but not PDGF-induced PDGF?? receptor activation. This is the first demonstration of the heterologous desensitization of an RTK via a transactivation pathway, and this phenomenon is specific for transactivation pathways because in all cases the PDGF?? receptor ligand PDGF-BB was able to directly stimulate receptor activity in spite of GPCR agonist pretreatment. Heterologous desensitization in transactivation signaling reveals a previously unknown short-term ???blackout??? period wherein no further transactivation signaling can occur to potentially exploit the mitogenic effects of RTK activation.

In zebrafish, the ventilatory response to hypoxia first develops at 3 days post-fertilization (d.p.f.) before O2-chemoreceptive neuroepithelial cells (NECs) of the gill appear at 7 d.p.f. This indicates the presence of extrabranchial chemoreceptors in embryos and a developmental transition to primarily gill O2 sensing. This thesis examined the skin NECs, which reach peak density in embryos but decline as gill NECs appear. Exposure of embryos and larvae to chronic hypoxia prevented the loss of skin NECs, shifted peak basal ventilation to a later developmental stage, and induced a hypoventilatory response to acute hypoxia. Chronic exposure to hyperoxia rapidly diminished skin NECs, shifted peak ventilation to earlier stages and eliminated the response to acute hypoxia. Administration of the neurotoxin 6-hydroxydopamine degraded nerve terminals that contact skin NECs and reduced both basal ventilation frequency and the hypoxic ventilatory response. Thus, skin NECs are candidates for extrabranchial O2 chemoreceptors in developing zebrafish.

In zebrafish, the ventilatory response to hypoxia first develops at 3 days post-fertilization (d.p.f.) before O2-chemoreceptive neuroepithelial cells (NECs) of the gill appear at 7 d.p.f. This indicates the presence of extrabranchial chemoreceptors in embryos and a developmental transition to primarily gill O2 sensing. This thesis examined the skin NECs, which reach peak density in embryos but decline as gill NECs appear. Exposure of embryos and larvae to chronic hypoxia prevented the loss of skin NECs, shifted peak basal ventilation to a later developmental stage, and induced a hypoventilatory response to acute hypoxia. Chronic exposure to hyperoxia rapidly diminished skin NECs, shifted peak ventilation to earlier stages and eliminated the response to acute hypoxia. Administration of the neurotoxin 6-hydroxydopamine degraded nerve terminals that contact skin NECs and reduced both basal ventilation frequency and the hypoxic ventilatory response. Thus, skin NECs are candidates for extrabranchial O2 chemoreceptors in developing zebrafish.

No description available.

Of the seven existing classes of serotonin receptors, the 5-HT7 receptors (5-HT7Rs) are the most recently discovered. Abundance of 5-HT7 in the central nervous system is suggestive of the receptor’s role in several physiological and pathophysiological functions. Existing research has afforded a number of compounds exhibiting specific affinity to the receptor. These selective ligands can provide structural information about the receptor and can serve as the foundation for pharmacological profiling . This thesis describes the synthesis of substituted pyrimidines and pyridines for affinity to the 5-HT7 receptor. Organometallic species are the cornerstone for sev-eral of the synthetic pathways.

Mucosal inflammation in conditions ranging from infective acute enteritis or colitis to inflammatory bowel disease (IBD) is accompanied by alteration in enterochromaffin (EC) cell numbers and serotonin (5-hydroxytryptamine; 5-HT) content in the gut. Previously we had shown that CD4+ T cells, via production of T helper (Th)2 cytokines, regulate EC cell biology in the Trichuris muris-infectious colitis model. I further examined the mechanisms of immuno-endocrine interactions in the context of intestinal inflammation. In chapter 3, utilizing human EC cell line and Trichuris muris-mouse model of infectious colitis we identified a critical role of interleukin (IL)-13, a key Th2 cytokine, in increasing EC cell numbers, tryptophan hydroxylase (TPH)1 expression (rate-limiting enzyme of mucosal 5-HT bio-synthesis), and 5-HT production. In chapter 4, we show that IL-13 driven intestinal inflammation is critically dependent on increased 5-HT production using dextran sulfate sodium (DSS) and dinitrobenzene sulphonic acid (DNBS) models of colitis. In DSS-induced colitis, we were the first to identify the increased production of IL-13 and its pathogenic role as IL-13 knockout (IL-13-KO) mice had less severe inflammation compared to wild-type, which was exacerbated following replenishment of 5-HT in IL-13-KO mice. In chapter 5, biopsy examination revealed, higher mucosal IL-13 expression accompanied inflammation in Crohn's disease (CD), which was additionally associated with increased TPH1, 5-HT receptor (5-HTR)3A, 5-HTR7 and decreased 5-HT transporter (5-HTT) expressions. Moreover, CD patients had elevated plasma and platelet-poor plasma 5-HT levels compared to healthy controls (HCs). Furthermore, 5-HTT polymorphism associated genotypes causing inefficiency in 5-HT re-uptake were more common in our patient cohort than HCs. The findings included in this thesis further emphasize the role of immuno-endocrine interactions in intestinal inflammation, which may be a step toward a better diagnosis or management or even a cure for a disease that is of growing concern, and in understanding IBD pathogenesis. / Dissertation / Doctor of Philosophy (PhD) / The gut produces most of the serotonin found in our body, where it regulates many normal functions. A group of special cells, named enterochromaffin cells, produces nearly all of the serotonin in the gut. In diseases of the gut, especially ones that involve inflammation resulting in symptoms like abdominal pain, diarrhea and bleeding, the number of these cells and serotonin concentration are different from that in the normal gut. I found that these changes are controlled by a particular protein produced by immune cells, called interleukin-13, and alteration in serotonin levels, in turn, contributes to the inflammatory process. Our laboratory experiments with cells and animals establish this connection between interleukin-13 and serotonin in gut inflammation. We further confirm this association between interleukin-13 and serotonin in human inflammatory bowel disease. Moreover, we identify a potential genetic cause of these changes in serotonin concentrations which may ultimately result in inflammatory bowel disease.

Dopamine (DA) receptor supersensitivity refers to the phenomenon of an enhanced physiological, behavioral or biochemical response to a DA agonist. Literature related to ontogenetic aspects of this process was reviewed. Neonatal 6-hydroxydopamine (6-OHDA) destruction of rat brain DA neurons produces overt sensitization to D1 agonist-induced oral activity, overt sensitization of some D2 agonist-induced stereotyped behaviors and latent sensitization of D1 agonist-induced locomotor and some stereotyped behaviors. This last process is unmasked by repeated treatments with D1 (homologous "priming") or D2 (heterologous "priming") agonists. A serotonin (5-HT) neurotoxin (5,7-dihydroxytryptamine) and 5-HT2C receptor antagonist (mianserin) attenuate some enhanced behavioral effects of D1 agonists, indicating that 5-HT neurochemical systems influence D1 receptor sensitization. Unlike the relative absence of change in brain D1 receptor number, DA D2 receptor proliferation accompanies D2 sensitization in neonatal 6-OHDA-lesioned rats. Robust D2 receptor supersensitization can also be induced in intact rats by repeated treatments in ontogeny with the D2 agonist quinpirole. In these rats quinpirole treatments produce vertical jumping at 3-5 wk after birth and subsequent enhanced quinpirole-induced antinociception and yawning. The latter is thought to represent D3 receptor sensitization. Except for enhanced D1 agonist-induced expression of c-fos, there are no changes in the receptor or receptor-mediated processes which account for receptor sensitization. Adaptive mechanisms by multiple "in series" neurons with different neurotransmitters may account for the phenomenon known as receptor supersensitivity.