Serotonergic neurons modulate a wide range of behaviors and functions, from mood and aggression to vital autonomic processes like heart rate, respiratory dynamics, and body temperature. We hypothesize that this broad scope reflects the collective actions of many functionally and molecularly distinct subtypes of serotonergic neurons, each with specialized roles in different neural processes. Supporting this idea are examples of heterogeneity among serotonergic neurons with respect to developmental origin, biophysical properties, and molecular expression; yet deciphering the functional and behavioral relevance of these differences has been challenging. In order to better understand serotonergic system organization, we have developed and applied a set of mouse genetic tools to subdivide serotonergic neurons into groups based on molecular criteria, and then to query these subtypes for differences with respect to biophysical properties, hodology, gene expression, and whole animal function. We applied these tools in a stage-wise fashion, from neural system en masse, as reference, and then to specific serotonergic neuron subtypes. From this, we have established that serotonergic neurons play key roles in at least two life-sustaining reflexes - the respiratory chemoreflex (breathing modulation to keep tissue PCO2/pH within physiological limits) and body temperature regulation. We found that chemoreflex modulation, but not body temperature regulation, maps to a specific serotonergic neuron subtype - that subtype with a developmental history of Egr2 gene expression. Further, in brain slice preparations, we found that this subtype is chemosensitive, increasing firing rate in response to conditions of hypercapnic acidosis. Thus, in vivo, Egr2-serotonergic neurons likely transduce chemosensory information into action potential firing to increase respiratory drive and ultimately breathing. Further, we found that Egr2-serotonergic neurons project selectively to respiratory nuclei involved in PCO2/pH sensory signal transduction, but not primary respiratory motor nuclei. This indicates that the serotonergic system has distinct sensory and motor divisions - another unexpected finding. In summary, these results establish a previously unappreciated functional modularity and organization to the serotonergic system, and open up potential for tailored function-specific therapeutic strategies, for example here as relates to disorders of respiratory homeostasis or thermoregulation.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

The goal of this dissertation was to evaluate genomic regulation during bovine mammary epithelial cell (BMEC) growth and differentiation. To accomplish this goal, a collagen gel cell culture system was developed that was capable of mimicking the prepartum stages of epithelial development and differentiation. In addition, a 4,600-cDNA bovine microarray was developed in order to profile gene expression. Analysis of BMEC in collagen cultures using various lactogenic conditions highlighted the critical importance of both hormonal and structural signals. The objective of the first study utilizing the microarray was to evaluate the contribution of the two prominent lactogenic factors in vitro, 1) prolactin and 2) gel release. Collectively, lactogenic stimulation appears to turn off genes associated with structural progression and morphogenesis, and turn on genes involved in alveolar MEC differentiation such as cell polarization, milk protein synthesis and ER/Golgi transport. The objective of the second study utilizing these resources was to evaluate the direct effects of thermal stress on BMEC growth and development. The structural response to thermal stress was characterized by morphogenic inhibition and dramatic regression of the ductal branches. Microarray analysis revealed an overall up-regulation of genes associated with stress response, DNA repair, protein degradation and cell death. In contrast, genes associated with cellular and MEC-specific biosynthesis, metabolism, and morphogenesis, were generally down-regulated. Subsequent to the analysis of BMEC differentiation was a targeted effort focusing on two small molecules hypothesized to be involved in regulating the BMEC secretory response: serotonin and prostaglandin E2. A pilot study suggested that serotonin is produced by bovine MEC and a model was proposed that describes serotonin's role as a feedback inhibitor during milk synthesis and secretion. A second pilot study demonstrated that PGE2 had a consistently positive influence on lumen diameter of alveolar structures in vitro. Overall, this dissertation provides new resources for studying bovine functional genomics, particularly within the mammary gland, and it provides a strong foundation for understanding genomic regulation of mammary epithelial structure and function. Furthermore, it establishes potential roles for local regulation of milk production by serotonin and PGE2.

No description available.

Biochemical theories postulate that deficient serotonergic functioning may be etiologically related to affective illness and aggressive behavior. In Study I mood and aggressivity were measured in thirty-six normal male subjects before and after ingestion of a Tryptophan Depleted, Tryptophan Loaded or Balanced amino acid mixture. While no differences in aggressivity were found, the Tryptophan Depleted group scored significantly higher at posttest on the MAACL Depression Scale than the control groups and demonstrated selective attention for dysphoric themes. In Study II a Balanced or Tryptophan Depleted amino acid mixture was administered to eighty normal male subjects prior to placing them in either a positive or negative environment, with or without instructions concerning the potential amino acid effects. The tryptophan depleted group became significantly more depressed than the control group regardless of environmental condition or instructional set. These findings suggest that lowered tryptophan may result in a central serotonergic dysfunction which is causally related to depressive affect and possibly to the pathogenesis of clinical forms of depression.

No description available.

The 5-HT7 receptor is the most recent addition to the 5-HT receptor family and has been linked to a variety of physiological and pathophysiological processes. Well established antide-pressant pharmaceuticals have recently been found to activate the 5-HT7 receptor, supporting the role of the 5-HT7 receptor in the antidepressant mechanism. The synthesis of potent selec-tive 5-HT7 receptor antagonists could afford a greater understanding of the 5-HT7 receptor function as well as lead to potential drug candidates. The synthesis of unfused biheteroaryl derivatives as 5-HT7 receptor ligands has been de-scribed within. These compounds have been tested for biological activity on the 5-HT6 and 5-HT7 receptors. 4-(3’-Furyl)-2-(N-substituted-piperazino)pyrimidines were found to be potent 5-HT7 receptor ligands. 4-(2’-Furyl)-2-(N-substituted-piperazino)pyrimidines have shown high se-lectivity for the 5-HT7 receptor over the 5-HT6 receptor.

This study assessed the in vivo effects of various serotonin (5-HT) receptor modulators on 5-HT neurotransmission in the rat hippocampus. Vortioxetine, humanized-vortioxetine, and escitalopram blocked the 5-HT transporter, but similar to ipsapirone did not dampen the sensitivity of postsynaptic 5-HT1A receptors. Long-term administration of all treatments increased the tonic activation of postsynaptic 5-HT1A heteroreceptors, an effect common to all antidepressants. Vortioxetine decreased the function of the terminal 5-HT1B autoreceptor under high but not a low degree of activation, thus showing that its partial agonism led to increased 5-HT release and that long-term administration results in the desensitization of terminal 5-HT1B autoreceptors. Vortioxetine overcame the effects of 5-HT1B and 5-HT3 receptor agonists. This study was unable to determine the involvement of 5-HT7 receptor antagonism exerted by vortioxetine affects 5-HT neurotransmission. Therefore, vortioxetine would appear to exert different actions, via transporter and receptor activity, on the serotonergic system in the hippocampus, consistent with its unique pharmacological profile.

Serotonin is a very important neuromodulatory agent that affects many physiological and behavioral responses of both vertebrates and invertebrates. In helminths, especially parasitic ones, not much is known about the biosynthesis and mode of action of serotonin or any of the related biogenic amine neurotransmitters, such as catecholamines (dopamine and noradrenaline). In this study, we cloned two full length cDNAs from Schistosoma mansoni encoding tryptophan hydroxylase (TPH) and tyrosine hydroxylase (TH). TPH and TH catalyze the rate limiting steps in the biosynthesis of serotonin and catecholamines, respectively. Both enzymes were expressed in Escherichia coli and the purified proteins were shown to have TPH and TH activities. This indicates that S. mansoni, and possibly other parasitic helminths, may be capable of synthesizing serotonin and catecholamines endogenously. In the second part of our studies, we looked at the mode of action of serotonin in helminths, in particular the molecular properties of serotonergic G protein-coupled receptors (GPCR). We cloned two helminth GPCRs, one from the free living nematode Caenorhabditis elegans and the second from S. mansoni. The C. elegans receptor (5-HT2Ce) was shown to encode a functional serotonin receptor with structural and signaling properties similar to those of mammalian 5-HT2 receptors. However, its agonist I antagonist binding profile differed from previously characterized serotonin receptors. The cloned S. mansoni receptor (SmGPCRx) was found to represent a new structural class of receptor, which shared about the same level of amino acid sequence homology with various biogenic amines receptors, such as serotonin, catecholamines, and octopamine receptors. Additional sequence analysis and immunolocalization studies confirmed that SmGPCRx possesses structural characteristics of a GPCR. SmGPCRx is the first GPCR ever cloned from a parasitic flatworm. Taken together, these studies mark an important first step to

Rats are nocturnal animals, their ingestive behaviours show circadian rhythms and the suprachiasmatic nuclei (SCN) of the hypothalamus are the primary pacemaker of these rhythms. Serotonin (5-HT) is one of the most abundant neurotransmitter in the SCN and it is involved in the regulation of ingestive behaviour. In this study, we compared food and water intakes of male adult Wistar rats infused during 7 days with serotonin (2.5 nmol/24h) or artificial cerebrospinal fluid (CSF) in the SCN in a three-way selection of macronutrients. Within 5-HT infusion, nocturnal and diurnal water intakes, and the nocturnal caloric intake from the protein diet were significantly lower than those of the CSF infused group. Decrease in water consumption was most significant in the early and middle dark phases. Besides, increased diurnal energy intake and decreased nocturnal energy intake were observed during 5-HT and CSF infusion. In conclusion, this first chronic and continuous infusion work on 5-HT in the SCN specifically disrupted the circadian rhythmicities in water and protein consumptions.