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Thesis / Master of Science (MS)

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Thesis (Ph.D. in Neuroscience) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 129-151). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (p. [110]-117).

Breathing is an essential homeostatic function and its disruption leads to disability, brain damage, and death. Serotonin (5-hydroxytryptamine; 5-HT) neurons in the brainstem play an important role in control of breathing. Medullary 5-HT neurons are stimulated by increased CO₂ and subsequently stimulate respiratory nuclei to increase ventilation and maintain normal blood gas levels. Anesthetic-induced breathing dysfunction is a serious concern in healthcare settings. In research settings, experiments are often performed under anesthesia, and therefore it is important to understand how these drugs affect animal physiology. Unfortunately, little is known about how anesthetics modulate 5-HT neurons, breathing, and CO₂ chemoreception in mice, as many of the previous studies have been performed in different species. Characterizing how anesthetics commonly used in both research and clinical settings affect 5-HT neurons, breathing and CO₂ chemoreception is valuable to the broader field of neuroscience since these drugs are so ubiquitously used in research. Breathing dysfunction and defects in the serotonergic system have been implicated in disorders, such as sudden unexpected death in epilepsy (SUDEP) and sudden infant death syndrome (SIDS), which means better characterizing the role of 5-HT neurons in breathing has translational impact as well. Here I examine whether halogenated inhalational anesthetics, which potentiate TWIK-related acid-sensitive K⁺ (TASK) currents and GABAA receptors, could mask an effect of CO₂ on 5-HT neurons. During in vivo plethysmography in mice, a therapeutic level of isoflurane (1%) markedly reduced the hypercapnic ventilatory response (HCVR) in all mouse strains tested. In dissociated cell cultures, isoflurane (1%) hyperpolarized 5-HT neurons and inhibited spontaneous firing. A subsequent decrease in pH from 7.4 to 7.2 depolarized 5-HT neurons, but that was insufficient to reach threshold for firing. Depolarizing current restored baseline firing and the firing frequency response to acidosis, indicating that isoflurane did not block the underlying mechanisms mediating chemosensitivity. These results demonstrate that isoflurane masks 5-HT neuron chemosensitivity in vitro, and markedly decreases the HCVR in vivo. Next, I demonstrate that ketamine-xylazine or urethane anesthesia also significantly reduced the HCVR in mice at both therapeutic and sub-therapeutic doses. However, mice treated with a sub-therapeutic dose of anesthesia decreased their O₂ consumption in parallel, and thus matched their ventilation to metabolic demands. Mice that were anesthetized with the therapeutic dose did not sufficiently match their breathing and metabolic demands, and thus anesthesia induced hypoventilation. Recordings from 5-HT neurons in culture indicated that neither ketamine nor urethane affected 5-HT neuron chemosensitivity. These data demonstrate that anesthetics with different molecular targets similarly reduce the HCVR in mice, but not all of their effects are mediated via 5-HT neurons. Moreover, both ketamine-xylazine and urethane anesthesia altered baseline breathing in different ways, suggesting they targeted different parts of the respiratory network. Finally I show that isoflurane anesthesia in neonatal mice caused depression of resting ventilation, which was different from isoflurane-anesthetized adults. This effect was more pronounced in wildtype mice compared to littermates with genetic deletion of 5-HT neurons. Isoflurane-induced breathing depression decreased and mice fully recovered following washout of isoflurane at P8. I observed that genetic deletion of 5-HT neurons in mice with a congenic C57Bl/6 background led to a more severe phenotype than previously described in mixed genetic background strains. These mice had decreased survival, severe growth retardation, and reduced baseline ventilation. These results indicate that 5-HT neurons have a different role during the neonatal period and that some mouse strains are more sensitive to genetic deletion of 5-HT neurons; thus, background genetics play an important role in phenotype presentation. In summary, different classes of anesthetics each strongly depress chemoreception. Isoflurane seems to affect breathing, in part, by hyperpolarizing 5-HT neurons and masking their chemosensitivity, whereas ketamine and urethane have less effect on 5-HT neurons. However, both ketamine-xylazine and urethane anesthesia alter baseline breathing. Isoflurane anesthesia decreases baseline ventilation in neonates, but this effect is absent in adults, which suggests that the effects of isoflurane on breathing changes as mice age. These data are important for the field of respiratory physiology because they highlight the sensitivity of breathing to the effects of anesthetics. These results are valuable to the broader field of neuroscience, because anesthetics are widely used during in vivo research. Additionally, some transgenic mouse strains are more sensitive to 5-HT neuron deletion depending on their genetic background. In the future it will be critical to characterize the molecular mechanisms that underlie these phenomena.

Methyl-accepting chemotaxis proteins MCPs can bind one or more receptor- specific ligands. In the case of the Tar MCP of Escherichia coli (TarEc), a primary attractant ligand is aspartate. Its binding to the periplasmic domain of Tar generates a conformational change that is transmitted via helix 4 transmembrane helix 2 (TM2). An inward movement of TM2 initiates a transmembrane signal to the cytoplasmic HAMP (histidine kinases, adenyl cyclases, methyl-accepting proteins, phosphatases) domain. Baseline CheA kinase-stimulating activity and ligand-induced responses are both strongly influenced by residues at the C-terminus of transmembrane helix 2 (TM2). The cytoplasmic aromatic anchor, composed of residues Trp-209 and Tyr-210 in TarEc, is of particular importance. These residues are not highly conserved among transmembrane receptors having a HAMP domain, although there are almost always some aromatic residues in this region. The question thus becomes what properties of this aromatic anchor are necessary for proper signal transduction. In this dissertation, I studied the effect on TarEc function by substituting all possible combinations of Ala, Phe, Tyr, and Trp at positions 209 and 210. This library of TarEc variants allowed the direct assessment of the effect of the residue composition of the aromatic anchor and led to a model of how the wild-type anchor maintains the base-line signaling state in TarEc. Additional receptor variants containing double aromatic tandems and Ala substitutions for the periplasmic Trp residue were created, and the aromatic residues were also shifted in position within the six residues 207-212. Trp, Tyr, and Phe, in that order, had the greatest effect on function when they were moved to novel positions. It was also discovered that Gly-211 plays a critical role in maintaining receptor function. A model was generated that proposes that Gly-211 plays a role in maintaining the flexibility of the TM2-HAMP domain connector. The results suggest that the signaling properties of the transmembrane sensor kinases of two-component systems can be predicted by the nature of their TM2-HAMP connections. It may also be possible to modulate their activity in a controlled way by manipulating the amino acid sequences that comprise those connections.

Cross-sectional studies have shown that well trained endurance athletes frequently have a lower peripheral and central chemoreceptor response (pRc and cRc) and a lower minute ventilation (Ve) during exercise compared to untrained individuals. Some recent prospective studies support these observations. We speculated that the reductions in chemoreceptor response and Ve may be the specific result of the high rates of ventilation occurring during endurance training. To test this idea, subjects performed voluntary eucapnic hyperpnea to simulate exercise hyperpnea while avoiding the metabolic consequences of physical exercise. We therefore examined the effects of respiratory muscle training (RMT: 20x30min sessions of voluntary eucapnic hyperpnea) on the pRc, cR, cycling performance, and Ve. Twenty endurance trained cyclists were randomized into RMT or control-groups. To indicate cRc both the hypercapnic ventilatory response at rest (HCVRr) and during light exercise (HCVRex) were measured in a background of 50% O2. The pRc was assessed by measuring the ventilatory response to a modified Dejours O2 test (4-6 trials of 10-12 breaths of 100% O2) during light exercise. Endurance performance and Ve were measured during a fixed-rate cycling endurance test, performed at 85% of the maximal workload until exhaustion. The RMT-group's cycling endurance improved significantly compared to controls (+3.26±4.98min versus -1.46±3.67min. p=0.027) but Ve was unchanged at all times analyzed. The pRc was significantly reduced in the RMT-group but unchanged in controls (-5.8±6.0% versus +O.1±4.6%, p=O.032). The cRc, both at rest and during exercise, was not significantly altered following RMT in either group. However, the X-intercept of HCVRex exhibited a significant shift to the left (-5.83±10.68mmHg, +O.38±2.48mmHg, p=O.047, RMT-group and controls respectively). The importance of this leftward shift and the reduced pRc, though statistically significant, is unclear because there were no significant changes in Ve during any test nor were there correlations between Ve or performance or the altered chemoreceptor responses. We conclude that exercise hyperpnea, as simulated by RMT in this study, is accompanied by a reduction in pRc and a leftward shift in the HCVRex, and improves cycling endurance; however, the altered chemoreceptor responses had little impact on Ve suggesting that their role in the control of ventilation during exercise is minor.

<p>A study of the tarsi and mouthparts of females in three representative genera of tabanids revealed that the ventro-lateral surface of the fore-tarsi and the aboral margin of the labella were the principal loci of contact chemical sensilla. Of the four main types of aboral labellar trichodes (A,B,C and D), two (B and C) proved to be gustatory and then only at the tip. Two other types of trichodes (E and F) were identified on the anterior aspect of the labella. These were different from the aboral setae (type A,B,C, and D) in shape, size and function. </p><p>Extension of the proboscis was taken as the index of positive tarsal stimulation and the criterion of positive labellar stimulation was the spreading of the labellar lobes in an extended proboscis. This was observed to be the same in free as well as attached experimental flies. Newly emerged females showed similar response.</p><p>The frequency method generally employed in psycho-physical studies for estimating thresholds in mammals was successfully extended to tabanids. Using this method the stimulative effectiveness and threshold of various sugars for the tarsal and labellar taste sensilla was determined. Comparison was made between the frequency method and the ascending method of estimating thresholds with regard to sucrose. The effect of starvation on responsiveness of tarsal and labellar contact chemoreceptors was determined. Newly emerged flies became increasingly sensitive to sucrose during the duration of tests when they were strictly maintained on water diet. </p><p>Intergenerlc differences and similarities were seen in the behaviour immediately before feeding. The flies fed on "dry" sucrose as well as on solutions of appropriate concentrations. By analysing the crop contents of wild-caught tabanids, it was established that they fed on sugars and these findings were related to tabanid feeding behaviour and adaptation in nature.</p><p>Using whole blood, sugars and blood-sugar mixtures at various concentrations and proportions (in the case of blood-sugar mixtures), their dispatch to crop and/or mid-gut was studied. In addition, select group of amino acids and nucleotides were also tested to determine if these acted as feeding stimulants for the deer-flies.</p><p>Wild-caught females of deer-flies lived on dry sucrose and water and on 1.OH glucose solution longer than on distilled water alone or without food and water.</p> / Thesis / Doctor of Philosophy (PhD)