DOI

10.17077/etd.7vdypc5l

Document Type

Dissertation

Date of Degree

Fall 2015

Degree Name

PhD (Doctor of Philosophy)

Degree In

Neuroscience

First Advisor

George B. Richerson

First Committee Member

William T. Talman

Second Committee Member

Timothy J. Brennan

Third Committee Member

Durga P. Mohapatra

Fourth Committee Member

Brian K. Gehlbach

Fifth Committee Member

Gordon F. Buchanan

Abstract

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.

Public Abstract

Breathing is one of the most important functions for life. Abnormal breathing is a major concern for health care providers and has been associated with human disorders such as sudden unexpected death in epilepsy (SUDEP) and sudden infant death syndrome (SIDS). Defects in serotonin neurons have been implicated in a variety of brain functions and diseases, including SUDEP and SIDS. One of the most important roles of serotonin neurons is breathing control. Serotonin neurons detect increased carbon dioxide in the body and signal respiratory control centers in the brainstem to stimulate breathing. This process is important for maintaining normal carbon dioxide levels in the body. Better understanding the role serotonin neurons play in breathing control is critically important for human health.

This study investigated the effects of anesthetics on respiration and the ability of animals to increase their breathing in response to increased carbon dioxide. Additionally, I describe the effects of anesthetics on serotonin neurons and how these effects could be responsible for abnormal breathing. I next describe how anesthetics affected breathing at different ages. Finally, I characterize the impaired breathing and growth of mice that lack serotonin neurons. Overall this work indicates that anesthetics reduce the increased breathing response to carbon dioxide and that, for some anesthetics, this is due in part to effects on serotonin neurons. My experiments also highlight that the role serotonin neurons play in control of breathing changes during development and that genetic background can alter the severity of breathing problems in some mouse models.

Keywords

anesthesia, chemoreceptor, raphe, respiration, serotonin

Pages

xv, 115 pages

Bibliography

Includes bibliographical references (pages 99-115).

Copyright

Copyright © 2015 Cory Allen Massey

Share

COinS