Molecular genetic studies on voltage-gated ion channels

Several different methods have been employed in the study of voltage-gated ion channels. Electrophysiological studies on excitable cells in vertebrates and molluscs have shown that many different voltage-gated potassium (K⁺) channels and sodium channels may coexist in the same organism. Parallel genetic studies in Drosophila have identified mutations in several genes that alter the properties of specific subsets of physiologically identified ion channels. Chapter 2 describes molecular studies that identify two Drosophila homologs of vertebrate sodium-channel genes. Mutations in one of these Drosophila sodium-channel genes are shown to be responsible for the temperature-dependent paralysis of a behavioural mutant para^ts. Evolutionary arguments, based on the partial sequences of the two Drosophila genes, suggest that subfamilies of voltage-gated sodium channels in vertebrates remain to be identified.

In Drosophila, diverse voltage-gated K⁺ channels arise from alternatively spliced mRNAs generated at the Shaker locus. Chapter 3 and the Appendices describe the isolation and characterization of several human K⁺-channel genes, similar in sequence to Shaker. Each of these human genes has a highly conserved homolog in rodents; thus, this K⁺-channel gene family probably diversified prior to the mammalian radiation. Functional K⁺ channels encoded by these genes have been expressed in Xenopus oocytes and their properties have been analyzed by electrophysiological methods. These studies demonstrate that both transient and noninactivating voltage-gated K⁺ channels may be encoded by mammalian genes closely related to Shaker. In addition, results presented in Appendix 3 clearly demonstrate that independent gene products from two K⁺-channel genes may efficiently co-assemble into heterooligomeric K⁺ channels with properties distinct from either homomultimeric channel. This finding suggests yet another molecular mechanism for the generation of K⁺-channel diversity.

The Regulation of Sleep and Circadian Rhythms: The Role of Melatonin and Adenosine in Zebrafish

Sleep is a highly conserved behavioral state whose regulation is still unclear. In this thesis I initially briefly introduce the known sleep circuitry and regulation in vertebrates, and why zebrafish is seen as a good model to study sleep-regulation. I describe the existing two-process model of sleep regulation, which posits that the two processes C (circadian) and S (homeostatic) control timing of sleep-wake behavior. I then study the role melatonin plays in the circadian regulation of sleep using zebrafish. Firstly, we find that the absence of melatonin results in a reduction of sleep at night, establishing that endogenous melatonin is required for sleep at night. Secondly, melatonin mutants show a reduction in sleep in animals with no functional behavioral rhythms suggesting that melatonin does not require intact circadian rhythms for its effect on sleep. Thirdly, melatonin mutants do not exhibit any changes in circadian rhythms, suggesting that the circadian clock does not require melatonin for its function. Fourthly, we find that in the absence of melatonin, there is no rhythmic expression of sleep, suggesting that melatonin is the output molecule of process C. Lastly, we describe a connection between adenosine signaling (output molecules of process S), and melatonin. Following this we proceed to study the role adenosine signaling plays in sleep-wake behavior. We find that firstly, adenosine receptor A1 and A2 are involved in sleep- wake behavior in zebrafish, based on agonist/antagonist behavioral results. Secondly, we find that several brain regions such as PACAP cells in the rostral midbrain, GABAergic cells in the forebrain and hindbrain, Dopamine and serotonin cells in the caudal hypothalamus and sox2 cells lining the hindbrain ventricle are activated in response to the A1 antagonist and VMAT positive cells are activated in response to the A2A agonist, suggesting these areas are involved in adenosine signaling in zebrafish. Thirdly, we find that knocking out the zebrafish adenosine receptors has no effect on sleep architecture. Lastly, we find that while the A1 agonist phenotype requires the zfAdora1a receptor, the antagonist and the A2A agonist behavioral phenotypes are not mediated by the zfAdora1a, zfAdora1b and zfAdoraA2Aa, zfAdora2Ab receptors respectively.