Pittman, Talia; (2024) The Role of Galanin Receptors in Zebrafish Sleep Homeostasis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Sleep is a highly conserved physiological state, vital for health and survival. The timing and structure of sleep are governed by two fundamental processes: the circadian clock and sleep homeostasis. Despite its evident fundamental importance, the specific mechanisms and neuronal circuits that underpin the homeostatic control of sleep remain largely unknown. Galanin has recently been identified as playing a pivotal role in homeostatic sleep regulation. Therefore, investigating the downstream molecular pathways and circuits mediating Galanin-dependent sleep regulation will advance our understanding of the mechanisms underpinning sleep homeostasis. The zebrafish model was employed—recognised for its genetic tractability, fecundity, rapid development, and the relevance of its sleep physiology to human biology—to explore the influence of Galanin's four putative receptors (Galr1a, Galr1b, Galr2a, and Galr2b) on sleep regulation. The expression of Galanin and its receptors were mapped using in situ-hybridisation chain reaction (HCR) techniques, revealing distinct neuroanatomical distribution within the larval zebrafish brain. Through CRISPR/Cas9 mutagenesis, novel zebrafish lines were generated with mutations in each of the four Galanin receptors and their sleep/wake phenotypes were characterised using a high-throughput behavioural monitoring system. This analysis implicated Galr2b in playing a vital role in mediating Galanin-dependent regulation of daily sleep and suggested Galr1a and Galr1b may exert negative feedback on Galanin’s sleep-promoting effects under baseline conditions. Sleep pressure assays, using pharmacologically-induced increases in neuronal activity to acutely drive sleep pressure, were employed to probe the homeostatic sleep phenotype of each Galnain receptor mutant. These revealed a significant reduction in the rebound sleep response of galr2a mutants, suggesting Galr2a plays a critical role in mediating Galanin-dependent homeostatic sleep. This research demonstrates distinct functions for each Galanin receptor and suggests independent pathways through which Galanin regulates daily sleep and homeostatic sleep regulation under high sleep pressure conditions.

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