Vertebrate circadian systems, composed of networks of endogenous clocks distributed across tissues and organs and synchronised to one another by one or several âmasterâ pacemakers, enable organisms to anticipate and exploit favourable conditions for daily activities. The epithalamic habenula has been identified as a component of the extended mammalian circadian system, where through integration of both intrinsic and extrinsic temporal cues, it exhibits circadian rhythms in neuronal activity. In lower vertebrates, such as zebrafish, the habenula contains a molecular clock and responds to changes in blue light via retinal ganglion cells, suggesting a possible effect of circadian effectors on its function. This thesis explores the circadian regulation of this highly conserved epithalamic structure as well as the role of its putative circadian oscillator in the regulation of vertebrate brain and behavioural states. To determine whether the zebrafish habenula, as in mammals, exhibits daily variations in cellular activity in vivo, three distinct approaches â monitoring of bioluminescent calcium (Ca2+) reporter, immunohistochemistry of phosphorylated extracellular signal-regulated kinase (pERK) and two-photon imaging of the Ca2+ indicator GCaMP6s â were applied. While the bioluminescent Ca2+ indicator failed to report any spontaneous Ca2+ events, the two other techniques produced consistent results, demonstrating circadian variation in the putative homologue of the mammalian lateral habenula (LHb), with increased levels of both cytosolic Ca2+ and pERK at night. The pERK levels, but not overall Ca2+ activity, also showed circadian differences in the putative homologue of the mammalian medial habenula (MHb), but these variations were of opposing polarities and occurred in different subregions of this structure. Secondly, in order to investigate the functional role of the habenula oscillator in regulation of zebrafish behaviour and brain neurotransmission, a transgenic line, in which the molecular clock was disrupted specifically in the habenula by a dominant-negative strategy, was generated. Circadian rhythms of locomotor activity under constant darkness and arousal-associated sensory responsiveness during the sleep-like state in the transgenic larvae were not affected. In contrast, in adult zebrafish as a result of habenula-specific clock disruption, brain levels of important neurotransmitters â dopamine, serotonin and acetylcholine â were significantly reduced during the day, but not during the night. Finally, circadian rhythmicity of habenula neuronal activity was monitored in mouse brain slices, by the simultaneous recording of extracellular multi-unit activity from multiple sites within the habenula. Both MHb and LHb exhibited elevated neuronal activity during the night, particularly in the ventral subregion of the MHb and lateral portion of the LHb. In addition, a putative output signal of the suprachiasmatic nucleus (SCN), the neuropeptide arginine vasopressin (AVP), induced excitation in the LHb. However, these results were recapitulated with the related neuropeptide oxytocin (OT) and a specific OT receptor (OTRs) agonist â results implying that the AVP- and OT-induced excitatory effects are likely mediated via OTRs. A more pronounced AVP-induced effect was observed during the day, while OT-induced excitation did not vary between day and night, implying that AVP, but not the OT, could contribute to circadian regulation of the rodent habenula. In conclusion, this methodologically diverse study contributes to our growing understanding of habenulaâs function in the processing and transmission of temporal context specific and the role of its circadian oscillator in the daily coordination of brain state and behaviour in vertebrate species.