Identification of neuron populations and circuits involved in sleep regulation in the bearded-dragon Pogona vitticeps

Sleep has traditionally been viewed as an offline behavioral state, initially believed to be exclusive to mammals and birds. However, extensive research over decades has revealed that sleep occurs in most if not all animal species, and that is an active process occurring at cellular, circuit, and organismal levels. Despite tremendous progress, the circuit mechanisms governing sleep remain poorly understood across species. Most of what we know about sleep regulation is based on studies in mammals. Our lab, using behavioral and electrophysiological approaches, identified a two-stage sleep pattern in bearded dragons (Pogona vitticeps), consisting of regularly alternating slow-wave (SW) and rapid-eye-movement (REM) activities. These findings, added to pre-existing ones about sleep in mammals and birds, thus suggest the existence of both REM and NREM sleep states in all amniotes, in turn suggesting their existence in a common ancestor. The circuits regulating mammalian sleep may thus be ancient, with a potential origin dating back at least 320 million years, and may be common (at least in part) to reptiles, birds, and mammals. This thesis aims to pinpoint the specific brain areas and neural populations responsible for sleep control in a reptilian model system, the Australian dragon Pogona vitticeps. This system was chosen because it may be the closest one (among the extant model systems for sleep) to a putative common ancestor. At the project's onset, a comprehensive brain map of Pogona was lacking. To facilitate the investigation of potential circuits and mechanisms governing sleep in Pogona, I used immunohistochemistry and fluorescent in situ hybridization, selecting markers for canonical neuromodulators and neurotransmitters. The goal of this approach was to identify the potential reptilian homologs of sleep regulatory areas known in mammals. To facilitate the visualization of these regions, I exploited 3D reconstructions and image-registration techniques. Using μCT scans as a reference for the Pogona brain, I successfully established a 3D, interactive atlas illustrating neuron populations that are good candidates to be associated with sleep control. Subsequently, to identify regions and neuron types related to sleep control in Pogona, I used immediate-early-gene mapping focusing on the gene, Early Growth Response 1 (EGR1). I compared EGR1 brain expression patterns between sleeping and awake animals. EGR1-positive cells associated with sleep and wakefulness were distributed throughout the brain, including the claustrum (CLA), tuberomammillary nucleus, optical tectum, subcoeruleus nucleus, and raphe areas. These cells were often intermingled or situated in distinct subregions within a particular area. In an electrophysiological study of sleep in Pogona, we discovered that the CLA is the origin of sharp-wave ripples, a characteristic feature of NREM sleep. Using virus tracing, we established direct connections between the CLA and numerous brain regions, including the forebrain, the diencephalon, the mesencephalon and the hindbrain, many of which are known to be associated with sleep in mammals. Our findings suggest that areas and neuronal types involved in sleep regulation in mammals also exist in Pogona, consistent with the common ancestry of these amniote lineages. These results imply that the mechanistic insights into sleep regulation gathered in Pogona may be applicable more broadly to amniote vertebrates, including mammals.