Title: Neuronal Circuit Dissection in the Drosophila Brain: the Role of Serotonin-Releasing Neurons in Arousal
Abstract: For animals, an optimal arousal state is essential for motivating or inhibiting a specific behavior. Arousal is regarded to be an internal factor that influences the initiation and execution of different behaviors, and is usually defined by three criteria: alertness to sensory stimuli, motor activity and emotional reactivity. A large amount of studies across many species have shown that different biogenic amines, like dopamine, serotonin or norepinephrine, contribute fundamentally to the neuronal substrate of arousal. In the first part of this study the potential modulatory effect of serotoninergic neurons on arousal in Drosophila was explored. To do so, a new transgenic line was generated that allows for both the thermogenetic activation of the serotonergic neurons and their visualization (i.e., identification). Furthermore, a behavioral assay was established to use walking velocity of individual flies as a readout of the animals’ arousal state. I could show that thermoactivation of serotoninergic neurons located in the brain region, but not in thoracic ganglia, led to a significant reduction in walking velocity. Furthermore, I could distinguish the behavioral consequence of activated serotoninergic neurons from those resulting from activated motor neurons by testing the startle-induced, reflexive response of the flies in several paradigms such as negative geotaxis, shock avoidance and flight. The reflexive stimulus reactivity of flies was unaffected by an activation of serotoninergic neurons, which means that the manipulation of these serotoninergic neurons did not lead to a locomotor deficit. In other words, the flies could move, they just would not do so. I further investigated the alertness of the flies to sensory stimuli as the second criterion of arousal. Serotoninergic neurons of starved flies (hunger representing an internal motivational factor) were activated in the presence of food (as an external stimulus). My results show that activation of serotoninergic neurons prevents starved flies from feeding. In addition, I investigated the modulatory effect of serotoninergic neurons on endogenous arousal (sleep-wake rhythms). I used temperature sensitive shibire expression to reversibly block serotoninergic neurons and found that this manipulation leads to shortened night sleep and increased activity during the night. In the second part of the study I asked whether the neuronal mechanism underlying arousal is a global function of the neurotransmitter serotonin in the brain, or whether it is localized to the function of specific circuits. Therefore, I applied different genetic strategies to manipulate small subsets of neurons. In the first step, using a stochastic approach, the locomotor phenotype of flies, in which different subsets of serotonergic neurons were activated, was analyzed. In this way, I was able to identify specific subdivisions of serotoninergic cells, namely those of the PMP cluster, as a candidate circuitry that underlies the modulation of arousal. In the next step, using an intersectional gene expression approach, I restricted transgene expression to candidate subsets of neurons and could, thereby, verify that two individual, serotoninergic cells of the PMP cluster are sufficient to modulate the arousal level in flies. Overall, I could show that serotoninergic neurons affect the arousal level of the fly, and that this modulatory effect can be elicited from a specific subset of serotoninergic neurons in PMP down cluster. In future studies, it will be essential to address potential combinatorial effects of the PMP down cluster with other subsets of serotoninergic neurons.