Electrophysiological Characterization of Insulin-Producing Cells in \(Drosophila\)

Insulin plays a critical role in maintaining metabolic homeostasis across species, regulating pathways underlying energy storage, reproduction, growth, and aging. In Drosophila melanogaster, insulin-producing cells (IPCs), located in the brain, are functionally analogous to mammalian pancreatic beta cells and secrete Drosophila insulin-like peptides (DILPs) in response to nutrient availability. While many insights into IPC activity have come from ex vivo studies, these approaches are limited in their ability to capture the complexity of intact sensorimotor and nutrient-sensing circuits. In vivo studies are crucial to understand how IPCs respond to physiological conditions such as starvation, nutrient ingestion, and refeeding. By retaining the complete nervous system and peripheral tissues, in vivo experiments enable the investigation of dynamic IPC activity in the context of intact and functional regulatory networks. This thesis leverages in vivo whole-cell patch- clamp electrophysiology, optogenetics, and calcium imaging to address these gaps. The first part of this thesis explores how nutritional states affect IPC activity and its impact on behavior. Using in vivo electrophysiological recordings, we demonstrate that IPC activity is strongly modulated by nutritional states. Nutritive sugars, such as glucose and fructose, robustly activate IPCs upon ingestion, whereas non-nutritive sugars and protein have no effect. Interestingly, glucose perfusion does not affect IPC activity, mirroring the mammalian incretin effect, where glucose ingestion amplifies insulin release compared to intravenous administration. IPCs also play a role in modulating starvation-induced locomotor hyperactivity, which is driven primarily by other modulatory neurons. These findings underscore the critical role of IPCs in regulating metabolic homeostasis and adaptive behavioral responses. The second part of the thesis delves into the neuromodulatory regulation of IPCs. Modulation of IPC activity is essential to regulate insulin release in response to the ever-changing energy demands. Using an integrative approach that includes single-nucleus transcriptomics, anatomical receptor mapping, connectomics, and optogenetics, we reveal that IPCs are a heterogeneous population with distinct receptor profiles. This heterogeneity enables differential responses to various aminergic and peptidergic modulatory inputs. Functional analyses uncover that certain modulators shift IPC population activity towards excitation or inhibition, possibly to tailor insulin release to the fly's metabolic demands. Notably, we identify at least three IPC sub-populations based on their distinct responses to neuromodulatory inputs, suggesting a mechanism for fine-tuned metabolic regulation. Overall, this thesis makes a significant contribution to our understanding of insulin signaling by demonstrating how IPCs integrate diverse nutritional and neuromodulatory inputs to regulate metabolic homeostasis. By employing advanced in vivo methodologies, this work highlights the complexity of IPC regulation and uncovers mechanisms that enable dynamic adaptations to changing physiological states. These findings advance our basic understanding of the neuronal and endocrine networks underlying metabolic homeostasis, shedding light on the intricate mechanisms that enable insulin regulation in Drosophila melanogaster.