Drosophila

The intricate processes governing cellular pH and its impact on protein and cellular function have been extensively explored. However, our understanding of the pH fluctuations that occur during routine cellular activities and their potential to modulate cell function remains, particularly within the highly dynamic pH landscape of a synapse. Investigating the scale, directionality, and temporal characteristics of these activity-dependent pH fluctuations at synapses is of paramount interest, as it carries profound implications for neurotransmitter release and signal transduction. Employing both empirical and computational modeling methods, our research explores the dynamic pH environment within the synaptic cleft of Drosophila glutamatergic motor neuron Ib terminals during synaptic activity and reveals its significance in modulating neurotransmission. Contrary to popular belief, we discovered that these terminals undergo activity-dependent extracellular alkalinization in response to both single action potentials and burst stimulation. This surprising phenomenon was also observed at the mouse calyx of Held. We found activity-dependent alkalinization to be predominantly driven by Ca2+ movement across the postsynaptic membrane, and by targeting pH indicators to subcellular domains, we identified alkalinization to primarily occur within the cleft.

Synaptogenesis is a requirement for cellular communication, but the specific molecular mechanisms underlying synaptogenesis are unclear. Here, we investigate and show the role of the protein Frazzled in synaptogenesis using the transheterozygous Frazzled loss-of-function (LOF) mutant in Drosophila.
Leveraging the UAS-GAL4 expression system, we drove expression of various Frazzled/DCC gene constructs in the Giant Fibers (GF) of flies and found changes to synaptogenesis and axon pathfinding.
We identified decreases in electrical synaptogenesis and distinct axon pathfinding errors in Frazzled LOF mutants. Strikingly, the expression of Frazzled intracellular domain (ICD) significantly rescues both phenotypes, while full-length Frazzled protein only partially rescues these phenotypes, prompting us to explore the role of different domains within the protein. Deleting the P1 and P2 domains of Frazzled does not rescue axon pathfinding but did partially rescue synaptogenesis while deleting the P3 domain failed to rescue either phenotype. Moreover, when we drive expression Frazzled with a point-mutated P3 domain, silencing its transcriptional activation domain, it fails to rescue both synaptogenesis and axon pathfinding.
These results strongly suggest that Frazzled regulates both synaptogenesis and axon pathfinding in the GFs and is necessary for synaptogenesis of the mixed electrochemical GF synapse. Our results provide novel insights into the molecular mechanisms governing neural circuit assembly and highlight Frazzled as a key player in axon guidance and synaptic development.

Proper formation of synapses in the developing nervous system is critical to the expected function and behavior of an adult organism. Neurons must project neurites, in the form of axons or dendrites, to target areas to complete synaptic circuits. The biochemical tool that cells use to interact with the external environment and direct the guidance of developing neurites are guidance receptors. One such guidance receptor that is extensively studied to uncover its roles in developmental disorders and disease is DSCAM (Down-Syndrome Cell Adhesion Molecule). To better understand the role of DSCAM in humans, a fly homolog Dscam1 was extensively characterized in the giant fiber system (GFS) of Drosophila to further explore its roles in axon guidance, synapse formation, and synapse function. The UAS-Gal4 system was used to alter the protein levels of Dscam1 within the giant fiber interneurons (GFs). A UAS-RNAi construct against Dscam1 was used to knockdown translation of all possible isoforms within the GFs. A UAS-Dscam1(TM2) construct was used to overexpress a single isoform of Dscam1 that is specifically trafficked to the axons. Confocal microscopy was used to determine the morphological changes associated with dysregulated Dscam1 levels. Visualization via fluorescent markers was accomplished of both pre- and post-synaptic cells, the GFs and tergotrochanteral motorneurons (TTMns), respectively, and synapse interface was determined as colocalization of the two cells. Additionally, the functional components of the GF-TTMn synapse, both gap-junctions, and presynaptic chemical active zones were tagged via fluorescent antibodies and quantified.

Acute pH sensitivity of many neural mechanisms highlights the vulnerability of neurotransmission to the pH of the extracellular milieu. The dogma is that the synaptic cleft will acidify upon neurotransmission because the synaptic vesicles corelease neurotransmitters and protons to the cleft, and the direct data from sensory ribbon-type synapses support the acidification of the cleft. However, ribbon synapses have a much higher release probability than conventional synapses, and it’s not established whether conventional synapses acidify as well. To test the acidification of the cleft in the conventional synapse, we used genetically encoded fluorescent pH reporters targeted to the synaptic cleft of Drosophila larvae. We observed alkalinization rather than acidification during activity, and this alkalinization was dependent on the exchange of protons for calcium at the postsynaptic membrane.
A reaction-diffusion computational model of the pH dynamics at the Drosophila larval neuromuscular junction was developed to leverage the experimental data. The model incorporates the release of glutamate, ATP, and protons from synaptic vesicles into the cleft, PMCA activity, bicarbonate, and phosphate buffering systems. By means of numerical simulations, we reveal a highly dynamic pH landscape within the synaptic cleft, harboring deep but exceedingly rapid acid transients that give way to a prolonged period of alkalinization.

Dopamine is an essential component in the neural pathway for attractive and aversive behavior. Dopaminergic (DA) neurons are known to have a key role in neurotransmission which can result in the modulation of different behaviors as well as the manifestation of different mental health disorders. Drosophila share similar genetics that are associated with several neurodegenerative diseases and disorders in humans. Furthermore, previous studies have shown conservation of DA neurons between humans and Drosophila which facilitate research using Drosophila as a model organism. In this study, we initially developed and tested a novel optogenetics system, which targeted neurons with spatial specificity, that activated or inhibited neurons through channelrhodopsin microbial opsins that are sensitive to red light. This system was then used to investigate the DA subsets that mediate attractive and aversive behavior. The activation of PPL1 clusters mostly resulted in aversive behavior as aligned with the literature, however activation of clusters with output neurons (PPL1 & PAM) concluded with different results.

Accumulating evidence points to a fundamental connection between sleep and feeding behavior. However, the temporal, genetic, and neuronal architecture that defines these relationships is poorly understood. Drosophila are amenable to high-throughput studies and offer numerous genetic tools which have advanced our understanding of the mechanistic relationships between these behaviors. However, certain features of the sleep-feeding axis have remained elusive, largely due to the separate measurement of sleep and feeding. Here, I develop a system which simultaneously measures sleep and feeding in individual animals by employing high resolution machine vision tracking and micro-controller interface functionality. Using this system, I show that food consumption drives a transient rise in sleep, which depends on food quality, quantity, and timing of a meal. The leucokinin system mediates these effects, particularly in response to protein ingestion. We further use the system to examine sleep homeostasis and demonstrate sleep dependence on energy expenditure and fat-brain communication. Collectively, these findings provide novel insight into the fundamental connections between sleep and feeding behavior.