Murphey, Rodney

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.

At the site of neuronal communication, multiple interacting components drive synapse structure and function. Synaptic vesicle pools, membrane proteins, mitochondria, and perisynaptic astrocyte processes (PAPs) are all structures that can be altered through naturally occurring plasticity mechanisms to modulate neurotransmission, and disruption of these structures can result in synapse dysfunction and disease. Due to the minute size of the synapse, electron microscopy (EM) remains the gold standard for ultrastructural characterization; however, due to the complexity of EM datasets, extraction of information has become a bottleneck which places limits on the amount of data that can be collected and analyzed. A need exists for easy-to-use workflows that automate and enhance analysis throughput, to keep up with the streams of image data that are able to be produced. Here, I develop the use of AI algorithms, correlative microscopy techniques, and novel structural analysis methods to characterize postsynaptic mitochondria, PAPs, synaptic vesicles, and integral membrane proteins and their impact on synapse structure and function. I show that both postsynaptic mitochondria and PAPs in the visual cortex are positioned to support synapse structure and function; cleavage of a synaptic adhesion molecule affects synaptic vesicle accumulation in the amygdala; and presynaptic voltage gated calcium channels aggregate near active zone machinery in the brainstem. In addition, I highlight the use of virtual reality as a fast and intuitive tool for the identification and isolation of individual neurites in 3D EM. Thus, my work establishes novel technical approaches for EM and advances our understanding of neuronal communication through original research of several synaptic components.

Insect grooming has various functions, including defense against parasites and pathogens, cleaning of dust particles, and maintenance of sensory receptors. The hierarchy of grooming behavior suggests that cleaning one body part is more crucial than the other, the priority order more specifically being eyes, antennae, abdomen, then wings, followed by the thorax. Histamine is an extensively studied neurotransmitter found in the central nervous system of many animals. In Drosophila, histamine is found in both the peripheral and central nervous systems and is necessary for visual and mechanosensory behaviors. Histamine-gated chloride channel 1 (HisCl1) and Ora transientless (Ort) are two characterized histamine receptors, both of which are vital for visual signaling in the fly.