Cellular and molecular mechanisms of neuronal remodelling at the neuromuscular junction

Abstract

Nervous system function depends on the coordinated activity of populations of neurons that are connected and communicate at junctions called synapses. Synapses are first established during embryogenesis using a highly precise genetic program from which neural circuits are defined, allowing processes such as perception, learning, memory, or locomotion. However, circuit wiring can be further remodeled using neuronal activity-dependent plasticity mechanisms. Therefore, synaptogenesis is a long-developmental process that involves synapse formation, maintenance, refinement, and elimination. The plasticity of neural circuits is critical because it allows for information storage and environmental adaptation in young individuals and throughout adulthood. Hence, it is not surprising that defects in synaptic connectivity and plasticity are characteristic of numerous neurodevelopmental and neurodegenerative diseases. Synapses are typically found in round varicosities formed at neuronal axons, called synaptic boutons, which are conserved structures from invertebrates to humans. Although boutons are important synaptic compartments where neurotransmission occurs, very little is known about the mechanism and dynamics of bouton formation, especially during the postembryonic period. Initial circuit formation is relatively well-characterized and requires intricate developmental events prior to synaptogenesis, including cell fate specification, cell migration, axon guidance, and synaptic target selection, all of which occur almost simultaneously in billions of neurons during vertebrate nervous system development. During this period, the mechanisms that give rise to synaptic boutons and synapses involve the formation of labile and dynamic growth cones (GCs), comprised of filopodia and lamellipodia, which guide axons towards their targets and differentiate into round boutons upon arrival at their destination. Once formed, the circuit architecture can change in response to developmental cues and external stimuli, such as synaptic activity, allowing for more selective remodeling, including bouton formation and elimination. However, how these and other structural changes are coordinated between synaptic partners and the mechanisms used for neuronal remodeling are still unknown. The aim of this PhD thesis was to address a fundamental question in neuroscience: How are synaptic boutons formed and integrated into wired neurons? The Drosophila neuromuscular junction (NMJ), a synapse formed between motor neurons (MNs) and skeletal muscle fibers that is essential for muscle contraction and movement, was adopted as a model to study this question. In addition to clearly discernible boutons, NMJs show robust structural plasticity during larval growth, with new boutons added to the neuronal arbor through developmental and activity-dependent processes. The analysis was performed mainly in 3rd instar larvae, in which developmental bouton addition is nearly completed, after induction of structural plasticity using depolarization protocols with a solution containing elevated K+ that mimics intense activity, and allows for the rapid addition of new boutons to the NMJ. This stimulation method was used and combined with live samples of Drosophila larvae to dissect the details of bouton outgrowth in real-time. Neuronal migration and growth are critical for proper synaptic wiring in the nervous system. To date, the mechanisms described for bouton formation have involved filopodia or lamellipodia structures. However, live imaging of unanesthetized Drosophila larval NMJs with high temporal resolution (in secs) sheds light on an unreported mechanism for bouton addition in wired neurons, which can potentially be used for rewiring of mature neurons. In the Drosophila larval NMJ, synaptic bouton addition in response to intense activity does not occur, as in the embryonic stage, where a GC differentiates into round boutons. Instead, new boutons emerge rapidly as round protrusions of the neuronal membrane resembling blebbing, a pressure-driven mechanism used by other cells in 3D migration and tissue invasion. Moreover, new boutons exhibit key characteristics of blebbing: a reduction in filamentous actin (F-actin) during their growth and the recruitment of non-muscle myosin II (MyoII), an essential regulator of blebbing. Because the NMJ is deeply inserted into the muscle, for the MN to attach new boutons to the muscle, it must further invade it, which is mechanistically similar to migration across other tissues. It is possible that MNs have adopted a strategy in which blebbing is combined with activity-dependent signaling pathways to modulate the formation of new boutons during intense neuronal activity. Interestingly, an association between muscle contraction and bouton formation was observed in live imaging movies, suggesting a role of the muscle during this process. Using several strategies, the muscle contraction potential was manipulated during NMJ stimulation, which showed that this mechanism of bouton formation requires muscle contraction around the neuronal membrane. One hypothesis is that the muscle plays a mechanical role in MN plasticity and bouton remodeling, possibly by compressing the MN during contractility and increasing its confinement, which is an important factor in promoting blebbing. These findings suggest that in addition to biochemical signaling, a balance of mechanical forces cooperates between MN and muscle cells to coordinate activitydependent bouton formation at the Drosophila NMJ. This research further expanded our understanding of the mechanisms that control synaptic bouton assembly in neurons and identified a mechanism, blebbing, by which wired neurons may form new boutons, allowing their structural expansion and plasticity, using transsynaptic physical interactions as the main driving force. Further investigation of this mechanism can contribute to a clear understanding of the normal neuronal structure and function, which is essential for studying responses to injury or disease.