bioRxiv Science⌕ Search

Biology subjects

Cunningham, K. L.

Publications and source records attributed to Cunningham, K. L..

5 recordsLinked to original sources

Loss of the extracellular matrix protein Perlecan disrupts axonal and synaptic stability during Drosophila development

Heparan sulfate proteoglycans (HSPGs) form essential components of the extracellular matrix (ECM) and basement membrane (BM) and have both structural and signaling roles. Perlecan is a secreted ECM-localized HSPG that contributes to tissue integrity and cell-cell communication. Although a core component of the ECM, the role of Perlecan in neuronal structure and function is less understood. Here we identify a role for Drosophila Perlecan in the maintenance of larval motoneuron axonal and synaptic stability. Loss of Perlecan causes alterations in the axonal cytoskeleton, followed by axonal breakage and synaptic retraction of neuromuscular junctions. These phenotypes are not prevented by blocking Wallerian degeneration and are independent of Perlecans role in Wingless signaling. Expression of Perlecan solely in motoneurons cannot rescue synaptic retraction phenotypes. Similarly, removing Perlecan specifically from neurons, glia or muscle does not cause synaptic retraction, indicating the protein is secreted from multiple cell types and functions non-cell autonomously. Within the peripheral nervous system, Perlecan predominantly localizes to the neural lamella, a specialized ECM surrounding nerve bundles. Indeed, the neural lamella is disrupted in the absence of Perlecan, with axons occasionally exiting their usual boundary in the nerve bundle. In addition, entire nerve bundles degenerate in a temporally coordinated manner across individual hemi-segments throughout larval development. These observations indicate disruption of neural lamella ECM function triggers axonal destabilization and synaptic retraction of motoneurons, revealing a role for Perlecan in axonal and synaptic integrity during nervous system development.

neuroscience↗

Molecular Logic of Synaptic Diversity Between Drosophila Tonic and Phasic Motoneurons

Although neuronal subtypes display unique synaptic organization and function, the underlying transcriptional differences that establish these features is poorly understood. To identify molecular pathways that contribute to synaptic diversity, single neuron PatchSeq RNA profiling was performed on Drosophila tonic and phasic glutamatergic motoneurons. Tonic motoneurons form weaker facilitating synapses onto single muscles, while phasic motoneurons form stronger depressing synapses onto multiple muscles. Super-resolution microscopy and in vivo imaging demonstrated synaptic active zones in phasic motoneurons are more compact and display enhanced Ca2+ influx compared to their tonic counterparts. Genetic analysis identified unique synaptic properties that mapped onto gene expression differences for several cellular pathways, including distinct signaling ligands, post-translational modifications and intracellular Ca2+ buffers. These findings provide insights into how unique transcriptomes drive functional and morphological differences between neuronal subtypes.

neuroscience↗

Regulation of presynaptic Ca2+ channel abundance at active zones through a balance of delivery and turnover

AbstractVoltage-gated Ca2+ channels (VGCCs) mediate Ca2+ influx to trigger neurotransmitter release at specialized presynaptic sites termed active zones (AZs). The abundance of VGCCs at AZs regulates neurotransmitter release probability (Pr), a key presynaptic determinant of synaptic strength. Although biosynthesis, delivery and recycling cooperate to establish AZ VGCC abundance, experimentally isolating these distinct regulatory processes has been difficult. Here we describe how the AZ levels of Cacophony (Cac), the sole VGCC mediating synaptic transmission in Drosophila, are determined. We also analyzed the relationship between Cac, the conserved VGCC regulatory subunit 2{delta}, and the core AZ scaffold protein Bruchpilot (BRP) in establishing a functional AZ. We find Cac and BRP are independently regulated at growing AZs, as Cac is dispensable for AZ formation and structural maturation, and BRP abundance is not limiting for Cac accumulation. Additionally, AZs stop accumulating Cac after an initial growth phase, whereas BRP levels continue to increase given extended developmental time. AZ Cac is also buffered against moderate increases or decreases in biosynthesis, whereas BRP lacks this buffering. To probe mechanisms that determine AZ Cac abundance, intravital FRAP and Cac photoconversion were used to separately measure delivery and turnover at individual AZs over a multi-day period. Cac delivery occurs broadly across the AZ population, correlates with AZ size, and is rate-limited by 2{delta}. Although Cac does not undergo significant lateral transfer between neighboring AZs over the course of development, Cac removal from AZs does occur and is promoted by new Cac delivery, generating a cap on Cac accumulation at mature AZs. Together these findings reveal how Cac biosynthesis, synaptic delivery, and recycling set the abundance of VGCCs at individual AZs throughout synapse development and maintenance.

neuroscience↗

The decoy SNARE Tomosyn sets tonic versus phasic release properties and is required for homeostatic synaptic plasticity

Synaptic vesicle release probability (Pr) is a key presynaptic determinant of synaptic strength established by cell intrinsic properties and further refined by plasticity. To characterize mechanisms that generate Pr heterogeneity between distinct neuronal populations, we examined glutamatergic tonic (Ib) and phasic (Is) motoneurons in Drosophila with stereotyped differences in Pr and synaptic plasticity. We found the decoy SNARE Tomosyn is differentially expressed between these motoneuron subclasses and contributes to intrinsic differences in their synaptic output. Tomosyn expression enables tonic release in Ib motoneurons by reducing SNARE complex formation and suppressing Pr to generate decreased levels of synaptic vesicle fusion and enhanced resistance to synaptic fatigue. In contrast, phasic release dominates when Tomosyn expression is low, enabling high intrinsic Pr at Is terminals at the expense of sustained release and robust presynaptic potentiation. In addition, loss of Tomosyn disrupts the ability of tonic synapses to undergo presynaptic homeostatic potentiation (PHP).

neuroscience↗

Examining molecular determinants underlying heterogeneity of synaptic release probability using optical quantal imaging

Neurons communicate through neurotransmitter release at specialized synaptic regions known as active zones (AZs). Using transgenic biosensors to image postsynaptic glutamate receptor activation following single vesicle fusion events at Drosophila neuromuscular junctions, we analyzed release probability (Pr) maps for a defined connection with ~300 AZs between synaptic partners. Although Pr was very heterogeneous, it represented a stable and unique feature of each AZ. Pr heterogeneity was not abolished in mutants lacking Synaptotagmin 1, suggesting the AZ itself is likely to harbor a key determinant(s). Indeed, AZ Pr was strongly correlated with presynaptic Ca2+ channel density and Ca2+ influx at single release sites. In addition, Pr variability was reflected in the postsynaptic compartment, as high Pr AZs displayed a distinct pattern of glutamate receptor clustering. Developmental analysis suggests that high Pr sites emerge from earlier formed AZs, with a temporal maturation in transmission strength occurring over several days.

neuroscience↗