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 are poorly understood. To identify molecular pathways that contribute to synaptic diversity, single-neuron Patch-seq 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 that synaptic active zones in phasic motoneurons are more compact and display enhanced Ca2+ influx compared with 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.

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 Perlecan's 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.

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 Ca 2+ 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 Ca 2+ buffers. These findings provide insights into how unique transcriptomes drive functional and morphological differences between neuronal subtypes.

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

Voltage-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 α2δ, and the core AZ scaffold protein Bruchpilot (BRP) in establishing a functional AZ. We find that 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 α2δ. 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.

Mechanisms controlling the trafficking, localization, and abundance of presynaptic Ca2+ channels.

Voltage-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. Given this functional significance, defining the processes that cooperate to establish AZ VGCC abundance is critical for understanding how these mechanisms set synaptic strength and how they might be regulated to control presynaptic plasticity. VGCC abundance at AZs involves multiple steps, including channel biosynthesis (transcription, translation, and trafficking through the endomembrane system), forward axonal trafficking and delivery to synaptic terminals, incorporation and retention at presynaptic sites, and protein recycling. Here we discuss mechanisms that control VGCC abundance at synapses, highlighting findings from invertebrate and vertebrate models.

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

Synaptic vesicle (SV) 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 soluble N-ethylmaleimide sensitive factor attachment protein receptor (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 SV 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.

Nerve cells transmit messages in the form of electrical and chemical signals. Electrical impulses travel along a neuron to the junction between two neighbouring cells, the synapse. There, chemical messengers called neurotransmitters are released from one cell and detected by the next, which can either excite or inhibit the recipient cell. Synapses differ in their ability to propagate signals and their signalling activity also fluctuates at times. Moreover, synaptic connections can be strengthened or weakened in a process called plasticity, which is a key part of learning new skills and recovering from a brain injury. It is thought that synaptic signalling might be amped up or dialled down to change the output of the connection between two cells, but exactly how this happens remains unclear. To investigate why synapses differ and how their signalling capabilities change, Sauvola et al. examined the connections between neurons and muscle cells in developing fruit flies. In fruit fly larvae, two types of neurons ­ called tonic Ib and phasic Is neurons ­ form synapses with muscle cells. But their synapses have different signalling properties: Ib synapses are weaker than Is synapses. Sauvola et al. hypothesised that a protein called Tomosyn ­ which is thought to restrict chemical signalling at the synapse ­ might be more active at weaker Ib synapses. Sauvola et al. found that Tomosyn was indeed more abundant at Ib synapses than at Is synapses, appearing to reflect their differences in signalling properties. In flies engineered to lack the Tomosyn protein, Ib synapses became four times stronger than usual, while Is synapses hardly changed. This supports the idea that Tomosyn restricts the release of neurotransmitters at typically weak Ib synapses. Further experiments showed Ib synapses in flies lacking Tomosyn also lost their malleability and ability to become strengthened during synaptic plasticity. Though the precise molecular interactions need further investigation, the findings suggest that Tomosyn is required for some forms of synaptic plasticity by controlling how much chemical signal neurons release. In summary, this work advances our understanding of synaptic signalling and brain plasticity, showing once again how the brain can change itself.

Synaptic Plasticity: Close Encounters of the Tonic and Phasic Kind.

Neuronal circuits have the capacity to maintain relatively constant activity in the face of perturbations that alter excitability or synaptic properties. A new study demonstrates that different classes of neurons co-innervating the same postsynaptic target express homeostatic plasticity with unique presynaptic features.

Neurons regulate synaptic strength through homeostatic scaling of active zones.

How neurons stabilize their overall synaptic strength following conditions that alter synaptic morphology or function is a key question in neuronal homeostasis. In this issue, Goel et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201807165) find that neurons stabilize synaptic output despite disruptions in synapse size, active zone number, or postsynaptic function by controlling the delivery of active zone material and active zone size.

Characterization of developmental and molecular factors underlying release heterogeneity at Drosophila synapses.

Neurons communicate through neurotransmitter release at specialized synaptic regions known as active zones (AZs). Using biosensors to visualize single synaptic vesicle fusion events at Drosophila neuromuscular junctions, we analyzed the developmental and molecular determinants of release probability (Pr) for a defined connection with ~300 AZs. Pr was heterogeneous but represented a stable feature of each AZ. Pr remained stable during high frequency stimulation and retained heterogeneity in mutants lacking the Ca2+ sensor Synaptotagmin 1. Pr correlated with both presynaptic Ca2+ channel abundance and Ca2+ influx at individual release sites. Pr heterogeneity also correlated with glutamate receptor abundance, with high Pr connections developing receptor subtype segregation. Intravital imaging throughout development revealed that AZs acquire high Pr during a multi-day maturation period, with Pr heterogeneity largely reflecting AZ age. The rate of synapse maturation was activity-dependent, as both increases and decreases in neuronal activity modulated glutamate receptor field size and segregation.