No abstract

SUMMARY Retrograde signaling systems are fundamental modes of communication that synapses utilize to dynamically and adaptively modulate activity. However, the inductive mechanisms that gate retrograde communication in the postsynaptic compartment remain enigmatic. We have investigated retrograde signaling at the Drosophila neuromuscular junction, where three seemingly disparate perturbations to the postsynaptic cell trigger a similar enhancement in presynaptic neurotransmitter release. We show that the same presynaptic genetic machinery and enhancements in active zone structure are utilized by each inductive pathway. However, all three induction mechanisms differ in temporal, translational, and CamKII activity requirements to initiate retrograde signaling in the postsynaptic cell. Intriguingly, pharmacological blockade of postsynaptic glutamate receptors, and not calcium influx through these receptors, is necessary and sufficient to induce rapid retrograde homeostatic signaling through CamKII. Thus, three distinct induction mechanisms converge on the same retrograde signaling system to drive the homeostatic strengthening of presynaptic neurotransmitter release.

Neuronal communication across synapses relies on neurotransmitter release from presynaptic active zones (AZs) followed by postsynaptic transmitter detection. Synaptic plasticity homeostatically maintains functionality during perturbations and enables memory formation. Postsynaptic plasticity targets neurotransmitter receptors, but presynaptic mechanisms regulating the neurotransmitter release apparatus remain largely enigmatic. By studying Drosophila neuromuscular junctions (NMJs) we show that AZs consist of nano-modular release sites and identify a molecular sequence that adds modules within minutes of inducing homeostatic plasticity. This requires cognate transport machinery and specific AZ-scaffolding proteins. Structural remodeling is not required for immediate potentiation of neurotransmitter release, but necessary to sustain potentiation over longer timescales. Finally, mutations in Unc13 disrupting homeostatic plasticity at the NMJ also impair short-term memory when central neurons are targeted, suggesting that both plasticity mechanisms utilize Unc13. Together, while immediate synaptic potentiation capitalizes on available material, it triggers the coincident incorporation of modular release sites to consolidate synaptic potentiation.

Summary We have interrogated the synaptic dialog that enables the bi-directional, homeostatic control of pre-synaptic efficacy at the glutamatergic Drosophila neuromuscular junction (NMJ). We find that homeo-static depression and potentiation use disparate genetic, induction, and expression mechanisms. Specifically, homeostatic potentiation is achieved through reduced CaMKII activity postsynaptically and increased abundance of active zone material presynaptically at one of the two neuronal subtypes innervating the NMJ, while homeostatic depression occurs without alterations in CaMKII activity and is expressed at both neuronal subtypes. Furthermore, homeostatic depression is only induced through excess presynaptic glutamate release and operates with disregard to the postsynaptic response. We propose that two independent homeostats modulate presynaptic efficacy at the Drosophila NMJ: one is an intercellular signaling system that potentiates synaptic strength following diminished postsynaptic excitability, while the other adaptively modulates presynaptic glutamate release through an autocrine mechanism without feedback from the postsynaptic compartment.

Neurons communicate through Ca 2+ -dependent neurotransmitter release at presynaptic active zones (AZs). Neurotransmitter release properties play a key role in defining information flow in circuits and are tuned during multiple forms of plasticity. Despite their central role in determining neurotransmitter release properties, little is known about how Ca 2+ channel levels are modulated to calibrate synaptic function. We used CRISPR to tag the Drosophila CaV2 Ca 2+ channel Cacophony (Cac) and investigated the regulation of endogenous Ca 2+ channels during homeostatic plasticity in males in which all endogenous Cac channels are tagged. We found that heterogeneously distributed Cac is highly predictive of neurotransmitter release probability at individual AZs and differentially regulated during opposing forms of presynaptic homeostatic plasticity. Specifically, Cac levels at AZ are increased during chronic and acute presynaptic homeostatic potentiation (PHP), and live imaging during acute expression of PHP reveals proportional Ca 2+ channel accumulation across heterogeneous AZs. In contrast, endogenous Cac levels do not change during presynaptic homeostatic depression (PHD), implying that the reported reduction in Ca 2+ influx during PHD is achieved through functional adaptions to pre-existing Ca 2+ channels. Thus, distinct mechanisms bi-directionally modulate presynaptic Ca 2+ levels to maintain stable synaptic strength in response to diverse challenges, with Ca 2+ channel abundance providing a rapidly tunable substrate for potentiating neurotransmitter release over both acute and chronic timescales.