Endogenous Tagging Reveals Differential Regulation of Ca2+ Channels at Single Active Zones during Presynaptic Homeostatic Potentiation and Depression

Abstract

Neurons communicate through Ca²⁺-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 Ca²⁺ channel levels are modulated to calibrate synaptic function. We used CRISPR to tag the Drosophila Ca_V2 Ca²⁺ channel Cacophony (Cac) and, in males in which all Cac channels are tagged, investigated the regulation of endogenous Ca²⁺ channels during homeostatic plasticity. 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, AZ Cac levels are increased during chronic and acute presynaptic homeostatic potentiation (PHP), and live imaging during acute expression of PHP reveals proportional Ca²⁺ channel accumulation across heterogeneous AZs. In contrast, endogenous Cac levels do not change during presynaptic homeostatic depression (PHD), implying that the reported reduction in Ca²⁺ influx during PHD is achieved through functional adaptions to pre-existing Ca²⁺ channels. Thus, distinct mechanisms bidirectionally modulate presynaptic Ca²⁺ levels to maintain stable synaptic strength in response to diverse challenges, with Ca²⁺ channel abundance providing a rapidly tunable substrate for potentiating neurotransmitter release over both acute and chronic timescales.SIGNIFICANCE STATEMENT Presynaptic Ca²⁺ dynamics play an important role in establishing neurotransmitter release properties. Presynaptic Ca²⁺ influx is modulated during multiple forms of homeostatic plasticity at Drosophila neuromuscular junctions to stabilize synaptic communication. However, it remains unclear how this dynamic regulation is achieved. We used CRISPR gene editing to endogenously tag the sole Drosophila Ca²⁺ channel responsible for synchronized neurotransmitter release, and found that channel abundance is regulated during homeostatic potentiation, but not homeostatic depression. Through live imaging experiments during the adaptation to acute homeostatic challenge, we visualize the accumulation of endogenous Ca²⁺ channels at individual active zones within 10 min. We propose that differential regulation of Ca²⁺ channels confers broad capacity for tuning neurotransmitter release properties to maintain neural communication.

Keywords: Drosophila; calcium channels; gene editing; homeostatic plasticity; neurotransmitter release; synapse.