Endogenous tagging reveals differential regulation of Ca²⁺ channels at single AZs 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 CaV2 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.

Homeostatic control of Drosophila neuromuscular junction function

“…New work has reported that endogenous synaptic protein levels of the active zone protein Bruchpilot (Brp, an ELKS/CAST homolog, (Kittel et al, 2006)) and Ca V 2/Cacophony do not change upon PHD induction (Gratz et al, 2019). These data are consistent with a model in which the deceased release associated with PHD is executed-at least in part-through functional modulations of existing active zone components.…”

“…Several labs confirmed increased Brp abundance during rapid or sustained PHP expression Goel, Li, & Dickman, 2017;Gratz et al, 2019). Intriguingly, acute and prolonged glutamate receptor perturbation also increases the fluorescence intensity of fluorescently-tagged presynaptic voltage-gated Ca 2+ channels ( Figure 4; Gratz et al, 2019;Li, Goel, Wondolowski, et al, 2018). Moreover, the fluorescence intensity of antibody stainings of several other presynaptic proteins-RBP, Unc13A, and Syntaxin-1A (Syx-1A)-increased after acute glutamate receptor inhibition .…”

“…Using STED and confocal microscopy, Weyhersmüller and colleagues (Weyhersmüller et al, 2011) provided evidence that the abundance of the presynaptic active zone (AZ) protein bruchpilot (Brp) is slightly increased after acute or chronic glutamate receptor perturbation (Figure 4a,b). Several labs confirmed increased Brp abundance during rapid or sustained PHP expression Goel, Li, & Dickman, 2017;Gratz et al, 2019). Intriguingly, acute and prolonged glutamate receptor perturbation also increases the fluorescence intensity of fluorescently-tagged presynaptic voltage-gated Ca 2+ channels ( Figure 4; Gratz et al, 2019;Li, Goel, Wondolowski, et al, 2018).…”

“…A recent study demonstrated that the vGlut overexpression-induced decrease in release during PHD correlates with reduced levels of a transgenically expressed GFP-tagged Ca V 2 Ca 2+ channel subunit (Cacophony-GFP) and decreased AP-induced presynaptic Ca 2+ influx (Gaviño, Ford, Archila, & Davis, 2015). As PHP requires enhanced presynaptic Ca 2+ influx (Frank et al, 2006;) that is correlated with increased levels of voltage-gated Ca 2+ channels (Gratz et al, 2019;Li, Goel, Wondolowski, Paluch, & Dickman, 2018), these data suggest that PHD could be implemented in an opposite type of mechanism as PHP. However, the same study (Gaviño et al, 2015), as well as another recent study (Li, Goel, Wondolowski, et al, 2018), uncovered that genes that are required for PHP are dispensable for PHD, implying-at least in part-different molecular pathways.…”

“…Unc18 or GFP-tagged Cacophony (Gratz et al, 2019;Li, Goel, Chen, et al, 2018), implying a sustained increase in the levels of these proteins during PHP. In this case, three of the above-mentioned genes that have been tested for PHP in the GluRIIA SP16 mutant background-cacophony (Frank et al, 2006), Brp Penney et al, 2012), and Srpk79D ( (2019), Arl8-dependent structural plasticity was dispensable for rapid PHP expression, but required for sustained PHP expression.…”