Endogenous tagging reveals differential regulation of Ca²⁺channels at single AZs during presynaptic homeostatic potentiation and depression

Abstract

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.

Rapid and Sustained Homeostatic Control of Presynaptic Exocytosis at a Central Synapse

“…Consistently, we uncovered elevated Ca 2+ -current density in the experimental conditions with increased pr, implying increased levels of presynaptic voltage-gated Ca 2+ channels. This observation is in line with previous light-microscopy based results from mammalian CNS synapses and the Drosophila NMJ (23,(66)(67)(68). 5…”

“…In addition to RRP size, we provide evidence for homeostatic pr modulation 30 under specific experimental conditions. Homeostatic pr changes have been described for the Drosophila NMJ (65,66) and in mammalian CNS cell culture (20,27), suggesting evolutionary conservation. A major factor that has been linked to homeostatic pr control is the modulation of presynaptic Ca 2+ influx (24,65).…”

A presynaptic spectrin network controls active zone assembly and neurotransmitter release

“…Differences between tnc KO/Df and control (w 1118 ) were analyzed using the Students T-test. This study N/A N/A Cac sfGFP-N Kate M. O'Connor-Giles (Gratz et al, 2019) N/A N/A…”

“…Cac is the poreforming subunit of the only CaV2 Ca 2+ channel required for neurotransmitter release at Drosophila NMJ (Macleod et al, 2006;Peng and Wu, 2007;Smith et al, 1996). Using an endogenously tagged cac allele, cac sfGFP (Gratz et al, 2019), we found that Ca 2+ channel levels were dramatically reduced at tnc NMJs (to 41.3  6.1% compared to control, p<0.0001) ( Figure 1E-G). Knockdown of neuronal Tnc similarly reduced the synaptic Cac sfGFP levels to 21.5  8.1% compared to the UAS-tnc RNAi control ( Figure 1H-J).…”

“…Other fly stocks were used in this study were: UAS-tnc, UAS-Dcr-2,UAS-tnc RNAi , UASmys RNAi , UAS-if RNAi , Df(3R)BSC655 (Wang et al, 2018); α-Spec rg41 (BSC 31999); UAS-Myc::α-Spec (from Claire H. Thomas ); Cac sfGFP-N (from Kate M. O'Connor-Giles) (Gratz et al, 2019). The BG380-Gal4, 24B-Gal4, elav-Gal4, and G14-Gal4 drivers were previously described.…”