A single-cross, RNA interference-based genetic tool for examining the long-term maintenance of homeostatic plasticity

Abstract

Citation:Brusich DJ, Spring AM and Frank CA (2015) A single-cross, RNA interference-based genetic tool for examining the long-term maintenance of homeostatic plasticity. Front. Cell. Neurosci. 9:107. doi: 10.3389/fncel.2015.00107 A single-cross, RNA interference-based genetic tool for examining the long-term maintenance of homeostatic plasticity Homeostatic synaptic plasticity (HSP) helps neurons and synapses maintain physiologically appropriate levels of output. The fruit fly Drosophila melanogaster larval neuromuscular junction (NMJ) is a valuable model for studying HSP. Here we introduce a genetic tool that allows fruit fly researchers to examine the lifelong maintenance of HSP with a single cross. The tool is a fruit fly stock that combines the GAL4/UAS expression system with RNA interference (RNAi)-based knock down of a glutamate receptor subunit gene. With this stock, we uncover important new information about the maintenance of HSP. We address an open question about the role that presynaptic Ca V 2-type Ca 2+ channels play in NMJ homeostasis. Published experiments have demonstrated that hypomorphic missense mutations in the Ca V 2 α1a subunit gene cacophony (cac) can impair homeostatic plasticity at the NMJ. Here we report that reducing cac expression levels by RNAi is not sufficient to impair homeostatic plasticity. The presence of wild-type channels appears to support HSP-even when total Ca V 2 function is severely reduced. We also conduct an RNAi-and electrophysiology-based screen to identify new factors required for sustained homeostatic signaling throughout development. We uncover novel roles in HSP for Drosophila homologs of Cysteine string protein (CSP) and Phospholipase Cβ (Plc21C). We characterize those roles through follow-up genetic tests. We discuss how CSP, Plc21C, and associated factors could modulate presynaptic Ca V 2 function, presynaptic Ca 2+ handling, or other signaling processes crucial for sustained homeostatic regulation of NMJ function throughout development. Our findings expand the scope of signaling pathways and processes that contribute to the durable strength of the NMJ.

α2δ-3 Is Required for Rapid Transsynaptic Homeostatic Signaling

“…EPSP amplitudes are decreased by ~30% in α2δ-3 k10814 /α2δ-3 106 and by ~50% in stj 1 /stj 2 (Figure S2), but homeostatic plasticity is completely blocked in both instances (Figure 1 and S2). Second, although missense mutations in the cac gene have been shown to block presynaptic homeostasis (Frank et al, 2009), RNAi-mediated depletion of the α1 subunit from the presynaptic terminal, sufficient to cause an ~80% reduction in EPSP amplitude compared to controls, did not impair presynaptic homeostasis (Brusich et al, 2015). An independent study made a similar observation, showing that reduced Ca V 2.1 channel abundance and a correlated ~50% decrease in single action potential (AP) induced calcium influx, did not impair the rapid induction of presynaptic homeostasis (Gavino et al, 2015).…”

“…Although we cannot rule out an action of α2δ-3 mutants on calcium current kinetics at the presynaptic terminal, our data strongly suggest that the primary effect of the α2δ-3 106 / α2δ-3 k10814 mutant is to reduce calcium current density. However, since reduced calcium influx alone does not disrupt presynaptic homeostatic plasticity (see above; (Brusich et al, 2015; Gavino et al, 2015)), there must be additional activities of α2δ-3 , in addition to its role in controlling Ca V 2.1 channel abundance, that are necessary for PHP.…”

“…However, not all evidence is consistent with this possibility. For example, RNAi-mediated depletion of Ca V 2.1 channels, sufficient to decrease release by ~80% does not prevent presynaptic homeostasis (Brusich et al, 2015). Thus, loss of α2δ-3 blocks PHP whereas loss of Ca V 2.1 α1 subunit does not.…”

Composition and Control of a Deg/ENaC Channel during Presynaptic Homeostatic Plasticity

“…Core molecular components of the signaling systems that achieve PHP have begun to emerge through the results of large-scale forward genetic screens at the Drosophila NMJ (Brusich et al, 2015; Dickman and Davis, 2009; Muller et al, 2012; Müller et al, 2015; Wang et al, 2014; Younger et al, 2013) and additional hypothesis-driven studies (Penney et al, 2012). A core finding has been the demonstration that an amiloride-sensitive Deg/ENaC channel functions presynaptically to drive the induction and sustained expression of PHP (Younger et al, 2013).…”

Homeostatic control of Drosophila neuromuscular junction function

“…The possibility of acute pharmacological induction and rapid expression of presynaptic homeostatic plasticity in the genetic model organism Drosophila (Frank et al, 2006) opened the door for genetic screens that are based on electrophysiological analysis of synaptic transmission (Brusich, Spring, & Frank, 2015;Dickman & Davis, 2009;Hauswirth et al, 2018;Kikuma et al, 2019;Müller, Pym, Tong, & Davis, 2011). At this point, we are able to take a retrospective view of these screens and the resulting characterized molecules.…”