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

Brusich DJ, Spring AM, Frank CA - Front Cell Neurosci (2015)

Bottom Line: The presence of wild-type channels appears to support HSP-even when total CaV2 function is severely reduced.We discuss how CSP, Plc21C, and associated factors could modulate presynaptic CaV2 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa Iowa City, IA, USA.

ABSTRACT
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 CaV2-type Ca(2+) channels play in NMJ homeostasis. Published experiments have demonstrated that hypomorphic missense mutations in the CaV2 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 CaV2 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 CaV2 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.

No MeSH data available.


The T15 stock induces robust homeostatic compensation. (A)T15 × WT crossing scheme. The genotype for T15 is (chromosomes X; II; III): elaV(C155)-GAL4; Scabrous-GAL4; BG57-GAL4, UAS-GluRIII[RNAi]. (B)T15 × WT larval NMJs have decreased quantal size (***p < 0.001, T-Test vs. WT). Evoked potentials are normal because of a homeostatic increase in QC (***p < 0.001). A control stock with only GAL4 drivers behaves similarly to WT. (C) Representative electrophysiological traces. Scale bars for EPSPs (mEPSPs): 5 mV (1 mV); 50 ms (1000 ms).
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Figure 2: The T15 stock induces robust homeostatic compensation. (A)T15 × WT crossing scheme. The genotype for T15 is (chromosomes X; II; III): elaV(C155)-GAL4; Scabrous-GAL4; BG57-GAL4, UAS-GluRIII[RNAi]. (B)T15 × WT larval NMJs have decreased quantal size (***p < 0.001, T-Test vs. WT). Evoked potentials are normal because of a homeostatic increase in QC (***p < 0.001). A control stock with only GAL4 drivers behaves similarly to WT. (C) Representative electrophysiological traces. Scale bars for EPSPs (mEPSPs): 5 mV (1 mV); 50 ms (1000 ms).

Mentions: When T15 females are crossed to wild-type males (herein T15 × WT, Figure 2A), the larval progeny exhibit starkly diminished NMJ quantal size and frequency, and a homeostatic increase in NMJ quantal content (Figures 2B,C). By contrast, when GAL4 driver control females are crossed to wild-type males (herein GAL4 Cont × WT), the NMJ electrophysiological profile of larval progeny is largely similar to that of wild-type NMJs (Figures 2B,C). The GAL4 drivers or genetic background may induce a slight increase in quantal size (Figure 2B). However, this increase is not statistically significant, and the data show that the presence of presynaptic GAL4 drivers exerts no adverse effects on evoked NMJ excitation (Figures 2B,C).


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

Brusich DJ, Spring AM, Frank CA - Front Cell Neurosci (2015)

The T15 stock induces robust homeostatic compensation. (A)T15 × WT crossing scheme. The genotype for T15 is (chromosomes X; II; III): elaV(C155)-GAL4; Scabrous-GAL4; BG57-GAL4, UAS-GluRIII[RNAi]. (B)T15 × WT larval NMJs have decreased quantal size (***p < 0.001, T-Test vs. WT). Evoked potentials are normal because of a homeostatic increase in QC (***p < 0.001). A control stock with only GAL4 drivers behaves similarly to WT. (C) Representative electrophysiological traces. Scale bars for EPSPs (mEPSPs): 5 mV (1 mV); 50 ms (1000 ms).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4374470&req=5

Figure 2: The T15 stock induces robust homeostatic compensation. (A)T15 × WT crossing scheme. The genotype for T15 is (chromosomes X; II; III): elaV(C155)-GAL4; Scabrous-GAL4; BG57-GAL4, UAS-GluRIII[RNAi]. (B)T15 × WT larval NMJs have decreased quantal size (***p < 0.001, T-Test vs. WT). Evoked potentials are normal because of a homeostatic increase in QC (***p < 0.001). A control stock with only GAL4 drivers behaves similarly to WT. (C) Representative electrophysiological traces. Scale bars for EPSPs (mEPSPs): 5 mV (1 mV); 50 ms (1000 ms).
Mentions: When T15 females are crossed to wild-type males (herein T15 × WT, Figure 2A), the larval progeny exhibit starkly diminished NMJ quantal size and frequency, and a homeostatic increase in NMJ quantal content (Figures 2B,C). By contrast, when GAL4 driver control females are crossed to wild-type males (herein GAL4 Cont × WT), the NMJ electrophysiological profile of larval progeny is largely similar to that of wild-type NMJs (Figures 2B,C). The GAL4 drivers or genetic background may induce a slight increase in quantal size (Figure 2B). However, this increase is not statistically significant, and the data show that the presence of presynaptic GAL4 drivers exerts no adverse effects on evoked NMJ excitation (Figures 2B,C).

Bottom Line: The presence of wild-type channels appears to support HSP-even when total CaV2 function is severely reduced.We discuss how CSP, Plc21C, and associated factors could modulate presynaptic CaV2 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa Iowa City, IA, USA.

ABSTRACT
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 CaV2-type Ca(2+) channels play in NMJ homeostasis. Published experiments have demonstrated that hypomorphic missense mutations in the CaV2 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 CaV2 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 CaV2 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.

No MeSH data available.