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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.


Postsynaptic GluRIII gene knock down induces robust homeostatic compensation. (A) Crossing scheme. NMJs from F1 larvae (genotype BG57-GAL4/UAS-GluRIII[RNAi]) are subjected to electrophysiological analyses. (B) Quantal size (miniature excitatory postsynaptic potentials, mEPSP) is decreased for BG57-GAL4/UAS-GluRIII[RNAi] larvae (***p < 0.001, Student's T-Test). Evoked potentials (excitatory postsynaptic potentials, EPSP) are normal because of a homeostatic increase in quantal content (QC) (**p < 0.01). (C) Representative electrophysiological traces. Scale bars for EPSPs (mEPSPs): 5 mV (1 mV); 50 ms (1000 ms).
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Figure 1: Postsynaptic GluRIII gene knock down induces robust homeostatic compensation. (A) Crossing scheme. NMJs from F1 larvae (genotype BG57-GAL4/UAS-GluRIII[RNAi]) are subjected to electrophysiological analyses. (B) Quantal size (miniature excitatory postsynaptic potentials, mEPSP) is decreased for BG57-GAL4/UAS-GluRIII[RNAi] larvae (***p < 0.001, Student's T-Test). Evoked potentials (excitatory postsynaptic potentials, EPSP) are normal because of a homeostatic increase in quantal content (QC) (**p < 0.01). (C) Representative electrophysiological traces. Scale bars for EPSPs (mEPSPs): 5 mV (1 mV); 50 ms (1000 ms).

Mentions: Partial knock down of GluRIII gene function should induce a significant decrease in NMJ quantal size and a homeostatic increase in NMJ quantal content (Marrus et al., 2004). Using the Drosophila pWiz vector (Lee and Carthew, 2003), we created a transgene to target the GluRIII gene for functional knock down by RNAi (see Materials and Methods). From a pWiz-UAS-GluRIII[RNAi] clone, we generated UAS-GluRIII[RNAi] transgenic Drosophila stocks and crossed them to stocks harboring the muscle-specific GAL4 driver, BG57-GAL4 (Budnik et al., 1996) (Figure 1A). We then analyzed the larval cross progeny by electrophysiology to assess the efficiency of glutamate receptor subunit knock down.


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)

Postsynaptic GluRIII gene knock down induces robust homeostatic compensation. (A) Crossing scheme. NMJs from F1 larvae (genotype BG57-GAL4/UAS-GluRIII[RNAi]) are subjected to electrophysiological analyses. (B) Quantal size (miniature excitatory postsynaptic potentials, mEPSP) is decreased for BG57-GAL4/UAS-GluRIII[RNAi] larvae (***p < 0.001, Student's T-Test). Evoked potentials (excitatory postsynaptic potentials, EPSP) are normal because of a homeostatic increase in quantal content (QC) (**p < 0.01). (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 1: Postsynaptic GluRIII gene knock down induces robust homeostatic compensation. (A) Crossing scheme. NMJs from F1 larvae (genotype BG57-GAL4/UAS-GluRIII[RNAi]) are subjected to electrophysiological analyses. (B) Quantal size (miniature excitatory postsynaptic potentials, mEPSP) is decreased for BG57-GAL4/UAS-GluRIII[RNAi] larvae (***p < 0.001, Student's T-Test). Evoked potentials (excitatory postsynaptic potentials, EPSP) are normal because of a homeostatic increase in quantal content (QC) (**p < 0.01). (C) Representative electrophysiological traces. Scale bars for EPSPs (mEPSPs): 5 mV (1 mV); 50 ms (1000 ms).
Mentions: Partial knock down of GluRIII gene function should induce a significant decrease in NMJ quantal size and a homeostatic increase in NMJ quantal content (Marrus et al., 2004). Using the Drosophila pWiz vector (Lee and Carthew, 2003), we created a transgene to target the GluRIII gene for functional knock down by RNAi (see Materials and Methods). From a pWiz-UAS-GluRIII[RNAi] clone, we generated UAS-GluRIII[RNAi] transgenic Drosophila stocks and crossed them to stocks harboring the muscle-specific GAL4 driver, BG57-GAL4 (Budnik et al., 1996) (Figure 1A). We then analyzed the larval cross progeny by electrophysiology to assess the efficiency of glutamate receptor subunit knock down.

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.