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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.


Knock down of cacophony gene function does not impair synaptic homeostasis. (A,B) mEPSP (black), QC (gray), NLS QC (white). (#p = 0.05; *p < 0.05; **p < 0.01; ***p < 0.001, T-test compared to control at 100% dotted line) (A) Diminished baseline neurotransmission for GAL4 control × UAS-cac[RNAi] larvae is consistent with cac gene knock down. Diminished release occurs across a range of extracellular [Ca2+]. (B)T15 × UAS-cac[RNAi] larvae show robust homeostatic compensation compared to GAL4 control × UAS-cac[RNAi] larvae. As expected, the T15 line induces a marked diminishment of quantal size (mEPSP). The NMJ responds with a robust increase in release. This response is observed across a range of extracellular calcium concentrations, and the same result holds whether or not QC is corrected for non-linear summation. (C) Representative electrophysiological traces. Scale bars for EPSPs (mEPSPs): 5 mV (1 mV); 50 ms (1000 ms).
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Figure 5: Knock down of cacophony gene function does not impair synaptic homeostasis. (A,B) mEPSP (black), QC (gray), NLS QC (white). (#p = 0.05; *p < 0.05; **p < 0.01; ***p < 0.001, T-test compared to control at 100% dotted line) (A) Diminished baseline neurotransmission for GAL4 control × UAS-cac[RNAi] larvae is consistent with cac gene knock down. Diminished release occurs across a range of extracellular [Ca2+]. (B)T15 × UAS-cac[RNAi] larvae show robust homeostatic compensation compared to GAL4 control × UAS-cac[RNAi] larvae. As expected, the T15 line induces a marked diminishment of quantal size (mEPSP). The NMJ responds with a robust increase in release. This response is observed across a range of extracellular calcium concentrations, and the same result holds whether or not QC is corrected for non-linear summation. (C) Representative electrophysiological traces. Scale bars for EPSPs (mEPSPs): 5 mV (1 mV); 50 ms (1000 ms).

Mentions: We crossed either the T15 stock or the aforementioned pre- and post-synaptic GAL4 driver control stock (Figure 2) to UAS-cac[RNAi]. For GAL4 Cont × UAS-cac[RNAi] progeny, NMJ evoked excitation and quantal content are markedly decreased compared to GAL4 Cont × WT controls (Figure 5A). This is true across a range of extracellular Ca2+ concentrations, though perhaps a bit less pronounced at physiological [Ca2+] (1.5 mM) (Figure 5A). As expected, for T15 × UAS-cac[RNAi] larval progeny, there is a significant decrease in NMJ quantal size (mEPSP) compared to GAL4 Cont × UAS-cac[RNAi] larval progeny (Figure 5B). However, T15 × UAS-cac[RNAi] NMJ EPSP amplitude is no different than control cross UAS-cac[RNAi] progeny (Figure 5C). This is due to a homeostatic enhancement in presynaptic quantal content (Figure 5B). This result holds for extracellular Ca2+ concentrations that permit a range of presynaptic release: 0.5, 1.0, and 1.5 mM. For each condition, homeostatic compensation of presynaptic release remains robust (Figure 5B).


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)

Knock down of cacophony gene function does not impair synaptic homeostasis. (A,B) mEPSP (black), QC (gray), NLS QC (white). (#p = 0.05; *p < 0.05; **p < 0.01; ***p < 0.001, T-test compared to control at 100% dotted line) (A) Diminished baseline neurotransmission for GAL4 control × UAS-cac[RNAi] larvae is consistent with cac gene knock down. Diminished release occurs across a range of extracellular [Ca2+]. (B)T15 × UAS-cac[RNAi] larvae show robust homeostatic compensation compared to GAL4 control × UAS-cac[RNAi] larvae. As expected, the T15 line induces a marked diminishment of quantal size (mEPSP). The NMJ responds with a robust increase in release. This response is observed across a range of extracellular calcium concentrations, and the same result holds whether or not QC is corrected for non-linear summation. (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
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Figure 5: Knock down of cacophony gene function does not impair synaptic homeostasis. (A,B) mEPSP (black), QC (gray), NLS QC (white). (#p = 0.05; *p < 0.05; **p < 0.01; ***p < 0.001, T-test compared to control at 100% dotted line) (A) Diminished baseline neurotransmission for GAL4 control × UAS-cac[RNAi] larvae is consistent with cac gene knock down. Diminished release occurs across a range of extracellular [Ca2+]. (B)T15 × UAS-cac[RNAi] larvae show robust homeostatic compensation compared to GAL4 control × UAS-cac[RNAi] larvae. As expected, the T15 line induces a marked diminishment of quantal size (mEPSP). The NMJ responds with a robust increase in release. This response is observed across a range of extracellular calcium concentrations, and the same result holds whether or not QC is corrected for non-linear summation. (C) Representative electrophysiological traces. Scale bars for EPSPs (mEPSPs): 5 mV (1 mV); 50 ms (1000 ms).
Mentions: We crossed either the T15 stock or the aforementioned pre- and post-synaptic GAL4 driver control stock (Figure 2) to UAS-cac[RNAi]. For GAL4 Cont × UAS-cac[RNAi] progeny, NMJ evoked excitation and quantal content are markedly decreased compared to GAL4 Cont × WT controls (Figure 5A). This is true across a range of extracellular Ca2+ concentrations, though perhaps a bit less pronounced at physiological [Ca2+] (1.5 mM) (Figure 5A). As expected, for T15 × UAS-cac[RNAi] larval progeny, there is a significant decrease in NMJ quantal size (mEPSP) compared to GAL4 Cont × UAS-cac[RNAi] larval progeny (Figure 5B). However, T15 × UAS-cac[RNAi] NMJ EPSP amplitude is no different than control cross UAS-cac[RNAi] progeny (Figure 5C). This is due to a homeostatic enhancement in presynaptic quantal content (Figure 5B). This result holds for extracellular Ca2+ concentrations that permit a range of presynaptic release: 0.5, 1.0, and 1.5 mM. For each condition, homeostatic compensation of presynaptic release remains robust (Figure 5B).

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.