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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.


Csp is required for long-term homeostatic compensation. (A)T15 × UAS-Csp[RNAi] shows a failure to upregulate quantal content compared to its GAL4-driven UAS-Csp[RNAi] control (ns, p = 0.15). Knock down of Csp shows a slight impairment in evoked neurotransmission (EPSP) compared to control (**p < 0.01). (B) Representative electrophysiological traces show the failure of T15 × UAS-Csp[RNAi] larvae to maintain evoked potentials at control levels. (C) Homozygosity for the CspDG29203 allele or heterozygosity for the CspEY22488 allele block homeostatic upregulation of quantal content compared to their respective non-GluRIIASP16genetic controls (ns, p = 0.44 and p = 0.14, respectively). (D) Representative traces show a failure of homeostatic compensation for GluRIIASP16, CspDG29203. (E) Postsynaptic knock down of Csp function leaves homeostatic plasticity intact. (F) Acute homeostatic compensation is intact in CspDG29203 as evidenced by the elevated quantal content in response to philanthotoxin-433 (PhTox) application (**p < 0.01). (G) A doubly heterozygous combination of CspDG29203/+ and cacS/+ shows a homeostatic block in the GluRIIASP16 background because of a failure to increase quantal content over cacS/+; CspDG29203/+ controls (ns, p = 0.24). By contrast, the single heterozygous mutations retain partial homeostatic compensatory capacity. Scale bars for EPSPs (mEPSPs): 5 mV (1 mV); 50 ms (2000 ms). (ns, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001).
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Figure 6: Csp is required for long-term homeostatic compensation. (A)T15 × UAS-Csp[RNAi] shows a failure to upregulate quantal content compared to its GAL4-driven UAS-Csp[RNAi] control (ns, p = 0.15). Knock down of Csp shows a slight impairment in evoked neurotransmission (EPSP) compared to control (**p < 0.01). (B) Representative electrophysiological traces show the failure of T15 × UAS-Csp[RNAi] larvae to maintain evoked potentials at control levels. (C) Homozygosity for the CspDG29203 allele or heterozygosity for the CspEY22488 allele block homeostatic upregulation of quantal content compared to their respective non-GluRIIASP16genetic controls (ns, p = 0.44 and p = 0.14, respectively). (D) Representative traces show a failure of homeostatic compensation for GluRIIASP16, CspDG29203. (E) Postsynaptic knock down of Csp function leaves homeostatic plasticity intact. (F) Acute homeostatic compensation is intact in CspDG29203 as evidenced by the elevated quantal content in response to philanthotoxin-433 (PhTox) application (**p < 0.01). (G) A doubly heterozygous combination of CspDG29203/+ and cacS/+ shows a homeostatic block in the GluRIIASP16 background because of a failure to increase quantal content over cacS/+; CspDG29203/+ controls (ns, p = 0.24). By contrast, the single heterozygous mutations retain partial homeostatic compensatory capacity. Scale bars for EPSPs (mEPSPs): 5 mV (1 mV); 50 ms (2000 ms). (ns, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001).

Mentions: Our screen found no elevation of NMJ quantal content when comparing T15 × UAS-Csp[RNAi] larval progeny vs. wild-type larvae (Figure 4). This result indicates a possible role for CSP in homeostatic plasticity, although it could also be consistent with expected Csp defects in neurotransmission. In a follow-up test, we found that T15 × UAS-Csp[RNAi] NMJ quantal content is not significantly different than GAL4 driver control × UAS-Csp[RNAi] NMJ quantal content (Figure 6A). This result suggests a bona fide defect in synaptic homeostasis (Figures 6A,B).


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)

Csp is required for long-term homeostatic compensation. (A)T15 × UAS-Csp[RNAi] shows a failure to upregulate quantal content compared to its GAL4-driven UAS-Csp[RNAi] control (ns, p = 0.15). Knock down of Csp shows a slight impairment in evoked neurotransmission (EPSP) compared to control (**p < 0.01). (B) Representative electrophysiological traces show the failure of T15 × UAS-Csp[RNAi] larvae to maintain evoked potentials at control levels. (C) Homozygosity for the CspDG29203 allele or heterozygosity for the CspEY22488 allele block homeostatic upregulation of quantal content compared to their respective non-GluRIIASP16genetic controls (ns, p = 0.44 and p = 0.14, respectively). (D) Representative traces show a failure of homeostatic compensation for GluRIIASP16, CspDG29203. (E) Postsynaptic knock down of Csp function leaves homeostatic plasticity intact. (F) Acute homeostatic compensation is intact in CspDG29203 as evidenced by the elevated quantal content in response to philanthotoxin-433 (PhTox) application (**p < 0.01). (G) A doubly heterozygous combination of CspDG29203/+ and cacS/+ shows a homeostatic block in the GluRIIASP16 background because of a failure to increase quantal content over cacS/+; CspDG29203/+ controls (ns, p = 0.24). By contrast, the single heterozygous mutations retain partial homeostatic compensatory capacity. Scale bars for EPSPs (mEPSPs): 5 mV (1 mV); 50 ms (2000 ms). (ns, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001).
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Figure 6: Csp is required for long-term homeostatic compensation. (A)T15 × UAS-Csp[RNAi] shows a failure to upregulate quantal content compared to its GAL4-driven UAS-Csp[RNAi] control (ns, p = 0.15). Knock down of Csp shows a slight impairment in evoked neurotransmission (EPSP) compared to control (**p < 0.01). (B) Representative electrophysiological traces show the failure of T15 × UAS-Csp[RNAi] larvae to maintain evoked potentials at control levels. (C) Homozygosity for the CspDG29203 allele or heterozygosity for the CspEY22488 allele block homeostatic upregulation of quantal content compared to their respective non-GluRIIASP16genetic controls (ns, p = 0.44 and p = 0.14, respectively). (D) Representative traces show a failure of homeostatic compensation for GluRIIASP16, CspDG29203. (E) Postsynaptic knock down of Csp function leaves homeostatic plasticity intact. (F) Acute homeostatic compensation is intact in CspDG29203 as evidenced by the elevated quantal content in response to philanthotoxin-433 (PhTox) application (**p < 0.01). (G) A doubly heterozygous combination of CspDG29203/+ and cacS/+ shows a homeostatic block in the GluRIIASP16 background because of a failure to increase quantal content over cacS/+; CspDG29203/+ controls (ns, p = 0.24). By contrast, the single heterozygous mutations retain partial homeostatic compensatory capacity. Scale bars for EPSPs (mEPSPs): 5 mV (1 mV); 50 ms (2000 ms). (ns, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001).
Mentions: Our screen found no elevation of NMJ quantal content when comparing T15 × UAS-Csp[RNAi] larval progeny vs. wild-type larvae (Figure 4). This result indicates a possible role for CSP in homeostatic plasticity, although it could also be consistent with expected Csp defects in neurotransmission. In a follow-up test, we found that T15 × UAS-Csp[RNAi] NMJ quantal content is not significantly different than GAL4 driver control × UAS-Csp[RNAi] NMJ quantal content (Figure 6A). This result suggests a bona fide defect in synaptic homeostasis (Figures 6A,B).

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.