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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.


Related in: MedlinePlus

Presynaptic Plc21C is required for homeostatic compensation. (A)T15-mediated RNAi knock down of Plc21C results in a failure to maintain the EPSP at control levels (***p < 0.001). Concerning homeostatic compensation, T15 × TRiP.JF01210 is the only cross to show increased quantal content compared to its GAL4 cross control (*p = 0.04), but even this quantal content was still significantly depressed compared to the T15 × WT control (*p < 0.05, One-Way ANOVA including all crosses in dataset, with Tukey's post-hoc). (B) Postsynaptic knock down of Plc21C does not affect homeostatic compensation: the EPSP is maintained (ns, p = 0.45) at control levels because the quantal content is upregulated (**p < 0.01). (C) Representative traces show normal neurotransmission upon postsynaptic knock down of GluRIII and Plc21C or a heterozygous loss of Plc21C via use of the deficiency chromosome Df(2L)BSC4. However, evoked release is not maintained upon T15-mediated knock down of Plc21C or the heterozygous loss of Plc21C in a GluRIIASP16 mutant background. (D) Evoked amplitudes (EPSPs) are unaltered upon heterozygous loss of Plc21C by use of the deficiency Df(2L)BSC4 or Plc21Cp60A allele though both do show a small, yet significant decrease in the amplitude of mEPSP events (***p < 0.001 and **p < 0.01, respectively). (E) Quantal content is minimally increased in the GluRIIASP16 background upon heterozygous loss of Plc21C compared to their respective genetic controls (*p < 0.05). For the Df(2L)BSC4 deficiency, the increased quantal content does not reach the level of increase found in GluRIIASP16 (***p < 0.001, One-Way ANOVA, Tukey's post-hoc). This is indicative of a partial impairment in homeostatic compensation. Scale bars for EPSPs (mEPSPs): 5 mV (1 mV); 50 ms (2000 ms).
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Figure 7: Presynaptic Plc21C is required for homeostatic compensation. (A)T15-mediated RNAi knock down of Plc21C results in a failure to maintain the EPSP at control levels (***p < 0.001). Concerning homeostatic compensation, T15 × TRiP.JF01210 is the only cross to show increased quantal content compared to its GAL4 cross control (*p = 0.04), but even this quantal content was still significantly depressed compared to the T15 × WT control (*p < 0.05, One-Way ANOVA including all crosses in dataset, with Tukey's post-hoc). (B) Postsynaptic knock down of Plc21C does not affect homeostatic compensation: the EPSP is maintained (ns, p = 0.45) at control levels because the quantal content is upregulated (**p < 0.01). (C) Representative traces show normal neurotransmission upon postsynaptic knock down of GluRIII and Plc21C or a heterozygous loss of Plc21C via use of the deficiency chromosome Df(2L)BSC4. However, evoked release is not maintained upon T15-mediated knock down of Plc21C or the heterozygous loss of Plc21C in a GluRIIASP16 mutant background. (D) Evoked amplitudes (EPSPs) are unaltered upon heterozygous loss of Plc21C by use of the deficiency Df(2L)BSC4 or Plc21Cp60A allele though both do show a small, yet significant decrease in the amplitude of mEPSP events (***p < 0.001 and **p < 0.01, respectively). (E) Quantal content is minimally increased in the GluRIIASP16 background upon heterozygous loss of Plc21C compared to their respective genetic controls (*p < 0.05). For the Df(2L)BSC4 deficiency, the increased quantal content does not reach the level of increase found in GluRIIASP16 (***p < 0.001, One-Way ANOVA, Tukey's post-hoc). This is indicative of a partial impairment in homeostatic compensation. Scale bars for EPSPs (mEPSPs): 5 mV (1 mV); 50 ms (2000 ms).

Mentions: When T15 is crossed to a UAS-Plc21C[RNAi] transgenic line (Dietzl et al., 2007), larval progeny have significantly lower NMJ quantal content than T15 × WT controls (Figures 4B,C, 7A). By contrast, when the muscle driver stock BG57-GAL4, UAS-GluRIII[RNAi] is crossed to the same UAS-Plc21C[RNAi] transgenic line, full homeostatic compensation occurs (Figures 7B,C). This latter result argues against Plc21C acting in the muscle to control synaptic homeostasis. A prior study demonstrated that presynaptic expression of the same UAS-Plc21C[RNAi] construct utilized in our screen (Plc21CGD11359) diminishes Plc21C mRNA levels by more than 50% (Dahdal et al., 2010). Moreover, Plc21C mRNA is found in the larval nervous system (Shortridge et al., 1991). Collectively, these data are consistent with a neuronal role for Plc21C in the homeostatic control of NMJ function.


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)

Presynaptic Plc21C is required for homeostatic compensation. (A)T15-mediated RNAi knock down of Plc21C results in a failure to maintain the EPSP at control levels (***p < 0.001). Concerning homeostatic compensation, T15 × TRiP.JF01210 is the only cross to show increased quantal content compared to its GAL4 cross control (*p = 0.04), but even this quantal content was still significantly depressed compared to the T15 × WT control (*p < 0.05, One-Way ANOVA including all crosses in dataset, with Tukey's post-hoc). (B) Postsynaptic knock down of Plc21C does not affect homeostatic compensation: the EPSP is maintained (ns, p = 0.45) at control levels because the quantal content is upregulated (**p < 0.01). (C) Representative traces show normal neurotransmission upon postsynaptic knock down of GluRIII and Plc21C or a heterozygous loss of Plc21C via use of the deficiency chromosome Df(2L)BSC4. However, evoked release is not maintained upon T15-mediated knock down of Plc21C or the heterozygous loss of Plc21C in a GluRIIASP16 mutant background. (D) Evoked amplitudes (EPSPs) are unaltered upon heterozygous loss of Plc21C by use of the deficiency Df(2L)BSC4 or Plc21Cp60A allele though both do show a small, yet significant decrease in the amplitude of mEPSP events (***p < 0.001 and **p < 0.01, respectively). (E) Quantal content is minimally increased in the GluRIIASP16 background upon heterozygous loss of Plc21C compared to their respective genetic controls (*p < 0.05). For the Df(2L)BSC4 deficiency, the increased quantal content does not reach the level of increase found in GluRIIASP16 (***p < 0.001, One-Way ANOVA, Tukey's post-hoc). This is indicative of a partial impairment in homeostatic compensation. Scale bars for EPSPs (mEPSPs): 5 mV (1 mV); 50 ms (2000 ms).
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Related In: Results  -  Collection

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Figure 7: Presynaptic Plc21C is required for homeostatic compensation. (A)T15-mediated RNAi knock down of Plc21C results in a failure to maintain the EPSP at control levels (***p < 0.001). Concerning homeostatic compensation, T15 × TRiP.JF01210 is the only cross to show increased quantal content compared to its GAL4 cross control (*p = 0.04), but even this quantal content was still significantly depressed compared to the T15 × WT control (*p < 0.05, One-Way ANOVA including all crosses in dataset, with Tukey's post-hoc). (B) Postsynaptic knock down of Plc21C does not affect homeostatic compensation: the EPSP is maintained (ns, p = 0.45) at control levels because the quantal content is upregulated (**p < 0.01). (C) Representative traces show normal neurotransmission upon postsynaptic knock down of GluRIII and Plc21C or a heterozygous loss of Plc21C via use of the deficiency chromosome Df(2L)BSC4. However, evoked release is not maintained upon T15-mediated knock down of Plc21C or the heterozygous loss of Plc21C in a GluRIIASP16 mutant background. (D) Evoked amplitudes (EPSPs) are unaltered upon heterozygous loss of Plc21C by use of the deficiency Df(2L)BSC4 or Plc21Cp60A allele though both do show a small, yet significant decrease in the amplitude of mEPSP events (***p < 0.001 and **p < 0.01, respectively). (E) Quantal content is minimally increased in the GluRIIASP16 background upon heterozygous loss of Plc21C compared to their respective genetic controls (*p < 0.05). For the Df(2L)BSC4 deficiency, the increased quantal content does not reach the level of increase found in GluRIIASP16 (***p < 0.001, One-Way ANOVA, Tukey's post-hoc). This is indicative of a partial impairment in homeostatic compensation. Scale bars for EPSPs (mEPSPs): 5 mV (1 mV); 50 ms (2000 ms).
Mentions: When T15 is crossed to a UAS-Plc21C[RNAi] transgenic line (Dietzl et al., 2007), larval progeny have significantly lower NMJ quantal content than T15 × WT controls (Figures 4B,C, 7A). By contrast, when the muscle driver stock BG57-GAL4, UAS-GluRIII[RNAi] is crossed to the same UAS-Plc21C[RNAi] transgenic line, full homeostatic compensation occurs (Figures 7B,C). This latter result argues against Plc21C acting in the muscle to control synaptic homeostasis. A prior study demonstrated that presynaptic expression of the same UAS-Plc21C[RNAi] construct utilized in our screen (Plc21CGD11359) diminishes Plc21C mRNA levels by more than 50% (Dahdal et al., 2010). Moreover, Plc21C mRNA is found in the larval nervous system (Shortridge et al., 1991). Collectively, these data are consistent with a neuronal role for Plc21C in the homeostatic control of NMJ function.

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.


Related in: MedlinePlus