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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

An RNAi- and electrophysiology-based screen for homeostatic factors. (A) Crossing scheme for screen T15 × UAS-yfg[RNAi] (“your favorite gene”). For UAS-RNAi lines on chromosomes II or III, male progeny are examined by electrophysiology because dosage compensated elaV(C155)-GAL4/Y male progeny should have a higher dose of presynaptic GAL4 than elaV(C155)-GAL4/+ female siblings. (B) Distribution of QC values from screened larvae. Eight T15 × UAS-RNAi crosses yield a QC smaller than two standard deviations below T15 × WT (red bars). (C) When QC is corrected for non-linear summation (NLS), twelve T15 × UAS-RNAi line crosses yield an NLS corrected QC (NLS QC) smaller than two standard deviations below WT (red bars). (D) Schematic to sort potential positives for follow-up analyses. (E) Negative data for some genes in the screen. Where a specific gene name is listed, the data represent the QC for T15 × UAS-yfg[RNAi]. Underlying data for displayed screen negatives has average evoked potentials >30 mV (not shown, but WT control EPSP = 33.2 ± 1.0 mV) and QC > 60.
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Figure 4: An RNAi- and electrophysiology-based screen for homeostatic factors. (A) Crossing scheme for screen T15 × UAS-yfg[RNAi] (“your favorite gene”). For UAS-RNAi lines on chromosomes II or III, male progeny are examined by electrophysiology because dosage compensated elaV(C155)-GAL4/Y male progeny should have a higher dose of presynaptic GAL4 than elaV(C155)-GAL4/+ female siblings. (B) Distribution of QC values from screened larvae. Eight T15 × UAS-RNAi crosses yield a QC smaller than two standard deviations below T15 × WT (red bars). (C) When QC is corrected for non-linear summation (NLS), twelve T15 × UAS-RNAi line crosses yield an NLS corrected QC (NLS QC) smaller than two standard deviations below WT (red bars). (D) Schematic to sort potential positives for follow-up analyses. (E) Negative data for some genes in the screen. Where a specific gene name is listed, the data represent the QC for T15 × UAS-yfg[RNAi]. Underlying data for displayed screen negatives has average evoked potentials >30 mV (not shown, but WT control EPSP = 33.2 ± 1.0 mV) and QC > 60.

Mentions: The prior electrophysiological data illustrate the potential utility of T15 as a genetic tool to study HSP. If T15 animals are crossed to animals bearing an effective UAS-RNAi transgene, the target gene should be knocked down in larval progeny both presynaptically (due to elaV(C155)-GAL4 and Sca-GAL4 driving UAS-RNAi of the target gene in neurons) and postsynaptically (due to BG57-GAL4 driving UAS-RNAi of the target gene in muscle)—all in the context of a homeostatic challenge to NMJ function due to GluRIII gene knock down. To employ T15 for this purpose, we executed a small-scale RNAi- and electrophysiology-based screen. We chose 62 UAS-RNAi stocks on chromosomes II and III (Dietzl et al., 2007; Ni et al., 2008, 2009). The screen was biased: we targeted genes encoding factors potentially required for proper presynaptic Ca2+ entry and handling, G-protein signals (which could impinge upon calcium channel function), factors known to regulate general synaptic functions, and possible trans-synaptic signaling molecules. To conduct the screen, we crossed T15 females with UAS-RNAi males and recorded from the NMJs of male larval progeny (Figure 4A). Male progeny were chosen because they should have a stronger dose of X-linked elaV(C155)-GAL4 than female progeny.


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)

An RNAi- and electrophysiology-based screen for homeostatic factors. (A) Crossing scheme for screen T15 × UAS-yfg[RNAi] (“your favorite gene”). For UAS-RNAi lines on chromosomes II or III, male progeny are examined by electrophysiology because dosage compensated elaV(C155)-GAL4/Y male progeny should have a higher dose of presynaptic GAL4 than elaV(C155)-GAL4/+ female siblings. (B) Distribution of QC values from screened larvae. Eight T15 × UAS-RNAi crosses yield a QC smaller than two standard deviations below T15 × WT (red bars). (C) When QC is corrected for non-linear summation (NLS), twelve T15 × UAS-RNAi line crosses yield an NLS corrected QC (NLS QC) smaller than two standard deviations below WT (red bars). (D) Schematic to sort potential positives for follow-up analyses. (E) Negative data for some genes in the screen. Where a specific gene name is listed, the data represent the QC for T15 × UAS-yfg[RNAi]. Underlying data for displayed screen negatives has average evoked potentials >30 mV (not shown, but WT control EPSP = 33.2 ± 1.0 mV) and QC > 60.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: An RNAi- and electrophysiology-based screen for homeostatic factors. (A) Crossing scheme for screen T15 × UAS-yfg[RNAi] (“your favorite gene”). For UAS-RNAi lines on chromosomes II or III, male progeny are examined by electrophysiology because dosage compensated elaV(C155)-GAL4/Y male progeny should have a higher dose of presynaptic GAL4 than elaV(C155)-GAL4/+ female siblings. (B) Distribution of QC values from screened larvae. Eight T15 × UAS-RNAi crosses yield a QC smaller than two standard deviations below T15 × WT (red bars). (C) When QC is corrected for non-linear summation (NLS), twelve T15 × UAS-RNAi line crosses yield an NLS corrected QC (NLS QC) smaller than two standard deviations below WT (red bars). (D) Schematic to sort potential positives for follow-up analyses. (E) Negative data for some genes in the screen. Where a specific gene name is listed, the data represent the QC for T15 × UAS-yfg[RNAi]. Underlying data for displayed screen negatives has average evoked potentials >30 mV (not shown, but WT control EPSP = 33.2 ± 1.0 mV) and QC > 60.
Mentions: The prior electrophysiological data illustrate the potential utility of T15 as a genetic tool to study HSP. If T15 animals are crossed to animals bearing an effective UAS-RNAi transgene, the target gene should be knocked down in larval progeny both presynaptically (due to elaV(C155)-GAL4 and Sca-GAL4 driving UAS-RNAi of the target gene in neurons) and postsynaptically (due to BG57-GAL4 driving UAS-RNAi of the target gene in muscle)—all in the context of a homeostatic challenge to NMJ function due to GluRIII gene knock down. To employ T15 for this purpose, we executed a small-scale RNAi- and electrophysiology-based screen. We chose 62 UAS-RNAi stocks on chromosomes II and III (Dietzl et al., 2007; Ni et al., 2008, 2009). The screen was biased: we targeted genes encoding factors potentially required for proper presynaptic Ca2+ entry and handling, G-protein signals (which could impinge upon calcium channel function), factors known to regulate general synaptic functions, and possible trans-synaptic signaling molecules. To conduct the screen, we crossed T15 females with UAS-RNAi males and recorded from the NMJs of male larval progeny (Figure 4A). Male progeny were chosen because they should have a stronger dose of X-linked elaV(C155)-GAL4 than female progeny.

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