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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.


Partial Gαq loss impairs homeostatic compensation. (A) Heterozygous loss of Gαq does not affect levels of evoked neurotransmission (ns, p > 0.05 for each allele compared to WT). However, as with loss of Plc21C, there is a small decrease in mEPSP amplitude (**p < 0.01 for each allele) and a small increase in QC (p-values vary for each allele, but all < 0.05). (B) When challenged with a loss of GluRIIA, Gαq28/+ and Gαq221c/+ NMJs show a complete block in homeostatic compensation (QC—ns, p > 0.05) while Gαq1370/+ and Gαqf04219/+ show partial compensation with quantal content elevated slightly compared to genetic control (***p < 0.001 and **p < 0.01, respectively, Student's T-Test) but not to the full extent seen in the GluRIIASP16 control (***p < 0.001 and *p < 0.05). (C,D) NMJ glutamate receptor subunit levels per unit of synapse area are not decreased in Gαq/+ mutants; if anything, they may be slightly enhanced (scale bar, 5 μm). Merged images include anti-GluRIII (red), -GluRIIA (green), and -HRP (blue) staining.
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Figure 8: Partial Gαq loss impairs homeostatic compensation. (A) Heterozygous loss of Gαq does not affect levels of evoked neurotransmission (ns, p > 0.05 for each allele compared to WT). However, as with loss of Plc21C, there is a small decrease in mEPSP amplitude (**p < 0.01 for each allele) and a small increase in QC (p-values vary for each allele, but all < 0.05). (B) When challenged with a loss of GluRIIA, Gαq28/+ and Gαq221c/+ NMJs show a complete block in homeostatic compensation (QC—ns, p > 0.05) while Gαq1370/+ and Gαqf04219/+ show partial compensation with quantal content elevated slightly compared to genetic control (***p < 0.001 and **p < 0.01, respectively, Student's T-Test) but not to the full extent seen in the GluRIIASP16 control (***p < 0.001 and *p < 0.05). (C,D) NMJ glutamate receptor subunit levels per unit of synapse area are not decreased in Gαq/+ mutants; if anything, they may be slightly enhanced (scale bar, 5 μm). Merged images include anti-GluRIII (red), -GluRIIA (green), and -HRP (blue) staining.

Mentions: Gαq-GTP and Gβγ G-proteins are classically known to activate PLCβ function (Tedford and Zamponi, 2006). A positive Gαq-Plc21C regulatory relationship appears conserved for Drosophila flight behavior (Banerjee et al., 2006). Therefore, we postulated that Plc21C could be playing a role in synaptic homeostasis downstream of G-protein signals. In the course of our screen, we discovered that genetic knock down of a Gαq-encoding gene only mildly impairs synaptic release compared to wild-type: T15 × UAS-Gαq[RNAi] (EPSP = 29.8 ± 1.6 mV; QC = 64.0 ± 3.7). This effect was not strong (possibly due to partial gene knock down by RNAi) and was not identified as a positive hit from the screen. However, given the results with Plc21C, we wished to probe Gαq with genetic mutants. We examined four strong Gαq loss-of-function alleles: Gαq1370, Gαqf04219, Gαq221c, and Gαq28. Each Gαq allele is homozygous lethal (well before the third instar larval stage), but Gαq/+ heterozygotes are viable. For baseline neurotransmission, Gαq/+ mutant NMJs show normal levels of postsynaptic excitation (EPSPs, Figure 8A). Interestingly, however, heterozygous Gαq/+ NMJs show partially impaired homeostatic plasticity in a GluRIIASP16 mutant background (Figure 8B).


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)

Partial Gαq loss impairs homeostatic compensation. (A) Heterozygous loss of Gαq does not affect levels of evoked neurotransmission (ns, p > 0.05 for each allele compared to WT). However, as with loss of Plc21C, there is a small decrease in mEPSP amplitude (**p < 0.01 for each allele) and a small increase in QC (p-values vary for each allele, but all < 0.05). (B) When challenged with a loss of GluRIIA, Gαq28/+ and Gαq221c/+ NMJs show a complete block in homeostatic compensation (QC—ns, p > 0.05) while Gαq1370/+ and Gαqf04219/+ show partial compensation with quantal content elevated slightly compared to genetic control (***p < 0.001 and **p < 0.01, respectively, Student's T-Test) but not to the full extent seen in the GluRIIASP16 control (***p < 0.001 and *p < 0.05). (C,D) NMJ glutamate receptor subunit levels per unit of synapse area are not decreased in Gαq/+ mutants; if anything, they may be slightly enhanced (scale bar, 5 μm). Merged images include anti-GluRIII (red), -GluRIIA (green), and -HRP (blue) staining.
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Figure 8: Partial Gαq loss impairs homeostatic compensation. (A) Heterozygous loss of Gαq does not affect levels of evoked neurotransmission (ns, p > 0.05 for each allele compared to WT). However, as with loss of Plc21C, there is a small decrease in mEPSP amplitude (**p < 0.01 for each allele) and a small increase in QC (p-values vary for each allele, but all < 0.05). (B) When challenged with a loss of GluRIIA, Gαq28/+ and Gαq221c/+ NMJs show a complete block in homeostatic compensation (QC—ns, p > 0.05) while Gαq1370/+ and Gαqf04219/+ show partial compensation with quantal content elevated slightly compared to genetic control (***p < 0.001 and **p < 0.01, respectively, Student's T-Test) but not to the full extent seen in the GluRIIASP16 control (***p < 0.001 and *p < 0.05). (C,D) NMJ glutamate receptor subunit levels per unit of synapse area are not decreased in Gαq/+ mutants; if anything, they may be slightly enhanced (scale bar, 5 μm). Merged images include anti-GluRIII (red), -GluRIIA (green), and -HRP (blue) staining.
Mentions: Gαq-GTP and Gβγ G-proteins are classically known to activate PLCβ function (Tedford and Zamponi, 2006). A positive Gαq-Plc21C regulatory relationship appears conserved for Drosophila flight behavior (Banerjee et al., 2006). Therefore, we postulated that Plc21C could be playing a role in synaptic homeostasis downstream of G-protein signals. In the course of our screen, we discovered that genetic knock down of a Gαq-encoding gene only mildly impairs synaptic release compared to wild-type: T15 × UAS-Gαq[RNAi] (EPSP = 29.8 ± 1.6 mV; QC = 64.0 ± 3.7). This effect was not strong (possibly due to partial gene knock down by RNAi) and was not identified as a positive hit from the screen. However, given the results with Plc21C, we wished to probe Gαq with genetic mutants. We examined four strong Gαq loss-of-function alleles: Gαq1370, Gαqf04219, Gαq221c, and Gαq28. Each Gαq allele is homozygous lethal (well before the third instar larval stage), but Gαq/+ heterozygotes are viable. For baseline neurotransmission, Gαq/+ mutant NMJs show normal levels of postsynaptic excitation (EPSPs, Figure 8A). Interestingly, however, heterozygous Gαq/+ NMJs show partially impaired homeostatic plasticity in a GluRIIASP16 mutant background (Figure 8B).

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.