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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.


GluRIII glutamate receptor subunits are dramatically decreased in the T15 line. (A-L) Immunostaining of wild type (WT), GAL4 Cont × WT, and T15 × WT NMJs with antibodies against GluRIII (red), Bruchpilot (Brp, green), and HRP (blue). A, E, and I show 40X images (scale bars, 10 μm) of muscle 6/7 NMJs from wandering third instar larvae. (B–D,F–H,J–L) Panels show various channels of 60X images (scale bars, 5 μm) of NMJs. (M) Quantification of the number of presynaptic active zones (marked by Brp) and GluRIII clusters at the muscle 6/7 synapse of segments A2 and A3 (*p < 0.05). (N) Calculation of total GluRIII levels per unit of synapse area. This measure takes into account both GluRIII cluster size and GluRIII intensity (see text for individual values; see Materials and Methods for details; *p < 0.05 compared to WT; ***p < 0.001). (O) Quantification of the number of boutons at segment A2 and A3 muscle 6/7 NMJs. n ≥ 6 NMJs stained for each condition.
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Figure 3: GluRIII glutamate receptor subunits are dramatically decreased in the T15 line. (A-L) Immunostaining of wild type (WT), GAL4 Cont × WT, and T15 × WT NMJs with antibodies against GluRIII (red), Bruchpilot (Brp, green), and HRP (blue). A, E, and I show 40X images (scale bars, 10 μm) of muscle 6/7 NMJs from wandering third instar larvae. (B–D,F–H,J–L) Panels show various channels of 60X images (scale bars, 5 μm) of NMJs. (M) Quantification of the number of presynaptic active zones (marked by Brp) and GluRIII clusters at the muscle 6/7 synapse of segments A2 and A3 (*p < 0.05). (N) Calculation of total GluRIII levels per unit of synapse area. This measure takes into account both GluRIII cluster size and GluRIII intensity (see text for individual values; see Materials and Methods for details; *p < 0.05 compared to WT; ***p < 0.001). (O) Quantification of the number of boutons at segment A2 and A3 muscle 6/7 NMJs. n ≥ 6 NMJs stained for each condition.

Mentions: We wished to examine the effects of these NMJ drivers and the UAS-GluRIII[RNAi] transgene on synapse development. We imaged larval NMJs by immunofluorescence microscopy. We utilized an anti-GluRIII antibody to examine glutamate receptors (Marrus et al., 2004). As expected, T15 × WT NMJs show a marked decrease in anti-GluRIII NMJ staining compared to wild-type controls or GAL4 × WT controls (Figures 3A–N). We note several aspects of anti-GluRIII staining that are diminished at T15 × WT NMJs. First, there is a 50% decrease in the number of anti-GluRIII puncta compared to wild-type control NMJs for muscles 6 and 7 (Figure 3M, p < 0.05, T-Test for both segments A2 and A3). In addition to this reduction in cluster number, we observed that individual GluRIII cluster size was greatly reduced (41.6 ± 3.5% compared to wild-type controls, p < 0.001, T-Test). Further analysis of digital immunofluorescence images reveals that the average intensity of each anti-GluRIII pixel is also decreased (82.3 ± 3.9% for the T15 × WT condition compared to wild-type controls, p = 0.03, T-Test). This intensity decrease is small. However, when considered in combination with the reductions in cluster number and size, we estimate an 88% decrease in GluRIII NMJ protein per unit of synapse area in T15 × WT larvae compared to wild-type larvae (Figure 3N, p < 0.001, T-Test; total fluorescence intensity, normalized for synapse area, see Materials and Methods). As expected with RNAi-mediated knock down, this is not a complete loss of GluRIII protein. Importantly these changes are consistent with our electrophysiological data for T15 × WT showing significantly decreased quantal size (reduced intensity/pixel and cluster size) and frequency (reduced cluster number).


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)

GluRIII glutamate receptor subunits are dramatically decreased in the T15 line. (A-L) Immunostaining of wild type (WT), GAL4 Cont × WT, and T15 × WT NMJs with antibodies against GluRIII (red), Bruchpilot (Brp, green), and HRP (blue). A, E, and I show 40X images (scale bars, 10 μm) of muscle 6/7 NMJs from wandering third instar larvae. (B–D,F–H,J–L) Panels show various channels of 60X images (scale bars, 5 μm) of NMJs. (M) Quantification of the number of presynaptic active zones (marked by Brp) and GluRIII clusters at the muscle 6/7 synapse of segments A2 and A3 (*p < 0.05). (N) Calculation of total GluRIII levels per unit of synapse area. This measure takes into account both GluRIII cluster size and GluRIII intensity (see text for individual values; see Materials and Methods for details; *p < 0.05 compared to WT; ***p < 0.001). (O) Quantification of the number of boutons at segment A2 and A3 muscle 6/7 NMJs. n ≥ 6 NMJs stained for each condition.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: GluRIII glutamate receptor subunits are dramatically decreased in the T15 line. (A-L) Immunostaining of wild type (WT), GAL4 Cont × WT, and T15 × WT NMJs with antibodies against GluRIII (red), Bruchpilot (Brp, green), and HRP (blue). A, E, and I show 40X images (scale bars, 10 μm) of muscle 6/7 NMJs from wandering third instar larvae. (B–D,F–H,J–L) Panels show various channels of 60X images (scale bars, 5 μm) of NMJs. (M) Quantification of the number of presynaptic active zones (marked by Brp) and GluRIII clusters at the muscle 6/7 synapse of segments A2 and A3 (*p < 0.05). (N) Calculation of total GluRIII levels per unit of synapse area. This measure takes into account both GluRIII cluster size and GluRIII intensity (see text for individual values; see Materials and Methods for details; *p < 0.05 compared to WT; ***p < 0.001). (O) Quantification of the number of boutons at segment A2 and A3 muscle 6/7 NMJs. n ≥ 6 NMJs stained for each condition.
Mentions: We wished to examine the effects of these NMJ drivers and the UAS-GluRIII[RNAi] transgene on synapse development. We imaged larval NMJs by immunofluorescence microscopy. We utilized an anti-GluRIII antibody to examine glutamate receptors (Marrus et al., 2004). As expected, T15 × WT NMJs show a marked decrease in anti-GluRIII NMJ staining compared to wild-type controls or GAL4 × WT controls (Figures 3A–N). We note several aspects of anti-GluRIII staining that are diminished at T15 × WT NMJs. First, there is a 50% decrease in the number of anti-GluRIII puncta compared to wild-type control NMJs for muscles 6 and 7 (Figure 3M, p < 0.05, T-Test for both segments A2 and A3). In addition to this reduction in cluster number, we observed that individual GluRIII cluster size was greatly reduced (41.6 ± 3.5% compared to wild-type controls, p < 0.001, T-Test). Further analysis of digital immunofluorescence images reveals that the average intensity of each anti-GluRIII pixel is also decreased (82.3 ± 3.9% for the T15 × WT condition compared to wild-type controls, p = 0.03, T-Test). This intensity decrease is small. However, when considered in combination with the reductions in cluster number and size, we estimate an 88% decrease in GluRIII NMJ protein per unit of synapse area in T15 × WT larvae compared to wild-type larvae (Figure 3N, p < 0.001, T-Test; total fluorescence intensity, normalized for synapse area, see Materials and Methods). As expected with RNAi-mediated knock down, this is not a complete loss of GluRIII protein. Importantly these changes are consistent with our electrophysiological data for T15 × WT showing significantly decreased quantal size (reduced intensity/pixel and cluster size) and frequency (reduced cluster number).

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.