A single-cross, RNA interference-based genetic tool for examining the long-term maintenance of homeostatic plasticity

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.

Keywords: CaV2 channels; Drosophila melanogaster; RNAi screening; cysteine string protein; homeostatic plasticity; neuromuscular junction; phospholipase C beta; synaptic transmission.

Figure 1

Postsynaptic GluRIII gene knock down induces robust homeostatic compensation. (A) Crossing scheme. NMJs from F1 larvae (genotype BG57-GAL4/UAS-GluRIII[RNAi]) are subjected to electrophysiological analyses. (B) Quantal size (miniature excitatory postsynaptic potentials, mEPSP) is decreased for BG57-GAL4/UAS-GluRIII[RNAi] larvae (***p < 0.001, Student's T-Test). Evoked potentials (excitatory postsynaptic potentials, EPSP) are normal because of a homeostatic increase in quantal content (QC) (**p < 0.01). (C) Representative electrophysiological traces. Scale bars for EPSPs (mEPSPs): 5 mV (1 mV); 50 ms (1000 ms).

Figure 2

The T15 stock induces robust homeostatic compensation. (A) T15 × WT crossing scheme. The genotype for T15 is (chromosomes X; II; III): elaV(C155)-GAL4; Scabrous-GAL4; BG57-GAL4, UAS-GluRIII[RNAi]. (B) T15 × WT larval NMJs have decreased quantal size (***p < 0.001, T-Test vs. WT). Evoked potentials are normal because of a homeostatic increase in QC (***p < 0.001). A control stock with only GAL4 drivers behaves similarly to WT. (C) Representative electrophysiological traces. Scale bars for EPSPs (mEPSPs): 5 mV (1 mV); 50 ms (1000 ms).

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.

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.

Figure 5

Knock down of cacophony gene function does not impair synaptic homeostasis. (A,B) mEPSP (black), QC (gray), NLS QC (white). (^#p = 0.05; *p < 0.05; **p < 0.01; ***p < 0.001, T-test compared to control at 100% dotted line) (A) Diminished baseline neurotransmission for GAL4 control × UAS-cac[RNAi] larvae is consistent with cac gene knock down. Diminished release occurs across a range of extracellular [Ca²⁺]. (B) T15 × UAS-cac[RNAi] larvae show robust homeostatic compensation compared to GAL4 control × UAS-cac[RNAi] larvae. As expected, the T15 line induces a marked diminishment of quantal size (mEPSP). The NMJ responds with a robust increase in release. This response is observed across a range of extracellular calcium concentrations, and the same result holds whether or not QC is corrected for non-linear summation. (C) Representative electrophysiological traces. Scale bars for EPSPs (mEPSPs): 5 mV (1 mV); 50 ms (1000 ms).

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 Csp^DG29203 allele or heterozygosity for the Csp^EY22488 allele block homeostatic upregulation of quantal content compared to their respective non-GluRIIA^SP16genetic controls (ns, p = 0.44 and p = 0.14, respectively). (D) Representative traces show a failure of homeostatic compensation for GluRIIA^SP16, Csp^DG29203. (E) Postsynaptic knock down of Csp function leaves homeostatic plasticity intact. (F) Acute homeostatic compensation is intact in Csp^DG29203 as evidenced by the elevated quantal content in response to philanthotoxin-433 (PhTox) application (**p < 0.01). (G) A doubly heterozygous combination of Csp^DG29203/+ and cac^S/+ shows a homeostatic block in the GluRIIA^SP16 background because of a failure to increase quantal content over cac^S/+; Csp^DG29203/+ 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).

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 GluRIIA^SP16 mutant background. (D) Evoked amplitudes (EPSPs) are unaltered upon heterozygous loss of Plc21C by use of the deficiency Df(2L)BSC4 or Plc21C^p60A 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 GluRIIA^SP16 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 GluRIIA^SP16 (***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).

Figure 8

Partial Gα_q 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 Gαq^221c/+ NMJs show a complete block in homeostatic compensation (QC—ns, p > 0.05) while Gαq¹³⁷⁰/+ and Gαq^f04219/+ 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 GluRIIA^SP16 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.