Fruit flies were reared in chambers with temperature control (29°C for RNAi screen; otherwise 25°C). w¹¹¹⁸ (Hazelrigg et al., 1984) is utilized as a wild-type control unless otherwise indicated. Drosophila stocks carrying various mutant alleles, GAL4 drivers, or UAS-driven transgenes were used. Stocks were either obtained from the Bloomington Drosophila Stock Center (BDSC, Bloomington, IN), the Vienna Drosophila RNAi Center (VDRC, Vienna, Austria), or directly from researchers who generated them. UAS-RNAi lines from VDRC (Dietzl et al., 2007) detailed in this manuscript include: UAS-cac[RNAi] (VDRC transformant #104168; cac^KK101478), UAS-Csp[RNAi] (VDRC #34168; Csp^GD10571), and UAS-Plc21C[RNAi] (VDRC #26558; Plc21C^GD11359). Plc21C TRiP UAS-RNAi lines (Ni et al., 2008, 2009) obtained from BDSC include: Plc21C^HMS0600, Plc21C^JF01210, Plc21C^JF01211. A transgenic wild-type cac expression construct is UAS-cac-eGFP^786C (Kawasaki et al., 2004). Classical mutant alleles and deficiency stocks include: GluRIIA^SP16 (Petersen et al., 1997), Csp^DG29203 (Wishart et al., 2012), Csp^EY22488 (BDGP gene disruption project, Bellen et al., 2004), cac^S (Smith et al., 1998), cac^HC129 (Kulkarni and Hall, 1987), Plc21C^p60A (Weinkove et al., 1999), Df(2L)BSC4 (J. Deal and K. Cook, BDSC), Gαq¹³⁷⁰ (Kain et al., 2008), Gαq^f04219 and Gαq^221c (Banerjee et al., 2006), and Gαq²⁸ (Yao and Carlson, 2010). GAL4 drivers include elaV(C155)-GAL4 (Lin and Goodman, 1994), Scabrous-GAL4 (Budnik et al., 1996), and BG57-GAL4 (Budnik et al., 1996).

We constructed UAS-GluRIII[RNAi] by PCR amplification from genomic DNA, introducing Xba I restriction sites, and cloning a tandem duplicated fold-back version of the PCR product into the pUAST pWiz vector (Lee and Carthew, 2003). For the PCR amplification step, the GluRIII gene PCR primers were as follows: CAF2L: 5′-TCGATCTAGAGATCCTCGAGCGAGGATGGACAGCGGA-3′ and CAF2R: 5′-TTATTCTAGATGATTATCTCGCCAATGATGC-3′. Standard injection procedures were used to generate UAS-GluRIII[RNAi] transgenic stocks. A UAS-GluRIII[RNAi] insertion on chromosome III was built into a screening stock with the full genotype: elaV(C155)-GAL4; Scabrous-GAL4/CyO-GFP; BG57-GAL4, UAS-GluRIII[RNAi]/TM6B. For simplicity, this screening stock is termed T15 in the manuscript, and balancer chromosomes are not included in its description in the Results Section of the text.

Wandering third instar larvae were selected for electrophysiological recordings. Sharp electrode recordings were taken from muscle 6 of abdominal segments 2 and 3, as previously described (Davis et al., 1998; Frank et al., 2006). Larvae were dissected in a modified HL3 saline: NaCl (70 mM), KCl (5 mM), MgCl2 (10 mM), NaHCO3 (10 mM), sucrose (115 mM = 3.9%), trehalose (4.2 mM = 0.16%), HEPES (5.0 mM = 0.12%), and CaCl₂ (0.5 mM, unless otherwise indicated). Data were collected using Axopatch 200B or Axoclamp 900A amplifiers (Molecular Devices, Sunnyvale, CA), digitized using a Digidata 1440A data acquisition system (Molecular Devices), and recorded with pCLAMP 10 acquisition software (Molecular Devices). For presynaptic nerve stimulation, a Master-8 pulse stimulator (A.M.P. Instruments, Jerusalem, Israel) and an ISO-Flex isolation unit (A.M.P. Instruments) were utilized to deliver 1 ms suprathreshold stimuli to the appropriate segmental nerve. The average spontaneous miniature EPSP (mEPSP) amplitude was quantified by measuring the amplitude of ~100–200 individual spontaneous release events per NMJ (unless mEPSP frequency was too low for ~1 min continuous recording, in which case all mEPSPs available were measured). The average per-NMJ mEPSP amplitudes were then averaged for each genotype. The average evoked EPSP amplitude was calculated for each NMJ. Quantal content (QC) was determined for each recording by calculating the ratio of average EPSP and average mEPSP amplitudes. Quantal contents were calculated for each recording and then averaged across NMJs of the indicated genotypes. Where indicated, quantal content was corrected for non-linear summation (Martin, 1955). Selected raw electrophysiological data are included as Supplementary Table 1.

Third instar larvae were fileted in HL3 saline. Dissected animals were fixed for 3 min in Bouin's fixative (Ricca Chemical Company, Arlington, TX), washed using standard procedures, and incubated in primary antibodies overnight at 4°C. This was followed by additional washes and a 2-hour incubation in secondary antibody at room temperature. Staining was performed using the following primary antibodies: mouse anti-Synapsin (3C11) 1:50 (Developmental Studies Hybridoma Bank, University of Iowa—DSHB) (Klagges et al., 1996); rabbit anti-Dlg 1:30,000 (Budnik et al., 1996); mouse anti-Brp (nc82) 1:250 (DSHB); rabbit anti-GluRIII 1:4000 (Marrus et al., 2004). The following fluorophore-conjugated antibodies were also used (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA): goat anti-mouse-488 1:1000 (DyLight); goat anti-rabbit-549 1:2000 (DyLight); Alexa-647 goat anti-HRP 1:500. Larval preparations were mounted in Vectashield (Vector Laboratories, Burlingame, CA) and imaged at room temperature using Zen software on a Zeiss 700 LSM mounted on an Axio Observer.Z1 using an EC Plan-Neofluar 40X Oil DIC Objective (aperture 1.30) or an EC Plan-Apochromat 63x Oil DIC Objective (aperture 1.40) (Carl Zeiss Microscopy, Jena, Germany). For each experiment, experimental and control larval preps were stained in the same container, mounted on the same slide, imaged using identical acquisition settings, and analyzed using the same procedure and thresholds.

Bouton numbers were quantified semi-automatically using the “Spots” function in Imaris x64 v7.6.0 (Bitplane, Zurich Switzerland). Boutons were counted using the anti-Synapsin channel with the XY diameter set at 3 μm. Active Zones were counted using the anti-Brp channel with an XY diameter of 0.3 μm. GluRIII clusters were counted using the anti-GluRIII channel with an XY diameter of 0.5 μm. The threshold was adjusted so that each bouton, active zone, or GluRIII cluster was recognized once. Any errors in automated counting were corrected by hand to arrive at the final value. GluRIII levels and area were assessed using ImageJ 1.48s/Java 1.6.0_24 (64-bit) with Fiji plugins. Z-stack images were compressed using the maximum projection function; an ROI was generated from a mask of the HRP channel and used to define the synapse; a second ROI was hand drawn to exclude any non-synaptic structures in the image; a minimum threshold was set for each channel to eliminate background fluorescence and held consistent within each experiment; the Measure function was used to assess fluorescence intensity and area for each channel [Brp(488), GluRIII(549), HRP(647)]. For each NMJ, total GluRIII fluorescence intensity and synapse coverage was normalized to the synaptic area of the HRP channel and compared between the genotypes analyzed. Cluster area was calculated by dividing the total synaptic area of GluRIII by the number of GluRIII clusters for each synapse imaged.

For each genotype, 25–30 wandering third instar, female larvae were sorted in HL3 saline, collected on ice and rinsed in HL3 with excess saline pipetted off before homogenization. Larvae were homogenized in 200 μL TRIzol reagent (Ambion, Life Technologies) using disposable plastic micropestles. Total homogenate was then brought to 1 mL by addition of 800 μL TRIzol, tubes were vigorously inverted, and the resultant homogenate was frozen at −80°C until all genotypes were prepared. Samples were allowed to thaw on ice and incubated for 5 min at room temperature before proceeding with RNA isolation using the manufacturer's instructions with the following changes: Following isopropanol addition, samples were placed at −20°C for precipitation, and two ethanol wash cycles were used. Samples were quantified using a Nanodrop (Thermo Scientific). Reverse transcription was done using iScript (Bio-Rad) according to manufacturer's instructions using 1 μg of total input. Quantitative PCR (Bio-Rad CFX96) was done using SYBR Green reagent (Bio-Rad) with a 10 μL final reaction volume and technical replicates in triplicate. Final primer concentrations were 250 nM. For cac, exon spanning primers were designed with the following sequences: cac-F 5′-cgggaacgagagttgtacg-3′ and cac-R 5′-actggagatggcagtacacg-3′ (this study), and RpL32-F 5′-atgaccatccgcccagcatac-3′ and RpL32-R 5′-ctgcatgagcaggacctccag-3′ (PK Geyer Lab, University of Iowa). Primer piloting was done using stepwise dilutions of iScript product to verify PCR efficiency. Final quantification was done using iScript product diluted to 1:444.4 in the final PCR. Melt curve analysis verified presence of a single product for all reactions.

Statistical significance was assessed either by Student's T-Test, comparing an experimental data set and a control data set, or One-Way ANOVA with Tukey's post-hoc test across multiple data sets, as appropriate. Specific p value ranges are given in the text, figures, and figure legends, with p < 0.05 marked as significant (^#p = 0.05; ^*p < 0.05; ^**p < 0.01; ^***p < 0.001). For some p-values that trend toward statistical significance (0.05 < p < 0.1), specific values are given as indicated. The values reported or plotted on bar graphs are mean ± SEM.

Partial knock down of GluRIII gene function should induce a significant decrease in NMJ quantal size and a homeostatic increase in NMJ quantal content (Marrus et al., 2004). Using the Drosophila pWiz vector (Lee and Carthew, 2003), we created a transgene to target the GluRIII gene for functional knock down by RNAi (see Materials and Methods). From a pWiz-UAS-GluRIII[RNAi] clone, we generated UAS-GluRIII[RNAi] transgenic Drosophila stocks and crossed them to stocks harboring the muscle-specific GAL4 driver, BG57-GAL4 (Budnik et al., 1996) (Figure 1A). We then analyzed the larval cross progeny by electrophysiology to assess the efficiency of glutamate receptor subunit knock down.

Postsynaptically driven BG57-GAL4 >> UAS-GluRIII[RNAi] yields a robust homeostatic challenge to NMJ function (Figures 1B,C) (See Supplementary Table 1 for selected raw electrophysiological data throughout the manuscript). NMJ effects are similar to the published GluRIII hypomorphic condition (Marrus et al., 2004). Compared to controls, BG57-GAL4/UAS-GluRIII[RNAi] NMJs show drastically decreased quantal amplitude (mEPSP = 0.81 ± 0.04 mV for wild-type control vs. 0.46 ± 0.05 mV for GluRIII knock down, p < 0.001, T-Test) and frequency (4.2 ± 0.2 Hz for control vs. 0.8 ± 0.1 Hz for GluRIII knock down, p < 0.001, T-Test). Despite these decreases in spontaneous miniature neurotransmission, evoked neurotransmission is normal (EPSP = 33.2 ± 1.0 mV for control vs. 33.6 ± 2.7 mV for GluRIII knock down) because of a homeostatic enhancement of quantal content (QC = 43.8 ± 2.1 for control vs. 76.6 ± 7.1 for GluRIII knock down, p < 0.01, T-Test) (Figures 1B,C).

By meiotic recombination, we placed a UAS-GluRIII[RNAi] transgene on chromosome III in cis with the BG57-GAL4 driver. To increase the potential of this stock as a genetic tool to study NMJ homeostasis, we augmented it with two presynaptic GAL4 drivers. We chose neuronal drivers elaV(C155)-GAL4 (Lin and Goodman, 1994) and Scabrous-GAL4 (Budnik et al., 1996). Multiple presynaptic drivers were used to enhance the efficiency of RNAi in neurons. For simplicity, we refer to the new stock incorporating all drivers as T15 (trans-synaptic). The genotype of T15 is elaV(C155)-GAL4; Sca-GAL4; BG57-GAL4, UAS-GluRIII[RNAi] (chromosomes X; II; III, balancer chromosomes omitted from this genotype). We also generated a control stock, containing the same GAL4 drivers, but not the UAS-GluRIII[RNAi] transgene.

When T15 females are crossed to wild-type males (herein T15 × WT, Figure 2A), the larval progeny exhibit starkly diminished NMJ quantal size and frequency, and a homeostatic increase in NMJ quantal content (Figures 2B,C). By contrast, when GAL4 driver control females are crossed to wild-type males (herein GAL4 Cont × WT), the NMJ electrophysiological profile of larval progeny is largely similar to that of wild-type NMJs (Figures 2B,C). The GAL4 drivers or genetic background may induce a slight increase in quantal size (Figure 2B). However, this increase is not statistically significant, and the data show that the presence of presynaptic GAL4 drivers exerts no adverse effects on evoked NMJ excitation (Figures 2B,C).

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).

Surprisingly, the presence of the GAL4 drivers (or the genetic background of the driver line stock) also appears to diminish GluRIII protein level (per unit of synapse area) in GAL4 × WT compared to wild-type controls (Figure 3N, p < 0.05). We are not certain why GluRIII levels are down in GAL4 × WT. Nevertheless, by electrophysiology, this depression is clearly not severe enough to diminish quantal size or evoked excitation (Figure 2B)—though it could explain a partial decrease in quantal frequency compared to WT (Supplementary Table 1). Nevertheless, the amount of GluRIII protein per unit synapse area in GAL4 × WT is about three times greater than the amount measured in T15 × WT (Figure 3N, p < 0.001).

We wished to examine other basic elements of synapse structure. To do this, we stained NMJs with an anti-Bruchpilot (Brp) antibody to count the number of presynaptic active zones (Wagh et al., 2006) or with anti-Discs Large (Dlg) (Budnik et al., 1996) and anti-Synapsin (Syn) (Klagges et al., 1996) antibodies to count the number of synaptic boutons. For synapses on both segments A2 and A3, neither active zone counts (Figure 3M, Brp puncta) nor bouton counts (Figure 3O) are significantly different when comparing T15 × WT NMJs vs. wild-type NMJs. These findings are interesting, considering the fact that T15 × WT NMJs have fewer GluRIII clusters apposed to the presynaptic active zone (Figure 3M). Collectively, our data show that T15 × WT NMJs have starkly decreased GluRIII levels, but otherwise grossly normal synapse growth and morphology.

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.

T15 × WT NMJs have significantly elevated quantal content compared to non-transgenic wild-type controls (Figures 2B, 4B). In analyzing the T15 × UAS-RNAi screen data, we considered quantal content as a measure of presynaptic release. We identified eight T15 × UAS-RNAi crosses that yielded progeny with a low NMJ quantal content (QC < 52, Figure 4B). For all eight, the level of presynaptic release is indistinguishable (or lower) than that of unchallenged, wild-type NMJs, potentially indicative of a block in homeostatic compensation. Considering the distribution of QC for all of the UAS-RNAi crosses from the screen, this level of presynaptic release is also about two standard deviations smaller than T15 × WT controls. We wished to ensure that our synaptic release analysis was accurate and restrictive. Therefore, we also applied a correction to our calculations of quantal content to account for effects of non-linear summation (NLS) when recording synaptic voltages (Martin, 1955). Considering the distribution of NLS corrected QC for all of the UAS-RNAi crosses from the screen, an NLS corrected QC < 85 is two standard deviations smaller than T15 × WT controls. Twelve T15 × UAS-RNAi crosses fall below this NLS QC threshold, including all eight identified by the prior cutoff (Figure 4C). We chose to focus initial follow-up efforts on the eight potential positives that showed a low quantal content by both criteria (Figure 4D).

We identified eight potential hits out of 62 UAS-RNAi stocks, which is a very high positive rate for a screen (12.9%). However, the screen is biased, and it is not surprising that several pre-selected factors could impair synapse function. Nevertheless, most T15 × UAS-RNAi crosses do not yield progeny with diminished NMJ QC. Several screen negatives with EPSP > 30 mV and QC > 60 are shown (Figure 4E). It is possible the corresponding target genes are not involved in synaptic homeostasis. However, it is also possible that conditions in this experiment do not yield sufficient gene knock down to reveal a homeostatic phenotype. One interesting example to consider in this regard is Drosophila ephrin. Prior work demonstrates that a presynaptic signaling system consisting of the Eph receptor tyrosine kinase and the cytoplasmic guanine exchange factor Ephexin is needed for the long-term maintenance of HSP (Frank et al., 2009). Drosophila Ephrin is a top candidate ligand for Drosophila Eph in this process, but the RNAi data from our screen do not support this conclusion (Figure 4E).

A low QC value from a screened T15 × UAS-RNAi line could reflect a genuine defect in synaptic homeostasis. It could also simply reflect a decrease in neurotransmission when the targeted gene is knocked down in NMJ tissues. We note that two of the eight UAS-RNAi lines identified appear to cause severe neurotransmission defects (Figures 4B–D), suggesting synapse dysfunction independent of possible defects in homeostatic plasticity (QC < 25 when crossed to T15; NLS QC < 35 when crossed to T15). One of these two RNAi lines targets Drosophila cacophony (cac)—a positive control RNAi line included in the screen that targets the α1a pore-forming subunit of Ca_V2 channels. Given previous studies examining Ca_V2 in neurotransmission and NMJ homeostasis (Frank et al., 2006, 2009; Tsurudome et al., 2010; Müller and Davis, 2012), this result is not surprising. However, using T15, we are able to garner new information about Cac/Ca_V2 and its role in synaptic homeostasis (see below). Finally, we also report initial characterizations of two novel homeostatic genes, Drosophila Csp and Plc21C (Figures 4B,C). Our screening and characterizations of other genes are ongoing, and we will report further characterizations elsewhere.

Ca_V2-type voltage-gated Ca²⁺ channels are critical for presynaptic neurotransmission. For many vertebrate and invertebrate synapses, Ca_V2 channels also gate homeostatic modulations of neurotransmission (Frank, 2014b). In Drosophila, homozygous, partial loss-of-function missense mutations like cac^S block the homeostatic potentiation of transmitter release at the NMJ after glutamate receptor impairment (Frank et al., 2006, 2009; Müller and Davis, 2012). By contrast, cac null mutants arrest as embryos (Kulkarni and Hall, 1987; Kurshan et al., 2009). Therefore, it is unknown if cac mutant impairment of HSP is due to partial loss of channel function—or if amino-acid substitutions such as the one encoded by cac^S (F1029I, transmembrane domain III S6) (Smith et al., 1998) impair specific functions or interactions critical to homeostatic signaling. Intriguingly, cac^S/+ heterozygotes display partial defects in courtship song behavior (Smith et al., 1998) and the execution of synaptic homeostasis (Frank et al., 2006, 2009). These data are consistent with the possibility that missense cac mutations may exert antimorphic effects on synaptic function.

Knocking down cac gene activity by RNAi allows one to test whether a simple reduction of Ca_V2 function suffices to impair synaptic homeostasis. Efficient RNAi-mediated cac knock down also circumvents possible antimorphic effects associated with genetic mutations (but not potential effects due to haploinsufficiency). For the screen and subsequent analyses here, we used a UAS-cac[RNAi] transgene to knock down cac gene function (cac^KK101478, see Materials and Methods) (Dietzl et al., 2007). By primary target sequence, cac^KK101478 is not predicted to induce off-target gene knock down. By quantitative RT-PCR (qPCR), we find that driving this transgene under simultaneous control of the neuronal drivers elaV(C155)-GAL4 and Sca-GAL4 diminishes total levels of cac mRNA by ~50% vs. a control utilizing just the drivers (See Materials and Methods).

We prepared mRNA for qPCR using whole larvae. Therefore, Cac protein loss in motor neurons and at synapses could be even greater than qPCR measurements. We tested the effectiveness of UAS-cac[RNAi] on cac gene function in two different ways. First, we crossed transgenically rescued cac null females elaV(C155)-GAL4, cac^HC129;; UAS-cac-eGFP^786c × UAS-cac[RNAi] males (or WT males for control). This UAS-cac[RNAi] cross (but not the WT control cross) kills all progeny, with only a small number of adults reaching the pupal eclosion stage and then either arresting during the process of eclosion or getting stuck in the food shortly after eclosing (not shown). Second, we find that presynaptic expression of cac^KK101478 starkly diminishes evoked neurotransmission (elaV(C155)-GAL4, Sca-GAL4 >> UAS-cac[RNAi]: EPSP = 8.5 ± 1.1 mV; QC = 8.4 ± 1.5, n = 8) compared to a wild-type control (WT: EPSP = 33.2 ± 1.0 mV, QC = 43.8 ± 2.1, n = 31). This diminishment of evoked neurotransmission is similar in severity to a cac^S mutant (Frank et al., 2006), and it suggests a significant loss of Cac protein at the NMJ. By contrast, muscle expression of cac^KK101478 (BG57-GAL4 >> UAS-cac[RNAi]) has no adverse effect on evoked neurotransmission (EPSP = 37.5 ± 2.0 mV; QC = 53.2 ± 2.9, n = 8).

We crossed either the T15 stock or the aforementioned pre- and post-synaptic GAL4 driver control stock (Figure 2) to UAS-cac[RNAi]. For GAL4 Cont × UAS-cac[RNAi] progeny, NMJ evoked excitation and quantal content are markedly decreased compared to GAL4 Cont × WT controls (Figure 5A). This is true across a range of extracellular Ca²⁺ concentrations, though perhaps a bit less pronounced at physiological [Ca²⁺] (1.5 mM) (Figure 5A). As expected, for T15 × UAS-cac[RNAi] larval progeny, there is a significant decrease in NMJ quantal size (mEPSP) compared to GAL4 Cont × UAS-cac[RNAi] larval progeny (Figure 5B). However, T15 × UAS-cac[RNAi] NMJ EPSP amplitude is no different than control cross UAS-cac[RNAi] progeny (Figure 5C). This is due to a homeostatic enhancement in presynaptic quantal content (Figure 5B). This result holds for extracellular Ca²⁺ concentrations that permit a range of presynaptic release: 0.5, 1.0, and 1.5 mM. For each condition, homeostatic compensation of presynaptic release remains robust (Figure 5B).

Our data suggest diminishment of cac gene function itself is not sufficient to block homeostatic potentiation of function. Nor is extracellular calcium concentration a factor. If Cac/Ca_V2 levels are simply diminished—but the copies of Cac α1a subunits present are wild-type copies—homeostatic plasticity is intact. By contrast, the presence of function-impairing Ca_V2 α1a subunits due to amino-acid substitution appears sufficient to block the induction and maintenance of homeostatic plasticity (Frank et al., 2006, 2009; Müller and Davis, 2012).

CSP is a conserved synaptic protein in the DnaJ family of chaperones. For both vertebrates and invertebrates, CSP executes functions that promote viability, coordinated locomotion, neuroprotection, and evoked neurotransmitter release (Zinsmaier, 2010). At the Drosophila NMJ, synaptic functions of CSP appear related to Ca²⁺ regulation. In Csp loss-of-function mutants, resting intra-terminal Ca²⁺ levels are altered (Dawson-Scully et al., 2000; Bronk et al., 2001; Dawson-Scully et al., 2007), and neurotransmission defects can be suppressed by high [Ca²⁺]_e or repetitive nerve stimulation (Dawson-Scully et al., 2000; Bronk et al., 2001). The fact that a UAS-Csp[RNAi] line emerged from our screen was somewhat surprising because a prior genetic screen demonstrated that a Csp mutation does not impair homeostatic plasticity when the NMJ is challenged with PhTox application (Goold and Davis, 2007; Dickman and Davis, 2009). However, this previous screen specifically examined factors for a role in the short-term induction of homeostatic plasticity, not its long-term maintenance.

Our screen found no elevation of NMJ quantal content when comparing T15 × UAS-Csp[RNAi] larval progeny vs. wild-type larvae (Figure 4). This result indicates a possible role for CSP in homeostatic plasticity, although it could also be consistent with expected Csp defects in neurotransmission. In a follow-up test, we found that T15 × UAS-Csp[RNAi] NMJ quantal content is not significantly different than GAL4 driver control × UAS-Csp[RNAi] NMJ quantal content (Figure 6A). This result suggests a bona fide defect in synaptic homeostasis (Figures 6A,B).

To follow up on the RNAi screen with genetic mutations, we examined two transposon insertion-induced Csp alleles, Csp^DG29203 and Csp^EY22488. Csp^DG29203 disrupts the 5′ UTR of Csp, and genetic evidence suggests that it is hypomorphic (Wishart et al., 2012). Csp^EY22488 is inserted in an intronic region of Csp. It causes significant homozygous lethality and appears to be a strong mutation genetically (data not shown). We generated two double mutant combinations: GluRIIA^SP16; Csp^DG29203, and GluRIIA; Csp^EY22488/+. The GluRIIA^SP16 deletion mutation induces a marked decrease in quantal size and a homeostatic increase in quantal content compared to wild-type controls (Figure 6C) (Petersen et al., 1997). By contrast, for the double mutant combinations, synaptic homeostasis is impaired: there is no significant increase in release compared to Csp mutants alone (Figure 6C). As a result, evoked postsynaptic excitation is stunted (Figure 6D). Interestingly, the Csp^EY22488/+ and Csp^DG29203 genetic conditions display somewhat different baseline electrophysiology, in terms of both quantal size and quantal content (Figure 6C). These differences could be due to different genetic backgrounds or Csp allelic strength. Nevertheless, both alleles block homeostatic compensation in response to GluRIIA gene loss (Figures 6C,D).

The vast majority of research published about Drosophila CSP demonstrates that it localizes to presynaptic terminals. However, it has also been reported that Drosophila muscles can express low levels of CSP (Eberle et al., 1998). To test whether CSP's function in synaptic homeostasis could reside in the postsynaptic compartment, we crossed the muscle driver stock BG57-GAL4, UAS-GluRIII[RNAi] to UAS-Csp[RNAi]. Knocking down Csp gene function in the muscle leaves synaptic homeostasis intact (Figure 6E); this result is consistent with a presynaptic role for CSP in synaptic homeostasis.

Collectively, our data demonstrate that Csp function is needed for the long-term maintenance of homeostatic plasticity. To double check whether Csp could also be required for the short-term induction of homeostatic plasticity, we applied PhTox to Csp^DG29203 mutant NMJs. By this assay, the rapid induction of homeostatic plasticity remains intact (Figure 6F), consistent with the previously reported results (Goold and Davis, 2007; Dickman and Davis, 2009). Therefore, CSP appears to be needed for the sustained expression of homeostatic plasticity throughout development, not its short-term induction.

cac^S/+; GluRIIA double mutant NMJs have partially impaired synaptic homeostasis (Frank et al., 2006) (and Figure 6G). As discussed above, this partial impairment could be due to an antimorphic allelic effect. The cac^S/+ genetic condition has been previously exploited to characterize second-site factors that could potentially execute homeostatic plasticity in conjunction with Ca_V2 by searching for genetic interactions with cac^S (Frank et al., 2009; Wang et al., 2014). In the sensitized cac^S/+ genetic background, we find that cac^S/+; GluRIIA; Csp^DG29203/+ NMJs have completely blocked synaptic homeostasis—i.e., there is no increase in quantal content compared to the cac^S/+;; Csp^DG29203/+ genetic control condition (Figure 6G). A strong double heterozygous phenotype could be consistent with Cac and CSP both functioning within synaptic homeostasis, either in a linear signaling pathway or in parallel processes. However, it could also reflect a summation of separate, partial defects in HSP for both cac^S/+; GluRIIA NMJs and GluRIIA; Csp^DG29203/+ NMJs (Figure 6G).

In designing our screen, we postulated that lipid signaling at the synapse could affect NMJ homeostasis. Canonically, Gαq-GTP (together with Gβγ G-proteins) activates PLCβ. In turn, PLCβ cleaves phosphatidylinositol 4,5-bisphosphase (PIP₂) into diacylglycerol (DAG) and inositol triphosphate (IP₃) (Tedford and Zamponi, 2006; Philip et al., 2010; Kadamur and Ross, 2013). These molecules are known to influence neurotransmission in several ways at many synapses, including the NMJ (Goni and Alonso, 1999; Cremona and De Camilli, 2001; Peters et al., 2001; Wu et al., 2002; Rohrbough and Broadie, 2005; Huang et al., 2006). To examine this pathway in the context of NMJ homeostasis, we considered a Drosophila PLCβ homolog gene, Plc21C—among other genes, some of which showed no phenotype (Figure 4E).

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 (Plc21C^GD11359) 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.

To address possible off-target effects of UAS-Plc21C[RNAi] expression, we acquired three additional UAS-Plc21C[RNAi] lines—Drosophila TRiP constructs (Transgenic RNAi Project) (Ni et al., 2009, 2008). Each TRiP line fails to robustly increase quantal content when crossed to T15, (Figure 7A). This result is consistent with an impairment of HSP. Additionally, we examined two heterozygous Plc21C deletion mutations: a chromosomal deficiency that removes one copy of Plc21C from the genome, Df(2L)BSC4, and a 5′ UTR and first exon deletion called Plc21C^p60A (Weinkove et al., 1999). Alone, neither heterozygous deletion diminishes evoked neurotransmission (Figures 7C,D). Heterozygous Plc21C/+ animals in a GluRIIA^SP16 mutant background do have a mild increase in quantal content compared to Plc21C/+ genetic controls (Figure 7E). However, this increase is not nearly as robust as one would expect for a GluRIIA condition (Figure 7E). This result indicates a partial impairment of NMJ homeostatic compensation (Figures 7C,E). Collectively, the data utilizing hypomorphic Plc21C conditions support the idea that presynaptic Plc21C gates the sustained expression of homeostatic plasticity.

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, Gαq^f04219, Gαq^221c, and Gαq²⁸. Each Gαq null 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 GluRIIA^SP16 mutant background (Figure 8B).

Each Gαq/+ mutant condition shows a small, but significant decrease in NMJ quantal size compared to wild-type controls (Figure 8A). We wondered if this decrease in quantal size could reflect a decrease in glutamate receptor expression levels and a postsynaptic role for Gαq. This possibility could potentially confound our analyses of homeostatic plasticity. Therefore, we stained Gαq/+ mutant NMJs with anti-GluRIII and anti-GluRIIA antibodies and quantified stained receptor levels per unit synapse area, as before (Figure 3). We find no significant decrease in glutamate receptor levels in Gαq/+ mutant NMJs (Figures 8C,D). In fact, for most measures, there appears to be a slight increase or a statistical trend toward a slight increase of glutamate receptor subunit expression (Figure 8C). This single experiment does not rule out possible postsynaptic functions for Gαq. However, our collective findings are consistent with the possibility that Gαq and Plc21C play concerted roles in the execution of homeostatic plasticity. Further experiments will be required to test if there is a direct regulatory relationship in the presynaptic nerve between the two proteins.

The concept of expressing GAL4-responsive UAS transgenes both pre- and post-synaptically at the NMJ is not novel, but it is useful. In our study we add the UAS-GluRIII[RNAi] transgene to provide a homeostatic challenge to the NMJ. We have utilized T15 by crossing it with UAS-RNAi transgenes and interrogating the synapse electrophysiologically. Variations on this genetic theme beyond RNAi screening are possible. For example, to test whether NMJ overexpression of a particular UAS-driven target gene impairs HSP, a single cross to T15 would be sufficient. In fact, an “overexpression screen” could be conducted by crossing T15 to Drosophila stocks carrying transposons with UAS insertions in orientations that drive gene expression (Rorth, 1996; Beinert et al., 2004; Bellen et al., 2004; Thibault et al., 2004; Staudt et al., 2005). For another example, viable loss-of-function mutations on the X-chromosome could be directly assayed for roles in homeostatic plasticity. This could be done by crossing mutant females to BG57-GAL4, UAS-GluRIII[RNAi] males and examining the mutant male larval progeny by electrophysiology. Chromosomal deficiency or duplication screens are also feasible by employing the BG57-GAL4, UAS-GluRIII[RNAi] chromosome in a similar crossing scheme. With GluRIIA genetic mutations, such screens or approaches require more extensive genetic work. The single-cross utility of a BG57-GAL4, UAS-GluRIII[RNAi] chromosome makes large-scale screens to isolate factors involved in developmental or long-term HSP possible. Finally, T15 does present hurdles. For example, with T15, RNAi-targeted genes are impaired both pre- and post-synaptically. Therefore, the screen itself provides limited information regarding tissue specificity of new homeostatic factors. Follow-up experiments examining positives in the pre- and/or post-synaptic compartments need to be done to garner this information. The advantage of being able to identify both pre- and post-synaptic regulators of HSP in a single-generation crossing scheme outweighs the disadvantage of follow-up work.

Ca_V2 channels help to evoke fast neurotransmission by allowing an influx of calcium into the presynaptic terminal upon neuronal depolarization (for comprehensive reviews on Ca_V channels, see Catterall et al., 2005; Zamponi, 2005). Considerable data from multiple synaptic preparations suggest that Ca_V2 channels also gate neurotransmitter release in a homeostatic fashion (Frank, 2014b). At the Drosophila NMJ, increased Ca_V2 function could be directly responsible for a homeostatic potentiation of presynaptic neurotransmitter release (Frank et al., 2006). Yet it is unclear precisely how this process works. It is not known if increased neurotransmission results from trafficking of new Ca_V2 channels to the synapse, a modulation of functional properties of Ca_V2 channels already present at the synapse—or even a modulation of Ca²⁺-triggered signaling processes downstream of Ca_V2 function.

Prior data examining cac loss-of-function mutants have not answered this question. Hypomorphic cac^S; GluRIIA double mutant NMJs have no significant increase in quantal content compared to cac^S mutant NMJs (Frank et al., 2006). Corroborating this finding, presynaptic Ca²⁺ imaging experiments demonstrate that postsynaptic glutamate receptor impairment correlates with an increase of presynaptic calcium transients (Müller and Davis, 2012)—and cac^S mutant NMJs are particularly resistant to potentiation of function after glutamate receptor impairment (Müller and Davis, 2012). A weaker hypomorphic allele, cac^TS2 can impair homeostatic compensation induced by GluRIIA deletion, but only if the cac^TS2; GluRIIA animals are reared at a high temperature (29°C)—likely lowering effective cac gene function (Frank et al., 2006). Together, these data suggest that absolute amount of Cac/Ca_V2 function may correlate with the homeostatic capacity of the synapse. Yet additional observations counter this idea. For instance, cac^S/+ heterozygous NMJs have nearly normal neurotransmission—significantly better than cac^TS2 homozygous NMJs—but cac^S/+; GluRIIA NMJs raised at 22°C have partially impaired synaptic homeostasis, while cac^TS2; GluRIIA animals reared at 22°C have intact homeostatic compensation (Frank et al., 2006). Additionally, heterozygous null cac^HC129/+ animals display normal homeostatic compensation (Frank et al., 2009). Taken altogether, it is possible that the presence of dysfunctional Ca_V2 channel subunits (e.g., Cac^S) throughout development—rather than the absolute degree of Ca_V2 dysfunction—could be the driving factor behind impairments of synaptic homeostasis.

Given these background data, it was important to check if severe diminishment of cac function is sufficient to impair homeostatic plasticity at the NMJ. Our analyses of T15 × UAS-cac[RNAi] NMJs demonstrate that synaptic homeostasis is still intact across multiple concentrations of [Ca²⁺]_e, even when there is severely defective baseline neurotransmission (Figure 5). It appears if the Ca_V2 channels present at the synapse are wild-type channels, the NMJ retains its homeostatic capacity—even if the number of functional Ca_V2 channels that successfully make it to the terminal is sharply reduced. This result is inconsistent with a model in which Ca²⁺-directed signaling downstream of Ca_V2 activity drives homeostatic plasticity. However, it does not resolve the question of whether the homeostatic potentiation of release normally proceeds through the addition of functional channels to the synapse or through modulation of channel properties. According to a “slot model” of Ca_V2.1 channel positioning, at presynaptic terminals there may exist only a limited number of channel-type-specific slots (Cao et al., 2004). If this were true at the Drosophila NMJ—and if all slots were normally occupied—it would point to modulation of Ca_V2 gating properties as a probable mechanism.

A recent paper corroborates this idea. Mutations in two Drosophila genes encoding epithelial sodium (ENaC) channels block both the rapid induction and long-term expression of synaptic homeostasis—importantly, by impairing necessary increases in presynaptic calcium influx (Younger et al., 2013). Interestingly, when the ENaC antagonist benzamil is acutely applied to GluRIIA mutant NMJs, presynaptic calcium influx is dramatically decreased. By contrast, benzamil has no effect on presynaptic calcium influx at wild-type NMJs (Younger et al., 2013). Collectively, these observations are consistent with a model in which Ca_V2 gating properties are enhanced during synaptic homeostasis, with ENaC channels mediating this enhancement (Younger et al., 2013).

CSP was originally identified in Drosophila from an antibody-based approach as a synapse-specific antigen (Zinsmaier et al., 1990). Follow-up work has demonstrated that CSP executes multiple synaptic functions (Zinsmaier, 2010; Donnelier and Braun, 2014). Interestingly, loss of CSP function appears to exacerbate synaptic problems over developmental time in organisms as varied as Drosophila (Zinsmaier et al., 1994), mice (Fernandez-Chacon et al., 2004; Garcia-Junco-Clemente et al., 2010), and humans (Benitez et al., 2011; Noskova et al., 2011; Velinov et al., 2012). These facts could be in line with data that CSP is required for the long-term maintenance of homeostatic plasticity—even though CSP is not required for its short-term induction (Dickman and Davis, 2009) (Figure 6F).

What is the specific role of CSP during the execution of HSP? This is unclear. Based on the literature, there are several plausible connections. As stated above, modulation of Ca_V2 functional properties could be key to synaptic homeostasis, and CSP has previously been implicated in the modulation of Ca_V2-type calcium channel function (Gundersen and Umbach, 1992). We observe a genetic interaction between Csp and cac loss-of-function mutations (Figure 6G)—however, other studies have failed to find evidence for direct Ca_V regulation by CSP, including studies at Drosophila synapses (Morales et al., 1999; Dawson-Scully et al., 2000). Therefore, other modes of regulation that genetically interact with cac are likely. For example, CSP has been implicated in the regulation of Ca²⁺-triggered exocytosis (Dawson-Scully et al., 2000; Bronk et al., 2001; Dawson-Scully et al., 2007), and recent studies have investigated possible CSP chaperone interactions with SNARE molecules. SNAP25 (t-SNARE) expression is significantly decreased in mice lacking CSPα (Chandra et al., 2005; Zhang et al., 2012). This represents a possible link to homeostatic plasticity because reduction of SNAP25 levels compromises homeostatic signaling at the Drosophila NMJ (Dickman et al., 2012).

Our data demonstrate that partial losses of Gαq and Plc21C gene function partially impair the homeostatic response (Figures 7, 8). The data open several possibilities for presynaptic control of neurotransmitter release and homeostatic plasticity. By one model, diminishment of PLCβ signaling could result in excess PIP₂ at the synapse at the expense of IP₃ and membrane-bound DAG. In other systems, it is well known that DAG activates protein kinase C (PKC), which in turn, activates Ca_V2 channels (Tedford and Zamponi, 2006). In this way, DAG-mediated regulation of Ca_V2 function could be a key determinant in homeostatic plasticity. However, by a second model, it has been reported that Plc21C gene function and DAG regulate the abundance of synaptic DUNC-13 at the Drosophila NMJ (Aravamudan and Broadie, 2003). DUNC-13 is a highly conserved protein critical for proper maintenance of SNARE-mediated vesicle exocytosis (Jahn and Fasshauer, 2012; Kasai et al., 2012; James and Martin, 2013), a process previously implicated in homeostatic plasticity at the NMJ (Dickman et al., 2012). There exists evidence that DUNC-13 family molecules could regulate the dynamics of the readily releasable pools (RRP) of synaptic vesicles (Chen et al., 2013). RRP size enhancement is another process implemented during synaptic homeostasis at the NMJ (Weyhersmüller et al., 2011; Müller et al., 2012). For a third model, depressed Gαq/PLCβ signaling could result in lower levels of synaptic IP₃. Canonically, intracellular IP₃ binds to IP₃ receptors and liberates Ca²⁺ from intracellular stores. In turn, Ca²⁺-dependent signaling cascades could permit the expression of homeostatic plasticity. Finally, PIP₂ itself is known to bimodally regulate Ca_V2 channels: Ca_V2 channels are stabilized by low levels of PIP₂, but inhibited by high levels of PIP₂ (Wu et al., 2002). Interestingly, an amino-acid substitution on the intracellular side of transmembrane domain III S6 on Ca_V2.1 alters its affinity for PIP₂. This is the same domain containing the amino-acid substitution in cac^S (Smith et al., 1998; Zhen et al., 2006). More experiments are required to define a precise mechanism of Gαq/PLCβ signaling in the execution of synaptic homeostasis at the NMJ. Regardless of mechanism, the results implicating Gαq and Plc21C in synaptic homeostasis suggest that a synaptic signal controls presynaptic excitability through a G-Protein Coupled Receptor (GPCR)/Gαq/PLCβ-mediated signaling pathway. This is an exciting possibility that is open to exploration via additional genetic approaches in Drosophila, taking advantage of tools like those we generated for this study.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.