Presynaptic homeostatic plasticity (PHP) is an evolutionarily conserved form of homeostatic control that is expressed in organisms ranging from fly to human (Cull-Candy et al., 1980; Plomp et al., 1992; Davis, 2013; Wang et al., 2011), at both central and peripheral synapses (Liu and Tsien, 1995; Davis and Goodman, 1998; Burrone et al., 2002; Thiagarajan et al., 2005; Kim and Ryan, 2010; Zhao et al., 2011; Davis, 2013; Henry et al., 2012; Jakawich et al., 2010). PHP can be induced in less than ten minutes and is expressed as a dramatic increase in synaptic vesicle fusion (≥200%) at a fixed number of presynaptic release sites.

An unexplained, emergent property of PHP is the preservation of short-term release dynamics during a stimulus train, referred to here as short-term plasticity or ‘STP’ (Figure 1; see Weyhersmüller et al., 2011; Müller et al., 2012b; Müller et al., 2015; Orr et al., 2017). STP is a fundamental property of neural coding, underlying behaviorally relevant circuit-level computations (Davis and Murphey, 1994). Indeed, STP is described as being ‘…an almost necessary condition for the existence of (short-lived) activity states in the central nervous system’ (Von der Malsburg, 1986; as recently quoted in Taschenberger et al., 2016). Thus, the fact that STP is held constant during the expression of PHP may be essential to the life-long stabilization of neural circuit function and animal behavior. But, it remains fundamentally unknown how presynaptic release can be rapidly doubled at a fixed number of active zones while maintaining constant short-term release dynamics.

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Two processes within the presynaptic terminal are known to be required for the homeostatic potentiation of vesicle release: 1) an increase in presynaptic calcium influx controlled by ENaC channel insertion in the presynaptic membrane (Younger et al., 2013; Orr et al., 2017) and 2) an increase in the readily releasable pool of synaptic vesicles that requires the presynaptic scaffolding proteins RIM and RBP (Müller et al., 2012b; Davis and Müller, 2015; Müller et al., 2015). It has been proposed that the combined effects of elevated presynaptic calcium and an increased supply of release ready vesicles are sufficient to achieve PHP. But, current models have yet to address how short-term release dynamics are stabilized.

STP can be strongly influenced by the partition of the readily releasable vesicle pool into two functional subclasses: (1) docked vesicles that have a low intrinsic calcium sensitivity of release that are referred to as ‘primed’ and (2) docked vesicles that have a relatively higher intrinsic calcium sensitivity and are referred to as ‘super-primed’. During a stimulus train, super-primed vesicles will dominate release during the first few action potentials and primed vesicle will dominate subsequent release (Taschenberger et al., 2016; Lee et al., 2013; Müller et al., 2010). Thus, a synapse harboring a large proportion of super-primed vesicles will favor a high initial release rate followed by synaptic depression while a synapse harboring a small proportion of super-primed vesicles will be prone to facilitation of release, followed by subsequent synaptic depression (Taschenberger et al., 2016; Lee et al., 2013). There is both pharmacological and genetic evidence in support of this model. Genetic mutations that impair vesicle super-priming convert depression-prone, high-release probability synapses into low-release probability synapses that express short-term facilitation (Frank et al., 2006; Deák et al., 2009; He et al., 2017). The calcium sensitivity of vesicle fusion is also highly sensitive to phorbol esters (PdBu), which lower the fusion barrier to release (Taschenberger et al., 2016; Lee et al., 2013). Application of PdBu, which is considered to drive the super-priming process, dramatically potentiates vesicle release and leads to enhanced short-term synaptic depression (Wang et al., 2016; Lee et al., 2013).

Here, we provide evidence that the stabilization of STP during PHP is achieved by maintaining a constant ratio of primed to super-primed synaptic vesicles. First, we confirm that STP is precisely preserved during the expression of PHP. Second, we document a PdBu sensitive release mechanism at the Drosophila NMJ and provide evidence that the fraction of super-primed vesicles is maintained during the full extent of PHP. Third, we provide molecular insight into how a constant fraction of super-primed vesicles is maintained during the expression of PHP. We demonstrate that Unc18 has an evolutionarily conserved function during the rapid induction of PHP. We show that Unc18 function during PHP is facilitated by the activity of presynaptic RIM and, remarkably, is antagonized by presynaptic Syntaxin. Based upon these and other data, we present a new model for the homeostatic control of synaptic vesicle release that is based upon the regulated control of Unc18 levels at the presynaptic release site, acting in concert with the priming activity of RIM to stabilize STP in the presence of a homeostatic doubling presynaptic neurotransmitter release.

We begin by documenting how the short-term dynamics of presynaptic release (short-term plasticity or STP) are held constant during the expression of PHP. We rapidly induce PHP by application of sub-blocking concentrations of the glutamate receptor antagonist philanthotoxin (PhTx, 10–20 µM). PhTx causes a ~ 50% decrease in miniature excitatory postsynaptic potential (mEPSP) amplitude and induces a homeostatic increase in presynaptic vesicle release that precisely counteracts the change in mEPSP amplitude, maintaining the amplitude of action potential evoked neurotransmitter release at baseline levels (Frank et al., 2006; Davis, 2013). As shown in Figure 1A, even at elevated extracellular calcium (3.0 mM [Ca²⁺]_e), excitatory postsynaptic currents (EPSC) are precisely maintained at control levels following application of PhTx (see Müller and Davis, 2012a; see below for further quantification).

Next, we characterize the expression of STP at two concentrations of external calcium (0.75 mM and 3.0 mM [Ca²⁺]_e), doing so in the presence and absence of PhTx to induce PHP (Figure 1A–C). Note that the effects of non-linear summation prevents accurate calculation of presynaptic release at calcium concentrations in excess of approximately 0.3 mM. As such, measurement of synaptic currents in two electrode voltage clamp configuration are essential for experiments where extracellular calcium exceeds 0.3 mM. As expected (Zucker and Regehr, 2002), STP is strongly dependent on the concentration of external calcium, showing facilitation at 0.75 mM [Ca²⁺]_e and depression at 3.0 mM [Ca²⁺]_e (Figure 1A,B). Application of PhTx causes an approximate doubling of presynaptic release during PHP that is similar in magnitude to the change in release observed when comparing wild type neurotransmission at 0.75 mM [Ca²⁺]_e and 3.0 mM [Ca²⁺]_e. However, the expression of PHP occurs without a change in release dynamics (Figure 1B,C). Specifically, there is no statistically significant change in paired pulse ratio comparing the presence and absence of PhTx, and this is true at both 0.75 mM [Ca²⁺]_e where facilitation dominates (p = 0.65; Student’s t-test, two tailed) and at 3.0 mM [Ca²⁺]_e where depression dominates (Figure 1C; p = 0.45; Student’s t-test, two tailed). Thus, PHP is achieved by doubling synaptic vesicle release without altering the expression of STP, confirming prior observations in this system (Weyhersmüller et al., 2011; Müller et al., 2012b; Müller et al., 2015; Orr et al., 2017). We sought to understand how this effect could be achieved.

As outlined in the introduction, STP is strongly influenced by the partition of the readily releasable vesicle pool into two functional subclasses: (1) docked vesicles that have a low intrinsic calcium sensitivity of release that are referred to as ‘primed’ and (2) docked vesicles that have a relatively higher intrinsic calcium sensitivity and are referred to as ‘super-primed’. A synapse with a large fraction of super-primed vesicles will show synaptic depression while a synapse with a small number of super-primed vesicles will facilitate. We hypothesize that the ratio of primed to super-primed vesicles is somehow maintained during the expression of PHP, allowing for a dramatic potentiation of vesicle release without altering the dynamics of release during a stimulus train. To test this hypothesis, we used phorbol esters to probe the ratio of primed to super-primed vesicles at the Drosophila NMJ.

It is well established that phorbol esters (PdBu) decrease the energy barrier to synaptic vesicle fusion, effectively converting the docked/primed vesicle pool into a super-primed, high release probability state (Lee et al., 2013; Taschenberger et al., 2016). Thus, magnitude of PdBu-dependent potentiation of presynaptic release is proportional to the size of the pool of synaptic vesicles that reside in a docked/primed, but not super-primed state. If PHP-dependent potentiation of presynaptic release preserves the ratio of primed to super-primed vesicles, then the effects of PdBu should be the same prior to and following application of PhTx to the synapse.

We first characterize the use of PdBu at the Drosophila NMJ. At 0.75 mM [Ca²⁺]_e, PdBu strongly potentiates both evoked and spontaneous vesicle fusion and converts STP from facilitation to depression (Figure 1A,D,E). Specifically, PdBu has no effect on mEPSP amplitude (Figure 1E), causes a significant increase in EPSC amplitude (Figure 1E) and a corresponding increase in quantal content (Figure 1E). We then express the effects of PdBu as a percent change compared to baseline in the absence of PdBu, observing a significant ~140% increase in release (Figure 1E, right; p<0.05). Three further effects were also quantified. First, application of PdBu causes a significant decrease in the paired-pulse ratio (Figure 1D; p<0.05). Second, we show that presynaptic release converges to a statistically similar steady state in the presence and absence of PdBu during a prolonged stimulus train (Figure 1—figure supplement 1; p>0.1, Student’s t-test, two-tailed; comparison of final three data points of stimulus train). Third, just as observed at the mammalian central synapses, we demonstrate that the effects of PdBu are dependent on the concentration of extracellular calcium (Lee et al., 2013; Taschenberger et al., 2016). When recording at elevated extracellular calcium (3 mM [Ca²⁺]_e), the effect of PdBu on EPSC amplitude is absent (Figure 1F). These data are consistent with the existence of a finite pool of docked vesicles that are uniformly accessed by action-potential induced release at elevated calcium, rendering PdBu without effect. Thus, PdBu functions comparably in Drosophila and at mammalian central synapses. And, by comparing the effects of PdBu on synaptic transmission at 0.75 mM [Ca²⁺]_e we can gain an estimate of the fraction of vesicles that exist within the primed versus super-primed state. Specifically, the magnitude of PdBu-mediated potentiation at 0.75 mM [Ca²⁺]_e is proportional to the number of vesicles that remain in the primed (not super-primed) state.

We next examined the effects of PdBu at synapses previously incubated in PhTx. Once again, at 0.75 mM [Ca²⁺]_e, PhTx causes a decrease in mEPSP amplitude, an increase in quantal content and no change in evoked EPSC amplitude (Figure 1E). When PdBu is applied after PhTx, there is no further change in mEPSP amplitude, compared to PhTx alone, as expected (Figure 1E). However, we find that application of PdBu enhances EPSC amplitudes in the presence of PhTx compared to controls with or without PhTx. The consequence is that quantal content is significantly increased compared to PdBu alone and compared to PhTx alone. Indeed, quantal content is potentiated 3-fold compared to baseline release in wild type (from approximately 200 vesicles per action potential to 600 vesicles per action potential). However, when we calculate the percent change in release caused by PdBu compared to PhTx alone, we find that release is potentiated by ~140%, the same percentage increase caused by PdBu applied to a wild type synapse (Figure 1E). Thus, the proportion of PdBu-sensitive vesicles remains constant following the induction of PHP. Since PHP in a wild type animal can be expressed without a change in short-term plasticity, we propose that the ratio of super-primed to primed vesicles remains constant during expression of PHP (Figure 1G).

Rop is required in the rapid induction of presynaptic homeostasis

In Drosophila, there is a single neuronally expressed unc-18 gene, termed Rop (Ras opposite). Throughout this paper, from this point onward, we refer to the Drosophila gene as Rop and orthologues in other species as unc-18. Prior genetic analyses isolated and characterized numerous mutations in the Rop gene, demonstrating that Rop is essential for spontaneous and evoked synaptic vesicle fusion, in agreement with data from other species (Harrison et al., 1994; Wu et al., 1998; Toonen and Verhage, 2007). We have taken advantage of previously characterized mutations in Rop to study the role of Unc-18 in presynaptic homeostatic plasticity (PHP).

We assayed PHP in two independent, heterozygous Rop loss-of-function mutants: 1) a previously characterized null allele (Rop^G27; Harrison et al., 1994; see Figure 2A) and 2) a small chromosomal deficiency that deletes the entire Rop gene locus (Df^(3L)BSC735, referred to hereafter as Df^Rop; Cook et al., 2012; Figure 2A). In both Rop^G27/+ and Df^Rop/+ heterozygous mutants, PHP is significantly suppressed compared to wild type (Figure 2B–2I). Specifically, upon application of PhTx, Rop^G27/+ mutants show an enhancement of quantal content (Figure 2H; p<0.01), but the magnitude of this enhancement is statistically significantly smaller than that observed in wild type (Figure 2I; p<0.001). Consistent with impaired homeostatic plasticity, EPSP amplitudes are significantly reduced in the presence of PhTx compared to baseline EPSP amplitudes (Figure 2C and G; p<0.05; p<0.0001). Notably, baseline release, recorded in the absence of PhTx, is unaltered in both Rop^G27/+ and Df^Rop/+ heterozygous mutants at the concentration of external calcium used in this experiment (0.3 mM [Ca²⁺]_e) (Figure 2C and G). Thus, two independently derived heterozygous loss-of-function mutants suppress PHP without an effect on baseline neurotransmission, arguing that PHP is highly sensitive to Rop gene dosage.

Figure 2

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Neuronally expressed rop is required for presynaptic homeostasis

The unc-18 gene is broadly expressed and has been shown to participate in membrane trafficking events outside the nervous system (Hata and Südhof, 1995; Riento et al., 2000; Toonen and Verhage, 2003). As such, Rop could function either pre- or postsynaptically during PHP. Therefore, we knocked down Rop expression specifically in the nervous system using the Gal4/UAS expression system. We took advantage of a previously characterized UAS-Rop-RNAi transgene (P{GD1523}v19696; Dietzl et al., 2007) to knock-down Rop specifically in neurons (c155-Gal4; Lin and Goodman, 1994). First, we demonstrate that PHP is strongly suppressed when UAS-Rop-RNAi is driven presynaptically (Figure 3A–E). Thus, Rop is necessary presynaptically for PHP. We note that presynaptic Rop knockdown also causes a decrease in baseline EPSP amplitude and quantal content by ~30% (Figure 3C; p<0.01), suggesting that presynaptic knockdown depletes Rop protein levels more substantially than that observed in the heterozygous Rop null mutant (Figure 2F–I). Indeed, when we compare wild type, Rop^G27/+ and presynaptic Rop knockdown, we find a progressively more severe decrease in mEPSP frequency that is consistent with progressively more severe depletion of Rop protein (Figure 3F). Thus, Rop knockdown can be considered a strong hypomorphic condition, supporting the conclusion that presynaptic Rop is essential for robust expression of PHP.

Figure 3

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Separable activity of rop during baseline transmission and PHP

Although PHP is selectively impaired in the Rop^G27/+ heterozygous mutant at 0.3 mM [Ca²⁺]_e, it remains formally possible that loss of Rop places a limit on the number of vesicles that can be released per action potential (quantal content) and, thereby, indirectly restricts the expression of PHP. To address this possibility, we asked whether baseline release and PHP change in parallel as a function of altered extracellular calcium in the Rop^G27/+ heterozygous mutations. Experiments were conducted at 0.3 mM [Ca²⁺]_e, 0.75 mM [Ca²⁺]_e, 1.5 mM [Ca²⁺]_e and 3.0 mM [Ca²⁺]_e. First, we find that baseline neurotransmitter release is impaired at 0.75 as well as 1.5 and 3.0 mM [Ca²⁺]_e in the Rop^G27 /+ heterozygous mutants compared to wild-type (Figure 4). But, there remains a highly cooperative relationship between extracellular calcium and vesicle release in the Rop^G27 /+ heterozygous mutants (Figure 4F). Next, we demonstrate that PHP is suppressed both at 1.5 mM (Figure 4A–C) and 3.0 mM [Ca²⁺]_e (Figure 4D–E), as evidenced by an inability of EPSCs to be restored to baseline values in the presence of PhTx (Figures 4C and 3E) (p<0.05). When we calculate the percent suppression of PHP at 0.3, 1.5, and 3.0 mM [Ca²⁺]_e we find a constant level of PHP suppression even though baseline release increases ~10 fold over this range of extracellular calcium concentrations (Figure 4F). From this we can make two conclusions. First, since release remains sensitive to changes in external calcium, a ceiling effect cannot explain the defect in PHP observed in the Rop^G27 /+ heterozygous mutants. More specifically, the increase in release in Rop^G27/+ comparing 0.3 mM [Ca²⁺]_e with 3.0 mM [Ca²⁺]_e vastly exceeds the change in vesicular release that would be expected for full expression of PHP at 0.3 mM [Ca²⁺]_e (see Figure 2). The fact that PHP is consistently inhibited by ~30%, irrespective of the concentration of external calcium, demonstrates that the magnitude of PHP expression correlates directly with the levels of Rop expression, not with the magnitude of evoked release or extracellular calcium concentration, arguing for an essential role of Rop in the mechanisms of PHP.

Figure 4

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Rop dependent vesicle priming correlates with expression of PHP

Unc-18 has been implicated in several different stages of the release process, including vesicle docking, priming, fusion pore formation and regulation of the readily releasable pool (RRP) of synaptic vesicles (Weimer et al., 2003; Toonen et al., 2006; Gulyás-Kovács et al., 2007; Fisher et al., 2001; Toonen and Verhage, 2007). Since we observe a significant disruption of PHP in the heterozygous Rop^G27/+ mutant background, we sought to define the parameters of presynaptic release that are particularly sensitive to the heterozygous Rop^G27/+ mutant, highlighting those actions of Rop that best correlate with impaired PHP. First, we observe a decrease in the frequency of spontaneous mEPSP events in Rop mutants (Figure 3F). mEPSP frequency is decreased by 36% and 59% compared to wild-type in Rop^G27/+ and UAS-Rop-RNAi, respectively (Figure 3F). This is consistent with prior reports in Drosophila and other systems (Wu et al., 1998; Toonen et al., 2006; Patzke et al., 2015).

It has previously been shown that a homeostatic modulation of the readily releasable pool (RRP) of vesicles is required for the expression of PHP (Müller et al., 2012b). RRP size can be estimated through quantification of the cumulative EPSC amplitude during a high frequency stimulus train (60 HZ, 30 stimuli) at elevated extracellular calcium (1.5 mM; Figure 5) and back extrapolation according to published protocols (Schneggenburger et al., 1999; Müller et al., 2015). We demonstrate that the cumulative EPSC amplitude in the Rop^G27/+ heterezogous mutant is unaltered at baseline compared to wild-type (Figure 5B). Then, we demonstrate that the Rop^G27/+ mutants show normal modulation of the RRP following application of PhTx, as demonstrated the maintenance of the cumulative EPSC amplitude in the presence and absence of PhTx, which diminishes the amplitude of underlying unitary release events by ~50% (Figure 5B). Thus, at the Drosophila NMJ, the RRP is not sensitive to Rop haplo-insufficiency and Rop is not limiting for the homeostatic potentiation of the RRP.

Figure 5

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It remains apparent, however, that the initial EPSC of the stimulus train in Rop^G27/+ mutants, recorded in the presence of PhTx, is smaller than that observed in wild type (Figure 5A–B see also Figure 3C for quantification). Thus, the homeostatic potentiation of the initial EPSC amplitude is disrupted in the Rop^G27/+ mutants, whereas the homeostatic potentiation of the RRP is normal. This is quantified by dividing the initial EPSC amplitude by the cumulative EPSC amplitude during the stimulus train, a parameter referred to as P_train (Figure 5C). It is apparent that release dynamics are altered as a consequence of decreased P_train in the Rop^G27/+ mutant in the presence of PhTx. Super-priming should contribute significantly to vesicle release in response to the first action potential of a stimulus train. Thus, one explanation for this result is that the super-primed vesicle pool has not been appropriately expanded in the Rop^G27/+ mutant. During the stimulus train, elevated intra-terminal calcium could overcome Rop^G27/+ haploinsufficiency by driving the normal priming process, leading to expansion of the RRP in the presence of PhTx. These data argue that Rop may be essential for the expansion of the super-primed vesicle pool during expression of PHP. It is worth noting that this is the first example of a significant alteration in presynaptic release dynamics that is specific to the induction of PHP.

A genetic interaction of rop and RIM during PHP

There has been considerable progress identifying presynaptic proteins that are necessary for presynaptic homeostatic plasticity (Davis, 2013; Müller et al., 2015). These genes include the active zone associated scaffolding proteins RIM (Rab3 Interacting Molecule) and RBP (RIM Binding Protein), both of which are components of a proposed molecular priming pathway within the presynaptic nerve terminal (Südhof, 2012a). If Rop is integrally involved in the mechanisms of PHP within the presynaptic terminal, potentially acting in the priming process during PHP, then we might expect genetic interactions with rim and RBP.

Genetic interactions were performed by assaying heterozygous, null mutations alone and in comparison to the effects observed in a double heterozygous condition (Frank et al., 2009; Müller et al., 2015). We first assayed baseline release. At 0.3 mM [Ca²⁺]_e, baseline release was normal in both Rop^G27/+ and rim¹⁰³/+ (Figure 6B). However, release (quantal content; 6C) was diminished by nearly 50% in the double heterozygous condition (WT QC = 34.8 ± 0.8; Rop^G27/+ QC = 30.1±1.6; rim¹⁰³/+ QC = 32.3±1.4; Rop^G27/rim¹⁰³ QC = 18.7 ± 1.3) (Figure 6C). This is a very strong genetic interaction for baseline release, even by comparison with previously published genetic interactions of other genes with rim (Wang et al., 2016; Orr et al., 2017; Hauswirth et al., 2018).

Figure 6

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Next, we assessed PHP. Upon application of PhTx, a heterozygous null mutation in rim (rim¹⁰³/+) causes a suppression of PHP (Figure 6D). This suppression is similar in magnitude to that observed in the Rop^G27/+ heterozygous null mutation (Figure 6D). However, when we examine a double heterozygous mutant with Rop^G27 /+ placed in trans to rim¹⁰³ /+, PHP is completely blocked (Figure 6C,D). To underscore the robustness of this genetic interaction, we plot the relationship between mEPSP amplitude and quantal content for individual recordings (Figure 6E). Each data point represents the average mEPSP and quantal content for a single NMJ recording (see also Hauswirth et al., 2018). In wild type, data can be fit with a line representing the homeostatic process. We also present dotted lines that encompass 95% of all of the data points in the wild type graph. This fit and 95% data interval are used to compare data distributions to other mutant backgrounds. In both of the single heterozygous mutants (Rop^G27 /+ and rim¹⁰³ /+) the data points lie below the best-fit line for wild type, but are largely retained within the 95% interval, consistent with a minor suppression of PHP when all data points are averaged (Figure 6C,D). However, in the double heterozygous animal (Rop^G27/rim¹⁰³) it is clear that PHP fails and the majority of data points in the presence of PhTx reside outside the interval that contains 95% of all wild type data points. More specifically, for heterozygous null Rop and rim recordings in the presence of PhTx, 25% and 16% of these individual data points in the presence of PhTx fall outside of the lines encompassing 95% of wild-type data (Figure 6E). In the double heterozygous mutants, 87% of PhTx data fall outside of the 95% confidence intervals (Figure 6E). This genetic interaction, referring specifically to PHP expression, is also conserved at elevated calcium levels (1.5 and 3.0 mM calcium; data not shown).

At baseline, there is a strong synergistic interaction between Rop and RIM, suggesting that both are functioning to control the same process relevant to vesicle release, perhaps synaptic vesicle priming (Gulyás-Kovács et al., 2007; Koushika et al., 2001). It remains unclear whether rop and rim function in the same genetic pathway based on this genetic interaction. The data are equally compatible with parallel pathways converging on the mechanism of PHP. Regardless, our data underscore that Rop has an essential function during the process of PHP. We consider this a particularly important line of reasoning, since it is not feasible to eliminate Rop and assess a block in PHP.

Next, we sought to understand the specificity of the genetic interaction between Rop and rim. To do so, we performed a series of additional genetic interactions with other genes previously implicated in the presynaptic synaptic vesicle priming process. First, we asked whether Rop shows a genetic interaction with RIM Binding Protein (RBP). RBP biochemically interacts with RIM and is thought to form an extended presynaptic scaffold (Wang et al., 2000; Liu et al., 2011). In Drosophila, RBP is also essential for both baseline release and PHP (Müller et al., 2015). But, the mechanism by which RBP participates in PHP is distinct from the mechanism by which RIM participates in PHP (Müller et al., 2015). The heterozygous null mutation in rbp/+ has no effect on baseline transmission at 0.3 mM external calcium, consistent with prior observations (Müller et al., 2015) (Supplementary file 1). We then demonstrate that PHP is also normal in the Rop^G27/rbpSTOP^STOP1 double heterozygous mutant (Figure 6F). This is evidence that there is specificity to the rop interaction with rim during PHP. It is worth noting that PHP is highly variable in the Rop^G27/rbpSTOP^STOP1 double heterozygous mutant, preventing us from making any additional conclusions regarding whether PHP is similar to, or different from wild type.

Next, we asked whether rim genetically interacts with Unc-13 during baseline neurotransmitter release and PHP. Munc-13 is known to biochemically interact with RIM (Betz et al., 2001). Alone, the dunc-13^P84200/+ heterozygous null mutation has no effect on baseline transmission or PHP (Supplementary file 1). Remarkably, the double heterozygous condition of rim and dunc-13 also has no effect on baseline transmission or PHP (Supplementary file 1 and Figure 6G). A similar set of findings is observed when we tested a double heterozygous condition of rim with rbp (Supplementary file 1 and Figure 6H). While baseline transmission is decreased in the double heterozygous combination of rim/+ and rbp/+, we find that PHP is normal, confirming previously published data (Müller et al., 2015). It is somewhat surprising that the rim/+; unc13/+ double heterozygous mutant has significantly more PHP than observed in rim/+ alone, perhaps relating to the functional importance of the RIM-Unc13 biochemical interaction during PHP. Regardless, when taken together, our results underscore the specificity and importance of the genetic interaction between rop and rim and the relevance of rop to the mechanisms of PHP.

Loss of rop and rim limits the super-primed vesicle pool

We note that heterozygous Rop^G27/+ mutants have a defect in PHP that is apparent on the first action potential of a stimulus train, but the homeostatic expansion of the RRP is apparent upon further stimulation (Figure 5). Both Rop and Rim are well-established molecular players that contribute to synaptic vesicle priming (Südhof, 2012a; Cook et al., 2012). We returned to the use of PdBu to assess whether the block of PHP in the Rop^G27/rim¹⁰³ double heterozygous mutant is correlated with a deficit in vesicle super-priming. As outlined above, the magnitude of PdBu-dependent potentiation in response to a single action potential should reflect the balance of primed to super-primed vesicles. A large PdBu effect argues for a smaller pool of super-primed vesicle pool. We compared wild type to the Rop^G27/rim¹⁰³ double heterozygous mutant, recording in the presence and absence of PdBu (Figure 7). Experiments were performed at 0.75 mM [Ca²⁺]_e, a condition in which PdBu potentiates wild type synapses by ~165% (Figure 7A–D). Next, we demonstrate that application of PdBu to each heterozygous mutant alone, either Rop^G27/+ or rim¹⁰³/+, is identical to wild type (Figure 7C,D; p>0.1 ANOVA). Then, we demonstrate that PdBu has a dramatically increased effect size when applied to the Rop^G27/rim¹⁰³ double heterozygous mutant, potentiating release by more than 350%, a dramatically increased effect size compared to application of PdBu to wild type and single heterozygous controls (Figure 7A–D, p<0.01; ANOVA). These data argue that the Rop^G27/rim¹⁰³ double heterozygous mutant limits the size of the super-primed vesicle pool, thereby impairing vesicle release in response to a single action potential, an effect that is strongly correlated with a block in the expression of PHP. Two other observations should be noted. First, the Rop^G27/rim¹⁰³ double heterozygous mutant recorded at 0.75 mM [Ca²⁺]_e has a greater effect on baseline release than observed at 0.3 mM [Ca²⁺]_e. The basis for this effect is unknown. Second, we show that the enhanced effects of PdBu on the Rop^G27/rim¹⁰³ double heterozygous mutant is consistent with the potentiation of release probability and an associated change in paired pulse ratio (Figure 7C). This simply confirms that PdBu is behaving as expected when applied to the Rop^G27/rim¹⁰³ double heterozygous mutant. Again, this type of synergistic genetic interaction is generally taken as evidence that two genes participate in the same process. It is not possible to make any conclusion regarding whether these genes are acting in a linear signaling pathway or in parallel signaling pathways. But, based on formal genetic argument, we are able to conclude that they likely converge to control the same processes at the presynaptic terminal, both the PdBu sensitive vesicle pool and PHP.

Figure 7

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Loss of syx1A rescues PHP in the heterozygous rop mutant background

Unc-18 is a well-established syntaxin binding protein (Hata et al., 1993; Pevsner et al., 1994; Halachmi et al., 1995). The Unc-18 interaction with Syntaxin is complex, including progressive interactions with closed and open conformations of Syntaxin. Ultimately, the Unc-18 interaction with open Syntaxin is thought to catalyze SNARE assembly, greatly decreasing the energy barrier to calcium-driven vesicle fusion. Indeed, Unc-18 binding to open Syntaxin is believed to be a prerequisite for efficient synaptic vesicle fusion (Dulubova et al., 2007; Deák et al., 2009). Based on the genetic interactions reported above, we expected that loss of syx1A would strongly enhance the loss of function phenotype of the heterozygous Rop^G27/+ mutant, resulting in diminished vesicle release and a block of PHP.

We acquired a previously published null mutation in syntaxin1A and examined baseline neurotransmitter release in a heterozygous syx1A mutant (syx1A^Δ229; Schulze et al., 1995). There is no change in baseline mEPSP amplitude, EPSP amplitude or quantal content in syx1A^Δ229/+ assayed at 0.3 mM [Ca²⁺]_e (Figure 8A–D). Thus, syntaxin is not haplo-insufficient for baseline release. Remarkably, the same is true for the double heterozygous condition, combining syx1A^Δ229/+ with Rop^G27/+. Baseline transmission is normal compared to wild type (Figure 8D). In other systems, it is estimated that Syntaxin1A is present in ~5 fold excess compared to the levels of synaptic Unc-18 (Graham et al., 2004). This could explain the lack of a genetic interaction at baseline.

Figure 8

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Next, we examined PHP. A heterozygous null mutation in syx1A (syx1A^Δ229/+) has normal PHP (Figure 8E), again consistent with a possible excess of Syntaxin protein at the release site. The heterozygous null mutation in Rop (Rop^G27/+) suppresses homeostatic potentiation (Figure 8E), confirming experiments presented earlier in this study. Remarkably, when we place Rop^G27 in trans to syx1A^Δ229 (double heterozygous condition), we find that homeostasis is fully expressed (Figure 8E). Impaired PHP caused by the Rop^G27/+ mutation is completely rescued to wild type levels. More specifically, in the double heterozygous mutants, EPSP amplitudes fully compensate in the presence of PhTx (Figure 8C) and there is a wild type level enhancement of quantal content (Figure 8E) in the presence of PhTx. Finally, the rescue of the Rop^G27/+ mutation by syx1A^Δ229/+ is not restricted to PHP. The rate of spontaneous vesicle fusion is also restored to wild type levels, underscoring the validity of this genetic rescue (Figure 8F). Note that there is, as yet, no indication that mEPSP frequency is directly responsible for the induction of PHP (Frank et al., 2006; Frank et al., 2009; Harris et al., 2015; Goold and Davis, 2007). Since we are examining heterozygous, null mutations, the most parsimonious conclusion is that Syntaxin normally functions to restrict the action of Rop that is required for PHP. Reducing the level of Syntaxin relieves a restriction on Rop activity and restores full expression of PHP. To provide further evidence for this surprising finding, we sought to disrupt the physical interaction of Rop and Syntaxin and assess whether this might also rescue the expression of PHP in the Rop^G27/+ mutant background.

Evidence that Syx1A restrains rop from participating in PHP

We performed an in vitro binding assay to confirm the biochemical interaction between Rop and Syntaxin1A. Recombinant wild-type Rop protein binds strongly to recombinant Syntaxin1A (GST-syx1A^ΔC) (Figure 9B–C) (K_D = 0.4 μM). Next, we surveyed previously characterized point mutations in the Rop gene, searching for candidate mutations that reside near the conserved Syntaxin binding interface (Figure 9A). Unc-18 binds to Syntaxin at two sites, an N-terminal region that interacts with the N-terminal peptide of Syntaxin and a helical region that represents a larger interaction surface. The Rop^G11 mutant harbors a point mutation (Asp 45→ Asn; Harrison et al., 1994) that resides within or directly adjacent to a predicted helical Syntaxin binding interface (Misura et al., 2000). Here, we demonstrate that this mutation completely abolishes in vitro binding between recombinant Rop^G11 mutant protein and recombinant GST-syx1A^ΔC protein (Figure 9B–C) (K_D = 36.1 μM).

Figure 9

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We next assayed baseline release in the Rop^G11 mutant, placed in trans to either a deficiency that removes the Rop gene locus, or the Rop^G27 null allele. To our surprise, both allelic combinations are viable to the third instar stage and we observe robust neurotransmitter release (Figure 9D). Specifically, we find that EPSC amplitudes are decreased by ~45% on average in both the Rop^G11/Df^Rop and Rop^G11/Rop^G27 allelic combinations compared to wild type (WT EPSC = 217.4 nA±7.3; Rop^G27/Rop^G11 EPSC = 134.2 nA±12.2; p<0.01; Df^Rop/Rop^G11 EPSC = 156.3 nA±17.2; p<0.01) (Figure 9F). This effect is more severe than the defect observed in the Rop^G11/+ heterozygous condition, consistent with loss of Rop function in both the Rop^G11/Df^Rop and Rop^G11/Rop^G27 allelic combinations. But, the presence of synchronous release is very surprising, since there is no wild type Rop protein at the synapse. From these data we conclude that the Rop^G11 mutation does not completely block all interactions between Rop and Syntaxin in vivo. The in vitro binding assay is predicted to test the binding of Rop to the closed conformation of Syntaxin. We speculate, based on work in other systems, that Rop^G11 could be localized to the SNARE complex through other molecular interactions, in vivo. Once at the release site, Rop^G11 mutant protein might still interact with the open conformation of Syntaxin and facilitate SNARE-mediated fusion.

Next, we assayed PHP in the Rop^G11/+ as well as the Rop^G11/Df^Rop and Rop^G11/Rop^G27 allelic combinations. In the presence of PhTx, EPSC amplitudes are restored to baseline values in the Rop^G11/+ mutant, indicative of fully functional PHP (Figure 9D–F). Remarkably, PHP is also fully expressed in both the Rop^G11/Df^Rop and Rop^G11/Rop^G27 allelic combinations (Figure 9D–F). Thus, the presence of the Rop^G11 allele fully rescues PHP in the presence of the Rop^G27/+ as well as the Df^Rop/+ alleles. While surprising, these data are entirely consistent with the observation that a heterozygous null mutation in syntaxin1A rescues PHP in the Rop^G27 mutant background (Figure 8). Taken together, our data are consistent with an emerging model in which Syx1A regulates the availability of Rop to participate in PHP (see discussion).

We have advanced our understanding of presynaptic homeostatic plasticity in several important ways. First, we demonstrate that rop (Unc-18) is essential for PHP, significantly extending prior evidence presented at the mouse NMJ (Sons et al., 2003). Thus, we argue that rop (Unc-18) is an evolutionarily conserved component of PHP signaling, enabling expression of PHP at the NMJ of Drosophila (this paper) and mice (Sons et al., 2003). Indeed, Unc-18 is the first molecular signaling component demonstrated to have a conserved, required function during PHP in both systems. Fly and mouse are separated by nearly 500 million years of evolution, suggesting that the molecular mechanisms of PHP may be as ancient as mechanisms that achieve action-potential induced, calcium-dependent, neurotransmitter release.

Second, our data demonstrate that Rop is limiting for the expression of PHP. Prior work at the mouse NMJ was the first to provide evidence that Unc-18 might participate in PHP (Sons et al., 2003). However, the prior work only included an analysis of Unc-18/+ heterozygous null mutant animals and, ultimately, could not rule out the formal possibility that Unc-18 was essential for baseline release and, as a secondary consequence, limited the expression of PHP. We provide several lines of evidence supporting the conclusion that Unc-18 has an activity that is necessary for PHP. For example, neurotransmitter release in the heterozygous Rop^G27 /+ mutant remains highly sensitive to changes in extracellular calcium. Yet, across a 10-fold range of extracellular calcium, PHP is suppressed by a constant fraction of ~30%. We also pursued a series of genetic interactions and provide evidence for a strong, specific, genetic interaction of Rop with rim, placing Unc-18 within a known PHP signaling framework.

Third, Unc-18 is the first integral component of the synaptic vesicle fusion apparatus to be linked to the expression of PHP. As such, our data provide a reasonable endpoint for the presynaptic homeostatic signaling system. In recent years, trans-synaptic signaling molecules have been shown to be required for PHP including Semaphorin/Plexin signaling (Orr et al., 2017), innate immune signaling (Harris et al., 2015) and signaling from the synaptic matrix (Wang et al., 2016). Many of these signaling systems interact with the presynaptic scaffolding protein RIM (Harris et al., 2015; Wang et al., 2016; Orr et al., 2017; Hauswirth et al., 2018). But, it was previously unknown what molecular mechanisms reside downstream of RIM, since RIM is a molecular scaffolding protein. We cannot formally conclude, based on our genetic data, that Rop functions downstream of RIM. However, it is clear that Rop is in a position to directly modulate the fusion apparatus and, as such, is very likely to mediate signaling that is localized to the active zone by the RIM-dependent cytomatrix.

The rescue of PHP by loss of Syntaxin or by disruption of Rop-Syntaxin binding is a surprise, but one that can be understood when placed in the context of work previously documented in other systems. A recent single molecule imaging study demonstrates that the majority of Unc-18 may reside outside of active zone with limited mobility (Smyth et al., 2013). It has also been shown that Syntaxin is present in large excess compared to Unc-18, and Syntaxin protein is broadly distributed beyond sites of synaptic vesicle fusion (Broadie et al., 1995; Graham et al., 2004). Thus, the majority of Unc-18 protein could interact with Syntaxin in the peri-active zone, presumably binding a closed Syntaxin conformation. Accordingly, Unc-18 would be in equilibrium, moving between a peri-active zone reservoir and fusion competent vesicles at the active zone. In this way, Syntaxin could restrict the amount of Unc18 available for participation in synaptic vesicle fusion at the release site. Thus, when we remove one copy of the syntaxin gene, or diminish the binding of Unc-18 to the closed confirmation of Syntaxin, we might be shifting the distribution of Unc-18 toward the release site and achieve a rescue PHP. This model is consistent with data in other systems, demonstrating that the levels of Unc18 at the release site can influence vesicle release rate. For example, over-expression of unc-18 in mice is sufficient to potentiate vesicle release (Voets et al., 2001), albeit to a limited extent (Toonen and Verhage, 2003). Thus, we propose that the expression of PHP involves an as yet unknown signaling event that mobilizes Unc-18 to the active zone where it is sufficient to promote vesicle priming as a final stage necessary for the full expression of PHP.

Data generate during this study are included in the manuscript and supporting files and tables.

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Author details

1. Jennifer M Ortega

   Department of Biochemistry and Biophysics, Kavli Institute for Fundamental Neuroscience, University of California San Francisco, San Francisco, California

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

2. Özgür Genç

   Department of Biochemistry and Biophysics, Kavli Institute for Fundamental Neuroscience, University of California San Francisco, San Francisco, California

   Contribution

   Data curation, Formal analysis, Writing—review and editing

   Competing interests

   No competing interests declared

3. Graeme W Davis

   Department of Biochemistry and Biophysics, Kavli Institute for Fundamental Neuroscience, University of California San Francisco, San Francisco, California

   Contribution

   Conceptualization, Funding acquisition, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   graeme.davis@ucsf.edu

   Competing interests

   Reviewing editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0003-1355-8401

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