Characterization of the role of the family as calcium sensors in facilitation and asynchronous neurotransmitter release

Sudipta Saraswati, Bill Adolfsen, and J. Troy Littleton*

The Picower Institute for Learning and Memory, Department of Biology and Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139

Communicated by Susumu Tonegawa, Massachusetts Institute of Technology, Cambridge, MA, July 17, 2007 (received for review March 13, 2007) Ca2؉ influx into presynaptic nerve terminals activates synaptic synapses is known. It has been widely postulated that other vesicle exocytosis by triggering fast synchronous fusion and a isoforms of the large Syt family may mediate asynchronous slower asynchronous release pathway. In addition, a brief rise in release. -Ca2؉ after consecutive action potentials has been correlated with Like baseline synaptic transmission, Ca2ϩ-regulated neuro a form of short-term synaptic plasticity with enhanced vesicle transmitter release is also required for a form of short-term fusion termed facilitation. Although the synaptic plasticity termed ‘‘facilitation.’’ Facilitation is an en- Synaptotagmin 1 (Syt1) has been implicated as the Ca2؉ sensor for hancement in synaptic transmission resulting from prior synaptic synchronous fusion, the molecular identity of the Ca2؉ sensors that activity that lasts on a millisecond time scale (8). When observed mediate facilitation and asynchronous release is unknown. To test with pairs of stimuli in which the second postsynaptic response whether the synchronous Ca2؉ sensor, Syt1, or the asynchronous is larger than the first, the phenomenon is termed ‘‘paired-pulse Ca2؉ sensor is involved in facilitation, we analyzed whether ge- facilitation.’’ This form of short-term synaptic plasticity has been netic elimination of Syt1 in Drosophila results in a concomitant correlated with elevated residual Ca2ϩ in presynaptic terminals impairment in facilitation. Our results indicate that Syt1 acts as a after an action potential (8). The molecular target on which Ca2ϩ redundant Ca2؉ sensor for facilitation, with the asynchronous Ca2؉ ions act to mediate facilitation is unknown, although it was sensor contributing significantly to this form of short-term plas- originally postulated that both evoked release and facilitation ticity. We next examined whether other members of the Drosoph- would share a common Ca2ϩ sensor (9). We tested this model by ila Syt family functioned in Ca2؉-dependent asynchronous release determining the role of the Drosophila panneuronal Syt or facilitation in vivo. Genetic elimination of other panneuronally in paired-pulse facilitation and basal synaptic transmission. Our expressed Syt proteins did not alter these forms of exocytosis, results indicate that a non-Syt Ca2ϩ sensor mediates asynchro- .indicating a non-Syt Ca2؉ sensor functions for both facilitation and nous release and contributes to presynaptic facilitation asynchronous release. In light of these findings, the presence of two presynaptic Ca2؉ sensors can be placed in a biological context, Results -a Syt1-based Ca2؉ sensor devoted primarily to baseline synaptic Both Syt1 and the Asynchronous Ca2؉ Sensor Contribute to Presyn transmission and a second non-Syt Ca2؉ sensor for short-term aptic Facilitation. To investigate whether the synchronous Ca2ϩ synaptic plasticity and asynchronous release. sensor, Syt1, is involved in facilitation, we examined paired-pulse facilitation by performing intracellular recordings from Dro- exocytosis ͉ synapse ͉ synaptic vesicle ͉ Drosophila ͉ synaptic plasticity sophila third-instar larval neuromuscular junctions (NMJs) of wild-type animals and -null mutants (sytAD4/sytN13). Evoked ast communication at synapses depends on Ca2ϩ-regulated neurotransmitter release in the absence of Syt1 is characterized Fneurotransmitter release at presynaptic nerve terminals (1). by a dramatic reduction in the overall number of vesicle fusion Ca2ϩ acts upon presynaptic Ca2ϩ-binding proteins that transduce events, compared with controls, and a shift from fast fusion to Ca2ϩ signals into a fusion of synaptic vesicles and a release of a slower and prolonged asynchronous release of vesicles (Fig. neurotransmitters. Three lines of evidence indicate the existence 1A). Given the reduced baseline synaptic transmission in syt1 2ϩ mutant animals, we examined facilitation in wild-type controls at of two kinetically distinct Ca sensors at most synapses, with 2ϩ one mediating a rapid, synchronous component of transmitter a reduced extracellular Ca concentration of 0.13 mM, which gave a comparable evoked response to syt1-null mutants in 1.5 release, and a second underlying a slower, asynchronous com- 2ϩ ponent of fusion. First, the kinetics of evoked neurotransmitter mM extracellular Ca . As shown in Fig. 1, a severe impairment of baseline synaptic transmission in the absence of the synchro- release exhibit a biphasic decay that can be fitted by a double- 2ϩ exponential curve, revealing fast and slow components of release nous Ca sensor did not result in a concomitant impairment of (2). Second, the divalent cation Sr2ϩ has been reported to have paired-pulse facilitation as measured by the ratio between two differential effects on the fast and slow components of neuro- postsynaptic responses. Indeed, paired-pulse facilitation is en- 2ϩ hanced in syt1 mutants compared with wild type across all transmitter release (2, 3), although differential Sr influx may 2ϩ also contribute to this effect (4). Third, in both mice and extracellular Ca levels examined (Fig. 1C), likely reflecting a Drosophila mutants lacking the synaptic vesicle Ca2ϩ-binding depletion of readily releasable vesicles in controls and the protein, Synaptotagmin 1 (Syt1), the fast synchronous compo- nent of release is abolished, whereas slow asynchronous release Author contributions: S.S. and B.A. contributed equally to this work; S.S., B.A., and J.T.L. is enhanced (5–7). Taken together, kinetic, pharmacological, designed research; S.S. and B.A. performed research; S.S., B.A., and J.T.L. analyzed data; 2ϩ and genetic evidence support a two-Ca sensor model for and S.S. and J.T.L. wrote the paper. neurotransmitter release, where: (i) Syt1 functions as the syn- The authors declare no conflict of interest. 2ϩ chronous Ca sensor and suppresses release through the asyn- Abbreviations: CFP, cyan fluorescent protein; CS, Canton S; EJP, excitatory junctional ϩ chronous pathway, and (ii) a residual Ca2 sensor remaining in potential; Mhc, myosin heavy chain; NMJ, neuromuscular junction; UAS, upstream activat- syt1-null mutants mediates asynchronous release. Although the ing sequence. ϩ asynchronous Ca2 sensor is kinetically distinct from Syt1, *To whom correspondence should be addressed. E-mail: [email protected]. neither its molecular identity nor its functional significance at © 2007 by The National Academy of Sciences of the USA

14122–14127 ͉ PNAS ͉ August 28, 2007 ͉ vol. 104 ͉ no. 35 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0706711104 Downloaded by guest on September 30, 2021 Fig. 1. Syt1 plays a redundant role in paired-pulse facilitation. Intracellular recordings of paired-pulse facilitation from Drosophila third-instar larval NMJs at muscle 6 of wild-type Canton S (CS) and syt1null mutants. (A) Representative traces at 1.5 mM extracellular Ca2ϩ in sytAD4/sytN13 and at 0.13 mM extracellular Ca2ϩ in wild type. (Calibration bar: 5 mV/5 ms.) (B) Quantification of quantal facilitation as measured by the difference between the numbers of quanta released in two postsynaptic responses at 25-ms interpulse intervals and 0.2 mM extracellular Ca2ϩ. The number of muscles examined and the average resting potentials (avg. RP) in millivolts Ϯ SEM for each genotype were as follows: CS (n ϭ 4; avg. RP ϭϪ62.88 Ϯ 3.68) and syt1 (n ϭ 6; avg. RP ϭϪ59 Ϯ 2.24). Data points are mean Ϯ SEM; statistical significance (*, P Ͻ 0.05) was determined by Student’s t test. (C) Quantification of facilitation, as measured by the ratio between two postsynaptic responses, at 25-ms interpulse intervals in control and syt1null mutants at 0.2, 0.4, and 1.5 mM extracellular Ca2ϩ. Data points are mean Ϯ SEM; statistical ϩ significance (*, P Ͻ 0.05) was determined by Student’s t test. The numbers of muscles examined for each genotype were as follows: CS (0.2 mM Ca2 , 6; 0.4 mM Ca2ϩ, 15; and 1.5 mM Ca2ϩ,8)andsyt1 (0.2 mM Ca2ϩ, 9; 0.4 mM Ca2ϩ, 27; and 1.5 mM Ca2ϩ, 17). Average muscle resting membrane potentials for each genotype were as follows: CS (0.2 mM Ca2ϩ, 54.58 Ϯ 1.34; 0.4 mM Ca2ϩ, 59.48 Ϯ 0.61; and 1.5 mM Ca2ϩ, 62.38 Ϯ 1.23) and syt1 (0.2 mM Ca2ϩ, 56.83 Ϯ 1.74; 0.4 mM Ca2ϩ, 56.74 Ϯ 0.69; and 1.5 mM Ca2ϩ, 61.19 Ϯ 1.37). (D) Quantification of facilitation, as measured by the ratio between two postsynaptic responses, at 25-, 50-, and 75-ms interpulse intervals in control at 0.13 mM extracellular Ca2ϩ and in syt1null mutant animals at 0.2, 0.4, and 1.5 mM extracellular Ca2ϩ. The numbers of muscles examined for each genotype were as follows: CS (25 ms, 19; 50 ms, 19; and 75 ms, 18), syt1 at 0.2 mM Ca2ϩ (25 ms, 9; 50 ms, 7; and 75 ms, 8), syt1 at 0.4 mM Ca2ϩ (25 ms, 27; 50 ms, 24; and 75 ms, 22), and syt1 at 1.5 mM Ca2ϩ (25 ms, 17; 50 ms, 14; and 75 ms, 11). Average muscle resting membrane potentials for each genotype were as follows: CS (25 ms, 56.08 Ϯ 0.54; 50 ms, 55.77 Ϯ 0.53; and 75 ms, 56 Ϯ 0.63), syt1 at 0.2 mM Ca2ϩ (25 ms, 56.83 Ϯ 1.74; 50 ms, 57.18 Ϯ 2.62; 75 ms, 57.75 Ϯ 2.76), syt1 at 0.4 mM Ca2ϩ (25 ms, 56.74 Ϯ 0.69; 50 ms, 56.46 Ϯ 0.79; and 75 ms, 57.31 Ϯ 0.81), and syt1 at 1.5 mM Ca2ϩ (25 ms, 61.19 Ϯ 1.37; 50 ms, 60.71 Ϯ 1.59; 75 ms, 58.56 Ϯ 1.68).

enhanced function of the asynchronous Ca2ϩ sensor previously asynchronous Ca2ϩ sensor and Syt1 can mediate presynaptic demonstrated in syt1 mutants (6, 7). Similar to wild type, the facilitation of neurotransmitter release. As such, both proteins extent of facilitation in syt1-null mutants also decreases with are likely to sense residual Ca2ϩ that occurs during paired increasing interpulse interval (Fig. 1D), indicating that the stimuli. kinetics of facilitation is unaltered in the absence of Syt1. As observed in syt1-null mutants in response to a single action Generation of Mutants Lacking Syt4 and Syt7. Given that the potential, release during paired-pulse stimulation remained asynchronous Ca2ϩ sensor contributes to facilitation, we tested asynchronous compared with controls. whether the two remaining Drosophila panneuronal Syt proteins, Although facilitation persists in syt1-null mutants, binding of Syt4 and Syt7 (10), function in presynaptic facilitation. It has residual Ca2ϩ to Syt1 also might contribute independently from been hypothesized that the asynchronous Ca2ϩ sensor at pre- the asynchronous Ca2ϩ sensor to promote facilitation. Given the synaptic terminals would be another member of the Syt family larger overall amount of release observed in controls compared (11). Syt proteins form a family of -containing NEUROSCIENCE with syt1 mutants, we tested whether Syt1 functions coopera- proteins with seven members in Drosophila and Ͼ14 members in tively with the asynchronous Ca2ϩ sensor to promote facilitation. mammals (12). Among the family members in Drosophila,Syt4 For this experiment, we measured paired-pulse facilitation as the and Syt7 are candidates for asynchronous Ca2ϩ sensors because difference between quantal content for the two postsynaptic they are the only isoforms of the family besides Syt1 to be responses at identical 0.2 mM Ca2ϩ concentrations in both panneuronally expressed and present in motor neurons (10). control and mutant animals. The syt1-null mutants showed a A P element (P{EPgy2}SytIVEY12073) inserted 100 bp upstream 5.6-fold reduction in the absolute number of vesicles released of the first exon of syt4 was used to generate syt4 mutations as during the second pulse, compared with controls at identical previously described (13). To determine whether imprecise extracellular Ca2ϩ (Fig. 1B). These results indicate that both the excisions removed the Syt4 protein, we performed Western blot

Saraswati et al. PNAS ͉ August 28, 2007 ͉ vol. 104 ͉ no. 35 ͉ 14123 Downloaded by guest on September 30, 2021 Fig. 3. Generation of animals lacking Syt7. (A) Syt7 knockdown construct. The initial genomic region of the locus, including exons and introns 1 and 2, was fused to the reverse cDNA coding for exons 1 and 2, maintaining the splice acceptor and donor sites in the two introns. (B)(Lower) The third-instar larval muscles from white and Mhc-Gal4;syt7 RNAi larvae stained with the Fig. 2. Generation of animals lacking Syt4. (A) Western blot of adult head ␣-Syt7 polyclonal antibody. (Upper) Muscle-specific expression of a syt7–CFP extracts from control (precise excision) and syt4BA1 mutants. One fly-head transgene with or without coexpression of the syt7 RNAi transgene. Larvae equivalent of protein was loaded into each lane and blotted by using the coexpressing the two transgenes have dramatically reduced Syt7–CFP levels in ␣-Syt4 polyclonal antibody. Although several nonspecific bands remain and muscles compared with control siblings. serve as loading controls, the syt4BA1 head extract is missing the abundant band corresponding to the predicted molecular weight of Syt4. (B) Immuno- staining for Syt4 at larval brains (Upper) and at muscles 6 and 7 of the third-instar larval NMJ (Lower) in control and syt4BA1 mutants. Control and controls (Fig. 3B), suggesting that the RNAi transgene could mutants were imaged by using identical confocal settings. efficiently target and eliminate syt7.

analysis and immunohistochemistry using a Syt4 polyclonal Paired-Pulse Facilitation Remains Intact in syt4 and syt7 Loss-of- antibody (10). Western blot analysis of adult head extract Function Animals. To test the hypothesis that Syt4 or Syt7 may isolated from one such deletion, syt4BA1, indicated that the Syt4 function in synaptic vesicle fusion, we performed electrophysi- protein was absent (Fig. 2A). Furthermore, immunostaining of ological analysis on loss-of-function syt4 and syt7 animals. We syt4BA1 revealed a loss of anti-Syt4 immunoreactivity in both the first investigated whether loss of Syt4 or Syt7 caused defects in CNS and muscles in wandering third-instar larvae (Fig. 2B), quantal content by measuring evoked excitatory junctional compared with precise excision controls (syt4pre1). potentials (EJPs) at the third-instar NMJ at 0.2 and 0.4 mM Because of the lack of P-element insertions in the vicinity of extracellular Ca2ϩ. As shown in Fig. 4 A–C, we did not observe the syt7 locus on the fourth , we generated syt7 any statistical differences in evoked EJP amplitudes between syt4 transgenic RNAi lines that express a genomic-cDNA syt7 RNAi or syt7 animals (syt4BA1 and C155; syt7 RNAi) and controls fusion under control of the Gal4/UAS system (Fig. 3A). To test (syt4pre1 and C155), indicating that robust neurotransmission is whether expression of the construct reduced Syt7 protein levels still intact in these mutants. Next, we investigated the possibility in vivo, we performed immunostaining experiments by using the that loss of Syt4 or Syt7 may have more subtle effects on quantal Syt7 polyclonal antibody (12). As shown in Fig. 3B, muscle- content by measuring evoked EJPs at a low 0.1 mM extracellular specific expression of the syt7 RNAi construct with the myosin Ca2ϩ concentration. Again, we did not detect any changes in the heavy chain (Mhc)-Gal4 driver reduced the levels of native Syt7 EJP amplitudes in syt4 or syt7 mutants (Fig. 4 B and C). These protein found on post-Golgi vesicles to below detection. To results indicate that loss of Syt4 or Syt7 does not affect basal further validate the syt7 RNAi construct, we constructed trans- synaptic transmission at the Drosophila third-instar NMJ. genic lines that express a syt7–cyan fluorescent protein [(CFP) Having identified paired-pulse facilitation as an assay for the fused to the C-terminal of syt7] transgene under the control of 2ϩ an upstream activating sequence (UAS). When driven by Mhc- asynchronous Ca sensor, we tested the possibility that syt4 or Gal4 driver, overexpression of this transgene mimicked the syt7 may function as the asynchronous sensor at synapses. We endogenous expression pattern of Syt7, with localization to a measured paired-pulse facilitation at larval NMJs in syt4-null post-Golgi vesicle compartment that costained with lysotracker mutants and syt7 RNAi knockdown animals (syt7 RNAi driven ϩ (Fig. 3B). We then tested for the ability of the syt7 RNAi by the panneuronal driver C155) at a 0.2 mM extracellular Ca2 construct to reduce the expression of the syt7-CFP transgene concentration. As shown in Fig. 5 A–D, the amount of facilitation when coexpressed within muscles. CFP fluorescence measured measured at 25-, 50-, and 75-ms interpulse intervals was statis- by confocal microscopy in muscles 6 and 7 in larvae carrying the tically the same for all genotypes tested, indicating that neither syt7 RNAi transgene was dramatically depleted compared with syt4 nor syt7 contributes to paired-pulse facilitation.

14124 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0706711104 Saraswati et al. Downloaded by guest on September 30, 2021 asynchronous compared with wild-type controls. These results confirm that Syt4 is not a residual asynchronous Ca2ϩ sensor and does not contribute to facilitation. We attempted similar exper- iments on syt1; syt7 double mutants. Although these larvae hatched and exhibited uncoordinated and slow movements, similar to syt1 mutant animals alone, they died during the first instar larval stage, preventing a detailed physiological analysis at the third-instar NMJ. However, the residual locomotion ob- served in double mutants suggests that Syt7, like Syt4, is not required for the asynchronous neurotransmitter release driving residual locomotion observed in syt1 mutants. Discussion Although the synaptic vesicle protein Syt1 has been implicated as the Ca2ϩ sensor for synchronous synaptic vesicle fusion, the molecular identity of the Ca2ϩ sensors that mediate facilitation and asynchronous release is unknown. It was originally postu- lated that both evoked release and facilitation would share the same Ca2ϩ sensor (9). We took a genetic approach to determine whether evoked release and facilitation are mediated by the same Ca2ϩ sensor, Syt1. Here we demonstrate that facilitation persists in Drosophila syt1 mutants, although the magnitude of facilitation is reduced. Our findings indicate that Syt1 acts as a redundant Ca2ϩ sensor, with a second asynchronous Ca2ϩ sensor to mediate facilitation. We also addressed whether other Syt family members act as the asynchronous Ca2ϩ sensor for facil- itation. Syt proteins have two well characterized Ca2ϩ-binding motifs known as C2 domains, which participate in multiple Ca2ϩ-dependent interactions, including lipid (14) and SNARE complex binding (15). The distinct Ca2ϩ affinities for individual Syt isoforms have made them attractive candidates for mediating asynchronous release. Fig. 4. Evoked neurotransmitter release in animals lacking Syt4 and Syt7. (A) Among the Syt family, two strong candidates for the asyn- ϩ Representative traces of evoked EJP at 0.4 mM extracellular Ca2 in control and chronous Ca2ϩ sensor are Syt4 and Syt7, which are conserved syt4BA1 mutants, as well as control (C155) and C155; syt7 RNAi knockdown 2ϩ through evolution (12) and highly expressed in the nervous animals. (B) Representative traces of evoked EJP at 0.1 mM extracellular Ca in system (10, 16). Syt7 localizes to the plasma membrane of PC12 control and syt4BA1 mutants, as well as control (C155) and C155; syt7 RNAi knockdown animals. (C) Quantification of evoked EJP amplitudes in control and cells when overexpressed, whereas endogenous Syt7 has been syt4null mutants, as well as control (c155) and C155; syt7 RNAi knockdown animals reported to be concentrated at presynaptic active zones of at 0.1, 0.2, and 0.4 mM extracellular Ca2ϩ. Data points are mean Ϯ SEM. The central synapses, where it was hypothesized to act as a plasma ϩ numbers of muscles examined and the average resting potential (avg. RP) in membrane Ca2 sensor in synaptic vesicle exocytosis (17). Syt7 ϩ millivolts Ϯ SEM for each genotype were as follows: control (0.1 mM Ca2ϩ, n ϭ 10, also exhibits the slowest disassembly kinetics of Ca2 –Syt mem- avg. RP ϭϪ64 Ϯ 1.27; 0.2 mM Ca2ϩ, n ϭ 14, avg. RP ϭϪ60.3 Ϯ 1.18; 0.4 mM Ca2ϩ, brane complexes (11), making it an attractive candidate for the ϩ n ϭ 9, avg. RP ϭϪ69 Ϯ 2), syt4null (0.1 mM Ca2 , n ϭ 9, avg. RP ϭϪ68 Ϯ 2.33; 0.2 asynchronous sensor. Similar to Syt7, several studies have sug- 2ϩ 2ϩ mM Ca , n ϭ 18, avg. RP ϭϪ59.43 Ϯ 0.92; 0.4 mM Ca , n ϭ 9, avg. RP ϭϪ66 Ϯ gested that Syt4 might function during synaptic vesicle exocyto- 2ϩ ϭ ϭϪ Ϯ 2ϩ ϭ 1.67), C155 (0.1 mM Ca , n 8, avg. RP 62 2.12; 0.2 mM Ca , n 30, avg. sis. Up-regulation of syt4 in PC12 cells alters fusion pore RP ϭϪ59 Ϯ 0.7; 0.4 mM Ca2ϩ, n ϭ 9, avg. RP ϭϪ75 Ϯ 2), and C155; syt7 RNAi (0.1 mM Ca2ϩ, n ϭ 8, avg. RP ϭϪ62 Ϯ 1.41; 0.2 mM Ca2ϩ, n ϭ 26, avg. RP ϭϪ59.02 Ϯ dynamics (18) and has been reported to influence the choice 0.82; 0.4 mM Ca2ϩ, n ϭ 9, avg. RP ϭϪ73 Ϯ 1). between ‘‘kiss and run’’ and full fusion (19). To test whether Syt4 or Syt7 is the asynchronous Ca2ϩ sensor, we generated loss-of-function mutants in Drosophila and char- Asynchronous Release and Paired-Pulse Facilitation Remain Intact in acterized their synaptic physiology at the NMJ. Neither null syt1;syt4 Double Mutants. To further test the hypothesis that syt4 mutations in syt4 nor RNAi knockdown of syt7 in neurons alters may encode the asynchronous Ca2ϩ sensor, we generated syt1; basic synaptic transmission at the Drosophila NMJ or paired- syt4 double-mutant animals and performed intracellular record- pulse facilitation. These results are consistent with our earlier in ings from larval NMJs to examine paired-pulse facilitation and vivo localization studies in Drosophila, which indicated that Syt4 evoked release. Like syt1 mutants alone, syt1; syt4 double mu- localizes postsynaptically and that Syt7 fails to target to larval tants displayed robust asynchronous release, indicating that Syt4 NMJ synapses (10). If neither Syt4 nor Syt7 participates in ϩ synaptic vesicle exocytosis in vivo, what are their endogenous is not the residual asynchronous Ca2 sensor remaining in functions? Recent experiments in our laboratory indicate that syt1-null mutants. To examine facilitation and reduce any pos- 2ϩ

Syt4 regulates the Ca -dependent exocytosis of retrograde NEUROSCIENCE sible effects of reduced baseline synaptic transmission in syt1; signals from the postsynaptic compartment, triggering changes in syt4 double-mutant animals, we recorded from wild-type con- presynaptic release properties and structural plasticity (13). The 2ϩ trols at a reduced 0.13 mM extracellular Ca concentration, localization of Syt7 to a putative lysosomal compartment raises which gave a comparable response to syt1; syt4 double mutants ϩ the possibility that Syt7 is required for a more ubiquitous role in in 1.5 mM extracellular Ca2 . As shown in Fig. 5E, paired-pulse vesicular trafficking, which is important for plasma membrane facilitation remains intact in syt1; syt4 double mutants, with repair processes, similar to its proposed role in mammals (20). enhanced facilitation as observed in syt1 mutants alone (Fig. 1C). Because Syt4 and Syt7 do not encode the asynchronous Ca2ϩ Similar to syt1 mutants alone, the release observed during sensor, it is difficult to conceive how any of the remaining four paired-pulse stimulation in syt1; syt4 double mutants remained Drosophila Syt proteins encoded in the genome could supply this

Saraswati et al. PNAS ͉ August 28, 2007 ͉ vol. 104 ͉ no. 35 ͉ 14125 Downloaded by guest on September 30, 2021 Fig. 5. Paired-pulse facilitation remains intact in syt4null mutants, syt7 RNAi knockdown animals, and syt1;syt4 double mutants. (A) Representative traces of facilitation at 0.2 mM extracellular Ca2ϩ in syt4BA1 mutants and control animals. (Calibration bar: 5 mV/20 ms; interpulse interval: 25 ms.) (B) Representative traces of facilitation at 0.2 mM extracellular Ca2ϩ in C155; syt7 RNAi knockdown and control (C155) animals. (Calibration bar: 5 mV/20 ms; interpulse interval: 25 ms.) (C) Quantification of facilitation at 25-, 50-, and 75-ms interpulse intervals in control and syt4null mutant animals at 0.2 mM extracellular Ca2ϩ. The numbers of muscles examined and the average resting potential (avg. RP) in millivolts Ϯ SEM for each genotype were as follows: control (25 ms, n ϭ 12, avg. RP ϭϪ59.84 Ϯ 1.31; 50 ms, n ϭ 12, avg. RP ϭϪ58.54 Ϯ 1.56; and 75 ms, n ϭ 12, avg. RP ϭϪ59.46 Ϯ 1.46) and syt4null (25 ms, n ϭ 16, avg. RP ϭϪ59.88 Ϯ 0.83; 50 ms, n ϭ 16, avg. RP ϭϪ60.08 Ϯ 0.86; and 75 ms, n ϭ 15, avg. RP ϭϪ59.3 Ϯ 0.8). (D) Quantification of facilitation at 25-, 50-, and 75-ms interpulse intervals in control (C155) and in C155; syt7 RNAi knockdown animals at 0.2 mM extracellular Ca2ϩ. The number of muscles examined and the avg. RP for each genotype were as follows: control (25 ms, n ϭ 26, avg. RP ϭϪ58.49 Ϯ 0.65; 50 ms, n ϭ 25, avg. RP ϭϪ58.07 Ϯ 0.69; and 75 ms, n ϭ 24, avg. RP ϭϪ58.41 Ϯ 0.76) and C155; syt7 RNAi (25 ms, n ϭ 20, avg. RP ϭϪ59.01 Ϯ 0.96; 50 ms, n ϭ 24, avg. RP ϭϪ59.34 Ϯ 0.85; and 75 ms, n ϭ 24, avg. RP ϭϪ59.3 Ϯ 0.83). (E) A representative trace from syt1; syt4 double mutants in 1.5 mM extracellular Ca2ϩ. (Calibration bar: 5 mV/5 ms.) Quantification of facilitation at 25-, 50-, and 75-ms interpulse intervals in control (CS) at 0.13 mM extracellular Ca2ϩ and in syt1; syt4 double mutants at 1.5 mM extracellular Ca2ϩ is shown. For comparison, facilitation in the syt1-null mutant alone at 1.5 mM extracellular Ca2ϩ is shown. The number of muscles examined and avg. RP for each genotype were as follows: CS (25 ms, n ϭ 19, avg. RP ϭ Ϫ56.08 Ϯ 0.54; 50 ms, n ϭ 19, avg. RP ϭϪ55.77 Ϯ 0.53; and 75 ms, n ϭ 18, avg. RP ϭϪ56 Ϯ 0.63) and syt1; syt4 (25 ms, n ϭ 11, avg. RP ϭϪ60.98 Ϯ 1.15; 50 ms, n ϭ 11, avg. RP ϭϪ60.32 Ϯ 0.97; and 75 ms, n ϭ 10, avg. RP ϭϪ59.7 Ϯ 1.16).

function. Syt-␣ and Syt-␤ are exclusively expressed in subsets of encoded in the Drosophila genome, Syt12 and Syt14, have putative neurosecretory cells and are not present in motor conserved only 2 and 3 of the 10 Ca2ϩ-coordinating aspartic acid neurons (10). Similar to mammals, the final two Syt proteins residues within their C2 domains, respectively, indicating that

14126 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0706711104 Saraswati et al. Downloaded by guest on September 30, 2021 these Syt proteins most likely function in Ca2ϩ-independent (including all splice donor and acceptor sites) of the syt7 locus trafficking pathways. In addition, their mRNAs are expressed at was PCR-amplified from genomic DNA and ligated into the levels below detection in Drosophila embryos, and antisera to the pUAST vector. The reverse cDNA sequence encoding the first proteins do not detect Syt12 or Syt14 protein at NMJ synapses two exons (Ϸ300 bp) of syt7 was then PCR-amplified from a (10). Our results complement a recent study that defined the cDNA library and directionally cloned into a pUAST-Syt7 protein content of mammalian synaptic vesicles (21), revealing genomic clone. Syt1 as the major synaptic vesicle Syt isoform. Besides Syt1, the authors also detected Syt2, -5, -12, and -17 by mass spectrometry Electrophysiological Analysis. Electrophysiological analysis of wan- on some synaptic vesicles. Syt2, -5, and -17 are mammalian- dering third-instar larva was performed in Drosophila saline [70 specific isoforms with no invertebrate homologs. Similar to mM NaCl/5 mM KCl/4 mM MgCl /10 mM NaHCO /5 mM Drosophila Syt12, mammalian Syt12 lacks most of the Ca2ϩ- 2 3 trehalose/115 mM sucrose/5 mM Hepes-NaOH (pH 7.2)] mod- coordinating aspartate residues in its C2 domains and does not bind Ca2ϩ in vitro (22). ified from HL3 by using an Axoclamp 2B amplifier (Axon Instruments, Foster City, CA) at 22°C as previously described In conclusion, our genetic studies in Drosophila argue against 2ϩ the idea that any member of the Syt family encodes the asyn- (23). Ca concentrations varied with experiment and are indi- chronous Ca2ϩ sensor, making a simplistic two-Syt Ca2ϩ sensor cated in the figure legends. Evoked EJPs were recorded intra- model of neurotransmitter release unlikely. While eliminating cellularly from muscle fiber 6 or 7 of segments A3–A5 under Syt1 as the only Ca2ϩ target for facilitation, our results indicate current clamp. Paired-pulse facilitation was measured by deter- that the asynchronous Ca2ϩ sensor plays a significant role in this mining the peak amplitude response occurring within a 20-ms form of presynaptic plasticity. Although kinetically distinct in its interval after stimulation. Stimulation pairs with no response to Ca2ϩ binding, experimental evidence demonstrating the func- the first stimulus were not included in the analysis. All error bars tional significance of the asynchronous Ca2ϩ sensor is lacking. indicate SEM. Statistical significance (P Ͻ 0.05) was determined ϩ The present study suggests a function for the asynchronous Ca2 by Student’s t test. sensor in the biology of short-term synaptic plasticity. In light of ϩ these findings, the presence of two presynaptic Ca2 sensors can Immunostaining and Western Blot Analysis. Immunostaining was ϩ now be placed in a biological context: a Syt1 Ca2 sensor devoted performed on third-instar larvae at wandering stage after rear- to baseline synaptic transmission and a second asynchronous ing at 25°C as described previously (24). The dilution of primary 2ϩ Ca sensor that contributes to short-term presynaptic plasticity. antibodies was as follows: Syt1 (1:1,000), Syt4 (1:500), and Syt7 Materials and Methods (1:1,000). Immunoreactive proteins were visualized on a Zeiss Pascal Confocal by using fluorescent secondary antibodies (Mo- Drosophila Genetics. Drosophila melanogaster was cultured on lecular Probes, Eugene, OR; Chemicon, Temecula, CA; Jackson standard medium at 25°C. syt4 pre1 is a precise excision used as ImmunoResearch, West Grove, PA). Western blots were done a control for syt4BA1. The syt7 RNAi is a transgenic RNAi knockdown for syt7. The C155 elav-GAL4 driver was used for by using standard laboratory procedures. All antibodies were neuronal expression of transgenes. used at a 1:1,000 dilution.

Generation of Syt7 Mutants by Transgenic RNAi. An Ϸ3.2-kb This work was supported by National Institutes of Health grants and the genomic fragment consisting of the first two exons and introns Packard Foundation (J.T.L.).

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