Wild-type mice are social animals and choose to spend more time in the chamber with another mouse (Figure 5A). This social interaction behavior was unaffected in MeCP2 S421A mice, demonstrating their ability to recognize other mice and their appropriate interest in their physical and social environment. Subsequently,

a second mouse that the test subject had never before encountered was placed within a small wire cage in the side-chamber opposite to the first, now familiar mouse. The wild-type test subjects spent the largest proportion of their time in the chamber containing the second, novel mouse and less time with the familiar choice (Figure 5B). By contrast, the MeCP2 S421A mice spent as much time with the familiar mouse as with the novel mouse. Because the MeCP2 S421A mice show appropriate interest in novel mice, it is unlikely that the increased time spent CHIR-99021 datasheet with familiar mice is due to a general deficit in social recognition upon VX 770 loss of MeCP2 S421 phosphorylation. Likewise, the MeCP2 S421A mice show no aversion to spending time alone and appear normal in tests of anxious behavior (Figure S2). Instead the increased interest in the familiar mouse suggests that the MeCP2 S421A mutants cannot distinguish between familiar and novel mice. This lack of discrimination between novel and familiar stimuli exhibited by the MeCP2 S421A mice was not limited to social behavior; when presented with both novel and

familiar inanimate objects, wild-type mice showed a behavioral preference for a novel object, whereas the MeCP2 S421A mice spent equal amounts of time investigating both familiar and novel objects (Figure 5C). This difference was evident at 30 min after the initial exposure to the familiar object, and persisted even after 24 hr had Dipeptidyl peptidase passed. Taken together, these findings support the conclusion that neuronal activity-dependent phosphorylation of MeCP2 at S421 is necessary to allow an animal to process novel experience and respond appropriately

to previously encountered objects or animals. This defect cannot be attributed to an absence of all learning and memory in these mice, as the performance of the MeCP2 S421A mice in spatial learning and memory tests is indistinguishable from wild-type (Figure S3). Instead the specific defect observed in the MeCP2 S421A mice suggests that the activity-dependent phosphorylation of MeCP2 S421 contributes to aspects of cognitive function underlying behavioral flexibility, and that the disruption of this aspect of MeCP2 regulation in RTT may be a factor in cognitive impairments observed in affected individuals. The abnormalities we observe in the MeCP2 S421A knockin animals demonstrate that this activity-dependent phosphorylation event is required for proper formation of the nervous system. We considered how the phosphorylation of S421 might modulate the molecular function of MeCP2 during neuronal development. Two distinct mechanisms have been proposed to explain how MeCP2 functions when bound to DNA.

To identify new molecules involved in neuromuscular signaling, we used RNAi to screen for cell adhesion molecules whose absence alters the responsiveness of Caenorhabditis elegans to the acetylcholinesterase

inhibitor aldicarb. Aldicarb treatment causes acute paralysis due to the accumulation of acetylcholine (ACh) in the synaptic cleft at the neuromuscular junction (NMJ). Gene inactivations that alter synaptic function can cause either resistance or hypersensitivity to aldicarb ( Miller et al., 1996, Sieburth et al., 2005 and Vashlishan selleck chemical et al., 2008). For this screen, we selected a collection of 216 putative cell adhesion molecules, based on the presence of protein domains found in CAMs (data not shown). A gene identified in this screen was rig-3, which encodes a GPI-anchored protein containing two Ig domains and a divergent fibronectin type III (FNIII) domain ( Figure 1A). RIG-3 has a pattern of protein domains that is similar to the Drosophila proteins Klingon and Wrapper, and to mammalian NCAMs ( Cox et al., 2004 and Yamagata check details et al., 2003). RIG-3 was previously implicated in axon guidance in C. elegans; however rig-3 single mutants do not show guidance defects ( Schwarz et al., 2009). Inactivation of rig-3 by RNAi caused significant hypersensitivity to aldicarb ( Figure 1B) and

a similar defect was Mannose-binding protein-associated serine protease observed in homozygous rig-3(ok2156) mutants ( Figure 1C). The ok2156 mutation deletes 1.5 kb of the rig-3 gene, spanning exons 2–5 (including most of the Ig domains and part of the FNIII domain); consequently, ok2156 is likely to cause a severe loss of gene function (www.wormbase.org) ( Figure 1A) ( Schwarz et al., 2009). The rig-3 aldicarb hypersensitivity defect was rescued by transgenes driving RIG-3 expression in all neurons (utilizing the snb-1 Synaptobrevin promoter, data not shown) and in cholinergic neurons (utilizing the unc-17 VAChT promoter) ( Figure 1C). By contrast, rig-3 transgenes expressed in GABA neurons, or in the intestine lacked rescuing

activity ( Figure 1C). None of these transgenes altered aldicarb responsiveness of wild-type animals (data not shown). These results suggest that RIG-3 functions in cholinergic neurons to regulate some aspect of neuromuscular function or development. Prior work showed that rig-3 is expressed in neurons and in the intestine (www.wormbase.org) ( Schwarz et al., 2009). A construct containing the full rig-3 genomic region, with mCherry inserted just after the signal sequence ( Figure 1A), was expressed in ventral cord motor neurons but not in body muscles ( Figure 2A and data not shown). To identify the rig-3 expressing motor neurons, we performed several double labeling experiments.

However, SIK2 levels were markedly reduced after OGD in parallel with the increase in CRE activity and TORC1 dephosphorylation and nuclear translocation. AMPK and SIK1 did not exhibit such temporal associations with TORC1 and CRE activity. These observations raised the possibility that SIK2 regulates CREB-dependent transcription through an effect on TORC1 phosphorylation and nuclear translocation. Consistent with this

hypothesis, Bcr-Abl inhibitor overexpression of a constitutively active SIK2 suppressed OGD-induced CRE activity and increased cell death, whereas an inactive SIK2 was neuroprotective. Similarly, RNAi downregulation or pharmacological inhibition of SIK2 increased CRE activity and neuronal survival. The demonstration that SIK2 overexpression reduced CRE activity in cells cotransfected with a wild-type (WT) TORC1 construct, but was unable to do so in the presence of a phosphorylation-resistant mutant of TORC1, clearly established that these effects of SIK2 were mediated by TORC1 phosphorylation. Next, the authors set out to investigate the upstream

mechanisms by which OGD Cell Cycle inhibitor modulates SIK2 levels and CREB transcriptional activity. After identifying CaMK I/IV as potential upstream mediators of TORC1-CREB activation, the authors explored how CaMK I/IV activity could lead to the reduction in SIK2 levels induced by OGD. They found that overexpression of dominant-negative CaMK I or CaMK IV constructs prevents the OGD-induced downregulation of SIK2. Ribonucleotide reductase In addition, SIK2 degradation was associated with an increase in the phosphorylation of a specific SIK2 residue (Thr484). The importance of this site for SIK2 degradation was demonstrated by the fact that a phosphorylation-resistant Thr484 did not result in SIK2 degradation. In contrast, phosphorylation of Ser587, a SIK2 site also known to negatively regulate TORC phosphorylation (Katoh et al., 2006), did not impact SIK2 protein levels. In support of this conclusion,

OGD increased SIK2 Thr484 phosphorylation, but not Ser587 phosphorylation, suggesting that Thr484, but not Ser587, is an important target of CaMK I/IV-dependent SIK2 degradation. Sasaki et al. (2011) provide further support that SIK2 is a principal regulator of neuronal survival by generating sik2−/− mice and investigating whether neurons from these mice are protected from OGD. They found that sik2−/− neurons display higher survival rates than WT following OGD, an effect associated with a concomitant increase in TORC1-CREB activity and induction of prosurvival genes such as Bdnf and Ppargc-1a. Importantly, to determine whether SIK2 is involved in the mechanisms of neuronal death in vivo, they examined ischemic lesions in WT and sik2−/− mice following occlusion of the middle cerebral artery, a well-established model of ischemic stroke.

(2007) (Figure 1D). To account for the variability of the firing rate in consecutive recordings under the same conditions (Hargreaves et al., 2005, Leutgeb et al., 2007 and Fyhn et al., 2007), we Selleckchem BAY 73-4506 emulated the effect of undersampling of the space, an unavoidable condition given the experimental protocols. To account for the effect of undersampling, we introduced a stochastic factor in every comparison with a variance dependent on the rate (see Experimental Procedures). The level of the correction was obtained by fitting to the experimental data (PV correlation) of two subsequent recordings

obtained under the same condition (Figure S3). We observed an exponential-like decay shape for the correlation curves with the global level of decorrelation monotonically and positively affected by the level of influence of the LEC input (regulated by α). A value of α = 0.32 (Figure 1D) gave the best fit. With the value of α determined, we could then examine how morphing affected rate remapping. First, we investigated whether the simulated place fields have properties that match those experimentally CHIR-99021 in vivo observed. We found that simulated granule cells have multiple place fields (average of 2.2 place fields) and have a mean place field size of 943 cm2. The distribution of the number of place fields in each active

cell was similar to experimental measurements (Figure 1E, t = 0.98, p < 0.0005). The place field size is also in accord with data (analysis of Leutgeb et al., 2007 by de Almeida et al., 2009a). We also tested whether the observed restricted diversity of grid cell activity (Barry et al., 2007) affects the results of our simulation. When the grid cell proprieties were limited to one orientation and three grades of spacing, no significant difference in the whatever distribution of the number of place fields (Wilcoxon, p = 0.65) or the PV

correlation (Student’s t test, two-tailed, p = 0.31) was found. These results are not unexpected given previous work showing that MEC input alone can account for these properties; what is added here is the demonstration that the LEC inputs, when included in the model, do not interfere with place cell formation in the DG by the MEC inputs. We next directly compared the remapping of individual place fields of our simulation of morphing with the results obtained by Leutgeb et al. (2007) (Figure 2A). The experimental results show that all place fields of the same cell remap and do so independently; thus, one field may increase its firing rate during morphing while the other decreases its rate. Figure 2B shows this to be similarly true in our simulated place fields. Moreover, the relative proportion of remapping patterns that exhibit a significant fit for linear, quadratic, and sigmoidal functions could not be distinguished from the experimental observations (Figure 2C, t = 0.93, not significant [n.s.]).

However, this analysis confirmed that the apparent volumes occupied by GlyRs and gephyrin scaffolds were linearly correlated DAPT supplier with a slope of 0.8. The strength of synaptic transmission is directly related to the number and activity of neurotransmitter receptors at synapses. Receptor numbers, in turn, depend on the number of available receptor binding sites. We therefore devised strategies for the quantification of densely packed synaptic proteins in fixed spinal cord neurons. Our first approach

was based on the sequential photoconversion of clustered Dendra2-gephyrin molecules and the counting of their photobleaching steps. This was validated with another, independent strategy of molecule counting, consisting in the bleaching of nonconverted Dendra2-gephyrin clusters and the calibration of their total fluorescence with the mean fluorescence http://www.selleckchem.com/products/cb-839.html intensity of single fluorophores. The advantage of the second approach is that it does not require photoconvertible probes,

meaning that it can be used for the quantification of conventional fluorophores (discussed later). Making use of the photoconversion of Dendra2-gephyrin, we first applied 100 ms pulses of 405 nm to convert small subsets of fluorophores, which were bleached by continuous illumination with a 561 nm laser (Figure 4A1). The pool of nonconverted Dendra2 was depleted by the end of these recordings. Dendra2 was chosen because it is less prone to blinking than mEos2 (Annibale et al., 2011). Of note, the decay traces exhibited steps of fluorescence intensity associated with single converted (red) Dendra2 fluorophores (Figure 4A2). The peak intensities of the pulses could

thus be translated into numbers of fluorophores. The sum of all the peak intensities then yielded the total number of Dendra2-gephyrin molecules within the cluster. This value was related to the fluorescence intensity of the nonconverted (green) Dendra2-gephyrin image taken with the mercury lamp prior to the recording, to obtain a conversion factor ϕ of fluorescence intensity per molecule (ϕ = 92 ± 12 arbitrary units [a.u.] of fluorescence per molecule; mean ± SEM, n = 14 clusters from nine fields of view and three independent experiments). This conversion was then used to quantify a large set of fluorescence ADAMTS5 images, which suggested that synaptic clusters contain Dendra2-gephyrin molecules numbering between tens and several hundreds, with an average of 218 ± 9 (mean ± SEM, n = 622 clusters from 42 cells and three experiments; Figure 4A3). As an alternative approach to quantify the number of gephyrin molecules at inhibitory synapses, we determined the single-molecule intensity and the lifetime of the nonconverted (green) Dendra2 fluorophores. First, synaptic Dendra2-gephyrin clusters were fully bleached with 491 nm laser illumination (Figure 4B1).

The brain was removed, postfixed for 2 hr at 4°C, cryoprotected in 30% (w/v) sucrose in PBS for 48 hr at 4°C, and frozen on dry ice. Cryostat sections (20 μm) were mounted on Superfrost Plus slides and stored at −80°C. For double staining with PTPσ and PSD-95, the brains were immediately

extracted and snap-frozen in Tissue-Tek OCT compound by using isopentane cooled in dry ice and ethanol. Transverse cryostat sections were cut at 12 μm and fixed in 100% methanol for 10 min at −20°C. Sections were incubated in blocking solution (PBS + 3% bovine serum albumin [BSA] and 5% normal goat serum) with 0.25% Triton X-100 and then incubated overnight at 4°C with anti-TrkC (1:500; C44H5; Cell Signaling) and anti-VGLUT1 (1:1000; NeuroMab N28/9) or anti-gephyrin Smad inhibitor (1:1000; mAb7a; Synaptic Systems), or anti-PTPσ (IgG1; 1:500; clone 17G7.2; MediMabs) SCH 900776 in vivo and anti-PSD-95

family (IgG2a; 1:500; clone 6G6-1C9; Thermo Scientific). DAPI (100 ng/ml) was included with appropriate secondary antibodies. Confocal images were captured sequentially at an optical thickness of 0.37 μm on a Fluoview FV500 using a 60 × 1.35 numerical aperture (NA) objective with 405, 488, and 568 nm lasers and custom filter sets. For testing binding of soluble Fc-fusion proteins, COS-7 cells on coverslips were transfected with the expression vectors and grown for 24 hr. The transfected COS cells were washed with extracellular solution (ECS) containing 168 mM NaCl, 2.4 mM KCl, 20 mM HEPES (pH 7.4), 10 mM D-glucose, 2 mM CaCl2, and 1.3 mM MgCl2 with 100 μg/ml BSA (ECS/BSA) and then

incubated with ECS/BSA containing 100 nM purified Thymidine kinase Fc-fusion protein for 1 hr at room temperature. The cells were washed in ECS, fixed with 4% paraformaldehyde, and incubated with blocking solution and then biotin-conjugated antibodies to human IgG Fc or human IgG (H+L) (donkey IgG; 1:1000; Jackson ImmunoResearch) and Alexa568-conjugated streptavidin (Invitrogen). Nonfluorescent NeutrAvidin-labeled FluoSpheres (Invitrogen; F-8777; aqueous suspensions containing 1% solids) with a diameter of 1 μm were rinsed in PBS containing 100 μg/ml BSA (PBS/BSA) and incubated with either biotin-conjugated anti-GFP (here called anti-YFP; Rockland Immunochemicals) or biotin-conjugated anti-human IgG Fc (Jackson ImmunoResearch) at ∼6 μg antibody per μl beads in PBS/BSA at RT for 2 hr and then rinsed in PBS/BSA. The anti-human IgG Fc-bound beads were further incubated in each soluble Fc protein at 1–2 μg Fc protein per μl beads in PBS/BSA at RT for 2 hr then rinsed in PBS/BSA. Beads were sprinkled onto hippocampal neuron cultures (1 μl per coverslip), and 1 day later the cells were fixed and stained. In utero electroporation was performed as described (Tabata and Nakajima, 2001). In brief, timed pregnant CD-1 mice at 15.5–16.0 days of gestation (E15.5–E16) were anesthetized, the uterine horns were exposed, and ∼1 μl DNA solution (1.5 μg/μl) mixed with Fast Green was injected into the lateral ventricle.

, 2009 and Law and Gold, 2009), a more prevalent framework to study perception has been the “Bayesian brain hypothesis” that the brain constructs and updates a generative

model of its sensory inputs (Doya et al., 2011). One particular formulation of this hypothesis is predictive coding (Friston, 2005 and Rao and Ballard, 1999) that postulates that PEs are weighted by their precision and are computed at any level of hierarchically organized information processing cascades, as in sensory systems. This has been examined by several fMRI studies that contrasted predictable versus unpredictable visual stimuli, finding PE responses in visual areas specialized for the respective stimuli used (Harrison et al., 2007 and Summerfield and Koechlin, 2008) and precision-weighting under attention (Kok et al., 2012). Other studies have used an explicit model of trial-wise PEs, using visual (Egner et al., Volasertib mouse 2010) or audio-visual associative learning (den Ouden et al., 2010 and den Ouden et al., ABT-888 solubility dmso 2009) paradigms. Notably,

these studies did not have explicit readouts of subjects’ predictions and used relatively simple modeling approaches: they either described implicit learning processes (in the absence of behavioral responses) using a delta-rule RL model (den Ouden et al., 2009 and Egner et al., 2010), or dealt with indirect measures of prediction (e.g., reaction times) using an ideal Bayesian observer with a fixed learning trajectory across subjects (den Ouden et al., 2010). Our

present study goes beyond these previous attempts by (1) requiring explicit trial-by-trial many predictions, and (2) characterizing learning via a hierarchical Bayesian model that provides subject- and trial-specific estimates of precision-weighted PEs at different hierarchical levels of computation. Based on these advances, the present study shows much more widespread sensory PE responses than previously reported. Replicated in two separate groups, these responses were not only found in the visual cortex, but also in many supramodal areas in prefrontal, cingulate, parietal, and insular cortex (Figure 2). Whereas a distribution of reward (Vickery et al., 2011) and value signals (FitzGerald et al., 2012) across the whole brain have recently been demonstrated in humans, this has not yet been shown, to our knowledge, for PEs; in this case, precision-weighted PEs about the sensory outcome (visual stimuli). Perhaps the most interesting aspect of our findings on sensory outcome PEs, ε2, was the significant activation of the midbrain. In humans, strong empirical evidence exists for DA involvement in processing reward PEs (Montague et al., 2004 and Schultz et al., 1997) and novelty (Bunzeck and Düzel, 2006).

, 1997), and these normalization parameters were applied to the functional volumes. The resulting functional volumes were then smoothed with an 8 mm FWHM Gaussian kernel. Analyses of restudy phase data were performed using a general linear model (GLM) in which a 4 s boxcar was convolved with the canonical hemodynamic response function (HRF) and its temporal and dispersion derivatives for each trial to model the Talazoparib datasheet BOLD response (Friston et al., 1998). For each restudy block, ten event types were modeled: subsequent associative/category hits (“hits”) for LD object, LD scene, SD

object, SD scene, and SS trials (collapsed across study pair category), and subsequent item only hits (“item only hits”) for LD object, LD scene, SD object, SD scene, and SS trials (collapsed across study pair category). Hit trials were defined as those restudy phase trials for which associative recognition was later successful on either memory test. Likewise, subsequent item only hit trials were defined as those trials for which associative memory was unsuccessful but item recognition was successful on either memory

test. Hit trials were utilized in analyses evaluating the relationships between brain activity, connectivity, and behavior in order to ensure that the relationships revealed actually relate to activity or connectivity associated with subsequent successful associative memory retrieval rather than due to some difference in the ratio of subsequently click here remembered to forgotten trials in a given

condition. While the previously described model was utilized in ROI identification, a second model was generated in which SS trials were segregated according to study pair category (objects, scenes). Isotretinoin For this model, we were unable to further segregate trials according to subsequent memory status given that few subjects contributed sufficient (9+) SS object hit trials to enable their inclusion in the analysis as such. Thus, for this model, all SS object trials were collapsed into a single event type, as were all SS scene trials, separately. The average number of trials in each of the LD object, LD scene, SD object, and SD scene hit conditions was 21, 20, 23, and 24, with minimum-maximum ranges of 11–36, 11–39, 10–43, and 9–41, respectively. Four of the 24 subjects analyzed did not have sufficient SS hit trials, so their data were not used in ROI specification involving the SS hit condition. The localizer blocks were also modeled using a GLM but here a 16 s boxcar was convolved with the canonical HRF and its temporal and dispersion derivatives to model the BOLD response. Three event types were modeled for the localizer blocks (scene, object, and scrambled object miniblocks). For each block and task, each model also included as covariates the across-scan mean and six regressors representing motion-related variance (three for rigid-body translation and three for rotation).

Furthermore, our study opens the possibility that changes in cell migration may more generally participate in the evolution of brain connectivity. Consistently, this website it was shown that the formation of the corpus callosum, a mammalian-specific tract, relies on the migration of guidepost neurons (Shu et al., 2003a and Niquille

et al., 2009). Indeed, the mammalian telencephalon is characterized by a complex choreography of tangential neuronal migrations originating in both dorsal and ventral regions, which is essential for its functioning because cell migration defects have been involved in the etiology of several pathologies (McManus and Golden, 2005). Thus, our study raises the intriguing possibility that tangential neuronal migrations may have promoted the evolution of the telencephalon at the expense of its developmental robustness. Wild-type and GFP-expressing transgenic mice (Hadjantonakis et al., 1998), maintained in Swiss OF1 background, were used for expression analysis and tissue culture. Slit1−/−, Slit2−/−, and Slit1−/−;Slit2−/− mutant embryos were obtained by crossing Slit1+/−, Slit2+/−, and Slit1−/−;Slit2+/− parents ( Plump et al., 2002) maintained in B6D2 background.

Robo1−/−, Robo2−/−, and Robo1−/−;Robo2−/− were obtained by crossing Robo1+/−, Robo2+/−, and Robo1+/−;Robo2+/− beta-catenin inhibitor parents ( Grieshammer et al., 2004, Long et al., 2004 and Ma and Tessier-Lavigne, 2007), which were maintained in CD1, C57BL/6, and mixed CD1–C57BL/6 backgrounds, respectively. Animals were kept under French and EU regulations. Chinese soft-shelled turtle embryos (Nagashima et al., 2009) and corn-snake embryos (Gomez et al., 2008) were fixed

in 4% paraformaldehyde (PFA) for 24–48 hr, kept in methanol, rehydrated, and cut into 100 μm thick sections on a vibratome. Sheep embryo was obtained by permission from a slaughterhouse in Cartagena (Spain), perfused with 4% PFA, Non-specific serine/threonine protein kinase postfixed overnight, embedded in paraffin, and cut into 8 μm sections. Human embryos were obtained from legal abortions (procedure approved by the French National Ethical Committee CCNESVS), staged, fixed in 4% PFA, cryoprotected, and cut into 12 μm thick sections as described previously (Verney et al., 2001). For in situ hybridization, mouse or chicken brains were fixed overnight in 4% PFA in PBS. Twenty micrometer frozen sections or 80 μm free-floating vibratome sections were hybridized with digoxigenin-labeled probes as described before (Lopez-Bendito et al., 2006). For immunohistochemistry, cultured slices/explants and mouse or chicken embryos were fixed in 4% PFA at 4°C for 30 min and for 2–3 hr, respectively. Immunohistochemistry was performed on culture slices, Matrigel pads, and 100 μm free-floating vibratome sections as previously described (Lopez-Bendito et al.

, 2013). Such acute manipulations severely impaired not only synchronous but also asynchronous release in hippocampal neurons (Figure 4). Consistent

with the studies in PC12 and primary chromaffin cells, this result suggests that effectively all Ca2+-triggered neurotransmitter release is mediated by a synaptotagmin. Moreover, this result agrees with studies indicating that Syt7 functions as a Ca2+ sensor in neuroendocrine secretion and in lysosome exocytosis (Shin et al., 2002, Chakrabarti et al., 2003, Fukuda et al., 2004, Tsuboi and Fukuda, 2007, Schonn et al., 2008, Gustavsson et al., 2008, Gustavsson et al., 2009, Enzalutamide chemical structure Li et al., 2009 and Segovia et al., 2010). Finally, a role for Syt7 as a Ca2+ sensor in synaptic exocytosis agrees well with the similar Ca2+-binding properties and Ca2+-dependent phospholipid- and

syntaxin-binding properties of Syt1 and Syt7 (Li et al., 1995 and Sugita et al., 2002). However, Syt7 exhibits two puzzling properties. First, in neurons Syt7 is not detectable in synaptic vesicles but is at least partly localized to the plasma membrane (Sugita et al., 2001 and Takamori et al., 2006). This is puzzling given the localization of Syt7 to secretory vesicles selleck chemical in nonneuronal cells (Chakrabarti et al., 2003, Fukuda et al., 2004, Schonn et al., 2008, Gustavsson et al., 2008 and Gustavsson et al., 2009). Second, whereas in all vesicular synaptotagmins tested up to date, the C2B domain Ca2+-binding sites are essential for Ca2+ stimulation of exocytosis and the C2A domain Ca2+-binding sites only assist in Ca2+ triggering of exocytosis (e.g., see Mackler et al., 2002, Nishiki and Augustine, 2004, Shin et al., 2009, Cao et al., 2011 and Lee

et al., 2013), in Syt7 the C2A domain Ca2+-binding sites were essential for asynchronous release and the C2B domain Ca2+-binding sites were dispensable (Bacaj et al., 2013). The differences in the localization and the relative C2 domain functions between Syt1 and Syt7 may be related to each other, and the plasma membrane localization Parvulin of Syt7 may also explain, at least in part, why Syt7 is generally less effective than Syt1 in triggering exocytosis. Alternatively, it is possible that a small amount of Syt7 is present on synaptic vesicles, and its relatively low Ca2+-triggering efficiency is due to its poor synaptic vesicle-sorting efficiency. The recent Syt7 results suggest that different synaptotagmins collaborate and compete with each other as Ca2+ sensors for release and expand the finding that Syt1 is also coexpressed with Syt2 or Syt9 in some synapses where these synaptotagmins also complement each other physiologically (Xu et al., 2007 and Pang et al., 2006b). In the nonphysiological situation of a Syt1 or Syt2 knockout synapse, the observed remaining Ca2+-dependent release may be more complex than simply allowing Syt7 function to become manifest (Figure 5).