Author response: Synaptotagmin 7 functions as a Ca2+-sensor for synaptic vesicle replenishment (2013)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Synaptotagmin (syt) 7 is one of three syt isoforms found in all metazoans; it is ubiquitously expressed, yet its function in neurons remains obscure. Here, we resolved Ca2+-dependent and Ca2+-independent synaptic vesicle (SV) replenishment pathways, and found that syt 7 plays a selective and critical role in the Ca2+-dependent pathway. Mutations that disrupt Ca2+-binding to syt 7 abolish this function, suggesting that syt 7 functions as a Ca2+-sensor for replenishment. The Ca2+-binding protein calmodulin (CaM) has also been implicated in SV replenishment, and we found that loss of syt 7 was phenocopied by a CaM antagonist. Moreover, we discovered that syt 7 binds to CaM in a highly specific and Ca2+-dependent manner; this interaction requires intact Ca2+-binding sites within syt 7. Together, these data indicate that a complex of two conserved Ca2+-binding proteins, syt 7 and CaM, serve as a key regulator of SV replenishment in presynaptic nerve terminals. https://doi.org/10.7554/eLife.01524.001 eLife digest Neurons communicate with one another at junctions called synapses. The arrival of an electrical signal called an action potential at the first neuron triggers the release of chemicals called neurotransmitters into the synapse. These chemicals then diffuse across the gap between the neurons and bind to receptors on the second cell. The neurotransmitter molecules are stored in the first cell in packages known as vesicles, which release their contents by fusing with the cell membrane. Following a fusion event, neurons must replenish their vesicle stocks to ensure that they are ready for the arrival of the next action potential. This replenishment process is known to involve a calcium-dependent pathway and a calcium-independent pathway. A protein called calmodulin, that binds calcium ions, has an important role in the first of these pathways. Now, Liu et al. have shown that another protein, synaptotagmin 7, also has a key role in the replenishment of synaptic vesicles, possibly as a sensor for calcium ions. Moreover, Liu et al. found that synaptotagmin 7 and calmodulin bind to each other to form a complex, which suggests that the calcium-dependent replenishment pathway is regulated by this complex. The synaptotagmins are a family of 17 proteins, three of which are present in all animals. Two of these were known to have roles in synapses, but the role of the third—synaptotagmin 7—had been unclear. In addition to providing a more complete understanding of the replenishment of synaptic vesicles, the work of Liu et al. also supplies the final piece of the jigsaw regarding the role of the synaptotagmins that are present in all animals. https://doi.org/10.7554/eLife.01524.002 Introduction Chemical communication at synapses in the central nervous system is subject to short- and long-term changes in strength (Malenka, 1994). For example, the replenishment of synaptic vesicles (SV) in the readily releasable pool (RRP), which can be exhausted during high frequency stimulation (HFS), plays a critical role in determining the rate and degree of short-term synaptic depression (Wang and Kaczmarek, 1998). It has been reported that Ca2+ plays an important role in the replenishment step (Dittman and Regehr, 1998; Wang and Kaczmarek, 1998; Hosoi et al., 2007; Sakaba, 2008). Thus, identification of Ca2+ sensors for SV replenishment has emerged as a critical issue, and the ubiquitous Ca2+-binding protein CaM has been implicated in this process (Sakaba and Neher, 2001; Hosoi et al., 2007), by interacting with Munc13-1 (Lipstein et al., 2013). Synaptotagmins represent another well-known family of Ca2+-binding proteins; some isoforms function as Ca2+-sensors for rapid SV exocytosis (Chapman, 2008). Of the 17 syt isoforms in the mammalian genome, only syt 1, 4, and 7 appear to be expressed in all metazoans (Barber et al., 2009), suggesting that they are involved in key, evolutionarily conserved, membrane trafficking pathways. In neurons, functions for syt 1 and 4 have been established. Syt 1 serves as a Ca2+ sensor for fast synchronous SV release (Geppert et al., 1994; Koh and Bellen, 2003; Nishiki and Augustine, 2004; Liu et al., 2009), and syt 4 modulates transmission by regulating neurotrophin release (Dean et al., 2009, 2012b) and also regulates exocytosis in the posterior pituitary (Zhang et al., 2009). One group reported that Syt7 knock-out (KO) mice (hereafter termed KO) had normal synaptic transmission (Maximov et al., 2008), but in a more recent study from the same group concluded that syt 7 functions as Ca2+ sensor for asynchronous release (Bacaj et al., 2013). It was proposed that this contradiction was due to the use of KO mice in the former study, as opposed to the more acute knock-down (KD) approach used in the latter study. However, KD of syt 7 in otherwise WT neurons had no effect (Bacaj et al., 2013), so the physiological function of syt 7 in the mammalian central nervous system remains an open issue, whereas there is evidence that syt 7 regulates asynchronous synaptic transmission at the Zebrafish neuromuscular junction (Wen et al., 2010). The apparent discrepancy regarding the physiological function of syt 7 between mice and Zebrafish is likely due to species differences, analogous to the different functions of syt 4 in Drosophila vs mice (Yoshihara et al., 2005; Dean et al., 2009; Wang and Chapman, 2010). Here, we used high frequency stimulation, and other methods, to study synaptic transmission in cultured hippocamal neurons from KO mice and discovered that syt 7, via a highly specific interaction with CaM, functions as a Ca2+-sensor that regulates Ca2+-dependent SV replenishment. Results Loss of syt 7 does not affect spontaneous release, or evoked release triggered by single action potentials We first carried out whole-cell voltage clamp recordings using primary hippocampal neurons obtained from wild-type (WT) and KO mice and found that spontaneous SV release, measured in the presence of TTX, was unaffected in the KO neurons (Figure 1A–E). We then carried out paired recordings by stimulating one neuron with a bipolar glass electrode and recording from a second synaptically connected neuron, as previously described (Liu et al., 2009), to examine evoked excitatory postsynaptic currents (EPSCs) triggered by single action potentials (AP). The EPSC amplitudes (Figure 1F–I,K–L), and the charge transfer kinetics (Figure 1J), were also unchanged in the KOs, confirming that syt 7 does not function as Ca2+ sensor for asynchronous release in hippocampal neurons (Maximov et al., 2008). We also examined the Ca2+-sensitivity of single AP-evoked SV release, by recording at different concentrations of extracellular Ca2+, and found that it was the same between WT and KO neurons (Figure 1K–M). Figure 1 Download asset Open asset Normal spontaneous and single AP evoked EPSCs in KO neurons. (A) Representative miniature EPSC (mEPSC; recorded in the presence of TTX) traces from WT and KO hippocampal neurons. (B) Representative mEPSCs from WT (grey) and KO (red) neurons. (C–E) mEPSC amplitude (C, WT: 20.78 ± 1.26 pA; KO: 20.46 ± 2.38 pA), charge (D, WT: 116 ± 12 fC; KO: 119 ± 18 fC) and frequency (E, WT: 2.32 ± 0.44 Hz; KO: 2.83 ± 0.63 pA) were the same between WT and KO neurons. (F–J) Analysis of single AP evoked EPSCs recorded from WT and KO neurons in the presence of 10 mM [Ca2+]o. (F) Representative EPSCs recorded from WT and KO neurons. (G–I) EPSC amplitude (G, WT: 0.25 ± 0.05 nA; KO: 0.24 ± 0.03 nA), peak latency (H, WT: 17.03 ± 2.22 ms; KO: 18.27 ± 2.29 ms) and rise time (I, WT: 4.8 ± 0.53 ms; KO: 5.21 ± 0.74 ms) were the same between WT and KO neurons. (J) The normalized cumulative total charge was fitted with a double exponential function (solid line). WT and KO neurons had identical charge transfer kinetics. (K) Representative single AP evoked EPSCs recorded from WT and KO neurons in the presence of the indicated [Ca2+]o. (L–M) EPSC amplitudes (L) and relative amplitudes (M) plotted vs [Ca2+]o. The n values indicate the number of neurons from two independent litters of mice; these values are provided in the bars in all figures in this study. All data represent mean ± SEM. Statistical significance was analyzed using the Student’s t test. https://doi.org/10.7554/eLife.01524.003 KO neurons exhibit enhanced synaptic depression We next examined short-term synaptic plasticity in WT and KO neurons by examining the paired pulse ratio (PPR), calculated by dividing the second EPSC (EPSC2) by the first EPSC (EPSC1) (50 ms interval; Figure 2A). We also measured synaptic depression during trains of HFS (20 Hz/2.5 s, Figure 3A). The PPR was the same between WT and KO neurons (Figure 2B), but KO neurons displayed faster depression during HFS (Figure 3B). Importantly, expression of WT syt 7, but not the 4D/N Ca2+-ligand mutant (which was properly folded but failed to bind Ca2+ (Figure 3—figure supplement 1)), rescued the depression phenotype (Figure 3B). We note that WT syt 7, and the 4D/N mutant, were both present in synapses but were not well colocalized with the SV marker, synapsin, as compared to syt 1 (Figure 3—figure supplement 2). The surface fractions, as well as the internal fractions, were similar between syt 1 and 7 measured by using a pHluorin tag (Figure 3—figure supplement 3), consistent with a previous report (Dean et al., 2012a). Figure 2 Download asset Open asset Loss of syt 7 does not affect the PPR. (A) Representative EPSCs recorded in paired-pulse experiments, with 50 ms inter-stimulus intervals, from WT, KO, or KO neurons expressing either WT (KO + syt 7) or the Ca2+ ligand mutant form (KO + syt 7 4D/N) of syt 7. (B) The PPRs were the same among all groups (WT: 0.83 ± 0.04; KO: 0.75 ± 0.03; KO + syt 7: 0.84 ± 0.05; and KO + syt 7 4D/N: 0.76 ± 0.03). The n values indicate the number of neurons, obtained from three independent litters of mice, used for these experiments. All data represent mean ± SEM. Statistical significance was analyzed by one-way ANOVA. https://doi.org/10.7554/eLife.01524.004 Figure 3 with 5 supplements see all Download asset Open asset SV replenishment is reduced in KO neurons. (A) Representative EPSC traces (20 Hz/2.5 s) recorded from WT (black), KO (red), or KO neurons expressing either WT (KO + syt 7, gray) or the 4D/N mutant form of syt 7 (KO + syt 7 4D/N, blue). The phasic and tonic components of transmission are indicated within the representative WT trace. (B) The peak amplitude of each EPSC was divided by the peak of the first response and plotted vs time. Inset: expansion of the last 10 responses. (C) Plot of cumulative total phasic charge transfer vs time. Data points from the last ten EPSCs were fitted with a linear function to calculate the RRP size (y-intercept in panel D), and SV replenishment rate (the slope divided by the RRP size; panel E). Inset: expansion of the last ten responses. (D) The RRP size is the same among all groups (WT: 38.70 ± 4.78 pC; KO: 33.49 ± 2.48 pC; KO + syt 7: 37.58 ± 3.02 pC; and KO + syt 7 4D/N: 34.16 ± 3.63 pC). (E) The SV replenishment rate was reduced in KO (0.26 ± 0.03 s−1) as compared to WT neurons (0.52 ± 0.06 s−1). WT (0.47 ± 0.05 s−1), but not 4D/N mutant syt 7 (0.24 ± 0.02 s−1), rescued SV replenishment. The n values indicate the number of neurons, obtained from three independent litters of mice, used for these experiments. All data shown represent mean ± SEM. ***p<0.001, analyzed by one-way ANOVA, compared to WT. https://doi.org/10.7554/eLife.01524.005 Five mechanisms could potentially contribute to the faster depression observed in KO neurons: changes in postsynaptic receptor density or kinetic properties; saturation or desensitization of postsynaptic receptors; a smaller RRP size which fails to supply sufficient SVs; defective SV replenishment; or slower SV recycling/refilling. Below, we address each of these possibilities and demonstrate that defective SV replenishment underlies the increased rate of depression in KO neurons. Loss of syt 7 impairs SV replenishment The unaltered quantal release properties in KO neurons (Figure 1A–E) rule out changes in postsynaptic components, such as the density or kinetic properties of AMPA receptors. To further exclude the role of desensitization or saturation of postsynaptic receptors during HFS, we monitored transmission, during HFS, in the presence of cyclothiazide (CTZ, 50 µM) and kynurenic acid (KYN, 100 µM), as described previously (Neher and Sakaba, 2001; Figure 3—figure supplement 4A). CTZ and KYN reduce AMPA receptor desensitization and saturation, respectively. KO neurons still displayed faster depression (Figure 3—figure supplement 4B). Next, we integrated the phasic charge from the HFS experiments (Figure 3A) and plotted the cumulative charge vs time (Figure 3C). The last ten EPSCs were best fitted by a linear function (solid line in Figure 3C; linearity is established in the inset), as described in previous reports utilizing hippocampal neurons (Stevens and Williams, 2007) and the calyx of Held (Schneggenburger et al., 1999; Hosoi et al., 2007), to calculate RRP size, release probability, and SV replenishment rates. The size of the RRP (the y-intercept) was similar between WT and KO neurons (Figure 3D). Since single AP evoked EPSCs were identical between WT and KO neurons (Figure 1F), SV release probability (calculated by dividing the EPSC charge by the RRP size) was unchanged in the KOs (0.21 ± 0.03 for KO vs 0.24 ± 0.02 for WT). The slope of the linear function was divided by the y-intercept in a given cell pair to calculate the SV replenishment rate. Strikingly, we found that the replenishment rate was reduced by ∼50% for phasic release in KO neurons (0.26 ± 0.03 s−1 for KO vs 0.52 ± 0.06 s−1 for WT) (Figure 3E). WT syt 7, but not the 4D/N mutant, fully rescued SV replenishment (Figure 3C,E). The size of the RRP was unchanged in the rescue experiments (Figure 3D). We also applied similar methods as described in Figure 3C to investigate SV replenishment during tonic transmission (Figure 3—figure supplement 5A,B); the replenishment rate was, again, reduced by ∼50% in KO neurons (2.19 ± 0.22 s−1 for KO vs 3.95 ± 0.61 s−1 for WT), and WT syt 7, but not the 4D/N mutant, rescued this defect (Figure 3—figure supplement 5C). These results are consistent with a previous report indicating that tonic transmission is maintained by Ca2+-dependent SV replenishment during activity (Otsu et al., 2004). Slower RRP recovery in KO neurons To further confirm the replenishment defect in KO neurons, we investigated the recovery kinetics of phasic release after HFS. Recovery was calculated by measuring the total charge transfer during phasic release at various time intervals, between bouts of HFS, where the second response was divided by the first response (Figure 4A). A double exponential function was used to fit the recovery time-course, revealing fast and slow components of recovery. Notably, fast recovery (Figure 4B) was delayed by ∼60% in KO neurons (1.32 ± 0.22 s) as compared to WT neurons (0.55 ± 0.17 s), while slow recovery (Figure 4C) was unaffected by loss of syt 7 (WT: 15.72 ± 2.85 s; KO: 14.13 ± 2.24 s). WT syt 7 (0.52 ± 0.12 s), but not the 4D/N mutant (1.28 ± 0.26 s), rescued fast recovery (Figure 4A,B), indicating that syt 7 regulates this process in a Ca2+-dependent manner during phasic release. Surprisingly, the recovery of total tonic transmission during HFS also displayed the same two recovery components (Figure 4—figure supplement 1A–C). WT syt 7, but not the 4D/N mutant, rescued fast recovery of tonic transmission (Figure 4—figure supplement 1A,B). Thus, recovery of phasic and tonic release might involve the same Ca2+-dependent pathway. Figure 4 with 1 supplement see all Download asset Open asset Impaired fast recovery of phasic transmission in KO neurons. (A) Time course for the recovery of phasic release (Δt = 0.5, 1, 3, 5, 9, 17, 33 and 65 s). Data were fitted using a double exponential function, yielding fast (B) and slow (C) recovery. The upper diagram is the stimulation protocol. Inset: expansion of the early phase of the plot. (B) The fast recovery time constant was increased in KO (1.32 ± 0.22 s) as compared to WT neurons (0.55 ± 0.17 s). WT syt 7 (0.52 ± 0.12 s), but not the 4D/N mutant (1.28 ± 0.26 s), rescued the SV replenishment rate in KO neurons. (C) The slow recovery time constant was invariant among all groups. (WT: 15.72 ± 2.85 s; KO: 14.13 ± 2.24 s; KO + syt 7: 17.03 ± 3.24 s; and KO + syt 7 4D/N: 18.88 ± 4.45 s). The n values indicate the number of neurons, obtained from three independent litters of mice, used for these experiments. All data shown represent mean ± SEM. ***p<0.001, analyzed by one-way ANOVA, compared to WT. https://doi.org/10.7554/eLife.01524.011 Enhanced synaptic depression in KO neurons during ‘first fusion’ events To address potential defects in SV recycling/refilling in KO neurons during ongoing transmission, we utilized synaptophysin-pHluorin (sypHy) to measure SV exocytosis driven by HFS (20 Hz) in the presence of bafilomycin (1 µM), an inhibitor of the vesicular proton pump (Figure 5A–C), to measure first fusion events. Under these conditions, in which SV recycling cannot play a role in ongoing synaptic transmission, KO neurons still displayed a significant reduction in SV release (Figure 5B). Importantly, KO neurons exhibited reductions in SV release as early as the second imaging time point (i.e., 1 sec, 20 Hz stimulation) (Figure 5C), consistent with electrophysiological recordings in Figure 3C. Moreover, we also examined first release events during HFS by recording synaptic currents in the presence of bafilomycin and, again, significantly faster depression (Figure 5D), reduced cumulative release (Figure 5E), unchanged RRP size (Figure 5F), and slower SV replenishment (Figure 5G), were observed in the KOs. These data indicate that SV recycling does not contribute to the observed reduction in SV release in KO neurons during HFS. Figure 5 Download asset Open asset The reduction in SV release in KO neurons during HFS is independent of vesicle recycling. (A) Representative image showing the sypHy signal from the same synapse upon stimulation in the absence, and presence, of bafilomycin. (B) Averaged traces of sypHy in WT (black) and KO (red) neurons in response to stimulation in the presence of bafilomycin. KO neurons displayed a significant reduction in sustained SV release as compared to WT neurons. Data were collected for ∼300 as from each from three independent litters of All data shown represent mean analyzed by ANOVA. The indicated by the is in panel (C) KO neurons reduced sypHy early in the analyzed using the Student’s t test. in response to HFS, in the presence of bafilomycin. (D) The peak amplitude of each EPSC was normalized to the peak of the first response and plotted vs time. (E) Plot of cumulative total phasic charge transfer vs time. Data were fitted and analyzed as described in Figure 3C. (F) The size of the RRP is the same in WT and KO neurons (WT: ± pC; KO: ± pC). in the SV replenishment rate in KO neurons in the presence of bafilomycin (WT: ± 0.06 KO: ± 0.03 s−1). The n values indicate the number of neurons, obtained from three independent litters of mice, used for these experiments. All data shown represent mean ± SEM. ***p<0.001, analyzed using the Student’s t test. Syt 7 functions as a Ca2+-sensor for Ca2+-dependent SV replenishment that Ca2+ plays a critical role in the replenishment of (Dittman and Regehr, 1998; Wang and Kaczmarek, 1998; Hosoi et al., 2007; Sakaba, 2008). The syt 7 4D/N mutant, which fails to bind Ca2+, was to rescue SV replenishment in KO neurons (Figure 3), suggesting that syt 7 functions as Ca2+-sensor during replenishment. To Ca2+ is for the function of syt 7, we measured recovery of the RRP by paired of each for 10 s), with time intervals, of with with and addition of acid to internal et al., (Figure For we also measured RRP recovery physiological Figure Figure Download asset Open asset Syt 7 functions as a Ca2+-sensor for Ca2+-dependent SV replenishment. (A) panel the (Δt = 7, and s). panel to of from different groups in 2 mM or (B) The RRP size, calculated from the first was same among the all groups 2 mM WT: ± KO: ± KO + syt 7: ± and KO + syt 7 4D/N: ± in conditions, WT: 0.83 ± KO: 0.76 ± (C) The time course of RRP recovery in 2 mM between of Data were fitted with a single exponential function to calculate the recovery time constant in panel (D) The recovery of the RRP in Data were fitted with a single exponential function to calculate the recovery time constant in panel (E) In 2 mM recovery of the RRP was significantly in KO ± s) neurons as compared to WT ± s) neurons, and was rescued by WT syt 7 ± s), but not the 4D/N mutant ± s). In conditions, RRP recovery in WT neurons ± s) was to the same as in KO neurons ± 3.95 s). The n values indicate the number of neurons, obtained from three independent litters of mice, used for these experiments. All data shown represent mean ± SEM. ***p<0.001, analyzed using one-way ANOVA, compared to WT. with the same RRP size measured by HFS among different groups (Figure the RRP size, calculated from the first response to was the same between WT, KO, and KOs expressing WT or the syt 7 4D/N mutant, in physiological (Figure a role for syt 7 in SV Moreover, the RRP size was the same between the and physiological (Figure Of there was a significant in the size of the RRP as measured using (Figure calculated by dividing the RRP charge by the quantal vs HFS (Figure ∼300 This was during electrical stimulation, only the synapses between the recorded neuron and neuron are In all of the synapses that on the recorded We then plotted the ratio of the second relative to the first as a function of the time to calculate the recovery time course of the RRP in physiological (Figure and (Figure these data were best fitted by a single exponential function (solid line in Figure In physiological recovery of the RRP was by ∼60% in the KOs ± for KO vs ± for WT) (Figure which is similar to the reported using HFS (Figure 4B). WT syt 7 ± s), but not the 4D/N mutant ± s), rescued this phenotype (Figure in (Figure the recovery of the RRP in WT neurons was reduced to the same as observed for KO neurons, with or expression of the 4D/N mutant in physiological (Figure Together, the results that there are Ca2+-dependent and Ca2+-independent for replenishment, and that syt 7 functions as a Ca2+-sensor that regulates the Ca2+-dependent pathway. SV release et al., the Ca2+-dependent for recovery of the observed in the experiments, is likely due to the In experiments that syt 7 to fusion at 100 = et al., it is that syt 7 can to Ca2+-dependent SV replenishment. Syt 7 with CaM to Ca2+-dependent SV replenishment The reduced SV replenishment phenotype in KO neurons is of the of CaM on vesicle replenishment in the calyx of Held (Sakaba and Neher, 2001; Hosoi et al., 2007), the that there are between CaM and syt syt 1 has been reported to bind CaM We using CaM and the of syt 1, 4, 7, and 10 (Figure Surprisingly, Ca2+-dependent to CaM was observed only for syt 7, the of the interaction of of syt 1, or other syt to CaM might have Moreover, the interaction between syt 7 and CaM was by the Ca2+-ligand in the syt 7 4D/N mutant (Figure and was specific for Ca2+ as was effect (Figure Together, these are consistent with a in which the Ca2+-sensor for replenishment is a complex between these two conserved Ca2+-binding proteins, syt 7 and Figure 7 with 1 supplement see all Download asset Open asset Syt 7 with CaM to Ca2+-dependent SV replenishment. (A) CaM the of syt 7, but not syt 1, 4, or in the presence, but not the absence, of the syt 7 4D/N mutant failed to 2 mM 1 mM between indicate that were to of the of the was no was observed CaM was from the not (B) fails to of syt 7 to 2 mM 1 mM 1 mM were as in panel (C) Representative EPSCs traces (20 Hz/2.5 s) recorded from WT (black) and KO (red) neurons in the presence of (D) Plot of cumulative total phasic release charge vs time in the presence of Data were analyzed as described in Figure 3C. (E) The size of the RRP was the same among all groups WT: 38.70 ± 4.78 pC; KO: 33.49 ± 2.48 pC; in neurons: WT: ± pC; KO: ± pC). (F) reduced SV replenishment in WT neurons (0.24 ± s−1 vs 0.52 ± 0.06 s−1 for but had no effect on KO neurons (0.21 ± 0.05 s−1 vs 0.26 ± 0.03 s−1 for The n values indicate the number of neurons, obtained from three independent litters of mice, used for these experiments. All data shown represent mean ± SEM. ***p<0.001, analyzed by one-way ANOVA, compared to WT. To address the potential of CaM to SV replenishment in hippocampal neurons, we next analyzed the SV replenishment rate in WT and KO neurons in the presence and of 20 µM), a CaM (Sakaba and Neher, 2001; Figure were for to recording to ensure that had and then HFS trains were WT neurons displayed faster depression (Figure and reduced cumulative phasic release (Figure in the presence of consistent with previous on the calyx of Held (Sakaba and Neher, 2001; Hosoi et al., cumulative tonic transmission in WT neurons was also reduced by (Figure supplement both cumulative phasic and tonic release in WT neurons were reduced to the same as in the KO neurons, which were not by (Figure Figure supplement These experiments also does not the size of the RRP (Figure and in WT neurons, the replenishment for phasic (Figure and tonic release (Figure supplement were both reduced to the same observed for the KOs, with or A CaM recovery of the RRP in WT neurons, loss of syt 7 We next examined the recovery of phasic (Figure and tonic release (Figure supplement as described in Figure 4, in the presence of during HFS, in WT and KO neurons. The time course for fast during phasic (Figure and tonic (Figure supplement transmission in WT neurons, was