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• W4313005778 abstract "Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Methods Data availability References Decision letter Author response Article and author information Metrics Abstract Synapses contain a limited number of synaptic vesicles (SVs) that are released in response to action potentials (APs). Therefore, sustaining synaptic transmission over a wide range of AP firing rates and timescales depends on SV release and replenishment. Although actin dynamics impact synaptic transmission, how presynaptic regulators of actin signaling cascades control SV release and replenishment remains unresolved. Rac1, a Rho GTPase, regulates actin signaling cascades that control synaptogenesis, neuronal development, and postsynaptic function. However, the presynaptic role of Rac1 in regulating synaptic transmission is unclear. To unravel Rac1’s roles in controlling transmitter release, we performed selective presynaptic ablation of Rac1 at the mature mouse calyx of Held synapse. Loss of Rac1 increased synaptic strength, accelerated EPSC recovery after conditioning stimulus trains, and augmented spontaneous SV release with no change in presynaptic morphology or AZ ultrastructure. Analyses with constrained short-term plasticity models revealed faster SV priming kinetics and, depending on model assumptions, elevated SV release probability or higher abundance of tightly docked fusion-competent SVs in Rac1-deficient synapses. We conclude that presynaptic Rac1 is a key regulator of synaptic transmission and plasticity mainly by regulating the dynamics of SV priming and potentially SV release probability. Editor's evaluation Keine et al. study the roles of the RhoGTPase Rac1 and actin in neurotransmitter release by ablating Rac1 at an age when synapses are essentially mature, thereby minimizing developmental compensations. They describe compelling findings supporting an increase in synaptic strength, interpreted as either an increase in release probability or priming of synaptic vesicles. Although direct support for Rac1-dependent altered presynaptic actin is not provided, the study delivers fundamental functional information on the role of Rac1 in regulating presynaptic release. https://doi.org/10.7554/eLife.81505.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Information encoding in the nervous system requires synaptic transmission to drive and sustain action potentials (APs) over rapidly changing and highly variable AP firing rates (Reinagel and Laughlin, 2001; Theunissen and Elie, 2014; Brette, 2015; Azarfar et al., 2018). However, the number of synaptic vesicles (SVs) available for fusion in response to an AP, the readily releasable pool (RRP), is limited (Alabi and Tsien, 2012). Therefore, tight regulation of SV release and RRP replenishment is required for synaptic reliability and temporally precise information encoding (Neher, 2010; Hallermann and Silver, 2013). Priming is the process that generates fusion-competent SVs. It is a critical step in the SV cycle that primarily regulates RRP size and SV pool replenishment. Priming also controls SV release probability (Pr) by determining SV fusogenicity (‘molecular priming’) and regulating the spatial coupling between docked SV and presynaptic Ca2+ entry (‘positional priming’) (Klug et al., 2012; Schneggenburger and Rosenmund, 2015; Neher and Brose, 2018). In some synapses, the SV priming kinetics are highly dependent on presynaptic cytosolic Ca2+ levels, which are activity-dependent. Importantly, human mutations in molecules that regulate priming are associated with neurological disorders (Waites et al., 2011; Wondolowski and Dickman, 2013; Torres et al., 2017; Bonnycastle et al., 2021). Therefore, elucidating the molecular mechanisms that regulate SV priming is critical to understanding the diversity of neuronal information encoding in health and disease. Actin is a central component of both the presynaptic and postsynaptic compartment, with diverse roles in regulating synaptic function and neuronal circuit development. Manipulation of actin dynamics or interference with presynaptic AZ proteins implicated in regulating actin dynamics affects transmitter release and SV replenishment, as well as Pr (Morales et al., 2000; Sakaba and Neher, 2003; Cingolani and Goda, 2008; Sun and Bamji, 2011; Waites et al., 2011; Lee et al., 2012; Lee et al., 2013; Montesinos et al., 2015; Rust and Maritzen, 2015). However, due to disparate results, the role of presynaptic actin signaling cascades in regulating transmitter release and SV pool replenishment is controversial. Finally, in contrast to the postsynaptic compartment, our understanding of how presynaptic regulators of actin signaling cascades control synaptic transmission and short-term plasticity (STP) is in the early stages. Rac1, a Rho GTPase, is a critical regulator of actin signaling cascades (Bosco et al., 2009; Yasuda, 2017), and human mutations in Rac1 are associated with neurological disorders (Bai et al., 2015; Reijnders et al., 2017; Zamboni et al., 2018). Rac1 is involved in multiple processes controlling synaptogenesis, axon guidance, neuronal development, and postsynaptic function to regulate neuronal circuit function (Bai et al., 2015). Although Rac1 is expressed in both the pre- and postsynaptic compartment (Threadgill et al., 1997; Doussau et al., 2000; Kumanogoh et al., 2001; O’Neil et al., 2021), its presynaptic role in regulating synaptic transmission is not well understood. Recent work using cultured hippocampal neurons proposed that presynaptic Rac1 is a negative regulator of SV pool replenishment (O’Neil et al., 2021), although at which steps in the SV cycle it exerts its regulatory role is unknown. Finally, how Rac1 regulates the temporal dynamics of transmitter release and pool replenishment in a native neuronal circuit remains elusive. To unravel the roles of presynaptic Rac1 in regulating transmitter release, we utilized the calyx of Held, a glutamatergic axosomatic presynaptic terminal in the auditory brainstem, which is the sole input to drive AP firing in the principal cells of the medial nucleus of the trapezoid body (MNTB) (Borst and Soria van Hoeve, 2012; Joris and Trussell, 2018). In the calyx of Held, RRP dynamics are tightly regulated to support a nearly failsafe synaptic transmission with high temporal precision, but the molecular machinery of transmitter release and SV pool replenishment and their regulation are similar to conventional presynaptic terminals in the central nervous system (Iwasaki and Takahashi, 1998; Iwasaki et al., 2000; Iwasaki and Takahashi, 2001; Sätzler et al., 2002; Taschenberger et al., 2002; Rollenhagen and Lübke, 2006; Neher and Sakaba, 2008; Alabi and Tsien, 2012; Eggermann et al., 2011; Hallermann and Silver, 2013; Schneggenburger and Rosenmund, 2015). In addition, molecular manipulations specific to only the calyceal terminal can be carried out at different developmental stages (Wimmer et al., 2006; Young and Neher, 2009; Chen et al., 2013; Lübbert et al., 2019). To elucidate the roles of presynaptic Rac1 in regulating transmitter release while avoiding interference with its role in synaptogenesis, axon guidance, and neuronal development, we selectively deleted Rac1 2 days after hearing onset in postnatal day (P) 14 mouse calyx of Held synapses. At this time point, the synapse is functionally mature, and neuronal properties of brainstem circuits are considered ‘adult-like’. Subsequently, we determined how the loss of Rac1 impacted calyx of Held – MNTB principal cell transmission at the adult stage (P28 onwards) (Sonntag et al., 2009; Crins et al., 2011; Sonntag et al., 2011). Presynaptic Rac1 deletion did neither affect the calyx of Held morphology nor active zone (AZ) ultrastructure but led to increased synaptic strength, faster SV pool replenishment, and augmented spontaneous SV release. Additionally, we found that the loss of Rac1 delayed EPSC onsets and potentiated asynchronous release during high-frequency train stimulation. Analysis of the experimental data with constrained STP models confirmed faster SV priming kinetics in Rac1-deficient synapses. Methods of quantal analysis, which assume a single and homogenous pool of readily releasable SVs (Neher, 2015; Schneggenburger and Rosenmund, 2015), reported an increased Pr and a tendency towards an increased RRP after Rac1 loss. Both experimental findings were corroborated in numerical simulation using a single-pool STP model (Weis et al., 1999). In contrast, simulations based on a sequential two-step priming scheme which assumes two distinct states of docked/primed SVs, a loosely docked (LS), immature priming state which is not fusion competent, and a tightly docked (TS), mature priming state which is fusion competent (Neher and Taschenberger, 2021; Lin et al., 2022), required only changes in SV priming kinetics but no change in Pr or the number of release sites to reproduce experimental data. Simulations based on the sequential two-step SV priming and fusion scheme fully accounted for the increased synaptic strength in Rac1−/− synapses by a larger abundance of tightly docked SVs. Therefore, we propose that presynaptic Rac1 is a key molecule that controls synaptic strength and STP primarily by regulating the SV priming dynamics and potentially Pr. Finally, we conclude that presynaptic Rac1 is a critical regulator of synaptic transmission and plasticity. Results Presynaptic deletion of Rac1 after hearing onset does not impact calyx of Held morphology or AZ ultrastructure Presynaptic terminals contain Rac1 (Doussau et al., 2000; O’Neil et al., 2021) which regulates synaptogenesis, axon guidance, and neuronal development (Xu et al., 2019; Zhang et al., 2021). In this study, we aimed to elucidate Rac1’s presynaptic function in controlling synaptic transmission and plasticity independent of its role in regulating synapse development and maturation at the calyx of Held synapse. To do so, we injected HdAd co-expressing Cre recombinase and EGFP into the cochlear nucleus (CN) of P14 Rac1flox/flox mice when the calyx of Held synapse is considered ‘adult-like’ and commenced synapse analysis at P28 onwards (Figure 1A; Sonntag et al., 2009; Crins et al., 2011; Sonntag et al., 2011). Since Rac1 controls synapse development and morphology, it was essential to determine whether the loss of Rac1 after hearing onset altered the calyx of Held morphology or AZ ultrastructure. We analyzed calyx morphology from 3D reconstructions of confocal z-stack images acquired from Rac1+/+ and Rac1−/− calyces at P28 and found no difference in calyx surface area or volume (Figure 1B). To determine if the loss of Rac1 impacted AZ ultrastructure, we performed ultrathin-section TEM and analyzed AZ length, SV distribution, and the number of docked SVs and found no difference between Rac1+/+ and Rac1−/− (Figure 1C). Therefore, we conclude that, after hearing onset, Rac1 does not regulate calyx of Held morphology or AZ ultrastructure. Figure 1 Download asset Open asset Loss of presynaptic Rac1 after hearing onset does not affect calyx of Held gross morphology or ultrastructure. (A) Cre recombinase-expressing HdAds were injected into the cochlear nucleus of Rac1fl/fl mice at P14, yielding Rac1−/− calyces of Held. All experiments were performed at around four weeks of age. Cre recombinase-expressing calyces could be visually identified by simultaneous expression of EGFP. (B1) Representative reconstruction of calyx terminals of Rac1+/+ (left) and Rac1−/− (right) animals. (B2) Calyx morphology assessed by surface area (left) and volume (right) was not affected by the loss of Rac1. (C1) Representative EM images of the active zone (yellow) and docked SV (blue) to assess synaptic ultrastructure. (C2) AZ length and number of docked SV were comparable between Rac1+/+ and Rac1−/−. (C3) SV distribution as a function of distance to AZ was not different between Rac1+/+ and Rac1−/−. Box plot whiskers extend to the minimum/maximum within the 1.5 interquartile range; open markers indicate individual data points. For EM data, the results of three independent investigators were averaged. All data shown in the figure and the detailed results of statistical tests are part of the supplementary file. Figure 1—source data 1 Excel file containing the data shown in Figure 1 and the results of statistical analysis. https://cdn.elifesciences.org/articles/81505/elife-81505-fig1-data1-v2.xlsx Download elife-81505-fig1-data1-v2.xlsx Loss of Rac1 increases synaptic strength and relative synaptic depression Perturbations of the presynaptic actin cytoskeleton impact synaptic transmission and plasticity in multiple model systems and synapses (Cole et al., 2000; Sakaba and Neher, 2003; Bleckert et al., 2012; Lee et al., 2012; Rust and Maritzen, 2015; Miki et al., 2016; Gentile et al., 2022; Wu and Chan, 2022). Since Rac1 is an actin cytoskeleton regulator, we examined how the loss of Rac1 impacted AP-evoked synaptic transmission and STP. Afferent fibers of calyx synapses were electrically stimulated with 50 APs at two stimulation frequencies (50 and 500 Hz), representing typical in-vivo firing rates at the calyx. AMPAR-mediated ESPCs were recorded in MNTB principal cells innervated by transduced (Rac1−/−) and non-transduced (Rac1+/+) calyces in 1.2 mM external Ca2+ and at 36–37°C to closely mimic in-vivo conditions (Figure 2A and B). By analyzing the initial response (EPSC1) of the EPSC trains, we found a robust increase in synaptic strength upon Rac1 deletion (Rac+/+ = 1.3 ± 0.4 nA vs. Rac1−/− = 3 ± 1.1 nA, p < 0.001, n = 15/15, Figure 2C and D1) with no change in EPSC waveform. Plotting EPSC amplitudes vs. stimulus number revealed substantial differences in STP between Rac1+/+ and Rac1−/− synapses. At 50 Hz stimulation, both Rac1+/+ and Rac1−/− showed short-term depression, which was more pronounced in Rac1−/− resulting in increased steady-state depression (EPSCss / EPSC1) (Figure 2A). Despite the stronger relative short-term depression, absolute steady-state EPSC amplitudes were almost twofold larger in Rac1−/− (Rac1+/+ = 0.47 ± 0.14 nA vs. Rac1−/− = 0.84 ± 0.22 nA, p < 0.001, n = 15/15, Figure 2D1). At 500 Hz stimulation, Rac1+/+ showed robust short-term facilitation, which was absent in Rac1−/− (Paired-pulse ratio PPR = EPSC2 / EPSC1: Rac1+/+ = 1.2 ± 0.1 vs. Rac1−/− = 1 ± 0.1, p < 0.001, n = 15/15, Figure 2B). Similar to 50 Hz stimulation, Rac1−/− showed stronger relative steady-state depression at 500 Hz stimulation. Notably, absolute steady-state EPSC amplitudes at 500 Hz stimulation frequency were similar between Rac1+/+ and Rac1−/− (Figure 2D1). Figure 2 Download asset Open asset Presynaptic Rac1 regulates synaptic vesicles release probability and synaptic strength. Synaptic transmission at the calyx of Held – MNTB synapse was studied using different stimulation frequencies at P28 after deletion of Rac1 at P14. (A1, B1) Representative evoked EPSCs for Rac1+/+ (black) and Rac1−/− (orange) at 50 Hz and 500 Hz stimulation frequency. Stimulus artifacts were blanked for clarity. (C) Magnification of the first EPSC (EPSC1). Ablation of presynaptic Rac1 resulted in increased EPSC1 amplitude with no change in EPSC dynamics. (A2–A4) At 50 Hz stimulation frequency, Rac1−/− showed stronger short-term depression despite larger steady-state EPSC amplitudes. (B2–B4) At 500 Hz stimulation frequency, loss of Rac1 resulted in a lack of short-term facilitation and increased synaptic depression with no change in steady-state EPSC amplitude. (D1) Population data showing an increase in EPSC1 amplitude in Rac1−/−. Steady-state EPSC amplitudes were increased in Rac1−/− at 50 Hz but not at 500 Hz stimulation frequency. (D2) Population data of the readily releasable pool (RRP) using three different estimation methods, suggesting little to no change in RRP size (D3) Population data indicating that EPSC1 release probability in Rac1−/− was elevated independent of estimation method. All data shown in the figure and the detailed results of statistical tests are part of the supplementary file. Figure 2—source data 1 Excel file containing the data shown in Figure 2 and the results of statistical analysis. https://cdn.elifesciences.org/articles/81505/elife-81505-fig2-data1-v2.xlsx Download elife-81505-fig2-data1-v2.xlsx Since the loss of Rac1 increased synaptic strength and altered STP, we aimed to identify the underlying mechanisms and evaluated RRP size and SV release probability (Pr) using established quantal analysis methods (Elmqvist and Quastel, 1965; Neher, 2015; Thanawala and Regehr, 2016). We estimated RRP size using 500 Hz stimulus trains which effectively depleted the RRP in both Rac1+/+ and Rac1−/− synapses by applying three conventional paradigms based on the common assumption of quantal release originating from a single and functionally homogenous pool of readily-releasable SVs (Neher, 2015; Schneggenburger and Rosenmund, 2015): EQ, NpRf, and SMN with correction (Elmqvist and Quastel, 1965; Neher, 2015; Thanawala and Regehr, 2016). All three methods reported a moderate increase in RRP size in Rac1−/− calyces (Figure 2D2), however, this was statistically significant only for the SMN analysis. The initial Pr of resting synapses was estimated by dividing the EPSC1 amplitude by the estimated RRP sizes. All three analysis methods revealed an approximately twofold increase in Pr (SMN: Rac1+/+ = 0.09 ± 0.02 vs. Rac1−/− = 0.15 ± 0.03, p < 0.001; NpRf: Rac1+/+ = 0.09 ± 0.02 vs. Rac1−/− = 0.17 ± 0.03, p < 0.001; EQ: Rac1+/+ = 0.09 ± 0.02 vs. Rac1−/− = 0.17 ± 0.04, p < 0.001, n = 15/15, Figure 2D3). Therefore, based on the assumption of a single and functionally homogenous RRP, this analysis indicates that presynaptic Rac1 deletion increases synaptic strength and short-term depression primarily by elevating initial Pr with little increase in RRP size. This suggests that Rac1 controls synaptic strength as a negative regulator of Pr. Loss of Rac1 increases mEPSC frequency but not amplitude The RRP size and the Pr of fusion-competent SVs is determined by the SV priming process, which involves the assembly of the molecular fusion apparatus, defined as ‘molecular priming’. In addition, Pr also depends on the spatial coupling between docked SVs and Ca2+ entry sites which may be adjusted by a distinct ‘positional priming’ step. Thus, ‘molecular priming’ encompasses the steps that render SVs fusion competent and regulate their intrinsic fusogenicity (Basu et al., 2007; Xue et al., 2010; Schneggenburger and Rosenmund, 2015; Schotten et al., 2015), while positional priming consists of the steps that place molecularly primed SVs close to voltage-gated calcium channels (VGCCs) (Neher, 2010). The spatial coupling between SV and VGCCs critically determines the speed and efficacy of AP-evoked release (Eggermann et al., 2011; Stanley, 2016). Spontaneous SV release is not or only little dependent on VGCCs (Schneggenburger and Rosenmund, 2015; Kavalali, 2020), thus the frequency of miniature EPSC (mEPSCs) can be interpreted as a readout of intrinsic SV fusogenicity at basal Ca2+ with increased SV fusogenicity causing higher mEPSC frequencies (Basu et al., 2007; Schotten et al., 2015; Dong et al., 2018). Therefore, to determine if an increased intrinsic SV fusogenicity caused the increase in Pr, we recorded mEPSCs from Rac1+/+ and Rac1−/− calyx synapses (Figure 3) and found a four-fold increase in mEPSC frequency (Rac1+/+ = 3.4 ± 4.4 Hz vs. Rac1−/− = 14.3 ± 6.5 Hz, p < 0.001, n = 15/11) with no change in mEPSC amplitude or waveform. To rule out that the increased mEPSC frequencies were due to changes in presynaptic Ca2+ currents, we recorded mEPSCs in the presence of 200 µM Cd2+, a non-specific VGCC blocker. Since Cd2+ did not affect mEPSC frequencies we conclude that Rac1 loss increases intrinsic SV fusogenicity at basal Ca2+. Figure 3 Download asset Open asset Presynaptic loss of Rac1 increases calcium-independent neurotransmitter release. (A) Representative recordings of mEPSCs for Rac1+/+ (left, black) and Rac1−/− (right, orange). (B1–B4) Rac1 deletion increased mEPSC frequency but did not affect mEPSC amplitude, rise time, or full width at half-maximal amplitude (FWHM). (C) The increased mEPSC rates at Rac1−/− were independent of presynaptic voltage-gated calcium channels (VGCC), as blocking VGCC with cadmium (Cd2+) did not affect mEPSC frequency. All data shown in the figure and the detailed results of statistical tests are part of the supplementary file. Figure 3—source data 1 Excel file containing the data shown in Figure 3 and the results of statistical analysis. https://cdn.elifesciences.org/articles/81505/elife-81505-fig3-data1-v2.xlsx Download elife-81505-fig3-data1-v2.xlsx Loss of Rac1 increases EPSC onset delays and decreases synchronicity of AP-evoked release Although we found an increase in SV fusogenicity, this does not rule out an additional role for Rac1 in regulating spatial coupling distances between molecularly primed SVs and VGCCs. In the mature calyx of Held, AP-evoked SV release is governed by local Ca2+ nanodomains, ensuring a fast onset and highly synchronous AP-evoked EPSCs to faithfully encode auditory information (Fedchyshyn and Wang, 2005). In addition to the gating kinetics of presynaptic VGCCs and postsynaptic AMPARs, the time between presynaptic AP and EPSC onset (EPSC onset delay) is determined by the coupling distance between SVs and VGCCs. The coupling distance defines the time for Ca2+ to diffuse and bind to the Ca2+ sensor and initiate SV fusion (Fedchyshyn and Wang, 2007; Nakamura et al., 2015). Thus, EPSC onset delays can serve as a readout of changes in spatial coupling distances, as increased onset delays are consistent with SVs being more loosely coupled to VGCCs and vice versa. Therefore, we measured EPSC onset delays during 50 Hz and 500 Hz stimulation (Figure 4A and B) and found them to become progressively larger during stimulation for Rac1+/+ and Rac1−/− calyces. At 50 Hz, the increase in EPSC onset delays during stimulus trains was comparable between Rac1+/+ and Rac1−/−, amounting to about 60 µs between the first and the last 10 EPSCs. At 500 Hz stimulation, however, EPSC onset delays increased more rapidly in Rac1−/−, with steady-state EPSC onset delays being significantly larger for Rac1−/− compared to Rac1+/+ (Rac1+/+ = 94 ± 32 µs vs. Rac1−/−= 131 ± 29 µs, p = 0.004, n = 15/15). Figure 4 Download asset Open asset Presynaptic loss of Rac1 decreases SV synchronicity and prolongs EPSC onset at high-frequency stimulation. (A) Experiments were performed at low (50 Hz, A1) and high (500 Hz, A2) stimulation frequencies. Representative recordings of first (EPSC1) and last (EPSC50) EPSC in the stimulus train. Traces are aligned at the EPSC onset of EPSC1. Stimulus artifacts are partially blanked for better visibility. Note the shift in the onset of EPSC50 in Rac1−/− compared to Rac1+/+ at 500 Hz but not 50 Hz. (B1) Absolute EPSC onset delay for 50 Hz (gray and light orange) and 500 Hz (black and orange) stimulation. (B2) EPSC onset delay relative to EPSC1 for 50 Hz (gray and light orange) and 500 Hz (black and orange). At 50 Hz, the EPSC onset delay was similar between Rac1+/+ and Rac1−/−. At 500 Hz, the EPSC onset delay was substantially larger at Rac1−/−. For better visualization, only every second data point is shown. (B3) EPSC onset delay of the last ten EPSCs relative to EPSC1 for 50 Hz and 500 Hz stimulation. EPSC delay of the last ten EPSCs in the stimulus train (EPSC41-50) was not different between Rac1+/+ and Rac1−/− at 50 Hz but increased for Rac1−/− at 500 Hz stimulation frequency. (C1) Analysis of ‘effective EPSC duration’ to estimate SV release synchronicity during 50 Hz and 500 Hz stimulation. Synchronicity was estimated from ‘effective EPSC duration’ by dividing the EPSC charge by the EPSC amplitude. Note the increase in effective EPSC duration for Rac1−/− at 500 Hz stimulation. For better visualization, only every second data point is shown (C2) EPSC duration was not different for EPSC1 but slightly longer for late EPSCs in Rac1−/− at 50 Hz and substantially longer at 500 Hz stimulation frequency. (C3) Effective EPSC duration of EPSC41-50 normalized to EPSC1. Note the progressive increase in effective EPSC duration in Rac1−/− with increasing stimulation frequency. All data shown in the figure and the detailed results of statistical tests are part of the supplementary file. Figure 4—source data 1 Excel file containing the data shown in Figure 4 and the results of statistical analysis. https://cdn.elifesciences.org/articles/81505/elife-81505-fig4-data1-v2.xlsx Download elife-81505-fig4-data1-v2.xlsx In addition to modulating EPSC onset delays, coupling distances between SV and VGCCs affect the time course of synchronous release and the relative contribution of synchronous vs. asynchronous release during AP trains (Wadel et al., 2007; Chen et al., 2015; Stanley, 2016; Yang et al., 2021). This is because synchronous release is dominated by tightly coupled SVs which rapidly fuse in response to high local [Ca2+], while asynchronous release likely represents a stronger contribution of more loosely coupled SVs (Sakaba, 2006; Schneggenburger and Rosenmund, 2015). An approximate measure for changes in the time course of AP-evoked release can be obtained by analyzing the EPSC charge over EPSC amplitude ratio (‘effective EPSC duration’) representing the width of a square current pulse with the same amplitude as the EPSC peak and same integral as the EPSC charge. Therefore, we calculated the effective EPSC duration for both 50 Hz and 500 Hz stimulation (Figure 4C) and found its value for EPSC1 comparable between Rac1+/+ and Rac1−/−. At steady-state during 50 Hz stimulation, however, the effective EPSC duration was slightly longer in Rac1−/− (Rac1+/+ = 0.32 ± 0.03 ms vs. Rac1−/− = 0.35 ± 0.03 ms, p = 0.006). At steady-state during 500 Hz stimulation, the effective EPSC duration in Rac1−/− calyces was prolonged further and increased by ~25% compared to Rac1+/+ (Rac1+/+ = 0.43 ± 0.04 ms vs. Rac1−/−= 0.55 ± 0.12 ms, p < 0.001). These findings are consistent with transmitter release being less synchronous in Rac1−/− synapses, especially during sustained activity at high AP firing rates. In summary, we found that Rac1−/− synapses had longer EPSC onset delays and showed more strongly increasing effective EPSC durations during stimulus trains, especially at high stimulation frequencies, implying less synchronous release. Since tighter SV to VGCCs coupling has the opposite effect, that is, generates shorter EPSC onset delays and more tightly synchronized release, we conclude that the increase of synaptic strength in Rac1−/− calyces is not due to tighter spatial coupling between SVs and VGCCs. Loss of Rac1 facilitates EPSC recovery and RRP replenishment The kinetics of molecular priming regulates RRP replenishment and determines steady-state release rates during high-frequency stimulation (Lipstein et al., 2013; Ritzau-Jost et al., 2018; Lipstein et al., 2021). Since Rac1 deletion increased steady-state release during 50 Hz stimulus trains, we hypothesized that SV pool replenishment proceeds faster in the absence of Rac1. To test how Rac1 loss influences RRP replenishment, we applied afferent fiber stimulation using a paired train protocol consisting of a 500 Hz conditioning train (50 APs) followed by a second 500 Hz test train at varying recovery intervals (Figure 5). Recovery was then measured for both the initial EPSC amplitude (EPSCtest) and the RRP estimate of the test trains. Recovery of the initial EPSC amplitude was quantified in terms of both its absolute (Figure 5A2) and its fractional value (Figure 5A3), with the latter being the ratio (EPSCtest − EPSCss) / (EPSC1 − EPSCss), where EPSCtest, EPSC1 and EPSCss are the initial amplitude of the test train, and the first and the steady-state amplitudes of the 500 Hz conditioning train, respectively. Recovery of absolute EPSCtest amplitude was significantly different between Rac1+/+ and Rac1−/− (Rac1+/+: A = 1.37, τfast = 28 ms, τslow = 2.7 s, fslow = 0.86, τw = 2.3 s vs. Rac1−/−: A = 3.4, τfast = 44 ms, τslow = 2.3 s, fslow = 0.83, τw = 1.9 s, p < 0.001, n = 15/15) and fractional EPSC recovery was over 50% faster in Rac1−/− (Rac1+/+: τfast = 40 ms, τslow = 2.8 s, fslow = 0.88, τw = 2.4 s vs. Rac1−/−: τfast = 47 ms, τslow = 2 s, fslow = 0.76, τw = 1.5 s, p < 0.001, Figure 5A). Next, we analyzed fractional RRP recovery by dividing the RRP estimate of the test train by the RRP estimate of the conditioning train and found that RRP recovery rates were about 40% faster in Rac1−/− (Rac1+/+: τfast = 22 ms, τslow = 1.9 s, fslow = 0.7, τw = 1.3 s vs. Rac1−/−: τfast = 40 ms, τslow = 1.4 s, fslow = 0.52, τw = 0.7 s, p < 0.001, Figure 5B). Finally, we compared the PPR (EPSC2 / EPSC1) of the test train after different recovery intervals. Independent of recovery interval, PPR was consistently lower in Rac1−/− (Rac1+/+ = 1.3 ± 0.1 vs. Rac1−/− = 1.1 ± 0.1, p < 0.001, Figure 5C), consistent with an increase in Pr. Figure 5 Download asset Open asset Loss of presynaptic Rac1 facilitates synaptic vesicle recovery. Recovery of single EPSC (EPSCtest) and RRP recovery was measured by two consecutive train stimuli (conditioning stimulus and recovery stimulus) at 500 Hz at varying recovery intervals. (A) Single EPSC recovery. (A1) Representative traces for Rac1+/+ (black) and Rac1−/− (orange) for recovery intervals ranging from 20 ms to 16 s. (A2) Recovery of absolute EPSC amplitudes as a function of recovery interval with a magnification of short intervals (right). (A3) Fractional EPSC r" @default.
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• W4313005778 title "Editor's evaluation: Presynaptic Rac1 controls synaptic strength through the regulation of synaptic vesicle priming" @default.
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