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J Physiol Volume 585, Number 3, 669-679, December 15, 2007 DOI: 10.1113/jphysiol.2007.137745
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SYMPOSIUM REPORT

Multiple vesicle recycling pathways in central synapses and their impact on neurotransmission

 Ege T. Kavalali1

1 Departments of Neuroscience and Physiology, The University of Texas Southwestern Medical Center, Dallas, TX 75390-9111, USA


   Abstract
Top
 Abstract
Functional significance of...
 Detection of synaptic vesicle...
 Molecular manipulations that...
 Chronic activity-dependent...
 Acute frequency-dependent...
 Conclusion
 References
 
Short-term synaptic depression during repetitive activity is a common property of most synapses. Multiple mechanisms contribute to this rapid depression in neurotransmission including a decrease in vesicle fusion probability, inactivation of voltage-gated Ca2+ channels or use-dependent inhibition of release machinery by presynaptic receptors. In addition, synaptic depression can arise from a rapid reduction in the number of vesicles available for release. This reduction can be countered by two sources. One source is replenishment from a set of reserve vesicles. The second source is the reuse of vesicles that have undergone exocytosis and endocytosis. If the synaptic vesicle reuse is fast enough then it can replenish vesicles during a brief burst of action potentials and play a substantial role in regulating the rate of synaptic depression. In the last 5 years, we have examined the impact of synaptic vesicle reuse on neurotransmission using fluorescence imaging of synaptic vesicle trafficking in combination with electrophysiological detection of short-term synaptic plasticity. These studies have revealed that synaptic vesicle reuse shapes the kinetics of short-term synaptic depression in a frequency-dependent manner. In addition, synaptic vesicle recycling helps maintain the level of neurotransmission at steady state. Moreover, our studies showed that synaptic vesicle reuse is a highly plastic process as it varies widely among synapses and can adapt to changes in chronic activity levels.

(Received 31 May 2007; accepted after revision 7 August 2007; first published online 9 August 2007)
Corresponding author E. T. Kavalali: Department of Neuroscience, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9111, USA. Email: ege.kavalali{at}utsouthwestern.edu


   Functional significance of synaptic vesicle recycling for neurotransmission
Top
 Abstract
 Functional significance of...
Detection of synaptic vesicle...
 Molecular manipulations that...
 Chronic activity-dependent...
 Acute frequency-dependent...
 Conclusion
 References
 
Synaptic vesicle recycling is essential for maintenance of neurotransmission in central synapses. However, it is yet uncertain if the only function of synaptic vesicle trafficking is to maintain fusion competence of synaptic vesicles and structural homeostasis of synapses in the long term. Or, in addition, can it modulate frequency dependence of synaptic responses during short-term synaptic plasticity (Li et al. 2005; Zenisek, 2005)? Clathrin-mediated endocytosis comprises a ubiquitous means for vesicle recycling in most cell types. This pathway typically possesses well-defined morphological markers (coated pits, endosomal intermediates, etc.) and adequate molecular tools are available to probe its properties in synapses. A rapid vesicle-recycling pathway, in contrast, may not employ the same molecular players and structural intermediates and is therefore harder to examine morphologically and molecularly. Therefore, most evidence in support of a fast retrieval and recycling mechanism for synaptic vesicles relies on electrophysiological and optical techniques with rapid time resolution (Harata et al. 2006a; Kavalali, 2006). Our results from experiments using a combination of fluorescence imaging of synaptic vesicle trafficking and electrophysiological detection of short-term synaptic plasticity collectively suggest that a rapid form of synaptic vesicle recycling strongly contributes to neurotransmission in a frequency-dependent manner in hippocampal synapses. This overview will summarize the experiments we conducted recently in four complementary studies to examine the impact of synaptic vesicle recycling on the kinetics of synaptic depression.


   Detection of synaptic vesicle recycling via comparison of electrophysiological and optical measures
Top
 Abstract
 Functional significance of...
 Detection of synaptic vesicle...
Molecular manipulations that...
 Chronic activity-dependent...
 Acute frequency-dependent...
 Conclusion
 References
 
During sustained activity most synapses show depression characterized by a rapid decrease in the postsynaptic responses down to a plateau level. Multiple mechanisms contribute to this rapid depression including a decrease in vesicle fusion probability, inactivation of voltage-gated Ca2+ channels or use-dependent inhibition of release machinery by presynaptic receptors. One of the underlying causes of this depression is thought to be a rapid reduction in the number of vesicles available for release in the synapse (Zucker & Regehr, 2002; Abbott & Regehr, 2004). The vesicles available for release can be supplied either by vesicle replenishment from a set of reserve vesicles or via reuse of vesicles that have undergone exocytosis and endocytosis. If the synaptic vesicle reuse is fast enough to replenish vesicles during a brief burst of action potentials then it can play a substantial role in regulating the rate of synaptic depression.

In order to determine the reliance of neurotransmission on synaptic vesicle endocytosis and reuse, one needs to estimate the time point when exocytosed vesicles become re-available for release. To measure this parameter we examined the kinetic difference between the rate of FM dye destaining and the time course of neurotransmitter release from a set of hippocampal synapses. The rationale behind these experiments stems from previous observations that during stimulation, FM2-10 (fast departitioning FM dye) can be cleared out of a fused vesicle within a second by departitioning into solution (Ryan et al. 1996; Klingauf et al. 1998; Kavalali et al. 1999; Pyle et al. 2000), or within milliseconds by lateral diffusion in the neuronal membrane (Zenisek et al. 2002). Both of these time frames are thought to be faster than the rate of fusion pore closure and endocytic retrieval (Klingauf et al. 1998; Sankaranarayanan & Ryan, 2001). Thus, FM dye destaining can report fusion of a particular vesicle only once as long as all FM dye leaves a vesicle upon fusion and recycled vesicles do not contain significant amounts of dye that could be detected as further destaining. In contrast, the same vesicles are rapidly refilled with neurotransmitter following endocytosis that could give rise to further synaptic responses (Fig. 1A and B ). Therefore, electrical recordings of neurotransmitter release report the sum of release events originating from fresh vesicles that have not fused before and recycled vesicles refilled with neurotransmitter. This key difference between the two reporters of vesicle mobilization results in a deviation between the kinetics of FM2-10 destaining and neurotransmitter release at the time when recycled vesicles start to be reused.


Figure 1
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  		Figure 1.  Estimation of the time course of vesicle reuse from simultaneous electrical recordings and fluorescence destaining
A, following fusion synaptic vesicles are retrieved and refilled with neurotransmitter (left). Therefore, electrical recordings of neurotransmitter release report the sum of release events originating from fresh vesicles that have not fused before and recycled vesicles refilled with neurotransmitter. In contrast, FM dye-filled vesicles cannot be refilled with dye during recycling (right). Therefore, FM dye destaining can report fusion of a particular vesicle only once as long as all FM dye leaves a vesicle upon fusion. B and C, whole-cell electrical recording of a sucrose response (B) and its instantaneous fluorescence counterpart from multiple boutons on the same neuron (C). D, average fluorescence response (F, grey line) was smoothed by curve fitting (dashed line) to reduce noise, which in turn helped obtain a smooth derivative of the fluorescence signal (dF/dt, smooth line). E, the difference between the rate of dye release and synaptic activity was assessed after alignment of the dF/dt and Current plots with respect to their peaks. Current plot was obtained by integrating current within 1 s intervals. The difference shown in the bottom graph was interpreted as the time course of vesicle reuse (from Sara et al. 2002; copyright 2002 by the Society for Neuroscience).

 
Figure 1 illustrates the procedure we used to analyse these experiments. In this particular experiment, we evoked neurotransmitter release by application of hypertonic sucrose solution onto a pyramidal cell (Fig. 1B). In the same region, we simultaneously recorded fluorescent destaining from synapses that were formed on the dendrites of the same cell (Fig. 1C). Hypertonic sucrose application was sustained for at least 25 s until destaining reached a plateau. In mature synapses this level corresponded to 30% of the total pool as determined by multiple applications of high K+ stimulation following a 1 min rest period (Pyle et al. 2000). Destaining profiles originating from all boutons were averaged and smoothed by fitting with multiple exponential functions (~4). The derivative of the smoothed destaining profile was calculated to obtain the time-dependent change in the rate of destaining (dF/dt plot in Fig. 1D). In order to correlate electrophysiological data at the same resolution with the rate of FM destaining acquired at 1 Hz, we calculated the total current during synaptic activity over 1 s intervals and normalized with respect to the maximum (Current plot in Fig. 1E), then we aligned the normalized dF/dt and Current plots with respect to their maxima (Fig. 1E). In all experiments there was significant agreement between the time courses of the two curves. The result of this analysis revealed a marked divergence between the rate of FM2-10 destaining and neurotransmitter release. After subtraction of the two curves, the difference was interpreted as the time course of recycled vesicles to join neurotransmission (Fig. 1E, lower panel). We followed the same procedure to estimate the time course of vesicle recycling in response to action potentials induced by extracellular field stimulation and found that after an initial round of exocytosis, vesicles were available for reuse with a delay of between 1 and 3 s during 30 Hz action potential or hypertonicity-induced stimulation. During these stimulation protocols, there was only limited mobilization of vesicles from the reserve pool. Furthermore, electrophysiological and ultrastructral analysis revealed that hippocampal synapses are significantly resilient to vesicle depletion under intense stimulation of neurotransmitter release induced by hyperosmolarity, high potassium or action potential firing at 30 Hz. Under these conditions, synaptic transmission rapidly depressed to a plateau level that was typically 10–40% of the initial response and persisted at this level for at least 5 min regardless of the developmental stage of synapses. This non-declining phase of transmission was partly sustained by fast recycling and reuse of synaptic vesicles even after minutes of stimulation.

In summary, these results suggested a role for fast vesicle recycling as a functional homeostatic mechanism that prevents vesicle depletion and maintains synaptic responses in the face of intense stimulation. By comparing the kinetics of FM2-10 destaining and pattern of neurotransmitter release through postsynaptic recordings, we could estimate the dynamic properties of vesicle recycling. This setting enabled us to show that fast recycling capability is an early emerging characteristic of central synapses during synapse maturation and empowers small synapses with few vesicles to cope with high demands on their limited vesicle supply (Sara et al. 2002).


   Molecular manipulations that alter the rate of synaptic vesicle recycling and synaptic depression
Top
 Abstract
 Functional significance of...
 Detection of synaptic vesicle...
 Molecular manipulations that...
Chronic activity-dependent...
 Acute frequency-dependent...
 Conclusion
 References
 
Rapid reuse of synaptic vesicles, as suggested by the experiments discussed above, requires that synaptic vesicle exocytosis and endocytosis are tightly coupled processes. Recent studies have extensively focused on synaptotagmins as potential mediators of this coupling. Synaptotagmins are characterized by an N-terminal transmembrane domain, a central linker and two C-terminal C2 domains (Sudhof, 2002). These proteins have been extensively studied as Ca2+ sensors for vesicle exocytosis, primarily through the characterization of synaptotagmin 1 (Geppert et al. 1994; Fernandez-Chacon et al. 2001). While synaptotagmin 1 and 2 are located on the synaptic vesicle, synaptotagmins 3, 6 and 7 are present on the synaptic plasma membrane (Sugita et al. 2001, 2002). The C2 domain of synaptotagmin has a high-affinity binding site for AP-2 and possibly stonin, two proteins believed to be important in clathrin-mediated endocytosis (Zhang et al. 1994; Li et al. 1995; Martina et al. 2001). In a recent study, rapid light-induced inactivation of synaptotagmin 1 in the Drosophila neuromuscular junction impaired delayed endocytosis supporting the biochemical results discussed above (Poskanzer et al. 2003).

Among the plasma membrane synaptotagmins, synaptotagmin 7 is of particular interest since the truncated splice variant of synaptotagmin 7 (syt7B), produced due to a conserved stop codon in the second exon of the alternatively spliced region (Sugita et al. 2001), inhibits receptor-mediated endocytosis in a number of non-neuronal cell lines (von Poser et al. 2000). Another splice variant of the same protein containing both C2 domains (syt7A) had no effect in the same system. The coincidence of two findings, namely the inhibition of receptor-mediated endocytosis by truncated synaptotagmin 7 variants in transfected fibroblasts (von Poser et al. 2000) and the discovery of the natural occurrence of such variants by alternative splicing in neurons (Sugita et al. 2001), raised the possibility that alternative splicing of synaptotagmin 7 may regulate synaptic vesicle recycling (Fig. 2A ). Therefore, we have utilized these molecules to isolate and study a potential clathrin-independent fast vesicle-recycling pathway in hippocampal synapses.


Figure 2
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  		Figure 2.  Effect of synaptotagmin 7 overexpression on synaptic responses detected electrophysiologically
A, the role of synaptotagmin 7 splice variants in directing vesicles towards kinetically distinct recycling pathways. The short splice variant of synaptotagmin 7 (Sy7-B) lacking the two C2 domains directs vesicles towards a fast recycling pathway. The full-length splice variant, synaptotagmin 7-A, targets vesicles towards slower trafficking pathways. These include classical clathrin-mediated endocytosis of individual vesicles, as well as slower pathways uncovered by synaptotagmin 7-A overexpression where vesicles bud off membrane infoldings and/or endosomal cisternae. B, representative whole-cell recordings in neurons from cultures transfected at high efficiency with regular synaptotagmin 7 (Syt7-A), short synaptotagmin 7 (Syt7-B) and two control proteins, synaptotagmin 1 (Syt 1) and ECFP alone. Recordings were made during 10 Hz stimulation from high-density cultures with each signal resulting from multiple synapses per neuron; only the first and last 20 responses during a total of 900 stimuli are shown. C, average normalized response amplitudes from cells stimulated at 10 Hz by field electrodes. Each point represents the average of 10 consecutive responses. Syt7-B overexpression results in a higher steady-state plateau of response amplitudes. In contrast, Syt7-A overexpression results in faster depression of the initial responses (P < 0.02; n = 12) as well as a lower response amplitude at steady-state compared with both controls (P < 0.03; n = 11–12) and Syt7-B (P < 0.001; n = 11, Student's two-tailed t test) overexpression (modified from Virmani et al. 2003; copyright 2003 by the authors).

 
We found that synapses containing the syt7B at high concentrations showed an increased rate of synaptic vesicle endocytosis after a brief stimulus pulse. These faster endocytosing vesicles also recycle faster as demonstrated by a 2-fold increase in the number of vesicles loaded with the styryl dye FM1-43 that become re-available for release within 30 s compared with synapses with control levels of protein. In contrast, the presence of syt7A at high levels results in no change in basal endocytosis, yet vesicles are targeted towards slower recycling pathways. These optical experiments were also in agreement with electrophysiological analysis of short-term depression. In these experiments, synapses expressing syt7B showed a slower rate of synaptic depression in response to repetitive stimulation whereas syt7A-expressing synapses depressed robustly (Fig. 2B and C) (Virmani et al. 2003). These two lines of evidence suggest that alternative splice forms of synaptotagmin 7 may target endocytosing vesicles to fast or slow recycling pathways, with the level of the various splice variants fine tuning this process dependent upon synapse type, function or activity experienced by the synapse. Interestingly, gene chip analysis of the mouse deficient in the Ca2+ channel {alpha}1A subunit showed that synaptotagmin 7 was up-regulated despite the fact that most synaptic proteins were unaltered. This suggests that synaptotagmin 7 contributes to the proper maintenance of synaptic vesicle recycling in these mice despite the absence of a key Ca2+ influx pathway (Piedras-Renteria et al. 2004). This finding supports the premise that synaptotagmin 7 levels and alternative splicing patterns can be altered by the activity history of a neuron thus rendering the vesicle recycling properties of its synaptic terminals plastic.


   Chronic activity-dependent regulation of synaptic vesicle recycling and synaptic depression
Top
 Abstract
 Functional significance of...
 Detection of synaptic vesicle...
 Molecular manipulations that...
 Chronic activity-dependent...
Acute frequency-dependent...
 Conclusion
 References
 
The experiments using splice variants of synaptotagmin 7 strongly suggest that synapses can potentially switch between distinct synaptic vesicle recycling pathways. Activity-dependent regulation of synaptotagmin 7 levels prompted us to examine whether the kinetics of synaptic vesicle recycling can be regulated by chronic changes in activity and if this regulation is reflected on the properties of short-term synaptic plasticity. To address this question, we took advantage of the different background activity levels in hippocampal and neocortical cultures to study the responsiveness of vesicle recycling to chronic alterations in activity (Fig. 3A and B ). The hippocampal and neocortical cultures also showed remarkably distinct kinetics of synaptic depression in response to sustained 10 Hz stimulation (Fig. 3C and D). The difference in the kinetics of synaptic depression was not due to differences in the desensitization of postsynaptic AMPA receptors as judged by its insensitivity to cyclothiazide (Fig. 3E). We found two parameters to be plastic in different synapses in response to changing chronic activity: mobilization of vesicles from the reserve pool and vesicle reuse (Fig. 3F–H). In hippocampal cultures a decrease in the normally high levels of network activity, with tetrodotoxin (TTX) incubation, increased synaptic depression by reducing the rate of vesicle mobilization from the reserve pool with little effect on the rate of vesicle reuse. In contrast, the rate of synaptic vesicle reuse in neocortical synapses responded robustly to changes in activity levels. Incubation with picrotoxin (PTX), slowed synaptic depression by significantly increasing synaptic vesicle reuse rate, whereas TTX treatment did not significantly change the rate of synaptic depression but slowed synaptic vesicle reuse. Interestingly, in neocortical cultures neither of these treatments had a major effect on synaptic vesicle mobilization from the reserve pool. Our results suggested that network activity is a critical determinant of synaptic vesicle trafficking and resulting synaptic response dynamics. Therefore, we proposed that different synapses not only rely on different pathways for maintaining neurotransmitter release but they also use different strategies to adapt and respond to alterations in network activity levels (Virmani et al. 2006).


Figure 3
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  		Figure 3.  Spontaneous network activity and the dynamics of presynaptic vesicle trafficking in neocortical and hippocampal cultures
A, spontaneous network activity in neocortical and hippocampal cultures. Sample traces of spontaneous network activity recorded from neurons in neocortical cultures (upper two traces) and hippocampal cultures (lower two traces). B, current-clamp analysis of spontaneous network activity in neocortical and hippocampal cultures. Sample traces of action potentials and excitatory postsynaptic potentials recorded from neurons in neocortical cultures (upper traces) and hippocampal cultures (lower traces). Arrows point to subthreshold synaptic activity. C, sample traces of synaptic depression recorded from hippocampal (upper traces) and neocortical (lower traces) neurons in culture during 10 Hz stimulation (the first 10 responses and the 100th to 110th response are shown). D, traces showing the average synaptic depression from a number of cells normalized to the amplitude of the first response (n = 21 cells and 8 cells for neocortex and hippocampus, respectively). Neocortical neurons depress significantly faster than hippocampal neurons in response to 10 Hz stimulation with significance (P < 0.01) emerging by the 5th response. E, treatment with cyclothiazide (CTZ) to block AMPA receptor desensitization slightly (grey symbols) decreased the time constant of rapid depression in both neocortical (n = 4) and hippocampal (n = 8) cultures but maintained the difference in the rates of depression between the two cultures. F, neocortical and hippocampal synapses have distinct kinetics of synaptic vesicle recycling. Synapses were loaded with FM2-10 using 1200 APs delivered at 10 Hz through field electrodes and after a brief wash were destained by 10 Hz stimulation for 90 s followed by multiple rounds of 90 mM K+ to maximally destain the synapses. The total recycling pool size was measured by loading synapses using 1200 APs delivered at 10 Hz and following extracellular dye washout, destaining the synapses using 90 mM K+. Hippocampal synapses showed a larger recycling pool size (P < 0.05, n = 5 samples each). G, average dye destaining curves from a number of experiments show that neocortical synapses destain slower than hippocampal synapses that was significant after 20 s of stimulation (P < 0.05 (two tailed t test), n = 5 samples each). The baseline by 90 s was not significantly different (P = 0.2). H, vesicle reuse was measured using a pulse chase experiment. The plot shows the average percentage of reused vesicles for neocortical and hippocampal synapses as a function of the time of continued stimulation ({Delta}t). With this protocol neocortical synapses show significantly faster vesicle reuse than hippocampal synapses (n = 550–800 synapses from 6 to 7 samples for each time point per condition; significance was determined by applying a stringent value of P < 10–8 using the Kolmogorov–Smirnov (K-S) test) (modified from Virmani et al. 2006; copyright 2006 by the Society for Neuroscience).

 
Recent findings suggest that synaptic vesicle recycling and short-term synaptic plasticity are extensively susceptible to alterations in temperature (Pyott & Rosenmund, 2002; Fernandez-Alfonso & Ryan, 2004; Micheva & Smith, 2005; Klyachko & Stevens, 2006; Kushmerick et al. 2006), and are dynamically modified during developmental maturation of synaptic terminals (Brenowitz & Trussell, 2001; Mozhayeva et al. 2002; Taschenberger et al. 2002) as well as acutely regulated by the levels of spontaneous activity (Johnson & Buonomano, 2007). Taken together with these recent results, our findings from dissociated neuronal cultures indicate that synaptic vesicle reuse is a highly plastic and dynamically regulated process, and its impact on neurotransmission varies among types of nerve terminals and depends on multiple acute and chronic regulatory factors.


   Acute frequency-dependent regulation of synaptic vesicle reuse in hippocampal synapses
Top
 Abstract
 Functional significance of...
 Detection of synaptic vesicle...
 Molecular manipulations that...
 Chronic activity-dependent...
 Acute frequency-dependent...
Conclusion
 References
 
As indicated in the previous sections, existing methods can only indirectly estimate the contribution and timing of vesicle reuse to neurotransmitter release during activity. To monitor synaptic vesicle reuse and re-availability of vesicles for consequent rounds of fusion after endocytosis, most studies have relied on the somewhat difficult comparison of presynaptic optical signals to postsynaptic electrical recordings. This comparison is complicated by the distinct physical nature, origin and complex kinetic correspondence of the two signals (Betz & Bewick, 1992, 1993; Sara et al. 2002; Li et al. 2005; Virmani et al. 2006). Therefore, in a recent study, we used an electrophysiological approach that took advantage of the neurotransmitter refilling process into vesicles (Ertunc et al. 2007). Using blockers of vacuolar ATPase (v-ATPase) such as folimycin, as well as small pH buffers that can be loaded into endocytosing vesicles, we impaired synaptic vesicle re-acidification and neurotransmitter refilling then tested the dependence of short-term synaptic depression on synaptic vesicle reuse. Electrophysiological detection of neurotransmission in such a setting has several advantages such as its millisecond time resolution, direct monitoring of the kinetics of neurotransmission and its applicability to complex tissues and neural circuitry from which optical signals are harder to detect over long periods. After folimycin treatment, we found that evoked synaptic response amplitudes recorded at low frequency (< 0.1 Hz) were relatively unaffected (Fig. 4A ). Under the same conditions, however, there was a use-dependent increase in the rate of synaptic depression in the CA1 region of hippocampal slices (Fig. 4). This effect was frequency dependent as higher stimulation frequencies resulted in an earlier onset of vesicle reuse (Fig. 4B–D). These findings provide evidence for the presence of a very fast pathway for vesicle reuse that operates with a time course of 300 ms to 1 s and reveal its impact on short-term synaptic plasticity in central synapses. This rapid time course is also consistent with the recent findings that used synaptic vesicles can be replenished with a rate less than 10 ms per vesicle (Griesinger et al. 2005; Saviane & Silver, 2006; Crowley et al. 2007).


Figure 4
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  		Figure 4.  Folimycin treatment induces frequency-dependent depression of synaptic responses at excitatory synapses in the CA1 region of hippocampal slices
A, bar graphs and representative traces of the average amplitude of the first evoked responses to the stimulation trains. There was a small but statistically insignificant decrease in the amplitudes of the first evoked responses recorded from folimycin-treated cells. B–D, blockade of vesicle refilling evaluated by synaptic depression at 1 Hz (B), 20 Hz (C) and 30 Hz (D) electrical stimulation after 10 min of folimycin exposure. Folimycin treatment hastened synaptic depression at excitatory synapses in higher frequencies with an early onset. Insets show the first and last 5 AP evoked EPSCs at 1 Hz, and the first and last 10 responses for 20 Hz and 30 Hz of continuous stimulation from individual recordings (the top trace for each experimental protocol represents controls and the bottom trace folimycin-treated cells). Folimycin-treated slices respond to stimulation with a continuous decline in neurotransmitter release with a faster rate of depression than that recorded in controls (P < 0.05, n = 5–7 cells for each experimental protocol and group). The decline was more prominent at higher frequencies in the folimycin-treated group. Data shown are mean ± S.E.M. (modified from Ertunc et al. 2007; copyright 2007 by the Society for Neuroscience).

 
These experiments also indicated that synaptic vesicle reuse contributes to neurotransmission in a frequency-dependent manner. We could account for our measurements at different frequencies with a single kinetic scheme (Fig. 5A ). These difference plots between synaptic depression profiles obtained from slices treated with the v-ATPase blocker folimycin and from vehicle-treated slices (control) report the extent of vesicle reuse at each frequency. The difference between folimycin-treated and control traces rises slowly at 1 Hz and is sustained throughout the duration of stimulation (Fig. 5B and C). In contrast, the difference between the two depression profiles displays a more transient nature at high frequencies (Fig. 5D and E). We used a simple model with three states connected by frequency-dependent rate constants to fit these data (Fig. 5A). In this model, the transition rates between the states ({alpha} and β) depend on stimulation frequency. This frequency dependence may arise from changes in intrasynaptic Ca2+ or the amount of vesicle fusion (the number of vesicles fused and to be retrieved). According to this model, synapses initially occupy the ‘rest’ state and transition to the ‘reuse’ state in response to stimulation. The rate constant ‘{alpha}’ from the rest to the reuse state can be taken as an upper boundary for the rate that the initial set of vesicles are reused after stimulation onset. Once in the reuse state, synapses transition into a state of ‘exhaustion’ with a rate of β in which synaptic vesicles cease to be reused rapidly, presumably due to saturation or depletion of the endocytic machinery that sustains rapid reuse. In Fig. 5 continuous lines depict the progression of the reuse state during stimulation estimated by fitting the model to the data by minimizing the mean square of the error. In the case of excitatory synapses, the rates {alpha} and β increase monotonically in response to the increase in the frequency of stimulation (Fig. 5F). In inhibitory synapses, the same rates plateau after 10 Hz (Fig. 5G). In both types of synapses, at 1 Hz reuse state appears to dominate at steady state when {alpha} and β are low. As stimulation frequency increases, the onset of reuse gets faster and the synapses rapidly transition into the exhaustion state thus limiting fast reuse to a transient period. This simple model satisfies the minimum requirements to account for the data presented in this study. In addition, frequency-dependent activation (rest -> reuse) and inactivation (reuse -> exhaustion) of synaptic vesicle reuse may potentially reconcile our results with earlier observations that synaptic vesicle reuse is more prominent at steady state at moderate frequencies around 1 Hz (Harata et al. 2006b). It is also possible to extend this model and include a second set of states with slower rates of transition between the states (a slow reuse mode). This extended model can account for our earlier finding that in hippocampal synapses synaptic vesicles can be reused under strong sustained stimulation at steady state albeit with a slower rate (Sara et al. 2002). It is important to note that in different synapses vesicle reuse may have different frequency-dependence characteristics and moreover these characteristics may shift in response to extrinsic factors such as chronic levels of network activity (Virmani et al. 2006).


Figure 5
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  		Figure 5.  A simple model for the frequency dependence of synaptic vesicle reuse
A, the figure depicts the kinetic model we used to account for our observations. In this model, synapses initially occupy the ‘rest’ state and transition to the ‘reuse’ state in response to stimulation with a rate {alpha}. Synapses transition from the reuse state to a state of ‘exhaustion’ with a rate β. The transition rates between the states ({alpha} and β) depend on stimulation frequency. B–E, the difference between folimycin-treated and control depression profiles at 1 and 30 Hz stimulations (including data from Fig. 4) plotted with respect to the number of action potentials for excitatory (B and D) and inhibitory (C and E) synapses ({circ}). Continuous lines depict the progression of the reuse state during stimulation estimated by fitting the model to the data by minimizing the mean square of the error. The occupancy of reuse state was calculated by solving the following first order equations: d(rest)/dn = –{alpha}(rest), d(reuse)/dn = {alpha}(rest) – β(reuse), d(exhaustion)/dn = β(reuse) where n denotes the number of action potentials. F, in the case of excitatory synapses the rates {alpha} and β increase monotonically in response to the increase in the frequency of stimulation. G, in inhibitory synapses, the same rates plateau after 10 Hz (modified from Ertunc et al. 2007; copyright 2007 by the Society for Neuroscience).

 

   Conclusion
Top
 Abstract
 Functional significance of...
 Detection of synaptic vesicle...
 Molecular manipulations that...
 Chronic activity-dependent...
 Acute frequency-dependent...
 Conclusion
References
 
Taken together, recent studies in our laboratory have aimed to address whether synaptic vesicle recycling can impact on rapid dynamics of neurotransmission during activity. Our findings indicate that synaptic vesicle trafficking is a dynamic and versatile regulator of neurotransmission. The rapid time course of the vesicle reuse proposed by our work may conflict with some of the recent measurements, which suggest slow kinetics for synaptic vesicle re-acidification (~5 s) and endocytosis (~15 s) (Atluri & Ryan, 2006; Granseth et al. 2006). However, these measurements do not necessarily preclude the presence of a rapid pathway for synaptic vesicle re-acidification and retrieval (Gandhi & Stevens, 2003; Ertunc et al. 2007). Indeed, our results are consistent with a large body of work examining the molecular mechanisms underlying synaptic vesicle endocytosis. For instance, studies using the Drosophila temperature-sensitive dynamin mutant shibire, have provided strong support for a relationship between vesicle recycling and synaptic release during stimulation. Dynamin is a GTPase that serves to pinch vesicles off the membrane during synaptic vesicle endocytosis. A direct comparison of the rate of synaptic depression in Drosophila neuromuscular junction from wild-type and shibire mutant flies showed that synapses from shibire mutant flies at non-permissive temperatures rapidly depressed without a plateau phase in response to high-frequency stimulation. The kinetic difference between this rate of depression and the depression observed with functional dynamin revealed a recycling rate of one to two vesicles per second per active zone (Delgado et al. 2000), a considerably fast rate in line with our estimations from hippocampal synapses (Sara et al. 2002). A recent study on a mouse knockout of dynamin-1 also revealed a similar rapid synaptic depression due to loss of vesicle endocytosis occurring during synaptic stimulation (Ferguson et al. 2007). The premise that synaptic vesicle recycling contributes to the maintenance of synaptic transmission on a rapid time scale is also supported by other molecular perturbations at the synapse. For instance, disruption of dynamin SH3 domain interactions (Shupliakov et al. 1997), genetic impairment of synaptojanin 1, an abundant presynaptic molecule which functions as a polyphosphoinositide phosphatase (Cremona et al. 1999; Luthi et al. 2001) or expression of differentially spliced isoforms of synaptotagmin 7 (Virmani et al. 2003) all led to frequency-dependent changes in the rate of short-term synaptic depression. Therefore, future molecular manipulations of the synaptic vesicle recycling machinery will not only help us uncover vesicle trafficking mechanisms but also provide an extremely valuable setting to study the kinetics and physiological significance of synaptic vesicle reuse during synaptic activity and how it contributes to information transfer in synaptic circuits.


   Footnotes
 
This report was presented at a symposium on Multiple synaptic vesicle retrieval pathways in neuronal physiology, which took place at the Life Sciences 2007 meeting, 9–12 July 2007, Glasgow, UK.


   References
Top
 Abstract
 Functional significance of...
 Detection of synaptic vesicle...
 Molecular manipulations that...
 Chronic activity-dependent...
 Acute frequency-dependent...
 Conclusion
 References
 
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   Acknowledgements
 
I would like to thank Deniz Atasoy, ChiHye Chung, Ferenc Deak, Mert Ertunc, Marina Mozhayeva, Yildirim Sara, Tuhin Virmani and Catherine Wasser for numerous invaluable discussions. I am grateful to Xinran Liu for our continuing collaboration on the electronmicroscopy of synapse structure. These studies were supported by grants from the National Institute of Mental Health and the American Heart Association.




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