Presynaptic plasticity, which predominantly consists of rapid changes in transmitter release induced during synaptic activity and results in enhancements or depressions of synaptic responses, is partly caused by changing the release efficacy of synaptic vesicles originating in distinct vesicle pools (Attwood and Karunanithi, 2002; Zucker and Regehr, 2002; Rizzoli and Betz, 2005; Neher, 2006). Brief bursts of afferent stimulation often give rise to an early frequency facilitation deriving from the readily releasable vesicle pool (RRP; e.g., Zucker and Regehr, 2002; Neher and Sakaba, 2008). In contrast, contributions of vesicles from the reserve or resting pools are less well defined (Wesseling and Lo, 2002; Li et al., 2005; Fernandez-Alfonso and Ryan, 2008). In the well-characterized excitatory and facilitating CA3-to-CA1 hippocampal glutamatergic synapse, where the presynaptic elements contain RRPs of 5–10 vesicles and a total population of 80–100 vesicles (Harris and Sultan, 1995; Schikorski and Stevens, 1997), we have recently described a functionally defined phase designated the delayed response enhancement (DRE), which appears to represent a vesicle pool which begins to release transmitter when stimulation has lasted approximately 3.5 s. Around 75 stimuli contributed to this enhancement phase, which was followed by response decays (Jensen et al., 2007; see, however, Garcia-Perez et al., 2008). The putative vesicle pool involved in this response, which appeared to be dependent on the vesicle-associated synapsin I/II proteins, was found to be sensitive to temperature and F-actin cycling, and to be developmentally regulated (Jensen et al., 2007; Bogen et al., 2009). The exact identity and localization of these vesicles, as well as their physiological importance, remain, however, unclear.

In order to define possible physiological relationships between the vesicles present in the RRP and those responsible for the DRE phase, we have here examined these response phases during activation of three well-known intraterminal signaling pathways, including those regulated by cyclic AMP (cAMP), diacylglycerols (DAG) and increased levels of Ca²⁺, all of which may change vesicle recruitment, docking, priming, and/or exocytotic fusion of releasable synaptic vesicles (e.g., Hilfiker et al., 1999; Leenders and Sheng, 2005; Neher, 2006). For this purpose, we have used hippocampal slice preparations from wild-type (WT) and synapsin I/II double knock-out (DKO) mice, the latter showing a significant decrease in synaptic vesicles but no change in glutamate receptor levels (Owe et al., 2005; Bogen et al., 2006). These preparations were either equilibrated in the absence or presence of the adenylate cyclase activator forskolin or the DAG analog β-phorbol-12,13-dibutyrate, or were examined at different levels of [Ca²⁺]_o (from 1 to 4 mM). Following equilibration, we examined responses obtained from the RRP vesicles by analyzing either fEPSPs obtained at 0.1 Hz stimulus frequency, paired-pulse facilitation (PPF) ratios obtained at 50 ms interstimulus intervals, or frequency facilitation magnitudes obtained during the initial part of prolonged 20 Hz stimulation trains. In contrast, responses from recruited vesicles giving rise to the delayed phases were examined by analyzing the DRE and subsequent responses during the final part of the 20 Hz stimulus trains. We show that stimulation-induced enhancements of synaptic responses induced by activation of the signaling pathways all gave comparable increases of fEPSPs during low-frequency stimulation, but that they gave different patterns of presynaptic modulation both during paired pulses, frequency facilitation and prolonged stimulation trains. Our data therefore indicate that in these prototypical glutamatergic excitatory synapses, different molecular stimuli which modulate temporally identical short-term plasticity phases may regulate functionally distinct vesicle pools.

Activations of the intracellular cAMP-, DAG-, and Ca²⁺-mediated second messenger systems in the glutamatergic CA3-to-CA1 synapse in mouse hippocampus induce similar increases in baseline responses. The major findings from this study show that during subsequent repetitive stimulations, where both vesicle docking and priming as well as recruitment from additional vesicle pools become more important, they induce different responses. These conclusions depend on experimentally obtained changes in postsynaptic responses caused by transmitter released from the RRP and/or other vesicle pools. Moreover, some but not all of the effects appear to depend on the synapsin I/II proteins, which may be involved both in response enhancements and depressions, depending on both the identity of the specific second messenger system activated and the vesicle pool involved.

Multiple effects of the 3 second messenger systems are known to occur throughout all synaptic compartments, and a variety of presynaptic and postsynaptic structures are well known as targets for the biochemical systems we examine (e.g., Walaas and Greengard, 1991). These include presynaptic and postsynaptic ion channels and postsynaptic receptors, the modification of which could change postsynaptic responses. However, given the exclusive presynaptic location of the synapsin I/II proteins, and the comparative analysis of the two genotypes, the most parsimonious explanation for the results obtained is that they represent putative modulations of endo- and exocytotic trafficking and recruitment of presynaptic vesicle pools. However, minor contributions from functional changes occurring elsewhere cannot be completely excluded, particularly those observed in the absence of synapsin I/II.

During stimulation at low frequency (0.1 Hz), where already primed vesicles are activated, both forskolin, PDBu and 4 mM Ca²⁺ induced increases in fEPSP magnitudes. Moreover, these changes in synaptic strength were essentially identical in WT and DKO preparations, demonstrating that despite the ability of, e.g., synapsin I to modulate exocytotic kinetics (Hilfiker et al., 1998; Coleman and Bykovskaia, 2009b), second messenger-induced modulations of single vesicle exocytosis were not targets for the synapsins I/II proteins. These data therefore tend to exclude that phosphorylation of domain A in the synapsin proteins, catalyzed by cAMP-or Ca²⁺-dependent protein kinases (Walaas and Greengard, 1991; Cesca et al., 2010), represents important regulators of the already primed vesicles that participate during Ca²⁺-induced membrane fusion and exocytosis (e.g., Lisman et al., 2007).

In contrast to the effect on single responses, the PPF patterns, which depend on the ability of the next RRP vesicles to be released upon the arrival of the second stimulus, were dependent on the distinct second messenger system, although to varying extents. Firstly, in the absence of any pharmacological treatment no effects were seen following removal of synapsin I/II, indicating that synapsin-dependent vesicle recruitment (Chi et al., 2001, 2003) is not a limiting factor. In contrast, the fEPSP enhancers had clear but different effects, with forskolin enhancing both the first and second fEPSP but leaving the PPF ratio unchanged, 4 mM [Ca²⁺]_o significantly decreasing and 1 mM Ca²⁺ significantly increasing the PPF ratio, and PDBu essentially abolishing the PPF and rather retaining the second fEPSP at baseline levels. An inverse relationship has been proposed to exist between release probability and subsequent changes in synaptic efficacy during chemical neurotransmission (Zucker and Regehr, 2002). Therefore, our PPF data suggest that in the CA1 synapse forskolin probably enhances synaptic responses without changing release probability (and therefore may enhance synaptic strength by increasing the number of functional RRP vesicles). Previous analyses of forskolin effects on PPF ratio in the same synapses are in essential agreement with our data, with minor PPF decreases of 4–10% being reported (Dumas and Foster, 1998; Kameyama et al., 1998). In contrast, other CNS synapses show highly significant decreases in PPF following forskolin treatment, including cerebellar parallel fiber-to Purkinje cell synapses (Chen and Regehr, 1997) and hippocampal mossy fiber-to CA3 pyramidal cell synapses (Weisskopf et al., 1994), both of which therefore appear to respond to increases in cAMP by increasing synaptic release probability. The different forskolin effects between these synapses and the synapses examined in this study indicate important differences in molecular or structural properties in different presynaptic populations (Bogen et al., 2006; Bragina et al., 2007). In contrast, the very low PPF ratio induced by PDBu treatment suggests that the enhanced responses in these synapses predominantly were caused by inducing major increases in vesicle release probability. Although this interpretation is at variance with previous studies performed in a number of species, where phorbol esters appeared to induce significant increases in RRP size (e.g., Gillis et al., 1996; Waters and Smith, 2000; Searl and Silinsky, 2003; Junge et al., 2004), our data are in agreement with recent studies (Basu et al., 2007; Lou et al., 2008), which conclude that PDBu, working through munc-13, preferentially induces a specific increase in release probability.

The PPF ratios induced by changes in [Ca²⁺]_o are in agreement with the proposed relation between facilitation and synaptic release probability (Zucker and Regehr, 2002). These changes may partly be due to initial dose-dependent effects on the vesicular Ca²⁺ sensor synaptotagmin, which determines the baseline fEPSP (e.g., Sun et al., 2007). Since no differences were seen between the second fEPSPs in the WT and DKO preparations, our data exclude synapsin I/II from participating in these changes. However, Ca²⁺ may have other modulatory effects on presynaptic vesicle functions, mediated by, e.g., Ca²⁺-calmodulin modulating a variety of protein phosphorylation systems (Walaas and Greengard, 1991; Jovanovic et al., 2001), the munc-13 priming proteins (Brose et al., 2004; Junge et al., 2004), and the vesicle-associated rab3A protein (Geppert et al., 1997; Park et al., 1997; Schlüter et al., 2006; Coleman and Bykovskaia, 2009a). In the present context, however, the contribution and importance of these separate modulatory systems on synaptic facilitation remain uncertain.

Analysis of the frequency facilitation induced by continuous afferent 20 Hz stimulation showed fEPSP enhancements which under standard conditions peaked after ∼12 stimuli, i.e., after 0.6 s stimulation (Hvalby et al., 2006; Jensen et al., 2007), with similar response magnitudes seen in the WT and DKO preparations. In contrast to the PPF responses, all three experimental manipulations induced substantial increases and decreased the time needed to reach the peak of this frequency facilitation. Hence, prolonged recruitment of RRP vesicles appeared to be both accelerated and enhanced by all treatments. However, subtle differences were observed. Forskolin-induced frequency facilitation showed a considerable magnitude increase and accelerated enhancement, in a manner not dependent on synapsin I/II, suggesting that the vesicles involved are not dependent on synapsin I/II proteins, despite these proteins being excellent substrates for cAMP-dependent protein kinase (Walaas and Greengard, 1991). Rather, other active zone components may be targets for cAMP-induced regulation, e.g., SNAP-25 (Nagy et al., 2002), the phosphorylation of which may be involved in regulating vesicle priming (Neher, 2006).

In contrast to the forskolin-induced effects, PDBu induced only a very brief, although accelerated increase in frequency facilitation, which also was independent on the synapsins I/II. Therefore, independently of the synapsin I/II proteins, PDBu induced a limited number of vesicles from the RRP to become available for exocytosis. Phorbol esters may mediate effects on release through activation of the munc-13 priming protein family (Rhee et al., 2002; Wierda et al., 2007; Lou et al., 2008), most probably by increasing release probability (Basu et al., 2007). The PDBu-induced small size and short duration of the frequency facilitation observed in this synapse appear consistent with the interpretation that DAG activation is restricted to recruitment of only a limited number of already primed, synapsin I/II-independent RRP vesicles.

Furthermore, increasing [Ca²⁺]_o from 1 to 4 mM in WT preparations also gave an increased frequency facilitation, which rapidly was transformed into a steep decay. However, in contrast to the frequency facilitations induced either under standard conditions, with forskolin or with PDBu, the 4 mM Ca²⁺-induced enhancements of the frequency facilitation were highly dependent on the synapsin I/II proteins. At this level of Ca²⁺, the frequency facilitation was enhanced by a factor of approx. 1.5 in the WT preparation, whereas without synapsins I/II, frequency facilitation in the DKO was increased by a factor of less than 1.3 (Figure 4). Hence, the presence of synapsins I/II led to almost a doubling of the frequency facilitation enhancement induced by the 20 Hz train. In contrast, at 1 mM Ca²⁺, the presence of synapsins I/II did not lead to significant differences in the frequency facilitation when compared to that seen in the DKO.

Given that frequency facilitations are thought to represent increased release of vesicles deriving from the RRP (Zucker and Regehr, 2002), these data indicate that a combination of high levels of Ca²⁺ and synapsins I/II proteins can modulate RRP vesicles. This unexpected conclusion is in contrast to the suggestion that synapsin-mediated functions would be restricted to reserve vesicles (Cesca et al., 2010), but appears to be partly supported by recent analysis of the postnatal development of these synapses (Bogen et al., 2009), where a transient synapsin I/II-induced enhancement of frequency facilitation induced at 2 mM Ca²⁺ occurred at developmental stages p18–27, following which it disappeared in adults. Apparently, in this synapse increasing the concentration of Ca²⁺ to 4 mM in adult mice appears to be sufficient to recreate this Ca²⁺- and-synapsin I/II-dependent response from RRP vesicles. Since neither forskolin (through cAMP) nor PDBu were able to mimic this effect, a unique relation between Ca²⁺-regulated biochemical mechanism(s) and synapsin I/II-dependent RRP vesicles clearly exists. Possible mechanisms may include Ca²⁺-induced activation of calcium-calmodulin-dependent protein kinase II (CaM kinase II) present in the active zone of excitatory synapses together with synapsin I/II (Tao-Cheng, 2006; Tao-Cheng et al., 2006), which, by phosphorylating synapsin I at sites 2,3, could induce vesicle–cytoskeletal dissociation and subsequent vesicle exocytosis (Llinas et al., 1991; Jovanovic et al., 2001; Chi et al., 2001, 2003; Hilfiker et al., 2005).

Finally, comparisons between the amplitudes of the frequency facilitations reached during the different pharmacological treatments indicate that forskolin, which led to the frequency facilitation reaching ∼2.6-fold increase of the baseline response, represents the most efficient modulator of the RRP vesicles in this synapse. The exact mechanisms for this cAMP-induced effect remain unclear, but could include both phosphorylation of multiple cAMP-dependent protein kinase substrates (Nagy et al., 2002, 2004; Leenders and Sheng, 2005), and activation of cAMP-induced release through mechanisms distinct from phosphorylation (e.g., Gekel and Neher, 2008).

The effects of second messenger modulations of other vesicle pools were somewhat unexpected, with both the DRE and the late response phases being potently regulated by some but not by other treatments. The DRE phase, which is restricted to WT preparations and may represent a pool of specific synapsin I/II-dependent (Jensen et al., 2007; Bogen et al., 2009) vesicles, responded to forskolin by being enhanced in a synapsin I/II-dependent manner, suggesting that phosphorylation of the synapsin domain A (Hilfiker et al., 2005) stimulated by cAMP (Walaas and Greengard, 1991) may be necessary for recruitment of vesicles involved in the DRE phase. In contrast, forskolin-induced enhancement of the previous or subsequent response phases occurred in a synapsin I/II-independent manner and would therefore rather be expected to be mediated through proteins distinct from the synapsins (Leenders and Sheng, 2005). In contrast to forskolin, PDBu showed essentially no effect on the DRE phase, but showed a small, synapsin-independent response enhancement during the later short-term plasticity phase. Indeed, following the major increase in single fEPSP and the small frequency facilitation induced by PDBu, the subsequent response patterns induced by PDBu mostly resembled the pattern induced in standard solution. Hence, treatment with PDBu may not activate major novel enhancement mechanisms, but rather activate and thereby occlude the mechanisms normally induced by 20 Hz stimulation. In contrast, the small but significant PDBu-induced final response enhancements which occurred in both genotypes were similar to the late response enhancement observed in forskolin-treated preparations (Figures 2A,B), suggesting the existence of distinct, late response enhancements, where both cAMP and DAGs may employ synapsin I/II-insensitive mechanisms. Finally, 4 mM [Ca²⁺]_o showed a tendency for decreasing the fEPSP during the DRE phase, followed by a significant Ca²⁺- and synapsin I/II-dependent fEPSP decrease. The latter may be related to the inverse effects of Ca²⁺-dependent enzymes on transmitter release observed during different stimulatory conditions (Jovanovic et al., 2001; Chi et al., 2003). Interestingly, this phase also coincided with the period during which these synapses showed both PDBu- and forskolin-induced response enhancements, raising the question of whether recruitment of one single pool of reserve vesicles can be enhanced by forskolin- or PDBu-dependent, synapsin I/II-independent mechanisms, and at the same time be decreased by Ca²⁺-synapsin I/II-dependent mechanisms.

In conclusion, our data demonstrate that in these small, prototypic excitatory glutamatergic synapses, different pools of synaptic vesicles are sensitive to a number of distinct regulatory mechanisms during continuous evoked glutamate release. In particular, we show that the RRP vesicles which mediate both single and paired synaptic responses as well as frequency facilitations induced in the absence or presence of forskolin or PDBu, are all essentially synapsin I/II-independent. In contrast, both the vesicles responsible for Ca²⁺-induced frequency facilitations, as well as vesicles involved in forskolin-induced response enhancements during the DRE phase were dependent on the presence of synapsin I/II. Further characterization of these response patterns remains an interesting challenge.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.