Synapsin-regulated synaptic transmission from readily releasable synaptic vesicles in excitatory hippocampal synapses in mice
1 Molecular Neurobiology Research Group, Institute of Basic Medical Sciences, University of Oslo, Norway
2 Nathan Kline Institute for Psychiatric Research, Orangeburg, NY 10962, USA
Abstract
The effects of synapsin proteins on synaptic transmission from vesicles in the readily releasable vesicle pool have been examined by comparing excitatory synaptic transmission in hippocampal slices from mice devoid of synapsins I and II and from wild-type control animals. Application of stimulus trains at variable frequencies to the CA3-to-CA1 pyramidal cell synapse suggested that, in both genotypes, synaptic responses obtained within 2 s stimulation originated from readily releasable vesicles. Detailed analysis of the responses during this period indicated that stimulus trains at 2–20 Hz enhanced all early synaptic responses in the CA3-to-CA1 pyramidal cell synapse, but depressed all early responses in the medial perforant path-to-granule cell synapse. The synapsin-dependent part of these responses, i.e. the difference between the results obtained in the transgene and the wild-type preparations, showed that in the former synapse, the presence of synapsins I and II minimized the early responses at 2 Hz, but enhanced the early responses at 20 Hz, while in the latter synapse, the presence of synapsins I and II enhanced all responses at both stimulation frequencies. The results indicate that synapsins I and II are necessary for full expression of both enhancing and decreasing modulatory effects on synaptic transmission originating from the readily releasable vesicles in these excitatory synapses.
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Introduction
Presynaptic plasticity in neurones (Hilfiker et al. 1999; Stevens & Wesseling, 1999; Zucker & Regehr, 2002; Stevens, 2004) is partly mediated by modulation of exocytotic probability both in a small, readily releasable vesicle pool (RRP) and in a reserve pool, the latter representing clusters of vesicles which must be recruited to the releasable pool prior to exocytosis (Dobrunz, 2002; Wesseling & Lo, 2002; Meinrenken et al. 2003). The vesicle-associated proteins synapsins I and II contribute to the regulation of the reserve pool, as indicated by a synaptic depression and a decrease in vesicle numbers and clusters in the absence of these proteins (Pieribone et al. 1995; Rosahl et al. 1995; Hilfiker et al. 1998, 2005; Gitler et al. 2004).
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Physiological analysis has indicated that the amplitudes of both mEPSPs and evoked EPSPs remain unchanged in cultured hippocampal neurones devoid of synapsins I, II and III (Gitler et al. 2004), indicating that the basic components for synaptic release remain intact in the complete absence of these proteins. In invertebrate neurones both exocytosis and rapid vesicle recycling appear to be sensitive to the presence of synapsins (Hilfiker et al. 1999, 2005; Fiumara et al. 2001; Humeau et al. 2001; Angers et al. 2002; but see Godenschwege et al. 2004). In contrast, when synaptic release was restricted to already docked vesicles in vertebrate synapses (Dobrunz, 2002; Wesseling & Lo, 2002), synaptic efficacy remained essentially intact in the absence of synapsins I and II under specific experimental conditions (Pieribone et al. 1995; Rosahl et al. 1995; Samigullin et al. 2004).
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In the present study, we have examined synaptic transmission deriving from the docked vesicles in mice lacking synapsin I and synapsin II. For this purpose, two physiologically distinct excitatory synapses in hippocampal slices from wild-type mice and mice devoid of synapsins I and II (double knock-out, DKO) have been examined. The en passant presynaptic boutons in the excitatory, glutamatergic CA3-to-CA1 pyramidal cell synapses exhibit release probabilities of around 0.2–0.5 (Dobrunz & Stevens, 1997). They have approximately 5–10 vesicles in the RRP and 5–10 times as many vesicles in the reserve pool (Schikorski & Stevens, 1997) and show synaptic facilitation upon repetitive stimulations (Cragg & Hamlyn, 1957; Andersen, 1960). In contrast, the excitatory synapses between the medial perforant path and the granule cells in the dentate gyrus, which have similar morphological characteristics, are characterized by a prominent synaptic depression, which possibly may be caused by a higher basal release probability (McNaughton, 1980; Rosahl et al. 1993; Dobrunz & Stevens, 1997). Our results suggest that synapsins I and/or II are involved in the modulation of transmission from the releasable vesicles in both synapses, but with distinct effects being observed in the two synapses.
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Some results have been presented in abstract form (Jensen et al. 2003).
Methods
Preparation of slices
Synapsin I and II DKO mice were generated as described previously (Ferreira et al. 1998). Experiments were performed on hippocampal slices (Li et al. 1995; Rosahl et al. 1995) prepared from adult (3–6 months old) DKO mice and wild-type control mice. The animals were killed in a glass container (5 l) containing Suprane (Baxter, 10 ml). Following circulatory arrest, the brains were removed. Transverse slices (400 μm) were cut from the middle portion of each hippocampus with a vibroslicer in artificial cerebrospinal fluid (ACSF, 4°C, bubbled with 95% O2–5% CO2, pH 7.4) containing (mM): 124 NaCl, 2 KCl, 1.25 KH2PO4, 2 MgSO4, 1 CaCl2, 26 NaHCO3 and 12 glucose. Slices were placed in a humidified interface chamber at 30 ± 1°C and perfused with ACSF containing 1 or 2 mM CaCl2. To block N-methyl-D-aspartic acid (NMDA) receptor-mediated synaptic plasticity, 50 μM 2-amino-5-phosphonovaleric acid (APV, Sigma-Aldrich, Oslo, Norway) was present throughout all experiments. In some experiments, the -amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-mediated responses were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 μM, Tocris, Bristol, UK) in order to reveal differences in amplitude of the presynaptic volley during 20 Hz stimulation.
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Animal experiments were conducted according to the Norwegian Animal Welfare Act and the European Union's Directive 86/609/EEC. Efforts were made to reduce the number of animals used.
Stimulation and recordings
Orthodromic synaptic stimuli (50 μs, < 280 μA, 0.1 Hz) were delivered alternately through two tungsten electrodes, either with one situated in the stratum radiatum and another in the stratum oriens of the CA1 region, or with electrodes in the outer and middle molecular layer of the dentate area. Extracellular synaptic responses were monitored by two glass electrodes (filled with ACSF) placed in the corresponding synaptic layers. After obtaining stable synaptic responses in both pathways (0.1 Hz stimulation) for at least 10–15 min, we stimulated either the radiatum pathway or the medial perforant path repetitively at a given frequency (2–20 Hz).
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In another set of experiments, the patch-clamp technique (current clamp) was used to record synaptic responses from single CA1 pyramidal cells (mice aged 36–39 postnatal days). The patch electrode solution contained (mM): 130 potassium gluconate, 10 Hepes, 1 MgCl2, 1 Mg2-ATP and 0.25 Na3-GTP (pH 7.3).
Analysis
We assessed the synaptic efficacy by measuring the maximal slope of the synaptic field potentials (fEPSPs) or the intracellular EPSP, and normalized the value of each response to the mean value recorded 1 min prior to the high frequency stimulation. In some experiments, we also measured the amplitude of the presynaptic volley in a similar manner. Data were pooled across animals of the same genotype and presented as mean ±S.E.M., and statistical significance was evaluated using a two-tailed unpaired t test.
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Results
Synapses in CA1
We first examined whether the Ca2+ dependencies of synaptic responses in the two genotypes were similar. Baseline fEPSPs were recorded in response to 0.1 Hz stimulation in ACSF containing either 1 or 2 mM[Ca2+]o, followed by equilibration for 60 min in either 2 or 4 mM CaCl2, respectively, in the two genotypes. Ca2+-dependent increases in the fEPSP were observed in accordance with a previous report (Huang et al. 1988). However, comparisons revealed no significant differences between the two genotypes, irrespective of whether Ca2+ was increased from 1 to 2 mM (Fig. 1A, left columns, P= 0.19) or from 2 to 4 mM (Fig. 1A, right columns, P= 0.63). Since [Ca2+]o represents such a strong determinant of baseline responses, we henceforth normalized all results to the baseline fEPSPs observed in 1 mM Ca2+.
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A, normalized increase in the mean field EPSP slope following equilibration (60 min) from 1 mM to 2 mM (left columns, 1–2) and from 2 mM to 4 mM Ca2+ (right columns, 2–4) in wild-type (WT; open columns, n= 12 and n= 19, respectively) and double knock-out (DKO; filled columns, n= 10 and n= 16, respectively) mice. Vertical bars indicate S.E.M.B, presynaptic volley amplitudes as a function of stimulus number during 20 Hz stimulation (2 mM Ca2+) in wild-type (, n= 13) and DKO (, n= 6) mice. Experiments were done in the presence of 50 μM APV and 10 μM CNQX. Vertical bars indicate S.E.M.
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To ascertain that the differences observed were not due to distinct changes in the presynaptic fibre volley in the two genotypes, the prevolley amplitude was analysed as a function of stimulus number during 20 Hz stimulation in the presence of 50 μM APV and 10 μM CNQX. No differences between the genotypes were observed (Fig. 1B).
The early synaptic responses induced by repetitive stimulations are thought to derive from exocytotic release of transmitter present in the already primed and docked vesicles in the RRP until this pool is depleted (Hilfiker et al. 1999; Wesseling & Lo, 2002). In order to restrict our analysis to the vesicles already present in the RRP, we first examined the early responses induced in the CA3-to-CA1 synapses by stimulation trains at 10–20 Hz (Dobrunz, 2002; Wesseling & Lo, 2002). Previous work has shown that stimulations either at 10 Hz in 4 mM Ca2+ (Dobrunz & Stevens, 1997) or at 20 Hz in 2.6 mM Ca2+ (Wesseling & Lo, 2002) will induce depletion of RRP within 1.5–3 s, and this is followed by release from vesicles recruited from recycling and/or reserve pools (Sakaba & Neher, 2001; Stevens, 2004; Sudhof, 2004).
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The normalized and pooled fEPSPs obtained in response to afferent stimulations given at different [Ca2+]o showed that the CA3-to-CA1 synapse responded to stimulation trains given at 5–20 Hz by initial enhancements in both wild-type (Fig. 2A, B and C) and DKO mice (Fig. 3A, B and C), all of which were dependent on frequency and [Ca2+]o. At 2 Hz, when compared to baseline fEPSP at the given [Ca2+]o (Fig. 1), only stimulation in 1 mM and 2 mM Ca2+, but not in 4 mM Ca2+, revealed similar enhancements (Figs 2D and 3D). The response enhancements could be further divided into an initial response increase which, irrespective of stimulation frequency, reached maximal values after 2–6 stimuli, which were followed by subsequent plateaus. The plateaus remained stable for 1–2 s during 10–20 Hz stimulation, and were then followed by decays in response, suggesting that under these conditions, the RRP approached depletion and that vesicles outside the RRP now became recruited (Wesseling & Lo, 2002). In contrast, the response plateaus seen in synapses stimulated at lower frequencies (2–5 Hz) remained almost unchanged, indicating that under these conditions these synapses were able to recruit sufficient vesicles during protracted stimulation (Kumashiro et al. 2005). These data indicate that, under high frequency stimulations (10–20 Hz), the initial response enhancement and subsequent plateau, which in this synapse are observed during the first 20–30 stimuli, represent release from already docked and primed vesicles in the RRP proper, while subsequent responses may include recycling/recruited vesicles from the reserve pool. In contrast, under low frequency conditions (2–5 Hz), we assume that only the initial 5–6 responses may represent release from already docked vesicles in the RRP.
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The graphs show the synaptic strength at different frequencies and at different [Ca2+]o. The inset in A shows superimposed synaptic responses in 2 mM Ca2+ at the times indicated by arrows and numbers. A, normalized and pooled field EPSP values in wild-type mice at 20 Hz stimulation in the presence of 1 mM (red circles, n= 12), 2 mM (blue circles, n= 22) and 4 mM Ca2+ (green triangles, n= 11). B–D, as in A but at 10 Hz (n= 13, n= 20 and n= 11), 5 Hz (n= 11, n= 12 and n= 10) and 2 Hz (n= 10, n= 10 and n= 10) stimulation, respectively. Vertical bars indicate S.E.M.
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The graphs show the synaptic strength at different frequencies and at different [Ca2+]o. The inset in A shows superimposed synaptic responses in 2 mM Ca2+ at the times indicated by arrows and numbers. A, normalized and pooled field EPSP values in wild-type mice at 20 Hz stimulation in the presence of 1 mM (red circles, n= 12), 2 mM (blue circles, n= 18) and 4 mM Ca2+ (green triangles, n= 10). B–D, as in A, but at 10 Hz (n= 10, n= 16 and n= 10), 5 Hz (n= 10, n= 9 and n= 10) and 2 Hz (n= 9, n= 12 and n= 9) stimulation, respectively. Vertical bars indicate S.E.M.
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Taking these response patterns into account, we analysed the responses in the two genotypes in more detail. As previously observed in both double (Rosahl et al. 1995) and triple knock-out (Gitler et al. 2004) preparations, the initial synaptic enhancements were generally similar in both genotypes. Closer comparisons showed, however, that during incubations at 2 and 4 mM Ca2+, slightly more prominent increases during the initial 4–5 synaptic responses appeared to be present in the DKO stimulated at 2–5 Hz when compared to the wild-type (Figs 2C and D and 3C and D). Following these initial phases, the responses seen at 2–5 Hz remained essentially stable in both genotypes when examined in 2 mM Ca2+, but decreased in the DKO when examined at 4 mM Ca2+. In contrast, during stimulation at 10–20 Hz, the initial responses appeared rather similar in both genotypes during the rising phase and subsequent plateau phases. After 2 s duration, subsequent response depressions were seen in both genotypes, with the DKO showing a particularly pronounced and rapid decay at 4 mM Ca2+.
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In a separate set of experiments, intracellular analysis was used to evaluate the validity of the results obtained by extracellular field recordings. The intracellularly recorded EPSPs obtained during 20 Hz stimulation from CA1 pyramids in medium containing 2 mM[Ca2+]o gave results in the two genotypes which substantiated the fEPSP results obtained under identical conditions (Fig. 4A and B).
Normalized and pooled intracellularly recorded EPSP values in wild-type mice (A) and DKO mice (B). Vertical bars indicate S.E.M., n= 17 (wild-type) and 11 (DKO).
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We characterized the genotype-dependent, synapsin I and II-mediated differences of these synapses in more detail by subtracting the results obtained in the DKO from those in the wild-type. The resulting graphs (Fig. 5) suggested that multiple Ca2+- and frequency-regulated synapsin-dependent components existed, some of which were occurring during release deriving from the RRP.
A, subtraction of the values obtained at different frequencies in DKO mice (Fig. 3) from those obtained in wild-type (Fig. 2) at 1 mM Ca2+. The frequency 20 Hz is symbolized by dark blue circles, 10 Hz by yellow circles, 5 Hz by orange circles and 2 Hz by turquoise circles. B–C, as in A, but at 2 mM and 4 mM Ca2+, respectively. D, subtraction of the intracellular EPSP values obtained by intracellular recordings at 20 Hz stimulation at 2 mM Ca2+ in DKO mice (Fig. 4B) from those in wild-type mice (Fig. 4A). Horizontal bars along the abscissa with colours corresponding to the respective frequencies indicate P < 0.05 when comparing the results from wild-type and DKO mice (">Figs 2 – 4).
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At 20 Hz stimulation, a synapsin-dependent effect which enhanced the responses deriving from the RRP as defined above could be seen to develop, which was statistically significant during incubation in ACSF containing 4 mM Ca2+ (Fig. 5C). This effect also occurred at lower release probabilities, i.e. at 2 mM Ca2+ and 1 mM Ca2+, albeit with a delayed statistical significance (Fig. 5A and B).
In contrast, at 2 Hz stimulation, a synapsin-dependent, apparently inhibitory effect decreasing the responses deriving from the RRP could be seen to develop, which was statistically significant during stimulation in ACSF containing 2 mM Ca2+, and remained stable for at least 80 stimuli. A similar tendency, albeit not statistically significant, was observed at 4 mM Ca2+, whereas this inhibitory synapsin-induced effect was not seen during incubations in 1 mM Ca2+ (Fig. 5A). It thus appears to represent a Ca2+-dependent phenomenon.
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These results suggest that under these conditions, the absence of synapsins I and II enhanced stimulus-induced exocytotic release, occurring both from the RRP and also, later, during release presumably originating outside the RRP. Hence, under these conditions expression of synapsins I and II may constrain release, irrespective of vesicle origin. Similar inhibitory tendencies in the early response patterns were also observed, although statistical comparison failed to reveal a significant difference, at 5 Hz in 2 mM Ca2+ and at 2 and 5 Hz in 4 mM Ca2+. Given the similarity to the significant changes observed at 2 Hz/2 mM Ca2+, these response patterns may represent a gradual transition towards the consistent enhancement seen at 20 Hz (see above).
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Synapses in area dentata
We also examined the synapsin dependence of evoked responses in the medial perforant path synapses. At a 50 ms interstimulus interval, the paired-pulse depression in these synapses was similar in the two genotypes (wild-type versus DKO mutants: 84 ± 1%, n= 16 versus 79 ± 2%, n= 16; P= 0.07); however, at a 500 ms interstimulus interval statistical evaluation revealed a significant difference between the two with a more pronounced depression seen in the DKO (wild-type versus DKO mutants: 87 ± 2%, n= 16 versus 80 ± 2%, n= 17; P= 0.01). Figure 6 shows normalized and pooled measurements of fEPSPs elicited in the middle molecular layer of the dentate area in the two genotypes in response to repetitive afferent stimulations given at 2 mM Ca2+. Stimulations at 20 Hz of both wild-type and DKO preparations led to an immediate depression of the fEPSPs, which after approximately 10 stimuli approached a steady state (Fig. 6A). At 2 Hz stimulations, already the second responses observed in the two genotypes were depressed to a steady state, and the latter was then maintained throughout the stimulation train (Fig. 6B). Quantification of the effects of synapsins I and II in these synapses showed that the presence of synapsins I and II led to less depressing effects already after two and three stimuli at both 2 Hz and 20 Hz (Fig. 6A and B). This indicates that in these synapses, the presence of synapsins I and II partly counteracts the decay of synaptic responses normally seen, irrespective of whether the response derives from early or late vesicle pools. Neither restraining nor bidirectional effects of synapsins were observed in these synapses.
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The graphs show synaptic strength in 2 mM Ca2+ at 20 Hz (upper panel) and 2 Hz (lower panel) in wild-type () and DKO mice (). Subtraction of the values obtained at 20 Hz and 2 Hz in DKO mice from those obtained in wild-type is represented by . A, normalized and pooled field EPSP values in wild-type (n= 16) and DKO (n= 17) mice at 20 Hz stimulation. The insets show synaptic responses in 2 mM Ca2+ superimposed at the stimulation times indicated by numbers. B, as in A, but at 2 Hz stimulation frequency. Vertical bars indicate S.E.M., n= 16 in both genotypes. Horizontal, filled bars along the abscissa indicate P < 0.05 when comparing the results from the two genotypes.
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Discussion
The present results demonstrate that, when examined in functionally intact synapses in adult rodent brain, synapsins I and II are necessary for short-term modulation of synaptic transmission as observed during the early synaptic responses elicited by a train of afferent stimulation. Some of the changes observed appear to be caused by modulatory effects on vesicles already present in the RRP, while others may be caused by changes in the recruitment of reserve vesicles, previously believed to be the main effect of synapsin proteins (Pieribone et al. 1995; Rosahl et al. 1995; Chi et al. 2001, 2003). In order to restrict the responses to the RRP, we have followed previous work which indicated that the RRP in these synapses will be emptied during electrophysiological experiments after 15 stimuli given at 10 Hz stimulations in 4 mM Ca2+ (Dobrunz & Stevens, 1997) or after 60 stimuli given at 20 Hz stimulations in 2.6 mM Ca2+ (Wesseling & Lo, 2002), while chemically induced exocytosis will empty the RRP during 2–3 s (Kumashiro et al. 2005; Ashton & Ushkaryov, 2005). Although age of animals, type of preparation and incubation temperatures may be different, these reports indicate that stimulatory trains lasting between 1.5 and 3 s would preferentially represent transmission deriving from the RRP. Our analysis, which indicates that responses during the initial 2 s derive from the RRP, irrespective of frequency, is in general agreement with these authors.
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Furthermore, we show that the synapsin I and II proteins can have different effects on RRP release in distinct synapses. In the CA3-to-CA1 synapse, which represents a prototypical small, facilitating excitatory CNS synapse, these include a synapsin I and II-dependent decrease in response to low (2 Hz) stimulation frequency at normal [Ca2+]o, and enhancing effects at a higher (20 Hz) stimulation frequency. In contrast, the medial perforant path-to-granule cell synapse represents a prototypical small, depressing excitatory CNS synapse, with high release probability and characterized by paired-pulse depression (McNaughton, 1980; Rosahl et al. 1993; Dobrunz & Stevens, 1997). Since these depressions were consistently smaller in the wild-type than in the DKO genotype, irrespective of low or high stimulation frequencies, the effects of synapsin I and II proteins in this synapse were strictly enhancing, as previously described in other synapses (Li et al. 1995; Rosahl et al. 1995; Pieribone et al. 1995; Hilfiker et al. 1998). These data are in contrast to the unidirectional enhancing effects recently observed in hippocampal neurones cultured from synapsin I, II and III triple knock-out mice (Gitler et al. 2004). Hence, it is possible that the dynamic, uni- and/or bi-directional synapsin effects on early release that we see remain restricted to adult, intact CNS synapses.
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Taken together, our results show that synapsins I and II can induce both decreases and enhancements of synaptic responses deriving from the initial stimulations in a stimulation train, with the direction of changes partly controlled by stimulation frequencies and levels of [Ca2+]o, and partly by the identity of the synapses studied. The mechanisms behind these distinct effects are not clear. Previous studies have generally concluded that the absence of synapsins decreases recruitment from reserve vesicles, with a profound change in the organization of the vesicle clusters partly being responsible (Pieribone et al. 1995; Rosahl et al. 1995; Walaas et al. 2000; Chi et al. 2001, 2003; Gitler et al. 2004). However, the present experimental protocol demonstrates that synapsin-dependent changes in synaptic response occur already during the initial stimuli, which would be difficult to reconcile with a recruitment from vesicle clusters, the latter apparently necessitating minimally 2–3 s stimulation (Wesseling & Lo, 2002; Kumashiro et al. 2005). Rather, dynamic molecular responses occurring in the releasable vesicle pool within the first few seconds may be responsible. Such changes would be consistent with dynamic biochemical effects on synapsins. Since administration of dephospho-synapsin into axons can decrease evoked synaptic transmission (Hackett et al. 1990; Llinas et al. 1991), stimulus-induced changes in synapsin phosphorylation may be involved (Schiebler et al. 1986; Hosaka et al. 1999; Jovanovic et al. 2001; Fiumara et al. 2001; Chi et al. 2003). Recent experiments have also indicated that prevention of protein phosphorylation by means of broad spectrum protein kinase inhibitors can mimic physiological effects of synapsin gene inactivation (. Hvalby, V. Jensen & S. I. Walaas, unpublished observations). However, to what degree such inhibitors work through modulation of synapsin phosphorylation remains unclear (Spillane et al. 1995; Samigullin et al. 2004). Recent studies have also indicated that distinct domains in the synapsin proteins may have enhancing or inhibiting effects on synaptic transmission, some of which are mediated by actin- and/or phosphorylation-dependent mechanisms (Hilfiker et al. 2005). Whether the synapsin I and II effects described here are dependent on such mechanisms is an interesting subject for further study.
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In conclusion, the results from this study indicate that synapsins I and II are necessary for full expression of modulatory effects on synaptic transmission originating from the readily releasable vesicles in various types of excitatory vertebrate neurones. Such results increase the possible functional importance and diversity of the synapsin proteins during synaptic transmission at vertebrate synapses.
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2 Nathan Kline Institute for Psychiatric Research, Orangeburg, NY 10962, USA
Abstract
The effects of synapsin proteins on synaptic transmission from vesicles in the readily releasable vesicle pool have been examined by comparing excitatory synaptic transmission in hippocampal slices from mice devoid of synapsins I and II and from wild-type control animals. Application of stimulus trains at variable frequencies to the CA3-to-CA1 pyramidal cell synapse suggested that, in both genotypes, synaptic responses obtained within 2 s stimulation originated from readily releasable vesicles. Detailed analysis of the responses during this period indicated that stimulus trains at 2–20 Hz enhanced all early synaptic responses in the CA3-to-CA1 pyramidal cell synapse, but depressed all early responses in the medial perforant path-to-granule cell synapse. The synapsin-dependent part of these responses, i.e. the difference between the results obtained in the transgene and the wild-type preparations, showed that in the former synapse, the presence of synapsins I and II minimized the early responses at 2 Hz, but enhanced the early responses at 20 Hz, while in the latter synapse, the presence of synapsins I and II enhanced all responses at both stimulation frequencies. The results indicate that synapsins I and II are necessary for full expression of both enhancing and decreasing modulatory effects on synaptic transmission originating from the readily releasable vesicles in these excitatory synapses.
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Introduction
Presynaptic plasticity in neurones (Hilfiker et al. 1999; Stevens & Wesseling, 1999; Zucker & Regehr, 2002; Stevens, 2004) is partly mediated by modulation of exocytotic probability both in a small, readily releasable vesicle pool (RRP) and in a reserve pool, the latter representing clusters of vesicles which must be recruited to the releasable pool prior to exocytosis (Dobrunz, 2002; Wesseling & Lo, 2002; Meinrenken et al. 2003). The vesicle-associated proteins synapsins I and II contribute to the regulation of the reserve pool, as indicated by a synaptic depression and a decrease in vesicle numbers and clusters in the absence of these proteins (Pieribone et al. 1995; Rosahl et al. 1995; Hilfiker et al. 1998, 2005; Gitler et al. 2004).
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Physiological analysis has indicated that the amplitudes of both mEPSPs and evoked EPSPs remain unchanged in cultured hippocampal neurones devoid of synapsins I, II and III (Gitler et al. 2004), indicating that the basic components for synaptic release remain intact in the complete absence of these proteins. In invertebrate neurones both exocytosis and rapid vesicle recycling appear to be sensitive to the presence of synapsins (Hilfiker et al. 1999, 2005; Fiumara et al. 2001; Humeau et al. 2001; Angers et al. 2002; but see Godenschwege et al. 2004). In contrast, when synaptic release was restricted to already docked vesicles in vertebrate synapses (Dobrunz, 2002; Wesseling & Lo, 2002), synaptic efficacy remained essentially intact in the absence of synapsins I and II under specific experimental conditions (Pieribone et al. 1995; Rosahl et al. 1995; Samigullin et al. 2004).
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In the present study, we have examined synaptic transmission deriving from the docked vesicles in mice lacking synapsin I and synapsin II. For this purpose, two physiologically distinct excitatory synapses in hippocampal slices from wild-type mice and mice devoid of synapsins I and II (double knock-out, DKO) have been examined. The en passant presynaptic boutons in the excitatory, glutamatergic CA3-to-CA1 pyramidal cell synapses exhibit release probabilities of around 0.2–0.5 (Dobrunz & Stevens, 1997). They have approximately 5–10 vesicles in the RRP and 5–10 times as many vesicles in the reserve pool (Schikorski & Stevens, 1997) and show synaptic facilitation upon repetitive stimulations (Cragg & Hamlyn, 1957; Andersen, 1960). In contrast, the excitatory synapses between the medial perforant path and the granule cells in the dentate gyrus, which have similar morphological characteristics, are characterized by a prominent synaptic depression, which possibly may be caused by a higher basal release probability (McNaughton, 1980; Rosahl et al. 1993; Dobrunz & Stevens, 1997). Our results suggest that synapsins I and/or II are involved in the modulation of transmission from the releasable vesicles in both synapses, but with distinct effects being observed in the two synapses.
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Some results have been presented in abstract form (Jensen et al. 2003).
Methods
Preparation of slices
Synapsin I and II DKO mice were generated as described previously (Ferreira et al. 1998). Experiments were performed on hippocampal slices (Li et al. 1995; Rosahl et al. 1995) prepared from adult (3–6 months old) DKO mice and wild-type control mice. The animals were killed in a glass container (5 l) containing Suprane (Baxter, 10 ml). Following circulatory arrest, the brains were removed. Transverse slices (400 μm) were cut from the middle portion of each hippocampus with a vibroslicer in artificial cerebrospinal fluid (ACSF, 4°C, bubbled with 95% O2–5% CO2, pH 7.4) containing (mM): 124 NaCl, 2 KCl, 1.25 KH2PO4, 2 MgSO4, 1 CaCl2, 26 NaHCO3 and 12 glucose. Slices were placed in a humidified interface chamber at 30 ± 1°C and perfused with ACSF containing 1 or 2 mM CaCl2. To block N-methyl-D-aspartic acid (NMDA) receptor-mediated synaptic plasticity, 50 μM 2-amino-5-phosphonovaleric acid (APV, Sigma-Aldrich, Oslo, Norway) was present throughout all experiments. In some experiments, the -amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-mediated responses were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 μM, Tocris, Bristol, UK) in order to reveal differences in amplitude of the presynaptic volley during 20 Hz stimulation.
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Animal experiments were conducted according to the Norwegian Animal Welfare Act and the European Union's Directive 86/609/EEC. Efforts were made to reduce the number of animals used.
Stimulation and recordings
Orthodromic synaptic stimuli (50 μs, < 280 μA, 0.1 Hz) were delivered alternately through two tungsten electrodes, either with one situated in the stratum radiatum and another in the stratum oriens of the CA1 region, or with electrodes in the outer and middle molecular layer of the dentate area. Extracellular synaptic responses were monitored by two glass electrodes (filled with ACSF) placed in the corresponding synaptic layers. After obtaining stable synaptic responses in both pathways (0.1 Hz stimulation) for at least 10–15 min, we stimulated either the radiatum pathway or the medial perforant path repetitively at a given frequency (2–20 Hz).
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In another set of experiments, the patch-clamp technique (current clamp) was used to record synaptic responses from single CA1 pyramidal cells (mice aged 36–39 postnatal days). The patch electrode solution contained (mM): 130 potassium gluconate, 10 Hepes, 1 MgCl2, 1 Mg2-ATP and 0.25 Na3-GTP (pH 7.3).
Analysis
We assessed the synaptic efficacy by measuring the maximal slope of the synaptic field potentials (fEPSPs) or the intracellular EPSP, and normalized the value of each response to the mean value recorded 1 min prior to the high frequency stimulation. In some experiments, we also measured the amplitude of the presynaptic volley in a similar manner. Data were pooled across animals of the same genotype and presented as mean ±S.E.M., and statistical significance was evaluated using a two-tailed unpaired t test.
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Results
Synapses in CA1
We first examined whether the Ca2+ dependencies of synaptic responses in the two genotypes were similar. Baseline fEPSPs were recorded in response to 0.1 Hz stimulation in ACSF containing either 1 or 2 mM[Ca2+]o, followed by equilibration for 60 min in either 2 or 4 mM CaCl2, respectively, in the two genotypes. Ca2+-dependent increases in the fEPSP were observed in accordance with a previous report (Huang et al. 1988). However, comparisons revealed no significant differences between the two genotypes, irrespective of whether Ca2+ was increased from 1 to 2 mM (Fig. 1A, left columns, P= 0.19) or from 2 to 4 mM (Fig. 1A, right columns, P= 0.63). Since [Ca2+]o represents such a strong determinant of baseline responses, we henceforth normalized all results to the baseline fEPSPs observed in 1 mM Ca2+.
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A, normalized increase in the mean field EPSP slope following equilibration (60 min) from 1 mM to 2 mM (left columns, 1–2) and from 2 mM to 4 mM Ca2+ (right columns, 2–4) in wild-type (WT; open columns, n= 12 and n= 19, respectively) and double knock-out (DKO; filled columns, n= 10 and n= 16, respectively) mice. Vertical bars indicate S.E.M.B, presynaptic volley amplitudes as a function of stimulus number during 20 Hz stimulation (2 mM Ca2+) in wild-type (, n= 13) and DKO (, n= 6) mice. Experiments were done in the presence of 50 μM APV and 10 μM CNQX. Vertical bars indicate S.E.M.
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To ascertain that the differences observed were not due to distinct changes in the presynaptic fibre volley in the two genotypes, the prevolley amplitude was analysed as a function of stimulus number during 20 Hz stimulation in the presence of 50 μM APV and 10 μM CNQX. No differences between the genotypes were observed (Fig. 1B).
The early synaptic responses induced by repetitive stimulations are thought to derive from exocytotic release of transmitter present in the already primed and docked vesicles in the RRP until this pool is depleted (Hilfiker et al. 1999; Wesseling & Lo, 2002). In order to restrict our analysis to the vesicles already present in the RRP, we first examined the early responses induced in the CA3-to-CA1 synapses by stimulation trains at 10–20 Hz (Dobrunz, 2002; Wesseling & Lo, 2002). Previous work has shown that stimulations either at 10 Hz in 4 mM Ca2+ (Dobrunz & Stevens, 1997) or at 20 Hz in 2.6 mM Ca2+ (Wesseling & Lo, 2002) will induce depletion of RRP within 1.5–3 s, and this is followed by release from vesicles recruited from recycling and/or reserve pools (Sakaba & Neher, 2001; Stevens, 2004; Sudhof, 2004).
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The normalized and pooled fEPSPs obtained in response to afferent stimulations given at different [Ca2+]o showed that the CA3-to-CA1 synapse responded to stimulation trains given at 5–20 Hz by initial enhancements in both wild-type (Fig. 2A, B and C) and DKO mice (Fig. 3A, B and C), all of which were dependent on frequency and [Ca2+]o. At 2 Hz, when compared to baseline fEPSP at the given [Ca2+]o (Fig. 1), only stimulation in 1 mM and 2 mM Ca2+, but not in 4 mM Ca2+, revealed similar enhancements (Figs 2D and 3D). The response enhancements could be further divided into an initial response increase which, irrespective of stimulation frequency, reached maximal values after 2–6 stimuli, which were followed by subsequent plateaus. The plateaus remained stable for 1–2 s during 10–20 Hz stimulation, and were then followed by decays in response, suggesting that under these conditions, the RRP approached depletion and that vesicles outside the RRP now became recruited (Wesseling & Lo, 2002). In contrast, the response plateaus seen in synapses stimulated at lower frequencies (2–5 Hz) remained almost unchanged, indicating that under these conditions these synapses were able to recruit sufficient vesicles during protracted stimulation (Kumashiro et al. 2005). These data indicate that, under high frequency stimulations (10–20 Hz), the initial response enhancement and subsequent plateau, which in this synapse are observed during the first 20–30 stimuli, represent release from already docked and primed vesicles in the RRP proper, while subsequent responses may include recycling/recruited vesicles from the reserve pool. In contrast, under low frequency conditions (2–5 Hz), we assume that only the initial 5–6 responses may represent release from already docked vesicles in the RRP.
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The graphs show the synaptic strength at different frequencies and at different [Ca2+]o. The inset in A shows superimposed synaptic responses in 2 mM Ca2+ at the times indicated by arrows and numbers. A, normalized and pooled field EPSP values in wild-type mice at 20 Hz stimulation in the presence of 1 mM (red circles, n= 12), 2 mM (blue circles, n= 22) and 4 mM Ca2+ (green triangles, n= 11). B–D, as in A but at 10 Hz (n= 13, n= 20 and n= 11), 5 Hz (n= 11, n= 12 and n= 10) and 2 Hz (n= 10, n= 10 and n= 10) stimulation, respectively. Vertical bars indicate S.E.M.
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The graphs show the synaptic strength at different frequencies and at different [Ca2+]o. The inset in A shows superimposed synaptic responses in 2 mM Ca2+ at the times indicated by arrows and numbers. A, normalized and pooled field EPSP values in wild-type mice at 20 Hz stimulation in the presence of 1 mM (red circles, n= 12), 2 mM (blue circles, n= 18) and 4 mM Ca2+ (green triangles, n= 10). B–D, as in A, but at 10 Hz (n= 10, n= 16 and n= 10), 5 Hz (n= 10, n= 9 and n= 10) and 2 Hz (n= 9, n= 12 and n= 9) stimulation, respectively. Vertical bars indicate S.E.M.
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Taking these response patterns into account, we analysed the responses in the two genotypes in more detail. As previously observed in both double (Rosahl et al. 1995) and triple knock-out (Gitler et al. 2004) preparations, the initial synaptic enhancements were generally similar in both genotypes. Closer comparisons showed, however, that during incubations at 2 and 4 mM Ca2+, slightly more prominent increases during the initial 4–5 synaptic responses appeared to be present in the DKO stimulated at 2–5 Hz when compared to the wild-type (Figs 2C and D and 3C and D). Following these initial phases, the responses seen at 2–5 Hz remained essentially stable in both genotypes when examined in 2 mM Ca2+, but decreased in the DKO when examined at 4 mM Ca2+. In contrast, during stimulation at 10–20 Hz, the initial responses appeared rather similar in both genotypes during the rising phase and subsequent plateau phases. After 2 s duration, subsequent response depressions were seen in both genotypes, with the DKO showing a particularly pronounced and rapid decay at 4 mM Ca2+.
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In a separate set of experiments, intracellular analysis was used to evaluate the validity of the results obtained by extracellular field recordings. The intracellularly recorded EPSPs obtained during 20 Hz stimulation from CA1 pyramids in medium containing 2 mM[Ca2+]o gave results in the two genotypes which substantiated the fEPSP results obtained under identical conditions (Fig. 4A and B).
Normalized and pooled intracellularly recorded EPSP values in wild-type mice (A) and DKO mice (B). Vertical bars indicate S.E.M., n= 17 (wild-type) and 11 (DKO).
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We characterized the genotype-dependent, synapsin I and II-mediated differences of these synapses in more detail by subtracting the results obtained in the DKO from those in the wild-type. The resulting graphs (Fig. 5) suggested that multiple Ca2+- and frequency-regulated synapsin-dependent components existed, some of which were occurring during release deriving from the RRP.
A, subtraction of the values obtained at different frequencies in DKO mice (Fig. 3) from those obtained in wild-type (Fig. 2) at 1 mM Ca2+. The frequency 20 Hz is symbolized by dark blue circles, 10 Hz by yellow circles, 5 Hz by orange circles and 2 Hz by turquoise circles. B–C, as in A, but at 2 mM and 4 mM Ca2+, respectively. D, subtraction of the intracellular EPSP values obtained by intracellular recordings at 20 Hz stimulation at 2 mM Ca2+ in DKO mice (Fig. 4B) from those in wild-type mice (Fig. 4A). Horizontal bars along the abscissa with colours corresponding to the respective frequencies indicate P < 0.05 when comparing the results from wild-type and DKO mice (">Figs 2 – 4).
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At 20 Hz stimulation, a synapsin-dependent effect which enhanced the responses deriving from the RRP as defined above could be seen to develop, which was statistically significant during incubation in ACSF containing 4 mM Ca2+ (Fig. 5C). This effect also occurred at lower release probabilities, i.e. at 2 mM Ca2+ and 1 mM Ca2+, albeit with a delayed statistical significance (Fig. 5A and B).
In contrast, at 2 Hz stimulation, a synapsin-dependent, apparently inhibitory effect decreasing the responses deriving from the RRP could be seen to develop, which was statistically significant during stimulation in ACSF containing 2 mM Ca2+, and remained stable for at least 80 stimuli. A similar tendency, albeit not statistically significant, was observed at 4 mM Ca2+, whereas this inhibitory synapsin-induced effect was not seen during incubations in 1 mM Ca2+ (Fig. 5A). It thus appears to represent a Ca2+-dependent phenomenon.
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These results suggest that under these conditions, the absence of synapsins I and II enhanced stimulus-induced exocytotic release, occurring both from the RRP and also, later, during release presumably originating outside the RRP. Hence, under these conditions expression of synapsins I and II may constrain release, irrespective of vesicle origin. Similar inhibitory tendencies in the early response patterns were also observed, although statistical comparison failed to reveal a significant difference, at 5 Hz in 2 mM Ca2+ and at 2 and 5 Hz in 4 mM Ca2+. Given the similarity to the significant changes observed at 2 Hz/2 mM Ca2+, these response patterns may represent a gradual transition towards the consistent enhancement seen at 20 Hz (see above).
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Synapses in area dentata
We also examined the synapsin dependence of evoked responses in the medial perforant path synapses. At a 50 ms interstimulus interval, the paired-pulse depression in these synapses was similar in the two genotypes (wild-type versus DKO mutants: 84 ± 1%, n= 16 versus 79 ± 2%, n= 16; P= 0.07); however, at a 500 ms interstimulus interval statistical evaluation revealed a significant difference between the two with a more pronounced depression seen in the DKO (wild-type versus DKO mutants: 87 ± 2%, n= 16 versus 80 ± 2%, n= 17; P= 0.01). Figure 6 shows normalized and pooled measurements of fEPSPs elicited in the middle molecular layer of the dentate area in the two genotypes in response to repetitive afferent stimulations given at 2 mM Ca2+. Stimulations at 20 Hz of both wild-type and DKO preparations led to an immediate depression of the fEPSPs, which after approximately 10 stimuli approached a steady state (Fig. 6A). At 2 Hz stimulations, already the second responses observed in the two genotypes were depressed to a steady state, and the latter was then maintained throughout the stimulation train (Fig. 6B). Quantification of the effects of synapsins I and II in these synapses showed that the presence of synapsins I and II led to less depressing effects already after two and three stimuli at both 2 Hz and 20 Hz (Fig. 6A and B). This indicates that in these synapses, the presence of synapsins I and II partly counteracts the decay of synaptic responses normally seen, irrespective of whether the response derives from early or late vesicle pools. Neither restraining nor bidirectional effects of synapsins were observed in these synapses.
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The graphs show synaptic strength in 2 mM Ca2+ at 20 Hz (upper panel) and 2 Hz (lower panel) in wild-type () and DKO mice (). Subtraction of the values obtained at 20 Hz and 2 Hz in DKO mice from those obtained in wild-type is represented by . A, normalized and pooled field EPSP values in wild-type (n= 16) and DKO (n= 17) mice at 20 Hz stimulation. The insets show synaptic responses in 2 mM Ca2+ superimposed at the stimulation times indicated by numbers. B, as in A, but at 2 Hz stimulation frequency. Vertical bars indicate S.E.M., n= 16 in both genotypes. Horizontal, filled bars along the abscissa indicate P < 0.05 when comparing the results from the two genotypes.
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Discussion
The present results demonstrate that, when examined in functionally intact synapses in adult rodent brain, synapsins I and II are necessary for short-term modulation of synaptic transmission as observed during the early synaptic responses elicited by a train of afferent stimulation. Some of the changes observed appear to be caused by modulatory effects on vesicles already present in the RRP, while others may be caused by changes in the recruitment of reserve vesicles, previously believed to be the main effect of synapsin proteins (Pieribone et al. 1995; Rosahl et al. 1995; Chi et al. 2001, 2003). In order to restrict the responses to the RRP, we have followed previous work which indicated that the RRP in these synapses will be emptied during electrophysiological experiments after 15 stimuli given at 10 Hz stimulations in 4 mM Ca2+ (Dobrunz & Stevens, 1997) or after 60 stimuli given at 20 Hz stimulations in 2.6 mM Ca2+ (Wesseling & Lo, 2002), while chemically induced exocytosis will empty the RRP during 2–3 s (Kumashiro et al. 2005; Ashton & Ushkaryov, 2005). Although age of animals, type of preparation and incubation temperatures may be different, these reports indicate that stimulatory trains lasting between 1.5 and 3 s would preferentially represent transmission deriving from the RRP. Our analysis, which indicates that responses during the initial 2 s derive from the RRP, irrespective of frequency, is in general agreement with these authors.
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Furthermore, we show that the synapsin I and II proteins can have different effects on RRP release in distinct synapses. In the CA3-to-CA1 synapse, which represents a prototypical small, facilitating excitatory CNS synapse, these include a synapsin I and II-dependent decrease in response to low (2 Hz) stimulation frequency at normal [Ca2+]o, and enhancing effects at a higher (20 Hz) stimulation frequency. In contrast, the medial perforant path-to-granule cell synapse represents a prototypical small, depressing excitatory CNS synapse, with high release probability and characterized by paired-pulse depression (McNaughton, 1980; Rosahl et al. 1993; Dobrunz & Stevens, 1997). Since these depressions were consistently smaller in the wild-type than in the DKO genotype, irrespective of low or high stimulation frequencies, the effects of synapsin I and II proteins in this synapse were strictly enhancing, as previously described in other synapses (Li et al. 1995; Rosahl et al. 1995; Pieribone et al. 1995; Hilfiker et al. 1998). These data are in contrast to the unidirectional enhancing effects recently observed in hippocampal neurones cultured from synapsin I, II and III triple knock-out mice (Gitler et al. 2004). Hence, it is possible that the dynamic, uni- and/or bi-directional synapsin effects on early release that we see remain restricted to adult, intact CNS synapses.
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Taken together, our results show that synapsins I and II can induce both decreases and enhancements of synaptic responses deriving from the initial stimulations in a stimulation train, with the direction of changes partly controlled by stimulation frequencies and levels of [Ca2+]o, and partly by the identity of the synapses studied. The mechanisms behind these distinct effects are not clear. Previous studies have generally concluded that the absence of synapsins decreases recruitment from reserve vesicles, with a profound change in the organization of the vesicle clusters partly being responsible (Pieribone et al. 1995; Rosahl et al. 1995; Walaas et al. 2000; Chi et al. 2001, 2003; Gitler et al. 2004). However, the present experimental protocol demonstrates that synapsin-dependent changes in synaptic response occur already during the initial stimuli, which would be difficult to reconcile with a recruitment from vesicle clusters, the latter apparently necessitating minimally 2–3 s stimulation (Wesseling & Lo, 2002; Kumashiro et al. 2005). Rather, dynamic molecular responses occurring in the releasable vesicle pool within the first few seconds may be responsible. Such changes would be consistent with dynamic biochemical effects on synapsins. Since administration of dephospho-synapsin into axons can decrease evoked synaptic transmission (Hackett et al. 1990; Llinas et al. 1991), stimulus-induced changes in synapsin phosphorylation may be involved (Schiebler et al. 1986; Hosaka et al. 1999; Jovanovic et al. 2001; Fiumara et al. 2001; Chi et al. 2003). Recent experiments have also indicated that prevention of protein phosphorylation by means of broad spectrum protein kinase inhibitors can mimic physiological effects of synapsin gene inactivation (. Hvalby, V. Jensen & S. I. Walaas, unpublished observations). However, to what degree such inhibitors work through modulation of synapsin phosphorylation remains unclear (Spillane et al. 1995; Samigullin et al. 2004). Recent studies have also indicated that distinct domains in the synapsin proteins may have enhancing or inhibiting effects on synaptic transmission, some of which are mediated by actin- and/or phosphorylation-dependent mechanisms (Hilfiker et al. 2005). Whether the synapsin I and II effects described here are dependent on such mechanisms is an interesting subject for further study.
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In conclusion, the results from this study indicate that synapsins I and II are necessary for full expression of modulatory effects on synaptic transmission originating from the readily releasable vesicles in various types of excitatory vertebrate neurones. Such results increase the possible functional importance and diversity of the synapsin proteins during synaptic transmission at vertebrate synapses.
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