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. 2007 Jun 14;583(Pt 1):129–143. doi: 10.1113/jphysiol.2007.131300

A delayed response enhancement during hippocampal presynaptic plasticity in mice

Vidar Jensen 1,2, S Ivar Walaas 1,3, Sabine Hilfiker 4, Arnaud Ruiz 5, Øivind Hvalby 1,2
PMCID: PMC2277251  PMID: 17569738

Abstract

High frequency afferent stimulation of chemical synapses often induces short-term increases in synaptic efficacy, due to increased release probability and/or increased supply of readily releasable synaptic vesicles. This may be followed by synaptic depression, often caused by vesicle depletion. We here describe an additional, novel type of delayed and transient response enhancement phase which occurred during prolonged stimulation at 5–20 Hz frequency of excitatory glutamatergic synapses in slices from the adult mouse CA1 hippocampal region. This second enhancement phase, which was most clearly defined at physiological temperatures and essentially absent at 24°C, was dependent on the presence of F-actin filaments and synapsins I and/or II, and could not be ascribed to changes in presynaptic action potentials, inhibitory neurotransmission or glutamate receptor desensitization. Time course studies showed that the delayed response phase interrupted the synaptic decay 3–4 s after stimulus train initiation and continued, when examined at 5–10 Hz frequencies, for approximately 75 stimuli before decay. The novel response enhancement, probably deriving from a restricted pool of synaptic vesicles, may allow maintenance of synaptic efficacy during prolonged periods of excitatory synaptic activity.

Neuronal information processing in the nervous system is profoundly influenced by changes in synaptic efficacy. Presynaptic modulation, which occurs during ongoing synaptic transmission and results in changes in transmitter release, may be mediated by changes in release probability and/or in recruitment of vesicles into the readily releasable vesicle pool (RRP) (Zucker & Regehr, 2002; Südhof, 2004; Schweizer & Ryan, 2006). Such changes often lead to an initial, short-lasting response enhancement (Magleby & Zengel, 1982; Dobrunz & Stevens, 1997), the presence of which is regulated, inter alia, by stimulation frequency, levels of [Ca2+]o and several synaptic proteins (see, e.g. Rosenmund et al. 2002; Sippy et al. 2003; Junge et al. 2004; Schlüter et al. 2006; Hvalby et al. 2006). This is usually followed by a monotonous decay, presumably due to decreased recruitment of vesicles from compartments of recruitable transmitter quanta (Wesseling & Lo, 2002). Previous work has indicated that the kinetics of this response decay, which appears to be sensitive to neuronal activity (Stevens & Wesseling, 1998), may be dependent on several vesicle-associated proteins, inter alia the GTP-binding rab3A protein (Geppert et al. 1994), the actin- and vesicle-binding synapsin proteins (Pieribone et al. 1995; Rosahl et al. 1995; Hilfiker et al. 2005; Sun et al. 2006), and Ca2+- and calmodulin-dependent protein kinases (Ryan, 1999; Hinds et al. 2003). Other studies have indicated that regulation of voltage-dependent Ca2+ channels and Ca2+-extrusion mechanisms may modulate synaptic depression by changing [Ca2+]i during stimulus trains (Regehr & Stevens, 2001; Zucker & Regehr, 2002). Variable efficacy of endocytic recycling of synaptic vesicles (Lee & Camilli, 2002; Evergren et al. 2007) may also modulate response magnitudes during trains. The decay usually leads to a steady state situation, the latter presumably representing an equilibrium between vesicle exocytosis and vesicle replenishment (Wesseling & Lo, 2002; Zucker & Regehr, 2002; Fernández-Alfonso & Ryan, 2006).

During studies on CA3-to-CA1 synapses in hippocampal slices from adult mice, we have observed a novel delayed response enhancement phase which we designate DRE and which displays a set of unique properties. Our results indicate that previous analyses have given an incomplete description of short-term plasticity in these synapses.

Methods

Preparation of slices

Experiments were performed on hippocampal slices (Hvalby et al. 2006) prepared from either wild-type mice or synapsin I and II double knock-out mice (DKO) (Ferreira et al. 1998); the animals were adult (3–6 months old). They were killed in a glass container (3 l) containing the general anaesthetic Suprane (Baxter, 10 ml). Following circulatory arrest, the brains were removed and 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 or 2 CaCl2, 26 NaHCO3 and 12 glucose. Slices were placed in a humidified interface chamber where the temperature was kept constant at 24°C, 29°C or 37°C. To avoid N-methyl-d-aspartic acid (NMDA) receptor-mediated synaptic plasticity, 50 μm dl-2-amino-5-phosphopentanoic acid (AP5; Sigma-Aldrich) was present throughout all experiments. To avoid contamination by the field excitatory post synaptic potentials (fEPSP) in experiments where we measured the amplitude of the compound action potential (prevolley, fibre volley), the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor-mediated responses were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 μm; Tocris) or by kynurenic acid (5 mm, Fluka).

The effects of cyclothiazide (100 μm; Sigma-Aldrich), Saclofen and Baclofen (both at 50 μm; Sigma-Aldrich), bicuculline methochloride (10 μm, Sigma-Aldrich), jasplakinolide (1 μm; Invitrogen) or cytochalasin B (20 μm; Sigma-Aldrich) were analysed at 29°C following incubation of the slices with the drugs for at least 90–120 min, a time span which allowed the drugs to induce functional effects (data not shown). When used as a solvent, the final concentrations of DMSO did not exceed 0.3%, which by itself had no effect on physiological responses (not shown). Separate experiments also confirmed that slices treated with cytochalasin B behaved similarly to those where stimulus-dependent actin cycling (Bernstein & Bamburg, 1989; Bernstein et al. 1998; Sankaranarayanan et al. 2003) had been induced, while treatment with the actin stabilizer jasplakinolide (Bubb et al. 1994) prevented such responses (data not shown).

Animal experiments were conducted according to the Norwegian Animal Welfare Act and the European Union's Directive 86/609/EEC.

Stimulation and recordings

Orthodromic synaptic stimuli (50 μs, < 300 μA, 0.1 Hz) were delivered alternately through two tungsten electrodes, one situated in the stratum radiatum and the other in the stratum oriens (control pathway) of the CA1 region. Extracellular synaptic responses were monitored by two glass electrodes (filled with ACSF) placed in the corresponding synaptic layers. Following the presence of stable synaptic responses in both pathways (0.1 Hz stimulation) for at least 10–15 min, selective repetitive stimulation of the radiatum pathway was performed at higher frequencies for a defined period of time.

Alternatively, in one set of experiments, the patch-clamp technique (current clamp) was used to record synaptic responses from single CA1 pyramidal cells (mice aged P36–P39). 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

The synaptic strength was assessed by measuring the maximal slope of the rising phase (V s−1) of the fEPSPs or the intracellular EPSPs, and normalizing the value of each response to the mean value recorded 1 min prior to the switch to a higher stimulation frequency. The maximal amplitude of the presynaptic fibre volley was measured by a similar approach. Early experiments indicated that while prolonged incubation of hippocampal slices with most of the pharmacological agents described led to stable responses, this was not the case with cytochalasin B, where a continuous decline was seen, in agreement with previous work (Kim & Lisman, 1999). In cytochalasin B-treated slices, we therefore first examined baseline fEPSP responses obtained during the last 5 min prior to and 10–15 min after application of the 20 Hz train in the stratum radiatum synapses, and compared them to responses obtained simultaneously in the control stratum oriens synapses. These experiments showed that both sets of synapses showed similar decreases in baseline responses during the experimental period (data not shown, difference between fEPSPs in stratum oriens and stratum radiatum was not significant, P = 0.65).

For every experiment, the paired pulse facilitation (PPF) ratio (50 ms interstimulus interval) was calculated as fEPSP2 slope/fEPSP1 slope, i.e. the value of the second response in the stimulation train was divided by the value of the first response. Three distinct time points were also used to characterize the time courses of the processes studied, i.e. a, the time needed to reach the maximal magnitude of the initial frequency facilitation; b, the transition point where the minimal value of the response during the subsequent decay period was observed; and c, the time point of the maximal value reached during the DRE phase (Fig. 1B). Time points were determined in each of the similarly treated experiments and data were pooled. Values are presented as means ±s.e.m., and statistical significance of differences was evaluated using Student's two-tailed, paired or unpaired t test.

Figure 1.

Figure 1

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A delayed response enhancement (DRE) at 20 Hz stimulation in hippocampal excitatory CA3-to-CA1 synapses A, normalized fEPSP slope measurements during afferent stimulation in stratum radiatum at 0.1 Hz, followed by 20 Hz for 60 s (indicated by black bar, note difference in time scales), and reversal to 0.1 Hz, obtained in an experiment performed at 29°C during NMDA receptor blockade (see Methods). The inset shows superimposed synaptic responses at the stimulation times indicated by arrows and numbers. B, normalized and pooled fEPSP slope measurements during the initial 30 s of 20 Hz stimulation (boxed area in A). Red circles indicate experimental results obtained at 29°C (n = 22); blue indicates a simulated second-order exponential decay based on the normalized and pooled fEPSP slope measurements from response number 20 to response number 1180; green indicates a similar decay based on measurements from stimulus number 20–40 and 1160–1180 (see Methods). a, the time point of the maximum magnitude of the initial frequency facilitation; b, time to the transition point between the initial frequency facilitation and the DRE; c, time needed to reach the peak of the DRE. The inset graph shows a comparison of normalized and pooled intracellular EPSP slope measurements from CA1 pyramidal cell recordings (blue circles, n = 17) and fEPSP data (red circles, n = 31), both obtained during 20 Hz stimulation at 29°C in slices from mice at postnatal day 36–39. The sweeps represent superimposed intracellular responses at the stimulation times indicated by arrows and numbers. Vertical bars indicate s.e.m.

In order to compare the extent to which the decay of the responses resembled second order response decays during the stimulus trains, in some of the experimental series we constructed a curve by employing a second order exponential decay (y =y0 + A1e−x/t1 + A2e−x/t2), based on the mean maximal slope measurements from stimulus number 20–40 and 1160–1180 (fitted by nonlinear regression, Origin 7.5).

Results

A delayed response enhancement (DRE) in CA3-to-CA1 synapses

Low frequency stimulation (0.1 Hz at 29°C) of the afferent fibres in the hippocampal CA1 region (stratum radiatum) in slices from wild-type mouse led to stable synaptic responses. When altered for 60 s by increasing stimulation frequency to 20 Hz, the synaptic efficacy changed, as indicated by a frequency-dependent enhancement followed by a rapid and discontinuous decay. Upon reversal to 0.1 Hz stimulation, the depressed responses reversed to baseline fEPSP values within a minute (Fig. 1A).

When a series of similar experiments, performed under standard conditions (20 Hz, 2 mm CaCl2, 29°C), were normalized and pooled, analysis of the responses observed during the first 30 s of the 20 Hz stimulation train showed that the response enhancements could be clearly separated into two parts (Fig. 1B, a and c). The early frequency facilitation comprised a rising phase which at 20 Hz reached a peak after approximately 12 stimuli (Table 1B) and was followed by a steep response decrease. Approximately 67 stimuli after train initiation, this phase was interrupted at a transition point (Table 1B, Fig. 1b) by a delayed response enhancement (DRE), which reached a peak magnitude 116 stimuli after train initiation (Table 1BFig. 1B,c). The response curve then reverted to a slowly decaying phase, which approximately 20 s after stimulus train initiation had decreased to 50% of the initial baseline level (Fig. 1B).

Table 1.

Characterization of response transformations during ongoing stimulation of CA1 synapses in mouse hippocampus

n a b c b – a c – b PPF ratio
A. Time (s)
    2 mm Ca2+ 20 Hz 22 0.62 ± 0.07 3.34 ± 0.06 5.81 ± 0.12 2.72 ± 0.08 2.47 ± 0.12 1.52 ± 0.05
    1 mm Ca2+ 20 Hz 12 0.73 ± 0.10 3.35 ± 0.13 5.99 ± 0.37 2.62 ± 0.13 2.64 ± 0.39 1.87 ± 0.20*
    4 mm Ca2+ 20 Hz 11 0.32 ± 0.07* 4.42 ± 0.19* 5.19 ± 0.23* 4.10 ± 0.22* 0.77 ± 0.13* 1.29 ± 0.07*
    2 mm Ca2+ 20 Hz DKO 18 0.55 ± 0.07 n.a. — — — 1.51 ± 0.06
    2 mm Ca2+ 20 Hz 37°C 24 0.86 ± 0.06* 2.70 ± 0.11* 4.45 ± 0.18* 1.90 ± 0.13* 1.72 ± 0.19* 1.48 ± 0.05
    2 mm Ca2+ 20 Hz 24°C 24 0.19 ± 0.03* n.a. — — — 1.59 ± 0.05
    2 mm Ca2+ 20 Hz saclofen 19 0.74 ± 0.09 3.38 ± 0.21 5.75 ± 0.27 2.64 ± 0.17 2.40 ± 0.26 1.40 ± 0.04
    2 mm Ca2+ 20 Hz baclofen 16 0.78 ± 0.09 4.18 ± 0.19* 6.38 ± 0.27* 3.40 ± 0.16* 2.20 ± 0.35 1.74 ± 0.10*
    2 mm Ca2+ 20 Hz bicuculline 21 0.83 ± 0.06* 3.49 ± 0.06 5.95 ± 0.16 2.66 ± 0.07 2.47 ± 0.18 1.36 ± 0.03
    2 mm Ca2+ 20 Hz cyclothiazide 18 0.59 ± 0.07 3.40 ± 0.14 5.77 ± 0.28 2.81 ± 0.16 2.37 ± 0.27 1.56 ± 0.08
    2 mm Ca2+ 20 Hz jasplakinolide 19 0.38 ± 0.05* 4.05 ± 0.07* 5.11 ± 0.15* 3.67 ± 0.08* 1.06 ± 0.15* 1.75 ± 0.07*
    2 mm Ca2+ 20 Hz cytochalasin B 14 1.07 ± 0.10* n.a. — — — 1.43 ± 0.06
    2 mm Ca2+ 10 Hz 20 0.79 ± 0.11 3.35 ± 0.16 10.36 ± 0.49* 2.63 ± 0.20 6.98 ± 0.55* —
    2 mm Ca2+ 5 Hz 12 0.98 ± 0.12* 3.30 ± 0.26 18.30 ± 1.94* 2.32 ± 0.29 15.00 ± 2.01* —
B. Stimulus number
    2 mm Ca2+ 20 Hz 22 12.45 ± 1.39 66.82 ± 1.15 116.18 ± 2.39 54.36 ± 1.66 49.36 ± 2.43 —
    2 mm Ca2+ 10 Hz 20 7.90 ± 1.11* 34.45 ± 1.63* 103.55 ± 4.91* 26.32 ± 2.03* 69.79 ± 5.47* —
    2 mm Ca2+ 5 Hz 12 4.92 ± 0.62* 16.50 ± 1.31* 91.50 ± 9.71* 11.58 ± 1.46* 75.00 ± 10.04* —
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A, time needed from stimulus initiation to reach: a, the peak magnitude of the initial frequency facilitation; b, the transition point between the initial frequency facilitation and DRE; and c, peak of the DRE. Subtractions (b – a) and (c – b) indicate the durations of the decaying phase of the initial frequency facilitation and the ascending parts of DRE, respectively. PPF ratio: pooled paired-pulse facilitation ratios (see Methods). Experiments were performed under the conditions indicated, and the results represent seconds (mean ±s.e.m.), derived from n experiments.

*

P < 0.05, compared to standard conditions (2 mm Ca2+, 20 Hz, 29°C). n.a., not applicable. B, number of stimuli (mean ±s.e.m.) needed to reach the time points and the intervals described in A. n indicates the number of experiments. *P < 0.05, compared to standard conditions (2 mm Ca2+, 20 Hz, 29°C).

Intracellular EPSPs obtained from the same preparation taken from younger animals (36–39 postnatal days) showed a pattern which was generally similar to the behaviour of the fEPSPs obtained at the same age (Fig. 1B, inset). Furthermore, the DRE was not restricted to a single species because we observed a similar response pattern under the same experimental conditions (20 Hz, 1 min, n = 24) in hippocampal slices from adult WKY rats (results not shown).

Previous studies have indicated that synaptic release during prolonged stimulation at the CA3-to-CA1 synapse can be described as originating from a two-compartment model, presumably representing the readily releasable vesicle pool (RRP) and a subsequent recruitment from reserve vesicles (Dobrunz & Stevens, 1997; Wesseling & Lo, 2002). In order to study the kinetics of the response changes, we fitted a second order decay to the whole curve based on mean fEPSP values (Fig. 1B, blue curved line). This approach, however, gave a fit which markedly deviated from the experimental data, indicating that the second order exponential curve fitting did not give acceptable approximations to the physiological situation. In order to avoid the DRE phase, we next fitted a decay function based on the mean maximal slope measurements from stimulus number 20–40 and from 1160 to 1180, and obtained a curve (Fig. 1B, green curved line) more in accordance with earlier reports (e.g. Wesseling & Lo, 2002). Individual curve fitting was attempted with the latter approach, but was discarded because the time window for the decay phase following frequency facilitation was too short to allow acceptable curve fitting in most tracings. The DRE phase was therefore further characterized without employing curve modelling.

Locus of DRE

The transient delayed response enhancement could reside pre- and/or postsynaptically. To elucidate the locus of DRE we examined mice devoid of synapsin I and II proteins (double knock-out mice; DKO), which are exclusively presynaptically localized (Camilli et al. 1990; Rosahl et al. 1995; Hilfiker et al. 1999). Comparison of slices from both genotypes subjected to a 20 Hz stimulation train showed that the peak magnitude of the initial frequency facilitation only showed a minor decrease in the DKO (Hvalby et al. 2006). Furthermore, the time point at which the peak occurred was not significantly different from wild-type mice (P = 0.48) (Table 1A). In contrast, the DRE phase was absent in the DKO mice (Fig. 2A), strongly supporting a presynaptic locus for this response.

Figure 2.

Figure 2

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The DRE depends on the presence of presynaptic synapsin molecules A, effects of the absence of synapsin I/II on fEPSP slope measurements in the CA3-to-CA1 synapse during 20 Hz stimulation. Experiments were performed on slices from synapsin I/II double knock-out mice (blue circles, n = 18). Red trace indicates a simulated second order exponential decay based on the normalized and pooled fEPSP slopes from stimulus number 20–40 and 1160–1180 (see Methods). The inset shows superimposed synaptic responses at the stimulation times indicated by arrows and numbers. Vertical bars indicate s.e.m.B, normalized and pooled presynaptic volley amplitudes as a function of time during 20 Hz stimulation in wild-type (red circles, n = 17) and synapsin DKO (blue circles, n = 13) mice. The prevolley amplitudes were binned in groups of two, and the experiments were done either in the presence of 50 μm APV and 10 μm CNQX, or in the presence of 5 mm kynurenic acid. The inset shows superimposed prevolley responses in slices from wild-type (left) or DKO mice (right) at the stimulation times indicated by arrows and numbers. Vertical bars indicate s.e.m.

Recent studies showed a decrease in presynaptic volley amplitude during a 4 s continuous stimulation at 20 Hz of the CA3-to-CA1 afferent fibres (Hvalby et al. 2006). Such release-independent depression of presynaptic function, presumably caused by failures in the generation or propagation of presynaptic action potentials, may contribute to synaptic depression (Muñoz-Cuevas et al. 2004). Figure 2B shows that the decrease in the maximum prevolley amplitude (red circles; n = 17) continued beyond the initial 4 s time point, reaching a 50% reduction at 20 s. It thus appears possible that the reduced synaptic transmission following the initial frequency facilitation could, at least partly, be caused by decreases in prevolley amplitude. The prevolley shape during the stimulus train indicated, however, that amplitude decreases also were accompanied by increases both in the latency to maximum amplitude and in the half-width of the prevolley (Fig. 2B, inset). This makes it unclear to what extent the observed changes in prevolley during a stimulation train would be directly translated into changes in transmitter release.

We also analysed to what extent the prevolley was dependent on the synapsin I/II proteins. Figure 2B shows that the time course of the changes in prevolley amplitude seen in the DKO mice (blue circles; n = 13) was almost identical to the time course observed in wild-type mice, suggesting that action potential generation and propagation during a stimulation train were not sensitive to synapsin gene expression. Interestingly, the decay of the synaptic responses which followed the initial frequency facilitation in synapsin DKO slices (Fig. 2A) also showed a high degree of dynamic similarity with the decay of the prevolley (Fig. 2B). Hence, the possibility that the synaptic response decay could be mediated, at least in part, by changes in the prevolley (Muñoz-Cuevas et al. 2004) cannot be excluded. However, since the prevolley changes remained similar in the two genotypes during continuous stimulation while the DRE was restricted to wild-type mice, a causal role for prevolley modifications in creating the DRE appears most unlikely.

Effects of actin modulators

The dependence of DRE on synapsin proteins suggests that intact vesicle clusters (Pieribone et al. 1995; Rosahl et al. 1995) may be important for the response enhancement. Although the mechanisms responsible for vesicle recruitment into the RRP, which may derive from both rapidly and slowly recycling pools of vesicles, remain incompletely understood (e.g. Ryan, 1999; Jordan et al. 2005; Gaffield et al. 2006; Tokuoka & Goda, 2006), interactions with actin microfilaments were previously suggested (Greengard et al. 1993; Benfenati et al. 1999; Hilfiker et al. 1999). Early work indicated that synaptic vesicle clusters and actin filaments were bound together in situ by synapsin proteins (Landis et al. 1988; Hirokawa et al. 1989; Greengard et al. 1993) by a mechanism which could be reversed by nerve terminal depolarization and synapsin phosphorylation (Chi et al. 2001). Moreover, stimulus-induced cycles of actin depolymerization–polymerization also occur in nerve terminals together with vesicle endo- and exocytosis (Bernstein et al. 1998; Shupliakov et al. 2002; Trifaro et al. 2002; Bloom et al. 2003; Sankaranarayanan et al. 2003). Finally, the polymerization state of actin per se may modulate transmitter release (Walaas, 1999; Morales et al. 2000; Doussau & Augustine, 2000). Taken together, these data suggest that changes in actin dynamics might be involved in stimulus-induced synaptic plasticity, including the DRE phenomenon. We therefore tested to what extent the response patterns seen in the CA1 synapses were dependent on actin polymerization.

Stabilization of F-actin filaments by preincubation with jasplakinolide (1 μm for 90–120 min) had no significant effects on either the baseline fEPSP slope (96 ± 12%, mean ± s.e.m.; n = 8) or the magnitudes of the responses seen during the 20 Hz train (Fig. 3A), but increased the paired pulse facilitation (PPF) ratio, suggesting a decrease in baseline release probability (Zucker & Regehr, 2002) being induced by F-actin stabilization (Table 1A). Furthermore, small but significant changes in the time course of responses were seen during 20 Hz stimulation trains, with the initial response peak being reached more rapidly, the transition point occurring later and the time period needed to reach the DRE peak being considerably shortened (Table 1A; Fig. 3A and B).

Figure 3.

Figure 3

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The DRE depends on an intact F-actin network A, fEPSP slope measurements from CA3-to-CA1 synapses during 20 Hz stimulation in slices treated with cytochalasin B (20 μm, n = 14, blue circles) or jasplakinolide (1 μm, n = 19, green circles) compared to experiments without these compounds (n = 22, red circles). Coloured horizontal bars along the abscissa, corresponding to the colours of the respective manipulations, indicate P < 0.05 when compared to the standard situation. Vertical bars indicate s.e.m. The inset shows simulated exponential decays based on the normalized and pooled fEPSP slope measurements from response number 30 to response number 1180 in slices from wild-type mice exposed to cytochalasin B (blue curved line) compared to slices from DKO mice in normal solution (black curved line; see Fig. 2A). Their respective time constants of decay are indicated. Ba, the time point of the maximum magnitude of the initial frequency facilitation (as indicated with arrow in A and with a colour code as in A); b, time to the transition point between the initial frequency facilitation and the DRE; c, time needed to reach the peak of the DRE. Vertical bars indicate s.e.m. *P < 0.05 when compared to the control situation.

In contrast, destabilization of F-actin filaments (Cooper, 1987) by preincubation of the slices with cytochalasin B (20 μm for 120 min) decreased the baseline fEPSP slope to 78 ± 7% of control (n = 11), similar to what has previously been reported (Kim & Lisman, 1999). This occurred without changes in the PPF (Table 1 A), arguing against a change in presynaptic release probability being responsible. Since a stable equilibrium was not reached during this period and the baseline fEPSP responses rather continued to decline as a function of time, we normalized the responses obtained by 20 Hz stimulations in the stratum radiatum in the presence of cytochalasin B to the baseline fEPSP obtained just prior to the stimulation train (see Methods). Using this approach, we found that cytochalasin B drastically changed the shape of the response patterns during the train (Fig. 3A). This change included a significant delay in the initial frequency facilitation peak (Table 1A; Fig. 3B), and, following this, a complete disappearance of both the subsequent steep decay, the interrupting transition point and the rising phase of the DRE. The response phases now rather merged into a single, more slowly decaying phase. Hence, disruption of F-actin with cytochalasin B had major effects on both the early response decay and the DRE phase.

The response pattern which followed cytochalasin B-induced actin destabilization indicated that vesicle exocytosis and/or vesicle trafficking barriers could have been modified by this treatment. Since endocytotic vesicle recruitment also appears to be dependent on actin and synapsin proteins (Shupliakov et al. 2002; Bloom et al. 2003; Evergreen et al. 2007), we compared the effects induced by actin modulation to those induced by inactivation of the synapsin I/II genes (Ferreira et al. 1998). For this purpose, we fitted decay curves based on the mean response values from stimulus number 30–1180, obtained either in wild-type animals in the presence of cytochalasin B or in synapsin I/II-deficient mice in the absence of cytochalasin B (Fig. 3A inset, blue and black curved lines, respectively). The response decay found in the synapsin DKO preparations showed a good fit to a double exponential decay, with fast and slow time constants of 1.37 s and 8.64 s, respectively, both of which fit well with a two-compartment model (Wesseling & Lo, 2002). In contrast, the response decay seen in the presence of cytochalasin B gave a good fit to a single exponential decay, with a time constant of 6.25 s. These data appear consistent with the interpretation that, while the absence of synapsins I/II affected neither the rapid nor the slow response decays, destabilization of F-actin with cytochalasin B selectively removed the initial, rapid response decay, but left the putative biological barrier(s) involved in the slow component of vesicle recruitment decay essentially intact.

Effects of temperature

We also observed strong temperature effects. Although PPF ratios were unchanged at both 24°C and 37°C when compared to control (Table 1A), indicating that baseline release probabilities were not strongly temperature dependent (Pyott & Rosenmund, 2002), at 24°C both the response magnitude and the time needed to reach the early peak were significantly decreased (Table 1A, Fig. 4A and B) and the subsequent DRE was essentially obliterated. In contrast, at 37°C the initial enhancement peak showed an unchanged magnitude, but a slower rise to the maximum level (Table 1A) when compared to that obtained at control temperature (Fig. 4A and B). However, this changed approximately 2.7 s after train initiation, from which time point the remaining responses magnitudes were significantly increased (Fig. 4A). Moreover, the responses also became accelerated, so that both the transition point and the peak of the DRE occurred significantly earlier at 37°C than at control temperature (Fig. 4A and B, Table 1A).

Figure 4.

Figure 4

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Modulation of the DRE in excitatory CA3-to-CA1 synapses by temperature and [Ca2+]o A, normalized and pooled experiments (20 Hz, 60 s, 2 mm CaCl2) as described in Figure 1. Results were obtained at 37°C (blue circles, n = 24), 29°C (red circles, n = 22) and 24°C (green circles, n = 24). Coloured horizontal bars along the abscissa, corresponding to the colours of the respective curves, indicate P < 0.05 when compared to fEPSP slope values at 29°C. Ba, the time point of the maximum magnitude of the initial frequency facilitation (as indicated with arrow and with a colour code as in A); b, time to the transition point between the initial frequency facilitation and the DRE; c, time needed to reach the peak of the DRE. Bars indicate s.e.m. *P < 0.05 when compared to the control situation. C, experiments (20 Hz, 60 s, 29°C) done after equilibration to different extracellular concentrations of [Ca2+]o. The results are presented relative to fEPSP slope values at 2 mm CaCl2. Coloured horizontal bars along the abscissa corresponding to the respective curves indicate P < 0.05 when compared to fEPSP slope values at 2 mm CaCl2. Green circles, 1 mm (n = 12), red circles, 2 mm (n = 22), and blue circles, 4 mm CaCl2 (n = 11), respectively. D, as B, but the coloured bars and analytical results correspond to the curves in different [Ca2+]o as shown in C. Bars indicate s.e.m. *P < 0.05 when compared to the results at 2 mm CaCl2. E, subtraction of the values obtained at 1 mm (green circles) and 4 mm CaCl2 (blue circles) from those obtained at 2 mm CaCl2.

Effects of [Ca2+]o

The effects of different [Ca2+]o levels on DRE were examined by equilibrating the slices in ACSF medium containing variable concentrations of CaCl2 (Hvalby et al. 2006). At 1 mm CaCl2, this treatment led to a decrease (49 ± 10% of control, n = 12) and at 4 mm CaCl2 to an increase (125 ± 4% of control, n = 19) of baseline fEPSP slope. Moreover, these treatments induced significant effects on PPF ratios (Table 1A), supporting the expected [Ca2+]o-mediated changes of release probability (Dobrunz & Stevens, 1997; Zucker & Regehr, 2002). To exclude major changes in axonal excitability, we also measured the maximum prevolley amplitude while keeping levels of [Mg2+]o constant (2 mm). Under these conditions, the prevolley amplitudes were unchanged at both 1 mm (105 ± 9%; n = 5) and 4 mm CaCl2 (97 ± 10%; n = 5), indicating that changes in [Ca2+]o led to major changes in synaptic release probability, but not in axonal excitability.

Interestingly, the initial frequency enhancement and decay periods showed distinct response patterns caused by changes in [Ca2+]o. As shown in Table 1A, during incubation at 4 mm CaCl2 the peak frequency facilitation response was reached significantly earlier while the decay period needed to reach the transition time point was significantly prolonged when compared to control. In contrast, at 4 mm CaCl2 the time needed to reach the maximum DRE magnitude was shorter than in the control (Fig. 4C and D, Table 1). Thus, whereas the response time patterns were similar at 1 mm and 2 mm CaCl2, at 4 mm CaCl2 the time parameters deviated significantly from the control situation, and rather showed a pattern which was similar to that obtained in the presence of jasplakinolide (Table 1A, Fig. 3).

To directly determine the impact of [Ca2+]o on the responses obtained during a 20 Hz stimulus train, we subtracted the results obtained at either 1 mm or 4 mm CaCl2 from results obtained during control conditions. Figure 4E shows that changing from 1 mm to 2 mm CaCl2 resulted in several distinct Ca2+-dependent responses during the stimulus train (green circles). These changes included an apparent doubling of the fEPSP slope during baseline equilibration (Hvalby et al. 2006) which was followed by a further increase during the frequency facilitation. In contrast, during the subsequent decay phase the differences between responses occurring at 1 mm and 2 mm CaCl2 decreased until the Ca2+-dependent effect represented not more than approximately 15% of baseline fEPSP. This effect remained constant until approximately 10 s after train initiation, thus including most of the DRE phase. It then decreased further until the effect of changing CaCl2 levels essentially disappeared after approximately 17–18 s (Fig. 4E).

In contrast, when changing from 2 mm to 4 mm CaCl2, the significant 25% increase in the baseline fEPSP which was induced during equilibration at high Ca2+ levels (Hvalby et al. 2006) was followed by an immediate response decay during the 20 Hz stimulus train. After approximately 12 stimuli, responses had decreased to below control levels, and this effect lasted for the remaining period of the train. Hence, when switching to 4 mm Ca2+, increases in synaptic strength were restricted to the initial equilibration phase, and were absent during the 20 Hz train (Fig. 4E). Taken together, these data suggest that a number of vesicle pools and/or replenishment processes with distinct Ca2+-dependencies were recruited during 20 Hz stimulation trains.

Effects of stimulation frequency

Application of variable stimulation frequencies led to distinct responses during the initial response enhancement (Hvalby et al. 2006). The magnitudes of the early peaks obtained at stimulation frequencies of 5 Hz and 10 Hz were both below the magnitudes seen at 20 Hz (Fig. 5A). Furthermore, the time needed to reach the initial peak was significantly prolonged at 5 Hz, but not at 10 Hz compared to control (Fig. 5B, Table 1A). In contrast, the times needed to reach the transition points were essentially similar at all frequencies tested (Table 1A), while the shapes of the subsequent DRE phase showed major frequency-dependent differences when examined as a function of time (Fig. 5A). Compared to the results obtained under standard conditions, the duration of DRE at 10 Hz was almost three times and at 5 Hz around six times the control values (Fig. 5B; Table 1A). When the DRE responses were analysed as a function of stimulus number, the latter differences disappeared (Fig. 5C and D, Table 1B), and the duration of the ascending part of the DRE was found to be maximal at both 5 Hz and 10 Hz, in both cases representing 70–75 stimuli. In contrast, at 20 Hz the ascending part of the DRE represented approximately 50 responses, and this was further decreased in the presence of 4 mm CaCl2, jasplakinolide or during incubation at 37°C (Table 1A and B). These data suggest that optimal numbers of synapsin-dependent DRE vesicles might be recruited into the RRP by a mechanism which predominantly depends on the stimulus frequency and polymerization state of F-actin.

Figure 5.

Figure 5

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Frequency modulation of the DRE in excitatory CA3-to-CA1 synapses A, normalized and pooled experiments (29°C, 2 mm CaCl2) as described in Fig. 1B. Results were obtained at a stimulation frequency of 20 Hz (red circles, n = 22), 10 Hz (blue circles, n = 20) and 5 Hz (green circles, n = 12). Coloured horizontal bars along the abscissa corresponding to the respective curves indicate P < 0.05 when compared to 20 Hz stimulation. Ba, the time point of the maximum magnitude of the initial frequency facilitation (as indicated with arrow and with a colour code as in A); b, time to the transition point between the initial frequency facilitation and the DRE; c, time needed to reach the peak of the DRE. Vertical bars indicate s.e.m. *P < 0.05 when compared to the control situation. C and D same as in A and B, but presented as a function of stimulus number. Bars indicate s.e.m. *P < 0.05 when compared to the results at 20 Hz.

The subsequent decaying phases of the delayed stimulus-induced response patterns also showed frequency dependencies, with the response magnitudes obtained during continuous stimulation being directly related to temperature and inversely related to stimulation frequencies and levels of [Ca2+]o. Stimulation at 20 Hz gave rapid response decays, stimulation at 5 Hz frequency gave slow response decays, and 10 Hz stimulation gave intermediate response decays (Figs 5A and C).

The final steady state situations indicated that at 5 Hz, the final response magnitudes were considerably above baseline, at 10 Hz they were approximately similar to baseline, while at 20 Hz they were depressed to approximately 50% of the baseline responses. These frequency data indicate that, following the initial facilitation and the DRE phases, the final response magnitudes occurring during continuous stimulation in these synapses were inversely related to stimulation frequencies and levels of [Ca2+]o, with prolonged stimulation at 10 Hz frequency inducing a steady response (Fernández-Alfonso & Ryan, 2004).

Effects of receptor manipulations

During stimulation of the CA3-to-CA1 fibres in the stratum radiatum, both feed-forward inhibition and eventually recurrent inhibition mediated by GABA will be activated. Therefore, we also examined whether GABAA or GABAB receptors had effects on the response patterns. Experiments done on slices equilibrated with the GABAB-receptor antagonist saclofen (50 μm) showed no significant changes in either the baseline fEPSP slopes (107 ± 12%; n = 5) or the response pattern during 20 Hz stimulation (Fig. 6A and B; Table 1A). GABAB-receptor-mediated presynaptic inhibition (Isaacson & Hille, 1997; Nicoll, 2004) was therefore hardly detectable in this experimental situation. In contrast, addition of the GABAB receptor agonist baclofen (50 μm) significantly decreased both the baseline fEPSP slope (to 34 ± 12%, n = 20; P < 0.05) and the stimulus-evoked responses during the entire 20 Hz train. The general shape of the response phases which occurred, including the DRE, remained essentially intact during GABAB-receptor activation, although close analysis revealed that both the transition point and the DRE peak occurred slightly later than in control (Fig. 6A and B; Table 1A). These data therefore appear to exclude GABAB-receptor-mediated modulations from being involved in the generation of DRE.

Figure 6.

Figure 6

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Neither GABA-mediated receptor modulations nor AMPA receptor desensitization interfere with the shape of the DRE A, normalized and pooled fEPSP slope measurements during 20 Hz stimulation in the presence of the GABAB receptor antagonist saclofen (50 μm, n = 19, blue circles) and the GABAB receptor agonist baclofen (50 μm, n = 16, green circles). The results are presented relative to fEPSP slope values without GABAB receptor interference (n = 22, red circles). Coloured horizontal bars along the abscissa correspond to the respective manipulations and indicate P < 0.05 when compared to the standard situation. Vertical bars indicate s.e.m.Ba, the time point of the maximum magnitude of the initial frequency facilitation (as indicated with arrow and with a colour code as in A); b, time to the transition point between the initial frequency facilitation and the DRE; c, time needed to reach the peak of the DRE. Bars indicate s.e.m. *P < 0.05 when compared to the control situation. C and D, as A and B, but experiments performed on slices treated with bicuculline (10 μm, n = 21, blue circles. E and F, as A and B, but experiments performed on slices treated with cyclothiazide (100 μm, n = 18, blue circles).

Similar experiments performed in the presence of the ionotropic GABAA receptor blocker bicuculline (10 μm; n = 21) also failed to substantiate a major GABAA-receptor mediated effect on the initial frequency facilitation and/or the DRE. Both the general shape of the response curves, the different time points and the durations of the response enhancements and decays were similar to those obtained in the absence of the GABAA-receptor antagonist (Fig. 6C and D; Table 1A).

AMPA-type glutamate receptors, which mediate the fEPSPs examined in this study, are subject to activity-dependent inactivation at high stimulation frequencies (Arai & Lynch, 1998a, b). Transient reversals of such inactivations may conceivably give rise to secondary response enhancements. However, experiments performed in the presence of cyclothiazide (100 μm), an inhibitor of stimulus-induced AMPA receptor desensitization which by itself has small effects on the synaptic potential (Diamond & Jahr, 1995; Arai & Lynch, 1998a), revealed no changes either in the frequency facilitation, the transition point or the DRE during 20 Hz stimulation (Fig. 6E and F; Table 1A). Following the DRE peak, however, there was a small response decrease induced by cyclothiazide during the late decaying phase (Fig. 6E). Despite this, AMPA receptor desensitization clearly did not contribute to the observed response patterns.

Discussion

The present study describes distinct patterns of presynaptic plasticity, including DRE, a novel form of delayed presynaptic short-term response enhancement which was observed in the hippocampal excitatory glutamatergic CA3-to-CA1 stratum radiatum synapse. Application of 5–20 Hz stimulation trains to this synapse resulted in two response enhancements which were separated by a period of rapidly decaying responses and were followed by a slow decay towards apparent equilibrium situations. This multiphasic pattern, which was observed in slices from both mouse and rat hippocampi and presumably was driven by several overlapping types of short-term plasticity (Thomson, 2000; Regehr & Stevens, 2001; Zucker & Regehr, 2002), showed a number of specific properties.

The initial response enhancement, which presumably derives from vesicles already present in the RRP (Junge et al. 2004; Hvalby et al. 2006; Schlüter et al. 2006), was manifested as a rapid frequency facilitation (Salin et al. 1996) with distinct durations and magnitude of the peak response. When preparations were equilibrated either in medium containing 4 mm Ca2+ or the F-actin stabilizer jasplakinolide, or when experiments were performed at low temperature (24°C), the time point of the maximal peak occurred earlier. Conversely, the peak was significantly delayed when examined in the presence of the actin destabilizer cytochalasin B or at higher temperature (37°C). Hence, the actin cytoskeleton may be importantly involved in this regulation. The mechanisms behind these changes, in particular whether they may reflect changes in intrinsic release probability or differences in the size of the RRP, remain uncertain. Previous studies have indicated inverse relations between release probability and paired pulse facilitation under a number of conditions (Dobrunz & Stevens, 1997; Zucker & Regehr, 2002). However, this study shows that treatments which accelerated the initial enhancement could be present together with both reduced, increased and unchanged PPF ratios. Similarly, treatments which led to delays of the initial enhancement could also be observed together with different PPF ratios. These discrepancies suggest that the durations of the initial frequency facilitations are not directly related to either initial release probabilities or the size of the RRP vesicle pool.

The subsequent decaying phase has previously been interpreted as following a depletion of RRP vesicles with compensatory vesicle replenishments originating from functionally and/or structurally distinct reserve or recycling vesicle pools (Dobrunz & Stevens, 1997; Wesseling & Lo, 2002; Schweizer & Ryan, 2006). We characterized this phase by measuring the duration of the decay from the initial peak to the transition point where maximal decay occurred. The only treatment which consistently shortened this decay consisted of increasing the incubation temperature to 37°C, while prolongation of the decay was obtained at 4 mm CaCl2 or in the presence of jasplakinolide (Table 1A). Unexpectedly, under the present experimental conditions the shape of this decay phase was not dependent on the presence of the presynaptic synapsin I/II proteins.

In contrast, destabilization of the F-actin microfilaments with cytochalasin B led to a disappearance of the rapid decay phase, leaving a slow decay which was not interrupted by a transition point. Examination by curve fitting showed that the latter decay curve could be mimicked by a single exponential decay, with a time constant comparable to that observed in the slow decay component of the synapsin DKO preparation. These results suggest that while F-actin is important, neither synapsin I/II-dependent vesicle clusters nor actin–vesicle interactions (Benfenati et al. 1999; Hilfiker et al. 2005) were important in determining the rapid decay kinetics. These results appear to be consistent with the interpretation that a putative functional ‘bottleneck’ responsible for the rapid response decay phase is dependent on the presence of intact actin microfilaments. Although not previously reported in hippocampal presynaptic elements, such F-actin-dependent structures would be functionally similar to the submembranous vesicle–actin barrier which has been shown to be physiologically important in e.g. chromaffin cells (Vitale et al. 1995). Interestingly, recent studies in cultured excitatory hippocampal synapses have indicated that the RRP and the apparent reserve vesicle clusters present in these cells appear not to be in dynamic equilibrium, but rather may represent functionally separate vesicle populations (Gitler et al. 2004).

The unexpected relations between the initial frequency facilitation duration and the subsequent response decays were of further interest. Irrespective of accelerated or retarded frequency facilitations and/or decay phases, the transition time point always occurred approximately 3–4 s after train initiation. The mechanism(s) responsible for such variations in decay durations together with a fixed transition time point remain(s) unclear. It appears, however, possible that the appearance of the fixed transition time point may reflect a mechanism which allows the recruitment of a novel group of quanta into the RRP to occur at a fixed time point, irrespective of the kinetics of the initial response enhancement and subsequent decay periods. Given that Ca2+-sensitive vesicle trafficking may be involved in such recruitment, stimulus-induced changes in the kinetics of Ca2+ channels (McAllister-Williams & Kelly, 1995), Ca2+-extrusion mechanisms (Khanansvili et al. 1995), vesicle motility driven by Ca2+/CaM-dependent protein kinase activities (Greengard et al. 1993; Ryan, 1999; Jordan et al. 2005; Sun et al. 2006; but see Tokuoka & Goda, 2006) and simple vesicle diffusion (Gaffield et al. 2006), all of which may be expected to be sensitive to variations in temperature and stimulation frequency, might be involved in this mechanism.

Following the transition point, the response decays underwent a reversal and led to the DRE phase, which showed a number of distinct characteristics. First, this reversal appeared to be exclusively caused by presynaptic mechanism(s). In support of this statement, our data indicate that synapsin-dependent vesicle clusters were obligatory for the appearance of DRE. Previous studies on synapsin effects, although not showing a delayed response enhancement, have indicated that absence of the synapsins I/II proteins will disrupt vesicle clusters and induce changes in synaptic vesicle density and numbers. Conversely, the presence of synapsin-containing synaptic vesicle clusters appears to counteract synaptic depression (Rosahl et al. 1995; Pieribone et al. 1995; Bogen et al. 2006; Kielland et al. 2006; Sun et al. 2006). Secondly, both the transition point and DRE phase were sensitive to temperature, known to be particularly important for presynaptic functions (Pyott & Rosenmund, 2002; Fernández-Alfonso & Ryan, 2004; Micheva & Smith, 2005; Kushmerick et al. 2006). Indeed, the prominent DRE response which occurred at 37°C was essentially obliterated at room temperature, at which temperature a number of experimental studies have been executed (e.g. Rosahl et al. 1995; Dobrunz & Stevens, 1997, 1999; Stevens & Wesseling, 1998; Wesseling & Lo, 2002).

In addition to the importance of the synapsin proteins, our results also strongly indicate that actin cycling may be essential for the presence of DRE. This statement is based on the observation that, although the DRE peak magnitude appeared to be unchanged, the duration of DRE was strongly decreased by F-actin stabilization. Conversely, F-actin destabilization led to an apparent disappearance of the DRE, and rather gave a novel, single exponential response decay pattern. These data are consistent with the interpretation that disruption of F-actin removed an actin-containing constraint and now led to vesicle recruitment from a large vesicle pool which was functionally merged with the RRP vesicles.

Irrespective of the exact mechanism involved in the apparent transformation of the exo/endocytotic vesicle cycling kinetics (Sankaranarayanan & Ryan, 2001), our data demonstrate that actin-modulating agents and the synapsin I/II proteins were involved. It is known that nerve terminal depolarization directly induces repetitive Ca2+-dependent cycles of actin polymerization and depolymerization on a time scale lasting for seconds (Bernstein & Bamburg, 1989; Bernstein et al. 1998; Sankaranarayanan et al. 2003). Since a variety of actin-modulating presynaptic proteins, including the synapsins, the MARCKS and the dynamin proteins (Camilli et al. 1990; Lee & Camilli, 2002; Calabrese & Halpain, 2005), are subject to depolarization-induced changes in phosphorylation in intact nerve terminals (Wang et al. 1988, 1989), we suggest that stimulus-induced changes in actin polymerization and vesicle trafficking may be partly mediated by stimulus-induced protein phosphorylation mechanisms, which thereby may represent the main factor determining the position of the transition point and the kinetics of the initial part of the subsequent DRE phase.

In contrast to the relatively stable position of the transition time point and start of the DRE phase, the duration of DRE was variable. Since the pool of recycling vesicles in cultured hippocampal synapses comprises approximately 80 vesicles (Ryan, 2001), it is interesting that the mature excitatory hippocampal synapses (Harris & Sultan, 1995; Schikorski & Stevens, 1997) examined in this study responded to stimulation trains with DRE phases which lasted for not more than 70–75 stimuli. Given a release probability of approximately 0.3–0.5 in these synapses (Dobrunz & Stevens, 1997), these results indicate that recruitment of DRE vesicles in the CA3-to-CA1 synapses during stimulation at 5–10 Hz may induce release of close to 50% of the total reserve vesicle pool. In contrast, at 20 Hz stimulation the DRE did not last for more than approximately 50 stimuli, and employing combinations of 20 Hz with either 4 mm CaCl2 or an F-actin stabilizator led to further decreases in the durations of DRE, which now were limited to 15–20 stimuli. These results therefore suggest that the number of releasable quanta originating from DRE vesicles was determined by a mechanism which is not directly dependent on interstimulus intervals or efficacy of Ca2+ removal, but rather on the dynamics of actin cycling and the number of recruitable vesicles present. Irrespective of the mechanisms responsible, our data suggest that the physiologically important 5–10 Hz theta frequency (Hasselmo, 2005) may give optimal duration of release from the DRE phase, while employment of higher [Ca2+]o and/or stimulation frequencies rather may decrease the duration of the DRE phase.

In conclusion, this study indicates that in addition to the well-known stimulus-induced frequency facilitation (Salin et al. 1996; Dobrunz & Stevens, 1997), which derives from the vesicles already present in the RRP, another group of transmitter-containing vesicles is clearly available in these excitatory synapses for generating a delayed response enhancement phase. The latter phase occurred 3–4 s after stimulation initiation, it was dependent on the presence of synapsins I/II proteins and intact F-actin filaments, and it lasted for approximately 75 stimuli under optimal conditions, a number which may reflect the size of the proposed recruitable vesicle pool. Although prolonged action potential trains rarely occur during hippocampal synaptic transmission (Dobrunz & Stevens, 1999), it is of interest that the 5–10 Hz hippocampal theta rhythm, which has been connected to, e.g. spontaneous movements and memory mechanisms (Hasselmo, 2005), is within the optimal frequency range for manifestation of the full extent of the DRE phase. The extent to which this response enhancement is physiologically involved in these synapses therefore represents an interesting subject for further study.

Acknowledgments

We thank P. Greengard and H.T. Kao for the gift of synapsin I and II-depleted animals, and D. Kullmann and P. O. Andersen for comments on an early version of the manuscript. MONERG was supported by the University of Oslo and the Jahre Foundation.

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