Bidirectional synaptic plasticity in response to single or paired pulse activation of NMDA receptors

Neuroscience Research 67 (2010) 108–116

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Bidirectional synaptic plasticity in response to single or paired pulse activation of NMDA receptors Fen-Sheng Huang ∗ , Abdul-Karim Abbas, Rui Li, Dzmitry Afanasenkau, Holger Wigström Department of Medical Biophysics, Institute of Neuroscience and Physiology, University of Gothenburg, Box 433, 40530 Gothenburg, Sweden

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Article history: Received 3 May 2009 Received in revised form 4 February 2010 Accepted 8 February 2010 Available online 16 February 2010 Keywords: Neuronal plasticity Long-term potentiation Long-term depression Synapse NMDA receptor Hippocampal slice

a b s t r a c t It is still incompletely known how NMDA receptors (NMDA-R) regulate bidirectional synaptic plasticity. We examined this issue by an experimental protocol in which paired pulse stimulation (PPS) with 50 ms interstimulus interval and basal frequency of 0.1 Hz was applied to CA1 area of rat hippocampal slices during low Mg2+ perfusion. Under blockade of NMDA-Rs by AP5, PPS for 12–60 min led to only a minor depression. In contrast, when PPS was applied in the absence of AP5, there was a prominent short-term potentiation (STP), mainly of AMPA-R mediated responses, with peak at 1 min and lasting 10–15 min. The STP was followed by a slowly developing long-term depression (LTD). Applying AP5 during the STP, converted it to a stable increase relative to the control pathway. Following peak STP, plasticity was controlled in a composite manner. Whereas the initial decay was counteracted by NMDA-R activation, the following LTD was dependent on such activation. Our data suggest that synaptic changes do not only depend on the instantaneous, NMDA-dependent Ca2+ concentration in the dendritic spine, but are also influenced by prior induction events. In addition to NMDA-R driven processes, passive relaxation contributes to the synaptic plasticity and in some cases outbalances the active control. © 2010 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

1. Introduction Activity-dependent strengthening and weakening of synapses is a central issue for learning and memory at the cellular level. Long-term potentiation (LTP) and long-term depression (LTD) are prominent examples of such plasticity. Both of them generally require NMDA receptor (NMDA-R) activation as an initial step, followed by postsynaptic influx of Ca2+ and triggering of Ca2+ dependent enzymes (Bliss and Collingridge, 1993; Malenka and Bear, 2004). According to an influential hypothesis, the direction of synaptic change is controlled by the induction strength such that small or moderate concentration of NMDA-dependent Ca2+ leads to LTD whereas larger activation causes LTP (Lisman, 1989; Dudek and Bear, 1992; Mulkey and Malenka, 1992). It has also been inferred that subpools of Ca2+ may be involved, considering that subunitspecific NMDA-R blockers affected LTP and LTD in a differential manner (Liu et al., 2004; Massey et al., 2004). Other studies support the idea of a unified triggering of LTP/LTD via a common Ca2+ level in the dendritic spine, independently of NMDA-R subtypes (Berberich et al., 2005; Li et al., 2007). According to the unified calcium model, Ca2+ in the dendritic spine is the controlling factor independently of how Ca2+ enters, considering NMDA-channels as well as several other sources (Artola and Singer, 1993; Shouval et al., 2002). How-

∗ Corresponding author. Tel.: +46 31 7863554; fax: +46 31 7863840. E-mail address: [email protected] (F.-S. Huang).

ever, while there is consensus that Ca2+ level is a main determinant for the direction of synaptic change, it is still unclear to what extent other factors contribute. In addition to the magnitude of the induction signal it has been considered that its temporal character may be involved. This is consistent with commonly used induction protocols, where LTP is typically induced by one or a few second-long high frequency trains of stimuli (Bliss and Collingridge, 1993), whereas LTD is induced by substantially longer stimulus trains, e.g. 10–15 min of lowfrequency stimulation, usually in the Hertz range (Kemp and Bashir, 2001). Nevertheless, it has also been demonstrated that relatively short trains (2–30 s, 10–100 Hz) can induce LTD under conditions of restricted Ca2+ influx (Mulkey and Malenka, 1992; Cummings et al., 1996). In contrast to these evidences for fast induction of LTD, a study with induction under NMDA-R unblocked conditions in Mg2+ -free solution reported that LTD was only induced with stimulation longer than about 3 min (Mizuno et al., 2001). A previous study in our lab examined bidirectional plasticity under such NMDA-R unblocked conditions, using a constant, basal frequency throughout the experiment (Dozmorov et al., 2003). In the present work, we have further elaborated this constant frequency design to explore the temporal character of LTP/LTD induction. A sudden increase in the level of NMDA-R activation was here accomplished by switching from single pulse stimulation (SPS) to paired pulse stimulation (PPS), while keeping slices in a low Mg2+ solution. We found that the direction of synaptic change was substantially influenced by the prior history of the synapse. Moreover, we observed

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contributions from both NMDA-dependent and -independent processes in the control of directionality. 2. Materials and methods 2.1. Hippocampal slice preparation Experiments were performed on Sprague–Dawley rats, 13–22 days old, including both males and females. The rats were sacrificed by decapitation after initial isoflurane anaesthesia. All measures were taken to minimize animal suffering and to reduce the number of animals used. The procedures conformed to the guidelines of the Swedish Council for Laboratory Animals and were approved by the Local Ethics Committee of Gothenburg University. The brain was removed and placed in an ice-cold artificial cerebrospinal fluid (ACSF) composed of (in mM): NaCl 119, KCl 2.5, CaCl2 0.5, MgCl2 6, NaHCO3 26, NaH2 PO4 1 and glucose 10, oxygenated by 95% O2 and 5% CO2 . Transverse hippocampal slices, 400 ␮m thick, were cut using a vibrating tissue slicer (Campden Instruments) and transferred to a holding chamber where they were stored at 22–24 ◦ C (room temperature) for at least 1 h in an ACSF solution similar to that above but with 2 mM CaCl2 . For the electrophysiological experiments, slices were transferred as needed to one or several “submerged type” recording chambers, perfused via an automated system (Biomux) at a rate of 2 ml/min (30–32 ◦ C) by the mentioned ACSF but with the concentration of Mg2+ lowered to 0.1 mM, Ca2+ being kept at 2 mM. The use of low Mg2+ allowed for expression of NMDA-R mediated responses. The perfusion ACSF also contained a low concentration (0.5–1 ␮M) of the AMPA receptor (AMPA-R) antagonist CNQX to partially block AMPA-R mediated responses, leading to a “balanced mixture” of AMPA and NMDA components. Experiments generally commenced in ACSF containing 50 ␮M of the specific NMDA-R antagonist AP5 to block NMDA-R mediated responses; AP5 was later removed, leading to unblock of NMDA-Rs. 2.2. Extracellular recording Field excitatory postsynaptic potentials (fEPSPs) were recorded from the CA1 apical dendritic layer using a glass micropipette; after filling with 1 M NaCl, the pipette resistance was 2–5 M. For all the experiments, 0.1 Hz stimulation was delivered alternately to two monopolar tungsten electrodes, positioned on either side of the recording electrode, providing access to two independent sets of synapses (successive stimuli being separated by 5 s). Pathway independence was verified by showing the absence of heterosynaptic as compared to homosynaptic paired pulse facilitation (PPF), using an interstimulus interval of 50 ms. Explicit calculation of the ratio PPFhetero /PPFhomo yielded an estimated overlap of 4.2 ± 2.0% (n = 12, p > 0.05). Stimulus strengths were initially adjusted for each slice to equalize the synaptic inputs of the two pathways (range 20–60 ␮A). Either single pulse stimulation (SPS) or paired pulse stimulation (PPS, interpulse interval 50 ms) was used. The aim of applying PPS was to quickly increase the level of NMDA-R activation under conditions of unblocked NMDA-Rs during perfusion with AP5-free ACSF. However, the effect of PPS was first tested in the presence of AP5 (50 ␮M) to examine the possible after-effects of PPS that might be independent of NMDA-Rs. Fig. 1(A) shows examples of isolated AMPA-R mediated fEPSPs recorded in the presence of AP5, evoked by either SPS or PPS (traces i and ii). Washing out AP5 led to composite fEPSPs (AMPA and NMDA components), evoked by SPS or PPS (traces iii and iv). 2.3. Data analysis Signals were amplified, filtered and transferred to a PC clone computer for on-line and off-line analyses by specially designed

Fig. 1. Experimental layout and key results. (A) Modes of activation illustrated by field EPSPs recorded under different experimental conditions, all in low Mg2+ solution (i)–(iv). Slices were subjected to single or paired pulse stimulation in combination with recording of either isolated AMPA (blockade of NMDA-R by AP5) or composite field EPSPs, yielding four combinations. The AMPA component was measured via an early time window (area marked AMPA) whereas the NMDA component was measured via a late time window (area marked NMDA). (B) Effect of paired pulse stimulation (PPS) under sequentially blocked and unblocked NMDA-R in a single experiment showing measurements of AMPA component (upper panel) and NMDA component (lower panel) for test pathway (gray symbols) and control pathway (black symbols). Periods of AP5 treatment and periods of paired pulse activation of the test pathway are indicated by bars. The control pathway received single pulse stimulation (SPS) throughout the experiment. The data show that both slow activation of NMDA-R by washout of AP5 and fast (additional) activation by turning on paired pulse stimulation triggered biphasic changes of the AMPA component.

electronic equipment (based on an Eagle Instruments Multifunction Board) and own developed computer software. Composite fEPSPs were analyzed with respect to AMPA and NMDA components using differently placed time windows, conforming with previous studies in our lab (Asztely et al., 1992; Xiao et al., 1995; Niu et al., 1999; Dozmorov et al., 2006). Basically, the AMPA component (and isolated AMPA EPSP) was measured via an early time window (first 1–2 ms after the fibre volley, see Fig. 1(A), black area) whereas the NMDA component was measured via a late time window (30–40 ms, see Fig. 1(A), gray area). In the case of PPS, the fEPSP evoked by the first pulse was generally analyzed; however, both fEPSPs were used in some experiments to establish PPF of the AMPA component. We quantified paired pulse ratio (PPR) as AMPA2 /AMPA1 , where indices 1 and 2 refer to the successive responses (also note that PPF = PPR − 1). In addition, measurements of both EPSPs were used to calculate the induction strength, defined as the sum of NMDA components of the two fEPSPs (NMDA1 + NMDA2 ). Measurements were calculated by

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paired pulse facilitation (not illustrated, but see Section 3.4). Longer periods of PPS, such as for 60 min, gave rise to a slowly developing, weak depression (91 ± 3% relative to control at 60 min of PPS, n = 5, p < 0.05), which persisted for 5–15 min after returning to single pulses (Fig. 2(B)). Because of the quick return to control level, the phenomenon is probably best described as a short-term depression (STD), even though it could be maintained for a long time when PPS was running. Additionally, Fig. 2 shows measurements of the interleaved responses of the control pathway receiving only SPS (black symbols). Comparison between test and control pathways shows that the STD was an input-specific phenomenon. 3.2. PPS causes initial STP followed by LTD under conditions of unblocked NMDA-R Fig. 2. Effect of PPS under NMDA-R block. Measurements of isolated AMPA EPSPs are shown for test (gray symbols) and control (black symbols) pathways, depicted as mean ± S.E.M. for 1 min periods. NMDA receptors were fully blocked due to the presence of 50 ␮M of the specific antagonist AP5. (A) Paired pulse stimulation (PPS) for 12 min delivered to the test pathway led to a minor, transient depression (n = 10). (B) A longer period of PPS (60 min) gave rise to a slowly developing, weak depression, which persisted for 5–15 min after returning to single pulses (n = 5). The control pathway received only single pulse stimulation (SPS) throughout the experiment. Horizontal bars indicate the time of applying PPS.

integrating the curve along the specified time window after substraction of the prestimulus baseline. For AMPA measurements of the second fEPSP, the level just before the second stimulation served as baseline. In most cases, values were corrected by substracting the corresponding measurements of the nonsynaptic potential obtained after total blockage of the fEPSPs by 50 ␮M AP5 and 10 ␮M CNQX. The final data were generally quantified as relative values compared to a reference level defining 100%. The results are presented as mean ± standard error (S.E.M.). Statistical comparisons were made using Student’s t-test. 2.4. Materials and drugs Drugs were obtained from Tocris Cookson (UK), Ascent Scientific (UK) or Sigma–Aldrich (St. Louis, MO, USA); prefabricated stimulating electrodes were obtained from World Precision Instruments, FL, USA, type TM33B. 3. Results PPS was delivered to hippocampal slices under two different experimental conditions, employing either blocked or unblocked NMDA-Rs. In the latter case, which is prime interest, turning on PPS was used to transiently increase the intensity of NMDA-R activation. Fig. 1 illustrates the experimental layout as well as data from a single experiment where the two types of test condition were applied in succession. It can be appreciated that during the second test period, plastic changes of AMPA responses occurred in response to both washout of AP5 and turning on PPS. These results are further quantified in a series of experiments detailed below. 3.1. PPS causes a minor STD under NMDA-R blockade We begin by considering the situation in which NMDA-Rs were blocked (first test period in Fig. 1(B)). As seen in Fig. 2(A), 12 min of PPS led to a minor depression of isolated AMPA fEPSPs (94 ± 2% relative to control at 12 min of PPS, n = 10, p < 0.05). After returning to SPS, responses recovered quickly back to control level. It should be kept in mind that the presented data refer to either single responses or to the first response during PPS. Additionally, PPS was also associated with a bigger second response, representing

Let us next consider the second test period in Fig. 1(B). The blockade of NMDA-Rs was here terminated by switching to AP5-free solution, leading to a gradual appearance of an NMDA component. There was also a concurrent growth of the AMPA component during the 15–20 min following the switch (Figs. 1(B) and, 3(A) and (B)) in conformity with prior work in our laboratory (Dozmorov et al., 2003; Li et al., 2007). In further agreement, sustained SPS caused a slowly developing depression of both fEPSP components (see control pathway data in Figs. 1 and 3). Interestingly, a period of PPS resulted in additional plasticity in the form of a short-term potentiation (STP). As illustrated by the average data in Fig. 3(A), PPS of the test pathway for 12 min resulted in a rapidly commencing potentiation, quickly turning into a decay and reaching back to near control values within 15 min. During the rest of the experiment, test and control pathways followed similar decaying trajectories, possibly an LTD-related phenomenon (Dozmorov et al., 2003). The PPS-induced STP was input-specific as revealed by comparing test and control pathways. On the average, the AMPA component was potentiated up to 145 ± 5% (p < 0.01, n = 6). In order to see whether longer induction periods might produce potentiation with other properties, perhaps longer lasting, we applied PPS for periods of 75–90 min. Fig. 3(B) shows that even PPS going on for such long time was unable to bring about a sustained potentiation but led to STP very similar to that obtained with only 12 min of PPS. The peak potentiation of the AMPA component averaged 146 ± 6% (p < 0.01, n = 5), which is close to the above-mentioned value. During the hour following STP, test and control responses decayed in near parallel, i.e. similar to the situation in Fig. 3(A), despite the fact that PPS was running all the time. However, there appeared to be a slight deviation between data from short and long PPS experiments. Accordingly, a tendency for “test > control” was present during the decaying phase in Fig. 3(A) (12 min PPS) compared to a tendency for “test < control” in Fig. 3(B) (75 min PPS). The statistical significance of this deviation was confirmed by a combined paired-difference test (1–2 = 11 ± 3, df = 9, p < 0.01, at 45 min). 3.3. STP peaks within 1 min and mostly affects the AMPA component To better quantify the onset of PPS-induced STP, the data in Fig. 3(A) and (B) were merged together and plotted in Fig. 3(C) using an expanded time scale, which allowed each response to be displayed. For completeness, both AMPA and NMDA components are shown. To illustrate the “induction strength”, we have additionally plotted the sum of NMDA components for the two EPSPs in each pair. It can be seen that STP was expressed predominantly as an increase of the AMPA component, developing gradually towards a peak within about 1 min (6 responses at 0.1 Hz). For the experiments in Fig. 3(C), the time to reach peak potentiation varied between 40 and 90 s with an average of 63 ± 5 s (n = 11). It can be noted that despite the fact that the induction strength was substan-

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Fig. 4. Changes of paired pulse facilitation (PPF) during PPS-induced plasticity. The paired pulse ratio (PPR) was determined for AMPA components of composite EPSPs during sustained PPS. Bars illustrate mean and S.E.M. of relative PPR changes, using the very first PPR as reference. (A) PPS-induced potentiation and depression are associated with changes of PPR. Labels “1, peak STP”; “2, after STP”; and “3, LTD” refer to measurements taken at 1 min, 8–10 min, and 60–75 min respectively after onset of PPS. The time points (1–3) are further depicted in the insert showing a schematic drawing of an experiment. Data were obtained from 8 experiments including those of Fig. 3(B). (B) and (C) Data from control experiments, demonstrating that 60 min of PPS under blockade of NMDA-R by AP5 caused no significant change of PPR ((B), data from Fig. 2(B), n = 5) whereas quick application of AP5, which abruptly abolished the NMDA component, had but a small effect on PPR ((C), n = 4). Taken together, these control data validate the PPR changes illustrated in (A) by showing that they were NMDA-dependent; still not due to simple crosstalk between AMPA and NMDA components.

Fig. 3. Effect of PPS under NMDA-R unblock. Measurements of AMPA component of composite EPSPs are shown for test (gray symbols) and control (black symbols) pathways, depicted as mean ± S.E.M. for 1 min periods ((A) and (B)). NMDA receptors were unblocked by starting perfusion with AP5-free solution shortly before the illustrated period. The curves in (C) are similar except that both AMPA (solid symbols) and NMDA components (open symbols) are shown, together with an additional induction strength curve, which equals the sum of the two NMDA components during PPS (or single NMDA component during SPS). In (C), all individual responses are depicted. (A) Applying PPS to the test pathway for 12 min resulted in a shortterm potentiation (STP), quickly reaching a peak and then starting to decay (n = 6). (B) Longer PPS (75 min) resulted in a similar STP as above (n = 5). In both (A) and (B), STP was followed by a slowly depeloping LTD, similar to the LTD of the control pathway which received only SPS. (C) Onset characteristics of PPS-induced STP. The graph is based on all of the experiments in (A) and (B) (n = 11). Note that the 100% level corresponds to the slow peak obtained after washing out AP5 (compare (A), (B) and Fig. 1(B)). The figure shows that the PPS-induced STP predominantly affected the AMPA component, reaching a peak in 50–60 s after beginning of PPS. The lower panel illustrates the induction strength. Horizontal bars indicate the time of applying PPS.

tially bigger than baseline at all positive time points, both AMPA and NMDA components started to decay after the first minute of PPS. The data were all obtained with a stimulus frequency of 0.1 Hz. In three other experiments with a lower frequency of 0.05 Hz, the time to peak appeared to be somewhat longer, around 2 min, suggesting a possible dependence on stimulus number (not illustrated).

five experiments in Fig. 3(B) plus three additional ones with slightly shorter recording periods. Values were calculated as PPR relative to the value of PPR obtained for the very first PPS response. This initial PPR amounted to 1.84 ± 0.10 (n = 8). LTD was found to be associated with a 24 ± 5% (p < 0.01) increase of PPR at 60–75 min after PPS onset. Conversely, there was a small decrease of PPR, 6.2 ± 2.0% (p < 0.05), at the peak of STP as measured 1 min after PPS onset. For comparison, when the NMDA component was blocked (Fig. 4(B), data from Fig. 2(B)), there was no significant change of PPR after 60 min of PPS, verifying that the changes of PPF illustrated in Fig. 4(A) were NMDA-dependent. To test whether the increased PPR during LTD might be a direct (trivial) consequence of the decrease of NMDA component (Fig. 1(B)), we carried out experiments where AP5 was added 20–30 min after commencing PPS, and assessing PPR just before and just after drug application. Application of AP5, which totally blocked the NMDA component, resulted in just a small increase of the PPR measurement (+3.2 ± 0.8%, p < 0.05, n = 4), illustrated in Fig. 4(C). As an additional control, we tested whether postsynaptic pharmacological depression of AMPA EPSPs might influence PPR. Decreasing the AMPA response about 3-fold by a 1–2 ␮M increase of the CNQX concentration—to simulate LTD—caused an increase of PPR by 8.1 ± 2.7% (p < 0.05, n = 8, not illustrated), possibly related to system nonlinearities. For comparison PPR changes were determined for an identically sized depression of presumably presynaptic origin. For this purpose, 10–20 ␮M adenosine was applied and washed out again prior to the CNQX test. In this case, PPR increased by 23 ± 4% (p < 0.01); more or less identical to the 24 ± 5% increase during LTD (see above).

3.4. LTD is associated with an increase of PPF 3.5. Stabilizing plasticity by NMDA-R blockade We further assessed PPF, an often used indicator of presynaptic changes, at different time points during the development of PPSinduced plasticity. Fig. 4(A) shows PPF data corresponding to the

As previously shown in Fig. 3, the SPS-induced potentiation was followed by a gradually developing LTD and fading of the differ-

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Fig. 5. NMDA-R block stabilizes PPS-induced plasticity. Measurements of AMPA component ((A), upper panel) and NMDA component ((B), lower panel) are shown for test (gray symbols) and control (black symbols) pathways, depicted as mean ± S.E.M. for 1 min periods. Under conditions of NMDA-R unblock, STP was induced by PPS as above but was here followed by reblock of NMDA-R by 50 ␮M of AP5 just after the peak. Although an early dip of AMPA responses occurred right after AP5 application, subsequent responses showed long-term stability together with a persistent difference between pathways. At 2 h after blockage of NMDA-R, the average value of fEPSPs in the test pathway was 123 ± 5% compared to the control pathway which did not receive PPS (n = 4, p < 0.05).

ence between pathways. To test whether these changes might be NMDA-dependent, we performed experiments where the NMDA-R antagonist AP5 was applied just after the peak of PPS-induced STP. As seen in Fig. 5, the NMDA component was quickly and effectively blocked by the antagonist. While the decay of the AMPA component was accentuated right after application of AP5, the later, slowly developing LTD was largely blocked. This stabilization of responses occurred for both test and control pathways. As a result, when comparing time courses of test and control responses, the initial PPS-induced difference remained throughout the experiment, at least in part (ratio of 124 ± 5% at 1 h, p < 0.05, n = 4). Adding AP5 after longer periods of PPS (1–2 h, n = 6) also blocked the slowly developing depression but at lower levels, verifying that it was properly described as LTD (compare Dozmorov et al., 2003). Despite the potentiation of test versus control responses in Fig. 5(A), there was no net potentiation when comparing with the initial baseline (102 ± 4% at 1 h, p > 0.05). To further address this issue, we did similar experiments but with additional stopping of stimulation for 14 min following peak STP. The idea was to prevent activity-dependent decay during the period after applying AP5 before reaching full block of NMDA-Rs. However, the stable levels attained in this case did not differ significantly from those above (test pathway 108 ± 3% of initial baseline, test/control ratio 132 ± 4%, n = 5, not illustrated). 3.6. NMDA-R activation counteracts the early decay To examine how NMDA-R activation contributes to the early temporal development of PPS-induced plasticity we superimposed 20 min of test pathway data from Figs. 3 and 5 into Fig. 6(A). Comparing the curves revealed that PPS-induced STP of the AMPA component actually decayed faster under blockade of NMDA-R by AP5 than it did under maintained NMDA-R activation. When measured 3–5 min after commencing PPS, the level of STP was 106 ± 3% (n = 4) in the presence of AP5 compared to 129 ± 5% (n = 6) in control slices that were lack of AP5 treatment (p < 0.01). However, due

Fig. 6. Activation of NMDA-Rs counteracts the early decay of STP. Measurements of AMPA responses during PPS-induced STP are shown under various manipulations affecting NMDA-dependent induction, depicted as mean ± S.E.M. for each successive response. The induction strength is also illustrated, defined as the sum of the two NMDA components during PPS (or single NMDA component during SPS). (A) Standard STP (black symbols, data from Fig. 3(A), n = 6) is compared to STP in slices where NMDA-Rs were blocked just after peak potentiation (gray symbols, data from Fig. 5(A), n = 4). Notably, blockade of NMDA-R fastened the early decay while preventing the following, slow LTD. In terms of active processes, NMDA-R activation thus counteracts early decay and promotes late decay. (B) Pathways are compared within the same slice (n = 7). While one pathway received PPS treatment for 12 min (standard PPS, black symbols), the other pathway received PPS for only 1.5 min, followed by return to SPS (minimal PPS, gray symbols). Comparison reveals that the higher level of NMDA-R activation (maintaining PPS instead of returning to SPS) slowered the early decay of STP, consistent with the above data. Note that the 100% levels in (A) and (B) correspond to the slow peak after washing out AP5.

to methodological difficulties in terms of a possible AMPA-NMDA crosstalk (NMDA-contamination of the AMPA measurement), the effect in Fig. 6(A) may be over-estimated, rendering the evidence from this comparison less convincing. We therefore designed an experiment less sensitive to crosstalk, which was carried out under NMDA-R unblocked conditions (Fig. 6(B)). STP was induced by PPS in both pathways, but with slightly different protocols, allowing for within-slice comparison. In one pathway (“standard PPS”), 12 min of PPS was applied as before whereas the other pathway (“minimal PPS”) received PPS for only 90 s, i.e. slightly beyond the peak of the STP curve. Consequently, in the minimal PPS pathway, the degree of NMDA-R activation was comparatively smaller during the period after the peak although it was not blocked as in experiments with AP5 application (see induction strength plots in Fig. 6). Our results in Fig. 6(B) show that AMPA responses of the two pathways reached similar peak values within about 1 min, as expected from the balanced experimental design (142 ± 5% and 140 ± 6%, respectively). In line with our conjecture above, the minimal PPS pathway was associated with a faster decay of AMPA responses in the early period after peak STP. Thus at 3–5 min after starting PPS (standard PPS pathway still receiving PPS but minimal PPS pathway receiving only SPS), the potentiation level of the minimal PPS pathway was 107 ± 7% as compared to

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119 ± 6% for the standard PPS pathway (n = 7). A paired difference test revealed that the effect was significant (12 ± 3%, p < 0.01). 4. Discussion It is still incompletely known how NMDA-Rs regulate bidirectional synaptic plasticity. The present results showed that, when these receptors were unblocked, increased activation by PPS caused an STP of AMPA-R mediated responses, lasting 10–15 min and being followed by a slowly developing LTD. The early decay of STP was counteracted by NMDA-R activation whereas the following LTD was dependent on such activation. The results emphasize the role of temporal factors in controlling the directionality of NMDAdependent synaptic plasticity. 4.1. Essential features of recording in low Mg2+ A major reason for using low Mg2+ was to obtain a situation in which NMDA receptor activation could be modulated without excessive changes in stimulus rate. Perfusion with low Mg2+ will substantially reduce the voltage-dependent block of NMDARs. Washout of the antagonist AP5 in the early part of experiments hence led to slow activation of NMDA-Rs. An additional, fast activation was obtained by switching from SPS to PPS under unblocked conditions. As a further result of NMDA-R unblocking, AMPA and NMDA components could be readily assessed in parallel, using suitably positioned time windows to measure early and late parts of the fEPSP. Previous work has shown that crosstalk (contamination of AMPA mesurement by the NMDA response and vice versa) is relatively small in this situation (Asztely et al., 1992; Xiao et al., 1995; Dozmorov et al., 2003). By studying changes of both AMPA and NMDA components, more information can be gained about synaptic mechanisms compared to standard AMPA EPSP recording. 4.2. Relation to prior work on PPS-induced plasticity The lack of a substantial effect under NMDA-R blockade is consistent with prior work in hippocampal slices where PPS applied at test stimulus rate was reported to produce no effect in 4–9week-old rats (Wasling et al., 2002). However, in animals aged only 6–12 days, a period of 20–30 min of PPS (50–200 ms interstimulus interval) was found to induce a heterosynaptic LTD, the induction of which involved activation of both NMDA-Rs and T-type VDCCs (Wasling et al., 2002). This result was observed under both normal conditions and in slices disinhibited by bicuculline. For comparison, our animals were 13–22 days old and experimental conditions differed to some extent by the use of low Mg2+ and AP5 in the bath. We did no experiments in disinhibited slices. It can be noted that PPS in disinhibited slices, together with a stimulus strength high enough to produce firing, led to initial potentiation followed by depression (Wasling et al., 2002), similar to the plasticity observed here. However, the plasticity in the referred study appeared to be a mixed phenomenon since the initial potentiation was homosynaptic whereas the LTD was heterosynaptic. In the present study, STP was evidently homosynaptic and this was most likely the case for the subsequent LTD as well, considering a prior investigation of ours (Dozmorov et al., 2003). While it appears that special conditions, such as very young animals, disinhibition or NMDA-R unblock, were needed to produce the above effects, special variants of PPS have been shown to be effective in inducing plasticity in more natural situations. Several studies have applied PPS in combination with low-frequency stimulation (LFS), yielding “PP-LFS” or “PP-1 Hz stimulation”, found to be effective in inducing of LTD. The induction of this LTD was generally found to be independent of NMDA receptors, being related instead to activation of mGluRs (Huber et al., 2000, 2001) and in

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one case additionally to AMPA/kainate receptors (Kemp and Bashir, 1999). These results are in contrast with our case of an NMDAdependent LTD. However, the outcome of PP-LFS treatment seemed to vary substantially depending on stimulation parameters. Thus, using a 200 ms instead of 50 ms interstimulus interval produced an NMDA-dependent instead of mGluR-dependent LTD (Kemp et al., 2000). 4.3. Mechanisms underlying PPS-induced plasticity LTP is generally considered to occur predominantly as an increase of the AMPA response, involving an increase of channel conductance and insertion of new AMPA-Rs (Muller and Lynch, 1988; Nicoll, 2003). Some prior work suggests that there is also an increase of NMDA-R mediated responses (Asztely et al., 1992; Clark and Collingridge, 1995), but developing more slowly during 1–2 h after induction (Xiao et al., 1996; Watt et al., 2004). The STP observed in our case as a selective increase of the AMPA component is thus in consistency with the general view of LTP mechanisms and the idea of STP as related to LTP. The later LTD, observed with both SPS and PPS, occurred via a depression of both AMPA and NMDA components, in line with previous results on LTD (Selig et al., 1995; Xiao et al., 1995). Our data therefore suggest that early and late parts of PPS-induced plasticity may involve different synaptic modifications. The slow LTD might then be presynaptic whereas the initial STP is most likely postsynaptic as we have argued. One could thus imagine that the postsynaptic Ca2+ accumulation could trigger the release of retrograde transmitter, such as endocannabinoids (OhnoShosaku et al., 2007), which may induce presynaptic inhibition and so depress both AMPA and NMDA components. The idea of a presynaptic locus for the slow LTD is in line with our observation of an increase of PPF, an indicator of transmitter release probability. However, the fact that a PPF change (a decrease) was also observed for the presumably postsynaptic STP throws doubt on the proposed pre- versus postsynaptic scenario. As an alternative, we may consider that the observed PPF changes (of both STP and LTD) would reflect postsynaptic processes. Thus, changes of PPF could result from altered weighting in a population of synapses with different release probabilities. Assuming that high release synapses (small PPF) are prone to undergo plasticity under the present low-frequency paradigm, the observed changes in PPF are expected to occur in terms of population averages. However, more research is needed to clarify the nature and location of the present PPS-induced plasticity. In a proposed model for LTP, the existence of two types of synaptic modification was postulated: one fast, implying that new AMPA receptors are inserted into existing “slots” in the postsynaptic membrane and one slow, implying a gradual widening of the synapse, giving space for more NMDA receptors (Lisman and Raghavachari, 2006). A reversal of the latter process could be responsible for LTD. One may speculate that such a distinction between receptor-related and morphology-related changes would apply to the present case as well. It can be noted that a change of spine morphology will affect both pre- and postsynaptic parts and so the LTD, but not STP, would contain a presynaptic component in this scenario. 4.4. Does long induction time promote LTD? Whereas LTP can be induced by short stimuli, down to a fraction of a second, the critical time range for LTD induction remains controversial, and different results have been obtained for different frequencies (Mulkey and Malenka, 1992; Cummings et al., 1996; Mizuno et al., 2001). Moreover, little is known about directionality control at very low frequencies such as below 1 Hz. As revealed here, continued stimulation at a constant low frequency (0.1 Hz)

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favoured depression over potentiation. The notion of a temporal factor for LTD is in general agreement with a previous study that tested different durations of 1 Hz LFS, using Mg2+ -free solution in combination with receptor antagonists to vary the induction strength (Mizuno et al., 2001). In that study, LTD was only obtained with long trains, exceeding about 3 min in the most favourable case, whereas LTP was induced by both short and long trains under strong induction. In contrast to our study, there was no “bivalent” induction strength able to cause potentiation in some cases and depression in others, depending on the stimulus duration. Regarding the role of stimulus duration, it is also relevant to consider the work on PP-LFS mentioned before. While most of these studies employed a constant, relatively large number of paired stimuli, which invariantly resulted in LTD, one such study used varying durations of PP-LFS (Huang and Kandel, 2006). It was found that shorter induction periods (1–3 min) led to LTP, whereas longer ones (10–15 min) gave LTD, much in line with the time dependency observed here. When taking measurements during PP-LFS, a potentiation–depression sequence was seen, similar to that in our case, with a peak at about 1 min; this behavior is also observed during LTD induction by standard LFS (Mulkey et al., 1993), but note the use of higher frequency (1 Hz versus 0.1 Hz in our case). 4.5. Possible mechanisms for bidirectional induction It is generally conceived that LTP is induced by strong activation of NMDA-Rs, leading to a large postsynaptic increase in Ca2+ , whereas LTD is induced by weaker stimulation, leading to a moderate postsynaptic increase in Ca2+ (Lisman, 1989; Shouval et al., 2002; Malenka and Bear, 2004). According to this view, Ca2+ alters the states of protein kinases and phosphatases in a concentrationdependent manner, leading to subsequent changes at the receptor level. However, this scenario is insufficient to explain the present situation where potentiation versus depression depended on stimulus duration. Hence, the plasticity induced via NMDA-R activation (by PPS or SPS) at a certain time was also influenced by prior induction events. Such plasticity of plasticity, generally referred to as metaplasticity (Abraham and Bear, 1996), has previously been described in terms of a sliding threshold for LTP induction (Bear, 1995; Stanton, 1996; Shouval et al., 2002). For instance, moving the threshold to a higher value will lead to depression for an induction strength that previously induced potentiation. As a biochemical explanation for such an increase of LTP threshold, it has been suggested that NMDA-R mediated currents undergo activity-dependent scaling via modification of the receptors, computationally equivalent to a sliding threshold (Shouval et al., 2002). In our case, this seems not enough to explain the induction of biphasic plasticity, arguing for involvement of other processes such as a change of Ca2+ buffer concentration (Gold and Bear, 1994; Jedlicka, 2002) or changes downstream of Ca2+ activation. The latter may involve altered enzyme dynamics or morphological changes of dendritic spines (Gold and Bear, 1994; Bear, 1995; Stanton, 1996). It may also be considered that Ca2+ amplification by calcium-induced calcium release (Chittajallu et al., 1998) could contribute to the dependence of the STP on a sudden increase of induction strength. 4.6. Active versus passive control of plasticity There are still some aspects of the plasticity considered here that are not accounted for by the suggested model. Interestingly, we discovered that, after the peak of PPS-induced STP, there were two types of decay, an early one (<10–15 min) and a late one, with different properties. While both displayed a dependence on NMDARs, the dependencies were in opposite directions. Thus, the early decay was counteracted by NMDA-R activation (negative effect compared to direction of change) whereas the later one was pro-

Fig. 7. NMDA-R-dependent and independent processes shaping bidirectional plasticity. (A) Schematic drawing of PPS-induced plasticity with a marked selection of cases at different time points: 1, onset of STP; 2, peak of STP; 3, early decay; 4, inversion point; and 5, late decay. Filled arrows show NMDA-dependent forces acting in potentiating (upward) direction at 1–3, or depressing (downward) direction at 5. Change in direction of driving force takes place at 4. These “active” forces are not necessarily aligned with the actual direction of change. Note that at 3, the fEPSP declines despite an active process in the potentiating direction. Hence, we hypothesize the existence of a “passive”, NMDA-independent, depressive force at 3, that overrides the NMDA-dependent potentiating effect. For symmmetry, we also hypothesize a late, passive force in the potentiating direction at 5; however, this is overridden by the active, NMDA-dependent one. (B) Sliding threshold model describing the relation between NMDA-dependent induction strength (thought of as Ca2+ concentration in the dendritic spine) and active potentiating/depressing drive. LTP threshold is thought to move depending on prior synaptic activity. Numbering of curves corresponds to time points in A. Two levels of NMDA-R activation are considered (somewhat simplified), representing SPS and PPS, respectively. (C) Block diagram with an initial “induction stage”, that creates an active driving force according to (A), followed by an “expression stage” where plasticity is formed. Feedforward circuit (left) represents sliding threshold control; feedback circuit (right) represents tendency for relaxation (passive force).

moted by such activation (positive effect compared to direction of change). As a solution, we propose that the early decay is mainly generated by an NMDA-independent mechanism, for simplicity, referred to as passive. This idea is also supported by the lack of LTP above initial baseline in the experiments with AP5 application after peak STP, with or without pausing of stimulation. However, in these experiments, there was a remaining difference between pathways, suggesting that NMDA-dependent processes were normally involved in the symmetrization of responses. It is also possible that the residual difference actually represented LTP but was masked by LTD. It remains to further determine the relation between the plasticities involved, both active and passive ones. The proposed components of PPS-induced plasticity are illustrated in Fig. 7(A), showing the rising and falling phases of STP which are influenced by a positive-going NMDA-dependent drive together with a passive, negative-going one which dominates after the peak. The later, slow LTD is considered to be largely an NMDAdependent process, but for completeness, we postulate that it also includes a relaxation in terms of a passive, upward-striving “decay”. This idea is consistent with unpublished data showing a return towards a higher level when blocking NMDA-Rs by AP5 after several hours of slow LTD. We imagine that active, NMDAdependent induction is a function of Ca2+ in the spine according to a U-shaped relation, similar to that predicted by BCM theory

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(Bienenstock et al., 1982; Artola and Singer, 1993). The set of such “BCM-curves” in Fig. 7(B) serves to illustrate the sliding of LTPthreshold. NMDA-dependent and independent drives may then interact via summation or in other ways to shape a final outcome in terms of a certain synaptic strength (Fig. 7(C)). While NMDA-dependent LTP and LTD are relatively well understood, less is known about relaxation mechanisms involved in reversal of these plasticities. The term STP is often used to indicate an initial, inherently unstable phase of LTP that is independent of NMDA-R activation once LTP is induced. How STP decays is still a controversial issue with respect to the need for synaptically activated processes, either presynaptically (Volianskis and Jensen, 2003) or postsynaptically (Abrahamsson et al., 2008). STP could be mediated via insertion of AMPA-Rs composed of GluR1/GluR2 subunits, compared to stable LTP thought to involve GluR2/GluR3 subunits (Shi et al., 2001). 4.7. Concluding remarks Our experiments with single or paired pulse activation under NMDA-unblocked conditions have significant implications for the biochemical machinery underlying bidirectional control of NMDAdependent synaptic plasticity. As implied by our data, the direction of synaptic change does not only depend on the instantaneous Ca2+ concentration in the dendritic spine but is substantially influenced by previous induction events. In terms of underlying mechanisms, the observed fast onset of potentiation and slow onset of depression probably reflect the kinetics of involved enzymes, such a certain kinases and phosphatases, or maybe molecular versus structural changes. In addition to an active, NMDA-dependent process, a passive relaxation also contributes and in some cases outbalances the active control. The possible functional significance of the results remains to be resolved. Perhaps, under normal conditions, the observed process could serve as a “novelty detector” to signal an increased level of presynaptic–postsynaptic correlation. Acknowledgements This study was supported by the Swedish Alzheimer Foundation and the Foundations of Åhlén, Gun & Bertil Stohne, Herbert & Karin Jacobsson, Sigurd & Elsa Golje, Ragnhild & Einar Lundström, Irma & Arvid Larsson-Röst, Wilhelm & Martina Lundgren, and handlanden Hjalmar Svensson; the Swedish Medical Society, the Gothenburg Medical Society and the Swedish Institute. References Abraham, W.C., Bear, M.F., 1996. Metaplasticity: the plasticity of synaptic plasticity. Trends Neurosci. 19, 126–130. Abrahamsson, T., Gustafsson, B., Hanse, E., 2008. AMPA silencing is a prerequisite for developmental long-term potentiation in the hippocampal CA1 region. J. Neurophysiol. 100, 2605–2614. Artola, A., Singer, W., 1993. Long-term depression of excitatory synaptic transmission and its relationship to long-term potentiation. Trends Neurosci. 16, 480–487. Asztely, F., Wigström, H., Gustafsson, B., 1992. The relative contribution of NMDA receptor channels in the expression of long-term potentiation in the hippocampal CA1 region. Eur. J. Neurosci., 681–690. Bear, M.F., 1995. Mechanism for a sliding synaptic modification threshold. Neuron 15, 1–4. Berberich, S., Punnakkal, P., Jensen, V., Pawlak, V., Seeburg, P.H., Hvalby, Ø., Köhr, G., 2005. Lack of NMDA receptor subtype selectivity for hippocampal long-term potentiation. J. Neurosci. 25, 6907–6910. Bienenstock, E.L., Cooper, L.N., Munro, P.W., 1982. Theory for the development of neuron selectivity: orientation specificity and binocular interaction in visual cortex. J. Neurosci. 2, 32–48. Bliss, T.V., Collingridge, G.L., 1993. A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361, 31–39. Chittajallu, R., Alford, S., Collingridge, G.L., 1998. Ca2+ and synaptic plasticity. Cell Calcium 24, 377–385.

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