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Instituto Cajal, CSIC, 28002-Madrid, Spain
Submitted 25 July 2002; accepted in final form 6 February 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Bolshakov and Siegelbaum 1994; Collingridge and Bliss 1987; Issac et al. 1998; Kullmann 1994; Kullmann et al. 1996; Malinow and Tsien 1990; Nicoll and Malenka 1999; Poncer and Malinow 2001; Stevens and Wang 1994).
Among the possible presynaptic mechanisms in LTP, extracellular adenosine triphosphate (ATP) has recently received considerable attention because of its suggested participation as an extracellular diffusible messenger (e.g., Fields and Stevens 2000). The regulation of synaptic transmission by ATP is through purinergic P2 receptors, both of the ionotropic P2X and metabotropic P2Y varieties (Fredholm et al. 1994). In the hippocampus, ATP released by SCs (Wieraszko et al. 1989) enhances the population spike (Nishimura et al. 1990) and contributes to the maintenance of LTP (Wieraszko and Ehrilch 1994). These effects of ATP have been suggested to be presynaptic (e.g., Robertson et al. 2001) and are blocked by extracellular ecto-protein kinase inhibitors (Chen et al. 1996).
In addition, the end product of ATP hydrolysis is adenosine that inhibits synaptic transmission through activation of presynaptic P1 adenosine receptors (Burnstock 1990; Daly et al. 1983; Fredholm et al. 1999; Nishimura et al. 1990). Therefore ATP may regulate synaptic transmission both directly and indirectly via activation of P1 and P2 receptors, respectively (e.g., Daly et al. 1983; Greene et al. 1985; Zimmermann 1994) and also by the simultaneous activation of P1 and P2 receptors (O'Kane and Stone 2000).
Our aim was to investigate the mechanisms that contribute to the LTP in CA3–CA1 pyramidal neuron synaptic contacts through the interactions with P1 and P2 receptors. We show that a brief caffeine application evoked a nondecremental LTP of SC excitatory postsynaptic currents (EPSCs; CAFLTP) that originates presynaptically. This CAFLTP does not require activation of postsynaptic NMDA receptors or an increase in postsynaptic cytosolic-free Ca2+ and is prevented by specific blockers of P1, P2, and ryanodine receptors. The results are consistent with the CAFLTP being caused by caffeine-mediated interactions with presynaptic P1, P2 purinoreceptors, and ryanodine receptors and originated by an increased probability of glutamate release at SC terminals.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Martín et al. 2001); therefore it suffices to briefly delineate them here. Young Wistar rats (13–17 days old) were anesthetized with ether and decapitated immediately after disappearance of the pinch reflex, and the brain was removed and submerged in artificial cerebrospinal fluid (ACSF; see following text) at 4°C and maintained at pH 7.4 by gassing with a 95% O2-5% CO2 mixture. All experiments conformed to International Guidelines on the ethical use of animals, and every effort was made to minimize the suffering and number of animals used. Transverse slices (350 µm) of the dorsal hippocampus were cut with a Vibratome (Pelco 1000, St. Louis, MO) and incubated in gassed ACSF (>1 h at 20–22°C). The ACSF contained (in mM) 124 NaCl, 2.69 KCl, 1.25 KH2PO4, 2 MgSO4, 26 NaHCO3, 2 CaCl2, and 10 glucose. Bicuculline or picrotoxin (50 µM) was added to block GABAA-mediated inhibitory synaptic transmission. Ryanodine (20 µM; from a stock solution in DMSO at 0.001%) and D-2-amino-5-phosphonovaleric acid (AP5; 50 µM) were added to the ACSF when necessary.
Slices were transferred to an immersion recording chamber placed in an inverted microscope stage (WPI, Sarasota, Fl) and superfused (2.5 ml/min) with gassed ACSF at room temperature (20–22°C). Bipolar nichrome wire (80 µm diam) electrodes were placed in the stratum radiatum near the border of CA1 pyramidal layer to stimulate SCs. Stimuli were single or paired pulses (50- to 100-ms delay) delivered at 0.3 s-1 via a stimulator/isolation unit (Cibertec, Madrid, Spain). Patch electrodes were fabricated from borosilicate glass capillaries (1B150F-4, WPI, Saratosa, FL) with a Brown-Flamming puller (Model P-80, Sutter Instruments, CA). Electrodes had resistance of 4–8 M
when filled with the internal solution that contained (in mM) 97.5 K-gluconate, 32.5 KCl 5 EGTA, 10 HEPES, 1 MgCl2, and 4 ATP (added immediately before recordings). In some experiments, K-gluconate was equimolarly substituted with CsCl in the pipette solution, and no differences were found in the responses evoked by stimulation of SCs. Occasionally, the Ca2+ chelator 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) was added to the internal pipette solution at a final concentration of 40 mM. In other experiments, ryanodine (stock solution in ethanol at 0.01%) was included in the internal pipette solution at a final concentration of 200 µM. All pipette solutions were adjusted to pH 7.2–7.3 with KOH or CsOH and had osmolarities between 280 and 290 mOsm/l.
Patch pipettes were positioned with a mechanical micromanipulator under visual control on the CA1 pyramidal layer. Whole cell recordings, either in the single-electrode current-clamp or voltage-clamp modes, were obtained with a PC-ONE amplifier (Dagan Corporation, MN). Pyramidal neurons were identified by the characteristic responses evoked by transmembrane current pulses under current clamp. Fast and slow capacitances were neutralized, and series resistance was compensated (
80%). Recordings were rejected when the series resistance (12.6 ± 4 M; n = 101) changed >20% during the experiment. The membrane potential (Vm) was held at -70 mV in voltage-clamp experiments except when indicated otherwise. Data were filtered at 3 kHz and transferred to the hard disk of a Pentium based computer using a DigiData 1200 interface and the pCLAMP 6.0 software (Axon Instruments, Foster City, CA).
Caffeine (anhydrous, stock solution in distilled water) was added directly to the chamber with an automatic calibrated microsyringe through a pipette (tip diameter: 400 µm) positioned with a mechanical micromanipulator close to the recording electrode tip. A single volume of 100 µl of the caffeine solution (10 mM) was delivered (total delivery time was in <1 s) 10–20 min after intracellular access was obtained. We estimated that the washout of caffeine from the recording chamber was in <10 min by measuring the spread and clearance, respectively, of similar volumes of concentrated solutions of methylene blue. The washout of caffeine from the bath was also evaluated during continuous superfusion with adenosine (50 µM, n = 4) by the decay to previous values (in <11.0 ± 0.5 min) of the EPSC amplitude increase caused by the antagonistic effect of the caffeine "pulse" on the presynaptic inhibition evoked by adenosine. Glutamate (dissolved in distilled water at 0.7 M; pH adjusted to 8.2 with NaOH) was iontophoretically applied through a micropipette (10–50 M), placed with a micromanipulator close to the recording electrode under visual guidance (<100 µm). A voltage pulse delivered by a stimulator (Cibertec, Madrid, Spain) to the voltage command input of a micro-electrode voltage-clamp amplifier (Biologic, Echirolles, France) provided the iontophoretic current. Drugs were purchased from Sigma except ryanodine, which was from Calbiochem (La Jolla, CA), suramin (stock solution in H2O), 8-cyclopentyltheophylline (CPT; stock solution in DMSO at 0.001%), L-glutamic acid, and AP5, which were from Tocris Cookson (Bristol, UK).
The pre- or postsynaptic origin of the observed regulation of EPSC amplitudes by caffeine was tested by both estimating changes in the paired-pulse facilitation (PPF), that are considered of presynaptic origin (e.g., Clark et al. 1994; Creager et al. 1980) and the modifications in the variance of EPSC amplitudes that reflect changes in the probability of transmitter release (e.g., Bekkers and Stevens 1990; Kullmann 1994; Malinow and Tsien 1990).
Changes in the PPF were estimated by calculating a PPF index from the expression (R2 - R1)/R1, where R1 and R2 were the peak amplitudes of the first and second EPSCs, respectively (Wang and Kelly 1996). To estimate the modifications in the variance of EPSC amplitudes that parallel the EPSC amplitude changes, we first calculated the noise-free coefficient of variation (NFCV) of the synaptic responses under potentiated and control conditions using the formalism NFCV = 
EPSC 2 - noise 2 /m, where EPSC 2 and noise 2 are the variance of the peak EPSC and baseline, respectively, and m is the mean EPSC amplitude. Assuming that a binomial process governs release probability (see following text), the variance of the EPSC should vary less than the square of the mean (e.g., Kullmann 1994). Therefore if the mean EPSC amplitude increases as a consequence of the number of quanta released, the NFCV should decrease. We also constructed plots comparing variation in the normalized m (termed M), to the change in response variance of the EPSC amplitude measured during the potentiation and normalized to the respective control values (i.e., 1/CV2) in each cell (Bekkers and Stevens 1990; Malinow and Tsien 1990). In these plots, that show the relationship between the 1/CV2 and M, values should follow the diagonal and the 1/CV2 values remain under 1.0 if the excitatory effect has a presynaptic origin. This method requires a binomial EPSC amplitude distribution, a condition that must be met for the synaptic variance to reflect the probability of transmitter release (i.e., the quantal variance). We could not directly test whether our data fits a binomial distribution, but synaptic fluctuations were always evident, and we assumed that synaptic release followed a binomial distribution.
Analysis of the spontaneous EPSC activity was performed with the ACSPLOUF software (obtained from Dr. Pierre Vincent, University of California, San Diego, CA). We estimated the cumulative probabilities of the amplitude and frequency of the spontaneous EPSCs recorded during 10 min in control conditions and 10 min after the caffeine challenge during the CAFLTP. Statistically significant differences were established at P < 0.05, using the Kolmogorov-Smirnov test. Data were compared using the Student's t-test and values are given as the mean ± SE.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Brief caffeine "pulse" evokes the CAFLTP
Control recordings of SC EPSCs evoked by paired-pulse stimulation (100-ms interval) were obtained after checking their stability during 10–20 min (Fig. 1A1). A single dose of 10 mM caffeine (100 µl) was then applied close to the recording pipette (see METHODS). After a short delay (<5 min) after the "pulse" of caffeine a marked potentiation of the peak EPSC amplitude was evoked that reached a maximum value in 15 min (a 229 ± 20% increase above control values; n = 7) and remained stable during the rest of the experiment (192 ± 26%; 70 min; n = 7; Fig. 1, A and B). Therefore this CAFLTP was not reverted by a prolonged washout in control ACSF.
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A marked persistent and significant reduction of the PPF index (-0.25 ± 0.06; P = 0.04; same cells) paralleled the CAFLTP (Fig. 1C). We also constructed plots of the 1/CV2 ratio as a function M (see METHODS) 10, 40, and 70 min after caffeine application. The 1/CV2 ratios revealed that values grouped following the diagonal and were consistently <1.0. The mean 1/CV2 ratios were 0.33 ± 0.07, 0.52 ± 0.2, and 0.51 ± 0.09 measured 10, 40, and 70 min after applying caffeine, respectively (Fig. 1D, same cells). Control recordings in the absence of caffeine were stable during >80 min without EPSC potentiation or rundown (Fig. 1E, n = 7).
The decreased PPF that paralleled the CAFLTP is consistent with an increased probability of transmitter release and thus suggests that a presynaptic mechanism is involved. Although the variance analysis suggest a presynaptic origin for the CAFLTP, other interpretations are also possible. The changes in the variance of EPSC amplitudes reduce the possibility of a postsynaptic participation but do not rule out the possible insertion of functional AMPA receptors at silent synapses that only express NMDA receptors. The freshly inserted AMPA receptors would signal an increased number of quanta by allowing the freshly incorporated receptors to sample release at terminals that were not tested before the potentiation (Kullmann 1994; Montgomery et al. 2001; Nicoll and Malenka 1999; Poncer and Malinow 2001).
Both AMPA and NMDA EPSC components are potentiated during the CAFLTP
A potentiation of the AMPA EPSC (AMPAEPSC) component, without changes in the NMDA EPSC (NMDAEPSC) component, characterizes the classical LTP of SC EPSCs in CA1 pyramidal neurons (e.g., Kullmann 1994; Nicoll and Malenka 1999; Poncer and Malinow 2001), which is thought to be triggered by the insertion of AMPA receptors at silent synapses (Isaac et al. 1995, 1998; Malenka and Nicoll 1999; Nicoll and Malenka 1999). However, if the CAFLTP has a presynaptic origin, the magnitude of the potentiation of both AMPAEPSC and NMDAEPSC should be similar. The AMPAEPSC and NMDAEPSC can be isolated because at -60 mV, only the fast AMPAEPSC is recorded due to the voltage-dependent block of the NMDA channel by extracellular Mg2+, whereas at +60 mV, there should also be a slower NMDAEPSC caused by the relief of the Mg2+ block of NMDA channels (e.g., Collingridge et al. 1983; Hestrin et al. 1990). At +60 mV and at delays >50 ms, the AMPAEPSC has completely vanished, whereas the slower NMDAEPSC is peaking and its amplitude can be estimated in isolation (e.g., Hestrin et al. 1990).
We found that the potentiation evoked by the caffeine pulse was similar at -60 and +60 mV (Fig. 2, A and B). The mean increases of peak AMPAEPSC from controls values were 255 ± 33, 217 ± 33, and 202 ± 21% 10, 40, and 70 min, respectively, after applying caffeine and the NMDAEPSC (measured at delays of 50 ms) increased 289 ± 47, 231 ± 25, and 207 ± 39% 10, 40, and 70 min (n = 4) after caffeine, respectively (Fig. 2, A and B).
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The "classical" SC LTP is paralleled by a reduction of the coefficient of variation (CV) of the AMPAEPSC without changes in the CV of the NMDAEPSC, a result that is also interpreted to indicate a postsynaptic LTP expression caused by the insertion of AMPA receptors at silent synapses (e.g., Kullmann 1994). However, the NFCV of AMPAEPSC and NMDAEPSC changed in similar proportions during the CAFLTP. The NFCV-AMPA decreased 0.23 ± 0.01, 0.19 ± 0.06, and 0.27 ± 0.05, 10, 40, and 70 min, respectively, after caffeine and NFCV-NMDA (measured at delays of 50 ms) decreased 0.10 ± 0.02, 0.11 ± 0.02, and 0.23 ± 0.06, 10, 40, and 70 min (same cells) after caffeine, respectively (Fig. 2C). These results are consistent with the CAFLTP being caused by a presynaptic increase in the number of quanta released (see METHODS and DISCUSSION).
Therefore the preceding results taken together are consistent with a presynaptic origin of the CAFLTP caused by an increased release probability. In addition, the results reduce the possible contribution of the insertion of AMPA receptors at silent synapses during the CAFLTP that characterizes the "classical" SC LTP.
A contribution of activity-dependent synaptic modifications to the CAFLTP was minimized because the selected cells did not fire action potentials during the experiments. However, evoked transmitter release and presynaptic action potentials were always present during stimulation and could be involved in the genesis of the CAFLTP. To verify or disprove the contribution of presynaptic activity evoked by stimulation, we tested the effects of a pulse of caffeine in the absence of SC stimulation. After a control recording of SC EPSCs, stimulation was stopped and caffeine was applied. The CAFLTP was present (213 ± 28%, n = 3) when synaptic stimulation was resumed after a 60-min waiting period (Fig. 3, A and B). Therefore neither synaptic activity evoked by SC stimulation or postsynaptic action potential activity are necessary for the induction of the CAFLTP.
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We also checked if caffeine was modifying the spontaneous synaptic activity by monitoring in voltage-clamp conditions the frequency and amplitude of spontaneous EPSCs in control conditions and from 10 min after the caffeine challenge (Fig. 3C) when the CAFLTP was growing to reach its maximum amplitude (see Fig. 1A). Pyramidal neurons in the CA1 region are known to display few spontaneous EPSCs in control conditions (e.g., Martín et al. 2001), and there were no significant differences from control values in the frequency and amplitude of spontaneous EPSCs and 10 min after caffeine application as indicated by comparing the corresponding cumulative probability plots of spontaneous EPSCs (Fig. 3D, n = 5). Therefore these results suggest that an increased "spontaneous" glutamate release is not contributing to the CAFLTP.
Caffeine may also modify the membrane conductance of the postsynaptic neuron and hence change the voltage control at the dendritic sites where EPSCs are generated, thus introducing possible artifacts in the recordings of EPSCs. However, there were no differences in the results obtained when K+ conductances were blocked with Cs+ in the pipette solution (n = 3). In addition, we found no significant changes in the rise and decay time constants (
) of EPSCs (fits to single exponential functions) that may reflect membrane conductance modifications at, or near, the sites of EPSC generation (e.g., Mainen et al. 1996). Rise and decay were 5.6 ± 0.6 and 17 ± 1 ms in control and 6.6 ± 1.2 and 13 ± 2 ms 80 min after caffeine application (n = 7), and these values were not statistically significant. We also checked the effects of caffeine on the Vm and Rin by injecting hyperpolarizing current pulses. The Vm depolarized 2.5 ± 0.3 mV and the Rin changed from 124 ± 14 to 139 ± 15 M in control and caffeine conditions, respectively (n = 15). These changes were not statistically significant. Therefore neither changes in voltage control nor in the postsynaptic K+ conductances can account for the observed effects of caffeine.
Classical LTP at SC-CA1 pyramidal neuron synapses does not occlude the CAFLTP
To further investigate the cellular mechanisms of the CAFLTP, we analyzed the effects of the caffeine pulse after evoking a classical LTP. After a brief control stabilization period, we induced the LTP either by tetanizing SCs (sequential delivery of 4 100-Hz 1-s-duration barrages repeated every 20 s; n = 2) or by simultaneously stimulating SCs at 2 Hz and depolarizing the recorded pyramidal cell to 0 mV during 60 s (n = 4). The LTP resulted in a 205 ± 68% increase of peak EPSC amplitude from controls 15 min after induction. Sixty minutes later we tested the effects of caffeine and a CAFLTP was evoked that resulted in a 228 ± 23% increase of peak EPSC amplitude (same cells) superimposed on the classical LTP (Fig. 4A). Therefore the preceding results suggest that different cellular mechanisms trigger both nondecremental potentiations.
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Caffeine does not potentiate postsynaptic glutamate currents
To further test the presynaptic nature of the CAFLTP, we examined the effects of caffeine on currents evoked by micro-iontophoretic glutamate applications. Control currents evoked by microiontophoresis of glutamate and control SC EPSCs were recorded in the same neurons (n = 6). Application of caffeine, evoked the CAFLTP of SC EPSCs (216 ± 21%) but did not significantly modify the glutamate-induced current (110 ± 13%; Fig. 4B). Therefore these results suggest that a rise in postsynaptic glutamate receptor sensitivity does not contribute to the CAFLTP.
Inhibition of NMDA receptors with AP5 does not prevent the CAFLTP
It is generally agreed that induction of the classical LTP at SC-CA1 pyramidal neuron synapses requires the activation of postsynaptic NMDA receptors (e.g., Collingridge and Bliss 1987; Kullmann et al. 1996; Nicoll and Malenka 1999; Poncer and Malinow 2001; Siegelbaum and Kandel 1991).
We therefore investigated the effects of blocking NMDA receptors with AP5 (50 µM) that prevented the classical LTP but did not modify CAFLTP in any of the cells tested (n = 3; Fig. 4C). Therefore the results exclude a contribution of postsynaptic NMDA receptors in the genesis of the CAFLTP.
CAFLTP is insensitive to intracellular BAPTA
The classical SC LTP requires an increase in the intracellular postsynaptic Ca2+ concentration (e.g., Malenka 1991; Nishiyama et al. 2000). To establish if changes in the cytosolic-free Ca2+ concentration due to Ca2+ inflow were involved in the genesis of the CAFLTP, we added the fast Ca2+ chelator BAPTA to the internal pipette solution (e.g., Williams and Johnston 1989; Yeckel et al. 1999). Intracellular BAPTA (40 mM), did not block the CAFLTP (269 ± 24%; n = 8) but prevented the classical SC LTP evoked by tetanization of SCs in the same neurons (Fig. 5). Therefore postsynaptic increases in the intracellular-free Ca2+ concentration due to Ca2+ inflow do not contribute to the CAFLTP.
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The preceding results are consistent with caffeine mediating the CAFLTP acting through presynaptic mechanisms, not depending on evoked synaptic activity, on postsynaptic action potentials, or on increases in the postsynaptic concentration of free Ca2+ and therefore supported by entirely different cellular mechanisms than the classical LTP at SC-CA1 pyramidal neuron synapses.
Block of adenosine P1 and purinergic P2 receptors inhibits the CAFLTP
Adenosine is the end product of ATP hydrolysis and blocks synaptic responses by interacting with specific presynaptic P1 receptors (e.g., Burnstock 1990; Miras-Portugal et al. 2000). Moreover, caffeine blocks P1 receptors (Daly et al. 1983; Fredholm et al. 1999; see following text), suggesting that interactions between caffeine and P1 receptors may contribute to the CAFLTP, by increasing Ca2+ influx during the action potential (see DISCUSSION). Therefore we blocked adenosine P1 receptors with CPT (10 µM; n = 5), which is an A1 type adenosine receptor antagonist, and applied the caffeine pulse 20 min latter. The CPT treatment inhibited the CAFLTP (Fig. 6A), suggesting that inhibition of adenosine P1 receptors by caffeine plays a major role in the induction of the CAFLTP. An increase of the EPSC peak amplitude was evoked by superfusion of 10 µM CPT (141 ± 16%; <40 min; n = 4) in the absence of the caffeine challenge, suggesting that in control conditions extracellular adenosine was inhibiting glutamate release (not shown).
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Caffeine may evoke ATP release from neurons and glia (see DISCUSSION), therefore we tested the effects of piridoxal-5'-phosphate-azophenyl 2',4'-disulphonate (PPADS) and of suramin, which are P2 purinergic receptor antagonists. Super-fusion with PPADS (20 µM) starting 10 min before the caffeine challenge abolished the late sustained phase of the CAFLTP without changing the amplitude of initial phase (Fig. 6B). The magnitude of the initial caffeine potentiation was 223 ± 22% (5 min; n = 5), and EPSCs amplitudes decreased to control values in <40 min. (Fig. 6B). Suramine, at a concentration of 10 µM that does not inhibit glutamate receptors (e.g., Nakatsuka and Gu 2001), reduced the magnitude and duration of the initial potentiation induced by caffeine (148 ± 29%; 5 min; n = 5) and also abolished the late phase of the CAFLTP (Fig. 6C).
To test if block of P2 purinoreceptors inhibited the late phase of the CAFLTP by interaction with processes occurring during the initial phase or conversely if the block occurred during the late phase of the CAFLTP, we first evoked the CAFLTP (248 ± 33%; 10 min) and 10 min after superfused suramin (10 µM; n = 4). The potentiated EPSCs gradually decreased to reach control values in <50 min (Fig. 6D). Therefore these results suggest that activation of P2 purinoreceptors is essential for the genesis of the late phase of the CAFLTP. Suramine did not modify the EPSC from control values (96.9 ± 5%; 40 min; n = 4) in the absence of caffeine.
The preceding results taken together suggest that the CAFLTP requires the inhibition of P1 and the subsequent activation of P2 purinoreceptors (see DISCUSSION).
Ryanodine superfusion blocks the CAFLTP
The preceding results suggest that caffeine acts presynaptically probably by increasing the release of Ca2+ from caffeine/ryanodine-sensitive Ca2+ stores. We tested the effects of blocking the release of Ca2+ from stores with extra- and intracellular-applied ryanodine. We reasoned that bath-applied ryanodine would block both pre- and postsynaptic stores, whereas ryanodine in the pipette solution would only block postsynaptic receptors. When ryanodine was superfused or added to the patch pipette cells were repeatedly depolarized (5 200-ms duration pulses to 0 mV) to induce Ca2+ influx through voltage-gated channels because it has been proposed that a rise in the intracellular Ca2+ concentration is required for ryanodine to interfere with release from intracellular Ca2+ stores (e.g., Caillard et al. 2000). To avoid possible activity-dependent potentiations, presynaptic stimulation was absent during these depolarizations, which were applied 5–10 min before the caffeine challenge.
We superfused with ACSF containing 20 µM ryanodine, a concentration that applied extracellulary irreversibly blocks the ryanodine-sensitive Ca2+ stores (McPherson et al. 1991), and >40 min later we tested the effects of the caffeine challenge. Caffeine induced a transient increase (136 ± 52%; 10 min.; n = 5) of the EPSC amplitude followed by a sustained decrease below control values (68 ± 18%) during the rest of the experiment, indicating that extracellular ryanodine blocked the CAFLTP (Fig. 7A).
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To test if the block of the late phase of the CAFLTP by extracellular ryanodine was due to interaction with presynaptic Ca2+ release from stores occurring during the early or the late phase of the CAFLTP, we superfused with ryanodine (20 µM) 10 min after inducing the CAFLTP (Fig. 7B). The potentiation reached values of 260 ± 24% immediately before the ryanodine challenge (n = 4) and then gradually decreased without reaching control values (209 ± 21, 130 ± 16, and 131 ± 25%, 30, 50, and 70 min, respectively, after caffeine; Fig. 7B). Therefore ryanodine totally inhibited the CAFLTP when super-fused before but not if applied after the caffeine challenge. Ryanodine did not modify the EPSC from control values (102 ± 6%; 40 min; n = 4) in the absence of caffeine.
The preceding results suggest that ryanodine blocks the CAFLTP by interacting with the release of Ca2+ from presynaptic stores during the early phase of the CAFLTP. However, the reduction of the CAFLTP by delayed application of ryanodine (Fig. 7B), may also suggest an important contribution of Ca2+ release intracellular presynaptic stores to the late phase.
However, intracellular ryanodine (200 µM in the pipette solution; see METHODS) applied >20 min after attaining access to the intracellular compartment, had no effect on the CAFLTP (240 ± 20%, n = 5; Fig. 7C).
Therefore this analysis suggests that presynaptic Ca2+ stores contribute to the CAFLTP, whereas postsynaptic Ca2+ stores are not involved.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Presynaptic origin of the CAFLTP
We show that the CAFLTP is associated with a nondecremental reduction of the PPF, a similar and persistent potentiation of both AMPAEPSC and NMDAEPSC, and comparable and sustained modifications in the variance of the amplitudes of both AMPAEPSC and NMDAEPSC. All the preceding effects have been linked with an increased probability of transmitter release (Atwood and Karunanithi 2002; Bekkers and Stevens 1990; Clark et al. 1994; Creager et al. 1980; Isaac et al. 1998; Kullmann 1994; Malinow and Tsien 1990; Poncer and Malinow 2001; Schulz et al. 1994; Stevens and Wang 1994). Therefore the increased probability of release during the CAFLTP reinforces the reliability of transmission between individual SC terminals and CA1 pyramidal neurons.
The PPF (1st point in the preceding paragraph) is a form of short-term presynaptic plasticity characterized by an increased peak amplitude of the second EPSC when it is elicited shortly after (<100 ms) a preceding EPSC (Clark et al. 1994; Creager et al. 1980; Fernández de Sevilla et al. 2002; Poncer and Malinow 2001). The increased second EPSC is thought to be caused by the raise in release probability that results from Ca2+ remaining in the terminal after the first action potential (Kamiya and Zucker 1994). Moreover, changes in the PPF, are considered of presynaptic origin (e.g., Creager et al. 1980; Clark et al. 1994).
Solid support in favor of a presynaptic origin of the CAFLTP was provided by the similar potentiation of both AMPAEPSC and NMDAEPSC (2nd point in the preceding paragraph) and by the similar reductions of the NFCV of both AMPAEPSC and NMDAEPSC (3rd point in the preceding paragraph) during the CAFLTP.
The parallel changes of both AMPAEPSC and NMDAEPSC during the CAFLTP contrast with the classical SC LTP of CA1 pyramidal neurons, where a potentiation of the AMPAEPSC and a reduction of the CV of the AMPAEPSC amplitude take place without changes in the NMDAEPSC. The isolated changes in the AMPAEPSC that parallel the classical SC LTP are interpreted to result from the insertion of functional AMPA receptors at silent synapses (e.g., Isaac et al. 1995, 1998; Kullmann 1994; Malenka and Nicoll 1999; Poncer and Malinow 2001).
The parallel potentiation and CV reductions of both AMPAEPSC and NMDAEPSC during the CAFLTP (present results) are consistent with a presynaptic increase in the number of quanta released because the variance of the signal must increase less than the square of the mean peak EPSC amplitude if the increment has a presynaptic origin (Kullamnn 1994) (see METHODS). Therefore taken together the preceding results can be explained by a presynaptic origin of the CAFLTP mediated by an increased release probability (e.g., Atwood and Karunanithi 2002). In addition, the findings reduce the possibility of a postsynaptic participation triggered by the insertion of functional AMPA receptors at silent synapses.
A CAFLTP can be superimposed on a previously evoked classical LTP (present experiments), suggesting different cellular mechanisms and sites of origin for both types of sustained potentiations. Activation of postsynaptic NMDA receptors is required to induce the classical SC LTP in CA1 pyramidal cells (e.g., Collingridge and Bliss 1987; Nicoll and Malenka 1999), an effect that we can exclude in our experiments because blocking NMDA receptors with AP5 did not modify the CAFLTP. In addition, the response evoked by glutamate iontophoresis is not potentiated by caffeine (present results), an effect that contrasts with the finding that responses evoked by exogenous AMPA are potentiated during the LTP in CA3 pyramidal neurons (e.g., Montgomery et al. 2001). Therefore an increased postsynaptic AMPA receptor sensitivity is not involved in the CAFLTP.
The classical LTP requires an increase in the postsynaptic intracellular concentration of free Ca2+ and is blocked by intracellular BAPTA that chelates Ca2+ and prevents the rise in cytosolic Ca2+ (Clark et al. 1994; Williams and Johnston 1989; Yeckel et al. 1999). However, BAPTA in the patch pipette did not block the CAFLTP but prevented the classical LTP (present results), ruling out a postsynaptic effect due to an increase in the cytosolic-free Ca2+ concentration in the genesis of the CAFLTP.
Purinergic receptors mediate the CAFLTP
Inhibition of A1 type adenosine receptors with CPT prevented the initial potentiation and the subsequent CAFLTP, suggesting that adenosine contributes to the initial induction phase of the CAFLTP. Adenosine inhibits synaptic transmission through the activation of presynaptic P1 adenosine receptors and caffeine acts via an antagonism of adenosine P1 receptors (e.g., Daly et al. 1983; Fredholm et al. 1999). Adenosine is released in the slice preparation and may attain extracellular concentrations that could inhibit glutamate release (Fredholm et al. 1984). Caffeine relieves the presynaptic inhibition by extracellular adenosine (Daly et al. 1983; Fredholm et al. 1999; Greene et al. 1985), thus increasing the inflow of Ca2+ and the probability of transmitter release. Consequently, superfusion with CPT would block P1 adenosine receptors at the presynaptic terminals of SCs, thus preventing the increased quantal release that caffeine would otherwise elicit by inhibiting presynaptic P1 adenosine receptors (see following text).
The late persistent expression phase of the CAFLTP was prevented by the block of P2 purinoreceptors with PPADS or suramin, implying that it requires activation of P2 purinergic receptors. However, to our knowledge there is no mention in the literature that caffeine directly interacts with P2 receptors. Therefore caffeine must evoke the release of ATP that activates P2 receptors from neurons and astrocytes. Evidence exists indicating that caffeine induces the release of ATP from neurons (e.g., Queiroz et al. 1999), and it is extremely likely that caffeine also evokes the release of ATP from astrocytes. Indeed, Ca2+ signals are evoked by the caffeine challenge in astrocytes and ATP is released and propagates the Ca2+ signal to other astrocytes (Cotrina et al. 1998; Golovina et al. 1996; Schipke et al. 2002). Therefore these results suggest that the induction of the CAFLTP requires inhibition of P1 adenosine receptors and the expression of the CAFLTP entails the activation of P2 purinoreceptors by the ATP released from neurons and astrocytes.
Release of Ca2+ from presynaptic stores contributes to the CAFLTP
We show that ryanodine in the intracellular pipette solution, at concentrations one order of magnitude higher than those that block Ca2+ release in CA3 pyramidal cells (Caillard et al. 2000), had no effect on the CAFLTP, excluding a contribution of the release of Ca2+ from postsynaptic caffeine/ryanodine-sensitive stores. However, extracellular ryanodine superfused before the caffeine challenge prevented the CAFLTP, suggesting an action mediated via modifications of the release of Ca2+ from presynaptic caffeine/ryanodine-sensitive stores (e.g., Caillard et al. 2000; Emptage et al. 2001; Krizaj et al. 1999; Liang et al. 2002; McPherson et al. 1991). The presence of a caffeine/ryanodine-sensitive intracellular Ca2+ pool has been well established in CA3 pyramidal neurons (e.g., McPherson et al. 1991; Miller et al. 1996), and the release of Ca2+ from intracellular stores may regulate glutamate release at the SC terminals in (Caillard et al. 2000; Emptage et al. 2001) mossy fiber terminals in the hippocampus (Liang et al. 2002) and hippocampal neurons in culture (e.g., Krizaj et al. 1999) via a Ca2+ -induced Ca2+ -release mechanism. Therefore present results are consistent with the view that an increased release of Ca2+ from presynaptic stores during the initial phase of the CAFLTP is required for the induction of the CAFLTP. In addition, we show that ryanodine applied after inducing the CAFLTP markedly reduced but did not prevent the CAFLTP, suggesting that intracellular Ca2+ release also contributes to the late expression phase of the CAFLTP. The release of Ca2+ would result in a higher intracellular Ca2+ concentration at presynaptic terminals and an increased probability of release by the arriving action potential. However, contrasting results showing no contribution of calcium-induced calcium release in short term plasticity have also been reported in CA1 pyramidal neurons (Carter et al. 2002), suggesting that different types of presynaptic plasticities may require diverse mechanisms.
Cellular mechanism of the caffeine potentiation
It is generally recognized that the Ca2+ concentration at the presynaptic terminal regulates transmitter release (e.g., Atwood and Karunanithi 2002). Caffeine concentrations in the millimolar range release Ca2+ from intracellular ryanodine-sensitive stores by increasing the affinity of the ryanodine receptor for cytosolic-free Ca2+ (e.g., Fredholm et al. 1999; Hernández-Cruz et al. 1995). Caffeine at millimolar concentrations also inhibits P1 adenosine receptors (Fredholm et al. 1999). At lower micromolar concentrations, caffeine acts exclusively via an antagonism of adenosine P1 receptors (e.g., Daly et al. 1983; Fredholm et al. 1999). Adenosine inhibits synaptic transmission by blocking presynaptic N-type Ca2+ channels (e.g., Wu and Saggau 1994). Adenosine is released in the slice preparation and may attain extracellular concentrations that could inhibit glutamate release (Fredholm et al. 1984, 1999). Caffeine by blocking P1 receptors relieves the presynaptic inhibition by extracellular adenosine (Daly et al. 1983; Fredholm et al. 1999; Greene et al. 1985), thus increasing the inflow of Ca2+ evoked by the action potential invading the terminal. The increased Ca2+ inflow triggers a larger release of Ca2+ from the caffeine/ryanodine sensitive stores through the sensitized (by caffeine) Ca2+-activated Ca2+-release mechanism. These effects of caffeine are initiated without the need of presynaptic action potential activity (present results), but they become manifest as an increased release probability when the action potential invades the terminal (present results).
Although the underlying molecular machinery of the CAFLTP remains to be discovered, the cooperative effects of a block of presynaptic adenosine receptors—that would increase Ca2+ influx through voltage-gated N channels—and the sustained increase in Ca2+ sensitivity of the presynaptic ryanodine-sensitive Ca2+stores could provide the rise in presynaptic Ca2+ that evokes the increased transmitter release.
In conclusion, the CAFLTP requires the interaction of caffeine with presynaptic P1, P2 purinoreceptors, and ryanodine receptors and is caused by an increased probability of glutamate release at SC terminals. Therefore this CAFLTP is mediated by entirely different mechanisms than those that have been proposed to underlie the "classical" LTP.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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This work was supported by Dirección General de Investigación Científica y Tecnológica, Ministerio de Educación y Ciencia, Spain (PM98-0113) and Comunidad Autónoma de Madrid (CAM) (08.5/00361998) grants to W. Buño. E. D. Martín was a CAM postdoctoral fellow.
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FOOTNOTES |
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Address for reprint requests: W. Buño, Instituto Cajal, Doctor Arce 37, Madrid 28002, Spain (E-mail: wbuno{at}cajal.csic.es).
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REFERENCES |
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) showing the time course of caffeine effects (
, as in other figures) on normalized (to control values) mean peak EPSCs amplitudes. Note the nondecremental potentiation (CAFLTP). C: summary data of the persistent reduction of the paired-pulse facilitation (PPF) index. D: plot of normalized 1/CV2 (CV = coefficient of variation of peak EPSC amplitudes; n = 7) vs. the normalized M (mean EPSC peak amplitudes: see METHODS) 10, 40, and 70 min after caffeine. E: in the absence of caffeine there were no significant changes in the average peak EPSC amplitude (n = 7). Holding potential was -70 mV and vertical bars show SE (as in all other cases).
) and NMDAEPSCs amplitude (+60 mV; 
5 cells.
