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MS 9594 Received 6 May 1999; accepted after revision 6 August 1999.
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ABSTRACT |
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INTRODUCTION |
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Long-term potentiation (LTP) is a form of activity-dependent increase in synaptic efficacy that has been proposed as the cellular and molecular basis for learning and memory. Although a vast array of molecules and processes have been reported to be involved in LTP induction and expression, the true nature and physiological relevance of this long-lasting form of synaptic plasticity remain uncertain. Over the past 20 years, the most extensive characterization of the molecular mechanisms involved in the induction and maintenance of LTP has been undertaken in the Schaffer collateral synapses onto CA1 pyramidal cells in the hippocampus (Bliss & Collingridge, 1993). In physiological conditions, the primary event in LTP induction is an influx of calcium ions into the postsynaptic cell (Bliss & Collingridge, 1993). This is normally produced by the activation of N-methyl-D-aspartate (NMDA) receptors under conditions of postsynaptic depolarization that remove the voltage-dependent blockade of the ion channel associated with those receptors by Mg2+ (Ascher & Nowak, 1987). The increase in intracellular Ca2+ concentration in turn triggers a cascade of events leading to the expression of LTP. Although the molecular events responsible for LTP expression and their cellular location are notoriously controversial at the present time, it is believed that the expression is due to increased transmitter release (Bekkers & Stevens, 1990; Malinow & Tsien, 1990) or is mediated by an upregulation of the 
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated response (Lynch et al. 1990; Mainen et al. 1998). Hence the induction and the expression of CA1 LTP are mediated by NMDA and AMPA receptors, respectively (Bliss & Collingridge, 1993).
Evidence is accumulating that several protein kinases are critically involved in the production of LTP. Included are Ca2+-calmodulin-dependent protein kinase II (CaMKII; Malenka et al. 1989; Malinow et al. 1989), protein kinase C (PKC; Malenka et al. 1986; Malinow et al. 1989), protein kinase A (PKA; Frey et al. 1993; Blitzer et al. 1995), protein kinase G (PKG; Zhuo et al. 1994) and protein tyrosine kinase (PTK; O'Dell et al. 1991; Grant et al. 1992; Cavus & Teyler, 1996; Lu et al. 1998). In the last few years, a great deal work has gone into elucidating the case for the involvement of CaMKII, PKC and PKA in the induction of LTP and has provided strong evidence that these protein kinase activities are required for the induction and maintenance of LTP (Bliss & Collingridge, 1993). Relatively little is known about the involvement of PTK in LTP induction and maintenance. The idea of the involvement of the PTK-related signalling cascade in mechanisms of a LTP induction was first presented by O'Dell et al. (1991), who found that pharmacological inhibitors of PTK produce diminished LTP at synapses in the CA1 area of the hippocampus. In addition, gene-knockout mice lacking the gene for the non-receptor tyrosine kinase Fyn but not Src show greatly impaired LTP induction and spatial learning (Grant et al. 1992; Kojima et al. 1997). Furthermore, a recent study also reported that the inhibitor that targets Src family kinases is able to inhibit the induction of LTP in the hippocampal CA1 neurons, suggesting that an increase in Src family kinase activity is also responsible for the induction of CA1 LTP (Lu et al. 1998). Taken together, these results strongly suggest that PTK activity could be required for LTP in the hippocampus. In light of these previous reports, the goal of the present study was to answer the following. (1) Where is the locus of the PTK inhibitors acting to block LTP? Is it pre- or postsynaptic? (2) How might PTK activity contribute to the induction of LTP? What is the possible target for PTK in the induction of LTP? (3) Besides tetanus-induced LTP, do PTK inhibitors also affect other forms of long-term synaptic plasticity in the hippocampus? The results provide further strong evidence that postsynaptic Src family PTK activity plays an essential role in the induction and the early stabilization processes of LTP at Schaffer collateral-CA1 synapses and that anoxia-induced LTP also requires PTK activity.
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METHODS |
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Slice preparation
Hippocampal slices (400 µm thick) were prepared from 28- to 35-day-old male Sprague-Dawley rats for intra- and extracellular synaptic recordings by the procedures described previously (Hsu & Huang, 1997). The rats were stunned and killed by cervical dislocation. The experiments were performed in accordance with the regulations of the University of Cheng-Kung and Cheng Kung Animal Care Committee. The transverse slices were cut from a tissue block of the brain using a Vibroslice (Campden Instruments, Silbey, UK). The slices were placed in a holding chamber of artificial cerebrospinal fluid (ACSF) oxygenated with 95 % O2-5 % CO2 and kept at room temperature for at least 1 h before recording. The composition of the ACSF was (mM): NaCl, 117; KCl, 4·7; CaCl2, 2·5; MgCl2, 1·2; NaHCO3, 25; NaH2PO4, 1·2; and glucose, 11; at pH 7·3-7·4, equilibrated with 95 % O2-5 % CO2. In some experiments, MgCl2 was reduced to 0·1 mM to enhance the NMDA receptor-channel conductance.
Electrophysiological recordings
A single slice was transferred to the recording chamber, in which it was held submerged between two nylon nets and maintained at 32 ± 1°C. The chamber consisted of a circular well of a low volume (1-2 ml) and was perfused constantly at a rate of 2-3 ml min-1. The CA1 region was surgically isolated from the CA3 region to prevent any epileptiform activity in CA3 cells from affecting the recordings in CA1. Bipolar stainless steel electrodes were placed in stratum radiatum to activate Schaffer collateral/commissural afferents at 0·033 Hz. The stimulus intensity was adjusted to subthreshold for action potential initiation. The strength of synaptic transmission was quantified by measuring the initial slope of EPSPs. LTP was elicited by applying a tetanus (100 Hz, 1 s) two times with an intertetanus interval of 20 s. Intracellular EPSPs were made from CA1 pyramidal neurons using glass microelectrodes filled with 4 M potassium acetate (80-100 M
). Extracellular field EPSPs (fEPSPs) were recorded in stratum radiatum with microelectrodes filled with 1 M NaCl (2-3 M). Microelectrodes were pulled from 1·0 mm microfibre capillary tubing on a Brown-Flaming electrode puller (Sutter Instruments, San Rafael, CA, USA). Electrical signals were amplified by an Axoclamp-2B amplifier (Axon Instruments, Foster City, CA, USA) and an Intel Pentium-based computer with pCLAMP software (versions 6.0.3 or 7.0, Axon Instruments) was used to on-line acquire and analyse the data. Lavendustin A, EPQ(pY)EEIPIA, or EPQYEEIPIA dissolved in 4 M potassium acetate solution were administered intracellularly by hyperpolarization current injection (0·1-0·3 nA) applied through the recording microelectrode for 30 min before tetanization, according to the methods described previously (Hsu & Huang, 1997).
Drug application
All drugs were applied by dissolving them to the desired final concentrations in ACSF and by switching the perfusion from control ACSF to drug-containing ACSF. Appropriate stock solutions of drugs were made and diluted with ACSF just before application. Genistein, lavendustin A, lavendustin B, diadzein, forskolin, 3-isobutyl-L-methylxanthine (IBMX) and phorbol 12,13-dibutyrate (PDBu) were dissolved in dimethylsulfoxide (DMSO) stock solutions and stored at -20°C until the day of experiment. The concentration of DMSO in the perfusion medium was 0·05-0·1 %, which alone had no effect on LTP (Hsu & Huang, 1997). The anoxic episodes were produced by switching to ACSF equilibrated with 95 % N2-5 % CO2. The exchange of solution produced a delay of 0·5 min before the new solution reached the recording chamber. Lavendustin A, lavendustin B, genistein, diadzein, PDBu, TEA and D-2-amino-5-phosphonopentanoic acid (D-APV) were purchased from Research Biochemicals International (Natick, MA, USA); forskolin, IBMX, picrotoxin and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were obtained from Sigma (St Louis, MO, USA). EPQ(pY)EEIPIA and EPQYEEIPIA were synthesized by Princeton Biomolecules (Columbus, OH, USA).
Statistical analysis
Results are expressed as means ± S.E.M. The significance of difference was evaluated by Student's two-tailed t test. Numbers of experiments are indicated by n. Probability values (P) of less than 0·05 were considered to be significant.
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RESULTS |
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Effects of PTK inhibitors on tetanic LTP induction
Orthodromic stimulation of the Schaffer collateral/commissural afferents evoked an excitatory postsynaptic potential (EPSP) in hippocampal CA1 neurons. The initial slope of the EPSP was used as a measure of synaptic strength. In the first series of experiments we tested whether PTK inhibitors could effectively block the induction of LTP. Following 15 min of stable baseline recording, a brief tetanic stimulation produced an immediate potentiation of EPSP that lasted for at least 40 min (Fig. 1A). The initial slope of the EPSP was 151·2 ± 12·5 % of baseline measured 30 min after tetanic stimulation (Fig. 1B, n = 10). These experiments showed that our tetanization protocol could effectively induce LTP at Schaffer collateral-CA1 synapses. In order to determine whether PTK activity is necessary for LTP, two specific PTK inhibitors, genistein and lavendustin A, were used. As shown in Fig. 1C, application of genistein (100 µM) alone in the bath caused a minor but significant reduction of the EPSP slope (8·9 ± 2·5 % of baseline, n = 8, P < 0·05) and prevented the subsequent induction of LTP. The average magnitude of the initial slope of the EPSP measured 30 min after the tetanic stimulation in the presence of genistein was 108·2 ± 11·9 % (n = 8, P < 0·05) of baseline (Fig. 1D). Lavendustin A (10 µM) had no significant effect on basal synaptic transmission (98·7 ± 1·7 % of baseline, n = 8, P = 0·67) and also prevented the subsequent LTP induction. The mean initial slope of the EPSP measured 30 min after the tetanic stimulation was 102 ± 6·9 % (n = 8, P < 0·05) of baseline (Fig. 1F). Daldzein is a structural analogue of genistein that does not inhibit PTKs. It had no significant effect on either the basal synaptic transmission or the induction of LTP. The mean potentiation in six neurons tested was 46·8 ± 8·9 % of baseline measured 30 min after tetanic stimulation (Fig. 1H). These results indicate that PTK activity plays an essential part in the induction of CA1 LTP.
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A, an example of the time course of homosynaptic LTP at Schaffer collateral-CA1 synapses. Superimposed traces were taken 5 min before (1) and 30 min after (2) tetanic stimulation (TS). The initial slope of the EPSP exhibited approximately 70 % potentiation following TS, which slowly decayed during the first 10 min and remained stable at 50 % increase thereafter. B, summary of data from ten experiments performed as in A. C, application of genistein (100 µM) alone caused a significant reduction of EPSP slope and prevented the subsequent LTP induction. Insets are superimposed traces taken at different times, as indicated. D, plots of the pooled data from eight experiments performed as in C. E, lavendustin A (10 µM) had no significant effect on the EPSP and also prevented the subsequent LTP induction. The superimposed EPSPs were taken at different times, as indicated. F, pooled data from eight experiments performed as in E. G, summary of all experiments in B, D and E. H, bar plots represent the percentage change of EPSP slope measured 30 min after TS at different manipulations, as indicated. Note that daldzein (100 µM), an inactive analogue of genistein, had no significant effect on the induction of LTP by TS (n = 6). Bars in C-G denote the period of delivery of genistein or lavendustin A. *P < 0·05 as compared with the control group. Calibration: 5 mV, 20 ms, applies to A, C and E. | ||
Recently, accumulating evidence has indicated that LTP induction in physiological conditions is reversed by a number of pharmacological manipulations. For example, antibodies to the neuronal cell adhesion molecules L1 and NCAM produce a marked decrease in the initial potentiation in vitro when administered within 1-2 min after delivery of LTP induction stimuli, but not thereafter (Lüthi et al. 1994; Muller et al. 1996). Because PTK activation is known to be part of the signalling cascade during L1 or NCAM activation (Beggs et al. 1994; Ignelzi et al. 1994), it was of particular interest to examine whether PTK inhibitors could also exert a time-dependent reversal of LTP similar to L1 and NCAM antibodies. Thus, the effect of lavendustin A administered after application of tetanic stimulation was investigated. Figure 2 summarizes experiments in which application of lavendustin A (10 µM) was initiated 3 min after (A), 10 min after (C), or 30 min after the induction of LTP (E). The application begun 3 min after tetanic stimulation revealed a significant reduction of the degree of LTP compared with the control (124·2 ± 8·2 % of baseline, n = 5, P < 0·05). However, application 10 or 30 min after tetanic stimulation showed no influence on the established LTP (Fig. 2C-F). The magnitude of LTP in slices exposed to lavendustin A 10 or 30 min after tetanic stimulation was 151·2 ± 8·6 % (n = 5) and 146·2 ± 7·6 % of baseline (n = 5), respectively, not significantly different from control values. The ability of lavendustin A to reverse the established LTP in a time-dependent manner may reflect that PTK influences LTP not only in the initial induction processes but also in the early stabilization processes.
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A-F, examples in which LTP was induced at Schaffer collateral-CA1 synapses. The specific PTK inhibitor lavendustin A (10 µM) was applied at various times after LTP induction: A, 3 min after; C, 10 min after; or E, 30 min after TS. B, D and F, summary of experiments similar to those shown in A, C and E, respectively. Note that lavendustin A erased potentiation when delivered 3 min after TS, but was without effect when applied 10 or 30 min after. The superimposed EPSPs in the inset of each graph illustrate representative recordings from example experiments taken at the times indicated by the numbers. Bars denote the period of delivery of lavendustin A. Calibration: 5 mV, 20 ms, applies to A, C and E. | ||
Since external superfusion of PTK inhibitors will affect both pre- and postsynaptic PTK activity, it was important to determine whether preventing the activation of postsynaptic PTK activity would affect the induction of LTP. To accomplish this, cells were impaled with microelectrodes filled with lavendustin A (1 mM). Figure 3A shows an example of an intracellular EPSP recording in response to tetanic stimulation in a lavendustin A-loaded cell. An extracellular fEPSP was monitored simultaneously with an intracellular EPSP to ensure that LTP was induced in the population of cells that were not subjected to lavendustin A. After a tetanic stimulation, transmission to the population of cells monitored with extracellular fEPSPs showed normal LTP. In contrast, transmission to the cell impaled with the lavendustin A-containing microelectrode showed no persistent enhancement of the EPSP. After a tetanic stimulation, the initial slope of the EPSP decayed to the baseline level within 20 min. The mean EPSP slope of the lavendustin A-loaded cells measured 30 min after tetanic stimulation was 103·2 ± 6·9 % of baseline (n = 5). In contrast, the slope of the fEPSP was significantly enhanced by tetanic stimulation (155·6 ± 8·2 % of baseline, n = 5; Fig. 3B). As a control for non-specific effects of lavendustin A, the inactive analogue lavendustin B (1 mM; Onoda et al. 1989) was also used in a separate experiment. In all three lavendustin B-loaded cells tested, tetanic stimulation resulted in normal LTP. The mean EPSP slope of lavendustin B-loaded cells measured 30 min after tetanic stimulation was 143·9 ± 8·6 % (n = 3) of baseline (data not shown). These results generally confirmed previous studies showing that the activation of postsynaptic PTK is necessary for the induction of CA1 LTP (O'Dell et al. 1991; Lu et al. 1998).
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A, example of an experiment in which lavendustin A (1 mM) in the recording microelectrode prevented LTP. In this experiment, an extracellular fEPSP ( | ||
Lavendustin A does not affect the PDBu-, forskolin-IBMX- or TEA-induced synaptic enhancement
The major problem with the use of pharmacological inhibitors to identify a biochemical event is their specificity. Because the induction of LTP has been reported to be disrupted by inhibitors of PKC (Malenka et al. 1989; Malinow et al. 1989), CaMKII (Malenka et al. 1989; Malinow et al. 1989) or PKA (Frey et al. 1993; Blitzer et al. 1995), it was important to check whether the blockade of the induction of LTP by genistein or lavendustin A is due to the non-specific effect of these two agents on serine/threonine kinase activity. To exclude this possibility, we performed three further experiments. First, we examined whether the long-term synaptic enhancement elicited by PDBu, a known activator of PKC, was antagonized by lavendustin A. As illustrated in Fig. 4A, application of PDBu (10 µM) caused a pronounced and long-lasting increase in the synaptic response. The average magnitude of the potentiation measured 10 min following washout of PDBu was 48·5 ± 5·8 % (n = 6). When the slices were perfused with lavendustin A at 10 µM, a concentration which could block the induction of tetanic LTP, there was no significant effect on PDBu's action. Following 10 µM PDBu, an increase of 49·3 ± 4·6 % (n = 6, P = 0·59) in the initial slope of the EPSP was found in the lavendustin A-pretreatment group (Fig. 4B). These data show that lavendustin A (10 µM) does not affect the PKC activity in the CA1 neurons.
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A, application of the PKC activator PDBu (10 µM) caused a pronounced and long-lasting increase in the initial slope of the EPSP (n = 6). Washing the slices with normal ACSF did not reverse the effect of PDBu. B, pretreatment of the hippocampal slices with lavendustin A (10 µM) had no effect on PDBu-induced LTP (n = 6). The superimposed EPSPs in the inset of each graph illustrate representative recordings from example experiments taken at the times indicated by the numbers. Bars denote the period of delivery of PDBu or lavendustin A. Calibration: 5 mV, 20 ms. | ||
Second, we examined the effect of lavendustin A on forskolin-induced synaptic enhancement in the hippocampal CA1 neurons. It is known that the increase in synaptic strength produced by forskolin results from an increase in adenylyl cyclase activity and in intracellular cAMP levels. cAMP then activates PKA. Thus, we can evaluate the effect of lavendustin A on forskolin's enhancement of the synaptic strength as a reflection of its action on PKA activity. In this set of experiments, 3-isobutyl-1-methylxanthine (IBMX) was used to potentiate the forskolin effect by protecting cAMP from hydrolysis. In agreement with a previous report (Chavez-Noriega & Stevens, 1992), application of forskolin (15 µM) and IBMX (50 µM) to the hippocampal slice preparation caused a pronounced increase in the initial slope of the EPSP. The average magnitude of potentiation measured 15 min following washout of forskolin-IBMX was 38·5 ± 5·2 % of baseline (n = 8; Fig. 5A). Application of lavendustin A (10 µM) had no discernible effect on the forskolin-IBMX-induced enhancement of synaptic response (39·2 ± 4·8 % of baseline , n = 6, P = 0·57; Fig. 5B). These results indicate that lavendustin A (10 µM) has no significant effect on the PKA activity.
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A, simultaneous application of forskolin (15 µM) and IBMX (50 µM) resulted in a long-lasting increase in the initial slope of the EPSP (n = 8). B, application of lavendustin A (10 µM) had no significant effect on the forskolin-IBMX-induced synaptic potentiation (n = 6). Insets are superimposed traces taken at different times, as indicated. Bars denote the period of delivery of forskolin-IBMX or lavendustin A. Calibration: 5 mV, 20 ms. | ||
Third, we tested whether the inhibition of LTP induction by PTK inhibitors is due to their non-specific blocking effect on CaMKII activity. Extracellular application of the potassium channel blocker TEA has been shown to elicit a long-lasting synaptic potentiation in the CA1 region of the hippocampus (Huang & Malenka, 1993) and this synaptic enhancement is due to activation of postsynaptic dihydropyridine-sensitive voltage-dependent Ca2+ channels (VDCCs). Moreover, it has been reported that the TEA-induced synaptic enhancement was also specifically attenuated by KN-62, a selective inhibitor of CaMKII (Huber et al. 1995), indicating that the activation of CaMKII is involved in TEA's action. Hence, changes in TEA-induced synaptic potentiation should be valuable as an assay for the modulation of CaMKII activity. We therefore attempted to examine the effect of lavendustin A on CaMKII by monitoring TEA's action. In agreement with a previous report (Huang & Malenka, 1993), a 7 min application of TEA (25 mM) was followed by a long-lasting increase in the initial slope of the EPSP. The average magnitude of potentiation measured 15 min following washout of TEA was 110·5 ± 5·4 % of baseline (n = 5; Fig. 6A). Pretreatment of the hippocampal slices with lavendustin A (10 µM) had no significant effect on the magnitude of TEA-induced synaptic potentiation (108·3 ± 5·7 % of baseline, n = 5, P = 0·69; Fig. 6B), indicating that the concentration of lavendustin A (10 µM) that blocks LTP induction does not affect the CaMKII activity.
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A, application of TEA (25 mM) caused a long-lasting increase in the initial slope of the EPSP (n = 5). B, summary of five experiments in which TEA was applied in the presence of lavendustin A (10 µM). Note that lavendustin A did not affect the TEA-induced LTP (n = 5). Insets are superimposed traces taken at different times, as indicated. Bars denote the period of delivery of TEA or lavendustin A. Calibration: 5 mV, 20 ms. | ||
Effects of PTK inhibitors on NMDA receptor- and AMPA receptor-mediated component of EPSPs
Because the induction and the expression of tetanic LTP at Schaffer collateral-CA1 synapses are mediated by NMDA and AMPA receptors, respectively, we next examined whether the blockade of tetanic LTP by PTK inhibitors is due to direct inhibition of the basal NMDA or AMPA receptor activation. To address this question, we examined the effect of PTK inhibitors on the pharmacologically isolated NMDA receptor- and AMPA receptor-mediated component of the EPSP. In the first series of experiments the effect of genistein and lavendustin A on a pure NMDA receptor-mediated EPSP (EPSPNMDA) was examined. To isolate the EPSPNMDA, ACSF containing a high concentration of the AMPA receptor antagonist CNQX (20 µM), to abolish the AMPA receptor-mediated component of the EPSP, and the 
-aminobutyric acid (GABAA) receptor antagonist picrotoxin (50 µM), to abolish the GABAA-mediated inhibitory postsynaptic potentials (IPSPs), was perfused. Under these conditions the remaining EPSP had a long rise time and was completely blocked by application of the NMDA receptor antagonist D-APV (25 µM), indicating that it was mediated by NMDA receptors. Synaptic responses were enhanced by maintaining slices in 0·1 mM magnesium and 10 µM glycine, a condition that significantly increases NMDA receptor-channel conductance. As shown in Fig. 7A, the isolated EPSPNMDA was not affected by either genistein (100 µM, 96·8 ± 2·9 % of baseline at 20 min, n = 4, P = 0·31) or lavendustin A (10 µM, 100·2 ± 1·5 % of baseline at 20 min, n = 4, P = 0·62).
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A, the NMDA receptor-mediated EPSP (EPSPNMDA) was recorded in the continuous presence of the non-NMDA receptor antagonist CNQX (20 µM) and the GABAA receptor antagonist picrotoxin (50 µM). The recording solution contained 0·1 mM Mg2+ and 10 µM glycine to enhance the NMDA receptor-channel conductance. Top, traces from example cells showing neither genistein (100 µM) nor lavendustin A (10 µM) had a significant effect on the EPSPNMDA. Bottom, plot of the percentage change of the amplitude of the EPSPNMDA in the presence of genistein ( | ||
To isolate the AMPA receptor-mediated EPSP (EPSPAMPA), experiments were carried out in the presence of both the NMDA receptor antagonist D-APV (50 µM) and the GABAA receptor antagonist picrotoxin (50 µM), leaving an isolated AMPA receptor-mediated EPSP. The remaining EPSP was completely blocked by application of the AMPA receptor antagonist CNQX (10 µM), indicating that it was mediated by the activation of AMPA receptors. As illustrated in Fig. 7B, genistein (100 µM) but not lavendustin A (10 µM) exerted a minor inhibition of the isolated EPSPAMPA. The mean EPSPAMPA amplitude measured 20 min after the application of genistein or lavendustin A was 92·6 ± 3·4 % (n = 4, P < 0·05) and 98·2 ± 2·1 % of baseline (n = 4, P = 0·41), respectively. These results imply that the basal NMDA or AMPA receptor-mediated synaptic transmission was not modulated tonically by basal PTK activity in the hippocampal CA1 neurons.
Non-receptor PTK Src participates in tetanic LTP
Having observed the inhibition of tetanic LTP induced by PTK inhibitors, we went on to identify the specific tyrosine kinase which is required in the postsynaptic cell during the induction of LTP. PTKs comprise two main classes, receptor-coupled and non-receptor PTKs (Ullrich & Schlessinger, 1990). The former are integral components of neurotrophin or growth factor receptors and regulate cell survival, growth and differentiation (Ullrich & Schlessinger, 1990). Non-receptor PTKs are components of intracellular signalling cascades, but their functions in mature neurons are poorly understood. It has been shown that Src family PTKs are widespread in the CNS with a high level of expression in the hippocampus (Sugrue et al. 1990), and may also play a role in the regulation of the NMDA receptor-channel activity (Yu et al. 1997). Because Src family PTKs could regulate the function of the NMDA receptor, which is necessary for the induction of tetanic LTP at Schaffer collateral-CA1 synapses, we set out to determine whether Src family PTKs participate in CA1 LTP. If Src is specifically associated with tetanic LTP, the sustained increase of Src activity should occlude the induction of LTP. To accomplish this, cells were impaled with microelectrodes filled with an activator of Src family kinases, EPQ(pY)EEIPIA (5 mM). Because EPQ(pY)EEIPIA has a high affinity for the SH2 domain of Src family kinases and the SH2 domain is known to be involved in negatively regulating Src kinase activity through intramolecular binding of the phosphorylated tail of this kinase, intracellular application of this peptide could compete for this intramolecular binding and therefore preclude the auto-inhibition of Src family kinases (Liu et al. 1993; Salter, 1998). Figure 8A shows an example of an intracellular EPSP recording in an EPQ(pY)EEIPIA-loaded cell. An extracellular fEPSP was monitored simultaneously with an intracellular EPSP to ensure that tetanic LTP was induced in the population of cells that were not subjected to Src activator. Application of EPQ(pY)EEIPIA produced a gradual and sustained enhancement of the slope of the EPSP. The mean initial slope of the EPSP of EPQ(pY)EEIPIA-loaded cells 30 min after the recordings was 186·7 ± 15·4 % of baseline (n = 4; Figs. 8A and B). Furthermore, the tetanic LTP induced by high frequency stimulation was not obtained in all four EPQ(pY)EEIPIA-loaded cells tested. In contrast, the fEPSP slope was significantly and persistently increased by 52·3 ± 11·3 % (n = 4) after tetanic stimulation (Fig. 8B). However, the non-phosphorylated form of the peptide, EPQYEEIPIA, which does not activate Src, had no effect on the basal synaptic transmission or the induction of tetanic LTP (Fig. 8C and D). The mean initial slope of the EPSP of the EPQYEEIPIA (5 mM)-loaded cells measured 30 min after a high frequency tetanic stimulation was 146·3 ± 14·5 % of baseline (n = 3, P > 0·05). These results indicate that the enhancement of EPSPs by the activation of Src family kinases and tetanus-induced LTP was mutually occluded and may share a common induction cellular mechanism.
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A, intracellular application of the Src family kinases activator EPQ(pY)EEIPIA (5 mM) resulted in a pronounced increase in the initial slope of the EPSP ( | ||
PTK inhibitors block the induction of anoxic LTP
Anoxic LTP is a long-lasting increase in synaptic effectiveness that follows a transient anoxic episode (Crépel et al. 1993; Hsu & Huang, 1997) and this phenomenon is found particularly at hippocampal Schaffer collateral-CA1 synapses and has been proposed as an early marker of delayed neuronal death induced by transient forebrain ischaemia (Crépel et al. 1993; Hammond et al. 1994). Because recent studies have shown that the neuronal loss following ischaemia and accompanying tyrosine phosphorylation can be alleviated by PTK inhibitors (Kindy, 1993; Ohtsuki et al. 1996), we have been prompted to examine whether PTK activity is required for the induction of anoxic LTP. To address this issue, we examined the effects of PTK inhibitors on the induction of anoxic LTP of the EPSPNMDA. A representative record, shown in Fig. 9A, illustrates the general sequence of changes in the initial slope of the EPSPNMDA of a hippocampal CA1 neuron due to an anoxic episode. In agreement with previous studies (Hsu & Huang, 1997; Huang & Hsu, 1997), a brief anoxic episode (3 min duration) strongly depressed the EPSPNMDA. The mean maximum depression in 15 slices tested was 94·3 ± 3·2 % after 3 min of anoxia (Fig. 9B). On return to reoxygenated medium the EPSPNMDA returned to control baseline within 10 min and was subsequently and progressively potentiated to reach a plateau 15-20 min after return to oxygen. This post-anoxic synaptic potentiation lasted for >1 h and was observed in 12 out of 15 slices tested. The degree of this long-lasting synaptic potentiation following an anoxic episode varied in different cells, ranging from 25 to 92 % (mean percentage of baseline: 65·2 ± 7·6 %, n = 15). As shown in Fig. 9C, superfusion of the hippocampal slices with genistein (100 µM) alone had no significant influence on baseline synaptic transmission or the reduction of the EPSPNMDA during the period of anoxia. However, the subsequent anoxia-induced LTP was completely blocked in 11 out of 12 slices tested. The mean initial slope of the EPSPNMDA measured 50 min after the return to reoxygenated medium was 106·4 ± 7·2 % of baseline (n = 12, P < 0·05; Fig. 9D). Similarly, when the slices were perfused with lavendustin A (10 µM), the anoxic LTP was also prevented. The initial slope of the EPSPNMDA was 105·7 ± 6·9 % of baseline (n = 8, P < 0·05) measured 50 min after return to oxygenated medium (Fig. 9E and F). However, the inactive genistein analogue daldzein (100 µM) had no significant effect on either the anoxia-induced synaptic depression or the anoxic LTP (159·7 ± 6·9 % of baseline, n = 6, P = 0·65; Fig. 9H). These data indicate that the induction of anoxic LTP requires the activity of PTKs in the hippocampal CA1 neurons.
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A, example of an experiment showing the effect of a short anoxic episode on the pharmacologically isolated EPSPNMDA. The percentage change in the initial slope of the EPSPNMDA of the hippocampal CA1 neurons due to the anoxic episode (3 min) was plotted as a function of time. Note that the synaptic response was progressively decreased during the anoxic episode. Following the switch from anoxic to reoxygenated medium, the EPSPNMDA returned to the control baseline within 10 min and was subsequently and persistently potentiated. B, summary of fifteen experiments similar to that shown in A. C and E, pretreatment of the hippocampal slices with the PTK inhibitors genistein (100 µM) or lavendustin A (10 µM) effectively blocked the subsequent potentiation of synaptic transmission induced by an anoxic episode. D and F, summary of twelve and eight experiments like that shown in C and E, respectively. However, neither genistein nor lavendustin A affected the anoxia-induced synaptic depression. G, summary of all experiments in B, D and F. H, bar plots representing the percentage change of the initial slope of the EPSPNMDA measured 50 min after the anoxic episode at different manipulations, as indicated. Note that daldzein (100 µM) had no effect on either anoxia-induced synaptic depression or anoxia-induced synaptic potentiation (n = 6). Insets in A, C and E are superimposed traces taken at different times, as indicated. Bars denote the period of the anoxic episode or the application of genistein or lavendustin A. *P < 0·05 as compared with the control group. Calibration: 5 mV, 50 ms, applies to A, C and E. | ||
Figure 10 summarizes all the experiments that we have performed to compare the effect of PTK inhibitors on the induction of multiple forms of long-lasting synaptic potentiation. PTK inhibitors, genistein and lavendustin A, specifically blocked the induction of tetanic LTP and anoxic LTP, but had no discernible effect on PDBu-, forskolin- IBMX- or TEA-induced LTP. Moreover, daldzein, an inactive structural analogue of genistein, failed to affect any forms of long-term synaptic plasticity tested in this study.
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Note that PTK inhibitors, genistein (100 µM) and lavendustin A (10 µM), specifically blocked the induction of tetanic LTP and anoxic LTP, but were without effect on the PDBu-, forskolin-IBMX- or TEA-induced LTP at Schaffer collateral-CA1 synapses. | ||
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DISCUSSION |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Through the use of specific inhibitors and an activator of PTKs, we have provided strong evidence that PTK activity is necessary for the induction of LTP produced by tetanic stimulation or an anoxic episode at Schaffer collateral-CA1 synapses. Microinjection of PTK inhibitor into postsynaptic cells significantly blocked LTP induction, suggesting that PTKs act postsynaptically. Directly activating Src family kinases in the postsynaptic cells produced a long-lasting synaptic potentiation and occluded tetanic LTP. Moreover, PTK inhibitors did not tonically affect the basal synaptic transmission mediated by either AMPA or NMDA receptors and affected none of the PDBu-, forskolin-IBMX- or TEA-induced synaptic potentiation.
Do PTKs have a signalling role in LTP?
The case for the involvement of PTK in LTP induction was first reported by O'Dell et al. (1991), who found that PTK inhibitors are able to inhibit the induction of LTP in area CA1 of rat hippocampal slices in vitro. Moreover, an in vivo study has also shown that i.c.v. injection of PTK inhibitors blocks the formation of LTP in dentate gyrus (Abe & Saito, 1993). In the present study, we used both the PTK inhibitors and an activator to identify more specifically the role of PTKs in long-term synaptic plasticity induced by a variety of manipulations. We have provided strong evidence that the induction of tetanic LTP requires PTK activity in the postsynaptic cells (Fig. 3). However, which type of PTK expressed in the CA1 neurons is essential for LTP induction? In the present study LTP induced by 100 Hz stimulation was blocked completely by the non-receptor PTK Src family kinase activator EPQ(pY)EEIPIA, strongly demonstrating that Src family kinases are the potential candidates for the PTKs involved in LTP induction. Because EPQ(pY)EEIPIA can bind one or more members of the Src family PTKs containing a SH2-binding domain (Liu et al. 1993; Salter, 1998), we do not know the identity of the Src family PTK(s) that are responsible for LTP induction. Further experiments are needed to resolve this issue.
Do PTKs have a signalling role in LTP stabilization processes?
A surprising finding in the present study is that PTK inhibitors could effectively yield a time-dependent reversal of LTP (Fig. 2). These data are, to our knowledge, the first evidence to support the idea that PTK activation is not only involved in the processes of LTP induction but also in the early stabilization processes occurring in the first minutes after induction. The links between PTK activation and the phenomenon of LTP reversal remain to be explored. Besides PTK inhibition, a variety of manipulations have been reported to effectively erase established LTP. These include brief anoxic events (Arai et al. 1990b), infusions of adenosine (Arai et al. 1990a), low-frequency synaptic stimulation (Fujii et al. 1991; Stäubli & Chun, 1996), antibodies to the neuronal cell adhesion molecules L1 and NCAM (Lüthi et al. 1994; Muller et al. 1996), and integrin antagonists (Bahr et al. 1997; Stäubli et al. 1998). Although these manipulations are all effective in reversing the established LTP, the duration of the 'vulnerable' period to disrupt LTP is strictly different. For example, the agents that dissociate the complex between L1 and NCAM or the antibodies to L1 or NCAM produce a marked decrease in the initial potentiation if applied within 1-2 min after delivery of LTP induction stimuli but are without effect if administered 10 min after (Lüthi et al. 1994; Muller et al. 1996); however, integrin antagonists still eliminate LTP even when applied 10 min after induction (Bahr et al. 1997; Stäubli et al. 1998). Based on these backgrounds, the neurochemical processes involved in the LTP stabilization can be separated into early and late phases. NCAMs participate in the early phase to stabilize the potentiated state of synapses. Integrins, on the other hand, contribute to the late phase to form new membrane configurations and then convert potentiation into a non-disruptive state (Bahr et al. 1997; Stäubli et al. 1998). It is of interest that the time period of PTK inhibition for reversal of LTP observed in the present study was indistinguishable from those with L1 and NCAM dissociating agents or antibodies. It is highly likely that PTKs are involved in neuronal cell adhesion molecule signalling which is required for the early stabilization processes of LTP. Further support for this view is the observation that c-Src and Fyn are the components of the L1 (Ignelzi et al. 1994) and NCAM (Beggs et al. 1994) signalling pathway stimulating neurite outgrowth, respectively. There is cumulative evidence that PTKs may come together to form a signalling complex (Pawson & Gish, 1992). It was found that two Src family kinases (c-Src and Fyn) stably associate with focal adhesion kinase (FAK) (Cobb et al. 1994) and there is reduced kinase activity and hypophosphorylation of FAK in fyn mutant mice (Grant et al. 1995), which have shown impairments in hippocampal CA1 LTP and spatial learning (Grant et al. 1992). Therefore, FAK may also be a component of PTK-related signal transduction pathways involved in the LTP stabilization.
Which PTK substrate participates in LTP induction?
The finding that PTKs contribute to LTP induction raises the question: How do they produce their effect? The effect of PTKs might be mediated through tyrosine phosphorylation of other proteins required for LTP induction. Although it is difficult, based on the present data, to estimate the exact molecular target for this process, a likely candidate is the NMDA receptors. At Schaffer collateral-CA1 synapses, the activation of NMDA receptors is well known to be involved in the induction of tetanic LTP. Recent observations indicate that the NMDA receptor-mediated response could be modulated by PTKs. It was found that currents through native NMDA receptors are increased by tyrosine phosphorylation (Wang & Salter, 1994), and this has also been observed with recombinant NMDA receptors (Chen & Leonard, 1996). Moreover, it was also reported that tetanus-induced LTP in the dentate gyrus of anaesthetized rats produces a sustained elevation in the tyrosine phosphorylation of NMDA receptor 2B subunits (Lau & Huganir, 1995). Based on these findings, it is possible that the NMDA receptor-channel is a potential substrate for PTKs which are involved in LTP generation. However, we could not exclude the possibility that PTKs might also act by enhancing the biochemical steps downstream of the activation of NMDA receptors, and this remains to be determined.
In agreement with a previous study (Lu et al. 1998) we find that PTK inhibitors exerted minor or no effect on the basal synaptic responses mediated by either AMPA or NMDA receptors (Fig. 7), suggesting that these inhibitors are unlikely to directly affect the postsynaptic glutamate receptors and the release of glutamate from the presynaptic nerve terminals under basal conditions. On the basis of these data, we assume that neither synaptic NMDA nor AMPA receptors are enhanced tonically by basal PTK function. Our results are consistent with recent findings of Lau & Huganir (1995), who, by protein phosphorylation assay, found that less than 5 % of the NR2B subunits of the NMDA receptor in rat brain were tyrosine phosphorylated under basal conditions, and also that under these conditions tyrosine phosphorylation of the AMPA receptor subunits GluR1-4 could not be observed.
PTK inhibitors prevent the induction of anoxic LTP
The signals involved in the generation of anoxic LTP have been extensively investigated but are not completely understood. Crépel et al. (1993) and Hammond et al. (1994) have shown that the induction of anoxic LTP was dependent on voltage, NMDA receptors and redox state. Recently, we have also demonstrated that the mechanism underlying the anoxic LTP is likely to be attributable to an enhancement of presynaptic glutamate release and a selective upregulation of postsynaptic NMDA receptor-mediated synaptic response through the Ca2+-dependent processes (Hsu & Huang, 1997; Huang & Hsu, 1997). Here, we have provided further evidence that the induction of anoxic LTP is prevented by PTK inhibitors, suggesting that PTK activation appears to be necessary for anoxic LTP induction (Fig. 9). Previous in vivo studies also demonstrated that the delayed neuronal death following cerebral ischaemia and accompanying tyrosine phosphorylation can be alleviated by PTK inhibitors (Kindy, 1993; Ohtsuki et al. 1996), implying the possibility that the activation of PTK is involved in the delayed neuronal death following ischaemia. Although the precise mechanisms which underlie the prevention of anoxic LTP or the delayed neuronal death following a brief anoxic episode by PTK inhibitors remain unknown, an inhibition of the anoxia-induced enhancement of postsynaptic NMDA receptor-mediated responses is an intriguing possibility. However, further experiments are necessary to test this possibility.
PTK inhibitors specifically block the development of NMDA receptor-dependent long-term synaptic plasticity
An interesting question derived from our present work is whether the activation of PTKs is a common signal contributing to the induction of long-term synaptic plasticity at Schaffer collateral-CA1 synapses. In the hippocampal CA1 region, long-term enhancement of synaptic strength can also be evoked by other means, for example by bath application of various chemical agents. An example of this is transient application of PKC activator phorbol esters. The phorbol ester-induced synaptic potentiation is observed in the presence of NMDA receptor antagonists, indicating that the NMDA receptor does not contribute to this form of synaptic potentiation (Malenka et al. 1986). A similar sustained potentiation of synaptic transmission was also observed by activation of the cAMP/PKA cascade and this form of synaptic potentiation was suggested to result from an increase of neurotransmitter release and thus the locus of action was presynaptic (Chavez-Noriega & Stevens, 1994). Moreover, NMDA receptor antagonists did not affect this increase in synaptic strength (Lu & Gean, 1999). Furthermore, recent studies have also shown that a brief application of the potassium channel blocker TEA was followed by a prolonged potentiation in the synaptic transmission and the TEA-induced synaptic transmission was dependent on VDCCs and CaMKII activation (Huang & Malenka, 1993; Huber et al. 1995). In the present study, we also examined the effects of PTK inhibitors on these three forms of long-term synaptic potentiation to determine whether PTKs also participate in an NMDA receptor-independent form of LTP. We found that lavendustin A fails to affect the synaptic enhancement produced by PDBu, forskolin-IBMX or TEA (Figs 4, 5 and 6). Taking together the effects of PTK inhibitors on tetanic LTP and anoxic LTP, we conclude that PTK activity is required for the development of NMDA receptor-dependent long-term synaptic plasticity but not NMDA receptor-independent forms.
In conclusion, the present study has provided experimental evidence that postsynaptic PTK activation is required for the induction and the early stabilization of tetanic LTP and the induction of anoxic LTP in area CA1 of the hippocampus. How can PTKs participate in LTP induction? An attractive possibility is that during induction of LTP, Src family kinases are activated rapidly, which leads to upregulation of NMDA receptor function (Wang & Salter, 1994; Lau & Huganir, 1995) and/or enhancement of some signalling cascades involved in the early stabilization processes of LTP (Salter, 1998). Our findings are further evidence to support the current hypothesis that PTKs are potential components of biochemical cascades that subserve induction of long-term synaptic plasticity at central synapses.
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This work was financially supported by a research grant from the Department of Health and the National Health Research Institute, Taiwan, R.O.C. (DOH88-HR-837) to K.-S.H.
Corresponding author
K.-S. Hsu: Department of Pharmacology, College of Medicine, National Cheng-Kung University, Tainan City, Taiwan 70101.
Email: richard{at}mail.ncku.edu.tw
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) was also monitored simultaneously with the intracellular EPSP (
). Note that long-lasting enhancement of the slope of the fEPSP was observed following TS, indicating that LTP could be induced in the population of cells that were not subjected to lavendustin A. B, summary of five experiments performed as in A. Insets in A are superimposed traces taken at different times, as indicated. Calibration: 5 mV, 20 ms for the EPSP and 1 mV, 20 ms for the fEPSP.