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Figure 1. Selective loading of presynaptic terminals with the Ca2+ indicator rhod-2 AM
A, schematic diagram showing the experimental arrangement. Membrane-permeable rhod-2 AM (0·2 mM) was pressure ejected into the stratum radiatum, resulting in selective loading of the presynaptic terminals through the Schaffer collateral-commissural (SCC) pathway. Fluorescence from the shaded area, which has a diameter of about 100 µm and is about 500 µm away from the ejection site, was recorded with a single photodiode. B, time courses of the presynaptic Ca2+ transient (pre[Ca2+]t, upper trace) and the field EPSP (lower trace) evoked by a single stimulus to the SCC pathway. Ca2+ signals were measured as relative fluorescence changes ( F/F), where F is the resting fluorescence level and F, the peak amplitude of fluorescence change caused by the stimulus. C, effects of a mixture of AMPA and NMDA antagonists (10 µM CNQX and 25 µM D-AP5) on the Ca2+ transient (upper traces) and field EPSP (lower traces). Two traces obtained before (control) and during application of the drugs are superimposed. D, effects of Ca2+-free solution containing 1 mM EGTA on pre[Ca2+]t and field EPSP.
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Whole-cell patch-clamp recordings
In separate experiments, voltage-clamp recordings were made from visually identified CA1 neurones in the stratum pyramidale using a whole-cell patch-clamp technique. Patch pipettes were filled with an internal solution (pH 7·2) containing (mM): caesium gluconate, 150; EGTA, 5·0; CaCl2, 1·0; Hepes, 10; Mg-ATP, 2·0; and lidocaine N-ethyl bromide quaternary salt (QX-314), 5·0. The liquid junction potential between the control external solution and the internal solution was -11 mV, and the membrane potential was corrected by this value. Electrode capacitance was compensated, but series resistance was not compensated in this study. The resistance of the pipette was 4-8 M
when filled with the internal solution. The access resistance was typically 15-40 M immediately after obtaining whole-cell recording, and was not allowed to vary by more than 10 % during the course of the experiment.
Data acquisition and analysis
The output of the photodiode was I-V converted and filtered at 500 Hz with an 8-pole Bessel filter (FLA-01, Cygnus Technology, Delaware Water Gap, PA, USA) to improve the signal-to-noise ratio. The extracellular field potentials and the whole-cell currents were recorded using an Axopatch-1D amplifier (Axon Instruments, Foster City, CA, USA) at 1 kHz. The fluorescence signals, the field potentials and the whole-cell currents were digitized with a 12-bit A/D converter (Digidata 1200A, Axon Instruments) and acquired at 10 kHz using Axoscope software (Axon Instruments). Analysis was done off-line with the pCLAMP system (Axon Instruments). All values are given as the mean ± S.E.M. Statistical analysis was performed using Student's t test, and P < 0·05 was accepted for statistical significance.
Drugs
Drugs used in this study were: kainate, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and D(-)-2-amino-5-phosphonopentanoic acid (D-AP5) all from Tocris Cookson, Bristol, UK; domoic acid, 5-nitro-6,7,8,9-tetrahydrobenzo-[
]-indole-2,3-dione-3-oxime (NS-102) and 1-(4-aminophenyl)-4-methyl-7,8-methylene-dioxy-5H-2,3-benzodiazepine (GYKI 52466) hydrochloride (RBI, Natick, MA, USA); tetrodotoxin (TTX) and glycine (Wako Pure Chemical Industries, Osaka, Japan); rhod-2 AM (Dojindo Laboratories, Kumamoto, Japan); Pluronic F-127 (Molecular Probes, Eugene, OR, USA); and QX-314 and DMSO from Sigma.
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RESULTS |
Simultaneous recordings of field EPSPs and presynaptic Ca2+ transients
With the loading technique described above, transient increases in rhod-2 fluorescence and field EPSPs evoked by single electrical stimulations to the Schaffer collateral- commissural (SCC) pathway were recorded simultaneously in hippocampal slice preparations. Figure 1B shows the time course of the representative Ca2+ transient and field EPSP. To verify the presynaptic origin of the observed Ca2+ transients, we examined the effects of the glutamate receptor antagonists CNQX (10 µM) and D-AP5 (25 µM), which block postsynaptic AMPA and NMDA receptors, respectively. As shown in Fig. 1C, the field EPSPs were blocked completely by a mixture of CNQX and D-AP5 whereas Ca2+ transients were not affected (n = 19), suggesting that the Ca2+ transients arose exclusively from the presynaptic structures. The Ca2+ transients were abolished in the Ca2+-free solution containing 1 mM EGTA (n = 3; Fig. 1D), suggesting that the increase in fluorescence intensity results from Ca2+ influx from extracellular space rather than Ca2+ release from intracellular stores. These results indicate that the transient fluorescence increase following a single SCC stimulation reflects Ca2+ influx into the presynaptic terminals during an action potential.
To assess that the rhod-2 fluorescence transients report faithfully the relative changes in Ca2+ influx into presynaptic terminals during an action potential, we examined whether there was a partial saturation of the indicator in our experimental conditions. As shown in Fig. 2, when a paired-pulse stimulation of the presynaptic fibres with 50 ms interstimulus interval was given, the increase in the fluorescence signal to the second stimulus was almost identical to that to the first stimulus (n = 4). This result suggests that each stimulus elicits the same amount of Ca2+ influx and that the fluorescence transients linearly report Ca2+ entry in our experimental conditions. This result differs from the previous study by Wu & Saggau (1994a), who reported that the magnitude of the second Ca2+ transient to paired-pulse stimulation (50 ms interstimulus interval) decreased to approximately 75 % of the first response at the same synapse when measured with the high-affinity indicator fura-2. The difference may be due to the relatively lower affinity of rhod-2 for Ca2+ (Kd = 1·0 µM; Minta et al. 1989) than fura-2 (Kd = 224 nM; Grynkiewicz, Poenie & Tsien, 1985), as well as the higher concentration of dye introduced to the presynaptic terminals in dye-loading conditions in this study.
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Figure 2. Rhod-2 fluorescence transients and field EPSPs induced by paired-pulse stimulation
A, superimposed traces of fluorescence transients (upper traces) and field EPSPs (lower traces) in response to single and paired-pulse stimulations (50 ms interstimulus interval). B, the fluorescence transient and field EPSP produced by the second stimulus (dotted traces) were extracted by subtracting the traces evoked by single stimulation from the paired responses, and are shown together with those evoked by single stimulation.
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Suppression of field EPSPs and presynaptic Ca2+ transients by KA
Next we examined the effects of low concentrations of KA on field EPSPs and pre[Ca2+]t. Bath application of 1 µM KA for 10 min reversibly suppressed both field EPSPs and pre[Ca2+]t (Fig. 3A and B). The amplitudes of field EPSPs and pre[Ca2+]t were decreased to 37·7 ± 4·0 % and 72·9 ± 2·4 % of control (n = 6), respectively. Time courses of inhibition and recovery of both the field EPSPs and pre[Ca2+]t were similar, but the relative magnitudes of the suppression were consistently larger for field EPSPs than for pre[Ca2+]t during and after application of KA (Fig. 3B). This difference may be due to the non-linear relationship between field EPSPs and pre[Ca2+]t (Dodge & Rahamimoff, 1967; Landò & Zucker, 1994; Wu & Saggau, 1997) and/or additional postsynaptic effects. To address this point, the quantitative relationships between field EPSPs and pre[Ca2+]t in our experimental conditions were determined by examining the effects of low Ca2+ (1·2 mM) and high Mg2+ (2·5 mM) solution which would suppress synaptic transmission exclusively by reducing pre[Ca2+]t. Application of the low Ca2+ solution for 10 min reduced field EPSPs and pre[Ca2+]t to 28·7 ± 2·7 % and 63·0 ± 1·1 % (n = 6), respectively (Fig. 4A, 
). The relationship between field EPSPs and Ca2+ concentration was non-linear and could be approximated by a power function: [EPSP] 
[Ca2+]m. The data from each experiment were plotted on the double-logarithmic scale in Fig. 4B. The linear regression line obtained with the least-squares fit in the graph shows a slope of 2·8, indicating that the exponent (m) in our experimental conditions was 2·8. The relationships between field EPSPs and pre[Ca2+]t during KA application (1 µm) for 10 min were almost similar to those obtained in the low Ca2+ solution (Fig. 4A and B, 
). Similar experiments were conducted using a low concentration of domoate (0·2 µM), which also acts as an agonist for the KA receptors. Domoate (0·2 µM for 10 min) reduced field EPSPs and pre[Ca2+]t to 52·5 ± 6·8 % and 80·1 ± 3·9 % of control (n = 6), respectively (Fig. 4A and B, 
). The quantitative relationships between them during domoate application are also similar to the data obtained in low Ca2+ conditions. The linear regression slopes for 1 µM KA and 0·2 µM domoate application were 3·1 and 2·7, respectively. These results suggest that KA and domoate suppress the synaptic transmission primarily by inhibiting the presynaptic Ca2+ influx during invasion of the action potential.
Recent reports have shown that KA receptors depress GABAergic transmission in the hippocampal CA1 region (Clarke et al. 1997; Cunha et al. 1997; Rodríguez-Moreno et al. 1997). These findings raise the possibility that this disinhibitory effect of KA might possibly influence the observed decreases in field EPSP and pre[Ca2+]t to some extent. To estimate the influence of a possible KA receptor-mediated depression of the GABAergic transmission, we have examined the effect of KA in the presence of 100 µM picrotoxin, a GABAA receptor blocker. In this experiment, the concentrations of both CaCl2 and MgSO4 were raised to 4 mM to prevent epileptiform activities. In such conditions, KA at 1 µM suppressed the field EPSPs and pre[Ca2+]t to 28·1 ± 5·8 % and 70·2 ± 2·6 %, respectively (n = 5). These values for the degree of suppression were similar to those observed in the absence of picrotoxin. This result suggests that the observed inhibition of field EPSPs and pre[Ca2+]t reflects predominantly the effect of KA on excitatory presynaptic terminals.
Enhancement of paired-pulse facilitation by KA
Presynaptic changes in transmitter release are often accompanied by changes in paired-pulse facilitation, a form of short-term synaptic plasticity (Manabe, Wyllie, Perkel & Nicoll, 1993). Therefore we next examined the effects of KA on paired-pulse facilitation. In this study, paired-pulse facilitation was expressed as a percentage facilitation of the second EPSP amplitude (i.e. 100 % indicates that the second response is twice as large as the first response; Baskys & Malenka, 1991) in response to paired stimuli with a 50 ms interstimulus interval. As shown in Fig. 5, the percentage facilitation was 47·4 ± 5·8 % in the control solution (n = 6). Application of 1 µM KA for 10 min reduced the first and second EPSPs to 32·9 ± 3·0 and 45·4 ± 3·7 % of those before KA application, respectively, but increased the percentage facilitation to 105·1 ± 10·6 % (n = 6). This effect of KA on paired-pulse facilitation was statistically significant (P < 0·05). Essentially similar results were obtained with domoate. Domoate (0·2 µM, 10 min) also suppressed the first and the second EPSPs to 38·0 ± 0·9 and 49·1 ± 1·1 % of those before domoate application, respectively (n = 6). Facilitation was 49·0 ± 3·8 % before domoate application, which increased to 92·5 ± 5·8 % in the presence of domoate (n = 6). This difference was also statistically significant (P < 0·05). These results are consistent with a presynaptic locus for the action of KA and domoate.
The KA receptor-mediated depression of GABAergic synaptic inhibition (Clarke et al. 1997; Cunha et al. 1997; Rodríguez-Moreno et al. 1997) may contribute to the observed increase in paired-pulse facilitation. Therefore we also examined the effect of KA on paired-pulse facilitation in the modified solution containing 4 mM CaCl2, 4 mM MgSO4 and 100 µM picrotoxin. In this solution, the paired-pulse facilitation was 36·6 ± 4·6 % before KA application, and this value was increased to 73·4 ± 4·7 % (n = 5) by a 10 min application of 1 µM KA. This difference was statistically significant (P < 0·05). Thus, KA at 1 µM increased the degree of the paired-pulse facilitation even in the presence of picrotoxin. This result strongly suggests that the increase in paired-pulse facilitation by KA is caused predominantly by action on the presynaptic site of the excitatory synapse.
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Figure 5. Enhancement of paired-pulse facilitation (PPF) by KA
A, specimen records of field EPSPs (mean of 10 consecutive responses) evoked by paired stimuli (50 ms interval) in control solution (upper trace) and during KA application (1 µM, 10 min; lower trace). B, superimposed traces of the scaled field EPSPs before and during KA application. The EPSP component of the field potentials was extracted by subtraction of presynaptic fibre volleys and stimulus artifacts recorded in the presence of 10 µM CNQX and 25 µM D-AP5. The first EPSP amplitude in the presence of KA was normalized to that in the control record. These traces clearly demonstrate that PPF is larger during KA application. C, percentage facilitation of the second EPSPs (100 % indicates that the second response is twice as large as the first response in this figure) in the absence (control,  ) and presence of 1 µM KA ( ). * Difference statistically significant (n = 6, t test, P < 0·05).
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Effects of KA on presynaptic fibre volley potentials
Next we addressed the question of how presynaptic Ca2+ influx during an action potential is reduced by KA. One obvious possibility is the depolarization block of action potentials in the presynaptic axons. In fact, depolarization of dorsal roots and the depression of primary afferent C-fibre volleys by KA application were reported (Agrawal & Evans, 1986). We therefore examined the effects of KA on presynaptic fibre volley potentials. We used low Ca2+ solution containing 0·1 mM Ca2+ and 3·6 mM Mg2+ to isolate presynaptic fibre volleys without contamination of the field EPSPs. As shown in Fig. 6A, introduction of the low Ca2+ solution completely abolished the field EPSPs, leaving only stimulus artifacts and presynaptic fibre volley potentials. The latter biphasic potentials were completely abolished by further addition of 0·5 µM TTX. This indicates that the biphasic potentials are presynaptic volleys. Application of 1 µM KA for 10 min affected neither the amplitude (to 100·9 ± 0·7 % of control, n = 6) nor waveform of the presynaptic volleys (see inset of Fig. 6B). These results suggest that the depolarization block of action potentials at the presynaptic axons by KA is less likely in the case of the hippocampal CA1 synapse.
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Figure 6. Effects of KA on presynaptic fibre potentials
A, field potentials shown on a faster time scale. In the control solution (a), the field potentials (mean of 10 consecutive responses) consist of stimulus artifacts, presynaptic fibre volley potentials ( ) and the following EPSP components. Replacement with low Ca2+ solution (0·1 mM Ca2+ and 3·6 mM Mg2+) abolished field EPSPs, leaving only artifacts and presynaptic volleys (b). The presynaptic volley potentials were abolished by the further addition of 0·5 µM tetrodotoxin (TTX; c). B, plot of presynaptic volley amplitude as a function of time (n = 6). Field potentials were recorded in the low Ca2+ solution, and KA (1 µM) and TTX (0·5 µM) were applied during the period as indicated by the open bars. Insets show the specimen records of presynaptic fibre volley potentials recorded in the absence and presence of 1 µM KA.
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Suppression of the NMDA-receptor mediated component of field EPSPs by KA
Since KA is a non-specific agonist activating both AMPA and KA receptors, the suppressing actions of KA on CA1 synaptic transmission could reflect the activation of AMPA as well as KA receptors. To evaluate the relative contribution of both receptors, we examined the effects of KA in the presence of the AMPA-selective antagonist GYKI 52466 (Donevan & Rogawski, 1993; Chittajallu et al. 1996). As shown in Fig. 7A, relatively slower field potentials were recorded in the Mg2+-free solution containing 100 µM GYKI 52466 and 10 µM glycine, with minimal changes in the presynaptic volleys recorded in the normal solution. These responses were field EPSPs mediated by NMDA receptor activation (field EPSPNMDA), since they were completely abolished by application of the NMDA antagonist D-AP5 (25 µM). By monitoring the field EPSPNMDA, the synaptic strength could be measured even if the AMPA receptors were completely blocked. As shown in Fig. 7B, application of 1 µM KA (10 min) reversibly suppressed the field EPSPNMDA to 53·9 ± 4·4 % of control (n = 6). This result indicates that a substantial part of the KA effect is mediated by activation of KA receptors, although the degree of inhibition was smaller than that observed in the absence of GYKI 52466 in normal solution (to 37·7 ± 4·0 %, n = 6, see Fig. 3, P < 0·05) (see Discussion).
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Figure 7. Effects of KA in the presence of the AMPA-selective antagonist GYKI 52466
A, recordings of field EPSPs mediated by NMDA receptor activation (field EPSPNMDA). To facilitate the recording of the NMDA component of EPSPs in isolation, a modified Mg2+-free solution containing glycine (10 µM) and GYKI 52466 (100 µM) was used. Switching to the Mg2+-free solutions resulted in isolation of the field EPSPNMDA of slower time course (b) than in normal solution (a). Further addition of D-AP5 (25 µM) abolished field EPSPNMDA, leaving presynaptic volleys unaffected (c). B, reversible inhibition of field EPSPNMDA by KA (1 µM, 10 min). C, comparison of the effects of 1 µM KA on field EPSPNMDA recorded in the Mg2+-free solution with GYKI 52466 (n = 6, ) and on field EPSPs recorded in the normal solution (EPSPAMPA, data in Fig. 3, n = 6, ). * Percentage inhibition slightly but significantly (P < 0·05) smaller for EPSPNMDA recorded in the presence of GYKI 52466.
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Partial blockade of the KA effects by KA antagonist NS-102
We tested the effect of the KA-selective antagonist NS-102 (Johansen, Drejer, Wätjen & Nielsen, 1993; Verdoorn, Johansen, Drejer & Nielsen, 1994; Chittajallu et al. 1996) on KA-induced suppression of pre[Ca2+]t and field EPSPs. Consistent with the report by Chittajallu et al. (1996) application of the 10 µM NS-102 alone (5 min) did not affect field EPSPs significantly (to 102·0 ± 0·8 % of control) and addition of KA (1 µM, 10 min) in the presence of 10 µM NS-102 suppressed field EPSPs to 80·1 ± 3·5 % of control (n = 6). As shown in Fig. 3, KA at 1 µM suppressed the field EPSPs to 37·7 ± 4·0 % in the absence of NS-102 (n = 6). Thus, the KA-induced suppression of the field EPSPs was significantly reduced by NS-102 (P < 0·05). The suppressive effect of KA on pre[Ca2+]t was also partially antagonized by NS-102. KA at 1 µM in the presence of 10 µM NS-102 reduced the pre[Ca2+]t to 89·3 ± 1·3 % of control (n = 6), whereas KA alone suppressed pre[Ca2+]t to 72·9 ± 2·4 % (n = 6, see Fig. 3). This difference was statistically significant (P < 0·05). These results are consistent with the involvement of KA receptors in the suppressive effects of KA on both pre[Ca2+]t and field EPSPs.
Suppression of EPSCs by KA
Finally, we examined the effects of KA on EPSCs using whole-cell patch-clamp recordings from the visually identified CA1 neurones in the stratum pyramidale. Prolonged depolarization of CA1 neurones caused by KA-induced activation of the AMPA receptor might reduce the field EPSPs by decreasing the driving force for the generation of the EPSPs. To determine how much this mechanism contributes to the observed suppression of field EPSPs, we compared the degree of KA-induced suppression of EPSCs with that of field EPSPs. As shown in Fig. 8, KA at 1 µM reversibly suppressed the evoked EPSCs to 42·6 ± 6·3 % of control while inducing small DC inward currents (50·9 ± 8·8 pA, n = 5) when CA1 neurones were voltage clamped at -71 mV. Thus, KA at 1 µM suppressed the EPSCs to almost the same extent as the field EPSPs (37·7 ± 4·0 %, Fig. 3). This result excludes the possibility that the suppressive effect of KA on field EPSPs is largely due to the postsynaptic depolarization.
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Figure 8. Effects of KA on EPSCs
A, representative EPSCs recorded with the use of the whole-cell patch-clamp technique in the control solution (a), during application of 1 µM KA (b), and after washout (c). These EPSCs were abolished almost completely by further addition of 10 µM CNQX (d). Each trace is the mean of six consecutive EPSCs. The cell was voltage clamped at -71 mV. B, time course of the effect of 1 µM KA on the EPSCs. The relative amplitudes of the EPSCs, with those before KA application as references, are plotted against time (n = 5). KA (1 µM) and CNQX (10 µM) were applied during the period as indicated by the open bars. a, b, c and d in the graph indicate the time points when traces Aa, b, c and d were recorded, respectively.
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DISCUSSION |
The present study has demonstrated the presynaptic inhibitory actions of prolonged application of low-dose KA, consistent with a previous study by Chittajallu et al. (1996). Furthermore, fluorescent Ca2+ measurement has revealed that the primary effect of KA is the suppression of Ca2+ influx into the presynaptic terminals during the invasion of an action potential. The involvement of KA receptors in the KA-induced suppression has also been demonstrated pharmacologically using drugs which interact specifically with KA receptors.
Suppression of presynaptic Ca2+ influx by KA at the hippocampal CA1 synapse
As for the mechanisms responsible for the presynaptic inhibition, two possibilities should be considered, i.e. reduction of the action potential-induced presynaptic Ca2+ influx and/or inhibition of the Ca2+-triggered exocytotic machinery. To differentiate these Ca2+-dependent and -independent mechanisms, we estimated quantitatively the relationship between decreases in pre[Ca2+]t and field EPSPs when slices were bathed in low Ca2+ solution. In such conditions, synaptic transmission was expected to be suppressed exclusively by reducing presynaptic Ca2+ influx. We confirmed the highly non-linear dependence of field EPSPs on pre[Ca2+]t as reported for many synapses (Dodge & Rahamimoff, 1967; Landò & Zucker, 1994; Wu & Saggau, 1997). Assuming the power-law relationship between presynaptic Ca2+ increase and transmitter release, the estimated value for the exponent m was 2·8 in our experimental conditions. A similar function with m = 3·5 was reported for the guinea-pig CA1 synapse measured with fura-2 signals (Wu & Saggau, 1994a, 1997). The slight difference in the m value may be due to different affinities and concentrations of the Ca2+ indicators within the terminals (rhod-2 vs. fura-2) as well as the different ages and species of the animals (rat vs. guinea-pig). Similar non-linear relationships during application of KA and domoate with low Ca2+ experiments suggest that KA suppresses the synaptic transmission mainly by presynaptic mechanisms, i.e. suppression of Ca2+ influx during an action potential.
Recent reports have shown that KA receptors depress GABAergic transmission in the hippocampal CA1 region (Clarke et al. 1997; Cunha et al. 1997; Rodríguez-Moreno et al. 1997). These findings raised the possibility that this effect of KA might possibly have some influence on the observed decreases in field EPSP and pre[Ca2+]t. We found, however, almost similar effects of KA on pre[Ca2+]t as well as paired-pulse facilitation, even in the presence of picrotoxin. These results suggest that the observed inhibition of field EPSPs and pre[Ca2+]t by KA mainly reflect the effect on excitatory terminals. The lower concentration of KA used in our experiments (1 µM) compared with previous reports (5-30 µM) may explain the lack of contribution of the KA-induced disinhibitory processes.
Mechanisms underlying inhibition of presynaptic Ca2+ influx by KA
How does KA reduce Ca2+ influx into the presynaptic terminal? We first considered the possibility that axonal depolarization blocks action potential conduction, as shown for the primary afferent C fibres (Agrawal & Evans, 1986). The absence of KA effects on presynaptic volley potentials in this study, however, suggests that this is not the case for the hippocampal CA1 synapse. However, the absence of obvious changes in presynaptic fibre volleys in our measurements may not necessarily mean the absence of 'localized' presynaptic depolarizing effects. If KA receptors are localized in the vicinity of presynaptic terminals, not in axons, small depolarizations would hardly be detectable with the extracellular recording used in this study. Such a highly strategic localization of KA receptors, as shown for other presynaptic autoreceptors (for example, see Yokoi et al. 1996), may decrease action potential-mediated electrotonic membrane depolarization at the presynaptic terminals, thereby reducing transmitter release evoked by afferent stimulation.
An alternative possibility is that KA-induced depolarization and subsequent Ca2+ accumulation within the terminals may inactivate the 'presynaptic' Ca2+ channels and thereby reduce Ca2+ influx during action potentials, since voltage-dependent as well as Ca2+-dependent mechanisms for inactivation of 'somatic' Ca2+ channels were reported in dissociated hippocampal CA1 neurones (Kay, 1991). Ca2+-dependent inactivation, however, seems unlikely, since the resting level of rhod-2 fluorescence (F) was not significantly increased by KA (data not shown).
Involvement of KA receptors in the presynaptic actions at CA1 synapse
The KA antagonist NS-102 at 10 µM only partially antagonized the suppressive effect of 1 µM KA. This may not necessarily mean the involvement of other receptors in the KA effect. Verdoorn et al. (1994) reported that 10 µM NS-102 suppressed the glutamate-evoked current at recombinant GluR6, which belongs to the family of KA receptor subunits, to approximately 50 % of control. Furthermore, Chittajallu et al. (1996) reported that 10 µM NS-102 antagonized the KA-induced suppression of NMDA receptor-mediated EPSCs in the presence of the AMPA-selective antagonist GYKI 52466 by 55 ± 4 %, an extent almost the same as in this study. These findings support the notion that the incomplete blockade is due to the weak antagonistic actions of NS-102 at 10 µM, the near-maximal concentration soluble in physiological solutions (Verdoorn et al. 1994), rather than involvement of other receptors in the KA effect.
It should be noted that the degree of suppression by KA was more prominent on the AMPA receptor-mediated than on the NMDA receptor-mediated EPSP recorded in the absence and presence of GYKI 52466, respectively (Fig. 7). This is possibly due to the partial suppression of presynaptic KA receptors by GYKI 52466. Consistent with this notion, Paternain et al. (1995) reported that GYKI 52466 at 100 µM weakly suppressed KA receptors in cultured hippocampal neurones (to approximately 70-80 % of control).
In both Xenopus oocytes and HEK 293 cells exclusively expressing recombinant KA receptor subunits, the continuous application of KA induces fast desensitizing responses (for review, see Bettler & Mulle, 1995). Furthermore the pharmacologically isolated native KA receptor responses in cultured embryonic hippocampal neurones also exhibit fast and complete desensitization (Lerma et al. 1993; Paternain et al. 1995). Such desensitization profiles may argue that the bath application method adopted in this study could hardly activate KA receptors. However, the recent demonstration of a substantial non-desensitizing component in native KA receptor responses in postnatal hippocampal cultures (
30 % of the peak responses; Wilding & Huettner, 1997) suggests the possible activation of KA receptors with bath application of KA. The relatively slow desensitization of the KA receptors was also demonstrated in certain dorsal root ganglion neurones (Huettner, 1990). Consistent with the slow desensitization component of native KA receptors, the decay of KA receptor-mediated EPSCs at the mossy fibre-CA3 synapse (
= 103 ms) was much slower than that of AMPA receptor-mediated EPSCs ( = 9·1 ms, Castillo et al. 1997; see also Vignes & Collingridge, 1997).
Is the KA-induced suppression due to direct action on presynaptic KA receptors? Several indirect mechanisms can be considered. Since similar Ca2+-dependent presynaptic inhibitory actions of adenosine and baclofen were reported for the hippocampal CA1 synapse (Wu & Saggau, 1994b, 1995), it is possible that activation of the KA receptors in postsynaptic neurones, interneurones, or glia would result in massive release of adenosine (Manzoni, Manabe & Nicoll, 1994) or GABA, and the released adenosine or GABA would act on the adenosine A1 or GABAB receptors on the presynaptic terminals of the CA1 synapse to suppress the glutamate release. Another possibility is that presynaptic metabotropic glutamate receptors (mGluR) at this synapse (Yoshino & Kamiya, 1995) are activated by glutamate that leaked out due to inhibition of glutamate uptake mechanisms by KA (Pocock, Murphie & Nicholls, 1988), which usually maintains the glutamate concentration at much lower levels in the synaptic clefts. All these possibilities, however, seem to be unlikely, since the presynaptic inhibitory effects of KA were not affected by the antagonists of mGlu, GABAB, and adenosine A1 receptors (Chittajallu et al. 1996).
Functional implications
Earlier studies have shown that prolonged application of a low dose of KA (1 µM), which should activate KA receptors preferentially, induces epileptiform activity in CA3 as well as in CA1 regions of hippocampal slice preparations (Westbrook & Lothman, 1983; Fisher & Alger, 1984). In contrast with the excitatory actions of KA, the evoked synaptic responses at CA1 synapses were markedly suppressed during application of 1 µM KA (Fisher & Alger, 1984; see also Collingridge, Kehl & McLennan, 1983b) although this concentration of KA caused minimal (Robinson & Deadwyler, 1981) or slight depolarization in CA1 neurones (5 mV; Fisher & Alger, 1984). It was also reported that KA suppressed field EPSPs in the CA1 region without affecting the presynaptic fibre volley (Collingridge, Kehl, Loo & McLennan, 1983a). The presynaptic inhibitory action of KA, shown by Chittajallu et al. (1996) and in this study, may explain this KA-induced suppression of the synaptic potentials. The absence of epileptiform activity during KA application in this study (see Figs 3A and 5A) may be due to the surgical disconnection of CA1 from CA2/3 regions, the lower recording temperature (22-26°C), and/or the relatively low [K+]o (2·7 mM) in our experimental conditions. The absence of epileptiform activity in our experimental conditions has facilitated the examination of presynaptic inhibitory action in isolation.
The physiological as well as pathophysiological functions of the presynaptic KA receptors in hippocampal CA1 synapses are currently unknown. The presence of multiple inhibitory autoreceptors at the CA1 synapse (KA receptor, Chittajallu et al. 1996; and mGluR, Baskys & Malenka, 1991) suggests that they limit the overexcitation of CA1 neurones and counteract neurotoxic actions of massive glutamate release in some pathological conditions, including brain ischaemia (Choi & Rothman, 1990). Involvement of the presynaptic KA receptor in activity-dependent synaptic plasticity, such as long-term potentiation and depression, is also tenable. Additional studies are clearly needed to establish the functions of the presynaptic KA receptors using specific pharmacological tools as well as gene targeting of KA receptor subunits.
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Acknowledgements
This work was supported by the Nissan Science Foundation, by CREST (Core Research for Evolutional Science and Technology) of the Japan Science and Technology Corporation (JST), and by Grants-in-Aid No. 09260206 for Scientific Research on Priority Areas on 'Functional Development of Neural Circuits' and No. 09780761 from the Ministry of Education, Science and Culture of Japan.
Corresponding author
H. Kamiya: Department of Physiology, Gunma University School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan.
Email: hkamiya{at}news.sb.gunma-u.ac.jp
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