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kainate and metabotropic glutamate receptors
Neuropharmacology 38 (1999) 495 – 504
Induction of LTD in the adult hippocampus by the synaptic activation of AMPA/kainate and metabotropic glutamate receptors Nicola Kemp, Zafar I. Bashir * Department of Anatomy, School of Medical Sciences, Uni6ersity of Bristol, Bristol, BS8 1TD, UK Accepted 27 November 1998
Abstract It has been suggested that the induction of long-term depression (LTD) may be developmentally regulated since LTD can be readily induced by LFS in slices from young but not adult animals. However, we have recently reported that paired pulse low frequency stimulation (PP-LFS) can reliably induce LTD in the CA1 region of adult hippocampal slices. We now describe the role of glutamate receptors in the induction of LTD in adult hippocampus. The induction of LTD was prevented by the combined application of AMPA/kainate and metabotropic glutamate (mGlu) receptor antagonists (CNQX and LY341495). However, LTD was not blocked by the co-application of NMDA and mGlu receptor antagonists nor by the co-application of NMDA and AMPA/kainate receptor antagonists. Taken together, the above results suggest that activation of either AMPA/kainate or mGlu receptors is sufficient to induce LTD. Therefore, these results suggest that PP-LFS can efficiently activate AMPA/kainate and mGlu receptors in order to induce long-lasting synaptic depression in the CA1 region of the adult hippocampus in vitro. © 1999 Elsevier Science Ltd. All rights reserved. Keywords: Long term depression; LTP; Synaptic plasticity; mGlu; N-methyl-D-aspartate; AMPA/kainate; Hippocampus
1. Introduction In recent years it has become apparent that homosynaptic long term depression (LTD) can be readily induced in the hippocampus and neocortex (Artola et al., 1990; Dudek and Bear, 1992; Mulkey and Malenka, 1992; Kirkwood and Bear, 1994). An understanding of the mechanisms involved in the induction and expression of LTD is important since decreases in synaptic efficacy are likely to be fundamental to the normal function and development of the CNS. A variety of different mechanisms have been described to underlie the induction of LTD in the hippocampus. For example, in neonatal animals (3 – 7 days) the induction of LTD has been shown to be dependent on metabotropic glutamate (mGlu) receptor activation (Bolshakov and Siegelbaum, 1994; Oliet et al., 1997) and voltage-gated calcium channel (VGCC) activation (Bolshakov and Siegelbaum, 1994). Furthermore, in young ( 14 days) * Corrresponding author. Tel.: + 44-117-9288392; fax: + 44-1179291687; e-mail: [email protected].
D-2-amino-5-phosphonopentanoate;
and aged ( 24 months) animals, LFS results in the induction of N-methyl-D-aspartate (NMDA) receptordependent LTD (Dudek and Bear, 1992; Mulkey and Malenka, 1992; Norris et al., 1996). Thus several different, and perhaps developmentally regulated, mechanisms of LTD induction appear to exist within the CA1 region of the hippocampus. The stimulus protocols generally used to induce LTD in young animals are relatively ineffective in inducing LTD in adult animals (Bashir and Collingridge, 1994; Errington et al., 1995; Wagner and Alger, 1995; Abraham et al., 1996; Norris et al., 1996; Kemp and Bashir, 1997a; but see Heynen et al., 1996; Staubli and Ji, 1996). Based on these findings a developmental downregulation of LTD has been inferred. Recently however, we demonstrated that a paired-pulse low frequency stimulation (PP-LFS) protocol, similar to that used previously in vivo (Doyere et al., 1996; Thiels et al., 1996), reliably induced LTD in the adult CA1 region (Kemp and Bashir, 1997a). Other protocols have also recently been demonstrated to induce LTD in adult hippocampus in vitro (Berretta and Cherubini,
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Fig. 1. The induction of LTD by PP-LFS in the CA1 region of the adult hippocampus. (A) Single experiment illustrating LTD. Two independent inputs (S0 and S1) were stimulated alternately (each at 0.033 Hz), except during PP-LFS when only one input was stimulated. Throughout the experiment each input received paired stimuli (interpulse interval of 50 ms; see illustrated traces) but only the slope of the 1st response is plotted. Each point is the average of four consecutive responses except during PP-LFS where 20 consecutive responses are averaged. LTD was induced by PP-LFS in S0 and subsequently in S1. (B) Pooled data (n =6) illustrating LTD. In this and subsequent figures the baseline periods are normalised to the 30 min immediately prior to PP-LFS. Where PP-LFS is subsequently delivered to S1 the baseline for this input is re-normalised. Traces are sample responses (average of four consecutive responses) taken from the time points indicated. For clarity, artifacts have been removed and replaced by an upward arrow. Periods of PP-LFS are indicated on the graphs by the two upward arrows joined horizontally.
1998). In the present study we have investigated the details of the mechanisms underlying paired-pulse LFSinduced LTD. We now demonstrate the finding that the induction of LTD in adult CA1 can be dependent on either the activation of AMPA/kainate or mGlu receptors. Some of this work has previously been published in abstract form (Kemp and Bashir, 1998).
2. Methods All experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act, 1986. Female albino rats (3– 4 months) were anaesthetised with halothane and decapitated. The brain was rapidly removed and placed in ice-cold artificial cerebrospinal fluid (aCSF; bubbled with 95% O2/5% CO2) which comprised (mM) NaCl, 124; KCl, 3; NaHCO3, 26; NaH2PO4, 1.25; CaCl2, 2; MgSO4, 1; D-glucose, 10. Transverse slices of hippocampus (400 mm) were cut on
a vibroslice (Campden Instruments) and stored in aCSF. After removing the CA3 region, individual slices were placed in a submerged recording chamber (28– 30oC, flow rate 2.5 ml/min). Standard techniques were used to record field excitatory postsynaptic potentials from the stratum radiatum in response to stimulation (20 ms, 3–10 V) of the Schaffer collateral-commissural pathway (SCCP). The stimulus intensity was set to evoke responses (between 0.5 and 1 mV amplitude) which were roughly 50% of the magnitude at which a population spike started to appear. Two independent sets of SCCP fibres (designated S0 and S1) were alternately stimulated (each at 0.033 Hz). Paired stimuli (50 ms interstimulus interval) were delivered throughout the experiment. Evoked responses were monitored on-line and re-analysed off-line using software developed by Dr W. Anderson (Anderson and Collingridge, 1997). PP-LFS; 900 paired stimuli (50 ms interstimulus interval), 1 Hz was delivered to induce LTD. Any changes in synaptic strength were expressed
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Fig. 2. The induction of LTD was not blocked by NMDA or mGlu receptor antagonists. (A) Pooled data illustrating the lack of effect of AP5 (50 mM) on LTD. PP-LFS was first delivered to S0 and then subsequently delivered to S1 in the presence of AP5. (B) MCPG (1 mM) did not block the induction of LTD.
relative to the normalised pre-LFS baseline (mean depression 9SEM) and significance tested (P B 0.05) using appropriate Student’s t-test at 30 min (unless otherwise indicated) following termination of LFS. Each experiment was carried out on a different slice from a different animal. When used, pharmacological agents were applied via the bathing medium. Drugs used were: D-2-amino-5-phosphonopentanoate (AP5), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), (S)-amethyl-4-carboxyphenylglycine (MCPG), 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) (all purchased from Tocris Neuramin) and LY 341495 (gift from Eli Lilly). Drugs were made up as stock solutions (100 – 1000× final concentration) in equimolar NaOH, except for AP5 (in H20) and CPCCOEt (in DMSO). DMSO at 0.1% had no effect on synaptic transmission nor on the induction of LTD.
3. Results
3.1. Paired pulse low frequency stimulation induces LTD Low frequency stimulation (LFS; 1 Hz, 900 stimuli) did not induce LTD in the CA1 region of the adult
hippocampus (not shown; see Bashir and Collingridge, 1994; Errington et al., 1995; Kemp and Bashir, 1997a). However, PP-LFS; 50 ms interpulse interval, 900 paired pulses at 1 Hz consistently resulted in the induction of homosynaptic long-term depression (S0; 289 4%; PB 0.05, n= 6, Fig. 1). When PP-LFS was subsequently delivered to the other input, LTD was induced (S1; 289 4%; PB 0.05, n =6), which was not different in magnitude to that in S0. This result does not depend on paired pulses being delivered throughout the experiment since delivering paired-pulses only during LFS results in LTD of a similar magnitude to that in the above experiments (data not shown).
3.2. Lack of effect of NMDA and mGlu receptor antagonists on LTD LTD induced by PP-LFS was not blocked by 50 mM AP5 (239 3%, n= 6, PB 0.05, Fig. 2A) nor by 1 mM MCPG (339 4%, n= 5, PB 0.05, Fig. 2B). In addition, LTD was not blocked by the combined presence of AP5 and MCPG (279 5%, n= 8; PB 0.05, Fig. 3A). However, MCPG is a relatively weak, competitive antagonist (Watkins and Collingridge, 1994) therefore we tested the recently described potent group I/II/III mGlu receptor antagonist LY341495 (Kingston et al., 1998;
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Fig. 3. The induction of LTD was not blocked by coapplication of mGlu and NMDA receptor antagonists. (A) In the presence of AP5 and MCPG the magnitude of LTD was comparable to control LTD. (B) In the presence of AP5 and the mGlu receptor antagonist LY341495 (100 mM) the magnitude of LTD was similar to that in the absence of LY341495.
Fitzjohn et al., 1998). This antagonist (100 mM), in the presence of AP5, did not prevent the induction of LTD (23 96%, n=5; PB 0.05, Fig. 3B). This suggests that the induction of LTD is not via activation of NMDA or mGlu receptors. We therefore tested whether there may be a role for AMPA/kainate receptors in LTD induction.
3.3. Lack of effect of NMDA and AMPA receptor antagonists on LTD PP-LFS was delivered in the presence of CNQX (20 mM) and AP5 to block ionotropic glutamate receptors (Fig. 4). After the complete blockade of evoked field potentials (no response to 1st or 2nd of the paired stimuli; see Fig. 4), PP-LFS was delivered to one of the two inputs. Following washout of the antagonists, LTD was observed specifically in the pathway that had received PP-LFS (S0: 339 2%, n =5, P B0.05). Transmission in the heterosynaptic pathway returned towards baseline levels (992%, n =5). Any small depression remaining in the heterosynaptic input is considered to be due to incomplete recovery; the magnitude of homosynaptic LTD expressed relative to the het-
erosynaptic input (re-normalised after antagonist washout) was 279 3%. This suggests that there is no role for AMPA or NMDA receptors in the induction of LTD.
3.4. Are glutamate receptors in6ol6ed in LTD? The above experiments might suggest that there is no role for the synaptic activation of glutamate receptors in the induction of LTD. Given the known functions of glutamate receptors in synaptic plasticity these results were unexpected. However, in the above experiments either AMPA/kainate receptors (Figs. 2 and 3) or mGlu receptors (Fig. 4) were not blocked. It remains possible that activation of either one of these receptors by itself is sufficient to induce LTD. Therefore, we delivered PP-LFS during a complete blockade of glutamatergic transmission (by CNQX, AP5 and LY341495). Under these conditions the induction of LTD was blocked reversibly (Fig. 5A). The magnitude of responses in the pathway that had received PP-LFS (S0: 199 3%, n =4) was not different (P\ 0.05) from those in the control pathway (139 3%, n=4). Expressed relative to the heterosynaptic input, there was no depression in the
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Fig. 4. The induction of LTD during combined antagonism of NMDA and AMPA/kainate receptors. Pooled data showing that perfusion of the antagonists AP5 and CNQX resulted in a complete blockade of postsynaptic responses, during which time PP-LFS was delivered to S0. Following washout of the antagonists there was a recovery of responses in the control input (S1) to baseline values. However, the pathway that received LFS (S0) remained significantly depressed below baseline levels. Inset shows that blockade of ionotropic receptors was complete; there was no response to either the 1st or the 2nd of the pair of stimuli in the presence of CNQX and AP5.
homosynaptic pathway (793%). PP-LFS delivered subsequently to S0 resulted in homosynaptic LTD (37 91%; n= 3, PB0.05). Thus glutamate receptors are involved in the induction of LTD. Since LY341495 is a broad-spectrum mGlu receptor antagonist we tested for an involvement of group I mGlu receptors in the induction of LTD using the selective antagonist CPCCOEt (Annoura et al., 1996; Casabona et al., 1997). PP-LFS was delivered in the combined presence of CPCCOEt (100 mM), AP5 and CNQX. Under these conditions the induction of LTD was blocked reversibly (Fig. 5B). Thus following washout of the antagonists, there was no significant difference (P\0.05; Fig. 5B) between the pathway that had received LFS (S0; 109 8%, n = 4; P \ 0.05) and the control pathway (S1; +4 96%, n = 4; P \ 0.05). PP-LFS was subsequently delivered to the same pathway (S0) and resulted in the induction of LTD (369 8%, n= 3, P B0.05). Thus group I mGlu receptors appear to be involved in LTD. In order to test whether antagonism of group I mGlu and AMPA/kainate receptors is sufficient to block LTD, experiments were carried out in the presence of CPCCOEt and CNQX (Fig. 6). Under these conditions
LTD was blocked reversibly (Fig. 6A). The synaptic strength in the test input (8 9 6%, n= 4; P \ 0.05) was not different (P\ 0.05) from the control input (59 4%, n = 4, Fig. 6B). Relative to the re-normalised heterosynaptic input there was no depression in the homosynaptic input (39 6%). Taken together all of the above results show that glutamate receptors are involved in LTD and that activation of either AMPA/kainate or mGlu receptors is sufficient to allow the induction of LTD. Furthermore, of the mGlu receptors, it appears that the group I subtypes are specifically involved in the induction of LTD.
3.5. Acti6ation of 6oltage-gated calcium channels are not in6ol6ed in LTD The role of AMPA/kainate receptors in the induction of LTD may be to provide the depolarisation necessary to activate voltage-gated calcium channels. In order to test this, experiments were carried out in the presence of AP5, CPCCOEt and nifedipine (10 mM, to block L-type calcium channels) or AP5, CPCCOEt and NiCl (50 mM, to block T-type calcium channels). Under neither
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Fig. 5. LTD is dependent on glutamatergic synaptic transmission. (A) Perfusion of the antagonists AP5, CNQX and LY341495 resulted in a complete blockade of synaptic transmission, during which time PP-LFS was delivered to S0. There was no difference in the recovery between the control (S1) and test pathway (S0) following washout of the antagonists (n =4). Following recovery, PP-LFS was delivered to S0 in three experiments and resulted in the induction of LTD on each occasion. (B) LTD was blocked by perfusion of CPCCOEt, AP5 and CNQX (n= 4). There was no difference in the recovery between the control (S1) and test (S0) pathways following washout of the antagonists. PP-LFS delivered subsequently to S0 (in three experiments) resulted in the induction of LTD on each occasion.
of these conditions was LTD blocked (339 7%, n= 3; Fig. 7A, and 24 91%, n =3; Fig. 7B). Therefore, neither T-type nor L-type calcium channels appear to be involved in the induction of LTD.
4. Discussion The results of this study show that homosynaptic LTD can be reliably induced in the CA1 region of the adult rat hippocampus by appropriate stimulus protocols. The combined antagonism of AMPA/kainate and mGlu receptors was the minimum required to prevent the induction of LTD. In addition, activation by PPLFS of just one of the above receptor types was sufficient to induce LTD. Thus, the induction of LTD was dependent on the synaptic activation of AMPA/kainate or mGlu receptors. Therefore, we have demonstrated two co-existing mechanisms each of which is able to trigger the induction and expression of LTD in the adult hippocampus.
Many previous reports have demonstrated a lack of LFS-induced LTD in the adult CA1 region of the hippocampus (e.g. Bashir and Collingridge, 1994; Errington et al., 1995; Wagner and Alger, 1995; Abraham et al., 1996; Norris et al., 1996; but see Heynen et al., 1996; Staubli and Ji, 1996). However, we (Kemp and Bashir, 1997b) and others (Kerr and Abraham, 1995; Wagner and Alger, 1995; Coussens et al., 1997) have previously demonstrated that appropriate pharmacological manipulations can facilitate LFS-induced LTD in the adult CA1 region. Thus, there is not an absolute developmental down-regulation of LTD. Furthermore, the present results show clearly that LTD can be induced by appropriate synaptic stimulation in the adult CA1 region. This suggests that the induction of LTD by LFS in adult slices may critically depend on experimental conditions. LTD was blocked by the combined antagonism of AMPA/kainate and mGlu receptors, suggesting that activation of NMDA receptors alone is insufficient to induce LTD. Furthermore, LTD was not blocked by
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Fig. 6. LTD was blocked by antagonism of mGlu and AMPA/kainate receptors. (A) Single example illustrating the reversible block of LTD by CPCCOEt and CNQX. (B) Pooled data (n =4) showing that LTD was blocked; control (S1) and test (S0) pathways both recovered to baseline following washout of the antagonists.
antagonism of NMDA receptors. Thus, there appears to be no role for NMDA receptors in LTD in the present study, using animals aged between 3 – 4 months. However, a role for NMDA receptors in the induction of LTD may be developmentally regulated since in young animals (14– 21 days) LTD induced by PP-LFS is blocked completely by AP5 (Kemp et al., 1998) whilst in animals of intermediate age (8 – 12 weeks) there is a partial effect of AP5 (Kemp and Bashir, 1997a). NMDA receptor-independent forms of LTD have been previously described in a number of different studies (Wickens and Abraham, 1991; Bashir and Collingridge, 1994; Christie et al., 1994; Wang et al., 1997; Berretta and Cherubini, 1998). The mechanisms by which CNQX-sensitive receptors may be involved in LTD are not clear. Voltage-gated calcium channel activity, presumably resulting from AMPA receptor-dependent depolarisation, has been shown to be necessary for the induction of LTD (Wickens and Abraham, 1991; Bolshakov and Siegelbaum, 1994; Coussens et al., 1997; Oliet et al., 1997). However, under the conditions of our experiments there appears to be no role for either T-type or L-type voltage gated calcium channels. Whilst calcium perme-
able AMPA receptors (Bettler and Mulle, 1995) are generally found on interneurones (Isa et al., 1996; Toth and McBain, 1998) their localisation on dendrites of pyramidal cells has also been suggested (Garaschuk et al., 1996; Yin et al., 1998). Thus, we cannot rule out that calcium permeating directly through AMPA receptor channels may play a role in the induction of LTD under the present experimental conditions. Synaptic activation of kainate receptors has been demonstrated recently in the hippocampus (Chittajallu et al., 1996; Castillo et al., 1997; Vignes and Collingridge, 1997) and it has been suggested that kainate receptors may be G-protein coupled (Rodriguez-Moreno and Lerma, 1998). Thus an involvement of kainate receptors in LTD remains an intriguing possibility. Given that LTD was blocked during complete antagonism of ionotropic and metabotropic glutamate receptors, but was induced during antagonism of ionotropic receptors alone, suggests a role for mGlu receptors in the induction of this form of plasticity. Many previous studies have implicated a role for mGlu receptors in LTD in, for example, the cerebellum (Linden et al., 1991; Aiba et al., 1994; Conquet et al., 1994; Shigemoto et al., 1994), visual cortex (Kato, 1993), CA1 (Bashir
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Fig. 7. The involvement of AMPA receptors in the induction of LTD is not via activation of voltage-gated calcium channels. Single examples (top) and pooled data (bottom) showing that (A) nifedipine and (B) nickel, in the presence of CPCCOEt and AP5, do not block the induction of LTD.
and Collingridge, 1994; Bolshakov and Siegelbaum, 1994; Yang et al., 1994; Oliet et al., 1997; Overstreet et al., 1997; Palmer et al., 1997; Otani and Connor, 1998) and CA3 regions of the hippocampus (Kobayashi et al., 1996). Of the mGlu receptors, group I (mGlu1/5) receptors which couple to intracellular calcium mobilisation (Pin and Duvoisin, 1995), are likely to be involved in the induction of this form of LTD, since LTD was blocked by the group I selective antagonist, CPCCOEt (Annoura et al., 1996). CPCCOEt has been reported to be 100 times more potent at mGlu1b than mGlu5a in cloned cell lines and at the concentrations (100 mM) used in this study CPCCOEt only slightly reduced mGlu5 receptor-dependent phosphoinositide turnover (Casabona et al., 1997). However, the evidence concerning the relative abundance of mGlu1 and 5 receptor subtypes in the CA1 (Masu et al., 1991; Lujan et al., 1996) would suggest that mGlu5 rather than mGlu1 is the subtype of most importance in this region. Thus, the relative roles of mGlu1 and mGlu5 receptors in LTD require further clarification. The induction of PP-LTD may be dependent on changes in intracellular calcium levels, as has been suggested for other forms of LTD (see Bear and Malenka, 1994; Bear and Abraham, 1996). However, in
the present experiments it appears that there is no role for calcium entry due to NMDA receptor activation, nor due to entry via L- or T-type voltage-gated calcium channels. Given our postulated role of mGlu receptors in the induction of LTD, an increase in intracellular calcium could be brought about via release from intracellular stores, as has been suggested previously for both LTP and LTD (Harvey and Collingridge, 1992; O’Mara et al., 1995). However, whether a rise in intracellular calcium is required for PP-LTD remains to be determined. In this present study, we find that PP-LFS can access the induction of LTD by different but concurrently active synaptic mechanisms. Similarly, an essential role for both mGlu receptors and voltage-gated calcium channels in the induction of LTD has been described in the cerebellum (Linden et al., 1991; Daniel et al., 1992; Conquet et al., 1994; Shigemoto et al., 1994), in neonatal CA1 (Bolshakov and Siegelbaum, 1994) and adult CA1 (Otani and Connor, 1998). In addition, NMDA and mGlu receptor-dependent forms of LTD have been reported to co-exist in young hippocampus (Oliet et al., 1997) and the mechanisms of expression of these two forms of LTD are proposed to be different. Whether each of the mechanisms that we have described in this
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study converge on the same or different expression mechanisms remains to be established. In the present study, the magnitude of LTD induced by activation of AMPA/kainate receptors alone (in the presence of NMDA and mGlu receptor antagonists) or by mGlu receptors alone (in the presence of NMDA and AMPA/ kainate receptor antagonists) was not significantly smaller than that induced by activation of both AMPA/ kainate and mGlu receptors. This suggests that LTD induced by each mechanism might be non-additive and therefore may converge on the same expression mechanisms. In conclusion we have demonstrated a stimulus protocol for the reliable induction of long-term synaptic depression in the adult CA1 region of the hippocampus. Surprisingly, this form of LTD was dependent on the activation of AMPA/kainate and/or mGlu receptors suggesting that PP-LFS may be an efficient means of synaptically activating these particular receptors in order to induce LTD.
Acknowledgements Supported by the Wellcome Trust. LY341495 generously provided by Eli Lilly.
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Kemp, N., Bashir, Z.I., 1997. NMDA receptor-dependent and -independent long-term depression in the CA1 region of the adult rat hippocampus in vitro. Neuropharmacology 36, 397–399. Kemp, N., Bashir, Z.I., 1997. A role for adenosine in the regulation of long-term depression in the adult rat hippocampus in vitro. Neurosci. Lett. 225, 189–192. Kemp, N., Bashir, Z.I., 1998. Glutamate receptor interactions required for the induction of LTD in the adult rat hippocampus in vitro. Forum Eur. Neurosci. 54.19. Kemp, N., Faulkes, S., Bashir, Z.I., 1998. Developmental changes in the mechanisms underlying induction of LTD in rat hippocampus in vitro. Soc. Neurosci. Abst. 425.7 Kerr, D.S., Abraham, W.C., 1995. Cooperative interactions among afferents govern the induction of homosynaptic long-term depression in the hippocampus. Proc. Natl. Acad. Sci. USA 92, 11637 – 11641. Kingston, A.E., Ornstein, P.L., Wright, R.A., Johnson, B.G., Mayne, N.G., Burnett, J.P., Belagaje, R., Wu, S., Schoepp, D.D., 1998. LY341495 is a nanomolar potent and selective antagonist at group II metabotropic glutamate receptors. Neuropharmacology 37, 1 – 12. Kirkwood, A., Bear, M.F., 1994. Homosynaptic long-term depression in the visual cortex. J. Neurosci. 14, 3404–3412. Kobayashi, K., Manabe, T., Takahashi, T., 1996. Presynaptic long term depression at hippocampal mossy fibre-CA3 synapse. Science 273, 648 – 650. Linden, D.J., Dickinson, M.H., Smeyne, M., Connor, J.A., 1991. A long-term depression of AMPA currents in cultured cerebellar Purkinje neurons. Neuron 7, 81–89. Lujan, R., Nusser, Z., Roberts, J.D., Shigemoto, R., Somogyi, P., 1996. Perisynaptic location of metabotropic glutamate receptors mGluR1 and mGluR5 on dendrites and dendritic spines in the rat hippocampus. Eur. J. Neurosci. 8, 1488–1500. Masu, M., Tanabe, Y., Tsuchida, K., Shigemoto, R., Nakanishi, S., 1991. Sequence and expression of a metabotropic glutamate receptor. Nature 349, 760–765. Mulkey, R.M., Malenka, R.C., 1992. Mechanisms underlying induction of homosynaptic long-term depression in area CA1 of the hippocampus. Neuron 9, 967–975. Norris, C.M., Korol, D.L., Foster, T.C., 1996. Increased susceptibility to induction of long-term depression and long-term potentiation reversal during aging. J. Neurosci. 16, 5382–5392. O’Mara, S.M., Rowan, M.J., Anwyl, R., 1995. Dantrolene inhibits long-term depression and depotentiation of synaptic transmission in the rat dentate gyrus. Neuroscience 68, 621–624. Oliet, S.H.R., Malenka, R.C., Nicoll, R.A., 1997. Two distinct forms of long-term depression coexist in CA1 hippocampal pyramidal cells. Neuron 18, 1294–1297. Otani, S., Connor, J.A., 1998. Requirement of rapid Ca2 + entry and synaptic activation of metabotropic glutamate receptors for the
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induction of long-term depression in adult rat hippocampus. J. Physiol. 511, 761 – 770. Overstreet, L.S., Pasternak, J.F., Colley, P.A., Slater, N.T., Trommer, B.L., 1997. Metabotropic glutamate receptor mediated longterm depression in developing hippocampus. Neuropharmacology 36, 831 – 844. Palmer, M.J., Irving, A.J., Seabrook, G.R., Jane, D.E., Collingridge, G.L., 1997. The group I mGlu receptor agonist DHPG induces a novel form of LTD in the CA1 region of the hippocampus. Neuropharmacology 36, 1517 – 1532. Pin, J.-P., Duvoisin, R., 1995. The metabotropic glutamate receptors: structure and functions. Neuropharmacology 34, 1 – 26. Rodriguez-Moreno, A., Lerma, J., 1998. Kainate receptor modulation of GABA release involves a metabotropic function. Neuron 20, 1211 – 1218. Shigemoto, R., Abe, T., Nomura, S., Nakanishi, S., Hirano, T., 1994. Antibodies inactivating mGluR1 metabotropic glutamate receptor block long-term depression in cultured Purkinje cells. Neuron 12, 1245 – 1255. Staubli, U.V., Ji, Z.X., 1996. The induction of homo- vs heterosynaptic LTD in area CA1 of hippocampal slices from adult rats. Brain Res. 714, 169 – 176. Thiels, E., Xie, X.P., Yeckel, M.F., Barrionuevo, G., Berger, T.W., 1996. NMDA receptor-dependent LTD in different subfields of hippocampus in vivo and in vitro. Hippocampus 6, 43 –51. Toth, K., McBain, C.J., 1998. Afferent-specific innervation of two distinct AMPA receptor subtypes on single hippocampal interneurones. Nat. Neurosci. 1, 572 – 578. Vignes, M., Collingridge, G.L., 1997. The synaptic activation of kainate receptors. Nature 388, 179 – 182. Wagner, J.J., Alger, B.E., 1995. GABAergic and developmental influences on homosynaptic LTD and depotentiation in rat hippocampus. J. Neurosci. 15, 1577 – 1586. Wang, Y., Rowan, M.J., Anwyl, R., 1997. Induction of LTD in the dentate gyrus in vitro is NMDA receptor independent, but dependent on Ca2 + influx via low-voltage-activated Ca2 + channels and release of Ca2 + from intracellular stores. J. Neurophysiol. 77, 812 – 825. Watkins, J., Collingridge, G., 1994. Phenylglycine derivatives as antagonists of metabotropic glutamate receptors. Trends. Pharmacol. Sci. 15, 333 – 342. Wickens, J.R., Abraham, W.C., 1991. The involvement of L-type calcium channels in heterosynaptic long-term depression in the hippocampus. Neurosci. Lett. 130, 128 – 132. Yang, X.D., Connor, J.A., Faber, D.S., 1994. Weak excitation and simultaneous inhibition induce long-term depression in hippocampal CA1 neurons. J. Neurophysiol. 71, 1586 – 1590. Yin, H.Z., Carriedo, S.G., Sensi, S.L., Claytor, K.J., Weiss, J.H., 1998. Dendritic expression of Ca2 + /Zn2 + permeable AMPA/ kainate channels on hippocampal pyramidal neurons. Soc. Neurosci. Abstr. 41.7.
Induction of LTD in the adult hippocampus by the synaptic activation of AMPA/kainate and metabotropic glutamate receptors Nicola Kemp, Zafar I. Bashir * Department of Anatomy, School of Medical Sciences, Uni6ersity of Bristol, Bristol, BS8 1TD, UK Accepted 27 November 1998
Abstract It has been suggested that the induction of long-term depression (LTD) may be developmentally regulated since LTD can be readily induced by LFS in slices from young but not adult animals. However, we have recently reported that paired pulse low frequency stimulation (PP-LFS) can reliably induce LTD in the CA1 region of adult hippocampal slices. We now describe the role of glutamate receptors in the induction of LTD in adult hippocampus. The induction of LTD was prevented by the combined application of AMPA/kainate and metabotropic glutamate (mGlu) receptor antagonists (CNQX and LY341495). However, LTD was not blocked by the co-application of NMDA and mGlu receptor antagonists nor by the co-application of NMDA and AMPA/kainate receptor antagonists. Taken together, the above results suggest that activation of either AMPA/kainate or mGlu receptors is sufficient to induce LTD. Therefore, these results suggest that PP-LFS can efficiently activate AMPA/kainate and mGlu receptors in order to induce long-lasting synaptic depression in the CA1 region of the adult hippocampus in vitro. © 1999 Elsevier Science Ltd. All rights reserved. Keywords: Long term depression; LTP; Synaptic plasticity; mGlu; N-methyl-D-aspartate; AMPA/kainate; Hippocampus
1. Introduction In recent years it has become apparent that homosynaptic long term depression (LTD) can be readily induced in the hippocampus and neocortex (Artola et al., 1990; Dudek and Bear, 1992; Mulkey and Malenka, 1992; Kirkwood and Bear, 1994). An understanding of the mechanisms involved in the induction and expression of LTD is important since decreases in synaptic efficacy are likely to be fundamental to the normal function and development of the CNS. A variety of different mechanisms have been described to underlie the induction of LTD in the hippocampus. For example, in neonatal animals (3 – 7 days) the induction of LTD has been shown to be dependent on metabotropic glutamate (mGlu) receptor activation (Bolshakov and Siegelbaum, 1994; Oliet et al., 1997) and voltage-gated calcium channel (VGCC) activation (Bolshakov and Siegelbaum, 1994). Furthermore, in young ( 14 days) * Corrresponding author. Tel.: + 44-117-9288392; fax: + 44-1179291687; e-mail: [email protected].
D-2-amino-5-phosphonopentanoate;
and aged ( 24 months) animals, LFS results in the induction of N-methyl-D-aspartate (NMDA) receptordependent LTD (Dudek and Bear, 1992; Mulkey and Malenka, 1992; Norris et al., 1996). Thus several different, and perhaps developmentally regulated, mechanisms of LTD induction appear to exist within the CA1 region of the hippocampus. The stimulus protocols generally used to induce LTD in young animals are relatively ineffective in inducing LTD in adult animals (Bashir and Collingridge, 1994; Errington et al., 1995; Wagner and Alger, 1995; Abraham et al., 1996; Norris et al., 1996; Kemp and Bashir, 1997a; but see Heynen et al., 1996; Staubli and Ji, 1996). Based on these findings a developmental downregulation of LTD has been inferred. Recently however, we demonstrated that a paired-pulse low frequency stimulation (PP-LFS) protocol, similar to that used previously in vivo (Doyere et al., 1996; Thiels et al., 1996), reliably induced LTD in the adult CA1 region (Kemp and Bashir, 1997a). Other protocols have also recently been demonstrated to induce LTD in adult hippocampus in vitro (Berretta and Cherubini,
0028-3908/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 9 8 ) 0 0 2 2 2 - 6
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Fig. 1. The induction of LTD by PP-LFS in the CA1 region of the adult hippocampus. (A) Single experiment illustrating LTD. Two independent inputs (S0 and S1) were stimulated alternately (each at 0.033 Hz), except during PP-LFS when only one input was stimulated. Throughout the experiment each input received paired stimuli (interpulse interval of 50 ms; see illustrated traces) but only the slope of the 1st response is plotted. Each point is the average of four consecutive responses except during PP-LFS where 20 consecutive responses are averaged. LTD was induced by PP-LFS in S0 and subsequently in S1. (B) Pooled data (n =6) illustrating LTD. In this and subsequent figures the baseline periods are normalised to the 30 min immediately prior to PP-LFS. Where PP-LFS is subsequently delivered to S1 the baseline for this input is re-normalised. Traces are sample responses (average of four consecutive responses) taken from the time points indicated. For clarity, artifacts have been removed and replaced by an upward arrow. Periods of PP-LFS are indicated on the graphs by the two upward arrows joined horizontally.
1998). In the present study we have investigated the details of the mechanisms underlying paired-pulse LFSinduced LTD. We now demonstrate the finding that the induction of LTD in adult CA1 can be dependent on either the activation of AMPA/kainate or mGlu receptors. Some of this work has previously been published in abstract form (Kemp and Bashir, 1998).
2. Methods All experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act, 1986. Female albino rats (3– 4 months) were anaesthetised with halothane and decapitated. The brain was rapidly removed and placed in ice-cold artificial cerebrospinal fluid (aCSF; bubbled with 95% O2/5% CO2) which comprised (mM) NaCl, 124; KCl, 3; NaHCO3, 26; NaH2PO4, 1.25; CaCl2, 2; MgSO4, 1; D-glucose, 10. Transverse slices of hippocampus (400 mm) were cut on
a vibroslice (Campden Instruments) and stored in aCSF. After removing the CA3 region, individual slices were placed in a submerged recording chamber (28– 30oC, flow rate 2.5 ml/min). Standard techniques were used to record field excitatory postsynaptic potentials from the stratum radiatum in response to stimulation (20 ms, 3–10 V) of the Schaffer collateral-commissural pathway (SCCP). The stimulus intensity was set to evoke responses (between 0.5 and 1 mV amplitude) which were roughly 50% of the magnitude at which a population spike started to appear. Two independent sets of SCCP fibres (designated S0 and S1) were alternately stimulated (each at 0.033 Hz). Paired stimuli (50 ms interstimulus interval) were delivered throughout the experiment. Evoked responses were monitored on-line and re-analysed off-line using software developed by Dr W. Anderson (Anderson and Collingridge, 1997). PP-LFS; 900 paired stimuli (50 ms interstimulus interval), 1 Hz was delivered to induce LTD. Any changes in synaptic strength were expressed
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Fig. 2. The induction of LTD was not blocked by NMDA or mGlu receptor antagonists. (A) Pooled data illustrating the lack of effect of AP5 (50 mM) on LTD. PP-LFS was first delivered to S0 and then subsequently delivered to S1 in the presence of AP5. (B) MCPG (1 mM) did not block the induction of LTD.
relative to the normalised pre-LFS baseline (mean depression 9SEM) and significance tested (P B 0.05) using appropriate Student’s t-test at 30 min (unless otherwise indicated) following termination of LFS. Each experiment was carried out on a different slice from a different animal. When used, pharmacological agents were applied via the bathing medium. Drugs used were: D-2-amino-5-phosphonopentanoate (AP5), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), (S)-amethyl-4-carboxyphenylglycine (MCPG), 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) (all purchased from Tocris Neuramin) and LY 341495 (gift from Eli Lilly). Drugs were made up as stock solutions (100 – 1000× final concentration) in equimolar NaOH, except for AP5 (in H20) and CPCCOEt (in DMSO). DMSO at 0.1% had no effect on synaptic transmission nor on the induction of LTD.
3. Results
3.1. Paired pulse low frequency stimulation induces LTD Low frequency stimulation (LFS; 1 Hz, 900 stimuli) did not induce LTD in the CA1 region of the adult
hippocampus (not shown; see Bashir and Collingridge, 1994; Errington et al., 1995; Kemp and Bashir, 1997a). However, PP-LFS; 50 ms interpulse interval, 900 paired pulses at 1 Hz consistently resulted in the induction of homosynaptic long-term depression (S0; 289 4%; PB 0.05, n= 6, Fig. 1). When PP-LFS was subsequently delivered to the other input, LTD was induced (S1; 289 4%; PB 0.05, n =6), which was not different in magnitude to that in S0. This result does not depend on paired pulses being delivered throughout the experiment since delivering paired-pulses only during LFS results in LTD of a similar magnitude to that in the above experiments (data not shown).
3.2. Lack of effect of NMDA and mGlu receptor antagonists on LTD LTD induced by PP-LFS was not blocked by 50 mM AP5 (239 3%, n= 6, PB 0.05, Fig. 2A) nor by 1 mM MCPG (339 4%, n= 5, PB 0.05, Fig. 2B). In addition, LTD was not blocked by the combined presence of AP5 and MCPG (279 5%, n= 8; PB 0.05, Fig. 3A). However, MCPG is a relatively weak, competitive antagonist (Watkins and Collingridge, 1994) therefore we tested the recently described potent group I/II/III mGlu receptor antagonist LY341495 (Kingston et al., 1998;
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Fig. 3. The induction of LTD was not blocked by coapplication of mGlu and NMDA receptor antagonists. (A) In the presence of AP5 and MCPG the magnitude of LTD was comparable to control LTD. (B) In the presence of AP5 and the mGlu receptor antagonist LY341495 (100 mM) the magnitude of LTD was similar to that in the absence of LY341495.
Fitzjohn et al., 1998). This antagonist (100 mM), in the presence of AP5, did not prevent the induction of LTD (23 96%, n=5; PB 0.05, Fig. 3B). This suggests that the induction of LTD is not via activation of NMDA or mGlu receptors. We therefore tested whether there may be a role for AMPA/kainate receptors in LTD induction.
3.3. Lack of effect of NMDA and AMPA receptor antagonists on LTD PP-LFS was delivered in the presence of CNQX (20 mM) and AP5 to block ionotropic glutamate receptors (Fig. 4). After the complete blockade of evoked field potentials (no response to 1st or 2nd of the paired stimuli; see Fig. 4), PP-LFS was delivered to one of the two inputs. Following washout of the antagonists, LTD was observed specifically in the pathway that had received PP-LFS (S0: 339 2%, n =5, P B0.05). Transmission in the heterosynaptic pathway returned towards baseline levels (992%, n =5). Any small depression remaining in the heterosynaptic input is considered to be due to incomplete recovery; the magnitude of homosynaptic LTD expressed relative to the het-
erosynaptic input (re-normalised after antagonist washout) was 279 3%. This suggests that there is no role for AMPA or NMDA receptors in the induction of LTD.
3.4. Are glutamate receptors in6ol6ed in LTD? The above experiments might suggest that there is no role for the synaptic activation of glutamate receptors in the induction of LTD. Given the known functions of glutamate receptors in synaptic plasticity these results were unexpected. However, in the above experiments either AMPA/kainate receptors (Figs. 2 and 3) or mGlu receptors (Fig. 4) were not blocked. It remains possible that activation of either one of these receptors by itself is sufficient to induce LTD. Therefore, we delivered PP-LFS during a complete blockade of glutamatergic transmission (by CNQX, AP5 and LY341495). Under these conditions the induction of LTD was blocked reversibly (Fig. 5A). The magnitude of responses in the pathway that had received PP-LFS (S0: 199 3%, n =4) was not different (P\ 0.05) from those in the control pathway (139 3%, n=4). Expressed relative to the heterosynaptic input, there was no depression in the
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Fig. 4. The induction of LTD during combined antagonism of NMDA and AMPA/kainate receptors. Pooled data showing that perfusion of the antagonists AP5 and CNQX resulted in a complete blockade of postsynaptic responses, during which time PP-LFS was delivered to S0. Following washout of the antagonists there was a recovery of responses in the control input (S1) to baseline values. However, the pathway that received LFS (S0) remained significantly depressed below baseline levels. Inset shows that blockade of ionotropic receptors was complete; there was no response to either the 1st or the 2nd of the pair of stimuli in the presence of CNQX and AP5.
homosynaptic pathway (793%). PP-LFS delivered subsequently to S0 resulted in homosynaptic LTD (37 91%; n= 3, PB0.05). Thus glutamate receptors are involved in the induction of LTD. Since LY341495 is a broad-spectrum mGlu receptor antagonist we tested for an involvement of group I mGlu receptors in the induction of LTD using the selective antagonist CPCCOEt (Annoura et al., 1996; Casabona et al., 1997). PP-LFS was delivered in the combined presence of CPCCOEt (100 mM), AP5 and CNQX. Under these conditions the induction of LTD was blocked reversibly (Fig. 5B). Thus following washout of the antagonists, there was no significant difference (P\0.05; Fig. 5B) between the pathway that had received LFS (S0; 109 8%, n = 4; P \ 0.05) and the control pathway (S1; +4 96%, n = 4; P \ 0.05). PP-LFS was subsequently delivered to the same pathway (S0) and resulted in the induction of LTD (369 8%, n= 3, P B0.05). Thus group I mGlu receptors appear to be involved in LTD. In order to test whether antagonism of group I mGlu and AMPA/kainate receptors is sufficient to block LTD, experiments were carried out in the presence of CPCCOEt and CNQX (Fig. 6). Under these conditions
LTD was blocked reversibly (Fig. 6A). The synaptic strength in the test input (8 9 6%, n= 4; P \ 0.05) was not different (P\ 0.05) from the control input (59 4%, n = 4, Fig. 6B). Relative to the re-normalised heterosynaptic input there was no depression in the homosynaptic input (39 6%). Taken together all of the above results show that glutamate receptors are involved in LTD and that activation of either AMPA/kainate or mGlu receptors is sufficient to allow the induction of LTD. Furthermore, of the mGlu receptors, it appears that the group I subtypes are specifically involved in the induction of LTD.
3.5. Acti6ation of 6oltage-gated calcium channels are not in6ol6ed in LTD The role of AMPA/kainate receptors in the induction of LTD may be to provide the depolarisation necessary to activate voltage-gated calcium channels. In order to test this, experiments were carried out in the presence of AP5, CPCCOEt and nifedipine (10 mM, to block L-type calcium channels) or AP5, CPCCOEt and NiCl (50 mM, to block T-type calcium channels). Under neither
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Fig. 5. LTD is dependent on glutamatergic synaptic transmission. (A) Perfusion of the antagonists AP5, CNQX and LY341495 resulted in a complete blockade of synaptic transmission, during which time PP-LFS was delivered to S0. There was no difference in the recovery between the control (S1) and test pathway (S0) following washout of the antagonists (n =4). Following recovery, PP-LFS was delivered to S0 in three experiments and resulted in the induction of LTD on each occasion. (B) LTD was blocked by perfusion of CPCCOEt, AP5 and CNQX (n= 4). There was no difference in the recovery between the control (S1) and test (S0) pathways following washout of the antagonists. PP-LFS delivered subsequently to S0 (in three experiments) resulted in the induction of LTD on each occasion.
of these conditions was LTD blocked (339 7%, n= 3; Fig. 7A, and 24 91%, n =3; Fig. 7B). Therefore, neither T-type nor L-type calcium channels appear to be involved in the induction of LTD.
4. Discussion The results of this study show that homosynaptic LTD can be reliably induced in the CA1 region of the adult rat hippocampus by appropriate stimulus protocols. The combined antagonism of AMPA/kainate and mGlu receptors was the minimum required to prevent the induction of LTD. In addition, activation by PPLFS of just one of the above receptor types was sufficient to induce LTD. Thus, the induction of LTD was dependent on the synaptic activation of AMPA/kainate or mGlu receptors. Therefore, we have demonstrated two co-existing mechanisms each of which is able to trigger the induction and expression of LTD in the adult hippocampus.
Many previous reports have demonstrated a lack of LFS-induced LTD in the adult CA1 region of the hippocampus (e.g. Bashir and Collingridge, 1994; Errington et al., 1995; Wagner and Alger, 1995; Abraham et al., 1996; Norris et al., 1996; but see Heynen et al., 1996; Staubli and Ji, 1996). However, we (Kemp and Bashir, 1997b) and others (Kerr and Abraham, 1995; Wagner and Alger, 1995; Coussens et al., 1997) have previously demonstrated that appropriate pharmacological manipulations can facilitate LFS-induced LTD in the adult CA1 region. Thus, there is not an absolute developmental down-regulation of LTD. Furthermore, the present results show clearly that LTD can be induced by appropriate synaptic stimulation in the adult CA1 region. This suggests that the induction of LTD by LFS in adult slices may critically depend on experimental conditions. LTD was blocked by the combined antagonism of AMPA/kainate and mGlu receptors, suggesting that activation of NMDA receptors alone is insufficient to induce LTD. Furthermore, LTD was not blocked by
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Fig. 6. LTD was blocked by antagonism of mGlu and AMPA/kainate receptors. (A) Single example illustrating the reversible block of LTD by CPCCOEt and CNQX. (B) Pooled data (n =4) showing that LTD was blocked; control (S1) and test (S0) pathways both recovered to baseline following washout of the antagonists.
antagonism of NMDA receptors. Thus, there appears to be no role for NMDA receptors in LTD in the present study, using animals aged between 3 – 4 months. However, a role for NMDA receptors in the induction of LTD may be developmentally regulated since in young animals (14– 21 days) LTD induced by PP-LFS is blocked completely by AP5 (Kemp et al., 1998) whilst in animals of intermediate age (8 – 12 weeks) there is a partial effect of AP5 (Kemp and Bashir, 1997a). NMDA receptor-independent forms of LTD have been previously described in a number of different studies (Wickens and Abraham, 1991; Bashir and Collingridge, 1994; Christie et al., 1994; Wang et al., 1997; Berretta and Cherubini, 1998). The mechanisms by which CNQX-sensitive receptors may be involved in LTD are not clear. Voltage-gated calcium channel activity, presumably resulting from AMPA receptor-dependent depolarisation, has been shown to be necessary for the induction of LTD (Wickens and Abraham, 1991; Bolshakov and Siegelbaum, 1994; Coussens et al., 1997; Oliet et al., 1997). However, under the conditions of our experiments there appears to be no role for either T-type or L-type voltage gated calcium channels. Whilst calcium perme-
able AMPA receptors (Bettler and Mulle, 1995) are generally found on interneurones (Isa et al., 1996; Toth and McBain, 1998) their localisation on dendrites of pyramidal cells has also been suggested (Garaschuk et al., 1996; Yin et al., 1998). Thus, we cannot rule out that calcium permeating directly through AMPA receptor channels may play a role in the induction of LTD under the present experimental conditions. Synaptic activation of kainate receptors has been demonstrated recently in the hippocampus (Chittajallu et al., 1996; Castillo et al., 1997; Vignes and Collingridge, 1997) and it has been suggested that kainate receptors may be G-protein coupled (Rodriguez-Moreno and Lerma, 1998). Thus an involvement of kainate receptors in LTD remains an intriguing possibility. Given that LTD was blocked during complete antagonism of ionotropic and metabotropic glutamate receptors, but was induced during antagonism of ionotropic receptors alone, suggests a role for mGlu receptors in the induction of this form of plasticity. Many previous studies have implicated a role for mGlu receptors in LTD in, for example, the cerebellum (Linden et al., 1991; Aiba et al., 1994; Conquet et al., 1994; Shigemoto et al., 1994), visual cortex (Kato, 1993), CA1 (Bashir
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Fig. 7. The involvement of AMPA receptors in the induction of LTD is not via activation of voltage-gated calcium channels. Single examples (top) and pooled data (bottom) showing that (A) nifedipine and (B) nickel, in the presence of CPCCOEt and AP5, do not block the induction of LTD.
and Collingridge, 1994; Bolshakov and Siegelbaum, 1994; Yang et al., 1994; Oliet et al., 1997; Overstreet et al., 1997; Palmer et al., 1997; Otani and Connor, 1998) and CA3 regions of the hippocampus (Kobayashi et al., 1996). Of the mGlu receptors, group I (mGlu1/5) receptors which couple to intracellular calcium mobilisation (Pin and Duvoisin, 1995), are likely to be involved in the induction of this form of LTD, since LTD was blocked by the group I selective antagonist, CPCCOEt (Annoura et al., 1996). CPCCOEt has been reported to be 100 times more potent at mGlu1b than mGlu5a in cloned cell lines and at the concentrations (100 mM) used in this study CPCCOEt only slightly reduced mGlu5 receptor-dependent phosphoinositide turnover (Casabona et al., 1997). However, the evidence concerning the relative abundance of mGlu1 and 5 receptor subtypes in the CA1 (Masu et al., 1991; Lujan et al., 1996) would suggest that mGlu5 rather than mGlu1 is the subtype of most importance in this region. Thus, the relative roles of mGlu1 and mGlu5 receptors in LTD require further clarification. The induction of PP-LTD may be dependent on changes in intracellular calcium levels, as has been suggested for other forms of LTD (see Bear and Malenka, 1994; Bear and Abraham, 1996). However, in
the present experiments it appears that there is no role for calcium entry due to NMDA receptor activation, nor due to entry via L- or T-type voltage-gated calcium channels. Given our postulated role of mGlu receptors in the induction of LTD, an increase in intracellular calcium could be brought about via release from intracellular stores, as has been suggested previously for both LTP and LTD (Harvey and Collingridge, 1992; O’Mara et al., 1995). However, whether a rise in intracellular calcium is required for PP-LTD remains to be determined. In this present study, we find that PP-LFS can access the induction of LTD by different but concurrently active synaptic mechanisms. Similarly, an essential role for both mGlu receptors and voltage-gated calcium channels in the induction of LTD has been described in the cerebellum (Linden et al., 1991; Daniel et al., 1992; Conquet et al., 1994; Shigemoto et al., 1994), in neonatal CA1 (Bolshakov and Siegelbaum, 1994) and adult CA1 (Otani and Connor, 1998). In addition, NMDA and mGlu receptor-dependent forms of LTD have been reported to co-exist in young hippocampus (Oliet et al., 1997) and the mechanisms of expression of these two forms of LTD are proposed to be different. Whether each of the mechanisms that we have described in this
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study converge on the same or different expression mechanisms remains to be established. In the present study, the magnitude of LTD induced by activation of AMPA/kainate receptors alone (in the presence of NMDA and mGlu receptor antagonists) or by mGlu receptors alone (in the presence of NMDA and AMPA/ kainate receptor antagonists) was not significantly smaller than that induced by activation of both AMPA/ kainate and mGlu receptors. This suggests that LTD induced by each mechanism might be non-additive and therefore may converge on the same expression mechanisms. In conclusion we have demonstrated a stimulus protocol for the reliable induction of long-term synaptic depression in the adult CA1 region of the hippocampus. Surprisingly, this form of LTD was dependent on the activation of AMPA/kainate and/or mGlu receptors suggesting that PP-LFS may be an efficient means of synaptically activating these particular receptors in order to induce LTD.
Acknowledgements Supported by the Wellcome Trust. LY341495 generously provided by Eli Lilly.
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