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Characterisation of the effects of ATPA, a GLUK5 receptor selective agonist, on excitatory synaptic transmission in area CA1 of rat hippocampal slices
Neuropharmacology 42 (2002) 889–902 www.elsevier.com/locate/neuropharm
Characterisation of the effects of ATPA, a GLUK5 receptor selective agonist, on excitatory synaptic transmission in area CA1 of rat hippocampal slices V.R.J. Clarke ∗, G.L. Collingridge MRC Centre for Synaptic Plasticity, Department of Anatomy, University of Bristol, University Walk, Bristol BS8 1TD, UK Received 22 November 2001; received in revised form 28 February 2002; accepted 4 March 2002
Abstract Kainate receptors are involved in a variety of synaptic functions in the CNS including the regulation of excitatory synaptic transmission. Previously we described the depressant action of the GLUK5 selective agonist (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid (ATPA) on synaptic transmission in the Schaffer collateral–commissural pathway of rat hippocampal slices. In the present study we report several new features of the actions of ATPA at this synapse. Firstly, the effectiveness of ATPA is developmentally regulated. Secondly, the effects of ATPA decline during prolonged or repeated applications. Thirdly, the effects of ATPA are not mediated indirectly via activation of GABAA, GABAB, muscarinic or adenosine A1 receptors. Fourthly, elevating extracellular Ca2+ from 2 to 4 mM antagonises the effects of ATPA. Some differences between the actions of ATPA and kainate on synaptic transmission in the Schaffer collateral–commissural pathway are also noted. 2002 Elsevier Science Ltd. All rights reserved. Keywords: Kainate receptors; ATPA; LY382884; GLUK5 receptors; EPSP; Hippocampus
1. Introduction Kainate receptors were originally identified as a separate family of L-glutamate receptors when it was found that kainate, but not 2-amino-3-(3-hydroxy-5-methyl-4isoxalolyl)propionic acid (AMPA) or N-methyl-Daspartic acid (NMDA) receptor ligands, depolarised isolated C-fibres (Davies et al., 1979; Agrawal and Evans, 1986) and that certain early glutamate receptor ligands differentially antagonised kainate versus other agonists (Davies and Watkins, 1979; McLennan and Lodge, 1979). Since these observations, considerable effort has been made in attempting to understand the functions of kainate receptors in the CNS (Lerma et al., 2001), aided by the recent development of more selective pharmacological tools (Bleakman and Lodge, 1998) and molecular biological techniques (Bettler and Mulle, 1995). Previously we reported that activation of kainate
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receptors was able to regulate excitatory synaptic transmission at CA1 synapses in the hippocampus (Chittajallu et al., 1996). We found that kainate could, in low doses, facilitate and, in high doses, depress synaptic transmission evoked by stimulation of Schaffer collateral– commissural fibres. In addition we reported that ATPA, a selective agonist for the GLUK5 (IUPHAR nomenclature of the receptor formerly known as GluR5 or iGlu5; Lodge and Dingledine (2001)) subtype of kainate receptor, also depressed excitatory synaptic transmission in this pathway (Vignes et al., 1998). Subsequent studies have further characterised the actions of kainate receptor agonists on synaptic transmission in the Schaffer collateral–commissural pathway (Kamiya and Ozawa, 1998; Frerking et al., 2001). In the present study, we provide a more detailed characterisation of the effects of ATPA on excitatory synaptic transmission at the Schaffer collateral–commissural pathway. These studies reveal several new features of the actions of ATPA: first, the effectiveness of ATPA is developmentally regulated; second, the effects progressively decrease during prolonged or repeated
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applications; third, they are not mediated indirectly via activation of GABAA, GABAB, muscarinic or adenosine A1 receptors; and fourth, the effects of ATPA are blocked by elevated Ca2+. In addition, the effects of ATPA and kainate on excitatory synaptic transmission differed in certain aspects.
2. Materials and methods
Wistar rats were either anaesthetised with halothane (3.5%) and decapitated or killed according to schedule 1, in accordance with UK Home Office legislation. The brain was removed rapidly and placed in ice-chilled, oxygenated artificial cerebrospinal fluid (aCSF) comprising of (in mM): NaCl 124, KCl 3, NaHCO3 26, CaCl2 2, MgSO4 1, D-glucose 10, NaH2PO4 1.25 saturated with 95% O2, 5% CO2. Parasaggital slices (400 µm thick) containing the hippocampal region were prepared in icechilled, oxygenated aCSF using a Campden vibroslicer. The hippocampus was dissected from these slices and area CA3 was removed surgically. These slices were then stored in aCSF at room temperature in a holding chamber for at least an hour, and for periods up to 14 h, prior to transfer to an interface recording chamber. Within the chamber, slices rested on a nylon mesh at the interface of warmed (28–32°C), perfusing (1.8–2.2 ml min⫺1) aCSF bubbled with 95% O2, 5% CO2 in a humidified atmosphere. Field excitatory postsynaptic potentials (fEPSPs) were recorded using glass microelectrodes containing 4 M NaCl placed within the stratum radiatum of area CA1 of the hippocampus. Responses were evoked by stimulation of the Schaffer collateral–commissural fibres every 30 s. Compounds were administered by addition to the superfusing medium and applied for a sufficient period to allow equilibration. Data were collected and analysed on-line using LTP software (Anderson and Collingridge, 2001; www.Ltp-program.com). All data are normalised with respect to a 20 minute baseline preceding the application of ATPA unless otherwise illustrated. Data are presented as mean ± S.E.M and statistical significance assessed using Student’s t-tests (P ⬍ 0.05 considered significant and indicated in the figures by ∗). (2S)-3-[[(1S)-1-(3,4-dichlorophenyl) ethyl] amino-2hydroxypropyl](phenylmethyl)-phoshinic acid (CGP 55845A) and (3S, 4aR, 6S, 8aR)-6-((4-carboxyphenyl) methyl- 1,2,3,4,4a,5,6,7,8,8a- decahydroisoquinoline-3carboxylic acid (LY382884) were kind gifts from Dr M. Pozza (Novartis, Basel, Switzerland) and Prof. D. Lodge (Lilly Research Centre Ltd., Erl Wood Manor, Windlesham, GU20 6PH), respectively. Other compounds were obtained from Tocris Cookson.
3. Results 3.1. ATPA depresses excitatory synaptic transmission in rat hippocampus We investigated the ability of ATPA to depress fEPSPs in the CA1 region of the hippocampus of rats within the age range 6–10 weeks, by applying 1 µM ATPA for 20 min (Fig. 1Ai). When ATPA was applied to naı¨ve slices under control conditions, it depressed fEPSPs. The effect varied considerably between slices with peak depressions between 1 and 80 % (mean peak depression 33 ± 3%; n ⫽ 36; p ⬍ 0.05). Concomitant with this depression there was an increase in the pairedpulse facilitation ratio (PPR) of 9 ± 2% (assessed by evoking 2 stimuli at an interval of 50 ms; n ⫽ 36; p ⬍ 0.05). Interestingly, during the application of 1 µM ATPA, the response recovered partially (e.g. peak depression, 33 ± 3%; depression immediately prior to washout, 29 ± 3%; n ⫽ 36; p ⬍ 0.05) (Fig. 1Ai). A range of concentrations (from 30 nM to 3 µM) was tested, with each application to a slice obtained from a different rat. The threshold concentration was approximately 100 nM, the EC50 was 0.57 µM and the maximum predicted depression was approximately 60 % (Fig. 1Aii). For comparison, we have included the nonlinear regression fit obtained from experiments performed on slices from 12–18 day old rats under identical experimental conditions (Vignes et al., 1998; Fig. 1); the values for threshold dose, EC50 and maximum depression were 100 nM, 0.60 µM and 70%, respectively. As noted previously (Bortolotto et al., 1999), the GLUK5 selective kainate receptor antagonist LY382884 blocked the effects of ATPA (Fig. 1B). In this series of experiments, LY382884 depressed fEPSPs slightly (by 12 ± 4%; n ⫽ 4; p ⬍ 0.05) without affecting the PPR ( ⫹ 2 ± 1%; n ⫽ 4). This is due to weak AMPA receptor antagonism (Bortolotto et al., 1999). Having corrected for the small effect of LY382884 per se, application of ATPA caused a depression of 6 ± 2% compared to 38 ± 5% when ATPA was applied to the same slices in the absence of LY382884 (n ⫽ 4; p ⬍ 0.05). 3.2. The effect of ATPA on fEPSPs is age-dependent In Fig. 2 a comparison of the effects of a 20 min application of ATPA (1 µM) in slices obtained from rats of different ages is illustrated. A peak depression of 52 ± 4% (n ⫽ 13; Fig. 2i) was observed for 2 week old rats. A comparable depression of 51 ± 3% (n ⫽ 7; Fig. 2ii) occurred for rats aged 4–6 weeks. However, depressions of 33 ± 3% (n ⫽ 36; see Fig. 1Ai) and 24 ± 3% (n ⫽ 16; Fig. 2iii) were observed as age increased to 6–10 and 10–16 weeks, respectively. The latter two values differ significantly (p ⬍ 0.05) from that
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Fig. 1. ATPA depresses fEPSPs in area CA1 of rats aged 6–10 weeks. (Ai) Graph of pooled experiments with single representative traces at the time points indicated (a–d) for an application of ATPA to a naı¨ve slice. Paired-pulse ratio was monitored by applying 2 stimuli at an inter-pulse interval of 50 ms. Each graph plots the mean ± s.e.m for 36 slices. (ii) Dose-response curve plotting peak synaptic depression versus ATPA concentration for rats aged 6–10 weeks. Each point plots the mean ± s.e.m for three slices except for 1 µM (n ⫽ 36). The solid line results from fitting the Hill equation with an EC50 of 0.57 µM and a Hill coefficient of 1.10. For comparison, we have superimposed the analogous fit obtained in rats aged 12–18 days (dotted line, EC50 0.60 µM, Hill coefficient 1.9; taken from Vignes et al. (1998)). (B) LY382884 antagonises the depressant effect of ATPA on fEPSPs. (i) A single representative example, with traces at the time points indicated (e–j), and (ii) graph of pooled experiments (n ⫽ 4) illustrating the effect of 10 µM LY382884. In this and subsequent figures scale bars represent 0.25 mV, 10 ms throughout and, unless otherwise stated, ATPA was applied for 20 min at a concentration of 1 µM and its effects expressed as a % depression relative to previous 20 min baseline.
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Fig. 2. The effects of ATPA on fEPSPs in area CA1 are age-dependent. (A) Graph of pooled experiments with single representative examples of the effects of ATPA on fEPSPs from rats (i) aged 2 weeks (n ⫽ 13), (ii) aged 4–6 weeks (n ⫽ 7) and (iii) 10–16 weeks (n ⫽ 16). (B) Summary histogram of the % peak depression of ATPA in rats aged 2 weeks and 4–6, 6–10 and 10–16 weeks, respectively (p ⬍ 0.05 versus 2 weeks; indicated by ∗). (C) Plots the % peak depression of fEPSPs by ATPA in a naı¨ve slice (1st slice, x axis) against that in a separate naı¨ve slice obtained from the same rat (2nd slice, y axis). The solid line results from linear regression using least squares fit and yields r2 ⫽ 0.952.
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observed at 2 weeks of age and indicate an age-dependent attenuation of the effects of ATPA on fEPSPs. Whilst the magnitude of depression varied between rats, it was consistent within single rats. We applied ATPA to two separate naı¨ve slices obtained from the same animal (n ⫽ 11). The peak depressions of fEPSP slope were similar (41 ± 7%; 1st slice and 44 ± 7%; 2nd slice, respectively) and the depressions correlated within animals (r2 ⫽ 0.952; Fig. 2C). 3.3. “Desensitisation” of the effects of ATPA We investigated further the ability of fEPSPs to recover in the continued presence of ATPA. When applied for 60 min, a peak depression of 50 ± 5% with an increase in paired-pulse facilitation of 17 ± 3% was observed (n ⫽ 5). Immediately preceding washout of ATPA, both parameters had recovered significantly to control values (⫺1 ± 9% and 0 ± 2%, respectively; n ⫽ 5; p ⬍ 0.05). During a 40 min washout period there was no significant effect on fEPSPs (Fig. 3A). In 3 slices, ATPA was re-applied (for 20 min), 60 min after commencing washout of the first application (Fig. 3B). This had a much diminished effect. In these experiments, the first application of ATPA caused a peak depression of 55 ± 6%, recovering to 14 ± 5% in the continued presence of ATPA. The second ATPA application resulted in a significantly attenuated peak depression of only 18 ± 2% (p ⬍ 0.05; n ⫽ 3). In another set of experiments, following washout of a 20 min application of ATPA, a subsequent application of ATPA produced a significantly smaller effect. Thus, the peak depressions observed were 42 ± 5% and 33 ± 4%, respectively (n ⫽ 6; p ⬍ 0.05; Fig. 3C). These experiments show that responses to ATPA display a pronounced “desensitisation” upon prolonged and repeated exposure. As a result of the “desensitisation” of the effects of ATPA, it was necessary to adopt specific protocols when investigating the sensitivity of the effects of ATPA to various experimental manipulations. When two ATPA applications were compared on the same slice then for 50% of slices, ATPA was first applied during the treatment and then again after washout, whilst for the other 50% of experiments the order was reversed (i.e. reversible manipulations; this includes the data obtained for the antagonist LY382884 in Fig. 1B). Alternatively, for irreversible manipulations (i.e. data obtained for irreversible antagonists such as CGP 55845A or atropine), a single application of ATPA was made under the two conditions, in interleaved experiments performed on two separate slices obtained from the same animal. 3.4. ATPA-mediated depression of fEPSPs is not affected by GABAA or GABAB receptor antagonists The GABAA receptor antagonist, bicuculline methochloride, did not antagonise the ATPA-induced
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depression of fEPSPs. Thus, ATPA caused a depression of 45 ± 5% under control conditions compared to 47 ± 8% in the presence of 10 µM bicuculline (p ⬎ 0.05; Fig. 4A). In these experiments, a second input was reset, by adjusting the stimulus intensity, such that the fEPSP slope was similar prior to addition of ATPA in either the presence or absence of bicuculline. Similar results were obtained; ATPA induced depressions of 45 ± 5% and 47 ± 6% under control conditions and in the presence of bicuculline, respectively (data not illustrated). Similarly, the GABAB receptor antagonist (2S)(+)5,5-dimethyl-2-morpholineacetic acid (SCH 50911) (20 µM) did not antagonise the ATPA-induced depression of fEPSPs. ATPA caused a depression of 28 ± 5% under control conditions compared to 28 ± 7 % in the presence of SCH 50911 (n ⫽ 6; p ⬎ 0.05; Fig. 4B). In contrast, SCH 50911 abolished the depressions of fEPSPs induced by 3 µM baclofen (n ⫽ 3; p ⬍ 0.05; data not shown). A second GABAB receptor antagonist, CGP 55845A (10 µM) was also without effect on ATPA-induced depressions of fEPSPs (n ⫽ 5; p ⬎ 0.05; data not shown). These results confirm the lack of any role for either GABAA or GABAB receptors in the ATPAmediated depression of fEPSPs at this synapse. 3.5. ATPA-mediated depression of fEPSPs is not affected by muscarinic or adenosine receptor antagonists We tested a variety of agonists and antagonists on evoked fEPSPs in order to determine whether the effects of ATPA could be explained via the indirect release of an, as yet undetermined, intermediary. The following agonists had no significant effect after a 20 min application (unless otherwise stated) in naı¨ve slices: cholecystokinin (CCK 8S) (1 µM; depression of ⫺1 ± 5%; n ⫽ 3); NPY13-36 (1 µM; ⫺4 ± 1%; n ⫽ 3); somatostatin (1 µM; 0 ± 1%; n ⫽ 3); substance P (3 µM; ⫺1 ± 1% ; n ⫽ 3); vasoactive intestinal peptide (VIP) (1 µM, 10 min; 1 ± 3%; n ⫽ 3); 4-[2-[[6-amino-9-(N-ethyl-b-Dribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid (CGS 21680) (adenosine A2A receptor agonist, 30 nM; 3 ± 3%; n ⫽ 3; 10 µM: 3 ± 2%; n ⫽ 4) (data not shown). The following agonists for muscarinic acetylcholine and adenosine A1 receptors, respectively, caused a depression of fEPSPs: carbachol (10 µM; 65 ± 3%; n ⫽ 3) and N6 cyclohexyladenosine (CHA) (300 nM; 68 ± 14%; n ⫽ 3) that could be reversed by the respective antagonists, atropine (10 µM; % recovery 110 ± 10%; n ⫽ 3; p ⬍ 0.05) and 8-cyclopentyl-1,3dipropylxanthine (DPCPX) (200 nM; 103 ± 5%; n ⫽ 3 ; p ⬍ 0.05; data not shown). Neither of these antagonists had any effect on the ATPA-mediated depression of fEPSPs: Thus, ATPA induced depressions of 28 ± 6%
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Fig. 3. ‘Desensitisation’ of the effects of ATPA. (A) Pooled data (n ⫽ 5) for a 60 min application of 1 µM ATPA on fEPSP slope and pairedpulse facilitation. (B) Pooled data (n ⫽ 3) illustrating the effect of a 20 min application of ATPA following washout of a 60 min application. (C) Pooled data (n ⫽ 7) illustrating the effects of two 20 min applications of ATPA. The second application occurred between 60–80 min after washout of the first. Again, the subsequent application of ATPA resulted in a peak depression that was reduced in magnitude (p ⬍ 0.05). Each pool is accompanied by traces from a single representative example at the time points indicated (a–q).
and 37 ± 11% under control conditions and in the presence of 10 µM atropine (n ⫽ 5; Fig. 5A) and 29 ± 5% and 27 ± 5% under control conditions and in the presence of 200 nM DPCPX (n ⫽ 10; Fig. 5B), respectively.
3.6. Ca2+ blocks ATPA-mediated depression of fEPSPs Increasing the extracellular bath concentration of Ca2+ from 2 to 4 mM greatly attenuated the ATPA-mediated
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Fig. 4. The ATPA induced depression of fEPSPs is not blocked by GABAA or GABAB receptor antagonists. (Ai) A single example of the effect of ATPA in the absence and presence of bicuculline. (Aii) Graph of pooled experiments (n ⫽ 4). (Bi) A single example of the effect of ATPA in the absence and presence of SCH50911. (Bii) Graph of pooled experiments (n ⫽ 6). Each single representative example is accompanied by traces taken at the time points indicated (a–l).
depression of fEPSPs (Fig. 6). Thus, ATPA caused a peak depression of 49 ± 5% in the presence of 2 mM Ca2+ compared to only 18 ± 6% in the presence of 4 mM Ca2+ (n ⫽ 8; p ⬍ 0.05). Since altering the Ca2+ concentration affected the size of the fEPSP, a second input was reset so that comparisons could be made on evoked fEPSPs of similar magnitude. Comparable results were
obtained for the reset input (with depressions of 48 ± 4% and 15 ± 6%, respectively; data not illustrated). In contrast to the effects of elevating Ca2+, lowering Ca2+ from 2 mM to 1 mM had no effect on the ATPAinduced depressions; these were 30 ± 8% and 33 ± 6% (n ⫽ 4), respectively (data not illustrated). Once again, comparable results were obtained for the reset input
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Fig. 5. The ATPA induced depression of fEPSPs is not blocked by muscarinic or adenosine A1 receptor antagonists. (A) A single example (i) and pooled data (n ⫽ 5; ii) illustrating the effects of ATPA in the absence and presence of atropine. (B) A single example (i) and pooled data (n ⫽ 10; ii) illustrating the effects of ATPA in the absence and presence of DPCPX. Each single representative example is accompanied by traces taken at the time points indicated (a–n).
(depressions of 34 ± 9% and 29 ± 8%, respectively; data not illustrated). Increasing the extracellular bath concentration of Mg2+ from 1 to 2 mM had no effect on ATPA-mediated depression of fEPSPs. ATPA caused a depression of 37 ± 11% in the presence of 1 mM Mg2+ compared to 33 ± 11% in the presence of 2 mM Mg2+ (n ⫽ 4), respectively. Again, similar results were obtained for the
reset input (depressions of 35 ± 9% and 33 ± 11%, respectively; data not illustrated) The changes in Ca2+ and Mg2+ concentrations altered the divalent cation ratio and consequently affected the size of the fEPSP. It was conceivable, therefore, that the effects of ATPA were dependent upon initial release probability rather than Ca2+ concentrations per se. Therefore, we doubled both divalent cation concentrations; a
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Fig. 6. The effects of ATPA are Ca2+-dependent. (A) A single example (i) and pooled data (n ⫽ 8; ii) in the presence of 2 and 4 mM Ca2+. (B) Summary of the effects of ATPA. The histograms plot the % inhibition for the ATPA-induced depressions under the various conditions indicated.
treatment which did not significantly alter the slope of the fEPSP or the paired-pulse facilitation ratio. Under these conditions ATPA-induced depressions were still sensitive to Ca2+, since it produced depressions of 39 ± 5% in the presence of 2 mM Ca2+, 1 mM Mg2+ but of only 24 ± 5% in the presence of 4 mM Ca2+, 2 mM Mg2+; n ⫽ 8, p ⬍ 0.05; Fig. 6). The effects of manipulating Ca2+ and Mg2+ concentrations and bicuculline, SCH 50911 and LY382884 are summarised in Fig. 6b.
3.7. A comparison of the effects of kainate Finally, we examined the effects of kainate in rats aged 6–10 weeks. Application of kainate (3 µM) for 20 min resulted in a depression of the fEPSP of between 13 and 76% (peak mean depression 52 ± 5%; n ⫽ 11; p ⬍ 0.05) with a concomitant increase in PPR (of 14 ± 5%; n ⫽ 11; p ⬍ 0.05) (Fig. 7A). Qualitatively, the effects of kainate were different to those of ATPA, since kainate caused a marked increase in excitability,
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Fig. 7. Summary of the effects of kainate on fEPSPs in area CA1 of rats aged 6–10 weeks. (A) Graph of pooled experiments for an application of kainate to a naı¨ve slice (n ⫽ 11). (B) A single example and pooled data (n ⫽ 4) for a 20 min application of kainate in the absence and presence of LY382884. (C) A single example and pooled data (n ⫽ 4) for a 20 min application of kainate in the presence of 2 and 4 mM Ca2+. Pooled data and single examples are accompanied by traces from a single representative example at the time points indicated (a–o).
manifest as the appearance of a population spike in response to the first, and of multiple population spikes in response to the second of the paired stimuli. The effects of kainate were little affected by LY382884 (10 µM; Fig. 7B). Having corrected for any effect of LY382884 per se, application of kainate caused a depression of 48 ± 6% compared to 54 ± 6% when
applied to the same slices in the absence of LY382884 (n ⫽ 6; p ⬎ 0.05). Increasing Ca2+ from 2 mM to 4 mM did not block the effects of kainate (peak depressions of 56 ± 7% and 48 ± 9%, respectively; n ⫽ 4) but did alter the time-course of the kainate-induced depression of fEPSPs (Fig. 7C).
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4. Discussion In the present study we have presented a characterisation of the effects of ATPA on excitatory synaptic transmission in the CA1 region of rat hippocampal slices. Previously we had reported that ATPA depresses the fEPSP, without affecting the presynaptic fibre volley, that it depresses both AMPA and NMDA receptormediated components of EPSCs and that these effects are associated with an alteration in paired-pulse facilitation (Vignes et al., 1998). Here we have extended our knowledge of this action of ATPA in several ways; we report (i) an age-dependence in the effectiveness of ATPA, (ii) that prolonged applications of ATPA result in pronounced “desensitisation” of its effects, (iii) that the effects of ATPA are unlikely to be mediated indirectly by GABA or other neurotransmitters/neuromodulators, and (iv) that the effects of ATPA are strongly Ca2+ dependent. We also present an initial comparison between the effects of ATPA and kainate. 4.1. ATPA as a GLUK5 receptor agonist ATPA is a selective GLUK5 receptor agonist. In ligand binding studies (Clarke et al., 1997) ATPA is a thousand fold more selective towards GLUK5 receptors than AMPA or other kainate receptor subunits (GLUA1-4, GLUK6, GLUK1, GLUK1/GLUK6 heteromers; see Lodge and Dingledine for IUPHAR nomenclature). Its potency at depressing fEPSPs in the present study (EC50 of 0.57 µM) is very similar to that reported previously (Vignes et al., 1998) for a different data set (0.60 µM) and is comparable with the EC50 values obtained for homomeric GLUK5 expressed in HEK293 cells (2.1 µM; Clarke et al. (1997) and Xenopus oocytes (0.66 µM; Stensbøl et al. (1999) and acutely isolated dorsal root ganglion (DRG) neurones (0.6 µM) (Clarke et al., 1997). Not surprisingly, ATPA is also potent on heteromeric kainate receptor assemblies that include the GLUK5 subunit (Cui and Mayer, 1999; Paternain et al., 2000). In higher concentrations than the standard used throughout the present study (i.e., 1 µM), ATPA acts on other receptors; it acts as a partial agonist on heteromeric assemblies of GLUK2 and GLUK6 subunits at concentrations above 10 µM (Paternain et al., 2000), consistent with its weak interaction with GLUK2 subunits (Clarke et al., 1997). It is also an AMPA receptor agonist (Lauridsen et al., 1985; Stensbøl et al., 1999), with a threshold for depolarisation of CA1 neurones of approximately 3 µM (Clarke and Collingridge, unpublished observations). However, the effects reported here correlate much more closely with an action on GLUK5 containing receptors. This conclusion is further supported by the sensitivity of the effect of ATPA to the GLUK5 selective antagonist LY382884 (Bortolotto et al., 1999). Furthermore, the
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ATPA induced depression of NMDA receptor-mediated EPSCs is blocked by (3SR, 4aRS, 6SR, 8aRS)-6-((((1Htetrazol-5-yl)methyl)oxy)methyl)-1,2,3,4,4a,5,6,7,8,8a decahydroisoquinoline-3-carboxylic acid (LY294486), another GLUK5 selective antagonist (Vignes et al., 1998). ATPA is less potent at depressing monosynaptically evoked inhibition in the CA1 region of the hippocampus (Clarke et al., 1997). In this system, the EC50 is 4.0 µM (Clarke and Collingridge, unpublished observations). One possibility is that this reflects different heteromeric assemblies of GLUK5-containing kainate receptors at different presynaptic locations. 4.2. Age-dependent actions of ATPA We observed a significant age-dependent reduction in the ability of ATPA to depress fEPSPs. This effect was not associated with an alteration in EC50, since the values obtained in the present study for 6–10 week old rats is similar to that obtained previously for 12–18 day old rats under otherwise identical experimental conditions (Vignes et al., 1998). Although the effectiveness of ATPA varied considerably between animals it was consistent in its effectiveness when tested on multiple slices from the same animal. Our results are therefore consistent with an observed decline in GLUK5 expression during development (Bahn et al., 1994). 4.3. “Desensitisation” and Ca2+-dependence of the effects of ATPA We observed that the effects of ATPA faded during prolonged application. This effect can be seen during the standard 20 min applications of ATPA but is even more striking during longer applications. Indeed, with applications of 1 h the response often returned to baseline values. The loss of the ability of ATPA to depress synaptic transmission showed little recovery even following prolonged wash with control medium. Indeed, a second application of ATPA tended to depress transmission to the level obtained at the end of the previous application. The time-course of the effect means that it is unlikely to be due to classical desensitisation but may involve some slow second messenger-induced regulation, perhaps involving G-protein signalling (Rodrı´guez-Moreno and Lerma, 1998; Schmitz et al., 2000; Frerking et al., 2001). The presence of the “desensitisation” is a potential complication when investigating the effects of ATPA. For example, “desensitisation” of its effects could be misconstrued as antagonism of its action. A surprising observation was the strong Ca2+ sensitivity of the effects of ATPA on fEPSPs. Other studies have also observed various degrees of Ca2+ dependence in the actions of kainate receptor ligands. Thus, raising extracellular Ca2+ occludes the facilitation of action potential-dependent Ca2+-dependent GABA release
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(Jiang et al., 2001) and the depression of evoked GABAergic transmission (Rodrı´guez-Moreno and Lerma, 1998) elicited by low and high concentrations of kainate, respectively. In addition, activation of GLUK6 homomers (Egebjerg and Heinemann, 1993) and GLUK6/GLUK2 heteromers (Fukushima et al., 2001) is modulated by extracellular Ca2+. The finding that 4 mM Ca2+ greatly inhibits ATPA mediated effects has implications for in vitro investigations, where divalent cation concentrations are often manipulated. For example, in studies in hippocampal slices the Ca2+ concentration is often raised to these levels as part of a strategy to minimise epileptiform activity. The consequences of this treatment for GLUK5 receptor function need to be taken into account. Further studies are required to determine the mechanism of the Ca2+-dependence of the actions of ATPA and its relationship to the phenomenon of ATPA “desensitisation”.
omodulators as the mediators of the actions of ATPA, including adenosine acting via A1 receptors and acetylcholine acting via muscarinic receptors. We also considered the possibility that neuropeptides, which might be co-released with GABA, could mediate the ATPA effects; however, none of the peptides tested, including those most commonly expressed within CA1 interneurones, mimicked the effects of ATPA. It seems unlikely, therefore, that the effects of ATPA are mediated indirectly via the release of a neuropeptide. In the absence of data to the contrary, the simplest explanation for the actions of ATPA is via GLUK5 containing receptors located presynaptically on Schaffer collateral–commissural fibres. We have recently reached a similar conclusion for the actions of ATPA on mossy fibres in the CA3 region of the hippocampus (Lauri et al., 2001).
4.4. Locus of the effects of ATPA
In area CA1 of 6–10 week old rats, 3 µM kainate caused a similar depression of the fEPSP as 1 µM ATPA. However, there were several differences in the actions of kainate. First, the kainate-induced, but not the ATPA-induced, depression of fEPSP amplitude was associated with an increase in excitability that was particularly pronounced in response to the second of paired stimuli. Second, the effects of kainate were only weakly antagonised by LY382884. Third, increasing Ca2+ to 4 mM had a much less pronounced effect on the actions of kainate. The effect of kainate to induce epileptiform activity at CA1 synapses can be attributed to a reduction in GABAergic inhibition (Collingridge et al., 1983; Kehl et al., 1984). ATPA, like kainate, also depresses evoked synaptic inhibition in this region (Clarke et al., 1997). The reason that its actions are not associated with increased excitability can be explained by a greater relative action on excitation versus inhibition, compared with kainate (Clarke and Collingridge, unpublished observations). The lower sensitivity of the effects of kainate to the actions of LY382884 and Ca2+ is presumably because its actions are predominantly via receptors lacking GLUK5 subunits. The concentration of kainate that we used (3 µM) is near the threshold for activating AMPA receptors in CA1 neurones. Therefore, part of the increase in excitability might be due to AMPA receptor-mediated depolarisation. However, under conditions where AMPA receptors are blocked pharmacologically, kainate can elicit a small inward current in CA1 neurones, suggesting that kainate receptors might also contribute to the postsynaptic action of kainate, possibly via activation of GLUK6 receptors (Chittajallu et al., 1996; Bureau et al., 1999). A small depolarisation, however mediated, would tend to depress the fEPSP indirectly. However, the majority of the depression can be attributed to the exist-
The findings that ATPA depresses both AMPA and NMDA receptor-mediated synaptic transmission and increases paired-pulse facilitation strongly suggests that the effects are mediated via a decrease in neurotransmitter release (Vignes et al., 1998). However, this does not necessarily mean that the GLUK5 receptors are present on Schaffer collateral–commissural axons or terminals. The effects could be mediated via the release of a neuromodulator due to the action of ATPA on other cellular elements. One recent study (Frerking et al., 1999) suggests that the kainate-induced depression of evoked GABAergic transmission is indirect, resulting from depolarisation of GABAergic interneurones (Cossart et al., 1998; Frerking et al., 1998) and subsequent increase in spontaneous GABA release (Fisher and Alger, 1984; Cossart et al., 1998; Frerking et al., 1998); GABA then acts (i) on presynaptic GABAB autoreceptors (Davies and Collingridge, 1993) to depress GABA release and (ii) on postsynaptic GABAA receptors to shunt the recorded response. A similar indirect mechanism might also explain the ability of ATPA to depress excitatory synaptic transmission. Such an argument seems plausible given excitatory terminals possess GABAB heteroreceptors that are activated by synaptically-released GABA (Isaacson et al., 1993; Davies and Collingridge, 1996). Furthermore, the activation of postsynaptic GABAA receptors would act to shunt EPSPs. However, our findings that the effects of ATPA are unaffected by the addition of GABAB or GABAA antagonists, applied alone or in combination (data not shown), dismisses this possibility, under the conditions of our experiments. Previously, we have shown that the ability of ATPA to depress GABAergic inhibition occurs independently of such a mechanism (Clarke et al., 1997). We can also exclude some of the more plausible neur-
4.5. A comparison of the effects of ATPA and kainate
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ence of presynaptic kainate receptors at these synapses (Chittajallu et al., 1996; Kamiya and Ozawa, 1998; Frerking et al., 2001). Therefore it is likely that activation of both LY382884-sensitive (i.e., GLUK5containing) and LY382884-insensitive (i.e., GLUK5lacking) kainate receptors can regulate synaptic transmission at CA1 synapses.
5. Concluding remarks The present work provides evidence that the ability of ATPA to depress fEPSPs at CA1 synapses is not due to some indirect mediator, such as GABA. However, in the course of this investigation, we have found some unexpected effects of ATPA on evoked excitatory synaptic transmission. First, responses to ATPA desensitise, as evident in the observed fade in the depression during the response and as a reduction of the size of successive responses. Second, the effects of ATPA are strongly Ca2+-dependent. Further investigations are required to determine the mechanisms responsible for, and the relationship between, these effects.
Acknowledgements Supported by the MRC. The authors would like to thank J. T. R. Isaac for his careful reading and helpful criticism of this manuscript.
References Agrawal, S.G., Evans, R.H., 1986. The primary afferent depolarising action of kainate in the rat. British Journal of Pharmacology 87, 345–355. Anderson, W.W., Collingridge, G.L., 2001. The LTP program: a data acquisition program for on-line analysis of long-term potentiation and other synaptic events. Journal of Neuroscience Methods 108, 71–83. Bahn, S., Volk, B., Wisden, W., 1994. Kainate receptor gene expression in the developing rat brain. Journal of Neuroscience 14, 5525–5547. Bettler, B., Mulle, C., 1995. AMPA and kainate receptors. Neuropharmacology 24, 123–139. Bleakman, D., Lodge, D., 1998. Neuropharmacology of AMPA and kainate receptors. Neuropharmacology 37, 1187–1204. Bortolotto, Z.A., Clarke, V.R.J., Delaney, C.M., Parry, M.C., Smolders, I., Vignes, M., Ho, K.H., Miu, P., Brinton, B.T., Fantaske, R., Ogden, A., Gates, M., Ornstein, P.L., Lodge, D., Bleakman, D., Collingridge, G.L., 1999. Kainate receptors are involved in synaptic plasticity. Nature 402, 297–301. Bureau, I., Bischoff, S., Heinemann, S.F., Mulle, C., 1999. Kainate receptor-mediated responses in the CA1 field of wild-type and GluR6-deficient mice. Journal of Neuroscience 19, 653–663. Chittajallu, R., Vignes, M., Dev, K.K., Barnes, J.M., Collingridge, G.L., Henley, J.M., 1996. Regulation of glutamate release by presynaptic kainate receptors in the hippocampus. Nature 379, 78–81. Clarke, V.R.J., Ballyk, B.A., Hoo, K.H., Mandelzys, A., Pellizzari, A.,
901
Bath, C.P., Thomas, J., Sharpe, E.F., Davies, C.H., Ornstein, P.L., Schoepp, D.D., Kamboj, R.K., Collingridge, G.L., Lodge, D., Bleakman, D., 1997. A GluR5 kainate receptor that regulates inhibitory synaptic transmission in the hippocampus. Nature 389, 599–603. Collingridge, G.L., Kehl, S.J., McLennan, H., 1983. Intracellularly recorded effects of kainic acid in rat hippocampal slices. Journal of Physiology 340, 43. Cossart, R., Esclapez, M., Hirsch, J.C., Bernard, C., Ben-Ari, Y., 1998. GluR5 kainate receptor activation in interneurons increases tonic inhibition of pyramidal cells. Nature Neuroscience 1, 470–478. Cui, C., Mayer, M.L., 1999. Heteromeric kainate receptors formed by the coassembly of GluR5, GluR6 and GluR7. Journal of Neuroscience 19, 8281–8291. Davies, C.H., Collingridge, G.L., 1993. The physiological regulation of synaptic inhibition by GABAB autoreceptors in rat hippocampus. Journal of Physiology 472, 245–265. Davies, C.H., Collingridge, G.L., 1996. Regulation of EPSPs by the synaptic activation of GABAB autoreceptors in rat hippocampus. Journal of Physiology 496, 451–470. Davies, J., Evans, R.H., Francis, A.A., Watkins, J.C., 1979. Excitatory amino acid receptors and synaptic excitation in the mammalian central nervous system. Journal of Physiology (Paris) 75, 641–654. Davies, J., Watkins, J.C., 1979. Selective antagonism of amino acidinduced and synaptic excitation in the cat spinal cord. Journal of Physiology 297, 621–635. Egebjerg, J., Heinemann, S.F., 1993. Ca2+ permeability of unedited and edited versions of the kainate receptor GluR6. Proceedings of the National Academy of Sciences USA 90, 755–759. Fisher, R.S., Alger, B.E., 1984. Electrophysiological mechanisms of kainic acid-induced epileptiform activity in the rat hippocampal slice. Journal of Neuroscience 4, 1312–1323. Frerking, M., Malenka, R.C., Nicoll, R.A., 1998. Synaptic activation of kainate receptors on hippocampal interneurons. Nature Neuroscience 1, 479–486. Frerking, M., Petersen, C.C.H., Nicoll, R.A., 1999. Mechanisms underlying kainate receptor-mediated disinhibition in the hippocampus. Proceedings of the National Academy of Sciences USA 96, 12917–12922. Frerking, M., Schmitz, D., Zhou, Q., Johansen, J., Nicoll, R.A., 2001. Kainate receptors depress excitatory synaptic transmission at CA3CA1 synapses in the hippocampus via a direct presynaptic action. Journal of Neuroscience 21, 2958–2966. Fukushima, T., Marshall, J., Sakata, K., Shingai, R., 2001. Calcium inhibits willardiine-induced responses in kainate receptor GluR6(Q)/KA-2. NeuroReport 12, 163–165. Isaacson, J.S., Solis, J.M., Nicoll, R.A., 1993. Local and diffuse synaptic actions of GABA in the hippocampus. Neuron 10, 165–175. Jiang, L., Xu, J., Nedergaard, M., Kang, J., 2001. A kainate receptor increases the efficacy of GABAergic synapses. Neuron 30, 503– 513. Kamiya, H., Ozawa, S., 1998. Kainate receptor mediated inhibition of presynaptic Ca2+ influx and EPSP in area CA1 of the rat hippocampus. Journal of Physiology 509, 833–845. Kehl, S.J., McLennan, H., Collingridge, G.L., 1984. Effects of folic and kainic acids on synaptic responses of hippocampal neurones. Neuroscience 11, 111–124. Lauridsen, J., Honore´ , T., Krosgaard-Larsen, P., 1985. Ibotenic acid analogues. Synthesis, molecular flexibility, and in vitro activity of agonists and antagonists at central glutamic acid receptors. Journal of Medicinal Chemistry 28, 668–672. Lauri, S.E., Delaney, C., Clarke, V.R.J., Bortolotto, Z.A., Ornstein, P.L., Isaac, J.T.R., Collingridge, G.L., 2001. Synaptic activation of a presynaptic kainate receptor facilitates AMPA receptor-mediated synaptic transmission at hippocampal mossy fibre synapses. Neuropharmacology 41, 907–915. Lerma, J., Paternain, A. V, Rodrı´guez-Moreno, A., Lo´ pez-Garcı´a, J.C.,
902
V.R.J. Clarke, G.L. Collingridge / Neuropharmacology 42 (2002) 889–902
2001. Molecular physiology of kainate receptors. Physiological Reviews 81, 971–998. Lodge, D., Dingledine, R., 2001. Ionotropic Glutamate Receptors. The IUPHAR Compendium of Receptor Characterization and Classification, 2nd ed. IUPHAR Media Ltd, London. McLennan, H., Lodge, D., 1979. The antagonism of amino acidinduced excitation of spinal neurones in the cat. Brain Research 169, 83–90. Paternain, A. V, Herrera, M.T., Nieto, M.A., Lerma, J., 2000. GluR5 and GluR6 kainate receptor subunits coexist in hippocampal neurons and coassemble to form functional receptors. Journal of Neuroscience 20, 196–205. Rodrı´guez-Moreno, A., Lerma, J., 1998. Kainate receptor modulation of GABA release involves a metabotropic function. Neuron 20, 1211–1218.
Schmitz, D., Frerking, M., Nicoll, R.A., 2000. Synaptic activation of presynaptic kainate receptors on hippocampal mossy fiber synapses. Neuron 27, 327–338. Stensbøl, T.B., Borre, L., Johansen, T.N., Egebjerg, J., Madsen, U., Ebert, B., Krogsgaard-Larsen, P., 1999. Resolution, absolute stereochemistry and molecular pharmacology of the enantiomers of ATPA. European Journal of Pharmacology 380, 153–162. Vignes, M., Clarke, V.R.J., Parry, M.C., Bleakman, D., Lodge, D., Ornstein, P.L., Collingridge, G.L., 1998. The GluR5 subtype of kainate receptor regulates excitatory synaptic transmission in areas CA1 and CA3 of the rat hippocampus. Neuropharmacology 37, 1269–1277.
Characterisation of the effects of ATPA, a GLUK5 receptor selective agonist, on excitatory synaptic transmission in area CA1 of rat hippocampal slices V.R.J. Clarke ∗, G.L. Collingridge MRC Centre for Synaptic Plasticity, Department of Anatomy, University of Bristol, University Walk, Bristol BS8 1TD, UK Received 22 November 2001; received in revised form 28 February 2002; accepted 4 March 2002
Abstract Kainate receptors are involved in a variety of synaptic functions in the CNS including the regulation of excitatory synaptic transmission. Previously we described the depressant action of the GLUK5 selective agonist (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid (ATPA) on synaptic transmission in the Schaffer collateral–commissural pathway of rat hippocampal slices. In the present study we report several new features of the actions of ATPA at this synapse. Firstly, the effectiveness of ATPA is developmentally regulated. Secondly, the effects of ATPA decline during prolonged or repeated applications. Thirdly, the effects of ATPA are not mediated indirectly via activation of GABAA, GABAB, muscarinic or adenosine A1 receptors. Fourthly, elevating extracellular Ca2+ from 2 to 4 mM antagonises the effects of ATPA. Some differences between the actions of ATPA and kainate on synaptic transmission in the Schaffer collateral–commissural pathway are also noted. 2002 Elsevier Science Ltd. All rights reserved. Keywords: Kainate receptors; ATPA; LY382884; GLUK5 receptors; EPSP; Hippocampus
1. Introduction Kainate receptors were originally identified as a separate family of L-glutamate receptors when it was found that kainate, but not 2-amino-3-(3-hydroxy-5-methyl-4isoxalolyl)propionic acid (AMPA) or N-methyl-Daspartic acid (NMDA) receptor ligands, depolarised isolated C-fibres (Davies et al., 1979; Agrawal and Evans, 1986) and that certain early glutamate receptor ligands differentially antagonised kainate versus other agonists (Davies and Watkins, 1979; McLennan and Lodge, 1979). Since these observations, considerable effort has been made in attempting to understand the functions of kainate receptors in the CNS (Lerma et al., 2001), aided by the recent development of more selective pharmacological tools (Bleakman and Lodge, 1998) and molecular biological techniques (Bettler and Mulle, 1995). Previously we reported that activation of kainate
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Corresponding author. Tel.: +44-117-9546474. E-mail address: [email protected] (V.R.J. Clarke).
receptors was able to regulate excitatory synaptic transmission at CA1 synapses in the hippocampus (Chittajallu et al., 1996). We found that kainate could, in low doses, facilitate and, in high doses, depress synaptic transmission evoked by stimulation of Schaffer collateral– commissural fibres. In addition we reported that ATPA, a selective agonist for the GLUK5 (IUPHAR nomenclature of the receptor formerly known as GluR5 or iGlu5; Lodge and Dingledine (2001)) subtype of kainate receptor, also depressed excitatory synaptic transmission in this pathway (Vignes et al., 1998). Subsequent studies have further characterised the actions of kainate receptor agonists on synaptic transmission in the Schaffer collateral–commissural pathway (Kamiya and Ozawa, 1998; Frerking et al., 2001). In the present study, we provide a more detailed characterisation of the effects of ATPA on excitatory synaptic transmission at the Schaffer collateral–commissural pathway. These studies reveal several new features of the actions of ATPA: first, the effectiveness of ATPA is developmentally regulated; second, the effects progressively decrease during prolonged or repeated
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applications; third, they are not mediated indirectly via activation of GABAA, GABAB, muscarinic or adenosine A1 receptors; and fourth, the effects of ATPA are blocked by elevated Ca2+. In addition, the effects of ATPA and kainate on excitatory synaptic transmission differed in certain aspects.
2. Materials and methods
Wistar rats were either anaesthetised with halothane (3.5%) and decapitated or killed according to schedule 1, in accordance with UK Home Office legislation. The brain was removed rapidly and placed in ice-chilled, oxygenated artificial cerebrospinal fluid (aCSF) comprising of (in mM): NaCl 124, KCl 3, NaHCO3 26, CaCl2 2, MgSO4 1, D-glucose 10, NaH2PO4 1.25 saturated with 95% O2, 5% CO2. Parasaggital slices (400 µm thick) containing the hippocampal region were prepared in icechilled, oxygenated aCSF using a Campden vibroslicer. The hippocampus was dissected from these slices and area CA3 was removed surgically. These slices were then stored in aCSF at room temperature in a holding chamber for at least an hour, and for periods up to 14 h, prior to transfer to an interface recording chamber. Within the chamber, slices rested on a nylon mesh at the interface of warmed (28–32°C), perfusing (1.8–2.2 ml min⫺1) aCSF bubbled with 95% O2, 5% CO2 in a humidified atmosphere. Field excitatory postsynaptic potentials (fEPSPs) were recorded using glass microelectrodes containing 4 M NaCl placed within the stratum radiatum of area CA1 of the hippocampus. Responses were evoked by stimulation of the Schaffer collateral–commissural fibres every 30 s. Compounds were administered by addition to the superfusing medium and applied for a sufficient period to allow equilibration. Data were collected and analysed on-line using LTP software (Anderson and Collingridge, 2001; www.Ltp-program.com). All data are normalised with respect to a 20 minute baseline preceding the application of ATPA unless otherwise illustrated. Data are presented as mean ± S.E.M and statistical significance assessed using Student’s t-tests (P ⬍ 0.05 considered significant and indicated in the figures by ∗). (2S)-3-[[(1S)-1-(3,4-dichlorophenyl) ethyl] amino-2hydroxypropyl](phenylmethyl)-phoshinic acid (CGP 55845A) and (3S, 4aR, 6S, 8aR)-6-((4-carboxyphenyl) methyl- 1,2,3,4,4a,5,6,7,8,8a- decahydroisoquinoline-3carboxylic acid (LY382884) were kind gifts from Dr M. Pozza (Novartis, Basel, Switzerland) and Prof. D. Lodge (Lilly Research Centre Ltd., Erl Wood Manor, Windlesham, GU20 6PH), respectively. Other compounds were obtained from Tocris Cookson.
3. Results 3.1. ATPA depresses excitatory synaptic transmission in rat hippocampus We investigated the ability of ATPA to depress fEPSPs in the CA1 region of the hippocampus of rats within the age range 6–10 weeks, by applying 1 µM ATPA for 20 min (Fig. 1Ai). When ATPA was applied to naı¨ve slices under control conditions, it depressed fEPSPs. The effect varied considerably between slices with peak depressions between 1 and 80 % (mean peak depression 33 ± 3%; n ⫽ 36; p ⬍ 0.05). Concomitant with this depression there was an increase in the pairedpulse facilitation ratio (PPR) of 9 ± 2% (assessed by evoking 2 stimuli at an interval of 50 ms; n ⫽ 36; p ⬍ 0.05). Interestingly, during the application of 1 µM ATPA, the response recovered partially (e.g. peak depression, 33 ± 3%; depression immediately prior to washout, 29 ± 3%; n ⫽ 36; p ⬍ 0.05) (Fig. 1Ai). A range of concentrations (from 30 nM to 3 µM) was tested, with each application to a slice obtained from a different rat. The threshold concentration was approximately 100 nM, the EC50 was 0.57 µM and the maximum predicted depression was approximately 60 % (Fig. 1Aii). For comparison, we have included the nonlinear regression fit obtained from experiments performed on slices from 12–18 day old rats under identical experimental conditions (Vignes et al., 1998; Fig. 1); the values for threshold dose, EC50 and maximum depression were 100 nM, 0.60 µM and 70%, respectively. As noted previously (Bortolotto et al., 1999), the GLUK5 selective kainate receptor antagonist LY382884 blocked the effects of ATPA (Fig. 1B). In this series of experiments, LY382884 depressed fEPSPs slightly (by 12 ± 4%; n ⫽ 4; p ⬍ 0.05) without affecting the PPR ( ⫹ 2 ± 1%; n ⫽ 4). This is due to weak AMPA receptor antagonism (Bortolotto et al., 1999). Having corrected for the small effect of LY382884 per se, application of ATPA caused a depression of 6 ± 2% compared to 38 ± 5% when ATPA was applied to the same slices in the absence of LY382884 (n ⫽ 4; p ⬍ 0.05). 3.2. The effect of ATPA on fEPSPs is age-dependent In Fig. 2 a comparison of the effects of a 20 min application of ATPA (1 µM) in slices obtained from rats of different ages is illustrated. A peak depression of 52 ± 4% (n ⫽ 13; Fig. 2i) was observed for 2 week old rats. A comparable depression of 51 ± 3% (n ⫽ 7; Fig. 2ii) occurred for rats aged 4–6 weeks. However, depressions of 33 ± 3% (n ⫽ 36; see Fig. 1Ai) and 24 ± 3% (n ⫽ 16; Fig. 2iii) were observed as age increased to 6–10 and 10–16 weeks, respectively. The latter two values differ significantly (p ⬍ 0.05) from that
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Fig. 1. ATPA depresses fEPSPs in area CA1 of rats aged 6–10 weeks. (Ai) Graph of pooled experiments with single representative traces at the time points indicated (a–d) for an application of ATPA to a naı¨ve slice. Paired-pulse ratio was monitored by applying 2 stimuli at an inter-pulse interval of 50 ms. Each graph plots the mean ± s.e.m for 36 slices. (ii) Dose-response curve plotting peak synaptic depression versus ATPA concentration for rats aged 6–10 weeks. Each point plots the mean ± s.e.m for three slices except for 1 µM (n ⫽ 36). The solid line results from fitting the Hill equation with an EC50 of 0.57 µM and a Hill coefficient of 1.10. For comparison, we have superimposed the analogous fit obtained in rats aged 12–18 days (dotted line, EC50 0.60 µM, Hill coefficient 1.9; taken from Vignes et al. (1998)). (B) LY382884 antagonises the depressant effect of ATPA on fEPSPs. (i) A single representative example, with traces at the time points indicated (e–j), and (ii) graph of pooled experiments (n ⫽ 4) illustrating the effect of 10 µM LY382884. In this and subsequent figures scale bars represent 0.25 mV, 10 ms throughout and, unless otherwise stated, ATPA was applied for 20 min at a concentration of 1 µM and its effects expressed as a % depression relative to previous 20 min baseline.
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Fig. 2. The effects of ATPA on fEPSPs in area CA1 are age-dependent. (A) Graph of pooled experiments with single representative examples of the effects of ATPA on fEPSPs from rats (i) aged 2 weeks (n ⫽ 13), (ii) aged 4–6 weeks (n ⫽ 7) and (iii) 10–16 weeks (n ⫽ 16). (B) Summary histogram of the % peak depression of ATPA in rats aged 2 weeks and 4–6, 6–10 and 10–16 weeks, respectively (p ⬍ 0.05 versus 2 weeks; indicated by ∗). (C) Plots the % peak depression of fEPSPs by ATPA in a naı¨ve slice (1st slice, x axis) against that in a separate naı¨ve slice obtained from the same rat (2nd slice, y axis). The solid line results from linear regression using least squares fit and yields r2 ⫽ 0.952.
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observed at 2 weeks of age and indicate an age-dependent attenuation of the effects of ATPA on fEPSPs. Whilst the magnitude of depression varied between rats, it was consistent within single rats. We applied ATPA to two separate naı¨ve slices obtained from the same animal (n ⫽ 11). The peak depressions of fEPSP slope were similar (41 ± 7%; 1st slice and 44 ± 7%; 2nd slice, respectively) and the depressions correlated within animals (r2 ⫽ 0.952; Fig. 2C). 3.3. “Desensitisation” of the effects of ATPA We investigated further the ability of fEPSPs to recover in the continued presence of ATPA. When applied for 60 min, a peak depression of 50 ± 5% with an increase in paired-pulse facilitation of 17 ± 3% was observed (n ⫽ 5). Immediately preceding washout of ATPA, both parameters had recovered significantly to control values (⫺1 ± 9% and 0 ± 2%, respectively; n ⫽ 5; p ⬍ 0.05). During a 40 min washout period there was no significant effect on fEPSPs (Fig. 3A). In 3 slices, ATPA was re-applied (for 20 min), 60 min after commencing washout of the first application (Fig. 3B). This had a much diminished effect. In these experiments, the first application of ATPA caused a peak depression of 55 ± 6%, recovering to 14 ± 5% in the continued presence of ATPA. The second ATPA application resulted in a significantly attenuated peak depression of only 18 ± 2% (p ⬍ 0.05; n ⫽ 3). In another set of experiments, following washout of a 20 min application of ATPA, a subsequent application of ATPA produced a significantly smaller effect. Thus, the peak depressions observed were 42 ± 5% and 33 ± 4%, respectively (n ⫽ 6; p ⬍ 0.05; Fig. 3C). These experiments show that responses to ATPA display a pronounced “desensitisation” upon prolonged and repeated exposure. As a result of the “desensitisation” of the effects of ATPA, it was necessary to adopt specific protocols when investigating the sensitivity of the effects of ATPA to various experimental manipulations. When two ATPA applications were compared on the same slice then for 50% of slices, ATPA was first applied during the treatment and then again after washout, whilst for the other 50% of experiments the order was reversed (i.e. reversible manipulations; this includes the data obtained for the antagonist LY382884 in Fig. 1B). Alternatively, for irreversible manipulations (i.e. data obtained for irreversible antagonists such as CGP 55845A or atropine), a single application of ATPA was made under the two conditions, in interleaved experiments performed on two separate slices obtained from the same animal. 3.4. ATPA-mediated depression of fEPSPs is not affected by GABAA or GABAB receptor antagonists The GABAA receptor antagonist, bicuculline methochloride, did not antagonise the ATPA-induced
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depression of fEPSPs. Thus, ATPA caused a depression of 45 ± 5% under control conditions compared to 47 ± 8% in the presence of 10 µM bicuculline (p ⬎ 0.05; Fig. 4A). In these experiments, a second input was reset, by adjusting the stimulus intensity, such that the fEPSP slope was similar prior to addition of ATPA in either the presence or absence of bicuculline. Similar results were obtained; ATPA induced depressions of 45 ± 5% and 47 ± 6% under control conditions and in the presence of bicuculline, respectively (data not illustrated). Similarly, the GABAB receptor antagonist (2S)(+)5,5-dimethyl-2-morpholineacetic acid (SCH 50911) (20 µM) did not antagonise the ATPA-induced depression of fEPSPs. ATPA caused a depression of 28 ± 5% under control conditions compared to 28 ± 7 % in the presence of SCH 50911 (n ⫽ 6; p ⬎ 0.05; Fig. 4B). In contrast, SCH 50911 abolished the depressions of fEPSPs induced by 3 µM baclofen (n ⫽ 3; p ⬍ 0.05; data not shown). A second GABAB receptor antagonist, CGP 55845A (10 µM) was also without effect on ATPA-induced depressions of fEPSPs (n ⫽ 5; p ⬎ 0.05; data not shown). These results confirm the lack of any role for either GABAA or GABAB receptors in the ATPAmediated depression of fEPSPs at this synapse. 3.5. ATPA-mediated depression of fEPSPs is not affected by muscarinic or adenosine receptor antagonists We tested a variety of agonists and antagonists on evoked fEPSPs in order to determine whether the effects of ATPA could be explained via the indirect release of an, as yet undetermined, intermediary. The following agonists had no significant effect after a 20 min application (unless otherwise stated) in naı¨ve slices: cholecystokinin (CCK 8S) (1 µM; depression of ⫺1 ± 5%; n ⫽ 3); NPY13-36 (1 µM; ⫺4 ± 1%; n ⫽ 3); somatostatin (1 µM; 0 ± 1%; n ⫽ 3); substance P (3 µM; ⫺1 ± 1% ; n ⫽ 3); vasoactive intestinal peptide (VIP) (1 µM, 10 min; 1 ± 3%; n ⫽ 3); 4-[2-[[6-amino-9-(N-ethyl-b-Dribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid (CGS 21680) (adenosine A2A receptor agonist, 30 nM; 3 ± 3%; n ⫽ 3; 10 µM: 3 ± 2%; n ⫽ 4) (data not shown). The following agonists for muscarinic acetylcholine and adenosine A1 receptors, respectively, caused a depression of fEPSPs: carbachol (10 µM; 65 ± 3%; n ⫽ 3) and N6 cyclohexyladenosine (CHA) (300 nM; 68 ± 14%; n ⫽ 3) that could be reversed by the respective antagonists, atropine (10 µM; % recovery 110 ± 10%; n ⫽ 3; p ⬍ 0.05) and 8-cyclopentyl-1,3dipropylxanthine (DPCPX) (200 nM; 103 ± 5%; n ⫽ 3 ; p ⬍ 0.05; data not shown). Neither of these antagonists had any effect on the ATPA-mediated depression of fEPSPs: Thus, ATPA induced depressions of 28 ± 6%
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Fig. 3. ‘Desensitisation’ of the effects of ATPA. (A) Pooled data (n ⫽ 5) for a 60 min application of 1 µM ATPA on fEPSP slope and pairedpulse facilitation. (B) Pooled data (n ⫽ 3) illustrating the effect of a 20 min application of ATPA following washout of a 60 min application. (C) Pooled data (n ⫽ 7) illustrating the effects of two 20 min applications of ATPA. The second application occurred between 60–80 min after washout of the first. Again, the subsequent application of ATPA resulted in a peak depression that was reduced in magnitude (p ⬍ 0.05). Each pool is accompanied by traces from a single representative example at the time points indicated (a–q).
and 37 ± 11% under control conditions and in the presence of 10 µM atropine (n ⫽ 5; Fig. 5A) and 29 ± 5% and 27 ± 5% under control conditions and in the presence of 200 nM DPCPX (n ⫽ 10; Fig. 5B), respectively.
3.6. Ca2+ blocks ATPA-mediated depression of fEPSPs Increasing the extracellular bath concentration of Ca2+ from 2 to 4 mM greatly attenuated the ATPA-mediated
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Fig. 4. The ATPA induced depression of fEPSPs is not blocked by GABAA or GABAB receptor antagonists. (Ai) A single example of the effect of ATPA in the absence and presence of bicuculline. (Aii) Graph of pooled experiments (n ⫽ 4). (Bi) A single example of the effect of ATPA in the absence and presence of SCH50911. (Bii) Graph of pooled experiments (n ⫽ 6). Each single representative example is accompanied by traces taken at the time points indicated (a–l).
depression of fEPSPs (Fig. 6). Thus, ATPA caused a peak depression of 49 ± 5% in the presence of 2 mM Ca2+ compared to only 18 ± 6% in the presence of 4 mM Ca2+ (n ⫽ 8; p ⬍ 0.05). Since altering the Ca2+ concentration affected the size of the fEPSP, a second input was reset so that comparisons could be made on evoked fEPSPs of similar magnitude. Comparable results were
obtained for the reset input (with depressions of 48 ± 4% and 15 ± 6%, respectively; data not illustrated). In contrast to the effects of elevating Ca2+, lowering Ca2+ from 2 mM to 1 mM had no effect on the ATPAinduced depressions; these were 30 ± 8% and 33 ± 6% (n ⫽ 4), respectively (data not illustrated). Once again, comparable results were obtained for the reset input
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Fig. 5. The ATPA induced depression of fEPSPs is not blocked by muscarinic or adenosine A1 receptor antagonists. (A) A single example (i) and pooled data (n ⫽ 5; ii) illustrating the effects of ATPA in the absence and presence of atropine. (B) A single example (i) and pooled data (n ⫽ 10; ii) illustrating the effects of ATPA in the absence and presence of DPCPX. Each single representative example is accompanied by traces taken at the time points indicated (a–n).
(depressions of 34 ± 9% and 29 ± 8%, respectively; data not illustrated). Increasing the extracellular bath concentration of Mg2+ from 1 to 2 mM had no effect on ATPA-mediated depression of fEPSPs. ATPA caused a depression of 37 ± 11% in the presence of 1 mM Mg2+ compared to 33 ± 11% in the presence of 2 mM Mg2+ (n ⫽ 4), respectively. Again, similar results were obtained for the
reset input (depressions of 35 ± 9% and 33 ± 11%, respectively; data not illustrated) The changes in Ca2+ and Mg2+ concentrations altered the divalent cation ratio and consequently affected the size of the fEPSP. It was conceivable, therefore, that the effects of ATPA were dependent upon initial release probability rather than Ca2+ concentrations per se. Therefore, we doubled both divalent cation concentrations; a
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Fig. 6. The effects of ATPA are Ca2+-dependent. (A) A single example (i) and pooled data (n ⫽ 8; ii) in the presence of 2 and 4 mM Ca2+. (B) Summary of the effects of ATPA. The histograms plot the % inhibition for the ATPA-induced depressions under the various conditions indicated.
treatment which did not significantly alter the slope of the fEPSP or the paired-pulse facilitation ratio. Under these conditions ATPA-induced depressions were still sensitive to Ca2+, since it produced depressions of 39 ± 5% in the presence of 2 mM Ca2+, 1 mM Mg2+ but of only 24 ± 5% in the presence of 4 mM Ca2+, 2 mM Mg2+; n ⫽ 8, p ⬍ 0.05; Fig. 6). The effects of manipulating Ca2+ and Mg2+ concentrations and bicuculline, SCH 50911 and LY382884 are summarised in Fig. 6b.
3.7. A comparison of the effects of kainate Finally, we examined the effects of kainate in rats aged 6–10 weeks. Application of kainate (3 µM) for 20 min resulted in a depression of the fEPSP of between 13 and 76% (peak mean depression 52 ± 5%; n ⫽ 11; p ⬍ 0.05) with a concomitant increase in PPR (of 14 ± 5%; n ⫽ 11; p ⬍ 0.05) (Fig. 7A). Qualitatively, the effects of kainate were different to those of ATPA, since kainate caused a marked increase in excitability,
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Fig. 7. Summary of the effects of kainate on fEPSPs in area CA1 of rats aged 6–10 weeks. (A) Graph of pooled experiments for an application of kainate to a naı¨ve slice (n ⫽ 11). (B) A single example and pooled data (n ⫽ 4) for a 20 min application of kainate in the absence and presence of LY382884. (C) A single example and pooled data (n ⫽ 4) for a 20 min application of kainate in the presence of 2 and 4 mM Ca2+. Pooled data and single examples are accompanied by traces from a single representative example at the time points indicated (a–o).
manifest as the appearance of a population spike in response to the first, and of multiple population spikes in response to the second of the paired stimuli. The effects of kainate were little affected by LY382884 (10 µM; Fig. 7B). Having corrected for any effect of LY382884 per se, application of kainate caused a depression of 48 ± 6% compared to 54 ± 6% when
applied to the same slices in the absence of LY382884 (n ⫽ 6; p ⬎ 0.05). Increasing Ca2+ from 2 mM to 4 mM did not block the effects of kainate (peak depressions of 56 ± 7% and 48 ± 9%, respectively; n ⫽ 4) but did alter the time-course of the kainate-induced depression of fEPSPs (Fig. 7C).
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4. Discussion In the present study we have presented a characterisation of the effects of ATPA on excitatory synaptic transmission in the CA1 region of rat hippocampal slices. Previously we had reported that ATPA depresses the fEPSP, without affecting the presynaptic fibre volley, that it depresses both AMPA and NMDA receptormediated components of EPSCs and that these effects are associated with an alteration in paired-pulse facilitation (Vignes et al., 1998). Here we have extended our knowledge of this action of ATPA in several ways; we report (i) an age-dependence in the effectiveness of ATPA, (ii) that prolonged applications of ATPA result in pronounced “desensitisation” of its effects, (iii) that the effects of ATPA are unlikely to be mediated indirectly by GABA or other neurotransmitters/neuromodulators, and (iv) that the effects of ATPA are strongly Ca2+ dependent. We also present an initial comparison between the effects of ATPA and kainate. 4.1. ATPA as a GLUK5 receptor agonist ATPA is a selective GLUK5 receptor agonist. In ligand binding studies (Clarke et al., 1997) ATPA is a thousand fold more selective towards GLUK5 receptors than AMPA or other kainate receptor subunits (GLUA1-4, GLUK6, GLUK1, GLUK1/GLUK6 heteromers; see Lodge and Dingledine for IUPHAR nomenclature). Its potency at depressing fEPSPs in the present study (EC50 of 0.57 µM) is very similar to that reported previously (Vignes et al., 1998) for a different data set (0.60 µM) and is comparable with the EC50 values obtained for homomeric GLUK5 expressed in HEK293 cells (2.1 µM; Clarke et al. (1997) and Xenopus oocytes (0.66 µM; Stensbøl et al. (1999) and acutely isolated dorsal root ganglion (DRG) neurones (0.6 µM) (Clarke et al., 1997). Not surprisingly, ATPA is also potent on heteromeric kainate receptor assemblies that include the GLUK5 subunit (Cui and Mayer, 1999; Paternain et al., 2000). In higher concentrations than the standard used throughout the present study (i.e., 1 µM), ATPA acts on other receptors; it acts as a partial agonist on heteromeric assemblies of GLUK2 and GLUK6 subunits at concentrations above 10 µM (Paternain et al., 2000), consistent with its weak interaction with GLUK2 subunits (Clarke et al., 1997). It is also an AMPA receptor agonist (Lauridsen et al., 1985; Stensbøl et al., 1999), with a threshold for depolarisation of CA1 neurones of approximately 3 µM (Clarke and Collingridge, unpublished observations). However, the effects reported here correlate much more closely with an action on GLUK5 containing receptors. This conclusion is further supported by the sensitivity of the effect of ATPA to the GLUK5 selective antagonist LY382884 (Bortolotto et al., 1999). Furthermore, the
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ATPA induced depression of NMDA receptor-mediated EPSCs is blocked by (3SR, 4aRS, 6SR, 8aRS)-6-((((1Htetrazol-5-yl)methyl)oxy)methyl)-1,2,3,4,4a,5,6,7,8,8a decahydroisoquinoline-3-carboxylic acid (LY294486), another GLUK5 selective antagonist (Vignes et al., 1998). ATPA is less potent at depressing monosynaptically evoked inhibition in the CA1 region of the hippocampus (Clarke et al., 1997). In this system, the EC50 is 4.0 µM (Clarke and Collingridge, unpublished observations). One possibility is that this reflects different heteromeric assemblies of GLUK5-containing kainate receptors at different presynaptic locations. 4.2. Age-dependent actions of ATPA We observed a significant age-dependent reduction in the ability of ATPA to depress fEPSPs. This effect was not associated with an alteration in EC50, since the values obtained in the present study for 6–10 week old rats is similar to that obtained previously for 12–18 day old rats under otherwise identical experimental conditions (Vignes et al., 1998). Although the effectiveness of ATPA varied considerably between animals it was consistent in its effectiveness when tested on multiple slices from the same animal. Our results are therefore consistent with an observed decline in GLUK5 expression during development (Bahn et al., 1994). 4.3. “Desensitisation” and Ca2+-dependence of the effects of ATPA We observed that the effects of ATPA faded during prolonged application. This effect can be seen during the standard 20 min applications of ATPA but is even more striking during longer applications. Indeed, with applications of 1 h the response often returned to baseline values. The loss of the ability of ATPA to depress synaptic transmission showed little recovery even following prolonged wash with control medium. Indeed, a second application of ATPA tended to depress transmission to the level obtained at the end of the previous application. The time-course of the effect means that it is unlikely to be due to classical desensitisation but may involve some slow second messenger-induced regulation, perhaps involving G-protein signalling (Rodrı´guez-Moreno and Lerma, 1998; Schmitz et al., 2000; Frerking et al., 2001). The presence of the “desensitisation” is a potential complication when investigating the effects of ATPA. For example, “desensitisation” of its effects could be misconstrued as antagonism of its action. A surprising observation was the strong Ca2+ sensitivity of the effects of ATPA on fEPSPs. Other studies have also observed various degrees of Ca2+ dependence in the actions of kainate receptor ligands. Thus, raising extracellular Ca2+ occludes the facilitation of action potential-dependent Ca2+-dependent GABA release
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(Jiang et al., 2001) and the depression of evoked GABAergic transmission (Rodrı´guez-Moreno and Lerma, 1998) elicited by low and high concentrations of kainate, respectively. In addition, activation of GLUK6 homomers (Egebjerg and Heinemann, 1993) and GLUK6/GLUK2 heteromers (Fukushima et al., 2001) is modulated by extracellular Ca2+. The finding that 4 mM Ca2+ greatly inhibits ATPA mediated effects has implications for in vitro investigations, where divalent cation concentrations are often manipulated. For example, in studies in hippocampal slices the Ca2+ concentration is often raised to these levels as part of a strategy to minimise epileptiform activity. The consequences of this treatment for GLUK5 receptor function need to be taken into account. Further studies are required to determine the mechanism of the Ca2+-dependence of the actions of ATPA and its relationship to the phenomenon of ATPA “desensitisation”.
omodulators as the mediators of the actions of ATPA, including adenosine acting via A1 receptors and acetylcholine acting via muscarinic receptors. We also considered the possibility that neuropeptides, which might be co-released with GABA, could mediate the ATPA effects; however, none of the peptides tested, including those most commonly expressed within CA1 interneurones, mimicked the effects of ATPA. It seems unlikely, therefore, that the effects of ATPA are mediated indirectly via the release of a neuropeptide. In the absence of data to the contrary, the simplest explanation for the actions of ATPA is via GLUK5 containing receptors located presynaptically on Schaffer collateral–commissural fibres. We have recently reached a similar conclusion for the actions of ATPA on mossy fibres in the CA3 region of the hippocampus (Lauri et al., 2001).
4.4. Locus of the effects of ATPA
In area CA1 of 6–10 week old rats, 3 µM kainate caused a similar depression of the fEPSP as 1 µM ATPA. However, there were several differences in the actions of kainate. First, the kainate-induced, but not the ATPA-induced, depression of fEPSP amplitude was associated with an increase in excitability that was particularly pronounced in response to the second of paired stimuli. Second, the effects of kainate were only weakly antagonised by LY382884. Third, increasing Ca2+ to 4 mM had a much less pronounced effect on the actions of kainate. The effect of kainate to induce epileptiform activity at CA1 synapses can be attributed to a reduction in GABAergic inhibition (Collingridge et al., 1983; Kehl et al., 1984). ATPA, like kainate, also depresses evoked synaptic inhibition in this region (Clarke et al., 1997). The reason that its actions are not associated with increased excitability can be explained by a greater relative action on excitation versus inhibition, compared with kainate (Clarke and Collingridge, unpublished observations). The lower sensitivity of the effects of kainate to the actions of LY382884 and Ca2+ is presumably because its actions are predominantly via receptors lacking GLUK5 subunits. The concentration of kainate that we used (3 µM) is near the threshold for activating AMPA receptors in CA1 neurones. Therefore, part of the increase in excitability might be due to AMPA receptor-mediated depolarisation. However, under conditions where AMPA receptors are blocked pharmacologically, kainate can elicit a small inward current in CA1 neurones, suggesting that kainate receptors might also contribute to the postsynaptic action of kainate, possibly via activation of GLUK6 receptors (Chittajallu et al., 1996; Bureau et al., 1999). A small depolarisation, however mediated, would tend to depress the fEPSP indirectly. However, the majority of the depression can be attributed to the exist-
The findings that ATPA depresses both AMPA and NMDA receptor-mediated synaptic transmission and increases paired-pulse facilitation strongly suggests that the effects are mediated via a decrease in neurotransmitter release (Vignes et al., 1998). However, this does not necessarily mean that the GLUK5 receptors are present on Schaffer collateral–commissural axons or terminals. The effects could be mediated via the release of a neuromodulator due to the action of ATPA on other cellular elements. One recent study (Frerking et al., 1999) suggests that the kainate-induced depression of evoked GABAergic transmission is indirect, resulting from depolarisation of GABAergic interneurones (Cossart et al., 1998; Frerking et al., 1998) and subsequent increase in spontaneous GABA release (Fisher and Alger, 1984; Cossart et al., 1998; Frerking et al., 1998); GABA then acts (i) on presynaptic GABAB autoreceptors (Davies and Collingridge, 1993) to depress GABA release and (ii) on postsynaptic GABAA receptors to shunt the recorded response. A similar indirect mechanism might also explain the ability of ATPA to depress excitatory synaptic transmission. Such an argument seems plausible given excitatory terminals possess GABAB heteroreceptors that are activated by synaptically-released GABA (Isaacson et al., 1993; Davies and Collingridge, 1996). Furthermore, the activation of postsynaptic GABAA receptors would act to shunt EPSPs. However, our findings that the effects of ATPA are unaffected by the addition of GABAB or GABAA antagonists, applied alone or in combination (data not shown), dismisses this possibility, under the conditions of our experiments. Previously, we have shown that the ability of ATPA to depress GABAergic inhibition occurs independently of such a mechanism (Clarke et al., 1997). We can also exclude some of the more plausible neur-
4.5. A comparison of the effects of ATPA and kainate
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ence of presynaptic kainate receptors at these synapses (Chittajallu et al., 1996; Kamiya and Ozawa, 1998; Frerking et al., 2001). Therefore it is likely that activation of both LY382884-sensitive (i.e., GLUK5containing) and LY382884-insensitive (i.e., GLUK5lacking) kainate receptors can regulate synaptic transmission at CA1 synapses.
5. Concluding remarks The present work provides evidence that the ability of ATPA to depress fEPSPs at CA1 synapses is not due to some indirect mediator, such as GABA. However, in the course of this investigation, we have found some unexpected effects of ATPA on evoked excitatory synaptic transmission. First, responses to ATPA desensitise, as evident in the observed fade in the depression during the response and as a reduction of the size of successive responses. Second, the effects of ATPA are strongly Ca2+-dependent. Further investigations are required to determine the mechanisms responsible for, and the relationship between, these effects.
Acknowledgements Supported by the MRC. The authors would like to thank J. T. R. Isaac for his careful reading and helpful criticism of this manuscript.
References Agrawal, S.G., Evans, R.H., 1986. The primary afferent depolarising action of kainate in the rat. British Journal of Pharmacology 87, 345–355. Anderson, W.W., Collingridge, G.L., 2001. The LTP program: a data acquisition program for on-line analysis of long-term potentiation and other synaptic events. Journal of Neuroscience Methods 108, 71–83. Bahn, S., Volk, B., Wisden, W., 1994. Kainate receptor gene expression in the developing rat brain. Journal of Neuroscience 14, 5525–5547. Bettler, B., Mulle, C., 1995. AMPA and kainate receptors. Neuropharmacology 24, 123–139. Bleakman, D., Lodge, D., 1998. Neuropharmacology of AMPA and kainate receptors. Neuropharmacology 37, 1187–1204. Bortolotto, Z.A., Clarke, V.R.J., Delaney, C.M., Parry, M.C., Smolders, I., Vignes, M., Ho, K.H., Miu, P., Brinton, B.T., Fantaske, R., Ogden, A., Gates, M., Ornstein, P.L., Lodge, D., Bleakman, D., Collingridge, G.L., 1999. Kainate receptors are involved in synaptic plasticity. Nature 402, 297–301. Bureau, I., Bischoff, S., Heinemann, S.F., Mulle, C., 1999. Kainate receptor-mediated responses in the CA1 field of wild-type and GluR6-deficient mice. Journal of Neuroscience 19, 653–663. Chittajallu, R., Vignes, M., Dev, K.K., Barnes, J.M., Collingridge, G.L., Henley, J.M., 1996. Regulation of glutamate release by presynaptic kainate receptors in the hippocampus. Nature 379, 78–81. Clarke, V.R.J., Ballyk, B.A., Hoo, K.H., Mandelzys, A., Pellizzari, A.,
901
Bath, C.P., Thomas, J., Sharpe, E.F., Davies, C.H., Ornstein, P.L., Schoepp, D.D., Kamboj, R.K., Collingridge, G.L., Lodge, D., Bleakman, D., 1997. A GluR5 kainate receptor that regulates inhibitory synaptic transmission in the hippocampus. Nature 389, 599–603. Collingridge, G.L., Kehl, S.J., McLennan, H., 1983. Intracellularly recorded effects of kainic acid in rat hippocampal slices. Journal of Physiology 340, 43. Cossart, R., Esclapez, M., Hirsch, J.C., Bernard, C., Ben-Ari, Y., 1998. GluR5 kainate receptor activation in interneurons increases tonic inhibition of pyramidal cells. Nature Neuroscience 1, 470–478. Cui, C., Mayer, M.L., 1999. Heteromeric kainate receptors formed by the coassembly of GluR5, GluR6 and GluR7. Journal of Neuroscience 19, 8281–8291. Davies, C.H., Collingridge, G.L., 1993. The physiological regulation of synaptic inhibition by GABAB autoreceptors in rat hippocampus. Journal of Physiology 472, 245–265. Davies, C.H., Collingridge, G.L., 1996. Regulation of EPSPs by the synaptic activation of GABAB autoreceptors in rat hippocampus. Journal of Physiology 496, 451–470. Davies, J., Evans, R.H., Francis, A.A., Watkins, J.C., 1979. Excitatory amino acid receptors and synaptic excitation in the mammalian central nervous system. Journal of Physiology (Paris) 75, 641–654. Davies, J., Watkins, J.C., 1979. Selective antagonism of amino acidinduced and synaptic excitation in the cat spinal cord. Journal of Physiology 297, 621–635. Egebjerg, J., Heinemann, S.F., 1993. Ca2+ permeability of unedited and edited versions of the kainate receptor GluR6. Proceedings of the National Academy of Sciences USA 90, 755–759. Fisher, R.S., Alger, B.E., 1984. Electrophysiological mechanisms of kainic acid-induced epileptiform activity in the rat hippocampal slice. Journal of Neuroscience 4, 1312–1323. Frerking, M., Malenka, R.C., Nicoll, R.A., 1998. Synaptic activation of kainate receptors on hippocampal interneurons. Nature Neuroscience 1, 479–486. Frerking, M., Petersen, C.C.H., Nicoll, R.A., 1999. Mechanisms underlying kainate receptor-mediated disinhibition in the hippocampus. Proceedings of the National Academy of Sciences USA 96, 12917–12922. Frerking, M., Schmitz, D., Zhou, Q., Johansen, J., Nicoll, R.A., 2001. Kainate receptors depress excitatory synaptic transmission at CA3CA1 synapses in the hippocampus via a direct presynaptic action. Journal of Neuroscience 21, 2958–2966. Fukushima, T., Marshall, J., Sakata, K., Shingai, R., 2001. Calcium inhibits willardiine-induced responses in kainate receptor GluR6(Q)/KA-2. NeuroReport 12, 163–165. Isaacson, J.S., Solis, J.M., Nicoll, R.A., 1993. Local and diffuse synaptic actions of GABA in the hippocampus. Neuron 10, 165–175. Jiang, L., Xu, J., Nedergaard, M., Kang, J., 2001. A kainate receptor increases the efficacy of GABAergic synapses. Neuron 30, 503– 513. Kamiya, H., Ozawa, S., 1998. Kainate receptor mediated inhibition of presynaptic Ca2+ influx and EPSP in area CA1 of the rat hippocampus. Journal of Physiology 509, 833–845. Kehl, S.J., McLennan, H., Collingridge, G.L., 1984. Effects of folic and kainic acids on synaptic responses of hippocampal neurones. Neuroscience 11, 111–124. Lauridsen, J., Honore´ , T., Krosgaard-Larsen, P., 1985. Ibotenic acid analogues. Synthesis, molecular flexibility, and in vitro activity of agonists and antagonists at central glutamic acid receptors. Journal of Medicinal Chemistry 28, 668–672. Lauri, S.E., Delaney, C., Clarke, V.R.J., Bortolotto, Z.A., Ornstein, P.L., Isaac, J.T.R., Collingridge, G.L., 2001. Synaptic activation of a presynaptic kainate receptor facilitates AMPA receptor-mediated synaptic transmission at hippocampal mossy fibre synapses. Neuropharmacology 41, 907–915. Lerma, J., Paternain, A. V, Rodrı´guez-Moreno, A., Lo´ pez-Garcı´a, J.C.,
902
V.R.J. Clarke, G.L. Collingridge / Neuropharmacology 42 (2002) 889–902
2001. Molecular physiology of kainate receptors. Physiological Reviews 81, 971–998. Lodge, D., Dingledine, R., 2001. Ionotropic Glutamate Receptors. The IUPHAR Compendium of Receptor Characterization and Classification, 2nd ed. IUPHAR Media Ltd, London. McLennan, H., Lodge, D., 1979. The antagonism of amino acidinduced excitation of spinal neurones in the cat. Brain Research 169, 83–90. Paternain, A. V, Herrera, M.T., Nieto, M.A., Lerma, J., 2000. GluR5 and GluR6 kainate receptor subunits coexist in hippocampal neurons and coassemble to form functional receptors. Journal of Neuroscience 20, 196–205. Rodrı´guez-Moreno, A., Lerma, J., 1998. Kainate receptor modulation of GABA release involves a metabotropic function. Neuron 20, 1211–1218.
Schmitz, D., Frerking, M., Nicoll, R.A., 2000. Synaptic activation of presynaptic kainate receptors on hippocampal mossy fiber synapses. Neuron 27, 327–338. Stensbøl, T.B., Borre, L., Johansen, T.N., Egebjerg, J., Madsen, U., Ebert, B., Krogsgaard-Larsen, P., 1999. Resolution, absolute stereochemistry and molecular pharmacology of the enantiomers of ATPA. European Journal of Pharmacology 380, 153–162. Vignes, M., Clarke, V.R.J., Parry, M.C., Bleakman, D., Lodge, D., Ornstein, P.L., Collingridge, G.L., 1998. The GluR5 subtype of kainate receptor regulates excitatory synaptic transmission in areas CA1 and CA3 of the rat hippocampus. Neuropharmacology 37, 1269–1277.