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Presynaptic group 1 metabotropic glutamate receptors may contribute to the expression of long-term potentiation in the hippocampal CA1 region
Neuroscience Vol. 94, No. 1, pp. 71–82, 1999 71 Copyright q 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00
Presynaptic group 1 mGlu receptors may regulate LTP
Pergamon PII: S0306-4522(99)00266-3
PRESYNAPTIC GROUP 1 METABOTROPIC GLUTAMATE RECEPTORS MAY CONTRIBUTE TO THE EXPRESSION OF LONG-TERM POTENTIATION IN THE HIPPOCAMPAL CA1 REGION ´ NCHEZ-PRIETO§ D. MANAHAN-VAUGHAN,*†‡ I. HERRERO,§ K. G. REYMANN k and J. SA *Leibniz Institute for Neurobiology, Department of Neurophysiology, Brenneckestr. 6, D-39008 Magdeburg, Germany †Institute for Physiology of the Charite, Humboldt University, Tucholskystr. 2, 10117 Berlin, Germany §Department of Biochemistry, Veterinary Faculty, Complutense University, Madrid 28040, Spain k Institute for Applied Neuroscience, Leipzigerstr 44, D-39118 Magdeburg, Germany
Abstract—In this study, we investigated the possible contribution of presynaptic group 1 metabotropic glutamate receptor activation to changes in synaptic efficacy by means of analysis of glutamate release in hippocampal synaptosomes. Data were interpreted in the context of group 1 metabotropic glutamate receptor involvement in synaptic plasticity in the CA1 region of freely moving rats. In synaptosomes, 3,5-dihydroxyphenylglycine enhanced diacylglycerol formation and facilitated vesicular Ca 21dependent glutamate release, whereas trans-azetidine-2,4-dicarboxylic acid had no effect on these processes. Trans-azetidine2,4-dicarboxylic acid enhanced glutamate release, but in a Ca 21-independent manner. This effect was mimicked by the l-glutamate uptake inhibitor l-trans-pyrrolidine-2,4-dicarboxylic acid. (R,S)-a-Methyl-4-carboxyphenylglycine blocked the effects of 3,5dihydroxyphenylglycine, but not trans-azetidine-2,4-dicarboxylic acid in synaptosomes. Short-term potentiation (100 Hz, three bursts of 10 stimuli, 0.1 ms stimulus duration, 10 s interburst interval) was induced in the CA1 region in vivo. The metabotropic glutamate receptor agonist 1S,3R-aminocyclopentane-2,3-dicarboxylic acid, or the group 1 metabotropic glutamate receptor agonists, 3,5-dihydroxyphenylglycine and trans-azetidine-2,4-dicarboxylic acid, dose-dependently facilitated short-term potentiation into long-term potentiation, which lasted . 24 h. The facilitation was inhibited by the metabotropic glutamate receptor antagonist, (R,S)-a-methyl-4-carboxyphenylglycine, and the group 1 metabotropic glutamate receptor antagonist, (S)-4-carboxyphenylglycine, but not by the group 2 metabotropic glutamate receptor antagonist, (R,S)-a-methylserine-O-phosphate monophenyl ester. l-Trans-pyrrolidine-2,4-dicarboxylic acid dose-dependently facilitated short-term potentiation into long-term potentiation, which lasted , 4 h. These data suggest that activation of group 1 metabotropic glutamate receptors results in presynaptic modulation of glutamate release. This effect may contribute to group 1 metabotropic glutamate modulation of the expression of long-term potentiation in vivo. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: mGlu, DHPG, ADA, ACPD, synaptosome, LTP.
and glutamate release studies. 12,14 In addition, synaptic transmission studies from area CA1 of the hippocampus have demonstrated that the locus of group 1 mGlu receptormediated inhibition is the presynaptic site. 12,26 Little is known, however, about the relative contribution of presynaptic group 1 mGlu receptors to synaptic transmission or LTP expression. In this study, we therefore attempted to characterize the role of presynaptic group 1 mGlu receptors in hippocampal synaptic transmission and to interpret our findings in the context of mGlu receptor involvement in LTP in vivo. An increase of presynaptic glutamate release has been reported to contribute to LTP expression. 10,12,20 The modulation by presynaptic group 1 mGlu receptors of glutamate release from nerve terminals was therefore investigated using hippocampal synaptosome preparations. Co-activation of N-methyl-d-aspartate receptors with group 1 mGlu receptors gives rise to LTP in the dentate gyrus in vivo. 25 The involvement of group 1 mGlu receptors in LTP in the anatomically and pharmacologically distinct CA1 region was thus investigated.
Long-term potentiation (LTP) in the CA1 region, both in vitro and in vivo, is believed to require the activation of both Nmethyl-d-aspartate receptors and metabotropic glutamate (mGlu) receptors. 2,5,22–25 However, the involvement of mGlu receptors in LTP is still a subject of controversy. 27,36 Recent evidence indicates that group 1 mGlu receptors, which characteristically couple to phospholipase C activation, may play a significant role in the expression of hippocampal LTP in vivo. 21,25 Group 1 mGlu receptors are mainly localized postsynaptically in the rat brain, 3,19,34,37 although a presynaptic role for these receptors has also been suggested by immunological 11,34 ‡To whom correspondence should be addressed at: the Leibniz Institute for Neurobiology. Tel.: 1 49 391 62 63 409; fax: 1 49 391 62 63 438. E-mail address: [email protected] (D. Manahan-Vaughan) Abbreviations: ACPD, 1S,3R-aminocyclopentane-2,3-dicarboxylic acid; ADA, trans-azetidine-2,4-dicarboxylic acid; 4-AP, 4-aminopyridine; BSA, bovine serum albumin; 4-CPG, (S)-4-carboxyphenylglycine; DHPG, 3,5-dihydroxyphenylglycine; EDTA, ethylenediaminetetra-acetate; EEG, electroencephalogram; EGTA, ethyleneglycolbis(aminoethyl ether)tetra-acetate; fEPSP, field excitatory postsynaptic potential; LTP, long-term potentiation; MCPG, (R,S)-a-methyl-4-carboxyphenylglycine; mGlu, metabotropic glutamate; MSOPPE, (R,S)-a-methylserineO-phosphate monophenyl ester; NADPH, reduced nicotinamide adenine dinucleotide phosphate; PDC, l-trans-pyrrolidine-2,4-dicarboxylic acid; STP, short-term potentiation; TES, N-Tris-(hydroxymethyl)methyl-2aminoethanesulfonic acid.
EXPERIMENTAL PROCEDURES
In vivo experiments Seven-week-old male Wistar rats (Schoenwalde, inbred strain from house stocks) underwent implantation of a monopolar recording and a 71
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bipolar stimulating electrode into the CA1 stratum radiatum and Schaffer collaterals, as described previously. 21 A hole was drilled (1 mm diameter) for the recording electrode (2.8 mm posterior to bregma, 1.8 mm lateral to the midline) and a second hole (1 mm diameter, 3.1 mm posterior to bregma, 3.1 mm lateral to the midline) made for the stimulating electrodes (coordinates based on Paxinos and Watson 32). The dura was pierced through both holes, and the recording and stimulating electrodes lowered into the CA1 stratum radiatum and the Schaffer collaterals, respectively. Recordings of evoked field potentials via the implanted electrodes were taken throughout surgery. A cannula was implanted in the lateral cerebral ventricle (0.08 mm posterior to bregma, 1.6 mm lateral to the midline). The animals were allowed between seven and 10 days to recover from surgery before experiments were conducted. The animals were allowed to move freely throughout experiments.
The baseline fEPSP data were obtained by averaging the response to stimulation, to obtain five sweeps at 0.1 Hz, every 5 or 15 min, as described above. The data were then expressed as mean percentage pre-injection baseline reading ^ S.E.M. The probability levels interpreted as statistically significant were P , 0.001, P , 0.01 and P , 0.05.
Measurement of evoked potentials
Preparation of synaptosomes
Responses were evoked by stimulation at low frequency (0.1 Hz). For each time-point, five evoked responses were averaged. The field excitatory postsynaptic potential (fEPSP) slope was measured as the maximal slope through the five steepest points obtained on the first negative deflection of the potential. The fEPSP slope was found by means of input/output curve determination, and all potentials employed as baseline criteria were evoked at a stimulus intensity which produced 40% of this maximum. Short-term potentiation (STP) was induced by a tetanus of 100 Hz (three bursts of 10 stimuli, 0.1 ms stimulus duration, 10 s interburst interval) and a stimulus intensity that was 20% of the maximum (determined by means of an input/ output curve). Hippocampal electroencephalogram (EEG) responses were measured via the recording electrode implanted in the CA1 stratum radiatum. EEG activity was discriminated from noise using a level-time function of the Spike-2 software package (Cambridge Electronic Design, Cambridge, U.K.). The spectral power of the EEG was measured after fast Fourier transformation to compare the characteristics of the EEGs measured before and after 100-Hz stimulation was given.
Hippocampi from male Wistar rats (eight weeks) were isolated and homogenized in a medium containing 320 mM sucrose, 0.5 mM EDTA and 5 mM N-Tris-(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES; pH 7.4). The homogenate was spun for 5 min at 900 × g and 48C, and the supernatants spun again for 10 min at 17,000 × g. From the pellets thus formed, the white, loosely compacted layer containing the majority of synaptosomes was gently resuspended in a medium containing 250 mM sucrose and 5 mM TES (pH 7.4; 3 ml/six hippocampi), and the protein determined by the Biuret method. Pellets containing 1 mg of protein were stored on ice. Synaptosomes remained fully viable, when stored as pellets, for at least 6 h after preparation, as judged by the extent of KCl- and 4aminopyridine (4-AP)-evoked glutamate release.
Compounds and drug treatment 1S,3R-1-Aminocyclopentane-1,3-dicarboxylic acid (ACPD), (R,S)a-methyl-4-carboxyphenylglycine (MCPG), (R,S)-a-methylserine-Ophosphate monophenyl ester (MSOPPE), trans-azetidine-2,4-dicarboxylic acid (ADA), (R,S)-3,5-dihydroxyphenylglycine (DHPG) and (S)-4-carboxyphenylglycine (4-CPG) were obtained from Tocris Cookson Ltd. l-Trans-pyrrolidine-2,4-dicarboxylic acid (PDC) was obtained from Research Biochemicals International. For injection, ADA was dissolved in 0.9% sodium chloride. All other drugs were first dissolved in 5 ml sodium hydroxide solution (1 mM), and then made up to a 100-ml volume with 0.9% sodium chloride. Due to the minute quantities of ACPD used, the drug was first made up as 20 nmol [drug dissolved in 5 ml sodium hydroxide solution (1 mM), and then made up to a 500-ml solution using 0.9% sodium chloride], and then further diluted to 0.2 or 0.4 nmol solutions with 0.9% sodium chloride. These respective solutions were used (in the absence of drugs) as the vehicle in the control experiments for each drug. The compounds were injected in a 5-ml volume over a 6-min period via a Hamilton syringe. Throughout the experiments, injections were administered following measurement of the baseline for 30 min. In LTP experiments, a tetanus was applied 30 min following injection, with measurements then taken at t 5, 10 and 15 min, and then 15-min intervals up to 4 h. Further measurements were taken at 24–25 h. In the case where a combination of two injections occurred in the same experiment, a 30min pause between the first and second injections occurred (during which baseline was measured); thereafter, the experimental protocol was followed as described above. The animals used in each study always served as their own controls,
i.e. prior to the commencement of each study an experiment was carried out where the stability of basal synaptic transmission was examined. If stability was seen over a 4-h period, then the effect of vehicle injection on STP was tested. In some animals, one week later, following an input/output test to confirm that the pre-tetanus values were re-established, the effect of drug injection on STP was evaluated. Data analysis
Glutamate release Glutamate release was assayed by on-line fluorimetry, as described previously. 31 Synaptosomal pellets were resuspended (0.67 mg/ml) in an incubation medium containing 122 mM NaCl, 3.1 mM KCl, 0.4 mM KH2PO4, 5 mM NaHCO3, 1.2 mM MgSO4, 10 mM glucose and 20 mM TES (pH 7.4), and preincubated at 378C for 1 h in the presence of 16 mM bovine serum albumin (BSA) to bind any free fatty acids released from synaptosomes during the preincubation. After preincubation, the synaptosomes were pelleted and resuspended in fresh incubation medium without BSA. An aliquot (1 ml) was transferred to a stirred cuvette containing 1 mM NADP 1, 50 units of glutamate dehydrogenase and 1.33 mM CaCl2 or 200 nM free Ca 21, and the fluorescence of NADPH followed in a Perkin–Elmer LS-50 luminescence spectrometer at excitation and emission wavelengths of 340 and 460 nm, respectively. Traces were calibrated by the addition of 2 nmol glutamate at the end of each assay. Data points were obtained at 2-s intervals. Determination of diacylglycerol Diacylglycerol was estimated as the amount of 32P incorporated from [t- 32P]ATP into phosphatidic acid in the presence of diacylglycerol kinase. Aliquots (0.5 ml) of the synaptosomal suspension (0.67 mg/ml) were added to 0.5 ml chloroform/methanol (95:5, v/v) and rapidly frozen at 2 808C, until all samples were collected. After thawing, the phases were separated by centrifugation, and the lower organic phase collected and evaporated in a water bath at 608C. The tubes containing diacylglycerol were incubated (final volume 100 ml) in a medium containing 70 mM NaCl, 1.4 mM b-mercaptoethanol, 35 mM MgCl2, 0.7% Nonidet P-40, 0.14 mg/ml phosphatidylserine and 35 mM piperazine-N,N-bis(2-ethanesulfonic acid) buffer (pH 6.8). The reaction, at 308C for 1 h, was started by the addition of 60 mU of diacylglycerol kinase, 25 mM ATP and 0.45 mCi of [t- 32P]ATP, and stopped with 100 ml of a mixture containing 5%
Fig. 1. The effect of the mGlu receptor agonist, ACPD, on tetanus-induced STP in the CA1 region. (A) EEG responses measured from the CA1 region of alert rats before and after 100-Hz tetanization. (i) EEG traces obtained immediately before and after tetanization. (ii) Fast Fourier transformation of EEG responses measured during a 5-min period immediately prior to, and for 5 min immediately after, tetanization indicates no significant difference in EEG activity due to tetanization. (B) ACPD (0.2 nmol) has no effect on STP of the fEPSP, when compared with vehicle-injected animals. (C) ACPD (0.4 nmol) significantly enhances the amplitude of STP with regard to fEPSP slope as compared with vehicle-injected STP controls. ACPD (0.4 nmol) has no effect on basal synaptic transmission. The mGlu receptor antagonist MCPG (1 mmol) significantly blocks the effect of 0.4 nmol ACPD from t 90 min post-tetanus. *P , 0.05. Line breaks indicate change in time-scale. (D) Original analog traces showing fEPSP responses in CA1, at three time-points: pre-injection, t 60 min and t 240 min post-tetanus in a vehicle/ACPD (0.2 nmol)-injected animal (i) and an animal injected with vehicle/ACPD (0.4 nmol) (ii).
Presynaptic group 1 mGlu receptors may regulate LTP
Fig. 1.
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perchloric acid, 10 mM ATP and 1 mM H3PO4. The phosphatidic acid was extracted with 0.7 ml chloroform/methanol (95:5, v/v), the solvent evaporated in the water bath at 608C and the radioactivity estimated after the addition of 1.2 ml scintillation liquid. 1,2-Dioctanoyl-snglycerol was used as standard. Statistical significance was determined using a two-tailed Student t-test. RESULTS
Effect of tetanic stimulation on hippocampal electroencephalogram In order to assess whether the stimulus protocol (100 Hz, three bursts of 10 stimuli, 0.1 ms stimulus duration, 10 s interburst interval) used for induction of STP had an effect on hippocampal EEG, EEG was monitored for 60 min before and at least 4 h following tetanization, through the same electrode used to monitor evoked potentials in the CA1 region (Fig. 1Ai). No apparent differences were noted in the appearance of the EEG, nor was there any significant difference in EEG as determined by fast Fourier transformation of the EEG responses measured pre-tetanization and posttetanization (Fig. 1Aii). Thus, one can assume that STP was not induced due to seizure activity in the hippocampus. Effect of 1S,3R-aminocyclopentane-2,3-dicarboxylic acid on tetanus-induced short-term potentiation When a “weak” tetanus was applied via the Schaffer collateral–commissural pathway to the stratum radiatum of the CA1 region (n 12), STP was generated which decayed gradually until approximately 2 h after tetanization. The same time-course for STP was found when the vehicle solution was applied 30 min before tetanus (n 9; Fig. 1B). ACPD (0.2 nmol, n 7) produced no effect on STP elicited by a weak tetanus, as fEPSP values returned to baseline levels by approximately t 120 min (Fig. 1B,D). When 0.4 nmol ACPD (n 5) was administered, STP was prolonged into LTP which lasted for over 24 h (P , 0.05 from t 120, compared to controls, n 9; Fig. 1C,D). ACPD (0.4 nmol) had no effect on basal synaptic transmission, which was monitored for 4 h following ACPD injection (n 4; Fig. 1C). MCPG (1 mmol/5 ml, n 7), when applied prior to 0.4 nmol ACPD and weak tetanus, significantly inhibited the facilitation of STP into LTP by ACPD (P , 0.05 from t 90 compared to controls, n 5; Fig. 1C). The response obtained was similar in profile to, and not statistically different from, that obtained in vehicle-injected controls. Effect of trans-azetidine-2,4-dicarboxylic acid on tetanusinduced short-term potentiation ADA (25 nmol, n 4) had no effect on STP, whereas 50 nmol induced a slight but statistically non-significant facilitation of LTP which returned to pre-tetanization values by approximately 4 h post-tetanus. A concentration of 100 nmol (n 5) enhanced STP into an LTP which lasted for over 24 h (P , 0.05 from t 90 compared to STP controls, n 9),
whereas vehicle-injected controls returned to baseline values by t 120 min (Fig. 2A,B). Increasing the concentration of ADA to 200 nmol (n 4) did not improve the amount of LTP seen (Fig. 2C). ADA (100 nmol) had no effect on basal synaptic transmission, which was monitored for 4 h following drug injection (n 6; Fig. 2A). MCPG (1 mmol), when applied 30 min prior to 100 nmol ADA, and weak tetanus completely inhibited ADA enhancement of STP into LTP (n 8, P , 0.05 from t 90 compared to ADA injected, n 5; Fig. 2D). A similar inhibition of LTP facilitation was obtained when 4-CPG (100 nmol, n 7) was applied prior to ADA (Fig. 2D). No effect on ADA-mediated LTP facilitation was seen when the group 2 mGlu receptor antagonist MSOPPE (200 nmol, n 6) was applied prior to ADA and weak tetanus (Fig. 2D). Neither 4-CPG nor MSOPPE were found previously to have an effect on basal synaptic transmission in the CA1 region. 25 Effect of 3,5-dihydroxyphenylglycine on tetanus-induced short-term potentiation No change in the profile of electrically induced STP was found when vehicle (n 8) or 10 nmol DHPG (n 4) was applied 30 min before tetanus (Fig. 3A,C). DHPG (20 nmol, n 7) produced an enhancement of STP into LTP (P , 0.05 from t 90 compared to controls; Fig. 3A, B). This potentiation was still present after 24 h (Fig. 3A, B). DHPG (20 nmol) had no effect on basal synaptic transmission, which was monitored for 4 h following injection (n 6; Fig. 3D). Increasing the concentration of DHPG to 50 nmol (n 4) did not improve the amount of LTP seen (Fig. 3C). MCPG (1 mmol), when applied prior to 20 nmol DHPG and weak tetanus, completely inhibited facilitation of LTP (n 5, P , 0.05 from t 90) compared to vehicle/DHPGinjected controls (n 6; Fig. 3D). The group 1 mGlu receptor antagonist 4-CPG produced a similar inhibition of DHPGmediated LTP facilitation (100 nmol, n 6; Fig. 3D). Effect of trans-azetidine-2,4-dicarboxylic acid and 3,5-dihydroxyphenylglycine on glutamate release from, and diacylglycerol production in, hippocampal synaptosomes To investigate the contribution of presynaptic group 1 mGlu receptors to synaptic transmission, we measured the ability of DHPG and ADA to modulate the release of glutamate in hippocampal nerve terminals. The depolarization of nerve terminals with a K 1 channel blocker, 4-AP, has been shown to open voltage-gated Ca 21 channels and to induce the release of glutamate. 39 Submaximal concentrations of 4-AP (50 mM), added in the presence of arachidonic acid, did not significantly alter the release of glutamate in the medium with EGTA, but significantly enhanced the release in the presence of CaCl2 (1.33 mM; Fig. 4A). Thus, the extent of the Ca 21dependent release induced by 50 mM 4-AP [which was calculated by subtracting the release obtained after 5 min of depolarization in 200 nM free Ca 21 (EGTA) from the release in
Fig. 2. The effect of the group 1 mGlu receptor agonist, ADA, on tetanus-induced STP in the CA1 region. (A) ADA (100 nmol) significantly enhances the amplitude of STP with regard to fEPSP slope as compared with vehicle-injected controls. ADA (100 nmol) has no effect on basal synaptic transmission. (B) Original analog traces showing fEPSP responses in CA1, at three time-points: pre-injection, t 60 min and t 240 min post-tetanus in an ADA (100 nmol)injected animal. (C) Dose–response curve for the facilitatory effect of ADA (10–200 nmol/5 ml) on STP in the CA1 region. The values represent the magnitude of LTP observed at 4 h post-tetanus. (D) Prior application of MCPG (1 mmol) or the group 1 mGlu receptor antagonist 4-CPG (100 nmol) completely inhibits the facilitation of STP into LTP by ADA (100 nmol). The group 2 mGlu receptor MSOPPE (200 nmol) has no effect on the facilitation of STP into LTP induced by ADA (100 nmol). *P , 0.05, **P , 0.01, ***P , 0.001. Line breaks indicate change in time-scale.
Presynaptic group 1 mGlu receptors may regulate LTP
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Presynaptic group 1 mGlu receptors may regulate LTP
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Fig. 4. The effect of DHPG and ADA on glutamate release from hippocampal nerve terminals. After preincubation of synaptosomes (0.67 mg/ml) at 378C for 1 h in the presence of BSA, the synaptosomes were washed and resuspended in BSA-free incubation medium. Glutamate release was estimated both in the presence of 1.33 mM CaCl2 (Ca 21), and in a medium where the free Ca 21 concentration was maintained at 200 nmol using EGTA. Synaptosomes were depolarized with 4-AP (50 mM) following addition of arachidonic acid (AA) either alone (A) or in combination with the mGlu receptor agonists DHPG (B) or ADA (C). Traces are computer-generated means of three to five data obtained from three to five preparations of synaptosomes. In each preparation, the synaptosomes from two rats were pooled.
1.33 mM CaCl2 (Ca 21)] was 1.1 ^ 0.2 nmol glutamate/mg protein (Fig. 4A). The Ca 21-dependent release was strongly potentiated (3.46 ^ 0.4 nmol glutamate/mg) by the addition of the group 1 mGlu receptor agonist DHPG (100 mM) prior to the depolarization (Fig. 4B), and this effect was abolished by the mGlu receptor antagonist MCPG at 2 mM. In contrast, ADA (100 mM) did not enhance the Ca 21-dependent release (1.0 ^ 0.2 nmol/mg) induced by a submaximal depolarization (Fig. 4C), although this putative mGlu receptor agonist dramatically enhanced the Ca 21-independent release observed in the medium with EGTA. This action was not antagonized by MCPG, however (Fig. 4C). Thus, both DHPG and ADA enhance the release of glutamate from hippocampal nerve terminals, albeit in different ways. Whereas DHPG potentiated the Ca 21-dependent release, which accounts for the release of vesicular glutamate in an MCPG-sensitive manner, on the other hand, the increase in glutamate release induced by ADA is due to the enhancement of cytoplasmic glutamate release, probably by reversal of a glutamate transporter via an MCPG-insensitive process. The facilitation of Ca 21-dependent glutamate release has been demonstrated to occur as a result of protein kinase C activation via diacylglycerol generated by phosphoinositidecoupled mGlu receptor stimulation. 40 Thus, the enhancement of the Ca 21-dependent release by DHPG (100 mM) parallels the ability of this compound to transiently generate diacylglycerol in hippocampal synaptosomes, an effect which was not observed with ADA (100 mM; Fig. 5). On the other hand, ADA induced a delayed and small increase in diacylglycerol levels, an action which was mimicked by the glutamate
transport inhibitor PDC (100 mM). For all three compounds (DHPG, ADA and PDC), the enhancement of diacylglycerol levels 120 s following drug application was significant (P , 0.01 in each case, compared to controls). The delayed response in diacylglycerol levels following ADA administration may be due to the accumulation of extracellular glutamate in the presence of ADA and PDC. These results indicate that ADA does not activate the presynaptic DHPG-sensitive mGlu receptor, which is involved in diacylglycerol production and the facilitation of glutamate exocytosis.
Effect of trans-azetidine-2,4-dicarboxylic acid, 3,5-dihydroxyphenylglycine and l-trans-pyrrolidine-2,4-dicarboxylic acid on Ca21 -independent glutamate PDC is a potent and selective l-glutamate transport inhibitor. However, since PDC is transportable, the expected action upon addition of this compound to synaptosomes is a heteroexchange, through the carrier, with cytoplasmic glutamate. Thus, it can be seen from Fig. 6A that PDC enhanced, in a concentration-dependent manner, the glutamate release from synaptosomes in an EGTA-containing medium. The finding that the group 1 mGlu receptor agonist ADA also enhanced the Ca 21-independent glutamate release in a concentration-dependent manner (10–100 mM; Fig. 6B), and the finding that this action was not antagonized by MCPG, raises the possibility that ADA, which is structurally related to PDC, also acts as a transportable inhibitor of the glutamate transporter.
Fig. 3. The effect of the group 1 mGlu receptor agonist, DHPG, on tetanus-induced STP in the CA1 region. (A) DHPG (20 nmol) significantly enhances the amplitude of STP with regard to fEPSP slope as compared with vehicle-injected controls. (B) Original analog traces showing fEPSP responses in CA1, at three time-points: pre-injection, t 60 min and t 240 min post-tetanus in a DHPG (20 nmol)-injected animal. (C) Dose–response curve for the facilitatory effect of DHPG (2–50 nmol/5 ml) on STP in the CA1 region. The values represent the magnitude of LTP observed at 4 h post-tetanus. (D) Prior application of either MCPG (1 mmol) or 4-CPG (100 nmol) significantly inhibits the effect of DHPG (20 nmol) from t 90 min post-tetanus. DHPG (20 nmol) has no effect on basal synaptic transmission. *P , 0.05, **P , 0.01, ***P , 0.001. Line breaks indicate change in time-scale.
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Fig. 5. DHPG but not ADA enhances diacylglycerol production. Timecourse of diacylglycerol formation induced by DHPG (A), ADA (B) and PDC (O), all added at 100 mM. At the times indicated, following addition of the agonist, 0.5-ml aliquots of synaptosomes (0.67 mg/ml) were taken and the diacylglycerol levels determined as indicated in the Experimental Procedures section. Control experiments show the diacylglycerol levels in the absence of any additions. The results are means ^ S.D. of three data from three preparations of synaptosomes. In each preparation, the synaptosomes from two rats were pooled.
Effect of l-trans-pyrrolidine-2,4-dicarboxylic acid on tetanus-induced short-term potentiation The glutamate transporter inhibitor PDC at a concentration of 20 (n 4) or 40 nmol/5 ml (n 5) had no effect when applied prior to tetanus-induced STP compared to controls (n 9). Application of 100 nmol PDC (n 6) facilitated
STP into LTP which lasted approximately 4 h (Fig. 7A, B). When basal synaptic transmission was monitored for 4 h in the presence of PDC (100 nmol), a transient increase in fEPSP slope function was seen (n 4) compared to controls (n 8). This effect became statistically significant at 30 min postinjection, but returned to pre-injection baseline values by 120 min post-injection. To investigate whether ADA facilitates STP into LTP in vivo through a presynaptic inhibition of glutamate transport, experiments were carried out where PDC was applied in combination with DHPG or ADA. Thus, a concentration of PDC which is subthreshold for facilitation of LTP (40 nmol) was applied together with a subthreshold concentration of ADA (25 nmol, n 4) prior to an STP-inducing tetanus. In this case, LTP occurred which declined to control levels (n 6) by 4 h post-tetanus (Fig. 7C). fEPSPs evoked in the PDC/ADA group were statistically significant from control levels from t 150 min until t 225 min post-tetanus (P , 0.05), whereupon evoked potentials returned to pre-tetanus levels. The combination of PDC (40 nmol) with ADA (25 nmol, n 4) had no significant effect upon basal synaptic transmission, which was monitored for 4 h following drug injection. When PDC (40 nmol) was applied with a concentration of DHPG which is subthreshold for facilitation of LTP (10 nmol, n 6), a facilitation of STP into LTP occurred which was still present 4 h post-tetanus, but had returned to control levels (n 5) by 24 h post-tetanus (Fig. 7C). fEPSPs evoked in the PDC/DHPG group were statistically significant from control levels from t 120 min until t 240 min post-tetanus (P , 0.01), but returned thereafter to pre-tetanus levels. The combination of PDC (40 nmol) with DHPG (10 nmol, n 4) had no significant effect upon basal synaptic transmission measured for 4 h following injection.
Fig. 6. The effect of the group 1 mGlu receptor agonists DHPG and ADA on glutamate transporter-mediated, Ca 21-independent glutamate release. After preincubation of synaptosomes (0.67 mg/ml) at 378C for 1 h in the presence of BSA, an aliquot (1 ml) was used to determine glutamate release. Glutamate release from polarized synaptosomes was determined in incubation medium, keeping the free Ca 21 concentration at 200 nM using EGTA, in the absence of any additions (control), or following the addition of ADA (10 or 100 mM; A), DHPG (100 mM; A) or PDC (10–100 mM; B). Traces are computer-generated means of three to five data obtained from three to five preparations of synaptosomes. In each preparation, the synaptosomes from two rats were pooled.
Presynaptic group 1 mGlu receptors may regulate LTP DISCUSSION
The findings of this study indicate that presynaptic group 1 mGlu receptor activation results in an enhancement of presynaptic glutamate release. This effect, in turn, may contribute to the alterations in synaptic efficacy evoked by group 1 mGlu receptor stimulation in vivo. Application of group 1 receptor agonists resulted in a facilitation of STP into LTP in the CA1 region in vivo. Taken together, the data of the current study suggest that not only postsynaptic, but also presynaptic group 1 mGlu receptors contribute to LTP facilitation in vivo. Group 1 mGlu receptors do not appear to contribute to basal synaptic transmission in vivo, as neither DHPG nor ADA influenced baseline measurements, in the current study, when applied at concentrations which were effective in converting STP into LTP. This observation may have a morphological explanation, as it has been shown that postsynaptic group 1 mGlu receptors are localized perisynaptically. 3,19 Thus, under conditions of basal synaptic transmission, insufficient glutamate may be released into the synaptic cleft to enable diffusion to the perisynaptic area and subsequent activation of group 1 mGlu receptors. It is not known whether presynaptic group 1 mGlu receptors are activated under basal synaptic conditions, although the lack of baseline effect of the agonists tested suggests that presynaptic group 1 mGlu receptors probably do not contribute to basal synaptic transmission. During LTP-inducing tetanization, however, increased presynaptic release of glutamate should result in both pre- and postsynaptic group 1 mGlu receptor activation. Presynaptic group 1 mGlu receptor activation at this time may play an important role in the elevation of presynaptic glutamate release, which is a characteristic of LTP, 10,13,19 whereas postsynaptic mGlu receptor activation gives rise to phospholipase C activation and subsequent release of calcium from intracellular stores. 30 In a physiological situation where group 1 mGlu receptors are activated (e.g., during LTP induction), it is therefore likely that both presynaptic and postsynaptic group 1 mGlu receptors participate concurrently in subsequent modifications of synaptic efficacy. In this study, the group 1 and 2 mGlu receptor agonist, ACPD, produced a similar facilitation of STP into LTP in the CA1 region, as was reported previously in vitro 1,4,6,28,29 and for the dentate gyrus in vivo. 23 It was shown previously that ACPD enhances Ca 21-dependent glutamate release from hippocampal synaptosomes in the presence of arachidonic acid, and that the mGlu receptor antagonist MCPG abolishes this effect. 40 This facilitatory effect of ACPD on glutamate release is probably due to the activation of a group 1 mGlu receptor, as it is mediated by the ACPD-induced formation of diacylglycerol and activation of presynaptic protein kinase C. 40 DHPG 15,35 is a specific group 1 mGlu receptor agonist. In this study, DHPG produced facilitations of STP into LTP which were similar in both magnitude and profile to that produced by ACPD, and which were consistent with previous reports that priming with DHPG facilitates LTP induction in the CA1 region in vitro 7 and the dentate gyrus in vivo. 25 These effects were also blocked by the mGlu receptor antagonist MCPG and by the group 1 mGlu receptor antagonist, 4CPG. In hippocampal synaptosomes, DHPG enhanced glutamate release in the presence of calcium, consistent with the activation of presynaptic group 1 mGlu receptors.
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A facilitation of LTP which is similar in profile to that induced by ACPD was stimulated through the putative group 1 mGlu receptor agonist ADA. This effect was inhibited by the mGlu receptor antagonist MCPG. ADA 9,18 is a structural analog of glutamate which stimulates phosphoinositide hydrolysis in hippocampal slices and activates inositol phosphate accummulation in transfected LLC-PK1 cells with an ec50 at mGluR1 of 189 mM and at mGluR5 of 32 mM. 25 By comparison, the ec50 of ACPD at mGluR1 is approximately 137 mM and is 50 mM at mGluR5. 33 It has been reported that ADA is an agonist at human group 2 mGlu receptors. 17 However, this effect has not been detected in rat tissue. 22 Furthermore, in the current study, application of the group 2 mGlu receptor antagonist MSOPPE 16,38 prior to ADA injection had no effect on the facilitation by ADA of STP into LTP. In addition, the finding that the group 1 mGlu receptor antagonist 4-CPG completely inhibited the facilitatory effects of ADA implies that it acts as an agonist of group 1 mGlu receptors. ACPD, DHPG and ADA produce facilitations of STP into LTP which are of similar magnitude and duration, whereas only ACPD 40 and DHPG, but not ADA, activate presynaptic group 1 mGlu receptors. However, ADA also potentiates glutamate release, albeit in an mGlu receptor-independent manner, by displacement of cytoplasmic glutamate. Thus, ADA mimicks the action of the glutamate uptake inhibitor, PDC, to which ADA is structurally related. It is possible that the activation of a presynaptic phosphoinositide-coupled mGlu receptor and the subsequent increase in glutamate release contributes to the conversion of STP into LTP, seen when ACPD or DHPG are given in the presence of “weak” tetanization, and that ADA mimicks this effect through the inhibition of the glutamate transporter. This possibility is further supported by the finding that the time-course for facilitation of glutamate release in the absence of calcium was similar to that observed with the Na 1-dependent glutamate transporter inhibitor PDC. 8,41 PDC itself was found to induce a dose-dependent facilitation of STP into LTP in this study, although the LTP produced did not persist much longer than 4 h, unlike LTP produced by the mGlu receptor agonist, ADA, which lasted for more than 24 h. The fact that ADAinduced conversion of STP into LTP was competely abolished by both MCPG and the group 1 mGlu receptor antagonist 4-CPG would indicate either a further action of ADA at postsynaptic group 1 mGlu receptors, or that the presynaptic increase in glutamate release provided by ADA enables the activation of postsynaptic group 1 mGlu receptors. Thus, the higher circulating levels of glutamate, which would occur in the synaptic cleft as a result of a possible ADA-mediated inhibition of glutamate transport, could in turn enable further activation of group 1 mGluRs which would thereby contribute to LTP facilitation. In order to try to ascertain whether mild activation of group 1 mGlu receptors together with inhibition of glutamate transport could facilitate LTP in a manner similar to that seen with ADA, concentrations of PDC and ADA, or PDC and DHPG, which were subthreshold for facilitation of LTP were tested. In each case, a facilitation of STP into LTP which lasted approximately 4 h occurred. This further suggests that, although a presynaptic non-mGlu receptor-mediated elevation of glutamate levels by ADA may contribute to LTP facilitation by this compound, postsynaptic group 1 mGlu receptor activation by ADA may account for the 24-h LTP
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Fig. 7. The effect of the glutamate uptake inhibitor, PDC, on tetanus-induced STP in the CA1 region. (A) PDC (100 nmol) significantly enhances the amplitude of STP with regard to fEPSP slope as compared with vehicle-injected controls. PDC (40 nmol) or vehicle injection have no effect on STP. (B) PDC (100 nmol), when applied in the absence of tetanization, induces a transient increase in basal synaptic transmission. (C) Application of PDC at a concentration which is subthreshold for LTP facilitation (40 nmol), together with a subthreshold concentration of ADA (25 nmol), facilitates STP into a short-lasting LTP. A similar short-lasting LTP was produced when PDC (40 nmol) was given at a subthreshold concentration of DHPG (10 nmol). *P , 0.05, **P , 0.01. Line breaks indicate change in time-scale.
Presynaptic group 1 mGlu receptors may regulate LTP
induced by application of the compound. Thus, the activation of postsynaptic group 1 mGlu receptors by ADA is consistent with its ability to activate inositol phosphate accumulation both in mGluR1 and mGluR5 transfected LLC-PK1 cells, 22 while the failure of ADA to activate diacylglycerol production and glutamate release facilitation would indicate that the facilitatory mGlu receptor at hippocampal nerve terminals is neither mGluR1 nor mGluR5. CONCLUSION
This study indicates that calcium-dependent presynaptic
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glutamate release is increased by activation of presynaptic group 1 mGlu receptors. In addition, group 1 mGlu receptor activation in the CA1 region facilitates the expression of LTP in vivo. These findings suggest a role for presynaptic mGlu receptors in LTP induction in vivo.
Acknowledgements—We would like to thank Ms Silvia Vieweg for valuable assistance in technical aspects of this work. We are indebted to Dr Thomas Seidenbecher and Mr Jurgen Buggert for expert advice and support in hippocampal EEG measurements.
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33. Pin J. P. and Duvoisin R. (1995) The metabotropic glutamate receptors: structure and function. Neuropharmacology 34, 1–26. 34. Romano C., Sesma M. A., McDonald C. T., O’Malley K., Van den Pol A. N. and Olney J. (1995) Distribution of metabotropic glutamate receptor mGlu receptor 5 immunoreactivity in rat brain. J. comp. Neurol. 355, 455–469. 35. Schoepp D., Goldsworthy J., Johnson B. G., Salhoff C. R. and Baker R. S. (1994) 3,5-Dihydroxyphenylglycine is a highly selective agonist for phosphoinositide-linked metabotropic glutamate receptors in the rat hippocampus. J. Neurochem. 63, 769–773. 36. Selig D. K., Lee H.-K., Bear M. F. and Malenka R. C. (1995) Reexamination of the effects of MCPG on hippocampal LTP, LTD and depotentiation. J. Neurophysiol. 74, 1075–1082. 37. Shigemoto R., Nakanishi S. and Mizuno N. (1992) Distribution of the mRNA for a metabotropic glutamate receptor (mGlu receptor 1) in the central nervous system: an in situ hybridization study in adult and developing rat. J. comp. Neurol. 322, 121–135. 38. Thomas N. K., Jane D. E., Tse H. W. and Watkins J. C. (1996) a-Methyl derivatives of serine-O-phosphate as novel selective competitive metabotropic glutamate receptor antagonists. Neuropharmacology 35, 637–642. 39. Tibbs G. R., Barrie A. P., Van Mieghem F. J., McMahon H. T. and Nicholls D. G. (1989) Repetitive action potentials in isolated nerve terminals in the presence of 4-aminopyridine: effects on cytosolic free Ca 21 and glutamate release. J. Neurochem. 53, 1693–1699. 40. Va´zquez E., Herrero I., Miras-Portugal M. T. and Sa´nchez-Prieto J. (1994) Facilitation of glutamate release by metabotropic glutamate receptors in hippocampal nerve terminals. Neurosci. Res. Commun. 15, 187–194. 41. Waldmeier P. C., Wicki P. and Feldtrauer J. J. (1993) Release of endogenous glutamate from rat cortical slices in presence of the glutamate uptake inhibitor l-trans-pyrrolidine-2,4-dicarboxylic acid. Naunyn-Schmiedeberg’s Arch. Pharmac. 348, 478–485. (Accepted 22 April 1999)
Presynaptic group 1 mGlu receptors may regulate LTP
Pergamon PII: S0306-4522(99)00266-3
PRESYNAPTIC GROUP 1 METABOTROPIC GLUTAMATE RECEPTORS MAY CONTRIBUTE TO THE EXPRESSION OF LONG-TERM POTENTIATION IN THE HIPPOCAMPAL CA1 REGION ´ NCHEZ-PRIETO§ D. MANAHAN-VAUGHAN,*†‡ I. HERRERO,§ K. G. REYMANN k and J. SA *Leibniz Institute for Neurobiology, Department of Neurophysiology, Brenneckestr. 6, D-39008 Magdeburg, Germany †Institute for Physiology of the Charite, Humboldt University, Tucholskystr. 2, 10117 Berlin, Germany §Department of Biochemistry, Veterinary Faculty, Complutense University, Madrid 28040, Spain k Institute for Applied Neuroscience, Leipzigerstr 44, D-39118 Magdeburg, Germany
Abstract—In this study, we investigated the possible contribution of presynaptic group 1 metabotropic glutamate receptor activation to changes in synaptic efficacy by means of analysis of glutamate release in hippocampal synaptosomes. Data were interpreted in the context of group 1 metabotropic glutamate receptor involvement in synaptic plasticity in the CA1 region of freely moving rats. In synaptosomes, 3,5-dihydroxyphenylglycine enhanced diacylglycerol formation and facilitated vesicular Ca 21dependent glutamate release, whereas trans-azetidine-2,4-dicarboxylic acid had no effect on these processes. Trans-azetidine2,4-dicarboxylic acid enhanced glutamate release, but in a Ca 21-independent manner. This effect was mimicked by the l-glutamate uptake inhibitor l-trans-pyrrolidine-2,4-dicarboxylic acid. (R,S)-a-Methyl-4-carboxyphenylglycine blocked the effects of 3,5dihydroxyphenylglycine, but not trans-azetidine-2,4-dicarboxylic acid in synaptosomes. Short-term potentiation (100 Hz, three bursts of 10 stimuli, 0.1 ms stimulus duration, 10 s interburst interval) was induced in the CA1 region in vivo. The metabotropic glutamate receptor agonist 1S,3R-aminocyclopentane-2,3-dicarboxylic acid, or the group 1 metabotropic glutamate receptor agonists, 3,5-dihydroxyphenylglycine and trans-azetidine-2,4-dicarboxylic acid, dose-dependently facilitated short-term potentiation into long-term potentiation, which lasted . 24 h. The facilitation was inhibited by the metabotropic glutamate receptor antagonist, (R,S)-a-methyl-4-carboxyphenylglycine, and the group 1 metabotropic glutamate receptor antagonist, (S)-4-carboxyphenylglycine, but not by the group 2 metabotropic glutamate receptor antagonist, (R,S)-a-methylserine-O-phosphate monophenyl ester. l-Trans-pyrrolidine-2,4-dicarboxylic acid dose-dependently facilitated short-term potentiation into long-term potentiation, which lasted , 4 h. These data suggest that activation of group 1 metabotropic glutamate receptors results in presynaptic modulation of glutamate release. This effect may contribute to group 1 metabotropic glutamate modulation of the expression of long-term potentiation in vivo. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: mGlu, DHPG, ADA, ACPD, synaptosome, LTP.
and glutamate release studies. 12,14 In addition, synaptic transmission studies from area CA1 of the hippocampus have demonstrated that the locus of group 1 mGlu receptormediated inhibition is the presynaptic site. 12,26 Little is known, however, about the relative contribution of presynaptic group 1 mGlu receptors to synaptic transmission or LTP expression. In this study, we therefore attempted to characterize the role of presynaptic group 1 mGlu receptors in hippocampal synaptic transmission and to interpret our findings in the context of mGlu receptor involvement in LTP in vivo. An increase of presynaptic glutamate release has been reported to contribute to LTP expression. 10,12,20 The modulation by presynaptic group 1 mGlu receptors of glutamate release from nerve terminals was therefore investigated using hippocampal synaptosome preparations. Co-activation of N-methyl-d-aspartate receptors with group 1 mGlu receptors gives rise to LTP in the dentate gyrus in vivo. 25 The involvement of group 1 mGlu receptors in LTP in the anatomically and pharmacologically distinct CA1 region was thus investigated.
Long-term potentiation (LTP) in the CA1 region, both in vitro and in vivo, is believed to require the activation of both Nmethyl-d-aspartate receptors and metabotropic glutamate (mGlu) receptors. 2,5,22–25 However, the involvement of mGlu receptors in LTP is still a subject of controversy. 27,36 Recent evidence indicates that group 1 mGlu receptors, which characteristically couple to phospholipase C activation, may play a significant role in the expression of hippocampal LTP in vivo. 21,25 Group 1 mGlu receptors are mainly localized postsynaptically in the rat brain, 3,19,34,37 although a presynaptic role for these receptors has also been suggested by immunological 11,34 ‡To whom correspondence should be addressed at: the Leibniz Institute for Neurobiology. Tel.: 1 49 391 62 63 409; fax: 1 49 391 62 63 438. E-mail address: [email protected] (D. Manahan-Vaughan) Abbreviations: ACPD, 1S,3R-aminocyclopentane-2,3-dicarboxylic acid; ADA, trans-azetidine-2,4-dicarboxylic acid; 4-AP, 4-aminopyridine; BSA, bovine serum albumin; 4-CPG, (S)-4-carboxyphenylglycine; DHPG, 3,5-dihydroxyphenylglycine; EDTA, ethylenediaminetetra-acetate; EEG, electroencephalogram; EGTA, ethyleneglycolbis(aminoethyl ether)tetra-acetate; fEPSP, field excitatory postsynaptic potential; LTP, long-term potentiation; MCPG, (R,S)-a-methyl-4-carboxyphenylglycine; mGlu, metabotropic glutamate; MSOPPE, (R,S)-a-methylserineO-phosphate monophenyl ester; NADPH, reduced nicotinamide adenine dinucleotide phosphate; PDC, l-trans-pyrrolidine-2,4-dicarboxylic acid; STP, short-term potentiation; TES, N-Tris-(hydroxymethyl)methyl-2aminoethanesulfonic acid.
EXPERIMENTAL PROCEDURES
In vivo experiments Seven-week-old male Wistar rats (Schoenwalde, inbred strain from house stocks) underwent implantation of a monopolar recording and a 71
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bipolar stimulating electrode into the CA1 stratum radiatum and Schaffer collaterals, as described previously. 21 A hole was drilled (1 mm diameter) for the recording electrode (2.8 mm posterior to bregma, 1.8 mm lateral to the midline) and a second hole (1 mm diameter, 3.1 mm posterior to bregma, 3.1 mm lateral to the midline) made for the stimulating electrodes (coordinates based on Paxinos and Watson 32). The dura was pierced through both holes, and the recording and stimulating electrodes lowered into the CA1 stratum radiatum and the Schaffer collaterals, respectively. Recordings of evoked field potentials via the implanted electrodes were taken throughout surgery. A cannula was implanted in the lateral cerebral ventricle (0.08 mm posterior to bregma, 1.6 mm lateral to the midline). The animals were allowed between seven and 10 days to recover from surgery before experiments were conducted. The animals were allowed to move freely throughout experiments.
The baseline fEPSP data were obtained by averaging the response to stimulation, to obtain five sweeps at 0.1 Hz, every 5 or 15 min, as described above. The data were then expressed as mean percentage pre-injection baseline reading ^ S.E.M. The probability levels interpreted as statistically significant were P , 0.001, P , 0.01 and P , 0.05.
Measurement of evoked potentials
Preparation of synaptosomes
Responses were evoked by stimulation at low frequency (0.1 Hz). For each time-point, five evoked responses were averaged. The field excitatory postsynaptic potential (fEPSP) slope was measured as the maximal slope through the five steepest points obtained on the first negative deflection of the potential. The fEPSP slope was found by means of input/output curve determination, and all potentials employed as baseline criteria were evoked at a stimulus intensity which produced 40% of this maximum. Short-term potentiation (STP) was induced by a tetanus of 100 Hz (three bursts of 10 stimuli, 0.1 ms stimulus duration, 10 s interburst interval) and a stimulus intensity that was 20% of the maximum (determined by means of an input/ output curve). Hippocampal electroencephalogram (EEG) responses were measured via the recording electrode implanted in the CA1 stratum radiatum. EEG activity was discriminated from noise using a level-time function of the Spike-2 software package (Cambridge Electronic Design, Cambridge, U.K.). The spectral power of the EEG was measured after fast Fourier transformation to compare the characteristics of the EEGs measured before and after 100-Hz stimulation was given.
Hippocampi from male Wistar rats (eight weeks) were isolated and homogenized in a medium containing 320 mM sucrose, 0.5 mM EDTA and 5 mM N-Tris-(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES; pH 7.4). The homogenate was spun for 5 min at 900 × g and 48C, and the supernatants spun again for 10 min at 17,000 × g. From the pellets thus formed, the white, loosely compacted layer containing the majority of synaptosomes was gently resuspended in a medium containing 250 mM sucrose and 5 mM TES (pH 7.4; 3 ml/six hippocampi), and the protein determined by the Biuret method. Pellets containing 1 mg of protein were stored on ice. Synaptosomes remained fully viable, when stored as pellets, for at least 6 h after preparation, as judged by the extent of KCl- and 4aminopyridine (4-AP)-evoked glutamate release.
Compounds and drug treatment 1S,3R-1-Aminocyclopentane-1,3-dicarboxylic acid (ACPD), (R,S)a-methyl-4-carboxyphenylglycine (MCPG), (R,S)-a-methylserine-Ophosphate monophenyl ester (MSOPPE), trans-azetidine-2,4-dicarboxylic acid (ADA), (R,S)-3,5-dihydroxyphenylglycine (DHPG) and (S)-4-carboxyphenylglycine (4-CPG) were obtained from Tocris Cookson Ltd. l-Trans-pyrrolidine-2,4-dicarboxylic acid (PDC) was obtained from Research Biochemicals International. For injection, ADA was dissolved in 0.9% sodium chloride. All other drugs were first dissolved in 5 ml sodium hydroxide solution (1 mM), and then made up to a 100-ml volume with 0.9% sodium chloride. Due to the minute quantities of ACPD used, the drug was first made up as 20 nmol [drug dissolved in 5 ml sodium hydroxide solution (1 mM), and then made up to a 500-ml solution using 0.9% sodium chloride], and then further diluted to 0.2 or 0.4 nmol solutions with 0.9% sodium chloride. These respective solutions were used (in the absence of drugs) as the vehicle in the control experiments for each drug. The compounds were injected in a 5-ml volume over a 6-min period via a Hamilton syringe. Throughout the experiments, injections were administered following measurement of the baseline for 30 min. In LTP experiments, a tetanus was applied 30 min following injection, with measurements then taken at t 5, 10 and 15 min, and then 15-min intervals up to 4 h. Further measurements were taken at 24–25 h. In the case where a combination of two injections occurred in the same experiment, a 30min pause between the first and second injections occurred (during which baseline was measured); thereafter, the experimental protocol was followed as described above. The animals used in each study always served as their own controls,
i.e. prior to the commencement of each study an experiment was carried out where the stability of basal synaptic transmission was examined. If stability was seen over a 4-h period, then the effect of vehicle injection on STP was tested. In some animals, one week later, following an input/output test to confirm that the pre-tetanus values were re-established, the effect of drug injection on STP was evaluated. Data analysis
Glutamate release Glutamate release was assayed by on-line fluorimetry, as described previously. 31 Synaptosomal pellets were resuspended (0.67 mg/ml) in an incubation medium containing 122 mM NaCl, 3.1 mM KCl, 0.4 mM KH2PO4, 5 mM NaHCO3, 1.2 mM MgSO4, 10 mM glucose and 20 mM TES (pH 7.4), and preincubated at 378C for 1 h in the presence of 16 mM bovine serum albumin (BSA) to bind any free fatty acids released from synaptosomes during the preincubation. After preincubation, the synaptosomes were pelleted and resuspended in fresh incubation medium without BSA. An aliquot (1 ml) was transferred to a stirred cuvette containing 1 mM NADP 1, 50 units of glutamate dehydrogenase and 1.33 mM CaCl2 or 200 nM free Ca 21, and the fluorescence of NADPH followed in a Perkin–Elmer LS-50 luminescence spectrometer at excitation and emission wavelengths of 340 and 460 nm, respectively. Traces were calibrated by the addition of 2 nmol glutamate at the end of each assay. Data points were obtained at 2-s intervals. Determination of diacylglycerol Diacylglycerol was estimated as the amount of 32P incorporated from [t- 32P]ATP into phosphatidic acid in the presence of diacylglycerol kinase. Aliquots (0.5 ml) of the synaptosomal suspension (0.67 mg/ml) were added to 0.5 ml chloroform/methanol (95:5, v/v) and rapidly frozen at 2 808C, until all samples were collected. After thawing, the phases were separated by centrifugation, and the lower organic phase collected and evaporated in a water bath at 608C. The tubes containing diacylglycerol were incubated (final volume 100 ml) in a medium containing 70 mM NaCl, 1.4 mM b-mercaptoethanol, 35 mM MgCl2, 0.7% Nonidet P-40, 0.14 mg/ml phosphatidylserine and 35 mM piperazine-N,N-bis(2-ethanesulfonic acid) buffer (pH 6.8). The reaction, at 308C for 1 h, was started by the addition of 60 mU of diacylglycerol kinase, 25 mM ATP and 0.45 mCi of [t- 32P]ATP, and stopped with 100 ml of a mixture containing 5%
Fig. 1. The effect of the mGlu receptor agonist, ACPD, on tetanus-induced STP in the CA1 region. (A) EEG responses measured from the CA1 region of alert rats before and after 100-Hz tetanization. (i) EEG traces obtained immediately before and after tetanization. (ii) Fast Fourier transformation of EEG responses measured during a 5-min period immediately prior to, and for 5 min immediately after, tetanization indicates no significant difference in EEG activity due to tetanization. (B) ACPD (0.2 nmol) has no effect on STP of the fEPSP, when compared with vehicle-injected animals. (C) ACPD (0.4 nmol) significantly enhances the amplitude of STP with regard to fEPSP slope as compared with vehicle-injected STP controls. ACPD (0.4 nmol) has no effect on basal synaptic transmission. The mGlu receptor antagonist MCPG (1 mmol) significantly blocks the effect of 0.4 nmol ACPD from t 90 min post-tetanus. *P , 0.05. Line breaks indicate change in time-scale. (D) Original analog traces showing fEPSP responses in CA1, at three time-points: pre-injection, t 60 min and t 240 min post-tetanus in a vehicle/ACPD (0.2 nmol)-injected animal (i) and an animal injected with vehicle/ACPD (0.4 nmol) (ii).
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perchloric acid, 10 mM ATP and 1 mM H3PO4. The phosphatidic acid was extracted with 0.7 ml chloroform/methanol (95:5, v/v), the solvent evaporated in the water bath at 608C and the radioactivity estimated after the addition of 1.2 ml scintillation liquid. 1,2-Dioctanoyl-snglycerol was used as standard. Statistical significance was determined using a two-tailed Student t-test. RESULTS
Effect of tetanic stimulation on hippocampal electroencephalogram In order to assess whether the stimulus protocol (100 Hz, three bursts of 10 stimuli, 0.1 ms stimulus duration, 10 s interburst interval) used for induction of STP had an effect on hippocampal EEG, EEG was monitored for 60 min before and at least 4 h following tetanization, through the same electrode used to monitor evoked potentials in the CA1 region (Fig. 1Ai). No apparent differences were noted in the appearance of the EEG, nor was there any significant difference in EEG as determined by fast Fourier transformation of the EEG responses measured pre-tetanization and posttetanization (Fig. 1Aii). Thus, one can assume that STP was not induced due to seizure activity in the hippocampus. Effect of 1S,3R-aminocyclopentane-2,3-dicarboxylic acid on tetanus-induced short-term potentiation When a “weak” tetanus was applied via the Schaffer collateral–commissural pathway to the stratum radiatum of the CA1 region (n 12), STP was generated which decayed gradually until approximately 2 h after tetanization. The same time-course for STP was found when the vehicle solution was applied 30 min before tetanus (n 9; Fig. 1B). ACPD (0.2 nmol, n 7) produced no effect on STP elicited by a weak tetanus, as fEPSP values returned to baseline levels by approximately t 120 min (Fig. 1B,D). When 0.4 nmol ACPD (n 5) was administered, STP was prolonged into LTP which lasted for over 24 h (P , 0.05 from t 120, compared to controls, n 9; Fig. 1C,D). ACPD (0.4 nmol) had no effect on basal synaptic transmission, which was monitored for 4 h following ACPD injection (n 4; Fig. 1C). MCPG (1 mmol/5 ml, n 7), when applied prior to 0.4 nmol ACPD and weak tetanus, significantly inhibited the facilitation of STP into LTP by ACPD (P , 0.05 from t 90 compared to controls, n 5; Fig. 1C). The response obtained was similar in profile to, and not statistically different from, that obtained in vehicle-injected controls. Effect of trans-azetidine-2,4-dicarboxylic acid on tetanusinduced short-term potentiation ADA (25 nmol, n 4) had no effect on STP, whereas 50 nmol induced a slight but statistically non-significant facilitation of LTP which returned to pre-tetanization values by approximately 4 h post-tetanus. A concentration of 100 nmol (n 5) enhanced STP into an LTP which lasted for over 24 h (P , 0.05 from t 90 compared to STP controls, n 9),
whereas vehicle-injected controls returned to baseline values by t 120 min (Fig. 2A,B). Increasing the concentration of ADA to 200 nmol (n 4) did not improve the amount of LTP seen (Fig. 2C). ADA (100 nmol) had no effect on basal synaptic transmission, which was monitored for 4 h following drug injection (n 6; Fig. 2A). MCPG (1 mmol), when applied 30 min prior to 100 nmol ADA, and weak tetanus completely inhibited ADA enhancement of STP into LTP (n 8, P , 0.05 from t 90 compared to ADA injected, n 5; Fig. 2D). A similar inhibition of LTP facilitation was obtained when 4-CPG (100 nmol, n 7) was applied prior to ADA (Fig. 2D). No effect on ADA-mediated LTP facilitation was seen when the group 2 mGlu receptor antagonist MSOPPE (200 nmol, n 6) was applied prior to ADA and weak tetanus (Fig. 2D). Neither 4-CPG nor MSOPPE were found previously to have an effect on basal synaptic transmission in the CA1 region. 25 Effect of 3,5-dihydroxyphenylglycine on tetanus-induced short-term potentiation No change in the profile of electrically induced STP was found when vehicle (n 8) or 10 nmol DHPG (n 4) was applied 30 min before tetanus (Fig. 3A,C). DHPG (20 nmol, n 7) produced an enhancement of STP into LTP (P , 0.05 from t 90 compared to controls; Fig. 3A, B). This potentiation was still present after 24 h (Fig. 3A, B). DHPG (20 nmol) had no effect on basal synaptic transmission, which was monitored for 4 h following injection (n 6; Fig. 3D). Increasing the concentration of DHPG to 50 nmol (n 4) did not improve the amount of LTP seen (Fig. 3C). MCPG (1 mmol), when applied prior to 20 nmol DHPG and weak tetanus, completely inhibited facilitation of LTP (n 5, P , 0.05 from t 90) compared to vehicle/DHPGinjected controls (n 6; Fig. 3D). The group 1 mGlu receptor antagonist 4-CPG produced a similar inhibition of DHPGmediated LTP facilitation (100 nmol, n 6; Fig. 3D). Effect of trans-azetidine-2,4-dicarboxylic acid and 3,5-dihydroxyphenylglycine on glutamate release from, and diacylglycerol production in, hippocampal synaptosomes To investigate the contribution of presynaptic group 1 mGlu receptors to synaptic transmission, we measured the ability of DHPG and ADA to modulate the release of glutamate in hippocampal nerve terminals. The depolarization of nerve terminals with a K 1 channel blocker, 4-AP, has been shown to open voltage-gated Ca 21 channels and to induce the release of glutamate. 39 Submaximal concentrations of 4-AP (50 mM), added in the presence of arachidonic acid, did not significantly alter the release of glutamate in the medium with EGTA, but significantly enhanced the release in the presence of CaCl2 (1.33 mM; Fig. 4A). Thus, the extent of the Ca 21dependent release induced by 50 mM 4-AP [which was calculated by subtracting the release obtained after 5 min of depolarization in 200 nM free Ca 21 (EGTA) from the release in
Fig. 2. The effect of the group 1 mGlu receptor agonist, ADA, on tetanus-induced STP in the CA1 region. (A) ADA (100 nmol) significantly enhances the amplitude of STP with regard to fEPSP slope as compared with vehicle-injected controls. ADA (100 nmol) has no effect on basal synaptic transmission. (B) Original analog traces showing fEPSP responses in CA1, at three time-points: pre-injection, t 60 min and t 240 min post-tetanus in an ADA (100 nmol)injected animal. (C) Dose–response curve for the facilitatory effect of ADA (10–200 nmol/5 ml) on STP in the CA1 region. The values represent the magnitude of LTP observed at 4 h post-tetanus. (D) Prior application of MCPG (1 mmol) or the group 1 mGlu receptor antagonist 4-CPG (100 nmol) completely inhibits the facilitation of STP into LTP by ADA (100 nmol). The group 2 mGlu receptor MSOPPE (200 nmol) has no effect on the facilitation of STP into LTP induced by ADA (100 nmol). *P , 0.05, **P , 0.01, ***P , 0.001. Line breaks indicate change in time-scale.
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Fig. 4. The effect of DHPG and ADA on glutamate release from hippocampal nerve terminals. After preincubation of synaptosomes (0.67 mg/ml) at 378C for 1 h in the presence of BSA, the synaptosomes were washed and resuspended in BSA-free incubation medium. Glutamate release was estimated both in the presence of 1.33 mM CaCl2 (Ca 21), and in a medium where the free Ca 21 concentration was maintained at 200 nmol using EGTA. Synaptosomes were depolarized with 4-AP (50 mM) following addition of arachidonic acid (AA) either alone (A) or in combination with the mGlu receptor agonists DHPG (B) or ADA (C). Traces are computer-generated means of three to five data obtained from three to five preparations of synaptosomes. In each preparation, the synaptosomes from two rats were pooled.
1.33 mM CaCl2 (Ca 21)] was 1.1 ^ 0.2 nmol glutamate/mg protein (Fig. 4A). The Ca 21-dependent release was strongly potentiated (3.46 ^ 0.4 nmol glutamate/mg) by the addition of the group 1 mGlu receptor agonist DHPG (100 mM) prior to the depolarization (Fig. 4B), and this effect was abolished by the mGlu receptor antagonist MCPG at 2 mM. In contrast, ADA (100 mM) did not enhance the Ca 21-dependent release (1.0 ^ 0.2 nmol/mg) induced by a submaximal depolarization (Fig. 4C), although this putative mGlu receptor agonist dramatically enhanced the Ca 21-independent release observed in the medium with EGTA. This action was not antagonized by MCPG, however (Fig. 4C). Thus, both DHPG and ADA enhance the release of glutamate from hippocampal nerve terminals, albeit in different ways. Whereas DHPG potentiated the Ca 21-dependent release, which accounts for the release of vesicular glutamate in an MCPG-sensitive manner, on the other hand, the increase in glutamate release induced by ADA is due to the enhancement of cytoplasmic glutamate release, probably by reversal of a glutamate transporter via an MCPG-insensitive process. The facilitation of Ca 21-dependent glutamate release has been demonstrated to occur as a result of protein kinase C activation via diacylglycerol generated by phosphoinositidecoupled mGlu receptor stimulation. 40 Thus, the enhancement of the Ca 21-dependent release by DHPG (100 mM) parallels the ability of this compound to transiently generate diacylglycerol in hippocampal synaptosomes, an effect which was not observed with ADA (100 mM; Fig. 5). On the other hand, ADA induced a delayed and small increase in diacylglycerol levels, an action which was mimicked by the glutamate
transport inhibitor PDC (100 mM). For all three compounds (DHPG, ADA and PDC), the enhancement of diacylglycerol levels 120 s following drug application was significant (P , 0.01 in each case, compared to controls). The delayed response in diacylglycerol levels following ADA administration may be due to the accumulation of extracellular glutamate in the presence of ADA and PDC. These results indicate that ADA does not activate the presynaptic DHPG-sensitive mGlu receptor, which is involved in diacylglycerol production and the facilitation of glutamate exocytosis.
Effect of trans-azetidine-2,4-dicarboxylic acid, 3,5-dihydroxyphenylglycine and l-trans-pyrrolidine-2,4-dicarboxylic acid on Ca21 -independent glutamate PDC is a potent and selective l-glutamate transport inhibitor. However, since PDC is transportable, the expected action upon addition of this compound to synaptosomes is a heteroexchange, through the carrier, with cytoplasmic glutamate. Thus, it can be seen from Fig. 6A that PDC enhanced, in a concentration-dependent manner, the glutamate release from synaptosomes in an EGTA-containing medium. The finding that the group 1 mGlu receptor agonist ADA also enhanced the Ca 21-independent glutamate release in a concentration-dependent manner (10–100 mM; Fig. 6B), and the finding that this action was not antagonized by MCPG, raises the possibility that ADA, which is structurally related to PDC, also acts as a transportable inhibitor of the glutamate transporter.
Fig. 3. The effect of the group 1 mGlu receptor agonist, DHPG, on tetanus-induced STP in the CA1 region. (A) DHPG (20 nmol) significantly enhances the amplitude of STP with regard to fEPSP slope as compared with vehicle-injected controls. (B) Original analog traces showing fEPSP responses in CA1, at three time-points: pre-injection, t 60 min and t 240 min post-tetanus in a DHPG (20 nmol)-injected animal. (C) Dose–response curve for the facilitatory effect of DHPG (2–50 nmol/5 ml) on STP in the CA1 region. The values represent the magnitude of LTP observed at 4 h post-tetanus. (D) Prior application of either MCPG (1 mmol) or 4-CPG (100 nmol) significantly inhibits the effect of DHPG (20 nmol) from t 90 min post-tetanus. DHPG (20 nmol) has no effect on basal synaptic transmission. *P , 0.05, **P , 0.01, ***P , 0.001. Line breaks indicate change in time-scale.
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Fig. 5. DHPG but not ADA enhances diacylglycerol production. Timecourse of diacylglycerol formation induced by DHPG (A), ADA (B) and PDC (O), all added at 100 mM. At the times indicated, following addition of the agonist, 0.5-ml aliquots of synaptosomes (0.67 mg/ml) were taken and the diacylglycerol levels determined as indicated in the Experimental Procedures section. Control experiments show the diacylglycerol levels in the absence of any additions. The results are means ^ S.D. of three data from three preparations of synaptosomes. In each preparation, the synaptosomes from two rats were pooled.
Effect of l-trans-pyrrolidine-2,4-dicarboxylic acid on tetanus-induced short-term potentiation The glutamate transporter inhibitor PDC at a concentration of 20 (n 4) or 40 nmol/5 ml (n 5) had no effect when applied prior to tetanus-induced STP compared to controls (n 9). Application of 100 nmol PDC (n 6) facilitated
STP into LTP which lasted approximately 4 h (Fig. 7A, B). When basal synaptic transmission was monitored for 4 h in the presence of PDC (100 nmol), a transient increase in fEPSP slope function was seen (n 4) compared to controls (n 8). This effect became statistically significant at 30 min postinjection, but returned to pre-injection baseline values by 120 min post-injection. To investigate whether ADA facilitates STP into LTP in vivo through a presynaptic inhibition of glutamate transport, experiments were carried out where PDC was applied in combination with DHPG or ADA. Thus, a concentration of PDC which is subthreshold for facilitation of LTP (40 nmol) was applied together with a subthreshold concentration of ADA (25 nmol, n 4) prior to an STP-inducing tetanus. In this case, LTP occurred which declined to control levels (n 6) by 4 h post-tetanus (Fig. 7C). fEPSPs evoked in the PDC/ADA group were statistically significant from control levels from t 150 min until t 225 min post-tetanus (P , 0.05), whereupon evoked potentials returned to pre-tetanus levels. The combination of PDC (40 nmol) with ADA (25 nmol, n 4) had no significant effect upon basal synaptic transmission, which was monitored for 4 h following drug injection. When PDC (40 nmol) was applied with a concentration of DHPG which is subthreshold for facilitation of LTP (10 nmol, n 6), a facilitation of STP into LTP occurred which was still present 4 h post-tetanus, but had returned to control levels (n 5) by 24 h post-tetanus (Fig. 7C). fEPSPs evoked in the PDC/DHPG group were statistically significant from control levels from t 120 min until t 240 min post-tetanus (P , 0.01), but returned thereafter to pre-tetanus levels. The combination of PDC (40 nmol) with DHPG (10 nmol, n 4) had no significant effect upon basal synaptic transmission measured for 4 h following injection.
Fig. 6. The effect of the group 1 mGlu receptor agonists DHPG and ADA on glutamate transporter-mediated, Ca 21-independent glutamate release. After preincubation of synaptosomes (0.67 mg/ml) at 378C for 1 h in the presence of BSA, an aliquot (1 ml) was used to determine glutamate release. Glutamate release from polarized synaptosomes was determined in incubation medium, keeping the free Ca 21 concentration at 200 nM using EGTA, in the absence of any additions (control), or following the addition of ADA (10 or 100 mM; A), DHPG (100 mM; A) or PDC (10–100 mM; B). Traces are computer-generated means of three to five data obtained from three to five preparations of synaptosomes. In each preparation, the synaptosomes from two rats were pooled.
Presynaptic group 1 mGlu receptors may regulate LTP DISCUSSION
The findings of this study indicate that presynaptic group 1 mGlu receptor activation results in an enhancement of presynaptic glutamate release. This effect, in turn, may contribute to the alterations in synaptic efficacy evoked by group 1 mGlu receptor stimulation in vivo. Application of group 1 receptor agonists resulted in a facilitation of STP into LTP in the CA1 region in vivo. Taken together, the data of the current study suggest that not only postsynaptic, but also presynaptic group 1 mGlu receptors contribute to LTP facilitation in vivo. Group 1 mGlu receptors do not appear to contribute to basal synaptic transmission in vivo, as neither DHPG nor ADA influenced baseline measurements, in the current study, when applied at concentrations which were effective in converting STP into LTP. This observation may have a morphological explanation, as it has been shown that postsynaptic group 1 mGlu receptors are localized perisynaptically. 3,19 Thus, under conditions of basal synaptic transmission, insufficient glutamate may be released into the synaptic cleft to enable diffusion to the perisynaptic area and subsequent activation of group 1 mGlu receptors. It is not known whether presynaptic group 1 mGlu receptors are activated under basal synaptic conditions, although the lack of baseline effect of the agonists tested suggests that presynaptic group 1 mGlu receptors probably do not contribute to basal synaptic transmission. During LTP-inducing tetanization, however, increased presynaptic release of glutamate should result in both pre- and postsynaptic group 1 mGlu receptor activation. Presynaptic group 1 mGlu receptor activation at this time may play an important role in the elevation of presynaptic glutamate release, which is a characteristic of LTP, 10,13,19 whereas postsynaptic mGlu receptor activation gives rise to phospholipase C activation and subsequent release of calcium from intracellular stores. 30 In a physiological situation where group 1 mGlu receptors are activated (e.g., during LTP induction), it is therefore likely that both presynaptic and postsynaptic group 1 mGlu receptors participate concurrently in subsequent modifications of synaptic efficacy. In this study, the group 1 and 2 mGlu receptor agonist, ACPD, produced a similar facilitation of STP into LTP in the CA1 region, as was reported previously in vitro 1,4,6,28,29 and for the dentate gyrus in vivo. 23 It was shown previously that ACPD enhances Ca 21-dependent glutamate release from hippocampal synaptosomes in the presence of arachidonic acid, and that the mGlu receptor antagonist MCPG abolishes this effect. 40 This facilitatory effect of ACPD on glutamate release is probably due to the activation of a group 1 mGlu receptor, as it is mediated by the ACPD-induced formation of diacylglycerol and activation of presynaptic protein kinase C. 40 DHPG 15,35 is a specific group 1 mGlu receptor agonist. In this study, DHPG produced facilitations of STP into LTP which were similar in both magnitude and profile to that produced by ACPD, and which were consistent with previous reports that priming with DHPG facilitates LTP induction in the CA1 region in vitro 7 and the dentate gyrus in vivo. 25 These effects were also blocked by the mGlu receptor antagonist MCPG and by the group 1 mGlu receptor antagonist, 4CPG. In hippocampal synaptosomes, DHPG enhanced glutamate release in the presence of calcium, consistent with the activation of presynaptic group 1 mGlu receptors.
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A facilitation of LTP which is similar in profile to that induced by ACPD was stimulated through the putative group 1 mGlu receptor agonist ADA. This effect was inhibited by the mGlu receptor antagonist MCPG. ADA 9,18 is a structural analog of glutamate which stimulates phosphoinositide hydrolysis in hippocampal slices and activates inositol phosphate accummulation in transfected LLC-PK1 cells with an ec50 at mGluR1 of 189 mM and at mGluR5 of 32 mM. 25 By comparison, the ec50 of ACPD at mGluR1 is approximately 137 mM and is 50 mM at mGluR5. 33 It has been reported that ADA is an agonist at human group 2 mGlu receptors. 17 However, this effect has not been detected in rat tissue. 22 Furthermore, in the current study, application of the group 2 mGlu receptor antagonist MSOPPE 16,38 prior to ADA injection had no effect on the facilitation by ADA of STP into LTP. In addition, the finding that the group 1 mGlu receptor antagonist 4-CPG completely inhibited the facilitatory effects of ADA implies that it acts as an agonist of group 1 mGlu receptors. ACPD, DHPG and ADA produce facilitations of STP into LTP which are of similar magnitude and duration, whereas only ACPD 40 and DHPG, but not ADA, activate presynaptic group 1 mGlu receptors. However, ADA also potentiates glutamate release, albeit in an mGlu receptor-independent manner, by displacement of cytoplasmic glutamate. Thus, ADA mimicks the action of the glutamate uptake inhibitor, PDC, to which ADA is structurally related. It is possible that the activation of a presynaptic phosphoinositide-coupled mGlu receptor and the subsequent increase in glutamate release contributes to the conversion of STP into LTP, seen when ACPD or DHPG are given in the presence of “weak” tetanization, and that ADA mimicks this effect through the inhibition of the glutamate transporter. This possibility is further supported by the finding that the time-course for facilitation of glutamate release in the absence of calcium was similar to that observed with the Na 1-dependent glutamate transporter inhibitor PDC. 8,41 PDC itself was found to induce a dose-dependent facilitation of STP into LTP in this study, although the LTP produced did not persist much longer than 4 h, unlike LTP produced by the mGlu receptor agonist, ADA, which lasted for more than 24 h. The fact that ADAinduced conversion of STP into LTP was competely abolished by both MCPG and the group 1 mGlu receptor antagonist 4-CPG would indicate either a further action of ADA at postsynaptic group 1 mGlu receptors, or that the presynaptic increase in glutamate release provided by ADA enables the activation of postsynaptic group 1 mGlu receptors. Thus, the higher circulating levels of glutamate, which would occur in the synaptic cleft as a result of a possible ADA-mediated inhibition of glutamate transport, could in turn enable further activation of group 1 mGluRs which would thereby contribute to LTP facilitation. In order to try to ascertain whether mild activation of group 1 mGlu receptors together with inhibition of glutamate transport could facilitate LTP in a manner similar to that seen with ADA, concentrations of PDC and ADA, or PDC and DHPG, which were subthreshold for facilitation of LTP were tested. In each case, a facilitation of STP into LTP which lasted approximately 4 h occurred. This further suggests that, although a presynaptic non-mGlu receptor-mediated elevation of glutamate levels by ADA may contribute to LTP facilitation by this compound, postsynaptic group 1 mGlu receptor activation by ADA may account for the 24-h LTP
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Fig. 7. The effect of the glutamate uptake inhibitor, PDC, on tetanus-induced STP in the CA1 region. (A) PDC (100 nmol) significantly enhances the amplitude of STP with regard to fEPSP slope as compared with vehicle-injected controls. PDC (40 nmol) or vehicle injection have no effect on STP. (B) PDC (100 nmol), when applied in the absence of tetanization, induces a transient increase in basal synaptic transmission. (C) Application of PDC at a concentration which is subthreshold for LTP facilitation (40 nmol), together with a subthreshold concentration of ADA (25 nmol), facilitates STP into a short-lasting LTP. A similar short-lasting LTP was produced when PDC (40 nmol) was given at a subthreshold concentration of DHPG (10 nmol). *P , 0.05, **P , 0.01. Line breaks indicate change in time-scale.
Presynaptic group 1 mGlu receptors may regulate LTP
induced by application of the compound. Thus, the activation of postsynaptic group 1 mGlu receptors by ADA is consistent with its ability to activate inositol phosphate accumulation both in mGluR1 and mGluR5 transfected LLC-PK1 cells, 22 while the failure of ADA to activate diacylglycerol production and glutamate release facilitation would indicate that the facilitatory mGlu receptor at hippocampal nerve terminals is neither mGluR1 nor mGluR5. CONCLUSION
This study indicates that calcium-dependent presynaptic
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glutamate release is increased by activation of presynaptic group 1 mGlu receptors. In addition, group 1 mGlu receptor activation in the CA1 region facilitates the expression of LTP in vivo. These findings suggest a role for presynaptic mGlu receptors in LTP induction in vivo.
Acknowledgements—We would like to thank Ms Silvia Vieweg for valuable assistance in technical aspects of this work. We are indebted to Dr Thomas Seidenbecher and Mr Jurgen Buggert for expert advice and support in hippocampal EEG measurements.
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