0 European Neuroscience Association European Journal of Neuroscience, Vol. 8, pp. 1182-1189, 1996 Differential Long-lasting Potentiation of the NMDA and Non-NMDA Synaptic Currents Induced by Metabotropic and NMDA Receptor Coactivation in Cerebellar Granule Cells Paola Rossi, Egidio D’Angelo and Vanni Taglietti lstituto di Fisiologia Generale, Via Forlanini 6, 1-27100, Pavia, Italy Keywords: cerebellum, LTP, metabotropic receptors, NMDA receptors, patch-clamp, rat Abstract Whole-cell patch-clamp recordings in rat cerebellar slices were used to investigate the effect of metabotropic glutamate receptor activation on mossy fibre-granule cell synaptic transmission. Transient application of 20 pM 1S,3R-1-aminocyclopentane-1,Sdicarboxylic acid simultaneously with low-frequency NMDA receptor activation induced long-lasting non-decrementalpotentiation of both NMDA and non-NMDA receptor-mediated synaptic transmission. Potentiation could be prevented by application of the metabotropic glutamate receptor antagonist (+)-Omethyl-4-carboxyphenyl-glycineat 500 pM. Characteristically, NMDA potentiation was two to three times as large as non-NMDA current potentiation, occurred only in a slow subcomponent, and was voltageindependent. This result demonstrates a pivotal role of NMDA receptors in the metabotropic potentiation of transmission, which may be important in regulating cerebellar information processing. Introduction Materials and methods Many central neurons express both ionotropic and metabotropic Slice preparation glutamate receptors. Metabotropic glutamate receptors (mGluRs) can Patch-clamp whole-cell recordings were carried out in granule cells be differentiated into several subtypes (mGluRI-mGluR8) based on of the internal granular layer of acutely isolated cerebellar slices. their structure, pharmacological sensitivity and signal transduction Cerebellar slices were obtained from 20- to 26-day-old rats (Wistar mechanism (Watkins and Collingridge, 1994; Pin and Duvoisin, strain, day of birth = postnatal day 1) as reported previously 1995). While N-methyl-o-aspartate (NMDA) and non-NMDA (or (D’Angelo et al., 1993, 1994, 1995). Rats were anaesthetized with AMPAkainate) ionotropic receptors mediate phasic transmission at halothane (Aldrich) before being killed by decapitation. Krebs solution excitatory synapses, activation of mGluRs regulates synaptic efficacy, for slice cutting and recovery contained (mh4): NaCl 120, KCI 2, contributing to the generation of long-term potentiation (LTP) or MgS04 1.2, NaHC03 26, KH2PO4 1.2, CaClz 2, glucose 11. This long-term depression of the excitatory postsynaptic currents (EPSCs) solution was equilibrated with 95% O2 and 5% C 0 2 (pH 7.4). Slices (Bliss and Collingridge, 1993; Linden and Connor, 1993). Of particular were maintained at room temperature before being transferred to the interest are the interaction of mGluRs and NMDA receptors in recording chamber (1.5 ml) mounted on the microscope stage. The generating plasticity and the possibility that NMDA receptors thempreparations were superfused at a rate of 2 4 mumin with Krebs selves are a target of potentiation (O’Connor er al., 1994). We solution to which 10 pM glycine and 10 pM bicuculline (Sigma) had investigated the actions of the mGluR agonist 1S,3R-1-aminocyclopenbeen added, and maintained at 30°C with a feed-back Peltier device. tane-l,3-dicarboxylic acid (t-ACPD) and of the antagonist (+)-0methyl-4-carboxyphenyl-glycine(MCPG) (Watkins and Collingridge, Data recording and analysis 1994) on NMDA and non-NMDA phasic synaptic transmission in Recordings were performed in the whole-cell configuration of the cerebellar granule cells (Garthwaite and Brodbelt, 1989; Silver er al., patch clamp (Edwards er al., 1989). Patch pipettes were pulled from 1992; D’Angelo et al., 1993, 1994). Coactivation of mGluRs and borosilicate glass capillaries (Hingelberg, Malsfeld, Germany) and NMDA, but not non-NMDA receptors, induced a long-lasting potentihad 7-11 MIR resistance before a seal was formed with a filling ation in an NMDA current subcomponent and, to a smaller extent, solution having the following composition (a CszS04 ):81, NaCl in the non-NMDA current. In view of the key role played by Nh4DA 4, MgS04 2, CaC120.02, BAPTA 0.1, glucose 10, ATP-Mg 3, HEPES receptors in regulating granule cell excitability (D’Angelo et al., 15 (pH was adjusted to 7.2 with CsOH). This solution buffered 1995), this form of potentiation may be important in regulating intracellular Ca2+at 100 nM, similar to the resting Ca2+concentration cerebellar information processing (Eccles et al., 1967; Linden and Connor, 1993). measured in granule cells (Irving et al., 1992). BAPTA (tetrapotassium Correspondence to: Egidio D’Angelo, as above Received 22 September 1995. revised 22 December 1995, accepted I0 January 1996 Potentiation of an NMDA current subcomponent -70 mV 1183 +60 mV t-ACPD mtr t-ACPD potentiation 1 1 20 ms An 1 .................. I Y I I L 20ms 1 FIG.1. EPSCs before and after t-ACPD potentiation. EPSCs (averages of four tracings) are shown at the holding membrane potentials of -70 and +60 mV. Vertical lines indicate the position at which amplitudes of the non-NMDA (A,) and NMDA currents (AN) were measured. 2 s 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E l salt) was obtained from Molecular Probes (Eugene, OR). Recordings were performed with an Axopatch 200A amplifier (Axon Instruments, Foster City, CA), and data were converted from analogue to digital at 20 or 2 kHz to resolve the different time courses of the nonNMDA and NMDA current components respectively. Electrical stimulation was performed using a bipolar tungsten electrode (Clark Instruments, Pangbourne, UK) placed over the mossy fibre bundle. A local flow of solution was delivered over the recording site through a four-barrel micropipette. The control solution was perfused before sealing; thereafter different compounds were applied by switching between the perfusion pathways, as specified for each experimental protocol. A typical experimental protocol is illustrated in Figures 1 and 2. Data were analysed with p-CLAMP software. Data are reported as mean ? SEM, and statistical comparison of means was made with paired Student’s r-tests [differences were considered not significant at P > 0.051. E 0 1 - 3 2 I , 0 6 4 t-ACPU wash , , I 10 / M 30 Time (min) FIG. 2. Metabotropic EPSC potentiation in a cerebellar granule cell. Representative EPSC tracings recorded at -70 mV or +60 mV at different times during the experiment are shown at the top. Relative A, values at -70 mV (open symbols) and A N values at +60 mV (filled symbols) are plotted at the bottom. Values of AN are reversed and scaled up to demonstrate the similar time course of potentiation in both EPSC components. In this and the following figures, (i) time is set to 0 at the beginning of t-ACPD perfusion; (ii) a horizontal bar represents perfusion, and application of 20 pM r-ACPD is indicated in black; and (iii) the inset shows superimposed passive current transients recorded at -70 mV (scale bars 50 PA, 0.5 ms). Here, four transients are shown in the control 10, 20 and 30 min after r-ACPD perfusion. Monitoring of recording conditions A major problem for the kinetic analysis of membrane currents over extended periods of time is the stability of the whole-cell configuration. In order to monitor the whole-cell condition, a passive current transient was elicited by a 10 mV hyperpolarizing voltage step just before each EPSC and sampled at 20 lcHz (D’Angelo et aZ., 1994). The constancy of the transient was assumed as a confirmation of stability, as shown in the insets in Figures 1-4. Assuming that the granule cell can be treated as a lumped somatodendritic compartment, these transients were also used to measure the passive properties of the electrode/cell system (Silver et al., 1992; D’Angelo et al., 1993, 1995). In the 42 cells covered in this study, at -70 mV membrane resistance was R, = 3.7 ? 0.3 GSl and membrane capacitance was C , = 3.4 ? 0.2 pF, reproducing typical values of cerebellar granule cells, and series resistance was R, = 15.5 % 0.9 MQ. From similar data, in each individual granule cell the cut-off frequency of the voltage-clamp system was estimated as fVc = (2x.RSC,)-’. The reliability of EPSC measurement was then assessed by comparison offvc with the maximum frequency content of the EPSC, which was estimated as fEPsc= 0.35/RT1&90(RTlCg0is the rise time between 10 and 90% of peak amplitude at -70 mV). The mean of values obtained in individual neurons were as follows: control conditions, fvc = 3.65 TL 0.52 ~ HandfEpsC Z = 0.5 5 0.07 ~ H(n Z = 35); 10 min after the beginning of t-ACPD perfusion, fVc = 3.56 ? 0.45 kHz and fEPsc = 0.6 ? 0.08 kHz (n = 29). indicating that the voltage-clamp was six times faster than the fastest (non-NMDA) EPSC component. EPSC recordings were considered in the present analysis for as long as fVcand fEPscremained stable. Another problem when modulatory processes are being investigated is the integrity of intracellular signal transduction pathways. In three experiments the perforated-patch technique was used (unpublished results), preventing wash-out of Ca2+ and organic cytoplasmic constituents (Horn and Marty, 1988). In the perforated-patch experiments, t-ACPD induced long-term EPSC changes similar to those obtained using the conventional whole-cell recording technique, indicating that modifications in cytoplasmic composition caused by the whole-cell pipette solution were not critical. However, the system response was slower in the perforated-patch than in the conventional whole-cell recording configuration (fvc = 1.2 -C 0.4 kHz, n = 3). The wholecell recording configuration was subsequently used for detailed analysis of EPSC changes. It should also be noted that no run-down of synaptic transmission has been reported in cerebellar granule cells in control recording conditions (Fig. 4B in D’Angelo et aZ., 1995). Drugs D-2-amino-5-phosphonovalericacid (APV),6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 7-chlorokynurenic acid (7-Cl-kyn), t-ACPD and MCPG were obtained from Tocris Cookson (Bristol, UK). 1184 Potentiation of an NMDA current subcomponent A 180 $ E z A 7 t60 mV -70 mV 3 2 4 B 1M)m B d+ 0 = =1 +MCffi 1 2 4NOX - 0 3 4 +(AW+ 7kyn) 5 1 0 1 5 2 0 2 5 r ) Tim (min) 0 10 20 30 Tim (min) FIG.3. General properties of t-ACPD potentiation. The time courses of EPSC amplitude changes (A, at -70 mV) are shown at the left (mean t SEM). Examples of EPSCs (averages of four traces) with the corresponding passive current transients (insets) are shown at the right. (A) Application of t-ACPD for 120 s (n = 7). (B) Protracted 1-ACPD application ( n = 5 ) . (C) Coapplication of f-ACPD and 500 p M MCPG for 120 s in slices which had been preincubated for at least 1 h with 500 p M MCPG ( n = 4). (D) Application of I-ACPD for 120 s simultaneously with interruption of mossy fibre stimulation (n = 5 ) . as indicated by a reference line (stim). Results The actions of the mGluR agonist r-ACPD on mossy fibre-granule cell synaptic currents were investigated in 42 granule cells of the internal granular layer of cerebellar slices in the whole-cell patchclamp recording configuration. The experiments were performed 2026 days after birth, when synaptic properties had developed mature characteristics (Garthwaite and Brodbelt, 1989; D'Angelo et al., 1993; Farrant et al., 1994; Monyer et al., 1994). EPSCs were activated by 0.1 Hz mossy fibre electrical stimulation of constant intensity. Inhibitory synaptic currents were blocked by the GABA-A receptor antagonist bicuculline (10 pM), and 10 pM glycine was added to saturate the glycine site on the NMDA receptor. The EPSCs showed a fast and a slow component (Fig. I ) , corresponding to the non-NMDA (AMPAkainate) and NMDA currents identified in previous work (Silver et al., 1992; D'Angelo et al., 1993). The EPSCs were depressed during application of 20 pM t-ACPD for 120 s; however, both the NMDA and the non-NMDA component showed marked enhancement during subsequent washing (Figs 1 and 2). NMDA and non-NMDA current potentiation followed a similar time course (Fig. 2). Amplitude estimates of the two EPSC components were obtained by measuring the non-NMDA current peak (A,) and the NMDA current 25 ms after the beginning of the EPSC (AN), as shown in Figure 1. Although changes in A, and A N were observed at both positive and negative membrane potentials, for convenience A, was usually measured at -70 mV and AN at +60 mV (Fig. 2). In all the reported experiments, the quality and stability of recordings were FIG.4. Effects of t-ACPD on the NMDA current. Following control EPSC recordings ( I ) , the NMDA EPSC was isolated by perfusing with 10 p M CNQX (2) and potentiated following application of 20 p M I-ACPD for 120 s (3). Finally. the NMDA-EPSC was blocked by 100 p M APV + 50 p M 7-CIkyn (4).( A ) EPSCs (averages of four tracings) in different phases of the experiment at -70 and +60 mV. The inset shows two passive current transients superimposed (phases I and 3). (B) Time course of potentiation in the NMDA current (n = 6, mean t SEM). The plot shows normalized AN values measured at +60 mV in the isolated NMDA EPSCs (filled circles) and in composite EPSCs (open symbols). monitored by activating passive current transients (inset), and by calculating the cut-off frequency of the voltage-clamp system cfvc) and the maximum frequency content of the EPSCs (fEpsc; see Materials and methods). General properties of t-ACPD potentiation As reported in Figure 2, following application of t-ACPD for 120 s, EPSC potentiation developed in 13 of the 14 neurons tested, attaining its maximum level within 10 min (Fig. 3A). The potentiation then lasted without any tendency to decrease for at least 45 min, i.e. the time during which a constant whole-cell current transient could be maintained in three cells. It should be noted that protracting the period of t-ACPD perfusion caused an enduring depression in both the NMDA and the non-NMDA EPSC component (n = 5/5; Fig. 3B). Application of t-ACPD with the mGluR antagonist MCPG (500 FM) (Watkins and Collingridge, 1994) for 120 s did not induce any subsequent potentiation (n = 4/4; Fig. 3C), the effect resembling that first reported at hippocampal synapses (Bashir et al., 1993; O'Connor et al., 1994). During t-ACPD and MCPG coapplication an EPSC depression was usually observed, as with t-ACPD alone. In these experiments, the slices were preincubated for at least 1 h with MCPG to improve MCPG blockade. Interestingly, potentiation did not arise when, after having interrupted synaptic stimulation during t-ACPD perfusion, stimulation was restarted (n = 5/5, Fig. 3D). On restimulation, however, the EPSCs were already maximally depressed. In summary, the induction of potentiation depended on specific MCPG-sensitive metabotropic stimulation and on simultaneous ionotropic receptor coactivation, while subsequent expression was independent of the presence of t-ACPD. Depression showed opposite Potentiation of an NMDA current subcomponent A mtr 120, 1 2 - 3 4 +(Aw+ W) 0 t-ACPDhashinp FIG. 6. Selective t-ACPD potentiation in a slow NMDA current subcomponent. The decay phase of NMDA EPSCs at +60 mV (averages of four traces) have been fitted to the sum of two exponential functions, which are shown superimposed. Control: q = 75.5 ms. T, = 210.7 ms, Af/(Af + A,) = 0.86. After r-ACPD potentiation: T~ = 61 ms, 7, = 438.4 ms. Af/(Af + A,) = 0.56. Note the increase in the slow. but not in the fast, component after I-ACPD potentiation. +CNOX 5 1 0 1 5 2 0 S m Tim (min) FIG.5. Effects of I-ACPD on the non-NMDA current. The non-NMDA-EPSC isolated with 100 pM APV + 50 FM 7-CI-kyn ( I ) was depressed during application of 20 pM t-ACPD for 120 s (2). and no switch to potentiation was observed during subsequent washing (3). Addition of 10 FM CNQX blocked the non-NMDA-EPSC (4). (A) EPSCs (averages of four traces) in different phases of the experiment at -70 mV. The inset shows three passive current transients superimposed (phases 1-3). ( B ) Time course of changes in the non-NMDA EPSC components (n = 8, mean f SEM). The plot shows normalized A, values measured at -70 mV. TABLE1. Potentiation of the NMDA and non-NMDA current NMDA EPSC Composite EPSC -70 mV +60 mV 1 1 1 85 A, (%) A N (%) +43.2 2 2.7 +38.1 f 7.7 n.s. +81.1 f 9.9 +110.6 % 27 n.s. P < 0.01 P < 0.01 + I 5 8 3 5 39.5 +I66 2 12.6 n.s. Amplitude changes induced by I-ACPD in composite EPSCs (n = 6) and in the pharmacologically isolated NMDA current ( n = 6). Reported values are the mean 2 SEM for increments in A, and A N relative to control measured 10 min after I-ACPD application and subsequent washing, at -70 and +60 mV. The statistical significance of paired Student’s 1-tests is reported (n.s., not significant). properties, implying the involvement of different receptors and mechanisms. Selective involvement of the NMDA current during induction In six experiments continuous perfusion of 10 pM CNQX was used to block non-NMDA receptors, obtaining the NMDA EPSC in isolation (Fig. 4). During t-ACPD application the NMDA EPSC was depressed, and during subsequent washing potentiation was observed both at -70 and at +60 mV (Fig. 4A). The NMDA current potentiation (evaluated as AN at -1-60 mV) developed following a similar time course both in NMDA EPSCs and in composite EPSCs (Fig. 4B). In eight experiments continuous perfusion of 100 pM APV + 50 pM 7-CI-kyn was used to block the NMDA receptors and obtain the non-NMDA EPSC in isolation (Fig. 5). Coapplication of the two drugs has been shown to enhance NMDA receptor blockade in the presence of 10 pM glycine in this preparation (D’Angelo ef al., 1995). During r-ACPD application the non-NMDA EPSC was depressed, but no potentiation was observed during subsequent washing in any of the eight cells tested (Fig. 5A, B). The absence of any potentiation after blocking of the NMDA receptors resembles the effect of suspending stimulation during t-ACPD application, in striking contrast to the nearly 100% success obtained both in control conditions and after blocking the nonNMDA receptors. Thus, simultaneous stimulation of metabotropic and NMDA, but not of non-NMDA receptors, was required to induce potentiation. Differential potentiation of the NMDA and non-NMDA current The analysis of A, and AN values in six cells allowed an evaluation of r-ACPD actions on the non-NMDA and NMDA components. As shown in Table I , following r-ACPD potentiation the NMDA component increased 2-3 times more than the non-NMDA component at both -70 and +60 mV (the differences were statistically significant, P < 0.01). Similar amplitude changes occurred in the NMDA current isolated pharmacologically (Table 1). On closer inspection, NMDA current decay kinetics proved biphasic both in composite EPSCs and in the NMDA current in isolation (Fig. 6; cf. D’Angelo et al., 1994). At +60 mV, a membrane potential at which the signal-to-noise ratio was favourable and the influence of the non-NMDA component on NMDA current decay was negligible, reliable fittings were obtained with the sum of two exponential functions (with mean time constants q = 41.5 ms and T> = 285.6 ms) in all six EPSC decays examined (Table 2). In potentiated EPSCs, neither zf nor 2, showed any significant change. Although amplitude in the faster subcomponent (Af) remained stable, amplitude in the slower subcomponent (A,) nearly doubled, changing the relative amplitude ratio Ad(Af + A,) from 0.46 to 0.23 (P < 0.01). Similar results were obtained in the six cells in which the NMDA current was isolated pharmacologically (Fig. 6; Table 2). These results indicate that following potentiation the NMDA current increased more than the non-NMDA current, and that the NMDA current increase was mostly related to a slow subcomponent. Voltage-dependence of NMDA EPSC potentiation In order to determine whether the NMDA current potentiation involved a voltage-dependent conductance change, we used AN measurements at different holding potentials. It should be noted that AN was sensitive to changes in the slow NMDA current subcomponent at both extremes of the membrane potential range examined (Fig. 5A and Table 1). In the control, A N showed a characteristic negative slope in currentvoltage plots at negative membrane potentials (Fig. 7A, open circles). Following t-ACPD potentiation, A N increased over the whole membrane potential range (Fig. 7A, filled circles), without any noticeable change in reversal potential. Measurements of A N and a current reversal potential of +10 mV (Fig. 7A) were then used to calculate conductance. Normalized conductance plots (Fig. 7B) were fitted to a Boltzmann equation of the form: 1186 Potentiation of an NMDA current subcomponent TABLE2. Bi-exponential decay kinetic\ of the NMDA current (ms) Ff Composite EPSC Control r-ACPD NMDA EPSC Control t-ACPD (ms) 5, At (PA) A, (PA) AJA, + A,) 41.5 f 6.4 42.1 2 6.8 n.s. 285.6 2 38.3 262.5 f 25 n.s. 6.9 f 0.6 47.9 2 1.5 n.s. 8 2 3.6 25.7 f 9.8 P < 0.02 0.46 t 0.09 0.23 -C 0.08 P < 0.01 44.7 2 9 51.3 f 5.8 n.s. 274 ? 38.5 317.4 2 63.1 ns. 14.7 2 2.8 13.4 2 2.2 n.s. 5.6 f 3.5 16.8 2 2.5 P < 0.02 0.72 2 0.08 0.44 f 0.05 P < 0.01 Changes in bi-exponential decay induced by r-ACPD in the NMDA current of composite EPSCs (n = 5) and in the NMDA current isolated pharmacologically ( n = 6). Values of ~ f T, ~ Af, , A, and AJAp + A,) at +60 mV are reported. The statistical significance of paired Student's t-tests is reported (n.s.. not significant). -104 ' , , , , , , ' , , , , , , , B A 7 150 CI FIG.7. Voltage-dependence of NMDA-EPSCs in control (open circles) and after f-ACPD potentiation (filled circles). (A) NMDA EPSC amplitude, measured as AN. at different holding potentials (n = 6, mean f SEM). (B)NMDA conductance plots normalized to maximum control conductance obtained from data shown in A. Boltzmann fits gave the following parameters: = I , g,i, = 0.025, V1/2 = 0.48 mV, K = 15 mV-' (R > in control g, 0.99). after t-ACPD potentiation g, = 2.06, g,, = 0.045, V1/2 = 0.23 mV, K = 15 mV-I (R > 0.99). (C) NMDA EPSC half-width at different holding potentials (n = 6, mean f SEM). Flc. 8. Hypothetical scheme illustrating the possible actions of glutamate and t-ACPD in the present experiments. Glutamate released during low-frequency mossy fibre transmission activates postsynaptic NMDA and non-NMDA receptors, while exogenous t-ACPD stimulates postsynaptic and probably also presynaptic mGluRs. On the postsynaptic side, coincidence of mGluR and NMDA stimulation causes long-term enhancement of NMDA and non-NMDA receptor-mediated transmission, which may involve a convergent action on intracellular Ca" release or protein kinase C (PKC) activity. The shaded areas indicate intracellular processes which, as well as presynaptic mCluRs. have been demonstrated in different studies but remain speculative here. The scheme is based on data in this work and in ( I ) Kinney and Slater (1993). Larson-Prior et a/. (1995); (2) Aronica et a/. (1993). Shigemoto ef a/. (1992). O'Connor et a/.(1995); (3) Irving et a/. (1992); (4) Bliss and Collingridge, (1993); ( 5 ) Glaum and Miller (1994). Ohishi et d.(1994). the membrane was depolarized (Fig. 7C, open circles), reflecting the intrinsic voltage-dependence of NMDA current kinetics (D' Angelo et al., 1994). Following t-ACPD potentiation, the half-width approximately doubled at all membrane potentials (Fig. 7C, filled circles). It should be noted that, since the decay rate of neither of the two NMDA EPSC subcomponents changed appreciably after potentiation, the half-width increase should reflect the higher slow-to-fast amplitude ratio between the two NMDA EPSC subcomponents. These results indicate that the mechanism of the t-ACPD NMDA EPSC potentiation was not voltage-dependent. Discussion where g,, and gminare the maximum and minimum conductances, V112is the half-activation potential and K is a slope factor (O'Connor et aL, 1994). Although both g,,, and gminrevealed the expected twofold increase in conductance, Vl12 and K did not change appreciably (Fig. 7B). To evaluate the changes in the time course of the NMDA EPSC before and after potentiation, we used half-width measures at different holding potentials. In the control, half-width became greater the more In this paper we first demonstrate that mGluR stimulation can induce a long-lasting potentiation of transmission at the mossy fibre-granule cell synapse of the rat cerebellum. Metabotropic potentiation was differentially expressed in the non-NMDA current and in a slow NMDA current subcomponent of the EPSCs, and depended on NMDA receptor coactivation. A hypothetical scheme illustrating the processes involved in this form of potentiation is illustrated in Figure 8 and is discussed below. Changes in the holding current [usually a reduction Potentiation of an NMDA current subcomponent at positive membrane potentials (Rossi and D’Angelo, unpublished observation)] suggested that non-synaptic membrane currents were also modulated by t-ACPD. The present results, however, do not provide evidence for slow metabotropic synaptic responses like those observed in hippocampal CA3 pyramidal neurons (Charpak and Gahwiler, 1991; Gerber et al., 1993) and in Purkinje neurons (Batchelor et al., 1995). General properties of metabotropic LTP The EPSC potentiation consisted of (i) an induction phase requiring coactivation of metabotropic and NMDA receptors, and (ii) an expression phase outlasting the presence of r-ACPD. EPSC potentiation (iii) could be prevented by the mGluR antagonist MCPG. Moreover, EPSC potentiation was (iv) long-lasting (at least 45 min) and (v) non-decremental over the recording time. Thus, the t-ACPD potentiation at the mossy fibre-granule cell synapse may be regarded as a ‘metabotropic’ form of LTP (Bliss and Collingridge, 1993). Two properties make the present t-ACPD potentiation more similar to that observed in dentate granule cells than to that in pyramidal neurons of the hippocampus. First, the present t-ACPD potentiation had a rapid onset ( < I 0 min), as in dentate granule cells (O’Connor et al., 1994), while that in CAI neurons is much slower (>30 min; Bortolotto and Collingridge, 1993). Secondly, mGluR stimulation was insufficient by itself to induce potentiation, unlike what occurs in hippocampal pyramidal cells (Bashir et al., 1993; Bortolotto and Collingridge, 1993), but specific NMDA receptor coactivation was also required, as recently observed in dentate granule cells (O’Connor et al., 1995). It is interesting to note that, in the turtle cerebellum, metabotropic potentiation in the NMDA receptor-dependent response of granule cells was observed in the continuous presence of t-ACPD (Kinney and Slater, 1993), a condition which induces depression in our experiments in the rat. Although there may be a genuine difference in the mechanism of mGluR potentiation, it should be noted that the enhancement of transmission in the turtle has been observed indirectly through the response of Purkinje cells to mossy fibre stimulation, so that t-ACPD could also change granule cell excitability and hence the response to mossy fibre stimulation. The induction phase During induction, a low-frequency (0.1 Hz) NMDA receptor activation, insufficient by itself to induce any potentiation, played a permissive role in the metabotropic potentiation of transmission. Induction may involve the convergence of NMDA receptors and mGluR actions on a common intracellular mechanism in the postsynaptic element (Otani et al., 1993). Influx of Ca2+ through the associated channel (Mayer and Westbrook, 1987) is the natural way of NMDA-receptor coupling to intracellular modulatory systems (Bliss and Collingridge, 1993). In the experiments reported in Figure 4, the NMDA EPSC charge at -70 mV was 103 5 31 fC (n = 4; see also D’Angelo et al., 1993). Supposing that three synapses are active, that just one-tenth of the NMDA channel charge is carried by Ca” (Mayer and Westbrook, 1987), and that Ca2+ diffuses in a dendritic terminal volume of 0.2 pm3 (Jakab and Hamory, 1987), Ca2+ would eventually attain a theoretical free concentration of 80 pM (conversion from charge to moles is calculated using Faraday’s constant). Thus, although the Ca2’ build-up is usually curtailed by intracellular Ca2+ buffering, low-frequency synaptic activation of the NMDA receptors at -70 mV may be sufficient to activate secondary Ca2+-dependent processes. It is possible, for instance, that Ca2+ influx through the NMDA channels augments the effectiveness of mGluRs in releasing Ca2+ from intracellular stores, as demonstrated 1187 in cerebellar granule cells in culture (Irving et al., 1992), eventually causing the cytoplasmic Ca2+ increase necessary for inducing potentiation (Bliss and Collingridge, 1993; Fig. 8). It is also possible that the inositol triphosphate elevation caused by mGluRl stimulation (Aronica et al., 1993) converges with Ca2+ elevation in activating protein kinase C and subsequent potentiation (Bliss and Collingridge, 1993; O’Connor et al., 1995; Fig. 8). During induction, EPSC depression tended to obscure the acute r-ACPD enhancement of the NMDA current observed using bath application of t-ACPD and NMDA in cerebellar granule cells (see fig. 5 by N. T. Slater in Glaum and Miller, 1994). The expression phase During the expression phase, potentiation occurred both in the NMDA and in the non-NMDA current (Fig. 8). That the NMDA current is a target for plasticity has been reported before (Berretta et al., 1991; Asztely et al., 1992; O’Connor et al., 1994; Weisskopf and Nicoll, 1995). In hippocampal granule cells the NMDA current potentiation is similar to the non-NMDA current potentiation (O’Connor et al., 1995). Here we demonstrate that, in cerebellar granule cells, NMDA current potentiation is two to three times as large as non-NMDA current potentiation, and that the NMDA current increases selectively in a slow subcomponent. The greater increase in the NMDA than in the non-NMDA current and the selective change in an NMDA current subcomponent, although not excluding presynaptic enhancement in glutamate release, suggest the involvement of postsynaptic mechanisms of channel modulation (Bliss and Collingridge, 1993). The enhancement in the slow NMDA current subcomponent is more likely to involve a change in NMDA channel deactivation (Jonas and Spruston, 1994) than a reduction in Mg2+ block (Ben-Ari et al., 1992; Chen and Huang, 1992), since potentiation was voltage-independent. The relationship of this finding with the expression of two NMDA receptor channel subunits, NR2A and NR-2C, in mature cerebellar granule cells (Monyer et al., 1994; see also Farrant et al., 1994) remains to be established. Relationship to glutamate receptor subtypes The observation that 500 pM MCPG prevented any further t-ACPD potentiation suggests that either mGluRl or mGluR2, but not mGluR4 (Watkins and Collingridge, 1994; Pin and Duvoisin, 1995), induces the EPSC potentiation. In granule cells in culture mGluRl stimulation determines activation of the intracellular phospholipase C/inositol triphosphate cascade and Ca2+ elevation (Aronica et al., 1993). Stimulation of postsynaptic MCPG-sensitive mGluRs (Bashir et al., 1993) linked to the phospholipase Chnositol triphosphate cascade is indeed considered an important step in the induction of potentiation in different preparations (Pin and Duvoisin, 1995). Thus, although evidence for mGluR, in siru does not seem definitive at present (Shigemoto et al., 1992). mGluRl is a likely candidate for the induction of potentiation observed here (Fig. 8). No evidence for mGluR2/mGluR~has been reported in granule cells either in in situ hybridization or immunolabelling studies (Ohishi et al., 1994). Differently from potentiation, EPSC depression during t-ACPD perfusion was not prevented by 500 pM MCPG and should act, therefore, through receptors other than mGluRl or mGluR2. Candidates for EPSC depression are receptors like m G l u h , which, as at other central synapses (Glaum and Miller, 1994), may cause presynaptic inhibition of mossy fibre glutamate release (Fig. 8). No direct evidence is currently available for any mGluRs in the mossy fibre, although neurons potentially projecting to the cerebellum contain the mGlu& mRNA transcript (Glaum and Miller, 1994). The m G l u h mRNA 1188 Potentiation of an NMDA current subcomponent transcript is also present in cerebellar granule cells (Tanabe et a/.. 1993), in which mGluR4 are known to mediate presynaptic inhibition of transmission at the parallel fibre-Purkinje cell synapse (LarsonPrior et al., 1995). Conclusions and functional implications Potentiation in the slow NMDA current subcomponent seems particularly suited to enhance the input-output function of the granule cell since, at high frequency, the NMDA current sustains temporal summation (D’Angelo et al., 1995). Potentiation in the non-NMDA current, by enhancing synaptic depolarization, favours the voltagedependent NMDA channel response (Fig. 7; D’Angelo er a / . , 1995). Moreover, since the NMDA receptor is an effector as well as a target of potentiation, the potentiated synapse may become highly susceptible to further plastic changes (Bortolotto et al., 1994). A similar mechanism may be called into play during high-frequency transmission, as reported at hippocampal synapses (Bashir et al., 1993; Bortolotto et al., 1994; O’Connor er al., 1994, 1995). At present, the relationship between this mechanism and the long-term enhancement in synaptic transmission caused by high-frequency mossy fibre stimulation (D’Angelo et al., 1995) remains to be established. There is evidence that potentiation at the mossy fibre-granule cell synapse is retransmitted to Purkinje cells through increased granule cell excitation (Kinney and Slater, 1993; Larson-Prior et al., 1995), suggesting an important role for the present form of potentiation in regulating information processing in the cerebellar cortex (Eccles et a/., 1967; Linden and Connor, 1993). Acknowledgements This work was supported by grants from the Minister0 della Ricerca Scientifica e Tecnologica and Consorzio Interuniversitario Nazionale di Fisica della Materia, Italy. Abbreviations AN A” APV CNQX EPSC 7-kyn MCPG NMDA r-ACPD NMDA current amplitude non-NMDA current amplitude D-2-amino-5-phosphonovalerate 6-cyano-7-nitroquinoxaline-2,3-dione excitatory postsynaptic current 7-chlorokynurenate ( + )-O-methyl-4-carboxyphenyl-glycine N-methyl-D-aspartate 1 S.3R- 1 aninocyclopentane- I ,3-dicarboxylic acid References Aronica, E., Condorelli, D. F., Nicoletti, F., Dell’Albani, P., Amico, C. and Balazs, R. ( 1993) Metabotropic glutamate receptors in cultured cerebellar granule cells: developmental profile. J. Neurochein., 60, 559-565. Asztely. F., Wigstrom. H. and Gustafsson, B. (1992) The relative contribution of NMDA receptor channels in the expression of long-term potentiation in the hippocampal CAI region. Eur: J. Neurosci., 4, 6 8 1 4 9 0 . Bashir, Z. I., Bortolotto, Z. 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