GABA receptor activation enhances mGluR-mediated ... - Nature

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GABAB receptor activation enhances mGluR-mediated responses at cerebellar excitatory synapses Moritoshi Hirono1, Tohru Yoshioka1,2 and Shiro Konishi3 1 Department of Molecular Neurobiology, Advanced Research Institute for Science and Engineering, Waseda University, Tokyo 169-8555, Japan 2 Department of Molecular Neurobiology, School of Human Sciences, Waseda University, Tokorozawa 359-1192, Japan 3 Laboratory of Molecular Neurobiology, Mitsubishi Kagaku Institute of Life Sciences and CREST (JST), 11-Minamiooya, Machida-shi, Tokyo 194-8511, Japan

Correspondence should be addressed to S.K. ([email protected])

Published online: 12 November 2001, DOI: 10.1038/nn764 Metabotropic γ-aminobutyric acid type B (GABAB) and glutamate receptors (mGluRs) are postsynaptically co-expressed at cerebellar parallel fiber (PF)–Purkinje cell (PC) excitatory synapses, but their functional interactions are unclear. We found that mGluR1 agonist-induced currents and [Ca2+]i increases in PCs were enhanced following co-activation of GABAB receptors. A GABAB antagonist and a G-protein uncoupler suppressed these effects. Low-concentration baclofen, a GABAB agonist, augmented mGluR1-mediated excitatory synaptic current produced by stimulating PFs. These results indicate that postsynaptic GABAB receptors functionally interact with mGluR1 and enhance mGluR1mediated excitatory transmission at PF–PC synapses. The interaction between the two types of metabotropic receptors provides a likely mechanism for regulating cerebellar synaptic plasticity.

GABA is the main inhibitory neurotransmitter in the central nervous system, and inhibitory GABAergic synapses are endowed with transmitter receptors including ionotropic GABAA and GABA C receptors and metabotropic GABA B receptors (GABABRs)1. Ionotropic GABA receptors exhibit considerable molecular diversity2, whereas recent cloning has revealed that the GABA BR gene encodes R1a and R2 subunits that form heterodimers to function3–6. GABABRs are coupled with Gi/o proteins either to inhibit neurotransmission presynaptically7,8 or to decrease the excitability postsynaptically by opening G-proteincoupled inwardly rectifying K + (GIRK) channels 9,10 . The GABABR-mediated Gi/o protein activation also depresses adenylyl cyclase and reduces Ca2+ channel currents11,12. However, correlations between these GABABR-mediated pharmacological actions and synaptic events are not completely understood. In the cerebellar cortex, radioautographic and immunocytochemical studies have shown that GABABR binding sites densely occur in the molecular layer, where the PCs extend their dendritic branches 13,14 . The postsynaptic localization of GABABRs on PCs was reported by in situ hybridization3–6. An electron microscopy study showed that GABABRs are present at the extra-postsynaptic sites of excitatory connections between parallel fibers (PFs) and PCs5,15. GABABRs also suppress a synaptic process called rebound potentiation of inhibitory transmission following PC depolarization 16 . However, it remains uncertain what physiological role the GABA BRs have at the PF–PC excitatory postsynaptic sites. Long-term depression (LTD) at PF–PC synapses has been proposed as a cellular mechanism of synaptic plasticity closely associated with motor learning17,18. Induction of LTD requires activation of type 1 metabotropic glutamate receptors (mGluR1), triggering a signaling cascade that nature neuroscience • volume 4 no 12 • december 2001

includes Gq protein-mediated activation of phospholipase C (PLC), hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) to inositol 1,4,5-triphosphate (IP3) and diacylglycerol, leading to intracellular Ca2+ concentration ([Ca2+]i) increase and protein kinase C (PKC) stimulation18–20. Interestingly, cellular localization of mGluR1 is similar to that of GABABRs: morphological studies have shown that mGluR1 is also expressed abundantly at the extra-postsynaptic sites of PF–PC synapses21–23. It is, therefore, reasonable to expect that GABABRs would interact with mGluR1 and modulate mGluR1-mediated physiological responses at these synaptic sites. Therefore, the aim of the present study was to explore the cross-talk between mGluR1 and GABABRs expressed by PCs in acute slices from the mouse cerebellum, using whole-cell recordings combined with Ca2+-signal imaging. We found that the activation of GABABRs by the exogenous agonist baclofen enhanced both mGluR1-mediated inward currents and Ca2+ signals in PCs. More importantly, we showed that endogenous GABA released by electrical stimulation in the cerebellar cortex mimicked the effect of the GABABR agonist, which augmented the mGluR1mediated slow excitatory synaptic current elicited by PF stimulation. Therefore, the cross-talk between mGluR1 and GABABR revealed in this study seems to call for a revision of our view that GABAB receptors serve an exclusively inhibitory role in chemical signaling at central synapses.

RESULTS GABAB activation enhanced mGluR current Iontophoretic application of the nonselective mGluR agonist 1S,3R-ACPD (1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid) produced an inward current in cerebellar PCs voltage-clamped 1207


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Fig. 1. Enhancement of 1S,3R-ACPD-induced inward current by GABA. (a) Inward current responses were induced by iontophoretic application of 1S,3R-ACPD to a Purkinje cell at a constant interval of 40 s. GABA (100 µM) was applied by perfusion for 3 min as indicated by horizontal bar. The record was obtained from a Purkinje cell held at –60 mV in the presence of bicuculline (30 µM) and TTX (0.5 µM). (b) 1S,3RACPD-induced responses indicated by 1, 2 and 3 in (a) are displayed on a fast time base.

at –60 mV, which is close to the resting potential24–26. The 1S,3RACPD-induced current was suppressed by (S)-4-carboxyphenylglycine (4CPG), a selective antagonist for group I mGluRs including mGluR1 and mGluR5 (ref. 26), whereas 1S,3R-ACPD did not produce any detectable current in PCs of mGluR1-deficient mice19, which indicates that the inward current resulted from activation of mGluR1. This mGluR1-mediated current increased markedly when GABA, an inhibitory transmitter substance, was co-applied by superfusion (Fig. 1). The increase of the 1S,3R-ACPD-current amplitude following 100 µM GABA co-application was 161 ± 8% of the control response (n = 4) in an artificial cerebrospinal fluid (ACSF) that contained tetrodotoxin (TTX, 0.5 µM) and the GABAA receptor

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antagonist bicuculline (30 µM). Therefore, it is likely that the enhancement of the 1S,3R-ACPD-induced current in the presence of GABA is mediated by GABABRs but not by GABAA receptors in PCs, which is consistent with the slow outward current observed during GABA application. We then examined whether the GABABR agonist baclofen reproduces the augmentation of the 1S,3R-ACPD response. When applied by perfusion, baclofen (3 µM) increased the amplitude of the 1S,3RACPD-induced current up to 226 ± 11% of the control response (n = 15; Fig. 2). To test whether the GABABR-induced enhancement is specific to the mGluR1-mediated response, we compared the effects of baclofen on inward current responses produced by 1S,3RACPD and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxasole propionic acid), an ionotropic GluR agonist. Baclofen selectively enhanced the 1S,3R-ACPD response without appreciably affecting the AMPA-induced current (Fig. 2a–c). Baclofen also produced an outward current (36 ± 4 pA, n = 15), as observed with GABA application. The baclofen-induced enhancement of the 1S,3R-ACPD response was reversible, subsiding over a period of approximately 20 minutes after the agonist was washed out. In a current-clamp mode, baclofen hyperpolarized PCs by –2.1 ± 2.2 mV (n = 4, Fig. 2d) and increased the amplitude of 1S,3R-ACPD-induced depolarization from 10.6 ± 0.7 mV to 14.8 ± 1.3 mV (Fig. 2e; n = 4, p < 0.05). The baclofen-induced enhancement of the mGluR1-mediated response was completely abolished by the selective GABABR antagonist CGP62349 (Fig. 3a) with a half-maximal inhibitory concentration IC50 of about 15 nM. The 1S,3R-ACPD-induced current remained almost unchanged in the presence of 100 nM CGP62349 (97 ± 2% of the control current, n = 4), which indicates that tonic activation of GABABRs by endogenous GABA released spontaneously is insufficient for a detectable enhancement of the mGluR1-mediated response. We then investigated whether activation of G-protein-coupled receptors other than GABABRs also enhances the mGluR1mediated inward current in PCs. The 1S,3R-ACPD-induced Fig. 2. Selective enhancement of mGluR1-mediated current following GABABR activation. (a) 1S,3R-ACPD- and AMPA-current responses were alternately induced by iontophoretic applications from separate microelectrodes to a single Purkinje cell. The GABABR agonist baclofen (3 µM) was applied by perfusion during the period indicated by the horizontal bar. (b) Sequential 1S,3R-ACPD- and AMPA-induced currents indicated as 1–3 in (a) are displayed on a fast time base. (c) Time course of the effect of GABABR activation on the 1S,3R-ACPD- and AMPA-induced current responses. The amplitude of both responses is given as a percentage of the control immediately before baclofen application. (d) A current-clamp recording of 1S,3RACPD-induced depolarization in a Purkinje cell and the effects of baclofen (3 µM) on the 1S,3RACPD-response and membrane potential. (e) Expanded and superimposed records of 1S,3R-ACPDinduced depolarizations before and during baclofen application. Each response was recorded as indicated by 1 and 2 in (d). nature neuroscience • volume 4 no 12 • december 2001



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Fig. 3. Blockade of baclofen-induced enhancement of the mGluR1mediated current response by the GABABR antagonist CGP62349. (a) 1S,3R-ACPD was iontophoretically applied at a time point indicated by arrows at a constant interval in the presence of CGP62349 (30 nM). Responses were recorded from a single PC before (left) and during perfusion of baclofen (3 µM, middle) and after washing out the GABABR agonist (right). (b) Comparison of the effects of Gi/o-coupled receptor agonists on the 1S,3R-ACPD-induced current response in PCs. The number in parentheses represents the number of PCs in which the effects of individual agonists were determined: baclofen (3 µM), serotonin (5-HT, 30 µM), carbachol (CCh, 100 µM) and adenosine (10 µM). ***P < 0.001, one-way ANOVA with Tukey’s post-test.

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current was not significantly altered by application of carbachol (CCh), serotonin (5-HT) or adenosine (Fig. 3b). However, muscarinic, 5-HT and adenosine receptors occur in the cerebellar cortex27–29, and 5-HT and adenosine elicit facilitation and inhibition of cerebellar GABAergic transmission, respectively, when applied by superfusion, as in this study30,31. It seems, therefore, that the enhancement of the mGluR1-mediated response requires selective activation of GABABRs in PCs. Characterization of GABABR-mediated enhancement First, the current–voltage relationship of the 1S,3R-ACPDinduced response was compared before and during GABABR activation. The 1S,3R-ACPD-induced current was obtained by subtracting current responses produced by a constant voltage ramp in the absence and presence of 1S,3R-ACPD (Fig. 4a and b). Baclofen increased the 1S,3R-ACPD-induced current, whereas its reversal potential was almost identical before and after baclofen application (–5.1 ± 4.2 mV and –2.8 ± 3.6 mV, respectively, n = 7, p = 0.68). The degree of the GABABR-mediated enhancement of the 1S,3R-ACPD current did not change in the membrane potential range examined (Fig. 4c), indicating that the GABABR-mediated enhancement of mGluR1-activated currents is independent of the membrane potential. Furthermore, the effect of GABABR activation did not depend on the amplitude of the 1S,3R-ACPD responses, as there was no correlation between the initial amplitude of 1S,3R-ACPD-induced currents (140 to 280 pA) and the extent of the baclofen-induced enhancement of 1S,3R-ACPD currents (Fig. 4d). Extrapolation of the baclofen-induced current response showed its reversal potential of –93.6 ± 5.0 mV (n = 5), which was close to the K+ equilibrium potential (–96.6 mV) predicted from the Nernst equation. In the presence of a GIRK channel inhibitor, Ba2+ (1 mM), baclofen still induced an outward current, the extent of which was almost comparable to that of the control response (43.3 ± 5.8 pA, n = 4, p = 0.46). Furthermore, Ba2+ had little effect on the GABABR-mediated enhancement of 1S,3R-ACPD-induced current (197 ± 22% of the control response, n = 4, p = 0.32; example, Fig. 4g). The GIRK current in principal neurons of the amygdala was, in fact, almost completely blocked by 1 mM Ba2+ when applied by superfusion to slice preparations (ref. 32 and unpublished observations). Therefore, it seems that the GABABR-mediated enhancement and outward currents are not due to the activation of GIRK channels. The baclofen-induced outward current and enhancement of mGluR1-mediated response were dose-dependent with half maximal effective concentrations (EC50) of approximately 1.01 and 0.94 µM, respectively (Fig. 4e), and pooled data showed that there was a modest correlation between both responses (Fig. 4f). In addition to the two effects, baclofen markedly inhibited PF-stimulation-evoked excitatory postsynaptic currents (EPSCs), and nature neuroscience • volume 4 no 12 • december 2001

the IC50 of PF-EPSC inhibition was approximately 0.77 µM, which is consistent with the value reported previously33,34. The effective concentration ranges of baclofen for inhibiting PF-EPSCs as well as causing the mGluR1-current enhancement and outward currents were very similar (Fig. 4e). Effect of baclofen on mGluR1-mediated [Ca2+]i transients Application of the mGluR agonist 1S,3R-ACPD increased [Ca2+]i in PCs as reported previously24–26. To determine the effects of baclofen on the mGluR1-induced [Ca2+]i increase, we performed simultaneous whole-cell recordings and intracellular Ca2+ imaging in PCs using a Ca 2+ indicator, fura-2. Baclofen (3 µM) enhanced not only the inward current but also the [Ca2+]i elevation produced in response to iontophoretic application of 1S,3R-ACPD (Fig. 5): the 1S,3R-ACPD-current and [Ca2+]i transients measured in the distal dendrites of PCs were increased to 180 ± 22% (n = 4, p < 0.01) and 321 ± 71% (n = 4, p < 0.05) of the control responses, respectively (Fig. 5c). 1S,3R-ACPD caused a larger increase in [Ca2+]i at distal dendrites than at proximal dendrites of the PC. Furthermore, baclofen increased the basal level of [Ca2+]i in three of four PCs tested. The averaged F340/F380 ratio reflecting the basal [Ca2+]i increased during the GABABR agonist application and recovered to the control level after the agonist was washed out (Fig. 5d). This effect was particularly significant in the recording site at proximal dendrites. The effect of baclofen on [Ca2+]i is not compatible with the previous observation that GABABR activation inhibits Ca2+ influx through P-type Ca2+ channels in PCs11. A possible involvement of P/Q-type Ca2+ channel inhibition in the modulation of mGluR1 response was excluded as the P/Q-type Ca2+ channel blocker ω-agatoxin IVA (100 nM) did not enhance the 1S,3R-ACPD-induced inward current but, rather, slightly reduced its amplitude to 82.8 ± 6.2% of the control response (n = 5, p < 0.05). Thus, it seems that GABABR activation increased the basal [Ca2+]i level by influencing internal Ca 2+ store through mGluR1-mediated and G-protein-coupled signaling pathways. An analogous mechanism has been proposed for the modulation of the basal [Ca2+]i level elicited by the activation of other metabotropic receptors, such as opioid receptors, and adenosine A1 and NPYY1 receptors35. Another possibility would be that baclofen enhances the [Ca2+]i 1209



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Fig. 4. Voltage-independent facilitation of GABABR-mediated enhancement of the 1S,3R-ACPD-induced currents, and comparisons of presynaptic and postsynaptic actions induced by baclofen. (a) 1S,3R-ACPD was iontophoretically applied to a Purkinje cell (PC) at 40-s intervals, and the holding potential of the PC (–60 mV) was shifted by constant voltage ramps from –130 to 0 mV for 1560 ms at the time points as indicated by (1, 1´) and (2, 2´) during and after the 1S,3R-ACPD-induced response, respectively. This sequence of ramp commands was repeated before (1, 2) and during baclofen (3 µM) application (1´, 2´). (b) 1S,3R-ACPD-induced currents determined by subtraction of current–voltage relationships produced by ramp commands as indicated in (a). (c) The amplitude ratios of 1S,3R-ACPD-induced currents before and during baclofen application calculated as (1´ – 2´)/(1 – 2) × 100 (%) are plotted against the membrane potential range determined in PCs (n = 5). (d) Relationship between the extent of baclofen-induced enhancement of mGluR1-mediated currents and the amplitude of 1S,3R-ACPD-currents before baclofen (3 µM) application in individual PCs. The straight line indicates a regression line with a correlation coefficient, r = –0.029, indicating no correlation between the two responses. (e) Comparison of dose–response relationships for the baclofen-induced enhancement of the mGluR1-mediated current, outward current and inhibition of PF-mediated EPSCs. The 50% effective doses(EC50) of baclofen were determined for the 3 responses (n = 3–15): the increase of the mGluR current expressed as a percentage of that induced by 100 µM baclofen (white circles and solid line, EC50 ≈ 0.94 µM), the amplitude of baclofen-induced outward current response expressed as a percentage of that induced by 100 µM baclofen (black circles and dashed line, EC50 ≈ 1.01 µM) and percentage inhibition of PF-EPSC by baclofen (white squares and dotted line, IC50 ≈ 0.77 µM). The Hill coefficient (n) determined from each dose–response curve was 0.84, 0.92 and 0.94, respectively. (f) Relationship between baclofen-induced enhancement of mGluR1mediated current and outward current response. The straight line indicates a regression line with a correlation coefficient was r = 0.558. (g) Effects of Ba2+ on the baclofen-induced outward current response and enhancement of mGluR1-mediated current. Ba2+ (1 mM) and baclofen (3 µM) were applied by perfusion during the period indicated by horizontal bars. Downward deflections represent the inward currents induced by iontophoretic application of 1S,3R-ACPD at a constant interval, as in (a).

rise via tonic activation of mGluR1 by glutamate released spontaneously from excitatory nerve terminals. We next examined the mechanisms underlying the baclofeninduced enhancement of the mGluR1 response. First, we investigated whether G i/o activation is required for the GABABR-mediated enhancement. Treatment of cerebellar slices with N-ethylmaleimide (NEM), a Gi/o inhibitor9, at a concentration of 50 µM for 15 minutes significantly reduced the extent of the baclofen-induced enhancement of the 1S,3R-ACPDinduced current (127 ± 17% of the baseline after NEM treatment, versus 226 ± 11% of the baseline in the control ACSF, n = 4, p < 0.001; Fig. 6a and b). However, the NEM treatment did not cause any significant effects on the 1S,3R-ACPD-current 1210

(89 ± 10% of the control response, n = 4, p = 0.39) and the AMPA-current (98 ± 6% of the control response, n = 3, p = 0.92). This finding suggests that G-proteins, presumably G i/o, are responsible for the GABABR-mediated enhancement. One target of Gi/o proteins linked with GABABRs might be adenylyl cyclase, as GABABR agonists reduce the level of intracellular cyclic AMP by inhibiting adenylyl cyclase12. However, the baclofen-induced increase in the 1S,3R-ACPD current was not significantly affected by treatment with either forskolin (30 µM) for 20 minutes or 8Br-cAMP (500 µM) for 35 minutes (data not shown). Treatment with protein kinase inhibitors H-7 (20 µM) or H-8 (20 µM) for 20 minutes also had no effect (data not shown). Another possible target of Gi/o proteins is PLC. Gi/o nature neuroscience • volume 4 no 12 • december 2001


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Fig. 5. Simultaneous recordings of GABABR activation-mediated enhancement of 1S,3R-ACPD-induced current and [Ca2+]i transients in a PC. (a) [Ca2+]i transients were fluorometrically measured at proximal (P, white) and distal dendrites (D, red) of the PC held at –60 mV starting 25 min after loading the Ca2+ indicator fura-2 (1 mM) via the recording electrode. The fura-2-loaded PC was viewed with a fluorescence image produced by 380 nm excitation wavelength (1). Pseudocolor ratio images were recorded before 1S,3R-ACPD application (2), during 1S,3R-ACPD application in the absence (3) and presence (4) of 3 µM baclofen. (b) Effects of baclofen on 1S,3R-ACPD-induced Ca2+ signals (top) and current responses (bottom). 1S,3R-ACPD was iontophoretically applied to a single PC at a constant interval as indicated by dots, and baclofen (3 µM) was applied by perfusion during the period indicated by a horizontal bar. The numbers 2–4 indicate the time points where the images in (a) were obtained, and the fluorescence ratio curves indicated by red and black were measures at distal and proximal dendritic sites, respectively. (c) Time courses of baclofen-induced enhancement of the mGluR1-mediated current (white circles) and [Ca2+]i transient, F340/F380 ratio (black circles) in PCs. Each response is expressed as a mean percentage of the control response determined immediately before baclofen (3 µM) application (n = 4). (d) Changes in basal [Ca2+]i level in PCs induced by baclofen. Each plot represents the mean ± s.e.m. of the F340/F380 ratios determined before (70–90 s) and during baclofen application (150–170 s) and after washing out of the drug (310–330 s), respectively, in each of four different PCs. *p < 0.05, ***p < 0.001, one-way ANOVA tested for the values before and during baclofen application.

proteins are linked to [Ca2+]i increases via activation of IP3 receptors35,36, and stimulation of GABABRs in the cerebellar cortex causes activation of PLC via G-proteins, thereby resulting in modulation of GABAA receptor fuctions37. We therefore tested the possible involvement of PLC and IP3 receptors in the GABABRmediated response. A selective PLC inhibitor, U73122 (10 µM), infused into PCs via a patch electrode, significantly suppressed the baclofen-induced enhancement of the 1S,3R-ACPD current when the effect was determined at least 30 minutes after intracellular application of the PLC inhibitor (n = 5, p < 0.05; Fig. 6d). Application of an IP3 receptor modulator, heparin (300 units/ml), also suppressed the GABABR-mediated enhancement (n = 4, p < 0.01). Furthermore, intracellular infusion of the Ca2+ chelator BAPTA (35 mM) significantly reduced the extent of the 1S,3R-ACPD current enhancement (n = 5, p < 0.05). The baclofen-induced outward current was also significantly reduced by all the three treatments (data not shown). The pharmacological manipulations used here caused only partial suppression of the mGluR1-mediated current response per se as previously reported 26,38 (Fig. 6c). Taken together, it is likely that the baclofen-induced basal [Ca2+]i rise resulting from Ca2+ release nature neuroscience • volume 4 no 12 • december 2001

from intracellular stores is responsible for the enhancement of the mGluR1 response. The baclofen-induced outward current might be attributable to a Ca2+-activated K+ current. Dual effects of baclofen on mGluR1-mediated EPSC In the presence of ionotropic glutamate and GABA receptor antagonists, repetitive stimulation of PFs produces in PCs a slow excitatory synaptic response that is mediated by group I mGluRs26,38–40. A crucial test is to determine whether GABABR activation enhances synaptically evoked mGluR-mediated responses (Fig. 7). Activation of PFs with 10 stimuli at 100 Hz produced fast EPSCs with a gradual increase in amplitudes that were followed by a slow inward EPSC. Fast and slow components of the synaptic responses were AMPA-receptor- and mGluR1mediated EPSCs, respectively, because the former was almost completely blocked by CNQX (30 µM) and the latter was abolished by the group I mGluR antagonist 4CPG (500 µM)26. Application of baclofen at a low concentration of 0.3 µM increased the amplitude of slow EPSCs to 118 ± 6% of the control (n = 9, p < 0.01; Fig. 7a–c). In contrast, baclofen at the same concentration decreased the amplitude of fast EPSCs to 89.2 ± 5.7% of the 1211



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Fig. 6. Effects of signal transduction a modulators on the GABABR-induced enhancement of the mGluR1-mediated current. (a) Effects of NEM treatment on 1S,3R-ACPDand AMPA-current responses and baclofen-induced enhancement of 1S,3R-ACPD-response. NEM (50 µM) was applied by perfusion. The responses were recorded at the time points indicated by 1 to 3 in (b). (b) Time courses of the NEM effects on 1S,3R-ACPD-current and GABABRinduced enhancement of 1S,3RACPD-response. The amplitude of c 1S,3R-ACPD-currents is expressed as a percentage of the controls immediately before NEM application (black squares) and baclofen application without NEM treatment (white circles), respectively. (c) Effects of the PLC inhibitor U73122 (10 µM) and the IP3 receptor inhibitor heparin (300 units/ml) infusions on the time course change of the 1S,3R-ACPDinduced current amplitudes. Each data point represents the mean ± s.e.m. of a percentage to the amplitude of initial 1S,3R-ACPD-current determined 2 min after obtaining whole-cell access in 5 to 12 PCs. (d) Effects of NEM (50 µM), U73122 (10 µM), heparin (300 units/ml) and BAPTA (35 mM) infusions on the baclofen-induced enhancement of 1S,3R-ACPD-induced current. Each compound except NEM was applied for at least 30 min by infusing into PCs via the recording electrode, and then the application of 3 µM baclofen was initiated. NEM was applied by superfusion for 15 min, and the effect of baclofen on 1S,3R-ACPD-induced current response was determined in the presence of NEM and expressed as a percentage of the control 1S,3RACPD-response before the GABABR agonist application. The number in parentheses represents the number of independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with Tukey’s post-test.

control (n = 9). When the concentration was increased to 1–30 µM, baclofen inhibited both fast and slow EPSCs to a similar degree (42.1 ± 8.1% and 43.9 ± 6.5% inhibition at 10 µM, respectively, n = 9; Fig. 7d and e). The dose–response relationships for the baclofen-induced enhancement and inhibition of the slow mGluR1-mediated EPSC are shown in Fig. 8a. The enhancement might be explained by postsynaptic interaction between GABABRs and mGluR1 activated by the excitatory transmitter released from PF terminals. The inhibitory action of baclofen might be due to a presynaptic mechanism of reducing the amount of transmitter released39. It is likely, therefore, that postsynaptic GABABRs are more susceptible to baclofen than are presynaptic GABABRs. In the following two series of experiments, we further determined whether endogenous GABA released from cerebellar interneurons can enhance the mGluR1-mediated slow EPSCs. First, we examined the effect of a GABA uptake inhibitor, SKF89976A, on the slow EPSCs. Application of SKF89976A at a concentration of 10 µM increased the amplitude of slow EPSCs in all PCs examined (112 ± 5% of the control, n = 4, p < 0.05), whereas the GABA uptake inhibitor at a higher concentration of 100 µM decreased the slow EPSC amplitude by 44.5 ± 6.8% (n = 4, data not shown). The observations suggest that endogenous GABA causes enhancement and inhibition of the mGluR1mediated transmission via activation of postsynaptic and 1212

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presynaptic GABABRs, respectively, depending on the concentration of GABA released into the synaptic cleft. Second, we examined the effect of the GABABR antagonist CGP62349 on the mGluR1-mediated EPSC. The slow EPSCs evoked by repetitive stimulation of PFs gradually decreased to 74.5 ± 5.5% of the control after application of CGP62349 at a concentration of 300 nM (n = 6, p < 0.01) and the inhibitory effect persisted over 10 minutes after the drug was washed out (Fig. 8b and c). In contrast, the GABABR antagonist caused a slight increase in amplitude of the fast EPSC (109 ± 7.9% of the control, n = 6). These observations are consistent with the possibility that the mGluR1-mediated slow EPSC could be enhanced by the activation of postsynaptic GABABRs due to endogenous GABA released during repetitive stimulation of PFs, as PFs form excitatory connections with GABA-containing interneurons to elicit GABA release. The modulation of fast EPSCs by endogenous GABA seems to be dominated by the inhibitory action of presynaptic GABABRs.

DISCUSSION G-protein-coupled GABABRs and mGluR1 display analogous distribution profiles at cerebellar PF–PC excitatory synapses with both receptors being localized mainly at extrasynaptic sites along the PC dendrites5,15,21–23. The physiological functions of presynaptic GABABRs in PF terminals have been well studied33,34, but relatively little is known about the functions of postsynaptic GABABRs on PCs. Therefore, we investigated functional interactions between postsynaptic GABABRs and mGluR1 in PCs. We found that activation of GABABRs by the exogenous agonist baclofen elicited a profound enhancement of the mGluR1 agonist 1S,3R-ACPD-induced current response and [Ca2+]i rise in PCs (Figs. 2 and 5) and that endogenous GABA synaptically released from cerebellar interneurons following PF stimulation could enhance the mGluR1-mediated synaptic response evoked by the PF excitatory transmitter (Fig. 8). Our data suggest that the GABA B R-mediated enhancement of mGluR1 response requires Ca2+ release from internal stores through a signaling nature neuroscience • volume 4 no 12 • december 2001


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Fig. 7. Enhancement by baclofen of mGluR1-mediated slow EPSCs produced in response to PF stimulation. (a) Fast and slow EPSCs were evoked by repetitive stimulation (100 Hz for 100 ms) of the PF at a constant interval of 60 s in the presence of CNQX (10 µM), AP5 (30 µM) and bicuculline (50 µM) and recorded from a PC. The synaptic responses were recorded before (top) and during baclofen (0.3 µM) application (middle), and after the drug was washed out (bottom). (b) Superimposed PF-mediated slow EPSCs in the control and 0.3 µM baclofen-containing ACSF are displayed on a fast time base. (c) Time course of the effects of baclofen on fast and slow EPSCs produced in PCs by PF stimulation. The amplitude of both EPSCs is expressed as a percentage of the control amplitude determined immediately before baclofen application (n = 9). (d) Inhibitory effects of a higher concentration of baclofen (10 µM) on PF-mediated slow EPSCs. (e) Time course of baclofen-induced inhibition of fast and slow EPSCs recorded from PCs. The amplitude of both responses was expressed as a percentage of the control determined immediately before 10 µM baclofen application (n = 4).

mechanism that involves G i/o-coupled PLC activation. This GABABR-mediated modulation of synaptic processes seemed to be specific to the mGluR1 responses, because the ionotropic AMPA-type GluR-mediated current response was not affected by the GABA BR agonist baclofen (Fig. 2). Furthermore, the enhancement of mGluR1-mediated responses is a unique property of metabotropic GABABRs, as other G-protein-linked receptors including serotonin, adenosine and muscarinic receptors were devoid of this capability in PCs. Two possible mechanisms may explain the cross-talk between GABABR and mGluR1 revealed in this study. First, because not only the mGluR1-activated current but also the mGluR1-induced [Ca2+]i increase in PCs were enhanced following GABABR activation, Ca2+ mobilization from internal stores through Gi/olinked PLC activation and IP3 formation might be critical in the GABABR-mGluR1 interaction. This notion was supported by the following observations: treatment with the Gi/o inhibitor NEM markedly attenuated the GABABR-mediated enhancement of the current response; infusion of the PLC inhibitor U73122 and the IP3 antagonist heparin into PCs suppressed these GABABRmGluR1 interactions; infusion of the Ca2+ chelator BAPTA also inhibited the effects of GABABR activation on mGluR1 responses; and application of the GABABR agonist baclofen increased basal [Ca2+]i (Fig. 5). Thus, it seems that GABABR activation may be linked to intracellular Ca2+ stores to modulate [Ca2+]i and enhance the mGluR1 functions including the current response and Ca2+ elevation, presumably through a cooperative upregulation of G-protein-coupled receptor signaling by Gβγ subunits as reported in β-adrenergic receptor-GABABR synergistic interactions41. This is compatible with the finding that mGluR1-mediated excitation at PF–PC synapses is markedly increased by activation of the climbing fiber input via a transient increase in nature neuroscience • volume 4 no 12 • december 2001

[Ca2+]i40. Studies have suggested that the regulation of Ca2+ release from internal stores in presynaptic and postsynaptic neurons profoundly influences short- and long-term plasticity at cerebellar synapses42–44. Another possibility is that the GABABR-mediated enhancement of the mGluR1-activated current and Ca 2+ signals are attributable to two independent mechanisms, although there is no direct evidence supporting this. GABABR activation produced a substantial outward current that was resistant to treatment with Ba2+, an inhibitor of GABABR-linked GIRK channels. A previous study using a genetic knockout of GIRK channels clearly demonstrated that presynaptic GABABR-mediated inhibition of neurotransmission is independent of GABABR-mediated GIRK channel activation10. In our study, GABABRs associated with the enhancement of synaptically evoked mGluR1 response exhibited the sensitivity higher than those associated with presynaptic inhibitory actions (Fig. 8), although the exogenous mGluR agonist 1S,3R-ACPD-induced response and PF–PC transmission were affected by baclofen in a similar concentration range. Therefore, it remains to be determined whether separate receptor subtypes with distinct signaling pathways are involved in the conventional GABABR-mediated presynaptic inhibition and postsynaptic GIRK channel activation, and in the mechanism of GABABR-mGluR1 cross-talk found in this study. Two possible physiological consequences might be associated with the GABABR–mGluR1 interaction at PF–PC synapses. First, the interaction may increase the excitability of PCs, as it is assumed that the GABABR activation enhances the slow depolarizing synaptic potential mediated through mGluR1 activation by the excitatory transmitter glutamate released from PF nerve terminals. If this is the case, GABA may serve dual functions at PF–PC synapses, either as an excitatory modulator caus1213


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b

c

Fig. 8. Dose dependency of baclofen-induced enhancement and inhibition of mGluR1-mediated slow EPSCs, and the effects of synaptic GABABR activation on fast and slow EPSCs tested by using the GABABR antagonist CGP62349. (a) Dose–response relationships between baclofen-induced facilitatory and inhibitory actions on mGluR1-mediated slow EPSCs following repetitive PF stimulation were determined in PCs. The number in parentheses represents the number of PCs in which effects of baclofen were tested. (b) CGP62349-induced increase in PF-mediated fast EPSC and decrease in slow EPSC amplitude. The synaptic responses following repetitive PF stimulation were recorded from a PC in the control (left) and in 300 nM CGP62349-containing ACSF (right). (c) Time courses of CGP62349-induced effects on fast and slow EPSCs. The amplitudes of both EPSCs are expressed as a percentage of the control amplitude determined immediately before application of the GABABR antagonist (n = 6).

ing the postsynaptic transient facilitation of PF excitatory inputs to PCs, or as a classical transmitter eliciting presynaptic inhibition of the ionotropic GluR-mediated fast excitatory transmission. Second, the GABABR-mGluR1 cross-talk may be critical in synaptic plasticity associated with the cerebellar function, as mGluR1 has been implicated as an important molecule in the induction of LTD at these synapses19,20, which is proposed as a key mechanism underlying motor coordination within in the cerebellar system17,18. Furthermore, studies have suggested that mGluR1-mediated Ca2+ release from internal stores in PCs acts as a coincidence detection mechanism for PF and CF activations, thereby leading to induction of LTD at PF–PC synapses44. Therefore, simultaneous activation of mGluR1 and GABABRs would enhance this mechanism. The physiological significance of GABABRs in synaptic plasticity has also been shown in other brain regions. Presynaptic GABABRs seem to be involved in longterm potentiation (LTP) in the hippocampus45,46. In addition, postsynaptic GABABRs contribute to long-term regulation of synaptic strength at GABAergic inhibitory synapses in the visual cortex47. In this case, GABABRs and adrenoceptors seem to act in concert to further enhance heterosynaptic monoaminergic LTP, possibly through GABA BR-mediated facilitation of monoamine-induced IP 3 formation. Negative regulation of synaptic plasticity, which involves postsynaptic GABABRs, has been reported at inhibitory synapses in the cerebellar cortex: the activation of GABABRs in PCs downregulates the long-lasting increase, or ‘rebound potentiation,’ of GABAA receptor sensitivity following depolarization of PCs 16 . The postsynaptic GABABR–mGluR1 interaction identified in this study provides a prominent example of the modulatory role of GABABRs in synaptic plasticity at excitatory PF–PC synapses. Thus, GABABRs localized in presynaptic and postsynaptic neurons seem to be significantly involved in long-term regulation of synaptic efficacy at various synapses in the central nervous system. 1214

Interaction between different transmitter receptor systems is one emerging feature of neurotransmission at central synapses. For instance, dopamine D and somatostatin SST5 receptors form heterodimers to create a novel receptor with augmented functional activity48. Another example has been demonstrated for dopamine D5 and GABAA γ2 receptors49: The GABAA-ligandgated channels complex with D5 receptors via direct binding, thereby enabling mutually inhibitory functional interactions between the two receptor systems. Cross-talk between neurotransmitter-gated cation channels has been reported for heterologous expression systems and cultured neurons, where structurally distinct nicotinic receptor and purinergic P2X2 receptor channels influence each other with regard to cross-inhibition between the two channels50. Conformational spread from one receptor to its neighbors is proposed as a possible mechanism for the cross-talk. As exemplified by this cross-talk, together with the GABABR-mGluR1 interaction identified in this study, the interplay between distinct receptor systems can provide a powerful mechanism to influence chemical signaling at central nervous system synapses.

METHODS Electrophysiology. BDF1 mice (4–5 weeks old) were anesthetised with pentobarbital, and sagittal slices (180–200 µm thick) of the cerebellar vermis were prepared using a vibrating microtome (Microslicer DTK1000, Dosaka, Kyoto, Japan). Whole-cell recordings were obtained from PCs visually identified under Nomarski optics using a water immersion objective (40×, NA 0.75, Zeiss, Germany). Slices were superfused with ACSF containing 138.6 mM NaCl, 3.35 mM KCl, 21 mM NaHCO3, 0.6 mM NaH2PO4, 9.9 mM glucose, 2.5 mM CaCl2, and 1 mM MgCl2 and were gassed with a mixture of 95% O2 and 5% CO2 (pH 7.4). TTX (0.5 µM) was added to the ACSF to block Na+ spikes and synaptic activity except in experiments investigating synaptic responses initiated by focal stimulation within the cerebellar cortex. Patch pipettes (3 to 6 MΩ) were filled with an internal solution containing 150 mM KCH3SO3, nature neuroscience • volume 4 no 12 • december 2001



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5 mM KCl, 0.1 mM K-EGTA, 5.0 mM Na-HEPES, 3.0 mM Mg-ATP and 0.4 mM Na-GTP (pH 7.4). KCH3SO3 in the patch pipette solution was replaced with an equimolar amount of the calcium chelator BAPTA (1,2bis-(2-aminophenoxy)ethane-N,N,N´,N´-tetraacetic acid; 35 mM) to examine the effects of the intracellular application of BAPTA on GABABR-mediated responses. The series resistance was 8–16 MΩ, which was monitored throughout the recording and was not compensated except in experiments investigating synaptic responses. Membrane currents were recorded in a whole-cell configuration using an EPC-7 amplifier (List Electronic, Darmstadt, Germany) and pCLAMP software (Axon Instruments, Foster City, CA), digitized, and stored on a computer disk for off-line analysis. 1S,3R-ACPD (100 mM) and AMPA (10 mM) were applied by iontophoresis (10–20 µA, 100–200 ms) through microelectrodes placed in the vicinity of PC soma from which recordings were obtained. As the 1S,3RACPD-induced current response tended to increase during the initial 15 min after breaking into the whole-cell configuration (Fig. 6c), the effect of GABABR activation was determined thereafter. Furthermore, the effect of a GABABR agonist, baclofen, on 1S,3R-ACPD-responses was tested only once in each slice, because the effect of the GABABR agonist on the mGluR1mediated responses attenuated with repeated applications. Synaptic responses were evoked by stimulation (30–50 V, 0.1–0.2 ms) via a glass microelectrode filled with ACSF and placed within the molecular layer. PFmediated EPSCs were identified based on paired-pulse facilitation. A PFand mGluR1-mediated slow EPSC was evoked by repetitive stimulation (10 pulses at 100 Hz) in an ACSF that contained 6-cyano-7-nitroquinoxaline2,3-dione (CNQX; 10 µM), D-2-amino-5- phosphonopentanoic acid (AP5; 30 µM) and bicuculline (50 µM). U73122, heparin and BAPTA were added to the patch pipette solution and infused into PCs during whole-cell recordings. Other inhibitors and receptor antagonists were applied by perfusion. Ca2+ imaging. The Ca2+ indicator fura-2 was added to the patch pipette solution at a concentration of 1 mM and loaded into the cells for 25–40 min. Fluorescence Ca2+ ratio imaging was done by excitation of the indicator at 340:380 nm and paired emission images were acquired using a cooled CCD camera (C4880, Hamamatsu Photonics, Hamamatsu, Japan) at 510 nm. Paired emission images were recorded every 2 s and ratio images were calculated using a digital image acquisition system and image processing software (ARGUS 50/CA, Hamamatsu Photonics). In all experiments, visual identification of cells and Ca2+ fluorescence imaging were done using a water immersion objective (LUMPlanFI 60×, NA 0.90, Olympus, Tokyo, Japan) capable of passing 340 nm light efficiently. Drugs. 1S,3R-ACPD and SKF89976A were obtained from Tocris Cookson (Bristol, UK); baclofen, heparin and TTX, from Wako (Osaka, Japan); ω-agatoxin IVA, from Peptide Institute (Osaka, Japan); and fura-2, from Dojindo (Kumamoto, Japan). CGP62349 was a gift from Novartis Pharma (Basel, Switzerland). All other chemicals were from Sigma (St. Louis, Missouri). Statistics. Each value is expressed as mean ± s.e.m. Unless otherwise stated, levels of significance were determined by unpaired Student’s t-test between groups.

ACKNOWLEDGEMENTS We thank J. Bockaert, T. Murakoshi, D. Rusakov, F. Saitow and K. Yoshioka for comments on the manuscript and Novartis Pharma (Basel, Switzerland) for the gift of CGP62349. This work was supported in part by a Grant-in-Aid 0727910 (T.Y.) from the Ministry of Education, Science, Sports and Culture of Japan, and Grant-in-Aids 96L00310 (T.Y.) and 12780603 (M.H.) from the Japan Society for Promotion of Science. S.K. is a research director of CREST, JST (Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation).

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