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Presynaptic modulation by group III metabotropic glutamate receptors (mGluRs) of the excitatory postsynaptic potential mediated by mGluR1 in rat cerebellar Purkinje cells
Neuroscience Letters 310 (2001) 61±65
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Presynaptic modulation by group III metabotropic glutamate receptors (mGluRs) of the excitatory postsynaptic potential mediated by mGluR1 in rat cerebellar Purkinje cells M.C. Miniaci a,b, P. Bonsi a,c, F. Tempia b,1, P. Strata b, A. Pisani a,c,* a Fondazione Santa Lucia- IRCCS- Rome, Italy Rita Levi Montalcini Center For Brain Repair, Dipartimento di Neuroscienze, UniversitaÁ di Torino, Torino, Italy c Clinica Neurologica, Dipartimento di Neuroscienze, UniversitaÁ di Roma ªTor Vergataº, Via di Tor Vergata 135, 00133 Rome, Italy b
Received 24 April 2001; received in revised form 4 July 2001; accepted 9 July 2001
Abstract Purkinje neurons were recorded from rat cerebellar slices. Parallel ®bres stimulation elicited a fast excitatory postsynaptic potential (EPSP) mediated by ionotropic glutamate (iGluR) a -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors followed by the inhibitory gamma-aminobutyric acidA (GABAA)-dependent postsynaptic potential. In the presence of antagonists for iGluRs and for GABAA receptors, brief tetanic activation evoked a slow metabotropic glutamate receptor (mGluR)-dependent EPSP (mGluR-EPSP). This mGluR-EPSP was blocked by the selective mGluR1 antagonists LY367385 and CPCCOEt, but not by the mGluR5 antagonist MPEP. Group II agonists affected neither iGluR-EPSP nor mGluR-EPSP. Conversely, L-AP4 and L-SOP, group III mGluR agonists, inhibited both iGluR- and mGluREPSPs. The depolarisations evoked by both AMPA and group I agonists were unaffected, indicating a presynaptic action of group III mGluRs. These data suggest that glutamate released by parallel ®bres activates group III mGluR autoreceptors, depressing both iGluR- and mGluR1-mediated EPSPs. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Electrophysiology; Cerebellum; Purkinje cell; Metabotropic glutamate receptors; Excitatory postsynaptic potential; LY367385; MPEP
Glutamatergic transmission involves both ionotropic (iGluRs) and metabotropic glutamate receptors (mGluRs) [5,13]. Purkinje cells of the cerebellar cortex have two distinct dendritic domains; the distal one is rich in spines and is innervated by a high number of parallel ®bres, while the proximal one has few spines innervated by a single climbing ®bre, the terminal arbor of the olivocerebellar neurons [19]. By immunocytochemistry, spines of both compartments display iGluRs and mGluRs [14], though no mGluR response to stimulation of the climbing ®bre has been found by electrophysiological recording [1,20]. Stimulation of the parallel ®bres evokes a fast excitatory postsynaptic potential (EPSP) mediated by a -amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subtype [10]. More recently, a slow mGluR-dependent EPSP has been described at these synapses [2,20]. To * Corresponding author. Tel.: 139-06-72596010; fax: 139-0672596006. E-mail address: [email protected] (A. Pisani). 1 Present address: Sezione di Fisiologia, Dipartimento di Medicina Interna, UniversitaÁ di Perugia, Perugia, Italy.
date, three different groups and eight distinct mGlu receptor subtypes have been cloned and grouped depending on their properties: group I (mGluR1 and 5); group II (mGluR2 and 3);and group III (mGluR4, 6, 7, 8) [5]. Experimental evidence indicates the involvement of group I mGluRs in the generation of mGluR-EPSP, most likely mGluR1 [3,20], since mGluR5 is not expressed in Purkinje cells [18]. The application of the group III agonist l-amino-4-phosphonobutanoic acid (L-AP4) causes a synaptic inhibition in a variety of brain regions, including the hippocampus, striatum, olfactory tract, spinal cord, thalamus [5] and also at the parallel ®bre-Purkinje cell synapse [16]. Here we extend the characterisation of the mGluR-EPSP by utilising new pharmacological tools selective for individual group I mGluR subtypes. In addition, we evaluated the modulatory role of group II and III mGluRs on both iGluR- and mGluR-EPSP. Male Wistar rats, 3±4 weeks of age were utilised for the experiments. The preparation and maintenance of cerebellar slices have been described in detail previously [20]. The animals were anaesthetised and decapitated. The cerebellar vermis was removed and placed in an ice-cold saline solu-
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 08 2- 1
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M.C. Miniaci et al. / Neuroscience Letters 310 (2001) 61±65
tion (08C, see text below). Sagittal slices, 200±300 mm thick were cut with a vibratome and kept for 30±60 min at room temperature. A single slice was transferred to a recording chamber mounted on the stage of an upright microscope (Axioskope FS, Zeiss) and fully submerged in a continuously ¯owing Krebs' solution (room temperature, 2.5±3 ml/ min) gassed with 95% O2 and 5% CO2. The composition of the solution was (in mM): 126 NaCl, 2.5 KCl, 1.3 MgCl2, 1.2 NaH2PO4, 2.4 CaCl2, 10 glucose, and 18 NaHCO3. For electrophysiological recordings sharp microelectrodes (35± 50 mOhm) were ®lled with a KCl (2 M) solution. Purkinje neurons were visually identi®ed and impaled. For synaptic stimulation, bipolar electrodes were placed on the molecular layer. An Axoclamp 2A ampli®er (Axon Instruments) was used for electrophysiological experiments. Traces of EPSPs were displayed on an oscilloscope, averaged and stored. Values reported in the text and in the ®gures are mean ^ SEM of changes in the respective cell population. Student's t-test (for paired and unpaired observations) was used to compare the means. Drugs were bath-applied by switching the solution to one containing known drug concentrations. Drugs were from Tocris Cookson (Bristol, U.K.) As previously described [10], in the presence of bicuculline (30 mM) in the perfusing solution, to block gammaaminobutyric acidA (GABAA) receptors, parallel ®bre
Fig. 1. (A,B) The traces show paired-pulse responses evoked by parallel ®ber stimulation in control condition (a), in the presence of L-AP4 and L-SOP (both 30 mM, b) and after washout of the drugs (c). (C) Bath-application of AMPA (a, 1 mM, 2 s) was not affected by L-AP4 (b, 30 mM). Note that the ®lled square does not conform to the time calibration bar, but represents the 2 s application of AMPA. (D) The graph shows the ratio of the second pulse response (black bar) to the ®rst pulse response (white bar) (EPSP2/EPSP1) in control condition and in the presence of L-AP4 and L-SOP (both 30 mM).
stimulation with a single shock elicited an EPSP (iGluREPSP) that was fully blocked by the AMPA receptor antagonist NBQX (10 mM). Bath-application of the selective group II mGluR agonist DCG-IV (1±3 mM, 5 min) did not affect the amplitude of the iGluR-EPSP (P . 0:05; n 6; not shown). Conversely, as previously shown [16], L-AP4, a group III mGluR agonist, caused a marked decrease in the amplitude of the recorded iGluR-EPSP (10±30 mM P , 0:005; n 9; not shown). We have con®rmed this ®nding by using another group III mGluR agonist, L-SOP (10± 30 mM, P , 0:001, n 11; not shown). Both drugs did not cause changes in the intrinsic membrane properties of Purkinje cells, implying a presynaptic effect of group III mGluRs. To support this view, we analysed synaptic responses to a pair of stimuli before (Fig. 1Aa,Ba) and during application of both L-AP4 and L-SOP (Fig. 1Ab,Bb). Changes in paired-pulse facilitation have been considered as an index of alterations in presynaptic transmitter release [11]; an increase in the ratio EPSP2/EPSP1 indicates a presynaptic site of action of the tested drug. In the presence of either 30 mM L-AP4 (Fig. 1Ab,D) or 30 mM L-SOP (Fig. 1Bb,D) (n 16, P , 0:005) a reversible increase in the ratio EPSP2/EPSP1 was observed (Fig. 1Ac,Bc). Moreover, the depolarising response to brief bath-applications of AMPA (Fig. 1Ca; 1 mM, 2 s) was unaltered in the presence of 30 mM L-AP4 (Fig. 1Cb, n 3) and L-SOP (30 mM, n 3, not shown), suggesting a presynaptic site of action of group III mGluR agonists. In the presence of NBQX (10 mM), bicuculline (30 mM) and d-APV (50 mM), a brief tetanic stimulation of the parallel ®bres (4±8 stimuli, 100 Hz, 100 ms duration) induced the appearance of a slow excitatory component which has been shown to depend upon the activation of group I mGluRs (Fig. 2) [3,20]. We
Fig. 2. (A) Tetanic activation of parallel ®bres in the presence of NBQX, D-APV and bicuculline methiodide (BMI) in the bathing solution evokes a slow mGluR-EPSP. LY367385 (50 mM) fully blocks the synaptic potential, but does not affect the AMPA component (white arrow). After 10±15 min washout, the mGluR-EPSP is restored. (B) Similarly, in the presence of CPCCOEt (100 mM) the control EPSP is signi®cantly reduced, and returns to control values upon 10±15 min washout. The ionotropic component is unaffected (white arrow). (C) Conversely, bath-applied MPEP, an mGluR5 antagonist, does not alter signi®cantly the control mGluR-EPSP.
M.C. Miniaci et al. / Neuroscience Letters 310 (2001) 61±65
took advantage of the currently available selective pharmacological tools to further de®ne the mGluR subtype involved. Bath-application of the competitive mGluR1 antagonist LY367385 (50 mM, 10±15 min) was able to fully block the mGluR-EPSP, whereas the residual AMPA-component was unaffected (Fig 2A; n 6, P , 0:005). The blockade by LY367385 was entirely reversed upon washout (Fig. 2A). Likewise, the non-competitive mGluR1 antagonist CPCCOEt (100 mM, 10±15 min) blocked the mGluR-EPSP in a reversible manner (Fig. 2B; n 5, P , 0:001). Conversely, MPEP (30 mM, 10±15 min), a non-competitive mGluR5 antagonist failed to affect this response (Fig. 2C; n 4, P . 0:05). In the presence of NBQX, APV and bicuculline, bath-application of DCG-IV (1±3 mM, 5 min) caused no signi®cant changes in the amplitude of the mGluR-EPSP (n 6, P . 0:05, not shown). Conversely, both group III agonists, L-AP4 (1±30 mM, n 8, P , 0:001) and L-SOP (1±30 mM, n 7, P , 0:001) were able to cause a dose-dependent reduction of the mGluR-EPSP amplitude (Fig. 3A,B). The doseresponse curve revealed an IC50 of 9.5 mM and 5.5 mM for L-AP4 and L-SOP, respectively (Fig. 3D). No effect on the intrinsic membrane properties of the recorded cells was observed. To support the idea of a presynaptic site of action of group III agonists on the mGluR-EPSP, we applied
Fig. 3. (A) The control mGluR-EPSP is elicited by a brief tetanic activation of parallel ®bres. Bath-applied L-AP4 (30 mM) largely reduces the mGluR-EPSP, returning to control values after washout. (B) A similar effect is observed in the presence of L-SOP (30 mM) compared to the control mGluR-EPSP. (C) Exogenous application of 3,5-DHPG (a, 30 mM, 25 s, in bicuculline and TTX) induces a membrane depolarisation of the recorded neuron, that is unaffected by 50 mM L-SOP (b). (D) Dose-response curve for the inhibitory effect of L-AP4 and L-SOP on the amplitude of the mGluR-EPSP. Each data point was obtained by averaging 4±8 single EPSPs. The IC50 values are indicated in the inset.
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exogenous (S)-3,5-dyhydroxyphenylglycine (3,5-DHPG), group I mGluR agonist. As expected, in a tetrodotoxin (TTX)-containing solution (0.5 mM), the membrane depolarisation induced by bath-applied 3,5-DHPG (Fig. 3Ca, 30 mM, 25 s) was unaffected in the presence of L-SOP (Fig. 3Cb) or L-AP4 (not shown). The slow time-course of the mGluR-EPSP did not allow to perform reliable paired-pulse facilitation experiments. In the present work we provide a further pharmacological demonstration that the mGluR-EPSP evoked by tetanic activation of parallel ®bres involves mGluR1. In addition, experimental evidence favours a modulatory action of presynaptic group III mGluRs on both the iGluR- and mGluR-EPSP, with no detectable effect following group II activation. Purkinje cells express high levels of the mGluR1 receptor subtype, with a subcellular distribution at postsynaptic somato-dendritic regions [4,14], while mGluR5, the other group I mGluR, is not expressed by these neurons [18]. It was suggested that mGluR1 is the receptor involved in the generation of the mGluR-EPSP in Purkinje cells [3,20]. Therefore, the inhibitory effect of both LY367385 and CPCCOEt shown in the present report is largely con®rmatory. However, it should be reminded that the work on the pharmacology of mGluRs has long been hampered by the lack of selective compounds able to discriminate unequivocally among receptor subtypes. Currently, LY367385, the 2-methyl analogue of (S)-4-carboxyphenylglycine (4CPG), an antagonist of mGluR1, is considered the competitive antagonist of choice for the study of this receptor subtype [17]. Likewise, CPCCOEt is considered a potent and selective non-competitive antagonist, acting at a site distinct from the agonist binding domain of mGluR1 [17]. Furthermore, the predicted lack of effect of MPEP and the negligible sensitivity to these antagonists for mGluR7, the other mGluR expressed by Purkinje cells [9], lead to the conclusion that the mGluR-EPSP is mediated by mGluR1. Compelling evidence supports an inhibitory effect of mGluRs at glutamatergic synapses, serving as presynaptic autoreceptors and thereby limiting glutamate release [5]. Morphological studies demonstrate that, at the parallel ®bre-Purkinje cell synapse, mGluR4 receptors are clustered along the presynaptic membrane [8,12]. In fact, the application of a group III mGluR agonist (L-AP4) produces a depression of AMPA-mediated transmission at parallel ®bre-Purkinje cell synapse, which has been shown to be mediated by the mGluR4 subtype, since this effect was lacking in mGluR4 knockout mice [16]. In agreement with these results, our data show that both group III agonists L-SOP and L-AP4 were able to increase the ratio EPSP2/EPSP1 in the paired pulse facilitation experiments, indicating a presynaptic site of action. The presynaptic localization of the action of group III agonists was con®rmed also by the lack of effect of L-AP4 on the membrane depolarisation induced by exogenously applied AMPA. These data, taken together, suggest that the release of glutamate from parallel ®bres is negatively modulated by mGluR4 autoreceptors.
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M.C. Miniaci et al. / Neuroscience Letters 310 (2001) 61±65
Our results extend this observation to the mGluR-EPSP, since the mGluR1-mediated EPSP was suppressed by group III agonists (L-AP4 and L-SOP), while the application of L-SOP did not affect the 3,5-DHPG-evoked postsynaptic membrane depolarisation. These results indicate that also glutamate released by parallel ®bres in response to the tetanic stimulation used to evoke the mGluR-EPSP is under an inhibitory control through mGluR4 autoreceptors. The involvement of the other mGluR expressed by Purkinje cells, mGluR7 appears unlikely since: (i) the potency of both these agonists and of endogenous glutamate at mGluR7 is much lower than at any other member of group III [5]; (ii) the postsynaptic location of mGluR7 immunoreactivity in Purkinje neurons [9]; (iii) the distribution of mGluR7 in the cerebellum is developmentally regulated, gradually decreasing with age [9]. The lack of effect of the group II agonist DCG-IV is also in agreement with the distribution of these receptors in the cerebellar cortex [15]. Although the functional role of the mGluR1-mediated EPSP is at present unclear, it is likely that this signal occurs in physiological conditions. In fact, repetitive high frequency activity is the physiological mode of mossy ®bre discharge in vivo. In response to mossy ®bre input at 100 Hz, cerebellar granule cells are able to discharge at more than 80 Hz [6], which is in the range that has been shown to evoke the mGluR1-EPSP [2,20]. A caveat in the interpretation of the physiological involvement of the mGluR1-EPSP are the experimental conditions utilized to elicit this signal, which included at least the pharmacological block of inhibitory synapses by application of a GABAA receptor antagonist [1,2,3,20]. However, the duration of the mGluR1-EPSP largely outlasts the feedforward inhibition of the Purkinje cell due to the activation of basket and stellate inhibitory interneurons by the same parallel ®bre burst which evokes the mGluR1-EPSP. An indication of the physiological relevance of mGluR1 for Purkinje cells are the motor performance and motor memory de®cits caused by the speci®c absence of this receptor in this cell type [7]. The physiological relevance of the inhibition exerted by parallel ®bre mGluR4 autoreceptors has already been shown by the appearance of a motor impairment in mGluR4 knock-out mice [16]. Our results show that parallel ®bre group III mGluRs, most likely mGluR4, depress the ®rst iGluREPSP but enhance the facilitation of following EPSP. In physiological conditions, the source of glutamate acting on presynaptic mGluR4 receptors is probably the amino acid released by parallel ®bre terminals. Therefore, a repetitive terminal activation, as by the parallel ®bre tetanus used to evoke the mGluR1-EPSP, will originate feedback auto-inhibition in the ®rst few pulses, but at the same time also a larger facilitation of following pulses. The detection of the mGluR1-EPSP can be considered as a test to assess the net effect of the activation of parallel ®bre mGluR4 on glutamate release. In this context, the depression produced by L-SOP on the mGluR1-EPSP indicates that, in spite of the enhanced facilitation, the amount of glutamate released
by a parallel ®bre tetanus is under a net inhibitory control through presynaptic mGluR4. We thank Drs G. Bernardi and P. Calabresi for critical reading of the manuscript and Mr M. Tolu for his technical assistance. This work was partially supported by a Ministero SanitaÁ (Progetto Finalizzato) grant to A.P. and by MURST and CNR grants to G.B and P.S. [1] Batchelor, A.M., Madge, D.J. and Garthwaite, J., Synaptic activation of metabotropic glutamate receptors in the parallel ®bre-Purkinje cell pathway in rat cerebellar slices, Neuroscience, 63 (1994) 911±915. [2] Batchelor, A.M. and Garthwaite, J., Frequency detection and temporally-dispersed synaptic signal association through a metabotropic glutamate receptor pathway, Nature, 385 (1997) 74±77. [3] Batchelor, A.M., Knoepfel, T., Gasparini, F. and Garthwaite, J., Pharmacological characterization of synaptic transmission through mGluRs in rat cerebellar slices, Neuropharmacology, 36 (1997) 401±403. [4] Baude, A., Nusser, Z., Roberts, J.D.B., Mulvihill, E., McIlhinnery, R.A.J. and Somogyi, P., The metabotropic glutamate receptor (mGluR1a) is concentrated at perisynaptic membrane of neuronal subpopulations as detected by immunogold reaction, Neuron, 11 (1993) 771±787. [5] Conn, P.J. and Pin, J.-P., Pharmacology and functions of metabotropic glutamate receptors, Annu. Rev. Pharmacol. Toxicol., 37 (1997) 205±237. [6] D'Angelo, E., De Filippi, G., Rossi, P. and Taglietti, V., Synaptic excitation of individual rat cerebellar granule cells in situ: evidence for the role of NMDA receptors, J. Physiol., 484 (1995) 397±413. [7] Ichise, T., Kano, M., Hashimoto, K., Yanagihara, D., Nakao, K., Shigemoto, R., Katsuki, M. and Aiba, A., mGluR1 in cerebellar Purkinje cells essential for long-term depression, synapse elimination, and motor coordination, Science, 288 (2000) 1832±1835. [8] Kinoshita, A., Ohishi, H., Nomura, S., Shigemoto, R., Nakanishi, S. and Mizuno, N., Presynaptic localization of mGluR4a, in the cerebellar cortex: a light and electron microscopy study in the rat, Neurosci. Lett., 207 (1996) 199±202. [9] Kinzie, J.M., Saugstad, J.A., Westbrook, G.L. and Segerson, T.P., Distribution of metabotropic glutamate receptor 7 messenger mRNA in the developing and adult brain, Neuroscience, 69 (1995) 167±176. [10] Konnerth, A., Llano, I. and Armstrong, C.M., Synaptic currents in cerebellar Purkinje cells, Proc. Natl. Acad. Sci. USA, 87 (1990) 2662±2665. [11] Manabe, T., Wyllie, D.J.I. and Nicoll, R.A., Modulation of synaptic transmission and long-term potentiation: effects on paired pulse facilitation and EPSC variance in the CA1 region of the hippocampus, J. Neurophysiol., 70 (1993) 1451±1459. [12] Mateos, J.M., Elezgarai, I., Benitez, R., Osorio, A., Bilbao, A., Azkue, J.J., Kuhn, R., Knopfel, T. and Grandes, P., Clustering of the group III metabotropic glutamate receptor 4a at parallel ®ber synaptic terminals in the rat cerebellar molecular layer, Neurosci. Res., 35 (1999) 71±74. [13] Nakanishi, S., Nakajima, Y., Masu, M., Ueda, Y., Nakahara, K., Watanabe, D., Yamaguchi, S., Kawabata, S. and Okada, M., Glutamate receptors: brain function and signal transduction, Brain Res. Rev., 26 (1998) 230±235. [14] Nusser, Z., Mulvihill, E., Streit, P. and Somogyi, P., Subsy-
M.C. Miniaci et al. / Neuroscience Letters 310 (2001) 61±65 naptic segregation of metabotropic and ionotropic glutamate receptors as revealed by immunogold localization, Neuroscience, 61 (1994) 421±427. [15] Ohishi, H., Ogawa-Meguro, R., Shigemoto, R., Kaneko, T., Nakanishi, S. and Mizuno, N., Immunohistochemical localization of metabotropic glutamate receptors, mGluR2 and mGluR3 in rat cerebellar cortex, Neuron, 13 (1994) 55±66. [16] Pekhletski, R., Gerlai, R., Overstreet, L.S., Huang, X.P., Agopyan, N., Traverse Slater, N., Abramow-Newerly, W., Roder, J.C. and Hampson, D.R., Impaired cerebellar synaptic plasticity and motor performance in mice lacking the mGluR4 subtype of metabotropic glutamate receptors, J. Neurosci., 16 (1996) 6364±6373.
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[17] Schoepp, D.D., Jane, D.E. and Monn, J.A., Pharmacological agents acting at subtypes of metabotropic glutamate receptors, Neuropharmacology, 38 (1999) 1431±1476. [18] Shigemoto, R., Nomura, S., Ohishi, H., Sugihara, H., Nakanishi, S. and Mizuno, N., Immunohistochemical localization of a metabotropic glutamate receptor, mGluR5, in the rat brain, Neurosci. Lett., 163 (1993) 53±57. [19] Strata, P. and Rossi, F., Plasticity of the olivocerebellar pathway, Trends Neurosci., 21 (1998) 407±413. [20] Tempia, F., Miniaci, M.C., Anchisi, D. and Strata, P.G., Postsynaptic current mediated by metabotropic glutamate receptors in cerebellar Purkinje cells, J. Neurophysiol., 80 (1998) 520±528.
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Presynaptic modulation by group III metabotropic glutamate receptors (mGluRs) of the excitatory postsynaptic potential mediated by mGluR1 in rat cerebellar Purkinje cells M.C. Miniaci a,b, P. Bonsi a,c, F. Tempia b,1, P. Strata b, A. Pisani a,c,* a Fondazione Santa Lucia- IRCCS- Rome, Italy Rita Levi Montalcini Center For Brain Repair, Dipartimento di Neuroscienze, UniversitaÁ di Torino, Torino, Italy c Clinica Neurologica, Dipartimento di Neuroscienze, UniversitaÁ di Roma ªTor Vergataº, Via di Tor Vergata 135, 00133 Rome, Italy b
Received 24 April 2001; received in revised form 4 July 2001; accepted 9 July 2001
Abstract Purkinje neurons were recorded from rat cerebellar slices. Parallel ®bres stimulation elicited a fast excitatory postsynaptic potential (EPSP) mediated by ionotropic glutamate (iGluR) a -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors followed by the inhibitory gamma-aminobutyric acidA (GABAA)-dependent postsynaptic potential. In the presence of antagonists for iGluRs and for GABAA receptors, brief tetanic activation evoked a slow metabotropic glutamate receptor (mGluR)-dependent EPSP (mGluR-EPSP). This mGluR-EPSP was blocked by the selective mGluR1 antagonists LY367385 and CPCCOEt, but not by the mGluR5 antagonist MPEP. Group II agonists affected neither iGluR-EPSP nor mGluR-EPSP. Conversely, L-AP4 and L-SOP, group III mGluR agonists, inhibited both iGluR- and mGluREPSPs. The depolarisations evoked by both AMPA and group I agonists were unaffected, indicating a presynaptic action of group III mGluRs. These data suggest that glutamate released by parallel ®bres activates group III mGluR autoreceptors, depressing both iGluR- and mGluR1-mediated EPSPs. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Electrophysiology; Cerebellum; Purkinje cell; Metabotropic glutamate receptors; Excitatory postsynaptic potential; LY367385; MPEP
Glutamatergic transmission involves both ionotropic (iGluRs) and metabotropic glutamate receptors (mGluRs) [5,13]. Purkinje cells of the cerebellar cortex have two distinct dendritic domains; the distal one is rich in spines and is innervated by a high number of parallel ®bres, while the proximal one has few spines innervated by a single climbing ®bre, the terminal arbor of the olivocerebellar neurons [19]. By immunocytochemistry, spines of both compartments display iGluRs and mGluRs [14], though no mGluR response to stimulation of the climbing ®bre has been found by electrophysiological recording [1,20]. Stimulation of the parallel ®bres evokes a fast excitatory postsynaptic potential (EPSP) mediated by a -amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subtype [10]. More recently, a slow mGluR-dependent EPSP has been described at these synapses [2,20]. To * Corresponding author. Tel.: 139-06-72596010; fax: 139-0672596006. E-mail address: [email protected] (A. Pisani). 1 Present address: Sezione di Fisiologia, Dipartimento di Medicina Interna, UniversitaÁ di Perugia, Perugia, Italy.
date, three different groups and eight distinct mGlu receptor subtypes have been cloned and grouped depending on their properties: group I (mGluR1 and 5); group II (mGluR2 and 3);and group III (mGluR4, 6, 7, 8) [5]. Experimental evidence indicates the involvement of group I mGluRs in the generation of mGluR-EPSP, most likely mGluR1 [3,20], since mGluR5 is not expressed in Purkinje cells [18]. The application of the group III agonist l-amino-4-phosphonobutanoic acid (L-AP4) causes a synaptic inhibition in a variety of brain regions, including the hippocampus, striatum, olfactory tract, spinal cord, thalamus [5] and also at the parallel ®bre-Purkinje cell synapse [16]. Here we extend the characterisation of the mGluR-EPSP by utilising new pharmacological tools selective for individual group I mGluR subtypes. In addition, we evaluated the modulatory role of group II and III mGluRs on both iGluR- and mGluR-EPSP. Male Wistar rats, 3±4 weeks of age were utilised for the experiments. The preparation and maintenance of cerebellar slices have been described in detail previously [20]. The animals were anaesthetised and decapitated. The cerebellar vermis was removed and placed in an ice-cold saline solu-
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 08 2- 1
62
M.C. Miniaci et al. / Neuroscience Letters 310 (2001) 61±65
tion (08C, see text below). Sagittal slices, 200±300 mm thick were cut with a vibratome and kept for 30±60 min at room temperature. A single slice was transferred to a recording chamber mounted on the stage of an upright microscope (Axioskope FS, Zeiss) and fully submerged in a continuously ¯owing Krebs' solution (room temperature, 2.5±3 ml/ min) gassed with 95% O2 and 5% CO2. The composition of the solution was (in mM): 126 NaCl, 2.5 KCl, 1.3 MgCl2, 1.2 NaH2PO4, 2.4 CaCl2, 10 glucose, and 18 NaHCO3. For electrophysiological recordings sharp microelectrodes (35± 50 mOhm) were ®lled with a KCl (2 M) solution. Purkinje neurons were visually identi®ed and impaled. For synaptic stimulation, bipolar electrodes were placed on the molecular layer. An Axoclamp 2A ampli®er (Axon Instruments) was used for electrophysiological experiments. Traces of EPSPs were displayed on an oscilloscope, averaged and stored. Values reported in the text and in the ®gures are mean ^ SEM of changes in the respective cell population. Student's t-test (for paired and unpaired observations) was used to compare the means. Drugs were bath-applied by switching the solution to one containing known drug concentrations. Drugs were from Tocris Cookson (Bristol, U.K.) As previously described [10], in the presence of bicuculline (30 mM) in the perfusing solution, to block gammaaminobutyric acidA (GABAA) receptors, parallel ®bre
Fig. 1. (A,B) The traces show paired-pulse responses evoked by parallel ®ber stimulation in control condition (a), in the presence of L-AP4 and L-SOP (both 30 mM, b) and after washout of the drugs (c). (C) Bath-application of AMPA (a, 1 mM, 2 s) was not affected by L-AP4 (b, 30 mM). Note that the ®lled square does not conform to the time calibration bar, but represents the 2 s application of AMPA. (D) The graph shows the ratio of the second pulse response (black bar) to the ®rst pulse response (white bar) (EPSP2/EPSP1) in control condition and in the presence of L-AP4 and L-SOP (both 30 mM).
stimulation with a single shock elicited an EPSP (iGluREPSP) that was fully blocked by the AMPA receptor antagonist NBQX (10 mM). Bath-application of the selective group II mGluR agonist DCG-IV (1±3 mM, 5 min) did not affect the amplitude of the iGluR-EPSP (P . 0:05; n 6; not shown). Conversely, as previously shown [16], L-AP4, a group III mGluR agonist, caused a marked decrease in the amplitude of the recorded iGluR-EPSP (10±30 mM P , 0:005; n 9; not shown). We have con®rmed this ®nding by using another group III mGluR agonist, L-SOP (10± 30 mM, P , 0:001, n 11; not shown). Both drugs did not cause changes in the intrinsic membrane properties of Purkinje cells, implying a presynaptic effect of group III mGluRs. To support this view, we analysed synaptic responses to a pair of stimuli before (Fig. 1Aa,Ba) and during application of both L-AP4 and L-SOP (Fig. 1Ab,Bb). Changes in paired-pulse facilitation have been considered as an index of alterations in presynaptic transmitter release [11]; an increase in the ratio EPSP2/EPSP1 indicates a presynaptic site of action of the tested drug. In the presence of either 30 mM L-AP4 (Fig. 1Ab,D) or 30 mM L-SOP (Fig. 1Bb,D) (n 16, P , 0:005) a reversible increase in the ratio EPSP2/EPSP1 was observed (Fig. 1Ac,Bc). Moreover, the depolarising response to brief bath-applications of AMPA (Fig. 1Ca; 1 mM, 2 s) was unaltered in the presence of 30 mM L-AP4 (Fig. 1Cb, n 3) and L-SOP (30 mM, n 3, not shown), suggesting a presynaptic site of action of group III mGluR agonists. In the presence of NBQX (10 mM), bicuculline (30 mM) and d-APV (50 mM), a brief tetanic stimulation of the parallel ®bres (4±8 stimuli, 100 Hz, 100 ms duration) induced the appearance of a slow excitatory component which has been shown to depend upon the activation of group I mGluRs (Fig. 2) [3,20]. We
Fig. 2. (A) Tetanic activation of parallel ®bres in the presence of NBQX, D-APV and bicuculline methiodide (BMI) in the bathing solution evokes a slow mGluR-EPSP. LY367385 (50 mM) fully blocks the synaptic potential, but does not affect the AMPA component (white arrow). After 10±15 min washout, the mGluR-EPSP is restored. (B) Similarly, in the presence of CPCCOEt (100 mM) the control EPSP is signi®cantly reduced, and returns to control values upon 10±15 min washout. The ionotropic component is unaffected (white arrow). (C) Conversely, bath-applied MPEP, an mGluR5 antagonist, does not alter signi®cantly the control mGluR-EPSP.
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took advantage of the currently available selective pharmacological tools to further de®ne the mGluR subtype involved. Bath-application of the competitive mGluR1 antagonist LY367385 (50 mM, 10±15 min) was able to fully block the mGluR-EPSP, whereas the residual AMPA-component was unaffected (Fig 2A; n 6, P , 0:005). The blockade by LY367385 was entirely reversed upon washout (Fig. 2A). Likewise, the non-competitive mGluR1 antagonist CPCCOEt (100 mM, 10±15 min) blocked the mGluR-EPSP in a reversible manner (Fig. 2B; n 5, P , 0:001). Conversely, MPEP (30 mM, 10±15 min), a non-competitive mGluR5 antagonist failed to affect this response (Fig. 2C; n 4, P . 0:05). In the presence of NBQX, APV and bicuculline, bath-application of DCG-IV (1±3 mM, 5 min) caused no signi®cant changes in the amplitude of the mGluR-EPSP (n 6, P . 0:05, not shown). Conversely, both group III agonists, L-AP4 (1±30 mM, n 8, P , 0:001) and L-SOP (1±30 mM, n 7, P , 0:001) were able to cause a dose-dependent reduction of the mGluR-EPSP amplitude (Fig. 3A,B). The doseresponse curve revealed an IC50 of 9.5 mM and 5.5 mM for L-AP4 and L-SOP, respectively (Fig. 3D). No effect on the intrinsic membrane properties of the recorded cells was observed. To support the idea of a presynaptic site of action of group III agonists on the mGluR-EPSP, we applied
Fig. 3. (A) The control mGluR-EPSP is elicited by a brief tetanic activation of parallel ®bres. Bath-applied L-AP4 (30 mM) largely reduces the mGluR-EPSP, returning to control values after washout. (B) A similar effect is observed in the presence of L-SOP (30 mM) compared to the control mGluR-EPSP. (C) Exogenous application of 3,5-DHPG (a, 30 mM, 25 s, in bicuculline and TTX) induces a membrane depolarisation of the recorded neuron, that is unaffected by 50 mM L-SOP (b). (D) Dose-response curve for the inhibitory effect of L-AP4 and L-SOP on the amplitude of the mGluR-EPSP. Each data point was obtained by averaging 4±8 single EPSPs. The IC50 values are indicated in the inset.
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exogenous (S)-3,5-dyhydroxyphenylglycine (3,5-DHPG), group I mGluR agonist. As expected, in a tetrodotoxin (TTX)-containing solution (0.5 mM), the membrane depolarisation induced by bath-applied 3,5-DHPG (Fig. 3Ca, 30 mM, 25 s) was unaffected in the presence of L-SOP (Fig. 3Cb) or L-AP4 (not shown). The slow time-course of the mGluR-EPSP did not allow to perform reliable paired-pulse facilitation experiments. In the present work we provide a further pharmacological demonstration that the mGluR-EPSP evoked by tetanic activation of parallel ®bres involves mGluR1. In addition, experimental evidence favours a modulatory action of presynaptic group III mGluRs on both the iGluR- and mGluR-EPSP, with no detectable effect following group II activation. Purkinje cells express high levels of the mGluR1 receptor subtype, with a subcellular distribution at postsynaptic somato-dendritic regions [4,14], while mGluR5, the other group I mGluR, is not expressed by these neurons [18]. It was suggested that mGluR1 is the receptor involved in the generation of the mGluR-EPSP in Purkinje cells [3,20]. Therefore, the inhibitory effect of both LY367385 and CPCCOEt shown in the present report is largely con®rmatory. However, it should be reminded that the work on the pharmacology of mGluRs has long been hampered by the lack of selective compounds able to discriminate unequivocally among receptor subtypes. Currently, LY367385, the 2-methyl analogue of (S)-4-carboxyphenylglycine (4CPG), an antagonist of mGluR1, is considered the competitive antagonist of choice for the study of this receptor subtype [17]. Likewise, CPCCOEt is considered a potent and selective non-competitive antagonist, acting at a site distinct from the agonist binding domain of mGluR1 [17]. Furthermore, the predicted lack of effect of MPEP and the negligible sensitivity to these antagonists for mGluR7, the other mGluR expressed by Purkinje cells [9], lead to the conclusion that the mGluR-EPSP is mediated by mGluR1. Compelling evidence supports an inhibitory effect of mGluRs at glutamatergic synapses, serving as presynaptic autoreceptors and thereby limiting glutamate release [5]. Morphological studies demonstrate that, at the parallel ®bre-Purkinje cell synapse, mGluR4 receptors are clustered along the presynaptic membrane [8,12]. In fact, the application of a group III mGluR agonist (L-AP4) produces a depression of AMPA-mediated transmission at parallel ®bre-Purkinje cell synapse, which has been shown to be mediated by the mGluR4 subtype, since this effect was lacking in mGluR4 knockout mice [16]. In agreement with these results, our data show that both group III agonists L-SOP and L-AP4 were able to increase the ratio EPSP2/EPSP1 in the paired pulse facilitation experiments, indicating a presynaptic site of action. The presynaptic localization of the action of group III agonists was con®rmed also by the lack of effect of L-AP4 on the membrane depolarisation induced by exogenously applied AMPA. These data, taken together, suggest that the release of glutamate from parallel ®bres is negatively modulated by mGluR4 autoreceptors.
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Our results extend this observation to the mGluR-EPSP, since the mGluR1-mediated EPSP was suppressed by group III agonists (L-AP4 and L-SOP), while the application of L-SOP did not affect the 3,5-DHPG-evoked postsynaptic membrane depolarisation. These results indicate that also glutamate released by parallel ®bres in response to the tetanic stimulation used to evoke the mGluR-EPSP is under an inhibitory control through mGluR4 autoreceptors. The involvement of the other mGluR expressed by Purkinje cells, mGluR7 appears unlikely since: (i) the potency of both these agonists and of endogenous glutamate at mGluR7 is much lower than at any other member of group III [5]; (ii) the postsynaptic location of mGluR7 immunoreactivity in Purkinje neurons [9]; (iii) the distribution of mGluR7 in the cerebellum is developmentally regulated, gradually decreasing with age [9]. The lack of effect of the group II agonist DCG-IV is also in agreement with the distribution of these receptors in the cerebellar cortex [15]. Although the functional role of the mGluR1-mediated EPSP is at present unclear, it is likely that this signal occurs in physiological conditions. In fact, repetitive high frequency activity is the physiological mode of mossy ®bre discharge in vivo. In response to mossy ®bre input at 100 Hz, cerebellar granule cells are able to discharge at more than 80 Hz [6], which is in the range that has been shown to evoke the mGluR1-EPSP [2,20]. A caveat in the interpretation of the physiological involvement of the mGluR1-EPSP are the experimental conditions utilized to elicit this signal, which included at least the pharmacological block of inhibitory synapses by application of a GABAA receptor antagonist [1,2,3,20]. However, the duration of the mGluR1-EPSP largely outlasts the feedforward inhibition of the Purkinje cell due to the activation of basket and stellate inhibitory interneurons by the same parallel ®bre burst which evokes the mGluR1-EPSP. An indication of the physiological relevance of mGluR1 for Purkinje cells are the motor performance and motor memory de®cits caused by the speci®c absence of this receptor in this cell type [7]. The physiological relevance of the inhibition exerted by parallel ®bre mGluR4 autoreceptors has already been shown by the appearance of a motor impairment in mGluR4 knock-out mice [16]. Our results show that parallel ®bre group III mGluRs, most likely mGluR4, depress the ®rst iGluREPSP but enhance the facilitation of following EPSP. In physiological conditions, the source of glutamate acting on presynaptic mGluR4 receptors is probably the amino acid released by parallel ®bre terminals. Therefore, a repetitive terminal activation, as by the parallel ®bre tetanus used to evoke the mGluR1-EPSP, will originate feedback auto-inhibition in the ®rst few pulses, but at the same time also a larger facilitation of following pulses. The detection of the mGluR1-EPSP can be considered as a test to assess the net effect of the activation of parallel ®bre mGluR4 on glutamate release. In this context, the depression produced by L-SOP on the mGluR1-EPSP indicates that, in spite of the enhanced facilitation, the amount of glutamate released
by a parallel ®bre tetanus is under a net inhibitory control through presynaptic mGluR4. We thank Drs G. Bernardi and P. Calabresi for critical reading of the manuscript and Mr M. Tolu for his technical assistance. This work was partially supported by a Ministero SanitaÁ (Progetto Finalizzato) grant to A.P. and by MURST and CNR grants to G.B and P.S. [1] Batchelor, A.M., Madge, D.J. and Garthwaite, J., Synaptic activation of metabotropic glutamate receptors in the parallel ®bre-Purkinje cell pathway in rat cerebellar slices, Neuroscience, 63 (1994) 911±915. [2] Batchelor, A.M. and Garthwaite, J., Frequency detection and temporally-dispersed synaptic signal association through a metabotropic glutamate receptor pathway, Nature, 385 (1997) 74±77. [3] Batchelor, A.M., Knoepfel, T., Gasparini, F. and Garthwaite, J., Pharmacological characterization of synaptic transmission through mGluRs in rat cerebellar slices, Neuropharmacology, 36 (1997) 401±403. [4] Baude, A., Nusser, Z., Roberts, J.D.B., Mulvihill, E., McIlhinnery, R.A.J. and Somogyi, P., The metabotropic glutamate receptor (mGluR1a) is concentrated at perisynaptic membrane of neuronal subpopulations as detected by immunogold reaction, Neuron, 11 (1993) 771±787. [5] Conn, P.J. and Pin, J.-P., Pharmacology and functions of metabotropic glutamate receptors, Annu. Rev. Pharmacol. Toxicol., 37 (1997) 205±237. [6] D'Angelo, E., De Filippi, G., Rossi, P. and Taglietti, V., Synaptic excitation of individual rat cerebellar granule cells in situ: evidence for the role of NMDA receptors, J. Physiol., 484 (1995) 397±413. [7] Ichise, T., Kano, M., Hashimoto, K., Yanagihara, D., Nakao, K., Shigemoto, R., Katsuki, M. and Aiba, A., mGluR1 in cerebellar Purkinje cells essential for long-term depression, synapse elimination, and motor coordination, Science, 288 (2000) 1832±1835. [8] Kinoshita, A., Ohishi, H., Nomura, S., Shigemoto, R., Nakanishi, S. and Mizuno, N., Presynaptic localization of mGluR4a, in the cerebellar cortex: a light and electron microscopy study in the rat, Neurosci. Lett., 207 (1996) 199±202. [9] Kinzie, J.M., Saugstad, J.A., Westbrook, G.L. and Segerson, T.P., Distribution of metabotropic glutamate receptor 7 messenger mRNA in the developing and adult brain, Neuroscience, 69 (1995) 167±176. [10] Konnerth, A., Llano, I. and Armstrong, C.M., Synaptic currents in cerebellar Purkinje cells, Proc. Natl. Acad. Sci. USA, 87 (1990) 2662±2665. [11] Manabe, T., Wyllie, D.J.I. and Nicoll, R.A., Modulation of synaptic transmission and long-term potentiation: effects on paired pulse facilitation and EPSC variance in the CA1 region of the hippocampus, J. Neurophysiol., 70 (1993) 1451±1459. [12] Mateos, J.M., Elezgarai, I., Benitez, R., Osorio, A., Bilbao, A., Azkue, J.J., Kuhn, R., Knopfel, T. and Grandes, P., Clustering of the group III metabotropic glutamate receptor 4a at parallel ®ber synaptic terminals in the rat cerebellar molecular layer, Neurosci. Res., 35 (1999) 71±74. [13] Nakanishi, S., Nakajima, Y., Masu, M., Ueda, Y., Nakahara, K., Watanabe, D., Yamaguchi, S., Kawabata, S. and Okada, M., Glutamate receptors: brain function and signal transduction, Brain Res. Rev., 26 (1998) 230±235. [14] Nusser, Z., Mulvihill, E., Streit, P. and Somogyi, P., Subsy-
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