- Email: [email protected]
Regulation of synaptic transmission in the mossy fibre-granule cell pathway of rat cerebellum by metabotropic glutamate receptors
Neuropharmacology 38 (1999) 805 – 815
Regulation of synaptic transmission in the mossy fibre-granule cell pathway of rat cerebellum by metabotropic glutamate receptors P. Vetter, J. Garthwaite, A.M. Batchelor * The Wolfson Institute for Biomedical Research, Uni6ersity College London, 1 Wakefield Street, London WC1N 1PJ, UK Accepted 23 December 1998
Abstract The role of metabotropic glutamate receptors (mGluRs) in the mossy fibre-granule cell pathway in rat cerebellum was studied using slice preparations and electrophysiological techniques. Application of the group I selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG) evoked, in a concentration-dependent manner (EC50 = 33 mM), a depolarising/hyperpolarising complex response from granule cells which was preferentially inhibited by the group I selective antagonist (S)-4-carboxyphenylglycine (4CPG). The group III selective agonist L-amino-4-phosphonobutyrate (AP4) evoked a hyperpolarising response (EC50 = 10 mM) which was inhibited by the group II/III selective antagonist (S)-a-methyl-4-phosphonophenylglycine (MPPG). The group II agonist (2S,2%R,3%R)-2-(2%,3%-dicarboxylcyclopropyl)glycine (DCG-IV) elicited no measurable voltage change. The amplitude of the synaptically-mediated mossy fibre response in granule cells was unaffected during application of AP4, was reduced by DHPG and was enhanced by DCG-IV (EC50 =80 nM). These effects were inhibited by the group selective antagonists 4CPG and (2S,1%S,2%S,3%R)-2-(2%-carboxy-3%-phenylcyclopropyl)glycine (PCCG-4), respectively. Further investigation using patch-clamp recording revealed that DCG-IV potently inhibited spontaneous GABAergic currents. We conclude that group I and III (but not group II) mGluRs are functionally expressed by granule cells, whereas unexpectedly group II or III mGluRs do not appear to be present presynaptically on mossy fibre terminals. Group II mGluRs are located on Golgi cell terminals; when activated these receptors cause disinhibition, a function which may be important for gating information transfer from the mossy fibres to the granule cells. © 1999 Elsevier Science Ltd. All rights reserved. Keywords: mGluRs; Golgi cell; Grease-gap; Disinhibition
1. Introduction The family of metabotropic glutamate receptors (mGluRs) presently consists of eight members, which have been divided into three groups (Pin and Duvoisin, 1995; Conn and Pin, 1997). When expressed heterologously, group I mGluRs (mGluR1 and 5) are positively coupled to phosphoinositide hydrolysis, while group II receptors (mGluR2 and 3) and group III receptors (mGluR4, 6-8) are negatively coupled to cyclic AMP. The manner in which these receptors function at central synapses in normal and disease states is unclear. In the cerebellum, at the synapse between parallel fibres and Purkinje cells, mGluRs are * Corresponding author. Tel.: +44-171-5044190; fax: + 44-1718371347. E-mail address: [email protected] (A.M. Batchelor)
present presynaptically (Crepel et al., 1991), postsynaptically (Batchelor and Garthwaite, 1997) and they play an essential role in the induction of one form of synaptic plasticity, long-term depression (Hartell, 1994). In contrast, little is known about the role of mGluRs at the upstream synapse formed between mossy fibres and granule cells. Glutamatergic mossy fibres, which provide more than 80% of the input to the cerebellar cortex, synapse onto granule cells in structures termed glomeruli. Granule cells, via their axons, the parallel fibres, synapse with Purkinje cells and inhibitory interneurones, including Golgi cells whose axons project into the glomerulus in close proximity to mossy fibregranule cell synapses (Eccles et al., 1967). Recently, mGluRs have been postulated to be involved in longterm potentiation at this synapse (Rossi et al., 1996), but which mGluR subtype(s) are involved and their location(s) are unclear.
0028-3908/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 9 9 ) 0 0 0 0 3 - 9
806
P. Vetter et al. / Neuropharmacology 38 (1999) 805–815
In situ hybridisation studies (Abe et al., 1992; Shigemoto et al., 1992; Ohishi et al., 1993a; Tanabe et al., 1993) indicate the existence of mRNA coding for subtypes of mGluRs in granule cells (group I/III) and in Golgi cells (group I/II). The distribution of the receptors within these cell types is unclear although there has been one immunocytochemical study at the electron microscopic level, which indicated that group II mGluRs are present on Golgi cell terminals, but not on granule cell dendrites (Ohishi et al., 1994). Whether or not mGluRs are present on mossy fibre terminals is not known, except they do not appear to express group II receptors (Ohishi et al., 1994). Studies of the physiology of mGluRs has been hampered by a lack of selective ligands. Recently, however, compounds have become available that exhibit selectivity between the three groups as assessed using receptors expressed in cell lines; however they have not been well characterised in neuronal tissue. The aim of this study was to identify the functional significance of mGluRs in the mossy fibre-granule cell pathway using electrophysiological recording techniques and group selective agonists and antagonists.
2. Methods
2.1. Slice preparation and electrophysiological recording techniques We used three types of cerebellar slice in this study: sagittal slices for patch-clamp recording of individual granule cells; pial slices for grease-gap recording of the effects of exogenous agonists on a population of granule cells, and biplanar slices, which are hybrid sagittal-pial slices, allowing grease-gap recording of the synaptic mossy fibre response in a population of granule cells. Slices were cut from the vermis of 3–4week-old (grease-gap) or 11 – 14-day-old (patch-clamp) Wistar rats in cold (4 – 8°C) artificial cerebrospinal fluid (aCSF) which contained (in mM): NaCl 120; KCl 2; CaCl2 2; NaHCO3 26; MgSO4 1.19; KH2PO4 1.18 and glucose 11, equilibrated with 95% O2, 5% CO2. Slices were allowed to recover at room temperature for at least one hour before recording. Pial slices were cut from the surface of the vermis in the longitudinal direction of the folium with a Vibroslice (Campden Instruments, Loughborough, UK). The slice was about 400 mm thick at the centre (therefore containing all the layers of the cerebellar cortex) and tapered at either end due to the curvature of the cerebellum (the tips contain only the molecular layer). A single folium of the pial slice was dissected out and installed in a two-compartment chamber with the bulk of the slice (containing the granule cell bodies) in the perfused compartment (vol.= 0.2 ml) and
one of the tapering ends passing through a greased hole in a 0.4 mm thick partition into the other compartment. The slice was perfused ( 1.5 ml/min; 30°C) with aCSF containing TTX (0.4 mM) in order to prevent possible indirect agonist effects. The potential difference between the two compartments was continuously monitored with Ag/AgCl bath electrodes connected to an amplifier (Grass P16D) which was interfaced (Digidata 1200, Axon Instruments, CA) to a personal computer running Axoscope or Axotape software (Axon Instruments). The viability of the slice was tested with application of AMPA (1 mM for 45 s). A depolarisation of greater than 700 mV was deemed acceptable. Applications of AMPA were repeated at intervals of 10 min until a stable response was achieved; generally this took two to three applications. For synaptic activation of the mossy fibre-granule cell pathway biplanar slices were cut from 3–4-weekold rat cerebellum using a Vibroslice with an additional cutting-guide as previously described in detail (Garthwaite and Batchelor, 1996). These slices incorporate the mossy fibres, which travel in the white matter in the sagittal plane, granule cells, and the parallel fibres projecting in a perpendicular plane. After recovery, a biplanar slice was trimmed to a single lobule (lobule VIa) and was positioned in a threecompartment chamber so that the central part of the slice containing the granule cell bodies was in the central perfused compartment (1.5 ml/min at 30°C). The white matter and parallel fibre sections passed through greased holes into the other two aCSF-filled compartments. The white matter was stimulated using a bipolar twisted-wire electrode (0.2 ms pulses, 70– 100 V repeated at 0.1 Hz). The signals generated between the other two compartments were recorded differentially as above. The grease-gap method of detecting a correlate of the membrane potential from a population of granule cells relies on the fact that their axons, the parallel fibres, project for 2–5 mm in the transverse plane. If a high extracellular resistance is placed across this portion, changes in membrane potential at one end due to the actions of exogenous compounds, or due to synaptic activity are electrotonically or actively (in the absence of TTX) propagated to the opposite end and this is detected as a change in bath potential between the two chambers. In all illustrations, upward and downward deflections are referred to as ‘depolarisation’ and ‘hyperpolarisation’, respectively. These terms, usually employed for intracellular recording, are justified both from a theoretical standpoint (polarity of the potential in the compartment containing granule cell bodies relative to the compartment into which their axons projected-rule negativity equated with depolarisation) and from the experimental observation that agents that normally depolarise
P. Vetter et al. / Neuropharmacology 38 (1999) 805–815
or hyperpolarise the membrane potential, as recorded intracellularly, also evoke the predicted signal in our system. Voltage changes in response to application of compounds to the pial slices were quantified by measuring the maximum or minimum value (for depolarising and hyperpolarising responses, respectively) relative to adjacent baseline. If there was no obvious response to a ligand the value was taken at the end of the application time. Because of slice to slice variation it was required to normalise the values. A full concentration-response curve was achieved on each slice and a sigmoidal curve was fitted (Origin 4.1, Microcal Software, Northampton, MA). The lower asymptote was fixed at zero and the higher (calculated) asymptote was taken as the 100% value for that slice and all the other data was normalised against it. For synaptic responses, the peak of the second slower wave (peaking at 25–30 ms) was measured relative to the prestimulus baseline. Whole cell recordings were performed on granule cells in 200 mm thick sagittal slices at room temperature (24–28°C). Cells were visualised using an upright microscope (Zeiss Axioskop) equipped with a water immersion lens (× 63). Whole cell currents were recorded using an Axopatch-200B amplifier (Axon Instruments). Patch electrodes had a resistance of 8–12 MV when filled with a solution containing (mM): CsCl 150, EGTA 10, HEPES 10 and Mg-ATP 4 (osmolality 284–289 mOsm; pH 7.35 – 7.40 with CsOH). Seal resistances were 3 – 8 GV and on breakthrough series resistance was 30 – 50 MV (60 – 70% compensated). The cells had an input resistance of 4 – 8 GV and a capacitance of 1.5 – 2.5 pF, consistent with published values for granule cells (Kaneda et al., 1995; Wall and Usowicz, 1997). Spontaneous GABAergic potentials were recorded in the presence of NBQX (10 mM) to block AMPA receptors.
2.2. Materials (S)-4-Carboxyphenylglycine (4CPG); (2S,2%R,3%R)-2(2%,3%-dicarboxylcyclopropyl)glycine (DCG-IV); (S)3,5-dihydroxyphenylglycine (DHPG); L-amino-4-phosphonobutyrate (AP4), (R,S)-a-cyclopropyl-4-phosphonophenylglycine (CPPG), ethyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate (EtCCC), 6-nitro-7sulphamoylbenzo[f]quinoxaline - 2,3 - dione (NBQX), (S)-a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and bicuculline methochloride were all from Tocris Cookson (Bristol, UK). (S)-a-Methyl-4phosphonophenylglycine (MPPG) and (2S,1%S,2%S,3%R)-2-(2%-carboxy-3%-phenylcyclopropyl) glycine (PCCG-4) were from Alexis Corporation (Nottingham, UK), tetrodotoxin (TTX) was from Latoxan (Rosans, France).
807
3. Results
3.1. Voltage changes elicited by mGluR ligands on granule cells Application of the group I mGluR agonist DHPG for 45 s evoked a reversible and concentration-dependent (EC50 = 33 mM; Fig. 1a, d) response in pial slices which comprised two components: an initial depolarisation, which desensitised during drug application (at 20–30 s), followed by a hyperpolarisation (Fig. 1a). The relative amplitude of the two components varied between preparations but it was constant for a particular slice. In contrast, the group III mGluR agonist AP4 caused a reversible, concentration-dependent (EC50 = 10 mM) hyperpolarisation (Fig. 1b, d) which did not exhibit desensitisation during the application (up to 15 min; not shown). The group II mGluR agonist DCG-IV had no noticeable effect during applications lasting up to 5 min at concentrations of up to 5 mM (Fig. 1c; n=3). The group I mGluR antagonist 4CPG (300 mM) evoked a small transient depolarisation and hyperpolarisation on application and washout, respectively (Fig. 2a). The compound abolished the effect of the group I agonist DHPG (40 mM) but had no effect on that of AP4 (12 mM; Fig. 2a, c). Another group I mGluR antagonist, EtCCC (100–300 mM) had effects on its own; it caused a progressive depolarisation amounting to 6% of the amplitude of an AMPA (1 mM) evoked depolarisation each minute, which diminished the utility of this compound in these particular studies. The depolarisation reversed on washout (not shown). MPPG (300 mM), a group II/III mGluR antagonist, consistently caused a small depolarisation on its own (Fig. 2b) and inhibited the amplitude of the AP4 (12 mM) response by more than 80% (Fig. 2b, c), whereas it had no significant effect on the response to DHPG (Fig. 2a, c). Another reported group III mGluR antagonist, CPPG (300 mM) (Toms et al., 1996), was somewhat less potent, inhibiting the AP4 response by 50% (n=2; not shown).
3.2. Effect of mGluR ligands on synaptic mossy fibre-granule cell transmission In order to test for effects on synaptic transmission in the mossy fibre pathway, we applied group selective mGluR ligands while stimulating mossy fibres at a low frequency (0.1 Hz). The synaptic response is composed of two depolarising components; a fast initial response and a slower second response (Fig. 3a, c, e). The response is almost entirely abolished by antagonists acting at AMPA receptors (Garthwaite and Brodbelt, 1989b). We predicted that activation of presynaptic mGluRs, if present, would result in a decreased effi-
808
P. Vetter et al. / Neuropharmacology 38 (1999) 805–815
ciency of synaptic transmission (Scanziani et al., 1997; but see Herrero et al., 1992). Application of the group I mGluR agonist DHPG reduced the amplitude of the synaptic potential in a concentration-dependent manner. At the highest concentration tested (100 mM) the response was decreased by about 50% (Fig. 3a, b, g). The effect of DHPG desensitised during the application (Fig. 3b). Full or partial recovery was observed on washout of the agonist for 20 min. Repeated applications of DHPG generally led to a diminishing response. The group I mGluR antagonist 4CPG (300 mM) applied alone increased the synaptic response amplitude to 157= 3% of control and subsequently reduced the response to DHPG to 9= 4% of control, n= 3, (Fig. 4a, b). The potentiation by 4CPG was not mimicked by another group I antagonist EtCCC (300 mM; n =3; not shown). The group III mGluR agonist AP4 (12 mM) had no significant effect on synaptic transmission (Fig. 3e, f, g). Exposure to the group II mGluR agonist DCG-IV, surprisingly, increased the response by about 50% (Fig. 3c, d, g). This effect was reversible and concentrationdependent (EC50 =80 nM; Fig. 4e). Application of the group II mGluR antagonist PCCG-4 (100 mM) had no effect on its own but inhibited the DCG-IV (300 nM)
induced potentiation by 839 6%, n = 3, (Fig. 4c, d).
3.3. Role of GABAA receptors in the DCG-IV induced potentiation of synaptic transmission The enhancement of the mossy fibre response by DCG-IV is reminiscent of the effect of the GABAA receptor antagonist bicuculline (Garthwaite and Brodbelt, 1989a). It was therefore hypothesised that DCGIV was acting by switching off GABAergic inhibition. In order to test this we first applied the GABAA antagonist bicuculline (30 mM) until a stable enhancement was established (Fig. 5a, c). Consistent with our hypothesis, application of DCG-IV (300 nM) had no additional potentiating effect in the presence of bicuculline (Fig. 5a–d).
3.4. Effect of DCG-IV on spontaneous GABAergic acti6ity in cerebellar granule cells To investigate the mechanism of the effect of DCGIV on GABAergic transmission more directly we performed whole-cell patch clamp recording of granule cells. As previously reported under similar recording conditions, large spontaneous inward currents were
Fig. 1. mGluR agonists elicit responses from a population of granule cells in cerebellar slices. (a) The group I selective mGluR agonist, DHPG, applied for 60 s (as indicated by solid bars) caused a depolarising/hyperpolarising complex response in a concentration-dependent manner. (b) Application of the group III selective mGluR agonist, AP4 (hollow bars), for 60 s caused a nondesensitising hyperpolarizing response in a concentration-dependent manner. (c) Application of the selective group II mGluR agonist DCG-IV for 120 s (grey bar) had no observable effect whereas in the same slice, AP4 was active. (d) Concentration response curves for DHPG (EC50 =33 mM) and AP4 (EC50 =12 mM). Data from a single slice were fitted with a sigmoidal function to obtain a maximal response which was used for normalisation (n = 3 – 4 slices). Horizontal scale bar in (a) also applies to (b) and (c).
P. Vetter et al. / Neuropharmacology 38 (1999) 805–815
809
Fig. 2. The mGluR antagonists 4CPG and MPPG selectively inhibit the responses to DHPG and AP4, respectively. Following 60 s control applications of DHPG (40 mM, solid bars) and AP4 (12 mM, hollow bars) slices were superfused for 5 min with (a) 4CPG or (b) MPPG before repeat applications of DHPG and AP4 in the continued presence of the inhibitors. 4CPG selectively inhibited the DHPG effect (a, c) whilst MPPG selectively inhibited the AP4 effect (b), the inhibition of the DHPG response by MPPG was not a consistent finding (c). After washing out the antagonists for approximately 15 min the responses returned to control levels. (c) Accumulated data showing mean 9 SE from n \3 slices.
present in 50% of recorded granule cells (Puia et al., 1994; Kaneda et al., 1995; Wall and Usowicz, 1997). These were inhibited by bicuculline (10 mM; Fig. 6a), TTX (1 mM; not shown) and they reversed direction around the calculated chloride reversal potential (5 mV; not shown), consistent with these events being spontaneous IPSCs (sIPSCs) mediated by GABAA receptors. The addition of DCG-IV (0.1 – 1 mM: 1 – 2 min) abolished the sIPSCs in a reversible manner (8/8 applications in five cells; Fig. 6a).
4. Discussion In this study, the actions of reportedly group selective mGluR agonists and antagonists were characterised in the mossy fibre pathway of rat cerebellar slices. The grease-gap recording technique (Harvey and Collingridge, 1993; Garthwaite and Batchelor, 1996) is well suited for such studies, since recordings are stable, thus allowing generation of concentration-response curves from a single slice, the population recording eliminates cell-to-cell variation, and lastly the technique is relatively non-invasive, which is likely to be of particular importance when studying second messenger-coupled receptors.
4.1. Pharmacology of mGluRs in the mossy fibre-granule cell pathway 4.1.1. Group I compounds DHPG selectively activates group I mGluRs (Ito et al., 1992; Kingston and Lodge, 1998) although it has also been reported to inhibit phospholipase-D activity perhaps mediated by a novel mGluR (Albani-Torregrossa et al., 1998). The complex response elicited by DHPG in pial slices was similar to those previously observed for the mixed group I/II agonist (1S,3R)-1aminocyclopentane-1,3-dicarboxylic acid (1S,3RACPD; East and Garthwaite, 1992) whereas a group II selective agonist DCG-IV had no observable effects in pial slices, consistent with the effects of DHPG observed being due to an action at a group I mGluR. However, DHPG did not mimic the oscillations observed during the washout of 1S,3R-ACPD (East and Garthwaite, 1992), which could be due to its combined action at group I and group II mGluRs. The EC50 (33 mM) value for DHPG is comparable with the values from heterologously expressed group I mGluRs (Ito et al., 1992; Kingston and Lodge, 1998). Consistent with reported selective action at group I mGluRs (Hayashi et al., 1994; Thomsen et al., 1994), the antagonist 4CPG blocked the effects of DHPG on
810
P. Vetter et al. / Neuropharmacology 38 (1999) 805–815
pial slices and on synaptic transmission in the biplanar slices at concentrations compatible with published data (see Kingston and Lodge, 1998). In addition, 4CPG caused a large enhancement of the mossy fibre response. This effect was not mimicked by another group I antagonist, EtCCC, but instead may have been due to reported group II agonist effects of 4CPG (Thomsen et al., 1994). In support of this explanation, application of 4CPG (300 mM) reversibly abolished the sIPSC’s recorded from granule cells with the patch-clamp technique (not shown, two applications to a single slice). EtCCC caused a slow progressive depolarisation in the pial slices of an unknown origin. This effect was not mimicked by 4CPG or the solvent (dimethylsulphoxide) applied alone (not shown) and was not observed when EtCCC was applied to Purkinje cells (Batchelor et al., 1997). In summary, both group I antagonists had additional actions.
4.1.2. Group II compounds Interestingly, DCG-IV, a potent group II mGluR agonist had no discernible depolarising or hyperpolarising effects on pial slices but was found to increase the
amplitude of the mossy fibre response recorded in granule cells (Fig. 3c, d, g). The EC50 value of approximately 80 nM is slightly lower than the EC50 values reported for rat mGluR2 (300 nM) and mGluR3 (200 nM) expressed in cell lines (Hayashi et al., 1993) but is similar to the concentrations reported to depress transmission via native receptors in the lateral perforant pathway of the hippocampus (88 nM; Bushell et al., 1996). The effects we observe are unlikely to be due to known effects at NMDA receptors since these only occur at higher concentrations of around 10 mM or more (Hayashi et al., 1993; Wilsch et al., 1994; Breakwell et al., 1997) and since its action was inhibited by a group II mGluR antagonist, PCCG-4 (Pellicciari et al., 1996; Thomsen et al., 1996).
4.1.3. Group III compounds A response to the group III agonist, AP4, has not been reported before in cerebellar granule cells. The EC50 value of 10 mM is higher than expected from heterologously expressed receptors (EC50 range 0.5–1 mM; except mGluR7 which is only activated by \100 mM; for references see Conn and Pin, 1997). Although,
Fig. 3. Effects of group selective mGluR agonists on the synaptically mediated mossy fibre response. (a, c, e) A single stimulation of the white matter (at upward arrowheads) evokes the characteristic dual-component mossy fibre response. Synaptic responses are shown before (A), during (B) and after (C) application of a group I (DHPG; a), group II (DCG-IV; c) or group III (AP4; e) mGluR agonist. (b, d, f) Timecourse of agonist effects on the peak amplitude (application times are represented by bars; responses were normalised to the baseline values before application of compounds). Traces in (a), (c) and (e) were taken at the times indicated with arrows. (g) Effects of mGluR agonists on the peak amplitude of the mossy fibre response from n= 3 slices. Timebars in (a) and (b) also apply to (c – f).
P. Vetter et al. / Neuropharmacology 38 (1999) 805–815
811
Fig. 4. 4CPG and PCCG-4 inhibit the effects of DHPG and DCG-IV on synaptic transmission, respectively. Sample traces (a) and timecourse (b) of mossy fibre responses from a single slice (A) before and (B) during application of DHPG (100 mM, solid bars). 4CPG (300 mM, hollow bars) enhanced transmission (C), and inhibited the effects of DHPG (D). On washout of the antagonist for 15 minutes there was partial recovery of the response to DHPG. (c) and (d) Mossy fibre response before (A) and during (B) application of DCG-IV (solid bars). Application of PCCG-4 (100 mM, hollow bar) had no effect alone (C) whereas it inhibited the response to DCG-IV (D). The response to DCG-IV recovered after washout of PCCG-4 for 8 min. (e) The effects of DCG-IV on the mossy fibre synaptic response were concentration-dependent (EC50 =80 nM, mean =SE, n =3). Traces in (a) and (c) are averages of four sequential sweeps.
consistent with our data, depression of synaptic transmission in slices by AP4 is generally only achieved with higher concentrations e.g. striatum EC50 10 mM (Pisani et al., 1997); hippocampus EC50 3 mM (Bushell et al., 1996). Application of MPPG (300 mM) inhibited the response to AP4 but not DHPG (Fig. 2b, c) in accordance with its reported antagonistic properties at group II/III mGluRs (Bedingfield et al., 1996; Bushell et al., 1996; Salt and Turner, 1996).
4.2. Functional rele6ance of mGluRs in the mossy fibre-granule cell pathway 4.2.1. Group I mGluRs The depolarisation/hyperpolarisation complex caused by activation of group I mGluRs in pial slices indicates that functional group I mGluRs exist on granule cells. Granule cells also respond to the mixed group I/II agonist ACPD in slices (East and Garthwaite, 1992; Rossi et al., 1996) and in cultures (Chavis et al., 1995),
812
P. Vetter et al. / Neuropharmacology 38 (1999) 805–815
which, according to our results, most likely represents the activation of group I rather than group II mGluRs. In situ hybridisation and immunohistochemical studies show low levels of mGluR1 in these cells (Shigemoto et al., 1992; Grandes et al., 1994) whereas mGluR5 is apparently absent (Abe et al., 1992; Shigemoto et al., 1993). We can therefore conclude that mGluR1 are the receptors most likely to be responsible for the effects of DHPG in pial slices. Application of ACPD during synaptic activation of NMDA receptors causes a long term potentiation of transmission which has been interpreted as being mediated by postsynaptic mGluR activation (Rossi et al., 1996). It has not yet been demonstrated that these receptors can be activated by synaptic stimulation (c.f. mGluR1 in Purkinje cells; Batchelor and Garthwaite, 1997). The depression of transmission induced by group I agonists could be due to the postsynaptic effects discussed above or, alternatively, there could be an additional group I mGluR presynaptic on mossy fibre terminals. Further experiments are necessary to distinguish these two possibilities.
4.2.2. Group II mGluRs The enhancement of the synaptic response by DCGIV is unlikely to be due to a direct postsynaptic mechanism, since group II receptors could not be detected in granule cells electrophysiologically, immunohistochemically or in in situ hybridisation experiments (Ohishi et al., 1993a,b; Tanabe et al., 1993; Ohishi et al., 1994, 1998 but see Chavis et al., 1994). A facilitation of transmitter release could potentially explain the effects of DCG-IV but this has only been reported for presynaptic group I mGluRs, not group II mGluRs (Herrero et al., 1992; Collins and Davies, 1993). We therefore considered it unlikely that the effect of DCG-IV was due to pre- or postsynaptic actions at the mossy fibre to granule cell synapse, implying that DCG-IV was acting in a less direct manner. A revealing observation was that DCG-IV caused an enhancement of the synaptic response similar to that caused by the GABAA receptor antagonist bicuculline. Moreover, bicuculline treatment occluded the effect of DCG-IV. These data suggest a shared mechanism, the simplest of which is that both bicuculline and DCG-IV are disabling the tonic in-
Fig. 5. Effects of bicuculline on the synaptic response and its potentiation by DCG-IV. Timecourse of mossy fibre synaptic response (c) and example traces (a) and (b) from a single experiment (roman numerals indicate where sample traces are extracted from). Application of bicuculline (bic, 30 mM, hollow bar) causes a 50% increase in the amplitude of the mossy fibre response (i) and (ii) co-perfusion of DCG-IV (300 nM, grey bar) has no effect (iii). On washout of bicuculline for 24 min the response amplitude decayed to control levels ((b)-iv ‘post-bic’) and (c). A second application of DCG-IV (v) causes the usual 40% potentiation. (d) Cumulative data from three slices indicating that DCG-IV-mediated potentiation (140 92%) of the mossy fibre response was reduced in the presence of bicuculline (101 9 1%).
P. Vetter et al. / Neuropharmacology 38 (1999) 805–815
813
Fig. 6. DCG-IV inhibits spontaneous inhibitory currents in cerebellar granule cells. (a) Patch-clamp recording of spontaneous inhibitory postsynaptic currents (sIPSC) in the presence of NBQX (10 mM) and AP5 (30 mM) to block excitatory currents. Application of DCG-IV (300 nM, time of application represented by solid bar) caused a rapid inhibition of the sIPSCs which reappeared upon washout of DCG-IV for 300 s. Application of the GABAA receptor antagonist bicuculline methochloride (‘BIC’) caused a similar reversible inhibition of the IPSCs. (b) Schematic diagram for the proposed role of group II mGluRs in the mossy fibre pathway. Firing of mossy fibres causes glutamate release, which diffuses to the terminals of nearby Golgi cells and activates group II mGluRs. The subsequent inhibition of GABA release from the axon terminals relieves the tonic GABAergic inhibition of the granule cells, thus leading to a net facilitation of glutamatergic transmission at the mossy fibre-granule cell synapse.
hibitory activity: bicuculline, by competitive blockade of the postsynaptic GABAA receptors and DCG-IV, by acting on Golgi cell terminals to inhibit GABA release. Further support for the hypothesis is provided by the finding that sIPSC’s recorded from granule cells using the patch-clamp technique were abolished when DCG-IV (0.1–1 mM) was applied (Fig. 6a). In situ hybridisation experiments suggest that expression of group II mGluRs is highest in the GABAergic Golgi cells (Ohishi et al., 1993a,b; Tanabe et al., 1993) and there is immunohistochemical evidence that these receptors are highly concentrated in the axon terminals (Ohishi et al., 1994, 1998). Similar disinhibition elicited by application of group II mGluR agonists has been reported in several other brain areas (Hayashi et al., 1993; Desai et al., 1994; Poncer et al., 1995). For this mechanism to be of physiological relevance, glutamate would have to diffuse from mossy fibre terminals to presynaptic group II mGluRs on adjacent
Golgi cell terminals (Barbour and Ha¨usser, 1997; Kullman and Asztely, 1998). This scenario is most likely to occur during high frequency firing of mossy fibres as observed in vivo (Lisberger and Fuchs, 1978), when large amounts of glutamate are released. Furthermore, Golgi cells terminals are in close proximity ( B1 mm), and group II mGluRs bind glutamate with relatively high affinity. It is poorly understood, how the high frequency firing of mossy fibres is transformed into the lower frequency patterns of firing observed in granule cells (Gabbiani et al., 1994). It has been postulated that the high levels of Golgi cell activity exert a strong inhibitory effect on transmission at mossy fibre-granule cell synapse (Gabbiani et al., 1994; Brickley et al., 1996). Group II mGluRs may switch this inhibition off, thus transiently enhancing communication at the mossy fibre-granule cell synapse, and thereby contributing to the signal modulation from mossy fibre
814
P. Vetter et al. / Neuropharmacology 38 (1999) 805–815
input to parallel fibre output in an activity dependent manner.
4.2.3. Group III mGluRs The hyperpolarisation in pial slices due to activation of group III mGluRs is most reasonably explained by the receptors being located on the granule cells. In situ hybridisation studies indicate that mRNA encoding for GluR4 is highly expressed in granule cells of adult animals whereas that for mGluR6-8 is not (Tanabe et al., 1993, 1993; Duvoisin et al., 1995; Kinzie et al., 1995). However, immunocytochemistry locates these receptors in the molecular layer of the cerebellar cortex, suggesting that they are inserted in the membrane of parallel fibre terminals rather than somato-dendritically. Consistent with this is data that AP4 depresses parallel fibre-Purkinje cell transmission (Pekhletski et al., 1996). The grease-gap recording from pial slices cannot differentiate between signals coming from the axon terminals or from the somato-dendritic end of the granule cells and so it is therefore possible that the hyperpolarisation that we observed could originate in the parallel fibre terminals.
5. Conclusions In this study, group selective mGluR ligands were characterised in rat cerebellar slices. Their actions provide evidence for the existence of group I (probably mGluR1) and group III mGluRs in granule cells. Synaptic transmission in the mossy fibre pathway was depressed by activation of group I mGluRs but was unaffected by AP4 and DCG-IV (in the presence of bicuculline), suggesting that both group III and group II mGluRs are absent from mossy fibre terminals. This is unusual for a glutamatergic synapse (Pin and Duvoisin, 1995; Conn and Pin, 1997). Activation of group II mGluRs facilitated synaptic transmission; it is proposed that this is due to inhibition of GABA release at nearby Golgi axon terminals, an effect which may play an important role in information transfer at mossy fibre-granule cell synapses.
Acknowledgements This study was supported by a Wellcome Trust project grant (JG and AMB) and a Wellcome Trust 4-year Neuroscience Prize Studentship to PV.
References Abe, T., Sugihara, H., Nawa, H., Shigemoto, R., Mizuno, N., Nakanishi, S., 1992. Molecular characterization of a novel
metabotropic glutamate receptor GluR5 coupled to inositol phosphate/Ca2 + signal transduction. J. Biol. Chem. 267, 13361– 13368. Albani-Torregrossa, S., Pellicciari, R., Moroni, F., Pellegrini-Giampietro, E., 1998. Metabotropic glutamate receptors coupled to phospholipase D. In: Moroni, F., Nicoletti, F., Pellegrini-Giampietro, D.E. (Eds.), Metabotropic Glutamate Receptors and Brain Function. Portland Press, London, pp. 251 – 260. Barbour, B., Ha¨usser, M., 1997. Intersynaptic diffusion of neurotransmitter. Trends Neurosci. 20, 377 – 384. Batchelor, A.M., Kno¨pfel, T., Gasparini, F., Garthwaite, J., 1997. Pharmacological characterization of synaptic transmission through mGluRs in rat cerebellar slices. Neuropharmacology 36, 401 – 403. Batchelor, A.M., Garthwaite, J., 1997. Frequency detection and temporally dispersed synaptic signal association through a metabotropic glutamate receptor pathway. Nature 385, 74–77. Bedingfield, J.S., Jane, D.E., Kemp, M.C., Toms, N.J., Roberts, P.J., 1996. Novel potent selective phenylglycine antagonists of metabotropic glutamate receptors. Eur. J. Pharmacol. 309, 71 –78. Breakwell, N.A., Huang, L., Rowan, M.J., Anwyl, R., 1997. DCG-IV inhibits synaptic transmission by activation of NMDA receptors in area CA1 of rat hippocampus. Eur. J. Pharmacol. 322, 173– 178. Brickley, S.G., Cull-Candy, S.G., Farrant, M., 1996. Development of a tonic form of synaptic inhibition in rat cerebellar granule cells resulting from persistent activation of GABAA receptors. J. Physiol. 497, 753 – 759. Bushell, T.J., Jane, D.E., Tse, H.-W., Watkins, J.C., Garthwaite, J., Collingridge, G.L., 1996. Pharmacological antagonism of the actions of group II and III mGluR agonists in the lateral perforant path of rat hippocampal slices. Br. J. Pharmacol. 117, 1457 – 1462. Chavis, P., Shinozaki, H., Bockaert, J., Fagni, L., 1994. The metabotropic glutamate receptor types 2/3 inhibit L-type calcium channels via a pertussis toxin-sensitive G-protein in cultured cerebellar granule cells. J. Neurosci. 14, 7067 – 7076. Chavis, P., Nooney, J.M., Bockaert, J., Fagni, L., Feltz, A., Bossu, J.L., 1995. Facilitatory coupling between a glutamate metabotropic receptor and dihydropyridine-sensitive calcium channels in cultured cerebellar granule cells. J. Neurosci. 15, 135 – 143. Collins, D.R., Davies, S.N., 1993. Co-administration of (1S,3R)-1aminocyclopentane-1,3-dicarboxylic acid and arachidonic acid potentiates synaptic transmission in rat hippocampal slices. Eur. J. Pharmacol. 240, 325 – 326. Conn, P.J., Pin, J.-P., 1997. Pharmacology and functions of metabotropic glutamate receptors. Ann. Rev. Pharmacol. Toxicol. 37, 205 – 237. Crepel, F., Daniel, H., Hemart, N., Jaillard, D., 1991. Effects of ACPD and AP3 on parallel-fibre-mediated EPSPs of Purkinje cells in cerebellar slices in vitro. Exp. Brain Res. 86, 402–406. Desai, M.A., McBain, C.J., Kauer, J.A., Conn, P.J., 1994. Metabotropic glutamate receptor-induced disinhibition is mediated by reduced transmission at excitatory synapses onto interneurons and inhibitory synapses onto pyramidal cells. Neurosci. Lett. 181, 78 – 82. Duvoisin, R.M., Zhang, C., Ramonell, K., 1995. A novel metabotropic glutamate receptor expressed in the retina and olfactory bulb. J. Neurosci. 15, 3075 – 3083. East, S.J., Garthwaite, J., 1992. Actions of a metabotropic glutamate receptor agonist in immature and adult rat cerebellum. Eur. J. Pharmacol. 219, 395 – 400. Eccles J., Ito M., Szentagothai J., 1967. The Cerebellum as a Neuronal Machine, Springer-Verlag, Berlin. Gabbiani, F., Midtgaard, J., Knopfel, T., 1994. Synaptic integration in a model of cerebellar granule cells. J. Neurophysiol. 72, 999– 1009.
P. Vetter et al. / Neuropharmacology 38 (1999) 805–815 Garthwaite, J., Batchelor, A.M., 1996. A biplanar slice preparation for studying cerebellar synaptic transmission. J. Neurosci. Methods 64, 189 – 197. Garthwaite, J., Brodbelt, A.R., 1989a. Glutamate as the principal mossy fibre transmitter in rat cerebellum: pharmacological evidence. Eur. J. Neurosci. 2, 177–180. Garthwaite, J., Brodbelt, A.R., 1989b. Synaptic activation of Nmethyl-D-aspartate and non-N-methyl-D-aspartate receptors in the mossy fibre pathway in adult and immature rat cerebellar slices. Neuroscience 29, 401 –412. Grandes, P., Mateos, J.M., Ru¨egg, D., Kuhn, R., Kno¨pfel, T., 1994. Differential cellular localization of three splice variants of the mGluR1 metabotropic glutamate receptor in rat cerebellum. NeuroReport 5, 2249 – 2252. Hartell, N.A., 1994. Induction of cerebellar long-term depression requires activation of glutamate metabotropic receptors. NeuroReport 5, 913 – 916. Harvey, J., Collingridge, G.L., 1993. Signal transduction pathways involved in the acute potentiation of NMDA responses by 1S,3RACPD in rat hippocampal slices. Br. J. Pharmacol. 109, 1085 – 1090. Hayashi, Y., Momiyama, A., Takahashi, T., Ohishi, H., Ogawa Meguro, R., Shigemoto, R., Mizuno, N., Nakanishi, S., 1993. Role of a metabotropic glutamate receptor in synaptic modulation in the accessory olfactory bulb. Nature 366, 687–690. Hayashi, Y., Sekiyama, N., Nakanishi, S., Jane, D.E., Sunter, D.C., Birse, E.F., Udvarhelyi, P.M., Watkins, J.C., 1994. Analysis of agonist and antagonist activities of phenylglycine derivatives for different cloned metabotropic glutamate receptor subtypes. J. Neurosci. 14, 3370 – 3377. Herrero, I., Miras Portugal, M.T., Sanchez Prieto, J., 1992. Positive feedback of glutamate exocytosis by metabotropic presynaptic receptor stimulation. Nature 360, 163–166. Ito, I., Kohda, A., Tanabe, S., Hirose, E., Hayashi, M., Mitsunaga, S., Sugiyama, H., 1992. 3,5-Dihydroxyphenyl-glycine: a potent agonist of metabotropic glutamate receptors. NeuroReport 3, 1013 – 1016. Kaneda, M., Farrant, M., Cull-Candy, S.G., 1995. Whole-cell and single-channel currents activated by GABA and glycine in granule cells of the rat cerebellum. J. Physiol. 485, 419–435. Kingston, A.E., Lodge, D., 1998. Pharmacology of group I metabotropic glutamate receptors. In: Moroni, F., Nicoletti, F., PellegriniGiampietro, D.E. (Eds.), Metabotropic Glutamate Receptors and Brain Function. Portland Press, London, pp. 261–269. Kinzie, J.M., Saugstad, J.A., Westbrook, G.L., Segerson, T.P., 1995. Distribution of metabotropic glutamate receptor 7 messenger RNA in the developing and adult rat brain. Neuroscience 69, 167 – 176. Kullman, D.M., Asztely, F., 1998. Extrasynaptic glutamate spillover in the hippocampus: evidence and implications. Trends Neurosci. 21, 8 – 14. Lisberger, S.G., Fuchs, A.F., 1978. Role of primate flocculus during rapid behavioral modification of vestibuloocular reflex II. Mossy fibre fring pattern during head rotation and eye movement. J. Neurophysiol. 41, 764–777. Ohishi, H., Shigemoto, R., Nakanishi, S., Mizuno, N., 1993a. Distribution of the mRNA for a metabotropic glutamate receptor (mGluR3) in the rat brain: an in situ hybridisation study. J. Comp. Neurol. 335, 252 – 266. Ohishi, H., Shigemoto, R., Nakanishi, S., Mizuno, N., 1993b. Distribution of the messenger RNA for a metabotropic glutamate receptor, mGluR2, in the central nervous system of the rat. Neuroscience 53, 1009–1018. Ohishi, H., Ogawa Meguro, R., Shigemoto, R., Kaneko, T., Nakanishi, S., Mizuno, N., 1994. Immunohistochemical localization of metabotropic glutamate receptors, mGluR2 and mGluR3, in rat cerebellar cortex. Neuron 13, 55–66. Ohishi, H., Neki, A., Mizuno, N., 1998. Distribution of a metabotropic glutamate receptor, mGluR2, in the central nervous system of the .
815
rat and mouse: an immunohistochemical study with a monoclonal antibody. Neurosci. Res. 30, 65 – 82. Pekhletski, R., Gerlai, R., Overstreet, L.S., Huang, X., Agopyan, N., Slater, N.T., Abramow-Newerly, W., Roder, J.C., Hampson, D.R., 1996. Impaired cerebellar synaptic plasticity and motor performance in mice lacking the mGluR4 subtype of metabotropic gluatamate receptor. J. Neurosci. 16, 6364 – 6373. Pellicciari, R., Marinozzi, M., Natalini, B., Costantino, G., Luneia, R., Giorgi, G., Moroni, F., Thomsen, C., 1996. Synthesis and pharmacological characterization of all sixteen stereoisomers of 2-(2%-carboxy-3%-phenylcyclopropyl)glycine. Focus on (2S,1%S,2%S,3%R)-2-(2%-carboxy-3%-phenylcyclopropyl)glycine, a novel and selective group II metabotropic glutamate receptors antagonist. J. Med. Chem. 39, 2259 – 2269. Pin, J.-P., Duvoisin, R., 1995. The metabotropic glutamate receptors: structure and functions. Neuropharmacology 34, 1 – 26. Pisani, A., Calabresi, P., Centonze, D., Bernardi, G., 1997. Activation of group III metabotropic glutamate receptors depresses glutamatergic transmission at corticostriatal synapse. Neuropharmacology 36, 845 – 851. Poncer, J., Shinozaki, H., Miles, R., 1995. Dual modulation of synaptic inhibition by distinct metabotropic glutamate receptors in the rat hippocampus. J. Physiol. 485, 121 – 134. Puia, G., Costa, E., Vicini, S., 1994. Functional diversity of GABA-activated Cl − currents in Purkinje versus granule neurons in rat cerebellar slices. Neuron 12, 117 – 126. Rossi, P., D’Angelo, E., Taglietti, V., 1996. Differential long-lasting potentiation of the NMDA and non-NMDA synaptic currents induced by metabotropic and NMDA receptor coactivation in cerebellar granule cells. Eur. J. Neurosci. 8, 1182 – 1189. Salt, T.E., Turner, J.P., 1996. Antagonism of the presumed presynaptic action of L-AP4 on GABAergic transmission in the ventrobasal thalamus by the novel mGluR antagonist MPPG. Neuropharmacology 35, 239 – 241. Scanziani, M., Salin, P.A., Vogt, K.E., Malenka, R.C., Nicoll, R.A., 1997. Use-dependent increases in glutamate concentration activate presynaptic metabotropic glutamate receptors. Nature 385, 630– 634. Shigemoto, R., Nakanishi, S., Mizuno, N., 1992. Distribution of the mRNA for a metabotropic glutamate receptor (mGluR1) in the central nervous system: an in situ hybridization study in adult and developing rat. J. Comp. Neurol. 322, 121 – 135. Shigemoto, R., Nomura, S., Ohishi, H., Sugihara, H., Nakanishi, S., Mizuno, N., 1993. Immunohistochemical localisation of a metabotropic glutamate receptor, mGluR5, in the rat brain. Neurosci. Lett. 163, 53 – 57. Tanabe, Y., Nomura, A., Masu, M., Shigemoto, R., Mizuno, N., Nakanishi, S., 1993. Signal transduction, pharmacological properties, and expression patterns of two rat metabotropic glutamate receptors, mGluR3 and mGluR4. J. Neurosci. 13, 1372–1378. Thomsen, C., Boel, E., Suzdak, P.D., 1994. Actions of phenylglycine analogs at subtypes of the metabotropic glutamate receptor family. Eur. J. Pharmacol. Mol. Pharmacol. 267, 77 – 84. Thomsen, C., Bruno, V., Nicoletti, F., Marinozzi, M., Pellicciari, R., 1996. (2S, 1%S, 2%S, 3%R)-2-(2%-carboxy-3%-phenylcyclopropyl)glycine, a potent and selective antagonist of type 2 metabotropic glutamate receptors. Mol. Pharmacol. 50, 6 – 9. Toms, N.J., Jane, D.E., Kemp, M.C., Bedingfield, J.S., Roberts, P.J., 1996. The effects of (RS)-alpha-cyclopropyl-4-phosphonophenylglycine ((RS)-CPPG), a potent and selective metabotropic glutamate receptor antagonist. Br. J. Pharmacol. 119, 851 – 854. Wall, M.J., Usowicz, M.M., 1997. Development of action potential-dependent and independent spontaneous GABAA receptor-mediated currents in granule cells of postnatal rat cerebellum. Eur. J. Neurosci. 9, 533 – 548. Wilsch, V.W., Pidoplichko, V.I., Opitz, T., Shinozaki, H., Reymann, K.G., 1994. Metabotropic glutamate receptor agonist DCG-IV as NMDA receptor agonist in immature rat hippocampal neurons. Eur. J. Pharmacol. 262, 287 – 291.
Regulation of synaptic transmission in the mossy fibre-granule cell pathway of rat cerebellum by metabotropic glutamate receptors P. Vetter, J. Garthwaite, A.M. Batchelor * The Wolfson Institute for Biomedical Research, Uni6ersity College London, 1 Wakefield Street, London WC1N 1PJ, UK Accepted 23 December 1998
Abstract The role of metabotropic glutamate receptors (mGluRs) in the mossy fibre-granule cell pathway in rat cerebellum was studied using slice preparations and electrophysiological techniques. Application of the group I selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG) evoked, in a concentration-dependent manner (EC50 = 33 mM), a depolarising/hyperpolarising complex response from granule cells which was preferentially inhibited by the group I selective antagonist (S)-4-carboxyphenylglycine (4CPG). The group III selective agonist L-amino-4-phosphonobutyrate (AP4) evoked a hyperpolarising response (EC50 = 10 mM) which was inhibited by the group II/III selective antagonist (S)-a-methyl-4-phosphonophenylglycine (MPPG). The group II agonist (2S,2%R,3%R)-2-(2%,3%-dicarboxylcyclopropyl)glycine (DCG-IV) elicited no measurable voltage change. The amplitude of the synaptically-mediated mossy fibre response in granule cells was unaffected during application of AP4, was reduced by DHPG and was enhanced by DCG-IV (EC50 =80 nM). These effects were inhibited by the group selective antagonists 4CPG and (2S,1%S,2%S,3%R)-2-(2%-carboxy-3%-phenylcyclopropyl)glycine (PCCG-4), respectively. Further investigation using patch-clamp recording revealed that DCG-IV potently inhibited spontaneous GABAergic currents. We conclude that group I and III (but not group II) mGluRs are functionally expressed by granule cells, whereas unexpectedly group II or III mGluRs do not appear to be present presynaptically on mossy fibre terminals. Group II mGluRs are located on Golgi cell terminals; when activated these receptors cause disinhibition, a function which may be important for gating information transfer from the mossy fibres to the granule cells. © 1999 Elsevier Science Ltd. All rights reserved. Keywords: mGluRs; Golgi cell; Grease-gap; Disinhibition
1. Introduction The family of metabotropic glutamate receptors (mGluRs) presently consists of eight members, which have been divided into three groups (Pin and Duvoisin, 1995; Conn and Pin, 1997). When expressed heterologously, group I mGluRs (mGluR1 and 5) are positively coupled to phosphoinositide hydrolysis, while group II receptors (mGluR2 and 3) and group III receptors (mGluR4, 6-8) are negatively coupled to cyclic AMP. The manner in which these receptors function at central synapses in normal and disease states is unclear. In the cerebellum, at the synapse between parallel fibres and Purkinje cells, mGluRs are * Corresponding author. Tel.: +44-171-5044190; fax: + 44-1718371347. E-mail address: [email protected] (A.M. Batchelor)
present presynaptically (Crepel et al., 1991), postsynaptically (Batchelor and Garthwaite, 1997) and they play an essential role in the induction of one form of synaptic plasticity, long-term depression (Hartell, 1994). In contrast, little is known about the role of mGluRs at the upstream synapse formed between mossy fibres and granule cells. Glutamatergic mossy fibres, which provide more than 80% of the input to the cerebellar cortex, synapse onto granule cells in structures termed glomeruli. Granule cells, via their axons, the parallel fibres, synapse with Purkinje cells and inhibitory interneurones, including Golgi cells whose axons project into the glomerulus in close proximity to mossy fibregranule cell synapses (Eccles et al., 1967). Recently, mGluRs have been postulated to be involved in longterm potentiation at this synapse (Rossi et al., 1996), but which mGluR subtype(s) are involved and their location(s) are unclear.
0028-3908/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 9 9 ) 0 0 0 0 3 - 9
806
P. Vetter et al. / Neuropharmacology 38 (1999) 805–815
In situ hybridisation studies (Abe et al., 1992; Shigemoto et al., 1992; Ohishi et al., 1993a; Tanabe et al., 1993) indicate the existence of mRNA coding for subtypes of mGluRs in granule cells (group I/III) and in Golgi cells (group I/II). The distribution of the receptors within these cell types is unclear although there has been one immunocytochemical study at the electron microscopic level, which indicated that group II mGluRs are present on Golgi cell terminals, but not on granule cell dendrites (Ohishi et al., 1994). Whether or not mGluRs are present on mossy fibre terminals is not known, except they do not appear to express group II receptors (Ohishi et al., 1994). Studies of the physiology of mGluRs has been hampered by a lack of selective ligands. Recently, however, compounds have become available that exhibit selectivity between the three groups as assessed using receptors expressed in cell lines; however they have not been well characterised in neuronal tissue. The aim of this study was to identify the functional significance of mGluRs in the mossy fibre-granule cell pathway using electrophysiological recording techniques and group selective agonists and antagonists.
2. Methods
2.1. Slice preparation and electrophysiological recording techniques We used three types of cerebellar slice in this study: sagittal slices for patch-clamp recording of individual granule cells; pial slices for grease-gap recording of the effects of exogenous agonists on a population of granule cells, and biplanar slices, which are hybrid sagittal-pial slices, allowing grease-gap recording of the synaptic mossy fibre response in a population of granule cells. Slices were cut from the vermis of 3–4week-old (grease-gap) or 11 – 14-day-old (patch-clamp) Wistar rats in cold (4 – 8°C) artificial cerebrospinal fluid (aCSF) which contained (in mM): NaCl 120; KCl 2; CaCl2 2; NaHCO3 26; MgSO4 1.19; KH2PO4 1.18 and glucose 11, equilibrated with 95% O2, 5% CO2. Slices were allowed to recover at room temperature for at least one hour before recording. Pial slices were cut from the surface of the vermis in the longitudinal direction of the folium with a Vibroslice (Campden Instruments, Loughborough, UK). The slice was about 400 mm thick at the centre (therefore containing all the layers of the cerebellar cortex) and tapered at either end due to the curvature of the cerebellum (the tips contain only the molecular layer). A single folium of the pial slice was dissected out and installed in a two-compartment chamber with the bulk of the slice (containing the granule cell bodies) in the perfused compartment (vol.= 0.2 ml) and
one of the tapering ends passing through a greased hole in a 0.4 mm thick partition into the other compartment. The slice was perfused ( 1.5 ml/min; 30°C) with aCSF containing TTX (0.4 mM) in order to prevent possible indirect agonist effects. The potential difference between the two compartments was continuously monitored with Ag/AgCl bath electrodes connected to an amplifier (Grass P16D) which was interfaced (Digidata 1200, Axon Instruments, CA) to a personal computer running Axoscope or Axotape software (Axon Instruments). The viability of the slice was tested with application of AMPA (1 mM for 45 s). A depolarisation of greater than 700 mV was deemed acceptable. Applications of AMPA were repeated at intervals of 10 min until a stable response was achieved; generally this took two to three applications. For synaptic activation of the mossy fibre-granule cell pathway biplanar slices were cut from 3–4-weekold rat cerebellum using a Vibroslice with an additional cutting-guide as previously described in detail (Garthwaite and Batchelor, 1996). These slices incorporate the mossy fibres, which travel in the white matter in the sagittal plane, granule cells, and the parallel fibres projecting in a perpendicular plane. After recovery, a biplanar slice was trimmed to a single lobule (lobule VIa) and was positioned in a threecompartment chamber so that the central part of the slice containing the granule cell bodies was in the central perfused compartment (1.5 ml/min at 30°C). The white matter and parallel fibre sections passed through greased holes into the other two aCSF-filled compartments. The white matter was stimulated using a bipolar twisted-wire electrode (0.2 ms pulses, 70– 100 V repeated at 0.1 Hz). The signals generated between the other two compartments were recorded differentially as above. The grease-gap method of detecting a correlate of the membrane potential from a population of granule cells relies on the fact that their axons, the parallel fibres, project for 2–5 mm in the transverse plane. If a high extracellular resistance is placed across this portion, changes in membrane potential at one end due to the actions of exogenous compounds, or due to synaptic activity are electrotonically or actively (in the absence of TTX) propagated to the opposite end and this is detected as a change in bath potential between the two chambers. In all illustrations, upward and downward deflections are referred to as ‘depolarisation’ and ‘hyperpolarisation’, respectively. These terms, usually employed for intracellular recording, are justified both from a theoretical standpoint (polarity of the potential in the compartment containing granule cell bodies relative to the compartment into which their axons projected-rule negativity equated with depolarisation) and from the experimental observation that agents that normally depolarise
P. Vetter et al. / Neuropharmacology 38 (1999) 805–815
or hyperpolarise the membrane potential, as recorded intracellularly, also evoke the predicted signal in our system. Voltage changes in response to application of compounds to the pial slices were quantified by measuring the maximum or minimum value (for depolarising and hyperpolarising responses, respectively) relative to adjacent baseline. If there was no obvious response to a ligand the value was taken at the end of the application time. Because of slice to slice variation it was required to normalise the values. A full concentration-response curve was achieved on each slice and a sigmoidal curve was fitted (Origin 4.1, Microcal Software, Northampton, MA). The lower asymptote was fixed at zero and the higher (calculated) asymptote was taken as the 100% value for that slice and all the other data was normalised against it. For synaptic responses, the peak of the second slower wave (peaking at 25–30 ms) was measured relative to the prestimulus baseline. Whole cell recordings were performed on granule cells in 200 mm thick sagittal slices at room temperature (24–28°C). Cells were visualised using an upright microscope (Zeiss Axioskop) equipped with a water immersion lens (× 63). Whole cell currents were recorded using an Axopatch-200B amplifier (Axon Instruments). Patch electrodes had a resistance of 8–12 MV when filled with a solution containing (mM): CsCl 150, EGTA 10, HEPES 10 and Mg-ATP 4 (osmolality 284–289 mOsm; pH 7.35 – 7.40 with CsOH). Seal resistances were 3 – 8 GV and on breakthrough series resistance was 30 – 50 MV (60 – 70% compensated). The cells had an input resistance of 4 – 8 GV and a capacitance of 1.5 – 2.5 pF, consistent with published values for granule cells (Kaneda et al., 1995; Wall and Usowicz, 1997). Spontaneous GABAergic potentials were recorded in the presence of NBQX (10 mM) to block AMPA receptors.
2.2. Materials (S)-4-Carboxyphenylglycine (4CPG); (2S,2%R,3%R)-2(2%,3%-dicarboxylcyclopropyl)glycine (DCG-IV); (S)3,5-dihydroxyphenylglycine (DHPG); L-amino-4-phosphonobutyrate (AP4), (R,S)-a-cyclopropyl-4-phosphonophenylglycine (CPPG), ethyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate (EtCCC), 6-nitro-7sulphamoylbenzo[f]quinoxaline - 2,3 - dione (NBQX), (S)-a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and bicuculline methochloride were all from Tocris Cookson (Bristol, UK). (S)-a-Methyl-4phosphonophenylglycine (MPPG) and (2S,1%S,2%S,3%R)-2-(2%-carboxy-3%-phenylcyclopropyl) glycine (PCCG-4) were from Alexis Corporation (Nottingham, UK), tetrodotoxin (TTX) was from Latoxan (Rosans, France).
807
3. Results
3.1. Voltage changes elicited by mGluR ligands on granule cells Application of the group I mGluR agonist DHPG for 45 s evoked a reversible and concentration-dependent (EC50 = 33 mM; Fig. 1a, d) response in pial slices which comprised two components: an initial depolarisation, which desensitised during drug application (at 20–30 s), followed by a hyperpolarisation (Fig. 1a). The relative amplitude of the two components varied between preparations but it was constant for a particular slice. In contrast, the group III mGluR agonist AP4 caused a reversible, concentration-dependent (EC50 = 10 mM) hyperpolarisation (Fig. 1b, d) which did not exhibit desensitisation during the application (up to 15 min; not shown). The group II mGluR agonist DCG-IV had no noticeable effect during applications lasting up to 5 min at concentrations of up to 5 mM (Fig. 1c; n=3). The group I mGluR antagonist 4CPG (300 mM) evoked a small transient depolarisation and hyperpolarisation on application and washout, respectively (Fig. 2a). The compound abolished the effect of the group I agonist DHPG (40 mM) but had no effect on that of AP4 (12 mM; Fig. 2a, c). Another group I mGluR antagonist, EtCCC (100–300 mM) had effects on its own; it caused a progressive depolarisation amounting to 6% of the amplitude of an AMPA (1 mM) evoked depolarisation each minute, which diminished the utility of this compound in these particular studies. The depolarisation reversed on washout (not shown). MPPG (300 mM), a group II/III mGluR antagonist, consistently caused a small depolarisation on its own (Fig. 2b) and inhibited the amplitude of the AP4 (12 mM) response by more than 80% (Fig. 2b, c), whereas it had no significant effect on the response to DHPG (Fig. 2a, c). Another reported group III mGluR antagonist, CPPG (300 mM) (Toms et al., 1996), was somewhat less potent, inhibiting the AP4 response by 50% (n=2; not shown).
3.2. Effect of mGluR ligands on synaptic mossy fibre-granule cell transmission In order to test for effects on synaptic transmission in the mossy fibre pathway, we applied group selective mGluR ligands while stimulating mossy fibres at a low frequency (0.1 Hz). The synaptic response is composed of two depolarising components; a fast initial response and a slower second response (Fig. 3a, c, e). The response is almost entirely abolished by antagonists acting at AMPA receptors (Garthwaite and Brodbelt, 1989b). We predicted that activation of presynaptic mGluRs, if present, would result in a decreased effi-
808
P. Vetter et al. / Neuropharmacology 38 (1999) 805–815
ciency of synaptic transmission (Scanziani et al., 1997; but see Herrero et al., 1992). Application of the group I mGluR agonist DHPG reduced the amplitude of the synaptic potential in a concentration-dependent manner. At the highest concentration tested (100 mM) the response was decreased by about 50% (Fig. 3a, b, g). The effect of DHPG desensitised during the application (Fig. 3b). Full or partial recovery was observed on washout of the agonist for 20 min. Repeated applications of DHPG generally led to a diminishing response. The group I mGluR antagonist 4CPG (300 mM) applied alone increased the synaptic response amplitude to 157= 3% of control and subsequently reduced the response to DHPG to 9= 4% of control, n= 3, (Fig. 4a, b). The potentiation by 4CPG was not mimicked by another group I antagonist EtCCC (300 mM; n =3; not shown). The group III mGluR agonist AP4 (12 mM) had no significant effect on synaptic transmission (Fig. 3e, f, g). Exposure to the group II mGluR agonist DCG-IV, surprisingly, increased the response by about 50% (Fig. 3c, d, g). This effect was reversible and concentrationdependent (EC50 =80 nM; Fig. 4e). Application of the group II mGluR antagonist PCCG-4 (100 mM) had no effect on its own but inhibited the DCG-IV (300 nM)
induced potentiation by 839 6%, n = 3, (Fig. 4c, d).
3.3. Role of GABAA receptors in the DCG-IV induced potentiation of synaptic transmission The enhancement of the mossy fibre response by DCG-IV is reminiscent of the effect of the GABAA receptor antagonist bicuculline (Garthwaite and Brodbelt, 1989a). It was therefore hypothesised that DCGIV was acting by switching off GABAergic inhibition. In order to test this we first applied the GABAA antagonist bicuculline (30 mM) until a stable enhancement was established (Fig. 5a, c). Consistent with our hypothesis, application of DCG-IV (300 nM) had no additional potentiating effect in the presence of bicuculline (Fig. 5a–d).
3.4. Effect of DCG-IV on spontaneous GABAergic acti6ity in cerebellar granule cells To investigate the mechanism of the effect of DCGIV on GABAergic transmission more directly we performed whole-cell patch clamp recording of granule cells. As previously reported under similar recording conditions, large spontaneous inward currents were
Fig. 1. mGluR agonists elicit responses from a population of granule cells in cerebellar slices. (a) The group I selective mGluR agonist, DHPG, applied for 60 s (as indicated by solid bars) caused a depolarising/hyperpolarising complex response in a concentration-dependent manner. (b) Application of the group III selective mGluR agonist, AP4 (hollow bars), for 60 s caused a nondesensitising hyperpolarizing response in a concentration-dependent manner. (c) Application of the selective group II mGluR agonist DCG-IV for 120 s (grey bar) had no observable effect whereas in the same slice, AP4 was active. (d) Concentration response curves for DHPG (EC50 =33 mM) and AP4 (EC50 =12 mM). Data from a single slice were fitted with a sigmoidal function to obtain a maximal response which was used for normalisation (n = 3 – 4 slices). Horizontal scale bar in (a) also applies to (b) and (c).
P. Vetter et al. / Neuropharmacology 38 (1999) 805–815
809
Fig. 2. The mGluR antagonists 4CPG and MPPG selectively inhibit the responses to DHPG and AP4, respectively. Following 60 s control applications of DHPG (40 mM, solid bars) and AP4 (12 mM, hollow bars) slices were superfused for 5 min with (a) 4CPG or (b) MPPG before repeat applications of DHPG and AP4 in the continued presence of the inhibitors. 4CPG selectively inhibited the DHPG effect (a, c) whilst MPPG selectively inhibited the AP4 effect (b), the inhibition of the DHPG response by MPPG was not a consistent finding (c). After washing out the antagonists for approximately 15 min the responses returned to control levels. (c) Accumulated data showing mean 9 SE from n \3 slices.
present in 50% of recorded granule cells (Puia et al., 1994; Kaneda et al., 1995; Wall and Usowicz, 1997). These were inhibited by bicuculline (10 mM; Fig. 6a), TTX (1 mM; not shown) and they reversed direction around the calculated chloride reversal potential (5 mV; not shown), consistent with these events being spontaneous IPSCs (sIPSCs) mediated by GABAA receptors. The addition of DCG-IV (0.1 – 1 mM: 1 – 2 min) abolished the sIPSCs in a reversible manner (8/8 applications in five cells; Fig. 6a).
4. Discussion In this study, the actions of reportedly group selective mGluR agonists and antagonists were characterised in the mossy fibre pathway of rat cerebellar slices. The grease-gap recording technique (Harvey and Collingridge, 1993; Garthwaite and Batchelor, 1996) is well suited for such studies, since recordings are stable, thus allowing generation of concentration-response curves from a single slice, the population recording eliminates cell-to-cell variation, and lastly the technique is relatively non-invasive, which is likely to be of particular importance when studying second messenger-coupled receptors.
4.1. Pharmacology of mGluRs in the mossy fibre-granule cell pathway 4.1.1. Group I compounds DHPG selectively activates group I mGluRs (Ito et al., 1992; Kingston and Lodge, 1998) although it has also been reported to inhibit phospholipase-D activity perhaps mediated by a novel mGluR (Albani-Torregrossa et al., 1998). The complex response elicited by DHPG in pial slices was similar to those previously observed for the mixed group I/II agonist (1S,3R)-1aminocyclopentane-1,3-dicarboxylic acid (1S,3RACPD; East and Garthwaite, 1992) whereas a group II selective agonist DCG-IV had no observable effects in pial slices, consistent with the effects of DHPG observed being due to an action at a group I mGluR. However, DHPG did not mimic the oscillations observed during the washout of 1S,3R-ACPD (East and Garthwaite, 1992), which could be due to its combined action at group I and group II mGluRs. The EC50 (33 mM) value for DHPG is comparable with the values from heterologously expressed group I mGluRs (Ito et al., 1992; Kingston and Lodge, 1998). Consistent with reported selective action at group I mGluRs (Hayashi et al., 1994; Thomsen et al., 1994), the antagonist 4CPG blocked the effects of DHPG on
810
P. Vetter et al. / Neuropharmacology 38 (1999) 805–815
pial slices and on synaptic transmission in the biplanar slices at concentrations compatible with published data (see Kingston and Lodge, 1998). In addition, 4CPG caused a large enhancement of the mossy fibre response. This effect was not mimicked by another group I antagonist, EtCCC, but instead may have been due to reported group II agonist effects of 4CPG (Thomsen et al., 1994). In support of this explanation, application of 4CPG (300 mM) reversibly abolished the sIPSC’s recorded from granule cells with the patch-clamp technique (not shown, two applications to a single slice). EtCCC caused a slow progressive depolarisation in the pial slices of an unknown origin. This effect was not mimicked by 4CPG or the solvent (dimethylsulphoxide) applied alone (not shown) and was not observed when EtCCC was applied to Purkinje cells (Batchelor et al., 1997). In summary, both group I antagonists had additional actions.
4.1.2. Group II compounds Interestingly, DCG-IV, a potent group II mGluR agonist had no discernible depolarising or hyperpolarising effects on pial slices but was found to increase the
amplitude of the mossy fibre response recorded in granule cells (Fig. 3c, d, g). The EC50 value of approximately 80 nM is slightly lower than the EC50 values reported for rat mGluR2 (300 nM) and mGluR3 (200 nM) expressed in cell lines (Hayashi et al., 1993) but is similar to the concentrations reported to depress transmission via native receptors in the lateral perforant pathway of the hippocampus (88 nM; Bushell et al., 1996). The effects we observe are unlikely to be due to known effects at NMDA receptors since these only occur at higher concentrations of around 10 mM or more (Hayashi et al., 1993; Wilsch et al., 1994; Breakwell et al., 1997) and since its action was inhibited by a group II mGluR antagonist, PCCG-4 (Pellicciari et al., 1996; Thomsen et al., 1996).
4.1.3. Group III compounds A response to the group III agonist, AP4, has not been reported before in cerebellar granule cells. The EC50 value of 10 mM is higher than expected from heterologously expressed receptors (EC50 range 0.5–1 mM; except mGluR7 which is only activated by \100 mM; for references see Conn and Pin, 1997). Although,
Fig. 3. Effects of group selective mGluR agonists on the synaptically mediated mossy fibre response. (a, c, e) A single stimulation of the white matter (at upward arrowheads) evokes the characteristic dual-component mossy fibre response. Synaptic responses are shown before (A), during (B) and after (C) application of a group I (DHPG; a), group II (DCG-IV; c) or group III (AP4; e) mGluR agonist. (b, d, f) Timecourse of agonist effects on the peak amplitude (application times are represented by bars; responses were normalised to the baseline values before application of compounds). Traces in (a), (c) and (e) were taken at the times indicated with arrows. (g) Effects of mGluR agonists on the peak amplitude of the mossy fibre response from n= 3 slices. Timebars in (a) and (b) also apply to (c – f).
P. Vetter et al. / Neuropharmacology 38 (1999) 805–815
811
Fig. 4. 4CPG and PCCG-4 inhibit the effects of DHPG and DCG-IV on synaptic transmission, respectively. Sample traces (a) and timecourse (b) of mossy fibre responses from a single slice (A) before and (B) during application of DHPG (100 mM, solid bars). 4CPG (300 mM, hollow bars) enhanced transmission (C), and inhibited the effects of DHPG (D). On washout of the antagonist for 15 minutes there was partial recovery of the response to DHPG. (c) and (d) Mossy fibre response before (A) and during (B) application of DCG-IV (solid bars). Application of PCCG-4 (100 mM, hollow bar) had no effect alone (C) whereas it inhibited the response to DCG-IV (D). The response to DCG-IV recovered after washout of PCCG-4 for 8 min. (e) The effects of DCG-IV on the mossy fibre synaptic response were concentration-dependent (EC50 =80 nM, mean =SE, n =3). Traces in (a) and (c) are averages of four sequential sweeps.
consistent with our data, depression of synaptic transmission in slices by AP4 is generally only achieved with higher concentrations e.g. striatum EC50 10 mM (Pisani et al., 1997); hippocampus EC50 3 mM (Bushell et al., 1996). Application of MPPG (300 mM) inhibited the response to AP4 but not DHPG (Fig. 2b, c) in accordance with its reported antagonistic properties at group II/III mGluRs (Bedingfield et al., 1996; Bushell et al., 1996; Salt and Turner, 1996).
4.2. Functional rele6ance of mGluRs in the mossy fibre-granule cell pathway 4.2.1. Group I mGluRs The depolarisation/hyperpolarisation complex caused by activation of group I mGluRs in pial slices indicates that functional group I mGluRs exist on granule cells. Granule cells also respond to the mixed group I/II agonist ACPD in slices (East and Garthwaite, 1992; Rossi et al., 1996) and in cultures (Chavis et al., 1995),
812
P. Vetter et al. / Neuropharmacology 38 (1999) 805–815
which, according to our results, most likely represents the activation of group I rather than group II mGluRs. In situ hybridisation and immunohistochemical studies show low levels of mGluR1 in these cells (Shigemoto et al., 1992; Grandes et al., 1994) whereas mGluR5 is apparently absent (Abe et al., 1992; Shigemoto et al., 1993). We can therefore conclude that mGluR1 are the receptors most likely to be responsible for the effects of DHPG in pial slices. Application of ACPD during synaptic activation of NMDA receptors causes a long term potentiation of transmission which has been interpreted as being mediated by postsynaptic mGluR activation (Rossi et al., 1996). It has not yet been demonstrated that these receptors can be activated by synaptic stimulation (c.f. mGluR1 in Purkinje cells; Batchelor and Garthwaite, 1997). The depression of transmission induced by group I agonists could be due to the postsynaptic effects discussed above or, alternatively, there could be an additional group I mGluR presynaptic on mossy fibre terminals. Further experiments are necessary to distinguish these two possibilities.
4.2.2. Group II mGluRs The enhancement of the synaptic response by DCGIV is unlikely to be due to a direct postsynaptic mechanism, since group II receptors could not be detected in granule cells electrophysiologically, immunohistochemically or in in situ hybridisation experiments (Ohishi et al., 1993a,b; Tanabe et al., 1993; Ohishi et al., 1994, 1998 but see Chavis et al., 1994). A facilitation of transmitter release could potentially explain the effects of DCG-IV but this has only been reported for presynaptic group I mGluRs, not group II mGluRs (Herrero et al., 1992; Collins and Davies, 1993). We therefore considered it unlikely that the effect of DCG-IV was due to pre- or postsynaptic actions at the mossy fibre to granule cell synapse, implying that DCG-IV was acting in a less direct manner. A revealing observation was that DCG-IV caused an enhancement of the synaptic response similar to that caused by the GABAA receptor antagonist bicuculline. Moreover, bicuculline treatment occluded the effect of DCG-IV. These data suggest a shared mechanism, the simplest of which is that both bicuculline and DCG-IV are disabling the tonic in-
Fig. 5. Effects of bicuculline on the synaptic response and its potentiation by DCG-IV. Timecourse of mossy fibre synaptic response (c) and example traces (a) and (b) from a single experiment (roman numerals indicate where sample traces are extracted from). Application of bicuculline (bic, 30 mM, hollow bar) causes a 50% increase in the amplitude of the mossy fibre response (i) and (ii) co-perfusion of DCG-IV (300 nM, grey bar) has no effect (iii). On washout of bicuculline for 24 min the response amplitude decayed to control levels ((b)-iv ‘post-bic’) and (c). A second application of DCG-IV (v) causes the usual 40% potentiation. (d) Cumulative data from three slices indicating that DCG-IV-mediated potentiation (140 92%) of the mossy fibre response was reduced in the presence of bicuculline (101 9 1%).
P. Vetter et al. / Neuropharmacology 38 (1999) 805–815
813
Fig. 6. DCG-IV inhibits spontaneous inhibitory currents in cerebellar granule cells. (a) Patch-clamp recording of spontaneous inhibitory postsynaptic currents (sIPSC) in the presence of NBQX (10 mM) and AP5 (30 mM) to block excitatory currents. Application of DCG-IV (300 nM, time of application represented by solid bar) caused a rapid inhibition of the sIPSCs which reappeared upon washout of DCG-IV for 300 s. Application of the GABAA receptor antagonist bicuculline methochloride (‘BIC’) caused a similar reversible inhibition of the IPSCs. (b) Schematic diagram for the proposed role of group II mGluRs in the mossy fibre pathway. Firing of mossy fibres causes glutamate release, which diffuses to the terminals of nearby Golgi cells and activates group II mGluRs. The subsequent inhibition of GABA release from the axon terminals relieves the tonic GABAergic inhibition of the granule cells, thus leading to a net facilitation of glutamatergic transmission at the mossy fibre-granule cell synapse.
hibitory activity: bicuculline, by competitive blockade of the postsynaptic GABAA receptors and DCG-IV, by acting on Golgi cell terminals to inhibit GABA release. Further support for the hypothesis is provided by the finding that sIPSC’s recorded from granule cells using the patch-clamp technique were abolished when DCG-IV (0.1–1 mM) was applied (Fig. 6a). In situ hybridisation experiments suggest that expression of group II mGluRs is highest in the GABAergic Golgi cells (Ohishi et al., 1993a,b; Tanabe et al., 1993) and there is immunohistochemical evidence that these receptors are highly concentrated in the axon terminals (Ohishi et al., 1994, 1998). Similar disinhibition elicited by application of group II mGluR agonists has been reported in several other brain areas (Hayashi et al., 1993; Desai et al., 1994; Poncer et al., 1995). For this mechanism to be of physiological relevance, glutamate would have to diffuse from mossy fibre terminals to presynaptic group II mGluRs on adjacent
Golgi cell terminals (Barbour and Ha¨usser, 1997; Kullman and Asztely, 1998). This scenario is most likely to occur during high frequency firing of mossy fibres as observed in vivo (Lisberger and Fuchs, 1978), when large amounts of glutamate are released. Furthermore, Golgi cells terminals are in close proximity ( B1 mm), and group II mGluRs bind glutamate with relatively high affinity. It is poorly understood, how the high frequency firing of mossy fibres is transformed into the lower frequency patterns of firing observed in granule cells (Gabbiani et al., 1994). It has been postulated that the high levels of Golgi cell activity exert a strong inhibitory effect on transmission at mossy fibre-granule cell synapse (Gabbiani et al., 1994; Brickley et al., 1996). Group II mGluRs may switch this inhibition off, thus transiently enhancing communication at the mossy fibre-granule cell synapse, and thereby contributing to the signal modulation from mossy fibre
814
P. Vetter et al. / Neuropharmacology 38 (1999) 805–815
input to parallel fibre output in an activity dependent manner.
4.2.3. Group III mGluRs The hyperpolarisation in pial slices due to activation of group III mGluRs is most reasonably explained by the receptors being located on the granule cells. In situ hybridisation studies indicate that mRNA encoding for GluR4 is highly expressed in granule cells of adult animals whereas that for mGluR6-8 is not (Tanabe et al., 1993, 1993; Duvoisin et al., 1995; Kinzie et al., 1995). However, immunocytochemistry locates these receptors in the molecular layer of the cerebellar cortex, suggesting that they are inserted in the membrane of parallel fibre terminals rather than somato-dendritically. Consistent with this is data that AP4 depresses parallel fibre-Purkinje cell transmission (Pekhletski et al., 1996). The grease-gap recording from pial slices cannot differentiate between signals coming from the axon terminals or from the somato-dendritic end of the granule cells and so it is therefore possible that the hyperpolarisation that we observed could originate in the parallel fibre terminals.
5. Conclusions In this study, group selective mGluR ligands were characterised in rat cerebellar slices. Their actions provide evidence for the existence of group I (probably mGluR1) and group III mGluRs in granule cells. Synaptic transmission in the mossy fibre pathway was depressed by activation of group I mGluRs but was unaffected by AP4 and DCG-IV (in the presence of bicuculline), suggesting that both group III and group II mGluRs are absent from mossy fibre terminals. This is unusual for a glutamatergic synapse (Pin and Duvoisin, 1995; Conn and Pin, 1997). Activation of group II mGluRs facilitated synaptic transmission; it is proposed that this is due to inhibition of GABA release at nearby Golgi axon terminals, an effect which may play an important role in information transfer at mossy fibre-granule cell synapses.
Acknowledgements This study was supported by a Wellcome Trust project grant (JG and AMB) and a Wellcome Trust 4-year Neuroscience Prize Studentship to PV.
References Abe, T., Sugihara, H., Nawa, H., Shigemoto, R., Mizuno, N., Nakanishi, S., 1992. Molecular characterization of a novel
metabotropic glutamate receptor GluR5 coupled to inositol phosphate/Ca2 + signal transduction. J. Biol. Chem. 267, 13361– 13368. Albani-Torregrossa, S., Pellicciari, R., Moroni, F., Pellegrini-Giampietro, E., 1998. Metabotropic glutamate receptors coupled to phospholipase D. In: Moroni, F., Nicoletti, F., Pellegrini-Giampietro, D.E. (Eds.), Metabotropic Glutamate Receptors and Brain Function. Portland Press, London, pp. 251 – 260. Barbour, B., Ha¨usser, M., 1997. Intersynaptic diffusion of neurotransmitter. Trends Neurosci. 20, 377 – 384. Batchelor, A.M., Kno¨pfel, T., Gasparini, F., Garthwaite, J., 1997. Pharmacological characterization of synaptic transmission through mGluRs in rat cerebellar slices. Neuropharmacology 36, 401 – 403. Batchelor, A.M., Garthwaite, J., 1997. Frequency detection and temporally dispersed synaptic signal association through a metabotropic glutamate receptor pathway. Nature 385, 74–77. Bedingfield, J.S., Jane, D.E., Kemp, M.C., Toms, N.J., Roberts, P.J., 1996. Novel potent selective phenylglycine antagonists of metabotropic glutamate receptors. Eur. J. Pharmacol. 309, 71 –78. Breakwell, N.A., Huang, L., Rowan, M.J., Anwyl, R., 1997. DCG-IV inhibits synaptic transmission by activation of NMDA receptors in area CA1 of rat hippocampus. Eur. J. Pharmacol. 322, 173– 178. Brickley, S.G., Cull-Candy, S.G., Farrant, M., 1996. Development of a tonic form of synaptic inhibition in rat cerebellar granule cells resulting from persistent activation of GABAA receptors. J. Physiol. 497, 753 – 759. Bushell, T.J., Jane, D.E., Tse, H.-W., Watkins, J.C., Garthwaite, J., Collingridge, G.L., 1996. Pharmacological antagonism of the actions of group II and III mGluR agonists in the lateral perforant path of rat hippocampal slices. Br. J. Pharmacol. 117, 1457 – 1462. Chavis, P., Shinozaki, H., Bockaert, J., Fagni, L., 1994. The metabotropic glutamate receptor types 2/3 inhibit L-type calcium channels via a pertussis toxin-sensitive G-protein in cultured cerebellar granule cells. J. Neurosci. 14, 7067 – 7076. Chavis, P., Nooney, J.M., Bockaert, J., Fagni, L., Feltz, A., Bossu, J.L., 1995. Facilitatory coupling between a glutamate metabotropic receptor and dihydropyridine-sensitive calcium channels in cultured cerebellar granule cells. J. Neurosci. 15, 135 – 143. Collins, D.R., Davies, S.N., 1993. Co-administration of (1S,3R)-1aminocyclopentane-1,3-dicarboxylic acid and arachidonic acid potentiates synaptic transmission in rat hippocampal slices. Eur. J. Pharmacol. 240, 325 – 326. Conn, P.J., Pin, J.-P., 1997. Pharmacology and functions of metabotropic glutamate receptors. Ann. Rev. Pharmacol. Toxicol. 37, 205 – 237. Crepel, F., Daniel, H., Hemart, N., Jaillard, D., 1991. Effects of ACPD and AP3 on parallel-fibre-mediated EPSPs of Purkinje cells in cerebellar slices in vitro. Exp. Brain Res. 86, 402–406. Desai, M.A., McBain, C.J., Kauer, J.A., Conn, P.J., 1994. Metabotropic glutamate receptor-induced disinhibition is mediated by reduced transmission at excitatory synapses onto interneurons and inhibitory synapses onto pyramidal cells. Neurosci. Lett. 181, 78 – 82. Duvoisin, R.M., Zhang, C., Ramonell, K., 1995. A novel metabotropic glutamate receptor expressed in the retina and olfactory bulb. J. Neurosci. 15, 3075 – 3083. East, S.J., Garthwaite, J., 1992. Actions of a metabotropic glutamate receptor agonist in immature and adult rat cerebellum. Eur. J. Pharmacol. 219, 395 – 400. Eccles J., Ito M., Szentagothai J., 1967. The Cerebellum as a Neuronal Machine, Springer-Verlag, Berlin. Gabbiani, F., Midtgaard, J., Knopfel, T., 1994. Synaptic integration in a model of cerebellar granule cells. J. Neurophysiol. 72, 999– 1009.
P. Vetter et al. / Neuropharmacology 38 (1999) 805–815 Garthwaite, J., Batchelor, A.M., 1996. A biplanar slice preparation for studying cerebellar synaptic transmission. J. Neurosci. Methods 64, 189 – 197. Garthwaite, J., Brodbelt, A.R., 1989a. Glutamate as the principal mossy fibre transmitter in rat cerebellum: pharmacological evidence. Eur. J. Neurosci. 2, 177–180. Garthwaite, J., Brodbelt, A.R., 1989b. Synaptic activation of Nmethyl-D-aspartate and non-N-methyl-D-aspartate receptors in the mossy fibre pathway in adult and immature rat cerebellar slices. Neuroscience 29, 401 –412. Grandes, P., Mateos, J.M., Ru¨egg, D., Kuhn, R., Kno¨pfel, T., 1994. Differential cellular localization of three splice variants of the mGluR1 metabotropic glutamate receptor in rat cerebellum. NeuroReport 5, 2249 – 2252. Hartell, N.A., 1994. Induction of cerebellar long-term depression requires activation of glutamate metabotropic receptors. NeuroReport 5, 913 – 916. Harvey, J., Collingridge, G.L., 1993. Signal transduction pathways involved in the acute potentiation of NMDA responses by 1S,3RACPD in rat hippocampal slices. Br. J. Pharmacol. 109, 1085 – 1090. Hayashi, Y., Momiyama, A., Takahashi, T., Ohishi, H., Ogawa Meguro, R., Shigemoto, R., Mizuno, N., Nakanishi, S., 1993. Role of a metabotropic glutamate receptor in synaptic modulation in the accessory olfactory bulb. Nature 366, 687–690. Hayashi, Y., Sekiyama, N., Nakanishi, S., Jane, D.E., Sunter, D.C., Birse, E.F., Udvarhelyi, P.M., Watkins, J.C., 1994. Analysis of agonist and antagonist activities of phenylglycine derivatives for different cloned metabotropic glutamate receptor subtypes. J. Neurosci. 14, 3370 – 3377. Herrero, I., Miras Portugal, M.T., Sanchez Prieto, J., 1992. Positive feedback of glutamate exocytosis by metabotropic presynaptic receptor stimulation. Nature 360, 163–166. Ito, I., Kohda, A., Tanabe, S., Hirose, E., Hayashi, M., Mitsunaga, S., Sugiyama, H., 1992. 3,5-Dihydroxyphenyl-glycine: a potent agonist of metabotropic glutamate receptors. NeuroReport 3, 1013 – 1016. Kaneda, M., Farrant, M., Cull-Candy, S.G., 1995. Whole-cell and single-channel currents activated by GABA and glycine in granule cells of the rat cerebellum. J. Physiol. 485, 419–435. Kingston, A.E., Lodge, D., 1998. Pharmacology of group I metabotropic glutamate receptors. In: Moroni, F., Nicoletti, F., PellegriniGiampietro, D.E. (Eds.), Metabotropic Glutamate Receptors and Brain Function. Portland Press, London, pp. 261–269. Kinzie, J.M., Saugstad, J.A., Westbrook, G.L., Segerson, T.P., 1995. Distribution of metabotropic glutamate receptor 7 messenger RNA in the developing and adult rat brain. Neuroscience 69, 167 – 176. Kullman, D.M., Asztely, F., 1998. Extrasynaptic glutamate spillover in the hippocampus: evidence and implications. Trends Neurosci. 21, 8 – 14. Lisberger, S.G., Fuchs, A.F., 1978. Role of primate flocculus during rapid behavioral modification of vestibuloocular reflex II. Mossy fibre fring pattern during head rotation and eye movement. J. Neurophysiol. 41, 764–777. Ohishi, H., Shigemoto, R., Nakanishi, S., Mizuno, N., 1993a. Distribution of the mRNA for a metabotropic glutamate receptor (mGluR3) in the rat brain: an in situ hybridisation study. J. Comp. Neurol. 335, 252 – 266. Ohishi, H., Shigemoto, R., Nakanishi, S., Mizuno, N., 1993b. Distribution of the messenger RNA for a metabotropic glutamate receptor, mGluR2, in the central nervous system of the rat. Neuroscience 53, 1009–1018. Ohishi, H., Ogawa Meguro, R., Shigemoto, R., Kaneko, T., Nakanishi, S., Mizuno, N., 1994. Immunohistochemical localization of metabotropic glutamate receptors, mGluR2 and mGluR3, in rat cerebellar cortex. Neuron 13, 55–66. Ohishi, H., Neki, A., Mizuno, N., 1998. Distribution of a metabotropic glutamate receptor, mGluR2, in the central nervous system of the .
815
rat and mouse: an immunohistochemical study with a monoclonal antibody. Neurosci. Res. 30, 65 – 82. Pekhletski, R., Gerlai, R., Overstreet, L.S., Huang, X., Agopyan, N., Slater, N.T., Abramow-Newerly, W., Roder, J.C., Hampson, D.R., 1996. Impaired cerebellar synaptic plasticity and motor performance in mice lacking the mGluR4 subtype of metabotropic gluatamate receptor. J. Neurosci. 16, 6364 – 6373. Pellicciari, R., Marinozzi, M., Natalini, B., Costantino, G., Luneia, R., Giorgi, G., Moroni, F., Thomsen, C., 1996. Synthesis and pharmacological characterization of all sixteen stereoisomers of 2-(2%-carboxy-3%-phenylcyclopropyl)glycine. Focus on (2S,1%S,2%S,3%R)-2-(2%-carboxy-3%-phenylcyclopropyl)glycine, a novel and selective group II metabotropic glutamate receptors antagonist. J. Med. Chem. 39, 2259 – 2269. Pin, J.-P., Duvoisin, R., 1995. The metabotropic glutamate receptors: structure and functions. Neuropharmacology 34, 1 – 26. Pisani, A., Calabresi, P., Centonze, D., Bernardi, G., 1997. Activation of group III metabotropic glutamate receptors depresses glutamatergic transmission at corticostriatal synapse. Neuropharmacology 36, 845 – 851. Poncer, J., Shinozaki, H., Miles, R., 1995. Dual modulation of synaptic inhibition by distinct metabotropic glutamate receptors in the rat hippocampus. J. Physiol. 485, 121 – 134. Puia, G., Costa, E., Vicini, S., 1994. Functional diversity of GABA-activated Cl − currents in Purkinje versus granule neurons in rat cerebellar slices. Neuron 12, 117 – 126. Rossi, P., D’Angelo, E., Taglietti, V., 1996. Differential long-lasting potentiation of the NMDA and non-NMDA synaptic currents induced by metabotropic and NMDA receptor coactivation in cerebellar granule cells. Eur. J. Neurosci. 8, 1182 – 1189. Salt, T.E., Turner, J.P., 1996. Antagonism of the presumed presynaptic action of L-AP4 on GABAergic transmission in the ventrobasal thalamus by the novel mGluR antagonist MPPG. Neuropharmacology 35, 239 – 241. Scanziani, M., Salin, P.A., Vogt, K.E., Malenka, R.C., Nicoll, R.A., 1997. Use-dependent increases in glutamate concentration activate presynaptic metabotropic glutamate receptors. Nature 385, 630– 634. Shigemoto, R., Nakanishi, S., Mizuno, N., 1992. Distribution of the mRNA for a metabotropic glutamate receptor (mGluR1) in the central nervous system: an in situ hybridization study in adult and developing rat. J. Comp. Neurol. 322, 121 – 135. Shigemoto, R., Nomura, S., Ohishi, H., Sugihara, H., Nakanishi, S., Mizuno, N., 1993. Immunohistochemical localisation of a metabotropic glutamate receptor, mGluR5, in the rat brain. Neurosci. Lett. 163, 53 – 57. Tanabe, Y., Nomura, A., Masu, M., Shigemoto, R., Mizuno, N., Nakanishi, S., 1993. Signal transduction, pharmacological properties, and expression patterns of two rat metabotropic glutamate receptors, mGluR3 and mGluR4. J. Neurosci. 13, 1372–1378. Thomsen, C., Boel, E., Suzdak, P.D., 1994. Actions of phenylglycine analogs at subtypes of the metabotropic glutamate receptor family. Eur. J. Pharmacol. Mol. Pharmacol. 267, 77 – 84. Thomsen, C., Bruno, V., Nicoletti, F., Marinozzi, M., Pellicciari, R., 1996. (2S, 1%S, 2%S, 3%R)-2-(2%-carboxy-3%-phenylcyclopropyl)glycine, a potent and selective antagonist of type 2 metabotropic glutamate receptors. Mol. Pharmacol. 50, 6 – 9. Toms, N.J., Jane, D.E., Kemp, M.C., Bedingfield, J.S., Roberts, P.J., 1996. The effects of (RS)-alpha-cyclopropyl-4-phosphonophenylglycine ((RS)-CPPG), a potent and selective metabotropic glutamate receptor antagonist. Br. J. Pharmacol. 119, 851 – 854. Wall, M.J., Usowicz, M.M., 1997. Development of action potential-dependent and independent spontaneous GABAA receptor-mediated currents in granule cells of postnatal rat cerebellum. Eur. J. Neurosci. 9, 533 – 548. Wilsch, V.W., Pidoplichko, V.I., Opitz, T., Shinozaki, H., Reymann, K.G., 1994. Metabotropic glutamate receptor agonist DCG-IV as NMDA receptor agonist in immature rat hippocampal neurons. Eur. J. Pharmacol. 262, 287 – 291.