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Complex interactions between mGluR1 and mGluR5 shape neuronal network activity in the rat hippocampus
Neuropharmacology 43 (2002) 131–140 www.elsevier.com/locate/neuropharm
Complex interactions between mGluR1 and mGluR5 shape neuronal network activity in the rat hippocampus Christophe Lanneau a, Mark H. Harries b,∗, Alison M. Ray a, Stuart R. Cobb c, Andrew Randall a, Ceri H. Davies a b
a Neurology CEDD, GlaxoSmithKline, Third Avenue, Harlow Essex CM19 5AW UK Psychiatry CEDD, GlaxoSmithKline Pharmaceuticals Plc, New Frontiers Science Park North, Third Avenue, Harlow, Essex CM19 5AW, UK c Neuroscience & Biomedical Systems, West Medical Building, University of Glasgow, Glasgow G12 8QQ, UK
Received 31 October 2001; received in revised form 31 May 2002; accepted 11 June 2002
Abstract Group I metabotropic glutamate receptors (mGluRs) cause increased neuronal excitability that can lead to epileptogenesis and neurodegeneration. Here we have examined how individual members of this subgroup of mGluRs affect synchronised hippocampal synaptic activity under normal and disinhibited conditions similar to those that occur during certain epileptic states. We demonstrate that activation of both mGluR1 and mGluR5 are important in increasing neuronal synaptic excitability by increasing synchrony between cells and driving correlated network activity in circuits that contain, or are devoid of, GABAA receptor-mediated synaptic inputs. The precise patterning of activity that occurs is complex and depends upon: (1) the existing pattern of ongoing network activity prior to mGluR activation; and (2) the relative extent of activation of each mGluR subtype. However, mGluR5 appears to be the principal mGluR subtype that initiates bursting activity irrespective of the inhibitory synaptic tone within the neuronal network. 2002 Elsevier Science Ltd. All rights reserved. Keywords: Metabotropic glutamate receptors; mGluR1; mGluR5, MPEP; LY367385; Theta rhythm; Multielectrode array
1. Introduction Synchronous discharges of groups of neurones in the vertebrate central nervous system play an important role in normal physiological processing of sensory information as well as in neuropathophysiological disease states such as epilepsy (Vanderwolf, 1969; Buzsa´ki et al., 1983, 1994; Singer, 1993; Jefferys et al., 1996). Numerous patterns of synchronous activity (e.g., β-, γand θ-rhythms, ripples, sharp waves and dentate spikes) have been recorded both in vitro and in vivo, often in association with particular patterns of behaviour. Whilst the mechanism of generation of many of these rhythmical discharges is not yet fully understood, there is substantial evidence to support the concept that each pattern of activity is critically dependent upon: (1) the intrinsic
Corresponding author. Tel.: +44-127-962-2124; fax: +44-127962-2555. E-mail address: [email protected] (M.H. Harries). ∗
membrane properties of each of the neurones within the synaptic circuit (Leung and Yim, 1991; Strata, 1998; Pike et al., 2000); and (2) the close integration of fast ionotropic and slower metabotropic receptor-mediated synaptic inputs (Whittington et al., 1995; Taylor et al., 1995; Boddeke et al., 1996; Konopacki et al., 1987; MacVicar and Tse, 1989; Williams and Kauer, 1997; Fisahn et al., 1998; McMahon et al., 1998; Cobb et al., 1999, 2000). Relevant to this last point, each neurotransmitter-mediated synaptic input to a neuronal network, whether it originates from within (intrinsic neuromodulatory input) or without the hippocampus (extrinsic neuromodulatory input), can shape the pattern of activity through pre- and post-synaptic mechanisms that change neuronal excitability as well as synaptic transmission (Buzsa´ki et al., 1994; Jefferys et al., 1996). Metabotropic glutamate receptor (mGluR)-mediated synaptic inputs are ideally equipped to fulfill these roles (Desai et al., 1992; Davies et al., 1995; Vignes et al., 1995) and, as such, there is a rapidly expanding literature on the effects of different mGluR subtypes on neuronal network func-
0028-3908/02/$ - see front matter 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 0 2 ) 0 0 0 8 6 - 2
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tion (Whittington et al., 1995; Taylor et al., 1995; Rutecki and Yang, 1997; Cobb et al., 2000). At least eight subtypes of mGluR have been cloned which can be subdivided into three classes on the basis of their pharmacology and coupling to signal transduction pathways (Nakanishi, 1992; Pin and Duvoisin, 1995; Schoepp et al., 1999). With reference to epilepsy, whilst all three groups of mGluR have modulatory actions (Sacaan and Schoepp, 1992; Merlin et al., 1995; Burke and Hablitz, 1995; Miyamoto et al., 1997; Rutecki and Yang, 1997) it is generally accepted that antagonists of group I mGluRs and agonists of group II and III mGluRs have the potential to be good anticonvulsants (Camo´ n et al., 1998; Wong et al., 1999). However, the lack of selective pharmacological agents with which to manipulate the function of individual mGluR subtypes (Watkins and Collingridge, 1994) has hampered investigations into the contribution that each subtype makes to shaping synaptic activity in neuronal networks. Given that in many brain regions, individual mGluR subtypes can be differentially restricted to specific populations of neurones which subserve specialized physiological roles (Romano et al., 1995; Lujan et al., 1996; Ferraguti et al., 1998; van Hooft et al., 2000) it is clear that such drugs are likely to have quite distinct actions to those which interfere with multiple subtypes. Until recently non-conditional transgenic knockout strategies have provided the principal approaches for studying the function of individual group I mGluR subtypes (Aiba et al., 1994; Conquet et al., 1994; Lu et al., 1997). However, these studies are subject to a range of potential genetic, developmental and compensatory limitations that can complicate interpretation of experimental results (Gerlai and Clayton, 1999). The recent development of selective antagonists for individual group I mGluR subtypes, however, now opens up the possibility of examining the functional roles of these mGluR subtypes in more detail, through acute manipulation of their function. The aim of the present study therefore was to use the selective mGluR1 and mGluR5 antagonists LY367385 (Clark et al., 1997) and MPEP (Gasparini et al., 1999) to investigate how these mGluR subtypes modify synchronised bursting activity in the hippocampus.
2. Methods 2.1. Slice preparation Experiments were performed on acutely prepared transverse hippocampal slices and organotypic hippocampal slice cultures. Organotypic slice cultures were prepared using methods similar to Stoppini et al. (1991). For acute slices, 3–4-week old male Wistar rats were sacrificed by dislocation of the neck and subsequent decapitation in accordance with UK Home Office guide-
lines. The brain was removed rapidly and 400 µm thick, transverse hippocampal slices were cut using a Campden vibroslicer. Slices were left to equilibrate in artificial cerebrospinal fluid (aCSF) at room temperature for at least 1 h prior to recording. 2.2. Electrophysiological recordings Synaptic activity was recorded using either whole cell patch clamp, intracellular sharp electrode or extracellular multielectrode array recording approaches. In all recording configurations, slices were bathed in warmed (35–37 °C), perfusing (2–4 ml.min⫺1) aCSF comprised of (mM): NaCl, 124; KCl, 3; NaHCO3, 26; NaH2PO4, 1.25; CaCl2, 2; MgSO4, 1; d-glucose, 10; and bubbled with 95% O2, 5% CO2. Intracellular current clamp recordings of CA3 pyramidal cells from acute hippocampal slices were made in bridge balanced current clamp mode (Axoclamp 2B amplifier) with glass microelectrodes (60–120 M⍀) filled with 2 M potassium methylsulphate. Whole cell patch clamp recordings from CA1 and CA3 pyramidal cells in organotypic hippocampal slices were made under visual guidance using an Olympus BX50WI upright microscope. Microelectrodes (5–8 M⍀) connected to an Axopatch 200B amplifier were filled with (mM): K-Gluconate, 135; MgCl2, 10; HEPES, 10; Mg ATP, 2; EGTA, 1; Na GTP, 0.3; CaCl2 , 0.1 at pH 7.2 and 290–295 mOsmol. Double patch clamp recordings were performed using the same approaches but with two Axopatch 200B amplifiers. Cell viability was assessed throughout the course of experiments via measurement of cell input resistance. Patch clamp data were filtered at 5 kHz, digitised at 20 kHz and stored on a PC hard disk drive for off-line analysis using Clampfit 8.2 (Axon Instruments Inc). Extracellular recordings of field potentials from organotypic hippocampal slices were made from all subfields using a planar multielectrode array (MEA) purchased from MultiChannel Systems (GmbH Reutlingen). A small piece of semiporous membrane bearing the hippocampal slice was cut out of the culture well using a scalpel blade. This was positioned slice side down onto an MEA, which had previously been coated using 5 µl of a solution comprising 1 cm2 cellulose nitrate filter paper dissolved in 10 ml methanol. Final orientation of the slice with respect to the electrodes was achieved with the aid of a Nikon SMZ 800 microscope. The MEA recording chamber was then immediately flooded with 1 ml of aCSF and a photographic image taken of the electrode positions beneath the hippocampal subfields (Nikon D1 digital camera). MEA extracellular recordings were made from all subfields of the slice using the procedures outlined by Egert et al. (1998). MEA data were sampled at 20 kHz per electrode, stored on hard disk or CD-R and
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analysed off-line using MCS and Neuroexplorer (Plexon Instruments Inc.) software. 2.3. Drugs In all experiments drugs were applied by addition to the perfusion medium. Bicuculline methiodide and tetrodotoxin citrate (TTX) were obtained from Sigma (Poole, Dorset UK). (S)-3,5-dihydroxyphenylglycine (DHPG), (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), [1-(S)-3,4-dichlorophenyl)ethyl]amino-2-(S)hydroxypropyl-p-benzyl-phosphonic acid (CGP55845A), 2-methyl-6-(phenylethynyl)pyridine (MPEP), (S)-(+)-α-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385), 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulphonamide disodium (NBQX) and d(⫺)-2-amino-5-phosphonopentanoate (AP5) were purchased from Tocris Cookson (Bristol, UK) and made up as concentrated stock solutions as suggested in accompanying data sheets. As such, all drugs were made up in water or equimolar sodium hydroxide which on their own produced no effects in any of the experiments undertaken as part of this study. Metabotropic glutamate receptor agonists were applied for 6– 12 min which was sufficient to enable changes in bursting activity to stabalize. mGluR antagonists were applied 6 min after DHPG induced changes in bursting activity had stabilized and their application sustained for such time as required to achieve maximal antagonism at each concentration tested. This study deals specifically with burst activity which was defined as excitatory postsynaptic potentials or currents (epsps or epscs) that have durations in excess of 150 ms. As such, all spontaneous stochastic events of shorter duration, such as those superimposed on bursting events illustrated in Figs. 2A and 3A, are excluded from the current analyses. All data are presented as means±standard error of the mean (S.E.M.) and statistical significance was assessed using ANOVA, paired or unpaired Student’s t-tests performed on raw data with P⬍0.05 being taken as indicating statistical significance.
3. Results 3.1. mGluR driven population activity In acutely prepared hippocampal slices bath application of the selective mGluR agonist ACPD (100–200 µM; n=6) induced a characteristic pattern of synchronised bursting activity recorded in CA3 pyramidal cells using either intracellular or whole cell patch clamp recording techniques. This pattern of activity consisted of 1–3 s long bursts of synchronous epsps occurring every 10–30 s that was abolished by the AMPA/kainate recep-
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tor antagonist NBQX (5 µM; n=3) and the voltage gated Na+ channel blocker TTX (1 µM; n=3) but not by the selective NMDA receptor antagonist AP5 (20 µM; n=3) illustrating that mGluR-induced bursting principally relies upon AMPA/kainate receptor subtype-mediated synaptic activity. This pattern of activity could be reproduced using the group I mGluR selective agonist DHPG (Fig. 1A) and autocorrelation analysis revealed that activity within bursting events was rhythmic at frequencies in the theta range of 4–12 Hz (mean frequency: 7.9±1.0 Hz for DHPG) (Fig. 1A). In some recordings there was the additional presence of some γ frequency (20–40 Hz) activity. Similar patterns of activity were recorded with dual patch-clamp recording techniques in organotypic hippocampal slices illustrating that this activity was independent of intracellular dialysis of individual neurones (which greatly reduces direct mGluR induced postsynaptic inward currents) and confirmed that it was a consequence of surrounding network activity (Fig. 1B). This was supported by multielectrode array (MEA) extracellular recording which clearly showed that DHPG-induced population bursts originated in area CA3 from where they propagated into area CA1 with a 2 ms delay (Fig. 1C). In all experiments brief drug applications were used (except where otherwise stated) to restrict the study to examination of the mechanisms responsible for the induction of mGluR-induced bursting rather than the prolonged maintenance of bursting described by Galoyan and Merlin (2000) that persists following the washout of mGluR agonists after hour long agonist exposures. To establish whether activation of individual mGluR subtypes could generate the bursting activity described in detail above we examined with the whole cell voltageclamp technique: (1) whether the mGluR5 selective agonist CHPG could initiate bursts of excitatory post synaptic currents (epscs) in CA3 pyramidal cells; and (2) how the mGluR1 and mGluR5 selective antagonists LY367385 and MPEP affected previously established DHPG-driven synchronised hippocampal activity. At 5 mM CHPG induced synchronised bursting activity which reoccurred at a frequency of 0.032±0.012 Hz (n=9) which was significantly lower than the 0.057±0.012 Hz induced by 20 µM DHPG (P⬍0.01, n=12) (Fig. 2A); a near maximal concentration of DHPG for inducing bursting activity (Fig. 2B). Higher concentrations of CHPG could not be tested because of the limited solubility of this compound. Despite this, the effectiveness of CHPG suggested that activation of mGluR5 alone was sufficient to induce synchronised bursting activity. However, it was still quite possible that mGluR1 contributed to the effects induced by DHPG. In this respect, we were not surprised to find that both LY367385 and MPEP reversed bursting activity induced by DHPG (Fig. 3A). In line with their reported affinities for mGluRs 1
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Fig. 1. Intraburst event frequency is rhythmic and occurs in the theta frequency range (4–8 Hz). (A) Current clamp traces from an intracellular recording of DHPG driven synchronised bursting activity in an acutely prepared hippocampal slice on a range of time bases and at the membrane potentials indicated. In the top most trace the cell was hyperpolarized during the period between the arrows in order to eliminate action potential firing. Below these traces are the autocorrelation and power spectrum plots generated from this synaptic activity. (B) This is similar to (A) except that activity recorded simultaneously from a CA3 and a CA1 pyramidal neurone in an organotypic hippocampal slice is illustrated. Note that in both preparations synaptic activity was rhythmic and that the predominant frequency of activity was approximately 5 Hz. (C) The uppermost panel is a micrograph of an organotypic hippocampal slice mounted on a multielectrode array. Subfield cell layers and the outline of the organotypic slice are highlighted with dashed lines. The squares indicate the position of two electrodes beneath CA3 and CA1 subfields. The gridded traces below are representative 1 s sweeps taken from 20 electrodes central to the slice in control medium (left hand trace) and in the presence of 20 µM DHPG (right hand trace). The traces in the lower left panel of (C) represent synaptic activity from the two electrodes boxed in above and which are in contact with areas CA3 and CA1. The time base has been expanded to illustrate that activity originated in CA3 and was propagated to area CA1. The event frequency histogram beside these traces illustrates the reproducible occurrence of bursts every 20 s.
and 5 multielectrode array analysis revealed that MPEP was more potent at blocking DHPG-induced bursting than LY367385 (Fig. 3C). The greater potency of MPEP was accentuated when these pharmacological studies were re-performed using longer exposure times to low concentrations of each antagonist. In particular, whereas the antagonist action of LY367385 was independent of exposure time, multielectrode array recordings revealed that MPEP, when applied at 1 µM for 1–2 h, produced greater antagonism than when applied for 20–30 min. As such, after 2 h its effects closely resembled those produced by 10–30 µM MPEP applied for 15–30 min (not illustrated); observations that may reflect the non competitive intra-transmembrane mechanism of action of this compound. Furthermore, in most experiments
MPEP actually abolished DHPG induced bursting whereas LY367385 never did (Fig. 3C). 3.2. Synchronised population activity in a disinhibited network Given that group I mGluRs had such pronounced effects on synchronised synaptic activity in normal aCSF we were interested to examine how mGluRs 1 and 5 affected coherent activity in a network in which fast synaptic inhibition was blocked using the competitive GABAA receptor antagonist bicuculline (10 µM) since this provides an alternative model which mimics patterns of activity observed during epileptiform activity in vivo. Bath application of bicuculline alone caused the appear-
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Fig. 2. Activation of group I mGluRs induces synchronised bursting. (A) Excerpts from a whole cell patch clamp recording of the activity of a single CA3 pyramidal neurone in control medium (top trace), in the presence of 5 mM CHPG, following washout of CHPG and in the presence of 20 µM DHPG (bottom trace). The smaller frequent events are asynchronous GABAergic and glutamatergic postsynaptic currents (pscs). Note that in the presence of both mGluR agonists the frequency and amplitude of these pscs increases and that superimposed on this activity there is the appearance of large bursts of activity. (B) The left hand graph is the concentration–response relationship for the effectiveness of DHPG to induce bursting activity. Scaling on the ordinate axis shows that data has been expressed relative to the concentration of DHPG which maximally potentiated bursting frequency in each experiment. The bar graph on the right shows the increases in burst frequency induced by ACPD and CHPG expressed relative to the increase induced by DHPG in five neurones. In this and all subsequent figures ∗ represents statistical significance at P⬍0.05. n numbers are denoted on individual bars.
ance of large bursting events (n=26; Fig. 4A) which increased in duration on subsequent application of DHPG (20 µM; n=18; Fig. 4A). CHPG (5 mM; n=8) also produced an increase in burst duration in disinhibited slices (Fig. 4). Consistent with the full and partial agonist activities of these compounds at group I mGluRs the mean duration of bursting events in the presence of the mGluR5 selective agonist CHPG (6.47±1.32 s) was significantly lower than that in the presence of the mGluR1/5 selective agonist DHPG (19.51±4.80 s). However, no significant difference in bursting frequency was observed (0.022±0.0041 Hz bicuculline, 0.018±0.0029 Hz DHPG and 0.025±0.0049 Hz CHPG) (Fig. 4B). Whilst these data again suggested that activation of
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mGluR5 was at least, in part, contributing to the effects of DHPG on disinhibition-induced bursting we attempted to establish a contribution of mGluR1 by examining how MPEP and LY367385 affected DHPG induced changes in bursting activity. MPEP (100 µM; n=5) produced a significant, yet still incomplete, reversal of the increase in burst duration induced by DHPG (Fig. 5A). These results were particularly interesting as bursts recorded in the presence of CHPG, in which mGluR5 alone should be activated, were very similar to those recorded in DHPG+MPEP, in which mGluR1 alone should be activated (Fig. 4A cf Fig. 5A). Even more perplexing was the effect of LY367385 (200 µM) on DHPG induced bursting. In particular, this antagonist had very variable effects on DHPG-induced increases in burst duration (Fig. 5A and B). This was because LY367385 consistently disrupted the coherence of bursts such that they became comprised of a complex series of events of variable duration and frequency (Fig. 5A and D). Interestingly, the selective NMDA receptor antagonist AP5 (20 µM; n=5) caused a similar splitting of bursting event waveform to that induced by LY367385 (Fig. 5D). These results were surprising in that MPEP, and not LY367385 (Faden et al., 2001), has previously been reported to exhibit NMDA receptor antagonist properties (O’Leary et al., 2000; Movsesyan et al., 2001). Irrespective of these results, coapplication of MPEP and LY367385 reversed DHPG–induced changes in bursting activity such that synaptic activity returned to a pattern that was not significantly different to that which occurred in bicuculline alone (Fig. 5B and C).
4. Discussion 4.1. Balanced inhibitory–excitatory networks The data generated here using a combination of complementary electrophysiological techniques confirm those of others (Taylor et al., 1995; Cobb et al., 1999, 2000) illustrating complex effects of group I mGluRs on the shaping of neuronal excitability within normal inhibitory–excitatory hippocampal networks. Here, synchronised bursting activity was induced by either the selective mGluR1/5 agonist, DHPG or the mGluR5 specific agonist CHPG. Notably, CHPG was always less effective than DHPG. Two possible explanations for these results are: (1) mGluR-induced bursting requires the activation of mGluR5 alone without mGluR1; the smaller effect of CHPG reflecting the partial agonist behaviour of CHPG at mGluR5 compared with the full agonist activity of DHPG at mGluR1/5 (Doherty et al., 1997); or (2) DHPG-induced changes in neuronal excitability require coactivation of mGluR1 and mGluR5; the effect of each receptor subtype being additive/synergistic to the other. To differentiate between these two possibilities we
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Fig. 3. Bursting activity depends upon the activation of both mGluR1 and mGluR5. (A) Traces are representative excerpts from an experiment in which 100 µM MPEP alone, 100 µM LY367385 alone and combined application of 100 µM MPEP and 100 µM LY367385 reversed 20 µM DHPG induced bursting activity. As in Fig. 2 the smaller frequent events are asynchronous GABAergic and glutamatergic postsynaptic currents (pscs). (B) Bar graph depicting pooled data from whole cell patch clamp studies as in (A) illustrating the burst frequency recorded under the experimental conditions described below each bar normalized to that recorded in the presence of 20 µM DHPG. ∗∗ represents statistical significance at P⬍0.01 (C) Concentration–response relationships for the antagonistic effects of MPEP and LY367385 on 20 µM DHPG induced bursting. Each data point has been normalised to the frequency of activity in DHPG alone. At 10, 30 and 100 µM MPEP reduced burst frequency significantly more than the respective concentration of LY 367385.
studied the effects of subtype selective group I mGluR antagonists on DHPG-induced changes in synaptic activity. Both mGluR1 and 5 selective antagonists inhibited DHPG induced bursting suggesting that both subtypes contribute to this phenomenon. In this respect, the concentrations of LY367385 necessary to cause antagonism of DHPG-driven bursting were generally in accord with its reported affinity at recombinant mGluR1 (LY367385=9 µM at mGluR1 and ⬎200 µM at mGluR5 (Bruno et al., 1999)). In contrast, relatively high concentrations of MPEP were required to block DHPG-induced effects despite its high affinity for cloned receptors (IC50 values against agonist induced PI hydrolysis: MPEP ⬎100 µM at mGluR1 and 36 nM at mGluR5; Gasparini et al. (1999). However, the effective concentration ranges of MPEP that have been used here are: (1) still below the concentrations required to antagonise mGluR1; and (2) are comparable to those required to block other electrophysiological and neurochemical effects of DHPG in other brain regions that are believed to be mediated by activation of mGluR5 (O’Leary et al., 2000; Pintor et al., 2000). The interesting observation that longer incubation times with MPEP increased the
apparent potency of this compound may be explained by slow kinetics of interaction between MPEP and mGluR5 because its binding site resides within transmembrane domains III and VII as opposed to the readily accessible N-terminal glutamate binding site (Pagano et al., 2000) where LY367385, a competitive antagonist, presumably binds to mGluR1. Irrespective of this, we are confident that the effects of LY367385 and MPEP are due to selective antagonism of mGluR1 and mGluR5, respectively and that both mGluR1 and mGluR5 contribute to synchronised population bursting. Interestingly, mGluR5 appeared to be particularly important in generating bursting activity since MPEP often abolished DHPG-induced bursting. In contrast, LY367385 produced only a 70– 80% inhibition suggesting that mGluR1 plays a secondary role to mGluR5 in initiating bursting activity. These differing roles may, in part, reflect coupling to distinct signal transduction pathways. For example, in human embryonic kidney cells mGluR1 and 5 induce distinct intracellular Ca2+ rises because of differential activation of intracellular store and receptor mediated release of Ca2+ (Kawabata et al., 1998). Furthermore, differential activation of distinct signal transduction pathways in
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Fig. 4. Activation of group I mGluRs alters burst waveform in disinhibited hippocampal networks. (A) Traces are whole cell patch clamp recordings in control medium, in the presence of 10 µM bicuculline , in the combined presence of bicuculline plus 20 µM DHPG, following washout back into 10 µM bicuculline and in the subsequent combined presence of bicuculline plus 5 mM CHPG. Note that whilst CHPG produced a widening of burst duration this effect was much smaller than that induced by DHPG. The holding potential in this experiment was ⫺70 mV. (B) Pooled data for the duration and frequency of bursts recorded in bicuculline in the presence and absence of mGluR agonists. Absolute values are given for burst duration and frequency under the experimental conditions indicated.
separate neuronal populations may further contribute to the observed differences (van Hooft et al., 2000). 4.2. Disinhibited networks In disinhibited networks activation of mGluR1 and mGluR5 produced complex changes in ongoing bursting activity. In particular, each subtype produced quite distinct changes in burst waveform architecture which differed from the situation in an inhibited network where both subtypes essentially supported almost identical burst waveforms. Most notably, in the disinhibited network, mGluR5 activation assessed under two distinct experimental conditions, induced quite distinct effects. Thus, CHPG caused a relatively small increase in burst duration whereas in the combined presence of DHPG and LY367385 burst duration was increased to a level not significantly different to that induced by DHPG alone. However, in the presence of LY367385 DHPGinduced burst durations were more variable than in DHPG alone possibly reflecting the more sporadic occurrence of intraburst events (Fig. 5D). Providing LY367385 is causing a complete block of mGluR1 activation the apparent differences between the effects of CHPG alone and DHPG plus LY367385 could potentially be explained by differences in the magnitude of
mGluR5 activation produced by these two drug treatments. Thus, the weak partial mGluR5 agonist activity of CHPG may provide just sufficient impetus to extend the burst a small amount without promoting the activation of a sustained period of secondary discharge, whereas the greater activation of mGluR5 in the combined presence of DHPG+LY367385 may more strongly drive secondary discharge generation. The natural extension of these findings is that activation of mGluR1 is necessary to convert this type of disjointed intraburst discharge into more of a continuum of intraburst discharge observed when DHPG alone induces burst widening. Alternatively, LY367385 and/or CHPG may have additional pharmacological effects, unrelated to mGluR activity, that may account for the differences in bursting pattern observed in the presence of these compounds. That said, it was interesting that antagonism of NMDA receptors also caused DHPG-induced bursts to be disrupted as observed when mGluR5 alone was activated by application of DHPG and LY367385. This observation might suggest that mGluR1 enhances NMDA receptor activity thereby prolonging the initial phase of the burst (see Fig. 5D) which in turn presumably impacts on subsequent events, such that in the presence of DHPG secondary events are closely packed together and are superimposed on a prolonged envelope of inward current
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Fig. 5. Antagonism of DHPG induced bursting in disinhibited hippocampal networks. (A) Differential antagonism of 20 µM DHPG-induced bursting by 100 µM MPEP and 200 µM LY367385. Note that MPEP almost completely reversed the DHPG induced increase in duration of bursts in bicuculline treated slices without significantly affecting their frequency of occurrence. In contrast, LY367385 did not significantly reduce overall burst duration but split bursts up into a series of much smaller bursts that followed each other in quick succession. Note that subsequent application of both antagonists returned bursting waveforms close to those recorded with bicuculline alone. (B) Bar graph plotting pooled data for the overall burst duration in the drug treatments illustrated in (A). (C) Bar graph plotting burst frequency under these drug regimes. (D) Comparison of the effects of 100 µM LY367385 and 20 µM AP5 on 20 µM DHPG-induced bursting activity in bicuculline treated slices. Note the simlarity of the effects of LY367385 and AP5.
(Fig. 5). The mGluR1/NMDA receptor interaction could be mediated directly through interplay between mGluR1 and NMDA receptors or arise through alterations in the integration of diverse neuronal circuits. Indeed: (1) mGluR1 has been shown to be closely associated with NMDA receptors in the so-called hebbosome complex (Husi et al., 2000); and (2) there is a precedent for potentiating interactions between group I mGluRs and NMDA receptors (Doherty et al., 1997; Awad et al., 2000). Consistent with this, activation of mGluR1, in the combined presence of DHPG+MPEP, clearly increased burst duration. Interestingly, however, CHPG had a similar effect to DHPG+MPEP (Fig. 5A cf Fig. 4A) indicating that in disinhibited networks maximal activation of mGluR1 fulfils a similar function to presumed partial activation of mGluR5. Thus activation of mGluR1 and mGluR5 may still synergize to promote burst duration through promotion of NMDA receptor activity and thereby bind together intraburst events.
Given these findings mGluR5 can be perceived to be the major driving force behind prolonging overall burst duration with activation of mGluR1 playing a secondary complementary role to mGluR5, forcing intraburst events into more of a continuum of events rather than the less organized patterns observed when only mGluR5 is activated. This is somewhat akin to the situation described for networks in which synaptic inhibition is intact, where once again mGluR5 takes the lead in generating bursting activity with mGluR1 promoting this activity when activated. The only difference here is that in the disinhibited network mGluR5 activates pronounced secondary discharges that prolong the burst waveform far beyond that recorded in strongly inhibited networks. The precise biochemical and cellular mechanisms by which both mGluR subtypes have their effects requires extensive further investigation, but likely involves effects that are transmitted through the network rather than just simple postsynaptic modifications in the recorded postsynaptic neurone.
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4.3. Concluding remarks In conclusion, we have shown that both mGluR1 and mGluR5 have major roles to play in the complex rearrangement of membrane properties, synaptic timing and magnitude of synaptic inputs that leads to the generation of synchronised bursting activity. The net excitatory influence of both subtypes supports the concept that antagonism of each mGluR subtype alone, or in combination, is a useful strategy for affording neuroprotection during neurodegenerative insults (Bruno et al., 1999, 2000). However, more extensive experimentation is required to define precisely the contribution made by individual subtypes on separate populations of principal and non principal neurones to each pattern of network activity. Generally though mGluR5 appears to play the dominant role in generating bursting activity irrespective of the inhibitory synaptic status of the neuronal network.
Acknowledgements CL is an EU funded Framework V funded postdoctoral research fellow.
References Aiba, A., Chen, C., Herrup, K., Rosenmund, C., Stevens, C.F., Tonegawa, S., 1994. Reduced hippocampal long-term potentiation and context-specific deficit in associative learning in mGluR1 mutant mice. Cell 79 (2), 365–375. Awad, H., Hubert, G.W., Smith, Y., Levey, A.I., Conn, P.J., 2000. Activation of metabotropic glutamate receptor 5 has direct excitatory effects and potentiates NMDA receptor currents in neurons of the subthalamic nucleus. Journal of Neuroscience 20 (21), 7871–7879. Boddeke, H.G.W.M., Best, R., Boeijinga, P.H., 1996. Synchronous 20Hz rhythmic activation in hippocampal networks induced by activation of metabotropic glutamate receptors in vitro. Neuroscience 76, 635–658. Bruno, V., Ksiazek, I., Battaglia, G., Lukic, S., Leonhardt, T., Sauer, D., Gasparini, F., Kuhn, R., Nicoletti, F., Flor, P.J., 2000. Selective blockade of metabotropic glutamate receptor subtype 5 is neuroprotective. Neuropharmacology 39, 2223–2230. Bruno, V., Battaglia, G., Kingston, A., O’Neill, M.J., Catania, M.V., DiGrezia, R., Nicoletti, F., 1999. Neuroprotective activity of the potent and selective mGluR1 antagonist, (+)-2-methyl-4carboxyphenylglycine (LY367385): comparison with LY357366, a broader spectrum equal affinity fore mGlu1a and mGlu5 receptors. Neuropharmacology 38, 199–207. Burke, J.P., Hablitz, J.J., 1995. Modulation of epileptiform activity by metabotropic glutomate receptors in immature rat neocortex. Journal of Neurophysiology 73, 205–217. Buzsa´ ki, G., Bragin, A., Chrobak, J.J., Nadasdy, Z., Sı´k, A., Hsu, M., Ylinen, A., 1994. Oscillatory and intermittent synchrony in the hippocampus: relevance to memory trace formation. In: Buzsa´ ki, G., Llinas, R., Singer, W., Berthroz, A., Christen, Y. (Eds.), Temporal Coding in the Brain. G. Springer-Verlag, Berlin, pp. 145–172. Buzsa´ ki, G., Leung, L-W.S., Vanderwolf, C.H., 1983. Cellular bases
139
of hippocampal EEG in the behaving rat. Brain Research Reviews 6, 139–171. Camo´ n, L., Vives, P., de Vera, N., Martinez, E., 1998. Seizures and neuronal damage induced in the rat by activation of group I metabotropic glutamate receptors with their selective agonist 3,5-dihydroxyphenylglycine. Journal of Neuroscience Research 51, 339–348. Clark, B.P., Baker, S.R., Goldsworthy, J., Harris, J.R., Kingston, A.E., 1997. 2-methyl-4-carboxyphenylglycine (LY367385) selectively antagonizes metabotropic glutamate mGluR1 receptors. Bioorganic and Medicinal Chemistry Letters 7, 2777–2870. Cobb, S.R., Bulters, D.O., Davies, C.H., 2000. Coincident activation of mGluRs and mAChRs imposes theta frequency patterning on synchronised network activity in the hippocampal CA3 region. Neuropharmacology 39, 1933–1942. Cobb, S.R., Bulters, D.O., Suchak, S., Riedel, G., Morris, R.G.M., Davies, C.H., 1999. Activation of nicotinic acetylcholine receptors patterns muscarinic acetylcholine receptor-mediated network activity in the rodent hippocampus. Journal of Physiology 518, 131–140. Conquet, F., Bashir, Z.I., Davies, C.H., Daniel, H., Ferraguti, F., Bordi, F., Franz-Bacon, K., Reggiani, A., Matarese, V., Conde´ , F., Collingridge, G.L., Cre´ pel, F., 1994. Motor deficit and impairment of synaptic plasticity in mice lacking mGluR1. Nature 372, 237–243. Davies, C.H., Clarke, V.R.J., Jane, D.E., Collingridge, G.L., 1995. Pharmacology of postsynaptic metabotropic glutamate receptors in rat hippocampal CA1 pyramidal neurones. British Journal of Pharmacology 116, 1859–1869. Desai, M.A., Smith, T.S., Conn, P.J., 1992. Multiple metabotropic glutamte receptors regulate hippocampal function. Synapse 12, 206– 213. Doherty, A.J., Palmer, M.J., Henley, J.M., Collingridge, G.L., Jane, D.E., 1997. (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) activates mGlu5, but not mGlu1, receptors expressed in CHO cells and potentiates NMDA responses in the hippocampus. Neuropharmacology 36, 265–267. Egert, U., Schlosshauer, B., Fennrich, S., Nisch, W., Fejtl, M., Knott, T., Muller, T., Hammerle, H., 1998. A novel organotypic long-term culture of the rat hippocampus on substrate-integrated multielectrode arrays. Brain Research Protocols 2 (4), 229–242. Faden, A.I., O’Leary, D.M., Fan, L., Bao, W., Mullins, P.G.M., Movsesyan, V.A., 2001. Selective blockade of the mGluR1 receptor reduces traumatic neuronal injury in bitro and improves outcome after brain trauma. Experimental Neurology 167, 435–444. Ferraguti, F., Conquet, F., Corti, C., Grandes, P., Kuhn, R., Knopfel, T., 1998. Immunohistochemical localization of the mGluR1beta metabotropic glutamate receptor in the adult rodent forebrain: evidence for a differential distribution of mGluR splice variants. Journal of Comparative Neurology 400, 391–407. Fisahn, A., Pike, F.G., Buhl, E.H., Paulsen, O., 1998. Cholinergic induction of network oscillations at 40Hz in the hippocampus in vitro. Nature 394, 186–189. Galoyan, S.M., Merlin, L.R., 2000. Long-lasting potentiation of epileptiform bursts by group I mGluRs is NMDA receptor-independent. Journal of Neurophysiology 83, 2463–2467. Gasparini, F., Lingenhohl, K., Stoehr, N., Flor, P.J., Heinrich, M., Vranesic, I., Biollaz, M., Allgeier, H., Heckendorn, R., Urwyler, S., Varney, M.A., Johnson, E.C., Hess, S.D., Rao, S.P., Sacaan, A.I., Santori, E.M., Velicelebi, G., Kuhn, R., 1999. R2-Methyl-6(phenylethynyl)-pyridine (MPEP), a potent, selective and systemically active mGlu5 receptor antagonist. Neuropharmacology 38 (10), 1493–1503. Gerlai, R., Clayton, N.S., 1999. Analysing hippocampal function in transgenic mice: an ethological perspective. Trends in Neurosciences 22 (2), 47–51. Husi, H., Ward, M.A., Choudhary, J.S., Blackstock, W.P., Grant, S.G.N., 2000. Proteomic analysis of NMDA receptor-adhesion protein signaling complexes. Nature Neuroscience 3, 661–669.
140
C. Lanneau et al. / Neuropharmacology 43 (2002) 131–140
Jefferys, J.G.R., Traub, R.D., Whittington, M.A., 1996. Neuronal networks for induced ‘40Hz’ rhythms. Trends in Neurosciences 19, 202–208. Kawabata, S., Kohara, A., Tsutsumi, R., Itahana, H., Hayashibe, S., Yamaguchi, T., Okada, M., 1998. Diversity of calcium signalling by metabotropic glutamate receptors. Journal of Biological Chemistry 273, 17381–17385. Konopacki, J., MacIver, M.B., Bland, B.H., Roth, S.H., 1987. Carbachol-induced EEG “theta” activity in hippocampal brain slices. Brain Research 405, 196–198. Leung, L-W.S., Yim, C-Y.C., 1991. Intrinsic membrane potentials in hippocampal neurons in vitro. Brain Research 553, 261–274. Lu, Y.-M., Jia, Z., Janus, C., Henderson, J.T., Gerlai, R., Wojtowicz, J.M., Roder, J.C., 1997. Mice lacking metabotropic glutamte receptor 5 show impaired learning and reduced CA1 long-term potentiation (LTP) but normal CA3 LTP. Journal of Neuroscience 17, 5196–5205. Lujan, R., Nusser, Z., Roberts, J.D.B., Shigemoto, R., Somogyi, P., 1996. Perisynaptic location of metabotropic glutamate receptors mGluR1 and mGluR5 on dendrites and dendritic spines in the rat hippocampus. European Journal of Neuroscience 8, 1488–1500. MacVicar, B.A., Tse, F.W.Y., 1989. Local neuronal circuitry underlying cholinergic rhythmic slow activity in the CA3 area of the rat hippocampus. Journal of Physiology 417, 197–212. McMahon, L.L., Williams, J.H., Kauer, J.A., 1998. Functionally distinct groups of interneurons idetified during rhythmic carbachol oscillations in hippocampus in vitro. Journal of Neuroscience 18, 5640–5651. Merlin, L.R., Taylor, G.W., Wong, R.K.S., 1995. Role of metabotropic glutamate receptor subtypes in the patterning of epileptiform activities in vitro. Journal of Neurophysiology 74, 896–900. Miyamoto, M., Ishida, M., Shinozaki, H., 1997. Anticonvulsive and neuroprotective actions of a potent agonist (DCG-IV) for group II metabotropic glutamate receptors against intraventricular kainate in the rat. Neuroscience 77, 131–140. Movsesyan, V.A., O’Leary, D.M., Fan, L., Bao, W., Mullins, P.G., Knoblach, S.M., Faden, A.I., 2001. mGluR5 antagonists 2-methyl6-(phenylethynyl)-pyridine and (E)-2-methyl-6-(2-phenylethenyl)pyridine reduce traumatic neuronal injury in vitro and in vivo by antagonizing N-methyl-d-aspartate receptors. Journal of Pharmacology and Experimental Therapeutics 296 (1), 41–47. Nakanishi, S., 1992. Molecular diversity of glutamate receptors and implications for brain function. Science 258, 597–603. O’Leary, D.M., Movsesyan, V., Vicini, S., Faden, A.I., 2000. Selective mGluR5 antagonists MPEP and SIB-1893 decrease NMDA or glutamate-mediated neuronal toxicity through actions that reflect NMDA receptor antagonism. British Journal of Pharmacology 131 (7), 1429–1437. Pagano, A., Ruegg, D., Litschig, S., Stoehr, N., Stierlin, C., Heinrich, M., Floersheim, P., Prezeau, L., Carroll, F., Pin, J.P., Cambria, A., Vranesic, I., Flor, P.J., Gasparini, F., Kuhn, R., 2000. The noncompetitive antagonists 2-methyl-6-(phenylethynyl)pyridine and 7hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester interact with overlapping binding pockets in the transmembrane region of group I metabotropic glutamate receptors. Journal of Biological Chemistry 275 (43), 33750–33758. Pike, F.G., Goddard, R.S., Suckling, J.M., Ganter, P., Kasthuri, N.,
Paulsen, O., 2000. Distinct frequency preferences of different types of rat hippocampal neurones in response to oscillatory input currents. Journal of Physiology 529 (1), 205–213. Pin, J.-P., Duvoisin, R., 1995. The metabotropic glutamate recptors: Structure and functions. Neuropharmacology 34, 1–26. Pintor, A., Pezzola, A., Reggio, R., Quarta, D., Popoli, P., 2000. The mGlu5 receptor agonist CHPG stimulates striatal glutamate release: possible involvement of A2A receptors. Neuroreport 11 (16), 3611–3614. Romano, C., Sesma, M.A., McDonald, C.T., O’Malley, K., Van den Pol, A., Olney, J.W., 1995. Distribution of metabotropic glutamte receptor mGluR5 immunorecativity in rat brain. Journal of Comparative Neurology 355, 455–469. Rutecki, P.A., Yang, Y., 1997. Metabotropic glutamate receptor activation modulates epileptiform activity in the hippocampus. Neuroscience 81, 927–935. Sacaan, A.I., Schoepp, D.D., 1992. Activation of hippocampal metabotropic excitatory amino acid receptors leads to seizures and neuronal damage. Neuroscience Letters 139, 77–82. Schoepp, D.D., Jane, D.E., Monn, J.A., 1999. Pharmacological agents acting at subtypes of metabotropic glutamte receptors. Neuropharmacology 38, 1431–1476. Singer, W., 1993. Synchronisation of cortical activity and its putative role in information processing and learning. Annual Reviews of Physiology 55, 349–374. Stoppini, L., Buchs, P.A., Muller, D., 1991. A simple method for organotypic cultures of nervous tissue. Journal of Neuroscience Methods 37 (2), 173–182. Strata, F., 1998. Intrinsic oscillations in CA3 hippocampal pyramids: physiological relevance to theta rhythm generation. Hippocampus 8, 666–679. Taylor, G.W., Merlin, L.R., Wong, R.K.S., 1995. Synchronised oscillations in hippocampal CA3 neurons induced by metabotropic glutamate receptor activation. Journal of Neuroscience 15, 8039–8052. Vanderwolf, C.H., 1969. Hippocampal electrical activity and voluntary movement in the rat. Electoencephalography and Clinical Neurophysiology 26, 407–418. van Hooft, J.A., Giuffrida, R., Blatow, M., Monyer, H., 2000. Differential expression of group I metabotropic glutamate receptors in functionally distinct hippocampal interneurons. Journal of Neuroscience 20, 3544–3551. Vignes, M., Clarke, V.R.J., Davies, C.H., Chambers, A., Jane, D.E., Watkins, J.C., Collingridge, G.L., 1995. Pharmacological evidence for an involvement of group II and group III mGluRs in the presynaptic regulation of excitatory synaptic responses in the CA1 region of rat hippocampal slices. Neuropharmacology 34, 973–982. Watkins, J., Collingridge, G.L., 1994. Phenylglycine derivatives as antagonists of metabotropic glutamate receptors. Trends in Pharmacological Sciences 15 (9), 333–342. Whittington, M.A., Traub, R.D., Jefferys, R.G.R., 1995. Synchronised oscillations in interneuron networks driven by metabotropic glutamate receptor activation. Nature 373, 612–615. Williams, J.H., Kauer, J.A., 1997. Properties of carbachol-induced oscillatory activity in rat hippocampus. Journal of Neuroscience 78, 2631–2640. Wong, R.K., Bianchi, R., Taylor, G.W., Merlin, L.R., 1999. Role of metabotropic glutamate receptors in epilepsy. Advances in Neurology 79, 685–698.
Complex interactions between mGluR1 and mGluR5 shape neuronal network activity in the rat hippocampus Christophe Lanneau a, Mark H. Harries b,∗, Alison M. Ray a, Stuart R. Cobb c, Andrew Randall a, Ceri H. Davies a b
a Neurology CEDD, GlaxoSmithKline, Third Avenue, Harlow Essex CM19 5AW UK Psychiatry CEDD, GlaxoSmithKline Pharmaceuticals Plc, New Frontiers Science Park North, Third Avenue, Harlow, Essex CM19 5AW, UK c Neuroscience & Biomedical Systems, West Medical Building, University of Glasgow, Glasgow G12 8QQ, UK
Received 31 October 2001; received in revised form 31 May 2002; accepted 11 June 2002
Abstract Group I metabotropic glutamate receptors (mGluRs) cause increased neuronal excitability that can lead to epileptogenesis and neurodegeneration. Here we have examined how individual members of this subgroup of mGluRs affect synchronised hippocampal synaptic activity under normal and disinhibited conditions similar to those that occur during certain epileptic states. We demonstrate that activation of both mGluR1 and mGluR5 are important in increasing neuronal synaptic excitability by increasing synchrony between cells and driving correlated network activity in circuits that contain, or are devoid of, GABAA receptor-mediated synaptic inputs. The precise patterning of activity that occurs is complex and depends upon: (1) the existing pattern of ongoing network activity prior to mGluR activation; and (2) the relative extent of activation of each mGluR subtype. However, mGluR5 appears to be the principal mGluR subtype that initiates bursting activity irrespective of the inhibitory synaptic tone within the neuronal network. 2002 Elsevier Science Ltd. All rights reserved. Keywords: Metabotropic glutamate receptors; mGluR1; mGluR5, MPEP; LY367385; Theta rhythm; Multielectrode array
1. Introduction Synchronous discharges of groups of neurones in the vertebrate central nervous system play an important role in normal physiological processing of sensory information as well as in neuropathophysiological disease states such as epilepsy (Vanderwolf, 1969; Buzsa´ki et al., 1983, 1994; Singer, 1993; Jefferys et al., 1996). Numerous patterns of synchronous activity (e.g., β-, γand θ-rhythms, ripples, sharp waves and dentate spikes) have been recorded both in vitro and in vivo, often in association with particular patterns of behaviour. Whilst the mechanism of generation of many of these rhythmical discharges is not yet fully understood, there is substantial evidence to support the concept that each pattern of activity is critically dependent upon: (1) the intrinsic
Corresponding author. Tel.: +44-127-962-2124; fax: +44-127962-2555. E-mail address: [email protected] (M.H. Harries). ∗
membrane properties of each of the neurones within the synaptic circuit (Leung and Yim, 1991; Strata, 1998; Pike et al., 2000); and (2) the close integration of fast ionotropic and slower metabotropic receptor-mediated synaptic inputs (Whittington et al., 1995; Taylor et al., 1995; Boddeke et al., 1996; Konopacki et al., 1987; MacVicar and Tse, 1989; Williams and Kauer, 1997; Fisahn et al., 1998; McMahon et al., 1998; Cobb et al., 1999, 2000). Relevant to this last point, each neurotransmitter-mediated synaptic input to a neuronal network, whether it originates from within (intrinsic neuromodulatory input) or without the hippocampus (extrinsic neuromodulatory input), can shape the pattern of activity through pre- and post-synaptic mechanisms that change neuronal excitability as well as synaptic transmission (Buzsa´ki et al., 1994; Jefferys et al., 1996). Metabotropic glutamate receptor (mGluR)-mediated synaptic inputs are ideally equipped to fulfill these roles (Desai et al., 1992; Davies et al., 1995; Vignes et al., 1995) and, as such, there is a rapidly expanding literature on the effects of different mGluR subtypes on neuronal network func-
0028-3908/02/$ - see front matter 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 0 2 ) 0 0 0 8 6 - 2
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tion (Whittington et al., 1995; Taylor et al., 1995; Rutecki and Yang, 1997; Cobb et al., 2000). At least eight subtypes of mGluR have been cloned which can be subdivided into three classes on the basis of their pharmacology and coupling to signal transduction pathways (Nakanishi, 1992; Pin and Duvoisin, 1995; Schoepp et al., 1999). With reference to epilepsy, whilst all three groups of mGluR have modulatory actions (Sacaan and Schoepp, 1992; Merlin et al., 1995; Burke and Hablitz, 1995; Miyamoto et al., 1997; Rutecki and Yang, 1997) it is generally accepted that antagonists of group I mGluRs and agonists of group II and III mGluRs have the potential to be good anticonvulsants (Camo´ n et al., 1998; Wong et al., 1999). However, the lack of selective pharmacological agents with which to manipulate the function of individual mGluR subtypes (Watkins and Collingridge, 1994) has hampered investigations into the contribution that each subtype makes to shaping synaptic activity in neuronal networks. Given that in many brain regions, individual mGluR subtypes can be differentially restricted to specific populations of neurones which subserve specialized physiological roles (Romano et al., 1995; Lujan et al., 1996; Ferraguti et al., 1998; van Hooft et al., 2000) it is clear that such drugs are likely to have quite distinct actions to those which interfere with multiple subtypes. Until recently non-conditional transgenic knockout strategies have provided the principal approaches for studying the function of individual group I mGluR subtypes (Aiba et al., 1994; Conquet et al., 1994; Lu et al., 1997). However, these studies are subject to a range of potential genetic, developmental and compensatory limitations that can complicate interpretation of experimental results (Gerlai and Clayton, 1999). The recent development of selective antagonists for individual group I mGluR subtypes, however, now opens up the possibility of examining the functional roles of these mGluR subtypes in more detail, through acute manipulation of their function. The aim of the present study therefore was to use the selective mGluR1 and mGluR5 antagonists LY367385 (Clark et al., 1997) and MPEP (Gasparini et al., 1999) to investigate how these mGluR subtypes modify synchronised bursting activity in the hippocampus.
2. Methods 2.1. Slice preparation Experiments were performed on acutely prepared transverse hippocampal slices and organotypic hippocampal slice cultures. Organotypic slice cultures were prepared using methods similar to Stoppini et al. (1991). For acute slices, 3–4-week old male Wistar rats were sacrificed by dislocation of the neck and subsequent decapitation in accordance with UK Home Office guide-
lines. The brain was removed rapidly and 400 µm thick, transverse hippocampal slices were cut using a Campden vibroslicer. Slices were left to equilibrate in artificial cerebrospinal fluid (aCSF) at room temperature for at least 1 h prior to recording. 2.2. Electrophysiological recordings Synaptic activity was recorded using either whole cell patch clamp, intracellular sharp electrode or extracellular multielectrode array recording approaches. In all recording configurations, slices were bathed in warmed (35–37 °C), perfusing (2–4 ml.min⫺1) aCSF comprised of (mM): NaCl, 124; KCl, 3; NaHCO3, 26; NaH2PO4, 1.25; CaCl2, 2; MgSO4, 1; d-glucose, 10; and bubbled with 95% O2, 5% CO2. Intracellular current clamp recordings of CA3 pyramidal cells from acute hippocampal slices were made in bridge balanced current clamp mode (Axoclamp 2B amplifier) with glass microelectrodes (60–120 M⍀) filled with 2 M potassium methylsulphate. Whole cell patch clamp recordings from CA1 and CA3 pyramidal cells in organotypic hippocampal slices were made under visual guidance using an Olympus BX50WI upright microscope. Microelectrodes (5–8 M⍀) connected to an Axopatch 200B amplifier were filled with (mM): K-Gluconate, 135; MgCl2, 10; HEPES, 10; Mg ATP, 2; EGTA, 1; Na GTP, 0.3; CaCl2 , 0.1 at pH 7.2 and 290–295 mOsmol. Double patch clamp recordings were performed using the same approaches but with two Axopatch 200B amplifiers. Cell viability was assessed throughout the course of experiments via measurement of cell input resistance. Patch clamp data were filtered at 5 kHz, digitised at 20 kHz and stored on a PC hard disk drive for off-line analysis using Clampfit 8.2 (Axon Instruments Inc). Extracellular recordings of field potentials from organotypic hippocampal slices were made from all subfields using a planar multielectrode array (MEA) purchased from MultiChannel Systems (GmbH Reutlingen). A small piece of semiporous membrane bearing the hippocampal slice was cut out of the culture well using a scalpel blade. This was positioned slice side down onto an MEA, which had previously been coated using 5 µl of a solution comprising 1 cm2 cellulose nitrate filter paper dissolved in 10 ml methanol. Final orientation of the slice with respect to the electrodes was achieved with the aid of a Nikon SMZ 800 microscope. The MEA recording chamber was then immediately flooded with 1 ml of aCSF and a photographic image taken of the electrode positions beneath the hippocampal subfields (Nikon D1 digital camera). MEA extracellular recordings were made from all subfields of the slice using the procedures outlined by Egert et al. (1998). MEA data were sampled at 20 kHz per electrode, stored on hard disk or CD-R and
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analysed off-line using MCS and Neuroexplorer (Plexon Instruments Inc.) software. 2.3. Drugs In all experiments drugs were applied by addition to the perfusion medium. Bicuculline methiodide and tetrodotoxin citrate (TTX) were obtained from Sigma (Poole, Dorset UK). (S)-3,5-dihydroxyphenylglycine (DHPG), (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), [1-(S)-3,4-dichlorophenyl)ethyl]amino-2-(S)hydroxypropyl-p-benzyl-phosphonic acid (CGP55845A), 2-methyl-6-(phenylethynyl)pyridine (MPEP), (S)-(+)-α-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385), 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulphonamide disodium (NBQX) and d(⫺)-2-amino-5-phosphonopentanoate (AP5) were purchased from Tocris Cookson (Bristol, UK) and made up as concentrated stock solutions as suggested in accompanying data sheets. As such, all drugs were made up in water or equimolar sodium hydroxide which on their own produced no effects in any of the experiments undertaken as part of this study. Metabotropic glutamate receptor agonists were applied for 6– 12 min which was sufficient to enable changes in bursting activity to stabalize. mGluR antagonists were applied 6 min after DHPG induced changes in bursting activity had stabilized and their application sustained for such time as required to achieve maximal antagonism at each concentration tested. This study deals specifically with burst activity which was defined as excitatory postsynaptic potentials or currents (epsps or epscs) that have durations in excess of 150 ms. As such, all spontaneous stochastic events of shorter duration, such as those superimposed on bursting events illustrated in Figs. 2A and 3A, are excluded from the current analyses. All data are presented as means±standard error of the mean (S.E.M.) and statistical significance was assessed using ANOVA, paired or unpaired Student’s t-tests performed on raw data with P⬍0.05 being taken as indicating statistical significance.
3. Results 3.1. mGluR driven population activity In acutely prepared hippocampal slices bath application of the selective mGluR agonist ACPD (100–200 µM; n=6) induced a characteristic pattern of synchronised bursting activity recorded in CA3 pyramidal cells using either intracellular or whole cell patch clamp recording techniques. This pattern of activity consisted of 1–3 s long bursts of synchronous epsps occurring every 10–30 s that was abolished by the AMPA/kainate recep-
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tor antagonist NBQX (5 µM; n=3) and the voltage gated Na+ channel blocker TTX (1 µM; n=3) but not by the selective NMDA receptor antagonist AP5 (20 µM; n=3) illustrating that mGluR-induced bursting principally relies upon AMPA/kainate receptor subtype-mediated synaptic activity. This pattern of activity could be reproduced using the group I mGluR selective agonist DHPG (Fig. 1A) and autocorrelation analysis revealed that activity within bursting events was rhythmic at frequencies in the theta range of 4–12 Hz (mean frequency: 7.9±1.0 Hz for DHPG) (Fig. 1A). In some recordings there was the additional presence of some γ frequency (20–40 Hz) activity. Similar patterns of activity were recorded with dual patch-clamp recording techniques in organotypic hippocampal slices illustrating that this activity was independent of intracellular dialysis of individual neurones (which greatly reduces direct mGluR induced postsynaptic inward currents) and confirmed that it was a consequence of surrounding network activity (Fig. 1B). This was supported by multielectrode array (MEA) extracellular recording which clearly showed that DHPG-induced population bursts originated in area CA3 from where they propagated into area CA1 with a 2 ms delay (Fig. 1C). In all experiments brief drug applications were used (except where otherwise stated) to restrict the study to examination of the mechanisms responsible for the induction of mGluR-induced bursting rather than the prolonged maintenance of bursting described by Galoyan and Merlin (2000) that persists following the washout of mGluR agonists after hour long agonist exposures. To establish whether activation of individual mGluR subtypes could generate the bursting activity described in detail above we examined with the whole cell voltageclamp technique: (1) whether the mGluR5 selective agonist CHPG could initiate bursts of excitatory post synaptic currents (epscs) in CA3 pyramidal cells; and (2) how the mGluR1 and mGluR5 selective antagonists LY367385 and MPEP affected previously established DHPG-driven synchronised hippocampal activity. At 5 mM CHPG induced synchronised bursting activity which reoccurred at a frequency of 0.032±0.012 Hz (n=9) which was significantly lower than the 0.057±0.012 Hz induced by 20 µM DHPG (P⬍0.01, n=12) (Fig. 2A); a near maximal concentration of DHPG for inducing bursting activity (Fig. 2B). Higher concentrations of CHPG could not be tested because of the limited solubility of this compound. Despite this, the effectiveness of CHPG suggested that activation of mGluR5 alone was sufficient to induce synchronised bursting activity. However, it was still quite possible that mGluR1 contributed to the effects induced by DHPG. In this respect, we were not surprised to find that both LY367385 and MPEP reversed bursting activity induced by DHPG (Fig. 3A). In line with their reported affinities for mGluRs 1
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Fig. 1. Intraburst event frequency is rhythmic and occurs in the theta frequency range (4–8 Hz). (A) Current clamp traces from an intracellular recording of DHPG driven synchronised bursting activity in an acutely prepared hippocampal slice on a range of time bases and at the membrane potentials indicated. In the top most trace the cell was hyperpolarized during the period between the arrows in order to eliminate action potential firing. Below these traces are the autocorrelation and power spectrum plots generated from this synaptic activity. (B) This is similar to (A) except that activity recorded simultaneously from a CA3 and a CA1 pyramidal neurone in an organotypic hippocampal slice is illustrated. Note that in both preparations synaptic activity was rhythmic and that the predominant frequency of activity was approximately 5 Hz. (C) The uppermost panel is a micrograph of an organotypic hippocampal slice mounted on a multielectrode array. Subfield cell layers and the outline of the organotypic slice are highlighted with dashed lines. The squares indicate the position of two electrodes beneath CA3 and CA1 subfields. The gridded traces below are representative 1 s sweeps taken from 20 electrodes central to the slice in control medium (left hand trace) and in the presence of 20 µM DHPG (right hand trace). The traces in the lower left panel of (C) represent synaptic activity from the two electrodes boxed in above and which are in contact with areas CA3 and CA1. The time base has been expanded to illustrate that activity originated in CA3 and was propagated to area CA1. The event frequency histogram beside these traces illustrates the reproducible occurrence of bursts every 20 s.
and 5 multielectrode array analysis revealed that MPEP was more potent at blocking DHPG-induced bursting than LY367385 (Fig. 3C). The greater potency of MPEP was accentuated when these pharmacological studies were re-performed using longer exposure times to low concentrations of each antagonist. In particular, whereas the antagonist action of LY367385 was independent of exposure time, multielectrode array recordings revealed that MPEP, when applied at 1 µM for 1–2 h, produced greater antagonism than when applied for 20–30 min. As such, after 2 h its effects closely resembled those produced by 10–30 µM MPEP applied for 15–30 min (not illustrated); observations that may reflect the non competitive intra-transmembrane mechanism of action of this compound. Furthermore, in most experiments
MPEP actually abolished DHPG induced bursting whereas LY367385 never did (Fig. 3C). 3.2. Synchronised population activity in a disinhibited network Given that group I mGluRs had such pronounced effects on synchronised synaptic activity in normal aCSF we were interested to examine how mGluRs 1 and 5 affected coherent activity in a network in which fast synaptic inhibition was blocked using the competitive GABAA receptor antagonist bicuculline (10 µM) since this provides an alternative model which mimics patterns of activity observed during epileptiform activity in vivo. Bath application of bicuculline alone caused the appear-
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Fig. 2. Activation of group I mGluRs induces synchronised bursting. (A) Excerpts from a whole cell patch clamp recording of the activity of a single CA3 pyramidal neurone in control medium (top trace), in the presence of 5 mM CHPG, following washout of CHPG and in the presence of 20 µM DHPG (bottom trace). The smaller frequent events are asynchronous GABAergic and glutamatergic postsynaptic currents (pscs). Note that in the presence of both mGluR agonists the frequency and amplitude of these pscs increases and that superimposed on this activity there is the appearance of large bursts of activity. (B) The left hand graph is the concentration–response relationship for the effectiveness of DHPG to induce bursting activity. Scaling on the ordinate axis shows that data has been expressed relative to the concentration of DHPG which maximally potentiated bursting frequency in each experiment. The bar graph on the right shows the increases in burst frequency induced by ACPD and CHPG expressed relative to the increase induced by DHPG in five neurones. In this and all subsequent figures ∗ represents statistical significance at P⬍0.05. n numbers are denoted on individual bars.
ance of large bursting events (n=26; Fig. 4A) which increased in duration on subsequent application of DHPG (20 µM; n=18; Fig. 4A). CHPG (5 mM; n=8) also produced an increase in burst duration in disinhibited slices (Fig. 4). Consistent with the full and partial agonist activities of these compounds at group I mGluRs the mean duration of bursting events in the presence of the mGluR5 selective agonist CHPG (6.47±1.32 s) was significantly lower than that in the presence of the mGluR1/5 selective agonist DHPG (19.51±4.80 s). However, no significant difference in bursting frequency was observed (0.022±0.0041 Hz bicuculline, 0.018±0.0029 Hz DHPG and 0.025±0.0049 Hz CHPG) (Fig. 4B). Whilst these data again suggested that activation of
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mGluR5 was at least, in part, contributing to the effects of DHPG on disinhibition-induced bursting we attempted to establish a contribution of mGluR1 by examining how MPEP and LY367385 affected DHPG induced changes in bursting activity. MPEP (100 µM; n=5) produced a significant, yet still incomplete, reversal of the increase in burst duration induced by DHPG (Fig. 5A). These results were particularly interesting as bursts recorded in the presence of CHPG, in which mGluR5 alone should be activated, were very similar to those recorded in DHPG+MPEP, in which mGluR1 alone should be activated (Fig. 4A cf Fig. 5A). Even more perplexing was the effect of LY367385 (200 µM) on DHPG induced bursting. In particular, this antagonist had very variable effects on DHPG-induced increases in burst duration (Fig. 5A and B). This was because LY367385 consistently disrupted the coherence of bursts such that they became comprised of a complex series of events of variable duration and frequency (Fig. 5A and D). Interestingly, the selective NMDA receptor antagonist AP5 (20 µM; n=5) caused a similar splitting of bursting event waveform to that induced by LY367385 (Fig. 5D). These results were surprising in that MPEP, and not LY367385 (Faden et al., 2001), has previously been reported to exhibit NMDA receptor antagonist properties (O’Leary et al., 2000; Movsesyan et al., 2001). Irrespective of these results, coapplication of MPEP and LY367385 reversed DHPG–induced changes in bursting activity such that synaptic activity returned to a pattern that was not significantly different to that which occurred in bicuculline alone (Fig. 5B and C).
4. Discussion 4.1. Balanced inhibitory–excitatory networks The data generated here using a combination of complementary electrophysiological techniques confirm those of others (Taylor et al., 1995; Cobb et al., 1999, 2000) illustrating complex effects of group I mGluRs on the shaping of neuronal excitability within normal inhibitory–excitatory hippocampal networks. Here, synchronised bursting activity was induced by either the selective mGluR1/5 agonist, DHPG or the mGluR5 specific agonist CHPG. Notably, CHPG was always less effective than DHPG. Two possible explanations for these results are: (1) mGluR-induced bursting requires the activation of mGluR5 alone without mGluR1; the smaller effect of CHPG reflecting the partial agonist behaviour of CHPG at mGluR5 compared with the full agonist activity of DHPG at mGluR1/5 (Doherty et al., 1997); or (2) DHPG-induced changes in neuronal excitability require coactivation of mGluR1 and mGluR5; the effect of each receptor subtype being additive/synergistic to the other. To differentiate between these two possibilities we
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Fig. 3. Bursting activity depends upon the activation of both mGluR1 and mGluR5. (A) Traces are representative excerpts from an experiment in which 100 µM MPEP alone, 100 µM LY367385 alone and combined application of 100 µM MPEP and 100 µM LY367385 reversed 20 µM DHPG induced bursting activity. As in Fig. 2 the smaller frequent events are asynchronous GABAergic and glutamatergic postsynaptic currents (pscs). (B) Bar graph depicting pooled data from whole cell patch clamp studies as in (A) illustrating the burst frequency recorded under the experimental conditions described below each bar normalized to that recorded in the presence of 20 µM DHPG. ∗∗ represents statistical significance at P⬍0.01 (C) Concentration–response relationships for the antagonistic effects of MPEP and LY367385 on 20 µM DHPG induced bursting. Each data point has been normalised to the frequency of activity in DHPG alone. At 10, 30 and 100 µM MPEP reduced burst frequency significantly more than the respective concentration of LY 367385.
studied the effects of subtype selective group I mGluR antagonists on DHPG-induced changes in synaptic activity. Both mGluR1 and 5 selective antagonists inhibited DHPG induced bursting suggesting that both subtypes contribute to this phenomenon. In this respect, the concentrations of LY367385 necessary to cause antagonism of DHPG-driven bursting were generally in accord with its reported affinity at recombinant mGluR1 (LY367385=9 µM at mGluR1 and ⬎200 µM at mGluR5 (Bruno et al., 1999)). In contrast, relatively high concentrations of MPEP were required to block DHPG-induced effects despite its high affinity for cloned receptors (IC50 values against agonist induced PI hydrolysis: MPEP ⬎100 µM at mGluR1 and 36 nM at mGluR5; Gasparini et al. (1999). However, the effective concentration ranges of MPEP that have been used here are: (1) still below the concentrations required to antagonise mGluR1; and (2) are comparable to those required to block other electrophysiological and neurochemical effects of DHPG in other brain regions that are believed to be mediated by activation of mGluR5 (O’Leary et al., 2000; Pintor et al., 2000). The interesting observation that longer incubation times with MPEP increased the
apparent potency of this compound may be explained by slow kinetics of interaction between MPEP and mGluR5 because its binding site resides within transmembrane domains III and VII as opposed to the readily accessible N-terminal glutamate binding site (Pagano et al., 2000) where LY367385, a competitive antagonist, presumably binds to mGluR1. Irrespective of this, we are confident that the effects of LY367385 and MPEP are due to selective antagonism of mGluR1 and mGluR5, respectively and that both mGluR1 and mGluR5 contribute to synchronised population bursting. Interestingly, mGluR5 appeared to be particularly important in generating bursting activity since MPEP often abolished DHPG-induced bursting. In contrast, LY367385 produced only a 70– 80% inhibition suggesting that mGluR1 plays a secondary role to mGluR5 in initiating bursting activity. These differing roles may, in part, reflect coupling to distinct signal transduction pathways. For example, in human embryonic kidney cells mGluR1 and 5 induce distinct intracellular Ca2+ rises because of differential activation of intracellular store and receptor mediated release of Ca2+ (Kawabata et al., 1998). Furthermore, differential activation of distinct signal transduction pathways in
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Fig. 4. Activation of group I mGluRs alters burst waveform in disinhibited hippocampal networks. (A) Traces are whole cell patch clamp recordings in control medium, in the presence of 10 µM bicuculline , in the combined presence of bicuculline plus 20 µM DHPG, following washout back into 10 µM bicuculline and in the subsequent combined presence of bicuculline plus 5 mM CHPG. Note that whilst CHPG produced a widening of burst duration this effect was much smaller than that induced by DHPG. The holding potential in this experiment was ⫺70 mV. (B) Pooled data for the duration and frequency of bursts recorded in bicuculline in the presence and absence of mGluR agonists. Absolute values are given for burst duration and frequency under the experimental conditions indicated.
separate neuronal populations may further contribute to the observed differences (van Hooft et al., 2000). 4.2. Disinhibited networks In disinhibited networks activation of mGluR1 and mGluR5 produced complex changes in ongoing bursting activity. In particular, each subtype produced quite distinct changes in burst waveform architecture which differed from the situation in an inhibited network where both subtypes essentially supported almost identical burst waveforms. Most notably, in the disinhibited network, mGluR5 activation assessed under two distinct experimental conditions, induced quite distinct effects. Thus, CHPG caused a relatively small increase in burst duration whereas in the combined presence of DHPG and LY367385 burst duration was increased to a level not significantly different to that induced by DHPG alone. However, in the presence of LY367385 DHPGinduced burst durations were more variable than in DHPG alone possibly reflecting the more sporadic occurrence of intraburst events (Fig. 5D). Providing LY367385 is causing a complete block of mGluR1 activation the apparent differences between the effects of CHPG alone and DHPG plus LY367385 could potentially be explained by differences in the magnitude of
mGluR5 activation produced by these two drug treatments. Thus, the weak partial mGluR5 agonist activity of CHPG may provide just sufficient impetus to extend the burst a small amount without promoting the activation of a sustained period of secondary discharge, whereas the greater activation of mGluR5 in the combined presence of DHPG+LY367385 may more strongly drive secondary discharge generation. The natural extension of these findings is that activation of mGluR1 is necessary to convert this type of disjointed intraburst discharge into more of a continuum of intraburst discharge observed when DHPG alone induces burst widening. Alternatively, LY367385 and/or CHPG may have additional pharmacological effects, unrelated to mGluR activity, that may account for the differences in bursting pattern observed in the presence of these compounds. That said, it was interesting that antagonism of NMDA receptors also caused DHPG-induced bursts to be disrupted as observed when mGluR5 alone was activated by application of DHPG and LY367385. This observation might suggest that mGluR1 enhances NMDA receptor activity thereby prolonging the initial phase of the burst (see Fig. 5D) which in turn presumably impacts on subsequent events, such that in the presence of DHPG secondary events are closely packed together and are superimposed on a prolonged envelope of inward current
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Fig. 5. Antagonism of DHPG induced bursting in disinhibited hippocampal networks. (A) Differential antagonism of 20 µM DHPG-induced bursting by 100 µM MPEP and 200 µM LY367385. Note that MPEP almost completely reversed the DHPG induced increase in duration of bursts in bicuculline treated slices without significantly affecting their frequency of occurrence. In contrast, LY367385 did not significantly reduce overall burst duration but split bursts up into a series of much smaller bursts that followed each other in quick succession. Note that subsequent application of both antagonists returned bursting waveforms close to those recorded with bicuculline alone. (B) Bar graph plotting pooled data for the overall burst duration in the drug treatments illustrated in (A). (C) Bar graph plotting burst frequency under these drug regimes. (D) Comparison of the effects of 100 µM LY367385 and 20 µM AP5 on 20 µM DHPG-induced bursting activity in bicuculline treated slices. Note the simlarity of the effects of LY367385 and AP5.
(Fig. 5). The mGluR1/NMDA receptor interaction could be mediated directly through interplay between mGluR1 and NMDA receptors or arise through alterations in the integration of diverse neuronal circuits. Indeed: (1) mGluR1 has been shown to be closely associated with NMDA receptors in the so-called hebbosome complex (Husi et al., 2000); and (2) there is a precedent for potentiating interactions between group I mGluRs and NMDA receptors (Doherty et al., 1997; Awad et al., 2000). Consistent with this, activation of mGluR1, in the combined presence of DHPG+MPEP, clearly increased burst duration. Interestingly, however, CHPG had a similar effect to DHPG+MPEP (Fig. 5A cf Fig. 4A) indicating that in disinhibited networks maximal activation of mGluR1 fulfils a similar function to presumed partial activation of mGluR5. Thus activation of mGluR1 and mGluR5 may still synergize to promote burst duration through promotion of NMDA receptor activity and thereby bind together intraburst events.
Given these findings mGluR5 can be perceived to be the major driving force behind prolonging overall burst duration with activation of mGluR1 playing a secondary complementary role to mGluR5, forcing intraburst events into more of a continuum of events rather than the less organized patterns observed when only mGluR5 is activated. This is somewhat akin to the situation described for networks in which synaptic inhibition is intact, where once again mGluR5 takes the lead in generating bursting activity with mGluR1 promoting this activity when activated. The only difference here is that in the disinhibited network mGluR5 activates pronounced secondary discharges that prolong the burst waveform far beyond that recorded in strongly inhibited networks. The precise biochemical and cellular mechanisms by which both mGluR subtypes have their effects requires extensive further investigation, but likely involves effects that are transmitted through the network rather than just simple postsynaptic modifications in the recorded postsynaptic neurone.
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4.3. Concluding remarks In conclusion, we have shown that both mGluR1 and mGluR5 have major roles to play in the complex rearrangement of membrane properties, synaptic timing and magnitude of synaptic inputs that leads to the generation of synchronised bursting activity. The net excitatory influence of both subtypes supports the concept that antagonism of each mGluR subtype alone, or in combination, is a useful strategy for affording neuroprotection during neurodegenerative insults (Bruno et al., 1999, 2000). However, more extensive experimentation is required to define precisely the contribution made by individual subtypes on separate populations of principal and non principal neurones to each pattern of network activity. Generally though mGluR5 appears to play the dominant role in generating bursting activity irrespective of the inhibitory synaptic status of the neuronal network.
Acknowledgements CL is an EU funded Framework V funded postdoctoral research fellow.
References Aiba, A., Chen, C., Herrup, K., Rosenmund, C., Stevens, C.F., Tonegawa, S., 1994. Reduced hippocampal long-term potentiation and context-specific deficit in associative learning in mGluR1 mutant mice. Cell 79 (2), 365–375. Awad, H., Hubert, G.W., Smith, Y., Levey, A.I., Conn, P.J., 2000. Activation of metabotropic glutamate receptor 5 has direct excitatory effects and potentiates NMDA receptor currents in neurons of the subthalamic nucleus. Journal of Neuroscience 20 (21), 7871–7879. Boddeke, H.G.W.M., Best, R., Boeijinga, P.H., 1996. Synchronous 20Hz rhythmic activation in hippocampal networks induced by activation of metabotropic glutamate receptors in vitro. Neuroscience 76, 635–658. Bruno, V., Ksiazek, I., Battaglia, G., Lukic, S., Leonhardt, T., Sauer, D., Gasparini, F., Kuhn, R., Nicoletti, F., Flor, P.J., 2000. Selective blockade of metabotropic glutamate receptor subtype 5 is neuroprotective. Neuropharmacology 39, 2223–2230. Bruno, V., Battaglia, G., Kingston, A., O’Neill, M.J., Catania, M.V., DiGrezia, R., Nicoletti, F., 1999. Neuroprotective activity of the potent and selective mGluR1 antagonist, (+)-2-methyl-4carboxyphenylglycine (LY367385): comparison with LY357366, a broader spectrum equal affinity fore mGlu1a and mGlu5 receptors. Neuropharmacology 38, 199–207. Burke, J.P., Hablitz, J.J., 1995. Modulation of epileptiform activity by metabotropic glutomate receptors in immature rat neocortex. Journal of Neurophysiology 73, 205–217. Buzsa´ ki, G., Bragin, A., Chrobak, J.J., Nadasdy, Z., Sı´k, A., Hsu, M., Ylinen, A., 1994. Oscillatory and intermittent synchrony in the hippocampus: relevance to memory trace formation. In: Buzsa´ ki, G., Llinas, R., Singer, W., Berthroz, A., Christen, Y. (Eds.), Temporal Coding in the Brain. G. Springer-Verlag, Berlin, pp. 145–172. Buzsa´ ki, G., Leung, L-W.S., Vanderwolf, C.H., 1983. Cellular bases
139
of hippocampal EEG in the behaving rat. Brain Research Reviews 6, 139–171. Camo´ n, L., Vives, P., de Vera, N., Martinez, E., 1998. Seizures and neuronal damage induced in the rat by activation of group I metabotropic glutamate receptors with their selective agonist 3,5-dihydroxyphenylglycine. Journal of Neuroscience Research 51, 339–348. Clark, B.P., Baker, S.R., Goldsworthy, J., Harris, J.R., Kingston, A.E., 1997. 2-methyl-4-carboxyphenylglycine (LY367385) selectively antagonizes metabotropic glutamate mGluR1 receptors. Bioorganic and Medicinal Chemistry Letters 7, 2777–2870. Cobb, S.R., Bulters, D.O., Davies, C.H., 2000. Coincident activation of mGluRs and mAChRs imposes theta frequency patterning on synchronised network activity in the hippocampal CA3 region. Neuropharmacology 39, 1933–1942. Cobb, S.R., Bulters, D.O., Suchak, S., Riedel, G., Morris, R.G.M., Davies, C.H., 1999. Activation of nicotinic acetylcholine receptors patterns muscarinic acetylcholine receptor-mediated network activity in the rodent hippocampus. Journal of Physiology 518, 131–140. Conquet, F., Bashir, Z.I., Davies, C.H., Daniel, H., Ferraguti, F., Bordi, F., Franz-Bacon, K., Reggiani, A., Matarese, V., Conde´ , F., Collingridge, G.L., Cre´ pel, F., 1994. Motor deficit and impairment of synaptic plasticity in mice lacking mGluR1. Nature 372, 237–243. Davies, C.H., Clarke, V.R.J., Jane, D.E., Collingridge, G.L., 1995. Pharmacology of postsynaptic metabotropic glutamate receptors in rat hippocampal CA1 pyramidal neurones. British Journal of Pharmacology 116, 1859–1869. Desai, M.A., Smith, T.S., Conn, P.J., 1992. Multiple metabotropic glutamte receptors regulate hippocampal function. Synapse 12, 206– 213. Doherty, A.J., Palmer, M.J., Henley, J.M., Collingridge, G.L., Jane, D.E., 1997. (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) activates mGlu5, but not mGlu1, receptors expressed in CHO cells and potentiates NMDA responses in the hippocampus. Neuropharmacology 36, 265–267. Egert, U., Schlosshauer, B., Fennrich, S., Nisch, W., Fejtl, M., Knott, T., Muller, T., Hammerle, H., 1998. A novel organotypic long-term culture of the rat hippocampus on substrate-integrated multielectrode arrays. Brain Research Protocols 2 (4), 229–242. Faden, A.I., O’Leary, D.M., Fan, L., Bao, W., Mullins, P.G.M., Movsesyan, V.A., 2001. Selective blockade of the mGluR1 receptor reduces traumatic neuronal injury in bitro and improves outcome after brain trauma. Experimental Neurology 167, 435–444. Ferraguti, F., Conquet, F., Corti, C., Grandes, P., Kuhn, R., Knopfel, T., 1998. Immunohistochemical localization of the mGluR1beta metabotropic glutamate receptor in the adult rodent forebrain: evidence for a differential distribution of mGluR splice variants. Journal of Comparative Neurology 400, 391–407. Fisahn, A., Pike, F.G., Buhl, E.H., Paulsen, O., 1998. Cholinergic induction of network oscillations at 40Hz in the hippocampus in vitro. Nature 394, 186–189. Galoyan, S.M., Merlin, L.R., 2000. Long-lasting potentiation of epileptiform bursts by group I mGluRs is NMDA receptor-independent. Journal of Neurophysiology 83, 2463–2467. Gasparini, F., Lingenhohl, K., Stoehr, N., Flor, P.J., Heinrich, M., Vranesic, I., Biollaz, M., Allgeier, H., Heckendorn, R., Urwyler, S., Varney, M.A., Johnson, E.C., Hess, S.D., Rao, S.P., Sacaan, A.I., Santori, E.M., Velicelebi, G., Kuhn, R., 1999. R2-Methyl-6(phenylethynyl)-pyridine (MPEP), a potent, selective and systemically active mGlu5 receptor antagonist. Neuropharmacology 38 (10), 1493–1503. Gerlai, R., Clayton, N.S., 1999. Analysing hippocampal function in transgenic mice: an ethological perspective. Trends in Neurosciences 22 (2), 47–51. Husi, H., Ward, M.A., Choudhary, J.S., Blackstock, W.P., Grant, S.G.N., 2000. Proteomic analysis of NMDA receptor-adhesion protein signaling complexes. Nature Neuroscience 3, 661–669.
140
C. Lanneau et al. / Neuropharmacology 43 (2002) 131–140
Jefferys, J.G.R., Traub, R.D., Whittington, M.A., 1996. Neuronal networks for induced ‘40Hz’ rhythms. Trends in Neurosciences 19, 202–208. Kawabata, S., Kohara, A., Tsutsumi, R., Itahana, H., Hayashibe, S., Yamaguchi, T., Okada, M., 1998. Diversity of calcium signalling by metabotropic glutamate receptors. Journal of Biological Chemistry 273, 17381–17385. Konopacki, J., MacIver, M.B., Bland, B.H., Roth, S.H., 1987. Carbachol-induced EEG “theta” activity in hippocampal brain slices. Brain Research 405, 196–198. Leung, L-W.S., Yim, C-Y.C., 1991. Intrinsic membrane potentials in hippocampal neurons in vitro. Brain Research 553, 261–274. Lu, Y.-M., Jia, Z., Janus, C., Henderson, J.T., Gerlai, R., Wojtowicz, J.M., Roder, J.C., 1997. Mice lacking metabotropic glutamte receptor 5 show impaired learning and reduced CA1 long-term potentiation (LTP) but normal CA3 LTP. Journal of Neuroscience 17, 5196–5205. Lujan, R., Nusser, Z., Roberts, J.D.B., Shigemoto, R., Somogyi, P., 1996. Perisynaptic location of metabotropic glutamate receptors mGluR1 and mGluR5 on dendrites and dendritic spines in the rat hippocampus. European Journal of Neuroscience 8, 1488–1500. MacVicar, B.A., Tse, F.W.Y., 1989. Local neuronal circuitry underlying cholinergic rhythmic slow activity in the CA3 area of the rat hippocampus. Journal of Physiology 417, 197–212. McMahon, L.L., Williams, J.H., Kauer, J.A., 1998. Functionally distinct groups of interneurons idetified during rhythmic carbachol oscillations in hippocampus in vitro. Journal of Neuroscience 18, 5640–5651. Merlin, L.R., Taylor, G.W., Wong, R.K.S., 1995. Role of metabotropic glutamate receptor subtypes in the patterning of epileptiform activities in vitro. Journal of Neurophysiology 74, 896–900. Miyamoto, M., Ishida, M., Shinozaki, H., 1997. Anticonvulsive and neuroprotective actions of a potent agonist (DCG-IV) for group II metabotropic glutamate receptors against intraventricular kainate in the rat. Neuroscience 77, 131–140. Movsesyan, V.A., O’Leary, D.M., Fan, L., Bao, W., Mullins, P.G., Knoblach, S.M., Faden, A.I., 2001. mGluR5 antagonists 2-methyl6-(phenylethynyl)-pyridine and (E)-2-methyl-6-(2-phenylethenyl)pyridine reduce traumatic neuronal injury in vitro and in vivo by antagonizing N-methyl-d-aspartate receptors. Journal of Pharmacology and Experimental Therapeutics 296 (1), 41–47. Nakanishi, S., 1992. Molecular diversity of glutamate receptors and implications for brain function. Science 258, 597–603. O’Leary, D.M., Movsesyan, V., Vicini, S., Faden, A.I., 2000. Selective mGluR5 antagonists MPEP and SIB-1893 decrease NMDA or glutamate-mediated neuronal toxicity through actions that reflect NMDA receptor antagonism. British Journal of Pharmacology 131 (7), 1429–1437. Pagano, A., Ruegg, D., Litschig, S., Stoehr, N., Stierlin, C., Heinrich, M., Floersheim, P., Prezeau, L., Carroll, F., Pin, J.P., Cambria, A., Vranesic, I., Flor, P.J., Gasparini, F., Kuhn, R., 2000. The noncompetitive antagonists 2-methyl-6-(phenylethynyl)pyridine and 7hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester interact with overlapping binding pockets in the transmembrane region of group I metabotropic glutamate receptors. Journal of Biological Chemistry 275 (43), 33750–33758. Pike, F.G., Goddard, R.S., Suckling, J.M., Ganter, P., Kasthuri, N.,
Paulsen, O., 2000. Distinct frequency preferences of different types of rat hippocampal neurones in response to oscillatory input currents. Journal of Physiology 529 (1), 205–213. Pin, J.-P., Duvoisin, R., 1995. The metabotropic glutamate recptors: Structure and functions. Neuropharmacology 34, 1–26. Pintor, A., Pezzola, A., Reggio, R., Quarta, D., Popoli, P., 2000. The mGlu5 receptor agonist CHPG stimulates striatal glutamate release: possible involvement of A2A receptors. Neuroreport 11 (16), 3611–3614. Romano, C., Sesma, M.A., McDonald, C.T., O’Malley, K., Van den Pol, A., Olney, J.W., 1995. Distribution of metabotropic glutamte receptor mGluR5 immunorecativity in rat brain. Journal of Comparative Neurology 355, 455–469. Rutecki, P.A., Yang, Y., 1997. Metabotropic glutamate receptor activation modulates epileptiform activity in the hippocampus. Neuroscience 81, 927–935. Sacaan, A.I., Schoepp, D.D., 1992. Activation of hippocampal metabotropic excitatory amino acid receptors leads to seizures and neuronal damage. Neuroscience Letters 139, 77–82. Schoepp, D.D., Jane, D.E., Monn, J.A., 1999. Pharmacological agents acting at subtypes of metabotropic glutamte receptors. Neuropharmacology 38, 1431–1476. Singer, W., 1993. Synchronisation of cortical activity and its putative role in information processing and learning. Annual Reviews of Physiology 55, 349–374. Stoppini, L., Buchs, P.A., Muller, D., 1991. A simple method for organotypic cultures of nervous tissue. Journal of Neuroscience Methods 37 (2), 173–182. Strata, F., 1998. Intrinsic oscillations in CA3 hippocampal pyramids: physiological relevance to theta rhythm generation. Hippocampus 8, 666–679. Taylor, G.W., Merlin, L.R., Wong, R.K.S., 1995. Synchronised oscillations in hippocampal CA3 neurons induced by metabotropic glutamate receptor activation. Journal of Neuroscience 15, 8039–8052. Vanderwolf, C.H., 1969. Hippocampal electrical activity and voluntary movement in the rat. Electoencephalography and Clinical Neurophysiology 26, 407–418. van Hooft, J.A., Giuffrida, R., Blatow, M., Monyer, H., 2000. Differential expression of group I metabotropic glutamate receptors in functionally distinct hippocampal interneurons. Journal of Neuroscience 20, 3544–3551. Vignes, M., Clarke, V.R.J., Davies, C.H., Chambers, A., Jane, D.E., Watkins, J.C., Collingridge, G.L., 1995. Pharmacological evidence for an involvement of group II and group III mGluRs in the presynaptic regulation of excitatory synaptic responses in the CA1 region of rat hippocampal slices. Neuropharmacology 34, 973–982. Watkins, J., Collingridge, G.L., 1994. Phenylglycine derivatives as antagonists of metabotropic glutamate receptors. Trends in Pharmacological Sciences 15 (9), 333–342. Whittington, M.A., Traub, R.D., Jefferys, R.G.R., 1995. Synchronised oscillations in interneuron networks driven by metabotropic glutamate receptor activation. Nature 373, 612–615. Williams, J.H., Kauer, J.A., 1997. Properties of carbachol-induced oscillatory activity in rat hippocampus. Journal of Neuroscience 78, 2631–2640. Wong, R.K., Bianchi, R., Taylor, G.W., Merlin, L.R., 1999. Role of metabotropic glutamate receptors in epilepsy. Advances in Neurology 79, 685–698.