Differential Effects of Metabotropic Glutamate Receptor Antagonists on Bursting Activity in the Amygdala

N. Bradley Keele, Volker Neugebauer, and Patricia Shinnick-Gallagher

Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 77555-1031


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Keele, N. Bradley, Volker Neugebauer, and Patricia Shinnick-Gallagher. Differential effects of metabotropic glutamate receptor antagonists on bursting activity in the amygdala. Metabotropic glutamate receptors (mGluRs) are implicated in both the activation and inhibition of epileptiform bursting activity in seizure models. We examined the role of mGluR agonists and antagonists on bursting in vitro with whole cell recordings from neurons in the basolateral amygdala (BLA) of amygdala-kindled rats. The broad-spectrum mGluR agonist 1S,3R-1-aminocyclopentane dicarboxylate (1S,3R-ACPD, 100 µM) and the group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG, 20 µM) evoked bursting in BLA neurons from amygdala-kindled rats but not in control neurons. Neither the group II agonist (2S,3S,4S)-alpha -(carboxycyclopropyl)-glycine (L-CCG-I, 10 µM) nor the group III agonist L-2-amino-4-phosphonobutyrate (L-AP4, 100 µM) evoked bursting. The agonist-induced bursting was inhibited by the mGluR1 antagonists (+)-alpha -methyl-4-carboxyphenylglycine [(+)-MCPG, 500 µM] and (S)-4-carboxy-3-hydroxyphenylglycine [(S)-4C3HPG, 300 µM]. Kindling enhanced synaptic strength from the lateral amygdala (LA) to the BLA, resulting in synaptically driven bursts at low stimulus intensity. Bursting was abolished by (S)-4C3HPG. Further increasing stimulus intensity in the presence of (S)-4C3HPG (300 µM) evoked action potential firing similar to control neurons but did not induce epileptiform bursting. In kindled rats, the same threshold stimulation that evoked epileptiform bursting in the absence of drugs elicited excitatory postsynaptic potentials in (S)-4C3HPG. In contrast (+)-MCPG had no effect on afferent-evoked bursting in kindled neurons. Because (+)-MCPG is a mGluR2 antagonist, whereas (S)-4C3HPG is a mGluR2 agonist, the different effects of these compounds suggest that mGluR2 activation decreases excitability. Together these data suggest that group I mGluRs may facilitate and group II mGluRs may attenuate epileptiform bursting observed in kindled rats. The mixed agonist-antagonist (S)-4C3HPG restored synaptic transmission to control levels at the LA-BLA synapse in kindled animals. The different actions of (S)-4C3HPG and (+)-MCPG on LA-evoked bursting suggests that the mGluR1 antagonist-mGluR2 agonist properties may be the distinctive pharmacology necessary for future anticonvulsant compounds.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Glutamate is an amino acid neurotransmitter that activates two broad classes of receptors: the ionotropic receptors, named N-methyl-D-aspartate (NMDA) and non-NMDA receptors, which are ligand-gated ionophores (Hollmann and Heinemann 1994), and the metabotropic glutamate receptors (mGluR), which are G-protein coupled to multiple effector systems. Eight genes are known to code these receptors, named mGluR1-mGluR8 (for review see Conn and Pin 1997; Pin and Duvoisin 1995). They have been classified into three main groups: group I receptors (mGluR1 and mGluR5) couple to phospholipase C (PLC) resulting in phosphoinositide (PI) hydrolysis and activation of protein kinase C (PKC) (Abe et al. 1992; Houamed et al. 1991; Masu et al. 1991); group II (mGluR2 and mGluR3) and group III (mGluR4, 6, 7 and 8) receptors inhibit adenylyl cyclase (Duvoisin et al. 1995; Okamoto et al. 1994; Saugstad et al. 1994; Tanabe et al. 1992, 1993). Certain phenylglycine derivatives have been shown to possess antagonist activity at mGluRs (Conn and Pin 1997; Watkins and Collingridge 1994). (+)-alpha -Methyl-4-carboxyphenylglycine [(+)-MCPG] is an antagonist for mGluR1 and mGluR2, whereas (S)-4-carboxy-3-hydroxyphenylglycine [(S)-4C3HPG] is an antagonist for mGluR1 but an agonist for mGluR2 (Hayashi et al. 1994; Thomsen et al. 1994a). The physiological effects of mGluR activation in epilepsy models are being described, although information about their functional role is slowly emerging. Early studies suggested that mGluRs participate in seizure activity because PI hydrolysis is lastingly up-regulated in the amygdala (Akiyama et al. 1992), and hippocampal PKC activity is enhanced (Akiyama et al. 1995; Chen et al. 1992) after amygdala-kindled seizures. However, there are inconsistent reports of the functional role of mGluRs in epileptiform activity. Some studies have shown that mGluRs can facilitate both bursting in vitro (Bianchi and Wong 1995; McBain 1994; Merlin and Wong 1997; Merlin et al. 1995; Zheng and Gallagher 1991) and seizures in vivo (McDonald et al. 1993; Tizzano et al. 1993, 1995a). Other studies have suggested that mGluRs are functionally inhibitory in seizure models (Attwell et al. 1995; Burke and Hablitz 1994; Suzuki et al. 1996). These conflicting results may possibly be explained on the basis of functional differences among different classes of receptors (Burke and Hablitz 1995; Dalby and Thomsen 1996; Tizzano et al. 1995a,b), where group I receptors play an excitatory role but group II and/or III receptors are inhibitory. This interpretation is supported by studies showing (S)-4C3HPG to have protective effects in seizure models (Bianchi and Wong 1995; Tang et al. 1997; Thomsen et al. 1994b) and models of excitotoxicity (Buisson and Choi 1995; Orlando et al. 1995). However, this view has been challenged recently. In situ hybridization with riboprobes for mGluR1 and mGluR5 have shown no lasting enhancement of receptor mRNA levels in hippocampus after amygdala-kindled seizures (Akbar et al. 1996) or after kainate-induced status epilepticus (Aronica et al. 1997). Furthermore, the group II and group III agonists and group III antagonists have been shown to have convulsant activity (Ghauri et al. 1996; Tang et al. 1997), whereas group III antagonists lack effect in the amygdala of kindled animals (Abdul-Ghani et al. 1997).

The purpose of this study was to characterize the mGluRs underlying bursting in amygdala neurons from kindled rats and to examine the functional changes in mGluR activation resulting from kindling. Previously this laboratory has described mGluR-mediated hyperpolarization resulting from opening of large-conductance, calcium-activated potassium channels (Holmes et al. 1996a; Rainnie et al. 1994) and depolarization caused by closing of potassium channels (Holmes et al. 1996b; Keele et al. 1997) in the amygdala. After amygdala kindling, the hyperpolarizing response is abolished, whereas the depolarization is enhanced (Holmes et al. 1996b). We have also shown an enhanced sensitivity to the presynaptic depressant effect of groups II and III agonists in kindled amygdala neurons (Neugebauer et al. 1997). We tested the hypothesis that the kindling-induced change in functional tone may be overcome by phenylglycine derivatives acting on mGluRs.

These data have appeared previously as an abstract (Keele and Shinnick-Gallagher 1996).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Slice preparation

Slices of control and kindled rat brain containing the basolateral amygdala (BLA) were prepared as previously described (Holmes et al. 1996a; Rainnie et al. 1994). Male Sprague-Dawley rats were decapitated, and the brains were rapidly removed and placed in cold (4°C) artificial cerebrospinal fluid (aCSF) of the following composition (in mM): 117 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 25 Na2HCO3, and 11 glucose (pH 7.4). aCSF was continuously aerated with a mixture of 95% O2-5% CO2. Coronal brain slices (500 µm thick) were prepared with a Vibroslice (Campden Instruments) and placed in a beaker of aCSF for >= 1 h before use. A single slice was then transferred to a recording chamber and submerged in aCSF that superfused the slice at ~2.5 ml/min.

Electrophysiological recording

Blind whole cell recordings were accomplished with the method of Blanton et al. (1989). Whole cell electrodes were fashioned from thin-wall (1.5 mm OD, 1.12 mm ID) borosilicate glass capillary (Drummond) pulled in two stages with a Flaming-Brown micropipette puller (Model P80, Sutter). Electrodes had tip resistances of 2-5 MOmega when filled with internal solution containing (in mM) 122 Kgluconate, 5 NaCl, 0.3 CaCl2, 2 MgCl2, 1 EGTA, 10 HEPES, 5 Na2ATP, and 0.4 Na3GTP. Electrode solutions were adjusted to pH 7.2 with KOH; osmolality was adjusted to 280 mosmol/kg with sucrose.

Recordings were performed in bridge mode of the amplifier. The seal-test function of the acquisition software (pCLAMP 6.0.2; Axon Instruments) was used to measure seal resistance, which typically ranged from 2 to 5 GOmega . On patch rupture, neurons were considered acceptable for experimentation if the resting membrane potential was less than or equal to -60 mV, and direct cathodal stimulation evoked action potentials (APs) overshooting 0 mV. Membrane current and voltage signals were low-pass filtered at 1 kHz with a four-pole Bessel filter (Warner Instrument) and digitized (Digidata 1200, Axon Instruments) at 5 kHz for computer storage (4DX2-66V, Gateway 2000). Analogue records of experiments were continuously acquired with both a pen chart recorder (Gould 2400) and a four-channel videotape recorder (A. R. Vetter).

Synaptic stimulation was delivered from an electrical stimulator (Grass S88) via a concentric bipolar electrode (SNE-100, Kopf Instruments) positioned in the lateral amygdala (LA). Each stimulation (150-µs duration) was given at a frequency of 0.2 Hz. Input-output relationships were constructed by delivering progressively greater stimulus intensity (in 0.5- to 1.0-V steps) from an intensity that evoked no synaptic response until the stimulus evoked AP firing. The threshold excitatory postsynaptic potential (EPSP) stimulus was defined as the lowest stimulus intensity that produced a measurable EPSP; AP threshold was defined as the lowest stimulus intensity capable of eliciting AP firing. In control neurons, an AP threshold stimulus evoked single spikes or spike doublets, whereas in kindled neurons an AP threshold stimulus evoked bursting.

In this report, the volley of APs evoked by applying exogenous substances is referred to simply as "bursting." Afferent stimulation that evokes bursting activity similar to that observed during ictal events is termed "epileptiform bursting."

Kindling

Rats were anesthetized with Equithesin (35 mg/kg pentobarbital and 145 mg/kg chloral hydrate), and tripolar electrodes (Plastics One) were implanted into the right BLA, as previously described (Holmes et al. 1996b; Rainnie et al. 1992). Electrode tips were positioned with the following coordinates from Paxinos and Watson (1986): anteroposterior -2.0 mm and lateral -4.5 mm relative to Bregma to a depth of 7.3 mm from the dural surface. Electrodes were secured to the skull with stainless steel screws and dental cement (Plastics One).

Kindling stimulation was initiated after a 5-day recovery period. Electrical stimulation of the BLA consisted of a 2-s train (60 Hz) of monophasic square waves each 2 ms in duration, twice a day for >= 8 h apart. The threshold current for evoking afterdischarge (AD) activity was determined on the first day of kindling stimulation. Afterward, stimulation intensity was 50-100 µA above the threshold to evoke ADs, which were monitored on a storage oscilloscope (Tektronix). Animals were typically kindled with current intensity between 300 and 500 µA. Behavioral seizure severity was rated according to the five-point ranking scale of Racine (1972). Animals received stimulation until three stage-five seizures were evoked. Three to 7 days after the last stage-five seizure, animals were killed, and brain slices were prepared for recording. Recordings were made in slices obtained from ipsilateral and contralateral hemispheres relative to the stimulation site. Control responses were obtained from both unoperated rats and sham (implanted, unstimulated) control rats.

Drug application

Drugs were applied via one of two methods, as described previously (Holmes et al. 1996a,b; Rainnie et al. 1994). (S)-4C3HPG and (+)-MCPG were superfused in the aCSF for >= 5-10 min before data collection to establish equilibrium in the tissue. Alternatively, 1S,3R-ACPD, DHPG, L-CCG-I, and L-AP4 were applied in a 10-µl drop from an Oxford pipetter to the inlet of the recording chamber. This method was validated by monitoring the ingress and egress of food dye drop applied to the recording chamber. The volume of aCSF in the chamber was kept constant (1 ml) across experiments, and the concentration of drug at the slice was estimated as 1% of the concentration of the drop. Drug concentration is reported as estimated final concentration in the bath. This method allows for rapid onset of drug-induced responses as well as quick offset, which helps minimize effects of desensitization. 1S,3R-ACPD, DHPG, L-CCG-I, L-AP4, (S)-4C3HPG, and (+)-MCPG were purchased from Tocris Cookson (Bristol, UK).

Data analysis

Data collected in the absence of drugs were compared with those collected during the presence of drugs with paired Student's t-test or, where appropriate, one-way ANOVA with post hoc Dunnett's multiple comparison test. Unpaired analyses were used to compare data collected from control and kindled neurons. Statistical significance was defined at the level of <= 0.05. Concentration-response relationships were evaluated with curve-fitting software (Prism 2.01, GraphPad Software, San Diego) and fitting the experimental data with the model equation
<IT>y</IT><IT>=</IT><IT>A</IT><IT>+</IT>(<IT>B</IT><IT>−</IT><IT>A</IT>)<IT>/</IT>[<IT>1+</IT>(<IT>10</IT><SUP><IT>C</IT></SUP><IT>/10</IT><SUP><IT>X</IT></SUP>)<SUP><IT>D</IT></SUP>]
where A = bottom plateau, B = top plateau, C = log (EC50), and D = Hill coefficient.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Pharmacology of mGluR agonist-evoked bursting in kindled BLA neurons

Whole cell recordings were obtained from neurons in the BLA of kindled rats as well as sham (unstimulated, n = 5) and unoperated (n = 10) controls. In neurons from control rats, application of 1S,3R-ACPD or (S)-3,5-dihydroxyphenylglycine (DHPG) did not evoke bursting (n = 11 and n = 8, respectively; data not shown). In agreement with earlier reports (Holmes et al. 1996a; Rainnie et al. 1994a), 1S,3R-ACPD (50-100 µM) evoked a hyperpolarization (2 mV, n = 2), a depolarization (3.5 ± 0.7 mV, n = 6), or a hyperpolarization followed by a depolarization (1.7 ± 0.7 mV and 3.0 ± 1.0 mV, respectively, n = 3) in control neurons. DHPG (20-50 µM) produced a membrane depolarization in only 50% (4/8) of control neurons tested (2.0 ± 0.4 mV, n = 4), and no response in the remaining neurons.

As previously reported (Holmes et al. 1996b), application of 1S,3R-ACPD to kindled neurons evokes bursting activity (Fig. 1A). To ascertain the type of mGluR involved in agonist-induced bursting observed in kindled animals, group-specific agonists were applied to BLA neurons under current-clamp conditions; typical responses are shown in Fig. 1. The 1S,3R-ACPD-induced bursting was concentration dependent; 50 µM evoked bursting activity in 67% (4/6) of neurons tested, whereas 100 µM produced bursting in 100% (8/8) of BLA neurons from kindled animals. The group I-specific mGluR agonist DHPG was also efficacious in evoking burst activity (Fig. 1B). DHPG (20 µM) produced bursts in all (9/9) neurons tested. In contrast to the effects of 1S,3R-ACPD and DHPG, 2S,3S,4S-alpha -carboxycyclopropylglycine (L,-CCG-I; Fig. 1C, left) at a concentration that activates group II receptors (10 µM) (Conn and Pin 1997) did not evoke bursting in any of four neurons examined; the group III agonist L-2-amino-4-phosphonobutyrate (L-AP4; 100 µM) also did not evoke bursts (n = 4; Fig. 1C, right). These data suggest that group I (mGluR1 or mGluR5), DHPG-sensitive mGluRs are involved in the agonist-induced bursting responses observed in kindled animals.



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Fig. 1. Metabotropic glutamate receptor (mGluR) agonist-evoked epileptiform bursting in kindled basolateral amygdala (BLA) neurons. Application of the broad-spectrum mGluR agonist 1S,3R-1-aminocyclopentane dicarboxylate (1S,3R-ACPD, 100 µM; A) or the group I receptor-selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG, 20 µM; B) evokes epileptiform bursting recorded in the same BLA neuron. In a different cell, the group II agonist (2S,3S,4S)-alpha -(carboxycyclopropyl)-glycine (10 µM; C, left) and the group III agonist L-2-amino-4-phosphonobutyrate (L-AP4, 100 µM; C, right) evoked no membrane responses. In all traces, arrows indicate application of agonist in this and subsequent figures. Holding potential was -60 mV for all traces, and downward deflections in A and B are electrotonic potentials evoked by 50-pA direct hyperpolarizing current (200-ms duration) to monitor membrane conductance. Scale is the same for all traces. Peak amplitudes of burst spikes have been attenuated by the chart recorder.

The effects of phenylglycine derivative antagonists on 1S,3RACPD- and DHPG-mediated bursting were also tested. (S)-4Carboxy-3-hydroxyphenylglycine [(S)-4C3HPG] has been shown to be an antagonist of mGluR1 (Hayashi et al. 1994; Joly et al. 1995; Kingston et al. 1995; Thomsen et al. 1994a) but an agonist for mGluR2 (Hayashi et al. 1994; Thomsen et al. 1994a), whereas (+)-alpha -methyl-4-carboxyphenylglycine [(+)-MCPG] is an antagonist of both mGluR1 (Hayashi et al. 1994; Joly et al. 1995; Kingston et al. 1995; Thomsen et al. 1994a) and mGluR2 (Hayashi et al. 1994; Thomsen et al. 1994a). We analyzed the effects of these two compounds to determine if the most commonly utilized phenylglycine compounds had functional anticonvulsant effect in amygdala neurons from kindled animals. Bath superfusion of (S)-4C3HPG (100-300 µM) at concentrations above the IC50 for antagonizing mGluR1 and above the EC50 for mGluR2 (Brabet et al. 1995; Hayashi et al. 1994; Joly et al. 1995; Kingston et al. 1995; Thomsen et al. 1994a) was sufficient to block the agonist-evoked epileptiform bursting in most cells tested. As shown in Fig. 2A, (S)-4C3HPG (300 µM) antagonized the 1S,3R-ACPD (50-100 µM)-evoked bursting in five of six cells tested. Likewise, superfusing (+)-MCPG (500 µM) at a concentration sufficient to antagonize mGluR1- and mGluR2-mediated actions (Brabet et al. 1995; Hayashi et al. 1994; Joly et al. 1995; Kingston et al. 1995; Thomsen et al. 1994a) blocked the 1S,3R-ACPD-evoked bursts (n = 5). The effects of (S)-4C3HPG (300 µM) and (+)-MCPG (500 µM) on DHPG (20 µM)-evoked bursts are illustrated in Fig. 2B. (S)-4C3HPG completely blocked bursting induced by DHPG in five of seven cells tested. Similarly, (+)-MCPG (500 µM) also blocked DHPG-induced bursting (n = 5). These data present a pharmacological profile consistent with group I mGluRs, possibly mGluR1, mediating the agonist-induced bursting in BLA neurons from kindled animals.



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Fig. 2. 1S,3R-ACPD- and DHPG-evoked bursting in kindled BLA neurons is blocked by phenylglycine "antagonists." A: epileptiform bursts were evoked by 1S,3R-ACPD (100 µM; top trace, Control) in a BLA neuron from a kindled rat. The agonist-induced bursting is inhibited in the presence of (S)-4C3HPG (300 µM; 2nd trace). This effect was reversible on washout (Wash; middle trace). Also, (+)-MCPG (500 µM; 4th trace) reversibly inhibited the 1S,3R-ACPD-evoked bursting. B: epileptiform bursts were evoked by DHPG (20 µM; top trace, Control) in the same BLA neuron illustrated in A. The agonist-induced bursting is inhibited in the presence of (S)-4C3HPG (300 µM; 2nd trace). This effect was reversible on washout (middle trace, Wash). Also, (+)-MCPG (500 µM; 4th trace) reversibly inhibited the DHPG-evoked bursting. All data shown are from the same BLA neuron. Phenylglycine compounds were superfused >= 5 min before testing agonists, and >15 min was allowed for washout. For all conditions, an interval of ~5 min was given between applications of 1S,3R-ACPD and DHPG. Membrane potential was held at -60 mV before addition of agonists and during superfusion of antagonists. Calibration bar is the same for all traces. Peak amplitudes of burst spikes have been attenuated by the chart recorder.

Synaptic activity in the BLA is enhanced by kindling

Previously, this laboratory has demonstrated that the efficacy of synaptic transmission in the BLA is greatly enhanced by kindling epileptogenesis (Asprodini et al. 1992; Neugebauer et al. 1997; Rainnie et al. 1992). We tested the hypothesis that mGluR subtypes that participate in bursting activity may be involved in the enhanced transmission observed in kindled animals. Figure 3A shows a typical response of a control BLA neuron to LA stimulation at progressively increasing stimulus intensity. There is a graded increase in the size of monosynaptic EPSPs, culminating in AP firing. In contrast, after kindling (Fig. 3B), only small EPSPs are elicited in a BLA neuron before epileptiform bursting occurs. The enhanced synaptic efficacy can be assessed by examination of the input-output function, constructed by plotting synaptic response amplitude as a function of the synaptic stimulation intensity (Fig. 3C). Kindling yields an increase in the slope of the input-output relationship. Stimulus intensity and the evoked synaptic responses in control and kindled neurons are compared in Table 1. The stimulus intensity for evoking threshold EPSPs is slightly, but not significantly, lowered in kindled animals compared with control animals. The threshold EPSP amplitudes were not changed in kindled animals. The stimulus intensity for evoking an EPSP just subthreshold for AP (burst) initiation was significantly decreased in kindled animals (P < 0.05, unpaired t-test). Also, the stimulus intensity necessary for spiking activity is significantly decreased as a result of kindling (P < 0.05, unpaired t-test). The enhanced synaptic activity observed on LA stimulation in kindled animals is consistent with previous reports (Asprodini et al. 1992; Rainnie et al. 1992) showing that synaptic activity in the BLA is enhanced by kindling. It is manifested in vitro as epileptiform bursting evoked at lower stimulus intensities than necessary to yield AP firing in neurons from control animals.



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Fig. 3. Kindling-induced changes in synaptic transmission. A: synaptic transmission from the lateral amygdala nucleus (LA) to the BLA in a control slice. Electrical stimulation of increasing intensity to the LA elicits graded monosynaptic excitatory postsynaptic potentials (EPSPs) followed by action potential (AP) firing at higher stimulation intensity. B: synaptic transmission from LA to BLA in a kindled slice. LA stimulation evokes only small amplitude EPSPs over a narrow voltage range followed by synaptically mediated bursting activity. In A and B, each trace shows a representative response to a single stimulation. C: input-output relationship (EPSP amplitude plotted as a function of stimulus intensity) obtained from the BLA cells shown in A (black-square---black-square, Control) and B (black-triangle---black-triangle, Kindled). After kindling, synaptic responses are enhanced in the BLA, indicated by the steeper slope of the input-output relationship. In C, the average EPSP amplitude resulting from 3-5 stimuli at each intensity (applied at 0.2 Hz) is plotted. Calibration in A is the same in B.


                              
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Table 1. Summary of input-output relationships in control and kindled animals

(S)-4C3HPG but not (+)-MCPG reverses the effects of kindling on synaptically evoked epileptiform bursting

The experiments described previously showed that the phenylglycines (S)-4C3HPG and (+)-MCPG effectively block the mGluR-agonist-evoked bursting in kindled neurons. On the basis of these data, it was hypothesized that these phenylglycines may also inhibit synaptically driven epileptiform bursting. As shown in Fig. 4, (S)-4C3HPG and (+)-MCPG had different effects on epileptiform bursting. In normal aCSF, burst threshold was determined (Fig. 4A). The same stimulus intensity was again tested after superfusing (S)-4C3HPG (300 µM; Fig. 4B). In response to stimulation that evoked epileptiform bursting in control aCSF, monosynaptic EPSPs were recorded in the presence of (S)-4C3HPG (300 µM). In a total of five neurons, EPSPs of 4.2 ± 2.0 mV (n = 5) were recorded at the predrug control burst threshold of 7.0 ± 0.6 V in the presence of (S)-4C3HPG (300 µM). After washout of (S)-4C3HPG (Fig. 4C), this stimulus intensity again evoked bursting. In contrast to the inhibitory effect of (S)-4C3HPG on epileptiform bursting, superfusing (+)-MCPG (500 µM) had little effect on evoked synaptic activity in kindled animals. As shown in Fig. 4D, superfusing (+)-MCPG (500 µM) did not inhibit epileptiform bursting in any neuron tested (n = 3). The anticonvulsant effect of (S)-4C3HPG or the lack of effect of (+)-MCPG cannot be explained by a change in membrane resistance. Hyperpolarizing current steps (50 pA, 100 ms; Fig. 4B, inset) preceding the application of the electrical stimulus are overlapping, suggesting that the tested compounds are not affecting membrane conductance. Taken together with the known relative agonist-antagonist properties of (S)-4C3HPG and (+)-MCPG (Brabet et al. 1995; Hayashi et al. 1994; Joly et al. 1995; Kingston et al. 1995; Thomsen et al. 1994a; Watkins and Collingridge 1994), these data suggest that the different functional effects of these compounds may be mediated by different mGluR subtypes.



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Fig. 4. The phenylglycine derivatives (S)-4C3HPG and (+)-MCPG affect synaptically evoked bursting differently in kindled animals. A: in a kindled BLA neuron, LA stimulation (6V) evokes epileptiform bursting before addition of any drugs (Control). B: after superfusion of the mixed mGluR1 antagonist-mGluR2 agonist, (S)-4C3HPG (300 µM, >5 min), the 6V stimulus evokes only an EPSP. C: after washout of (S)-4C3HPG (Wash), the electrical stimulation again evokes bursting. D: superfusing the antagonist of mGluR1 and mGluR2, (+)-MCPG (500 µM, >5 min), had no effect on synaptically evoked bursting. However, (+)-MCPG did inhibit the agonist-evoked bursting in this neuron (same as in Fig. 2). In all traces, a hyperpolarizing current step (50 pA, 100 ms) preceded the electrical stimulus to monitor input resistance. B, inset, shows overlaid electrotonic potentials from traces A-D. These data suggest that the effect of the phenylglycine compounds on bursting is independent of any change in postsynaptic input resistance. Calibration in A is the same for B-D. All data are from the same kindled BLA neuron, and each trace shows a single representative response to LA stimulation.

The concentration dependence of the reversible anticonvulsant effect of (S)-4C3HPG is illustrated in Fig. 5 and summarized in Fig. 6. As the concentration of (S)-4C3HPG increases, there is an inhibition of epileptiform bursting activity and a broader range of EPSP amplitudes is measured (Fig. 5, D and E). One-way ANOVA revealed a significant effect of concentration on the increase in burst threshold (Fig. 6; P < 0.0001). The EC50 for the (S)-4C3HPG-induced increase in threshold for AP (burst or spike) initiation was calculated as 345 µM (see METHODS). When the epileptiform bursting is inhibited by application of (S)-4C3HPG, increasing the stimulus intensity above the previous burst threshold, now produces spiking that resembles that recorded from control animals (cf. Figs. 5E and Fig. 3A). Figure 7 shows the input-output relationships obtained before (Control), during, and after (Wash) superfusion of (S)-4C3HPG (300 µM), in the same cell as Fig. 5. (S)-4C3HPG decreased the slope of the input-output relationship. Stimulus intensity and evoked synaptic responses recorded in kindled neurons before and during superfusion of (S)-4C3HPG are compared in Table 2. The presence of (S)-4C3HPG (300 µM) did not significantly affect the threshold for eliciting EPSPs or the amplitude of threshold EPSPs. However, the presence of (S)-4C3HPG (300 µM) did significantly increase the stimulus intensity necessary for eliciting maximum amplitude EPSPs (P < 0.05, n = 5). Also, (S)-4C3HPG significantly elevated the stimulus intensity necessary for AP initiation (P < 0.05, n = 5). These data suggest that the inhibitory effect of (S)-4C3HPG on epileptiform bursting may be due to a reduction in the functional gain of the synapse, preventing BLA neurons from reaching threshold for AP (bursting) generation.



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Fig. 5. Concentration-response dependence of (S)-4C3HPG-induced inhibition of synaptically evoked bursting. A: electrical stimulation of the LA at increasing intensities (6, 6.5, and 7 V overlaid in this example) in normal artificial cerebrospinal fluid (Control) evokes only a small monosynaptic EPSP (6.5 V) followed by bursting (7 V). B-E: superfusion of (S)-4C3HPG at progressively increasing concentrations blocks bursting and reveals a larger range of stimulus intensities that elicit EPSPs. The EPSPs recorded in the presence of 300 µM (S)-4C3HPG (E) are evoked by stimulus intensities of 7, 8, 9, and 10 V, with synaptically evoked AP firing occurring at 12 V. Moreover, these synaptic potentials closely resemble those recorded from control animals (cf. Fig. 3A) than those recorded in neurons from kindled animals. F: LA stimulation at 6.5- and 7-V evoked bursting after >15 min washout of (S)-4C3HPG. Calibration in A applies to all traces. All data are from the same kindled BLA neuron, and each trace shows a single representative response to LA stimulation.



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Fig. 6. Concentration-response relationship of (S)-4C3HPG-induced increase in threshold for AP initiation. Increasing concentrations of (S)-4C3HPG result in progressively higher stimulus intensities required to evoke AP (burst or spike) firing (EC50 = 345 µM). At low concentrations of (S)-4C3HPG (e.g., 10-30 µM) there was a tendency toward epileptiform bursting (see Fig. 6, B and C). At higher concentrations of (S)-4C3HPG (e.g., 100-1,000 µM) AP firing resembled the spiking observed in control slices (see Fig. 3A). Data were collected for 13 different kindled BLA neurons, and each point represents the average response of n = 5 neurons (except 1,000 µM, n = 2). One-way ANOVA revealed a significant effect of (S)-4C3HPG concentration on burst/spike threshold [F(5,29) = 55.2, P < 0.0001]. *** P < 0.001 relative to predrug control (post hoc Dunnett's multiple comparison test).



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Fig. 7. The input-output relationship of the kindled LA-BLA synapse in the presence and absence of (S)-4C3HPG. In a kindled BLA neuron, electrical stimulation of the LA elicits threshold EPSP and bursting at similar stimulus intensity (Control, ---open circle ---). After superfusion of (S)-4C3HPG (300 µM; ------), EPSPs can be elicited over a broader range of stimulus intensities. (S)-4C3HPG decreases the slope of the input-output relationship, suggesting that (S)-4C3HPG restores normal synaptic transmission at the kindled LA-BLA synapse. Points on the graph represent the average of 3-5 EPSPs at each stimulus intensity under all 3 conditions. All data were collected from the same neuron, illustrated in Fig. 6.


                              
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Table 2. Summary of effect of (S)-4C3HPG* on input-output relationship in kindled animals


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The data of this study show that 1) group-I mGluRs (mGluR1 and/or mGluR5) participate in bursting evoked in kindled amygdala neurons, 2) synaptic transmission at the LA-BLA synapse is enhanced by kindling-induced epileptogenesis, and 3) activation of group II receptors in conjunction with inhibition of group I receptors has anticonvulsant actions on kindled BLA neurons by blocking epileptiform bursting and restoring synaptic transmission in the amygdala to that similar to control neurons. Moreover, these data suggest that the combination of group I antagonism and group II receptor activation by substances similar to (S)-4C3HPG may be potential future targets for treatment of seizure disorders.

It was shown that the broad-spectrum mGluR agonist 1S,3R-ACPD evokes bursting in kindled BLA neurons but not neurons from control animals, in agreement with our previous report (Holmes et al. 1996b). Other in vitro evidence supports a facilitatory role of 1S,3R-ACPD in bursting activity. 1S,3R-ACPD produces burst firing in dorsolateral septal neurons (Zheng and Gallagher 1991, 1995), and in thalamic neurons, firing mode is changed from single-spike to burst firing by 1S,3R-ACPD (McCormick and von Krosigk 1992). In hippocampal CA3 neurons, mGluR agonists increase the frequency of picrotoxin-induced bursts (Merlin and Wong 1997; Merlin et al. 1995). In vivo, limbic seizures are produced by intrahippocampal injection (Sacaan and Schoepp 1992), systemic administration (McDonald et al. 1993), or intrathalamic injection (Tizzano et al. 1993) of 1S,3R-ACPD. In contrast, others have shown in vitro an inhibitory effect of 1S,3R-ACPD on epileptic activity in cortical neurons (Burke and Hablitz 1994; Sheardown 1993). Similarly, intra-amygdala injections of L-AP4 (Abdul-Ghani et al. 1997) or 1S,3R-ACPD in amygdala-kindled rats depress seizures in vivo (Suzuki et al. 1996).

The conflicting evidence of the role of mGluRs in seizure models may result from functional effects of activating different receptor subtypes. We further characterized the receptors involved in bursting by testing group-selective agonists. Only the group I-specific ligand DHPG caused bursting similar to 1S,3R-ACPD, suggesting group I receptors are involved in bursting in kindled BLA neurons. Some evidence suggests that group I receptors are functionally excitatory, both in control and epileptic neurons. The group I agonist DHPG, but not group II or III agonists, has been shown to mediate the excitatory effects of mGluRs in hippocampal CA1 neurons (Gereau and Conn 1995) from control animals. In kindled animals, biochemical data have shown that mGluR agonist-mediated PI hydrolysis is lastingly enhanced in the amygdala (Akiyama et al. 1992), suggesting PLC-coupled (group I) receptors are up-regulated in kindled animals, and hippocampal PKC activity is enhanced by amygdala kindling (Akiyama et al. 1995; Chen et al. 1992), further supporting a role for group I receptors in epileptiform activity. Intracerebral administration of DHPG has been shown to evoke seizure activity (Tizzano et al. 1995b) and to convert short picrotoxin-induced bursts into prolonged bursts with increased frequency (Merlin and Wong 1997; Merlin et al. 1998). Other evidence suggests that group II and group III receptors may have inhibitory actions on epileptiform activity. The group II agonist L-CCG-I suppresses bicuculline-induced bursts in cortical neurons (Burke and Hablitz 1995) and both L-CCG-I and L-AP4 protect against DHPG-induced seizures in vivo (Tizzano et al. 1995b). In amygdala-kindled rats, injection of L-AP4 into the amygdala depresses kindled seizures in vivo (Abdul-Ghani et al. 1997; Suzuki et al. 1996). Similarly, the presynaptic mGluR agonist 1S,3S-ACPD (Watkins and Collingridge 1994) inhibited seizures in amygdala-kindled animals (Attwell et al. 1995). L-CCG-I and L-AP4 block evoked bursting in kindled BLA neurons in vitro by presynaptically depressing transmission (Neugebauer et al. 1997). However, recent evidence suggests that group II and group III receptors may have both convulsant and anticonvulsant effects (Ghauri et al. 1996; Tang et al. 1997). The results presented here are consistent with group I, but not group II or III, mGluRs facilitating bursting in kindled BLA neurons.

The agonist-evoked bursting in kindled BLA neurons was blocked by both phenylglycine compounds (+)-MCPG and (S)-4C3HPG, and both are antagonists of mGluR1 in expression systems (Brabet et al. 1995; Hayashi et al. 1994; Joly et al. 1995; Thomsen et al. 1994a). These data together with the agonist pharmacology suggest that group I receptors are involved in bursting in the BLA. In high K+ epileptiform activity in hippocampal neurons is blocked by (+)-MCPG (McBain 1994). Also, (+)-MCPG decreased picrotoxin-induced burst frequency in hippocampal CA3 neurons (Merlin et al. 1995), and both (+)-MCPG and (S)-4C3HPG blocked 4-aminopyridine (4-AP)-induced bursting in CA1 neurons (Bianchi and Wong 1995). Other studies, however, have shown contradictory results. For example, (S)-4C3HPG increased the frequency of picrotoxin-induced bursting (Merlin et al. 1995), and MCPG did not block the maintenance of 4-AP bursting in amygdala neurons, although the induction of bursting was inhibited (Arvanov et al. 1995). Also, it has been recently shown that mRNA levels for mGluR1 and mGluR5 are differentially altered in hippocampus by amygdala kindling but are not lastingly changed (Akbar et al. 1996). Additionally, changes in mGluR2 and 4 mRNAs were observed in kainate-evoked status epilepticus (Aronica et al. 1997), but changes of mRNA levels may not reflect alterations in the functional state of the receptor. Together the results of this study are consistent with a facilitatory action of group I mGluR on bursting in kindled BLA neurons because the group I selective agonist DHPG mimicked the bursting evoked by 1S,3R-ACPD, and the agonist-induced bursting was blocked by the mGluR1 antagonists (+)-MCPG and (S)-4C3HPG. Further, these results suggest that activation mGluR1 can result in bursting activity in kindled BLA neurons.

The agonist-evoked bursting and epileptiform synaptic bursting may be functionally related. One possibility is that the enhanced effects of group I receptor activation directly contributes to the epileptiform bursting resulting from synaptic stimulation. The results of this study suggest and previous reports from this lab have shown that group I mGluR agonist-evoked inward currents are enhanced in kindled amygdala neurons (Holmes et al. 1996b; Keele and Shinnick-Gallagher 1997) and that synaptic transmission is also enhanced (Rainnie et al. 1992). Thus it is likely that the stimulus-evoked epileptiform burst results, at least in part, from enhanced activity of the postsynaptic group I receptors. Alternatively, the agonist-evoked bursting may be a consequence of hyperexcitability in the neuronal circuitry that produces the epileptiform bursting such that the small depolarizations mediated by mGluR agonists act on hyperexcited epileptic network circuits to evoke bursting. However, kindling-induced up-regulation of agonist-evoked inward currents suggests that the former may play a role in stimulus-induced epileptiform bursts.

The block of the DHPG- and 1S,3R-ACPD-evoked bursting by the phenylglycine compounds suggested that there may be an enhanced contribution of group I mGluRs to intrinsic kindling-induced synaptic bursting and that the tested phenylglycines may also have inhibitory effects on synaptically driven bursting in kindled animals. In the presence of (S)-4C3HPG, synaptic transmission from the LA to the BLA closely resembled that of control animals. EPSPs could be evoked with a broader range of stimulus intensities in (S)-4C3HPG, similar to control animals, and electrical stimulation of the LA, which originally gave rise to epileptiform bursting, induced EPSPs in the presence of (S)-4C3HPG in kindled BLA neurons. Furthermore, (S)-4C3HPG increased the stimulation intensity necessary for spike generation. These data are similar to those of Neugebauer et al. (1997), who showed that that L-CCG-I and L-AP4 inhibit LA-evoked epileptiform bursting. However, in contrast to findings with group II and III selective agonists (Neugebauer et al. 1997), increasing the stimulation intensity in the presence of (S)-4C3HPG did not produce epileptiform bursting but elicited only AP spiking that resembled synaptic transmission in control animals.

The inhibitory effect of (S)-4C3HPG may result from greater efficacy at group II receptors because Neugebauer et al. (1997) reported an enhanced inhibitory effect of presynaptic group II receptors in the amygdala after kindling. However, activating group II receptors seems insufficient to explain the profound inhibitory effect of (S)-4C3HPG on epileptiform bursting described here. Neugebauer et al. (1997) reported that epileptiform bursting was not abolished by group II mGluR agonist because a larger stimulus intensity still evoked epileptiform bursting. Here we show that epileptiform bursting was completely abolished in the presence of (S)-4C3HPG. Also, with (S)-4C3HPG there was no significant alteration of threshold EPSPs, which would be expected if (S)-4C3HPG inhibited epileptiform bursting caused by a selective presynaptic group II mechanism. In addition, (S)-4C3HPG inhibited group I agonist-evoked bursting. Thus our data suggest that (S)-4C3HPG may exert its inhibitory action on epileptiform bursting via a combination of antagonism of the excitatory group I and activation of inhibitory group II receptors.

(+)-MCPG did not inhibit LA-evoked epileptiform bursting in kindled animals. It was previously shown in this laboratory (Arvanov et al. 1995) that (RS)-MCPG did not inhibit established bursting evoked by the convulsant 4-AP but did prevent the transition from normal to epileptic neural activity when administered before addition of the convulsant. Dalby and Thomsen (1996) also reported that (+)-MCPG had no effect on pentylenetetrazol-induced seizures in mice. In contrast to these studies, (+)-MCPG was able to block long bursts evoked by 4-AP (Bianchi and Wong 1995) as well as to decrease the frequency of picrotoxin-induced spontaneous bursts in rat hippocampal CA3 cells (Merlin et al. 1995), and the maintenance of mGluR agonist mediated increase of picrotoxin-induced burst duration (Merlin and Wong 1997). (+)-MCPG also suppressed bicuculline-induced epileptiform activity in neocortex but was ineffective in antagonizing the effects of 1S,3R-ACPD on bicuculline bursting (Burke and Hablitz 1995).

The lack of inhibition of LA-evoked bursting by (+)-MCPG is probably not due to insufficient concentration. (+)-MCPG at a concentration of 500 µM blocked agonist-evoked bursting, even in neurons where afferent-evoked epileptiform bursting was unaffected. Data obtained in cell systems expressing mGluRs also suggest that lower concentrations are sufficient to observe pharmacological effects of (+)-MCPG (Hayashi et al. 1994; Kingston et al. 1995; Thomsen et al. 1994a). However, Joly et al. (1995) and Brabet et al. (1995) showed that (+)-MCPG antagonized glutamate-induced PI hydrolysis in mGluR1alpha -expressing cells but not in mGluR5-expressing cells. The lack of effect of (+)-MCPG may be due to its low potency at mGluR5 (Brabet et al. 1995; Joly et al. 1995). In the amygdala (+)-MCPG (200 µM) itself reduced synaptic transmission in control neurons (Keele et al. 1995); the inhibition could be due to a reduction in tonic group I mGluR activation or, as suggested for LTP in the hippocampus, to an agonist action at a group II mGluR (Breakwell et al. 1998). Either of these actions would be expected to reduce epileptiform bursting.

The (S)-4C3HPG-mediated inhibition of epileptiform bursting in kindled BLA neurons suggests it may have anticonvulsant properties. (S)-4C3HPG has been shown to have anticonvulsant efficacy in animal models of epilepsy and neuroprotective action against ischemia. Intracerebral injection of (S)-4C3HPG inhibits sound-induced seizures in DBA/2 mice (Thomsen et al. 1994b) and genetically epilepsy-prone rats (Tang et al. 1997) as well as pentylenetetrazol-induced seizures in mice (Dalby and Thomsen 1996). In guinea pig hippocampal CA3 neurons, (S)-4C3HPG blocked 4-AP-induced bursting (Bianchi and Wong 1995). (S)-4C3HPG protected cortical cultures from oxygen and glucose deprivation as well as NMDA-induced excitotoxic cell death (Buisson et al. 1996) and protected striatal neurons from quinolinic acid lesions (Orlando et al. 1995). These reports strongly support our conclusion that (S)-4C3HPG has anticonvulsant efficacy, and furthermore this study extends previous findings in acute in vitro seizure models by showing the anticonvulsant effects of the (S)-4C3HPG on epileptiform bursting in vitro from chronic in vivo kindled animals.

The neuroprotective and antiepileptic actions of (S)-4C3HPG reported here and in other studies (Buisson and Choi 1995; Dalby and Thomsen 1996; Orlando et al. 1995; Tang et al. 1997; Thomsen et al. 1994b) may be due to its mixed action of a mGluR1 antagonist and mGluR2 agonist. However, (S)-4C3HPG is reported to be a partial agonist at mGluR5 (EC50 > 300 µM), whereas (+)-MCPG is ineffective at this receptor subtype (Brabet et al. 1995; Joly et al. 1995). This distinct pharmacology of (S)-4C3HPG may also contribute to its anticonvulsant effects on kindling-induced epileptiform activity in BLA neurons.

In summary, this study has shown that group I mGluRs (mGluR1 or mGluR5) participate in agonist-induced bursting in amygdala neurons from kindled animals and suggests that the enhanced efficacy of synaptic transmission seen after amygdala kindling may involve these receptors. These results lend further support to the concept that group I receptors have facilitatory effects that may increase neuronal excitability in seizure models. In contrast, group II receptors in BLA neurons are inhibitory and may act as a braking mechanism to control hyperexcitability. Finally, it is suggested, based on cumulative evidence for the opposing roles of group I and II mGluRs, that compounds with a distinct agonist and antagonist pharmacological profile similar to (S)-4C3HPG may be useful for future treatment of seizure disorders.


    ACKNOWLEDGMENTS

The authors thank Drs. Joel P. Gallagher and Kei Yamada for critical reading of the manuscript.

This research was supported by National Institute of Neurological Disorders and Stroke Grant NS-24643 to P. Shinnick-Gallagher.

Present addresses: N. B. Keele, Dept. of Psychology and Neurosciences, Baylor University, Box 97334, Waco, TX 76798-7334; V. Neugebauer, Dept. of Anatomy and Neuroscience, Marine Biomedical Institute, University of Texas Medical Branch, Galveston, TX 77555-1069.


    FOOTNOTES

Address for reprint requests: P. Shinnick-Gallagher, Dept. of Pharmacology and Toxicology, University of Texas Medical Branch, Route 1031, Galveston, TX 77555-1031.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 28 May 1998; accepted in final form 26 January 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society