Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 77555-1031
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ABSTRACT |
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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)--(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 (+)-
-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.
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INTRODUCTION |
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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
).
(+)-
-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).
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METHODS |
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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 M
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 G. 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
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RESULTS |
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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-
-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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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
(+)-
-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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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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(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.
|
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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DISCUSSION |
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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 mGluR1
-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.
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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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.
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REFERENCES |
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