Department of Pharmacology and Toxicology, The University of Texas Medical Branch, Galveston, Texas 77555-1031
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ABSTRACT |
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Keele, N. Bradley,
Fatiha Zinebi,
Volker Neugebauer, and
P. Shinnick-Gallagher.
Epileptogenesis Up-Regulates Metabotropic Glutamate Receptor
Activation of Sodium-Calcium Exchange Current in the Amygdala.
J. Neurophysiol. 83: 2458-2462, 2000.
Postsynaptic metabotropic glutamate (mGlu) receptor-activated inward
current mediated by Na+-Ca2+ exchange was
compared in basolateral amygdala (BLA) neurons from brain slices of
control (naïve and sham-operated) and amygdala-kindled rats. In
control neurons, the mGlu agonist, quisqualate (QUIS; 1-100 µM),
evoked an inward current not associated with a significant change in
membrane slope conductance, measured from current-voltage relationships
between 110 and
60 mV, consistent with activation of the
Na+-Ca2+ exchanger. Application of the group I
selective mGlu receptor agonist
(S)-3,5-dihydroxyphenylglycine
[(S)-DHPG; 10-1000 µM] or the endogenous agonist,
glutamate (10-1000 µM), elicited the exchange current. QUIS was more
potent than either (S)-DHPG or glutamate (apparent
EC50 = 19 µM, 57 µM, and 0.6 mM, respectively) in
activating the Na+-Ca2+ exchange current. The
selective mGlu5 agonist,
(R,S)-2-chloro-5-hydroxyphenylglycine [(R,S)-CHPG; apparent EC50 = 2.6 mM]
also induced the exchange current. The maximum response to
(R,S)-DHPG was about half of that of the
other agonists suggesting partial agonist action. Concentration-response relationships of agonist-evoked inward currents
were compared in control neurons and in neurons from kindled animals.
The maximum value for the concentration-response relationship of the
partial agonist (S)-DHPG- (but not the full agonist-
[QUIS or (R,S)-CHPG]) induced inward
current was shifted upward suggesting enhanced efficacy of this agonist
in kindled neurons. Altogether, these data are consistent with a
kindling-induced up-regulation of a group I mGlu-, possibly mGlu5-,
mediated responses coupled to Na+-Ca2+ exchange
in BLA neurons.
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INTRODUCTION |
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Eight metabotropic glutamate (mGlu) receptors have
been cloned and are subdivided into three groups, group I (mGlu1 and
mGlu5), group II (mGlu2 and mGlu3), and group III (mGlu4, 6, 7, and 8; Conn and Pin 1997; Pin and Duvoisin
1995
). Activation of mGlu receptors can facilitate epileptiform
activity in vitro (Merlin and Wong 1997
; Merlin
et al. 1995
) and in vivo (Tizzano et al. 1995
),
can be functionally inhibitory (Rainnie et al. 1994
;
Suzuki et al. 1996
), or can both enhance and inhibit
epileptiform activity (Burke and Hablitz 1995
;
Ghauri et al. 1996
; Tizzano et al. 1995
). These differing effects of mGlu receptor activation in epilepsy may be
dependent on mGlu receptor subtype, synapse, brain area, or model.
In control basolateral amygdala (BLA) neurons mGlu receptor activation
elicits a hyperpolarization (Rainnie et al. 1994) due to
opening of large-conductance Ca2+-activated
K+ channels (Holmes et al. 1996a
)
followed by a depolarization/inward currents mediated by inhibition of
potassium channels or activation of
Na+-Ca2+ exchange
(Holmes et al. 1996b
; Keele et al. 1997
).
Kindling abolishes the mGlu-evoked hyperpolarization and enhances the
depolarization mediated by inhibition of potassium channels, resulting
in epileptiform bursting (Holmes et al. 1996b
;
Keele et al. 1999
). The purpose of this study was to
test whether mGlu receptor-activation of Na+-Ca2+ exchange current
is enhanced by kindling-induced seizure activity and to analyze the
possible mGlu subtype underlying activation of the exchange current.
Here, we report that kindling-induced epilepsy up-regulates the
Na+-Ca2+ exchange current
induced by activation of a group I, possibly mGlu5.
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METHODS |
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Amygdala slices from control and kindled male Sprague-Dawley
rats (90-200 g) were prepared as described (Keele et al.
1997). Rats were decapitated; the brains rapidly removed,
placed in cold (4°C) artificial cerebrospinal fluid (aCSF) of the
following composition (in mM): NaCl, 117; KCl, 4.7;
CaCl2, 2.5; MgCl2, 1.2;
NaH2PO4, 1.2;
Na2HCO3, 25; and glucose,
11 (pH = 7.4), and aerated with a mixture of
O2/CO2 (95/5%). Coronal
brain slices (500-µm-thick) were prepared by using a vibroslice and
kept in aCSF at room temperature for
1 h. Slices were submerged in a
recording chamber superfused with aCSF (2.5 ml/min, 31 ± 1°C).
Blind whole-cell recordings were performed as described previously
(Keele et al. 1997
) by using patch electrodes (2-5
M
, pH = 7.2, 280 mosmol/kg) when filled with internal solution
containing (in mM): Cs-gluconate, 122; NaCl, 5;
CaCl2, 0.3; MgCl2, 2; EGTA,
1; HEPES, 10; Na2-ATP, 5;
Na2-GTP, 0.4. Currents were acquired with
an Axoclamp 2A amplifier with a switching frequency of 5-6 kHz (30%
duty cycle; gain, 3-8 nA/mV; time constant, 20 ms). Signals were
low-pass filtered at 1 kHz with a 4-pole Bessel filter and digitized at
5 kHz. Resting membrane potential was more negative than
60 mV and
direct cathodal stimulation evoked action potentials overshooting 0 mV.
Analogue records were continuously acquired with a pen chart recorder.
Current-voltage (I-V) relationships of mGlu
agonist-induced currents were obtained by either 1)
applying voltage ramp commands from a holding potential
(Vh) of 60 to
110 mV (80 mV/s) before and during the peak of the drug-induced current, or
2) applying voltage step commands (500 ms) from
Vh =
60 to
110 mV in 10 mV intervals
before and during the maximum evoked current. No difference between the
two I-V protocols was noted.
Rats were anesthetized with Equithesin (35 mg/kg pentobarbital
and 145 mg/kg chloral hydrate) and the right BLA implanted with
tripolar electrodes, as previously described (Holmes et al. 1996b; Keele et al. 1999
). Electrode tips were
positioned at anteroposterior
2.0 mm and lateral
4.5 mm relative to
Bregma at a depth of 7.3 mm from the dural surface (Paxinos and
Watson 1986
) and secured to the skull with stainless steel
screws and dental cement. After 5 days, kindling stimulation (50-100
µA above the afterdischarge threshold) was initiated and consisted of
a 2 s train (60 Hz) of monophasic square waves (2 ms), twice a
day. Behavioral seizure severity was rated according to Racine
(1972)
. Brain slices were prepared 3-7 days after the last of
three consecutive stage five seizures. Control drug responses were
obtained from naïve unoperated rats and confirmed in
sham-operated animals (n = 9) which have been shown
to exhibit mGlu responses similar to those in unoperated control
animals (Holmes et al. 1996b
).
(S)-3,5-dihydroxyphenylglycine
[(S)-DHPG],
(R,S)-2-chloro-5-hydroxyphenylglycine
[(R,S)-CHPG], glutamate, and quisqualate (QUIS) were
applied to the inlet of the recording chamber in a 10-µL drop from an
Oxford pipetter as described previously (Keele et al.
1997, 1999
). Drop entry and emergence were
monitored with food dye. The final concentration in the bath (1 ml) was
estimated as 1% of that of the drop. Rapid onset/offset of drug
responses minimized effects of desensitization.
(S)-DHPG, (R,S)-CHPG,
(R,S)-1-aminoindan-1,5-dicarboxylic acid (AIDA),
D-aminophosphonovaleric acid (D-APV), and
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were purchased from Tocris
Cookson (St. Louis). Tetrodotoxin (TTX), QUIS, and glutamate were
obtained from Sigma.
Data were compared by using a paired Student's t-test,
a one-way analysis of variance (ANOVA), or unpaired analysis (two-way ANOVA). Concentration-response relationships were plotted and fitted
with the equation y = A + (B A)/[1 + (10C/10X)D]
where A is bottom plateau, B is top
plateau, C is log (EC50), and
D is Hill coefficient.
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RESULTS |
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Agonist pharmacology of mGlu receptor-activated inward current in control animals
In basolateral amygdala neurons, QUIS evokes an inward current
mediated by Na+-Ca2+
exchange (Keele et al. 1997). Figure
1 compares typical exchange currents
activated by mGlu agonists in BLA neurons in the presence of
D-APV (50 µM), CNQX (30 µM), and TTX (1 µM).
Drop-application of QUIS (30 µM; Fig. 1A) induces a peak
inward current amplitude of 81 ± 13 pA (n = 14),
whereas the group I selective agonist (S)-DHPG (300 µM)
elicits an inward current of 45 ± 6 pA (n = 17, Fig. 1C). The endogenous ligand, glutamate (300 µM), at a 10-fold higher concentration than QUIS, evokes an equivalent current of
88 ± 18 pA (n = 8; Fig. 1E). The
selective mGlu5 agonist, (R,S)-CHPG (4 mM), induces a peak
inward current of 103 ± 15 pA (Fig. 1G), suggesting
mediation of the exchange current by the mGlu5 subtype.
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The QUIS inward current was accompanied by a parallel inward shift in the I-V relationship indicating that membrane slope conductance was not increased (Fig. 1B). Membrane conductance during the peak of the current was 96 ± 2% (n = 11) that of control. Membrane slope conductance was 7.2 ± 0.7 nS before and 6.9 ± 0.7 nS (P > 0.05; paired t-test; n = 11) during the QUIS (30 µM) inward current. Similarly, membrane conductance during the (S)-DHPG current (Fig. 1D) was 98 ± 1% (n = 18) of control conductance [control: 8.6 ± 0.6 nS; (S)-DHPG (300 µM): 8.4 ± 0.6 nS; P > 0.05, n = 18], whereas that of the glutamate- (300-100 µM) induced current (Fig. 1F) was 94 ± 5% (n = 6) of control [control: 8.0 ± 1.2 nS; glutamate (300 µM): 7.4 ± 1.2 nS; P > 0.05, n = 6]. The (R,S)-CHPG-induced inward current was also not accompanied by a change in slope conductance (Fig. 1, G and H; n = 7). Both phenylglycine agonists were blocked by group I antagonist, AIDA (n = 2).
Concentration-response relationships for group I mGlu agonists
Concentration-response curves obtained for QUIS,
(S)-DHPG, (R,S)-CHPG, and glutamate are
illustrated in Fig. 2. Comparison of the
curves for these agonists showed a rank order of potency of QUIS > (S)-DHPG GLU > (R,S)-CHPG.
Nonlinear (sigmoid) regression analyses of the agonist-evoked current
amplitudes show apparent EC50s of 19 µM
(n = 3-14), 57 µM (n = 4-17), 0.6 mM (n = 3-9), and 2.6 mM (n = 5-9)
with maximum current amplitudes of 109 ± 30 pA (100 µM),
54 ± 6 pA (1 mM), 157 ± 15 pA (1 mM), and 103 ± 15 pA
(4 mM) for QUIS, (S)-DHPG, glutamate, and
(R,S)-CHPG, respectively. These results also suggest that
QUIS, glutamate, and (R,S)-CHPG are full agonists
for this current, whereas (S)-DHPG may be a partial agonist.
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Changes in mGlu receptor-activated inward currents recorded in kindled animals
The concentration-response relationship for (S)-DHPG-induced inward currents (Fig. 3A) shows that the effect of this agonist is enhanced by kindling-induced epileptogenesis. The curve for the (S)-DHPG inward current in kindled animals is shifted upward relative to that obtained from control animals. In kindled neurons, (S)-DHPG has an apparent EC50 of 160 µM. Two-way ANOVA indicated a significant effect due to the kindling treatment (P < 0.01) as well as a significant effect (P < 0.0001) of (S)-DHPG concentration in both control and kindled conditions. (R,S)-CHPG also induced activation of the Na+-Ca2+exchanger (Fig. 3B) with an apparent EC50 in kindled neurons of 1.8 mM; a two-way ANOVA indicated a significant effect due to the (R,S)-CHPG concentration (P < 0.0001) in control and kindled animals but no significant effect due to the kindling treatment (P < 0.26). A similar lack of effect was observed with QUIS (kindled EC50 = 19 µM). The enhanced response to (S)-DHPG is consistent with up-regulation of an mGlu receptor-activated Na+-Ca2+-exchange current in kindled BLA neurons.
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DISCUSSION |
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The main findings of this study are 1) the Na+-Ca2+ exchange current is produced by activation of a group I mGlu receptor, possibly mGlu5, and 2) kindling-induced epilepsy up-regulates the mGlu-activated exchange current.
Pharmacology of mGlu receptor-activation of Na+-Ca2+ exchange
The pharmacology of the mGlu agonist response suggests that the
receptor underlying the inward current is a group I mGlu receptor, possibly an mGlu5. Glutamate and (R,S)-DHPG reportedly
activate mGlu1a and mGlu5a expressing cells with similar potency
(Brabet et al. 1995) and (R,S)-CHPG is
selective agonist for mGlu5 (Doherty et al. 1997
).
Previous evidence in ventral medial hypothalamic neurons suggested that
group I mGlu receptors mediate activation of the exchange current
(Lee and Boden 1997
). Agonist potency in BLA neurons was
QUIS > (S)-DHPG
glutamate > (R,S)-CHPG, data consistent with activation of mGlu5 subtype.
QUIS and glutamate activate the
Na+-Ca2+ exchange current
in a manner consistent with that of a complete agonist, whereas
(S)-DHPG in control BLA neurons evoked currents with a
maximum amplitude about 50% of that produced by QUIS, glutamate, or
(R,S)-CHPG. These data suggest that (S)-DHPG may
be acting as a partial agonist at the group I receptor mediating the
inward exchange current. This characteristic of DHPG has been reported
by others (Brabet et al. 1995; Joly et al.
1995
; Schoepp et al. 1994
).
mGlu receptor activation of
Na+-Ca2+ exchange is also
observed in cerebellar Purkinje cells (Linden et al.
1994; Staub et al. 1992
) and in ventral medial
hypothalamus (Lee and Boden 1997
) where DHPG is an
agonist for the exchange current. In the BLA, more mGlu5 than mGlu1 is
expressed (Romano et al. 1995
) and the mGlu5 selective
agonist (R,S)-CHPG activates the exchange current albeit at
low potency. Thus receptor localization and response to CHPG support
mGlu5 as the subtype underlying activation of the exchanger in BLA neurons.
The inward current evoked by (S)-DHPG is enhanced in kindled animals
In the BLA, (S)-DHPG behaves as a partial agonist with
a peak current amplitude that was markedly increased in kindled animals relative to control. The mechanisms underlying partial agonism are not
known and are beginning to be defined (Clark and Bond 1998; Clarke et al. 1999
; Kenakin
1996
). Several possible mechanisms may account for the reported
observations in kindled animals. In ventral medial hypothalamus
neurons, the mGlu-activated
Na+-Ca2+ exchange current
has been shown to be inhibited by guanosine 5'-O-(2-thiodiphosphate)
(GDP-
-S) and potentiated by guanosine 5'-O-(3-thiotriphosphate)
(GTP-
-S), indicating mediation by G proteins (Lee and
Boden 1997
). If the maximum response of a partial agonist is
dependent on the efficiency of the coupling mechanism, then kindling
may induce a more efficient second-messenger coupling, e.g., between
the receptor and G protein or between the G protein and the effector
system, and larger response amplitude to (S)-DHPG would
be observed; this effect would not be obvious with agonists in which
coupling is already highly efficient (Kenakin 1996
). Group I mGlus can also dimerize (Robbins et al. 1999
;
Romano et al. 1996
). If kindling increases dimerization,
partial agonists may detect increases in the dimerized state, whereas
full agonists may not. Some data suggests that partial agonists in one
system may be full agonists in another due to the environment extrinsic to the receptor and may involve the stoichiometry/complement of G-proteins or organization/composition of the lipid bilayer
(Ghanekar et al. 1997
). A kindling-induced alteration in
similar constituents could account for the selective enhancement of
(R,S)-DHPG compared with the other agonists.
Alternatively, it has been shown that in expression systems increasing
the amount of mGlu density can increase the maximum response
(Hermans et al. 1999
). If the result of kindling is an
increase in the number of mGlus, a partial agonist like
(S)-DHPG that requires occupancy of all available
receptors to produce its maximum response (Kenakin 1996
)
in normal animals would produce a larger response in kindled animals
because of the increased number of receptors. Highly efficacious
agonists may not be able to detect increased receptors except perhaps
with excessive transmitter release that can be attained during epilepsy.
The mGlu receptor activation of Na+-Ca2+ exchange may be a mechanism for removal of the excess intracellular Ca2+ that occurs during periods of high glutamate release; the Ca2+ extrusion and Na+ entry may contribute further to increasing cellular excitability and ictal-like bursting activity. Additionally, up-regulation of Na+-Ca2+ exchange may reduce intracellular Ca2+ concentration such that inhibitory Ca2+-activated potassium channels in the amygdala are unable to contribute to regulation of membrane excitability.
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ACKNOWLEDGMENTS |
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We thank Drs. K. M. Johnson and O. S. Steinsland for helpful discussion and Dr. Kris Tokarski for help with the CHPG experiments.
This work 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 Neuroscience, Baylor University, PO Box 97334, Waco, TX 76798-7334; V. Neugebauer, Dept. of Anatomy and Neurosciences, Medical Research Bldg. 2.130, The University of Texas Medical Branch, Galveston, TX 77555-1069.
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FOOTNOTES |
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Address reprint requests to P. Shinnick-Gallagher.
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 27 September 1999; accepted in final form 13 December 1999.
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REFERENCES |
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