Epileptogenesis Up-Regulates Metabotropic Glutamate Receptor ...

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Epileptogenesis Up-Regulates Metabotropic Glutamate Receptor Activation of Sodium-Calcium Exchange Current in the Amygdala N. BRADLEY KEELE, FATIHA ZINEBI, VOLKER NEUGEBAUER, AND P. SHINNICK-GALLAGHER Department of Pharmacology and Toxicology, The University of Texas Medical Branch, Galveston, Texas 77555-1031 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 (naı¨ve and sham-operated) and amygdala-kindled rats. In control neurons, the mGlu agonist, quisqualate (QUIS; 1–100 ␮M), 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 ␮M] or the endogenous agonist, glutamate (10 –1000 ␮M), elicited the exchange current. QUIS was more potent than either (S)-DHPG or glutamate (apparent EC50 ⫽ 19 ␮M, 57 ␮M, and 0.6 mM, respectively) in activating the Na⫹-Ca2⫹ exchange current. The selective mGlu5 agonist, (R,S)-2-chloro-5hydroxyphenylglycine [(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 upregulation of a group I mGlu-, possibly mGlu5-, mediated responses coupled to Na⫹-Ca2⫹ exchange in BLA neurons.

INTRODUCTION

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 recepThe 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. 2458

tor 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 kindlinginduced epilepsy up-regulates the Na⫹-Ca2⫹ exchange current induced by activation of a group I, possibly mGlu5. METHODS

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-␮m-thick) were prepared by using a vibroslice and kept in aCSF at room temperature for ⱖ1 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

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FIG. 1. Inward currents evoked by application (arrows) of mGlu receptor agonists to basolateral amygdala (BLA) neurons in the presence of D-2-amino-5-phosphovaleric acid [D-APV (50 ␮M)], 6-cyano-7-nitroquinoxaline-2,3-dione [CNQX (30 ␮M)], and tetrodotoxin [TTX (1 ␮M)]. A: quisqualate [QUIS (30 ␮M)] inward current is associated with inward shift (I-V) curve but no change in slope conductance (B). Group I agonist, (S)-3,5-dihydroxyphenylglycine [(S)DHPG], (C), glutamate (300 ␮M, E), and the mGlu 5 agonist, (R,S)-2-chloro-5-hydroxyphenylglycine [(R,S)-CHPG] (2 mM, G) induce inward currents without a change in slope conductance (D, F, H). B, D, and F: current-voltage (I-V) relationships obtained with voltage ramp commands; H: the I-V curve obtained by applying voltage step commands. Long downward deflections numbered in A, C, and E correspond to the I-V relationships shown in B, D, and F, respectively. Repetitive downward deflections result from 5 mV (600-ms duration) hyperpolarizing voltage steps as a monitor of input conductance. VH ⫽ ⫺60 mV. Calibration (A, C, and E) ⫽ 50 pA, 20 s, and 60 pA, and 26 s (G). Agonists were applied to different neurons.

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 ␮A 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 naı¨ve 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-5hydroxyphenylglycine [(R,S)-CHPG], glutamate, and quisqualate

(QUIS) were applied to the inlet of the recording chamber in a 10-␮L 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 (DAPV), 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 fit-

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ted 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. RESULTS

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 ␮M), CNQX (30 ␮M), and TTX (1 ␮M). Drop-application of QUIS (30 ␮M; Fig. 1A) induces a peak inward current amplitude of 81 ⫾ 13 pA (n ⫽ 14), whereas the group I selective agonist (S)-DHPG (300 ␮M) elicits an inward current of 45 ⫾ 6 pA (n ⫽ 17, Fig. 1C). The endogenous ligand, glutamate (300 ␮M), 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. 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 ␮M) 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 ␮M): 8.4 ⫾ 0.6 nS; P ⬎ 0.05, n ⫽ 18], whereas that of the glutamate- (300 –100 ␮M) induced current (Fig. 1F) was 94 ⫾ 5% (n ⫽ 6) of control [control: 8.0 ⫾ 1.2 nS; glutamate (300 ␮M): 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).

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 ␮M. 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 ␮M). The enhanced response to (S)-DHPG is consistent with up-regulation of an mGlu receptor-activated Na⫹-Ca2⫹-exchange current in kindled BLA neurons. DISCUSSION

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 ⱖ glutamate ⬎ (R,S)-CHPG, data consistent with activation of mGlu5 subtype.

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 ⱖ GLU ⬎ (R,S)CHPG. Nonlinear (sigmoid) regression analyses of the agonistevoked current amplitudes show apparent EC50s of 19 ␮M (n ⫽ 3–14), 57 ␮M (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 ␮M), 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. 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

FIG. 2. Concentration-response relationships for inward currents activated by mGlu receptor agonists in control animals. Increasing concentrations of QUIS (●), glutamate (f), (S)-DHPG (E), and (R,S)-CHPG (䡺) applied to BLA neurons results in inward currents of increasing magnitude (Vh ⫽ ⫺60 mV). Points on the curve represent mean current amplitudes (⫾SE) of 3–18 different BLA neurons. Lines, least-squares nonlinear (sigmoid) regression fit to the individual data points for each agonist. All currents obtained in D-APV (50 ␮M), CNQX (30 ␮M), and TTX (1 ␮M), except those with 100 ␮M QUIS, which were recorded in 100 ␮M CNQX.

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FIG. 3. Concentration-response relationship for (S)-DHPG but not (R,S)-CHPG activated-inward current is enhanced in BLA neurons from kindled animals. Concentration-response relationship for inward currents evoked by the group I mGlu agonist (S)-DHPG (A), and the mGlu5 selective agonist (R,S)CHPG (B) are shown in control (●) and kindled (f) brain slices. The concentration-response curves in control are the same as in Fig. 2 and compared with data from kindled animals. Data points, mean (⫾SE) of 4 –17 different BLA neurons.

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 ago-

nists 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. 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. Address reprint requests to P. Shinnick-Gallagher. Received 27 September 1999; accepted in final form 13 December 1999.

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