The Journal of Neurophysiology Vol. 78 No. 6 December 1997, pp. 3475-3478
Copyright ©1997 by the American Physiological Society

Loss of Long-Lasting Potentiation Mediated by Group III mGluRs in Amygdala Neurons in Kindling-Induced Epileptogenesis

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

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

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Neugebauer, Volker, N. Bradley Keele, and Patricia Shinnick-Gallagher. Loss of long-lasting potentiation mediated by group III mGluRs in amygdala neurons in kindling-induced epileptogenesis. J. Neurophysiol. 78: 3475-3478, 1997. Long-lasting modifications of synaptic transmission can be induced in the amygdala by electrical stimulation as done in the long-term potentiation (LTP) model of learning and memory and the kindling model of epilepsy. The present study reports for the first time a long-lasting potentiation (LLP) of synaptic transmission that is induced pharmacologically by the activation of group III metabotropic glutamate receptors (mGluRs) in basolateral amygdala (BLA) neurons. In whole cell voltage-clamp mode, BLA neurons were recorded in brain slices from control rats and rats with amygdala-kindled seizures. The group III mGluR agonist L-2-amino-4-phosphonobutyrate (L-AP4, 10 µM) induced LLP of monosynaptic excitatory postsynaptic currents (EPSCs) evoked by electrical stimulation in the lateral amygdala (maximum 258 ± 50% of predrug control; means ± SE) in control (n = 7) but not in kindled neurons(n = 6). LLP was measured 15 min after the superfusion of L-AP4, lasted for >45 min, and was not accompanied by postsynaptic membrane changes. L-AP4 induced LLP was prevented by the group III mGluR antagonist (S)-2-methyl-2-amino-4-phosphonobutyrate (MAP4; 100 µM, n = 6) but not the group II mGluR antagonist (2S,3S,4S)-2-methyl-2-carboxycyclopropylglycine (MCCG; 100 µM, n = 3). LLP was not observed after superfusion of the group II mGluR agonist (2S,3S,4S)-2-(carboxycyclopropyl)glycine (L-CCG; 1.0 and 10 µM) in either control (n = 13) or kindled (n = 10) neurons. If the underlying mechanisms and the functional significance of pharmacologically induced LLP are similar to those of LTP, the loss of L-AP4 induced LLP in kindled neurons may be a neurobiological correlate of learning and memory deficits in kindled animals and long-term alterations of brain functions in patients with epilepsies.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

The amygdala plays a role in learning and memory associated with emotion (McGaugh et al. 1990) and was shown to undergo modification of synaptic transmission in the long-term potentiation (LTP) model of learning and memory (Chapman and Bellavance 1992; Gean et al. 1993; Watanabe et al. 1995). The amygdala also is one of the brain areas most sensitive to kindling-induced neuroplasticity (Löscher et al. 1995), another form of long-lasting enhancement of synaptic transmission. In the kindling model of temporal lobe epilepsy, repeated initially subconvulsive electrical stimuli to certain brain areas result in the progressive development of partial and generalized seizures (Goddard et al. 1969; Racine 1978). Understanding the cellular mechanisms underlying the kindling-induced long-term alterations in brain function may initiate therapeutic strategies to prevent long-term effects of seizures, including intractable epilepsies and memory disorders (Sutula et al. 1995).

The G-protein coupled metabotropic glutamate receptors (mGluRs) consist of eight subtypes, which, on the basis of sequence homology, signal transduction mechanisms, and pharmacological profile, are classified into groups I, II, and III (Knöpfel et al. 1995; Pin and Duvoisin 1995). Postsynaptically, group I mGluRs are up-regulated in kindled basolateral amygdala (BLA) neurons whereas group II mGluRs are downregulated (Holmes et al. 1996). Presynaptically, activation of groups II and III mGluRs depresses synaptic transmission in control BLA neurons; in kindled neurons this inhibitory effect is enhanced 30-fold (Neugebauer et al. 1997).

Here we report a novel pharmacologically induced form of long-lasting potentiation (LLP) of excitatory synaptic transmission in the BLA after the activation of group III but not group II mGluRs. Importantly, this pharmacologically induced LLP is lost in kindled neurons. Although it is presently unclear how this form of LLP relates to the established LTP model of learning and memory, the loss of pharmacologically induced LLP in kindled neurons may be relevant for the learning and memory deficits in kindled animals and patients with epilepsies.

	METHODS

Abstract Introduction Methods Results Discussion References

Amygdala brain slices were obtained from control and kindled male Sprague-Dawley rats (90-200 g) as previously described (Neugebauer et al. 1997). Rats were decapitated and the brains were rapidly placed in cold oxygenated (95% O₂-5% CO₂) artificial cerebrospinal fluid (ACSF) containing (in mM) 117 NaCl, 4.7 KCl, 1.2 NaH₂PO₄, 2.5 CaCl₂, 1.2 MgCl₂, 25 NaHCO₃, and 11 glucose. Coronal brain slices (500 µm) were prepared with a vibroslice. After incubation in ACSF at room temperature for 1 h, a single brain slice was transferred to the submerged recording chamber and superfused with ACSF [31 ± 1°C].

Blind whole cell recordings (Blanton et al. 1989) were obtained from BLA neurons by using patch electrodes with tip resistances of 3-5 M. Two internal solutions (pH 7.2-7.3; 280 mOsm/kg) were used. The first contained (in mM) 122 K-gluconate, 5 NaCl, 0.3 CaCl₂, 2 MgCl₂, 1 ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA), 10 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 5 Na₂-ATP, and 0.4 Na₃-guanosine 5-triphosphate(GTP). The second contained (in mM) 140 KMeSO₄, 10 HEPES, 2 Na₂-ATP, and 0.3 Na₃-GTP. Because no difference of drug effects was found with either internal solution, the data were pooled. Discontinuous single-electrode voltage clamp recordings were acquired by using an Axoclamp-2A amplifier with a switching frequency of 5-6 kHz (30% duty cycle; gain of 5-8 nA/mV; time constant 20 ms). Signals were low-pass filtered at 1 kHz with a 4-pole Bessel filter (Warner Instrument), digitized at 5 Hz (Digidata 1200), acquired, and analyzed with pCLAMP 6.03 software.

Monosynaptic excitatory postsynaptic currents (EPSCs) were evoked in BLA neurons by electrical stimulation (150-µs square-wave pulses at <0.25 Hz) of afferents from the lateral amygdala with a concentric bipolar stimulating electrode. Test stimuli were adjusted to ~80% of the intensity required for orthodromic spike generation.

The following pharmacological agents (Tocris Cookson) were tested and superfused in the ACSF for ~14 min: (2S,3S,4S)-2-(carboxycyclopropyl)glycine (L-CCG), L(+)-2-amino-4-phosphonobutyrate (L-AP4), (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine (MCCG), and (S)-2-methyl-2-amino-4-phosphonobutyrate (MAP4).

For the kindling procedure (see Neugebauer et al. 1997), rats were anesthetized with Equithesin (35 mg/kg pentobarbital sodium, 145 mg/kg chloral hydrate) and implanted with tripolar electrodes into the right BLA by using the coordinates (A/P 2.0 mm; L 4.5 mm; and D/V 7.3 mm) from Paxinos and Watson (1996). Kindling stimulation began 5 days after implantation and consisted of a 2-s train of 60-Hz monophasic square waves (2 ms) at 50-100 µA above afterdischarge threshold, administered twice daily. Brain slices for the electrophysiological experiments were obtained 5.1 ± 0.3 days after the last of three consecutive stage 5 (fully kindled) seizures. Control slices were obtained from both unoperated and sham-operated rats.

Data were compared using the unpaired t-test. Values are given as the means ± SE and statistical significance accepted at P < 0.05. Slope conductances were calculated from the current-voltage relationships by using the linear curve fit function of pCLAMP software.

	RESULTS

Abstract Introduction Methods Results Discussion References

Long-lasting potentiation of synaptic transmission by L-AP4 in control but not in kindled neurons

BLA neurons were recorded in whole cell voltage-clamp mode and held at 60 mV. In a previous study, we reported that application of the group III mGluR agonist L-AP4 induced a concentration-dependent inhibition of EPSC amplitude (Neugebauer et al. 1997). After the superfusion of L-AP4 (10 µM, 14 ± 0.8 min, n = 7) in the same control neurons a LLP of monosynaptic EPSCs was observed. LLP in control neurons was concentration-dependent (not shown): L-AP4 (1 µM, n = 12) induced a potentiation of 151 ± 34% and no significant potentiation (108 ± 15%) was observed with L-AP4 (0.1 µM, n = 9). Fig. 1B shows that LLP induced by L-AP4 (10 µM) reached a maximum of 258 ± 50% of control and was significantly different(P < 0.05, unpaired t-test) from the synaptic responses of neurons in brain slices from fully kindled animals (METHODS). The kindled neurons did not exhibit LLP after equimolar concentrations of L-AP4 (10 µM, 14 ± 0.5 min, n = 6) or concentrations equipotent in depressing synaptic transmission (1.0 µM, 14 ± 1 min, n = 5). On the other hand, in the same kindled neurons, the concentration-dependent inhibition of EPSC amplitude induced by L-AP4 was enhanced as reported previously (Neugebauer et al. 1997). L-AP4 induced LLP was not accompanied by changes in slope conductance (Fig. 1C) and postsynaptic membrane currents (not shown).

View larger version (27K): [in this window] [in a new window]   FIG. 1. L-2-amino-4-phosphonobutyrate (L-AP4) induces long-lasting potentiation (LLP) in control but not kindled neurons. A: traces recorded from a representative control neuron show average of 8-10 monosynaptic excitatory postsynaptic currents (EPSCs) at times indicated (a-d) in B. B: after superfusion of indicated concentrations of L-AP4 (at 0 min) a LLP was recorded in control () but not in kindled BLA neurons ( and ). EPSC peak amplitudes obtained for each neuron before, during, and after L-AP4 were averaged and expressed as percent of predrug control values (100%). Symbols and error bars represent means ± SE. * P < 0.05; ** P < 0.01; *** P < 0.001, unpaired t-test. C: L-AP4 had no consistent effects on membrane conductance (GL-AP4). Right: difference of slope conductance during and after superfusion of L-AP4 compared with predrug control values (left). For each control (open bars, n = 7) and kindled neuron (filled bars: 10 µM L-AP4, n = 6; 1 µM L-AP4: n = 5) slope conductance was calculated from I-V relation before, during, and after L-AP4. Membrane voltage was held at 60 mV.

MAP4 but not MCCG antagonizes L-AP4 induced LLP

LLP is blocked in the presence of the group III mGluR antagonist MAP4 (100 µM) but not the group II mGluR antagonist MCCG (100 µM, Fig. 2A). Figure 2B shows that L-AP4 (10 µM, 14 ± 0.9 min) induces a slight synaptic potentiation in the presence of MAP4 (100 µM; n = 6). This effect is significantly (P < 0.05-0.001, unpaired t-test) different from the L-AP4 induced LLP in the presence of MCCG (100 µM, n = 3). MAP4 and MCCG themselves did not consistently affect baseline synaptic transmission, neither in this study nor in a previous study (Neugebauer et al. 1997). L-AP4 had no effect on slope conductance (Fig. 2C) and membrane currents (not shown).

View larger version (22K): [in this window] [in a new window]   FIG. 2. (S)-2-methyl-2-amino-4-phosphonobutyrate (MAP4) but not (2S,3S,4S)-2-methyl-2-carboxycyclopropylglycine (MCCG) antagonizes L-AP4 induced LLP. A: traces recorded from a representative control neuron show average of 8-10 monosynaptic EPSCs at times indicated (a-c) in B. B: L-AP4 (10 µM) induced LLP was significantly (P < 0.05-0.001, unpaired t-test) suppressed in presence of the group III mGluR antagonist MAP4 () but not group II mGluR antagonist MCCG (); display as in Fig. 1. C: L-AP4 had no consistent effects on slope conductance (GL-AP4) in presence of MCCG (open bars, n = 3) or MAP4 (filled bars, n = 6); symbols as defined in Fig. 1.

L-CCG does not induce LLP in control and in kindled neurons

Consistent with our previous findings (Neugebauer et al. 1997), L-CCG (1 µM, 14 ± 0.6 min) was 10 times more potent in depressing monosynaptic EPSCs than L-AP4 (cf. Fig. 1B). As opposed to L-AP4 however, L-CCG did not induce LLP in control (n = 13) or kindled (n = 5) neurons (Fig. 3A). L-CCG had no effect on slope conductance (Fig. 3B) and postsynaptic membrane current (not shown). Higher concentrations of L-CCG (10 and 100 µM) did not induce LLP but increased slope conductance and evoked a small outward current (~20 pA) in control neurons (n = 7), suggesting that these concentrations are not selective for presynaptic group II mGluRs.

View larger version (24K): [in this window] [in a new window]   FIG. 3. A: L-CCG (1 µM) did not induce LLP in control () or kindled neurons (). B: L-CCG did not change slope conductance (GL-CCG) in control (open bars, n = 13) or kindled neurons (filled bars, n = 5); symbols as in Fig. 1.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

The main findings of this study are that: 1) the group III mGluR agonist L-AP4 induces LLP of synaptic transmission in control but not in kindled BLA neurons; 2) L-AP4 induced LLP is not accompanied by apparent postsynaptic membrane changes; 3) L-AP4 induced LLP is blocked by the group III mGluR antagonist MAP4 but not by the group II antagonist MCCG; and 4) a group II mGluR agonist, L-CCG, does not induce LLP.

Our data suggest that this pharmacologically induced LLP in the amygdala is mediated through group III mGluRs. L-CCG and L-AP4 at the low concentrations used in this study can discriminate between group II and group III mGluRs, respectively (Knöpfel et al. 1995; Pin and Duvoisin 1995). LLP was selectively antagonized by MAP4 but not by MCCG, novel second generation group III and group II mGluR antagonists, respectively (Knöpfel et al. 1995; Pin and Duvoisin 1995). Neither L-AP4 (<50 µM) nor L-CCG (<10 µM) changed postsynaptic membrane properties, which is consistent with the presynaptic action described in our previous studies (Neugebauer et al. 1997; Rainnie and Shinnick-Gallagher 1992) and the presynaptic receptor-specific agonist concentrations reported in the literature (Knöpfel et al. 1995; Pin and Duvoisin 1995). The mechanisms and site of action of the L-AP4 induced LLP have not been determined in this study and, if similar to LTP, may well be an issue as complex and controversial as whether or not the mechanisms underlying LTP are pre- or postsynaptic.

Neuroplastic changes in the amygdala are well documented both in the kindling model of epilepsy (Holmes et al. 1996; Neugebauer et al. 1997) and in LTP phenomena associated with learning and memory (Chapman and Bellavance 1992; Gean et al. 1993; Watanabe et al. 1995). Interestingly, a recent study has found impaired cerebellar plasticity and ability to learn complex motor tasks in mice lacking the group III mGluR4 subtype (Pekhletski et al. 1996). The loss of L-AP4 induced LLP in kindled neurons observed in the present study is not the result of a loss or down-regulation of presynaptic group III mGluRs because in kindled amygdala neurons the potency of groups II and III mGluR agonists in depressing synaptic transmission is enhanced ~30-fold (Neugebauer et al. 1997). The ability of other agonists to produce a pharmacologically induced LLP of synaptic transmission in control or kindled amygdala neurons has not yet been examined. L-AP4 induced LLP may be specific for the lateral amygdala-BLA synapse because at the BLA-central amygdala synapse no LLP is recorded in control neurons, although a similar increase in sensitivity to the presynaptic depressant action of L-AP4 is measured in neurons from kindled animals (unpublished observations).

It is not clear at the moment how L-AP4 induced LLP relates to the established LTP model of learning and memory. The important role of the amygdala in emotional learning and memory may link the enhancement of synaptic transmission in L-AP4 induced LLP to memory associated processes. The loss of pharmacologically induced LLP in kindled neurons may then reflect the disruptive effect of kindling on learning behavior in animals and altered cognitive and neuropsychological functions in patients with epilepsies (Racine 1978; Sutula et al. 1995). It should be noted in this context that kindling and LTP show both mutual facilitation as well as suppression (cf. Caine 1989). Compensatory and adaptive changes in kindling could override LLP phenomena (cf. Post and Weiss 1996). Alternatively, kindling producing a long-lasting neurobiological trace could remove elements required for memory formation from the available pool, leaving an inadequate number for subsequent formation of learning and memory associated with LLP phenomena (Racine 1978).

	ACKNOWLEDGEMENTS

  Present addresses: V. Neugebauer, Dept. of Anatomy and Neurosciences, University of Texas Medical Branch, Galveston, TX 77555-1069; N. B. Keele, NIH/NINDS/ERB/NES, 9000 Rockville Pike, Building 10, Room 5N-250, Bethesda, MD 20892.

	FOOTNOTES

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

  Received 21 April 1997; accepted in final form 22 August 1997.

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