Metabotropic glutamate receptor agonist-induced hyperpolarizations in rat basolateral amygdala neurons: receptor characterization and ion channels.

1. Metabotropic glutamate receptor (mGluR)-agonist-induced hyperpolarizations and corresponding outward currents were analyzed in basolateral amygdala (BLA) neurons in rat brain slice preparations with current-clamp and single-electrode voltage-clamp recording to characterize the mGluR subtype(s) and the ion channel(s) mediating this response. 2. The mGluR agonist (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) induced a membrane hyperpolarization or outward current in BLA neurons in a concentration-dependent manner (median effective concentration = 34 microM; range = 10-200 microM); the 1S,3R-ACPD hyperpolarizations are recorded in 89% of neurons that accommodate or cease firing in response to a 400-ms depolarizing current injection (0.5 nA). 3. mGluR agonists elicited hyperpolarizations or outward currents in a concentration-dependent manner in the following rank order of potency: (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine (L-CCG-I) > 1S,3R-ACPD > (s)-4-carboxyphenylglycine = (RS)-4-carboxy-3-hydroxyphenylglycine (4C3HPG) > L-aminophosphonobutyric acid > (1S,3S)-1-amino-cyclopentane-1,3-dicarboxylic acid. In contrast, the mGluR agonists quisqualate and ibotenate induced only depolarizations in the presence of D-2-amino-5-phosphonovalerate and 6-cyano-7-nitroquinoxaline-2,3-dione in BLA neurons. 4. The 1S,3R-ACPD-induced outward current is mediated through a large-conductance calcium-dependent potassium (BK) conductance. The BK channel blockers iberiotoxin and charybdotoxin blocked the response, as did the potassium channel blockers tetraethylammonium and 4-aminopyridine; the small-conductance calcium-activated potassium channel blocker apamin did not affect the response. 5. The mGluR-agonist-induced hyperpolarization is blocked in amygdala slices from animals pretreated with pertussis toxin (PTX). 1S,3R-ACPD hyperpolarizations were recorded in neurons contralateral but not ipsilateral to the site of PTX injection. 6. The antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG, 500 microM) reduced significantly the 1S,3R-ACPD-induced hyperpolarization. 7. In conclusion, the relative potency of L-CCG-I and 4C3HPG in evoking only hyperpolarizations (outward currents) in accommodating neurons, and the observation that MCPG (500 microM) reduces the hyperpolarization, suggest that a group-II-like mGluR underlies the hyperpolarizing response. The mGluR-induced response is sensitive to iberiotoxin and to pretreatment with PTX, suggesting activation of BK channels through a group II mGluR linked to a PTX-sensitive G protein in BLA neurons.

Loss of mGluR-mediated hyperpolarizations and increase in mGluR depolarizations in basolateral amygdala neurons in kindling-induced epilepsy.

1. Intracellular recordings were made from neurons of the basolateral amygdala (BLA) in in vitro slice preparations to determine long-term differences in metabotropic glutamate receptor (mGluR) agonist-induced membrane responses in control and amygdala-kindled rats. 2. (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine-1 (L-CCG-I; 100 microM) typically evoked a hyperpolarization/outward current in control BLA neurons; the hyperpolarization is mediated through a group-II-like mGluR subtype of receptor and is recorded in accommodating neurons that cease firing in the presence of a long (400 ms) depolarizing current injection (0.5 nA). In amygdala-kindled slices, L-CCG-I (100 microM) hyperpolarized only 1 of 13 BLA neurons. 3. 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) (100 microM) elicited a hyperpolarization/depolarization (outward/inward current) in control neurons and evoked only a membrane depolarization (inward current) in kindled BLA neurons; this depolarization is similar to that mediated by group I mGluR activation in other neurons. 4. In control nonaccommodating neurons the concentration-response relationship for the 1S,3R-ACPD-induced inward current had a median effective concentration (EC50) of 49 microM and a maximum amplitude of 182 +/- 30 (mean +/- SE) pA. In kindled nonaccommodating neurons the EC50 of the concentration-response relationship for 1S,3R-ACPD was shifted to 29 microM and the maximum value increased to 265 +/- 15 pA, reflecting an increase in efficacy. 5. These data suggest that amygdala kindling causes lasting changes in mGluR responses in the BLA reflecting a downregulation of a group-II-like mGluR subtype mediating the hyperpolarizing response and an upregulation of a group I mGluR1 or 5 subtype. The hyperpolarizing response reduced by kindling and the increase in the group I mGluR response may reflect an alteration in the balance between inhibition and excitation and may contribute to the transition to epileptiform bursting in kindled neurons.

The functional role of metabotropic glutamate receptors in epileptiform activity induced by 4-aminopyridine in the rat amygdala slice.

The metabotropic glutamate receptor (mGluR) antagonist, (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG; 500 microM), was tested on intracellularly recorded epileptiform activity induced by 4-aminopyridine (4-AP) in amygdala neurons. Superfusing 4-AP (1 mM) produced interictal spiking followed by ictal bursting. MCPG prevented the progressive transition from interictal spiking to ictal bursting but affected neither induction of interictal spiking nor maintenance of ongoing ictal bursting. These data suggest that mGluRs may be involved in the induction of ictal seizure events.

Activation of postsynaptic metabotropic glutamate receptors by trans-ACPD hyperpolarizes neurons of the basolateral amygdala.

Glutamate has traditionally been regarded as an excitatory neurotransmitter. Synaptic activation of ionotropic glutamate receptors mediates fast EPSPs in the CNS. Moreover, activation of metabotropic glutamate receptors (mGluRs), which are coupled to second messenger effector systems via GTP-binding proteins (G-proteins), results in the expression of slow EPSPs. We have now examined the response of basolateral amygdala (BLA) neurons to activation of postsynaptic mGluRs. In approximately 78% of BLA neurons examined, activation of postsynaptic mGluRs results in membrane hyperpolarization and an associated decrease in membrane input resistance or a hyperpolarization followed by a depolarization associated with an increase in input resistance. The purpose of this study was to address the mechanisms underlying the membrane hyperpolarization. Here, we report that the ACPD-induced hyperpolarization is insensitive to TTX, is dependent on extracellular K+ concentrations, and has a reversal potential (-84 mV) close to that estimated from the Nernst equation for an increase in a K+ conductance. In addition, the ACPD response is resistant to (1) intracellular chloride loading, (2) the GABAB receptor antagonist CGP55845A, (3) the ACh receptor antagonist atropine, and (4) the ionotropic glutamate receptor antagonists CNQX and APV. These data suggest that the hyperpolarization results from a direct activation of postsynaptic mGluRs on neurons of the BLA. Furthermore, we performed studies that suggest that the hyperpolarization is G-protein mediated and results from activation of a TEA-sensitive, calcium-dependent potassium conductance. The sensitivity of this conductance to thapsigargin further suggests that this response requires the release of calcium from intracellular stores. In summary, these data suggest a role for glutamate as an inhibitory transmitter in the BLA during periods of metabotropic glutamate receptor activation. In nuclei such as the BLA that are exquisitely sensitive to seizure induction, an inhibitory response to glutamate may act to delay the onset of epileptogenesis.

The infusion of an NMDA antagonist into perirhinal cortex suppresses amygdala-kindled seizures.

The seizure-modulating role of N-methyl-D-aspartate (NMDA) receptors located in several limbic areas was investigated. Amygdala-kindled rats were microinfused with the selective NMDA-receptor antagonist 2-amino-5-phosphonovalerate (APV, 1 microliter, 70 nmol) or artificial cerebrospinal fluid (ACSF) applied through a cannula located in either the amygdala or perirhinal, pyriform or deep prepyriform cortices. APV infused into the stimulation site raised the threshold for seizure generation. Surprisingly, APV infused into perirhinal cortex, but not into other regions, also dramatically suppressed behavioural seizures and afterdischarges (AD) elicited 5 min after the infusion. If stimulus intensities were markedly elevated however, the seizure suppression was overcome. This latter effect was reversible and repeatable, as seizures and AD were reliably reinstated when these animals were stimulated after infusion with ACSF. A similar effect, whereby perirhinal infusions blocked seizure activity, was also demonstrated in an animal kindled from the olfactory bulb and in one kindled from the perforant path. These results suggest that NMDA receptors located in the perirhinal cortex may play a major role in the modulation of AD activity elicited from more distal brain regions. Furthermore, activation of perirhinal cortex may be a critical requirement for the generation of amygdala-stimulated AD in the kindled animal.

The N-methyl-D-aspartate antagonists aminophosphonovalerate and carboxypiperazinephosphonate retard the development and expression of kindled seizures.

To investigate the possible role of N-methyl-D-aspartate (NMDA) receptors in the development and expression of amygdaloid-kindled seizures, rats were either chronically infused with 2-amino-5-phosphonovalerate (APV, 20-40 mM) or pre-injected with carboxypiperazine-phosphonate (CPP, 1-10 mg/kg), both selective NMDA-receptor antagonists, and then kindled from the amygdala. At the higher dose (40 mM), APV blocked the induction of long-term potentiation in the dentate gyrus. APV also retarded clinical seizure development dose-dependently and increased seizure thresholds without affecting afterdischarge (AD) duration. These same doses of APV had only small anticonvulsant effects on established kindled seizures. Although CPP (1-10 mg/kg) had no effect when rats were kindled 45 min after injection it dose-dependently retarded focal and generalization stages at the 150 min injection-kindling interval. Once relieved of drug, animals proceeded to develop stage 5 seizures with shorter duration ADs than saline-control animals. When the previously-kindled, saline groups were crossed to CPP a small depressant effect on seizure expression was observed. These results suggest that NMDA receptors are primarily involved in kindling development rather than in maintaining the kindled state.

Kindling-induced changes in EEG recorded during stimulation from the site of stimulation. III. Direct pharmacological manipulations of the kindled amygdala.

In our previous studies, we hypothesized that activation and subsequent collapse of GABA-mediated inhibition during tetanus is an important seizure-triggering mechanism in the kindled epileptogenic focus. To examine this hypothesis, in the present study, we investigated the effects of pharmacological manipulations of the kindled amygdala with several drugs, and measured the kindled seizures as well as the EEG events during tetanus. The results obtained were: (i) The selective GABA-A agonist, muscimol (1 and 5 nM/1 microliter), suppressed kindled seizures in a dose-dependent fashion, and the 5 nM muscimol significantly prolonged EEG suppression and reduced the number of oscillations in the subsequent rhythmic synchronous discharge. Similar effects followed systemic injection of diazepam (2 mg/kg). (ii) The selective GABA-B agonist, baclofen (5 nM), had no effect on kindled seizures nor on the EEG events during tetanus. (iii) The NMDA antagonist, 2-amino-5-phosphonovaleric acid (80 nM), significantly reduced the afterdischarge duration and significantly delayed the appearance of the rhythmic synchronous discharge. However, these effects were not observed immediately, but 24 to 72 h after microinjection. (iv) The muscarinic cholinergic antagonist, atropine (40 and 80 nM), suppressed kindled seizures in a dose-dependent fashion, but the atropine caused marked synchronous discharge both in the awake resting EEG and during tetanic stimulation. We conclude that the GABA-A system, including the benzodiazepine system, is more involved in the seizure-triggering mechanism of amygdala kindling than the GABA-B system, that there is an interaction between the GABA-A and NMDA system, and that the cholinergic participation is independent of the primary seizure-triggering mechanisms.