The objective of this project is to explore new strategies for the rational development of antiepileptic drugs based upon their interaction with neuronal ion channel systems. Cellular electrophysiological recording techniques are used to study drug modulation of neurotransmitter-gated and voltage-activated ion channels in brain slices, cultured neurons and heterologous cells transfected with cloned ion channel subunit genes. Correlative studies are carried out in animal models. Recent studies have focused on kainate-type glutamate receptors. We have demonstrated that a component of the excitatory synaptic response evoked in basolateral amygdala neurons by external capsule stimulation is mediated by kainate receptors containing the GluR5 subunit. In the present reporting period, we continued to examine GluR5 kainate receptor mediated neurotransmission and synaptic plasticity mechanisms in the amygdala, a key brain site for epileptogenesis in animal models and a common primary focus for seizures in human epilepsy. We also began studies examining the role of GluR5 kainate receptors in seizures and epileptogenesis in the rat amygdala in vivo and in the amygdala slice preparation. Using in situ hybridization histochemistry and RT-PCR, we had previously demonstrated a high density of GluR5 kainate receptor mRNA expression in the basolateral amygdala. We now have confirmed the presence of GluR5 kainate receptor subunits using immunocytochemistry with an antibody directed against the C-terminal region of the GluR5 subunit. Immunoelectron microscopy demonstrated labeling of many synapses in the basolateral amygdala. The labeling was largely postsynaptic, compatible with our observation that synaptic activation of GluR5 kainate receptors induces a fast excitatory synaptic response in basolateral neurons. To determine the extent of editing of GluR5 kainate receptors, mRNA was extracted from microdissections and subjected to RT-PCR followed by restriction endonuclease cleavage with BbVI31. A proportion of the GluR5 mRNAs were unedited, indicating that they can form calcium permeable kainate receptors. Accordingly, whole cell patch clamp recordings from principal neurons in the basolateral amygdala revealed that GluR5 kainate receptor mediated synaptic responses are inwardly rectifying, as expected if the GluR5 kainate receptors that contribute to the response are unedited and calcium permeable. We previously observed that GluR5 kainate receptor activation induces a novel form of NMDA receptor-independent enduring synaptic facilitation that requires calcium entry through calcium-permeable (unedited) GluR5 kainate receptors. This novel synaptic plasticity is induced by low frequency activation of excitatory afferents to basolateral amygdala neurons in the external capsule. A unique characteristic of this novel synaptic plasticity is that it is not input pathway specific. Induction of GluR5 kainate receptor-mediated synaptic facilitation by external capsule stimulation results in enhanced strength of a distinct non-stimulated excitatory input from the basal amygdala. In contrast, we have shown through both field potential and intracellular recordings that a conventional form of long-term potentiation (LTP) can be induced in basolateral amygdala neurons by high-frequency stimulation of the external capsule input; this LTP is input-specific. The demonstration that GluR5 kainate receptors mediate a novel form of activity-dependent synaptic enhancement raises the possibility that GluR5 kainate receptors could play a role in epileptic hyperexcitability and epileptogenesis, such as occurs in response to kainate receptor agonists including the neurotoxins kainate and domoate. The work further suggests that pharmacological blockade of GluR5 kainate receptors could be useful in the prevention and treatment of some forms of epilepsy. To investigate the possibility that selective activation of GluR5 kainate receptors mediates epileptogeneis, we used the GluR5 agonist ATPA. Microinjection of ATPA into the rat amygdala resulted in acute seizures. ATPA at low concentrations also induced epileptiform discharges recorded extracellularly in the amygdala slice. These discharges could be eliminated by decahydroisoquinolone antagonists of GluR5 kainate receptors. Our studies therefore demonstrate for the first time that selective activation of GluR5 kainate receptors induces epileptic activity. To examine the physiological basis of this epileptiform activity, we explored the possibility that ATPA affects the strength of GABA-A receptor-mediated synaptic inhibition in the basolateral amygdala. In recordings of field responses evoked by external capsule stimulaion, ATPA was found to diminish GABA-A receptor-mediated inhibition as assessed in a paired-pulse protocol. In addition, in whole cell voltage clamp recordings, inhibitory synaptic currents (IPSCs) evoked by local stimulation were depressed by ATPA. We conclude that kainate receptor activation induces epileptiform activity in the basolateral amygdala, in part, by suppressing GABA mediated inhibition. Activation of other excitatory amino acid receptors, including AMPA receptors, does not cause the same effect. These results likely explain the unique ability of kainate receptor agonists, such as kainate and domoate, to induce epileptiform activity and suggest that selective antagonists of kainate receptors could have activity as anticonvulsants under some circumstances.
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