REPEATED TETANIC STIMULATION IN PIRIFORM CORTEX IN-VITRO - EPILEPTOGENESIS AND PHARMACOLOGY

1. Focal cortical epilepsy was investigated by applying tetanic stimul ation repeatedly (100 Hz, 2 s in duration, once every 10 min, 10 episo des) to layer III association fibers in rat piriform cortex slices and recording both extracellular and intracellular responses from the end opiriform nucleus. To promote excitability, piriform slices were incub ated in artificial cerebrospinal fluid (ACSF) containing 0.9 mM Mg2+ a nd 5 mM K+, at an initial temperature of 10-12 degrees C, which was al lowed to warm passively to room temperature. 2. Responses recorded ext racellularly in the endopiriform nucleus consisted of two types: weak stimulation evoked an early-occurring, small-amplitude, negatively def lecting potential; strong stimulation evoked a more complex response c omprising both an early potential of maximal amplitude and a later-occ urring epileptiform potential of greater amplitude and longer duration . Late-occurring epileptiform potentials were not observed in slices i ncubated in ACSF at room temperature. 3. Both the early potential and the late-occurring epileptiform responses were abolished by the non-N- methyl-D-aspartic acid (non-NMDA) subtype of glutamate receptor blocke r, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 mu M). Application o f D(-)-2-amino-5-phosphonopentanoic acid (APV; 50 mu M) to block NMDA receptors was without effect on the early potential but diminished the late-occurring epileptiform potential. The late-occurring potential w as unable to follow stimulation delivered at a frequency of 1 Hz. Thes e results suggest that the early potential was generated monosynaptica lly and dependent solely on the activation of non-NMDA receptors, wher eas the late-occurring epileptiform potential was polysynaptic in orig in and possessed both a CNQX- and an APV-sensitive component. 4. Respo nses increased progressively in both amplitude and duration after teta nic stimulation. The threshold intensity required to evoke the complex dual-component potential was reduced by tetanic stimulation. An incre ase in multiunit spiking activity, indicating an increase in synchrono us discharges, was also observed. A residual potential could be evoked in the presence of CNQX (10 mu M) after the tetanic stimulation proce dure. 5. Spontaneous discharges occurred as early as after the first e pisode of tetanic stimulation and persisted for the duration of the ex periment. Spontaneous discharges were abolished by either CNQX or by a fourfold increase in extracellular Mg2+ concentration, the latter rev ersibly. APV reduced the frequency of spontaneous discharges by 38.6 /- 9.3% (mean +/- SE). The conventional anticonvulsant drug 5,5-diphen ylhydantoin, the benzodiazepine receptor agonist midazolam, and the be nzodiazepine receptor antagonist flumazenil were without effect on the frequency of spontaneous discharges. Evoked responses were also unaff ected by either 5,5-diphenylhydantoin or midazolam. Slices not exposed to cold ACSF, although demonstrating potentiation of evoked responses after tetanization did not produce spontaneous epileptiform discharge s. 6. Intracellular recordings from endopiriform neurons revealed the cellular correlates of the extracellular responses. Weak stimulation e voked a small-amplitude depolarizing potential. Increasing the intensi ty of stimulation increased the amplitude of this response and also ev oked a second depolarizing potential of greater amplitude occurring at variable latencies. Maximal stimulation evoked an action potential. A fter tetanic stimuli, responses resembling a paroxysmal depolarizing s hift consisting of a depolarizing potential with superimposed multiple action potentials were evoked reliably. Passive membrane properties a fter repeated tetanic stimulation were not different when compared wit h control. 7. This novel model of in vitro focal cortical epilepsy has many features characteristic of conventional kindling including 1) pr ogressive nature; 2) reduced threshold to evoke discharges; and 3) per sistence. The piriform cortex slice preparation should contribute to t he elucidation of basic mechanisms of focal cortical epileptogenesis. Because of the intractability of spontaneous discharges and evoked res ponses to conventional anticonvulsants, this preparation might be usef ul in the assessment of novel antiepileptic drugs.