1. Gamma-Aminobutyric acid (GABA) withdrawal syndrome (GWS) represents a particular model of focal epilepsy consecutive to the interruption of a chronic intracortical GABA infusion and is characterized by the appearance of focal epileptic electroencephalographic (EEG) discharges and localized clinical signs on withdrawal of GABA. Effects of Ca2+ channel blockers and N-methyl-D-aspartate (NMDA) antagonists were evaluated in living rats presenting a GWS after interruption of a 5-day GABA infusion into the somatomotor cortex and in neocortical slices obtained from such rats. Bursting properties and morphology of neurons were also analyzed in slices. 2. In living rats, the noncompetitive NMDA antagonist phencyclidine [1-(1-phenylcyclohexyl) piperidine] and the Ca2+ antagonist flunarizine [E-1 (bis(4fluorophenyl)methyl)-4(3phenyl2-propenyl)-piperazine] were administered systemically to two groups of rats. Rats in the first group (n = 12) were injected with the drug 30-60 min before discontinuation of the GABA infusion. In this case, phencyclidine (10 mg/kg ip) prevented the development of GWS (n = 5), whereas flunarizine (40 mg/kg ip) had no consistent effect on the GWS appearance and characteristics (n = 7). Rats in the second group (n = 12) were injected 60-90 min after GABA discontinuation, i.e., during a fully developed GWS. In that case, neither drug suppressed GWS. 3. Neuronal activities in the epileptic focus were studied in slices with conventional intracellular recording and stimulation techniques. From the 65 neurons recorded, 29 responded with EPSPs and paroxysmal depolarization shifts (PDSs) to white matter stimulation (synaptic bursting or SB cells). Nineteen other neurons presented, in addition to synaptically induced PDSs, bursts of action potentials (APs) induced by intracellular depolarizing current injection (intrinsic bursting or IB cells). The remaining 17 neurons presented no bursting properties to either synaptic stimulation or depolarizing current injection (nonbursting or NB cells). 4. The recorded neurons were located 0.7-1.2 mm distant from the lesion because of the penetration of the GABA infusion cannula. Intracellular injection of neurons (n = 4) with biocytin or Lucifer yellow revealed that both SB and IB neurons were large, spiny pyramidal neurons localized in layer V of the sensorimotor cortex. 5. Bath application of the selective antagonist of NMDA receptors DL-2amino-5 phosphonovalerate or DL-2amino-7phosphonoheptanoate (10-50-mu-M) reversibly reduced the amplitude (by 25-50%) and the duration (by 20-25%) of PDSs in all cases (n = 17). At the same doses, NMDA antagonists did not affect 1) the EPSPs induced by a stimulation at low intensity in SB (n = 13), IB (n = 4), or NB (n = 6) neurons or 2) the firing patterns induced by intracellular depolarizing current injection in SB, IB and NB cells. 6. Intrinsic bursts induced in IB neurons were abolished by organic (n = 4) and inorganic (n = 8) Ca2+ antagonists, unmasking a slow depolarization (3-6 mV in amplitude and 60-100 ms in duration) that underlies these bursts. Both the Ca2+-sensitive and the slow Ca2+-independent depolarization were resistant to tetrodotoxin (TTX) application (0.5-mu-M). The organic Ca2+ antagonist verapamil (20-30-mu-M) reduced the amplitude of PDSs by 40-70% and suppressed the bursts of APs riding on the depolarizing wave in SB (n = 5) and IB cells (n = 4). The inorganic Ca2+ antagonists Co2+ (2 mM), Mg2+ (2 mM), and Cd2+ (0.5 mM) also reduced the amplitude of PDSs by > 50% and blocked the associated bursts in SB (n = 7) and IB cells (n = 8). None of the Ca2+ antagonists affected EPSPs induced in SB, IB, or NB cells by stimulation at low intensity or the EPSP-single AP sequences evoked in NB cells (n = 5) by stronger stimuli. 7. The effects of Ca2+ antagonists on IB and SB cells were followed by a transitory enhancement of bursting activity as a rebound of excitability at the beginning of the washout of the Ca2+ antagonists (n = 10). Intracellular application of ethylene glycolbis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), a Ca2+ chelator, increased the amplitude and duration of PDSs and favored the appearance of additional APs in both SB (n = 2) and IB neurons (n = 1). 8. In conclusion, voltage-dependent Ca2+ currents are responsible for bursting of IB pyramidal neurons but not for epileptogenesis. Moreover, NMDA-associated cationic channels are involved in epileptogenesis and at least partly in the induction of PDSs observed during GWS. Simultaneous involvement of several factors in the genesis of bursts explains the refractoriness of GWS to administration of Ca2+ blockers, NMDA antagonists, or GABA(A) agonists in living rats. The results also emphasize the important role of network interconnections between GABA-treated neurons and neurons located outside the infusion site.
