Abstracts

Rationale: Epileptogenesis is a process in which normal cerebral cortex is permanently transformed to express spontaneous seizure discharges. This transformation may be induced by any neural process that evokes long-lasting changes in network excitability. Protein kinase C (PKC) is a common intracellular second messenger for many such processes, phosphorylating substrates such as channels, receptors and other enzymes. Others have previously reported long-lasting effects on cellular properties and synaptic responses induced by PKC activation, but none have looked at network effects. We used phorbol-12,13-dibutyrate (PDBu) to activate PKC and report herein the induced network effects.Methods: Transverse (400 μm) hippocampal slices from 2-4 week old guinea pigs were placed in an interface chamber maintained at 35°C, bubbled with 95% O₂–5% CO₂ and perfused with artificial CSF containing (in mM): 124 NaCl, 26 NaHCO₃, 5 KCl, 2 CaCl₂, 1.6 MgCl₂, and 10 D-glucose. Intracellular recordings were obtained from CA3 stratum pyramidale; synchronous field potentials were recorded from CA1 stratum pyramidale. Unless otherwise indicated, experiments were performed in the presence of 50 μM picrotoxin, a GABA_A receptor antagonist, to elicit baseline interictal activity (i.e. rhythmic synchronized bursts <500 ms long). Data are reported as means ± SE; n = number of slices; P<0.05 was deemed significant.Results: In the absence of picrotoxin (PTX), continuous bath application of 1 μM PDBu elicited spontaneous bursts >1 s in duration in CA3 pyramidal cells, with simultaneous field recordings in CA1 revealing these to be synchronized ictaform bursts generated by the local population of neurons. These discharges progressively increased in length: bursts reached 1 s in length by 67.8 ± 13.1 min, and reached a maximum length of 7.6 ± 0.6 s by 89.7 ± 13.1 min (n=5). In the presence of PTX, PDBu initially increased the interictal burst frequency from 0.11 to 0.18 Hz by 20.2 ± 3.4 min of PDBu exposure, followed by the appearance of ictal-length bursts (i.e. >1 s) by 33.6 ± 3.6 min (n=8). 4α-PDBu, an analog of PDBu with no effect on PKC, failed to elicit any change in PTX burst frequency or duration. As PKC activation can enhance neurotransmission, and activation of muscarinic, NMDA, or group I metabotropic glutamate receptors (mGluRs) can elicit ictaform activity in the hippocampal slice, we tested the involvement of these receptor systems in producing PDBu-induced bursts. Neither NMDA blocker (50 μM APV), muscarinic antagonist (10 μM atropine), nor mGluR1 antagonist (25 μM LY367385) had any effect on PDBu-induced network activities. By contrast, 25 μM MPEP, an mGluR5 antagonist, significantly blunted maximal burst lengthening by PDBu; the peak burst length was 1.2 ± 0.1 s (n=9, P<0.0001)Conclusions: These data are the first to reveal that (1) PKC activation enhances network excitability, potentially contributing to epileptogenesis, and (2) mGluR5 activation contributes to the expression of PKC-induced bursts, with no apparent contribution from mGluR1, NMDA or muscarinic receptors. (Funded by NIH grant NS40387 to LRM)