Abstracts

Activation of metabotropic receptors in the CNS elicits long-lasting effects that may participate in epileptogenesis. Many such receptors are linked to phosphoinositide or phosphatidylcholine metabolism, and the resultant DAG production activates protein kinase C (PKC). We used a phorbol ester to activate PKC to determine its effect on picrotoxin-induced network activities [italic]in vitro[/italic]., 400 [mu]m transverse guinea pig hippocampal slices were placed in an interface chamber and intracellular recordings were obtained from CA3 stratum pyramidale. Picrotoxin (50 [mu]M), a GABA[sub]A[/sub] antagonist, was bath-applied to elicit baseline interictal activity (synchronized discharges [lt]500 ms long), and changes in burst length were monitored following the application of phorbol-12,13-dibutyrate (PDBu). Bursts were considered ictaform if they met the following three criteria: (1) unaffected by hyperpolarization, (2) the oscillations within each burst slowed in frequency toward the end of the burst, and (3) length of the burst exceeded 1 s, the presumed minimum length for a clinically-correlated experience to potentially occur if the discharge were expressed [italic]in vivo[/italic]. Data are reported as means [plusmn] SE; significance was determined using the Student[apos]s t-test, with [italic]P[/italic][lt]0.05 deemed significant., As phorbol esters activate second messengers, preincubation for at least 30 min is recommended. Yet within 40-60 min, bath application of 1 [mu]M PDBu typically increased the length of picrotoxin-induced interictal bursts to ictaform events 2-8 s long (mean peak burst length 5.6 [plusmn] 0.5 s; latency to produce 1 s ictal-length burst 48 [plusmn] 4 min; n=7 out of 8 slices); this effect persisted for over 1 hr following washout of PDBu. When PDBu was applied in the presence of 10 [mu]M chelerythrine, a PKC inhibitor, the burst prolongation was either completely prevented (n=4) or significantly suppressed (peak burst length 2.8 [plusmn] 0.7 s; time to ictal-length 68 [plusmn] 13 min; n=6; [italic]P[/italic] [lt] 0.05). Bursts could be blocked by application of either 1 [mu]M tetrodotoxin (n=3) or a combination of NMDA and AMPA receptor antagonists (50 [mu]M D-AP5 and 20 [mu]M CNQX, respectively; n=2), suggesting the discharges were synchronized events; no intrinsic oscillatory activity was uncovered during these manipulations. NMDA antagonist alone could not prevent the induction of these bursts, but did reversibly shorten the length of expressed bursts, as did 25 [mu]M MPEP, an mGluR5 antagonist. While PDBu has been reported previously to enhance glutamate release, bath application of the glutamate reuptake inhibitor L-[italic]trans[/italic]-2,4-PDC (100-200 [mu]M) was unable to simulate the response seen with PDBu (n=2). Application of PDBu in the absence of picrotoxin also elicited burst prolongation, but a higher concentration was necessary to result in a similar peak burst length and time to ictal response (2-10 [mu]M; n=4)., Generalized PKC activation in the hippocampal slice has powerful excitatory effects on network activity with implications toward epileptogenesis., (Supported by NIH NS40387 (L.R.M.).)