SPREAD OF EPILEPTIFORM ACTIVITY IN THE IMMATURE RAT NEOCORTEX STUDIEDWITH VOLTAGE-SENSITIVE DYES AND LASER-SCANNING MICROSCOPY

1. Adult rats and rats with a postnatal age of 3-29 days (PN 3-29) wer e used for the preparation of in vitro slices of the frontal neocortex . Epileptiform activity was induced by bath application of the gamma-a minobutyric acid-A (GABA(A)) receptor antagonists bicuculline or picro toxin. 2. The voltage-sensitive dye RH 414 and a laser scanning micros cope were used for multiple-she optical recordings of membrane potenti al changes associated with epileptiform activity. Optical signals were compared with simultaneously measured extracellular field potentials. 3. Optical signals could be reliably recorded for the duration of the experiments (2-4 h). Extracellular recordings of convulsant-induced p aroxysmal depolarizing shifts (PDSs) in slices stained with RH 414 wer e comparable with those obtained in unstained slices. Changes in dye s ignals in response to reductions in extracellular calcium, addition of tetrodotoxin (TTX), or application of excitatory amino acid receptor antagonists indicate that the fluorescence changes correlate well with established electrophysiological measures of epileptiform activity. 4 . In slices from adult animals, dye signals were observed at all recor ding sites. The response with the shortest latency occurred invariably at the site of stimulation, and activity spread rapidly in both verti cal and horizontal directions. Spread was significantly faster in the vertical than in the horizontal direction. 5. Epileptiform activity wa s absent or only weakly expressed in slices from PN 3-9 animals. Activ ity was detectable predominantly in upper cortical layers. 6. Dye sign als were observed at all measurement points in slices from PN 10-19 an imals. In this age group, peak amplitude increased with spread of acti vity from lower to upper cortical layers. There was no significant dif ference between the speed of propagation in the vertical and in the ho rizontal directions. Spontaneous epileptiform activity occurred at a h igh rate in the PN 10-19 age group, and signals associated with sponta neous epileptiform events were largest in upper layers. 7. In the PN 1 0-19 age group, optical signals were characterized by the repetitive o ccurrence of PDS discharges superimposed on a sustained response. The amplitude of the sustained response decreased with increasing distance from the site of stimulation. Analysis of the latencies revealed that the superimposed PDS-like events were generated at multiple sites wit hin the scanning area. Amplitude and rate of rise were largest in slic es from PN 10-19 animals. These values declined with ongoing developme nt. 8. The N-methyl-D-aspartate (Nh IDA)-mediated component of optical ly recorded epileptiform activity was determined by either subtraction of responses recorded in the presence of D-2-amino-5-phosphonovaleric acid (APV) from those taken under control conditions or by applicatio n of the non-NMDA receptor antagonist 6-cyano-7-nitraquinoxaline-2,3-d ione (CNQX). The NMDA components obtained by these two procedures disp layed slow rates of rise and different patterns of spatial distributio n. 9. Our results demonstrate that the voltage-sensitive dye RH 414 ca n be used to reliably monitor the initiation, distribution, and spread of convulsant-induced epileptiform activity in the rat neocortex in v itro. In PN 10-19 animals, epileptiform activity displays the highest degree of synchronization, which declines with ongoing development. Th e observation that activity can start from multiple sites in the tissu e to form long-lasting ictal-like events that are not observed in adul t animals suggests that the threshold for the initiation of epileptifo rm activity may be lower in the immature neocortex.