The results of this study demonstrate the presence of endogenous SREDs in HEC slices prepared from a large group of epileptic animals. While all HEC slices isolated from epileptic animals exhibit SREDs, HEC slices from control animals had no evidence of SREDs. We have shown that there are two main variants of SREDs that occur naturally in the HEC slices prepared from epileptic animals; one with a slow (0.25 Hz) bursting frequency, and the other with a much faster (3.2 Hz) bursting frequency. Both patterns of activity persisted uninterrupted for >30 min, and SREDs were synchronous throughout the hippocampal-parahippocampal circuit. Longer recordings in selected HEC slices from epileptic animals demonstrated that the SREDs persisted indefinitely and were recorded for up to 4 hours of recording time. Animals with fast SRED bursting were from animals with a higher seizure frequency of greater than 3 seizures per day. Animals with slow SRED bursting were from animals with less than 1 seizure per day. Although this suggests that the seizure frequency in epileptic animals relates to the presence of slow vs. fast SRED bursting, it is not possible to clearly establish this relationship between the in vitro and in vivo situations. The facts that hippocampal slices from control animals did not manifest SREDs indicate that these epileptiform events in the slices from epileptic animals represent the altered “epileptic phenotype. With simultaneous whole cell current clamp recordings in CA3 pyramidal cells, it was evident that the pattern of depolarization varied between the two types of recording patterns. The patch slice recordings indicated that the slower type of epileptiform activity was characterized by having a gradual buildup in the degree of spontaneous depolarization, while the faster type expressed a recurrent increase in number of spikes with each PDS event. The results of this study provide a methodology to conduct continuous long-term monitoring of HEC slices in vitro prepared from epileptic animals and provides a unique model to study endogenous epileptiform activity.
Hippocampal slices in normal Mg2+
concentrations have similar findings as the slices in the slightly lower concentrations except that the frequency of SREDs and SRSs is reduced by 3 fold. The concentration of Mg2+
used in this study was the same concentration used by our laboratory and others to conduct hippocampal slice physiology [36
]. The presence or absence of SREDs and SRSs was not affected by the concentration of Mg2+
, only the frequency of discharges. Since hippocampal slices can only be maintained for limited periods of time in the recording chamber, the concentrations of Mg2+
used provide optimal electrographic activity to conduct sophisticated experiments to evaluate epileptiform discharges in vitro. This model offers a viable alternative to intact animal models to study epileptiform activity in vitro from control and epileptic animals.
The data reported here provide an analysis of the presentations of endogenous SREDs in epileptic HEC slices and demonstrate the existence of two major variants of epileptiform activity with regards to the frequency of SREDs. In all recordings of SREDs, the extracellular field discharge recordings correlated with whole cell current clamp recordings. These results indicate that large populations of neurons are interacting and participating to generate the spontaneous bursting activity observed during SREDs in HEC slices from epileptic animals. Earlier studies also demonstrated two types of interictal activity (slow and fast) in hippocampal slices treated with acute application of either pilocarpine or 4-aminopyridine [21
]. In addition, previously published studies have noted that hippocampal slice preparations from chronically seizing pilocarpine-treated rats can express epileptiform activity upon electrical stimulation [38
]. The data presented in this study confirm the presence of endogenous epileptiform discharges at a chronic stage of seizure expression and provide direct evidence that essentially all HEC slices from epileptic animals in this model manifest SREDs in vitro. Our data also demonstrate that the expression of all types of interictal activity is generalized throughout the hippocampal-parahippocampal circuit. This is suggestive of a recruitment phenomenon among the interconnecting regions of the hippocampus in this model of endogenous epileptiform discharges from chronic epileptic animals.
The high frequency pattern of SREDs observed in this study had a different rhythmic bursting pattern in comparison to the slower pattern of SREDs. In the high frequency pattern the whole cell current clamp recordings of CA3 pyramidal cells presented a larger number of PDS events, each with multiple action potentials that were preceded by spiking events consisting of a minimal number of bursts, progressing to multiple complex PDS events. Immediately following the expression of a complex PDS there was an immediate return to single spike PDS events (). This is in contrast to studies with acutely induced epileptic events. In such instances interictal spikes have been followed by refractory periods suggestive of inhibitions preventing a transition to ictal activity [39
]. The use of the epileptic HEC slice preparation to evaluate and study the properties of epileptic events offers a powerful tool to investigate the basis for neuronal excitability.
] has suggested that following kindling there are three components contributing to the epileptiform activity: 1) clusters of localized bursts 2) synchronous discharges of high amplitude spikes and 3) random components. However, extracellular recordings have pointed to enhanced EPSPs and population spikes as being the underlying mechanism [41
]. The SREDs in HEC slices from pilocarpine-induced epileptic animals provide evidence that there is a reciprocal interaction between the regions of the intact hippocampal-parahippocampal circuit. These results from our study indicate that in the chronic epilepsy condition the SREDs are not exclusive to any one region of the hippocampus. One might argue about site of origin of discharges in this preparation. Using titanic stimulation of Schaeffer collaterals in the hippocampal-EC slice, our previous study demonstrated that DG lead in generating individual bursts within the secondary discharge, followed by CA3, CA1, and EC. Similarly, using lesion studies, we also showed that severing the mossy fibers between DG and CA3 abolished the secondary event without affecting the immediate after-discharge. This data along with in vivo observations that DG may have its “gate” function reduced following maximal activation [42
] implicate DG as an important area in the generation of discharges.
One might argue that to prove NMDA involvement in epileptogenesis, the ideal experiment would be to block this receptor system after the course of SE and then monitor for development of SRS. We have dealt with this issue in depth in some of our previous studies [9
]. Similar to this study, NMDA receptor blockade prior to or during SE prevented development of epilepsy. However, we have routinely observed that MK-801 administered after pilocarpine-SE does not prevent development of epilepsy (unpublished observations). In fact, it has also shown that N-methyl-D-aspartate receptor blockade after status epilepticus protects against limbic brain damage but not against epilepsy in the kainate model of temporal lobe epilepsy [43
]. Thus, treating animals with MK-801 during SE prevents the development of SRSs and SREDs. However, giving MK-801 after SE in a single dose or over a 2 week treatment regimen does not prevent the development of SRSs or SREDs. MK-801 administration after the injury has occurred does not block epileptogenesis.
In summary, these results demonstrate that HEC slices isolated from epileptic animals manifested SREDs and indicate that the epileptic phenotype is associated with hyperexcitability in the hippocampus that can be recorded and studied using continuous recording techniques in HEC slice preparations. The use of epileptic HEC slices for studying the pathophysiology and electrophysiological properties of the epileptic hippocampus offers a unique approach to studying the epileptic phenotype. When developing models for anticonvulsant efficacy and for studying the pathophysiology of epilepsy, it is important to employ epileptic tissue [5
]. The use of the HEC slice preparation from epileptic animals characterized in this study provides a novel model for studying epileptic tissue in vitro and offers a potent tool for developing new therapeutic agents for the treatment of epilepsy and for the study of the epileptic condition.