Epileptiform Activity In Organotypic Slice Cultures
Potentially the best suited preparations for studies on seizure generation and seizure-induced cell death are organ-otypic slice cultures. Organotypic cultures maintain some of the intrinsic properties of the tissue, including the most important aspects of connectivity. However, the preparations are usually made from pups (P6-P10) and then studied after 7 to 30 days in vitro; therefore these preparations do not necessarily represent adult tissue. The preparations are usually maintained in an artificial growth medium, which may include serum, so at least some of their properties are probably not comparable to those of adult tissue. Finally, as the preparations are more or less deafferentate and de-efferented, abnormal connectivity may develop (Gutierrez and Heinemann, 1999). The degree of abnormal connectivity depends on the age of the animal at preparation, the brain area that is prepared (with or without adjacent tissue), and the culture medium. Unfortunately, no systematic studies have yet been performed to control for such alterations. In isolated organotypic hippocampal slice cultures in which the entorhinal cortex has been removed, the development of the dentate gyrus (DG) is usually incomplete. There is evidence for some mossy fiber sprouting, even when the entorhinal cortex is left attached to the hippocampus (Routbort et al., 1999). Aberrant connectivity between area CA1 and the DG has also been reported (Gutierrez and Heinemann, 1999). Because some of this abnormal connectivity mimics aberrant connectivity in epileptic brain, organotypic slice cultures may be viewed as good models of chronically epileptic tissue, where abnormal connectivity is one of the hallmarks of tissue reorganization.
In spite of the limitations mentioned here, organotypic slice cultures offer some advantages over dissociated cultures and acute slices. First, they possess a limited extracellular space and contain many, if not all, of the neuron types present in the comparable area in vivo (i.e., they are
"organotypic"). Second, they are not acutely damaged. Third, they are thinner (150-200 mm) than acute slice preparations (350-500 mm) and therefore do not require abnormally high oxygen and glucose levels for maintenance of excitability. In fact, high oxygen levels may damage the slices (Pomper et al., 2000; 2001). Finally, they can be put back in the incubator after a series of seizures, a manipulation that gives the investigator an opportunity to explore long-term effects of seizure-like events.
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