Abstracts

Rationale: The study of epileptogenesis in vitro requires a platform that is capable of monitoring neural activity in brain slices over a period of several days or weeks. Changes in activity, either due to development of the neural circuit in the slice, or due to long-term application of drugs, can then be evaluated in the same slice. This will shed light on the mechanisms underlying the synchronization of neural firing, appearance of seizures, and changes in spontaneous activity due to seizure-induced plasticity. Methods: The interface method for maintaining organotypic slices was modified to culture p4-p7 rat hippocampus slices on glass multiple electrode arrays (MEAs). Mini-culture wells with a diameter slightly larger than a neonatal rat hippocampus slice were cut from a 150 μm Sylgard membrane and sealed against a glass substrate printed with gold microelectrodes. The presence of the Sylgard well contributed to the long-term stability of slice cultures by minimizing the movement of culture medium and improving the adhesion of slices to the MEA surface. Recordings were carried out every 3 days from the same set of electrodes over the time of culture, typically 21-42 days. Recording medium lacked horse serum and was buffered with HEPES rather than bicarbonate; it was otherwise identical to the culture medium. Artificial cerebrospinal fluid with low magnesium concentration was used to generate seizures in organotypic slices. Upon completion of a recording session, MEAs were re-filled with culture medium and returned to a standard tissue culture incubator. Culture and recording media changes were carried out in a sterile flow hood, and MEA chambers were sealed for recording and incubation to avoid contamination. Results: Sylgard mini-wells ensured good adhesion of slices to the MEA, permitting recordings from the same region of the slice for up to 42 days or longer. The activity recorded from hippocampal slices evolved over time. Sporadic activity and few bursts were seen up to the first week in vitro, while by the end of second week, the activity mainly consisted of regular bursts. By third week, the inter-burst intervals became shorter, and in low magnesium ACSF, seizures were observed. We are also evaluating the effects of the age of the animal at the time of sacrifice on the development of the activity pattern. In addition, we are examining whether induced seizures have a long-term effect on the spontaneous activity exhibited by the slices. Conclusions: We have developed a platform that is capable of noninvasive, reliable long-term recording of the electrical activity in organotypic slices. This platform will be useful in examining the relationships between epilepsy, applied pharmaceuticals, and neural network development.