Organotypic hippocampal slice cultures (OHSCs) are widely used for anatomical, molecular and electrophysiological studies of the development of neuronal networks. Electrophysiological recordings are usually limited to a single time point during development, and recording conditions differ greatly based on culture conditions. Consequently, little is known about the maturation of neuronal network activity in vitro. Here, we describe a simple method that allows long-term electrophysiological recordings during culture maintenance in a CO2 incubator. We compared the occurrence of spontaneous network activity, including epileptiform activity, in OHSCs (maintained in Neurobasal/B27 serum-free medium) prepared at different postnatal days and investigated the effects of changes in osmolality and pH. Recordings over 48 h revealed spontaneous network activity culminating in seizure-like events (SLEs) in 65.4% of the OHSCs (n=78). SLE incidence peaked during the first 6h following implantation of the microelectrodes and a secondary increase in SLE-incidence began after 9h of recording and averaged 2.65SLEs/h. The initial peak was likely initiated by transient alkalosis induced by the low pCO2 during the positioning of the electrodes, whereas successive changes in the composition of the culture medium might explain the secondary increase in SLE incidence. Notably, changes in osmolality had no effect on SLE induction. In conclusion, long-term recordings in OHSCs will help to reveal changes in spontaneous network activity during maturation. The extent to which the axonal reorganization known to occur in OHSCs contributes to the susceptibility to epileptogenesis remains to be determined.
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