A new indirect co-culture set up of mouse hippocampal neurons and cortical astrocytes on microelectrode arrays.

Microelectrode arrays (MEAs) are widely used to investigate neuronal network activity in vitro at multiple sites. While this system has been successfully used with primary embryonic rat hippocampal or cortical neurons, its applicability for mouse hippocampal neurons has so far not been reported in detail. As mouse genetics offer a large variety of models, it is highly desirable to close this gap. For that purpose, we established and characterized an indirect co-culture assay of mouse hippocampal neurons in the presence of astrocytes on MEAs. Embryonic day 15.5 (E15.5) mouse hippocampal neurons were cultivated on MEAs in completely defined medium. We show, that the co-culture with postnatal primary mouse astrocytes allows the establishment and the maintenance of neuronal networks under these conditions. We were able to cultivate the neurons for at least 28 days in vitro (DIV) and observed the first neuronal network activity around 7 DIV. Hippocampal neurons showed early bursting behavior and synchronous activity that evolved further with increasing time in culture. The application of bicuculline increased network activity, which revealed the presence of active GABAergic interneurons. Taken together, this study provides a novel MEA-based assay for investigating the activity in neuronal networks in an indirect neuron-astrocyte co-culture setting, and leads to first insights into the physiological development of mouse hippocampal neurons under these conditions in vitro.