Higher cognitive functions are accompanied by complex patterns of distributed activity in neuronal networks that synchronize and orchestrate active neurons into neuronal assemblies. In the hippocampus, such assemblies represent the spatial locations and paths an animal is or has been walking through, respectively. The latter implies that such assemblies have been formed and stabilized and can be reactivated afterwards. Important questions in this context are: i) how many assemblies (i.e. memory traces) can co-exist in the hippocampal network? ii) How many individual cells of an assembly can be omitted without disturbing the specificity of the output? iii) How much overlap can exist between two assemblies without resulting in the same output?

To approach such questions, we developed a low cost and easy-to-implement imaging setup, which allows to simultaneously monitor single cell activity and field potentials of spontaneously occurring oscillations. We mounted a custom-built epi-fluorescence microscope to a Haas-type interface chamber, where spontaneous oscillations can best be studied. To measure the activity of single neurons in hippocampal slice cultures from P6 (C57BL6 mice), we used a recombinant adeno-associated virus (AAV) to express a calcium-sensitive fluorescent protein (GCaMP3.NES) in CA1 and CA3 neurons. After 14–21 days in culture, field potential recordings revealed spontaneous occurrence of sharp wave-ripple network events during which a fraction of local neurons is coherently activated. With our custom-built setup we could monitor a field of view of 410 mm x 410 mm with single-neuron optical resolution (20x objective, 0.4 NA). We developed a highly sensitive and specific wavelet-based method of cell identification allowing simultaneous observation of more than 150 neurons at frame rates of up to 60 Hz.

Our approach provides a new tool to investigate correlated assembly activity and cognition-related oscillation patterns in the hippocampus and other brain regions.