Details

Autor(en) / Beteiligte

• Reichinnek, Susanne
• von Kameke, Alexandra
• Hagenston, Anna M.
• Freitag, Eckehard
• Roth, Fabian C.
• Bading, Hilmar
• Hasan, Mazahir T.
• Draguhn, Andreas
• Both, Martin

Titel

Reliable optical detection of coherent neuronal activity in fast oscillating networks in vitro

Ist Teil von

• NeuroImage (Orlando, Fla.), 2012-03, Vol.60 (1), p.139-152

Ort / Verlag

United States: Elsevier Inc

Erscheinungsjahr

2012

Quelle

MEDLINE

Beschreibungen/Notizen

• Cognitive and behavioral functions depend on the activation of stable neuronal assemblies, i.e. distributed groups of co-active neurons within neuronal networks. It is therefore crucial to monitor distributed patterns of activity in real time with single-neuron resolution. Microelectrode recordings allow detection of coincidence between discharges of identified units at high temporal resolution, but are not able to reveal the full spatial pattern of activity in multi-cellular assemblies. Therefore, observation of such distributed sets of neurons is a stronghold of optical techniques, but the required resolution, sensitivity, and speed are still challenging current technology. Here, we report a new approach for monitoring neuronal assemblies, using memory-related network oscillations in rodent hippocampal circuits as a model. The cytosolic calcium-sensitive fluorescent protein GCaMP3.NES was expressed using recombinant adeno-associated viral (rAAV)-mediated gene transfer in CA3 pyramidal neurons of cultured mouse hippocampal slices. After 14–21days in culture, field potential recordings revealed spontaneous occurrence of sharp wave-ripple network events during which a fraction of local neurons is coherently activated. Using a custom-built epi-fluorescence microscope we could monitor a field of view of 410μm×410μm with single-neuron optical resolution (20× 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 60Hz. Our recording configuration and image analysis provide a tool to investigate cognition-related activity patterns in the hippocampus and other circuits. ► We observe spontaneous calcium transients in cultured hippocampal slices. ► Protein-based calcium dyes are expressed by viral gene transfer. ► Signal-to-noise ratio is optimized by wavelet-based filtering. ► Single cells can be distinguished by a rigid classifying algorithm. ► Our method allows analysis of co-active assemblies of multiple neurons.