Fast three-dimensional imaging of neuronal and dendritic spine assemblies in the
mouse visual cortex using GCaMP6 and two-photon microscopy
Szalay, G.1, Judák, L.1, Spitzer, K.1, Nyitrai, G.1, Katona, G.1, Maák. P.2, Veress, M.2,
Chiovini, B.1,3, Pálfi, D.1,3, Rózsa, B.1,3
1
Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary
Department of Atomic Physics, Budapest University of Technology and Economics, Budapest, Hungary
3
The Faculty of Information Technology, Pázmány Péter Catholic University, Budapest, Hungary
2
Understanding the cooperated activity of neuronal networks and subcellular computational
units requires measurement methods that read out neural activity on different spatial and
temporal scales. Advances in imaging techniques and fluorescent marker synthesis enable the
investigation of these principles in better and better detail. Of the different Ca2+ sensors,
genetically-encoded Ca2+ sensors have the advantages that they 1) are genetically targetable to
different neuronal subpopulations, 2) allow the simultaneous labeling and measurement of
dendritic spines and neuronal somata, and 3) provide a long measurement window.
Furthermore, the latest GCaMP6 variants have comparable onset kinetics and signal-to-noise
ratio to synthetic dies. On the imaging side, the most powerful tool for large-depth spineresolution real-time 3D measurement of the intact brain is acousto-optical (AO) two-photon
microscopy. This technology can restrict scanning to regions containing actual information
and avoid unnecessary background areas, increasing the measurement speed and signal-tonoise ratio with several orders of magnitude.
We developed a novel AO system for GCaMP6 measurements which can operate in two
modes: 1) random access mode (near-cubic-millimeter scanning range, 100 Hz sampling rate),
or 2) trajectory scanning mode (contiguous linear scanning regions, kHz sampling rate). The
system also allows the compensation of spine-level motion artifacts during in vivo
measurements. In this work, we use this microscope to measure visual stimulation-induced
Ca2+ responses from GCaMP6 labeled dendritic spines and neuronal networks from the V1
visual cortex. We investigated the variability of neuronal coding in assemblies of up to 500
neurons and, separately, up to 100 dendritic spines during successive stimuli or behavioral
paradigm. In both cases, the obtained data revealed largely variable response patterns. The
source of this versatility is still under investigation.