Functional imaging of long neuronal processes and network in behaving animals in any arbitrarily tilted plane in 3D

01/30/2020

Tamas Tompa¹, Katalin Ocsai¹, Mate Marosi¹, Zsofia Medveczky¹, Gergely Szalay¹, Gergely Katona¹, Balazs Rozsa¹

¹ 

The neural network is a 3 dimensional complicated mesh of elements like cell bodies, dendrites, dendritic spines, axons and others, forming connections by extending processes in all directions and in different shapes. To understand the computation performed by this network (ie. dendritic integration, somatic processing etc), one needs to record near-simultaneously the activity of these elements in a possibly large volume of tissue. Conventional functional microscopy methods are generally limited to 2-dimensional planes parallel with the front lens of the microscope and the cortical surface. Imaging through cortical layers was so far only possible either non-simultaneously or with highly invasive methods (ie: microprisms). Using two-photon acusto-optical (AO) microscopy we presented already a method to overcome the limitation of the 2-dimensional scanning, by a random-access point scanning method (Katona et al 2012), that we later complemented with the 3D drift AO scanning (Szalay et al 2016). 3D drift AO scanning can extend each scanning point to small 3D lines, surface or volume elements, preserving fluorescence information for fast off-line motion correction. With this method in vivo motion artifacts can be effectively eliminated, allowing fast 3D measurement of over 100 dendritic spines with 3D lines; hundreds of somata with squares and cubes; multiple spiny dendritic segments with surface and volume elements. However, this earlier solution contained some limitation regarding the scanning patterns and acquisition speed. Here we present a complex scanning solution which can exploit the full capabilities of the 3D scanning options, providing virtually compromise-free ROI scanning options. This technique allows us to measure various scanning patterns on any arbitrary tilted frame, with 40 Hz scanning speed for a full field imaging and up to 3Khz on special scanning patterns. This feature makes it possible to record raster videos of all cortical layers simultaneously or record individual neurons with their entire apical dendrites, or obliquely running neural elements with high speed. Here we present our results from the visual cortex (V1) of awake behaving mice involved in a visual task and show calcium events occurring along the entire length of the cortical pyramidal neuron’s soma and apical dendrites and within a group of neurons. On these elements we record local dendritic spikes, and attempt to demonstrate instances of signal integration at the level of the calcium signals.