Custom-access serial holography (CASH) is a new method for optical recording of neuronal activity in 3D at high speed in-vivo. Our implementation allows random access of 20 cells at 1 kHz up to 200 cells at 0.1 kHz in head-fixed behaving mice across a cortical space of 500 x 500 x 500 m3 size. Using fast acousto-optic spatial light modulation, every single laser pulse of a 40 kHz regenerative amplifier is individually patterned to serially access a selection of target cells with a square 5x5 spot excitation volume covering the cell body and, for the prevention of recording artefacts, the surrounding space in anticipation of the cell displacements during animal behavior. The recorded activity is corrected for neuropil signaling by weighted subtraction of a neuropil reference signal obtained by interleaved sampling of neuropil activity close to each cell.
We performed 3D-CASH recordings of GCaMP6f expressing neurons in layer 2/3 and 5 of mouse primary visual cortex in response to moving contrast gratings and applied deconvolution to estimate spikes. Thanks to the fast recording permit by 3D-CASH, the cortical laminar structure is revealed in the temporal organization of the activity: pairwise correlation was higher between intralaminar vs. interlaminar neuron pairs; principal component analysis of the correlation matrix revealed a component assigning weights of opposite sign to neurons in different layers; closest follower spikes occurred with higher probability in a neuron of the same layer. 3D-CASH allows also following the response to the temporal periodicity of the stimulus, which features a phasic (R1) and a non-phasic component (R0). R1/R0 values are broadly distributed with weak bimodality resembling the transition between pure non-phasic response (complex receptive field) to phasic response (simple receptive field).
Our data validate thus 3D-CASH as a method for assessing neuronal activity in 3D-distributed cortical circuits at high sampling rate.
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