Multi-layer Cortical Ca²⁺ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

Abstract

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical neurons with two-photon microscopy has provided unique insights into cortical structure, function and plasticity. However, these studies are limited to head fixed animals, greatly reducing the behavioral complexity available for study. In this paper, we describe a procedure for performing chronic fluorescence microscopy with cellular-resolution across multiple cortical layers in freely behaving mice. We used an integrated miniaturized fluorescence microscope paired with an implanted prism probe to simultaneously visualize and record the calcium dynamics of hundreds of neurons across multiple layers of the somatosensory cortex as the mouse engaged in a novel object exploration task, over several days. This technique can be adapted to other brain regions in different animal species for other behavioral paradigms.

A role for astroglial calcium in mammalian sleep

“…Imaging frames were captured at 10.1 frames per second with an exposure time of 49.664 ms at a gain of 2.0. LED power ranged from 50 -80% to adjust the upper tail of the histogram to be as close to a pixel value of 1500 to ensure good signal-to-noise ratio (33). The LED power was set at the beginning of the experiment for each mouse and did not change for the remainder of the experiment.…”

“…For each animal, movies acquired during baseline and during SD and recovery were concatenated into a timeseries. This timeseries was spatially downsampled by a factor of 2 from 1440 x 1080 pixels to 720 x 540 pixels and temporally downsampled by a factor of 5 to 2 Hz to reduce the data footprint (33). Defective pixels were also rectified (33).…”

“…This timeseries was spatially downsampled by a factor of 2 from 1440 x 1080 pixels to 720 x 540 pixels and temporally downsampled by a factor of 5 to 2 Hz to reduce the data footprint (33). Defective pixels were also rectified (33). Small lateral displacements were then corrected for by registering all frames to a single reference frame free of movement using the motion correction algorithm in the Data Processing Software.…”

“…Small lateral displacements were then corrected for by registering all frames to a single reference frame free of movement using the motion correction algorithm in the Data Processing Software. To identify individual cells, 28 images were converted to ΔF/F images by expressing each frame as the relative change from the mean image obtained from the entire movie (ΔF/Fmean = F -Fmean/Fmean) (33,34). A spatial bandpass filter was also applied using default settings (low cut-off = 0.005; high cut-off = 0.500) to help delineate individual cells.…”

“…EEG and EMG data were collected with a Grass 7 polygraph system (Natus Medical Incorporated, Pleasanton, CA) via a light-weight cable for sleep phenotyping experiments in GSTIM1 WT and cKO mice. EEG & EMG signals were amplified and digitized at 256 Hz using VitalRecorder acquisition software (SleepSign for Animal, Kissei Comtec Co., LTD, Nagano, 33 Japan). EEG and EMG data were high-and low-pass filtered at 0.3 and 100 Hz and 10 and 100 Hz, respectively.…”