Fig. 4. High-speed translaminar imaging reveals region-specific sensory and motor-evoked activity in behaving GFAP-GCaMP6f mice.

a Schematic showing the translaminar imaging approach. An 0.7 mm × 0.7 mm × 0.7 mm glass reflective microprism was inserted at the lateral interface between the spinal gray and white matter at the L4-L5 spinal level without tissue removal (Methods). The microprism’s reflective hypotenuse tilts the microscope’s imaging plane by 90 degrees allowing high-speed measurements across laminae. Imaging was performed at 75 μm or 125 μm focal depth from the vertical microprism face. Scale bar, 0.5 mm. b Predicted extent and borders of spinal laminae across the 700 μm field of view (FOV) for 75 μm (top) and 125 μm focal depths (bottom). c Example fluorescence images showing the translaminar blood vessel pattern in a GFAP-GCaMP6f mouse at 75 μm and 125 μm focal depths four weeks after microprism implantation. Scale bars, 100 μm. d Average intensity projection image from a different GFAP-GCaMP6f mouse at 75 μm focal depth overlaid with 10 μm × 10 μm ROIs. Only ROIs with at least 50% (yellow) or 75% ∆F/F (orange) in response to a noxious tail pinch (p > 500 g; duration: 1.5 s ± 0.5 s) are shown. In this example, the tail pinch did not evoke a locomotor response. Scale bar, 100 μm. e Noxious tail pinch evoked activity across tissue depth and time for the example recording shown in d. Each row depicts the percent of active ROIs (≥50% ∆F/F) for a given tissue depth. The corresponding pressure stimulus, locomotor activity, and average calcium transient across the FOV are shown above the activity heat map. Running speed was recorded by placing the animal on a spherical treadmill (Supplementary Movie 3). The vertical dashed line indicates pinch onset. f Population data showing the average percent of active ROIs across tissue depths. Significant activity occurred in central FOV regions, corresponding to the upper spinal laminae. g Population data showing average calcium response onset latency for pinch-only and run-only trials. h Population data showing individual ROI and average calcium transient duration for pinch-only trials. i Activity map from the same animal as in d for a spontaneous run trial without pressure stimulus application. Same focal depth as in d. Scale bar, 100 μm. j Running-evoked astrocyte activity across tissue depth and time (Supplementary Movie 4). The pressure sensor readout, locomotor activity, and average calcium signal across the FOV are shown above the activity heat map. The vertical dashed lines indicate run onset (Methods). k Population data showing the average percent of active ROIs across tissue depths for run trials. Significant activity occurred in FOV regions corresponding to deep dorsal horn laminae involved in motor processing. l Population data showing individual ROI and average calcium transient duration for run-only trials (2–4 s-long runs with ≥25 s rest period before the run). The data in f, g (left), h are from 1105 ROIs, 18 recordings, and 5 mice. The data in g (right), k, l are from 855 ROIs, 8 recordings, and 2 mice. All shown data were acquired four weeks after microprism implantation. Two-sided paired t-tests determined P values, and all bar plots are presented as mean ± s.e.m. The box and whisker plots mark the median and the 25th and 75th percentiles, and the whiskers cover the minimum and maximum of the data. All images are representatives from one sample. Images with similar properties were obtained across multiple independent samples. Source data are provided as a Source Data file. Panel 4a image reproduced with permission from ref. ³⁵.