Figure 4.
Two-Photon GCaMP6f Imaging of Motor Cortex Layer 2/3 Neurons Reveals Direction-Selective Activity in the Directional Reaching Task
(A) Left: side-view schematics of a head-fixed mouse under a two-photon microscope during the directional reaching task. Center: photomicrograph of the cranial window on top of the frontal cortex with AAV1-hSyn-GCaMP6f infected neurons (green fluorescence) in spots A and B. White cross, bregma; FL-ISI, forelimb intrinsic signal. Detailed field of view (FOV) of layer 2/3 neurons recorded during directional reaching task. Color-shaded ROIs correspond to task-related neurons and their trial type preference (color coding on the top: L, left; C, center; R, right). Right: normalized GCaMP6f fluorescence corresponding to the same ROIs; green, yellow, and pink shades correspond to the trial type (L, C, and R). Neurons were sorted according to their trial type preference.
(B) Percentage of task-related ROIs per FOV in spots A and B showing a higher proportion of related neurons in spot A. Light blue shades, 25th and 75th quartiles; horizontal line, median. ∗p = 0.004, Wilcoxon rank test.
(C) Calcium fluorescence traces (ΔF/F0) were triggered at the time of reaching onset for left, center, and right trials (time = 0). Left: four example ROIs showing different directional selectivity responses. Right: all reach-related ROIs projected in a three-dimensional space of normalized fluorescence response amplitude for left, center, and right trials. Each dot corresponds to one ROI with color-coded directional response.
(D) Number of ROIs per FOV with responses selective for one, two, or three directions showing that the majority of the imaged ROIs showed reach-related response to a single trial type. Light blue shades, 25th and 75th quartiles; horizontal line, median. ∗p < 0.05, Tukey post hoc test, Friedman RM ANOVA on ranks.
(E) Number of ROIs per FOV with direction selectivity preference to left, center, and right trials showing that, on average, all three direction-selective neurons are equally represented in each FOV. Black line represents the population mean; error bars represent SD.