GoKawiila, Histological image of jRGECO1a expression in pons-projecting premotor cortical neurons, which are enriched in the deeper part of layer V around 700 µm below the surface of the brain. RFA, rostral forelimb area of the premotor cortex. b, For the example VR–reach matched imaging session pair in Fig. 1, mean two-photon images show spatial filters for all L5PTs and GrCs with detected activity in both tasks (138 L5PT and 368 GrCs). Traces show 50 example neurons from each imaging session and cell type with colours corresponding to the cell maps above. c, Cross-day cell tracking retention rates. For each session pair, dots indicate the fraction of cells in session 1 that also had detected activity in session 2. While overall retention was lower for GrCs than L5PT due to optical constraints, there was no significant difference between cross-task and same-task retention rates within either cell type (L5PT: p = 0.5; GrCs: p = 1, two-sided Mann–Whitney U-test), demonstrating that tracking attrition was not driven by context-switching (18 cross-task and 9 same-task session pairs from 9 mice). d, Bars show fractions of cells in each cross-task session pair that were reliable across both tasks, which did not differ by cell type (p = 0.3, Wilcoxon signed-rank test; 18 cross-task session pairs). e,f, Custom dual-site microscope mechanics enabling cross-day registration. To accommodate the challenges of tracking dual-region brain activity while alternating between two distinct behavioural apparatuses, we redesigned a dual-site two-photon microscope28 to equip both the cortex arm and the mouse platform with joystick-controlled motorized 3D translation. e, Isolated view of the redesigned cortex arm. Two high-load, long-travel (75 mm), high-accuracy Zaber stages provided motorized x and z control (X-LRQ075HP-DE51). A third compact motorized Zaber stage provided 25 mm of y travel (LSA25A). f, Overall view of both the motorized cortex arm and the cerebellum arm with 16×0.8 NA Nikon and 40×0.8 NA Olympus objectives positioned over the imaging sites on a model mouse skull. Below, a motorized 3D translation platform with 150 mm x/y travel (Zaber X-LRQ150AP-DE51C) and 40 mm z travel (Zaber X-VSR40A) allowed positioning the cerebellum under the cerebellar imaging objective for both behavioural apparatuses. Registration followed a four-step sequence: (1) Align the cerebellar objective optical axis to the window using the cerebellar arm pitch/yaw rotational axes. (2) Position the cerebellar imaging site using the motorized translation platform; fine-tune depth via objective z-piezo; (3) Align the cortical objective optical axis using the left arm pitch/yaw rotational axes; (4) Position the cortex imaging site using the motorized cortex arm; fine-tune depth via objective z-piezo. g, Single-trial behavioural data from a representative matched reach–VR session pair. Traces (top) show single position trajectories and Rasters (bottom) show binary lick sensor contacts with trials grouped into rewarded and omitted reward blocks for ease of visualization. h, Brain motion. Dots show sessions, quantified as standard deviation across all frames of the lateral motion correction computed by the image registration algorithm. Brain motion did not differ between tasks, but was slightly higher for L5PT, probably because the primary skull fixation plate encased the cerebellar window while the cortical window was stabilized by a smaller auxiliary plate (GrCs: p = 0.08; L5PT: p = 0.3, two-sided Wilcoxon signed-rank test; 18 cross-task session pairs).
Source data