Fig. 3: System for three-dimensional optogenetic stimulation and laminar recordings in freely moving mice.

A Fiber-bundle probe used in the experiments for light emission at different depths in the brain tissue. Scale bar: 1 mm. Schematic side view (left panel); sequentially illuminated fibers lead to defined depth illumination (right panel). B Multifiber polishing holder and ~70 simultaneously polished fibers illuminated with green light for visualization. Scale bar: 4 mm. C Bilateral ChR2 injection locations, fiber bundle implantation locations, and BE probe implantation illustrated in three exemplary coronal sections. D Optical set-up for flexible three-dimensional optogenetic stimulation (fiber-bundle shown as a blue line and a photodetector signal cable shown as a yellow line) and laminar recordings (ephys-cable shown as a green line) in a freely moving mouse. A fiber bundle transmits the galvo scanning position to the animal. The fiber bundle (Schott imaging fiber bundle; see methods) was 2 m long and attenuated the light by 75%. E Implantation schematic. The locations of the fibers in the fiber-matrix connector were detected using an implanted photodetector. The fibers emit stray light that can be detected by the photodiode in the transparent cement. This photodiode signal was sent via the commutator to the recording system. F Connector behind an aligning glass plate during implantation. To make the fibers visible, a white light source was directed toward the mouse skull. The deep 30 μm fibers (Fiber 1) exhibited a red emission with a low intensity (leftmost fiber in each row). The superficial 30 μm fibers (Fiber 6) exhibited a yellowish emission with a higher intensity (middle fiber in each row). The 11.8 μm fibers absorbed a higher percentage of the white light above the brain and therefore emitted white light (scale bar: 500 μm). Some fibers were not covered by the fiber bundle because the connector was aligned by manual adjustments of the original silicon plate. For the fiber bundle, the silicon plate is not necessary, and the connector on the animal side can have hexagonal constraining walls (3D-printed or CNC machined) to ensure alignment with the hexagonal fiber bundle. G Readout of the implanted photodiode during X/Y galvo scanning (scale bar: 500 μm). Voltage is linearly mapped to the jet pseudocolor scale in MATLAB. Source data are provided as a Source Data file.