Mo 10 manipulator

All procedures were performed according to the guidelines set by the Janelia Farm Research Campus Institutional Animal Care and Use Committee and Institutional Biosafety Committee. Animals (adult C57/BL6J, Charles River; either sex) mice were anesthetized under isoflurane and AAV virus encoding smFPs, serotype 2/1 was injected with a custom-made volumetric injection system (based on a Narishige MO-10 manipulator). Glass pipettes (Drummond) were pulled and beveled to a sharp tip (30 µm outer diameter), back-filled with mineral oil and front-loaded with viral suspension immediately before injection.

For in vivo two-photon calcium imaging experiments, before attaching the head-post to the skull, we performed a circular craniotomy over the left visual cortex (diameter, 4 to 5 mm). The dura was left intact. For imaging activity during behavior (Figures 5), V1 neurons were labeled with virus expressing GCaMP6f AAV1-Syn-GCaMP6f-WPRE-SV40 (University of Pennsylvania Gene Therapy Program Vector Core). Virus was injected in four to seven different locations in the visual cortex (20 nl per location, 10 nl/min, 200 - 315 μm deep) using a custom volumetric injection system based on a Narishige MO-10 manipulator [47 (link)]. To prevent backflow during withdrawal, the pipette was left in the brain for over 5 minutes before withdrawal. For the V1 anesthetized recordings (Figure 3 and S5), the transgenic mouse line Thy1-GCaMP6s[29 (link)] (line GP4.3) was used and no virus was injected. An imaging window was constructed from two layers of microscope cover glass (Fisher Scientific no. 1 and no. 2) joined with a UV curable optical glue. The window was embedded into the craniotomy and secured in place using black dental cement and then the head-post was attached with the same dental cement. The glass window gently pressed against the brain, minimizing movement artifacts during behavior[52 (link)].