All procedures were approved by the Janelia Farm Research Campus Institutional Animal Care and Use Committee. We used adult (> P60) male PV-IRES-cre mice (B6;129P2-Pvalbtm1(cre)Arbr/J, The Jackson Laboratory). All surgeries were conducted under isoflurane anesthesia (1.5–2%). Additional drugs reduced potential inflammation (Ketofen, 5mg/kg, subcutaneously) and provided local (Marcaine, 0.5% solution injected under the scalp) and general analgesia (Buprenorphine, 0.1 mg/kg, intraperitoneal). A circular piece of scalp was removed and the underlying bone was cleaned and dried. The periostium was removed with a dental drill and the exposed skull was covered with a thin layer of cyano-acrylic primer (Crazy glue). A custom-machined titanium frame was cemented to the skull with dental acrylic (Lang Dental).
Afferents from the somatosensory cortex were labeled with virus expressing tdTomato 33 (rAAV-CAG-tdTomato, serotype 2/1; 20 nl at 300 and 550 um depths). The C2 barrel was targeted based on intrinsic signal imaging 28 (link). The virus was injected with a custom, piston-based, volumetric injection system (based on a Narishige, MO-10, manipulator) 46 (link). Glass pipettes (Drummond) were pulled and beveled to a sharp tip (30 um outer diameter). Pipettes were back-filled with mineral oil and front-loaded with viral suspension immediately prior to injection.
A craniotomy was made over vM1 (size, 3×2mm; center relative to Bregma: lateral, 0.8 mm; anterior, 1 mm, left hemisphere,Fig. 2a–d ). These coordinates were previously determined using intracortical microstimulation 8 (link),16 (link),18 (link), mapping axonal projections from vS1 in vM1 8 (link),47 (link), and trans-cellular labeling with pseudorabies virus (data not shown). Neurons underlying the craniotomy were labeled by injecting virus expressing GCamP3 (rAAV-syn-GCaMP3, serotype 2/1, produced by the University of Pennsylvania Gene Therapy Program Vector Core). The brain was covered with agar (2%). 4–8 sites (10–15 nl/site; depth, 150–210 um; rate, 10 nl/minute) were injected per craniotomy.
The imaging window was constructed from two layers of standard microscope coverglass (Fisher; # 2, thickness, 170 – 210 um), joined with a UV curable optical glue (NOR-61, Norland): a larger piece was attached to the bone; a smaller insert fit snugly into the craniotomy (Fig. 2b, d ). The bone surrounding the craniotomy was thinned to allow for a flush fit between insert and the underlying dura.
After virus injection, the glass window was lowered into the craniotomy. The space between the glass and the bone was sealed off with a thin layer of agar (2%), and the window was cemented in place using dental acrylic (Lang Dental). At the end of the surgery, all whiskers on the right side of the snout except row C were trimmed. The mice recovered for 3 days before starting water restriction. Imaging sessions started 14–21 days after the surgery.
Afferents from the somatosensory cortex were labeled with virus expressing tdTomato 33 (rAAV-CAG-tdTomato, serotype 2/1; 20 nl at 300 and 550 um depths). The C2 barrel was targeted based on intrinsic signal imaging 28 (link). The virus was injected with a custom, piston-based, volumetric injection system (based on a Narishige, MO-10, manipulator) 46 (link). Glass pipettes (Drummond) were pulled and beveled to a sharp tip (30 um outer diameter). Pipettes were back-filled with mineral oil and front-loaded with viral suspension immediately prior to injection.
A craniotomy was made over vM1 (size, 3×2mm; center relative to Bregma: lateral, 0.8 mm; anterior, 1 mm, left hemisphere,
The imaging window was constructed from two layers of standard microscope coverglass (Fisher; # 2, thickness, 170 – 210 um), joined with a UV curable optical glue (NOR-61, Norland): a larger piece was attached to the bone; a smaller insert fit snugly into the craniotomy (
After virus injection, the glass window was lowered into the craniotomy. The space between the glass and the bone was sealed off with a thin layer of agar (2%), and the window was cemented in place using dental acrylic (Lang Dental). At the end of the surgery, all whiskers on the right side of the snout except row C were trimmed. The mice recovered for 3 days before starting water restriction. Imaging sessions started 14–21 days after the surgery.
