The surgery protocol for paper 'geometry of sequence working memory in macaque prefrontal cortex'

Experimental model preparation
 

Subjects

   Two rhesus monkeys M1 and M2 (Macaca mulatta, male, 5.0–6.1 kg, 4 years old) were used in the study. To express the Ca2+ indicator GCaMP6s , we injected AAV1.Syn.GCaMP6s.WPRE.SV40 (titer 2.3e13 GC/ml, Penn Vector Core) into the prefrontal cortex in each monkey. All experimental protocols followed the Guide of the Institutional Animal Care and Use Committee (IACUC) of Peking University Laboratory Animal Center and were approved by the Peking University Animal Care and Use Committee.
 

Surgeries

    Three sequential surgeries were performed on each animal under general anesthesia. In the first surgery, head posts were implanted on each animal’s skull. Two head posts were implanted on the back of the head and one on the forehead. A Y-shaped steel frame was fixed to these head posts for head stabilization during training and recording. Both monkeys were then trained on the behavioral task over 7 months. After reaching the performance criteria, the second surgery was performed. In the second surgery, a 20 mm craniotomy was made on the skull over the region of lateral prefrontal cortex located along the principal sulcus (PS) until the arcuate sulcus (AS), roughly corresponding to Walker’s areas 46 and 8, and the dura was opened. Then, the virus injections were performed. Around 200 nL AAV1.Syn.GCaMP6S.WPRE.SV40 (Penn Vector Core, titer 2.7e13 GC/ml) was injected at a depth of around 400 μm. The injection sites were located alongside the PS, roughly corresponding to Walker’s area 46 (see fig. S2 for details). After virus injection, the dura was sutured, and the removed skull bone was replaced. In the first week of recovery, ceftriaxone sodium antibiotic was administered.
The last surgery was performed 8–9 weeks later to implant an imaging window. The skull was reopened and the subjacent dura was cut off (diameter 16 mm) to expose the cortex. A glass coverslip (15 mm in diameter and 0.17 mm in thickness) was glued with a titanium ring (12 mm outer diameter and 10 mm inner diameter). A ring-shape GORE membrane (12 mm inner diameter and 20 mm outer diameter) was mounted onto the titanium ring and glued on the outer part of the glass coverslip. The coverslip, titanium ring, and membrane constituted a window unit. The window unit was gently pushed onto the cortical surface. The GORE membrane was inserted under the dura. The titanium ring was glued to the skull with dental acrylic. The entire imaging window was then covered with a steel shell to protect the coverslip.

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