Nanoz

Animals were implanted with microdrive arrays (Versadrive-8 Neuralynx) in either AC (eight animals) or FR2 (seven animals) after reaching behavioral criteria of d’≥1.0. For surgery, animals were anesthetized with ketamine (40 mg/kg) and dexmedetomidine (0.125 mg/kg). Stainless steel screws and dental cement were used to secure the microdrive to the skull, and one screw was used as ground. Each drive consisted of eight independently adjustable tetrodes. The tetrodes were made by twisting and fusing four polyimide-coated nichrome wires (Sandvik Kanthal HP Reid Precision Fine Tetrode Wire; wire diameter 12.5 μm). The tip of each tetrode was gold-plated to an impedance of 300–400 kOhms at 1 kHz (NanoZ, Neuralynx).

Skin impedance of the ESS electrodes was measured over seven hours to evaluate the stability of electrode-skin contact. Impedance measurements were collected using the NanoZ (NeuraLynx, Bozeman, MT, USA). The impedance between the electrode and the skin was measured for each ESS electrode contact for all muscles. At each time point, impedances were collected at logarithmically equal frequency intervals between 4 Hz and 2 kHz for a total of 20 frequencies, with each measurement the result of averaging 40 measurements. The impedance was averaged over subjects and muscle groups for each frequency to obtain an impedance versus frequency graph at each time point. These frequencies were chosen for our measurements because the majority of sEMG power lies between 5–500 Hz and this range has been used in prior research [10 (link),19 (link),27 (link)]. Preliminary testing showed that impedance measurements change over hours, not minutes, so measurements were taken every 30 min for 2 h, then every hour for the next 5 h. Impedance measurements were collected only for the ESS electrodes, as the rigid, boxed design of the Delsys electrodes do not allow the user to directly evaluate contact impedance [41 ].

After completion of behavioral testing, mice were deeply anesthetized with a mixture ketamine/xylazine/acepromazide (100, 16, 3 mg/kg, respectively, intraperitoneal injection). For mice implanted with electrodes, microlesions were performed by passing a current of ~ 10 µA for 10–20 s through each electrode using a Nano Z (Neuralynx, USA) to identify electrode tips. Mice were then perfused transcardially with 4% paraformaldehyde in PBS (PFA). The brains were extracted and postfixed overnight in PFA at 4°C and subsequently washed in PBS for an additional 24 h at 4°C. Brains and sections were cryoprotected in solution of 30% ethylene glycol, 30% glycerol, and 40% PBS until used. Each brain was then sectioned at 50 µm using a vibratome: every section was sequentially collected in four different 1.5 mL tubes to allow different analyses (electrode location, plaque analyses, immunohistochemistry).