Isoflurane

All procedures involving animals were approved by the Institutional Animal Care and Use of Committee of the University of Pittsburgh. Male Sprague-Dawley rats (250–350 g, Hilltop, Scottsdale, PA) were anesthetized with isoflurane (5% v/v induction, 2.5% v/v maintenance, Henry Schein Animal Health, Elizabethtown, PA), and 2:1 N₂O:O₂ (Matheson Tri-Gas, Bernards, NJ). Rats were wrapped in a heating blanket (37°C) and placed in a stereotaxic frame. The incisor bar was adjusted so the dorsalventral measurements at lambda and bregma were no more than 0.01 mm apart (flat skull). A small craniotomy (3 × 5 mm) was made over the dorsal striatum. Microdialysis probes, continuously perfused, were lowered slowly into the dorsal striatum (1.6 mm anterior and 2.5 mm lateral from bregma) or the ventral striatum (1.6 mm anterior and 1.4 mm lateral from bregma) over the course of approximately 30 min to final position of 7 mm or 8mm (respectively) below dura (Figure 1A). Probes were secured with bone screws and acrylic cement. Anesthesia was removed and animals were placed in a BASi Raturn chamber (MD-1404, Bioanalytical Systems Inc., West Lafayette, IN) for housing.
As the goal of these animal studies was to demonstrate the MD-ROOC’s capabilities, we used three animals with the probe in the dorsal striatum and one animal with the probe in the ventral striatum. Data from one of each are shown.

100min after the start of the testing session, rats were briefly anaesthetized with isoflurane (Henry Schein, Pittsburgh, PA), and then intraperitoneally injected with tribromoethanol (375mg/kg; Sigma Aldrich, St.Louis, MO) and transcardially perfused with 0.9% saline followed by 4% paraformaldehyde in 0.1M borate buffer. The brains were stored for 20–24h at 4°C in a paraformaldehyde and 12% sucrose mixture and then rapidly frozen in hexanes cooled with dry ice and stored at −80°C. Brains were cut into 30μm coronal sections using a microtome and were collected into four adjacent series. One tissue series was processed with ORX immunohistochemistry followed by Fos immunohistochemistry, described below. Another series was mounted and stained with thionin for identification of cytoarchitectonic borders, and the remaining two were stored in cryoprotectant in a −20°C freezer.

Six pairs (n=6) of matched Ts2 and wild-type disomic (2N) littermates were analyzed at 12 months of age for a total of 12 animals (3 mice per group for EM and 3 mice per group for endosomal isolation). All animals were anesthetized with isoflurane (Henry Schein, Dublin, OH, US) inhalation before being sacrificed. Both females and males were used for EM and biochemical analyses; specifically, 2 matched pairs of females and 1 pair of males were used for EM and 2 pairs of males and 1 pair of females for the organelle isolation. All animal procedures were performed following the National Institutes of Health guidelines with approval from the Institutional Animal Care and Use Committee at the Nathan S. Kline Institute for Psychiatric Research.