Md2262

We performed microdialysis in awake and freely moving animals while they were tested in the operant chamber. We used either a concentric microdialysis probe of 2 mm length, 0.5 mm outer diameter, cutoff 30,000 kDa (BASi, Lafayette, IN, United States), or a combination probe (BASi, MD2262) that allowed muscimol infusion in addition to performing microdialysis. The probe was lowered 2 mm below the cannulae guide tip into the ILC. The probe was continuously perfused at a rate of 2 μl/min with artificial cerebrospinal fluid (ACSF) using a microperfusion pump (KDS100L model from KD Scientific, Inc., Holliston, MA, United States). The composition of the ACSF solution was 147 mM NaCl, 1.2 mM CaCl₂, 4 mM KCl, 2 mM Na₂HPO₄, and 0.2 mM NaH₂ PO₄ (pH 7.4), freshly prepared with sterile distilled water. After a stabilization period of 120 min, three basal samples of 10 min each were collected in separate tubes containing 2.0 μl of 0.2 N perchloric acid, after which samples were collected every 10 min during all experimental protocols.

Animals were anaesthetized with intra-parenchymal (i.p.) injections of ketamine (70 mg/kg) and xylazine (6 mg/kg) and submitted to standard stereotaxic procedures. One set of rats was implanted with a single 23 gauge stainless steel cannula aimed at 2 mm above the left insular cortex (1.2 mm anterior, 5.5 mm lateral and 3 mm ventral to Bregma; injector protruded 3 mm from the cannula) and a microdialysis guide cannula with an infusion tube (BASi MD 2262) in the right IC (corresponding coordinates for opposite side, and 4.9 mm ventral to Bregma; probe protruded 2 mm from the cannula) [46] (Fig. 1A). Another set of rats was implanted with two (bilateral) stainless steel cannulae aimed at 2.5 mm above the NBM (1.5 mm posterior, 2.5 mm lateral and 4.9 mm ventral to Bregma; injectors protruded 2.5 mm from the cannulae) and one microdialysis guide cannula (BASi MD 2200) in the right IC (coordinates above) (Paxinos and Watson, 2004) (Fig. 1B). The infusion cannulae and microdialysis guide cannula were fixed to the skull with two surgical screws and dental acrylic cement. Stylets were inserted into the guide cannulae to prevent clogging.