Brain slicing

For all in vitro experiments, 15- to 18-day-old mice were initially anesthetized with ketamine (100 mg/ kg) and xylazine (3 mg/kg) intraperitoneally and transcardially perfused with chilled (4°C) sucrose-based slicing solution containing the following (in mM): 234 sucrose, 11 glucose, 26 NaHCO₃, 2.5 KCl, 1.25 NaH₂PO₄, 10 MgCl₂, 0.5 CaCl₂. After the brain was removed from the skull, it was cut to obtain auditory colliculo-thalamocortical brain slice (aCTC) as shown (Figure 2—figure supplement 1) and as described before (Llano et al., 2014; Slater et al., 2015). 600 μm thick horizontal brain slices were obtained to retain the connectivity between IC, MGB, TRN and AC. All slices were incubated for 30 min at 33°C in a solution composed of (in mM: 26 NaHCO₃, 2.5 KCl, 10 glucose, 126 NaCl, 1.25 NaH₂PO₄, 3 MgCl₂, and 1 CaCl₂). After incubation, all slices were transferred to a perfusion chamber coupled to an upright Olympus BX51 microscope, perfused with artificial cerebrospinal fluid (ACSF) containing (in mM) 26 NaHCO₃, 2.5 KCl, 10 glucose, 126 NaCl, 1.25 NaH₂PO₄, 2 MgCl₂, and 2 CaCl₂. Another set of experiments was done in a different laboratory to exclude any experimental factors related to our laboratory environment, chemicals, or anesthesia. As reported previously (Krause et al., 2014), following full anesthesia by isoflurane, a C57BL/6J mouse was immediately decapitated without cardiac perfusion, the animal’s brain was extracted and immersed in cutting artificial CSF [cACSF; composed of (in mM) 111 NaCl, 35 NaHCO₃, 20 HEPES, 1.8 KCl, 1.05 CaCl₂, 2.8 MgSO₄, 1.2 KH₂PO₄, and 10 glucose] at 0–4°C. Slices were maintained in cACSF at 24°C for >1 hr before transfer to the recording chamber, which was perfused at 3–6 ml/min with ACSF [composed of (in mM) 111 NaCl, 35 NaHCO₃, 20 HEPES, 1.8 KCl, 2.1 CaCl₂, 1.4 MgSO₄, 1.2 KH2PO₄, and 10 glucose]. All of the solutions were bubbled with 95% oxygen/5% carbon dioxide and all experiments were done at room temperature.