| OCR Text |
Show 66 (Gee et al., 2014). Brain slices were prepared using established techniques (Kispersky et al., 2012; Netoff et al., 2005; Smeal et al., 2012). Briefly, mice of either sex were deeply anesthetized using isoflurane and decapitated at various time points spanning P56 through P120. Brains were rapidly dissected and placed in ice-cold (4°C) oxygenated sucrose Ringer's solution containing (in mM): 200 sucrose, 26 NaHCO3, 10 D-glucose, 3 KCl, 3 MgCl2, 1.4 NaH2PO4 and 1 CaCl2. The pH was maintained at 7.4 by saturation with 95% O2-5% CO2. A vibrating microtome (VT1200; Leica, Wetzlar, Germany) with the bath chilled to 1.5°C (Minichiller; Huber, Offenburg, Germany), was used to prepare 400 μm thick coronal slices in oxygenated sucrose Ringer's solution (4°C). Following sectioning, slices were transferred to a holding chamber filled with oxygenated artificial cerebrospinal fluid (ACSF; room temperature) containing (in mM): 126 NaCl, 26 NaHCO3, 10 Dglucose, 3 KCl, 2 MgCl2, 2 CaCl2 and 1.4 NaH2PO4 for at least 30 min. During imaging, slices were placed in a heated (32°C) immersion style recording chamber (Warner Instruments, Hamden, CT, USA) mounted on a microscope stage and perfused with ACSF. Slice were given 10 min in the slice chamber to equilibrate. Two-photon imaging All controlled imaging parameters are listed in Table 4.1. Two-photon imaging was performed using a Prairie Technologies Ultima Multi-Photon Microscopy System (Bruker, Billerica, MA, USA) built around a mode-locked Ti:Sapphire laser source emitting 140 fs pulses at an 80 MHz repetition rate with a wavelength pulses at an 80 MHz repetition rate with a wavelength adjustable from 690-1040 nm (Chameleon Ultra I; Coherent, Santa Clara, CA, USA). GCaMP5G and tdTomato were simultaneously excited with a laser emission wavelength of 940 nm (Akerboom et al., 2012; Drobizhev et al., 2011). Laser |
