FM1-43FX-loaded slices were fixed using rapid microwave fixation in 6% gluteraldehyde, 2% formaldeahyde in PBS as described previously (Jensen and Harris, 1989). After fixation, the samples were transferred into 100 mM glycine in PBS (1 hr), then rinsed in 100 mM ammonium chloride (1 min) and washed in PBS. For photoconversion, the slices were incubated in an oxygen-bubbled diaminobenzidine solution (DAB, 1 mg/ml, Kem
En Tec diagnostics). The DAB solution was refreshed after 10 min and the region of interest was illuminated with intense blue light (<500 nm from a Mercury lamp) for 22–25 min. After photoconversion, the GABA receptor signaling samples were prepared for electron microscopy using an established protocol (Jensen and Harris, 1989). Briefly, the samples were placed in 1% osmium tetroxide (Agar Scientific) and 1.5% potassium
ferrocyanide (Sigma) in cacodylate buffer and, after osmication, stained en block in uranyl acetate and dehydrated for embedding in EPON resin (TAAB). Sectioned samples were laid on bare mesh or formvar-coated slot grids and sections collected between ∼5 and ∼15 μm from the photoilluminated surface (see also Figure S1) were viewed using a Hitachi-7100 transmission electron microscope. Digital images were acquired using a 2,048 × 2,048 charge-coupled device camera (Gatan). Wild-type C57/blk6 mice (24–56 days old) were anesthetized with isoflurane (5% for induction, 1.5%–2.5% for surgery, and 0%–0.5% during recording), augmented with chlorprothixene first (0.5–2 mg/kg, intraperitoneally). A 2–3 mm diameter craniotomy was opened over visual cortex. The dura mater was Selleckchem Afatinib left intact. A thin layer of agar (1.5%) dissolved in aCSF (150 mM NaCl, 2.5 mM KCl, 10 mM HEPES, 2 mM CaCl2, and 1 mM MgCl2; pH adjusted with NaOH to 7.3; 300 mOsm) and placed on top of the brain helped dampen movement. A homeothermic heat pad maintained body temperature within the physiological range. Water-based opthalmic ointment maintained eye health. Visual stimulus presentation was controlled by routines written in MATLAB using the Psychophysics Toolbox extensions (Brainard, 1997; Kleiner et al., 2007, Perception
36 ECVP, abstract; Pelli, 1997). Square-wave gratings (0.04 cycles/deg, 2 cycles/s) of black (2 cd/m2) and white (86 cd/m2) bars in eight different orientations were displayed on an LCD screen (ESAW 7 inch VGA TFT, set at 1,024 × 768 resolution and 60 Hz refresh rate) to map orientation selectivity. For control gray screen stimulation, the total luminance was matched to that of the grating stimulus. The screen was shrouded with a cone up to the eye of the mouse to prevent contamination of the imaging pathway with light from the visual stimulus. The visual stimulus extended from +20° to +124° in azimuth and from −10° to +42° in elevation. A custom-built two-photon microscope using galvanometer-based scan mirrors (6 mm diameter, Cambridge Technologies) with a 16× magnification and 0.