8 NA, Olympus, Tokyo, Japan). Scanning and image acquisition were controlled by Fluoview software (Olympus); the average power delivered
to the brain was <50 mW. Imaging was carried out 72 hr before and after a retinal lesion (Figure 2A) at high resolution (1024 × 1024 pixels, 0.08 μm per pixel, 0.5 μm z step size). Imaging regions were repeatedly found by aligning the blood vessel pattern on the surface of the brain. Lower-resolution image stacks (512 × 512 pixels, 2.5 μm z step size) were acquired to visualize the dendritic and axonal branching pattern and the position of the soma, which, for the cells analyzed here, was confirmed to be located in cortical layer 1 or 2/3. Cells in the LPZ were chosen such that they were located at least 50 μm from the borders to avoid CP-673451 order any ambiguity. Cells located outside of the LPZ had cell bodies that were located >50 μm from the edge of the LPZ, as determined using intrinsic imaging within selleck products 3 days after the lesion. Distance from the border of the LPZ was calculated based on the position of the axon or, in the case of dendritic measurements, the cell body. High-resolution images were used for the analysis of dendritic spines and axonal boutons.
Image analysis was carried out using ImageJ (US National Institutes of Health, Bethesda, MD) and performed in three dimensional z stacks. Analysis of spines and boutons were restricted to cortical layers 1 and 2/3 (0–200 μm below the cortical surface). All protrusions, spines and filopodia were counted, including those that extended in the z axis. Spines and boutons were counted without the knowledge of experimental condition. Survival fraction is calculated at each time point as the number of boutons or spines still present that Casein kinase 1 were present at the first time point as a fraction of the
total number of initial boutons or spines. In total, we analyzed 16,259 boutons and 9633 spines over 9–12 time points. GAD65-GFP animals were deeply anaesthetized and cardially perfused first with saline (0.9% NaCl solution with 2.8 mg/liter heparin and 5 mg/liter lidocaine) and then with chilled 4% paraformaldehyde (4°C) for 30 min. Perfused brains were transferred to 30% sucrose solution for 2 days, after which they were sectioned coronally at 30 μm thickness. For analysis of excitatory and inhibitory synapses and inhibitory neuron cell type, the GFP signal in GABAergic neurons was amplified by immunofluorescence staining (chicken antibody to GFP, 1:1,000, Chemicon). GABAergic synapses were visualized by fluorescent labeling of VGAT (rabbit antibody to VGAT, 1:200, Synaptic Systems, Göttingen, Germany) and gephyrin (mouse antibody to gephyrin, 1:400, Synaptic Systems), as described previously (Wierenga et al., 2008). Glutmatergic synapses were visualized by fluorescent labeling of VGlut (rabbit α-VGlut1, 1:400, Synaptic Systems), as described previously (Becker et al., 2008).