, 2007). In each section, stacks of ∼1.5 mm × 1.5 mm × 0.05 mm were imaged using optimized mosaic optical-sectioning microscopy (Oberlaender et al., 2009) and an oil-immersion objective

(Olympus 100× UPLAN S APO, NA 1.4), yielding a voxel size of 0.184 μm × 0.184 μm × 0.5 μm. Manual postprocessing of individual sections (Dercksen et al., 2012), as well as automated alignment of reconstructed branches across sections (Dercksen et al., 2009), was performed using a custom-designed three-dimensional (3D) editing environment based on ZIBamira visualization software v2010.06 (Zuse Institute Berlin). Pia and barrel outlines were manually traced in each section at low resolution (Olympus 4× UPLAN S APO, NA 0.16) and added to the tracings in Neurolucida software (MicroBrightfield). Reconstructions INCB28060 ic50 were placed into a standardized coordinate system. The origin was defined as the center of the L4 barrel that INK1197 molecular weight contained the majority of the neuron’s axon (“the principal barrel”). The z axis was set to point dorsally, parallel to the vertical axis of the principal barrel. The x axis was defined as the line joining the centers of the principal barrel and the first rostrally neighboring barrel within the same row. Measurements were performed in ZIBamira and double checked in NeuroExplorer v9.03 (MicroBrightfield). Axon length per

individual column was determined by extrapolating the respective L4 barrel contours, rather than idealized barrels, along the vertical axis toward the pia and white matter. Supragranular, granular, and infragranular projections (i.e., above, within, and below the principal barrel) were measured for each column individually because barrel height

varied between columns. Average interbouton distances were obtained from high-resolution image stacks (100× objective, 0.2 μm optical sections). Horizontally projecting axonal segments were randomly selected for analysis because varicosities are difficult to unambiguously identify when an axon travels along the optical axis (vertically). Interbouton distances were determined by manually marking the 3D location of each bouton along the reconstructed axons. much Custom-written ZIBamira routines were used to measure distance along the axons between these markers. Measurements were performed for 1,835 boutons from axonal segments in ten different rats (six control and four deprived). IgorPro (WaveMetrics) was used for statistical analysis of morphological data. All reconstructions, analyses, and bouton counting were performed double blind (i.e., control and deprived groups were only known after reconstructions and analyses were finalized). An additional 11 adult rats were used for physiology experiments. Prior to surgery, whiskers were trimmed (n = 6) or sham trimmed (n = 5) for 8–25 days. Rats were initially anesthetized with isoflurane. A single craniotomy was made over a thin region of skull overlying the left barrel cortex (0.2 × 1.0 mm; centered 2.5 mm posterior to bregma and 5.