A role for casein kinase 2a in the Drosophila circadian clock

Abstract

). The number and location of dendritic protrusions (protrusion length was more than one-third of dendritic shaft diameter) were identified in each view without prior knowledge of the animal’s age, the interval between views or the order of the views. The total number of spines (N) was pooled from dendritic segments of different animals. Data throughout the text are presented as means^ s:d: Filopodia were identified as long thin structures (generally larger than twice the average spine length, ratio of head diameter to neck diameter ,1.2:1 and ratio of length to neck diameter .3:1). The remaining protrusions were classified as spines. Three-dimensional stacks were used to ensure that tissue movements and rotation between imaging intervals did not influence spine identification. Spines or filopodia were considered the same between two views on the basis of their spatial relationship to adjacent landmarks and their relative position to immediately adjacent spines (Figs 2 and 3). Spines in the second view were considered different if they were more than 0.7 mm from their expected positions based on the first view. We chose 0.7 mm as a cut-off distance because apparent spine position can shift by up to ,0.3 mm in either direction along the axis of dendritic shafts owing to changes in spine morphology, slight tissue rotation and movements related to brain pulsation. We estimate the imaging resolution of our two-photon microscope (60 £ , numerical aperture 0.9, at 920 nm) to be ,0.7 mm. We found no significant changes in adult spine stability with three different cut-off distances (0.5, 0.7 and 0.9 mm), showing that our conclusion of spine stability is not dependent on minor changes in these criteria. Even though slight rotations of shafts and protrusions could either obscure existing spines (suggesting spine elimination) or reveal previously hidden spines (suggesting spine addition), such rotational artefacts would only underestimate the amount of stability observed. For quantification of changes in spine morphology, we minimized the possibility of rotational artefacts by preselecting from three-dimensional image stacks only spines parallel to the imaging plane in both views. Dendrites containing saturated pixels were excluded. Imaging software (NIH ImageJ) was used to measure the spine length and head diameter of identical spines between views. The edges of spines were detected with a Sobel detector algorithm (ImageJ). Curve fitting was done with the user-defined fitting function on Igor Pro (WaveMetrics, Lake Oswego, Oregon, USA). The single-exponential function was fitted to the data by using the built-in iterative Levenberg‐Marquardt function to minimize x 2