Primary antibodies were visualized with the appropriate secondary antibodies conjugated to either FITC or rhodamine (Jackson Immunoresearch Laboratories, West Grove, PA). Coverslips of fixed mouse neurons or rat neurons cotransfected with various GFP-tagged tau constructs and DsRed (or GFP alone) were photographed on an inverted Nikon epifluorescent microscope with a 60× oil lens and a computerized focus motor at 21–35 DIV. All digital images were photographed and processed

with MetaMorph Imaging System (Universal Imaging Corporation, West Chester, PA). All images of fixed and Z VAD FMK live neurons were taken as stacks (15 planes at 0.5 micron increments) and processed by deconvolution analyses using the MetaMorph software with the nearest planes and averaged into one single image. A dendritic protrusion with an expanded head that was 50% wider than its find more neck was defined as a spine. The number of spines from one neuron was counted manually and normalized per 100 μm dendritic length. To measure the dendritic fluorescence intensity of individual rat hippocampal neurons,

living neurons were photographed and processed with MetaMorph software as described above. Then, using Image J software (Image J 1.42q Software, National Institutes of Health, http://rsb.info.nih.gov/ij), the fluorescent pixel intensity along a user-defined line drawn at three different random positions across a GFP htau-transfected dendrite was measured as distance along the x axis plotted against pixel gray value on the y axis and expressed as area under a curve. The area under the curve above baseline was measured and represented as fluorescent pixel intensity using Image J software. To estimate the amount

of glutamate receptors in dendritic spines, fixed mouse neurons immunoreactive for PSD95 and a GluR antibody (N-GluR1, GluR1 detected with a C terminus antibody, GluR2/3, NR1) were photographed and processed with MetaMorph software as described above. Then, immunoreactive clusters of PSD95 were autoselected using the MetaMorph software and the location of these clusters was transferred to images displaying glutamate receptor immunoreactivity Carnitine palmitoyltransferase II on the same neuron. PSD95 immunoreactivity was used to identify dendritic spines. A cursor was placed in the center of the glutamate receptor clusters in dendritic spines to estimate glutamate receptor immunoreactivity as fluorescent pixel intensity in the spines (value Y1). Another cursor was placed in an adjacent dendritic shaft to measure glutamate receptor fluorescent pixel intensity (value Y2) and the ratio of glutamate receptor immunoreactive fluorescence intensity in spines/dendrites (Y1/Y2) was plotted on the y axis.