We present new methods for segmentation of hippocampal dendritic spines in 3D confocal light microscopy and computation of 3-D morphological attributes characterizing dendritic spine plasticity. The methods are applied on 3D confocal light microscopy images of dendritic spines from dissociated hippocampal cultures. The segmentation method is based on the principle of multi-scale opening which uses a set of user-specified seeds and iteratively segments structures at different scales starting at a large scale and progressing toward finer scales. The accuracy of the segmentation method is evaluated by comparing its results with the gold standard obtained by manual labelling. The reproducibility of the overall method involving segmentation as well as computation of structural measures is assessed by comparing the values of structural measures derived from segmentation results generated using seeds from three mutually-blinded users. Finally, the performance of the overall method is examined in terms of its ability to characterize spine morphological changes after chemically induced long-term potentiation.