Methods for three-dimensional analysis of dendritic spine dynamics.

Dendritic spines are small bulbous expansions that receive input from a single excitatory synapse. Although spines are often characterized by a mushroom-like morphology, they come in a wide range of sizes and shapes, even within the same dendrite. In a developing brain, spines exhibit a high degree of structural and functional plasticity, reflecting the formation and elimination of synapses during the maturation of neuronal circuits. The morphology of spines in developing neurons is affected by synaptic activity, hence contributing to the experience-dependent refinement of neuronal circuits, learning, and memory. Thus, understanding spine dynamics and its regulation is of central importance to studies of synaptic plasticity in the brain. The challenge has been to develop a computer-based assay that will quantitatively assess the three-dimensional change in spine movements caused by various stimuli and experimental conditions. Here, we provide detailed protocols for cell plating, transient transfections, and time-lapse imaging of dendritic spines. For the analysis of dendritic spine dynamics, we present two methods based on quantitative three-dimensional measurements.