Analysis Of Actin Turnover And Spine Dynamics In Hippocampal Slice Cultures

Activity-dependent remodeling of dendritic spines is essential for neural circuit development and synaptic plasticity. Indeed, a tight correlation has been described between activity-dependent synaptic plasticity and structural plasticity in the CNS. Actin is highly enriched within spine heads, and changes in actin cytoskeleton dynamics have been show to mediate changes in the size and shape of spines. However, most studies so far either analyzed the structural changes and the role of the actin cytoskeleton separately or inferred the spine shape changes from changes in fluorescently labeled actin. The latter approach leaves open the possibility that actin changes in spines occur independently from the morphological changes. The method we present here involves the preparation of an adequate in vitro model allowing the transfection of single pyramidal neurons using single-cell electroporation. Moreover, it allows the simultaneous time-lapse imaging of spine size/shape using a membrane-targeted or cytoplasmatic fluorochrome and monitoring of actin dynamics using fluorescence recovery after photobleaching for eGFP tagged actin. Applying this protocol provides the possibility to simultaneously image dendritic spine shape and actin and thereby directly connect these two phenomena at the level of individual spines.