The function of dendritic spines, postsynaptic sites of excitatory input in the mammalian central nervous system (CNS), is still not well understood. Although changes in spine morphology may mediate synaptic plasticity, the extent of basal spine motility and its regulation and function remains controversial. We investigated spine motility in three principal neurons of the mouse CNS: cerebellar Purkinje cells, and cortical and hippocampal pyramidal neurons. Motility was assayed with time-lapse imaging by using two-photon microscopy of green fluorescent protein-labeled neurons in acute and cultured slices. In all three cell types, dendritic protrusions (filopodia and spines) were highly dynamic, exhibiting a diversity of morphological rearrangements over short (<1-min) time courses. The incidence of spine motility declined during postnatal maturation, but dynamic changes were still apparent in many spines in late-postnatal neurons. Although blockade or induction of neuronal activity did not affect spine motility, disruption of actin polymerization did. We hypothesize that this basal motility of dendritic protrusions is intrinsic to the neuron and underlies the heightened plasticity found in developing CNS. Dendritic spines are the major sites of excitatory input in mammalian central nervous system (CNS) neurons (1, 2), but their function is still not well understood. In recent years, dendritic spines have been shown to act as biochemical compartments (3–5) that could mediate synapse-specific plasticity (6, 7), perhaps through rapid changes in spine morphology (8). Indeed, recent studies have demonstrated that activity can elicit new spine-like protrusions (9, 10). Nevertheless, the question of whether spines are inherently motile remains controversial. In developing hippocampal neurons from both dissociated and slice cultures, dendritic filopodia, proposed to be precursors of mature spines, are highly dynamic (11, 12). However, after synapse formation in culture and in acute slices of mature hippocampus, spines appear to be relatively immotile (12, 13). In contrast, spines on hippocampal neurons in long-term cultures are extremely dynamic, even though these neurons bear synaptic contacts (14). To determine the extent of motility of dendritic spines, its developmental regulation and mechanisms, we imaged three major classes of spiny neurons of the CNS: cerebellar Purkinje neurons, and pyramidal neurons from cortex and hippocampus. Acute or cultured slices were transfected with enhanced green fluorescent protein DNA, expressed under a cytomegalovirus promoter (CMV-EGFP), by using biolistic gene transfer (15). Labeled cells were then imaged by using a custom-built two-photon microscope (16). We find that in the cells examined at mid- to late-postnatal stages, the majority of dendritic spines are motile structures over time scales as short as a minute, and that motility subsides, although does not disappear, with increasing age of the cell. Spines are motile in both cultured and acute cortical slices of the same chronological age, and this motility is actin based, suggesting that motility may occur in vivo. Finally, spine motility is surprisingly unaffected by global changes in activity. Our results imply that spine motility is an intrinsic feature of CNS neurons
