Dendritic spines are micron-sized membrane protrusions of neuronal dendrites that are the major sites of contact for glutamatergic presynaptic inputs in the mammalian central nervous system (Hering and Sheng, 2001
). Spines are motile and assume diverse shapes, which are correlated with synaptic strength (Matsuzaki et al., 2001
). Further, spine morphology is subject to rapid alteration by patterns of neuronal activity and postsynaptic glutamate receptor activation (Lang et al., 2004
; Matsuzaki et al., 2004
). Recent studies have found that new spines form and existing spines grow in response to stimuli that induce long-term potentiation (LTP) in the hippocampus (Engert and Bonhoeffer, 1999
; Maletic-Savatic et al., 1999
; Lang et al., 2004
; Matsuzaki et al., 2004
), the dominant cellular model for learning and memory (Malenka and Nicoll, 1999
). Such structural growth and expansion of dendritic spines has served as a paradigmatic cellular model for physical alteration of neural circuitry during learning-related plasticity. Along with spine growth, morphological changes associated with LTP include enlargement of the postsynaptic density (PSD) (Toni et al., 1999
), remodeling of spine actin (Okamoto et al., 2004
), redistribution of polyribosomes (Ostroff et al., 2002
) and mitochondria (Li et al., 2004
), and the appearance of intraspinous vesicles (Toni et al., 2001
). The latter observation points to the possibility for spatially regulated membrane trafficking events during spine growth. However, to date, emphasis has been placed on spine morphological plasticity through remodeling of the actin cytoskeleton (Tada and Sheng, 2006
). Interestingly, linkage of the actin cytoskeleton to the plasma membrane through the protein kinase C target MARCKS maintains spine morphology (Calabrese and Halpain, 2005
). However, the source of membrane for increased spine volume and new spine formation during LTP has been unknown.
Membrane for spine expansion could be provided by lateral plasma membrane of the dendritic shaft or by fusion of intracellular membrane compartments. Dendrites are rich in internal membrane compartments including extensive smooth endoplasmic reticulum (Spacek and Harris, 1997
), sparse Golgi elements (Horton et al., 2005
), and endosomes (Cooney et al., 2002
) whose function and molecular identity remain poorly characterized. In nonneuronal cells, membrane traffic through endosomes contributes to cell morphogenesis. In migrating chick embryo fibroblasts, the transferrin (Tf) receptor (TfR), a representative fast and constitutive recycling transmembrane protein, is loaded into perinuclear recycling endosomes and then trafficked to the surface of the leading lamella (Hopkins et al., 1994
). In addition, membrane accumulation for cleavage furrow growth during the terminal phase of cytokinesis in the early Caenorhabditis elegans embryo is dependent on Rab11 (Skop et al., 2001
), a small GTPase required for membrane transport from the recycling endosome to the plasma membrane (Ullrich et al., 1996
). Finally, disruption of the Drosophila exocyst components sec5, sec6, and sec15 in epithelial cells causes accumulation of E-cadherin in an enlarged recycling endosomal compartment and inhibits its delivery to the plasma membrane (Langevin et al., 2005
). Such studies suggest that cellular membrane microdomain composition and shape is governed by rates of endocytosis to and exocytosis from endosomes aligned through the endocytic recycling pathway.
At excitatory synapses on dendritic spines, postsynaptic membrane composition is subject to continuous regulation by endocytic cycling of neurotransmitter receptors (Ehlers, 2000
). Continuous endocytosis of postsynaptic molecules occurs at specialized endocytic zones on spines lateral to the PSD (Blanpied et al.,2002
). Given the small size of dendritic spines, endocytic or exocytic events could have a significant impact on spine size. Yet, in the absence of plasticity-inducing stimuli, most spines are structurally stable (Grutzendler et al., 2002
), suggesting precise spatial control over membrane recycling. Notably, recycling endosomes are highly dynamic in hippocampal neuron dendrites (Prekeris et al., 1999
). Moreover, ultrastructural analysis has revealed a widespread pool of recycling compartments and vesicles that serve multiple dendritic spines (Cooney et al., 2002
), potentially positioning endosomes to provide membrane and molecular material to specific spine microdomains.
During LTP, the number of AMPA-type glutamate receptors at the plasma membrane increases due to enhanced transport from recycling endosomes (Park et al., 2004
). This increase in postsynaptic AMPA receptors produces an increase in AMPA receptor-mediated transmission (Shi et al., 1999
). In addition, LTP-inducing stimuli increase the abundance of coated vesicles in spines (Toni et al., 2001
) and increase the rate of recycling endosome transport (Park et al., 2004
). These results point to the possibility that enhanced recycling not only ensures enhanced synaptic efficacy by providing AMPA receptors but also mediates spine growth and structural remodeling by supplying lipid membrane and other unknown proteins, thereby coupling membrane remodeling with synaptic potentiation. Yet, little is known about where the relevant endosomal compartments reside in dendrites and spines, whether exocytic events from recycling endosomes occur within spines, and how membrane trafficking from endosomes is regulated during plasticity-associated spine growth.
In this study, we have investigated the effect of recycling endosome transport on spine growth and maintenance and the regulation of such transport by LTP-inducing stimuli. Using a combination of live-cell imaging, electron microscopy, and direct visualization of exocytosis from dendritic endosomes, we demonstrate that transport from recycling endosomes bidirectionally regulates spine formation and loss. During LTP, recycling endosomes and endosomal vesicles are rapidly mobilized into spines. Disruption of recycling endosome transport leads to acute spine collapse and prevents LTP-induced spine formation. Exocytosis of cargo from recycling endosomes occurs locally within dendritic spines, is trigged by activation of synaptic NMDA receptors, and occurs concurrently with spine enlargement. These results demonstrate a novel requirement for intracellular membrane trafficking in spine morphogenesis, provide direct evidence for local exocytosis in spines, and identify recycling endosomes as the source of membrane material for activity-dependent spine growth. Activity-dependent establishment of local endosomal recycling provides a potential mechanism linking structural and functional plasticity of highly compartmentalized dendritic spines.