SPATIAL ORGANIZATION OF COFILIN IN DENDRITIC SPINES B. RACZ AND R. J. WEINBERG* Department of Cell and Developmental Biology, and Neuroscience Center, University of North Carolina, Chapel Hill, NC 27599, USA Abstract—Synaptic plasticity is associated with morpho- logical changes in dendritic spines. The actin-based cy- toskeleton plays a key role in regulating spine structure, and actin reorganization in spines is critical for the main- tenance of long term potentiation. To test the hypothesis that a stable pool of F-actin rests in the spine “core,” while a dynamic pool lies peripherally in its “shell,” we per- formed immunoelectron microscopy in the stratum radia- tum of rat hippocampus to elucidate the subcellular distri- bution of cofilin, an actin-depolymerizing protein that me- diates reorganization of the actin cytoskeleton. We provide direct evidence that cofilin in spines avoids the core, and instead concentrates in the shell and within the postsyn- aptic density. These data suggest that cofilin may link synaptic plasticity to the actin remodeling that underlies changes in spine morphology. © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: cytoskeleton, anatomy, synaptic plasticity, NMDA, actin, electron microscopy. Most glutamatergic fibers in the mammalian forebrain make synaptic contacts onto dendritic spines. Spines can move and change shape; this motility is activity-dependent in both the developing and mature brain, especially via gluta- mate acting at N-methyl-D-aspartate receptor (NMDARs, Fischer et al., 2000; Star et al., 2002). It was proposed more than 20 years ago that movement of dendritic spines mediated by reorganization of the actin cytoskeleton might play a role in memory (Crick, 1982; Fifkova and Delay, 1982; Matus et al., 1982). Considerable work since that time suggests that long-term synaptic plasticity is tightly coupled to actin remodeling (Fukazawa et al., 2003; Oka- moto et al., 2004). Actin remodeling in dendritic spines is mediated by a complex interplay among actin-binding pro- teins (dos Remedios et al., 2003; Carlisle and Kennedy, 2005; Ethell and Pasquale, 2005), but little is yet known about their organization. Actin in spines is highly dynamic, cycling between assembled filamentous (F-actin) and soluble monomeric globular (G-actin) forms (Star et al., 2002; Okamoto et al., 2004). Proteins of the actin-depolymerizing factor/cofilin family regulate the actin cycle, enhancing turnover of actin both by increasing the rate of depolymerization at filament ends, and by cutting long filaments into shorter ones (Car- lier and Pantaloni, 1997; Bamburg, 1999; Chen et al., 2000). Cofilin, a 20 kDa protein that binds cooperatively to two longitudinally-associated actin subunits of F-actin, is enriched at sites of motility associated with rapid actin reorganization, such as the leading edge and ruffling mem- branes of cultured cells, and in corresponding regions of neuronal growth cones (Gungabissoon and Bamburg, 2003; DesMarais et al., 2004, 2005); it is also expressed at high levels in adult brain (Moriyama et al., 1990). Recent work examining the relationship between long term de- pression (LTD) and spine shrinkage in hippocampal slices implicates cofilin also in this highly-specialized form of motility (Zhou et al., 2004). Phosphorylation inactivates cofilin, whose activity is tightly regulated by an interplay between (Lin-11/Isl-1/Mec- 3)-domain-containing protein kinase (LIMK), and the phos- phatase Slingshot (Meng et al., 2004; Wang et al., 2005). Cofilin has been identified as a downstream target for Rho family GTPases (Meyer and Feldman, 2002), known to exert a profound effect on spine morphology, at least in part via LIMK (Gungabissoon and Bamburg, 2003). LIMK is mutated in Williams syndrome, a genetic disease asso- ciated with mental retardation and visuo-spatial cognitive deficits (Frangiskakis et al., 1996; Bellugi et al., 1999), suggesting that dysregulation of neuronal cofilin may have clinical consequences. Cofilin has been detected in brain by immunoblot (Yon- ezawa et al., 1987; Shaw et al., 2004), but virtually nothing is yet known about its distribution within the brain. In the present study, we used immunocytochemistry to verify that cofilin is expressed in dendritic spines. Pharmacological experiments have led to the hypothesis that actin in the central core of spines is relatively stable, whereas that lying in its periphery or “shell” is highly dynamic (Halpain, 2000). Studies showing enhanced motility at the tip of the spine provide only weak support for this “two-pool” hypoth- esis, because of the limited resolution of light microscopy (Roelandse et al., 2003). Reasoning that cofilin provides a high-resolution probe for sites of dynamic actin, we here use immuno-electron microscopy to determine whether this actin binding protein concentrates in the shell as im- plied by the two-pool hypothesis. Our results reveal novel features of the internal organization of the spine. *Correspondence to: R. J. Weinberg, Department of Cell and Devel- opmental Biology, University of North Carolina, 108 Taylor Hall, CB#7090, Chapel Hill, NC 27599, USA. Tel: +1-919-966-1277; fax: +1-919-966-1856. E-mail address: rjw@med.unc.edu (R. J. Weinberg). Abbreviations: AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropi- onic acid; DAB, 3,3=-diaminobenzidine tetrahydrochloride; LIMK, (Lin- 11/Isl-1/Mec-3)-domain-containing protein kinase; LTD, long term de- pression; NDS, normal donkey serum; NMDAR, N-methyl-D-aspartate receptor; PB, phosphate buffer; PBS, phosphate-buffered saline; PFA, paraformaldehyde; PSD, postsynaptic density; VGLUT1, vesicular glu- tamate transporter type 1. Neuroscience 138 (2006) 447– 456 0306-4522/06$30.00+0.00 © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2005.11.025 447