ARTICLE IN PRESS Synaptic activity and F-actin coordinately regulate CaMKIIa localization to dendritic postsynaptic sites in developing hippocampal slices Raheel Ahmed, Xiang-ming Zha, Steven H. Green, and Michael E. Dailey * Department of Biological Sciences, 369 Biology Building, University of Iowa, Iowa City, IA 52242-1324, USA Received 3 June 2005; revised 6 August 2005; accepted 29 August 2005 We examined the timing and mechanisms of CaMKIIA recruitment to nascent synapses of developing rat hippocampal pyramidal neurons in slice culture. Time-lapse confocal imaging shows that GFP-CaMKIIA in transfected neurons accumulates in spines as they are forming, and loss of CaMKIIA coincides with spine destabilization. Immunolabeling shows that endogenous CaMKIIA is concentrated at postsynaptic sites in spines under ambient slice culture conditions, and this is not disrupted by short-term (3 h) synaptic activity blockade or Latruncu- lin-induced F-actin depolymerization. However, the combination of activity blockade and F-actin depolymerization significantly reduces synaptic CaMKIIA. Conversely, postsynaptic activation induces syn- aptic recruitment of CaMKIIA even in the presence of F-actin depolymerizing drugs. Thus, synaptic-activity-dependent mechanisms and (synaptic activity-independent) F-actin-based mechanisms are individually sufficient and act in parallel to localize CaMKIIA to the dendritic spine compartment. Moreover, the timing of CaMKIIA recruitment to developing spines suggests a role for CaMKIIA in spine assembly and maintenance. D 2005 Elsevier Inc. All rights reserved. Introduction CNS synaptogenesis involves an orchestrated assembly of proteins at postsynaptic sites (Ahmari et al., 2000; Hering and Sheng, 2001; Scannevin and Huganir, 2000; Zhai et al., 2001). One of the most abundant synaptic proteins, Ca 2+ /calmodulin- dependent protein kinase II (CaMKII), is concentrated at postsynaptic densities (PSDs) in spines (Kennedy et al., 1983), along with downstream substrates, including NMDA receptors (NMDA-Rs) and AMPA-receptors (AMPA-Rs) (Yoshimura et al., 2000). Expression of CaMKIIa is developmentally regulated, and CaMKIIa increase coincides with dendritic arbor stabiliza- tion and synapse formation (Kelly and Vernon, 1985; Scheetz et al., 1996; Wu and Cline, 1998; Zou and Cline, 1999). Spines appear to form through maturation of existing dendritic protrusive structures, such that some dynamic filopodia convert to intermediate protospines that eventually stabilize into mature spines bearing synapses (Dailey and Smith, 1996; Ziv and Smith, 1996). Concomitantly, PSDs form in or translocate into these dendritic protrusions (Marrs et al., 2001; Okabe et al., 2001; Prange and Murphy, 2001). The dynamics of CaMKIIa recruitment during synaptic spine formation and maturation remain unknown as are the roles of CaMKIIa in dendritic spine and synapse formation and remodeling in the mammalian CNS. In principle, dynamic changes in the subcellular localization of CaMKIIa during synaptic development and plasticity may regulate CaMKIIa activity and determine its downstream targets (Fink and Meyer, 2002; Kelly and Vernon, 1985; Shen et al., 2000). CaMKIIa accumulation at postsynaptic sites in hippo- campal neurons increases during long-term potentiation (LTP) induced by cAMP elevation in slice preparations (Otmakhov et al., 2004). Biochemical analysis and experimental observations in cell cultures suggest mechanisms by which CaMKIIa may be localized to synapses. In low-density dissociated hippocampal cultures, NMDA-R stimulation can induce translocation of GFP- CaMKIIa to ‘‘punctuate structures’’ along dendrites (Shen and Meyer, 1999), and biochemical studies indicate that postsynaptic CaMKIIa accumulation following NMDA-R stimulation can occur through direct binding to NMDA-R subunits, NR2B and NR1 (Leonard et al., 1999). F-actin in spines also can serve as docking sites for CaMKIIa by tethering CaMKIIa/h hetero- oligomers through interaction with CaMKIIh (Shen et al., 1998). F-actin-associated a-actinin and densin-180 also may anchor CaMKIIa within actin-rich spines (Walikonis et al., 2001). Thus, the respective roles of activity- and actin-dependent mechanisms of CaMKIIa localization to developing spine synapses in situ are still unclear. To investigate these questions, we have examined the spatio- temporal patterns of CaMKIIa localization during developmental 1044-7431/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2005.08.020 * Corresponding author. Fax: +1 319 335 1069. E-mail address: michael-e-dailey@uiowa.edu (M.E. Dailey). Available online on ScienceDirect (www.sciencedirect.com). www.elsevier.com/locate/ymcne YMCNE-01736; No. of pages: 15; 4C: 3, 4, 8 DTD 5 Mol. Cell. Neurosci. xx (2005) xxx – xxx