Subcellular Organization of CaMKII in Rat Hippocampal Pyramidal Neurons
Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a key role in N-methyl-D-aspartate (NMDA) receptor-dependent long-term synaptic plasticity; its location is critical for signal transduction, and may provide clues that further elucidate its function. We therefore examined the subcellular localization of CaMKII in CA1 stratum radiatum of adult rat hippocampus, by using immuno-electron microscopy after chemical fixation. When tissue was fixed quickly, the concentration of CaMKIIα (assessed by pre-embedding immunogold) was significantly higher in dendritic shafts than in spine heads. However, when tissue was fixed 5 minutes after perfusion with normal saline, the density of labeling decreased in dendritic shaft while increasing in spine heads, implying rapid translocation into the spine during brief perimortem stress. Likewise, in quickly fixed tissue, CaMKII within spine heads was found at comparable concentrations in the "proximal" half (adjacent to the spine neck) and the "distal" half (containing the postsynaptic density [PSD]), whereas after delayed fixation, label density increased in the distal side of the spine head, suggesting that CaMKII within the spine head moves toward the PSD during this interval. To estimate its distribution at the synapse in vivo, we performed postembedding immunogold staining for CaMKII in quick-fixed tissue, and found that the enzyme did not concentrate primarily within the central matrix of the PSD. Instead, labeling density peaked ∼40 nm inside the postsynaptic membrane, at the cytoplasmic fringe of the PSD. Labeling within 25 nm of the postsynaptic membrane concentrated at the lateral edge of the synapse. This lateral "PSD core" pool of CaMKII may play a special role in synaptic plasticity.
© 2013 Wiley Periodicals, Inc. Received March 5, 2013; Revised May 23, 2013; Accepted for publication May 23, 2013. Article first published online: 23 Aug. 2013. Grant sponsor: National Institutes of Health; grant numbers: P01 NS44306 (to M.B.K.) and R01 NS35527 (to R.J.W.). We thank Susan Burette and Kristen D. Phend for histological assistance; Alain Burette for confocal analysis of double-labeling immunofluorescence; and Alain Burette, Ayse Dosemeci, Johannes Hell, and Sridhar Raghavachari for comments on the manuscript text. Conflict of Interest Statement: The authors declare there is no conflict of interest. Role of Authors: All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: RJW, JDD, and MBK. Acquisition and analysis of data: JDD and RJW. Interpretation of data: JDD, MBK, and RJW. Drafting of the manuscript: JDD and RJW. Obtained funding to support the research: MBK and RJW.
Accepted Version - nihms681187.pdf