Calcium/calmodulin-dependent protein kinase II (CaMKII) is a leading candidate for a synaptic memory molecule because it is persistently activated after long-term potentiation (LTP) induction and because mutations that block this persistent activity prevent LTP and learning. Previous work showed that synaptic stimulation causes a rapidly reversible translocation of CaMKII to the synaptic region. We have now measured green fluorescent protein (GFP)-CaMKII␣ translocation into synaptic spines during NMDA receptor-dependent chemical LTP (cLTP) and find that under these conditions, translocation is persistent. Using red fluorescent protein as a cell morphology marker, we found that there are two components of the persistent accumulation. cLTP produces a persistent increase in spine volume, and some of the increase in GFP-CaMKII␣ is secondary to this volume change. In addition, cLTP results in a dramatic increase in the bound fraction of GFP-CaMKII␣ in spines. To further study the bound pool, immunogold electron microscopy was used to measure CaMKII␣ in the postsynaptic density (PSD), an important regulator of synaptic function. cLTP produced a persistent increase in the PSD-associated pool of CaMKII␣. These results are consistent with the hypothesis that CaMKII␣ accumulation at synapses is a memory trace of past synaptic activity.

Postsynaptic densities (PSDs) contain proteins that regulate synaptic transmission. We determined the positions of calcium/calmodulin-dependent protein kinase II (CaMKII) and PSD-95 within the three-dimensional structure of isolated PSDs using immunogold labeling, rotary shadowing, and electron microscopic tomography. The results show that all PSDs contain a central mesh immediately underlying the postsynaptic membrane. Label for PSD-95 is found on both the cytoplasmic and cleft sides of this mesh, averaging 12 nm from the cleft side. All PSDs label for PSD-95. The properties of CaMKII labeling are quite different. Label is virtually absent on the cleft sides of PSDs, but can be heavy on the cytoplasmic side at a mean distance of 25 nm from the cleft. In tomograms, CaMKII holoenzymes can be visualized directly, appearing as labeled, tower-like structures reflecting the 20 nm diameter of the holoenzyme. These towers protrude from the cytoplasmic side of the central mesh. There appears to be a local organization of CaMKII, as judged by fact that the nearest-neighbor distances are nearly invariant over a wide range of labeling density for CaMKII. The average density of CaMKII holoenzymes is highly variable, ranging from zero to values approaching a tightly packed state. This variability is significantly higher than that for PSD-95 and is consistent with an information storage role for CaMKII.

Depolarization of rat hippocampal neurons with a high concentration of external potassium induces a thickening of postsynaptic densities (PSDs) within 1.5-3 min. After high-potassium treatment, PSDs thicken 2.1-fold in cultured neurons and 1.4-fold in hippocampal slices compared with their respective controls. Thin-section immunoelectron microscopy of hippocampal cultures indicates that at least part of the observed thickening of PSDs can be accounted for by an accumulation of Ca 2؉ ͞calmodulin-dependent protein kinase II (CaMKII) on their cytoplasmic faces. Indeed, PSD-associated gold label for CaMKII increases 5-fold after depolarization with potassium. The effects of high-potassium treatment on the composition and structure of the PSDs are mimicked by direct application of glutamate. In cultures, glutamate-induced thickening of PSDs and the accumulation of CaMKII on PSDs are reversed within 5 min of removal of glutamate and Ca 2؉ from the extracellular medium. These results suggest that PSDs are dynamic structures whose thickness and composition are subject to rapid and transient changes during synaptic activity. P ostsynaptic densities (PSDs) are specialized protein complexes apposed to the postsynaptic membrane. These complexes contain and organize various components of the postsynaptic response to neurotransmitter release such as receptors and signal transduction molecules. A structural change in the PSD is likely to reflect an organizational change in the postsynaptic molecular machinery and, therefore, a functional modification of the synapse.PSDs can thicken after cerebral ischemia (1, 2) and in cultured hippocampal neurons when ischemic energy depletion is mimicked by the inclusion of a mitochondrial uncoupler in the absence of glucose (3). The thickening of PSDs is accompanied by an increase in certain proteins, including Ca 2ϩ ͞calmodulin-dependent protein kinase II (CaMKII), in the PSD fraction (1), suggesting that these may have accumulated on the PSD structure. Immunogold labeling of CaMKII in hippocampal cultures confirms that CaMKII accumulates on the PSD under ischemialike conditions (3). Ischemia is known to result in excessive glutamate release (4, 5), so it is possible that glutamate mediates the above-mentioned modification of PSDs.Early studies (6) revealed that PSDs in unanesthetized animals are thicker than those in anesthetized animals, suggesting an influence of synaptic activity on PSD thickness. Thickening of the PSD also occurs in conditions expected to promote activation of glutamate receptors, such as rhythmic stimulation of the dentate gyrus in the guinea pig hippocampus (7), combined application of glutamate and acetylcholine in the sensorimotor region of the rat cerebral cortex (8), or chronic N-methyl-Daspartate treatment of doubly innervated optic tecta in the frog (9). However, it is not clear whether the PSD thickening under these nonpathological conditions is also accompanied by an accumulation of CaMKII on the PSDs. Observations at the light microscopic level (10) do indi...

Postsynaptic density protein 95 (PSD-95), a specialized scaffold protein with multiple protein interaction domains, forms the backbone of an extensive postsynaptic protein complex that organizes receptors and signal transduction molecules at the synaptic contact zone. Large, detergentinsoluble PSD-95-based postsynaptic complexes can be affinity-purified from conventional PSD fractions using magnetic beads coated with a PSD-95 antibody. In the present study purified PSD-95 complexes were analyzed by LC/MS/MS. A semiquantitative measure of the relative abundances of proteins in the purified PSD-95 complexes and the parent PSD fraction was estimated based on the cumulative ion current intensities of corresponding peptides. The affinity-purified preparation was largely depleted of presynaptic proteins, spectrin, intermediate filaments, and other contaminants prominent in the parent PSD fraction. We identified 525 of the proteins previously reported in parent PSD fractions, but only 288 of these were detected after affinity purification. We discuss 26 proteins that are major components in the PSD-95 complex based upon abundance ranking and affinity co-purification with PSD-95. This subset represents a minimal list of constituent proteins of the PSD-95 complex and includes, in addition to the specialized scaffolds and Nmethyl-D-aspartate (NMDA) receptors, an abundance of ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, small G-protein regulators, cell adhesion molecules, and hypothetical proteins. The identification of two Arf regulators, BRAG1 and BRAG2b, as copurifying components of the complex implies pivotal functions in spine plasticity such as the reorganization of the actin cytoskeleton and insertion and retrieval of proteins to and from the plasma membrane. Another copurifying protein (Q8BZM2) with two sterile ␣ motif domains may represent a novel structural core element of the PSD.Molecular & Cellular Proteomics 6:1749 -1760, 2007. The postsynaptic density (PSD)1 is a disk-shaped protein complex lining the postsynaptic membrane. In a recent study its total mass was estimated to be around 1 million kDa (1). The function of this massive protein complex appears to be anchoring and organizing postsynaptic neurotransmitter receptors and corresponding signaling molecules at the active zone. Thus, it is expected that the extent and type of the postsynaptic response to neurotransmitter release will largely depend on the molecular composition and organization of the PSD.The first tentative identification of PSD components was done in the late 1970s when several laboratories developed the methodology to isolate PSD fractions and started analyzing them by biochemical methods (2, 3). The general strategy, still applied today, was treatment of synaptosomal fractions with detergents that solubilize membranes but leave the PSD relatively intact and subsequent separation of membrane-free PSDs by further centrifugation. Analysis of PSD fractions continued to reveal additional putative PSD components in l...

Cytoskeletal and postsynaptic density (PSD) fractions from forebrain contain discrete spherical structures that are immunopositive for Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Spherical structures viewed by rotary shadow electron microscopy have an average diameter of approximately 100 nm and, in distinction to postsynaptic densities, do not immunolabel for PSD-95. These structures were purified to near homogeneity by extraction with the detergent N-lauryl sarcosinate. Biochemical analysis revealed that CaMKII accounts for virtually all of the protein in the purified preparation, suggesting that spherical structures are clusters of self-associated CaMKII. Exposure of cultured hippocampal neurons to a mitochondrial uncoupler in glucose-free medium promotes the formation of numerous CaMKII-immunopositive structures identical in size and shape to the CaMKII clusters observed in subcellular fractions. Clustering of CaMKII would reduce its kinase function by preventing its access to fixed substrates. On the other hand, clustering would not affect the ability of the large cellular pool of CaMKII to act as a calmodulin sink, as demonstrated by the Ca(2+)-dependent binding of gold-conjugated calmodulin to CaMKII clusters. We propose that the observed clustering of CaMKII into spherical structures is a protective mechanism preventing excessive protein phosphorylation upon loss of Ca(2+) homeostasis, without compromising calmodulin regulation.

The postsynaptic density (PSD), apparent in electron micrographs as a dense lamina just beneath the postsynaptic membrane, includes a deeper layer, the “pallium”, containing a scaffold of Shank and Homer proteins. Though poorly defined in traditionally prepared thin-section electron micrographs, the pallium becomes denser and more conspicuous during intense synaptic activity, due to the reversible addition of CaMKII and other proteins. In this Perspective article, we review the significance of CaMKII-mediated recruitment of proteins to the pallium with respect to both the trafficking of receptors and the remodeling of spine shape that follow synaptic stimulation. We suggest that the level and duration of CaMKII translocation and activation in the pallium will shape activity-induced changes in the spine.

We recently showed by electron microscopy that the postsynaptic density (PSD) from hippocampal cultures undergoes rapid structural changes after ischemia-like conditions. Here we report that similar structural changes occur after delay in transcardial perfusion fixation of the mouse brain. Delay in perfusion fixation, a condition that mimics ischemic stress, resulted in 70%, 90%, and 23% increases in the thickness of PSDs from the hippocampus (CA1), cerebral cortex (layer III), and cerebellar cortex (Purkinje spines), respectively. In step with PSD thickening, the amount of PSD-associated alpha-calcium calmodulin-dependent protein kinase II (alpha- CaMKII) label increased more in cerebral cortical spines than in Purkinje spines. Although the Purkinje PSDs thickened only slightly after delayed fixation, they became highly curved, and many formed sub-PSD spheres approximately 80 nm in diameter that labeled for CaMKII. Delayed perfusion fixation also produced more cytoplamic CaMKII clusters ( approximately 110 nm in diameter) in the somas of pyramidal cells (from hippocampus and cerebral cortex) than in Purkinje cells. Thus a short delay in perfusion fixation produces cell-specific structural changes at PSDs and neuronal somas. Purkinje cells respond somewhat differently to delayed perfusion fixation, perhaps owing to their lower levels of CaMKII, and CaMKII binding proteins at PSDs. We present here a catalogue of structural changes that signal a perfusion fixation delay, thereby providing criteria by which to assess perfusion fixation quality in experimental structural studies of brain and to shed light on the subtle changes that occur in intact brain following metabolic stress.

Spinules found in brain consist of small invaginations of plasma membranes which enclose membrane evaginations from adjacent cells. Here, we focus on the dynamic properties of the most common type, synaptic spinules, which reside in synaptic terminals. In order to test whether depolarization triggers synaptic spinule formation, hippocampal slice cultures (7 day-old rats, 10–14 days in culture) were exposed to high K+ for 0.5–5 min, and examined by electron microscopy. Virtually no synaptic spinules were found in control slices representing a basal state, but numerous spinules appeared at both excitatory and inhibitory synapses after treatment with high K+. Spinule formation peaked with ~1 min treatment at 37 ° C, decreased with prolonged treatment, and disappeared after 1–2 min of washout in normal medium. The rate of disappearance of spinules was substantially slower at 4 °C. NMDA treatment also induced synaptic spinule formation, but to a lesser extent than high K+ depolarization. In acute brain slices prepared from adult mice, synaptic spinules were abundant immediately after dissection at 4ºC, extremely rare in slices allowed to recover at 28 °C, but frequent after high K+ depolarization. High pressure freezing of acute brain slices followed by freeze-substitution demonstrated that synaptic spinules are not induced by chemical fixation. These results indicate that spinules are absent in synapses at low levels of activity, but form and disappear quickly during sustained synaptic activity. The rapid turnover of synaptic spinules may represent an aspect of membrane retrieval during synaptic activity.