IKK regulates the deubiquitinase CYLD at the postsynaptic density.

K63-linked polyubiquitination of proteins regulates their trafficking into specific cellular pathways such as endocytosis and autophagy. CYLD, a deubiquitinase specific for K63-linked polyubiquitins, is present in high quantities at the postsynaptic density (PSD). It was previously shown that, under excitatory conditions, CaMKII activates CYLD in a Ca(2+)-dependent manner. The observation that CYLD can also be phosphorylated in the absence of Ca(2+) in isolated PSDs led us to further explore the regulation of CYLD under basal conditions. A possible involvement of the autonomous form of CaMKII and IKK, both kinases known to be localized at the PSD, was examined. A CaMKII inhibitor CN21 had no effect on CYLD phosphorylation in the absence of Ca(2+), but two different IKK inhibitors, IKK16 and tatNEMO, inhibited its phosphorylation. Immuno-electron microscopy on hippocampal cultures, using an antibody for CYLD phosphorylated at S-418, revealed that the phosphorylated form of CYLD is present at the PSD under basal conditions. Phosphorylation of CYLD under basal conditions was inhibited by IKK16. NMDA treatment further promoted phosphorylation of CYLD at the PSD, but IKK16 failed to block the NMDA-induced effect. In vitro experiments using purified proteins demonstrated direct phosphorylation and activation of CYLD by the beta catalytic subunit of IKK. Activation of IKK in isolated PSDs also promoted phosphorylation of CYLD and an increase in endogenous deubiquitinase activity for K63-linked polyubiquitins. Altogether, the results suggest that in the absence of excitatory conditions, constitutive IKK activity at the PSD regulates CYLD and maintains basal levels of K63-linkage specific deubiquitination at the synapse.

NMDA-induced accumulation of Shank at the postsynaptic density is mediated by CaMKII.

Shank is a specialized scaffold protein present in high abundance at the postsynaptic density (PSD). Using pre-embedding immunogold electron microscopy on cultured hippocampal neurons, we had previously demonstrated further accumulation of Shank at the PSD under excitatory conditions. Here, using the same experimental protocol, we demonstrate that a cell permeable CaMKII inhibitor, tatCN21, blocks NMDA-induced accumulation of Shank at the PSD. Furthermore we show that NMDA application changes the distribution pattern of Shank at the PSD, promoting a 7-10 nm shift in the median distance of Shank labels away from the postsynaptic membrane. Inhibition of CaMKII with tatCN21 also blocks this shift in the distribution of Shank. Altogether these results imply that upon activation of NMDA receptors, CaMKII mediates accumulation of Shank, preferentially at the distal regions of the PSD complex extending toward the cytoplasm.

CYLD, a deubiquitinase specific for lysine63-linked polyubiquitins, accumulates at the postsynaptic density in an activity-dependent manner.

Polyubiquitin chains on proteins flag them for distinct fates depending on the type of polyubiquitin linkage. While lysine48-linked polyubiquitination directs proteins to proteasomal degradation, lysine63-linked polyubiquitination promotes different protein trafficking and is involved in autophagy. Here we show that postsynaptic density (PSD) fractions from adult rat brain contain deubiquitinase activity that targets both lysine48 and lysine63-linked polyubiquitins. Comparison of PSD fractions with parent subcellular fractions by Western immunoblotting reveals that CYLD, a deubiquitinase specific for lysine63-linked polyubiquitins, is highly enriched in the PSD fraction. Electron microscopic examination of hippocampal neurons in culture under basal conditions shows immunogold label for CYLD at the PSD complex in approximately one in four synapses. Following depolarization by exposure to high K+, the proportion of CYLD-labeled PSDs as well as the labeling intensity of CYLD at the PSD increased by more than eighty percent, indicating that neuronal activity promotes accumulation of CYLD at the PSD. An increase in postsynaptic CYLD following activity would promote removal of lysine63-polyubiquitins from PSD proteins and thus could regulate their trafficking and prevent their autophagic degradation.

Modification of the synaptic cleft under excitatory conditions.

The synaptic cleft is the extracellular part of the synapse, bridging the pre- and postsynaptic membranes. The geometry and molecular organization of the cleft is gaining increased attention as an important determinant of synaptic efficacy. The present study by electron microscopy focuses on short-term morphological changes at the synaptic cleft under excitatory conditions. Depolarization of cultured hippocampal neurons with high K+ results in an increased frequency of synaptic profiles with clefts widened at the periphery (open clefts), typically exhibiting patches of membranes lined by postsynaptic density, but lacking associated presynaptic membranes (18.0% open clefts in high K+ compared to 1.8% in controls). Similarly, higher frequencies of open clefts were observed in adult brain upon a delay of perfusion fixation to promote excitatory/ischemic conditions. Inhibition of basal activity in cultured neurons through the application of TTX results in the disappearance of open clefts whereas application of NMDA increases their frequency (19.0% in NMDA vs. 5.3% in control and 2.6% in APV). Depletion of extracellular Ca2+ with EGTA also promotes an increase in the frequency of open clefts (16.6% in EGTA vs. 4.0% in controls), comparable to that by depolarization or NMDA, implicating dissociation of Ca2+-dependent trans-synaptic bridges. Dissociation of transsynaptic bridges under excitatory conditions may allow perisynaptic mobile elements, such as AMPA receptors to enter the cleft. In addition, peripheral opening of the cleft would facilitate neurotransmitter clearance and thus may have a homeostatic and/or protective function.

Trafficking of AMPA receptors at plasma membranes of hippocampal neurons.

The number of AMPA receptors at synapses depends on receptor cycling. Because receptors diffuse rapidly in plasma membranes, their exocytosis and endocytosis need not occur near synapses. Here, pre-embedding immunogold electron microscopy is applied to dissociated rat hippocampal cultures to provide sensitive, high-resolution snapshots of the distribution of surface AMPA receptors in spines, dendrites, and cell bodies that will be informative about trafficking of AMPA receptors. The density of the label for GluR2 varies, but is consistent throughout cell body and dendrites in each individual neuron, except at postsynaptic densities (PSDs), where it is typically higher. Glutamate receptor 2 (GluR2) labels at PSDs significantly increase after synaptic activation by glycine treatment and increase further upon depolarization by high K(+). Islands of densely packed labels have consistent size and density but vary in frequency under different experimental conditions. These patches of label, which occur on plasma membranes of cell bodies and dendrites but not near PSDs, are taken to be the aftermath of exocytosis of AMPA receptors. A subpopulation of clathrin-coated pits in cell bodies and dendrites label for GluR2, and the number and amount of label in individual pits increase after NMDA treatment. Coated pits near synapses typically lack GluR2 label under basal conditions, but ∼40% of peri-PSD pits label for GluR2 after NMDA treatment. Thus, exocytosis and endocytosis of AMPA receptors occur mainly at extrasynaptic locations on cell bodies and dendrites. Receptors are not preferentially exocytosed near PSDs, but may be removed via endocytosis at peri-PSD locations after activation of NMDA receptors.

Optimization of protocols for pre-embedding immunogold electron microscopy of neurons in cell cultures and brains.

Immunogold labeling allows localization of proteins at the electron microscopy (EM) level of resolution, and quantification of signals. The present paper summarizes methodological issues and experiences gained from studies on the distribution of synaptic and other neuron-specific proteins in cell cultures and brain tissues via a pre-embedding method. An optimal protocol includes careful determination of a fixation condition for any particular antibody, a well-planned tissue processing procedure, and a strict evaluation of the credibility of the labeling. Here, tips and caveats on different steps of the sample preparation protocol are illustrated with examples. A good starting condition for EM-compatible fixation and permeabilization is 4% paraformaldehyde in PBS for 30 min at room temperature, followed by 30 min incubation with 0.1% saponin. An optimal condition can then be readjusted for each particular antibody. Each lot of the secondary antibody (conjugated with a 1.4 nm small gold particle) needs to be evaluated against known standards for labeling efficiency. Silver enhancement is required to make the small gold visible, and quality of the silver-enhanced signals can be affected by subsequent steps of osmium tetroxide treatment, uranyl acetate en bloc staining, and by detergent or ethanol used to clean the diamond knife for cutting thin sections. Most importantly, verification of signals requires understanding of the protein of interest in order to validate for correct localization of antibodies at expected epitopes on particular organelles, and quantification of signals needs to take into consideration the penetration gradient of reagents and clumping of secondary antibodies.

Stimulation-induced differential redistributions of clathrin and clathrin-coated vesicles in axons compared to soma/dendrites.

Clathrin-mediated endocytosis plays an important role in the recycling of synaptic vesicle in presynaptic terminals, and in the recycling of transmitter receptors in neuronal soma/dendrites. The present study uses electron microscopy (EM) and immunogold EM to document the different categories of clathrin-coated vesicles (CCV) and pits (CCP) in axons compared to soma/dendrites, and the depolarization-induced redistribution of clathrin in these two polarized compartments of the neuron. The size of CCVs in presynaptic terminals (~ 40 nm; similar to the size of synaptic vesicles) is considerably smaller than the size of CCVs in soma/dendrites (~ 90 nm). Furthermore, neuronal stimulation induces an increase in the number of CCV/CCP in presynaptic terminals, but a decrease in soma/dendrites. Immunogold labeling of clathrin revealed that in presynaptic terminals under resting conditions, the majority of clathrin molecules are unassembled and concentrated outside of synaptic vesicle clusters. Upon depolarization with high K+, label for clathrin became scattered among de-clustered synaptic vesicles and moved closer to the presynaptic active zone. In contrast to axons, clathrin-labeled CCVs and CCPs were prominent in soma/dendrites under resting conditions, and became inconspicuous upon depolarization with high K+. Thus, EM examination suggests that the regulation and mechanism of clathrin-mediated endocytosis differ between axon and dendrite, and that clathrin redistributes differently in these two neuronal compartments upon depolarization.

Activity-dependent redistribution of CaMKII in the postsynaptic compartment of hippocampal neurons.

Calcium/calmodulin-dependent protein kinase II (CaMKII), an abundant protein in neurons, is involved in synaptic plasticity and learning. CaMKII associates with multiple proteins located at or near the postsynaptic density (PSD), and CaMKII is known to translocate from cytoplasm to PSD under excitatory conditions. The present study examined the laminar distribution of CaMKII at the PSD by immunogold labeling in dissociated hippocampal cultures under low calcium (EGTA or APV), control, and stimulated (depolarization with high K+ or NMDA) conditions. The patterns of CaMKII distribution are classified with particular reference to the two layers of the PSD: (1) the PSD core, a layer within ~ 30-40 nm to the postsynaptic membrane, and (2) the PSD pallium, a deeper layer beyond the PSD core, ~ 100-120 nm from the postsynaptic membrane. Under low calcium conditions, a subpopulation (40%) of synapses stood out with no CaMKII labeling at the PSD, indicating that localization of CaMKII at the PSD is sensitive to calcium levels. Under control conditions, the majority (~ 60-70%) of synapses had label for CaMKII dispersed evenly in the spine, including the PSD and the nearby cytoplasm. Upon stimulation, the majority (60-75%) of synapses had label for CaMKII concentrated at the PSD, delineating the PSD pallium from the cytoplasm. Median distance of label for CaMKII to postsynaptic membrane was higher in low calcium samples (68-77 nm), than in control (59-63 nm) and stimulated samples (49-53 nm). Thus, upon stimulation, not only more CaMKII translocated to the PSD, but they also were closer to the postsynaptic membrane. Additionally, there were two relatively infrequent labeling patterns that may represent intermediate stages of CaMKII distribution between basal and stimulated conditions: (1) one type showed label preferentially localized near the PSD core where CaMKII may be binding to NR2B, an NMDA receptor concentrated at the PSD core, and (2) the second type showed label preferentially in the PSD pallium, where CaMKII may be binding to Shank, a PSD scaffold protein located in the PSD pallium. Both of these distribution patterns may portray the initial stages of CaMKII translocation upon synaptic activation. In addition to binding to PSD proteins, the concentrated CaMKII labeling at the PSD under heightened excitatory conditions could also be formed by self-clustering of CaMKII molecules recruited to the PSD. Most importantly, these accumulated CaMKII molecules do not extend beyond the border of the PSD pallium, and are likely held in the pallium by binding to Shank under these conditions.

Immunogold labeling of synaptic vesicle proteins in developing hippocampal neurons.

Synaptic vesicles (SV) contain high concentrations of specific proteins. How these proteins are transported from soma to synapses, and how they become concentrated at SV clusters at presynaptic terminals were examined by immunogold electron microscopy in dissociated rat hippocampal neurons at 3-6 days in culture, a developmental stage when axonal transport of SV proteins is robust. In neuronal somas, labels for the SV integral membrane proteins (synaptophysin, SV2, VAMP/synaptobrevin, and synaptotagmin) were localized at Golgi complexes and other membranous structures that were dispersed in the cytoplasm as individual vesicle/vacuoles. These vesicles/vacuoles became aggregated in axons, with the size of aggregates ranging from 0.2 to 2 µm in length. Pleomorphic vesicle/vacuoles within the aggregate were typically larger (50-300 nm) than SVs, which were uniform in size at ~ 40 nm. These pleomorphic vesicles/vacuoles are probably transport cargos carrying SV integral membrane proteins from the soma, and then are preferentially sorted into axons at early developmental stages. Serial thin sections of young axons indicated that many labeled aggregates were not synaptic, and in fact, some of these axons were without dendritic contacts. In contrast, labels for two SV-associated proteins, synapsin I and α-synuclein, were not localized at the Golgi complexes or associated with membranous structures in the soma, but were dispersed in the cytoplasm. However, these SV-associated proteins became highly concentrated on clusters of SV-like vesicles in axons, and such clusters were already distinctive in axons as early as 3 days in culture. These clusters consisted of ~ 4-30 vesicles in single thin sections, and the vesicles were of a uniform size (~ 40 nm). Serial sectioning analysis showed that these clusters could be part of nascent synapses or exist in axons without any dendritic contact. Importantly, the vesicles were intensely labeled for SV integral membrane proteins as well as SV-associated proteins. Thus, these EM observations reveal that the two groups of proteins, SV integral membrane and SV-associated, proceed through different routes of biosynthesis and axon transport, and are only sorted into the same final compartment, SV clusters, when they are in the axons.

Stimulation induces gradual increases in the thickness and curvature of postsynaptic density of hippocampal CA1 neurons in slice cultures.

Activity can induce structural changes in glutamatergic excitatory synapses, including increase in thickness and curvature of the postsynaptic density (PSD); these structural changes can only be documented by electron microscopy. Here in organotypic hippocampal slice cultures where experimental conditions can be easily manipulated, increases in thickness and curvature of PSDs were noticeable within 30 s of stimulation and progressed with time up to 3 min. These structural changes were reversible upon returning the samples to control medium for 5-10 min. Thus, the postsynaptic density is a very dynamic structure that undergoes rapid reorganization of its components upon stimulation, and recovery upon cessation of stimulation. The gradual increase in thickness of PSD could result from a gradual translocation of some PSD proteins to the PSD, and the increase in curvature of the PSD is likely led by postsynaptic elements.

Postsynaptic densities fragment into subcomplexes upon sonication.

Postsynaptic density (PSD) fractions were isolated from rat forebrain and sonicated. Pellets from sonicated samples examined by electron microscopy revealed particles with an electron density similar to PSDs that appeared to be fragments of PSDs. Immuno-gold labeling confirmed that some of these contained PSD-95 and/or SynGAP. Biochemical analysis of supernatant and pellet fractions from sonicated samples showed almost complete recovery of several major PSD components (SynGAP, PSD-95, Shank3, Homer and Glutamate receptors) in the pellet, while the supernatant contained known contaminants of PSD fractions, such as glial acidic fibrillary protein and neurofilament protein, as well as actin and α-actinin, indicating susceptibility of these cytoskeletal elements to mechanical disruption. Size distributions of particulate material in control and sonicated samples were clearly different, with particles in the 40-90 nm range observed only in sonicated samples. Fragmentation of the PSD into subcomplexes containing major constituents suggests a patchwork structure consisting of weakly bound modules, that can be readily dissociated from each other through mechanical disruption. Modular organization and weak association between modules would endow the PSD with lateral structural flexibility.

FAM81A protein, a novel component of the postsynaptic density in adult brain.

Analysis of affinity-purified PSD-95 complexes had previously identified a 'hypothetical protein', product of the gene FAM81A [1]. The present study examined the tissue and subcellular distribution of FAM81A protein and its expression levels during development. Comparison of different organs indicates selective expression of FAM81A protein in brain. FAM81A is expressed late in development, with a post-natal gradual increase in brain levels that parallels the expression of PSD-95. Comparison of subcellular fractions from adult brain shows that the distribution of FAM81A protein is similar to that of PSD-95, with a drastic enrichment in the postsynaptic density fraction. Immuno-electron microscopy of adult brain tissue reveals specific immunogold labeling for FAM81A protein at postsynaptic densities in the forebrain. The label for FAM81A protein is concentrated at the cytoplasmic edge of the electron-dense core of the postsynaptic density, with a mean distance of ∼33 nm from the postsynaptic membrane. These observations firmly establish FAM81A protein as a component of the postsynaptic density in the adult brain, suggesting a role in synaptic function.

Stimulation-induced structural changes at the nucleus, endoplasmic reticulum and mitochondria of hippocampal neurons.

Neurons exhibit stimulation-induced ultrastructural changes such as increase of thickness and curvature of the postsynaptic density, decrease in contact area between subsurface cistern and plasma membrane, and formation of CaMKII clusters and synaptic spinules. These structural characteristics help in identifying the activity state of the neuron and should be taken into consideration when interpreting ultrastructural features of the neurons. Here in organotypic hippocampal slice cultures where experimental conditions can be easily manipulated, two additional features are documented in forebrain neurons as reliable benchmarks for stimulation-induced structural changes: (1) The neuronal nucleus showed conspicuous clustering of dark chromatin, and (2) the endoplasmic reticulum formed stacks with a uniform gap of ~ 13 nm filled with dark materials. Both structural changes progressed with time and were reversible upon returning the slice cultures to control medium. These stimulation-induced structural changes were also verified in dissociated hippocampal neuronal cultures and perfusion-fixed brains. In hippocampal slice cultures, the neuronal chromatin clustering was detectable within 30 s of depolarization with high K+ (90 mM) or treatment with NMDA (50 µM). In contrast, the formation of ER cisternal stacks did not become apparent for another 30 s. Importantly, in dissociated neuronal cultures, when the extracellular calcium was chelated by EGTA, treatment with high K+ no longer induced these changes. These results indicate that the stimulation-induced chromatin clustering and formation of ER stacks in neurons are calcium-dependent. Additionally, mitochondria in neuronal somas of tissue culture samples consistently became swollen upon stimulation. However, swollen mitochondria were also present in some neurons of control samples, but could be eliminated by blocking basal activity or calcium influx. This calcium-dependent structural change of mitochondria is specific to neurons. These structural changes may bring insights to the neuron's response to intracellular calcium rise upon stimulation.

Activity-dependent decrease in contact areas between subsurface cisterns and plasma membrane of hippocampal neurons.

Subsurface cistern (SSC) in neuronal soma and primary dendrites is a specialized compartment of endoplasmic reticulum (ER) that is in close apposition (10 nm) with the plasma membrane (PM). ER-PM contact areas are thought to be involved in intracellular calcium regulation. Here, structural changes of SSC in hippocampal neurons were examined by electron microscopy upon depolarization with high K+ (90 mM) or application of NMDA (50 µM) in rat dissociated cultures as well as organotypic slice cultures. The number and average length of SSC-PM contact areas in neuronal somas significantly decreased within 30 s under excitatory condition. This decrease in SSC-PM contact area progressed with time and was reversible. These results demonstrate a structural decoupling between the SSC and the PM upon stimulation, suggesting that there may be a functional decoupling of the calcium regulation. Because SSC-PM contact areas may mediate calcium influx, the decrease in contact area may protect neurons from calcium overload upon heightened stimulation.

Distribution of densin in neurons.

Densin is a scaffold protein known to associate with key elements of neuronal signaling. The present study examines the distribution of densin at the ultrastructural level in order to reveal potential sites that can support specific interactions of densin. Immunogold electron microscopy on hippocampal cultures shows intense labeling for densin at postsynaptic densities (PSDs), but also some labeling at extrasynaptic plasma membranes of soma and dendrites and endoplasmic reticulum. At the PSD, the median distance of label from the postsynaptic membrane was ~27 nm, with the majority of label (90%) confined within 40 nm from the postsynaptic membrane, indicating predominant localization of densin at the PSD core. Depolarization (90 mM K+ for 2 min) promoted a slight shift of densin label within the PSD complex resulting in 77% of label remaining within 40 nm from the postsynaptic membrane. Densin molecules firmly embedded within the PSD may target a minor pool of CaMKII to substrates at the PSD core.

Neural precursor cells form integrated brain-like tissue when implanted into rat cerebrospinal fluid.

There is tremendous interest in transplanting neural precursor cells for brain tissue regeneration. However, it remains unclear whether a vascularized and integrated complex neural tissue can be generated within the brain through transplantation of cells. Here, we report that early stage neural precursor cells recapitulate their seminal properties and develop into large brain-like tissue when implanted into the rat brain ventricle. Whereas the implanted cells predominantly differentiated into glutamatergic neurons and astrocytes, the host brain supplied the intact vasculature, oligodendrocytes, GABAergic interneurons, and microglia that seamlessly integrated into the new tissue. Furthermore, local and long-range axonal connections formed mature synapses between the host brain and the graft. Implantation of precursor cells into the CSF-filled cavity also led to a formation of brain-like tissue that integrated into the host cortex. These results may constitute the basis of future brain tissue replacement strategies.

Structural changes at synapses after delayed perfusion fixation in different regions of the mouse brain.

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.

A novel synaptic junction preparation for the identification and characterization of cleft proteins.

Identification of synaptic cleft components has been hampered by the lack of a suitable preparation enriched in synaptic junctions devoid of adjoining peripheral membranes. Prior strategies for the isolation of synaptic junctions, relying on detergents for the removal of peripheral membranes, resulted in substantial loss of membranes lining the cleft. Here, a novel, detergent-free method is described for the preparation of a synaptic junction (SJ) fraction, using phospholipase A2. Limited digestion of synaptic plasma membrane (SPM) fraction with phospholipase A2 followed by centrifugation over a sucrose cushion results in selective removal of membranes peripheral to the cleft while junctional membranes remain relatively intact as observed by electron microscopy. Enrichment in synaptic junctional structures and loss of membranes peripheral to the junctional area are further verified by demonstrating enrichment in PSD-95 and loss in mGluR5, respectively. The SJ fraction is enriched in neuroligins and neurexins, in agreement with immuno-electron microscopy data showing their selective localization to the junctional area. Among additional cell adhesion molecules tested, N-cadherin and specific isoforms of the SynCAM and SALM families also show marked enrichment in the SJ fraction, suggesting preferential localization at the synaptic cleft while others show little enrichment or decrease, suggesting that they are not restricted to or concentrated at the synaptic cleft. Treatment of the SJ fraction with glycosidases results in electrophoretic mobility shifts of all cell adhesion molecules tested, indicating glycosylation at the synaptic cleft. Biochemical and ultrastructural data presented indicate that the novel synaptic junction preparation can be used as a predictive tool for the identification and characterization of the components of the synaptic cleft.

IRSp53 accumulates at the postsynaptic density under excitatory conditions.

IRSp53 (BAIAP2) is an abundant protein at the postsynaptic density (PSD) that binds to major PSD scaffolds, PSD-95 and Shanks, as well as to F-actin. The distribution of IRSp53 at the PSD in cultured hippocampal neurons was examined under basal and excitatory conditions by immuno-electron microscopy. Under basal conditions, label for IRSp53 is concentrated at the PSD. Upon depolarization by application of a medium containing 90 mM K+, the intensity of IRSp53 label at the PSD increased by 36±7%. Application of NMDA (50 µM) yielded 53±1% increase in the intensity of IRSp53 label at the PSD compared to controls treated with APV, an NMDA antagonist. The accumulation of IRSp53 label upon application of high K+ or NMDA was prominent at the deeper region of the PSD (the PSD pallium, lying 40-120 nm from the postsynaptic plasma membrane). IRSp53 molecules that accumulate at the distal region of the PSD pallium under excitatory conditions are too far from the plasma membrane to fulfill the generally recognized role of the protein as an effector of membrane-bound small GTPases. Instead, these IRSp53 molecules may have a structural role organizing the Shank scaffold and/or linking the PSD to the actin cytoskeleton.

The Postsynaptic Density: There Is More than Meets the Eye.

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.