The Postsynaptic Density

16
Rochelle S. Cohen

Contents
Brief History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 476
The Postsynaptic Density Is a Dense Submembranous Structure Containing an Array
of Molecules Important in Synaptic Transmission and Plasticity . . . . . . . . . . . . . . . . . . . . . . . . . . 476
The Structure of the PSD Reflects Its Complex Molecular Organization . . . . . . . . . . . . . . . . . . 480
The Arrangement of Molecular Components of the PSD Mediates Communication
of Signaling Events in Space and Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487
Environment, Hormones, and Electrophysiological Input Influence PSD Structure . . . . . . . 502
The PSD in Neurological Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 503
Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 504
Future Directions Combine State-of-the Art Imaging Techniques, Electrophysiology,
Molecular Approaches, and Genetic Animal Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 504
Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509

Abstract
The postsynaptic density is a dense, submembranous filamentous structure
beneath the postsynaptic membrane. It is a disklike structure that may or may
not display perforations. The intimacy of its association with the postsynaptic
membrane is reflective of the distribution of some proteins, which are shared
between the PSD and postsynaptic membrane, e.g., receptors, ion channels, and
adhesion proteins. The PSD displays a complex array of proteins, which are
arranged in a hierarchical manner: (1) receptors, ion channels, and adhesion
proteins shared with the postsynaptic membrane; (2) scaffold proteins connecting

R.S. Cohen (*)

Department of Anatomy and Cell Biology (MC 512), University of Illinois at Chicago
College of Medicine, Chicago, IL, USA
e-mail: rscohen@uic.edu

# Springer Science+Business Media New York 2016 475

D.W. Pfaff, N.D. Volkow (eds.), Neuroscience in the 21st Century,
DOI 10.1007/978-1-4939-3474-4_17
476 R.S. Cohen

the receptors to each other, to other membrane components, and to the actin
cytoskeleton; and (3) the actin-based cytoskeleton, itself. The core structure of the
PSD appears to be the scaffold protein postsynaptic density-95 (PSD-95).
Another key component of the PSD is the enzyme Ca2+/calmodulin-dependent
protein kinase II (CaMKII), which is capable of autophosphorylation and, thus,
has been implicated in long-term processes. The PSD has been shown to undergo
morphological alterations in various parameters with behavioral, hormonal, and
electrophysiological inputs. The PSD is important in signal transduction events at
the synapse and may be involved in information storage.

Keywords
AMPA receptor (AMPAR) • CaMKII • Chemical LTP (cLTP) • MAGUK family
scaffolding proteins • Metabotropic glutamate receptors • NMDA receptor
(NMDAR) • Postsynaptic density (PSD) • PSD. See Postsynaptic density
(PSD) • Synapses

Brief History

The Postsynaptic Density Is a Dense Submembranous Structure

Containing an Array of Molecules Important in Synaptic
Transmission and Plasticity

As described in ▶ Chap. 14, “Cell Biology of the Synapse,” excitement was at a

pitch in the late 1950s when Sanford Palay first viewed the ultrastructure of the
synapse by electron microscopy. In addition to prominent synaptic vesicles,
Dr. Palay saw pre- and postsynaptic thickenings in close apposition to the pre- and
postsynaptic membranes, respectively. Subsequently, several investigators, includ-
ing Alan Peters, presented detailed ultrastructural views of the postsynaptic thick-
ening, which then became known as the postsynaptic density (PSD) (Fig. 1).
The PSD varies in thickness and diameter, and some have one or more perfora-
tions (Fig. 1). Other variations in PSD structure include differences in length,
curvature, and the presence of specializations underneath the PSDs; some of these
variations and their proposed significance are discussed below.
Attention to other aspects of the PSD lay dormant until the 1970s when the
enrichment and biochemical characterization of PSD fractions attracted the interest
of several groups, notably those of Philip Siekevitz, Carl Cotman, Andrew Matus
and James W. Gurd. The ability to isolate PSDs was based on subcellular fraction-
ation procedures developed earlier by Victor P. Whittaker and others, who were able
to obtain fractions enriched in nerve endings called synaptosomes. The synaptosome
consists of pinched-off pre- and postsynaptic elements, including synaptic vesicles
and the presynaptic membrane, as well as the attached postsynaptic membrane and
its underlying PSD.
16 The Postsynaptic Density 477

Fig. 1 An electron
micrograph of a section of a
synapse cut normal to the
plane of the synaptic junction.
Asterisks postsynaptic density
(PSD), s synaptic vesicles,
v vacuole. Double arrows
indicate a hole in the PSD and
the absence of cleft material at
this site. Single arrows
indicate postsynaptic cell
filamentous material attached
to the central mass of the PSD
(Adapted from Cohen RS,
Siekevitz P (1978) The form
of the post-synaptic density: a
serial section study. J Cell
Biol 78:35–46)

Lysing synaptosomes with water or dilute buffer released synaptic vesicles and
other internal presynaptic organelles. A synaptosomal membrane fraction (Fig. 2)
was then able to be separated from the vesicles and other presynaptic organelles of
the synaptosome fraction by density gradient centrifugation. Application of deter-
gents to the synaptosome or synaptosomal membrane fraction dissolved many of
their membranous elements, with important exceptions described below, and subse-
quent subcellular fractionation led to the enrichment of a PSD fraction.
The Siekevitz and Cotman, Matus, and Walter E. Mushynski groups used
non-ionic and ionic detergents, respectively, to obtain an enriched PSD (Fig. 3) or
synaptic junctional complex fraction. Caution is used with the word “isolate,” as
“pure” PSD fractions were unattainable. PSDs by their nature are sticky, and some
non-PSD proteins adhere to the structures. For example, an iodinated myelin fraction
added at the beginning of the subcellular fractionation procedure, stuck to the PSD
fraction. Use of the two different types of detergents resulted in yields of PSDs that
had slightly different chemical compositions. The non-ionic detergent Triton X-100
left more proteins in the fraction, which were likely to be membrane proteins that are
shared with the PSD structure. On the other hand, ionic detergents, such as n-lauroyl
sarcosinate and deoxycholate to solubilize membranes, may have removed some
proteins that are shared between the postsynaptic membrane and PSD. This issue
became a prescient indicator of the current view of an important function of the PSD
in tethering and organizing receptors in the membrane.
An exciting extension of the PSD preparation is reviewed by Ursula Wyneken,
Juan José Marengo, and Fernando Orrego, who developed a system by which
isolated PSDs can be incorporated into giant liposomes. In this way, the
478 R.S. Cohen

Fig. 2 Synaptosomal
membrane fraction. This
fraction shows synaptosomal
plasma membranes, some
with synapses. Arrows
indicate recognizable
postsynaptic densities
(Cohen et al. 1977)

II 0.5 mm

characterization of ionotropic glutamate receptors (iGluRs) in the steady state can be

achieved by the patch clamp technique, which is used to record the currents of single
ion channels. Liposomes are artificially made vesicles composed of the same
material as cell membranes and iGluRs maintain their specific physiological and
pharmacological properties within the liposome. In this way, the receptors can be
studied within the context of the PSD environment. This is advantageous because
determination can be made as to whether or not they are modulated by virtue of their
being anchored in the PSD and if the modulations depend on the influence of
neighboring regulatory proteins, which are likely to be absent in extrasynaptic
glutamate receptors, the type usually used for electrophysiological recordings.
Specific iGluRs can be recorded, as well as the effects of their regulation by
modifications on the cytoplasmic surface of the PSD, where regulatory enzymes,
such as kinases and phosphatases, are located.
In the cell biological climate of the 1970s, there was a strong focus on cytoskel-
etal proteins in non-muscle cells. The filamentous nature of parts of the PSD led
16 The Postsynaptic Density 479

Fig. 3 PSD preparation after

treatment with 0.5 % Triton
X-100 75 mM KCl.
Membrane profiles are
removed by washing the
final pellet of PSDs with
0.5 % Triton X-100 75 mM
KCl. Arrows indicate
subsynaptic bodies or
subsynaptic web material.
Inset shows PSD fraction
at lower magnification
(Cohen et al. 1977)

investigators to look to actin as a candidate for a PSD protein, particularly, since

about 10 % of the enriched PSD fraction contains a protein of 45 kDa, the molecular
weight of actin, as seen by gel electrophoresis. Concurrently, immunocytochemical
techniques were being developed, whereby specific antibodies were tagged with
fluorescent molecules visible with light microscopy, or electron-dense gold particles
or enzymes that catalyzed a reaction resulting in an electron-dense reaction product,
visible with light and electron microscopy. In this way, actin was subsequently
localized within the PSD, filaments emanating from it, and within the dendritic
spine cytoskeletal network in situ. It was then intuitively proposed that actin was
responsible for movement of the PSD, possibly in regard to its shape changes,
evident, at the time, only in the various forms in which the PSD appeared in static
electron microscopic views.
The advent of molecular biology and sophisticated imaging techniques enabled
hypotheses about PSDs, such as its compartmentalized morphology, molecular
composition, dynamism, and role in higher brain functions, to come to fruition.
Here, current structural, biochemical, and functional views of PSDs are described.
480 R.S. Cohen

The Structure of the PSD Reflects Its Complex Molecular

Organization

In thin sections, the ultrastructure of the PSD appears as a thick or thin dense
structure underneath the postsynaptic membrane (Fig. 1). Because of its density,
conventional electron microscopy of thin sections does not provide optimal visual-
ization of the internal complexity of PSDs, except for some of the filamentous
structures emanating from them. Essentially, the PSD is a disklike structure of
various shapes and dimensions, and some of them possess one or more perforations,
also of varying shapes and sizes, which may be centrally or peripherally placed.
Figure 4 shows drawings of en face views (i.e., parallel to the plane of the synaptic
junction) of PSDs from several synapses by Alan Peters and Ita R. Kaiserman-
Abramof, demonstrating the variability of PSD size, shape, and perforations. Other
modifications include the presence of subsynaptic bodies (Fig. 3a, ▶ Chap. 14, “Cell
Biology of the Synapse”) and a subsynaptic web (Fig. 3b, ▶ Chap. 14, “Cell Biology
of the Synapse”).
PSDs have a mean diameter of 360 nanometers (nm) and range in width from
33 to 58 nm. Thicker PSDs have been isolated from cerebral cortex and midbrain,
and thinner ones have been isolated from cerebellum. All of these brain areas contain
both thick and thin PSDs, and the reason for the differential enrichment during the
fractionation procedure is unknown. The two types may represent two different
classes of synapses characterized by E. G. Grey: type I having a more obvious band
of dense-staining material at the postsynaptic side and type II having a less prom-
inent and thinner band. Type I synapses are thought to be excitatory and type II,
inhibitory; however, the validity of this distinction is unclear.

PSDs Display Perforations

A salient feature of some PSDs is the presence of one or more perforations (Figs. 1, 4
and 5). Whereas their functional significance is not known, they appear in larger
PSDs. Perforations display various shapes, including macular-shaped (Fig. 4) and
completely segmented PSDs with separate release zones. Because perforations
appear in PSDs of animals that have undergone physiological or behavioral alter-
ations, some perforations are thought to result from these challenges (section
“Environment, Hormones, and Electrophysiological Input Influence PSD Struc-
ture”). However, time-lapse studies described in section “Cytoskeletal and Scaffold-
ing Proteins Contribute to Structural Dynamics of the PSD” below indicate that
PSDs may, also, exhibit structural changes spontaneously.

The Presence of Particulate Components in PSDs and Some Proteins

in PSD Fractions Depends on Brain Area and PSD Surface
Structural components of PSDs are obvious in unidirectional and rotary-shadowed
electron microscopic replicas. Replicas are made by coating subcellular structures
with carbon and platinum and then floating the original material away. The film that
is retained is the replica, which permits the detailed visualization of the surface
features of a structure. PSDs isolated from cerebral cortex (Fig. 5) and midbrain
16 The Postsynaptic Density 481

1 2 3 4

5 6 7 8

9 10 11 12

13 14 15 16

17 18 19 v

1 micron

Fig. 4 (continued)
482 R.S. Cohen

21 22 23 24 25

26 27 28 29 30

31 32 33 34 35

36 37 38 39 40

41 42 43 44 45

46 47 48 49 50

51 52 53 54 55

1 micron

Fig. 4 (a, b) Reconstructions from serial sections of the patterns formed by 55 different postsyn-
aptic densities. The stippled areas represent the densities seen en face and the broken lines the extent
of the apposition between axon terminals and the dendritic spines (Reprinted with kind permission
from Springer Science + Business Media: Cell and Tissue Research, The small pyramidal neuron of
the rat cerebral cortex, 100(4), 1969, page 498, Alan Peters and Ita R. Kaiserman-Abramof, Fig. 7)
16 The Postsynaptic Density 483

Fig. 5 Electron microscopic examination of a PSD preparation isolated from cerebral cortex. The
picture is an en face view of PSD (arrow), with a perforation in the middle of the disk. A cross-
sectional view of a PSD is seen in the lower right of the micrograph (asterisk). Bar, 200 nm
(Adapted from Carlin RK, Grab DJ, Cohen RS, Siekevitz P (1980) Isolation and characterization of
postsynaptic densities from various brain regions: enrichment of different types of postsynaptic
densities from various brain regions. J Cell Biol 86:831–843)

exhibit particulate bodies in thin sections. The en face replica views of PSDs isolated
from midbrain (Fig. 6a) and cerebral cortex (Fig. 6c, d) show PSDs with particle-like
aggregates 20–30 nm in diameter; these appear to be lacking in the thinner PSDs
from the cerebellum (Fig. 6b). The latter may be considered a multiperforated disk,
composed of proteins which are not in the form of aggregates and some of which
attach the PSD to the membrane. On the other hand, the PSDs obtained from
midbrain (Fig. 6a) and cerebral cortex (Fig. 6c, d) preserve this basic structure
with the addition of the aforementioned particulate aggregates. In regard to protein
composition of the two disparate fractions, the main difference is the presence of the
51 kDa protein, Ca2+/calmodulin-dependent protein kinase II (CaMKII), and the
Ca2+-binding protein, calmodulin, in the thicker PSDs. As discussed below, CaMKII
is the major protein of midbrain and cerebral cortex PSDs. The thin PSDs do not
contain any protein that is more abundant than any of the others.
Detailed studies from the laboratories of John E. Lisman and Thomas S. Reese
have revealed the structure of both the cleft and cytoplasmic surfaces of PSDs and
pinpointed the positions of CaMKII and the important PSD protein postsynaptic
density protein-95 (PSD-95) at the PSD using techniques of immunogold labeling,
replicas, and electron microscopic tomography. Electron microscopic tomography is
advantageous because it can generate computed slices of tissues that are thinner than
those prepared using conventional thin sectioning techniques. PSD-95 is a protein
involved in the organization of other PSD and postsynaptic membrane proteins and
484 R.S. Cohen

a b

c d

Fig. 6 Isolated PSDs from midbrain (a), cerebellum (b), and cerebral cortex (c and d) as viewed
through high magnification by the replica method with rotary shadowing. In (a), single arrows point
to possible particulate bodies, and the double arrows point to the large central hole. In (c), the PSD
is apparently broken up; the single arrows point to 6–9-nm filaments, and the double arrows point
to 12–15-nm filaments. (d) shows a PSD with extensions of 6–9-nm filaments (arrows) apparently
arising from one surface of the PSD. Bar, 200 nm (Carlin et al. 1980)
16 The Postsynaptic Density 485

is further described below (section “The NMDA Receptor (NMDAR) Associates

with the MAGUK Family Scaffolding Proteins”). Replicas appeared as circular disks
from 180 to 750 nm in diameter and showed two different surfaces: one a raised,
smooth plateau, representing the surface contiguous to the postsynaptic membrane
(designated as the cleft side); the other displaying a convoluted contour, representing
the surface facing the underlying cytoplasm (designated as the cytoplasmic side)
(Fig. 7). Each of these surfaces showed a unique surface texture. The cleft surface
displayed a dense layer of 5–15 nm surface particles. These particles were positioned
on top of another layer made up of 2 nm diameter filaments, which formed a central
mesh. Openings of 40–80 nm in diameter pierced the central mesh. Membrane
patches of 50–100 nm in diameter were often seen on the smoother surfaces. On
the other hand, the cytoplasmic surfaces displayed a convoluted appearance due to
irregular protuberances from a layer of material on top of the central mesh.
All PSDs are labeled with PSD-95 on both of its faces, i.e., the cytoplasmic side
and the cleft side (Fig. 7). The distribution of CaMKII labeling is different; the label
for this protein is virtually absent on the side facing the cleft, but it is prevalent on the
cytoplasmic side. CaMKII holoenzymes were visualized directly in electron micro-
scopic tomograms and seen as labeled, towerlike structures, protruding from the
cytoplasmic face of the central mesh. In addition, CaMKII, itself, appears to have a
local organization. Nearest neighbor distances are virtually consistent over a wide
range of CaMKII labeling density. However, there is variability in the average
density of CaMKII holoenzymes with values ranging from zero to that of a highly
packed state and is higher than that for PSD-95. This variability is suggestive of a
role for information storage for CaMKII. In addition, Shank, a scaffolding protein
(section “Shank Organizes Other PSD Scaffolding Proteins”), was mainly seen on
the cytoplasmic, convoluted face of the PSD.

Filamentous Components of the PSD Are Present Within

and on the Cytoplasmic Face of the PSD
Electron microscopic views of PSDs, either in thin sections of synapses in situ, in
PSD fractions, or in replicas of isolated PSDs (Fig. 6c, d), display filaments within
and emanating from the PSD. The size of the filaments emanating from the PSD is
6 nm in diameter, the same dimension as actin filaments, and actin was one of the
first proteins to be identified in PSDs by immunocytochemical and immunochemical
methods. PSD actin is critical to the dynamic structural plasticity of PSDs; depoly-
merization of actin with latrunculin A abruptly halts shape changes in the structure,
as discussed below. Other actin-associated proteins are discussed in section “Cyto-
skeletal and Scaffolding Proteins Contribute to Structural Dynamics of the PSD.”
Reese and colleagues used electron microscopic tomography combined with
immunogold labeling to reveal the nature of the interactions of other filaments
within the PSD (Fig. 8). They demonstrated that vertically oriented filaments (with
respect to the postsynaptic membrane), 5 nm in diameter and 20 nm long, which
contact the postsynaptic membrane, are present throughout the core of the PSD and
constitute a major structural element of PSDs and confer its characteristic dense
appearance, as seen in conventional electron microscopy. Most of these filaments
486 R.S. Cohen

Fig. 7 Cleft and cytoplasmic surfaces of PSDs are different. (a) Cleft surface of PSD immunogold-
labeled for PSD-95. Immunogold particles appear as white dots (small arrow). Counts of gold label
on PSD are shown at the top right. The opening in the central mesh is indicated by the large arrow.
(b) Cytoplasmic surface of PSD immunogold-labeled for PSD-95. The opening in the central mesh
is indicated by an arrow. (c) Enlarged area from (a). Granular particles are indicated by arrowheads.
Underlying filaments of the central mesh are indicated by an arrow. (d) Enlarged area of (b).
Underlying filaments of the central mesh are indicated by an arrow. (e) Cleft surface of PSD
immunogold-labeled for Shank. Any label present is typically at edge of PSD (arrow). PSD
contains a patch of membrane (enlarged in (g)). (f) Cytoplasmic surface of PSD immunogold-
labeled for Shank. Material protruding above the central mesh is indicated by an arrow.
16 The Postsynaptic Density 487

belong to the important postsynaptic density protein-95 (PSD-95) family of proteins

(section “The NMDA Receptor (NMDAR) Associates with the MAGUK Family
Scaffolding Proteins”). The vertical filaments contact two kinds of transmembrane
structures, the sizes and positions of which correspond to those of glutamate
receptors. The vertical filaments intermingle with a meshwork of long and short
horizontal filaments, 5–6 nm in diameter and 30–35-nm long, which are positioned
10–20 nm from the postsynaptic membrane. The short horizontal filaments lie
somewhat closer to the postsynaptic membrane than the longer horizontal filaments.
The longer horizontal filaments appear to link adjacent N-methyl-D-aspartic acid
receptor (NMDAR)-type structures, and the smaller horizontal filaments appear to
link both NMDAR- and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
receptor (AMPAR)-type structures.

The Arrangement of Molecular Components of the PSD Mediates

Communication of Signaling Events in Space and Time

The dense appearance of the PSD belies its complex organization, which allows both
temporal and spatial communication of signaling events at the membrane with signal
transduction pathways in the cytoplasm, as well as for modulation of these events by
other regulatory proteins, including kinases and phosphatases. These proteins have a
wide variety of functions, making the PSD central to synaptic function and plasticity.
Adhesion proteins are likely to link and align the PSD to presynaptic components of
the synapse. PSD proteins involved in signal transduction include receptors and ion
channels, kinases and phosphatases, and guanosine triphosphatases (GTPases). Pro-
teins involved in organization of the PSD may include cytoskeletal and associated
proteins, membrane-trafficking proteins, molecular motors, and scaffold proteins.
Others may be involved in energy demands, protein synthesis, and processing.

Alterations in Scaffold Proteins May Influence Excitatory Synaptic

Transmission Through Their Interactions with Neurotransmitter
Receptors, Regulatory Enzymes, and Ion Channels
Whereas the PSD is a dynamic structure, its constituent proteins are organized in a
precise and hierarchical manner. Ionotropic and metabotropic glutamate receptors,
for example, have distinct locations within the postsynaptic membrane and PSD.
The ionotropic NMDA and non-NMDA (AMPA) glutamate receptors are predom-
inant centrally along the PSD, whereas the metabotropic glutamate receptors

Fig. 7 (continued) (g) Membrane patch (arrowhead) has typical rim of particles. (h) Enlarged area
of (f). Underlying filaments of central mesh are indicated by an arrow. Scale bars, 100 nm
(Reprinted with kind permission from Society for Neuroscience Jennifer D. Petersen, Xiaobing
Chen, Lucia Vinade, Ayse Dosemeci, John E. Lisman, and Thomas S. Reese The Journal of
Neuroscience, December 3, 2003, 23(35):11270–11278. Page 11272. Figure 2. PMID: 14657186;
http://www.jneurosci.org/)
488 R.S. Cohen

Fig. 8 Vertical filaments at the PSD. (a) Electron micrograph (EM) of a PSD in a mushroom-
shaped dendritic spine. Structural details are obscured by overlap within this 120-nm-thick section.
(b) Vertical filaments (green arrows) are apparent in a 1.5-nm-thick virtual section derived from the
tomographic reconstruction of the section in (a). The synaptic vesicle is indicated by an asterisk
(Scale bar: 100 nm). (c) Rendering of vertical filaments (red) from the tomographic reconstruction.
Vertical filaments, 5 nm in diameter and 20 nm long, contact the postsynaptic membrane (yellow).
(Insets) Virtual sections from which particular vertical filaments (green) are segmented (Scale bar:
20 nm). (e) En face view showing uniform distribution of vertical filaments at the PSD. (f) Overlap
of vertical filaments contributes to the typical thickened appearance of a PSD viewed in cross
section (Chen et al. 2008) (Reprinted with permission from Proceedings of the National Academy
of Sciences, U.S.A. “Copyright (2008) National Academy of Sciences, U.S.A.”)

(mGluRs) are peripheral in location (Fig. 9). Scaffold proteins bind to these receptors
and preserve the overall relationships among signaling proteins, as well as interact
with each other. The presence of the organizing scaffold, thereby, allows signaling
proteins to be placed in deliberate positions for efficiency of signal transduction
proteins in regulating important processes, including alterations in synaptic strength,
16 The Postsynaptic Density 489

Fig. 9 Possible relationships of some of the proteins in the PSD to scaffolding proteins at a
hypothetical excitatory synapse. The edges of the PSD are depicted on the right and left of the
diagram at angles to the extrasynaptic membrane. The scaffold protein PDS-95 is shown in red.
Sites of palmitoylation emanating from them extend into the postsynaptic membrane (not shown).
The NMDA receptor (NMDAR) and the AMPA receptor (AMPAR) are localized to the synapse,
itself, whereas the metabotropic glutamate receptor (mGluR) is positioned at extrasynaptic sites.
The AMPAR is complexed with the scaffold protein Stargazin, which, in turn, may interact with
NMDARs or other proteins, such as the adhesion molecule neuroligin via PSD-95; the AMPARs
also bind GRIP, a protein involved in the clustering of AMPARs. The adhesion proteins neuroligin
and N-cadherin link the pre- (not shown) and postsynaptic processes. SynGAP also interacts with
PSD-95. Whereas neuroligin can interact with other proteins, such as GKAP, via PSD-95,
N-cadherin interacts with GKAP via β-catenin. GKAP can interact with Shank, which, itself,
organizes other scaffolds. Homer can interact with Shank, as well as the membrane of the spine
apparatus (not shown). PSD-95, GKAP, Shank, and Homer proteins interact with each other and
form the centerpiece of the scaffold. Ion channels may be present at excitatory synapses, including
the small-conductance Ca2+ -activated K+ (SK) channels and the voltage-independent G-protein-
coupled inward rectifying K+ (GIRK) channels. Ca2+ influx through the NMDAR activates many
molecules including kinases, such as CaMKII; it may also activate the SK channels at PSDs. The
GIRK channels, on the other hand, are predominantly located at extrasynaptic sites and are activated
by the metabotropic GABAB receptor via Gβγ. CaMKII is located in the PSD, as well as the
underlying dendritic spine cytoplasm, and can be translocated from the spine into the PSD. Actin
and/or its binding proteins (e.g., α-actinin, cortactin, profilin) are located near the postsynaptic
membrane (not shown) and at the cytoplasmic face of the PSD. Thus, the PSD contains three major
tiers of proteins: (1) receptors, ion channels, and adhesion proteins shared with the postsynaptic
membrane; (2) scaffold proteins connecting the receptors to each other, to other membrane
components, and to the actin cytoskeleton; and (3) the actin-based cytoskeleton, itself (Figure by
Zoe Asta)

such as long-term potentiation (LTP), a persistent enhancement of synaptic strength

resulting from high-frequency synchronous stimulation of the synapse, and long-
term depression (LTD), a persistent reduction in synaptic efficacy resulting from
either high- or low-frequency stimulation.
490 R.S. Cohen

The following subsections describe some examples of the main types of postsyn-
aptic scaffold proteins and their interaction with the membrane and cytoplasmic
faces of ionotropic and metabotropic glutamate receptors at excitatory synapses. The
description below of some of the scaffolding proteins is organized according to the
receptors with which they associate within the PSD. Mary Kennedy, one of the
pioneers in elucidating the molecular architecture of PSDs, enumerated seven major
classes of scaffold proteins: (1) the MAGUK family (membrane-associated
guanylate kinase-like proteins, such as PSD-95); (2) the GRIP/ABP (AMPAR-
binding protein) and GRIP-like family (those proteins displaying a string of PDZ
domains (section “The NMDA Receptor (NMDAR) Associates with the MAGUK
Family Scaffolding Proteins”), such as protein interacting with C kinase [PICK]);
(3) The GKAP/SAPAP (guanylate kinase-associated proteins) family; (4) the Shank/
proSAP family; (5) the Homer family; (6) TARPs (transmembrane AMPAR regula-
tory proteins); and (7) the cytosolic tails of transmembrane proteins specific to PSDs,
such as the NR2 subunits of the NMDAR.
Scaffold proteins, themselves, are organized into their own hierarchy. For exam-
ple, some scaffold proteins that bind directly to receptors, such as PSD-95 and other
members of the MAGUK family, may be located higher up in the hierarchy. These
associate, in turn, with other more distally located scaffold proteins, such as GKAP,
Shank, and the Homer family, which, in turn, link up protein complexes associated
with the various classes of glutamate receptors.

The NMDA Receptor (NMDAR) Associates with the MAGUK Family Scaffolding
Proteins
NMDARs are ligand-gated Ca2+ permeable channels that are involved in synaptic
plasticity, including LTP and LTD. They are tetrameric complexes consisting of NR1
and NR2 subunits. The latter display long cytoplasmic tails, which extend into the
cytoplasm, where they may bind to scaffold proteins and enzymes involved in
signaling. The C-termini of the tails end in the conserved sequence -ESDV or
-ESEV, which, in turn, binds the receptor to one of the members of the MAGUK
family, PSD-95. PSD-95 molecules contribute 2.3 % of the mass of the PSD. The
molecule exhibits a sequence of three repeated 90-residue domains, called PDZ
domains, an SH3 domain, and a guanylate kinase-like (GK) domain, which acts as a
protein binding domain. A PDZ domain is a domain common to many proteins and
is an acronym for the first letters of three proteins: PSD-95, Drosophila disc large
tumor suppressor, and zona occludens-1 protein. PDZ domains can bind specific
sequences positioned at the carboxyl end of a membrane protein. Alternatively, PDZ
domains can associate via PDZ-PDZ interactions. Palmitoylation is a posttransla-
tional lipid modification of neuronal protein, and PSD-95 is a major palmitoylated
protein in the brain. Enzymatic activity that palmitoylates PDS-95 targets this
molecule to cellular membranes.
In regard to NMDARs, the C-terminal consensus sequence in NR2 subunits binds
to the first and second of the PDZ domains of PSD-95. It can also bind to the other
PDZ domains of other MAGUKs. PSD-95 appears to be involved in several
functions, including targeting of the receptors to the postsynaptic membrane,
16 The Postsynaptic Density 491

AMPAR trafficking, coupling of the receptors to signaling proteins, and/or in

anchoring of receptors to the cytoskeleton. The extended carboxyl tails of the NR2
subunits also serve a scaffolding function in their ability to bind signaling proteins.
For example, the tails bind directly to the signaling enzymes CaMKII and rasGRF1,
the actin-associated protein α-actinin, as well as the scaffold protein yotiao, which, in
turn, binds protein phosphatase 1 and protein kinase A.

The AMPA Receptor (AMPAR) Associates with TARP, GRIP, PICK1, and NSF
AMPARs regulate a channel permeable to Na+ and K+ and are involved in the
mediation of fast excitatory glutaminergic transmission. They consist of four homol-
ogous subunits, which assemble in different combinations to form specific AMPAR
subtypes. AMPAR trafficking and insertion or subtraction into or from the postsyn-
aptic membrane, respectively, form the basis of activity-dependent alterations in
synaptic strength. Glutamate receptor (GluR) subunits directly interact with five
scaffold proteins, which participate in AMPAR trafficking: the TARP protein
Stargazin; GRIP (GRIP1)/ABP (GRIP2); PICK1 (protein interacting with C kinase
1); and NSF (N-ethylmaleimide-sensitive factor). PSD-95 is also involved in
AMPAR trafficking, albeit indirectly, by way of its interaction with Stargazin.
PSD-95 also binds to some subunits of kainate receptors.
The cytosolic tail of the AMPAR subunit GluR2 can associate with the fourth or
fifth PDZ domain of GRIP (GRIP1) and ABP (GRIP2). The palmitoylated form of
either GRIP or ABP is directed toward the postsynaptic membrane; on the other
hand, the non-palmitoylated form is directed to intracellular membranes. Regulation
of the palmitoylation of these scaffold proteins fixes AMPARs to either of these
membranous compartments. Dissociation of the GluR2-GRIP/ABP complex is
necessary for insertion and removal of AMPARs at the synapse. PICK1 binds to
protein kinase C-α (PKCα) and is thought to position it near the tail of GluR2 for a
phosphorylation reaction, which subsequently releases GluR2 from GRIP/ABP.
Another interesting example of an AMPAR scaffolding protein is the TARP
Stargazin (Fig. 9). Its cytoplasmic tail controls receptor trafficking, whereas the
outer part of the molecule that projects into the extracellular space regulates channel
gating. The C-terminal consensus binding motif of TARPs can bind to the first two
PDZ domains of PSD-95, a process necessary for activity-dependent translocation of
AMPARs to postsynaptic sites.

Metabotropic Glutamate Receptors Associate with Members of the Homer

Family
Two groups of G protein metabotropic receptors activated by glutamate regulate
neuronal excitability and synaptic strength. In group I, localized at the periphery of
PSDs (Fig. 9), mGluR1 and mGluR5 regulate phospholipase C, which results in the
production of the second messengers inositol triphosphate (IP3) and diacylglycerol.
Their association with the PSD is mediated by the Homer family, Homer 1, 2, and
3, which are alternatively spliced into long isoforms (contains a C-terminal coiled-
coil [CC] domain, which promotes self-association into tetramers) and short
isoforms (does not contain the CC domain and are monomeric isoforms [called
492 R.S. Cohen

“a” isoforms]). The expression of the short isoform is rapidly induced by synaptic
activity, whereas the long isoform is constitutively expressed. Homers display an
N-terminal EVH1 domain, which binds to a proline-rich motif in mGluRs and other
proteins. This domain also binds to the IP3 receptor (IP3R) and the scaffold protein
Shank. The tetrameric forms are centers for cross-linking the disparate EVH1
domain-interacting proteins, whereas the monomeric forms act counter to this
process.
The IP3R is an IP3-sensitive Ca2+ channel, which is present in smooth endoplas-
mic reticulum (SER) of dendritic spines (▶ Chap. 14, “Cell Biology of the
Synapse”). SERs store and release Ca2+ and appear to be located near the spine
periphery. When group 1 mGluRs are activated, IP3 is generated and subsequently
binds to the IP3R, resulting in the release of Ca2+ from the SER. Therefore, the
mGluR/Homer/IP3 couples activation of group 1 mGluRs to Ca2+ in dendritic
spines. Neural activity results in the rapid upregulation of the short isoforms of
Homer, which is thought to compete with the long form, thereby interfering with the
relationship between mGluRs and the IP3R and, consequently, the release of Ca2+.
The competition between these two forms of Homer may also be involved with other
Homer targets including Shank (section “Shank Organizes Other PSD Scaffolding
Proteins”), with expression of the short isoforms of Homer resulting in a decreased
synaptic targeting of Shank and other PSD proteins and an inhibition of synaptic
transmission.

Shank Organizes Other PSD Scaffolding Proteins

The aforementioned scaffolds, themselves, are organized into a higher-order scaffold
by the protein Shank (Fig. 9); Kennedy called Shank “the scaffold of scaffolds.” The
disposition of scaffold proteins appears to be laminar in nature, as determined by
measuring distances of various scaffold proteins from the postsynaptic membrane.
PSD-95 is present at an average peak distance of 12 nm from the membrane; GKAP-
Shank, on the other hand, is located 24–26 nm from the membrane. The cytoskeletal-
associated proteins, CRIPT and dynein, are located 29–31 nm from the membrane.
Thus, Shank is located in a position to link PSD-95 and associated proteins to the
more distal actin cytoskeleton. Shank displays ankyrin repeats, an SH3 domain, a
PDZ domain, a proline-rich domain, and an SM domain. The SM domain can
interact with each other and with sharpin to form homomultimers. The PDZ domain
can also dimerize in an antiparallel configuration, adding to the multimerization. It is
thought that Shank multimer may reinforce PSD organization and may bind Homer
and GKAP which, in turn, links to PSD-95. In this way, the Shank platform could
cross-link the NMDAR and mGluR complexes, as well as associate with AMPAR
complexes through direct binding to the GluR1 subunit via interactions between the
TARP proteins and PSD-95.
Importantly, the Shank scaffold acts as an intermediary for the PSD and the
underlying actin cytoskeleton of dendritic spines. The Shank scaffold can interact
with actin-associated proteins, including cortactin, actin-binding protein 1, and
spectrin/fodrin. Shank is, therefore, central to communicating the events occurring
16 The Postsynaptic Density 493

in the postsynaptic membrane and PSD to alterations in the underlying spine

cytoskeleton.

Some Ion Channels Are Present in PSDs of Excitatory Synapses

Ion channels are also located in PSDs of excitatory synapses in central neurons
(Fig. 9). For example, the voltage-independent G-protein-coupled inward rectifying
K+ (GIRK) and small-conductance Ca2+-activated K+ (SK) channels bring about
specificity of signaling by their localization to these synapses. SK channels are
enriched at PSDs and are coupled to NMDARs. Ca2+ influx through NMDARs
and R-type Ca2+ channels increases the channel activity of SK2, with an ultimate
effect of decreasing synaptic signaling and induction of synaptic plasticity. GIRK
channels are activated by GABABRs via Gβγ; both are primarily extrasynaptic, but
also located at PSDs. At excitatory synapses of dendrites and dendritic spines, these
channels assemble and bind particular sets of proteins to form macromolecular
signaling complexes which, in turn, alter neurotransmission and synaptic plasticity.

Scaffold Proteins Are Also Present at Inhibitory Synapses

The above discussion focused on the PSD of excitatory synapses. Postsynaptic
specializations of inhibitory synapses also contain molecules, which regulate the
organization and function of neurotransmitter receptors. The ionotropic gamma
aminobutyric acid (GABA) receptors (GABARs) (mainly GABAA receptors
[GABAARs]) are chloride channels consisting of distinct subunits from at least six
disparate gene families. They are usually separated from NMDARs and AMPARs,
are present at inhibitory synapses, and may be located in the PSD and at
extrasynaptic sites of excitatory synapses. The intracellular domains of GABAA
Rs interact with cytoplasmic proteins, albeit in a different manner than that for
glutamate receptors. The C-termini of GABAAR subunits are exposed on the
extracellular side of the postsynaptic membrane. A cytoplasmic domain binds to a
GABAAR-associated protein (GABARAP). GABARAP has similarities in its
sequence to microtubule-associated proteins (MAPs) 1A and 1B and, therefore,
may be a part of some MAP complexes. GABAC receptors also interact with
MAPs. Ionotropic glycine receptors bind to the microtubule-binding protein
gephyrin. Inhibitory synapses are present dendritic shafts, which contain abundant
microtubules and MAPs; it is, therefore, not surprising that MAPs may secure some
GABARs and glycine receptors at specific sites along the dendrite.

CaMKII Is a Major PSD Protein that Modulates Signal Transduction at

Synapses
In addition to scaffolding molecules, PSDs possess a wealth of signaling molecules.
One of the most fascinating and important signaling molecules in the PSD is a
dodecameric CaMKII, a protein also present in many other neuronal subcellular
compartments. CaMKII is the major component of forebrain PSDs, constituting 6 %
of the mass of PSDs. Because CaMKII occupies such a large proportion of the PSD,
it is likely to be a key player in the determination of PSD structure. This supposition
is supported by the observation that the CaMKIIβ isoform can bundle actin filaments
494 R.S. Cohen

in dendritic spines and is necessary for preserving dendritic spine morphology.

A fundamental characteristic of CaMKII is its ability to phosphorylate its target
substrates, one of which is itself in a process called autophosphorylation. Because of
the complex nature of the molecule and its association with NMDAR subunits,
CaMKII has been implicated in synaptic and behavioral memory and learning.
The primary structure of CaMKII has several domains: a catalytic (kinase)
domain; a regulatory (autoinhibitory) domain, in turn consisting of an inhibitory
domain, a calmodulin-binding domain, and threonine (Thr)286, Thr305, and Thr306
residues; and a self-association domain, which also contains variable inserts
(Fig. 10). The self-association domain at the carboxyl end of the kinase is respon-
sible for assembling a large, non-dissociable holoenzyme, consisting of 12 subunits,
which are organized into two hexameric rings. The basic unit of assembly is a dimer
of association domains, with one subunit contributed by both the upper and lower
rings. The association domain, in turn, is linked to the catalytic and regulatory
domains by a variable region. In the inactive state, individual pairs of catalytic
subunits bind to each other via a coiled-coil interaction between their calmodulin-
binding domains and the holoenzyme appears as six dimers. This configuration
blocks peptide and ATP binding to the catalytic kinase domains, ensuring Ca2+
dependence for its initial activation. In its autoinhibited or regulatory state, the
regulatory domain is situated so as to sterically hinder both the substrate-binding
site and catalytic domain, thereby precluding ATP binding to the Thr286 residue
(Fig. 10). It is thought that the binding of Ca2+/calmodulin results in the dissociation
of the members of an inactive dimer pair. In this way, the active sites of the enzymes
can be exposed, allowing autophosphorylation events to take place between
neighbors.
During the initial activation, Ca2+/calmodulin binding displaces the
autoregulatory segment from the catalytic segment, thereby freeing the catalytic
activity of the kinase domain. The Ca2+/calmodulin-activated kinase can then
phosphorylate its own neighboring subunit that is also bound to Ca2+/calmodulin
(Fig. 10). The minimum requirement to activate the entire molecule is, therefore, the
binding of Ca2+/calmodulin to two neighboring subunits. That is, the first is required
for activation of one subunit and the second is needed for the conformational change
to expose the phosphorylation site Thr286 to its neighbor.
The phosphorylated subunit of CaMKII is active even when Ca2+ is no longer
present because in the presence of the phosphorylated Thr286, the regulatory, i.e.,
autoinhibitory, segment can no longer bind to its catalytic, kinase domain. Also, the
affinity of CaMKII for calmodulin is increased 13,000-fold, thereby extending the
length of time that the enzyme is active. The phosphorylated, active subunit can then
phosphorylate another neighboring subunit in the absence of Ca2+/calmodulin.
When Ca2+ is low, the Thr286 residue is dephosphorylated before the entire holo-
enzyme is activated because the incidence of adjacent subunits with bound Ca2+/
calmodulin is correspondingly low. When Ca2+ is present for a short while, short-
term persistent activity can occur in the absence of Ca2+ but is extinguished if
Thr286 becomes dephosphorylated. On the other hand, the enzyme becomes auton-
omous and exhibits Ca2+-independent activity when the rate of autophosphorylation
16 The Postsynaptic Density 495

Catalytic Regulatory Self-

(Kinase) (Autoinhibitory) association
Calmodulin
lnhibitory binding
+
NH3 COO−

1 286 305 306 478

Autophosphorylation
1

+ ATP
+ ATP
2+
+ Ca / Calmodulin
+ Ca2+ / Calmodulin 2
3 + ATP

Fig. 10 CaMKII and autophosphorylation. (Top figure) Functional domains within the Ca2+/
calmodulin-dependent kinase primary structure. The structure consists of a catalytic (kinase)
domain (green); a regulatory (autoinhibitory) domain (red); and a self-association domain (blue).
The calmodulin-binding domain is located in the regulatory part of the molecule. Phosphorylation
occurs on the Thr286 site of the regulatory domain. Thr305 and Thr306 are also important
phosphorylation sites in the regulation of synaptic depression. Isoform differences in the CaMKII
molecule are determined by variable inserts (multiple alternatively spliced sequences) in the self-
association domain (not shown). (Bottom figure) Autophosphorylation of CaMKII. One half of a
CaMKII hexamer is shown on the left. This part of the molecule is inactive because the regulatory
(autoinhibitory) domain (red) is obstructing the catalytic (kinase) domain (green). Ca2+/calmodulin
(orange dot) binding to the regulatory domain of subunit 1, as illustrated in the right part of the
diagram, releases the binding of the regulatory and catalytic domains, the catalytic domain becomes
exposed, and the subunit is activated (yellow dot). This subunit can then catalyze the phosphory-
lation (black dot) of subunit 2, its clockwise neighbor, in the presence of Ca2+/calmodulin. The
phosphorylation of subunit 2 on Thr286 keeps the subunit in an opened conformation and it can
then phosphorylate the next clockwise subunit, subunit 3, which is active even in the absence of Ca2
+
/calmodulin. Because subunit 3 is phosphorylated and remains opened, it can phosphorylate the
next clockwise neighbor and so on. This process is called autophosphorylation. The reaction
persists as long as a phosphatase, such as protein phosphatase 1 (PP1), does not interfere with the
progression. The process works because the subunits are arranged in a circle and not a line; that is, if
any subunit becomes dephosphorylated, a neighboring subunit can rephosphorylate it. The attach-
ment of the subunits by the self-association domain is advantageous because they can act indepen-
dently of each other for activity and Ca2+/calmodulin binding and, also, interact within the
holoenzyme for intramolecular autophosphorylation (Figure by Zoe Asta)

is greater than the rate of dephosphorylation. It is this latter state that may form the
basis of CaMKII as a molecule involved in synaptic plasticity.
A well-studied example of the correlation of the activation states of CaMKII with
other synaptic events is LTP, whereby Ca2+ entry into the postsynaptic neuron
triggers an activity-dependent strengthening of synapses. CaMKII is thought to be
activated by Ca2+ entry via the NMDAR during LTP. Interestingly, CaMKII is
496 R.S. Cohen

a b

c
1.0
Cumulative probability

0.8

0.6

0.4

0.2

0.0
0 10 20 30 40 50
Particles/µm of PSD

Fig. 11 cLTP induction results in persistent accumulation of CaMKIIα at the PSD. (a, b) Electron
micrographs of hippocampal synapses labeled for CaMKIIα under control conditions (a) and 1 h
after induction of cLTP (b). Silver-enhanced gold particles appear as irregular black grains.
Asterisks indicate grains counted as PSD-associated CaMKII labeling. Scale bar, 100 nm. (c)
Cumulative distribution of densities of gold label for CaMKIIα at individual PSDs from slice
cultures under control conditions (▴) and after induction of chemical LTP (•) (Reprinted with kind
permission from Society for Neuroscience Nikolai Otmakhov, Jung-Hwa Tao-Cheng, Stephen
Carpenter, Brent Asrican, Ayse Dosemeci, Thomas S. Reese and John Lisman 2004, Page 9329.
PMID: 15496668; http://www.jneurosci.org/)

necessary and sufficient for LTP induction. There are actually several mechanisms
through which CaMKII may enhance synaptic transmission. These include enhanc-
ing transmission by phosphorylating the AMPAR, by binding to the NMDAR and
organizing new scaffolding assemblies for more AMPARs, and by enhancing the
delivery of additional AMPARs to the membrane. John Lisman and colleagues have
shown that NMDAR-dependent chemical LTP (cLTP) (induction of potentiation by
chemical means) results in persistent accumulation of CaMKII at the PSD (Fig. 11).
16 The Postsynaptic Density 497

Whereas many studies on the role of CaMKII in synaptic plasticity have focused
on its role in LTP, emerging data have made the picture even more complex and
intriguing. Recent evidence by Lisman and colleagues indicate that autonomous
CaMKII can promote either LTP or LTD, depending upon the phosphorylation state
of other threonine residues (T305/T306) on the molecule. The T305/T306 site is
located within the calmodulin-binding domain, and its phosphorylation prevents
activation of CaMKII by Ca2+/calmodulin. Also, when this site is phosphorylated, it
is more difficult to generate LTP induction, but less difficult to generate LTD
induction.
Lisman and colleagues performed a series of elegant experiments using CaMKII
constructs containing mutations. Neurons were transfected with the complete holo-
enzyme, which contained the mutation, T286D. This form of the enzyme is
“pseudophosphorylated” on the autoregulatory site, maintaining the activity of the
enzyme in an active state, independent of Ca2+/calmodulin binding; in other words,
it is a functional “mimicry” of phosphorylation. The investigators anticipated that the
expression of the phosphorylated mutant molecule CaMKIIT286D would enhance
synaptic transmission in a manner similar to a truncated, constituently active form,
where the amplitude of the AMPAR excitatory postsynaptic currents (EPSCs) was
larger than that seen in adjacent nontransfected neurons. Instead, the investigators
saw a decrease in the amplitude of spontaneous AMPAR-mediated EPSCs. T286D
failed to potentiate synaptic strength and, also, produced synaptic depression
through an LTD-like (dependent upon NMDAR function and on phosphatase activ-
ity) process. This finding was surprising, given the wealth of data implicating
CaMKII in LTP. Overexpression of the mutant, CaMKIIT286D:T305D:T306D,
which mimics phosphorylation at all three residues (T286, T305, and T306), resulted
in synaptic depression. On the other hand, expression of the triple mutant
CaMKIIT286D:T305A:T306A, which cannot be phosphorylated at T305/T306,
but is phosphorylated at T286, precluded the development of LTD, but led to the
expression of LTP. These data indicate that the phosphorylated mutant molecule
CaMKIIT286D can produce synaptic depression when T305/T306 is phosphory-
lated. Interestingly, phosphorylation at T305/T306 decreases the binding of CaMKII
to the PSD, which may affect synaptic plasticity by decreasing the strengthening of
synapses. Also, phosphorylation of T305/T306 precludes CaMKII interaction with
α-actinin, an actin-bundling protein.
To distinguish the role of CaMKII in LTP induction versus LTP maintenance,
K. Ulrich Bayer and colleagues used the cell-penetrating fusion peptide inhibitor
tatCN21. LTP induction takes place when the Ca2+ concentration inside the post-
synaptic cells is greater than a critical threshold and involves a transient activation of
CaMKII and phosphokinase C (PKC) and other kinases. Maintenance of LTP, on the
other hand, involves the persistent activation of these molecules. Expression of LTP
involves the long-lasting cellular changes of the maintenance signal. The inhibitor
tatCN21 is highly CaMKII selective in terms of the kinase compared to other
CaMKII inhibitors, which inhibit more than one type of kinase. tatCN21 blocks
498 R.S. Cohen

both stimulated and autonomous CaMKII activity. This inhibitor also has the
advantage of distinguishing LTP induction versus maintenance or in memory acqui-
sition compared to storage and retrieval. Under the conditions of the experiment,
tatCN21 inhibited LTP induction, but not LTP maintenance. Likewise, tatCN21
inhibited learning, but not memory storage or retrieval in a hippocampus-dependent
contextual fear conditioning paradigm in mice. The results suggest that the function
of CaMKII autonomy is a computational device necessary for LTP induction and
learning, but not as a long-term storage mechanism involved in LTP maintenance or
memory retention.

Cytoskeleton and Associated Proteins Constitute a Large Proportion

of PSD Proteins
The cytoskeletal protein actin is another key to PSD structure and function.
Proteonomic analysis revealed that actin and its associated proteins account for
12 % of the isolated PSD fraction. Proteonomics (prote from protein + ome from
genome) permits large-scale analysis of entire sets of proteins and their modifica-
tions. As described below, actin polymerization is essential for the “morphing” of the
PSD into different configurations. Actin-associated proteins include α-actinin,
adducin, ankyrins, Arp2/3 complex, contactin, drebin, LIM, neurabin, and α/β
spectrins and fodrin. Other cytoskeletal proteins at or in association with the PSD
include the following: MAP1A, MAP1B, and MAP2, septin, and tubulin; and the
motor proteins, dynein and myosin IIb, myosin V, and myosin VI.
Actin and its binding proteins appear to be present throughout the extent of the
PSD. At the postsynaptic membrane face, it has been implicated in receptor anchor-
ing, and depolymerization results in dispersion of NMDARs and AMPARs at
excitatory synapses and decreases in the clustering of gephyrin (a glycine receptor
scaffolding protein) at inhibitory synapses. NMDARs attach to actin by way of
single actin-binding proteins, such as α-actinin, whereas AMPARs contact the
cytoskeleton via PSD scaffold proteins. Also, the actin cytoskeleton may control
postsynaptic receptor mobility in and out of the boundary of the synaptic area (i.e.,
synaptic and extrasynaptic receptors). It has been proposed that actin may regulate
the organization of receptors and their associations within the PSD. On the cyto-
plasmic face of the PSD, actin is associated with scaffold proteins. Depolymerization
of actin by latrunculin treatment abolishes half of the PSD-bound Shank, GKAP, and
Homer 1c. Importantly, the remaining actin is unaffected, and there is only a partial
loss of AMPARs. The PSD may be organized into functional subsets, which permit
some molecules to separate from subcompartments of the PSD, or it may be
composed of interlinked layers with interfaces that require continuous maintenance
of their integrity by actin regulation. Since larger and more complex PSDs correlate
with increased numbers of receptors and larger dendritic spine heads, the PSD may
participate in the organization of the dendritic spine cytoskeleton. Some of the
functions of actin and its binding proteins in relation to the dynamics of PSD
structure are discussed below.
16 The Postsynaptic Density 499

Cytoskeletal and Scaffolding Proteins Contribute to Structural

Dynamics of the PSD
Interaction of F-actin and scaffolding proteins may regulate PSD maintenance and
remodeling. Studies from Shigeo Okabe and colleagues demonstrated both actin-
dependent and actin-independent fractions of scaffolding molecules and regulatory
mechanisms for actin dynamics that are specific to individual molecules. Acute
pharmacological disruption of F-actin with latrunculin A prevented activity-
dependent redistribution of GKAP (binds to PSD-95 and Shank family proteins),
Shank2 (also known as ProSAP1 and cortactin-binding protein 1), and PSD-ZIP45
(Homer 1c; a direct binding partner of type 1 mGluRs); however, the localization of
PSD-95 was unaffected. Actin disassembly also resulted in the dissociation of
PSD-95, GKAP, and Homer from the Shank-containing protein complex isolated
by immunoprecipitation. F-actin is, therefore, essential to the integration and main-
tenance of several PSD scaffolding proteins. However, there are other fractions of
PSD proteins, also including PSD-95, GKAP, Shank, and Homer, which are immo-
bile and may be tightly associated to a stable structure resistant to actin depolymer-
ization. Elimination of NMDARs or mGluRs by using cultured neurons from mutant
mice had no effect on the mobility of their binding scaffolds, even though scaffold-
ing proteins have direct binding motifs to the receptors.
Other studies indicate the interaction of Shank and Homer proteins with other
cytoskeletal proteins involved in the actin network. Shank can interact with α-fodrin,
cortactin, and actin-binding protein 1, and fodrin and actin-binding protein 1 can also
interact with F-actin. Cortactin can bind F-actin and can also turn on the Arp2/3 actin
nucleation mechanism, creating branch points in preexisting filaments; it can also
interact with Shank and control spine morphogenesis; α-actinin can interact with
NR2B and CaMKII. Homer family proteins have been shown to bind F-actin and to
the actin-binding protein debrin. Fodrin binds to subunits of NMDARs in an
activity-dependent manner, and the interaction is regulated by calcium and several
protein kinases. In this way, movement of the NMDAR within the membrane is
regulated by synaptic activity.
As discussed below (section “Environment, Hormones, and Electrophysiological
Input Influence PSD Structure”), the PSD has proven to be a dynamic structure that
changes its conformation upon physiological and behavioral challenges within a
range of time scales. In an elegant study by Thomas A. Blanpied, Justin M. Kerr, and
Michael D. Ehlers, the internal dynamics of the scaffold protein PSD-95 were
viewed using time-lapse imaging of single PSDs in dendritic spines of hippocampal
neurons expressing green fluorescent protein (GFP)-tagged PSD-95. The investiga-
tors showed that individual PSDs underwent wide-ranging structural changes spon-
taneously within minutes (Fig. 12) or even seconds. Moreover, coexpression of
PSD-95 with other PSD proteins indicated that the same changes occurred at the
same time for multiple PSD proteins. These include Stargazin-green fluorescent
protein (GFP), Shank-GFP, and GKAP1-GFP, all colocalizing with PSD-95-
mCherry (Fig. 12). The protein pairs showed rapid and virtually identical reshaping.
500 R.S. Cohen

PSD-95-GFP
0 min 1 2 3 4 5 6 7
0 min 2 4 6 8 10
Stg-GFP

PSD-95-
12 14 16 18 20 22 mCh

Overlay

Fig. 12 Individual PSDs undergo continuous morphological plasticity. The figure at the left shows
time-lapse imaging of a single PSD in the spine of a hippocampal neuron. Images acquired at
several z planes were maximally projected and interpolated for display (Scale bar, 1 μm). The figure
at the right shows simultaneous and coordinate reshaping of PSD-95 mCherry with Stargazin-GFP.
(Scale bar: 1 μm) (Blanpied et al. 2008) (Reprinted with permission from Proceedings of the
National Academy of Sciences, U.S.A. “Copyright (2008) National Academy of Sciences, U.S.A.”)

Analyses of PSD elliptical form indicated that spontaneous alterations in PSD

morphology came about incrementally, rather than abruptly, which would have
been the case if proteins were added or removed as larger subunits. In fact, the
number of PSD-95 molecules did not change during the time observed. PSD changes
at neighboring synapses were not synchronous, indicating a local and synapse-
specific regulation of PSD structure.
To determine if the actin cytoskeleton regulates the abovementioned alterations in
PSD structure, latrunculin A was used to depolymerize actin. This treatment resulted
in the cessation of PSD dynamics (Fig. 13). Moreover, it is unlikely that the actin-
dependent restructuring depends on the subtraction or addition of molecules at
particular sites within in the PSD, as the molecular exchange of PSD-95 appears
to be actin independent. Of particular interest was the observation that there was no
correlation between spine dynamics, which are regulated by actin, and PSD
restructuring. It was suggested that changes in PSD morphology, rather than in
spine morphology, may represent altered synaptic function. Moreover, changes in
scaffold positioning regulated by actin may control the local density of signaling
molecules bound to PSD scaffolds, activating or ending signaling within subregions
of the PSD.
The scaffolding protein Shank is localized to the convoluted or cytoplasmic face
of the PSD. The distribution of members of some of the Shank family of proteins
appears to depend on activity in the case of hippocampal synapses. Reese and his
colleagues have shown that Shanks are localized predominantly at PSDs and in the
filamentous network near it, in a range up to 120 nm from the postsynaptic
membrane. Different Shanks have a disparate distribution. Shank2 is usually local-
ized at or near PSDs, whereas Shank1 is also present throughout the dendritic spine
head. In general, Shank proteins translocate toward the PSD upon depolarization
with K+ for 2 min (Fig. 14). The translocation is reversible (for Shank1) (Fig. 14) and
dependent upon extracellular Ca2+ (Fig. 15). Shank1 displays a greater activity-
induced redistribution than Shank2. Shank1, therefore, may play a role in activity-
induced structural changes, whereas Shank2 may be a more stable protein located at
the junction of the PSD and spine cytoplasm and may contribute to longer-lasting
16 The Postsynaptic Density 501

a Variation between synapses

20%

0%
b Baseline
0 min 2 4 6 8 10 12 14
15%

0%

c After latrunculin A
15%

0%

Fig. 13 Actin drives spatial fluctuations of PSD-95 molecular density in individual PSDs.
(a) Fractional protein density maps of single PSDs from several cells. The color scale shows the
proportion of total molecules per 1,000 nm2 (Spatial scale bar: 1 μm). (b) Time-lapse density maps
of a PSD over time (Spatial scale bar: 1 μm). (c) Density maps 1 min after application of 2 μm
latrunculin A (Spatial scale bar: 1 μm). Color scale as in (a) (Blanpied et al. 2008) (Reprinted with
permission from Proceedings of the National Academy of Sciences, U.S.A. “Copyright (2008)
National Academy of Sciences, U.S.A.”)

a b c

Fig. 14 Immunolabeling with pan-Shank antibody in cultured hippocampal neurons showing

representative distribution of label at rest (a), after 2 min of high K+ (b), and after 30 min of
recovery from high K+ (c). The density of label in the PSD and contiguous network (Zone 1)
appears to increase with stimulation and returns to resting level with 30 min rest. Scale bar = 100
nm (Tao-Cheng et al. 2010) (Copyright Elsevier (2010))

structural changes. Importantly, concomitant with the movement of Shanks toward

the PSD, there was an increase in PSD curvature, which may indicate recent synaptic
activity. An increase in the number of synapses with positive synaptic curvature was
also seen following treatment with the hormone estrogen (section “Environment,
Hormones, and Electrophysiological Input Influence PSD Structure”).
502 R.S. Cohen

a b

Fig. 15 Representative electron micrographs showing distributions of label for pan-Shank in zone
1 following 2-min high K+ either in the presence (a) or absence of external Ca2+. (b) The
stimulation-induced clustering of label for Shank in zone 1, and the PSD curvature change appears
to depend on external Ca2+. Scale bar = 100 nm (Tao-Cheng et al. 2010) (Copyright Elsevier
(2010))

Environment, Hormones, and Electrophysiological Input Influence

PSD Structure

The concept that the PSD is, indeed, a structure that undergoes structural alterations
with various inputs came from pioneering studies by William Greenough, Roger
W. West, and Timothy J. DeVoogd in 1978. These investigators used a model,
whereby rats were reared from weaning in either a complex environment (EC) or
social environment (SC) and compared them to those raised in the impoverished or
isolated conditions (IC). Rats in the EC environment were housed in a large cage
filled with toys that were changed daily. These rats were also permitted to explore a
different toy-filled field daily for 30 min. The IC environment consisted of a standard
laboratory cage. The SC environment was a standard laboratory cage containing one
other rat and permitted the distinction between environmental complexity versus
social condition. Rats from the EC and SC groups displayed approximately 25 %
more perforated PSDs in their occipital cortex (the center for processing visual
information in the mammalian brain) than those raised in the IC environment.
Further statistical analysis revealed a significant effect of rearing environment,
although there was no statistical difference between the EC and SC environments.
Interestingly, when a subpopulation of larger synapses (larger than 0.25 μm in
diameter) was examined, there was about a 35 % difference in frequency in perfo-
rated PSDs in EC and SC versus IC rats. Moreover, the relative frequency of
perforated synapses more than tripled between 10 and 60 days of age. Nevertheless,
this phenomenon is not restricted to early brain development following weaning. In
another experiment, rats were placed in the SC or IC environments for 130 days
16 The Postsynaptic Density 503

starting at weaning. The SC animals displayed significantly more synapses with

perforated PSDs than that seen in the IC animals. In this experiment, alterations in
the frequency of perforated PSDs were independent of PSD size, which remained
constant in rats in both conditions.
PSD structure is also influenced by hormones. Sookja K. Chung, Donald
W. Pfaff, and Rochelle S. Cohen showed that estrogen altered the morphology of
PSDs in the midbrain central gray region of the brain of ovariectomized rats; this
region is part of the neural circuit for rodent female reproductive behavior. These
synaptic parameters include an increase in the length of PSDs, an increase in the
number of PSDs with perforations, an increase in the number of synapses showing
positive curvature, and an increase in the number of synapses, themselves. Changes
in the curvature of PSDs may be an index of synaptic efficacy or maturity and may
reflect long-lasting plastic changes.
In regard to LTP and behaviors related to memory and learning, most of the
research has focused on changes in dendritic spine structure, as described in
▶ Chap. 14, “Cell Biology of the Synapse.” However, studies on hippocampal
PSDs by Kristen M. Harris and colleagues indicate that they appear larger and
tend to have more perforations following the induction of LTP, suggesting that the
perforations are transient responses to activation. It has also been shown that an
NMDAR antagonist caused a decrease in PSD volume, either reflecting a decline in
the process of receptor insertion into the membrane, the removal of receptors by the
antagonist, or that the insertion did not occur or occurred outside the time frame of
the experiment.

The PSD in Neurological Disorders

Because of the multitude of proteins in the PSD, the complexity of their interactions,
and their central role in neurotransmission and synaptic plasticity, the PSD is
vulnerable to deleterious alterations in structure and function brought about by
genetic or environmental factors. There is an intimate relationship between dendritic
spines and PSDs via the PSD scaffolds and actin-based cytoskeleton. Perturbation of
these PSD proteins can alter dendritic spine morphology and, therefore, dynamics
and lead to various disorders, which depend on specific spine shapes.
Atypical spine shapes and/or distribution has been seen in humans with Down’s
syndrome and mouse models of this disorder, epilepsy, and in animals undergoing
the natural aging process, for example, and these are discussed in ▶ Chap. 14, “Cell
Biology of the Synapse.”
Scaffolding and other PSD proteins have been implicated in several neurological
disorders. One that is specifically linked to scaffolding proteins is the 22q13.3
deletion syndrome. In this disorder, there is a generalized developmental delay,
normal or accelerated growth, hypotonia (low muscle tone), delays in expressive
speech, and somewhat dysmorphic facial features. The disorder has been directly
associated with the ProSAP2/Shank3 gene, localized on the 22q13.3 chromosome.
Mutations in the Shank3 and in the postsynaptic adhesion molecules neuroligin-3
504 R.S. Cohen

and neuroligin-4 have been identified as causative factors in autism spectrum

disorder. Mutations in SAPAP3/GKAP3 have been linked to obsessive compulsive
disorder. Deletion of the gene for δ-catenin, which encodes a cadherin-associated
protein in PSDs, results in severe mental retardation.

Outlook

Future Directions Combine State-of-the Art Imaging Techniques,

Electrophysiology, Molecular Approaches, and Genetic Animal
Models

Whereas a tremendous amount of knowledge regarding the structure and function of

PSDs has been amassed since Sanford Palay’s first look in the 1950s, this structure
remains an enigma in many ways and the avenues for future research are plentiful.
The development of molecular probes and animal models, as described in the studies
of the Lisman and Bayer groups, allows precise analysis of structure-function
relationships of molecules and their role in electrophysiological events and behav-
iors. These tools may further clarify the role of molecules, such as CaMKII, in LTP
and learning and memory. Cutting-edge imaging technology, such as electron
microscopic tomography, as seen in studies by the Reese group, permits visualiza-
tion of relationships among PSD proteins and a molecular dissection of its subunits.
The remarkable, dynamic views of PSDs as seen in the studies by the Ehlers and
Okabe groups with time-lapse imaging and other imaging techniques give us insight
into the PSD in its natural state and provide a basis for visualizing PSD structure with
different physiological inputs. Several proteomic studies are now emerging; not only
will this approach give us insight into the relationship of proteins within the PSD but
also the relationship of the PSD with other synaptic structures. Seth Grant and
colleagues have now isolated PSDs from adult human neocortical biopsies, and
1,461 proteins were identified using proteonomic profiling, i.e., comparisons of
protein levels in multiple samples. Moreover, the genes that code for these proteins
comprise more than 7 % of the 20,000 protein-coding genes of humans,
underscoring the importance and intricacy of the PSD. The team linked their catalog
into the human genome sequence, relating each of the proteins to a specific gene.
They were able to make evolutionary comparisons with Neanderthals and other
mammals, such as mice, which serve as an important animal model in neuroscience.
Furthermore, they noted that mutations in human PSD genes are involved in the
etiology of 133 neurological and psychiatric diseases and these mutations were
enriched in cognitive, affective, and motor phenotypes involving sets of genes.
Neuroscientists are, therefore, on the precipice of understanding the roles of the
PSD in higher brain functions and complex behaviors and disorders resulting from
the structural and functional disruption of this complex and multifaceted synaptic
structure.
16 The Postsynaptic Density 505

Glossary

α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid (AMPA)

Receptor A glutamate receptor that regulates a channel permeable to Na+ and
K+.
Actin A cytoskeletal protein 6 nm in diameter and found in both monomeric
(G-actin) and filamentous (F-actin) forms. It is found in muscle and non-muscle
cells.
Adenosine Triphosphate (ATP) A nucleotide produced by phosphorylation and
cellular respiration used as an energy source and as a substrate in signal trans-
duction pathways by kinases and adenylate cyclase. Adenylate cyclase uses ATP
to produce cyclic AMP a second messenger molecule.
Adhesion Proteins Proteins involved in the binding of cells to each other usually
consisting of an intracellular domain that may interact with the cytoskeleton, a
transmembrane domain and an extracellular domain.
AMPA Receptor Binding Protein (ABP) An AMPA receptor binding protein that
interacts with glutamate receptor 2/3 (GluR2/3) is homologous to glutamate
receptor-interacting protein (GRIP) and is localized to the postsynaptic density.
It contains seven PDZ domains and may be involved in AMPA receptor regula-
tion and localization.
Ca2+/Calmodulin-Dependent Protein Kinase II (CaMKII) An enzyme with an
ability to phosphorylate its target substrates one of which is itself in a process
called autophosphorylation.
Chemical Long-Term Potentiation (cLTP) Induction of potentiation by chemical
means such as with application of the adenyl cyclase activator forskolin and the
phosphodiesterase inhibitor rolipram.
Complex Environment (EC) A large cage filled with toys for example.
Electron Microscopic Tomography An advanced electron microscopic technique
whereby computed slices of tissues that are thinner than those prepared using
conventional thin sectioning techniques reveal relationships of structures to each
other.
Excitatory Postsynaptic Currents (EPSCs) The flow of ions into a postsynaptic
cell due to the opening of ligand-sensitive channels. It is a synaptic current that
increases the probability of an action potential firing and is mediated by an
increase in Na+ or Ca2+ conductance or a decrease in K+ conductance.
Gamma Aminobutyric Acid (A) Receptor-Associated Protein (GABARAP) A
member of a small family of ubiquitin homologous proteins. It clusters neuro-
transmitter receptors by mediating interaction with the cytoskeleton.
Gamma Aminobutyric Acid (GABA) An inhibitory neurotransmitter. Inhibitory
transmitters are likely to block the events that cause an action potential in the
responding neuron.
Gamma Aminobutyric Acid (GABA) Receptors (GABARs) GABAA receptors
are ligand-gated chloride ion channels and GABAB receptors are G-protein-
coupled receptors.
506 R.S. Cohen

Glutamate Receptor-Interacting Protein (GRIP) A scaffold protein containing

PDZ domains. It is an AMPA receptor binding protein and appears to be involved
in the targeting of AMPA receptors.
G-Protein-Coupled Inward Rectifying K+ (GIRK) Channels A family of inward
rectifier potassium ion channels that open via a signal transduction cascade.
Pertussis toxin-sensitive G-protein-coupled receptors lead the cascade releasing
activated G protein βγ-subunits (Gβγ) from inactive G protein complexes (Gαβγ).
The Gβγ protein then interacts with G-protein-coupled inward rectifying K+
(GIRK) channels, which then open and become permeable to potassium ion.
Hyperpolarization of the cell results. The channels regulate slow postsynaptic
inhibitory signaling and hormone secretion.
Green Fluorescent Protein (GFP) A protein derived from the jellyfish Aequorea
victoria. The jellyfish contains a photoprotein called aequorin, which releases
blue light when bound to calcium. The blue light is absorbed by green fluorescent
protein, which then gives off a green light. The green fluorescent protein gene
may be joined to a gene for a protein of interest, and when the protein is
translated, it will have green fluorescent protein associated with it. Following
excitation with blue light, it will exhibit green fluorescence.
Guanosine Triphosphate (GTP) A purine nucleotide important in signal trans-
duction particularly with G proteins.
Guanylate Kinase-Associated Protein (GKAP) A protein that links Shank to the
PSD-95 protein complex including NMDA-type glutamate receptors. It is a
synaptic protein that binds directly to the guanylate kinase-like (GK) domain of
the four members of the PSD-95 family, and it appears to be a major component
of the postsynaptic density. GKAP may link ion channel/PSD-95 clusters to the
subsynaptic cytoskeleton or downstream signal transduction cascades. It
undergoes activity-dependent ubiquitination and degradation, thereby playing a
role in activity-dependent synaptic remodeling via the ubiquitin-proteosome
system.
Homer A postsynaptic scaffolding protein involved in maintenance and activity-
induced synaptic plasticity.
Impoverished or Isolated Conditions (IC) A standard laboratory cage.
Inositol Triphosphate (IP3) and Inositol Triphosphate Receptor (IP3R) IP3 is
involved in signal transduction. IP3 and diacylglycerol are generated when an
extracellular regulatory molecule binds to a membrane receptor that activates a G
protein. The alpha subunit of the G protein subsequently activates the enzyme
phospholipase C which cleaves off IP3 from a membrane phospholipid with
diacylglycerol remaining in the membrane. Specifically, IP3 binds to its receptor
IP3R on the membrane of the rough endoplasmic reticulum and sarcoplasmic
reticulum of muscle and opens a calcium channel, resulting in the release of
calcium into the cytoplasm and sarcoplasm of muscle, respectively. The calcium
then activates various cellular processes.
Ionotropic Glutamate Receptors (iGLURs) Heteromeric ligand-gated ion chan-
nels which open in response to glutamate and play a role in the mediation of
16 The Postsynaptic Density 507

excitatory synaptic transmission. They display four hydrophobic regions within

its central portion.
Latrunculin A toxin derived from sponges which binds to actin and prevents its
polymerization.
Long-Term Depression (LTD) A persistent reduction in synaptic efficacy resulting
from either high- or low-frequency stimulation.
Long-Term Potentiation (LTP) Alterations in synaptic strength that persists in
brain slices and in intact animals when high-frequency tetani is applied to the
perforant pathway of the hippocampus a brain region involved in memory
storage.
Membrane-Associated Guanylate Kinase-Like Proteins (MAGUK) Family A
family of scaffolding proteins which are involved in the regulation and formation
of cell junctions.
Metabotropic Glutamate Receptors (mGLURs) Types of glutamate receptors
which are members of the G-protein-coupled receptors. They have seven trans-
membrane domains, which span the cell membrane. They do not function as ion
channels like ionotropic glutamate receptors but, rather, activate biochemical
cascades which, in turn, lead to the modification of other proteins and, ultimately,
processes.
Microtubule-Associated Proteins (MAPs) Accessory proteins associated with
microtubules that are involved in assembly and stabilization of microtubules
and binding to other microtubules or other filaments or cell organelles.
Nanometer (nm) One billionth (10 9) of a meter.
N-Cadherin A molecule meaning “calcium-dependent adhesion.” It is calcium
dependent and plays a role in cell adhesion.
N-Ethylmaleimide-Sensitive Factor (NSF) A scaffold protein which participates
in AMPA receptor trafficking.
Neuroligins Adhesion molecules involved in trans-synaptic signaling.
N-Methyl-D-Aspartic Acid (NMDA) Receptor A glutamate receptor that regu-
lates a channel permeable to Ca2+ K+, and Na+.
Palmitoylation A posttranslational lipid modification of neuronal proteins.
PDZ Domain A structural domain of 80–90 amino acids. PDZ is an acronym for
the first letters of the three proteins: PSD-95; Drosophila disc large tumor
suppressor; and zona occludens-1 protein.
Postsynaptic Density (PSD) Dense area behind the postsynaptic membrane
containing receptors ion channels, scaffolding proteins, and cytoskeletal proteins.
Postsynaptic Density Protein (PSD-95) A molecule displaying a sequence of three
repeated 90 residue domains called PDZ (PSD-95; Drosophila disc large tumor
suppressor; and zona occludens-1 protein) domains an SH3 domain, and a
guanylate kinase-like (GK) domain, which acts as a protein binding domain. It
is a member of the core scaffold complex of excitatory synapses.
Protein Interacting with Kinase C (PICK) Scaffold proteins displaying a string of
PDZ domains. PICK1 interacts with glutamate receptor subunits and participates
in AMPA receptor trafficking.
508 R.S. Cohen

Protein Kinase C α (PKCα) A family of serine- and threonine-specific protein

kinases that can be activated by calcium and diacylglycerol. It is involved in
AMPA receptor trafficking.
Proteonomic Profiling Proteomics subject to quantitative analysis revealing dif-
ferences in protein expression across samples.
Proteonomics The study of an organism’s complete array of proteins
encompassing those modifications made to specific sets of proteins, including
phosphorylation, ubiquitination, and others. The word proteome, itself, is derived
from proteins and genome. Various techniques contribute to the study of
proteonomics, including those which ascertain protein-protein interactions, such
as protein microarrays and mass spectrometry.
Pseudophosphorylation A process whereby a mutated form of calcium/calmodu-
lin-dependent protein kinase II (CaMKII) maintains the enzyme in its active state
independent of Ca2+/calmodulin binding. It is a functional “mimicry” of
phosphorylation.
Shank A family of scaffold proteins containing many sites for protein-protein
interactions, located at postsynaptic sites of brain excitatory synapses.
Small-Conductance Ca2+-Activated K+ (SK) Channels A subfamily of K+-acti-
vated Ca2+ channels. Activated by an increase in intracellular Ca2+ concentration
they permit K+ movement across the cell membrane. When activated, the firing
frequency of action potentials is limited and the resultant hyperpolarization
decreases the firing frequency of action potentials in several neurons. It is
important in regulating after hyperpolarization and also plays a role in the
regulation of dendritic excitability, synaptic transmission, and synaptic plasticity.
Social Environment (SC) A standard laboratory cage containing one other rat.
Stargazin A transmembrane protein specific to brain, its cytoplasmic tail influences
AMPA receptor trafficking, i.e., clustering and regulation of AMPA receptors,
and the domain projecting into the extracellular space regulates channel gating.
Subcellular Fractionation A method for enriching cell fractions. First differential
centrifugation separates nuclear, cytoplasmic, and membrane components based
on the size and density of particular organelles. Following differential centrifu-
gation, enrichment of other organelles and/or membranes is achieved by density
gradient centrifugation, whereby a continuous or stepped gradient of sucrose or
cesium chloride is formed in a centrifuge tube. The relevant sample is placed on
top of the gradient, and following further centrifugation at high relative centrif-
ugal forces (g-force), particular organelles will settle at the position in the gradient
at which their density corresponds to that of the surrounding sucrose or cesium
chloride.
Synapse-Associated Protein 90/Postsynaptic Density-95-Associated Protein
(SAPAP) A protein concentrated in the postsynaptic density and binds to the
guanylate cyclase domain of PSD-95.
Synapses Sites of contacts between nerve cells. Chemical neurotransmission takes
place at these sites.
16 The Postsynaptic Density 509

Synaptosomal Membrane Fraction A fraction of membranes enriched in mem-

branes from the presynaptic and postsynaptic terminals. They are obtained after
lysing the synaptosome the pinched-off nerve ending, with water or dilute buffer,
and enriched upon density gradient centrifugation.
Synaptosomes Nerve endings pinched off from axons. They are formed during
homogenization of brain tissue.
SynGAP A Ras-GTPase activating protein that interacts with the PDZ domains of
PSD-95 and synapse-associated protein (SAP) 102. It associates with NMDA-
type glutamate receptor.
Transmembrane Receptor AMPA Regulating Proteins (TARPs) Proteins
involved in the trafficking and regulation of AMPA receptors. Stargazin is a
member of the TARP family.

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