Glutamatergic Synapses: Molecular Organization

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1 Glutamatergic Synapses: Molecular Organization Morgan Sheng, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA Jerry W Lin, Harvard Medical School, Boston, MA, USA Glutamatergic synapses, which comprise the majority of excitatory synapses in the mammalian central nervous system, function by presynaptic release of glutamate onto postsynaptic glutamate receptors. At the postsynaptic site, the molecular interactions between glutamate receptors and various intracellular proteins suggest an intimate relationship between synaptic structure and function, and may provide insight into the mechanisms of synaptic plasticity that underlie learning and memory. Glutamate as the Transmitter at Excitatory Synapses in the Central Nervous System Synapses are specialized cell cell junctions that allow neurons to communicate with each other and with nonneuronal targets. Upon presynaptic stimulation, neurotransmitters are released from the presynaptic terminal into the synaptic cleft. Neurotransmitters diffuse across this intercellular space and bind to neurotransmitter receptors on the postsynaptic membrane to effect a postsynaptic response. Synapses can be classified as excitatory or inhibitory depending on the effect of presynaptic stimulation on the postsynaptic potential. Excitatory synapses induce an excitatory postsynaptic potential (EPSP) that depolarizes the membrane toward the threshold required for activation of an action potential. Conversely, inhibitory synapses induce an inhibitory postsynaptic potential that hyperpolarizes the membrane away from the threshold potential. Glutamate is the major excitatory neurotransmitter in the vertebrate central nervous system (CNS). Glutamate was first identified as an excitatory neurotransmitter when it was observed that glutamate applied topically to the motor cortexcaused tonic convulsions, and that high concentrations of glutamate existed in mammalian brain. Anatomical evidence for glutamate as a neurotransmitter came with the development of specific antibodies to glutamate. Immunocytochemistry combined with anterograde labelling of primary afferent terminals demonstrated an accumulation of glutamate in nerve terminals, an anatomical prerequisite for any neurotransmitter. In the hippocampus, lesions of the major afferents to the dentate gyrus from the entorhinal cortexled to decreased glutamate release in the dentate gyrus, suggesting that glutamate acts as the neurotransmitter for the entorhinal input to the dentate gyrus. Furthermore, studies in the Secondary article Article Contents. Glutamate asthe Transmitterat Excitatory Synapsesin the Central Nervous System. Three Classes of Glutamate Receptors in the Postsynaptic Membrane. Dendritic Spines as the Postsynaptic Target for Glutamatergic Synapses. A Prominent Postsynaptic Density Associated with Glutamatergic Synapses. Clustering of Glutamate Receptors at the Postsynaptic Membrane by PDZ Domain-containing Proteins. Postsynaptic Signal Transduction Molecules that Respond to Calcium and their Roles in Learning and Memory. Synthesis of Certain Postsynaptic Membrane Proteins within the Dendrite. Summary hippocampus showed that glutamate is released in a calcium-dependent manner upon depolarization. Glutamate demonstrates all the cardinal features of an excitatory neurotransmitter. It is stored in synaptic vesicles in the presynaptic terminal where it can be released in a calcium-dependent manner into the synapse. Glutamatecontaining vesicles are specifically associated with excitatory synapses, and are not found in the same synaptic terminals as g-aminobutyric acid (GABA), the major inhibitory transmitter of the CNS. High-affinity glutamate transporters in the presynaptic membrane as well as in neighbouring glial cells provide a mechanism for rapid glutamate uptake and signal termination. Exogenously applied glutamate induces an excitatory postsynaptic response identical to that elicited by presynaptic stimulation. And finally, compelling indirect evidence for glutamate being the major excitatory neurotransmitter comes from the specific concentration of glutamate receptors at the postsynaptic site in excitatory synapses. Three Classes of Glutamate Receptors in the Postsynaptic Membrane Glutamate receptors are transmembrane proteins that bind specifically to glutamate on the extracellular side of the membrane. Upon binding of glutamate, glutamate receptors are activated and transduce this signal into intracellular responses. Several classes of glutamate receptors can be distinguished by their functional and pharmacological properties. In addition, the development of selective agonists and antagonists has led to the further subdivision of these classes of glutamate receptors. Most recently, molecular cloning of glutamate receptors has ENCYCLOPEDIA OF LIFE SCIENCES 2001, John Wiley & Sons, Ltd. 1

2 confirmed the existence of the major types, and revealed an even greater genetic heterogeneity than was predicted from pharmacological studies. The diversity of glutamate receptor subtypes along with the neuron s capability to combine different receptors at a single synapse allows for a variety of postsynaptic responses to a given presynaptic input. Glutamate receptors can be grouped into two main categories: ionotropic receptors (which are glutamategated cation channels) and metabotropic receptors (which are glutamate activated G protein-coupled receptors). The ionotropic receptors can be further categorized into N- methyl-d-aspartate (NMDA) receptors and non-nmda receptors. NMDA receptors NMDA receptors are ligand-gated ion channels that are selectively permeable to divalent cations, particularly Ca 2 1. They consist of multimeric complexes of homologous subunits from two subfamilies: NR1 and NR2. NR1 is essential for NMDA receptor function and is able to form weakly functional homomeric channels in Xenopus oocytes. Four NR2 subunits (NR2A D), each encoded by its own gene, are unable to form functional channels on their own, but greatly enhance NMDA receptor function when coexpressed with NR1. Each NR2 subunit confers distinct pharmacological and electrophysiological properties on the heteromeric NMDA receptor complex. Further NMDA receptor diversity arises from alternative splicing at three sites in the NR1 messenger RNA (mrna) that generates eight distinct NR1 splice variants. Though still controversial, it has been suggested that each NMDA receptor represents a tetramer of two NR1 and two NR2 subunits. Topologically, each subunit contains an extracellular N-terminus, followed by three membrane-spanning domains (M1, M3 and M4) and one membrane loop (M2) resulting in an intracellular C-terminal tail. Pharmacologically, the NMDA receptor has sixdistinct sites: (1) a principal ligand site that opens the channel when bound by glutamate; (2) a glycine site which also must be occupied for the channel to open; (3) a polyamine-binding site that can potentiate NMDA receptor activity when both the glutamate and glycine sites are saturated; (4) a voltage-dependent magnesium-binding site within the channel that blocks the NMDA receptor pore at membrane resting potential; (5) a voltage-independent zincbinding site that inhibits NMDA receptor activity; and (6) a site within the channel that binds open channel or usedependent blockers of NMDA receptor activity such as phencyclidine, MK-801, or ketamine. These multiple sites contribute to the pharmacological complexity of NMDA receptors. NMDA receptors have been implicated in the induction of certain forms of synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD) in the hippocampus. LTP and LTD are long-lasting, activitydependent increases and decreases in synaptic efficacy, respectively. Interestingly, the voltage-dependent magnesium block of NMDA receptors permits receptor activation only at depolarized membrane potentials. This allows NMDA receptors to act as coincidence detectors that activate exclusively during simultaneous presynaptic release of glutamate and postsynaptic depolarization (arising presumably from an alternative input). Given NMDA receptor involvement in synaptic potentiation, coincidence detection by NMDA receptors offers an attractive mechanism to support a Hebbian model of synaptic plasticity. Non-NMDA: a-amino-3-hydroxy-5- methylisoxazole-4-propionic acid receptors (AMPARs) and kainate (KA) receptors Non-NMDA receptors are responsible for most of the short and fast excitatory transmission in the vertebrate CNS. They are voltage-independent ion channels, permeable to Na 1 and K 1, that permit a net depolarizing influx of cations upon activation by glutamate. They share similar membrane topology with NMDA receptors, and like NMDA receptors, non-nmda receptors represent heteromeric complexes of various subunits. There are two classes of non-nmda receptors based on pharmacology: AMPA receptors and KA receptors. AMPA receptors are composed of four possible subunits (GluR1 4) whereas KA receptors consist of five possible subunits (GluR5 7 and KA1 2). Each subunit can form homomers or heteromers with other subunits within its class but no complexes are formed between subunits from different classes. NMDA and non-nmda receptors can be distinguished not only pharmacologically, but also in a number of functional respects. NMDA receptors mediate a relatively slow and long-lasting excitatory postsynaptic current (EPSC) while AMPA receptors generate fast, short-lasting EPSCs. Whereas NMDA receptors can initiate signalling pathways via Ca 2 1 influx, AMPA receptors are relatively impermeable to calcium. This low Ca 2 1 permeability is imparted by an RNA editing mechanism whereby a crucial glutamine in the pore-forming region of the GluR2 subunit is changed to arginine. This substitution renders the GluR2 subunit impermeable to Ca 2 1 ; moreover, all AMPA receptors that contain an edited GluR2 subunit are relatively impermeable to Ca 2 1. Conversely, when the other three AMPA subunits (GluR1, GluR3 and GluR4) are expressed as homo- or heteromers among themselves, the resultant channels are found to be permeable to Ca 2 1. Such Ca 2 1 -permeable AMPA receptors, however, are rarely found in vivo and exist only in a few specialized populations of neurons such as inhibitory interneurons. 2

3 Until recently, AMPA and KA receptors have been difficult to distinguish due to the lack of specific pharmacological agents. Initial evidence for separate receptors came from observation of differential spinal cord C-fibre sensitivities to AMPA and KA stimulation. Closer examination of the receptors revealed that KA receptors undergo rapid desensitization after activation while AMPA receptors undergo relatively little desensitization. Recently, a specific AMPA receptor antagonist (GYKI 53655) has enabled the functional isolation of KA receptors. Studies using GYKI have revealed that KA receptors contribute to postsynaptic currents and antagonize GABAergic inhibition of CA1 neurons in the hippocampus. These results suggest a role for KA receptors in pathologies resulting from neuronal hyperexcitability such as epilepsy and excitotoxicity. Metabotropic glutamate receptors (mglurs) Metabotropic glutamate receptors are coupled to G proteins (which in turn stimulate second messenger signalling pathways in cells), and as such, they mediate slower synaptic responses (occurring over seconds and minutes, rather than milliseconds for ionotropic glutamate receptors). The mglur family contains seven members (mglur1 7), each encoded by its own gene. Each mglur has a large extracellular domain that binds glutamate and seven membrane-spanning domains typical of G proteincoupled receptors. The seven family members can be separated into three subgroups based on primary sequence similarity, mode of intracellular signal transduction, and pharmacology. mglur1 and mglur5 respond strongly to AMPA and are coupled to phosphoinositol hydrolysis and subsequent release of Ca 2 1 from intracellular stores. mglur2 and mglur3 are activated by ACPD (trans-1- amino-cyclopentane-1,3-dicarboxylate) and are negatively coupled to adenylate cyclase to reduce levels of camp (cyclic adenosine monophosphate). mglur4, mglur6 and mglur7 are also negatively coupled to adenylate cyclase but prefer as agonist L-AP4 (l-2-amino-4-phosphonobutyrate). Due to a paucity of specific agonists and antagonists for each of the three subgroups, the mglurs have defied precise functional characterization. Presently, phenylglycine derivatives are being identified as mglur subtype-specific antagonists. As the pharmacology of mglurs matures, the specific functions of each subgroup should become more apparent. Dendritic Spines as the Postsynaptic Target for Glutamatergic Synapses In neurons, many glutamate receptors are uniquely located on spines, small protrusions from the surface of neuronal dendrites. Glutamate receptors are less often found on dendritic shafts and neuronal cell bodies, in contrast to inhibitory receptors such as GABA receptors. Dendritic spines are believed to contain exclusively excitatory receptors and represent the major postsynaptic target of excitatory synaptic input. Morphologically, a prototypical spine has a terminal varicosity, the head, connected to the dendritic shaft by a neck of various length and width. Necks can vary in length between 0.08 and 1.58 mm and in diameter between 0.04 and 0.46 mm. The total volume of spines ranges between and 0.56 mm 3, with the average volume being mm 3. The small size and unique shape of the dendritic spine allows it to function as a specialized compartment within neurons. Initially, spines were hypothesized to function as electrically isolated compartments, but more recently, the view has gained favour that they operate as tiny biochemical compartments regulated by synaptic activity. Because of the small volume of the dendritic spine, a large increase in postsynaptic calcium concentration can be obtained even after a small influxof calcium ions. This may facilitate NMDA receptor-mediated responses, which typically arise from postsynaptic increases in calcium mediated by calcium influxthrough NMDA receptors. The density and activity of calcium pumps in the membrane of the spine neck can then modulate the amount of calcium that leaks from the spine head to the dendritic shaft. Properties of the spine neck could determine the amount of synaptic signal (in this case, an increase in calcium concentration) that reaches the dendrite. These very same properties would also serve to insulate the spine head from dramatic changes in calcium concentrations in the dendritic shaft or in neighbouring dendritic spines. Thus, dendritic spines are anatomical specializations that probably allow for highly localized (i.e. synapse-specific) postsynaptic signal processing. A Prominent Postsynaptic Density Associated with Glutamatergic Synapses Particularly prominent in dendritic spines are structures known as postsynaptic densities (PSDs). PSDs, typically several hundred nanometres in diameter, were first identified by electron microscopy in the 1950s. They were described as amorphous electron-dense structures immediately adjacent to the cytoplasmic face of the postsynaptic membrane. PSDs are found at the postsynaptic site directly apposed to the active zones in the presynaptic terminal (where synaptic vesicles fuse with the presynaptic membrane to effect exocytotic release of neurotransmitter into the synaptic cleft). Isolated PSDs appear to be a network of filamentous material holding together particulate components. In addition to their localization at axospinous 3

4 synapses, PSDs have also been observed at asymmetric (presumably excitatory) axodendritic synapses. PSDs are not always continuous structures and may be perforated. In cross-section, perforated PSDs look merely like discontinuous linear structures; en face, however, perforated PSDs appear as discs with punched-out holes. Biochemical characterization of the PSD became possible when procedures were developed that allowed for their purification. Fractions highly enriched in PSDs are typically obtained by detergent extraction of centrifugally purified synaptosomes. A wide variety of PSD components have been identified which can be categorized into five functional groups: (1) Cytoskeletal proteins such as actin, tubulin, fodrin and a-actinin-2. The PSD can be considered in part a local specialization of the submembrane cytoskeleton that supports the architecture of the postsynaptic site. (2) Cell adhesion molecules such as N- cadherin, neuroligin and densin-180. The presence of cell adhesion molecules is consistent with a proposed role for the PSD in the adherence of pre- and postsynaptic membranes and in the apposition of pre- and postsynaptic machinery. (3) Glutamate receptors, such as NMDA receptors and AMPA receptors. As mentioned, ionotropic receptors that are activated by the excitatory neurotransmitter glutamate are concentrated in the PSD. (4) Signal transduction proteins, including the calcium-binding protein calmodulin (CaM); serine/threonine protein kinases such as Ca 2 1 /calmodulin-dependent kinase II (CaMKII), protein kinase C (PKC), and camp-dependent kinase (PKA); tyrosine kinases like src and fyn; and phosphatases such as protein phosphatase 1 (PP1) and calcineurin (CaN). These proteins are involved in transducing signals from the postsynaptic membrane receptors into the cell. In addition, these signalling molecules may also act directly on glutamate receptors at the PSD to modulate their function. Thus, the PSD can be considered a localized signalling complexspecialized for reception and transmission of excitatory synaptic signals. Postsynaptic signal transduction is made more efficient and more specific because the relevant proteins are concentrated in the PSD and brought into physical proximity of the receptors that activate them, possibly even placing the intracellular signalling components directly at the mouth of the receptor/channel. (5) Scaffolding proteins, such as the PSD-95 family of membrane-associated guanylate kinases (MAGUKs), and the glutamate receptor interacting protein (GRIP). These proteins embody the essence of the PSD in that they mediate the formation of specific multiprotein complexes and organize the structure and function of the postsynaptic specialization. Clustering of Glutamate Receptors at the Postsynaptic Membrane by PDZ Domain-containing Proteins Multimeric complexes of proteins at the PSD represent functional interactions between intracellular proteins and receptors and ion channels, and the PSD-95 family of PDZ domain-containing proteins may form the scaffolding upon which these multiprotein complexes are constructed. PSD-95 family members contain three N-terminal PDZ domains followed by an SH3 domain and a guanylate kinase-like (GK) domain. The first and second PDZ domains of PSD-95 bind the cytoplasmic C-terminal tails of NMDA receptors and Shaker-type K 1 channels (Kornau et al., 1995; Niethammer et al, 1996). This marked the initial demonstration that PDZ domains are modular protein interaction motifs that bind to specific short peptide sequences at the very C-termini of interacting proteins. Except for one protein, SAP97, other PSD-95 family members are predominantly postsynaptic and appear to behave in vitro identically to PSD-95 (also known as SAP90). The interaction between PSD-95 family members and receptor/channel proteins may subserve four major functions: (1) aggregation or clustering of the receptor/channel; (2) targeting of the receptor/channel to specific membrane domains; (3) immobilization of the receptor/channel by anchoring to the cortical cytoskeleton; and (4) coupling of the receptor/channel to downstream signalling molecules (see Figure 1). NMDA PSD-95 AMPA GRIP Figure 1 Interactions between glutamate receptors and PDZ-containing scaffolding proteins may subserve several functions, notably aggregation or clustering of the receptor/channel; targeting of the receptor/channel to specific membrane domains; immobilization of the receptor/channel by anchoring to the cortical cytoskeleton; and coupling of the receptor/ channel to downstream signalling molecules. 4

5 Clustering function of PSD-95 family proteins PSD-95 can induce clustering of binding partners (such as NMDA receptors and Shaker K 1 channels) when coexpressed with them in heterologous cells (Kim et al., 1995; Kim et al., 1996). PSD-95 cannot form clusters on its own, but rather induces coclustering of binding partners via a mechanism that depends on a head-to-head multimerization of PSD-95. This head-to-head interaction is mediated by the conserved N-terminal regions of PSD-95 family proteins which contain a pair of cysteines that appear to be sites of palmitoylation and/or disulfide bond formation. Whether or not this clustering mechanism occurs in vivo has yet to be determined. Synaptic targeting/localization function of PSD family proteins Evidence for a targeting function for the PSD-95 family proteins comes from fly genetics. PSD-95 exists in Drosophila as a single known homologue called discs large (Dlg), which has the same domain organization and binding specificities as PSD-95. Dlg proteins are concentrated in Drosophila neuromuscular junctions (NMJs) where they are colocalized with both Shaker K 1 channels and glutamate receptors. Dlg mutant flies have abnormal NMJ morphology and Dlg loss-of-function mutants are unable to target Shaker K 1 channels to the NMJ (Tejedor et al., 1997). Furthermore, hybrids of heterologous proteins with the Shaker K 1 channel C-terminal tail are found to be targeted to the NMJ in wild-type but not Dlg mutant flies. The role of Dlg in glutamate receptor targeting has not yet been defined, but Dlg appears not to interact with glutamate receptors in flies as PSD-95 interacts with NMDA receptors in mammals. Anchoring of NMDA receptors to cytoskeleton NMDA receptors are tightly associated with the PSD and are insoluble in all but strong detergents such as sodium dodecyl sulfate (SDS). These characteristics suggest that NMDA receptors are anchored at the postsynaptic site to insoluble proteins that are components of the cytoskeleton. One mechanism of anchoring is likely to involve PSD-95 family proteins. Recently, a novel postsynaptic protein called CRIPT has been shown to bind specifically to the third PDZ domain of PSD-95 family members and to recruit them to microtubules in heterologous cells. These findings suggest that CRIPT may link the PSD-95- associated complex, which contains NMDA receptors, to a tubulin-based cytoskeleton at the postsynaptic site. PSD-95-mediated links between NMDA receptors and the cytoskeleton necessarily involve NMDA receptor subunit NR2 because only NR2 can bind to PDZ domains of PSD-95 family members. The essential NR1 subunit of NMDA receptors (at least its common splice variants) does not bind PSD-95 but may interact directly with the cytoskeleton. The membrane proximal region, C0, found in the cytoplasmic tail of all NR1 splice variants, binds a- actinin-2, an actin crosslinking protein. Ca 2 1 /CaM can compete with a-actinin-2 for binding to NR1, and the interaction between Ca 2 1 /CaM and NR1 has been shown to downregulate NMDA receptor function, perhaps by dissociating it from the actin-based cytoskeleton (Wyszynski et al., 1997; Krupp et al., 1999). An alternatively spliced exon segment, C1, in the NR1 cytoplasmic tail harbours the major PKC phosphorylation sites of NR1. The existence of C1 and its phosphorylation state can regulate the clustering of NR1 proteins expressed in heterologous cells. Recent studies have identified two proteins that bind directly to this C1 segment: neurofilament light chain (NF-L) and yotiao, a novel putative cytoskeletal protein. The functional significance of these interactions remains to be determined. Interaction of AMPA receptors with GRIP Despite the fact that they are localized postsynaptically in glutamatergic synapses with NMDA receptors, AMPA receptors do not interact with PSD-95 family members. Instead, GluR2 and GluR3 subunits bind to domains PDZ4 and PDZ5 of a novel protein, GRIP, that has seven PDZ domains. Interestingly, overexpression of a competing GluR2 C-terminal peptide inhibits synaptic localization of AMPA receptors, suggesting a possible involvement of GRIP binding in the synaptic targeting of AMPA receptors (Dong et al., 1997). The biochemical and function characterization of GRIP lags behind PSD-95. Nevertheless, the identification of a distinct multivalent PDZ protein that probably anchors AMPA receptors suggests that the glutamatergic postsynaptic site can be divided into NMDA receptor- and AMPA receptorassociated microdomains, each with a distinct set of proteins and each linked to a specific intracellular signalling pathway. Postsynaptic Signal Transduction Molecules that Respond to Calcium and their Roles in Learning and Memory Among glutamate receptors, the NMDA receptor is unique in its high permeability to calcium. Postsynaptic signalling molecules that respond to calcium are therefore likely to mediate many of the intracellular events following NMDA receptor activation. Morever, NMDA receptor activation and the ensuing rise in intracellular calcium are critical for the induction of LTP and LTD, forms of synaptic plasticity that may embody the cellular basis for memory formation in the brain. Thus, calcium-dependent 5

6 signalling molecules in the PSD are likely to play major roles in the plasticity of synapses thought to underlie learning and memory. Ca 2 1 /calmodulin-dependent kinase II Ca 2 1 /calmodulin dependent kinase II (CaMKII) is abundant in the PSD and can cause phosphorylation of many PSD proteins including AMPA receptors. In fact, CaMKII has been shown to potentiate AMPA receptor function by direct phosphorylation of the GluR1 subunit. Once activated by calcium/calmodulin, CaMKII undergoes autophosphorylation in its regulatory domain that renders it relatively calcium-independent. This autophosphorylation can be thought of as a molecular switch that translates a transient rise in calcium concentration into a prolonged increase in kinase activity. CaMKII can also control gene expression by phosphorylating cyclic AMP response element-binding protein (CREB), a transcription factor that may activate a programme of gene expression necessary for the maintenance of LTP and memory. Consistent with a role in synaptic plasticity, mice deficient in CaMKII exhibit impaired hippocampal LTP (Silva et al., 1992). Furthermore, introduction of CaMKII into neurons has been shown to enhance postsynaptic responses and to occlude further induction of LTP by synaptic stimulation. Taken together, the evidence strongly supports an important role for CaMKII in synaptic plasticity and possibly learning and memory. Neuronal nitric oxide synthase The Ca 2 1 /CaM-dependent enzyme neuronal nitric oxide synthase (nnos) is partially localized to the PSD, possibly by binding to PDZ2 of PSD-95. Interestingly, rather than via the typical C-terminus-binding mechanism, this interaction occurs by a PDZ PDZ interaction involving the single PDZ domain at the N-terminus of nnos (Brenman et al., 1996). Since PSD-95 binds directly to NMDA receptors, its interaction with nnos could bring this calcium-regulated enzyme under the influence of calcium influxthrough activated NMDA receptors. nnos catalyses the conversion of l-arginine into l-citrulline and the signalling gas nitric oxide (NO). NO can inhibit NMDA receptors by s-nitrosylation and thus may function in negative feedback inhibition of NMDA receptors. NO may also play a role as a retrograde messenger in LTP. Although it has been established that induction of LTP involves the NMDA receptor and postsynaptic Ca 2 1 influx, the maintenance of LTP involves presynaptic mechanisms, at least in part. NO produced in the postsynaptic cell may diffuse to the presynaptic terminal to enhance transmitter release, but the mechanisms for such an action have yet to be established. Currently, the role of NO in synaptic plasticity and/or learning and memory remains controversial. Calcineurin Calcineurin (also known as PP2B) is a Ca 2 1 /CaMdependent serine/threonine protein phosphatase located not only at the PSD but throughout the entire neuron. LTP and LTD have been hypothesized to represent opposite manifestations of a common mechanism of synaptic plasticity, in which synaptic efficacy depends on the phosphorylation state of a set of synaptic proteins. As a phosphatase, calcineurin opposes the activity of kinases such as CaMKII and can be expected to have an inhibitory effect on synaptic potentiation. Indeed, calcineurin decreases the mean open time and open probability of NMDA receptors whereas calcineurin inhibitors prolong NMDA receptor opening times and block NMDA receptor desensitization. Inhibitors of calcineurin have also been shown to enhance Ca 2 1 -dependent glutamate release from presynaptic terminals, suggesting that calcineurin activity can suppress glutamate release. In hippocampal slices, inhibition of calcineurin enhances basal synaptic transmission and lowers the threshold for LTP induction while blocking LTD induction. Calcineurin activity is thought to enhance the activity of protein phosphatase 1 (PP1) by dephosphorylating and inactivating the specific PP1 inhibitor protein. Interestingly, PP1 can dephosphorylate and inactivate PSD-associated CaMKII, providing an alternative mechanism by which calcineurin can oppose synaptic potentiation. Furthermore, PP1 may dephosphorylate and inactivate CREB, a CaMKII substrate. It has been hypothesized that prolonged synaptic activity, such as that which stimulates LTP induction, inactivates calcineurin and increases the lifetime of phosphorylated CREB, thus promoting the transcription of genes necessary to maintain LTP. Calpain Calpain is a Ca 2 1 -dependent protease found at postsynaptic sites that appears to be activated specifically by NMDA receptor stimulation. NMDA receptor blockers limit the activity of calpain, and inhibitors of calpain have been shown to block LTP induction in the CA1 region of the hippocampus. This evidence suggests a role for calpain in NMDA receptor-mediated synaptic plasticity. Calpain activity may facilitate induction of LTP by proteolytically generating fragments of CaMKII or PKC that are constitutively active. Calpain also selectively degrades actin and fodrin, a form of spectrin found only in the brain. The degradation of these cytoskeletal proteins may mediate calpain-induced changes in spine architecture, although whether or how this would affect synaptic 6

7 strength is unclear. Aside from their effect on spine morphology, cytoskeletal remodelling may also redistribute and modulate AMPA and NMDA receptors. Interestingly, calpain has recently been suggested to proteolyse NMDA receptor subunit NR2 and AMPA receptor subunit GluR1 close to their C-termini. If other AMPA subunits such as GluR2 or GluR3 can be processed in a similar manner, then calpain proteolysis may represent a common means by which NMDA and AMPA receptors can be freed from anchoring proteins such as PSD-95 and GRIP. Redistribution of these receptors may then contribute to alterations in synaptic strength. Ras Ras is a small guanine nucleotide-binding protein that localizes to the inner face of the plasma membrane, including at postsynaptic sites. Ras is activated at the membrane by receptor tyrosine kinases and by calcium influxand transmits these signals to intracellular mitogenactivated protein kinase (MAPK) cascades via successive phosphorylation of raf and mitogen-activated/extracellular receptor-regulated kinases (MEKs). Activated MAPK can phosphorylate Elk-1, a transcription factor that acts in conjunction with serum response factor to activate a programme of gene expression that promotes neuronal differentiation, neurite outgrowth and neural plasticity. Concomitantly, MAPK can phosphorylate and inactivate CREB2, a transcription repressor, thereby further stimulating the same set of immediate early genes activated by Elk-1. The role of MAPK in synaptic plasticity has been well documented in two systems: the generation of longterm facilitation in the neurons that control the Aplysia gill-withdrawal reflex(martin et al., 1997; Bailey et al., 1997) and the increases in synaptic strength observed in Drosophila mutants that have constitutively high synaptic activity or camp levels (the camp-dependent kinase PKA can phosphorylate MAPK) (Schuster et al., 1996a; Schuster et al., 1996b). Clearly, ras activation can lead to synaptic enhancement, but how do NMDA receptor activation and subsequent increases in calcium lead to ras activation? Increased calcium concentrations have been shown to promote the tyrosine phosphorylation of PYK2, a postsynaptic tyrosine kinase. PYK2 is hypothesized to activate ras by recruiting Shc and the Grb2 SOS complexto phosphotyrosine residues on the activated PYK2 protein. SOS is a known guanine nucleotide exchange factor (GEF) that activates ras by exchanging its bound GDP for GTP. Alternatively, calcium may bypass tyrosine kinases altogether and activate ras by means of a Ca 2 1 -sensitive ras- GEF called ras-grf. Although all the mechanisms of ras activation are, in theory, possible, they have yet to be demonstrated in neuronal synapses. Recently, a novel ras GTPase-activating protein called SynGAP has been shown to be concentrated at postsynaptic sites via interaction with PSD-95. Although the synaptic function of SynGAP is not known, its presence implies that a ras or similar signalling pathway does indeed operate at the postsynaptic density. Synthesis of Certain Postsynaptic Membrane Proteins within the Dendrite Synaptic specificity is a hallmark of synaptic plasticity. For instance, LTP is restricted to stimulated synapses and not manifested at other unstimulated synapses made on the same neuron. Since the long-term maintenance of synaptic plasticity is thought to involve gene expression, a vexing question is how newly synthesized proteins are targeted specifically to a subset of (potentiated) synapses in a neuron. In neurons, the targeting of newly synthesized proteins to particular synapses is a poorly understood process. One attractive hypothesis is that newly expressed proteins involved in synaptic plasticity are synthesized locally in the vicinity of their designated synapse, as occurs at the vertebrate neuromuscular junction. The first suggestion that protein synthesis might occur in the postsynaptic compartment of neurons came with the discovery of polyribosome complexes in dendrites. Subsequently, other components of the translation machinery such as rough endoplasmic reticulum and golgi apparati have been found in dendrites. Recently, it has been possible to demonstrate directly protein synthesis in isolated dendrites. Consistent with the idea of synapse-associated protein synthesis is the presence of certain mrnas in dendrites. However, in situ hybridization techniques have localized only a handful of mrnas at high levels in dendrites; the vast majority of mrnas are concentrated in the cell body. Presumably, the dendritic mrnas contain localization signals that allow them to be delivered from the cell soma to the dendrites. The nature of such a signal and the mechanisms for mrna sorting have yet to be determined. More sensitive polymerase chain reaction (PCR)-based methods have detected many more mrnas in dendritic compartments, but the significance of these findings remains to be established. The identified dendritic mrnas encode a variety of proteins. One notable example is the mrna for CaMKIIa, a subunit of CaMKII, an enzyme that is abundant in the PSD. Intriguingly, emerging evidence indicates that synaptic activity may regulate the translation of dendritic mrnas. Neurotrophin-induced LTP appears to rely on local protein synthesis within dendrites, and NMDA receptor activation induces phosphorylation of the eukaryotic translation elongation factor 2 within restricted dendritic regions. These results suggest that local synthesis of specific proteins may play an important role in synaptic 7

8 function and plasticity. However, much remains to be done to understand the functional significance of dendritic mrnas and their role in postsynaptic gene expression. Summary Molecular characterization of the postsynaptic site of glutamatergic synapses has revealed a diverse set of receptors each exhibiting distinct properties and likely to be associated intracellularly with a specific multiprotein complexthat constitutes a structural and functional microdomain. The PSD represents the subsynaptic accumulation of such microdomains that govern how glutamate receptors are clustered together, anchored to the cytoskeleton, and linked to specific intracellular signalling pathways. The combination of different receptors and their associated proteins at a single synapse permits various postsynaptic responses to the same presynaptic input. Furthermore, biochemical compartmentalization by synaptic spines allows for a synapse-specific response. The identification of PSD components responsible for synaptic organization has shown an intimate relationship between synaptic structure and function. Complete molecular characterization of the glutamatergic synapse will probably elucidate the mechanisms of plasticity of synapses and provide insight into learning and memory. References Bailey CH, Kaang BK, Chen M, Martin KC, Lim CS, Casadio A and Kandel ER (1997) Mutation in the phosphorylation sites of MAP kinase blocks learning-related internalization of apcam in Aplysia sensory neurons. Neuron 18: Brenman JE, Chao DS, Gee SH, McGee AW, Craven SE, Santillano DR, Wu Z, Huang F, Xia H, Peters MF, Froehner SC and Bredt DS (1996) Interaction of nitric oxide synthase with the postsynaptic density protein PSD-95 and alpha1-syntrophin mediated by PDZ domains. Cell: 84: Dong H, O Brien RJ, Fung ET, Lanahan AA, Worley PF and Huganir RL (1997) GRIP: a synaptic PDZ domain-containing protein that interacts with AMPA receptors. Nature 386: Kim E, Niethammer M, Rothschild A, Jan YN and Sheng M (1995) Clustering of shaker-type K 1 channels by interaction with a family of membrane-associated guanylate kinases. Nature 378: Kim E, Cho K-O, Rothschild A and Sheng M (1996) Heteromultimerization and NMDA receptor-clustering activity of chapsyn-110, a member of the PSD-95 family of proteins. Neuron 17: Kornau H-C, Schenker LT, Kennedy MB and Seeburg PH (1995) Domain interaction between NMDA receptor subunits and the postsynaptic density protein PSD-95. Science 269: Krupp JJ, Vissel B, Thomas CG, Heinemann SF and Westbrook GL (1999) Interactions of calmodulin and alpha-actinin with the NR1 subunit modulate Ca 2 1 -dependent inactivation of NMDA receptors. Journal of Neuroscience 19: Martin KC, Michael D, Rose JC, Barad M, Casadio A, Zhu H and Kandel ER (1997) MAP kinase translocates into the nucleus of the presynaptic cell and is required for long-term facilitation in Aplysia. Neuron 18: Niethammer M, Kim E and Sheng M (1996) Interaction between the C terminus of NMDA receptor subunits and multiple members of the PSD-95 family of membrane-associated guanylate kinases. Journal of Neuroscience 16: Schuster CM, Davis GW, Fetter RD and Goodman CS (1996a) Genetic dissection of structural and functional components of synaptic plasticity. I. Fasciclin II controls synaptic stabilization and growth. Neuron 17: Schuster CM, Davis GW, Fetter RD and Goodman CS (1996b) Genetic dissection of structural and functional components of synaptic plasticity. II. Fasciclin II controls presynaptic structural plasticity. Neuron 17: Silva AJ, Stevens CF, Tonegawa S and Wang Y (1992) Deficient hippocampal long-term potentiation in alpha-calcium-calmodulin kinase II mutant mice. Science 257: Tejedor FJ, Bokhari A, Rogero O, Gorczyca M, Zhang J, Kim E, Sheng M and Budnik V (1997) Essential role for dlg in synaptic clustering of Shaker K 1 channels in vivo. Journal of Neuroscience 17: Wyszynski M, Lin J, Rao A, Nigh E, Beggs AH, Craig AM and Sheng M (1997) Competitive binding of human alpha-actinin and calmodulin to the NMDA receptor. Nature 385: Further Reading Bettler B and Mulle C (1995) AMPA and kainate receptors. Neuropharmacology 34: Ehlers MD, Mammen AL, Lau L-F and Huganir RL (1996) Synaptic targeting of glutamate receptors. Current Opinions in Cell Biology 8: Kennedy MB (1997) The postsynaptic density at glutamatergic synapses. Trends in Neuroscience 20: Kornau H-C, Seeburg PH and Kennedy MB (1997) Interactions of ion channels and receptors with PDZ domain proteins. Current Opinions in Neurobiology 7: Milner B, Squire LR and Kandel ER (1998) Cognitive neuroscience and the study of memory. Neuron 20: Mori H and Mishina M (1995) Structure and function of the NMDA receptor channel. Neuropharmacology 34: Nakanishi S (1994) Metabotropic glutamate receptors: synaptic transmission, modulation, and plasticity. Neuron 13: Ziff EB (1997) Enlightening the postsynaptic density. Neuron 19: