Modulatory effects of L-DOPA on D2 dopamine receptors in rat striatum, measured using in vivo microdialysis and PET

Putative modulatory effects of L-3,4-dihydroxyphenylalanine (L-DOPA) on D2 dopamine receptor function in the striatum of anaesthetised rats were investigated using both in vivo microdialysis and positron emission tomography (PET) with carbon-11 labelled raclopride as a selective D2 receptor ligand. A single dose of L-DOPA (20 or 100mg/kg i.p.) resulted in an increase in [11C]raclopride binding potential which was also observed in the presence of the central aromatic decarboxylase inhibitor NSD 1015, confirming that the effect was independent of dopamine. This L-DOPA evoked D2 receptor sensitisation was abolished by a prior, long-term administration of L-DOPA in drinking water (5 weeks, 170mg/kg/day). In the course of acute L-DOPA treatment (20mg/kg), extracellular GABA levels were reduced by approximately 20% in the globus pallidus. It is likely that L-DOPA sensitising effect on striatal D2 receptors, as confirmed by PET, may implicate striato-pallidal neurones, hence a reduced GABA-ergic output in the projection area. Since the L-DOPA evoked striatal D2 receptor supersensitivity habituates during long-term treatment, the effects reported here may contribute to the fluctuations observed during chronic L-DOPA therapy in Parkinson's disease.     

Antiparkinsonian actions of glutamate antagonists--alone and with L-DOPA: a review of evidence and suggestions for possible mechanisms

There has been much speculation of late as to whether antagonists of glutamate receptors can be used to combat the motor difficulties of Parkinson's disease, either as monotherapy, or as polytherapy to boost the effects of conventional L-DOPA treatment. The latter seems to be the more practical approach and the therapeutic implications of such treatment have been discussed in some detail. However, the mechanisms by which glutamate antagonists potentiate the antiparkinsonian actions of L-DOPA, remain cryptic. In this review we have explored the evidence and considered the practicality of using NMDA and non-NMDA receptor blockers to treat parkinsonism, as well as focusing on the ways in which the behavioural synergy between dopamine and glutamate systems could conceivably arise at the cellular level. Particular attention has been paid to the differential interaction between glutamate antagonists and postsynaptic dopamine D1 and D2 receptory mechanisms, since these are currently believed to reflect the activity of the two major basal ganglia output circuits: the so-called direct pathway to the substantia nigra and the indirect pathway to the globus pallidus. Finally, we have considered the new proposal, that inhibiting glutamate transmission in the basal ganglia accelerates the enzymic conversion of L-DOPA to dopamine at presynaptic sites.     

Distribution and mobility of lectin receptors on synaptic membranes of identified neurons in the central nervous system

The distribution and mobility of concanavalin A (Con A) and Ricinus communis agglutinin (RCA) receptors (binding sites) on the external surfaces of Purkinje, hippocampal pyramidal, and granule cells and their attached boutons were studied using ferritin-lectin conjugates. Dendritic fields of these cells were isolated by microdissection and gently homogenized. Cell fragments and pre- and postsynaptic membranes were labeled with the ferritin-lectin conjugates at a variety of temperatures, and the distribution of lectin receptors was determined by electron microscopy. Both classes of these lectin receptors were concentrated at nearly all open and partially open postsynaptic junctional membranes of asymmetric-type synapses on all three neuron types. Con A receptors were most concentrated at the junctional membrane region, indicating that the mature neuron has a specialized nonrandom organization of carbohydrates on its outer surface. Lectin receptors located on postsynaptic junctional membranes appeared to be restricted in their mobility compared to similar classes of receptors on extrajunctional membrane regions. Labeling with ferritin-RCA and -Con A at 37 degrees C produced clustering of lectin receptors on nonjunctional surfaces; however, Con A and RCA receptors retained their nonrandom topographic distribution on the postsynaptic junctional surface. The restricted mobility of lectin receptors was an inherent property of the postsynaptic membrane since the presynaptic membrane was absent. It is proposed that structures in the postsynaptic density may be transmembrane-linked to postsynaptic receptors and thereby determine topographic distribution and limit diffusion of specialized synaptic molecules. Speicalized receptor displays may play an important role in the formation and maintenance of specific synaptic contacts.     

Putting occupation into practice: occupation as ends, occupation as means

We have investigated the effect of 5-HT2 receptor agonist or antagonist administration on postsynaptic 5-HT1A receptor sensitivity assessed by two behavioral measures, reciprocal forepaw treading or hypothermia induced by acute injection of the 5-HT1A receptor agonist 8-OH-DPAT. The effectiveness of these drug treatments to downregulate 5-HT2A receptors was confirmed by measuring the binding of [3H]-ketanserin in cortical homogenates, because all of these drug treatments have been shown to result in the downregulation of 5-HT2A receptor sites. Acute or chronic treatment of rats with the 5-HT2 receptor antagonist mianserin, or chronic administration of the 5-HT2A receptor antagonist ketanserin, did not alter 8-OH-DPAT-induced hypothermia or forepaw treading. These data indicate that downregulation of 5-HT2A receptors is not sufficient to alter these postsynaptic 5-HT1A receptor-mediated responses. Chronic treatment of rats with the 5-HT2 receptor agonist DOI, however, resulted in the attenuation of both 5-HT1A receptor-mediated responses measured in separate experimental groups. The apparent desensitization of 5-HT1A receptors following chronic DOI treatment was not accompanied by a change in either the number or affinity of 5-HT1A receptor sites as measured by the binding of [3H]-8-OH-DPAT in hippocampal homogenates. Chronic activation of 5-HT2 receptors may be one mechanism by which the sensitivity postsynaptic 5-HT1A receptors can be regulated.     

Heterogeneous distributions of histamine H3, dopamine D1 and D2 receptors in rat brain

The changes of the histamine H3 and dopamine D1 or D2 receptor binding sites induced by quinolinic acid treatment were studied in order to discriminate the comparative distribution. This treatment resulted in similar decreases in histamine H3 and dopamine D1 receptor binding sites in the striatum and ipsilateral substantia nigra. Dopamine D2 receptor binding sites were relatively well conserved, whereas H3 receptors decreased considerably. These results suggest that histamine H3 and dopamine D1 receptor binding sites are localized on the striatonigral projection neurones which are together sensitive to quinolinic acid, and that the distributional compartment of dopamine D2 receptor binding sites is quite different from those of histamine H3 and dopamine D1 receptors.     

The region of the herpes simplex virus type 1 LAT gene involved in spontaneous reactivation does not encode a functional protein

A family of muscarinic acetylcholine receptor proteins mediates diverse pre- and postsynaptic functions in the hippocampus. However the roles of individual receptors are not understood. The present study identified the pre- and postsynaptic muscarinic acetylcholine receptors at the perforant pathway synapses in rat brain using a combination of lesioning, immunocytochemistry and electron microscopic techniques. Entorhinal cortex lesions resulted in lamina-specific reductions of m2, m3, and m4 immunoreactivity in parallel with the degeneration of the medial and lateral perforant pathway terminals in the middle and outer thirds of the molecular layer, respectively. In contrast, granule cell lesions selectively reduced m1 and m3 receptors consistent with degeneration of postsynaptic dendrites. Direct visualization of m1-m4 by electron microscopic immunocytochemistry confirmed their differential pre- and postsynaptic localizations. Together, these findings provide strong evidence for both redundancy and spatial selectivity of presynaptic (m2, m3 and m4) and postsynaptic (m1 and m3) muscarinic acetylcholine receptors at the perforant pathway synapse.     

Changes in rat brain muscarinic receptors after inhibitory avoidance learning

It is widely accepted that cerebral acetylcholine is necessary for learning and memory, but little is known about the type of muscarinic receptors involved in these functions. To investigate this problem, [3H]-N-methyl-scopolamine which binds to different types of muscarinic receptors, [3H]-Pirenzepine an M1 receptor antagonist, and [3H]-Oxotremorine-M which binds mainly to M2 receptors, were used as ligands to look for possible changes in muscarinic receptor density in neostriatum (NEO), hippocampus (HIP), amygdala (AMY), and temporo-parietal neocortex (CTX), after testing for retention of inhibitory avoidance, trained with high or low footshock intensities. After low reinforcement there was an M1 postsynaptic receptor up-regulation in NEO, HIP, and CTX, and an M2 presynaptic receptor down-regulation in HIP, which suggests a concerted pre- and postsynaptic cholinergic activation in this area. An up-regulation of both M1 and M2 receptors was detected in CTX of low and high footshocked animals, which indicates the presence of a cortical postsynaptic M2 receptor.     

A postsynaptic interaction between dopamine D1 and NMDA receptors promotes presynaptic inhibition in the rat nucleus accumbens via adenosine release

The mechanism underlying dopamine D1 receptor-mediated attenuation of glutamatergic synaptic input to nucleus accumbens (NAcc) neurons was investigated in slices of rat forebrain, using whole-cell patch-clamp recording. The depression by dopamine of EPSCs evoked by single-shock cortical stimulation was stimulus-dependent. Synaptic activation of NMDA-type glutamate receptors was critical for this effect, because dopamine-induced EPSC depressions were blocked by the competitive NMDA receptor antagonist D/L-2-amino-5-phosphonopentanoate (AP5). Application of NMDA also depressed the EPSC, and both this effect and the dopamine depressions were blocked by the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), implicating adenosine release in the EPSC depression. A1 receptor agonists also depressed EPSCs by a presynaptic action, causing increased paired-pulse facilitation, but this was insensitive to AP5. Activation of D1 receptors enhanced both postsynaptic inward currents evoked by NMDA application and the isolated NMDA receptor-mediated component of synaptic transmission. The biochemical processes underlying the dopamine-induced EPSC depression did not involve either protein kinase A or the production of cAMP and its metabolites, because this effect was resistant to the protein kinase inhibitors H89 and H7 and the cAMP-specific phosphodiesterase inhibitor rolipram. We conclude that activation of postsynaptic D1 receptors enhances the synaptic activation of NMDA receptors in nucleus accumbens neurons, thereby promoting a transsynaptic feedback inhibition of glutamatergic synaptic transmission via release of adenosine. Unusually for D1 receptors, this phenomenon occurs independently of adenylyl cyclase stimulation. This process may contribute to the locomotor stimulant action of dopaminergic agents in the NAcc.     

Evidence that agrin directly influences presynaptic differentiation at neuromuscular junctions in vitro

The synaptic protein agrin is required for aspects of both pre- and postsynaptic differentiation at neuromuscular junctions. Although a direct effect of agrin on postsynaptic differentiation, presumably through the MuSK receptor, is established, it is not clear whether agrin directly affects the presynaptic nerve. To provide evidence on this point, we used anti-agrin IgG to disrupt agrin function in chick ciliary ganglion (CG) neuron/myotube cocultures. In cocultures grown in the presence of 200 microg/ml anti-agrin IgG, clustering of acetylcholine receptors (AChRs), extracellular matrix proteins, and the synaptic vesicle protein synaptotagmin (syt) at nerve-muscle contacts was inhibited. Syt clustering was still inhibited in the presence of 100 microg/ml blocking antibody, while the postsynaptic clustering of AChRs, heparan sulphate proteoglycan, and s-laminin was retained. Additionally, in CG neurons cultured with COS cells expressing agrin A0B0, which lacks the ability to signal postsynaptic differentiation, syt clustering was induced and this clustering was also blocked by anti-agrin IgG. Our results demonstrate that agrin function is acutely required for pre- and postsynaptic differentiation in vitro, and strongly suggest that agrin is directly involved in the induction of presynaptic differentiation.     

Evidence of multiple mechanisms of avermectin resistance in haemonchus contortus--comparison of selection protocols

The ginsenosides Rb1 and Rg1, the major components of ginseng saponin, inhibited not only methamphetamine-induced hyperactivity but also conditioned place preference (CPP) in mice following a single or repeated administration. Dopamine (DA) receptor supersensitivity, which developed in methamphetamine-induced CPP mice, was also inhibited by both Rb1 and Rg1. Therefore, the present results suggest that Rb1 and Rg1 may be the active components of ginseng saponin in the modulation of methamphetamine-induced dopaminergic behaviors such as hyperactivity and CPP, supporting our previous conclusion that ginseng saponin might modulate methamphetamine-induced dysfunction at both the pre- and postsynaptic DA receptors.     

Modulation of dopamine release from rat striatum by protein kinase C: interaction with presynaptic D2-dopamine-autoreceptors

1. Interactions between dopamine receptors and protein kinase C (PKC) have been proposed from biochemical studies. The aim of the present study was to investigate the hypothesis that there is an interaction between protein kinase C and inhibitory D2-dopamine receptors in the modulation of stimulation-induced (S-I) dopamine release from rat striatal slices incubated with [3H]-dopamine. Dopamine release can be modulated by protein kinase C and inhibitory presynaptic D2 receptors since phorbol dibutyrate (PDB) and (-)-sulpiride, respectively, elevated S-I dopamine release. 2. The protein kinase C inhibitors polymyxin B (21 microM) and chelerythrine (3 microM) had no effect on stimulation-induced (S-I) dopamine release. However, when presynaptic dopamine D2 receptors were blocked by sulpiride (1 microM), an inhibitory effect of both PKC inhibitors on S-I dopamine release was revealed. Thus, sulpiride unmasks an endogenous PKC effect on dopamine release which suggests that presynaptic D2 receptors normally suppress endogenous PKC activity. This is supported by results in striatal slices which were pretreated with PDB to down-regulate PKC. In this case the facilitatory effect of sulpiride was completely abolished. 3. The inhibitory effect of the dopamine D2/D3 agonist quinpirole on S-I dopamine release was partially attenuated by PKC down-regulation. Since the effect of sulpiride was completely abolished under the same conditions, this suggests that exogenous agonists may target a PKC-dependent as well as a PKC-independent pathway. The inhibitory effect of apomorphine was not affected by either polymyxin B or PKC down-regulation, suggesting that it operated exclusively through a PKC-independent mechanism. 4. These results suggest that there are at least two pathways involved in the inhibition of dopamine release through dopamine receptors. One pathway involves dopamine receptor suppression of protein kinase C activity, perhaps through inhibition of phospholipase C activity and this is preferentially utilized by neuronally-released dopamine. The other pathway which seems to be utilized by exogenous agonists does not involve PKC.     

Modulation of pre- and postsynaptic calcium dynamics by ionotropic glutamate receptors at a plastic synapse

This study was conducted to assess the role of ionotropic glutamate receptors in the modulation of calcium dynamics on both sides of a vertebrate plastic synapse. Retrograde labeling of neuronal elements with high-affinity calcium-sensitive dyes was used in conjunction with confocal imaging techniques in an in vitro lamprey brain stem preparation. A prolonged calcium transient was measured both pre- and postsynaptically in response to a period of high-frequency ("tetanic") stimulation to the vestibulospinal-reticulospinal synapse. The ionotropic glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM) and D,L-2-amino-5-phosphonopentanoate (D,L-AP5; 100 microM) reduced the calcium signal in both compartments of the synapse. The presynaptic D,L-AP5-sensitive component was enhanced markedly by the removal of Mg2+ from the superfusate. Increasing the extracellular stimulus intensity progressively augmented the presynaptic calcium signal, suggesting the recruitment of excitatory axo-axonic inputs onto these fibers. Further, the presence of an excitatory amino acid-mediated presynaptic potential underlying a component of the Ca2+ signal was demonstrated by electrophysiological recordings from vestibulospinal axons. Bath application of agonist, in the presence of tetrodotoxin (1 microM), confirmed the existence of N-methyl-D-aspartate receptors at the presynaptic element capable of modulating calcium levels. The postsynaptic Ca2+ response, which is known to be necessary for long-term potentiation (LTP) induction at this synapse, was localized to areas of the dendritic tree that correlated with the location of known synaptic inputs; thus the synaptically activated rise in postsynaptic calcium may confer the synapse specificity of LTP induction previously demonstrated. In summary, we have demonstrated the existence of physiologically activated presynaptic ionotropic glutamate receptors that are capable of modulating levels of intracellular calcium and have highlighted the importance of receptor-mediated increases in postsynaptic calcium for neuronal plasticity in the lamprey.     

SAP97 is associated with the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor GluR1 subunit

Rapid glutamatergic synaptic transmission is mediated by ionotropic glutamate receptors and depends on their precise localization at postsynaptic membranes opposing the presynaptic neurotransmitter release sites. Postsynaptic localization of N-methyl-D-aspartate-type glutamate receptors may be mediated by the synapse-associated proteins (SAPs) SAP90, SAP102, and chapsyn-110. SAPs contain three PDZ domains that can interact with the C termini of proteins such as N-methyl-D-aspartate receptor subunits that carry a serine or threonine at the -2 position and a valine, isoleucine, or leucine at the very C terminus (position 0). We now show that SAP97, a SAP whose function at the synapse has been unclear, is associated with alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors. AMPA receptors are probably tetramers and are formed by two or more of the four AMPA receptor subunits GluR1-4. GluR1 possesses a C-terminal consensus sequence for interactions with PDZ domains of SAPs. SAP97 was present in AMPA receptor complexes immunoprecipitated from detergent extracts of rat brain. After treatment of rat brain membrane fractions with the cross-linker dithiobis(succinimidylpropionate) and solubilization with sodium dodecylsulfate, SAP97 was associated with GluR1 but not GluR2 or GluR3. In vitro experiments with recombinant proteins indicate that SAP97 specifically associates with the C terminus of GluR1 but not other AMPA receptor subunits. Our findings suggest that SAP97 may be involved in localizing AMPA receptors at postsynaptic sites through its interaction with the GluR1 subunit.     

Modifications of platelet phospholipid fatty acid composition in streptozocin-induced diabetic rats

Mesulergine (N,N-dimethylsulphamide-N'-1,6-dimethyl-ergoline-8 alpha-yl) is an active semisynthetic ergot derivative with lower antiprolactin potency compared with bromocriptine or pergolide. Since no data are yet available on the effects of mesulergine on pituitary dopamine receptors, the present study has been designated to elucidate the influence of this drug on prolactin secretion in vivo and in vitro and 3H-spiperone binding by the anterior pituitary gland in female Wistar rats with experimentally induced hyperprolactinemia. Three weeks after bilateral ovariectomy and subcutaneous implantation of silastic tubes, containing 10 mg of diethylstilbestrol, a dramatic rise in serum prolactin levels was observed (1.67 +/- 0.23 vs. 80.82 +/- 3.80 ng/ml; P less than 0.001). Mesulergine attenuated the stimulatory effect of diethylstilbestrol on serum prolactin level in a time- and dose-dependent fashion. At concentration range between 10(-5) and 10(-7) M it also inhibited prolactin secretion from cultured rat pituitary cells to the medium during 180 min incubation in a dose-dependent manner. Scatchard analyses performed on the in vitro 3H-spiperone binding kinetics in a dispersed anterior pituitary cell culture, prepared from the pituitaries from rats treated for four weeks with diethylstilbestrol, showed that chronic mesulergine treatment (in dose of 3.0 mg/kg injected s.c. for 10 days) induced a significant decrease in the number of dopamine D2-binding sites (Bmax 28.00 +/- 4.20 vs. 42.80 +/- 4.76 fmol/10(6) cells; P less than 0.01) without any changes in D2-receptor affinity. Our results suggested that antiprolactin activity of mesulergine in vivo and in vitro is probably associated with agonistic effect of this drug on D2-dopamine receptors.     

Ultrastructural localization of glutamate receptor subunits (NMDAR1, AMPA GluR1 and GluR2/3) and spinothalamic tract cells

The associations of glutamate receptor subunits (NMDAR1, AMPA GluR1 and GluR2/3) and spinothalamic tract neurons in the rat lumbar spinal cord dorsal horn were investigated. Staining for NMDAR1 and AMPA GluR1 and GluR2/3 receptor subunits was observed throughout the spinothalamic tract soma and dendrites, particularly in association with the rough endoplasmic reticulum and some postsynaptic membrane sites. Immunostaining for NMDAR1 and AMPA GluR2/3 was also noted in presynaptic membrane sites. Localization of both NMDA and AMPA glutamate receptor subunits in association with spinothalamic tract neurons provides anatomical evidence in support of the various interactions reported for glutamate receptors in nociception. Presynaptic localization of the AMPA GluR2/3 receptor subunit suggests that spinothalamic tract cells may also be affected presynaptically by AMPA glutamate receptor interactions.     

Small structural changes of chromosome 8. Two cases with evidence for deletion

The degeneration of the substantia nigra that characterises Parkinson's disease may cause an alteration in sensitivity of striatal dopamine receptors. The development of denervation supersensitivity has been held to be responsible for some of the effects of chronic levodopa therapy. The rotating rodent is an animal model commonly used to study the phenomenon of striatal dopamine receptor supersensitivity, and to investigate drugs which may prove to be beneficial in the treatment of Parkinson's disease. We have investigated as to whether long-term oral administration of levodopa to mice with unilateral destruction of striatal dopaminergic nerve terminals influences dopaminergic receptor denervation supersensitivity as judged by the circling response following systemically administered levodopa. It does not do so and the relevance of these findings to the treatment of Parkinson's disease is discussed.     

Recruitment of functional GABA(A) receptors to postsynaptic domains by insulin

Modification of synaptic strength in the mammalian central nervous system (CNS) occurs at both pre- and postsynaptic sites. However, because postsynaptic receptors are likely to be saturated by released transmitter, an increase in the number of active postsynaptic receptors may be a more efficient way of strengthening synaptic efficacy. But there has been no evidence for a rapid recruitment of neurotransmitter receptors to the postsynaptic membrane in the CNS. Here we report that insulin causes the type A gamma-aminobutyric acid (GABA[A]) receptor, the principal receptor that mediates synaptic inhibition in the CNS, to translocate rapidly from the intracellular compartment to the plasma membrane in transfected HEK 293 cells, and that this relocation requires the beta2 subunit of the GABA(A) receptor. In CNS neurons, insulin increases the expression of GABA(A) receptors on the postsynaptic and dendritic membranes. We found that insulin increases the number of functional postsynaptic GABA(A) receptors, thereby increasing the amplitude of the GABA(A)-receptor-mediated miniature inhibitory postsynaptic currents (mIPSCs) without altering their time course. These results provide evidence for a rapid recruitment of functional receptors to the postsynaptic plasma membrane, suggesting a fundamental mechanism for the generation of synaptic plasticity.     

Dopamine/neuroleptic receptors in basal hypothalamus and pituitary

In order to determine whether the basal hypothalamus or the pituitary (or both) is the likely locus of action of the tuberoinfundibular (TI) dopamine neurons, these regions were examined for dopamine and neuroleptic receptors. High affinity receptors for haloperidol and dopamine were found in the rat pituitary while none were detected in rat basal hypothalamus. The relative ability of two neuroleptics, chlorpromazine and haloperidol, to displace (3H)haloperidol from the receptor in monkey pituitary is similar to that for rat striatum. The lack of receptors capable of binding (3H)haloperidol or (3H)dopamine in the basal hypothalamus strongly suggests that the TI neurons do not produce postsynaptic effects in this region. The pituitary receptors for (3H)haloperidol and (3H)dopamine have the characteristics of a functional system. The presence of neuroleptic/dopamine receptors in the pituitary and lack of such receptors in the basal hypothalamus supports the hypothesis that dopamine may act directly as a prolactin release inhibiting factor (PIF) rather than releasing PIF from adjacent nerve terminals in the median eminence.     

Heterogeneity in the molecular composition of excitatory postsynaptic sites during development of hippocampal neurons in culture

To determine their roles in the assembly of glutamatergic postsynaptic sites, we studied the distributions of NMDA- and AMPA-type glutamate receptors; the NMDA receptor-interacting proteins alpha-actinin-2, PSD-95, and chapsyn; and the PSD-95-associated protein GKAP during the development of hippocampal neurons in culture. NMDA receptors first formed nonsynaptic proximal dendrite shaft clusters within 2-5 d. AMPA receptors were diffuse at this stage and began to cluster on spines at 9-10 d. NMDA receptor clusters remained partially nonsynaptic and mainly distinct from AMPA receptor clusters until after 3 weeks in culture, when the two began to colocalize at spiny synaptic sites. Thus, the localization of NMDA and AMPA receptors must be regulated by different mechanisms. alpha-Actinin-2 colocalized with the NMDA receptor only at spiny synaptic clusters, but not at shaft nonsynaptic or synaptic clusters, suggesting a modulatory role in the anchoring of NMDA receptor at spines. PSD-95, chapsyn, and GKAP were present at some, but not all, nonsynaptic NMDA receptor clusters during the first 2 weeks, indicating that none is essential for NMDA receptor cluster formation. When NMDA receptor clusters became synaptic, PSD-95 and GKAP were always present, consistent with an essential function in synaptic localization of NMDA receptors. Furthermore, PSD-95 and GKAP clustered opposite presynaptic terminals several days before either NMDA or AMPA receptors clustered at these presumptive postsynaptic sites. These results suggest that synapse development proceeds by formation of a postsynaptic scaffold containing PSD-95 and GKAP in concert with presynaptic vesicle clustering, followed by regulated attachment of glutamate receptor subtypes to this scaffold.