Localization of acetylcholine receptors in central synapses

The localization of cholinergic receptors in brain synaptosomes and in synapses of the midbrain reticular formation and hypothalamic preoptic nucleus has been demonstrated by means of a horseradish peroxidase- alpha-bungarotoxin (HRP-alpha-Btx) conjugate. Only a small proportion of the total number of synapses was reactive. Axon terminals of reactive synapses contained primarily small clear vesicles, while synapses characterized by large numbers of dense core vesicles were unreactive. Toxin-binding sites were found to occur in a thickened zone of the postsynaptic surface. This procedure can be employed to study the regional distribution and localization of nicotinic receptor sites in the central nervous system.     

Mobile NMDA receptors at hippocampal synapses

Glutamate receptors are concentrated in the postsynaptic complex of central synapses. This implies a highly organized and stable postsynaptic membrane with tightly anchored receptors. Recent reports of rapid AMPA receptor insertion and removal at synapses have challenged this view. We examined the stability of synaptic NMDA receptors on cultured hippocampal neurons using the open-channel blockers (+)-MK-801 and ketamine to tag synaptic NMDA receptors. NMDA receptor-mediated EPSCs showed an anomalous recovery following "irreversible" MK-801 block. The recovery could not be attributed to MK-801 unbinding or insertion of new receptors, suggesting that membrane receptors had moved laterally into the synapse. At least 65% of synaptic NMDA receptors were mobile. Our results indicate that NMDA receptors can move laterally between synaptic and extrasynaptic pools, providing evidence for a dynamic organization of synaptic NMDA receptors in the postsynaptic complex.     

Diffusion barriers constrain receptors at synapses

The flux of neurotransmitter receptors in and out of synapses depends on receptor interaction with scaffolding molecules. However, the crowd of transmembrane proteins and the rich cytoskeletal environment may constitute obstacles to the diffusion of receptors within the synapse. To address this question, we studied the membrane diffusion of the γ-aminobutyric acid type A receptor (GABA(A)R) subunits clustered (γ2) or not (α5) at inhibitory synapses in rat hippocampal dissociated neurons. Relative to the extrasynaptic region, γ2 and α5 showed reduced diffusion and increased confinement at both inhibitory and excitatory synapses but they dwelled for a short time at excitatory synapses. In contrast, γ2 was ～3-fold more confined and dwelled ～3-fold longer in inhibitory synapses than α5, indicating faster synaptic escape of α5. Furthermore, using a gephyrin dominant-negative approach, we showed that the increased residency time of γ2 at inhibitory synapses was due to receptor-scaffold interactions. As shown for GABA(A)R, the excitatory glutamate receptor 2 subunit (GluA2) of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) had lower mobility in both excitatory and inhibitory synapses but a higher residency time at excitatory synapses. Therefore barriers impose significant diffusion constraints onto receptors at synapses where they accumulate or not. Our data further reveal that the confinement and the dwell time but not the diffusion coefficient report on the synapse specific sorting, trapping and accumulation of receptors.     

Eph receptors at synapses: implications in neurodegenerative diseases

Precise regulation of synapse formation, maintenance and plasticity is crucial for normal cognitive function, and synaptic failure has been suggested as one of the hallmarks of neurodegenerative diseases. In this review, we describe the recent progress in our understanding of how the receptor tyrosine kinase Ephs and their ligands ephrins regulate dendritic spine morphogenesis, synapse formation and maturation, as well as synaptic plasticity. In particular, we discuss the emerging evidence implicating that deregulation of Eph/ephrin signaling contributes to the aberrant synaptic functions associated with cognitive impairment in Alzheimer's disease. Understanding how Eph/ephrin regulates synaptic function may therefore provide new insights into the development of therapeutic agents against neurodegenerative diseases.     

Recruitment of activation receptors at inhibitory NK cell immune synapses

Natural killer (NK) cell activation receptors accumulate by an actin-dependent process at cytotoxic immune synapses where they provide synergistic signals that trigger NK cell effector functions. In contrast, NK cell inhibitory receptors, including members of the MHC class I-specific killer cell Ig-like receptor (KIR) family, accumulate at inhibitory immune synapses, block actin dynamics, and prevent actin-dependent phosphorylation of activation receptors. Therefore, one would predict inhibition of actin-dependent accumulation of activation receptors when inhibitory receptors are engaged. By confocal imaging of primary human NK cells in contact with target cells expressing physiological ligands of NK cell receptors, we show here that this prediction is incorrect. Target cells included a human cell line and transfected Drosophila insect cells that expressed ligands of NK cell activation receptors in combination with an MHC class I ligand of inhibitory KIR. The two NK cell activation receptors CD2 and 2B4 accumulated and co-localized with KIR at inhibitory immune synapses. In fact, KIR promoted CD2 and 2B4 clustering, as CD2 and 2B4 accumulated more efficiently at inhibitory synapses. In contrast, accumulation of KIR and of activation receptors at inhibitory synapses correlated with reduced density of the integrin LFA-1. These results imply that inhibitory KIR does not prevent CD2 and 2B4 signaling by blocking their accumulation at NK cell immune synapses, but by blocking their ability to signal within inhibitory synapses.     

Kainate receptors differentially regulate release at two parallel fiber synapses

Presynaptic kainate receptors (KARs) facilitate or depress transmitter release at several synapses in the CNS. Here, we report that synaptically activated KARs presynaptically facilitate and depress transmission at parallel fiber synapses in the cerebellar cortex. Low-frequency stimulation of parallel fibers facilitates synapses onto both stellate cells and Purkinje cells, whereas high-frequency stimulation depresses stellate cell synapses but continues to facilitate Purkinje cell synapses. These effects are mimicked by exogenous KAR agonists and eliminated by blocking KARs. This differential frequency-dependent sensitivity of these two synapses regulates the balance of excitatory and inhibitory input to Purkinje cells and therefore their excitability.     

High-resolution quantitative visualization of glutamate and GABA receptors at central synapses

Glutamate and GABA are the main transmitters in the central nervous system and their effects are mediated by ionotropic and metabotropic receptors. Immunogold electron microscopy has revealed the quantitative localization of these receptors at 20-30nm resolution. SDS-digested freeze-fracture replica labeling (SDS-FRL), a newly developed immunogold method, provides an accurate estimate of molecule numbers. Here, we summarize the recent advances in quantitative receptor localization, including use of SDS-FRL analyses to determine numbers of AMPA-type glutamate receptors in the cerebellum. The two-dimensional view and high sensitivity of SDS-FRL have revealed small, irregularly shaped AMPA receptor clusters within cerebellar synapses.     

Synaptic drive at developing synapses: Transient upregulation of kainate receptors

At the onset of a period of intense synaptic refinement initiated by synchronized eye opening (EO), rapid changes in postsynaptic NMDA receptor and AMPA receptor currents (NMDARcs and AMPARcs) occur within the superficial visual layers of the rodent superior colliculus (sSC; Lu and Constantine‐Paton [ 2004 ]: Neuron 43:237–249). Subsequently, evoked non‐NMDARc amplitudes increase, but by 2 weeks after EO (AEO) they decrease significantly. Here, using whole‐cell patch‐clamp recording, we demonstrate that small, slowly desensitizing excitatory kainate receptor currents (KARcs) are responsible for the rise and subsequent fall in non‐NMDARcs. The increase in KAR transmission parallels inhibitory GABA_A responses that plateau at 7 days AEO. By 2 weeks AEO, KARcs are gone. AMPARcs remain unchanged during the appearance and disappearance of the KARcs, despite increases in sSC neuropil activity and continued refinement of inputs to individual sSC neurons. We suggest that in the interval of heightened activity, before SC inhibition matures, many AMPARcs desensitize and are relatively ineffective at relieving the Mg²⁺ block on NMDARs. This transient appearance of slowly desensitizing, long‐duration KARcs may provide increased membrane depolarization necessary for NMDAR function and continuation of synaptic refinement. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 737–750, 2010     

Neurexins and neuroligins: new partners for GABAA receptors at synapses

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain. As one of several types of endogenous receptors, GABA_A receptors have been shown to be essential in most, if not all, aspects of brain functioning, including neural development and information processing. Mutations in genes encoding GABA_A receptors and alterations in the function of GABA_A receptors are associated with many neurologic diseases, and GABA_A receptors have been clinically targeted by many drugs, such as benzodiazepines and general anesthetics. Extensive studies have revealed a number of intracellular chaperons/interactions for GABA_A receptors, providing a protein-protein network in regulating the trafficking and location of GABA_A receptors in the brain. Recently, neurexins and neuroligins, two families of transmembrane proteins present at neurological synapses, are implicated as new partners to GABA_A receptors. These works shed new light on the synaptic regulation of GABA_A receptor activity. Here, we summarized the proteins that were implicated in the function of GABA_A receptors, including neurexins and neuroligins.     

A new affinity matrix for mineralocorticoid receptors

The behavior of mineralocorticoid and glucocorticoid receptors of rabbit kidney cytosol was investigated on two affinity gels: a new affinity matrix prepared with a 3-O-derivative of carboxymethyloxime deoxycorticosterone (deoxycorticosterone gel) and a gel linked to a 17 beta-dexamethasone derivative (dexamethasone gel). Deoxycorticosterone gel was highly specific, since it retained mineralocorticoid but not glucocorticoid receptors, and dexamethasone gel exhibited high selectivity for glucocorticoid receptors since it did not bind mineralocorticoid receptors. The use of these two matrices allowed separation of mineralocorticoid and glucocorticoid receptors and further characterization of each type of cytosolic receptors after its isolation. Cytosolic mineralocorticoid and glucocorticoid receptors stabilized by tungstate were found to have a Stokes radius of approximately 6 nm, as determined by high performance size exclusion chromatography and a sedimentation coefficient of approximately 9 S, determined on a glycerol density gradient containing tungstate, under either high or low salt conditions. The hydrodynamic parameters, binding characteristics, and specificity of mineralocorticoid receptors were the same in the untreated and dexamethasone gel-treated cytosol. Similarly glucocorticoid receptor characteristics remained unchanged after deoxycorticosterone gel treatment, indicating biochemical independence of cytosolic mineralocorticoid and glucocorticoid receptors. The [3H]aldosterone receptor complex eluted from deoxycorticosterone gel was recovered with a 30-40% yield and a purification factor of about 1000. Purified mineralocorticoid receptors had the same sedimentation coefficient as cytosolic mineralocorticoid receptors (9 S) but a different Stokes radius (4 versus 6 nm). The decrease in the Stokes radius of the purified mineralocorticoid receptors was probably due to the gel filtration method. These results indicate that the newly synthesized matrix specific for mineralocorticoid receptors constitutes a powerful tool for their extensive purification.     

Reciprocal stabilization of glycine receptors and gephyrin scaffold proteins at inhibitory synapses

Postsynaptic scaffold proteins immobilize neurotransmitter receptors in the synaptic membrane opposite to presynaptic vesicle release sites, thus ensuring efficient synaptic transmission. At inhibitory synapses in the spinal cord, the main scaffold protein gephyrin assembles in dense molecule clusters that provide binding sites for glycine receptors (GlyRs). Gephyrin and GlyRs can also interact outside of synapses, where they form receptor-scaffold complexes. Although several models for the formation of postsynaptic scaffold domains in the presence of receptor-scaffold interactions have been advanced, a clear picture of the coupled dynamics of receptors and scaffold proteins at synapses is lacking. To characterize the GlyR and gephyrin dynamics at inhibitory synapses, we performed fluorescence time-lapse imaging after photoconversion to directly visualize the exchange kinetics of recombinant Dendra2-gephyrin in cultured spinal cord neurons. Immuno-immobilization of endogenous GlyRs with specific antibodies abolished their lateral diffusion in the plasma membrane, as judged by the lack of fluorescence recovery after photobleaching. Moreover, the cross-linking of GlyRs significantly reduced the exchange of Dendra2-gephyrin compared with control conditions, suggesting that the kinetics of the synaptic gephyrin pool is strongly dependent on GlyR-gephyrin interactions. We did not observe any change in the total synaptic gephyrin levels after GlyR cross-linking, however, indicating that the number of gephyrin molecules at synapses is not primarily dependent on the exchange of GlyR-gephyrin complexes. We further show that our experimental data can be quantitatively accounted for by a model of receptor-scaffold dynamics that includes a tightly interacting receptor-scaffold domain, as well as more loosely bound receptor and scaffold populations that exchange with extrasynaptic pools. The model can make predictions for single-molecule data such as typical dwell times of synaptic proteins. Taken together, our data demonstrate the reciprocal stabilization of GlyRs and gephyrin at inhibitory synapses and provide a quantitative understanding of their dynamic organization.     

Distinct NMDA receptors provide differential modes of transmission at mossy fiber-interneuron synapses

Dentate gyrus granule cells innervate inhibitory interneurons via a continuum of synapses comprised of either Ca(2+)-impermeable (CI) or Ca(2+)-permeable (CP) AMPA receptors. Synapses at the extreme ends of this continuum engage distinct postsynaptic responses, with activity at CI synapses being strongly influenced by NMDA receptor activation. NMDARs at CI synapses have a lower NR2B subunit composition and a higher open probability, which generate larger amplitude and more rapid EPSCs than their CP counterparts. A novel form of NMDAR-dependent long-term depression (iLTD) is associated with CI-mossy fiber synapses, whereas iLTD at CP synapses is dependent on Ca(2+)-permeable AMPA receptor activation. Induction of both forms of iLTD required elevation of postsynaptic calcium. Thus mossy fibers engage CA3 interneurons via multiple synapse types that will act to expand the computational repertoire of the mossy fiber-CA3 network.     

Distribution of glycine receptors at central synapses: an immunoelectron microscopy study

The distribution of receptors for a neurotransmitter was investigated cytochemically for the first time in the central nervous system, at synapses established on cells of the ventral horn of the rat cervical spinal cord. Three monoclonal antibodies (mAb's) raised against glycine receptors were used. Immunofluorescent staining already showed discontinuous labeling at the surface of neurons, and immunoenzymatic electron microscopy further revealed that the antigenic determinants were confined to the postsynaptic membrane and concentrated at the level of the synaptic complex. More specifically, one mAb directed against the receptive subunit of the oligomeric receptor recognized an epitope on the extracellular side of the plasma membrane, whereas two other mAb's bound to the cytoplasmic face. Epitopes for the last two mAb's were more accurately localized with protein A-colloidal gold, using an intermediate rabbit anti-mouse immunoglobulin serum. (a) In addition to the presence of gold particles in areas facing the presynaptic active zone (visualized with ethanolic phosphotungstic acid), the labeling extended beyond this zone for approximately 50-60 nm, which corresponds to the width of one presynaptic dense projection. (b) The distances between the mid membrane and the gold particles were different for the two mAb's (with means of 21.7 +/- 8.5 nm and 29.8 +/- 10.4 nm, respectively). The data suggest that one of the recognized epitopes is close to the plasma membrane, whereas the second protrudes into the cytoplasm. Our results indicate that the receptor is a transmembrane protein which has a restricted spatial distribution on the postsynaptic neuronal surface.     

Molecular physiology of P2X receptors and ATP signalling at synapses

ATP is found in every cell, where it is a major source of energy. But in the nervous system, ATP also has additional actions, which include its role in fast synaptic transmission and modulation. Here I discuss the 'fast' actions of ATP at synapses, the properties of the receptors that are activated by ATP and the physiology of ATP signalling, with emphasis on its role in pain processing.