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.     

Localization of mineralocorticoid receptors at mammalian synapses

In the brain, membrane associated nongenomic steroid receptors can induce fast-acting responses to ion conductance and second messenger systems of neurons. Emerging data suggest that membrane associated glucocorticoid and mineralocorticoid receptors may directly regulate synaptic excitability during times of stress when adrenal hormones are elevated. As the key neuron signaling interface, the synapse is involved in learning and memory, including traumatic memories during times of stress. The lateral amygdala is a key site for synaptic plasticity underlying conditioned fear, which can both trigger and be coincident with the stress response. A large body of electrophysiological data shows rapid regulation of neuronal excitability by steroid hormone receptors. Despite the importance of these receptors, to date, only the glucocorticoid receptor has been anatomically localized to the membrane. We investigated the subcellular sites of mineralocorticoid receptors in the lateral amygdala of the Sprague-Dawley rat. Immunoblot analysis revealed the presence of mineralocorticoid receptors in the amygdala. Using electron microscopy, we found mineralocorticoid receptors expressed at both nuclear including: glutamatergic and GABAergic neurons and extra nuclear sites including: presynaptic terminals, neuronal dendrites, and dendritic spines. Importantly we also observed mineralocorticoid receptors at postsynaptic membrane densities of excitatory synapses. These data provide direct anatomical evidence supporting the concept that, at some synapses, synaptic transmission is regulated by mineralocorticoid receptors. Thus part of the stress signaling response in the brain is a direct modulation of the synapse itself by adrenal steroids.     

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.     

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.     

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.     

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.     

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.     

Diffusion in lipid bilayers containing barriers

In epithelial cells, a barrier or tight junction restricts the diffusion of lipid probes from the apical to the basolateral side of the outer membrane bilayer. This phenomenon is studied theoretically with the diffusion equation on planar and spherical surfaces. Two models for the tight junction are considered: a penetrable barrier embedded in a monolayer and an impenetrable obstacle in the outer membrane of a bilayer than must be bypassed by flip-flopping between inner and outer membranes. The rate of passing from one side of the cell to the other is calculated for each of these models under steady state conditions. The results are compared with recent fluorescent photobleaching recovery experiments. The theoretical interpretation indicates that it would be difficult to distinguish experimentally between the flip-flop case and the barrier crossing case. Assuming a flip-flop model, large differences in the magnitude of the flip-flop rates of probes are necessary to explain the experimental results as suggested by Dragsten et al. (Dragsten, P. R., R. Blumenthal, and J. S. Handler, 1981, Nature [Lond.], 294:718--722).     

Long-term potentiation selectively expressed by NMDA receptors at hippocampal mossy fiber synapses

The mossy fiber to CA3 pyramidal cell synapse (mf-CA3) provides a major source of excitation to the hippocampus. Thus far, these glutamatergic synapses are well recognized for showing a presynaptic, NMDA receptor-independent form of LTP that is expressed as a long-lasting increase of transmitter release. Here, we show that in addition to this "classical" LTP, mf-CA3 synapses can undergo a form of LTP characterized by a selective enhancement of NMDA receptor-mediated transmission. This potentiation requires coactivation of NMDA and mGlu5 receptors and a postsynaptic calcium rise. Unlike classical LTP, expression of this mossy fiber LTP is due to a PKC-dependent recruitment of NMDA receptors specifically to the mf-CA3 synapse via a SNARE-dependent process. Having two mechanistically different forms of LTP may allow mf-CA3 synapses to respond with more flexibility to the changing demands of the hippocampal network.     

gamma-Aminobutyric acid-containing terminals can be apposed to glycine receptors at central synapses

The distributions of terminals containing gamma-aminobutyric acid (GABA) and of endings apposed to glycine receptors were investigated cytochemically in the ventral horn of the rat spinal cord. For this purpose, a polyclonal antibody raised to recognize glutamic acid decarboxylase (GAD), a synthetic enzyme for GABA, and three monoclonal antibodies (mAb's) directed against the glycine receptor were used. Double immunofluorescence showed that, surprisingly, GAD-positive terminals are closely associated in this system with glycine receptors at all the investigated cells, most of which were spinal motoneurons. Furthermore, double labeling was performed with immunoenzymatic recognition of GAD and indirect marking of mAb's with colloidal gold. With this combined approach, it was found, at the electron microscopic level, that all GAD-positive terminals are in direct apposition with glycine receptors while, on the other hand, not all glycine receptors are in front of GABA-containing boutons. This result is not due to a cross-reactivity of mAb's with GABA receptors as shown by using as a control synapses known to use GABA as a neurotransmitter in the cerebellar cortex. Indeed, no glycine receptor immunoreactivity was detected on Purkinje cells facing basket axon terminals. However, Purkinje neurons can express glycine receptor immunoreactivity at other synaptic contacts. Assuming that the presence of postsynaptic receptors for glycine indicates that this amino acid is used for neurotransmission at a given synapse, our results strongly support the notion that GABA and glycine, two classical inhibitory transmitters, coexist at some central connections. However, such is not always the case; in the cerebellum, Golgi terminals impinging on the dendrites of granule cells are either GAD-positive or face glycine receptors, in a well-segregated manner.     

Presynaptic NMDA receptors mediate IPSC potentiation at GABAergic synapses in developing rat neocortex

Background

NMDA receptors are traditionally viewed as being located postsynaptically, at both synaptic and extrasynaptic locations. However, both anatomical and physiological studies have indicated the presence of NMDA receptors located presynaptically. Physiological studies of presynaptic NMDA receptors on neocortical GABAergic terminals and their possible role in synaptic plasticity are lacking.

Methodology/Principal Findings

We report here that presynaptic NMDA receptors are present on GABAergic terminals in developing (postnatal day (PND) 12-15) but not older (PND21-25) rat frontal cortex. Using MK-801 in the recording pipette to block postsynaptic NMDA receptors, evoked and miniature IPSCs were recorded in layer II/III pyramidal cells in the presence of AMPA/KA receptor antagonists. Bath application of NMDA or NMDA receptor antagonists produced increases and decreases in mIPSC frequency, respectively. Physiologically patterned stimulation (10 bursts of 10 stimuli at 25 Hz delivered at 1.25 Hz) induced potentiation at inhibitory synapses in PND12-15 animals. This consisted of an initial rapid, large increase in IPSC amplitude followed by a significant but smaller persistent increase. Similar changes were not observed in PND21-25 animals. When 20 mM BAPTA was included in the recording pipette, potentiation was still observed in the PND12-15 group indicating that postsynaptic increases in calcium were not required. Potentiation was not observed when patterned stimulation was given in the presence of D-APV or the NR2B subunit antagonist Ro25-6981.

Conclusions/Significance

The present results indicate that presynaptic NMDA receptors modulate GABA release onto neocortical pyramidal cells. Presynaptic NR2B subunit containing NMDA receptors are also involved in potentiation at developing GABAergic synapses in rat frontal cortex. Modulation of inhibitory GABAergic synapses by presynaptic NMDA receptors may be important for proper functioning of local cortical networks during development.     

Incorporation of inwardly rectifying AMPA receptors at silent synapses during hippocampal long-term potentiation

Despite decades of study, the mechanisms by which synapses express the increase in strength during long-term potentiation (LTP) remain an area of intense interest. Here, we have studied how AMPA receptor subunit composition changes during the early phases of hippocampal LTP in CA1 pyramidal neurons. We studied LTP at silent synapses that initially lack AMPA receptors, but contain NMDA receptors. We show that strongly inwardly rectifying AMPA receptors are initially incorporated at silent synapses during LTP and are then subsequently replaced by non-rectifying AMPA receptors. These findings suggest that silent synapses initially incorporate GluA2-lacking, calcium-permeable AMPA receptors during LTP that are then replaced by GluA2-containing calcium-impermeable receptors. We also show that LTP consolidation at CA1 synapses requires a rise in intracellular calcium concentration during the early phase of expression, indicating that calcium influx through the GluA2-lacking AMPA receptors drives their replacement by GluA2-containing receptors during LTP consolidation. Taken together with previous studies in hippocampus and in other brain regions, these findings suggest that a common mechanism for the expression of activity-dependent glutamatergic synaptic plasticity involves the regulation of GluA2-subunit composition and highlights a critical role for silent synapses in this process.     

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.     

Analysis of relations between NMDA receptors and GABA release at olfactory bulb reciprocal synapses

In the mammalian olfactory bulb, signal processing is mediated by synaptic interactions between dendrites. Glutamate released from mitral cell dendrites excites dendritic spines of granule cells, which in turn release GABA back onto the mitral cell dendrites, forming a reciprocal synaptic pair. This feedback synaptic circuit was shown to be mediated predominantly by NMDA receptors. We further utilized caged Ca2+ compounds to obtain insight into the mechanism that couples NMDA receptor activation to GABA release. Feedback inhibition elicited by photo-release of caged Ca2+ in mitral cell secondary dendrites persisted when voltage-gated Ca2+ channels were blocked by cadmium (Cd2+) and nickel (Ni2+). These results indicate that Ca2+ influx through NMDA receptors can directly trigger presynaptic GABA release for local dendrodendritic feedback inhibition.