cGMP-induced presynaptic depression and postsynaptic facilitation at glutamatergic synapses in visual cortex

This study was aimed to examine the neuronal and glial response in the hypothalamus and neurohypophysis of rats with streptozotocin-induced diabetes. At various time intervals after induction of diabetes the neurons in the paraventricular- (PVN) and supraoptic- (SON) nucleus showed upregulated arginine vasopressin (AVP) and oxytocin (OXT) immunoexpression, being most pronounced at 2 weeks. Concomitant to this was the hypertrophy of PVN and SON neurons. NMDAR1, which was constitutively and moderately expressed in normal rats, was markedly augmented, being most intense at 4 months. This coincided with the expression of neuronal nitric oxide synthase (nNOS). Contrary to this, the expression of GluR2/3 was progressively downregulated, so that it was hardly detected at 4 months. Both astrocytes and microglia marked by anti-GFAP and OX-42, respectively, appeared activated. In pars nervosa, the projection target of the axon terminals of PVN and SON neurons, massive axons and terminals (Herring bodies) laden with neurosecretions were observed in diabetic rats. Colocalization study showed that the neurosecretions were internalized by activated pituicytes and microglia associated with the axons. The present results suggest that the neurosecretion of PVN and SON neurons is enhanced in diabetes. This is coupled by upregulation of NMDAR1 and nNOS but downregulation of GluR2/3. It is speculated that the glutamate receptors and NO are linked to overactivation of PVN and SON neurons leading ultimately to cell death of some of them. The pituicytes and microglia in pars nervosa would help to modulate the release of neurosecretion.     

Shaping excitation at glutamatergic synapses.

Glutamatergic synapses vary, exhibiting EPSCs of widely different magnitudes and timecourses. The main contributors to this variability are: presynaptic factors, including release probability, quantal content and vesicle composition; factors that modulate the concentration and longevity of glutamate in the cleft, including diffusion and the actions of glutamate transporters; and postsynaptic factors, including the types and locations of ionotropic glutamate receptors, their numbers, and the nature and locations of associated intracellular signalling systems.     

Internalization at glutamatergic synapses during development

Glutamate receptors are internalized from the cell membrane via clathrin-coated pits. However, little is known about where this occurs - whether at or near the synapse or at some distance from it. In this study we used immunogold localization in the rat brain (mainly hippocampus) to show that clathrin-coated pits are found both at the edge of the synaptic active zone and at further postsynaptic distances, including on the sides of the spine; we also localize these pits specifically to glutamatergic synapses. In addition, we show that clathrin-coated pits can internalize both N-methyl-d-aspartate (in vivo) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (in vitro data only) receptors at extrasynaptic sites not associated directly with synapses. Also, caveolin might be prevalent at excitatory synapses, although it is not known whether it is involved in receptor internalization, receptor stabilization, or some other function.     

L-phenylalanine selectively depresses currents at glutamatergic excitatory synapses

To explore the hypothesis that L-phenylalanine (L-Phe) depresses glutamatergic synaptic transmission and thus contributes to brain dysfunction in phenylketonuria (PKU), the effects of L-Phe on spontaneous and miniature excitatory postsynaptic currents (s/mEPSCs) in rat and mouse hippocampal and cerebrocortical cultured neurons were studied using the patch-clamp technique. L-Phe depressed the amplitude and frequency of both N-methyl-D-aspartate (NMDA) and non-NMDA components of glutamate receptor (GluR) s/mEPSCs. The IC(50) of L-Phe to inhibit non-NMDAR mEPSC frequency was 0.98 +/- 0.13 mM, a brain concentration seen in classical PKU. In contrast, D-Phe had a significantly smaller effect, whereas L-leucine, an amino acid that competes with L-Phe for brain transporter, had no effect on mEPSCs. Unlike GluR s/mEPSCs, GABA receptor mIPSCs were not attenuated by L-Phe. A high extracellular concentration of glycine prevented the attenuation by L-Phe of NMDAR current, activated by exogenous agonist, and of NMDAR s/mEPSC amplitude, but not of NMDAR s/mEPSC frequency. On the other hand, L-Phe significantly depressed non-NMDAR current activated by low but not high concentrations of exogenous agonists. Glycine-independent attenuation of NMDAR s/mEPSC frequency suggests decreased presynaptic glutamate release caused by L-Phe, whereas decreased amplitudes of NMDAR and non-NMDAR s/mEPSCs are consistent with competition of L-Phe for the glycine- and glutamate-binding sites of NMDARs and non-NMDARs, respectively. The finding that GluR activity is significantly depressed at conditions characteristic of classical PKU indicates a potentially important contribution of impaired GluR function to PKU-related mental retardation and provides important insights into the potential physiological consequences of impaired GluR function.     

Microglia shape presynaptic properties at developing glutamatergic synapses

Deficient neuron–microglia signaling during brain development is associated with abnormal synaptic maturation. However, the precise impact of deficient microglia function on synaptic maturation and the mechanisms involved remain poorly defined. Here we report that mice defective in neuron-to-microglia signaling via the fractalkine receptor (Cx3cr1 KO) show reduced microglial branching and altered motility and develop widespread deficits in glutamatergic neurotransmission. We characterized the functional properties of CA3–CA1 synapses in hippocampal slices from these mice and found that they display altered glutamatergic release probability, maintaining immature properties also at late developmental stages. In particular, CA1 synapses of Cx3cr1 KO show (i) immature AMPA/NMDA ratio across developmental time, displaying a normal NMDA component and a defective AMPA component of EPSC; (ii) defective functional connectivity, as demonstrated by reduced current amplitudes in the input/output curve; and (iii) greater facilitation in the paired pulse ratio (PPR), suggesting decreased release probability. In addition, minimal stimulation experiments revealed that excitatory synapses have normal potency, but an increased number of failures, confirming a deficit in presynaptic release. Consistently, KO mice were characterized by higher number of silent synapses in comparison to WT. The presynaptic deficits were corrected by performing experiments in conditions of high release probability (Ca²⁺/Mg²⁺ratio 8), where excitatory synapses showed normal synaptic multiplicity, AMPA/NMDA ratio, and proportion of silent synapses. These results establish that neuron–microglia interactions profoundly influence the functional maturation of excitatory presynaptic function.     

RNA binding proteins accumulate at the postsynaptic density with synaptic activity

Neuronal activity elicits changes in synaptic composition that play an important role in experience-dependent plasticity (Choquet and Triller, 2003; Lisman and Raghavachari, 2006; Bourne and Harris, 2008; Holtmaat and Svoboda, 2009). We used a modified version of stable isotope labeling by amino acids in cell culture to identify activity-dependent modifications in the composition of postsynaptic densities (PSDs) isolated from rat primary neuronal cultures. We found that synaptic activity altered ～2% of the PSD proteome, which included an increase in diverse RNA binding proteins (RNABPs). Indeed, 12 of the 37 identified proteins whose levels changed with synaptic activity were RNABPs and included the heterogeneous nuclear ribonucleoproteins (hnRNPs) G, A2/B1, M, and D. Knockdown of hnRNPs M and G using shRNAs resulted in altered numbers of dendritic spines, suggesting a crucial role for these proteins in spine density. Synaptic activity also resulted in a concomitant increase in dendritic and synaptic poly(A) mRNA. However, this increase was not affected by knockdown of hnRNPs M or G. Our results suggest that hnRNP proteins regulate dendritic spine density and may play a role in synaptodendritic mRNA metabolism.     

Organization of postsynaptic density proteins and glutamate receptors in axodendritic and dendrodendritic synapses of the rat olfactory bulb

Glutamate neurotransmission in the olfactory bulb involves both axodendritic synapses and dendrodendritic reciprocal synapses and possibly also extrasynaptic receptors. By using a sensitive immunogold procedure, we have investigated the organization of two synaptic scaffolding molecules, PSD-95 and PSD-93, as well as N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) receptors, at these heterogeneous glutamate signaling sites. Immunolabeling for PSD-95 and PSD-93 was present in all major types of putative glutamatergic synapse, suggesting that these proteins are essential components of the synaptic signaling apparatus. The linear density and the subsynaptic distribution of PSD-95/PSD-93 gold particles did not differ significantly between axodendritic and dendrodendritic synapses. Antibodies recognizing NMDA and AMPA receptor subunits also labeled asymmetric synapses throughout the olfactory bulb. Immunolabeling for the AMPA receptor subunits GluR2/3 was similar in all types of synapse. In contrast, immunogold signals for the NR1 subunit of NMDA receptors varied significantly among different synapse populations, with olfactory nerve synapses in the glomerular layer showing the lowest labeling intensity. Although the lateral dendrites of mitral and tufted cells have been reported to respond to glutamate, they did not display significant plasma membrane labeling for the NR1 subunit or for PSD-95, suggesting that the physiological effects of glutamate at these sites are mediated by NMDA autoreceptors that are not clustered and occur only at a low density on the dendritic surface. Our quantitative analysis of olfactory bulb synapses indicates that the density of NMDA receptors is not determined by the complement of PSD-95/PSD-93. The latter molecules appear to be expressed in an all-or-none fashion and may form a standard lattice common to different types of glutamatergic synapse.     

Quantification of postsynaptic density proteins: glutamate receptor subunits and scaffolding proteins

The postsynaptic density (PSD) protein complex has long been a major target of proteomics in neuroscience. As the number of glutamate receptors on a synapse is one of the main determinants of synaptic efficacy, determining the absolute numbers of receptors in the PSD is necessary for estimating the amplitude of the excitatory postsynaptic current (EPSC) in individual synapses. Moreover, as the receptor molecules are embedded in a macromolecular complex within the PSD, stoichiometry between the receptors and other PSD proteins could help explain the functional and regional specialization of the synapses and their possible roles in synaptic plasticity. Here, I review various studies concerned with the quantification of PSD proteins.     

Neuroligin-3 is a neuronal adhesion protein at GABAergic and glutamatergic synapses

Synaptic adhesion molecules are thought to play a critical role in the formation, function and plasticity of neuronal networks. Neuroligins (NL1-4) are a family of presumptive postsynaptic cell adhesion molecules. NL1 and NL2 isoforms are concentrated at glutamatergic and GABAergic synapses, respectively, but the cellular expression and synaptic localization of the endogenous NL3 and NL4 isoforms are unknown. We generated a panel of NL isoform-specific antibodies and examined the expression, developmental regulation and synaptic specificity of NL3. We found that NL3 was enriched in brain, where NL3 protein levels increased during postnatal development, coinciding with the peak of synaptogenesis. Subcellular fractionation revealed a concentration of NL3 in synaptic plasma membranes and postsynaptic densities. In cultured hippocampal neurons, endogenous NL3 was highly expressed and was localized at both glutamatergic and GABAergic synapses. Clustering of NL3 in hippocampal neurons by neurexin-expressing cells resulted in coaggregation of NL3 with glutamatergic and GABAergic scaffolding proteins. Finally, individual synapses contained colocalized NL2 and NL3 proteins, and coimmunoprecipitation studies revealed the presence of NL1-NL3 and NL2-NL3 complexes in brain extracts. These findings suggest that rodent NL3 is a synaptic adhesion molecule that is a shared component of glutamatergic and GABAergic synapses.     

The metabolic turnover of the major proteins of the postsynaptic density

We have used the method of Austin, Lowry, Brown and Carter, to measure the steady-state metabolic half-life of tubulin (alpha and beta individually) and actin (beta and gamma together) in the total cytosolic (S3), microsomal (P3), synaptic plasma membrane (SPM) and synaptic junction (SJ) subcellular fractions from 6-day-old and adult chicken forebrain. In the SPM and SJ fractions we also measured the steady-state metabolic half-life of the major postsynaptic density protein (mPSDp). In SPM and SJ fractions from 6-day-old chickens tubulin and actin turned over approximately twice as slowly (t1/2 approximately equal to 24 days) as tubulin and actin in the S3 fraction (t1/2 approximately equal to 13 days). This difference was unlikely merely to be due to association with membranes since the t1/2 values for the proteins were the same in P3 and S3. The estimated t1/2 values for mPSDp were similar to that for tubulin and actin in SPM and SJ fractions. Similar results were obtained in adult chickens except that all t1/2 values in all fractions were approximately 30% larger. The calculated t1/2 values did not change between labelling periods of 4 and 6.5 h suggesting that the lag phase of incorporation of newly synthesized PSD proteins is sufficiently rapid to not produce this result artefactually. When the brain from a non-labelled chicken was homogenized in the presence of the S3 fraction from a labelled chicken and sub-fractionated the relative specific activities of the SPM and SJ fractions produced were 1-2% of those from the labelled brain. These results support the notion that tubulin and actin are intrinsic components of the PSD.     

The role of postsynaptic density proteins in neural degeneration and regeneration

正The structure,function and plasticity of excitatory glutamatergic synapses in the brain are significantly altered by neurodevelopmental and neurodegenerative disorders(Grabrucker et al.,2011;Fourie et al.,2014;Musardo et al.,2014;Lee et al.,2015),resulting in impairments in cognition,learning,memory,motor and sensory function.Revers-     

Postsynaptic muscarinic m2 receptors at cholinergic and glutamatergic synapses of mouse brainstem motoneurons

In many brain areas, few cholinergic synapses are identified. Acetylcholine is released into the extracellular space and acts through diffuse transmission. Motoneurons, however, are contacted by numerous cholinergic terminals, indicating synaptic cholinergic transmission on them. The muscarinic m2 receptor is the major acetylcholine receptor subtype of motoneurons; therefore, we analyzed the localization of the m2 receptor in correlation with synapses by electron microscopic immunohistochemistry in the mouse trigeminal, facial, and hypoglossal motor nuclei. In all nuclei, m2 receptors were localized at the membrane of motoneuronal perikarya and dendrites. The m2 receptors were concentrated at cholinergic synapses located on the perikarya and most proximal dendrites. However, m2 receptors at cholinergic synapses represented only a minority (<10%) of surface m2 receptors. The m2 receptors were also enriched at glutamatergic synapses in both motoneuronal perikarya and dendrites. A relatively large proportion (20–30%) of plasma membrane–associated m2 receptors were located at glutamatergic synapses. In conclusion, the effect of acetylcholine on motoneuron populations might be mediated through a synaptic as well as diffuse type of transmission. J. Comp. Neurol. 521:2008–2024, 2013. © 2012 Wiley Periodicals, Inc.     

Protective cap over CA1 synapses: extrasynaptic glutamate does not reach the postsynaptic density

Numerous data indicate that nonsynaptic release of glutamate occurs both in normal and pathophysiological conditions. When reaching receptors in the postsynaptic density (PSD), glutamate (Glu) could affect the synaptic transmission. We have tested this possibility in the hippocampal CA1 synapses of rats, either by applying exogenous Glu to the CA1 neurons or by disruption of Glu transporter activity. L-Glu (400 microM) was directly applied to the hippocampal slices acutely isolated from the rats. It produced a strong inhibition of both ortho- and antidromically elicited action potentials fired by CA1 neurons while the excitatory postsynaptic current (EPSC) measured in these neurons remained totally unaffected. The optical isomer D-Glu which is not transported by the systems of Glu uptake inhibited not only orthodromic and antidromic spikes, but also EPSC. Non-specific glutamate transporter inhibitor DL-threo-beta-hydroxyaspartic acid (THA, 400 microM) mimicked the effects of exogenous Glu and produced strong inhibition of both orthodromic and antidromic spikes, without any influence on the amplitude of EPSCs. Dihydrokainate (DHK, 300 microM), selective inhibitor of GLT-1 subtype of glutamate transporter, exerted a significant inhibitory action on the orthodromically evoked spikes and also on the EPSC. Our results indicate that extrasynaptic and PSD membranes of CA1 neurons form separate compartments differing in the mechanisms and efficiency of external Glu processing: the protection of PSD markedly prevails.     

Pre- and postsynaptic GABA receptors at reciprocal dendrodendritic synapses in the olfactory bulb

Presynaptic ionotropic receptors are important regulators of synaptic function; however, little is known about their organization in the presynaptic membrane. We show here a different spatial organization of presynaptic and postsynaptic GABA(A) receptors at reciprocal dendrodendritic synapses between mitral and granule cells in the rat olfactory bulb. Using postembedding electron microscopy, we have found that mitral cell dendrites express GABA(A) receptors at postsynaptic specializations of symmetric (GABAergic) synapses, as well as at presynaptic sites of asymmetric (glutamatergic) synapses. Analysis of the subsynaptic distribution of gold particles revealed that in symmetric synapses GABA(A) receptors are distributed along the entire postsynaptic membrane, whereas in asymmetric synapses they are concentrated at the edge of the presynaptic specialization. To assess the specificity of immunogold labelling, we analysed the olfactory bulbs of mutant mice lacking the alpha1 subunit of GABA(A) receptors. We found that in wild-type mice alpha1 subunit immunoreactivity was similar to that observed in rats, whereas in knockout mice the immunolabelling was abolished. These results indicate that in mitral cell dendrites GABA(A) receptors are distributed in a perisynaptic domain that surrounds the presynaptic specialization. Such presynaptic receptors may be activated by spillover of GABA from adjacent inhibitory synapses and modulate glutamate release, thereby providing a novel mechanism regulating dendrodendritic inhibition in the olfactory bulb.     

ZD7288 inhibits postsynaptic glutamate receptor-mediated responses at hippocampal perforant path-granule cell synapses

Hyperpolarization-activated channels (Ih) are widely expressed in the nervous system and believed to play an important role in the regulation of membrane excitability and rhythmic activity. Recent evidence suggests that Ih may be involved in long-term potentiation (LTP) in the hippocampus; however, the results are controversial. To explore the possible causes of these differing results, the effects of Ih blockers on synaptic activity were evaluated in mouse hippocampal slices. ZD7288 (20 micro m), a selective Ih blocker, apparently prevented the induction of LTP, while Cs+ (1 mm), a commonly used Ih blocker, had no effect on LTP at hippocampal perforant path-dentate granule cell synapses. In addition, ZD7288 but not Cs+ abolished basal synaptic transmission. Results from voltage-clamp experiments showed that ZD7288 produced a very little inhibition on hyperpolarization-activated currents, indicating a weak expression of the Ih in granule neurons. Outside-out patch recordings revealed that ZD7288 inhibited glutamate receptor-mediated responses, while Cs+ had no effect on them. Meanwhile, ZD7288 reduced both alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate and N-methyl-d-aspartate receptor-mediated excitatory postsynaptic currents. The results suggest that ZD7288-induced reduction of synaptic transmission may result from its inhibition of the postsynaptic glutamate receptors on dentate granule neurons.     

Amyloid fibrils induce dysfunction of hippocampal glutamatergic silent synapses

Silent glutamatergic synapses lacking functional AMPA (α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazoleproprionate) receptors exist in several brain regions including the hippocampus. Their involvement in the dysfunction of hippocampal glutamatergic transmission in the setting of Alzheimer's disease (AD) is unknown. This study demonstrated a decrease in the percentage of silent synapses in rats microinjected with amyloid fibrils (Aβ_1–40) into the hippocampal CA1. Also, pairing low‐frequency electric stimuli failed to induce activation of the hippocampal silent synapses in the modeled rats. Immunoblotting studies revealed a decreased expression of GluR1 subunits in the hippocampal CA1 synaptosomal preparation, indicating a potential reduction in the GluR1 subunits anchoring in postsynaptic density in the modeled rats. We also noted a decreased expression of phosphorylated cofilin, which regulates the function of actin cytoskeleton and receptor trafficking, and reduced expression of the scaffolding protein PSD95 in the hippocampal CA1 synaptosome in rats injected with Aβ_1–40. Taken together, this study illustrates dysfunction of hippocampal silent synapse in the rodent model of AD, which might result from the impairments of actin cytoskeleton and postsynaptic scaffolding proteins induced by amyloid fibrils.     

Disruption of striatal glutamatergic transmission induced by mutant huntingtin involves remodeling of both postsynaptic density and NMDA receptor signaling

We study the striatal susceptibility to NMDA receptor (NMDAR)-mediated injury of two Huntington’s disease (HD) transgenic mice: R6/1 and R6/1:BDNF^+/−. We found that R6/1:BDNF^+/− mice – which express reduced levels of BDNF – were more resistant than R6/1 mice to intrastriatal injection of quinolinate. This increased resistance is related to a differential reduction in expression of NMDAR scaffolding proteins, MAGUKs (PSD-95, PSD-93, SAP-102 and SAP-97) but not to altered levels or synaptic location of NMDAR. A robust reorganization of postsynaptic density (PSD) was detected in HD transgenic mice, shown by a switch of PSD-93 by PSD-95 in PSD. Furthermore, NMDAR signaling pathways were affected by different BDNF levels in HD mice; we found a reduction of synaptic αCaMKII (but not of nNOS) in R6/1:BDNF^+/− compared to R6/1 mice. The specific regulation of MAGUKs and αCaMKII in striatal neurons may reflect a protective mechanism against expression of mutant huntingtin exon-1.     

Endocannabinoid-dependent plasticity at GABAergic and glutamatergic synapses in the striatum is regulated by synaptic activity

Long-term depression (LTD) at striatal synapses is mediated by postsynaptic endocannabinoid (eCB) release and presynaptic cannabinoid 1 receptor (CB₁R) activation. Previous studies have indicated that eCB mobilization at excitatory synapses might be regulated by afferent activation. To further address the role of neuronal activity in synaptic plasticity we examined changes in synaptic strength induced by the L-type calcium channel activator 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methyl ester (FPL 64176, FPL) at glutamatergic and γ-aminobutyric acid (GABA)ergic synapses in the striatum. We found that the basic mechanisms for FPL-mediated eCB signaling are the same at glutamatergic and GABAergic synapses. FPL-induced LTD (FPL-LTD) was blocked in slices treated with the CB₁R antagonist AM251 (2 μ m ), but established depression was not reversed by AM251. FPL-LTD was temperature dependent, blocked by protein translation inhibitors and prevented by intracellular loading of the anandamide transporter inhibitor VDM11 (10 μ m ) at both glutamatergic and GABAergic synapses. FPL-LTD at glutamatergic synapses required paired-pulse afferent stimulation, while FPL-LTD at GABAergic synapses could be induced even in the absence of explicit afferent activation. By evaluating tetrodotoxin-insensitive spontaneous inhibitory postsynaptic currents we found that neuronal firing is vital for eCB release and LTD induction at GABAergic synapses, but not for short-term depression induced by CB₁R agonist. The data presented here suggest that the level of neuronal firing regulates eCB signaling by modulating release from the postsynaptic cell, as well as interacting with presynaptic mechanisms to induce LTD at both glutamatergic and GABAergic synapses in the striatum.     

The postsynaptic scaffold proteins ProSAP1/Shank2 and Homer1 are associated with glutamate receptor complexes at rat retinal synapses

The postsynaptic density (PSD) at glutamatergic synapses is a macromolecular complex of various molecules that organize the different glutamate receptors spatially and link them to their appropriate downstream signaling pathways and to the cytoskeleton. Recently, a new family of multidomain proteins called Shanks or ProSAPs (proline-rich synapse-associated proteins) has been identified. They are suggested to be central adaptor proteins of the PSD of glutamatergic synapses, bridging different types of glutamate receptor complexes. With immunocytochemistry and light and electron microscopy, we examined the cellular, synaptic, and postnatal developmental expression of ProSAP1/Shank2 at the synapses of rat retina. With double-labeling experiments and confocal microscopy, we analyzed the association of ProSAP1/Shank2 with proteins specific for glutamatergic, glycinergic, and gamma-aminobutyric acid (GABA)ergic synapses and with proteins known to be involved in the structural and functional organization of PSDs containing N-methyl-D-aspartate receptors [95-kDa postsynaptic density protein (PSD-95)], group I metabotropic glutamate receptors (Homer1), and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors [glutamate receptor-interacting protein (GRIP)]. ProSAP1/Shank2 was present postsynaptically at the glutamatergic ribbon synapses of photoreceptor and bipolar cells, and it was absent from glycinergic and GABAergic amacrine cell synapses. The double-labeling experiments revealed a high rate of colocalization of ProSAP1/Shank2 with Homer1 and PSD-95, and little colocalization with GRIP. These data suggest that ProSAP1/Shank2 acts as an organizer at PSDs of different glutamatergic retinal synapses.