Synaptic Membrane Fractions Research Articles

Effect of H2O2 on Na,K-ATPase.

Na,K-ATPase is a member of the P-type ATPase family, which transforms the energy of ATP to the transmembrane Na/K gradient that is used to create membrane potential, support the excitability of neurons and myocytes, control pH, and transport substances. The regulation of the Na,K-ATPase function by physiological regulators also comprises a central role in the adaptation of organisms to different conditions.H2O2is one of the main signaling molecules in redox metabolism and plays important function in cellular physiology. H2O2 also regulates signaling pathways via the specific oxidation of proteins harboring redox-sensitive moieties, like metal centers or cysteine residues, which control their activity. The Na,K-ATPase is redox-sensitive with an "optimal redox potential range," where the reactive oxygen species (ROS), levels beyond this "optimal range" are responsible for enzyme inhibition. Thus reactive oxygen species manifest a hermetic effect, which is expressed by biphasic action; stimulation by low doses and inhibition by high doses. This study was aimed to reveal redox-sensitivity of brain synaptic membrane fractions Na,K-ATPase via H2O2 effects. Different concentrations of H2O2 change the kinetic parameters of the enzyme system for MgATP complex, Na+, and K+ differently. Moreover, H2O2 changes p-chloromercuribenzoic acids (PCMB) affinity. H2O2 targets thiols of the Na,K-ATPase - low and high concentrations of H2O2 change the oxidative state of thiolate (S-) from Cys differently, resulting in the corresponding activation or inhibition of the enzyme. Targeting thiols of the Na,K-ATPase tunes the activity of the Na,K-ATPase to the cellular demands and sustains the enzyme activity at the "optimal" level.

Leucine-Rich Repeats and Transmembrane Domain 2 Controls Protein Sorting in the Striatal Projection System and Its Deficiency Causes Disturbances in Motor Responses and Monoamine Dynamics.

The striatum is involved in action selection, and its disturbance can cause movement disorders. Here, we show that leucine-rich repeats and transmembrane domain 2 (Lrtm2) controls protein sorting in striatal projection systems, and its deficiency causes disturbances in monoamine dynamics and behavior. The Lrtm2 protein was broadly detected in the brain, but it was enhanced in the olfactory bulb and dorsal striatum. Immunostaining revealed a strong signal in striatal projection output, including GABAergic presynaptic boutons of the SNr. In subcellular fractionation, Lrtm2 was abundantly recovered in the synaptic plasma membrane fraction, synaptic vesicle fraction, and microsome fraction. Lrtm2 KO mice exhibited altered motor responses in both voluntary explorations and forced exercise. Dopamine metabolite content was decreased in the dorsal striatum and hypothalamus, and serotonin turnover increased in the dorsal striatum. The prefrontal cortex showed age-dependent changes in dopamine metabolites. The distribution of glutamate decarboxylase 67 (GAD67) protein and gamma-aminobutyric acid receptor type B receptor 1 (GABABR1) protein was altered in the dorsal striatum. In cultured neurons, wild-type Lrtm2 protein enhanced axon trafficking of GAD67-GFP and GABABR1-GFP whereas such activity was defective in sorting signal-abolished Lrtm2 mutant proteins. The topical expression of hemagglutinin-epitope-tag (HA)-Lrtm2 and a protein sorting signal abolished HA-Lrtm2 mutant differentially affected GABABR1 protein distribution in the dorsal striatum. These results suggest that Lrtm2 is an essential component of striatal projection neurons, contributing to a better understanding of striatal pathophysiology.

Some Kinetic Features of Na,K-ATPase and Sensitivity to Noradrenaline

A comparative kinetic analysis of albino rat brain synaptic and kidney plasma membrane fraction Na,K-ATPase was performed to comprehend the different levels of sensitivity of these fractions to the neurotransmitter noradrenaline. Noradrenaline (NA) inhibits the rat brain synaptic membrane Na,K-ATPase, changes the stoichiometry of Na+ and K+ and shifts the enzyme system from an MgATP to an Mg2+ dependent cycle. While the kidney plasma membrane fraction Na,K-ATPase is not sensitive to noradrenaline. To investigate the mechanism underlying this difference, we studied enzyme velocity dependence on the concentration of Mg2+. The 1/V = f(Mg2+) function has shown different kinetic features for the synaptic and kidney plasma membrane Na,K-ATPase. With the addition of ethylene glycol tetraacetic acid (EGTA) to the reaction medium the geometric form of 1/V = f(Mg2+) function is affected differently. We thereafter measured the essential activator number for Na+ and K+ with, in excess Mg2+. The results of these experiments reveal that, contrary to the synaptic membrane Na,K-ATPase, the kidney plasma membrane fraction Na,K-ATPase does not possess an Mg2+ dependent cycle and noradrenaline exhibits different modulatory effects on the enzyme system.

AMPA Receptor Expression Requirement During Long-Term Memory Retrieval and Its Association with mTORC1 Signaling.

Recently, it was reported that mechanistic/mammalian target of rapamycin complex 1 (mTORC1) activity during memory retrieval is required for normal expression of aversive and non-aversive long-term memories. Here we used inhibitory-avoidance task to evaluate the potential mechanisms by which mTORC1 signaling pathway participates in memory retrieval. First, we studied the role of GluA-subunit trafficking during memory recall and its relationship with mTORC1 pathway. We found that pretest intrahippocampal infusion of GluR23ɣ, a peptide that selectively blocks GluA2-containing AMPA receptor (AMPAR) endocytosis, prevented the amnesia induced by the inhibition of mTORC1 during retrieval. Additionally, we found that GluA1 levels decreased and GluA2 levels increased at the hippocampal postsynaptic density subcellular fraction of rapamycin-infused animals during memory retrieval. GluA2 levels remained intact while GluA1 decreased at the synaptic plasma membrane fraction. Then, we evaluated the requirement of AMPAR subunit expression during memory retrieval. Intrahippocampal infusion of GluA1 or GluA2 antisense oligonucleotides (ASO) 3 h before testing impaired memory retention. The memory impairment induced by GluA2 ASO before retrieval was reverted by GluA23ɣ infusion 1 h before testing. However, AMPAR endocytosis blockade was not sufficient to compensate GluA1 synthesis inhibition. Our work indicates that de novo GluA1 and GluA2 AMPAR subunit expression is required for memory retrieval with potential different roles for each subunit and suggests that mTORC1 might regulate AMPAR trafficking during retrieval. Our present results highlight the role of mTORC1 as a key determinant of memory retrieval that impacts the recruitment of different AMPAR subunits.

Dissociated Role of D-Serine in Extinction During Consolidation vs. Reconsolidation of Context Conditioned Fear.

Extinction-based exposure therapy is widely used for the treatment of anxiety disorders, such as post-traumatic stress disorder (PTSD). D-serine, an endogenous co-agonist at the glycine-binding site of the N-methyl-D-aspartate-type glutamate receptor (NMDAR), has been shown to be involved in extinction of fear memory. Recent findings suggest that the length of time between the initial learning and an extinction session is a determinant of neural mechanism involved in fear extinction. However, how D-serine is involved in extinction of fear memory at different timings remains unclear. In the present study, we investigated the role of D-serine in immediate, delayed and post-retrieval extinction (P-RE) of contextual fear memory using wild-type (WT) and serine racemase (SRR) knockout (KO) mice that exhibit 90% reduction in D-serine content in the hippocampus. We found that SRR disruption impairs P-RE, facilitates immediate extinction (IE), but has no effect on delayed extinction (DE) of contextual fear memories. The impaired P-RE of contextual fear memory in SRRKO mice was associated with increased expression of the GluA1 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptor (AMPAR) in the hippocampal synaptic membrane fraction after P-RE, and this increase of AMPAR and impaired P-RE were rescued by the administration of D-serine to SRRKO mice. Our findings suggest that D-serine is differentially involved in the regulation of contextual fear extinction depending on the timing of behavioral intervention, and that combining D-serine or other drugs, enhancing the NMDAR function, with P-RE may achieve optimal outcomes for the treatment of PTSD.

The Functional Amyloid Orb2A Binds to Lipid Membranes

Lipid membranes interact with and influence the aggregation of many amyloid-forming proteins. Orb2 is a cytoplasmic polyadenylation element-binding protein homolog in Drosophila melanogaster that forms functional amyloids necessary for long-term memory. One isoform, Orb2A, has a unique N-terminus that has been shown to be important for the formation of amyloid-like aggregates and long-term memory in vivo. Orb2A is also found enriched in the synaptic membrane fraction. Our sequence and hydropathy analysis suggests that it can form an amphipathic helix, which is ideal for lipid membrane interaction. We used circular dichroism and site-directed spin labeling coupled with electron paramagnetic resonance to test the first 88 amino acids of Orb2A for lipid interaction. We show that Orb2A1-88 interacts with anionic lipid membranes using an amphipathic helix at its unique N-terminus. This interaction depends on the charge of the lipid membrane and the degree of membrane curvature. We used transmission electron microscopy and electron paramagnetic resonance to show that the presence of anionic small unilamellar vesicles inhibits amyloid fibril formation by Orb2A. This inhibition by anionic membranes could be a potential mechanism regulating Orb2A amyloid formation in vivo.

A novel synaptic junction preparation for the identification and characterization of cleft proteins.

Identification of synaptic cleft components has been hampered by the lack of a suitable preparation enriched in synaptic junctions devoid of adjoining peripheral membranes. Prior strategies for the isolation of synaptic junctions, relying on detergents for the removal of peripheral membranes, resulted in substantial loss of membranes lining the cleft. Here, a novel, detergent-free method is described for the preparation of a synaptic junction (SJ) fraction, using phospholipase A2. Limited digestion of synaptic plasma membrane (SPM) fraction with phospholipase A2 followed by centrifugation over a sucrose cushion results in selective removal of membranes peripheral to the cleft while junctional membranes remain relatively intact as observed by electron microscopy. Enrichment in synaptic junctional structures and loss of membranes peripheral to the junctional area are further verified by demonstrating enrichment in PSD-95 and loss in mGluR5, respectively. The SJ fraction is enriched in neuroligins and neurexins, in agreement with immuno-electron microscopy data showing their selective localization to the junctional area. Among additional cell adhesion molecules tested, N-cadherin and specific isoforms of the SynCAM and SALM families also show marked enrichment in the SJ fraction, suggesting preferential localization at the synaptic cleft while others show little enrichment or decrease, suggesting that they are not restricted to or concentrated at the synaptic cleft. Treatment of the SJ fraction with glycosidases results in electrophoretic mobility shifts of all cell adhesion molecules tested, indicating glycosylation at the synaptic cleft. Biochemical and ultrastructural data presented indicate that the novel synaptic junction preparation can be used as a predictive tool for the identification and characterization of the components of the synaptic cleft.

Prefrontal Gamma-Aminobutyric Acid Type A Receptor Insertion Controls Cue-Induced Relapse to Nicotine Seeking

Current smoking cessation therapies offer limited success, as relapse rates remain high. Nicotine, which is the major component of tobacco smoke, is thought to be primarily responsible for the addictive properties of tobacco. However, little is known about the molecular mechanisms underlying nicotine relapse, hampering development of more effective therapies. The objective of this study was to elucidate the role of medial prefrontal cortex (mPFC) glutamatergic and gamma-aminobutyric acid (GABA)ergic receptors in controlling relapse to nicotine seeking. Using an intravenous self-administration model, we studied glutamate and gamma-aminobutyric acid receptor regulation in the synaptic membrane fraction of the rat mPFC following extinction and cue-induced relapse to nicotine seeking. Subsequently, we locally intervened at the level of GABAergic signaling by using a mimetic peptide of the GABA receptor associated protein-interacting domain of GABA type A (GABAA) receptor subunit γ2 (TAT-GABAγ2) and muscimol, a GABAA receptor agonist. Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid and N-methyl-D-aspartate receptors were not regulated after the 30-min relapse test. However, GABAA receptor subunits α1 and γ2 were upregulated, and interference with GABAA receptor insertion in the cell membrane using the TAT-GABAγ2 peptide in the dorsal mPFC, but not the ventral mPFC, significantly increased responding during relapse. Increasing GABAA transmission with muscimol in the dorsal and ventral mPFC attenuated relapse. These data indicate that cue-induced relapse entails a GABAergic plasticity mechanism that limits nicotine seeking by restoring inhibitory control in the dorsal mPFC. GABAA receptor-mediated neurotransmission in the dorsal mPFC constitutes a possible future therapeutic target for maintaining smoking abstinence.

Phosphorylation and Assembly of Glutamate Receptors After Brain Ischemia

Overassembly of synaptic glutamate receptors leads to excitotoxicity. The goal of this study is to investigate phosphorylation and assembly of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid and N-methyl-D-aspartate receptors after brain ischemia with reperfusion (I/R). Rats were subjected to 15 minutes of global ischemia followed by 0.5, 4, and 24 hours of reperfusion. Phosphotyrosine peptides of glutamate receptors in synaptosomal fraction after I/R were identified and quantified by state-of-the-art immuno-affinity purification of phosphotyrosine peptides followed by liquid chromatography/mass spectrometry/mass spectrometry analysis (immunoaffinity purification-coupled liquid chromatography/mass spectrometry/mass spectrometry). Glutamate receptor phosphorylation and synaptic assembly after I/R were studied by biochemical methods. Numerous phosphotyrosine-sites of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid and N-methyl-D-aspartate were upregulated by approximately 2- to 37-fold after I/R. A core glutamate receptor kinase, Src kinase, was significantly activated. GluR2/3 and NR2A/B were rapidly clustered from extrasynaptic to synaptic membrane fractions after I/R. GluR2/3 was then translocated into the intracellular pool, whereas NR2A/B remained in the synaptic fraction for as long as 24 hours. Consistently, trafficking-related phosphorylation of GluR2/3-S880 was significantly but transiently upregulated, whereas NR2A/B-Y1246 and NR2A/B-Y1472 were significantly and persistently upregulated after I/R. Phosphorylation of glutamate receptors at synapses may lead to overassembly of glutamate receptors, probably via activation of Src family kinases, after I/R. This study provides global proteomic information about glutamate receptor tyrosine phosphorylation after brain ischemia.

Biochemical and morphological characterization of MAGI‐1 in neuronal tissue

The membrane-associated guanylate kinase with inverted organization (MAGI) proteins consist of three members, MAGI-1, MAGI-2 (also known as S-SCAM), and MAGI-3. Although MAGI-2 has been analyzed and shown to interact with a variety of postsynaptic proteins, functional analyses and characterization of MAGI-1 in neuronal tissues have been rare. In this study, we prepared a specific antibody against MAGI-1, anti-MAGI-1, and carried out biochemical and morphological analyses of MAGI-1 in rat neuronal tissues. By Western blotting, a high level of MAGI-1 was detected in nervous tissues, especially in olfactory bulb. Biochemical fractionation clarified that MAGI-1 was relatively enriched in the synaptosomal vesicle and synaptic plasma membrane fractions, whereas MAGI-2 and MAGI-3 appeared to be in the synaptic plasma membrane and postsynaptic density fractions. Immunofluorescent analyses revealed diffuse distribution of MAGI-1 in the cell body and processes of primary cultured rat hippocampal neurons, whereas MAGI-2 and MAGI-3 were likely to be enriched at synapses. Immunohistochemical analyses demonstrated that MAGI-1 was expressed in Purkinje cells, in hypocampal neurons in CA1 region, in the glomerulus region of olfactory bulb, and at the dorsal root entry zone in embryonic rat spinal cord. These results suggest neuronal roles of MAGI-1 different from those of MAGI-2/3.

Critical Role of Amyloid-like Oligomers of Drosophila Orb2 in the Persistence of Memory

A long-standing question in the study of long-term memory is how a memory trace persists for years when the proteins that initiated the process turn over and disappear within days. Previously, we postulated that self-sustaining amyloidogenic oligomers of cytoplasmic polyadenylation element-binding protein (CPEB) provide a mechanism for the maintenance of activity-dependent synaptic changes and, thus, the persistence of memory. Here, we found that the Drosophila CPEB Orb2 forms amyloid-like oligomers, and oligomers are enriched in the synaptic membrane fraction. Of the two protein isoforms of Orb2, the amyloid-like oligomer formation is dependent on the Orb2A form. A point mutation in the prion-like domain of Orb2A, which reduced amyloid-like oligomerization of Orb2, did not interfere with learning or memory persisting up to 24hr. However the mutant flies failed to stabilize memory beyond 48hr. These results support the idea that amyloid-like oligomers of neuronal CPEB are critical for the persistence of long-term memory.

Proteomics, Ultrastructure, and Physiology of Hippocampal Synapses in a Fragile X Syndrome Mouse Model Reveal Presynaptic Phenotype

Fragile X syndrome (FXS), the most common form of hereditary mental retardation, is caused by a loss-of-function mutation of the Fmr1 gene, which encodes fragile X mental retardation protein (FMRP). FMRP affects dendritic protein synthesis, thereby causing synaptic abnormalities. Here, we used a quantitative proteomics approach in an FXS mouse model to reveal changes in levels of hippocampal synapse proteins. Sixteen independent pools of Fmr1 knock-out mice and wild type mice were analyzed using two sets of 8-plex iTRAQ experiments. Of 205 proteins quantified with at least three distinct peptides in both iTRAQ series, the abundance of 23 proteins differed between Fmr1 knock-out and wild type synapses with a false discovery rate (q-value) <5%. Significant differences were confirmed by quantitative immunoblotting. A group of proteins that are known to be involved in cell differentiation and neurite outgrowth was regulated; they included Basp1 and Gap43, known PKC substrates, and Cend1. Basp1 and Gap43 are predominantly expressed in growth cones and presynaptic terminals. In line with this, ultrastructural analysis in developing hippocampal FXS synapses revealed smaller active zones with corresponding postsynaptic densities and smaller pools of clustered vesicles, indicative of immature presynaptic maturation. A second group of proteins involved in synaptic vesicle release was up-regulated in the FXS mouse model. In accordance, paired-pulse and short-term facilitation were significantly affected in these hippocampal synapses. Together, the altered regulation of presynaptically expressed proteins, immature synaptic ultrastructure, and compromised short-term plasticity points to presynaptic changes underlying glutamatergic transmission in FXS at this stage of development.

Effects of Different Types of Anesthetic Agents on Cellular Protein Kinase C-γ Dynamics in Mouse Brain

Background/Aims: Although protein kinase C-γ (PKC-γ) is a target for the effects of volatile anesthetics, the molecular mechanisms of the kinase function during their action remain unclear. We examined the effects of different types of anesthetics on PKC-γ knockout mice. Furthermore, we investigated the dynamics of the kinase in brain cells obtained from mice anesthetized with these anesthetics. Methods: We measured the required times for loss of righting reflex (rtfLORR) after administration of isoflurane, sevoflurane, or propofol on PKC-γ knockout mice and compared the times with those of wild-type mice. We also used immunoblotting to investigate the intracellular distribution of PKC-γ and phosphorylated PKC-γ (p-PKC-γ) in brain cell fractions obtained from wild-type mice during the loss of righting reflex induced by these anesthetics. Results: Isoflurane (2.6%) and sevoflurane (3.4%) used at twice the minimum alveolar concentration significantly prolonged the rtfLORRs in PKC-γ knockout mice compared to those in wild-type mice. On the other hand, no significant difference was observed between knockout and wild-type mice treated with propofol (200 mg/kg). Examination of the cellular fractions isolated from volatile anesthetic-treated mouse brains showed that PKC-γ was significantly decreased in the synaptic membrane fraction (P2), whereas p-PKC-γ was significantly increased in P2. There was no significant change in the supernatant fraction (S). In propofol-treated mice, PKC-γ and p-PKC-γ showed no significant changes in P2 or S. Conclusion: Our results provide new evidence to support the possibility of the involvement of PKC-γ in the actions of volatile anesthetics.

DGKι regulates presynaptic release during mGluR-dependent LTD

Diacylglycerol (DAG) is an important lipid second messenger. DAG signalling is terminated by conversion of DAG to phosphatidic acid (PA) by diacylglycerol kinases (DGKs). The neuronal synapse is a major site of DAG production and action; however, how DGKs are targeted to subcellular sites of DAG generation is largely unknown. We report here that postsynaptic density (PSD)-95 family proteins interact with and promote synaptic localization of DGKι. In addition, we establish that DGKι acts presynaptically, a function that contrasts with the known postsynaptic function of DGKζ, a close relative of DGKι. Deficiency of DGKι in mice does not affect dendritic spines, but leads to a small increase in presynaptic release probability. In addition, DGKι-/- synapses show a reduction in metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) at neonatal (∼2 weeks) stages that involve suppression of a decrease in presynaptic release probability. Inhibition of protein kinase C normalizes presynaptic release probability and mGluR-LTD at DGKι-/- synapses. These results suggest that DGKι requires PSD-95 family proteins for synaptic localization and regulates presynaptic DAG signalling and neurotransmitter release during mGluR-LTD.

Changes in molecular composition of rat medial prefrontal cortex synapses during adolescent development

Postnatal brain development continues throughout adolescence into young adulthood. In particular, synapse strengthening and elimination are prominent processes during adolescence. However, molecular data of this relatively late stage of synaptic development are sparse. In this study, we used iTRAQ (isobaric tag for relative and absolute quantification)-based proteomics and electron microscopy to investigate the molecular composition of a synaptic membrane fraction from adolescent postnatal day (P)34 and P44 and adult (P78) rat medial prefrontal cortex. Differential expression of proteins was most prominent between early adolescence and young adulthood (35%, P34-P78), with an over-representation of cell-membrane proteins during adolescent development (between P34 and P44), and synaptic vesicle proteins between late adolescence and young adulthood (P44-P78). Indicative of the critical period of development, we found that, between P34 and P44, a substantial number of proteins was differentially expressed (14%), much more than during the period after adolescence, i.e. between P44 and P78 (5%). A striking observation was the developmental non-stoichiometric regulation of distinct classes of proteins from the synaptic vesicle and the presynaptic release machinery. Electron microscopy demonstrated a small change in the number of docked vesicles between P34 and P44, but not in the total number of synaptic vesicles and in the size of the vesicle cluster. We conclude that the molecular composition of synapses, and more specifically the synaptic release machinery, of the medial prefrontal cortex changes drastically during adolescent development.

V-ATPase Membrane Sector Associates with Synaptobrevin to Modulate Neurotransmitter Release

Acidification of synaptic vesicles by the vacuolar proton ATPase is essential for loading with neurotransmitter. Debated findings have suggested that V-ATPase membrane domain (V0) also contributes to Ca(2+)-dependent transmitter release via a direct role in vesicle membrane fusion, but the underlying mechanisms remain obscure. We now report a direct interaction between V0 c-subunit and the v-SNARE synaptobrevin, constituting a molecular link between the V-ATPase and SNARE-mediated fusion. Interaction domains were mapped to the membrane-proximal domain of VAMP2 and the cytosolic 3.4 loop of c-subunit. Acute perturbation of this interaction with c-subunit 3.4 loop peptides did not affect synaptic vesicle proton pump activity, but induced a substantial decrease in neurotransmitter release probability, inhibiting glutamatergic as well as cholinergic transmission in cortical slices and cultured sympathetic neurons, respectively. Thus, V-ATPase may ensure two independent functions: proton transport by a fully assembled V-ATPase and a role in SNARE-dependent exocytosis by the V0 sector.

Extracellular Matrix Plasticity and GABAergic Inhibition of Prefrontal Cortex Pyramidal Cells Facilitates Relapse to Heroin Seeking

Successful treatment of drug addiction is hampered by high relapse rates during periods of abstinence. Neuroadaptation in the medial prefrontal cortex (mPFC) is thought to have a crucial role in vulnerability to relapse to drug seeking, but the molecular and cellular mechanisms remain largely unknown. To identify protein changes that contribute to relapse susceptibility, we investigated synaptic membrane fractions from the mPFC of rats that underwent 21 days of forced abstinence following heroin self-administration. Quantitative proteomics revealed that long-term abstinence from heroin self-administration was associated with reduced levels of extracellular matrix (ECM) proteins. After extinction of heroin self-administration, downregulation of ECM proteins was also present in the mPFC, as well as nucleus accumbens (NAc), and these adaptations were partially restored following cue-induced reinstatement of heroin seeking. In the mPFC, these ECM proteins are condensed in the perineuronal nets that exclusively surround GABAergic interneurons, indicating that ECM adaptation might alter the activity of GABAergic interneurons. In support of this, we observed an increase in the inhibitory GABAergic synaptic inputs received by the mPFC pyramidal cells after the re-exposure to heroin-conditioned cues. Recovering levels of ECM constituents by metalloproteinase inhibitor treatment (FN-439; i.c.v.) prior to a reinstatement test attenuated subsequent heroin seeking, suggesting that the reduced synaptic ECM levels during heroin abstinence enhanced sensitivity to respond to heroin-conditioned cues. We provide evidence for a novel neuroadaptive mechanism, in which heroin self-administration-induced adaptation of the ECM increased relapse vulnerability, potentially by augmenting the responsivity of mPFC GABAergic interneurons to heroin-associated stimuli.

The effects of aging on N-methyl-d-aspartate receptor subunits in the synaptic membrane and relationships to long-term spatial memory

There are declines in the protein expression of the NR2B (mouse ε2) and NR1 (mouse ζ1) subunits of the N-methyl-d-aspartate (NMDA) receptor in the cerebral cortex and hippocampus during aging in C57BL/6 mice. This study was designed to determine if there is a greater effect of aging on subunit expression and a stronger relationship between long-term spatial memory and subunit expression within the synaptic membrane than in the cell as a whole. Male, C57BL/6JNIA mice (4, 11 and 26 months old) were tested for long-term spatial memory in the Morris water maze. Frontal cortex, including prefrontal regions, and hippocampus were homogenized and fractionated into light and synaptosomal membrane fractions. Western blots were used to analyze protein expression of NR2B and NR1 subunits of the NMDA receptor. Old mice performed significantly worse than other ages in the spatial task. In the frontal cortex, the protein levels of the NR2B subunit showed a greater decline with aging in the synaptic membrane fraction than in the whole homogenate, while in the hippocampus a similar age-related decline was observed in both fractions. There were no significant effects of aging on the expression of the NR1 subunit. Within the middle-aged mouse group, higher expression of both NR2B and NR1 subunits in the synaptic membrane of the hippocampus was associated with better memory. In the aged mice, however, higher expression of both subunits was associated with poorer memory. These results indicate that aging could be altering the localization of the NR2B subunit to the synaptic membrane within the frontal cortex. The correlational results suggest that NMDA receptor functions, receptor subunit composition, and/or the environment in which the receptor interacted in the hippocampus were not the same in the old animals as in younger mice and this may have contributed to memory declines during aging.

The Post-Synaptic Density of Human Postmortem Brain Tissues: An Experimental Study Paradigm for Neuropsychiatric Illnesses

Recent molecular genetics studies have suggested various trans-synaptic processes for pathophysiologic mechanisms of neuropsychiatric illnesses. Examination of pre- and post-synaptic scaffolds in the brains of patients would greatly aid further investigation, yet such an approach in human postmortem tissue has yet to be tested. We have examined three methods using density gradient based purification of synaptosomes followed by detergent extraction (Method 1) and the pH based differential extraction of synaptic membranes (Methods 2 and 3). All three methods separated fractions from human postmortem brains that were highly enriched in typical PSD proteins, almost to the exclusion of pre-synaptic proteins. We examined these fractions using electron microscopy (EM) and verified the integrity of the synaptic membrane and PSD fractions derived from human postmortem brain tissues. We analyzed protein composition of the PSD fractions using two dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) and observed known PSD proteins by mass spectrometry. Immunoprecipitation and immunoblot studies revealed that expected protein-protein interactions and certain posttranscriptional modulations were maintained in PSD fractions. Our results demonstrate that PSD fractions can be isolated from human postmortem brain tissues with a reasonable degree of integrity. This approach may foster novel postmortem brain research paradigms in which the stoichiometry and protein composition of specific microdomains are examined.

Synaptic removal of diacylglycerol by DGKζ and PSD-95 regulates dendritic spine maintenance

Diacylglycerol (DAG) is an important lipid signalling molecule that exerts an effect on various effector proteins including protein kinase C. A main mechanism for DAG removal is to convert it to phosphatidic acid (PA) by DAG kinases (DGKs). However, it is not well understood how DGKs are targeted to specific subcellular sites and tightly regulates DAG levels. The neuronal synapse is a prominent site of DAG production. Here, we show that DGKzeta is targeted to excitatory synapses through its direct interaction with the postsynaptic PDZ scaffold PSD-95. Overexpression of DGKzeta in cultured neurons increases the number of dendritic spines, which receive the majority of excitatory synaptic inputs, in a manner requiring its catalytic activity and PSD-95 binding. Conversely, DGKzeta knockdown reduces spine density. Mice deficient in DGKzeta expression show reduced spine density and excitatory synaptic transmission. Time-lapse imaging indicates that DGKzeta is required for spine maintenance but not formation. We propose that PSD-95 targets DGKzeta to synaptic DAG-producing receptors to tightly couple synaptic DAG production to its conversion to PA for the maintenance of spine density.