Early events in the trafficking of N-methyl-D-aspartate (NMDA) receptors.

The N-methyl-D-aspartate (NMDA) receptor plays a central role at excitatory synapses where it has been implicated in multiple functions associated with synaptic plasticity. While this receptor has been intensely studied with respect to its physiology and pharmacology, its cell-biological properties, such as subunit assembly, post-translational processing and trafficking in neurons, are only beginning to be addressed. Critical to many of the functions of the NMDA receptor are the multiple proteins with which it interacts. While these interactions have been most thoroughly studied with respect to the receptor at the synapse, the same proteins may also interact with the receptor much earlier in its biosynthetic pathway and play important roles in receptor trafficking from the endoplasmic reticulum to the synapse.

Molecular determinants of NMDA receptor internalization.

Although synaptic AMPA receptors have been shown to rapidly internalize, synaptic NMDA receptors are reported to be static. It is not certain whether NMDA receptor stability at synaptic sites is an inherent property of the receptor, or is due to stabilization by scaffolding proteins. In this study, we demonstrate that NMDA receptors are internalized in both heterologous cells and neurons, and we define an internalization motif, YEKL, on the distal C-terminus of NR2B. In addition, we show that the synaptic protein PSD-95 inhibits NR2B-mediated internalization, and that deletion of the PDZ-binding domain of NR2B increases internalization in neurons. This suggests an involvement for PSD-95 in NMDA receptor regulation and an explanation for NMDA receptor stability at synaptic sites.

The role of glycosylation in ionotropic glutamate receptor ligand binding, function, and trafficking.

Members of the ionotropic glutamate receptor (iGluR) family have between 4 and 12 consensus asparagine (N)-linked glycosylation sites. They are localized on the extracellular N-termini, and the loop between the penultimate and last transmembrane domains. These regions also contain the essential elements for formation of the ligand binding site. N-linked glycosylation does not appear to be essential for formation of the ligand binding site per se, but there are demonstrated interactions between glycosylation state and ligand binding affinity, receptor physiology, susceptibility to allosteric modulation and, in some cases, trafficking. There is no indication of a general role for N-linked glycosylation in iGluRs; instead the effects of glycosylation vary among glutamate receptor subtypes and splice variants, with specific effects on structure or function with different subunits.

PDZ domain suppression of an ER retention signal in NMDA receptor NR1 splice variants.

The NMDA receptor NR1 subunit has four splice variants that differ in their C-terminal, cytoplasmic domain. We investigated the contribution of the C-terminal cassettes, C0, C1, C2, and C2', to trafficking of NR1 in heterologous cells and neurons. We identified an ER retention signal (RRR) in the C1 cassette of NR1, which is similar to the RXR motif in ATP-sensitive K(+) channels (Zerangue et al., 1999). We found that surface expression of NR1-3, which contains C1, is due to a site on the C2' cassette, which includes the terminal 4 amino acid PDZ-interacting domain. This site suppresses ER retention of the C1 cassette and leads to surface expression. These findings suggest a role for PDZ proteins in facilitating the transition of receptors from an intracellular pool to the surface of the neuron.

Rapid effects of kainate administration on alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor properties in rat hippocampus.

We investigated changes in AMPA receptor properties in rat hippocampus 5 h after systemic kainate administration. Quantitative [3H]AMPA autoradiography and Western blot analysis of receptor subunits GluR1-3 in different subcellular fractions were used to evaluate possible alterations in binding characteristics and immunological properties of the receptors in synaptic and nonsynaptic fractions. Both ligand-binding and Western blots revealed significant changes in binding and immunological properties of nonsynaptic receptors but relatively smaller changes in synaptic receptors 5 h after kainate administration. GluR2/3 showed a greater relative change in the synaptic receptor population compared to GluR1, suggesting either a shift in subunit composition of AMPA receptors or the formation of a synaptic subpopulation of AMPA receptors with truncated C-terminal domain of GluR1 subunits. The effects of kainic acid were blocked by cycloheximide treatment indicating that the changes were due at least in part to increased synthesis of AMPA receptor subunits. The results indicate that excessive synaptic activity produces rapid changes in both synaptic and nonsynaptic AMPA receptor properties.

Developmental changes in subcellular AMPA/GluR receptor populations in rat forebrain.

Forebrains from rats of postnatal days (PND) 2, 7, 14, 21, and 30-40 were subjected to subcellular fractionation and samples from crude mitochondrial (P2, which contain synaptic plasma membranes) and microsomal (P3) fractions were used for SDS-PAGE and Western blotting with antibodies against GluR1, and GluR2/3 subunits of AMPA/GluR receptors. GluR immunoreactivity in P2 fractions increased gradually from PND 2 to PND 30. In contrast, GluR immunoreactivity in P3 fractions increased sharply at early postnatal ages, and was higher than in adults as early as at PND 7. Data were compared to postnatal changes in 3H-AMPA binding reported in various studies. Significant correlations were observed between changes in GluR immunoreactivity in P3 fractions and changes in high-affinity binding on one hand and between changes in GluR immunoreactivity in P2 fractions, and changes in low affinity binding. These data further establish that glutamate receptors present in different subcellular compartments represent different maturational states of the receptors, and suggest that changes in GluR populations could participate in mechanisms of synaptic plasticity.

High- and low-affinity alpha-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([3H]AMPA) binding sites represent immature and mature forms of AMPA receptors and are composed of differentially glycosylated subunits.

Quantitative alpha-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([3H]AMPA) binding autoradiography was performed on frozen-thawed sections from rat brain after preincubation at 0 or 35 degrees C for 1 h. Preincubation at 35 degrees C instead of 0 degrees C resulted in a selective decrease of [3H]AMPA binding assayed at a low concentration of [3H]AMPA (50 nM) and an enhancement of binding at a high concentration (500 nM). The decrease in [3H]AMPA binding after preincubation at 35 degrees C was accompanied with the loss of the lighter organelles of P3 (microsomal) fractions. These organelles were found to contain a small subpopulation of AMPA/GluR receptors exhibiting a high affinity for [3H]AMPA (K(D) approximately 14 nM), whereas heavier organelles exhibited lower affinity for AMPA (K(D) approximately 190 nM). This small subpopulation of AMPA/GluR receptors contained almost exclusively a structurally distinct species of GluR2/3 subunits with an apparent molecular mass of 103.5 kDa (assessed with anti-GluR2/3, C-terminal antibodies). Experiments using two deglycosylating enzymes, N-glycopeptidase F and endoglycosidase H, clearly indicated that the 103.5-kDa species represented a partially unglycosylated form of GluR2/3 subunits containing the high-mannose type of oligosaccharide moiety, whereas receptors present in synaptosomal fractions were composed of subunits with complex oligosaccharides. A similar result was obtained by using an antibody recognizing the N-terminal domain of GluR2(4). The same enzymatic treatment indicated that GluR1 subunits also exhibited a partially glycosylated form. These data indicate that high-affinity [3H]AMPA binding sites represent nonsynaptic, intracellular membrane-bound AMPA receptors that differ from synaptic receptors by at least the glycosylation state of GluR2 (and GluR1) subunits. In addition, our results provide a relatively simple way of assessing changes in two spatially and structurally distinct [3H]AMPA binding/GluR sites.

Developmental changes in alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor properties and expression in the rat hippocampal formation.

The developmental changes in alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor properties in rat hippocampus were evaluated with quantitative autoradiography of ligand binding and in situ hybridization performed in adjacent sections with antisense oligonucleotides for AMPA receptor subunits (GluR1-3, flip and flop splice variants). Specific 3H-AMPA binding in different hippocampal subfields increased between postnatal day 7 and 15 and was higher in CA3 during the postnatal period when compared to adult levels. This effect was mostly due to high levels of high affinity binding sites in cell body layers during the developmental period. By contrast, autoradiograms of 3H-AMPA binding predominantly to the low affinity binding sites indicated an absence of these sites in cell body layers and the overall levels of binding exhibited little overshoot compared to adult levels during the developmental period. The changes in binding of the antagonist of the AMPA receptor, 6-nitro-7-cyanoquinoxaline-2,3-dione were markedly different from those for the high affinity AMPA binding sites but quite similar to those for the low affinity sites. The binding was extremely low at postnatal day 7 and increased rapidly between postnatal day 7 and 15 and slowly between postnatal day 15 and adult. Low levels of binding were observed in the cell body layer at every postnatal age. The changes in expression of messenger RNAs for the different subunits of the AMPA receptors were well correlated with the modifications in high affinity AMPA binding sites measured in the cell body layers also exhibiting an increased expression of the receptors at the transcriptional level during the developmental period as compared to adult levels. The relative expression of the GluR2 subunits decreased during the postnatal period and the time course for this reduction paralleled that for the increased vulnerability of hippocampal pyramidal neurons to a variety of insults. The results indicate that both the messenger RNAs for the subunits and the AMPA receptors exhibit increased levels of expression during the postnatal period compared to adult levels. They also suggest that nascent receptors might bind AMPA with high affinity before their insertion in membranes into functional receptors that have low affinity for agonists and high affinity for antagonists. The changes in subunit composition of the receptors during the postnatal period may have important implications for mechanisms of plasticity as well as of neuropathology.

Differential subcellular localization of two populations of glutamate/AMPA receptors in the rat telencephalon.

The distribution of glutamate AMPA receptors in the synaptosomal and microsomal fractions of neonatal and adult rat telencephalon was studied by determining the saturation kinetics at equilibrium of 3H-AMPA and 3H-CNQX binding. At both ages, synaptosomal preparations exhibited two populations of 3H-AMPA binding sites with a small number of high affinity sites and a large number of low affinity sites. 3H-AMPA binding to microsomal preparations from both neonatal and adult rat telencephalon exhibited a much higher proportion of high affinity relative to low affinity sites. 3H-CNQX binding to the same fractions did not parallel 3H-AMPA binding, but was correlated with the low affinity 3H-AMPA binding and with a marker of plasma membranes. The results suggest that nonsynaptic glutamate/AMPA receptors have a high affinity for agonist and become low affinity when inserted into postsynaptic membranes and that 3H-CNQX binds synaptic but not nonsynaptic glutamate/AMPA receptors with high affinity.

Postsynaptic factors in the expression of long-term potentiation (LTP): increased glutamate receptor binding following LTP induction in vivo.

Several lines of evidence indicate that LTP in the hippocampus is associated with a change in the properties of postsynaptic glutamate receptors. In the present study, we used quantitative autoradiography to examine the binding properties of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) and N-methyl-D-aspartate subclasses of glutamate receptors in frozen brain sections obtained from rats in which perforant-path LTP was induced in vivo. Induction of LTP resulted in a selective increase in [3H]AMPA binding in those hippocampal subfields receiving perforant-path axons. Increases in [3H]AMPA binding in dentate gyrus (stratum moleculare) were highly correlated with the magnitude of LTP recorded in this structure. Scatchard analyses of [3H]AMPA and 6-cyano-7-nitro-[3H]quinoxaline-2,3-dione (an AMPA receptor antagonist) binding in the dentate gyrus indicated that LTP induction resulted in an increase in the number of AMPA receptor binding sites. No changes in the binding of 3H-labeled N-[1-(thienyl)cyclohexyl]piperidine (an N-methyl-D-aspartate receptor antagonist) were observed in any hippocampal subfield. These results suggest that a modification in postsynaptic AMPA receptors plays a role in the expression of synaptic enhancement following LTP induction in the hippocampus.

Effect of Temperature and Calcium on the Binding Properties of the AMPA Receptor in Frozen Rat Brain Sections.

The elucidation of the mechanisms regulating the properties of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) subtype of glutamate receptors is important for understanding glutamatergic transmission. Here we report that qualitative as well as quantitative analysis of tritiated ligand binding to the AMPA receptor on thin frozen rat brain tissue sections reveals the existence of several mechanisms regulating the binding properties of AMPA receptors. Preincubation of tissue sections at 35 degrees C results in a decreased amount of [3H]AMPA binding as compared to that measured following preincubation at 0 degrees C. The decrease in binding appears to be mainly localized to cell bodies as evaluated by autoradiography, and could be due to proteolysis. Preincubation with calcium at 35 degrees C produces increased levels of [3H]AMPA binding. The effect of calcium is mimicked by manganese and to a lesser extent by magnesium; it is concentration-dependent with a 50% effective concentration for calcium of approximately 150 microM, time-dependent and temperature-dependent. The calcium-induced increase in [3H]AMPA binding is different among various brain structures, being larger in area CA1 of the hippocampus and in the superficial layers of the cerebral cortex. The effect of calcium is partly reduced by preincubation with the calpain inhibitor leupeptin and potentiated by preincubation with purified calpain II. The calcium-induced increase in [3H]AMPA binding is associated with a decrease in the binding of an antagonist of AMPA receptors, [3H]6-nitro-7-cyanoquinoxaline-2,3-dione. The results indicate that the binding properties of the AMPA receptor are rapidly regulated by calcium-dependent processes, and possibly by calcium-dependent proteases. They suggest that modulation of the binding properties involves changes in the configuration of the receptor, producing opposite changes in the affinities of the receptor for agonists and antagonists. Finally, these results strengthen the hypothesis that changes in the properties of AMPA receptors might underlie various forms of synaptic plasticity.

Phospholipase A2-induced changes in AMPA receptor: an autoradiographic study.

The expression of long-term potentiation and learning of a classical conditioning task increase [3H]-AMPA binding in hippocampus. Phospholipase A2 (PLA2) has been proposed to underly these changes, as PLA2 treatment of membrane preparations increases the affinity of AMPA receptors for agonists. We demonstrate here that preincubation of thin (10 microns) frozen rat brain sections with exogenous PLA2 and calcium at physiological temperature changes the binding properties of AMPA receptors. Quantitative autoradiography reveals that PLA2-treatment produces a differential increase in [3H]-AMPA binding across brain regions. The same treatment also decreases the binding of an antagonist ([3H]-CNQX) throughout the brain. We propose that PLA2 treatment results in a modification of the AMPA receptors which is regionally specific, probably due to different AMPA receptor subunit compositions.

Time-Temperature Conditions of Gyros.

Four gyro operations in foodservice establishments were examined for the possibility that pathogenic foodborne bacteria could survive and/or grow during each step of these operations. Gyros cooked on broilers attained temperatures lethal to vegetative pathogenic bacteria on the surface of the meat and in the thin layer just below the surface, but nowhere else. However, only meat sliced from the surface was normally put in gyro sandwiches or otherwise served. The temperatures of gyros as they cooled were such that bacterial growth could occur, both on the surfaces and within the mass. After gyros had been cooked and cooled, as many as 10,000 Clostridium perfringens per gram were recovered from samples taken just under the surface. Temperatures of gyro meat during reheating varied with the method of reheating, and they were in safe ranges when slices of meat were reheated in microwave ovens and steam chambers. When gyros were reheated on broilers, however, temperatures lethal to vegetative pathogenic bacteria occurred at and near the surfaces only. Recommendations for procedures to use for cooking, slicing, hot holding, cooling, and reheating gyros to prevent this product from becoming a vehicle of foodborne illness are given. Emphasis is on using the entire gyro the day it is originally cooked, rapid cooling of any leftover portions, and thorough reheating of leftover gyros.