Alpha-amino-3-hydroxy-5-methyl-4-isoxazole Proprionic Acid Receptor Research Articles

Investigation of neuronal auto-antibodies in systemic lupus erythematosus patients with epilepsy

PurposeEpilepsy is an important feature for neuropsychiatric involvement in systemic lupus erythematosus (SLE) with unknown mechanism. Our aim was to investigate the presence of neuronal auto-antibodies (NAbs) in neuropsychiatric SLE (NPSLE). MethodsEighteen SLE patients (17 females, 1 male) experiencing recurrent seizures were enrolled to this study. Their clinical characteristics, EEG and MRI findings and follow-up information were evaluated from their files. Antibodies against voltage-gated potassium channel (VGKC)-complex antigens, contactin-associated protein-like 2 (CASPR-2), leucine-rich, glioma inactivated 1 (LGI1), glutamic acid decarboxylase (GAD), N-methyl-d-aspartate receptor (NMDA-R), alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor (AMPA-R) and type B gamma aminobutyric acid receptors (GABAB-R) were screened in the sera of these patients. Moreover, indirect immunohistochemistry and immunocytochemistry tests were performed to reveal neuropil antibodies. ResultsSix out of 18 patients (33.3%) had various forms of NAbs. Among them, one patient had antibodies against GAD, one patient with hippocampal sclerosis on MRI was CASPR-2 antibody positive, whereas the remaining four patients showed hippocampal neuropil staining. We could not find a significant difference between seropositive and seronegative groups, regarding the clinical characteristics, EEG and MRI findings. ConclusionThis study is the first to show hippocampal neuronal staining (4/18) reflecting antibodies against unknown neuronal cell surface antigens in SLE patients with epilepsy, besides the rare occurrence of GAD and CASPR2 antibodies. Further prospective studies are needed to search for new NAbs and uncover their pathogenic role in SLE associated with epilepsy.

Effects of GYKI 52466 on Early Vasospasm in Rats

The pathogenesis and treatment of cerebral vasospasm (CV) after subarachnoid hemorrhage (SAH) remains a matter of discussion. The authors investigated the efficacy of GYKI 52466, a 2,3-benzodiazepine that is a selective and potent alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) receptor antagonist, in a rat femoral artery vasospasm model. Twenty-seven Wistar albino rats were used in this study. The animals were divided into 3 groups of 9 animals each: sham-operated control (group 1), vasospasm group (group 2), and vasospasm-plus-treatment group (group 3). In groups 2 and 3, autologous blood (0.1 mL) was applied to a 1-cm segment of the femoral artery, which was then wrapped with a silicone cuff. One minute after blood application, the rats in group 3 received an intraperitoneal injection of 15 mg/kg GYKI 52466 every 12 h for 24 h. Responses to blood application and treatment were evaluated with light and electron microscopy examinations of femoral artery specimens at 72 h. On light microscope examination, the mean diameters of the arterial lumens in groups 1, 2, and 3 were 514.47+/-15.3, 317.63+/-12.1, and 503.91+/-9.6 microm, respectively. At 24 h, the mean arterial wall thickness in group 1 was 77.69+/-4.2 microm. This mean thickness in group 2 increased to 164.82+/-9.1 microm. After GYKI treatment in group 3, the mean arterial wall thickness measured 95.37+/-5.3 microm. In group 2 rats, electron microscopy demonstrated various changes including marked luminal narrowing and increased wall thickness in the femoral arterial wall. The most striking finding were the degenerative changes in the endothelium, which presented as a corrugated appearance of the internal elastic lamina. Rats in group 3 had endothelia that were slightly constricted and smooth muscle cells that were relaxed; changes in the vessel wall and internal elastic lamina were less prominent in these rats than in the rats of group 2. The results of the present study suggest that GYKI 52466 inhibited AMPA receptors and induced relaxation of smooth muscle cells in the wall of the femoral artery in a rat model. This substance may be a protective and therapeutic agent in the treatment of cerebral vasospasm.

DECREASED AMPA GLuR2, BUT NOT GLuR3, mRNA EXPRESSION IN RAT AMYGDALA AND DORSAL HIPPOCAMPUS FOLLOWING MORPHINE‐INDUCED BEHAVIOURAL SENSITIZATION

1. Repeated administration of psychostimulants and micro-opioid receptor agonists elicits a progressive enhancement of drug-induced behavioural responses, a phenomenon termed behavioural sensitization. These changes in behaviour may reflect plastic changes requiring regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) receptor function. 2. In the present study, rats were treated for 7 days with saline or morphine (10 mg/kg). After a washout period of either 24 h or 7 days, locomotion, oral stereotypy and state-dependent memory in a passive avoidance test were measured in the presence or absence of 6-cyano-7-nitroquinoxaline-2,3-dione disodium salt (CNQX; 3 mg/kg), an AMPA receptor antagonist. In order to evaluate the mechanism underlying the behavioural responses, quantitative real-time reverse transcription-polymerase chain reaction was used to evaluate mRNA expression of the AMPA receptor subunits GluR2 and GluR3 in the striatum, prefrontal cortex, hippocampus, hypothalamus and amygdala of animals treated repeatedly with morphine. 3. The results indicate that repeated morphine treatment followed by 7 days (but not 24 h) washout produces behavioural sensitization, as determined by locomotion, oral stereotypy and state-dependent memory. Blockade of AMPA receptors with CNQX on the test day did not alter these behavioural responses. In addition, repeated morphine treatment followed by 7 days (but not 24 h) washout decreased GluR2 mRNA expression in both the amygdala (by 50%) and hippocampus (by 35%). Repeated morphine treatment did not alter GluR3 mRNA expression in any brain area assessed. 4. These data imply that AMPA receptors are involved in the development (but not expression) phase of behavioural sensitization. The decreases in GluR2 mRNA expression in the amygdala and hippocampus may result in the formation of calcium-permeable AMPA receptors, which are believed to play an important role in behavioural sensitization.

Contributions of nucleus accumbens core and shell GluR1 containing AMPA receptors in AMPA- and cocaine-primed reinstatement of cocaine-seeking behavior

Glutamate signaling in the nucleus accumbens influences reinstatement of previously extinguished cocaine-seeking behavior in rats. Whether or not region specific glutamate signaling in the nucleus accumbens contributes to reinstatement of cocaine-seeking behavior is not known. We investigated whether directly stimulating ionotropic glutamate receptors (GluRs) within the nucleus accumbens core or shell would differentially influence renewed cocaine-seeking behavior following extinction training. We also tested the hypothesis that GluR1 subunit (GluR1) containing alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) receptors in the nucleus accumbens core and not the shell regulate reinstatement of previously extinguished cocaine-seeking behavior. Microinjection of AMPA into the nucleus accumbens shell and the nucleus accumbens core dose-dependently elicited significant cocaine-seeking behavior. Administration of antisense oligonucleotides (AS) directed against GluR1 subunit mRNA into the core and shell disrupted AMPA- and cocaine-primed reinstatement—with the most pronounced effects seen in the nucleus accumbens shell. These results demonstrate that GluRs in the nucleus accumbens core and shell influence AMPA- and cocaine-primed reinstatement, yet the nucleus accumbens shell exerts a prepotency over the nucleus accumbens core.

Selective estrogen receptor‐α but not ‐β agonist treatment modulates brain α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptors

Estradiol was previously reported to decrease brain alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)-receptor-specific binding. The contributions of estrogen receptor subtypes in the estradiol modulation of AMPA receptors and its predominant subunit GluR2 are unknown. These experiments investigated whether an estrogenic receptor subtype is involved in the estradiol effect on AMPA-receptor-specific binding and GluR2 mRNA levels. Ovariectomized Sprague-Dawley rats were treated 2 days after ovariectomy for 2 weeks with 17beta-estradiol, an agonist for ERalpha 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), or an agonist for ERbeta 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) and compared with intact control rats. Uterus weights, used as aperipheral measure of estrogenic activity, were decreased after ovariectomy and increased by estradiol and PPT but not DPN treatments. In prefrontal and cingulate cortices, the striatum, and the nucleus accumbens, ovariectomy increased [3H]AMPA-specific binding compared with intact controls, which was corrected by estradiol treatment. In all these brain regions, PPT, but not DPN, mimicked the estradiol decrease of AMPA-receptor-specific binding; in the cingulate cortex, the effect of PPT did not reach statistical significance. GluR2 mRNA levels of vehicle-treated ovariectomized rats remained unchanged compared with intact rats in the brain regions investigated. Estradiol and PPT treatment but not DPN decreased GluR2 subunit mRNA levels in the prefrontal cortex and the striatum of ovariectomized rats, whereas no significant change was observed in the cingulate cortex or the nucleus accumbens. The present results suggest that an ERalpha is involved in the estradiol modulation of AMPA receptors in the cortex, striatum, and nucleus accumbens.

Hippocampal Long-Term Synaptic Plasticity and Signal Amplification of NMDA Receptors

The direction of plasticity at CA3-CA1 hippocampal synapses is determined by the strength of afferent stimulation. Weak stimuli lead to long-term depression (LTD) and strong stimuli to long-term potentiation (LTP), but both require activation of synaptic N-methyl-D-aspartate receptors (NMDARs). These receptors are therefore necessary and required for the induction of plasticity at CA3-CA1 synapses even though they carry little of the current responsible for the basal excitatory post-synaptic potential (EPSP). The influx of Ca(2+) via NMDARs triggers the subsequent and persistent changes in the expression of alpha-amino-3-hydroxy-5 methylisoxazole-4-proprionic acid receptors (AMPARs) and these receptors are responsible for the major part of the basal EPSP. The degree of activity of NMDARs is determined in part by extracellular Mg(2+) and by the co-agonists for this receptor, glycine and D-serine. During strong stimulation, a relief of the voltage-dependent block of NMDARs by Mg(2+) provides a positive feedback for NMDAR Ca(2+) influx into postsynaptic CA1 spines. In this review, we discuss how the induction of LTP at CA3-CA1 synapses requires further signal amplification of NMDAR activity. We discuss how the regulation of NMDARs by protein kinases and phosphatases is brought into play. Evidence is presented that Src family kinases (SFKs) play a "core" role in the induction of LTP by enhancing the function and expression of NMDARs. At CA3-CA1 synapses, NMDARs are largely composed of NR1 (NMDA receptor subunit 1)-NR2A or NR1-NR2B containing subunits. Recent, but controversial, evidence has correlated NR1-NR2A receptors with the induction of LTP and NR1-NR2B receptors with LTD. However, LTP can be induced by activation of either subtype of NMDAR and the ratio of NR2A:NR2B receptors has been proposed as an alternative determinant of the direction of synaptic plasticity. Many transmitters and signal pathways can modify NMDAR function and expression and, for a given stimulus strength, they can potentially lead to a change in the balance between LTP and LTD. As opposed to the "core" mechanisms of LTP and LTD, the resulting alterations in this balance underlie "meta-plasticity." Thus, in addition to their contribution to core mechanisms, we will also discuss how Src-family kinases could preferentially target NR1-NR2A or NR1-NR2B receptors to alter the relative contribution of these receptor subtypes to synaptic plasticity.

Resistance of human adult oligodendrocytes to AMPA/kainate receptor-mediated glutamate injury

Multiple sclerosis is an inflammatory disease of the CNS leading to the destruction of oligodendrocytes (OLs), myelin sheaths and axons. The mediators of tissue injury remain unknown. Glutamate, which can be released by activated immune cells or produced within the CNS, has been implicated as a potential mediator of tissue injury in multiple sclerosis. alpha-Amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) and kainate are highly toxic when added to rodent OL cultures. Using OLs derived from human adult surgical specimens, we investigated AMPA/kainate receptor expression and the effects of receptor stimulation on the viability of human OLs. We find that human adult OLs in vitro express low levels of ionotropic glutamate receptors and are resistant to excitotoxicity mediated by high and sustained doses of AMPA or kainate, even when receptor desensitization is blocked. In contrast, rat OLs show strong AMPA receptor expression and are susceptible to excitotoxicity, as previously demonstrated. Furthermore, we show in human brain sections that OLs do not express AMPA receptors in situ and that glial expression of AMPA receptors is limited to astrocytes. The apparent lack of glutamate receptor expression on human OLs and their resistance to AMPA/kainate toxicity should be considered when postulating mechanisms of tissue injury in multiple sclerosis.

Striatal glutamatergic mechanisms and extrapyramidal movement disorders.

The nonphysiologic stimulation of striatal dopaminergic receptors, as a result of disease- or drug-related denervation or intermittent excitation, triggers adaptive responses in the basal ganglia which contribute to the appearance of parkinsonian symptoms and later to the dyskinesias and other alterations in motor response associated with dopaminergic therapy. Current evidence suggests that these altered responses involve activation of signal transduction cascades in striatal medium spiny neurons linking dopaminergic to coexpressed ionotropic glutamatergic receptors of the N-methyl-D-aspartate (NMDA) and Alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) classes. These intraneuronal signaling pathways appear capable of modifying the phosphorylation state of NMDA and AMPA receptor subunits; resultant sensitization enhances cortical glutamatergic input which in turn modifies striatal output in ways that compromise motor behavior. The regulation of these spiny neuron glutamate receptors can also be affected by the activation state of coexpressed nondopaminergic receptors as well as by changes associated with Huntington's disease. These observations lend new insight into molecular mechanisms contributing to the integration of synaptic inputs to spiny neurons. They also suggest novel approaches to the pharmacotherapy of extrapyramidal motor dysfunction.

Brain-derived Neurotrophic Factor Regulates Surface Expression of α-Amino-3-hydroxy-5-methyl-4-isoxazoleproprionic Acid Receptors by Enhancing the N-Ethylmaleimide-sensitive Factor/GluR2 Interaction in Developing Neocortical Neurons

In hippocampal neurons, the exocytotic process of alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)-type glutamate receptors is known to depend on activation of N-methyl-d-aspartate channels and its resultant Ca(2+) influx from extracellular spaces. Here we found that brain-derived neurotrophic factor (BDNF) induced a rapid surface translocation of AMPA receptors in an activity-independent manner in developing neocortical neurons. The receptor translocation became evident within hours as monitored by [(3)H]AMPA binding and was resistant against ionotropic glutamate receptor antagonists as evidenced with surface biotinylation assay. This process required intracellular Ca(2+) and was inhibited by the blockers of conventional exocytosis, brefeldin A, botulinum toxin B, and N-ethylmaleimide. To explore the translocation mechanism of individual AMPA receptor subunits, we utilized the human embryonic kidney (HEK) 293 cells carrying the BDNF receptor TrkB. After the single transfection of GluR2 cDNA or GluR1 cDNA into HEK/TrkB cells, BDNF triggered the translocation of GluR2 but not that of GluR1. Subsequent mutation analysis of GluR2 carboxyl-terminal region indicated that the translocation of GluR2 subunit in HEK293 cells involved its N-ethylmaleimide-sensitive factor-binding domain but not its PDZ-interacting site. Following co-transfection of GluR1 and GluR2 cDNAs, solid phase cell sorting revealed that GluR1 subunits were also able to translocate to the cell surface in response to BDNF. An immunoprecipitation assay confirmed that BDNF stimulation can enhance the interaction of GluR2 with N-ethylmaleimide-sensitive factor. These results reveal a novel role of BDNF in regulating the surface expression of AMPA receptors through a GluR2-NSF interaction.

N-Methyl-d-aspartate-induced α-Amino-3-hydroxy-5-methyl-4-isoxazoleproprionic Acid (AMPA) Receptor Down-regulation Involves Interaction of the Carboxyl Terminus of GluR2/3 with Pick1: LIGAND-BINDING STUDIES USING Sindbis VECTORS CARRYING AMPA RECEPTOR DECOYS

The dynamics of alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)-type glutamate receptors, as represented by their exocytosis, endocytosis and cytoskeletal linkage, has often been implicated in N-methyl-d-aspartate (NMDA)-dependent synaptic plasticity. To explore the molecular mechanisms underlying the AMPA receptor dynamics, cultured hippocampal neurons were stimulated with 100 microm NMDA, and the biochemical and pharmacological changes in the ligand binding activity of AMPA receptor complexes and its subunits, GluR1 and GluR2/3, were investigated. The NMDA treatment reduced the total amount of bound [(3)H]AMPA on the surface of the neurons but not in their total membrane fraction. This process was mimicked by a protein kinase C activator, phorbol ester, but blocked by an inhibitor of the same kinase, calphostin C. The NMDA-induced down-regulation of the ligand binding activity was also reflected by the decreased AMPA-triggered channel activity as well as by the cells' reduced immunoreactivity for GluR1. In parallel, the NMDA treatment markedly altered the interaction between the AMPA receptor subunits and their associating molecule(s); the association of PDZ molecules, including Pick1, with GluR2/3 was enhanced in a protein-kinase-C-dependent manner. Viral expression vectors carrying GluR1 and GluR2 C-terminal decoys, both fused to enhanced green fluorescent protein, were transfected into hippocampal neurons to disrupt their interactions. The overexpression of the C-terminal decoy for GluR2 specifically and significantly blocked the NMDA-triggered reduction in [(3)H]AMPA binding, whereas that for GluR1 had no effects. Co-immunoprecipitation using anti-Pick1 antibodies revealed that the overexpressed GluR2 C-terminal decoy indeed prevented Pick1 from interacting with the endogenous GluR2/3. Therefore, these observations suggest that the NMDA-induced down-regulation of the functional AMPA receptors involves the interaction between GluR2/3 subunits and Pick1.

Kainate-induced epilepsy alters protein expression of AMPA receptor subunits GluR1, GluR2 and AMPA receptor binding protein in the rat hippocampus.

Kainic acid induces seizures with consecutive degeneration of highly vulnerable hippocampal CA3 neurons in adult rats. An abnormal influx of calcium through newly synthesized alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) receptors lacking the GluR2 subunit, which normally renders AMPA receptors calcium impermeable, is thought to play a pivotal role for postictal neuronal death (GluR2 hypothesis). Using a specific GluR2 antiserum, postictal hippocampal GluR2 protein expression was investigated and compared to GluR1 between 6 and 96 h after seizure induction. In addition, postictal protein expression of a recently cloned AMPA receptor binding protein (ABP), which anchors AMPA receptors in the plasma membrane was also analyzed, to address the question of whether its protein expression is associated with neuronal death or survival. At 6 h after seizure induction, GluR2 immunoreactivity (IR) in CA3 was more markedly reduced compared to GluR1, but at 24 h GluR2 IR reattained control levels. More importantly, GluR2 IR was also markedly, but transiently decreased between 6 and 48 h in hippocampal CA1 neurons, but no significant cell loss was observed. These findings modify the GluR2 hypothesis in so far as only a subset of, but not all, hippocampal CA1 and CA3 pyramidal neurons may die due to reduced GluR2 levels with consecutive calcium overload through calcium-permeable AMPA receptors. ABP was induced postictally in presumed CA2 and a subpopulation of CA3 neurons and seems not to be involved in mechanisms of delayed neuronal death.

AMPA receptor function is altered in GLUR2-deficient mice.

The GluR2 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor determines many of the biophysical properties of native AMPA receptors, including Ca++ permeability. Genetically engineered mice unable to edit the Q to R site of the GluR2 subunit die within 3 wk postpartum, presumably due to toxicity associated with enhanced Ca++ influx through AMPA receptors. In contrast, disruption of the gene encoding GluR2 is not necessarily lethal. The objective of this study was to explore potential mechanisms that permit survival of GluR2 (-/-) mice despite AMPA receptors that are highly Ca++ permeable. Whole-cell, patch-clamp recording of AMPAreceptor responses in cortical pyramidal cells revealed that the kinetics of recovery from desensitization were significantly slower for receptors from GluR2 (-/-) mice compared to receptors from GluR2 (+/+) mice. The recovery time constants for AMPA receptors from GluR2 (-/-) and GluR2 (+/+) mice were 109.8 +/- 17 ms and 54.4 +/- 7.1 ms, respectively. The slower recovery kinetics would be expected to reduce Ca++ influx during repetitive stimulation. Because both RNA editing at the R/G site and alternative splicing of the flip and flop module affect AMPA receptor desensitization recovery rates, the possibility that these mechanisms were changed in GluR2 (-/-) mice was investigated. On a macroscopic level, neither editing nor splicing of the GluR-1, 3 or 4 subunits were changed in GluR2 (-/-) mice compared to GluR2 (+/+) mice. In summary, an increase in the time constant for recovery from desensitization may contribute to the ability of GluR2 (-/-) to survive.

Altered pattern of light transmittance and resistance to AMPA-induced swelling in the dentate gyrus of the epileptic hippocampus.

Glutamate receptor-mediated changes in light transmittance were imaged in the dentate gyri of the epileptic hippocampi, taken from patients with temporal lobe epilepsy and the rat pilocarpine model, to investigate epilepsy-associated alterations in activity-induced cell swelling. A static pattern of light transmittance corresponded to the layered structure of dentate gyrus and reflected epilepsy-associated alterations. Hypoosmotic stress produced more than 35% of dynamic changes in the increase of light transmittance as a reflection of osmotic swelling in the epileptic dentate gyri. This degree of increase was not different from the increase observed in control dentate gyri, suggesting that the capability of osmotically regulating cell volume was preserved in the epileptic dentate gyri. In contrast, alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) induced activity-dependent swelling and an increase in light transmittance by 60.5% in the control dentate gyri, whereas the degree of increase in the epileptic dentate gyri remained 17.9% in response to AMPA. Selective attenuation of light transmittance in response to AMPA in the epileptic but not control dentate gyri suggested a possible alteration in the swelling properties of the epileptic dentate gyri that are linked to the AMPA receptor activation. Surviving cells in the epileptic hippocampus may have a mechanism of downregulating neuronal activity-dependent swelling to maintain optimal cell volume during repeated network hyperexcitation in epilepsy.

Speaking out of turn: a role for silent synapses in pain.

Severe tissue or nerve injury can result in a chronic and inappropriate sensation of pain, mediated in part by the sensitization of spinal dorsal horn neurons to input from primary afferent fibers. Synaptic transmission at primary afferent synapses is mainly glutamatergic. Although a functioning excitatory synapse contains both alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors in the postsynaptic membrane, recent evidence suggests that dorsal horn neurons contain some "silent" synapses, which exhibit purely NMDA receptor-mediated evoked postsynaptic currents and do not conduct signals at resting membrane potential. Serotonin, which is released onto dorsal horn neurons by descending fibers from the rostroventral medulla, potentiates sensory transmission by activating silent synapses on those neurons, i.e., by recruiting functional AMPA receptors to the postsynaptic membrane. This phenomenon may contribute to the hyperexcitability of dorsal horn neurons seen in chronic pain conditions.

A selective AMPA antagonist, LY293558, suppresses morphine withdrawal-induced activation of locus coeruleus neurons and behavioral signs of morphine withdrawal

The glutamate receptor subtype that mediates the morphine withdrawal-induced activation of locus coeruleus (LC) neurons was examined in this study using in vitro and in vivo single-unit electrophysiologic recordings. For LC neurons recorded in vitro in rat brain slices, the selective alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) antagonist, LY293558, showed a greater than 10-fold selectivity for inhibiting the excitatory effects of AMPA vs kainate, and a greater than 30-fold selectivity for inhibiting the excitatory effects of AMPA vs NMDA. LY293558 also greatly reduced the response of LC neurons to glutamate in a concentration-dependent manner. In in vivo recordings in anesthetized rats, pretreatment with LY293558 (0.1 to 10 mg/kg, i.p.) dose dependently suppressed the morphine withdrawal-induced activation of LC neurons. In unanesthetized, morphine-dependent animals, pretreatment with LY293558 (1 to 30 mg/kg, i.p.) dose dependently suppressed naltrexone-precipitated morphine withdrawal signs. These results indicate: (1) AMPA receptors mediate a large component of the excitatory effects of glutamate on LC neurons; (2) activation of AMPA receptors plays an important role in the morphine withdrawal-induced activation of LC neurons; (3) AMPA antagonists can suppress many signs of morphine withdrawal in awake animals; and (4) AMPA antagonists may have therapeutic effects in humans for the treatment of opiate withdrawal.

Glutamate receptor regulation of rat nucleus accumbens neurons in vivo.

Extracellular single cell recording and microiontophoretic techniques were used to characterize the roles of ionotropic and metabotropic glutamate receptors (iGluRs and mGluRs) in glutamate-induced excitation of rat nucleus accumbens (NAc) neurons in vivo. Pulse-ejected glutamate (16-128 nA) induced a current-dependent increase in the firing of quiescent NAc neurons. A stronger excitatory response to alpha-amino-3-hydroxy-5-methyl-4-isoxazole-proprionic acid (AMPA) was observed at much lower ejection currents (0.1-6.4 nA). Compared to AMPA and glutamate, N-methyl-D-aspartate (NMDA) induced a much less potent excitation in a narrow current range (1-4 nA) and only when neurons were previously "primed" with other excitatory amino acids (EAAs). Higher ejection currents of all three EAA agonists drove NAc neurons into a state of apparent depolarization block. AMPA-evoked firing was selectively blocked by the AMPA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) whereas NMDA-induced activity was selectively prevented by the NMDA receptor antagonist 2-amino-5-phosphonovalerate (D-AP5). DNQX, but not D-AP5, significantly attenuated glutamate-evoked activity. The mGluR receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-t-ACPD) failed to evoke activity of NAc neurons, but significantly reduced the excitatory effects of other EAAs. This modulatory effect of 1S,3R-t-ACPD was consistently blocked by the selective mGluR antagonist L(+)-2-amino-3-phopsonopropionic acid (L-AP3) whereas another mGluR antagonist (RS)-4-carboxy-3-hydroxy phenylglycine (4C3HPG) was inconsistent in this regard. These results indicate that the excitatory effects of glutamate on rat NAc neurons in vivo are primarily mediated by non-NMDA iGluRs and that mGluRs function to dampen excessive glutamate transmission through iGluRs.

Chronic effects of typical and atypical neuroleptics on glutamate receptor subtypes

Epilepsy is an important feature for neuropsychiatric involvement in systemic lupus erythematosus (SLE) with unknown mechanism. Our aim was to investigate the presence of neuronal auto-antibodies (NAbs) in neuropsychiatric SLE (NPSLE).Eighteen SLE patients (17 females, 1 male) experiencing recurrent seizures were enrolled to this study. Their clinical characteristics, EEG and MRI findings and follow-up information were evaluated from their files. Antibodies against voltage-gated potassium channel (VGKC)-complex antigens, contactin-associated protein-like 2 (CASPR-2), leucine-rich, glioma inactivated 1 (LGI1), glutamic acid decarboxylase (GAD), N-methyl-d-aspartate receptor (NMDA-R), alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor (AMPA-R) and type B gamma aminobutyric acid receptors (GABAB-R) were screened in the sera of these patients. Moreover, indirect immunohistochemistry and immunocytochemistry tests were performed to reveal neuropil antibodies.Six out of 18 patients (33.3%) had various forms of NAbs. Among them, one patient had antibodies against GAD, one patient with hippocampal sclerosis on MRI was CASPR-2 antibody positive, whereas the remaining four patients showed hippocampal neuropil staining. We could not find a significant difference between seropositive and seronegative groups, regarding the clinical characteristics, EEG and MRI findings.This study is the first to show hippocampal neuronal staining (4/18) reflecting antibodies against unknown neuronal cell surface antigens in SLE patients with epilepsy, besides the rare occurrence of GAD and CASPR2 antibodies. Further prospective studies are needed to search for new NAbs and uncover their pathogenic role in SLE associated with epilepsy.

Biochemical and assembly properties of GluR6 and KA2, two members of the kainate receptor family, determined with subunit-specific antibodies.

To examine subunit assembly and biochemical properties of two members of the kainate family of glutamate receptors (GluR), antibodies were made to synthetic peptides corresponding to the carboxyl termini of GluR6 and KA2. Immunoblot analysis of membranes from human embryonic kidney cells transfected with glutamate receptor cDNAs showed that these antibodies are selective for their respective receptor subunit except that the antibody to GluR6 also recognizes GluR7, which is expected due to the sequence homology between the two subunits at the carboxyl terminus. In transfected cell membranes, immunoblot analysis with the antibody to GluR6 showed a major immunoreactive band at 118 kDa and minor bands at 103 and 28 kDa. The 103-kDa band appears to be a deglycosylated form of GluR6 since deglycosylation eliminates staining at 118 kDa and increases staining of the 103-kDa band. Immunoblot analysis of KA2 transfected cell membranes shows a major band at 123 kDa and minor bands at 109 and 37 kDa. Deglycosylation converts the 123-kDa band into a 109-kDa band. Analysis of brain tissues shows that both antibodies label single major bands which migrate at the same molecular masses as those from transfected cell membranes, 118 and 123 kDa for GluR6 and KA2, respectively. Immunoprecipitation studies showed that antibodies to GluR6 and KA2 selectively immunoprecipitated [3H]kainate binding activity, but not 3H-labeled alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) binding activity, from Triton X-100-solubilized rat brain membranes. Furthermore, each antibody coimmunoprecipitated GluR6 and KA2 from cells co-transfected with GluR6 and KA2 cDNAs and from detergent-solubilized rat brain membranes, indicating that these two subunits can coassemble into a molecular complex. Interestingly, GluR1 and GluR2, subunits of the AMPA receptor, also co-immunoprecipitated with GluR6 in cells co-transfected with GluR6 and GluR1 or GluR2 cD-NAs. Such complexes appear to be present to a limited extent in the brain.

Neurotoxic effects of tumor necrosis factor alpha in primary human neuronal cultures are mediated by activation of the glutamate AMPA receptor subtype: implications for AIDS neuropathogenesis.

Human immunodeficiency virus type 1 (HIV) infection of the central nervous system is characterized by neuronal loss in discrete areas of the central nervous system. We have previously demonstrated that HIV-infected monocytes in culture with astroglial cells produce high levels (> or = 200 pg/ml) of the cytokine tumor necrosis factor-alpha (TNF alpha). We now demonstrate that TNF alpha (> or = 200 pg/ml) is neurotoxic to cultured primary human fetal cortical neurons at both light and electron microscopic levels. Subtoxic doses of TNF alpha (50 pg/ml) are neurotoxic in combination with the glutamate (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) subtype receptor agonist AMPA (100 microM). The neurotoxic effects of TNF alpha (200 pg/ml) are blocked in part by the AMPA receptor antagonist, 6-cyano-7-nitroquinoxaline-2, 3-dione (10 microM). This suggests that TNF alpha may exert neurotoxic effects on human neurons by indirect activation of AMPA receptors, which may be important in the pathogenesis and treatment of HIV-mediated encephalopathy.