Glutamate Receptor Binding Research Articles

The effect of pentylenetetrazol kindling on synaptic mechanisms of interacting glutamatergic and opioid system in the hippocampus of rats

Endogenous opioids modulate processes of central excitability such as long-term potentiation and electrical kindling. Little is known about the neurochemical alterations in the interaction of the glutamatergic and opioid system in the development of pentylenetetrazol (PTZ) kindling in rats. Therefore, in the present study we investigated glutamate, DAMGO and naltrindole receptor binding, receptor protein expression by Western blot and ex vivo glutamate transmitter release in PTZ kindled rats. The specific 3 H -DAMGO and -naltrindole binding to hippocampal membranes displayed no significant changes in kindled rats compared to controls. In contrast, the 3 H - l-glutamate binding was significantly enhanced after completion of PTZ kindling. The expression of receptor protein for glutamate as well as the naloxone- and naltrindole-induced 3 H - d-aspartate release from hippocampal slices did not alter in any case as a consequence of PTZ kindling. The PTZ induced enhancement of the glutamate binding sites in the hippocampus was downregulated to control level by natrindole treatment of rats prior to each PTZ application. Furthermore, naltrindole pretreatment of rats significantly inhibited the development of seizure susceptibility. In contrast, naloxone was not able to alter the seizure activity induced by PTZ as well as the transmitter receptor binding. The results are discussed in the light of a modulating role of δ-opioid receptors in PTZ kindling.

Melatonin plays a protective role in quinolinic acid-induced neurotoxicity in the rat hippocampus

The neuroprotective effect of melatonin against the quinolinic acid-induced degeneration of rat hippocampal neurons was investigated. Three groups of rats were given intrahippocampal injections of either; saline, quinolinic acid or i.p. injections of melatonin prior to and after being injected with quinolinic acid. On the fifth day after the intrahippocampal injections the brains were removed and the hippocampi either sectioned and stained for microscopic examination or used in glutamate receptor binding studies. The results show that melatonin protects hippocampal neurons from quinolinic acid-induced neurodegeneration and partially prevents the decrease in glutamate receptor numbers caused by quinolinic acid. Thus, melatonin has the potential to reduce hippocampal neuronal damage induced by neurotoxins such as quinolinic acid.

Altered brain neurotransmitter receptors in transgenic mice expressing a portion of an abnormal human huntington disease gene.

Loss of neurotransmitter receptors, especially glutamate and dopamine receptors, is one of the pathologic hallmarks of brains of patients with Huntington disease (HD). Transgenic mice that express exon 1 of an abnormal human HD gene (line R6/2) develop neurologic symptoms at 9-11 weeks of age through an unknown mechanism. Analysis of glutamate receptors (GluRs) in symptomatic 12-week-old R6/2 mice revealed decreases compared with age-matched littermate controls in the type 1 metabotropic GluR (mGluR1), mGluR2, mGluR3, but not the mGluR5 subtype of G protein-linked mGluR, as determined by [3H]glutamate receptor binding, protein immunoblotting, and in situ hybridization. Ionotropic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainate receptors were also decreased, while N-methyl-D-aspartic acid receptors were not different compared with controls. Other neurotransmitter receptors known to be affected in HD were also decreased in R6/2 mice, including dopamine and muscarinic cholinergic, but not gamma-aminobutyric acid receptors. D1-like and D2-like dopamine receptor binding was drastically reduced to one-third of control in the brains of 8- and 12-week-old R6/2 mice. In situ hybridization indicated that mGluR and D1 dopamine receptor mRNA were altered as early as 4 weeks of age, long prior to the onset of clinical symptoms. Thus, altered expression of neurotransmitter receptors precedes clinical symptoms in R6/2 mice and may contribute to subsequent pathology.

Sulphur-containing amino acids are agonists for group 1 metabotropic receptors expressed in clonal RGT cell lines

Comparison of the pharmacological effects of a range of sulphur-containing amino acids on human mGluR1 α and mGluR5a has been undertaken. cDNAs of each mGluR were transfected into a Syrian hamster tumour cell line AV12-664 that was previously transfected with the rat glutamate–aspartate transporter protein (GLAST). The l-isomers of cysteine sulphinic acid (CSA), homocysteine sulphinic acid (HCSA), cysteic acid (CA) and serine- O-sulphate (SOS) stimulated PI hydrolysis in human mGluR1 α and mGluR5a cells with full agonist effects. d-CSA, the only active d-isomer, was a partial agonist for mGluR5a whereas l-sulphocysteine ( S-CYS) showed weak agonist-like effects at high concentrations on both mGluR1 α and mGluR5a. l-Homocysteic acid was inactive on both mGluR1 α and mGluR5a cells. Treatment of mGluR cultures with glutamate pyruvate transaminase did not alter the potencies of the S-amino acids on PI hydrolysis responses. Inhibitor constants ( K i) obtained for l-HCSA, l-CSA, l-CA and l-SOS in [ 3H]glutamate receptor binding studies with mGluR1 α cells indicated that l-HCSA, l-CSA, l-CA and l-SOS can bind specifically to mGluR1 with l-HCSA showing the highest affinity. These results confirm that certain endogenously produced S-amino acids may interact directly with group 1 mGluRs.

Memory Formation: The Sequence of Biochemical Events in the Hippocampus and Its Connection to Activity in Other Brain Structures

Recent data have demonstrated a biochemical sequence of events in the rat hippocampus that is necessary for memory formation of inhibitory avoidance behavior. The sequence initially involves the activation of three different types of glutamate receptors followed by changes in second messengers and biochemical cascades led by enhanced activity of protein kinases A, C, and G and calcium–calmodulin protein kinase II, followed by changes in glutamate receptor subunits and binding properties and increased expression of constitutive and inducible transcription factors. The biochemical events are regulated early after training by hormonal and neurohumoral mechanisms related to alertness, anxiety, and stress, and 3–6 h after training by pathways related to mood and affect. The early modulation is mediated locally by GABAergic, cholinergic, and noradrenergic synapses and by putative retrograde synaptic messengers, and extrinsically by the amygdala and possibly the medial septum, which handle emotional components of memories and are direct or indirect sites of action for several hormones and neurotransmitters. The late modulation relies on dopamine D1, β-noradrenergic, and 5HT1A receptors in the hippocampus and dopaminergic, noradrenergic, and serotoninergic pathways. Evidence indicates that hippocampal activity mediated by glutamate AMPA receptors must persist during at least 3 h after training in order for memories to be consolidated. Probably, this activity is transmitted to other areas, including the source of the dopaminergic, noradrenergic, and serotoninergic pathways, and the entorhinal and posterior parietal cortex. The entorhinal and posterior parietal cortex participate in memory consolidation minutes after the hippocampal chain of events starts, in both cases through glutamate NMDA receptor-mediated processes, and their intervention is necessary in order to complete memory consolidation. The hippocampus, amygdala, entorhinal cortex, and parietal cortex are involved in retrieval in the first few days after training; at 30 days from training only the entorhinal and parietal cortex are involved, and at 60 days only the parietal cortex is necessary for retrieval. Based on observations on other forms of hippocampal plasticity and on memory formation in the chick brain, it is suggested that the hippocampal chain of events that underlies memory formation is linked to long-term storage elsewhere through activity-dependent changes in cell connectivity.

Microinfusion of the metabotropic glutamate receptor agonist 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid into the nucleus accumbens induces dopamine-dependent locomotor activation in the rat.

Although the striatum has one of the highest densities of metabotropic glutamate receptor (mGluR) binding sites in the brain, little is known about their physiological role. In this study we characterized the contribution of mGluRs located in the ventral part of the striatum (the nucleus accumbens) to the control of extrapyramidal motor function. Activation of mGluRs by local infusion of the selective agonist 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD; 25, 50 and 100 nmol/0.5 microl) into the nucleus accumbens induced a dose-dependent increase in locomotor activity in rats. Intra-accumbens infusion of a selective antagonist of mGluRs, alpha-methyl-4-carboxyphenylglycine (MCPG) did not modify spontaneous locomotion but decreased the locomotor response to 1S,3R-ACPD. This effect appeared to be mediated by dopamine, since blockade of dopamine receptors with haloperidol (0.05 and 0.1 mg/kg i.p.) dose-dependently reduced 1S,3R-ACPD-induced locomotor activation. Furthermore, D-amphetamine (0.5 mg/kg, i.p.) combined with intra-accumbens infusion of 1S,3R-ACPD (100 nmol) potentiated the locomotor hyperactivity response to a higher level than that seen with a single treatment with either drug. In contrast, D-amphetamine-induced hypermotility was abolished by infusion of MCPG (100 nmol) into the nucleus accumbens. These results demonstrate that glutamate may control extrapyramidal motor function through metabotropic receptors. Furthermore, activation of metabotropic glutamate receptors appears to act in synergy with the dopamine system at the level of the nucleus accumbens to produce a motor stimulant response.

Hippocampal N-methyl- d-aspartate and kainate binding in response to entorhinal cortex aspiration or 192 IgG-saporin lesions of the basal forebrain

Lesion models in the rat were used to examine the effects of removing innervation of the hippocampal formation on glutamate receptor binding in that system. Bilateral aspiration of the entorhinal cortex was used to remove the cortical innervation of the hippocampal formation and the dentate gyrus. The subcortical input to the hippocampus from cholinergic neurons of the basal forebrain was lesioned by microinjection of the immunotoxin 192 IgG-saporin into the medial septum and vertical limb of diagonal band. After a 30-day postlesion survival, the effects of these lesions on N-methyl- d-aspartate-displaceable [ 3H]glutamate and [ 3H]kainate binding in the hippocampus were quantified using in vitro autoradiography. The bilateral entorhinal lesion induced a sprouting response in the dentate gyrus, measured by an increase in the width of [ 3H]kainate binding. It also induced an increase in the density of [ 3H]kainate binding in CA3 stratum lucidum and an increase in N-methyl- d-aspartate binding throughout the hippocampus proper and the dentate gyrus. The selective lesion of cholinergic septal input did not have any effect on hippocampal [ 3H]kainate binding and induced only a moderate decrease in N-methyl- d-aspartate binding that was not statistically reliable. The entorhinal and cholinergic lesions were used as in vivo models of the degeneration of hippocampal input that occurs in normal aging and Alzheimer's disease. The results from the present lesion study suggest that some, but not all, of the effects on hippocampal [ 3H]kainate and N-methyl- d-aspartate binding induced by the lesions are consistent with the status of binding to these receptors in aging and Alzheimer's disease. Consistent with the effects of aging and Alzheimer's disease is an altered topography of [ 3H]kainate binding after entorhinal cortex lesion and a modest decline in N-methyl- d-aspartate binding after lesions of the cholinergic septal input to the hippocampus.

In Vitro Metal Inhibition of N-Methyl-D-Aspartate Specific Glutamate Receptor Binding in Neonatal and Adult Rat Brain

ABSTRACTThe in vitro effect of methyl mercury (MM) and lead (Pb) on N-methyl-D-aspartate (NMDA)-specific glutamate receptor binding in neonatal (10 days old) and adult rat brain was investigated. The cerebral cortex was isolated from the neonatal and adult male Sprague-Dawley rats and the synaptic plasma membranes were prepared to study the NMDA-specific glutamate receptor binding by using (3H)-glutamic acid. The metal salts such as methyl mercury chloride and lead acetate were used to study the effect of MM and Pb. Both MM and Pb significantly inhibited the receptor binding in neonatal and adult rat brain in a concentration-dependent manner. MM (IC50:0.95 ±0.08μM) was more potent in inhibiting the receptor binding than Pb (IC50 60 ±7μM) in neonatal rat brain. A similar high potency was observed for MM than Pb in adult rat brain but the IC50 values are very high (70 ± 6μM and 300 ± 24 μM respectively) indicating less effect compared to neonatal brain. The data suggest that NMDA-receptor binding was more sensitive to MM and Pb in neonatal brain than in adult. MM was more effective than Pb because of its more lipophilicity.

Gp120, a human immunodeficiency virus-1 coat protein, augments excitotoxic hippocampal injury in perinatal rats.

Recent data suggest that gp120, a human immunodeficiency virus-1 (HIV-1) coat glycoprotein that is secreted by HIV-infected cells, is neurotoxic, and that this toxicity is mediated, at least in part, by activation of N-methyl-D-aspartate-type excitatory amino acid receptors. To test this hypothesis in vivo, we examined the neurotoxicity of gp120 injected intrahippocampally, alone or co-injected with the selective excitatory amino acid agonist N-methyl-D-aspartate, in seven-day-old rats. Severity of injury in the lesioned hippocampus was assessed five days later, using three outcome measures: histopathology, hippocampal atrophy (derived from regional cross-sectional area measurements) and loss of [3H]glutamate receptor binding (based on in vitro autoradiography assays). To confirm that any observed effects were attributable to gp120 bioactivity, each group of experiments included controls that received equal amounts of heat-treated gp120. Gp120 (200 ng) elicited minimal focal pyramidal cell loss immediately adjacent to the injection track; there was no hippocampal atrophy or loss of [3H]glutamate binding. Co-injection of 50 ng gp120 with N-methyl-D-aspartate (5 nmol, threshold excitotoxic dose) increased the severity of hippocampal injury; hippocampal atrophy was greater in animals that received injections of 5 nmol N-methyl-D-aspartate in combination with 50 ng gp120 than in those that received either N-methyl-D-aspartate alone (5 nmol) or 5 nmol N-methyl-D-aspartate+50 ng heat-treated gp120 (mean+/-S.E.M. percentage reduction in injected hippocampal volume vs contralateral: N-methyl-D-aspartate, -19+/-3; N-methyl-D-aspartate+gp120, -26.8+/-2.1; N-methyl-D-aspartate+heat-treated gp120, -14.0+/-2.2; P<0.001, ANOVA). Treatment with the competitive N-methyl-D-aspartate antagonist 3-((RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (20mg/kg) markedly reduced the severity of injury elicited by the combination of gp120 with N-methyl-D-aspartate. These data support the hypothesis that locally secreted gp120 could exert neurotoxic effects, mediated by N-methyl-D-aspartate receptor activation, in vivo in the immature brain.

P-645 - Repeated noise stress alters metabotropic glutamate receptor binding in rat brain

P-645 - Repeated noise stress alters metabotropic glutamate receptor binding in rat brain

Unilateral eyeball enucleation differentially alters AMPA-, NMDA- and kainate glutamate receptor binding in the newborn rat brain

The aim of the present work was to evaluate the neurochemical effects of early unilateral visual deprivation as a model of impaired visual maturation. For this purpose, binding to the different ionotropic glutamate receptor subtypes was quantified in vision-related and vision-unrelated brain structures of control and unilaterally deprived newborn rats. At post-natal (PN) day 10, male Sprague-Dawley rats underwent either unilateral eyeball enucleation (enucleation group, n = 12) or sham operation (control group, n = 12). In each group, brains were obtained either post-natal day 20 ( n = 6) or post-natal day 30 ( n = 6) and processed for quantitative in vitro autoradiography selective for NMDA, kainate, and AMPA glutamate-binding sites, as well as for the presynaptic adenosine A1 receptor as a control of the deafferentation efficacy. In control animals, quantitative autoradiography revealed an increase in NMDA (e.g. +45% in superior colliculus) and kainate receptor binding (e.g. +55% in visual cortex, layer IV) from post-natal day 20 to post-natal day 30, associated with stable levels of AMPA receptor binding, in the vision-related structures. In the deafferented visual structures, monocular enucleation induces a marked decrease in A1 site density (e.g. −38 to − 52%, in the superficial layer of superior colliculi, at PN day 20 and PN day 30, respectively) in parallel with a mild increase in both NMDA (e.g. + 8 to 9%, in superior colliculi and visual cortex, layer IV at PN day 30, respectively) and AMPA (e.g. +16%, in layer IV of the visual cortex at PN day 30). Superimposed on marked bilateral decreases at PN day 30 in the enucleated rats, kainate receptor binding also revealed a slight but significant decrease (−5%) in the deafferented superior colliculus as compared to the non-deafferented side. The present findings (different time-courses of, and differential effects of deafferentation on, the NMDA, kainate and AMPA glutamate receptor subtypes throughout the visual brain structures) further support the involvement of these receptors in distinctive roles during maturation of the visual system.

Non-NMDA glutamate receptor binding in canine brain after global cerebral ischemia and reperfusion.

We employed a canine model to test the effects of global cerebral ischemia and reperfusion on binding to alpha-amino-3-hydroxy-5-methyl- 4-isoxazole proprionate (AMPA), kainate (KA), and metabotropic glutamate receptors. Ischemia was induced by 10 min of cardiac arrest, followed by restoration of spontaneous circulation for periods of 0, 0.5, 2, 4, and 24 h. Frozen sections were prepared from parietal and temporal cortex, hippocampus, and striatum, and in vitro autoradiography was performed with one of three radioligands: [3H]AMPA, [3H]KA, or [3H] glutamate (using conditions allowing specific labeling of the metabotropic binding site). In striatum, metabotropic binding was unchanged, whereas AMPA and KA binding decreased by 20-30% at 30 min postischemia, remaining depressed through 24 h. In cortex, AMPA and metabotropic binding were decreased at several time-points after ischemia and recirculation, particularly in parietal cortex, whereas KA binding was unaffected in this tissue. Binding to hippocampal regions was largely unchanged, except for a decrease in KA binding at 2 and 4 h postischemia. These findings contrast with results from parallel studies showing increased striatal binding to NMDA receptors following ischemia. Decreased binding to non-NMDA glutamate receptors in striatum and parietal cortex may serve to protect against damage mediated through these receptors.

NMDA and non-NMDA sensitive [ l- 3H] glutamate receptor binding in the brain of the Naples high- and low-excitability rats: an autoradiographic study

The Naples high-excitability (NHE) and low-excitability (NLE) rat lines, selectively bred for high and low activity in a Làt maze, respectively, are used as an animal model in the study of hippocampal functions. The aim of this study was to investigate the anatomical distribution of N-methyl- d-aspartate (NMDA) and non-NMDA sensitive [ 3H]glutamate receptor binding by quantitative autoradiography in the brain of the NHE and NLE rats with a randomly bred line (NRB) as controls. Twenty-micron-thick cryostat sagittal sections were incubated at 4°C with 150 nM [ l- 3H]glutamate alone or in the presence of 100 μM NMDA or 2.5 μM quisqualate (QA). Non-specific binding was determined in the presence of 1 mM of non-labeled glutamate. The sections were exposed to tritium-sensitive films for 3 weeks at 4°C. Quantitative analysis revealed: (1) higher levels of total binding in NHE than in NRB and NLE rats in all areas but the cerebellum; (2) fewer binding sites for both NMDA and QA receptors and larger binding sites for QA receptors in the hippocampus of NLE and NHE rats, respectively; (3) a positive correlation between total binding sites and activity level in a Làt maze in all areas, except the cerebellar molecular layer with NLE < NHE, which was due to a differential contribution from NMDA and non-NMDA types. Thus, the brain of the NHE rats shows an imbalance between NMDA and non-NMDA sensitive [ l- 3H]glutamate receptors.

Glutamate Receptor Binding in the Human Hippocampus and Adjacent Cortex During Development and Aging

Glutamate Receptor Binding in the Human Hippocampus and Adjacent Cortex During Development and Aging

Glutamate receptor binding in the human hippocampus and adjacent cortex during development and aging

Distinct patterns of age-related alterations in NMDA (MK801 binding) and non-NMDA, AMPA (CNQX), and kainate binding have been identified in human hippocampus and parahippocampal gyrus in normal individuals with no evidence of degenerative brain disease ranging in age from 24 gestational weeks to 94 years. Whereas MK801 binding did not alter substantially over this age range, CNQX binding rose from low levels in the fetus to maximum levels between neonate and middle age, andkainate binding declined extensively from the perinatal to adult stage. Following maturity, there were no significant changes in kainate binding, although MK801 binding increased in CA1 and CA3 and CNQX binding declined in several regions, particularly CA2 and subiculum. For each receptor binding the timing of these fluctuations occurring during development and aging varied within different regions of the dentate gyrus, hippocampus proper, subicular complex, and entorhinal cortex examined. The transient peaks receptor binding are likely to reflect processes of synaptogenesis and pruning and may provide clues regarding the role of the different glutamate receptor subtypes in various pathologies of the hippocampus and adjacent cortex associated with developmental disorders (of genetic origin or due to perinatal trauma or insult). The absence of substantial changes in any subtype examined from middle to old age suggests alterations in transmitter binding to these glutamate receptors are not involved in senescent neurodegeneration.

Beta-amyloidosis in normal aging and transmitter signaling in human temporal lobe.

Interactions between abnormal amyloid precursor protein metabolism and cholinergic dysfunction are increasingly apparent. Both of these major features of Alzheimer's disease occur in restricted loci in normal aging--a potential model for early Alzheimer type pathology. Entorhinal cortex is particularly vulnerable to beta-amyloidosis and compared with other cortical areas is remarkable for the relatively high density of nicotinic (3H-nicotine) but not other cholinergic or glutamate receptor binding. With increasing age, post-maturity, there is a persistent decline in nicotinic receptor binding in entorhinal cortex whereas muscarinic M1 and non-M1, glutamate NMDA and non-NMDA receptors are spared. Normal elderly individuals, distinguished by the absence of beta A4 immunoreactive plaques in this area, are differentiated from those with plaques by higher nicotine binding. Amongst individuals with an established history of smoking tobacco, nicotinic receptor binding and hippocampal choline acetyltransferase were elevated compared with non-smokers and preliminary evidence indicates a reduced density of cortical plaques. These findings are consistent with the hypothesis that down regulation of the nicotinic cholinergic receptor--a ligand gated calcium channel known to control the expression of neurotrophins--plays a role in the evolution of Alzheimer-type pathology.

L-glutamate-induced changes in intracellular calcium oscillation frequency through non-classical glutamate receptor binding in cultured rat myocardial cells

The effects of L-glutamate, N-methyl-D-aspartate (NMDA), kainate (KA) and L-alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate (AMPA) on intracellular Ca 2+ oscillation frequency were studied in cultured rat myocardial cells. Ca 2+ oscillations per minute were increased as compared to control by L-glutamate (100 μM) from 3.8 ± 2.2 to 25.7 ± 4.3 (p < 0.001) and the NMDA-receptor agonist, NMDA (100 μM), from 1.2 ± 0.8 to 34.8 ± 10.1 (p < 0.011). Increases over control frequency were also seen in response to the non-NMDA receptor agonists KA (100 μM) from 5.8 ± 2.3 to 25.6 ± 3.2 ( p < 0.001) and AMPA (10 μM) from 3.8 ± 1.2 to 13.3 ± 1.8 ( p < 0.001). The non-competitive NMDA receptor antagonist, (+)-5-methyl-10,ll-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-iminemaleate (MK801) (10 μM), decreased the Ca 2+ oscillation frequency induced by NMDA (100 μM from 36.8 ± 12.2 to 7.2 ± 7.2 ( p < 0.05). (±)-2-Amino-7-phosphonoheptanoic acid (AP7), a competitive inhibitor at the NMDA receptor inhibited the increase in frequency induced by KA (100 μM) at all concentrations tested (0.1, 1.0, 10 and 100 μM) . 6,7-Dinitroquinoxaline-2,3-dione (DNQX), a competitive inhibitor at non-NMDA receptors, also decreased the oscillation frequency elicited by KA (100 μM) from 35.4 ± 9.4 to 28.2 ± 9.8, 24.8 ± 9.8 and 11 ± 9.5 at concentrations of 0.1, 1.0 and 10 μM respectively. The peak amount of intracellular Ca 2+ as expressed as the fluo 3 ratio, F F rest , was not increased by l-glutamate, NMDA or KA. These results suggest the presence of a novel glutamate receptor composed of both non-NMDA and NMDA subunits on cultured rat myocardial cells, and receptor stimulation leads to an increase in intracellular Ca 2+ oscillation frequency.

Developmental rearrangements of cortical glutamate-NMDA receptor binding sites in late human gestation

NMDA-preferring glutamate receptor binding sites were characterized using the site-selective ligand [ 3H]MK801, in synaptic membranes prepared from cerebral cortex tissue obtained postmortem from human infants who had died with minimal neurological and neuropathological impairment between 22 and 42 weeks' gestation. It proved necessary to modify the assay protocol used with adult tissue before reliable data could be obtained. In the four cortical region studied (prefrontal, motor, occipital, temporal), [ 3HIMK801 bound to a single class of sites which showed significant variations in affinity only in motor cortex. The density of [ 3H]MK801 binding sites (calculated at constant affinity) showed marked increases in all cortical regions over this period. The extent to which glutamate could enhance [ 3HIMK801 binding became significantly lower in prefrontal and motor cortex as gestation progressed, so that at term, little activation was apparent. In occipital and temporal cortex, this parameter was low throughout late gestation. The evidence suggests that Glutamate-NMDA binding sites may undergo structural rearrangements which alter their ability to interact with ligands during the later stages of human gestation, and that such changes are regionally variable.

Purification and characterization of a low molecular weight endogenous glutamate binding inhibitor (LGBI) in porcine brain

One of the endogenous substances which modulate glutamate receptor binding was isolated and highly purified from porcine brain. The purification involved extraction of brain tissue with doubled distilled water, followed by gel filtration, anion exchange, cation exchange, and several steps of C18 reverse-phase high performance liquid chromatography (HPLC). A low molecular weight glutamate binding inhibitor (LGBI) was purified to apparent homogeneity as judged from the elution profile of an HPLC column, in which a symmetrical peak was obtained when the eluate was monitored at 220 nm. The LGBI appears to be a small molecule (< 2 kD) that is heat- and acid/base-stable. The highly purified LGBI has no effect on GABAA and benzodiazepine receptor binding. The LGBI is not L-glutamate, L-aspartate or other negatively charged endogenous substances, since they are clearly separated from the LGBI in anion exchange chromatography. The inhibitory effect of the LGBI on [3H]L-glutamate binding is reversible, and it only changes the Bmax while the Kd remains the same. Since the membrane preparations used for [3H]L-glutamate binding assays for the detection of LGBI activity were enriched with quisqualate (QA)-sensitive subtypes, it was suggested that the LGBI could be a modulator of the QA receptor. Some amino acids which produce significant inhibition of glutamate binding activity were also compared with the LGBI, and they all showed no resemblance to the LGBI. The chemical structure of the LGBI remains to be determined.

MK801 induces late regional increases in NMDA and kainate receptor binding in rat brain.

We have previously shown that a single dose of PCP produces a dose-related increase in NMDA-sensitive 3H-glutamate binding in CA1 of hippocampus 24 hours later, and some regional changes in kainate binding. Here we report that dizocilpine (MK 801) (0.1 mg/kg and 1 mg/kg), a selective agonist at the PCP receptor and a noncompetitive antagonist of NMDA, produces a similar increase in NMDA-sensitive glutamate and kainate receptor binding in hippocampus 24 hours after a dose. These observations support the conclusion that blockade of glutamate-mediated transmission at the NMDA receptor selectively increases NMDA-sensitive glutamate receptor binding in CA1 of hippocampus and kainate binding in CA3 and dentate gyrus at putatively delayed time points. Several additional areas outside of hippocampus also showed receptor changes at 24 hours after MK801.