Kiyokazu Ogita

Age-related decreases of the N-methyl-d-aspartate receptor complex in the rat cerebral cortex and hippocampus

Binding activities of central excitatory amino acid receptors were examined in Triton-treated membrane preparations of the cerebral cortex and hippocampus from brains of rats at 2, 7 and 29 months after birth. Aged rats exhibited a significant reduction of [3H]glutamate (Glu) binding displaceable by N-methyl-D-aspartate (NMDA), as well as strychnine-insensitive [3H]glycine binding in both central structures, as compared with those in young rats. Binding of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne maleate (MK-801), a non-competitive NMDA antagonist used to label the activated state of ion channels linked to NMDA-sensitive receptors, also decreased with aging irrespective of the experimental conditions employed. Scatchard analysis revealed that reduction of both [3H]Glu binding and [3H]MK-801 binding were due to a significant decrease in the densities of binding sites with aging, with their affinities being unaltered. Binding of [3H]D,L-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), which is a specific agonist for quisqualate-sensitive receptors, was unchanged with aging when determined in the absence of 100 mM potassium thiocyanate (KSCN). However, AMPA binding determined in the presence of added KSCN was about 25% reduced in both brain regions of aged rats. Binding of [3H]kainate to kainate-sensitive receptors was unchanged with aging. These results suggest that glutaminergic neurotransmission mediated by NMDA-sensitive receptors may be selectively impaired with aging in the hippocampus and cerebral cortex among 3 different subclasses of excitatory amino acid receptors in the brain.

Glutathione and signal transduction in the mammalian CNS

Abstract : The tripeptide glutathione (GSH) has been thoroughly investigated in relation to its role as antioxidant and free radical scavenger. In recent years, novel actions of GSH in the nervous system have also been described, suggesting that GSH may serve additionally both as a neuromodulator and as a neurotransmitter. In the present article, we describe our studies to explore further a potential role of GSH as neuromodulator/neurotransmitter. These studies have used a combination of methods, including radioligand binding, synaptic release and uptake assays, and electrophysiological recording. We report here the characteristics of GSH binding sites, the interrelationship of GSH with the NMDA receptor, and the effects of GSH on neural activity. Our results demonstrate that GSH binds via its γ‐glutamyl moiety to ionotropic glutamate receptors. At micromolar concentrations GSH displaces excitatory agonists, acting to halt their physiological actions on target neurons. At millimolar concentrations, GSH, acting through its free cysteinyl thiol group, modulates the redox site of NMDA receptors. As such modulation has been shown to increase NMDA receptor channel currents, this action may play a significant role in normal and abnormal synaptic activity. In addition, GSH in the nanomolar to micromolar range binds to at least two populations of binding sites that appear to be distinct from all known excitatory amino acid receptor subtypes. GSH bound to these sites is not displaceable by glutamatergic agonists or antagonists. These binding sites, which we believe to be distinct receptor populations, appear to recognize the cysteinyl moiety of the GSH molecule. Like NMDA receptors, the GSH binding sites possess a coagonist site(s) for allosteric modulation. Furthermore, they appear to be linked to sodium ionophores, an interpretation supported by field potential recordings in rat cerebral cortex that reveal a dose‐dependent depolarization to applied GSH that is blocked by the absence of sodium but not by lowering calcium or by NMDA or (S)‐2‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate antagonists. The present data support a reevaluation of the role of GSH in the nervous system in which GSH may be involved both directly and indirectly in synaptic transmission. A full accounting of the actions of GSH may lead to more comprehensive understanding of synaptic function in normal and disease states.

Selective Potentiation of DNA Binding Activities of Both Activator Protein 1 and Cyclic AMP Response Element Binding Protein Through In Vivo Activation of N-Methyl-d-Aspartate Receptor Complex in Mouse Brain

Abstract: DNA binding activities of several transcription factors were evaluated in nuclear extracts from brains of mice that were injected intracerebroventricularly with N‐methyl‐d‐aspartic acid (NMDA) using gel‐shift assays. An injection of saline transiently increased binding of both probes for activator protein 1 (AP1) and cyclic AMP response element binding protein (CREB) 30 min after the injection, and NMDA was effective in inducing a more potent increment of binding of both probes 1–5 h after the injection than did saline. However, no significant alterations were found in binding of probes for other transcription factors tested up to 4 h following the injection of NMDA. The potentiation by NMDA was prevented in a dose‐dependent manner by administration of the non‐competitive NMDA antagonist (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine, the NMDA antagonist d,l‐(E)‐2‐amino‐4‐propyl‐5‐phosphono‐3‐pentenoic acid, and the glycine antagonist 5,7‐dichlorokynurenic acid, whereas administration of the proposed polyamine antagonist ifenprodil was rather ineffective in protecting against the potentiation by NMDA. These results support the proposal that an intracerebroventricular injection of NMDA may selectively potentiate DNA binding activities of both AP1 and CREB through activation of the NMDA receptor complex in mouse brain.

A possible role of glutathione as an endogenous agonist at the N-methyl-D-aspartate recognition domain in rat brain

Abstract: Glutathione, both reduced (GSH) and oxidized (GSSG), was effective in displacing binding of l‐[3H]‐glutamic acid (l‐[3H]Glu) and dl‐(E)‐2‐[3H]amino‐4‐propyl‐5‐phosphono‐3‐pentenoic acid ([3H]CGP‐39653) in rat brain synaptic membranes, with less potent displacement of binding of dl‐α‐amino‐3‐hydroxy‐5‐[3H]‐methylisoxazole‐4‐propionic and [3H]kainic acids. Liquid chromatographic analysis revealed that both GSH and GSSG were contaminated with l‐Glu by <1%. Both GSH and GSSG potentiated (+)‐5‐[3H]methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine ([3H]MK‐801) binding in a manner similar to that found with l‐Glu. Pre‐treatment with glutamate dehydrogenase (GDH) induced a marked rightward shift of the concentration‐response curve for l‐Glu in the presence of NAD without affecting that in its absence, whereas GDH was ineffective in affecting the potentiation by both GSH and GSSG even in the presence of NAD. In the presence of GSH at a maximally effective concentration, both glycine (Gly) and spermidine potentiated [3H]MK‐801 binding to a somewhat smaller extent than that found in the presence of l‐Glu at a maximally effective concentration. The potentiation of [3H]MK‐801 binding by GSH was invariably attenuated by addition of CGP‐39653, d‐2‐amino‐5‐phosphonovaleric acid (d‐AP5), and 5,7‐dichlorokynurenic acid (DCKA), whereas GSH was effective in diminishing potencies of CGP‐39653, d‐AP5, DCKA, and 6,7‐dichloroquinoxaline‐2,3‐dione to inhibit [3H]MK‐801 binding when determined in the presence of both l‐Glu and Gly. These results suggest that glutathione may be an endogenous agonist selective for the N‐methyl‐d‐aspartate (NMDA) recognition domain on the NMDA receptor ionophore complex.

Neurochemical aspects of the N-methyl-d-aspartate receptor complex

The N-methyl-D-aspartic acid (NMDA)-sensitive subclass of brain excitatory amino acid receptors is supposed to be a receptor-ionophore complex consisting of at least 3 different major domains including an NMDA recognition site, glycine (Gly) recognition site and ion channel site. Biochemical labeling of the NMDA domain using [3H]L-glutamic acid (Glu) as a radioactive ligand often meets with several critical methodological pitfalls and artifacts that cause a serious misinterpretation of the results. Treatment of brain synaptic membranes with a low concentration of Triton X-100 induces a marked disclosure of [3H]Glu binding sensitive to displacement by NMDA with a concomitant removal of other several membranous constituents with relatively high affinity for the neuroactive amino acid. The NMDA site is also radiolabeled by the competitive antagonist (+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid that reveals possible heterogeneity of the site. The Gly domain is sensitive to D-serine and D-alanine but insensitive to strychnine, and this domain seems to be absolutely required for an opening of the NMDA channels by agonists. The ionophore domain is radiolabeled by a non-competitive type of NMDA antagonist that is only able to bind to the open but not closed channels. The binding of these allosteric antagonists is markedly potentiated by NMDA agonists in a manner sensitive to antagonism by isosteric antagonists in brain synaptic membranes and additionally enhanced by further inclusion of Gly agonists through the Gly domain. Furthermore, physiological and biochemical responses mediated by the NMDA receptor complex are invariably potentiated by several endogenous polyamines, suggesting a novel polyamine site within the complex. At any rate, activation of the NMDA receptor complex results in a marked influx of Ca2+ as well as Na+ ions, which subsequently induces numerous intracellular metabolic alterations that could be associated with neuronal plasticity or excitotoxicity. Therefore, any isosteric and allosteric antagonists would be of great benefit for the therapy and treatment of neurodegenerative disorders with a risk of impairing the acquisition and formation process of memories.

Inhibition of vesicular glutamate storage and exocytotic release by Rose Bengal

It had been thought that quantal size in synaptic transmission is invariable. Evidence has been emerging, however, that quantal size can be varied under certain conditions. We present evidence that alteration in vesicular [3H]l‐glutamate (Glu) content within the synaptosome (a pinched‐off nerve ending preparation) leads to a change in the amount of exocytotically released [3H]Glu. We found that Rose Bengal, a polyhalogenated fluorescein derivative, is a quite potent membrane‐permeant inhibitor (Ki = 19 nm) of glutamate uptake into isolated synaptic vesicles. This vesicular Glu uptake inhibition was achieved largely without affecting H+‐pump ATPase. We show that various degrees of reduction elicited by Rose Bengal in [3H]Glu in synaptic vesicles inside the synaptosome result in a corresponding decrease in the amount of [3H]Glu released in a depolarization‐ (induced by 4‐aminopyridine) and Ca2+‐dependent manner. In contrast, fluorescein, the halogen‐free analog of Rose Bengal, which is devoid of inhibitory activity on vesicular [3H]Glu uptake, failed to change the amount of exocytotically released [3H]Glu. These observations suggest that glutamate synaptic transmission could be altered by pharmacological intervention of glutamate uptake into synaptic vesicles in the nerve terminal, a new mode of synaptic manipulation for glutamate transmission.

Rapid and selective enhancement of DNA binding activity of the transcription factor AP1 by systemic administration of N-methyl-d-aspartate in murine hippocampus

Brain nuclear extracts of ddY mice contained 3 different transcription factors with leucine-zipper domains, including activator protein-1 (AP1), cyclic AMP responsive element binding protein (CREB) and Myc. An intraperitoneal injection of N-methyl-D-aspartate (NMDA) was effective in selectively inducing 6-fold enhancement of DNA binding activity of AP1 in the hippocampus 2 h after the administration. Furthermore, NMDA induced less than 2-fold potentiation of the AP1 binding in the striatum, hypothalamus, medulla-pons and cerebral cortex in a rank order of decreasing magnitude. However, the AP1 binding was not significantly affected by the systemic injection of NMDA in the midbrain and cerebellum. In contrast, NMDA virtually did not alter DNA binding activities of both CREB and Myc in discrete structures of murine brain under similar experimental conditions. These results suggest that the systemic administration of NMDA may induce rapid and selective enhancement of DNA binding activity of AP1 in murine hippocampus.

Rapid potentiation of DNA binding activities of particular transcription factors with leucine-zipper motifs in discrete brain structures of the gerbil with transient forebrain ischemia

Binding of radiolabeled double stranded oligonucleotide probes for nuclear transcription factors with leucine-zipper motifs, such as activator protein-1 (AP1), cyclic AMP response element binding protein (CREB) and Myc, was unevenly distributed in gerbil brain in a manner peculiar to each factor. Among 3 different hippocampal subfields examined, the dentate gyrus had the highest basal DNA binding activities of AP1 with progressively less potent binding in the CA3 and CA1 subfields. Similarly, the dentate gyrus was highest in the basal binding of probes for both CREB and Myc among the 3 distinct hippocampal subregions. However, transient forebrain ischemia for 5 min induced more potent enhancement of the AP1 binding in the CA1 subfield 4 h after the insult than in the CA3 subfield and dentate gyrus. In contrast, the ischemic injury similarly tripled DNA binding activities of CREB without markedly affecting those of Myc in hippocampal CA1 and CA3 subfields. Binding of the probe for AP1 was also markedly potentiated following ischemia in the thalamus, caudate putamen, frontal cortex and cerebellar cortex in a rank order of decreasing magnitude, while the ischemic insult induced slight but statistically significant potentiation of both CREB and Myc binding in the thalamus without affecting that in other discrete brain regions. These results suggest that expression of AP1 may be a determinant of unique vulnerability and/or resistance to an ischemic insult in the gerbil hippocampus.

Positive correlation between prolonged potentiation of binding of double-stranded oligonucleotide probe for the transcription factor AP1 and resistance to transient forebrain ischemia in gerbil hippocampus

Gel retardation electrophoresis revealed that binding of a radiolabelled double-stranded oligonucleotide probe for the nuclear transcription factor activator protein-1 was markedly potentiated in the CA1 and CA3 subfields and the dentate gyrus of the hippocampus of the gerbils with transient forebrain ischemia for 5 min, which is known to induce delayed death of pyramidal neurons exclusively in the CA1 subfield. The potentiation was transient in the vulnerable CA1 subfield, but persistent up to 18 h in the resistant CA3 subfield and dentate gyrus. However, no significant alteration was detected in endogenous levels of cyclic AMP response element binding protein phosphorylated at serine133 in these three different hippocampal structures 3 h after the reperfusion. On the other hand, hypothermia during ischemia which is known to protect the CA1 subfield against ischemic damages, led to a prolonged elevation of the activator protein-1 binding up to 9 h after the reperfusion in this vulnerable subfield at least in part through expression of c-Fos protein. Moreover, activator protein-1 binding was significantly elevated in the CA1 subfield up to 12 h after forebrain ischemia for 2 min which is shown not to induce marked damages to the vulnerable subfield. These results suggest that prolonged elevation of DNA binding activity of activator protein-1 may be responsible for molecular mechanisms underlying the unique vulnerability and/or resistance of particular subfields to a transient ischemic insult in the gerbil hippocampus.

Sustained potentiation of AP1 DNA binding is not always associated with neuronal death following systemic administration of kainic acid in murine hippocampus.

Mice were intraperitoneally injected with kainic acid (KA), followed by dissection of frozen coronal sections and subsequent punching out of the pyramidal and granular cell layers in the hippocampus under a binocular microscope. Systemic administration of KA resulted in marked and sustained potentiation of binding of a radiolabeled double stranded oligonucleotide probe for the nuclear transcription factor activator protein-1 (AP1) in the pyramidal cell layers of the CA1 and CA3 subfields and the granule cell layers of the dentate gyrus 2-18 h later. Morphological evaluation using cresyl violet revealed marked losses of neuronal layers in the pyramidal CA1 and CA3 subfields, but not in the granular dentate gyrus, within 6 weeks after administration. Supershift analysis using antibodies against different Jun and Fos family members differentiated between AP1 DNA binding in hippocampal nuclear extracts obtained 2 and 18 h after the administration of KA. These results suggest that neuronal death may not always follow modulation of de novo synthesis of particular proteins through sustained potentiation of AP1 DNA binding which involves expression of different Jun and Fos family members in response to systemic administration of KA in murine hippocampus.