Michel Baudry

A glycine site associated with N-methyl-D-aspartic acid receptors: characterization and identification of a new class of antagonists.

Abstract Membranes from rat telencephalon contain a single class of strychnine‐insensitive glycine sites. That these sites are associated with N‐methyl‐d‐aspartic acid (NMDA) receptors is indicated by the observations that [3H]glycine binding is selectively modulated by NMDA receptor ligands and, conversely, that several amino acids interacting with the glycine sites increase [3H]N‐[1‐(2‐thienyl)cyclohexyl]piper‐idine ([3H]TCP) binding to the phencyclidine site of the NMDA receptor. The endogenous compound kynurenate and several related quinoline and quinoxaline derivatives inhibit glycine binding with affinities that are much higher than their affinities for glutamate binding sites. In contrast to glycine, kynurenate‐type compounds inhibit [3H]TCP binding and thus are suggested to form a novel class of antagonists of the NMDA receptor acting through the glycine site. These results suggest the existence of a dual and opposite modulation of NMDA receptors by endogenous ligands.

Development of glutamate binding sites and their regulation by calcium in rat hippocampus

The postnasal development of the Na-independent [3H]glutamate binding sites, which exhibit some characteristics of postsynaptic glutamate receptors, has been studied in rat hippocampal membranes. The amount of binding sites (expressed in pmol/hippocampus) represents 4% of the adult level at postnatal day (PND) 4, increases very rapidly until PND 9, and then increases at a slower rate reaching 80% of the adult value at PND 23. In contrast, the density of binding sites (expressed in pmol/mg protein) exhibits a maximum at PND 9 and slowly decreases to reach the adult value at PND 23. These changes seen to be only quantitative since the affinity (about 450nM) and Hill coefficient (about 1.0) of these binding sites remain constant throughout development. Calcium ions have been shown to markedly stimulate [3H]glutamate binding in adult hippocampal membranes. This effect appears on PND 9--10 and increases rapidly until PND 16 when it is similar to that seen in the adult rat. We also determined the minimum age at which long-term potentiation (LTP) of synaptic transmission could be detected in the CA1 field of hippocampal slice preparations following repetitive electrical stimulation of the Schaffer-commissural pathways. LTP was only rarely detected at PND 8 whereas it could be reliably obtained after PND 9. These results indicate that the postnatal development of Na-independent glutamate binding sites closely parallels synapse formation in the hippocampus, further supporting the idea that the binding sites are associated with a physiological receptor. They also show that the appearance of the stimulatory effect of calcium on glutamate binding occurs at a time when several forms of synaptic plasticity appear in the hippocampus. In particular the correlation of the development of LTP with the calcium-stimulation of glutamate binding suggests that these phenomena have similar cellular mechanisms.

Postnatal development of inflammation in a murine model of Niemann-Pick type C disease: immunohistochemical observations of microglia and astroglia.

Niemann-Pick type C (NPC) is a rare and fatal neurovisceral storage disorder that is currently untreatable. In most cases, NPC is caused by mutations of the NPC1 gene, which encodes a glycoprotein playing an important role in cholesterol transport. Mice lacking the NPC1 gene exhibit several pathological features of NPC patients and have been widely used to provide insights into the mechanisms of the disease. In the present study, we analyzed the postnatal development of pathological manifestations of inflammation in several brain regions of NPC1-/- mice. Brain sections from NPC1-/- and wild-type (NPC1+/+) mice were immunostained with the MAC1 antibody, which recognizes microglia, with antibodies against glial fibrillary acidic protein (GFAP), which recognize astrocytes, and with antibodies against the cytokine interleukin-1beta (IL-1beta). Numbers of MAC1 immunopositive cells were markedly increased in several brain regions of NPC1-/- mice as early as 2 weeks of age. This effect was particularly evident in globus pallidus, ventral lateral thalamus, medial geniculate nucleus, and cerebellum. MAC1-immunopositive cells had enlarged cell bodies and shorter processes, suggesting they were in an active state. By 4 weeks, most brain structures exhibited enhanced microglial activation in NPC1-/- mice, and this was maintained at 12 weeks. At 2 weeks, reactive astrocytes were only observed in the ventral lateral thalamus while they were present throughout the brain of NPC1-/- mice at 4 weeks of age. Moreover, the astroglial reaction coincided with up-regulation of the cytokine, interleukin-1beta, in most, but not all brain regions. In particular, no interleukin-1beta up-regulation was observed in regions devoid of neuronal degeneration. These results suggest that microglial activation precedes and might be causally related to neuronal degeneration, while astrocyte activation might be a consequence of neuronal degeneration.

Brain spectrin, calpain and long-term changes in synaptic efficacy

This chapter discusses the possibility that proteolytic digestion of cytoskeletal proteins, in particular spectrin, is part of the mechanisms through which physiological activity elicits structural and chemical changes in brain synapses. Recent work from several laboratories has produced a description of the initial events that trigger the long-term potentiation (LTP) of synaptic responses that appears in hippocampus after brief episodes of high frequency electrical stimulation. A likely sequence is as follows: suppression of IPSPs, prolongation of EPSPs, activation of N-methyl-D-aspartate (NMDA) receptors, influx of calcium into target cells. After briefly describing the evidence for this triggering sequence, the review takes up the question of what types of calcium sensitive chemistries are available to synaptic region that could produce functional changes lasting for weeks (i.e., for LTP). It is argued that the partial degradation of spectrin by a calcium-activated protease (calpain) provides a mechanism of this type. Spectrin is a substrate for calpain and both it and a breakdown product comparable to that produced by calpain are found in postsynaptic densities. Moreover, there is substantial evidence that spectrin regulates the surface chemistry and morphology of cells and thus its partial degradation would be expected to produce pronounced and persistent modifications in synapses. To reinforce this point, the review discusses recent findings suggesting that calpain mediated proteolysis of spectrin and other cytoskeletal proteins produces substantial changes in the shape of blood-borne cells and the distribution of their surface receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of two [3H]glutamate binding sites in rat hippocampal membranes.

Abstract: The specific binding of l‐[3H]glutamate was investigated in the presence and the absence of sodium ions in freshly prepared membranes from rat hippocampus. Sodium ions were found to have a biphasic effect; low concentrations induced a marked inhibition of the binding (in the range 0.5‐5.0 mM), whereas higher concentrations resulted in a dose‐dependent stimulation of binding (in the range 10‐ 150 mM). These results permit the discrimination of two binding sites in hippocampal membranes. Both Na+‐independent and Na+‐dependent binding sites were saturable, exhibiting dissociation constants at 30°C of 750 nM and 2.4 μM, respectively, with Hill coefficients not significantly different from unity, and maximal number of sites of 6.5 and 75 pmol/mg protein, respectively. [3H]Glutamate binding to both sites reached equilibrium between 5 and 10 min and was reversible. The relative potencies of a wide range of compounds, with known pharmacological activities, to inhibit [3H]glutamate binding were very different for the Na+‐independent and Na+‐ dependent binding and suggested that the former sites were related to postsynaptic glutamate receptors, whereas the latter were related to high‐affinity uptake sites. This conclusion was also supported by the considerable variation in the regional distribution of the Na+‐dependent binding site, which paralleled that of the high‐affinity glutamate uptake; the Na+‐independent binding exhibited less regional variation.

Stimulation of NMDA receptors induces proteolysis of spectrin in hippocampus

Stimulation of N-methyl-D-aspartate (NMDA) receptors was found to induce proteolysis of brain spectrin in hippocampal slices. The effect was dependent upon extracellular calcium, blocked by the antagonist 2-amino-5-phosphonovalerate (AP5), and was not reproduced by potassium-induced depolarization. These results are consistent with the hypothesis that the involvement of NMDA receptors in plasticity and excitotoxicity is at least partially mediated by calcium-activated proteolysis of cytoskeletal proteins.

Quinoxaline derivatives are high-affinity antagonists of the NMDA receptor-associated glycine sites

Membranes from rat telencephalon contain strychnine-insensitive glycine binding sites associated with NMDA receptors. Three quinoxaline derivatives, among them the high-affinity AMPA receptor antagonists CNQX and DNQX, were found to inhibit [3H]glycine binding to these sites with micromolar affinities. Binding of these compounds to the glycine site also inhibited glutamate-stimulated association and dissociation of [3H]TCP. This suggests that these AMPA antagonists, like the structurally related compound kynurenate, act as glycine site antagonists.

Chronic administration of a thiol-proteinase inhibitor blocks long-term potentiation of synaptic responses

It has been proposed that activation of a calcium-sensitive protease (calpain) is a crucial step in the induction of long-term potentiation (LTP). To test this hypothesis, we used chronic recording techniques to measure the effects of intraventricular infusion of leupeptin, a calpain inhibitor, on LTP in the hippocampus. Rats implanted bilaterally with stimulating electrodes in the Schaffer-commissural system and one recording electrode in the apical dendrites of field CA1 were fitted with osmotic mini-pumps delivering either leupeptin (20 mg/ml) or saline at a rate of 0.5 microliter/h into the lateral ventricle. Short bursts of high-frequency stimulation with the bursts delivered at 5/s were used to induce LTP in those animals which had stable responses for several days. Rats in the saline group (n = 11) exhibited an immediate LTP effect that remained in place over successive days of testing, while only 3 of 13 leupeptin treated animals showed evidence of LTP 24 h after high-frequency stimulation, and in only one of those was a sizeable effect recorded over several days. The average change in responses at the 24-h test point was +33% for the controls and +4% for the leupeptin group (P less than 0.01). The block of LTP induction was reversible, since high-frequency stimulation applied after disconnecting the pumps led to a robust LTP effect that lasted for several days in 6 of 7 animals tested. There were no detectable differences in baseline responses in the presence and absence of leupeptin.

The protease inhibitor leupeptin interferes with the development of LTP in hippocampal slices

The effect of leupeptin, an inhibitor of thiol-proteases, was tested on the induction of long-term potentiation (LTP) in field CA1 of hippocampal slices. Two h of drug application did not produce substantial changes while a greater than 3-h application caused a sizeable reduction in the degree of LTP induced. Leupeptin had no obvious effects on the facilitation of postsynaptic responses occurring within or between the short high frequency bursts used to induce LTP, suggesting that the drug acted on cellular chemistries occurring after the initial physiological events that normally trigger LTP. These results are consistent with the hypothesis that a calcium-activated thiol protease (calpain) is involved in the induction of LTP.

Calcium dependent aspects of synaptic plasticity, excitatory amino acid neurotransmission, brain aging and schizophrenia: A unifying hypothesis

(1) The functional and structural reorganization of dendritic spines by calcium activated proteases is postulated to play a causal role in the production of the phenomenology of brain aging and in particular in the development of pathology and degeneration. Excitatory neurotransmission appears to be essential for the development of irreversible synaptic changes. (2) One of the genes modified in schizophrenia is postulated to be directly or indirectly linked to the control of excitatory neurotransmission; possibly the normal switching on of the expression of the adult form of the NMDA receptor is altered, resulting in an inappropriate functioning of this receptor. This genetic characteristic might explain the apparent resistance of schizophrenic brains to aging.