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The Journal of Neuroscience, December 3, 2003, 23(35):11094-11103
Previous Article | Next Article
Behavioral/Systems/Cognitive
Modulation of the Innate Immune Response by NMDA Receptors Has Neuropathological Consequences
Isaias Glezer,1,2 Hakima Zekki,1 Cristoforo Scavone,2 andSerge Rivest1 1Laboratory of Molecular Endocrinology, Centre Hospitalier Université Laval Research Center, and Department of Anatomy and Physiology, Laval University, Québec, Canada G1V 4G2, and 2Department of Pharmacology, Institute of Biomedical Science, University of São Paulo, 05508-900 São Paulo, Brazil
Abstract
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Abstract
Introduction
The aim of this study was to determine whether glutamate receptors modulate the innate immune response in the brain of C3H/HeN and C3H/HeJ mice; the latter bear a loss of function in the toll-like receptor (TLR) 4 gene. Mice received an intrastriatal (IS) infusion of lipopolysaccharide (LPS), the exogenous ligand for TLR4, and were killed at several times thereafter. This treatment activated the transcription of a wide variety of genes involved in the control of the innate immune response. MK-801, an antagonist of NMDA glutamate receptor subtype, exacerbated the effects of the endotoxin in the brain of C3H/HeN mice but not in TLR4-deficient animals. The ipsilateral side of C3H/HeN mice exhibited stronger hybridization signals for the mRNA encoding TLR2, CD14, tumor necrosis factor-, and inhibitory factor-
B
Key words: innate immune response; in situ hybridization histochemistry; inflammation; lipopolysaccharide; proinflammatory cytokines; microglia; macrophages; glutamate; demyelination
Introduction
Mammalian toll-like receptors (TLRs) are receptors involved in the recognition of pathogen-associated molecular patterns (PAMPs) and also molecules responsible for mounting appropriate responses against microorganisms (Anderson, 2000). Lipopolysaccharide (LPS) is a well characterized inducer of innate immune response recognized by monocytes/macrophages through its binding to membrane CD14 receptors, which transfer LPS to TLR4 via myeloid differentiation protein 2 (Akira et al., 2001
Despite the beneficial role of microglial activation in immune-mediated host defense, microglial-derived proinflammatory molecules have been associated with neurodegenerative disorders. Activated microglia and astrocytes are found in the brain of Alzheimer's, Parkinson's, and Huntington's disease patients as well as individuals suffering of multiple sclerosis (MS) and amyotrophic lateral sclerosis (Pasinetti, 1998
, and tumor necrosis factor
Glutamate is the major excitatory neurotransmitter in the CNS, and the NMDA subtype of glutamate ionotropic receptors has been implicated in neurodegenerative diseases (Beal, 1995
The aim of this study was, therefore, to determine whether the NMDAR antagonist MK-801 has the ability to alter the inflammatory response in the brain of C3H/HeN and C3H/HeJ mice in response to an intracerebral bolus of the endotoxin LPS. We also investigated the potential consequences of such response in the cerebral tissue via different approaches.
Materials and Methods
Animals
Adult male C3H/HeN mice (body weight, 25-29 gm; Charles River Canada, St. Constant, Québec, Canada), C3H/HeJ mice (Jax Mice; Jackson Laboratory, Bar Harbor, ME), or Sprague Dawley rats (body weight,250 gm; Charles River Canada) were acclimated to standard laboratory conditions (14/10 hr light/dark cycle; lights on at 6:00 A.M. and off at 8:00 P.M.) with ad libitum access to rodent chow and water. Animal breeding and experiments were conducted according to Canadian Council on Animal Care guidelines, as administered by the Laval University Animal Care Committee.
Experimental protocols
Acute intraparenchymal injections. Mice receiving intrastriatal (IS) injections were anesthetized with an intraperitoneal injection of avertin (2,2,2 tribromoethanol; Sigma-Aldrich, St. Louis, MO) and placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA). The right caudate putamen was reached using a small cannula (33 gauge) at the coordinates 0.0 mm anteroposterior, -2.0 mm lateral, and -3.0 mm dorsoventral according to a mouse brain atlas (Paxinos and Franklin, 2001At different time points, after intraparenchymal injections (6, 24, and 72 hr and 2 weeks), animals were deeply anesthetized via an intraperitoneal injection of a mixture of ketamine hydrochloride and xylazine and then rapidly perfused transcardially with 0.9% saline, followed by 4% paraformaldehyde in 0.1 M borax buffer, pH 9.5, at 4°C. Brains were removed rapidly from the skulls, postfixed for 2-4 d, and then placed in a solution containing 10% sucrose diluted in 4% paraformaldehyde-borax buffer overnight at 4°C. The frozen brains were mounted on a microtome (Reichert-Jung; Cambridge Instruments Company, Deerfield, IL) and cut into 20 µm coronal sections from the olfactory bulb to the end of the medulla. The slices were collected in cold cryoprotectant solution (0.05 M sodium phosphate buffer, pH 7.3, 30% ethylene glycol, and 20% glycerol) and stored at -20°C.
Long-term intraparenchymal infusions. A chronic indwelling cannula was implanted as described previously (Nadeau and Rivest, 2003
cRNA probes and in situ hybridization
Plasmids were linearized, and the sense and antisense riboprobes were synthesized as described in Table 1. Radioactive cRNA copies were synthesized by incubation of 250 ng of linearized plasmid in 6 mM MgCl2, 40 mM Tris, pH 7.9, 2 mM spermidine, 10 mM NaCl, 10 mM DTT, 0.2 mM ATP/GTP/CTP, 100 µCi of
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Table 1. Plasmids and enzymes used for the synthesis of the cRNA probes
Hybridization histochemical localization of TLR2, inhibitory factor, and NMDAR1 mRNA was performed on every 12th section of the entire rostrocaudal extent of each brain, as described previously (Laflamme et al., 1999
Detection of neuronal cell death, demyelination, and histological analysis
The presence of neuronal cell death was investigated with the Fluoro-Jade B (FJB) method. Briefly, every 12th section of the whole rostrocaudal extent of each brain was mounted onto poly-L-lysine-coated slides, dried under vacuum for 2 hr, dehydrated through graded concentrations of alcohol (50, 70, and 100%; 1 min), rehydrated through graded concentrations of alcohol (100, 70, and 50%; 1 min), and rinsed for 1 min in distilled water. Then, the sections were dipped and shacked in potassium permanganate solution (0.06%) for 10 min and rinsed for 1 min in distilled water. Slides were next dipped and shacked into a solution containing a mixture of 0.0004% FJB (Histochem, Jefferson, AR) plus 0.1% acetic acid (Sigma-Aldrich) plus 0.0002% 4',6'-diamidino-2-phenylindole (Molecular Probes, Eugene, OR) for 20 min. The slides were thereafter rinsed three times in distilled water (1 min each), dried, dipped in xylene three times (2 min each), and coverslipped with DPX.Demyelination was determined via Luxol Fast Blue (LFB) staining. The brain sections mounted onto poly-L-lysine-coated slides were dehydrated through graded concentrations of alcohol (50, 70, and 95%; 1 min each) and incubated at 60°C for 6 hr in LFB solution [1% Solvent Blue 38 (Sigma) in 95% ethanol and 0.5% acetic acid]. The sections were then rinsed in 95% ethanol (1 min), 0.05% lithium carbonate (1-5 min; Sigma), and 70% alcohol (two dips). Thereafter, the slides were stained in 1% eosine Y solution (EM Diagnostic System, Gibbstown, NJ) for 30 sec, rinsed in water, incubated in 0.25% cresyl violet (Sigma) for 30 sec, rinsed in water, dehydrated through graded concentrations of alcohol (50, 70, 95, and 100%; 1 min each), cleared in xylene for 1 min (two times), and coverslipped with DPX.
Nissl stain was used as a general index of cellular morphology that may be altered in response to the different treatments.
Quantitative analysis
Quantitative analyses were performed as described previously (Nadeau and Rivest, 2003The dorsal basal ganglia and hippocampus were selected for the semiquantification of NMDAR1 mRNA. These structures exhibited high constitutive NMDAR1 expression levels and strong transcriptional activation of immune-related genes in response to intracerebral LPS/MK-801 administration. Measurements were performed in 10 C3H/HeN and 10 C3H/HeJ mice, and data are reported as mean OD values (±SEM).
Demyelination was evaluated on digitalized LFB-stained sections within the corpus callosum and caudoputamen as described previously (Nadeau and Rivest, 2003
The statistical analysis was performed by a one- or two-way ANOVA, followed by a Bonferroni/Dunn test procedure as post hoc comparisons or Student's t test for NMDAR1 subunit mRNA levels.
Results
LPS-induced expression of TLR2 and CD14 is modulated by MK-801 in a TLR4-dependent manner
Figure 1 depicts representative hybridization signals in the brain of mice treated with saline, LPS, LPS/MK-801, or MK-801 alone. As expected, implantation of the cannula and the stress of infusion caused a low expression of TLR2 mRNA. Such a localized pattern of positive TLR2-expressing cells was found in the brain of both saline- and MK-801-infused mice (Fig. 1). This low signal contrasts with the intense and widespread transcriptional activation of TLR2 ipsilateral to the side of LPS infusion (Fig. 1Ab, LPS). MK-801 exacerbated the effects of endotoxin injected directly within the basal ganglia. At 24 hr, the signal was more intense in the brain of mice treated with the NMDAR antagonist and endotoxin (Fig. 1Ac). Semiquantitative analyses performed on x-ray films revealed a significant difference between the LPS- and LPS/MK-801-treated groups (Fig. 1C).
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Figure 1. TLR2 mRNA expression in the brain of C3H/HeN and C3H/HeJ mice after an intraparenchymal bolus of saline, LPS, or the antagonist of the NMDAR MK-801. A, Representative hybridization signals in emulsion-dipped coronal sections (20 µm) taken from C3H/HeN mice that received only saline solution (a), LPS (b; 0.5 µg), a mixture containing LPS (0.5 µg) and MK-801 (c; 1 µg, LPS/MK-801), or MK-801 (d; 1 µg) in the dorsal basal ganglia. B, Emulsion-dipped coronal sections of C3H/HeJ mice, which bear a loss of function in the TLR4, killed 24 hr after a single bolus of saline solution (a), LPS (b), LPS combined with MK-801 (c), and MK-801 (d). C, Semiquantitative analysis of TLR2 mRNA levels (OD) in the ipsilateral side of C3H/HeN and C3H/HeJ mice 24 hr after the intracerebral insults. Please note the robust hybridization signal across the ipsilateral side of mice that received LPS combined with the antagonist of the NMDARs. Results represent means ± SEM of three to four mice per group. Statistical analysis was performed by using a two-way ANOVA, followed by a Bonferroni's multiple comparison test. **Significantly different (p < 0.01) from saline-injected group; ***significantly different (p < 0.001) from saline-injected group; #significantly different (p < 0.05). For more details, see Materials and Methods. CPu, Caudate putamen; LV, lateral ventricle. Scale bar, 1250 µm.
This amplification by MK-801 was essentially abolished in C3H/HeJ mice, because these mice were much less sensitive to either LPS alone or combined with the NMDAR antagonist (Fig. 1B). It is interesting to note that despite the lack of functional TLR4, a single intracerebral bolus of LPS was able to increase TLR2 expression in regions ipsilateral to the injection site. However, the signal was, to a great extent, lower in C3H/HeJ mice than in their wild-type controls, and MK-801 remained without significant effect in TLR4-mutant mice (Fig. 1B,C). These data indicate that the ability of glutamate and NMDARs to modulate the innate immune response in the brain is dependent on the prior binding of LPS to its receptor TLR4.The same phenomenon took place for the gene encoding CD14 (Fig. 2). The endotoxin caused a profound increase in the transcriptional activation of the gene encoding LPS receptor in the ipsilateral side of mice killed 6 hr after the single intraparenchymal infusion. The cells lining the leptomeninges and few blood vessels exhibited a strong hybridization signal for CD14 transcript 6 hr after the cerebral injection with LPS alone or combined with MK-801 (Fig. 2b,c). As for TLR2, the expression wave across the brain parenchyma increased in intensity 24 hr after the treatment combining LPS with the antagonist of the NMDAR (Fig. 2k). This effect of MK-801 persisted up to 72 hr after injection, but the differences in the hybridization signal were not as marked as those found at 24 hr (Fig. 2j,k,r s). Once again, C3H/HeJ mice were relatively resistant to the treatments, but the endotoxin was capable of causing an increase in CD14 gene expression in a localized area ipsilateral to the injection site at 6 hr (Fig. 2f,g). The signal largely decreased in the brain of these mice 24 hr after the intracerebral administration of the endotoxin that was combined or not with MK-801 (Fig. 2n,o). Seventy-two hours after the different treatments, very few positive CD14-expressing cells were found in the CNS of CH3/HeJ mice (Fig. 2v,w).
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Figure 2. Time-related expression of CD14 mRNA in the brain of C3H/HeN and C3H/HeJ mice after a single intraparenchymal bolus of LPS and MK-801. These dark-field photomicrographs were taken from coronal sections hybridized with radioactive CD14 cRNA probe and dipped into nuclear emulsion milk. These images are representative examples of the expression pattern of CD14 transcript 6 hr (a-h), 24 hr (i-p), and 72 hr (q-x) after the infusion with saline, LPS, LPS plus MK-801, or MK-801 alone. Please note the robust signal in the area ipsilateral to the injection site of C3H/HeN mice challenged with LPS and MK-801. Although this effect was impaired in HeJ mice, the endotoxin was still capable of causing a significant increase in CD14 transcription in the brain of this strain (f, g). bv, Blood vessels; cc, corpus callosum; CPu, caudate putamen; Cx, cortex. Scale bar, 1000 µm.
TLR4-dependent induction of proinflammatory signaling and gene transcription
The next series of assays evaluated whether expression of receptors involved in the innate immune response is associated with an increase in NF-
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Figure 3. Expression wave of the proinflammatory cytokine TNF-
De novo induction of I
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Figure 4. TLR4-dependent effects of MK-801 on LPS-induced I
Unlike the other transcripts, no positive signal was found for the gene encoding iNOS in the brain of C3H/HeJ mice in response to the endotoxin injected either alone or together with MK-801 (Fig. 4B). In C3H/HeN mice, the message significantly increased over the background level 6 hr after the intracerebral infusion of LPS alone or combined with MK-801 (Fig. 4Bj,k). At 24 hr, numerous clusters of iNOS-expressing cells were detected in the region infused with endotoxin, and the antagonist of the NMDARs failed to modulate the pattern of iNOS expression that was nonuniform and restricted to isolated clusters of positive cells (data not shown). The signal returned to background levels 72 hr after the IS infusion with the bacterial cell wall component combined or not with MK-801 (data not shown).NMDAR1 subunit expression in the brain of C3H/HeN and C3H/HeJ mice
To verify whether the NMDA antagonist failed to modulate LPS-induced immune response in C3H/HeJ mice because of a major difference in receptor expression, NMDAR1 subunit mRNA was measured via in situ hybridization histochemistry. As shown in Figure 5, the pattern and intensity of NMDAR1 gene expression were similar in the brain of both mouse strains. The distribution of the gene encoding this critical NMDAR was also similar to that already described in other studies (Laurie and Seeburg, 1994
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Figure 5. Expression of NMDAR1 subunit mRNA in the brain of C3H/HeN and C3H/HeJ mice. A, Representative signals in coronal sections hybridized with sense or antisense riboprobe and exposed to x-ray films (Biomax; Kodak). B, Dark-field photomicrographs taken from coronal sections hybridized with radioactive NMDAR1 cRNA probe and dipped into nuclear emulsion milk. Intensity and pattern of the hybridization signal were similar across the brains of C3H/HeN and C3H/HeJ mice. C, Semiquantitative analysis of relative NMDAR1 mRNA levels (OD) in the caudate putamen (CPu) and hippocampus (Hip) of C3H/HeN and C3H/HeJ mice. Scale bar, 1000 µm.
Integrity of the neuronal elements
The consequences of altering the inflammatory response by MK-801 and TLR4 on the cerebral tissue were determined via two different approaches. FJB is a polyanionic fluorescein derivative that has been shown to be a sensitive and reliable marker for histochemical localization of neuronal degeneration (Schmued et al., 1997
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Figure 6. Neuronal integrity in the brain of C3H/HeN and C3H/HeJ mice after intrastriatal infusion of LPS and the antagonist of NMDARs. A, The fluorochrome FJB was used as a marker for histochemical localization of degenerative neurons. Degenerating neurons (positive control) were found in hippocampus of a rat killed 6 d after being adrenalectomized (a, inset). Such positive neurons were never observed in regions depicting the robust expression of the different transcripts, except along the needle track because of the mechanical injury (white arrowheads). Small non-neuronal cells (probably infiltrating cells) were also found in the CNS of C3H/HeN mice treated with LPS alone or combined with MK-801 (white arrows). B, Nissl-stained sections at the level of dorsal basal ganglia. Neuronal density and morphology were similar among all the groups included in this study. Black arrows point to the cannula track. Cx, Cortex; cc, corpus callosum; contra, contralateral side; CPu, caudate putamen; ipsi, ipsilateral side; ct, cannula track. Scale bars, 100 µm; insets, 50 µm.
Nissl stain was also used to evaluate the morphology and histological integrity of the cerebral tissues. Figure 6B shows examples of brain sections taken from saline-, LPS-, or LPS plus MK-801-infused mice, and no convincing changes were observed 72 hr after the different treatments (Fig. 6Ba-c). The sections remained intact, and ipsilateral sides were similar to the contralateral areas at 2 weeks after LPS infusion in both mouse strains (Fig. 6Bd-f).Genes involved in the transfer from the innate to adaptive immunity
The gene encoding TLR2 was still expressed in the brains of C3H/HeN mice 2 weeks after the single bolus of LPS (Fig. 7a) but not in those of C3H/HeJ mice (Fig. 7b). Few positive and small scattered I
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Figure 7. The innate immune response is not associated with a transfer to the adaptive immunity. Representative examples of nuclear emulsion-dipped coronal sections depicting expression of genes involved in the transfer from the innate to adaptive immune response 2 weeks after LPS injection in the dorsal striatum. TLR2 mRNA was still expressed in the ipsilateral side of C3H/HeN mice 14 d after a single bolus of LPS (a), whereas such signal was not present in the infused regions of C3H/HeJ mice (b). Few I
Brain damage caused by a long-term infusion
To verify the relevance of innate immune response modulated by glutamate to brain physiopathology, an experimental protocol was designed to expose the cerebral tissue to long-term stimulation. Figure 8 depicts LFB-stained sections from animals treated with saline, LPS, LPS combined with MK-801, or MK-801 alone during 72 hr. Animals that received combined treatment of LPS plus MK-801 exhibited a striking degree of demyelination compared with the other groups, which was confirmed by semiquantitative analysis of differential OD with the contralateral side (Fig. 8A,B). LPS infusion for a period of 3 d was also able to cause demyelination, but differential OD failed to reach statistical significance because of the high degree of variability among animals of this group (Fig. 8A,B). It is possible that variable diffusions throughout the brain parenchyma contribute to different degrees of demyelination between animals and dorsoventral-rostrocaudal levels. However, demyelination was clear in all animals that received the double treatment, which was not the case in the cerebral tissue of rats that were infused only with the endotoxin. Demyelination was apparently not associated with neuronal cell death, because no positive FJB neurons were found in the brain of chronically treated rats. This pattern of demyelination was never observed in the brain of C3H/HeN or C3H/HeJ mice that received an acute bolus with the different solutions (data not shown).
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Figure 8. Demyelination in response to long-term infusion of the endotoxin LPS and NMDAR antagonist MK-801 in ipsilateral sides of adult male SD rats. A, Luxol Fast Blue-stained coronal sections from animals infused with saline, LPS (0.5 µg/d), LPS combined with MK-801 (1 µg/d), or MK-801 alone and killed 72 hr after implantation of mini-osmotic pumps. B, Differential OD measured in several rostrocaudal sections of each rat included in this experiment. Statistical analysis was performed by using a one-way ANOVA, followed by a Bonferroni's multiple comparison test. *Significantly different (p < 0.01) from the saline-injected group. For more details on image analysis, see Materials and Methods. Black arrowheads indicate the tip of the cannula. Scale bar, 1250 µm.
Discussion
Innate immune response is crucial for protecting the brain and maintaining its integrity against invading microorganisms. Characterization of TLRs highlighted the existence of receptors for recognizing specific PAMPs and mounting an organized response to eliminate pathogens. TLR4 is believed to be an essential receptor for proper response to LPS but not other PAMPs (Anderson, 2000Although C3H/HeJ mice were relatively insensitive to LPS, the endotoxin was still capable of triggering gene expression in the CNS of these mice. However, strong differences were observed in the time of induction and between the different transcripts. For example, TNF-
In contrast to most genes analyzed here, in situ hybridization failed to detect any positive signal for iNOS in the cerebral tissue of C3H/HeJ mice challenged with the endotoxin combined with vehicle or MK-801. The gene encoding iNOS has been identified recently as one of a class of genes dependent of IFN-I
In this regard, innate immune response in the CNS is the result of a coordinated expression of cytokines and other inflammatory mediators in response to infection and insults. It remains, however, difficult to analyze the exact consequences of this response during pathological conditions. There is now compelling evidence of a bilateral talk between glia and neurons, and molecules involved in such interaction may play key roles in neurodegenerative processes. Glutamate is a putative mediator, because it is a major excitatory neurotransmitter and its metabolism and synaptic concentrations are regulated by astrocytes. Various glutamate receptor subtypes are expressed in astrocytes, oligodendrocytes, as well as in microglia (Noda et al., 2000
The coadministration of LPS and noncompetitive antagonist MK-801 increased the expression levels of TLR2 mRNA and other inflammatory genes, which supports the concept that glutamatergic pathways can modulate immune reaction in the brain. It is interesting to note that MK-801 did not exacerbate the effects of LPS on all the genes assayed here, and the role of glutamate cannot be generalized to all the transcriptional processes that take place in response to an intracerebral bolus of LPS. However, the genes that were significantly modulated by the antagonist of the NMDARs were clearly dependent on the previous binding of LPS to its receptor TLR4. Although the impaired response to the endotoxin in C3H/HeJ mice may explain the failure of MK-801 to modulate the effects of LPS, inflammation is still induced in the brain of these animals. The ability of MK-801 to exacerbate the immune response to LPS should, therefore, be detectable with such mild microglial reactivity. However, this was not the case, and the interaction between the endotoxin and NMDARs was observed only in C3H/HeN mice, which indicates that TLR4 plays a critical role in this process. It is also possible that MK-801 acts only in presence of a robust immune reaction, such as in LPS-treated C3H/HeN mice. This led us to propose that a full response to LPS, which is TLR4 dependent, could be essential for the ability of NMDARs to modulate proinflammatory signaling and gene transcription.
MK-801 has been found recently to be toxic for microglial cells in culture, which underscores the existence of NMDARs in these cells (Hirayama and Kuriyama, 2001
Despite the robust inflammatory response and changes in the signal intensity by MK-801, no signs of neurodegeneration were observed via FJB and Nissl stain techniques. This was also the case in the brain of C3H/HeJ mice that exhibited impairment of the immune reaction after IS infusions with the different solutions. Although TLR2 mRNA signal was still positive 2 weeks after LPS infusion in C3H/HeN mice, the brain of these animals failed to show any signs of altered neuronal morphology and neurodegeneration. This is further supported by the negative signal for the cytokine IFN-
In disagreement with this study and others (Szczepanik et al., 1996
In conclusion, TLR4 is crucial for MK-801 to modulate the innate immune reaction in response to intraparenchymal LPS bolus. The robust and transient inflammatory wave induced by the bacterial cell wall component and NMDAR antagonist was not associated with cell death in the CNS. In contrast, long-term exposure to the same treatment provokes demyelination. There is, therefore, a fine line between beneficial and detrimental properties of immune-related compounds in the brain, and NMDARs may play a determinant role in this equilibrium.
Footnotes
Received Aug 29, 2003; revised October 1, 2003; accepted October 7, 2003.This work was supported by the Canadian Institutes of Health Research [CIHR; the former Medical Research Council of Canada (MRCC)]. S.R. is an MRCC Scientist and holds a Canadian Research Chair in Neuroimmunology. I.G. is supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (200842/01-3), Fundação de Amparo à Pesquisa do Estado de São Paulo (00/10679-0), and CIHR. H.Z. is supported by a studentship from the K. M. Hunter/CIHR, and C.S. is a research fellow of CNPq. We thank Dr. A. Israel (Institut Pasteur, Paris, France) for the mouse I
Correspondence should be addressed to Dr. Serge Rivest, Laboratory of Molecular Endocrinology, Centre Hospitalier Université Laval Research Center, and Department of Anatomy and Physiology, Laval University, 2705 Boulevard Laurier, Québec, Canada G1V 4G2. E-mail: Serge.Rivest{at}crchul.ulaval.ca.
Copyright © 2003 Society for Neuroscience 0270-6474/03/2311094-10$15.00/0
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