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Reversal of age-related oxidative stress prevents hippocampal synaptic plasticity deficits by protecting d-serine-dependent NMDA receptor activation

Authors

  • Coline Haxaire,

    1. Centre de Psychiatrie et Neurosciences, Université Paris Descartes, Sorbonne Paris Cité, UMR 894, Paris, 75014, France
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  • Fabrice R Turpin,

    1. Centre de Psychiatrie et Neurosciences, Université Paris Descartes, Sorbonne Paris Cité, UMR 894, Paris, 75014, France
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    • Present address: Synaptic plasticity laboratory, Queensland Brain Institute, Saint Lucia, Australia

  • Brigitte Potier,

    1. Centre de Psychiatrie et Neurosciences, Université Paris Descartes, Sorbonne Paris Cité, UMR 894, Paris, 75014, France
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  • Myriam Kervern,

    1. Centre de Psychiatrie et Neurosciences, Université Paris Descartes, Sorbonne Paris Cité, UMR 894, Paris, 75014, France
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  • Pierre-Marie Sinet,

    1. Centre de Psychiatrie et Neurosciences, Université Paris Descartes, Sorbonne Paris Cité, UMR 894, Paris, 75014, France
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  • Gérard Barbanel,

    1. Institut des Biomolecules Max Mousseron, UMR 5247 CNRS-UM1-UM2, Université Montpellier II, Montpellier, 34095, France
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  • Jean-Pierre Mothet,

    1. Equipe ‘gliotransmission et synaptopathies’, Centre de Recherche en Neurobiologie et Neurophysiologie de Marseille, UMR6231-CNRS-Universite Aix-Marseille, Faculté de Médecine secteur Nord 51, Marseille, 13344, France
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  • Patrick Dutar,

    1. Centre de Psychiatrie et Neurosciences, Université Paris Descartes, Sorbonne Paris Cité, UMR 894, Paris, 75014, France
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  • Jean-Marie Billard

    1. Centre de Psychiatrie et Neurosciences, Université Paris Descartes, Sorbonne Paris Cité, UMR 894, Paris, 75014, France
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Jean-Marie Billard, Centre de Psychiatrie et Neurosciences, Université Paris Descartes, Sorbonne Paris Cité, UMR 894, 2 ter rue d’Alésia, Paris, 75014, France. Tel.: + 33 1 40 78 86 47; fax +33 1 45 80 72 93; e-mail: jean-marie.billard@inserm.fr

Summary

Oxidative stress (OS) resulting from an imbalance between antioxidant defenses and the intracellular accumulation of reactive oxygen species (ROS) contributes to age-related memory deficits. While impaired synaptic plasticity in neuronal networks is thought to underlie cognitive deficits during aging, whether this process is targeted by OS and what the mechanisms involved are still remain open questions. In this study, we investigated the age-related effects of the reducing agent N-acetyl-L-cysteine (L-NAC) on the activation of the N-methyl- d-aspartate receptor (NMDA-R) by its co-agonist d-serine, because alterations in this mechanism contribute greatly to synaptic plasticity deficits in aged animals. Long-term dietary supplementation with L-NAC prevented oxidative damage in the hippocampus of aged rats. Electrophysiological recordings in the CA1 of hippocampal slices indicated that NMDA-R-mediated synaptic potentials and theta-burst-induced long-term potentiation (LTP) were depressed in aged animals, deficits that could be reversed by exogenous d-serine. Chronic treatment with L-NAC, but not acute application of the reducing agent, restored potent d-serine-dependent NMDA-R activation and LTP induction in aged rats. In addition, it is also revealed that the age-related decrease in d-serine levels and in the expression of the synthesizing enzyme serine racemase, which underlies the decrease in NMDA-R activation by the amino acid, was rescued by long-term dietary treatment with L-NAC.

 Our results indicate that protecting redox status in aged animals could prevent injury to the cellular mechanisms underlying cognitive aging, in part by maintaining potent NMDA-R activation through the d-serine-dependent pathway.

Introduction

Brain aging is generally accompanied by cognitive deficits, in particular by learning and memory impairments, which have been intensively studied in animal models including rodents [for a review see (Billard, 2006)]. Whereas obvious structural changes occur within the central nervous system (CNS) in dementia-associated memory decline, normal aging is not associated with a significant loss of neurons (Gallagher et al., 1996), indicating that the brain alterations involved are much more subtle (Foster, 2006; Burke & Barnes, 2010). It has therefore been postulated that changes in the functional properties of neuronal networks could play a critical role in the induction of age-related memory impairments (Rosenzweig & Barnes, 2003). Because memory formation is now viewed as being closely dependent on the capacity of the brain to regulate long-lasting changes in neuronal communication (Martin et al., 2000; Lisman & McIntyre, 2001), age-related deficits in learning and memory could occur in parallel with the impairment of functional plasticity at central synapses. Experimental data confirm this assumption, because the expression of synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD) of synaptic transmission, is altered in the brain of memory-deficient aged animals (Clayton et al., 2002; Foster, 2006; Potier et al., 2010).

The N-methyl-D-aspartate subtype of glutamate receptors (NMDA-R) is known to play a major role in the induction of synaptic plasticity as well as in the acquisition of memory traces (Morris et al., 1990; Izquierdo, 1991). In addition to glutamate, NMDA-R activation requires the binding of the amino acid d-serine at the glycine binding site, at least in brain areas involved in memory processing [for reviews see (Martineau et al., 2006; Wolosker, 2006; Billard, 2008)]. Impaired LTP expression at hippocampal synapses of the aging brain has recently been linked to weaker NMDA-R activation by endogenous d-serine (Mothet et al., 2006; Turpin et al., 2011), and the administration of exogenous d-serine or the related compound d-cycloserine rescues both synaptic plasticity and spatial memory in aged rodents (Baxter et al., 1994; Billard & Rouaud, 2007; Potier et al., 2010). In the recent ‘d-serine shuttle’ hypothesis, it has been suggested that the precursor l-serine synthesized by astrocytes is converted to the d-isomer in neurons through direct racemization by serine racemase (SR), after which the amino acid is returned to astrocytes where it accumulates (Wolosker, 2011). During synaptic activation, d-serine is released into the synaptic cleft to act on NMDA-R and then taken up again by astrocytes and neurons to be degraded by the flavoprotein d-amino acid oxidase (dAAO) (see the study by Martineau et al., 2006 for a review). Convincing clues indicate that the lower availability of d-serine in the hippocampus of aged rats is attributed to an alteration in the biosynthetic pathway rather than to an increased degradation of the amino acid, because the expression of SR, but not dAAO, is significantly altered (Mothet et al., 2006; Turpin et al., 2011). Interestingly, the targeted disruption of SR in mice affects memory abilities as well as the expression of synaptic plasticity, confirming a pivotal role for this gene in cognition (Basu et al., 2009; Labrie et al., 2009). These results thus strongly point to SR as a key component of learning and memory processes, and moreover, one that could be preferentially impacted during aging.

Oxidative stress (OS), characterized by the intracellular accumulation of reactive oxygen species (ROS), has been linked to a large range of insults affecting bodily functions during aging. The functioning of neuronal networks within the CNS needs high levels of oxygen, and comparative studies indicate that antioxidant levels in the brain are low compared to other organs (Halliwell, 1992). It is therefore expected that the CNS is particularly sensitive to the oxidative stress associated with age (Droge & Schipper, 2007). Accordingly, a substantial body of data indicates that ROS concentrations increase in aged neurons, inducing lipid peroxidation and the oxidation of proteins and nucleic acids (Sohal & Weindruch, 1996). Among the broad spectrum of intracellular mechanisms that regulate redox status in living cells, glutathione (GSH) is a potent endogenous ROS scavenger that prevents the occurrence of oxidative stress (Reid & Jahoor, 2001; Cruz et al., 2003). Interestingly, long-term dietary manipulations that increase GSH content are helpful in alleviating the age-related neuronal alterations induced by compromised antioxidative defense systems, as demonstrated, for example, by treatment with N-acetyl-L-cysteine (L-NAC) (Martinez et al., 2000; Cocco et al., 2005). This prophylactic agent is a thiol compound that serves mainly as a precursor of GSH synthesis, but can also act directly as a free-radical scavenger (Ziment, 1988).

In light of the protective function of L-NAC, we asked whether it could prevent synaptic plasticity deficits in aged rats, by maintaining the functionality of the d-serine-dependent pathway and the expression of SR.

Results

Chronic treatment with L-NAC prevents the age-related impairment of redox status

We first checked for the occurrence of OS, as well as for the effects of long-term dietary treatment with L-NAC, in hippocampal tissues of the aged animals used for functional investigations. OS during aging is best illustrated by the characteristic increase in carbonylated proteins. Using an oxyblot approach (Fig. S1), we evaluated the levels of carbonylated proteins, in young adults (n = 5), in aged control rats (n = 5) and in aged animals supplemented with L-NAC for 10 months (n = 5) (Fig. 1). A very large 167% increase (from 0.095 ± 0.018 to 0.254 ± 0.079, arbitrary units) in the mean content of carbonylated proteins occurred as the animals matured, and this increase was prevented by L-NAC treatment [F2,12 = 5.86, P < 0.01]. In fact, this treatment even resulted in a substantial 80% reduction down to 0.034 ± 0.007 in the mean amount of carbonylated proteins when compared to young adult animals. Post hoc Student–Newman–Keuls analysis of the data evidenced significant differences (P < 0.05) between aged and young adult animals, and between aged and L-NAC-treated animals. By contrast, L-NAC-treated rats did not significantly differ from young adult animals.

Figure 1.

 Long-term dietary treatment with L-NAC prevents age-related oxidative stress. Bar graphs representing the percentage change in the carbonylated proteins and in the GSH/GSSG ratio in control and L-NAC-treated aged rats, normalized to young adult animals. (anova, Student–Newman–Keuls post hoc test, **P < 0.01). Note that the decreased ratio in aged control animals, indicating that oxidative stress has occurred, is completely prevented by long-term dietary supplementation with L-NAC.

As L-NAC is a precursor of GSH, we next checked whether the oxidized status of GSH was modified in our conditions. Mean levels of GSH and its oxidized form GSSG were estimated by HPLC for each experimental group. The GSH/GSSG ratio was then calculated to serve as an index of redox status, a decrease in the ratio being indicative of the occurrence of OS (Griffith, 1999). A comparison of young adult and aged control animals showed a 19% decrease in mean GSH/GSSG ratio (from 5.5 ± 0.9 to 4.4 ± 0.4). Interestingly, a potent increase in the ratio (+58%, P < 0.001) was seen in hippocampal extracts from aged animals supplemented with L-NAC (6.9 ± 0.9) compared to those from aged control rats (Fig. 1). The GSH/GSSG ratio calculated for the L-NAC-treated group was in fact even higher (+28%) than that in young control adults. Our results thus indicate that senescent animals chronically treated with L-NAC from middle age onwards do not display OS in hippocampal tissues, unlike nontreated rats of the same age.

L-NAC treatment protects against age-related impairment of NMDA-R–mediated synaptic plasticity

In control aCSF, theta-burst stimulation (TBS) induced a significant and long-lasting potentiation of synaptic transmission in slices from young adult rats [F1,20 = 23.9, P < 0.0001, n = 10] whereas potentiation was not maintained in slices from aged control rats [F1,14 = 0.9, NS, n = 8] (Fig. 2 a). Thus, an age-related impairment of TBS-dependent LTP occurred [F1,16 = 4.9, P < 0.05], similar to that shown before (Mothet et al., 2006; Turpin et al., 2011). In slices from aged rats treated with L-NAC, however, a persistent increase in synaptic transmission was recorded after the same conditioning stimulation [F1,18 = 30.9, P < 0.0001, n = 10] (Fig. 2 a). Interestingly, not only was LTP generated by TBS in these animals, its magnitude was not significantly different from levels obtained in slices from young adult rats [F1,18 = 0.3, NS]. These results thus show that long-term dietary treatment with L-NAC is capable of preventing the age-related impairment of TBS-induced plasticity in the hippocampal slices.

Figure 2.

 Long-term dietary treatment with L-NAC prevents age-related deficits in NMDA-R-dependent synaptic plasticity. (a) Time course of mean theta-burst stimulation (TBS)-induced long-term potentiation (LTP), calculated from slices of young adults (n = 10), aged controls (n = 8) and aged animals treated with L-NAC (n = 8), in control medium (repeated measures anova, *P < 0.05). In insert, representative traces of fEPSPs recorded before and 60 min after TBS in a young adult (i), an aged control (ii) and an aged rat treated with L-NAC (iii), are superimposed. Bars: 10 ms and 0.5 mV. (b) Superimposed sample traces of evoked NMDA-R-mediated fEPSPs recorded from slices of a young adult, an aged control and an aged rat treated with L-NAC. (c) Comparison of NMDA-R-mediated fEPSP/PFV ratio calculated at a stimulus intensity of 300, 400 and 500 μA in young adult (n = 11), aged control (n = 10) and aged rats treated with L-NAC (n = 10). (Repeated measures anova, *P < 0.05).

In slices from young adult animals bathed in aCSF containing the selective NMDA-R antagonist d-APV (80 μm, n = 7), TBS enhanced synaptic transmission but only for a few minutes (not illustrated), indicating that the maintenance of TBS-induced LTP requires the activation of NMDA-R [see also (Mothet et al., 2006; Yang et al., 2010)]. We therefore looked for L-NAC-related effects that act directly on NMDA-R activation by isolating and comparing NMDA-R-mediated field excitatory postsynaptic potentials (fEPSPs) in the different groups of animals.

In low Mg2+ medium supplemented with the AMPA-R antagonist NBQX (10 μm), fEPSPs were generated by electrical stimulation (Fig. 2 b) and were blocked at the end of the recording by d-APV (30 μm). In young adult rats (11 slices/6 animals), fEPSP magnitude was higher than in aged control animals (10 slices/7 animals) [F1,18 = 5.4, P < 0.05] whereas the magnitude of presynaptic fiber volleys (PFVs) was unchanged [F1,18 = 0.1, NS]. Consequently, the fEPSP/PFV ratio, an index of synaptic efficacy, was significantly higher in younger rats [F1,18 = 4.8, P < 0.05], confirming that postsynaptic NMDA-R activation is impaired in aged animals (Fig. 2 c).

In L-NAC-treated aged rats (10 slices/6 animals), the fEPSP/PFV ratio was significantly higher than in aged controls [F1,18 = 5.9, P < 0.05] but was not statistically different from the ratio in slices from young adult animals [F1,19 = 0.2, NS]. These data therefore indicate that potent NMDA-R activation in aged rats can be preserved by preventing OS through chronic L-NAC administration (Fig. 2 c).

L-NAC treatment protects against age-related deficits in d-serine-dependent NMDA-R activation

In addition to glutamate, the binding of the co-agonist d-serine at the glycine binding site is a necessary condition for NMDA-R activation in the hippocampus (Mothet et al., 2000, 2006). We have previously shown that d-serine-dependent NMDA-R activation is attenuated in aged rats (Junjaud et al., 2006; Mothet et al., 2006). We therefore asked whether L-NAC could lead to the maintenance of potent NMDA-R activation in aged animals by targeting this mechanism.

With respect to isolated NMDA-R-dependent synaptic potentials, d-serine added at 100 μm to saturate glycine binding sites (Junjaud et al., 2006) enhanced the fEPSP/PFV ratio by 41.6 ± 7.4% in 10 of the 11 slices tested from young adult animals (when determined for a 400 μA stimulus intensity). This potentiation effect of the co-agonist on isolated NMDA-R-dependent fEPSPs was significantly higher in aged rats (P < 0.05), where the increase reached 79.2 ± 18.1% (in 8 of 10 slices tested). As a consequence, the age-related decrease in the fEPSP/PFV ratio was totally reversed by d-serine (Fig. 3 a), as we have recently shown (Turpin et al., 2011). Interestingly, when exogenous co-agonist was added to slices from L-NAC-treated aged animals, the fEPSP/PFV ratio increased by 63.3 ± 14.5% (in 8 of 10 slices), a level slightly higher but not significantly different from the effect in young adult animals. Altogether, we evidence that long-term dietary supplementation with L-NAC prevents the impairment of NMDA-R activation in aged rats by maintaining the activation of glycine binding sites by endogenous d-serine.

Figure 3.

 Long-term dietary treatment with L-NAC protects against age-related deficits of d-serine-dependent NMDA-R activation. (a) Comparison of NMDA-R-mediated fEPSP/PFV ratio calculated at a stimulus intensity of 300, 400 and 500 μA from slices of young adult (n = 11), aged control (n = 10) and aged rats treated with L-NAC (n = 10), superfused with aCSF supplemented with d-serine (100 μm). (b) Time course of mean TBS-induced LTP calculated from slices of young adult (n = 9), aged controls (n = 11) and aged animals treated with L-NAC (n = 8), in aCSF supplemented with d-serine (100 μm).

Next, we checked whether this protective effect of L-NAC on NMDA-R-dependent potentials through d-serine could account for the preservation of TBS-induced LTP. In the presence of saturating d-serine (100 μm), TBS-induced LTP was significantly enhanced when compared with that induced with control aCSF, in slices from both young adult [F119,1904 = 2.47, P < 0.0001, n = 9) and aged rats [F81,1377 = 1.9, P < 0.001, n = 11]. Interestingly, under these conditions, in which the glycine binding site of NMDA-R was saturated by the co-agonist (Junjaud et al., 2006), the age-related impairment of TBS-induced LTP was occluded, because the magnitude of LTP was not statistically different between the two groups of animals [F1,20 = 0.4, NS] (Fig. 3 b).

On contrary, adding d-serine to slices from L-NAC-treated aged animals did not significantly affect the amplitude or time course of LTP [F119,1904 = 0.3, NS, n = 8]. Under these conditions, LTP expression in L-NAC-treated animals was comparable to that recorded in slices from young adult [F1,14 = 0.2, NS] (Fig. 3b) and aged rats [F1,16 = 0.4, NS] (Fig. 3 b).

Acute GSH but not L-NAC application enhances TBS-induced LTP in aged rats

As previously mentioned, L-NAC serves as a precursor for GSH synthesis, but evidence suggests that it could also act directly as a free-radical scavenger (Ziment, 1988). We therefore determined the effects of an acute application (30 min) of either L-NAC (100–500 μm) or GSH (50 μm) on TBS-induced LTP in young adult and aged rats. In aged animals, L-NAC failed to affect the magnitude or time course of TBS-induced LTP, either at 500 μm [F1,15 = 0.7, NS; n = 8] (Fig. 4 a) or 100 μm (n = 6). In young adult rats, notably, the expression of TBS-induced LTP was not altered in the presence of 100 μm L-NAC (n = 5) but was significantly reduced at 500 μm, at least during the last 15 min. of recording [F1,18 = 6.9, P < 0.05; n = 10] (Fig. 4 b). As the same concentration of L-NAC did not alter the magnitude of NMDA-R-mediated synaptic potentials in these animals (Fig. S2), it seems likely that the LTP impairment reflected nonspecific effects of the drug.

Figure 4.

 Acute application of GSH, but not L-NAC, enhances TBS-induced LTP in aged animals. (a) Time course of mean TBS-induced LTP calculated from slices of aged animals in control aCSF (n = 9) and in the presence of L-NAC (500 μm, n = 8). (b) Similar recordings obtained from slices of young adult rats (repeated measures anova, *P < 0.05). (C) Time course of mean TBS-induced LTP calculated from slices of aged animals in control aCSF (n = 9) and in the presence of glutathione (50 μm, n = 9) (repeated measures anova, *P < 0.01). (b) Similar recordings obtained from slices of young adult rats.

In contrast, TBS-induced LTP in aged rats was significantly greater when GSH (50 μm) was added to aCSF than under control conditions [F1,16 = 7.2, P < 0.01; n = 9] (Fig. 4 c), whereas the molecule had no effect on young adult animals [F1,14 = 0.1, NS; n = 6] (Fig. 4 d).

These results strongly suggest that long-term dietary treatment with L-NAC preserves LTP expression in aged animals, most probably through the elevation of GSH levels rather than direct scavenging activity.

L-NAC treatment prevents the decrease in d-serine levels and serine racemase expression but not the increase in GFAP expression in the hippocampus of aged rats

The age-related impairment of d-serine-dependent NMDA-R activation has been found to reflect the reduced availability of the amino acid, due at least in part to the weaker expression of the synthesizing enzyme serine racemase (SR) (Mothet et al., 2006; Turpin et al., 2011). We also know that astrocytes by furnishing d-serine are necessary for the induction of synaptic plasticity in the hippocampus (Henneberger et al., 2010). Finally, reactive gliosis, characterized by a significant increase in the levels of glial fibrillary acidic protein (GFAP), is a hallmark of aging (Finch, 2003) then affecting diffusion parameters in the extracellular space (Sykova et al., 2002). This structural alteration could modify the accessibility of d-serine to NMDAR glycine binding sites. We therefore asked whether L-NAC treatment could prevent the age-related deficits of d-serine-dependent synaptic plasticity through the regulation of d-serine levels and SR expression and could also impact age-related astrogliosis.

In the hippocampus of aged control rats (Fig. 5 a), semi-quantitative Western blotting indicated that protein levels of SR were reduced by 25.1% (P < 0.01, n = 5) as compared to young adult controls (n = 5), whereas GFAP content was significantly enhanced by 50.9% (P < 0.01). Interestingly, SR levels in the hippocampus of L-NAC-treated aged rats (n = 5) were similar to those estimated in tissue from young adult animals (n = 6; Fig. 5 b), indicating that long-term treatment with the reducing agent was able to protect against the age-related downregulation of SR expression. In contrast, GFAP levels remained 49.9% higher (P < 0.001) in L-NAC-treated aged rats, indicating that astrogliosis was not affected by the treatment (Fig. 5 b), a finding confirmed by immunohistochemical analysis for GFAP. Indeed, there was substantial labeling for astrocytes with elongated processes in the hippocampus of young adult rats (Fig. S3 A). In aged control individuals, these glial cells were hypertrophied, with enhanced GFAP labeling and thick, highly ramified processes (Fig. S3 B). A similar phenotype was observed in the hippocampus of aged rats treated with L-NAC, confirming the persistence of astrogliosis in these animals (Fig. S3 C).

Figure 5.

 Long-term dietary treatment with L-NAC prevents the decrease in serine racemase expression but not the increase in GFAP levels in aged rats. (a, top) Immunoblots for serine racemase (SR, upper band), glial fibrillary acidic protein (GFAP, middle band) and β-actin (lower band) in young adult (Y) and aged (A) control rats. (a, bottom) Bar graph depicting quantification of immunoreactivity for SR and GFAP in young adult (n = 5) and aged control (n = 5) rats, normalized to β-actin protein levels (**P < 0.01). (b, top) Immunoblots for SR, GFAP and β-actin in young adult rats and aged animals treated with L-NAC. (b, bottom) Bar graph depicting quantification of immunoreactivity for SR and GFAP in young adult (n = 7) and aged L-NAC-treated (n = 6) rats, normalized to β-actin protein levels (**P < 0.01).

Knowing that L-NAC prevents age-related downregulation of SR, we then asked whether these positive modulations by NAC could at the end impact the levels of d-serine in aged rats. We evaluated hippocampal levels of d-serine in young adults (n = 5), in aged control rats (n = 5) and in aged animals supplemented with L-NAC (n = 5) (Fig. S4). In the hippocampus of aged rats, levels of d-serine were significantly reduced by 77.9% (from 5.12 ± 0.92 to 1.13 ± 0.23 nmole mg−1 of proteins, P < 0.001) as previously reported (Mothet et al., 2006; Turpin et al., 2011). In the aged rats treated with L-NAC, levels of d-serine are only decreased by 31.1% (3.53 ± 0.48 nmole mg−1 of proteins), which illuminates the positive rescue effect of L-NAC (Fig. S4) because L-NAC-treated animals significantly differ from aged controls (P < 0.01) but not from young adult animals (Fig. S4). These results therefore fully parallel the effects of L-NAC on the expression of SR.

Discussion

This study shows that the impairment of NMDA-R-dependent LTP in slices from aged control rats is rescued in those from senescent animals chronically treated with the reducing agent L-NAC, and in which oxidative stress has been prevented. Acute application of the reducing agent has no significant effect in these animals, whereas the L-NAC-derived thiol compound GSH enhances LTP expression. In addition, long-term dietary treatment with L-NAC leads to the preservation of potent NMDA-R activation by its endogenous co-agonist d-serine in aged animals. This effect is associated with the maintenance of the expression of the d-serine synthesizing enzyme SR and of d-serine levels in hippocampal tissues. Our results further strengthen the role of the d-serine-dependent pathway in cognitive aging and pinpoint SR as a prime target for the deleterious effects of age-related OS.

Since it was initially formulated in the late fifties (Harman, 1956), the free-radical theory of aging, which postulates that aging processes could be slowed down by reinforcing antioxidant defenses, has been supported by experimental evidence. As antioxidant levels are low in the brain as compared to non-neural tissues (Halliwell, 1992), while high levels of oxygen are required for cerebral functions, the progressive accrual of oxidative damage in the brain throughout life has been proposed to underlie cognitive aging, a notion that has been widely reviewed (Watson et al., 2006). Indeed, changes in redox regulation in the CNS are accompanied by neuronal dysfunction, in particular by the alteration of synaptic plasticity (Bernard et al., 1997; Kamsler & Segal, 2007). As synaptic plasticity is believed to be the essential neuronal substrate for learning and memory (Martin et al., 2000; Lisman & McIntyre, 2001), it could be viewed as a preferred mechanism by which OS could alter memory functions.

Our study shows that long-term dietary treatment with L-NAC protects senescent animals against changes in redox status, thus preventing the occurrence of oxidative damage, as previously reported (Cocco et al., 2005). Importantly, this treatment prevents the well-characterized LTP deficits displayed by nontreated aged animals [reviewed in (Billard, 2006)], confirming the view that synaptic plasticity is indeed targeted by OS for the modulation of age-related cognitive impairment. L-NAC exerts its antioxidant properties mostly as a precursor of intracellular cysteine and GSH, but can also act directly as an ROS scavenger (Ziment, 1988). However, we show here that the acute application of L-NAC does not affect LTP expression in aged rats. In contrast, GSH is able to rescue this process [see also (Yang et al., 2010)]. These results therefore indicate that, at least in our preparation, L-NAC does not act directly on ROS-dependent by-products but rather through GSH levels or through the stimulation of cytosolic enzymes that participate in GSH generation.

It is now well documented that NMDA-R activation plays a major role in the induction of synaptic plasticity (Morris et al., 1990; Izquierdo, 1991). The age-related decrease in NMDA-R activation recorded here [see also (Barnes et al., 1997; Potier et al., 2000; Clayton et al., 2002)] is mainly responsible for LTP deficits, because both alterations are alleviated by boosting NMDA-R function with saturating concentrations of the co-agonist d-serine, which acts at the glycine binding site (Junjaud et al., 2006; Mothet et al., 2006). These results also confirm that the dysfunction of this modulatory site is a relevant mechanism underlying the impairment of synaptic plasticity associated with age, as we have recently suggested elsewhere (Potier et al., 2010; Turpin et al., 2011). Interestingly, our results indicate that long-term dietary treatment with L-NAC in aged rats leads to the maintenance of potent NMDA-R activation. In addition, they show that the susceptibility of NMDA-R activation to exogenous d-serine in these animals is comparable to that in young adults and significantly lower than in aged control rats. Taken together, these results indicate that the potent activation of NMDA-R by the association of endogenous d-serine binding with its glycine binding site is preserved by chronic treatment with the reducing agent, providing evidence that it is a key mechanism by which OS alters synaptic plasticity during aging. Interestingly, in aged LOU/C/Jall rats, a model of healthy aging that does not exhibit a deficit in d-serine-dependent NMDA-R activation (Kollen et al., 2010; Turpin et al., 2011), OS does not occur, as illustrated by the absence of changes in the GSH/GSSG ratio (J. M. Billard, and G. Barbanel, unpublished data). However, the occurrence of deleterious effects of OS on NMDA-R activation independent of d-serine regulation cannot be totally ruled out. Indeed, it has been shown that the exogenous application of GSSG inhibits responses mediated by NMDA-R in cultures of rat cortical and retinal ganglion cells, through the activation of a specific redox modulatory site (Aizenman et al., 1990; Lipton et al., 1998). As the GSH/GSSG ratio decreases with age, as confirmed in this study, it is likely that some changes at the redox modulatory site attributed to GSSG elevation could also contribute to the age-related alteration of NMDA-R activation [see (Bodhinathan et al., 2010)]. Accordingly, we found that reversing the decrease in the GSH/GSSG ratio in slices from aged animals by the exogenous application of GSH facilitated LTP expression in these animals [see also (Yang et al., 2010)].

We have recently shown that the expression of serine racemase (SR) decreases during aging, resulting in lower d-serine availability in hippocampal tissues and, consequently, the impairment of NMDA-R activation (Mothet et al., 2006; Turpin et al., 2011). Interestingly, we show here that this decrease in d-serine levels and the downregulation of SR expression does not occur in senescent animals chronically treated with L-NAC. On the other hand, SR activity is inhibited by nitric oxide-mediated S-nitrosylation (Mustafa et al., 2007) and by sulfhydryl oxidation (Wolosker et al., 1999). Taken together, these results suggest that L-NAC may alleviate age-related changes in d-serine availability by preserving both the expression and the activity of its synthesizing enzyme.

A change in the astrocytic network with aging could represent another possible target for amelioration of impaired d-serine-dependent NMDA-R activation by L-NAC. Indeed, it has been proposed that d-serine is synthesized mainly in neurons and then transferred to the astrocytic compartment where it accumulates, and from which it is released upon synaptic activation (Wolosker, 2011). This view is supported by another recent finding that astrocyte-derived d-serine is necessary for the induction of synaptic plasticity (Henneberger et al., 2010). A wealth of evidence indicates that aging is associated with reactive astrogliosis, characterized by cellular hypertrophy and an increase in GFAP levels (Finch, 2003). This astrogliosis impairs the diffusion of molecules through the extracellular space, affecting neuron-glial interactions (Sykova et al., 2002). Nevertheless, we found that aged L-NAC-treated animals, which demonstrated potent d-serine-dependent activation of NMDA-R function, still displayed astrogliosis with enhanced GFAP levels in hippocampal tissues. This result strongly indicates that L-NAC mainly acts through mechanisms independent of the accessibility of NMDA-R to d-serine.

In conclusion, the present investigation provides additional evidence demonstrating that the SR-dependent pathway is critical to mechanisms underlying cognitive aging. It also suggests that the chronic administration of precursors of reducing compounds such as L-NAC holds promise in the treatment of memory deficits in the elderly.

Experimental procedures

All experiments were carried out in accordance with the European Communities Council Directive (86/809/EEC) regarding the care and use of animals for experimental procedures and were approved by the local ethics committee (Comité regional d’éthique en expérimentation animale Paris Descartes, Université Paris 5). The experiments were conducted with 4–6 month-old ‘adult’ (n = 18) and 25–27 month-old ‘aged’ (n = 31) male Wistar rats purchased from Charles River (France). Animals were housed in Plexiglas cages and maintained on a controlled light-dark cycle at constant temperature (22 ± 2 °C), with ad libitum access to food. Aged animals were divided into two groups. One group (n = 10) received 500 mg of L-NAC dissolved in 500 mL of tap water while the control group (n = 21) received the same volume of plain tap water. Water bottles were changed once a day because L-NAC is expected to be stable at room temperature and to remain active when dissolved in water for at least 24 h (Davis et al., 2007). To minimize differences between individuals in the ingestion of L-NAC, rats were only housed two to a cage. L-NAC treatment was initiated at 14 months of age and maintained until the day of experimental procedures.

Evaluation of oxidative status

Tissues were sonicated in 2% SDS containing PMSF as a protease inhibitor and centrifuged. Protein amounts were determined using the Bio-Rad DC protein assay kit, with bovine serum albumin as a standard, and adjusted to 1 mg mL−1. The global level of carbonylated proteins was semiquantitatively evaluated via a simplified system derived from the Oxyblot analysis. Briefly summarized, carbonyl groups were reacted with a dinitrophenyl hydrazine solution in an acidic medium. After neutralization, the corresponding hydrazones were spotted onto a nitrocellulose membrane and labeled with a rabbit anti-DNP antiserum (1:150, Millipore, USA). After extensive washing, the membrane was reacted with a peroxidase-conjugated goat anti-rabbit IgG (1:300, Millipore, USA) and developed by enhanced chemiluminescence (ECL, Amersham Biosciences, UK) (Fig. S1). The relative amount of carbonylated protein was directly proportional to the chemiluminescence detected and analyzed on an Odyssey Li-Cor instrument. Tissue concentrations of GSH, either in its reduced form (GSH) or the oxidized form glutathione disulfide (GSSG), were evaluated by reverse-phase high-performance liquid chromatography (HPLC) using fluorescence detection and precolumn derivatization with the specific reagent mono-bromo-bimane. The ratio between GSH and GSSG was then determined.

Ex vivo electrophysiology

Acute slice preparation

Transverse hippocampal slices (400 μm) were obtained as previously described (Potier et al., 2000) from rats anesthetized with halothane before decapitation. Slices were prepared in ice-cold artificial cerebrospinal fluid (aCSF) and placed in a holding chamber for at least 1 h. The composition of aCSF was as follows (in mm): NaCl, 124; KCl, 3.5; MgSO4, 1.5; CaCl2, 2.3; NaHCO3, 26.2; NaH2PO4, 1.2; and glucose, 11; pH, 7.4. A single slice was transferred to the recording chamber at a time and continuously submerged with aCSF pregassed with 95% O2/5% CO2.

Recordings

Extracellular recordings were obtained at 25–28 °C from the apical dendritic layer of the CA1 area using micropipettes filled with 2 m NaCl. Presynaptic fiber volleys (PFVs) and field excitatory postsynaptic potentials (fEPSPs) were evoked by electrical stimulation of Schaffer collaterals and commissural fibers located in the stratum radiatum. NMDA-R-mediated fEPSPs were recorded in slices perfused with low-Mg2+ (0.1 mm) aCSF supplemented with the AMPA/kainate receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzoquinoxaline-7-sulfonamide (NBQX, 10 μm). CA3 and CA1 were separated by a knife cut to prevent the propagation of epileptiform discharges.

Synaptic transmission

The averaged slope of three PFVs and fEPSPs was measured using Win LTP software (Anderson & Collingridge, 2001). To evaluate the level of receptor activation, the fEPSP/PFV ratio was plotted against stimulus intensity (300, 400 and 500 μA). The effects of exogenous d-serine added at 100 μm to saturate NMDA-R glycine binding sites (Junjaud et al., 2006) were assessed by determining the fEPSP/PFV ratio 15 min after the addition of the co-agonist to the aCSF.

Synaptic plasticity

To investigate LTP of synaptic transmission, a test stimulus was applied every 10 s in control medium and adjusted to get an fEPSP with a baseline slope of 0.1 V s−1. The averaged slope of three fEPSPs was measured for 15 min before theta-burst stimulation (TBS), consisting of five trains of four 100 Hz pulses each, separated by 200 ms and delivered at the test intensity. This sequence was repeated four times with an interburst interval of 10 s. Testing with a single pulse was then resumed for 60 min to determine the level of LTP. In pharmacological experiments, d-2-amino-5-phosphonovalerate (d-APV, 50 μm) or d-serine (100 μm) was added to the aCSF 10 min before the establishment of the baseline and maintained throughout recording.

Drugs

The following drugs were used in the present study: N-acetyl-L-cysteine (L-NAC) (Sigma Aldrich, Saint Quentin Fallavier, France); the NMDA receptor antagonist d-2-amino-5-phosphonovalerate (d-APV) (Tocris Cookson, Bristol, UK); the AMPA receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzoquinoxaline-7-sulfonamide (NBQX) (Tocris Cookson), the NMDA receptor co-agonist D-serine (Tocris Cookson) and the reductant L-glutathione reduced (GSH) (Sigma Aldrich). All pharmacological agents were diluted directly in the aCSF from stock solutions prepared in distilled water or in dimethylsulfoxide.

Semi-quantitative immunoblotting analysis

Western blot analysis was performed as described previously (Mothet et al., 2006). Briefly, after cell lysis, protein extracts were subjected to electrophoresis (12% SDS–polyacrylamide gel) and electroblotted onto polyvinylidene fluoride membranes (Immobilon-P, Millipore). Membranes were probed with polyclonal antibodies to serine racemase (1:200, Santa Cruz Biotechnology, Wiltshire, UK), β-actin (1:5000, Santa Cruz Biotechnology) and monoclonal GFAP (1:6000, Chemicon International, UK) for 1 h at room temperature. After washing, they were incubated with peroxidase-conjugated anti-rabbit, anti-mouse, or anti-goat immunoglobulin G secondary antibodies (1:2000, Vector Laboratories, Burlingame, USA). Immunoblots were developed by enhanced chemiluminescence (ECL, Amersham Biosciences, UK) using X ray film (Hyperfilm ECL, Amersham Biosciences, UK). Molecular sizes were estimated by separating prestained molecular weight markers (6.5–175 kDa) in parallel (New England BioLabs, UK). Protein bands of interest were analyzed by measuring optical density (OD) using scanning densitometry. Densitometric results for serine racemase and GFAP were normalized to that of β-actin.

Immunohistochemistry

Each animal was perfused through the heart under deep anesthesia (pentobarbital) with 600 mL of 4% paraformaldehyde (PAF) in phosphate buffer (PB, 0.1M; pH 7.4) at 4 °C. Brains were dissected, postfixed for 2 h in 4% PAF at 4 °C and cryoprotected in 30% sucrose in PB (4 °C).

To block nonspecific antigen recognition by the donkey secondary antibody, coronal sections (40 μm thick) were preincubated for 2 h at room temperature in NDST blocking solution composed of PB 0.1M, 0.3% Triton X-100 0.9% NaCl, and 10% normal donkey serum. Sections were then incubated overnight at 4 °C with a monoclonal GFAP antibody (GA5, Chemicon, Millipore, France), diluted 1/2500 in 1% NDST. Sections were rinsed three times in NDST and incubated for 45 min in 1:500 Alexa Fluor 488-conjugated donkey anti-mouse IgG (Molecular probes, Eugene, OR, USA). Sections were rinsed three times in PB 0.1M, mounted on glass slides, and coverslipped with Fluoromount (Southern Biotechnology Associates, CliniSciences, France). Sections were examined using a confocal laser scanning microscope (Leica TCS SP2 confocal imaging system, Leica Microsystems, Heidelberg, Germany) equipped with an Ar 488 nm laser.

Determination of D-serine levels in hippocampal tissues

Tissues were sonicated in 2% SDS containing PMSF as a protease inhibitor and centrifuged. Protein amounts were determined using the Bio-Rad DC protein assay kit, with bovine serum albumin as a standard, and adjusted to 1 mg mL−1. Levels of D-serine were determined on the liquid-phase using a Amplex Red–based fluorimetric assay adapted from Mothet et al. (2005). The amounts of D-Ser in samples were measured by quantification of generated hydrogen peroxide during its degradation by RgDAAO (Mothet et al., 2005). Actually, 10-μL sample were added to 200 μL of media containing 100 mm Tris–HCl, pH 8.8, 10 U mL−1 peroxidase, and 400 μm Amplex Red (Invitrogen) and completed with 20 μL of RgDAAO (75 U mL−1). The fluorescence signal was then detected in a microplate reader (Mithras, Berthold Technologies) with excitation at 544 nm and emission at 590 nm. The concentration of D-serine in each sample was estimated by comparison with a standard curve.

Analysis of data

All results are expressed as means ± SEM. Statistical analysis was carried out using analyses of variance (anova) followed by post hoc tests: Student–Newman–Keuls (SNK) when comparing the three (young, aged, and L-NAC-treated aged rats) groups, and by post hoc (paired or unpaired) t tests when comparing only aged with L-NAC-protected aged rats. In all cases, differences were considered significant when P < 0.05.

Acknowledgments

The author thanks A. Cougnon and D. Bergerot for their care of the animals and for the daily treatment of the rats with NAC. This manuscript was prepared with editorial help from Gap Junction (http://www.gap-junction.com).

Author contributions

C.H., F.R.T., M.K., P-M.S., G.B., B.P., and J-M.B. designed experiments. J-M.B. wrote the article. C.H., F.R.T., M.K., and J-M.B. performed and analyzed electrophysiological recordings in slices. P-M.S performed and analyzed Western blotting. G.B. performed and analyzed measurements as well as the global levels of carbonylated proteins. J-P.M performed and analyzed measurements relative to D-serine. B.P performed and analyzed immunohistochemical experiments. All authors discussed the results and implications and commented on the manuscript at all stages.

Disclosure

The authors declare that they have no actual or potential conflict of interest, financial or otherwise, related to the present work. The data contained in the present manuscript have not been submitted elsewhere and will not be submitted elsewhere while under consideration at Aging Cell. Procedures and the final version of the manuscript were approved by all authors.

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