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. 2014 Aug 20;36(5):9698. doi: 10.1007/s11357-014-9698-0

Circuit-specific changes in d-serine-dependent activation of the N-methyl-D-aspartate receptor in the aging hippocampus

M Labarrière 1, F Thomas 1, P Dutar 1, L Pollegioni 2,3, H Wolosker 4, J-M Billard 1,✉
PMCID: PMC4453931  PMID: 25138794

Abstract

Age-related memory deficits have recently been associated with the impaired expression of d-serine-dependent synaptic plasticity in neuronal networks of the hippocampal CA1 area. However, whether such functional alterations are common to the entire hippocampus during aging remains unknown. Here, we found that d-serine was also required for the induction of N-methyl-D-aspartate receptor (NMDA-R)-dependent long-term potentiation (LTP) at perforant path-granule cell synapses of the dentate gyrus. LTP as well as isolated NMDA-R synaptic potentials were impaired in slices from aged rats, but in contrast to the CA1, this defect was not reversed by exogenous d-serine. The lower activation of the glycine-binding site by the endogenous co-agonist does not therefore appear to be a critical mechanism underlying age-related deficits in NMDA-R activation in the dentate gyrus. Instead, our data highlight the role of changes in presynaptic inputs as illustrated by the weaker responsiveness of afferent glutamatergic fibers, as well as changes in postsynaptic NMDA-R density. Thus, our study indicates that although NMDA-R-dependent mechanisms driving synaptic plasticity are quite similar between hippocampal circuits, they show regional differences in their susceptibility to aging, which could hamper the development of effective therapeutic strategies aimed at reducing cognitive aging.

Electronic supplementary material

The online version of this article (doi:10.1007/s11357-014-9698-0) contains supplementary material, which is available to authorized users.

Keywords: Dentate gyrus, Synaptic plasticity, LTP, NMDA receptors, Glycine-binding site, Learning and memory

Introduction

The decline in memory abilities commonly encountered during normal aging, especially with regard to newly acquired information, significantly alters the quality of life of the elderly (Luo and Craik 2008). Because its integrity is critical for normal memory function, the hippocampal formation has been the focus of much experimental interest, and many structural and functional changes have been characterized throughout the aging hippocampus. Among the different mechanisms proposed to explain cognitive aging are deficits in synaptic plasticity such as those in the long-term potentiation (LTP) of glutamate transmission (see Barnes 2003; Billard 2006; Burke and Barnes 2010; Foster 2006; Lynch 2010; Rosenzweig and Barnes 2003 for reviews), a property of neuronal networks thought to trigger memory formation (Izquierdo 1991; Kim and Linden 2007; Lisman and McIntyre 2001; Lynch 2004; Martin et al. 2000). Although experimental evidence shows that LTP is impaired in the CA1 area (Barnes et al. 1996; Clayton et al. 2002; Foster 2002; Moore et al. 1993; Shankar et al. 1998; Turpin et al. 2011) as well as in the dentate gyrus of aged rodents (Barnes and McNaughton 1985; Barnes et al. 2000; Griffin et al. 2006; Lynch 1998), whether the underlying mechanisms in these different hippocampal regions share common features or are specific to each area is not well understood (Barnes 1994). This is a moot point considering that age-related changes in the number of synaptic contacts have been found in the dentate gyrus and CA3 but not in the CA1 (see Burke and Barnes 2010 for review), suggesting the differential susceptibility of glutamatergic circuits within the hippocampus to aging processes.

The activation of the N-methyl-D-aspartate (NMDA-R) subtype of glutamate receptors is a critical step for LTP (Collingridge and Bliss 1995; Malenka and Nicoll 1993). The amino acid d-serine, by binding to the NMDA-R glycine-binding site, regulates the expression of functional plasticity in numerous brain areas including the hippocampal CA1 (reviewed in Billard 2012). Importantly, d-serine levels are reduced in the aging hippocampus due to the weaker expression of its synthesizing enzyme, serine racemase (Potier et al. 2010). In the CA1 area, this decrease impairs NMDA-R activation and LTP expression, indicating the critical role of d-serine in the mechanisms underlying hippocampus-related cognitive aging (Haxaire et al. 2012; Mothet et al. 2006; Turpin et al. 2011). In the dentate gyrus also, NMDA-R activation and LTP expression are affected by age (Barnes et al. 2000; Diana et al. 1994; Dieguez and Barea-Rodriguez 2004; Sierra-Mercado et al. 2008), but whether d-serine is involved in these dysfunctions remains unknown. Determining if the d-amino acid significantly contributes to the age-related deterioration of functional plasticity in the different hippocampal circuits is an important issue in the search for therapeutic strategies targeting cognitive aging.

In light of the critical role of d-serine in the functional plasticity of CA1 circuits throughout life span, we therefore asked whether the d-amino acid also drives age-related changes at perforant pathway-granular cell synapses of the dentate gyrus.

Methods

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. The experiments were conducted with 4- to 6-month-old “adult” (n = 45) and 23- to 26-month-old “aged” (n = 15) male Sprague-Dawley rats purchased from Charles River (France). Rats were housed by threes in Plexiglas cages and maintained on a controlled light-dark cycle at constant temperature (22 ± 2 °C), with ad libitum access to food and water.

Electrophysiology

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 perfused with aCSF pregassed with 95 % O2/5 % CO2.

Extracellular recordings were obtained at 28–30 °C. Bipolar tungsten electrodes were used for stimulation, and 2–6-MΩ glass micropipettes were filled with 2 M NaCl for recording. Stimulation and recording electrodes were placed in the middle one third of the dentate gyrus molecular layer (500 μm apart). Presynaptic fiber volleys (PFVs) and field excitatory postsynaptic potentials (fEPSPs) were evoked, and the averaged slope of three PFVs and fEPSPs was measured in the linear part using Win LTP software (Anderson and Collingridge 2001). To evaluate the level of receptor activation, an index of synaptic efficacy (ISE) corresponding to the fEPSP/PFV slopes ratio was plotted against stimulus intensity (500 to 900 μA). In some experiments, NMDA-R-mediated fEPSPs were isolated in slices perfused with low-Mg2+ (0.1 mM) aCSF supplemented with the non-NMDA receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzoquinoxaline-7-sulfonamide (NBQX, 10 μM). 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 changes in ISE 15 min after the addition of the co-agonist to the aCSF.

In order to investigate LTP of synaptic transmission, a test stimulus was applied every 15 s in aCSF supplemented for at least 10 min with 50 μM bicuculline methiodide (Tocris, Illkirch, France). This was necessary to shut down the powerful inhibition occurring in the dentate gyrus, which counteracts the induction of synaptic plasticity (Arima-Yoshida et al. 2011). The intensity was adjusted to obtain a fEPSP with a baseline slope of 0.1 V/s. The averaged slope of three fEPSPs was measured for 15 min before the delivery of a 100 Hz conditioning stimulus (1-s duration at the test intensity). Testing with a single pulse was then resumed for 60 min to determine the level of LTP.

In pharmacological experiments, all drugs were applied to the aCSF 10 min before the establishment of the baseline and maintained throughout the recording. The drugs applied included d-2-amino-5-phosphonovalerate (d-APV, 30–50 μM), d-serine (100 μM), glycine (500 μM) and the specific antagonist of the NMDA-R glycine-binding site L689,560 (10 μM) (all from Tocris, France).

In some experiments performed in adult animals, LTP was monitored in slices preincubated for at least 90 min with 0.4 U/ml (0.4 μg/ml) recombinant pure d-amino acid oxidase (dAAO, EC 1.4.3.3), the d-serine degrading enzyme, or with 20 μg/ml purified recombinant d-serine deaminase (DsdA, EC 4.3.1.18) to remove endogenous d-serine. The purified recombinant enzymes were prepared as previously described (Fantinato et al. 2001; Shleper et al. 2005).

Data analysis

All results are expressed as means ± SEM. The significance of LTP expression was determined by comparing the 15 min of baseline recordings with values recorded between 45 and 60 min after the conditioning stimulation. The significance of changes in LTP magnitude between groups was determined by comparing the last 15 min of recordings. In order to take into account the correlations inherent in repeated measures (LTP and input/output curves), p values were calculated using multivariate analyses of variance followed by Tukey’s post hoc tests. A paired t test was used to evaluate the significance of d-serine effects on NMDA synaptic transmission. In all cases, differences were considered significant when p ≤ 0.05.

Results

d-serine contributes to NMDA-R-dependent LTP in the dentate gyrus

The action of d-serine on the NMDA-R glycine-binding site is required for the expression of synaptic plasticity in the hippocampal CA1 (Henneberger et al. 2010; Mothet et al. 2006; Yang et al. 2003), but its contribution to NMDA-R-dependent LTP processes in the dentate gyrus remains to be characterized (see Balu et al. 2013). In control aCSF containing bicuculline (see “Methods”), the 1 × 100 Hz conditioning stimulation delivered to slices from adult animals (23 slices from 21 rats) induced a significant potentiation of synaptic transmission [F1,48 = 33.7, p < 0.0001] that persisted until the end of the recording (Fig. 1). This LTP was dependent on the activation of NMDA-R glycine-binding sites since it was abolished in slices bathed with 50 μM of the selective competitive antagonist d-APV (nine slices from six rats) or with 10 μM of the selective antagonist of the glycine-binding site L689,560 (nine slices from five rats) (Fig. 1), which did not affect basal synaptic transmission (Fig. S1A). The role of d-serine was then determined in slices preincubated with the d-serine degrading enzyme dAAO to deplete endogenous d-serine content. While basal neurotransmission was not modified (Fig. S1B), LTP was completely blocked in dAAO-pretreated tissues (nine slices from six rats) but was rescued by adding saturating concentrations of d-serine to the aCSF (eight slices from five rats) (Fig. 2a, c). These results suggest that LTP deficits in dAAO-treated slices could result from the lower occupancy of glycine-binding sites by the endogenous co-agonist. However, treatment with dAAO generates hydrogen peroxide, known to affect LTP generation (Klann and Thiels 1999; Massaad et al. 2009; Serrano and Klann 2004). We therefore recorded LTP expression in slices preincubated with DsdA, a bacterial enzyme that catalyzes the deamination of d-serine into pyruvate and ammonia without generating H2O2 (Shleper et al. 2005). Similar to dAAO, treatment with DsdA did not affect basal synaptic transmission (Fig. S1C) but abolished LTP (nine slices from seven rats), and this was reversed by supplementing the aCSF with 500 μM glycine (11 slices from seven rats) (Fig. 2b). In these experiments, glycine was preferred to d-serine to reactivate the glycine-binding site to avoid fast degradation of added D-serine by DsdA.

Fig. 1.

Fig. 1

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Activation of NMDA-R glycine-binding site is necessary for LTP induction in the dentate gyrus. a Time course of mean 1 × 100 Hz-induced LTP calculated from slices (n = 23) of young rats in a control aCSF and in medium supplemented with the NMDA-R competitive antagonist d-APV (n = 9). b Time course of mean 1 × 100 Hz-induced LTP calculated from 23 slices of young rats in a control aCSF and from nine slices in medium supplemented with the competitive antagonist of the glycine binding site L689,560. For both a and b, in inserts are representative traces of fEPSPs recorded before and 60 min after the conditioning stimulation for each pharmacological condition. Bars: 10 ms and 0.5 mV. c Bar graphs illustrating the mean increase (±SEM) in fEPSP slope averaged from 45 to 60 min after the conditioning stimulation in each pharmacological condition (***p < 0.0001)

Fig. 2.

Fig. 2

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d-serine is required for LTP induction in the dentate gyrus of adult rats. a Time course of mean 1 × 100 Hz-induced LTP expressed in slices preincubated with the d-serine degrading enzyme d-amino acid oxidase (dAAO) with (n = 9) or without (n = 9) supplementation with d-serine (100 μM). b Time course of mean 1 × 100 Hz-induced LTP expressed in slices preincubated with the d-serine degrading enzyme d-serine deaminase (DsdA) with (n = 11) or without (n = 9) supplementation with glycine (500 μM). For both a and b, in inserts are representative traces of fEPSPs recorded before and 60 min after the conditioning stimulation for each enzymatic and pharmacological conditions. Bars: 10 ms and 0.5 mV. c Bar graphs illustrating the mean increase (±SEM) in fEPSP slope averaged from 45 to 60 min after the conditioning stimulation in each pharmacological condition (*p < 0.05 and ** p < 0.01)

Taken together, these results indicate that d-serine is necessary for LTP expression in the dentate gyrus, of adult animals as previously reported for the CA1 area (Balu et al. 2013; Henneberger et al. 2010; Mothet et al. 2006; Yang et al. 2003).

NMDA-R-dependent LTP is impaired in aged rats

In the next step, we attempted to determine whether LTP expression was impaired in the dentate gyrus of slices from aged animals under our experimental conditions.

In response to the conditioning stimulation, synaptic transmission immediately increased in slices from these animals (12 slices from 11 rats). However, this potentiation then slowly returned to normal, and 1 h after the tetanus, there was no statistical difference from baseline values [F1,20 = 0.86, p = 0.23]. Consequently, the magnitude of LTP was significantly decreased in aged animals when compared to adult controls [F1,36 = 17.5, p = 0.0002] (Fig. 3a).

Fig. 3.

Fig. 3

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NMDA-R-dependent synaptic activation is impaired in the dentate gyrus of aged rats. a Time course of mean 1 × 100 Hz-induced LTP expressed in slices from adult (n = 23) or aged rats (n = 12). In inserts are representative traces of fEPSPs recorded before and 60 min after the conditioning stimulation (**p < 0.01). Bars: 10 ms and 0.5 mV. b Superimposed samples traces of evoked NMDA-R-mediated fEPSPs recorded from a slice of an adult (left) and an aged rat (right) using a 600 μA (1) or 800 μA (2) current intensities and after addition of 30 μM of the NMDA-R antagonist d-APV (3). c Comparison of NMDA-R-mediated synaptic efficacy defined as the fEPSP/PFV ratio calculated at stimulus intensity of 500 to 900 μA in slices from adult (n = 11) and aged rats (n = 12) (*p < 0.05)

We have shown above that LTP induction is closely dependent on NMDA-R activation. We therefore checked whether this activation was impaired in aged animals by isolating synaptic potentials specifically generated by these ionotropic glutamate receptors (see “Methods”).

In slices from adult animals (11 slices from eight rats), PFVs and d-APV-sensitive fEPSPs were elicited by electrical stimulation (Fig. 3b). Interestingly, ISE, determined by the fEPSP/PFV ratio, was significantly reduced in slices from aged animals (12 slices in six rats) regardless of the stimulus intensity [F1,21 = 6.1, p = 0.02] (Fig. 3c). Further analysis of the ISE showed that the age difference in PFV magnitude was not statistically significant [F1,21 = 3.3, p = 0.08] whereas fEPSPs were significantly diminished [F1,21 = 15.1, p = 0.0008], indicating a predominant contribution of postsynaptic mechanisms to the age-related impairment of NMDA-R activation.

Age-related deficits in NMDA-R activation and LTP are independent of changes at the glycine-binding site

Since the impaired activation of glycine-binding sites by endogenous d-serine, previously seen in the CA1 area (Mothet et al. 2006; Potier et al. 2010), could be a putative postsynaptic mechanism, we determined the responsiveness of isolated NMDA-R-mediated synaptic responses to the exogenous application of a 100 μM saturating concentration of d-serine (Junjaud et al. 2006) in slices from both groups of animals.

In slices from adult rats (11 tested slices from seven rats), d-serine significantly increased the ISE by around 30 % (paired t test, p < 0.01), regardless of the stimulus intensity (Fig. 4a), indicating that glycine-binding sites are not saturated in dentate gyrus slice preparations. In contrast, recordings performed in slices from aged animals (12 slices from six rats) showed that the exogenous application of the NMDA-R co-agonist was unable to significantly enhance the ISE, except at the highest intensities of stimulation (Fig. 4a), suggesting that glycine-binding sites become saturated by endogenous d-serine with age in this particular hippocampal area. An age-related alteration of the NMDA-R subunit composition could also contribute to this saturation effect (Bai et al. 2004) that seems unlikely since the affinity of the GluN2 versus GluN1 subunits for d-serine is quite similar (Matsui et al. 1995; Priestley et al. 1995). Since the ISE remains significantly weaker in aged animals than in adults under conditions of saturating d-serine (Fig. 4b), one could argue that changes in glycine-binding-site occupancy do not underlie the age-related deficits in NMDA-R-dependent synaptic potentials in the dentate gyrus.

Fig. 4.

Fig. 4

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Age-related deficits of NMDA-R synaptic activation do not depend on d-serine. a Effects of d-serine (100 μM) on NMDA-R-mediated synaptic efficacy calculated at stimulus intensity of 500 to 900 μA in slices from adult (left, n = 11) and aged rats (right, n = 12) (**p < 0.01). b Comparison of NMDA-R-mediated synaptic efficacy in slices from adult and aged rats calculated in the presence of d-serine (100 μM) (**p < 0.01). c Time course of mean 1 × 100 Hz-induced LTP expressed in slices from adult (n = 14) or aged rats (n = 11) supplemented with d-serine (100 μM). In inserts are representative traces of fEPSPs recorded before and 60 min after the conditioning stimulation (**p < 0.01). Bars: 10 ms and 0.5 mV

We next checked the effects of exogenous d-serine on the aged-related impairment of LTP expression. When delivered in the presence of 100 μM d-serine, significant LTP [F1,25 = 12.6, p = 0.001] was induced in slices from adult rats (14 slices from nine rats), reaching a magnitude similar to that obtained with control aCSF [F1,40 = 0.1, p = 0.77]. In aged rats (11 slices from eight rats), providing d-serine to slices did not modify the response to the tetanus and no long-lasting potentiation was generated (Fig. 4c). Consequently, the age-related LTP impairment was maintained in the dentate gyrus even after saturating NMDA-R glycine-biding sites with d-serine [F1,22 = 8.5, p = 0.008]. A similar result was obtained by using exogenous glycine (500 μM) to activate glycine-binding sites (six slices from three animals) (not illustrated).

Presynaptic mechanisms participate in age-related deficits of basal glutamatergic synaptic transmission

We have postulated above that the age-related impairment of NMDA-R activation occurs predominantly through postsynaptic mechanisms, based on the fact that PFVs elicited by the stimulation of glutamate afferents are not statistically modified in aged animals whereas fEPSPs generated at the postsynaptic level are significantly reduced. However, the possibility of a presynaptic contribution could not be definitively ruled out, considering that extracellular recordings of isolated NMDA-R-dependent potentials require low-Mg2+ aCSF, which greatly alters fiber excitability. This experimental constraint could therefore have masked a putative age-related difference in PFVs, which, although not significant, was close to the threshold for biological relevance (p = 0.08). We therefore attempted to overcome this bias by recording synaptic potentials in the dentate gyrus in control aCSF.

In adult rat slices (35 slices from 17 rats), PFVs and fEPSPs were elicited by electrical stimulation (Fig. 5a), the latter being blocked by NBQX at the end of the recording. Under the same conditions, PFV slopes were significantly reduced in aged animals [F1,73 = 12.6, p = 0.0008] (33 slices in 12 rats), indicating that presynaptic responsiveness is indeed altered in the aged rat dentate gyrus (Fig. 5b). Interestingly, non-NMDA-mediated fEPSPs were reduced to a similar extent [F1,73 = 11.6, p = 0.0008] (Fig. 5c), and the ISE was maintained in aged individuals (Fig. 5d). These results strongly suggest that the principal contribution to the age-related decrease in basal glutamate transmission in the dentate gyrus comes from a presynaptic defect.

Fig. 5.

Fig. 5

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Changes in pre-synaptic activity underlies the age-related decrease in basal synaptic transmission. a Superimposed samples traces of evoked non-NMDA-R-mediated fEPSPs recorded in a slice from an adult (top) and an aged rat (bottom) before and after supplemenatation with the non-NMDAR antagonist NBQX (10 μM). b Plot of the mean PFV slope against the stimulus intensity calculated from slices of adult (n = 35) and aged (n = 35) rats. (**p < 0.01). c Plot of the mean fEPSP slope against the stimulus intensity calculated from slices of the same animals. (**p < 0.01). d Comparison of non-NMDA-R-mediated synaptic efficacy (IES) determined in slices from adult and aged rats for 300, 400, and 500 μA stimulus intensities.

Discussion

The present study provides new and additional evidence showing that the relative importance of the various cellular mechanisms underlying age-related deficits in hippocampus-dependent memory presents clear-cut regional differences, at least with respect to the functional properties of neuronal networks in the CA1 and dentate gyrus. Conceptually, this is not surprising, considering the specificity of the cell types and connectivity of each hippocampal region. However, the fact that glutamate transmission and NMDA-R activation mainly underlie neuronal communication and synaptic plasticity within both areas raises the possibility of common alterations during the process of aging. Our results indicate that the d-serine-dependent regulation of NMDA-R activation is necessary for LTP expression in the dentate gyrus but is not a crucial mechanism underlying age-related deficits, which depend more on changes in presynaptic afferent activity and postsynaptic receptor density. These results are in clear contrast to the predominant role of the d-serine-related pathway in the LTP deficits that take place during aging in the CA1 area (Haxaire et al. 2012; Junjaud et al. 2006; Mothet et al. 2006; Turpin et al. 2012), in which a significant contribution of presynaptic factors has been ruled out (see Burke and Barnes 2010 for review). These results therefore highlight the importance of the regional specificity of the cellular and functional alterations underlying age-related memory deficits, even at the level of a single structure such as the hippocampal formation, a major point to be taken into consideration in therapeutic strategies aimed at reducing cognitive aging.

After several decades of intensive investigations, there is now no doubt that the weaker capacity of aging neuronal networks to adapt their synaptic communication to changes in environmental conditions underlies cognitive defects, particularly memory deficits (Rosenzweig and Barnes 2003). In view of its pivotal role in memory formation, most aging studies have investigated changes in activity in the hippocampus, focusing more intensively on the CA1 area than the dentate gyrus (see Barnes 2003; Billard 2006; Burke and Barnes 2010; Foster 2006; Lynch 2010 for reviews). Taken together, these studies show that age-related deficits in synaptic plasticity affect the entire hippocampus, but whether the different hippocampal areas share the same pathways has remained unresolved (Barnes 1994). This is particularly the case with the impaired LTP seen in the CA1 area as well as in the dentate gyrus of the aging hippocampus, since changes in receptor density or pharmacological properties have mostly been studied in the former (Barnes et al. 1996, 1997; Potier et al. 2000) while the impact of cytokines has mainly been investigated in the latter (see Lynch 2010).

It has recently been found that the decrease in levels of the endogenous NMDA-R co-agonist d-serine is a critical mechanism driving LTP impairment in the CA1 of aged rats (Haxaire et al. 2012; Mothet et al. 2006; Turpin et al. 2011), indicating a possible role for the amino acid in the age-related deficits occurring in the dentate gyrus also. We show in this report that degrading endogenous d-serine through tissue incubation with dAAO impairs LTP expression, although this could be due to the production of reactive oxygen species (ROS) during degradation. However, LTP impairment by dAAO can be rescued by providing d-serine to the external medium. In addition, LTP is also compromised by DsdA, which selectively degrades d-serine without ROS production (Shleper et al. 2005). From these results, we can conclude that the activation of NMDA-R by d-serine is indeed required for LTP expression in the dentate gyrus. Such a critical role for the amino acid at this particular hippocampal location has also been recently characterized in mice knocked out for the synthesizing enzyme serine racemase, which display a 90 % reduction in cortical d-serine (Balu et al. 2013). However, a contribution of glycine cannot be definitively ruled out, since this amino acid may be significantly involved when synaptic activation is forced, such as in the LTP paradigm, as recently demonstrated in the amygdala (Li et al. 2013). Indeed, preliminary results from our group indicate that LTP is also impaired in the dentate gyrus in slices incubated with glycine oxidase, which specifically removes glycine (Billard and Wolosker, unpublished results). Nevertheless, a role for this amino acid in age-related LTP deficits is unlikely, since glycine levels do not change with age in hippocampal tissues (Mothet et al. 2006). In addition, saturating NMDA-R co-agonist binding sites with exogenous glycine does not rescue the impaired LTP displayed by aged animals. Importantly, our results also indicate that d-serine does not play a major role in these aged-related deficits, since neither impaired LTP nor reduced NMDA-R activation is reversed by providing the d-amino acid to tissues, in striking contrast to what has been found in the CA1 area (Potier et al. 2010). These results therefore indicate that the putative consequences of age-related changes in the activation of NMDA-R co-agonist binding sites are minimal in the dentate gyrus, compared to the importance of these alterations in CA1 neuronal networks.

Ours result indicate that isolated NMDA-R-dependent synaptic responses decrease with age in the dentate gyrus, as reported by previous extracellular and patch-clamp recording studies (Krause et al. 2008; Yang et al. 2008). This impairment was observed under specific experimental conditions of exacerbated excitability (through Mg2+ omission from the aCSF) in which the activation of presynaptic glutamate afferents was similar in adult and aged subjects, as well as under conditions of maximal NMDA-R activation where co-agonist binding sites were saturated with d-serine. Thus, the decrease in NMDA-R density, previously reported in the dentate gyrus of aged rodents and monkeys (Gazzaley et al. 1996; Magnusson et al. 2002; Morrison and Gazzaley 1996; Newton et al. 2007), significantly contributes to the reduction in NMDA-R activation. On the other hand, our results also indicate an age-related decrease in the magnitude of presynaptic afferent activity when synaptic responses are recorded under control conditions, as previously reported (Barnes et al. 1992; Burke et al. 2012; Yang et al. 2008). Therefore, in addition to a lower postsynaptic receptor density, the age-related decrease in NMDA-R activation also reflects weaker glutamate release consecutive to a reduction in synaptic contacts from axons originating in the entorhinal cortex (Geinisman et al. 1992; Nicholson et al. 2004; Smith et al. 2000). It is interesting to note that in our study, the decrease in the size of PFVs paralleled a similar reduction in postsynaptic potentials. This result differs from earlier data indicating that the loss of afferent synapses in the dentate gyrus during senescence is compensated for by an increase in the quantal size of the remaining synaptic inputs such that the PFV/fEPSP ratio is larger in aged animals (Barnes and McNaughton 1980; Foster et al. 1991; Yang et al. 2008). While this discrepancy could reflect differences in rat strains and/or technical protocols, our present results are validated by patch-clamp recordings showing that dendritic integration is altered in the granule cells of aged rats, resulting in weaker excitatory postsynaptic currents (Krause et al. 2008).

Besides the role of presynaptic inputs, the results of our present study also suggest that the reduced NMDA-R density could mask a possible role for d-serine. Indeed, levels of the amino acid are reported to be reduced in extracts of the whole aging hippocampus (Mothet et al. 2006; Turpin et al. 2011), and preliminary data indicating that the expression of the d-serine synthesizing enzyme serine racemase is also reduced in the dentate gyrus during aging (Latour and Denis, personal communication). We could thus speculate that despite their age-related decrease, d-serine levels are still elevated enough to saturate the low density of NMDA-R expressed in the dentate gyrus at this time (Gazzaley et al. 1996; Magnusson et al. 2002; Morrison and Gazzaley 1996; Newton et al. 2007). This could explain why providing exogenous d-serine does not enhance NMDA-R activation at this location as it does in the CA1 of aged animals (Haxaire et al. 2012; Junjaud et al. 2006; Mothet et al. 2006; Turpin et al. 2011)

To conclude, this study provides additional viewpoints concerning the complexity of changes in circuit connectivity and functionality underlying hippocampal aging and particularly the relative contribution of each of these changes with regard to the subregion considered (see also Burke and Barnes 2010). We are still far from understanding the respective importance of the various specific alterations and how they interact, but the fact that relieving each of these deficits individually is capable of reversing age-related memory impairment, as described in the literature for different processes, indicates that they all affect cognitive performance.

Electronic supplementary material

Figure S1 (93.5KB, gif)

Basal synaptic transmission is not affected by pharmacological and enzymatic manipulations of the slices. (A) Plot of the mean non-NMDA-R-mediated synaptic efficacy (IES) determined in slices from adult rats for 300, 400 and 500μA stimulus intensities before and after application of the specific antagonist of the NMDA-R glycine binding site L689,560 (10 μM). (B) Plot of the mean IES determined in control slices and in slices pretreated with the d-serine degrading enzyme d-amino acid oxidase (dAAO). (C) Plot of the mean IES determined in control slices and in slices pretreated with the degrading enzyme d-serine deaminase (DsdA). In each condition are presented superimposed samples traces of evoked non-NMDA-R-mediated fEPSPs recorded in control condition and in the presence of the drug using a 400μA current intensity. (GIF 93 kb)

High Resolution image (TIFF 159 kb) (159.9KB, tif)

Acknowledgment

This manuscript was prepared with editorial help from Gap Junction (www.gap-junction.com). This work was supported by the National Institute of Health and Medical research (J-M.B) and by the Israel Science Foundation, Legancy Heritage Fund and the Allen and Jewel Prince Center for Neurodegenerative Disordres of the brain (H.W)

Conflict of interest

All authors state that they have no actual or potential conflicts of interest, including financial, personal, or other relationships with concerned people or organizations within 3 years of beginning the work reported here.

Abbreviations

aCSF

Artificial cerebrospinal fluid

dAAO

d-amino acid oxidase

fEPSP

Field excitatory postsynaptic potential

d-APV

d-2-amino-5-phosphonovalerate

DsdA

d-serine deaminase

IES

Index of synaptic efficacy

KO

Knockout

LTP

Long-term potentiation

NBQX

2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzoquinoxaline-7-sulfonamide

NMDA-R

N-methyl-d-aspartate receptor

PFV

Presynapic fiber volley

SR

Serine racemase

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1 (93.5KB, gif)

Basal synaptic transmission is not affected by pharmacological and enzymatic manipulations of the slices. (A) Plot of the mean non-NMDA-R-mediated synaptic efficacy (IES) determined in slices from adult rats for 300, 400 and 500μA stimulus intensities before and after application of the specific antagonist of the NMDA-R glycine binding site L689,560 (10 μM). (B) Plot of the mean IES determined in control slices and in slices pretreated with the d-serine degrading enzyme d-amino acid oxidase (dAAO). (C) Plot of the mean IES determined in control slices and in slices pretreated with the degrading enzyme d-serine deaminase (DsdA). In each condition are presented superimposed samples traces of evoked non-NMDA-R-mediated fEPSPs recorded in control condition and in the presence of the drug using a 400μA current intensity. (GIF 93 kb)

High Resolution image (TIFF 159 kb) (159.9KB, tif)

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