Subunit contribution to NMDA receptor hypofunction and redox sensitivity of hippocampal synaptic transmission during aging

Decreased NMDAR synaptic currents during aging

Whole-cell patch-clamp recordings of synaptically evoked NMDAR-mediated EPSCs were obtained from CA1 pyramidal cells of hippocampal slices obtained from young (11/4 cells/animals) and aged (9/4 cells/animals) animals. For a subset of cells, isolation of NMDA currents was confirmed by bath application of AP-5 (100 µM) (Suppl. Fig. 1). The EPSCs were recorded at holding voltages between -60 and +60 mV in 20 mV steps. No age-related difference was observed for intrinsic properties, including access resistance, membrane resistance, and capacitance (Table 1). For negative voltages, currents were inward with reduced amplitudes, consistent with Mg²⁺ blockade of the NMDAR channel. The reversal potential was calculated from a regression of responses through -20, 0, and +20 mV. The reversal potential of the synaptically evoked current was near 0 mV, consistent with NMDAR permeability to multiple cations, and no age-related difference was observed for the reversal potential (young: -6.9 ± 3.1 mV; aged: -3.4 ± 3.2 mV). A repeated measures Analysis of variance (ANOVA) on EPSC amplitudes across the voltage steps indicated a tendency (p = 0.051) for an interaction of age and membrane potential due to an increase in outward currents for young animals at positive potentials (Fig. 1 A, Suppl. Fig. 2 A & B).

To examine synaptic input-output relationships, cells were held at +40 mV and NMDAR EPSCs were recorded across a range of stimulation intensities (5, 10, 15, 20, 25, 30, 40, and 60 V). For each stimulation intensity, the peak amplitude of the EPSC was obtained from CA1 cells of aged (n = 24/14 cells/animals) and young (n = 25/11 cells/animals) animals. A repeated measures ANOVA across stimulation intensities indicated an interaction of age and stimulation intensity [F(7,329) = 6.58, p< 0.0001] and the age difference was due to larger peak EPSC in young animals (Fig. 1 B, C). The results confirm that NMDAR synaptic responses are reduced during aging [15–18].

The GluN2A/GluN2B subunit composition is not altered with advanced age

The subunit composition can be studied by examining the time course of NMDAR synaptic responses. An increase in the ratio of GluN2A/GluN2B subunits results in a more rapid decay, producing a shortened synaptic response [25,33]. To examine age differences in the decay of the synaptic response, the EPSC responses for 30, 40, and 60 V stimulation were normalized to the peak of the response, and the time to decay to 50% was calculated. A small, nonsignificant, decrease in the decay rate was observed for aged animals, suggesting that aging is not associated with a large shift in the GluN2A/ GluN2B ratio (Fig. 1 D).

Next, we employed GluN2 selective antagonists to examine age differences in the contribution of GluN2 subtypes to the EPSC peak synaptic response. Again, cells were held at +40 mV. Following at least five-minutes of stable baseline recording of the isolated NMDAR EPSC, the GluN2B selective antagonist, ifenprodil (5 µM), was bath applied. Ifenprodil decreased the peak EPSC by ~30%, and an ANOVA on percent of baseline for the peak EPSC, measured 15 min following application of ifenprodil, indicated no age difference (young: 70.59 ± 8.14 mean ± SEM% of baseline, n = 4/4 cells/animals; aged: 71.58 ± 10.39%, n = 6/5 cells/animals) (Fig. 2 A & B). The decrease was specific to ifenprodil, as no effect was observed following application of vehicle alone.

Similarly, bath application of the GluN2A selective antagonist, NVP (0.4 µM), reduced the peak NMDAR EPSC by ~45% over the next 15 min. No change in the peak EPSC was observed in the absence of NVP, recorded over the same duration. An ANOVA on percent of baseline for the peak EPSC, measured 15 min following application of NVP, indicated no age difference in the reduction of the EPSC (young: 52.10 ± 7.28% of baseline, n = 4/2 cells/animals; aged: 57.14 ± 9.2%, n = 6/3 cells/animals) (Fig. 2 C & D).

The DTT-mediated potentiation in NMDAR currents involves an increased contribution of GluN2B

Following bath application of the reducing agent DTT (0.5 mM), input-output curves of the peak NMDAR EPSC were again obtained (aged: n = 7/3 cells/animals, young: n = 8/2 cells/animals) and compared to input-output curves for the respective control condition for young and aged animals (Fig. 1C). A repeated measures ANOVA across stimulation intensities for the control condition and DTT conditions, within each age group, indicated an effect of treatment on the peak EPSC for aged animals [F(1,203) = 53.24, p < 0.0001], but not for young animals [F(1,217) = 0.49, p > 0.05] (Fig. 3A & B), confirming an age-related difference in the DTT-induced potentiation of the NMDAR response.

To determine if DTT influenced the decay rate of the NMDAR response, the NMDAR EPSC in response to 30, 40, and 60 V stimulation was normalized to the peak of the response at each stimulation intensity, and the time to decay to 50% of the peak was calculated in the presence of DTT and compared to the control condition. Bath application of DTT increased the time to half-decay ~4 fold in aged animals and ~2 fold in young animals (Fig. 3 C). An ANOVA repeated across stimulation intensities indicated a treatment effect [F(1,106) = 22.76, p < 0.0001] in the absence of an age difference or an interaction of age and treatment. ANOVAs within each age group confirmed that DTT increased the time to half-decay of the EPSC for aged [F(1,52) = 12.61, p < 0.005] and young [F(1,54) = 11.55, p < 0.005] animals.

To examine the contribution of GluN2A/GluN2B subunit to DTT-induced potentiation in NMDAR function, subunit selective antagonists were applied to slices obtained from aged animals in the presence of DTT. Figure 3D shows input-output curves collected from cells of aged animals exposed to DTT alone (n = 7/3 cells/animals), or in the presence of DTT followed by application of NVP (n = 5/2 cells/animals) or ifenprodil (n = 5/3 cells/animals). A repeated measures ANOVA across stimulation intensities indicated an interaction of stimulation intensities and treatment [F(12,84) = 5.01, p < 0.0001]. Post hoc comparisons for treatment effects indicated NVP tended (p = 0.055) to decrease the peak response to ~65%, relative to the DTT alone condition. The effect of ifenprodil was more robust, decreasing the peak response (p < 0.005) to ~45% of the DTT alone condition (Fig. 3D). Accordingly, in contrast to the greater effect of NVP under controls conditions, ifenprodil reduced the peak response to a greater extent in the presence of DTT. Thus, DTT application increased decay time and increased sensitivity to ifenprodil in aged animals, suggest an increase contribution of GluN2B to the NMDAR synaptic response.

Redox regulation of NMDAR function depends on NMDAR activity

Next, we sought to determine if the DTT-mediated growth of the NMDAR response in aged animals represents an activity-dependent NMDAR plasticity. An influx of Ca²⁺ following NMDAR activation induces long-term modifications in NMDAR function in young animals [34,35]. Due to the inhibition of Ca²⁺ entry for cells held at +40 mV, we employed extracellular fEPSP recordings of isolated NMDAR synaptic responses to examine the role of NMDAR activity and Ca²⁺ on the DTT-mediated potentiation of the NMDAR synaptic response in slices from aged animals. Control recordings, examining the effects of DTT alone, were interleaved between recordings in which the GluN2A or GluN2B selective antagonist were applied, prior to DTT application. For control slices (n = 31slices/26 aged animals), DTT (0.5 mM) induced an increase (161.2 ± 6.2% mean ± SEM of baseline) in the NMDAR-mediated synaptic response, similar to previous reports [6,8,9,13] (Fig. 4).

The GluN2B antagonists, ifenprodil (5 µM, n = 8/8 slices/animals) or Ro 25-6981 (5 µM, n = 5/5 slices/animals) were added to the bath at least 45 min prior to application of DTT. In another set of slices (8 slices from 4 animals), the concentration of Ca²⁺ in the bath was increased from 2 mM to 3 mM, and ifenprodil was added to the bath at least 45 min prior to application of DTT. An ANOVA on the percent increase in the synaptic response, 60 min following application of DTT indicated a treatment effect [F(3,48) = 5.7, p = 0.02]. Post hoc tests indicated that, in 2 mM Ca²⁺ recording medium, ifenprodil (120.0 ± 2.5%) and Ro 25-6981 (120.8 ± 10.0%) inhibited the DTT-induced growth of the NMDAR-mediated synaptic responses, relative to the control condition (Fig. 4 A & B). In contrast, when the level of Ca²⁺ was increased to 3 mM, ifenprodil failed to attenuate the DTT-induced potentiation of NMDAR-mediated synaptic responses (155.2 ± 8.7%) (Fig. 4 A & B).

To determine the contribution of GluN2A to the DTT-induced potentiation of the NMDAR response, NVP (0.4 µM, n = 7/6 slices/animals) was added to the bath 45 min prior to application of DTT. For some slices (n = 8/4 slices/animals), the concentration of Ca²⁺ in the bath was increased from 2 mM to 3 mM, and NVP was added to the bath 45 min prior to application of DTT. An ANOVA on the percent change in the response, 60 min after DTT application, indicated a difference across treatment groups [F(3,45) = 4.48, p = 0.0078]. Post hoc tests indicated that NVP in 2 mM Ca²⁺ significantly attenuated the DTT-mediated growth (113.3 ± 6.4%) relative to controls (Fig. 4 C & D). Again, raising the level of Ca²⁺ was able to overcome the NMDAR antagonist inhibition of the DTT-mediated growth in the synaptic response. Together, the results indicate that the DTT-mediated growth of the NMDAR response depends on the level of NMDAR activation and Ca²⁺ entry.

One possibility is that DTT increased Ca²⁺ entry through GluN2A containing receptors by chelating Zn²⁺ [24,36–38]. However, addition of excess Zn²⁺ (ZnCl₂ 1 µM, n = 3/3 slices/animals) failed to block the effects of DTT, which increased the response (160.3 ± 8.4%) (Fig. 5A). In addition, bath application of the Zn²⁺ chelating agent ZX1 (100 µM, n = 11 slices/6 animals) failed to enhance the NMDAR synaptic response (103.5 ± 3.99%) (Fig. 5B). In contrast, when DTT was subsequently applied 60 min following application of ZX1, a significant (p < 0.0001) increase (143.6 ± 8.79%, n = 11 slices/6 animals) in NMDAR response was observed, suggesting that DTT effects are not due to Zn²⁺ chelation (Fig. 5C).

In young animals, influx of Ca⁺² through NMDARs results in CaMKII-mediated trafficking and insertion of NMDAR subunits into the postsynaptic membrane [27,28], and the DTT-mediated increase in the NMDAR response in older animals is blocked by inhibition of CaMKII [6]. In addition, CaMKII activity determines the long-term maintenance of synaptic strength [32,39]. Oxidizing agents decrease the NMDAR synaptic response, specifically in young animals [6,14,40]. To determine whether ongoing CaMKII activity is involved in the decrease of NMDAR responses under oxidizing conditions, slices from young animals were exposed to the CaMKII inhibitor KN-62 (10 µM) or dimethyl sulfoxide (DMSO) vehicle for at least 60 min before application of the oxidizing agent, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) (0.5 mM). Bath application of DTNB in the presence of vehicle decreased the NMDAR-fEPSP (66.3 ± 4.9%, 7/7 slices/animals) 60 min following drug application. The DTNB-induced decrease in the NMDAR-fEPSP was blocked by the CaMKII selective antagonist, KN-62 (101.6 ± 2.9%, 7/7 slices/animals) [F(1,12) = 37.97, p < 0.0001], suggesting that the basal level of kinase activity determines the effectiveness of redox modulators on the NMDAR response (Fig. 6 A & B).

Animals

Procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of University of Florida and were in accordance with guidelines established by the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals. Male Fischer 344 rats, young (4–6 months, n = 30) and aged (24-26 months, n = 95), were obtained from the National Institute on Aging colony at Harlan (Indianapolis, IN, USA).

Whole Cell Patch Clamp Recordings

Rats were anaesthetized with intraperitoneal injection of xylazine (10 mg/kg) and ketamine (100 mg/kg). Isoflurane (5%) was administered for 5 min, and following no indication of a withdrawal reflex, animals were perfused transcardially with an ice-cold sucrose-cutting solution (~50 ml) containing (in mM): 206 sucrose, 2 KCl, 25 NaHCO₃, 1.2 NaH₂PO₄, 1 CaCl₂, 1 MgSO₄, 0.01 glycine, and 10 D-glucose saturated with 95% O₂/5% CO₂. Following decapitation, brains were removed and placed in ice-cold sucrose-cutting solution for 2 minutes. Horizontal hippocampal sections of 350 µm were generated using a Lecia vibratome 3000 (Buffalo Grove, IL, USA) and sections were immediately transferred to a holding chamber with artificial cerebrospinal fluid, at 32-36ºC, containing (in mM): 124 NaCl, 2.5 KCl, 25 NaHCO₃, 1.23 NaH₂PO₄, 1 CaCl, 3 MgSO₄, and 10 D-glucose saturated with 95% O₂/5% CO₂. Sections were allowed to equilibrate for at least 30 min prior to being transferred into the recording chamber. The recording chamber was perfused (2ml/min) with recording medium (in mM): 126 NaCl, 3 KCl, 25 NaHCO₃, 1.2 NaH₂PO₄, 2.4 CaCl₂, 1.5 MgSO₄, and 11 D-glucose, saturated with 95% O₂/5% CO₂, warmed to 30ºC. Prior to patch-clamp recording, sections were allowed to equilibrate for at least 20-minute in the recording chamber.

The recording chamber was mounted on a Burleigh-Gibarltar stage (Thor labs, Newton, NJ, USA) attached to an Olympus BX51WI microscope with infrared differential interference contrast optics (Shinjuku, Tokyo, Japan). Sections were visualized at 10x using a Hamamatsu C4742-95 digital camera (Hamamatsu City, Shizuoka Pref., Japan) attached to a Dell Optiplex 7010 running HCImage Live software (Hamamatsu) on a Windows 7 OS. Glass micropipettes (Sutter, Novato, CA, USA) were filled with a CsMeS0₃- based solution containing (in mM): 140 CsMeS0₃, 8 NaCl, 1 MgCl₂, 0.2 EGTA, 10 HEPES, 2 Mg-ATP, 0.3 Na-GTP, 5 QX-314, pH 7.3 (CsOH). NMDAR EPSCs were isolated by addition of 6,7-dinitroquinoxaline-2,3-dione (DNQX, 30 μM, Cayman Chemical) and picrotoxin (PTX, 20 μM, Tocris) to the recording medium. Open-tip glass pipette resistances ranged from 3-6 MΩ. Pipettes were placed directly over CA1 pyramidal cell bodies at 10x (~0.2mm from the stimulator electrode) and then a 40x water-emersion objective was used to visualize contact with cell membranes. Whole-cell voltage clamp recordings were obtained with an Axon Multiclamp700B (Molecular Devices, Sunnyvale, CA, USA). Signals were sampled at 10kHz, filtered at 1kHz, and digitized with a Digidata 1440A (Molecular Devices) using Clampex (v.10.2, Molecular Devices). Cells were held at -60mV while access resistance, membrane resistance, and whole-cell capacitance were measured using a -10mV step protocol. Average whole-cell parameters are presented in Table 1. Cells were not included in the final analyses if access resistance changed more than ~35% during the experiment. A concentric bipolar stainless steel electrode was positioned in stratum radiatum and 50 µsec bipolar pulses (0.033 Hz) from a Grass stimulator (SD9) were used to evoke EPSCs and generate stimulation voltage-peak EPSC curves. For pharmacological studies, stimulation intensity was adjusted in order to obtain ~50% maximum EPSC and a baseline response was recorded prior to drug application.

Extracellular field potential recordings

Methods for hippocampal slice preparation and electrophysiological recording of total and NMDAR-mediated synaptic responses have been published previously [6,8]. Briefly, hippocampi were harvested and slices (~400 µm) cut parallel to the alvear fibers. Slices were placed in a recording chamber and bathed in 30 ± 0.5^oC oxygenated recording medium (in mM): NaCl 124, KCl 2, KH₂PO₄ 1.25, MgSO₄ 2, CaCl₂ 2, NaHCO₃ 26, and glucose 10. Extracellular fEPSP from stratum radiatum of CA1 were recorded with glass micropipettes (4-6 MΩ) filled with recording medium. A concentric bipolar stainless steel electrode was positioned ~1 mm away in the middle of the stratum radiatum. Field potentials (0.033 Hz) were evoked by biphasic stimulus pulses (100 μsec). Signals were amplified, filtered (1 Hz and 1 kHz), and stored on computer for off-line analysis. For analysis, two cursors were placed around the initial descending phase of the waveform and the maximum slope (mV/ms) of the fEPSP was determined by a computer algorithm that found the maximum change across all sets of consecutively recorded points (20 kHz sampling rate) between the 2 cursors.

The NMDAR-mediated component of synaptic transmission (NMDAR-fEPSP) was isolated by incubating slices in recording medium containing low Mg²⁺ (0.5 mM), DNQX (30 μM), and PTX (10 μM) for at least 60 minutes. For pharmacological studies, the stimulation intensity was adjusted to evoke a response ~50% of maximum and baseline NMDAR-mediated synaptic responses were recorded for at least 10 min prior to and for 60 min after drug application.

Pharmacological agents

Other pharmacological agents were bath applied at final concentrations of: DTT (0.5 mM, Sigma), DTNB (0.5 mM, Sigma), 2-amino-5-phosphonopentanoic acid (AP5, Sigma, 100 µM), NVP-AAM077 (NVP, 0.4 µM, Sigma), ifenprodil (5 µM, Sigma), Ro 25-6981 (5 µM, Tocris), ZnCl₂ (1 µM, Sigma), ZX1 (100 µM, Strem Chemicals), and 4-[(2S)-2-[(5-isoquinolinylsulfonyl)-methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl)propyl] phenyl isoquinolinesulfonic acid ester (KN-62, 10 µM, Tocris). DTT, Ro 25-6981, NVP, ZnCl₂, and ZX1 were directly dissolved in the recording medium. DNQX was initially dissolved in DMSO (Sigma) and diluted in recording medium to a final DMSO concentration of <0.01%. PTX, DTNB, and ifenprodil were dissolved in ethanol and diluted in recording medium to a final ethanol concentration of 0.0001%.

Statistical analysis

ANOVAs were carried out using StatView 5.0 (SAS Institute) in order to determine significant main effects and interactions. Post hoc ANOVAs and Fisher’s protected least significant difference comparisons, with the p-value set at 0.05, were used to further localize significant differences. In cases of multiple comparisons (e.g. baseline responses relative to DTT or NMDAR antagonists), Bonferroni corrections were applied.