Coronal brain slices containing the hippocampus were obtained from 21- to 28-day-old Sprague–Dawley rats. Prior to decapitation, animals were anesthetized with isofluorane (4%, 2 L/min O₂ flow rate), in conformity with the guidelines of the Canadian Council of Animal Care. The brain was removed and placed in an oxygenated (95% O₂/5% CO₂) physiological solution, artificial cerebrospinal fluid (ACSF), at 4°C containing (mM) 126 NaCl, 2.5 KCl, 1 MgCl₂, 26 NaHCO₃, 1.25 NaH₂PO₄, 2 CaCl₂, 1 ascorbic acid, and 10 glucose (300 mOsm, pH = 7.3).

A tissue block containing the hippocampal region of interest was sectioned into 300 μm thick slices using a vibrating microtome (Vibratome Series 300, Vibratome, Bannockburn, IL, USA). Slices were incubated in an oxygenated submersion chamber at room temperature for a minimum of 1 h prior to recording.

Voltage-clamp experiments were performed at room temperature using borosilicate pipettes filled with a pipette solution containing (in mM) 130 Cs⁺-methanesulfonate, 10 N-2-hydroxy-ethylpiperazine- N′-2-ethanesulfonic acid (HEPES), 10 CsCl, 2 MgCl₂, 2 ATP-Mg, 0.2 GTP-tris(hydroxy-methyl)aminomethane, 5 lidocaine N-ethylbromide (QX-314), 5 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; BAPTA). The pH was adjusted to 7.2 with CsOH and osmolarity was adjusted to 280–290 mOsm. The liquid junction potential was measured (∼10 mV) and membrane potential (Vm) was corrected accordingly. Pipettes had a resistance of 3–5 MΩ. Whole-cell access resistance ranged from 5 to 15 MΩ and was monitored throughout the experiments.

Recordings were obtained from PCs and Ints in the stratum pyramidale and stratum radiatum of the CA1 region of the hippocampus, respectively (Figure 1; also see Discussion). We recorded from Ints located ∼250–300 μm from the stratum pyramidale. Following the interneuron classification (Klausberger, 2009), we suggest that the Ints we recorded were Schaffer collateral-associated cells and/or apical dendrite innervating cells which express cholecystokinin (i.e., CCK-expressing Ints; Klausberger, 2009). The soma of Schaffer collateral-associated cells is located mainly in stratum radiatum with dendrites spanning all layers (Figure 1). The axons of these cells innervate the oblique and to a lesser extent basal dendrites of CA1 PCs and Ints mainly in stratum radiatum (Klausberger, 2009). Apical dendrite innervating cells have soma, dendritic, and axonal distributions very similar to those of Schaffer collateral-associated cells (Figure 1). However, electron microscopic investigations have indicated that the apical dendrite-targeting cells innervate preferentially the main apical shaft of CA1 PCs (Klausberger et al., 2005).

Whole-cell patch-clamp recordings were acquired using a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA, USA) under visual control using differential interference contrast and infrared video microscopy (IR-DIC; Olympus BX50WI; Olympus Canada, Inc., Markham, ON, Canada). Whole-cell currents were recorded from individual PCs and Ints voltage-clamped at −70 and −30 mV.

Post-synaptic responses were evoked by electrical stimulation of the Schaffer collaterals with a bipolar microelectrode positioned in the stratum radiatum. Stimulation, consisting of 300 μs uration current pulses (0.1–1 mA; 0.1 Hz), was adjusted to evoke EPSC amplitudes in the range of 60–120 pA at Vm = −70 mV in ACSF.

To isolate the NMDAR-mediated component of evoked responses we used normal ACSF (at −30 mV) or ACSF containing a low concentration of MgCl₂ (0.1 mM) with osmolarity maintained by CaCl₂ (at −70 mV), and the 2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl)propanoic acid receptor (AMPAR) antagonist 1,2,3,4-tetrahydro-6-nitro-2,3-dioxobenzo[f]quinoxaline-7-sulfonamide (NBQX, 20 μM), the GABA_A receptor antagonist picrotoxin (50 μM), and the GABA_B receptor antagonist 3-[[(3,4-dichlorophenyl)methyl]amino]propyl]diethoxymethyl)phosphinic acid (CGP 52432, 10 μM; Martina et al., 2003).

One pitfall with the patch-clamp technique is the distortion of voltage-gated currents from neurons in brain slices due to non-uniform space-clamp control. To reduce current attenuation due to space-clamp that can occur when recording voltage-gated currents from neurons in brain slices, we recorded the CA1 neurons using a pipette solution containing Cs²⁺ and QX-314, to block K⁺ and Na²⁺ conductances. The additional blockage of AMPAR, GABA_A receptor, and the GABA_B receptor further reduced space-clamp attenuation. We also tried to obtain recordings with very low series resistances and compensate them as much as possible (90%). Attention was also given to the shape of the evoked responses during off-line analysis (Williams and Mitchell, 2008). Collectively, these measures ensured that space-clamp errors were minimal.

To isolate the AMPAR-mediated component of evoked responses, we used a pipette solution containing (in mM) 130 K⁺-gluconate, 10 HEPES, 10 KCl, 2 MgCl₂, 2 ATP-Mg, 0.2 GTP, and normal ACSF containing the NMDAR antagonist dl-2-amino-5-phosphonovaleric acid (AP5; 50 μM), picrotoxin (50 μM), and CGP 52432 (10 μM).

A paired-pulse stimulation paradigm was used to evaluate the locus of action of memantine. It is known that alterations in the paired-pulse ratio of evoked post-synaptic currents (EPSCs) following drug application are an indication of a pre-synaptic action of the drug. We delivered two stimuli with an inter-stimuli interval of 100 ms. The paired-pulses were repeated 30 times with an interval of 10 s, and then averaged for analysis. The paired-pulse ratio was determined by dividing the peak amplitude of the second evoked NMDAR current by the peak amplitude of the first (peak 2/peak 1; Thomson, 2000).

Kinetic analysis was performed on averaged evoked EPSCs (30 consecutive traces). The rise-times of NMDAR currents were measured at 10–90%. Their decays were fitted with the exponential functions: y = A_f exp(−t/τ_f) + A_s exp(−t/τ_s) for double- and y = A1exp(−t/τ) for single exponential decay, where A is the amplitude, τ is the decay time-constant, and the subscript f and s denote fast and slow components, respectively. Weighted time-constants (τ_mean) were calculated using the equation: τ_mean = [A_f/(A_f + A_s)]τ_f + [A_s/(A_s + A_f)]τ_s (Stocca and Vicini, 1998). The dose-response data for memantine on PCs and Ints were fitted with the Hill equation: Y = Bottom + [(Top + Bottom)/1 + 10^{(LogIC50−X) × HillSlope}]. The variable Bottom is the Y value at the bottom plateau (0), Top is the Y value at the top of the plateau (100), and LogIC₅₀ is the X value when the response is halfway between Bottom and Top. The variable HillSlope (Hill coefficient) describes the steepness of the curve. Data were collected using pClamp 10 software (Molecular Devices, Sunnyvale, CA, USA). Analyses were performed off-line with IGOR software (WaveMetrics, Inc., Lake Oswego, OR, USA). Statistical significance of the results was determined with paired Student’s t-tests (two-tailed). All values are expressed as means ± SEM, and a p-value of <0.05 was considered significant.

All drugs were obtained from Sigma-Aldrich (St Louis, MO, USA), with the exception of NBQX and GCP 52432 which were purchased from Tocris Bioscience (Minneapolis, MN, USA), and NVP-AMM077 which was a kind gift from Dr. Yves Auberson (Novartis, Basel, Switzerland). NVP-AAM077, Ro25-6981, and memantine were applied until the amplitude of the NMDAR currents reached a stable plateau. To establish that a plateau for the effect of memantine was attained (i.e., that equilibrium blockade was achieved), the average responses of two consecutive recordings of 5 min of evoked NMDAR responses needed to display the same amplitude and shape. For every neuron, memantine was applied until a plateau was reached. Longer times were required for low memantine concentrations to attain this plateau (i.e., see Figure A1 in Appendix for 1 μM memantine).

The electro-responsive properties of PCs and Ints were studied in current-clamp by applying 1000 ms current pulses from resting membrane voltage (Vm) in normal ACSF. Whole-cell recordings were obtained using pipettes filled with a solution containing (mM): 130 K⁺-gluconate, 10 HEPES, 10 KCl, 2 MgCl₂, 2 ATP, and 0.2 GTP. The amplitude of current pulses was varied in increments of 10 pA. The input resistance (Rin) was estimated in the linear portion of current–voltage plots. The membrane time-constant (τ) was derived from single exponential fits to voltage responses in the linear portion of current–voltage relations. The spike amplitude and the spike amplitude at half-duration were measured from the first action potential evoked by a current pulse.

In some experiments, recorded neurons were identified by including Lucifer Yellow (2 mM) in the pipette. The slices were removed from the chamber and fixed for 1–3 days in 0.1 M phosphate-buffered saline, pH 7.4, containing 4% paraformaldehyde. Slices were washed in dimethyl-sulfoxide (DMSO) for 1 h and visualized with a Fluo-View FV1000 Olympus confocal microscope (Olympus Canada, Inc., Markham, ON, Canada) using 10× and 40× water immersion objectives. Three-dimensional reconstructions of the neurons were made from z-series optical sections using the Olympus FV10-ASW Viewer.

Cells were visually identified in hippocampal slices with IR-DIC and selected for recordings on the basis of their morphology and localization in specific layers of the CA1 region (see Materials and Methods). PCs and Ints were recorded in the pyramidal layer and stratum radiatum, respectively (Figure 1). A total of 11 Ints and 10 PCs were morphologically identified using Lucifer Yellow (see Materials and Methods). In the experiments designed to record Ints in the stratum radiatum, pipettes were positioned over the ovoid somatic profile. All cells chosen in this way (n = 11) were found to be Ints with a-spiny, very sparsely spiny, or varicose dendrites (Figure 1C; Freund and Buzsáki, 1996). Whole-cell recordings were obtained in current-clamp mode to examine the intrinsic membrane properties of PCs (n = 10) and Ints (n = 11). The PCs generated spike trains that exhibited frequency adaptation when depolarized (Figure 1D). The Ints could sustain high firing rates without or with various degrees of accommodation (Figure 1E). In addition, they had significantly pronounced after-hyperpolarization (I_{ahp fast}, 10 vs. 2 mV in PCs, p = 0.001; I_{ahp slow}, 14 vs. 3 mV in PCs, p = 0.00001; Table 1 and Figure 1D inset) and I_h current (sag at −100 pA current step: 13.5 ± 1.7 vs. 2.9 ± 0.4 mV in PCs, p = 0.0007; Figures 1D,E). A summary of the electrophysiological properties of the PCs and Ints recorded in this study is provided in Table 1. In Ints, resting membrane potential (Vm) was significantly more depolarized (−57 vs. −69 mV in PCs, p = 0.00078), action potential amplitude (spike amplitude) was significantly smaller (69 vs. 96 mV in PCs, p = 0.00023), and input resistance (Rin) was significantly larger (457 vs. 96 MΩ in PCs, p = 0.0000012), while the duration of the action potential at half-amplitude was not significantly different (1.7 vs. 1.6 ms in PCs, p = 0.59). These data are in agreement with that previously reported by Martina et al. (2003).

To test the effect of memantine on neurons regulating excitation-inhibition balance in the hippocampal neuronal circuitry, we recorded NMDAR currents in PCs and Ints (Figure 2), and measured the effect of the bath application of memantine at different concentrations. To evoke post-synaptic glutamatergic currents in PCs and Ints, the Shaffer collaterals were stimulated with a bipolar electrode. The NMDAR-mediated component of the post-synaptic current was pharmacologically isolated at Vm = −70 mV in a low Mg²⁺ACSF (see Materials and Methods). The isolated NMDAR currents displayed an averaged amplitude of 57.46 ± 9.10 pA (n = 62) in PCs and 28.00 ± 2.65 pA (n = 54) in Ints. Memantine attenuated NMDAR current amplitudes in both PCs (Figure 2A) and Ints (Figure 2B); see Figure 2 legend for percentages of reduction). Memantine antagonism was concentration-dependent, with IC₅₀ values of (LogIC₅₀ = −5.11) 7.7 μM and (LogIC₅₀ = −4.77) 17 μM for PCs and Ints, respectively (Figure 2C). The two IC₅₀ values were significantly different (p = 0.01326; non-parametric Wilcoxon Rank Test). In addition to the higher affinity of memantine for NMDARs in PCs vs. Ints, the fitting parameters of the Hill equation showed significantly different Hill coefficients (see Materials and Methods) in PCs (Hill coefficient = 0.6101 ± 0.07) and Ints (Hill coefficient = 1.106 ± 0.16; p = 0.0086; Figure 2C). A Hill coefficient lower than one indicates positive cooperativity, suggesting that the PCs may possess at least two subtypes of NMDAR, one of which appears to have a higher affinity for memantine, whereas Ints (negative cooperativity) may express a single NMDA receptor subtype with a lower affinity for memantine.

To rule out the possibility that these differences were attributable to memantine-mediated changes in neurotransmitter release, we recorded NMDAR currents in PCs and delivered paired-pulse stimulation (100 ms) to the Schaffer collaterals in the absence and presence of memantine (10 and 300 μM; see Materials and Methods). In the absence and presence of memantine (10 and 300 μM) the second response showed facilitation. The evoked currents had peak 2/peak 1 ratios of 2.32 ± 0.15 (n = 8) in the absence of memantine and did not change in presence of memantine at 10 μM 2.24 ± 0.07 (n = 4) and 300 μM 2.12 ± 0.09 (n = 4), respectively (Figure A2 in Appendix). These values were not significantly different (p = 0.69 and p = 0.36 for 10 and 300 μM memantine, respectively), demonstrating that memantine does not alter neurotransmitter release by acting on pre-synaptic afferents.

To further explore the possibility of a memantine effect on neurotransmitter release we used the NMDA open-channel blocker MK-801. Rosenmund et al. (1993) showed that the rate of progressive MK-801 block of NMDAR currents reflects the probability of pre-synaptic neurotransmitter release. If the probability of release is high, more terminals will release neurotransmitter, more post-synaptic NMDAR channels will open, and the MK-801 progressive block will be more rapid. Consistent with this hypothesis, we measured the progressive MK-801 (5 μM) block of NMDAR currents before and after application of memantine (10 μM) in PCs and Ints to estimate the effect of memantine on the probability of pre-synaptic release. NMDAR currents were evoked every 5 s and MK-801 time-course measured by the fitting of a single exponential [y = A1exp(−t/τ)] of the NMDAR current peak amplitude plotted against stimulation number (Figure A3 in Appendix). The MK-801 block rate was 250 stimuli in PCs (n = 8) and 318 stimuli in Ints (n = 7), suggesting a higher probability of release in PCs compared to Ints (Figure A3 in Appendix). This is not surprising considering that the non-uniform size and structure of synaptic terminals in the central nervous system results in variable probabilities of release at terminals within a single synapse, and is not the same for different terminals on different types of neurons. Next, memantine (10 μM) was applied until NMDAR currents attenuated to a plateau, then MK-801 was co-applied (Figure A3 in Appendix). The block rate of MK-801 + memantine was 214 stimuli in PCs (n = 4) and 275 stimuli in Ints (n = 8). These values were not significantly different from those obtained with MK-801 alone (PCs: p = 0.35; Ints: p = 0.91). These results support the previous finding that memantine had no effect on pre-synaptic release probability.

To ensure that the observed effects of memantine were specific to NMDARs, we evoked AMPAR currents in the absence and presence of memantine (10 μM; see Materials and Methods). Evoked AMPAR currents had an activation time-constant of 2.47 ± 0.73 ms and a deactivation kinetic of 20.18 ± 2.7 ms (n = 4). After 25 min of continuous memantine exposure, the activation time-constant (2.38 ± 0.54 ms), the deactivation kinetic (21.60 ± 2.7 ms, n = 4), and the current amplitude (51.42 ± 13.33 pA control, 53.75 ± 12.6 pA memantine, n = 4; Figure A4 in Appendix) did not change significantly (p = 0.9). These observations indicate that memantine’s effects were limited to NMDAR currents on Schaffer collateral-CA1 synapses.

The primary binding site of memantine on NMDARs overlaps that of Mg²⁺ (Kashiwagi et al., 2002; Chen and Lipton, 2005), however, memantine is thought to act as a more effective surrogate for the divalent Mg²⁺ (Parsons et al., 2007). In the hippocampal network, the Shaffer collaterals release glutamate that generates post-synaptic currents (PSCs), which depend largely on the activation of AMPARs. AMPARs depolarize the post-synaptic membrane causing the Mg²⁺ block to be relieved and NMDARs to open.

To study the effect of memantine on NMDAR currents in PCs and Ints in physiological Mg²⁺, we recorded NMDAR-mediated component of the post-synaptic current as described above but in normal ACSF (see also Materials and Methods). The neurons were held at −30 mV to release the Mg²⁺ block and memantine was added to the bath solution at different concentrations. The cells were depolarized to −30 mV since, in the NMDAR IV curve, the larger inward NMDAR currents are evoked at this potential (Nowak et al., 1984). As in low Mg²⁺ ACSF, memantine attenuated NMDAR current amplitudes in both PCs and Ints (Figure 3). In PCs, bath application of 100 nM, 1 μM, 10 μM, 100 μM, 300 μM, 500 μM, and 1 mM memantine reduced the amplitude of the NMDAR currents 0.7 ± 1.15% (n = 4), 22.73 ± 2.66% (n = 4), 60.97 ± 9.72% (n = 4), 77.15 ± 4.77% (n = 5), 97.52 ± 0.63% (n = 2), 100% (n = 3), and 100% (n = 3), respectively. In Ints, bath application of 100 nM, 1 μM, 10 μM, 100 μM, 300 μM, 500 μM, and 1 mM memantine reduced the amplitude of the NMDAR currents 0% (n = 3), 5.48 ± 3.09% (n = 4); 38.97 ± 6.96% (n = 7), 76.70 ± 3.38% (n = 4), 91.99 ± 2.80% (n = 5), 100% (n = 3); 100% (n = 3). As in low Mg²⁺ ACSF, memantine antagonism was concentration-dependent in both PCs and Ints. At −30 mV in presence of Mg²⁺, IC₅₀ values were (LogIC₅₀ = −5.37) 4.2 μM and (LogIC₅₀ = −4.64) 22.45 μM for PCs and Ints, respectively, further supporting a higher affinity of memantine for PCs’ NMDAR currents. The two IC₅₀ values were significantly different (p = 0.000232; non-parametric Wilcoxon Rank Test).

In addition to the higher affinity of memantine for NMDARs in PCs vs. Ints, the fitting parameters of the Hill equation showed significantly different Hill coefficients (see Materials and Methods) in PCs (Hill coefficient = 0.617 ± 0.02) and Ints (Hill coefficient = 0.879 ± 0.08; p = 0.003; Figure 3). Hill coefficients lower than one indicate positive cooperativity, suggesting that, in the virtual absence of Mg²⁺, both PCs and Ints possess at least two subtypes of NMDAR; in contrast, the Hill coefficient values obtained in presence of Mg²⁺ suggested that Ints (negative cooperativity) may express a single NMDA receptor subtype with a lower affinity for memantine.

N-Methyl-d-aspartate receptor are composed of NR1 and NR2 subunits. The expression of NR1/NR2A subunits produces channels with faster deactivation (tens of milliseconds) than NR1/NR2B or NR1/NR2C (hundreds of milliseconds; Stocca and Vicini, 1998), whereas the NMDARs containing NR1/NR2D subunits display very slow kinetics (seconds; Stocca and Vicini, 1998; Misra et al., 2000). In the hippocampus PCs express mainly NR2A and NR2B subunits, while Ints have a larger percentage of NR2C and NR2D subunits (Monyer et al., 1994; Martina et al., 2003). To verify the difference in subunit composition in the two types of neurons, we observed the biophysical properties of the NMDAR currents in PCs and Ints. The NMDAR current decay in both PCs and Ints was best-fitted with a bi-exponential function (Figure 4). The best-fitted decay values for PCs and Ints are shown in Table 2. The values of τ_{deact f} and τ_{deact s} between PCs and Ints were not significantly different. However, the difference in relative portions (A_f and A_s) of decay time-constants in PCs and Ints was significant and hence the τ_mean (Table 2; see Materials and Methods), confirming the different molecular make-up of NMDARs in PCs and Ints.

To determine the effect of memantine on different components of NMDAR currents, we recorded currents in presence of the NR2B-containing NMDARs antagonist Ro25-6981 (500 nM; Fischer et al., 1997) and the NR2A-containing NMDARs antagonist NVP-AAM077 (50 nM; Auberson et al., 2002; Weitlauf et al., 2005). NMDAR currents were evoked every 10 s by releasing pre-synaptic glutamate via electrical stimulation and the antagonists were added to the bath solution via perfusion (see above).

Ro25-6981 is a potent and selective activity-dependent blocker of NMDARs containing the NR2B subunit. IC₅₀ values are 0.009 and 52 μM for cloned receptor subunit combinations NR1C/NR2B and NR1C/NR2A, respectively (Fischer et al., 1997). However, NR2B antagonists are influenced by Mg²⁺ concentration and NR2B-directed NMDAR antagonists in the hippocampal slice are more potent in the absence of Mg²⁺ (Williams, 1993; Mott et al., 1998; Rauner and Köhr, 2011). Consequently, in this experimental series, NMDAR currents were recorded in the virtual absence of Mg²⁺ (low Mg²⁺ ACSF). Under these conditions, Ro25-6981 (500 nM) reduced the amplitude of the NMDAR currents by 24.45 ± 6.71% (n = 9) in PCs and by 35.07 ± 6.98% (n = 8) in Ints, respectively (Figure 6A).

NVP-AAM77 is a competitive antagonist of NR2A-containing NMDARs. It has been shown that for glutamate-evoked currents, the IC₅₀ values for NVP-AAM077 acting at NR1/NR2A and NR1/NR2B NMDA receptors were 16 ± 2 nM (NR1/NR2A) and 302 ± 18 nM (NR1/NR2B; Frizelle et al., 2006), respectively. NVP-AAM077 (50 nM) reduced the amplitude of the NMDAR currents by 45.5 ± 3.40% (n = 7) in PCs and by 21.00 ± 9.49% (n = 7) in Ints, respectively. However, caution is warranted when interpreting NVP-AAM077 data since NVP-AAM077 (400 nM) has been reported to target non-NR2A-containing NMDARs when applied 30 min prior to an agonist (Weitlauf et al., 2005). However, if antagonism of NVP-AAM077 on NMDAR currents is similar at 50 and 400 nM, then the specificity of NVP-AAM077 for NR2A was conserved under the experimental conditions employed here. Bath application of NVP-AAM077 (400 nM) reduced the amplitude of the NMDAR currents by 50.54 ± 3.90% (n = 15) in PCs and by 21.50 ± 16.3% (n = 8) in Ints, respectively. The plateau effect of NVP-AAM077 at 50 and at 400 nM was reached after 10 min of perfusion (see Figure 6A) and remained constant, indicating that NVP-AAM077’s specificity for NR2A was conserved even at 400 nM. The reductions in current caused by NVP-AAM077 (400 nM) and (50 nM) were not significantly different (p = 0.223 for PCs and p = 0.428 for Ints), suggesting that only NR2A-containing NMDARs were antagonized by NVP-AAM077.

The difference in the percentage of NMDARs containing the NR2A and NR2B subunit in PCs and Ints (Monyer et al., 1994; Martina et al., 2003) can also account for the faster deactivation decay observed in PCs (Figure 4C; see Discussion).

We then applied memantine and measured its effect on different components of NMDARs (Figure 5E). Memantine was used at 10 μM since this concentration was close to the IC₅₀ for PCs and Ints.

In the presence of NVP-AAM077, the residual NMDAR currents (see decay time-constants in Tables 3 and 4) were reduced by memantine by 42.02 ± 4.13% (n = 15; in the presence of 400 nM NVP-AAM077) and 42.90 ± 3.54% (n = 7; in the presence of 50 nM NVP-AAM077) in PCs and by 64.76 ± 2.76% (n = 8 in the presence of 400 nM NVP-AAM077) and 58.00 ± 9.96% (n = 5; in the presence of 50 nM NVP-AAM077) Ints, respectively (Figures 5A–D). The values for NVP-AAM077 used at 400 and 50 nM were not significantly different (p = 0.442 for PCs and p = 0.258 for Ints), while the differences between PCs and Ints were (400 nM: p = 0.026; 50 nM: p = 0.039).

In the presence of Ro25-6981, the residual NMDAR currents (see decay time-constants in Tables 3 and 4) were reduced by memantine by 61.49 ± 7.04% (n = 9) and 33.77 ± 5.91% (n = 8) in PCs and Ints, respectively (Figures 5B,C). The difference between PCs and Ints was significantly different (p = 0.0047).

To further explore the specific effects of memantine on the NR2C and 2D subunit component of NMDARs in PCs and Ints, we co-applied NVP-AAM077 and Ro25-6981. This reduced NMDAR current amplitudes by 74.81 ± 3.11% (n = 4) in PCs and 62.18 ± 4.16% (n = 8) in Ints (Figure 6). The subsequent application of memantine (10 μM) further reduced the residual NMDAR currents by 27.76 ± 7.67% (n = 4; p = 0.0429) and 28.43 ± 6.79% (n = 8; p = 0.018) in PCs and in Ints, respectively (Figures 5C and 6).