Abstract
The dentate gyrus is a key relay station that controls information transfer from the entorhinal cortex to the hippocampus proper. This process relies heavily on dendritic integration by dentate granule cells (GCs) of excitatory synaptic inputs from the medial and lateral entorhinal cortex via medial and lateral perforant paths (MPP and LPP, respectively). Inputs from the entorhinal cortex onto GCs exhibit activity-dependent long-term plasticity of N-methyl-D-aspartate receptor (NMDAR)-mediated synaptic transmission. However, the properties, underlying mechanisms, and input-specificity of this plasticity remain poorly understood. Here, we examined NMDAR plasticity rules at MPP-GC and LPP-GC synapses using physiologically relevant stimulation patterns in acute hippocampal slices from rats and mice. Unlike MPP-GC synapses, LPP-GC synapses did not express homosynaptic NMDAR-LTP. Additionally, inducing NMDAR-LTP at MPP-GC synapses potentiated NMDAR transmission at distal LPP-GC synapses. The same stimulation protocol induced homosynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-LTP at MPP-GC synapses but heterosynaptic AMPAR-LTD at distal LPP synapses, indicating that NMDAR and AMPAR plasticity are controlled by different plasticity rules. Notably, heterosynaptic but not homosynaptic NMDAR-LTP required Ca2+ release from intracellular, ryanodine receptor-dependent Ca2+ stores. Lastly, genetic deletion of the GluN2D subunit from GCs or selective antagonism of GluN2D-containing NMDARs abolished heterosynaptic LTP. Heterosynaptic NMDAR-mediated LTP may have important consequences for the dendritic integration of functionally distinct excitatory inputs by dentate granule cells.
Introduction
Activity-dependent changes in synaptic strength are widely regarded as a key mechanism underlying experience-driven refinement of neuronal circuits and memory formation (, ). Synaptic plasticity can manifest in multiple forms, including homo- and heterosynaptic plasticity. Homosynaptic plasticity is expressed only at stimulated synapses, whereas heterosynaptic plasticity involves changes in strength at neighboring unstimulated synapses. Although it is well-established that glutamate NMDA receptors (NMDARs) mediate excitatory synaptic transmission and can undergo long-term potentiation (LTP) and long-term depression (LTD) (, ), most studies have focused on homosynaptic (, ) and heterosynaptic (, , ) plasticity of the AMPA receptor (AMPAR)-mediated component of glutamatergic excitatory transmission. However, whether NMDAR-mediated transmission undergoes heterosynaptic plasticity remains to be investigated.
The dentate gyrus is the main input area of the hippocampus (, ). Dentate gyrus granule cells (GCs) receive prominent cortical projections from the medial and lateral entorhinal cortex, which convey context and content-related information, through the medial and lateral perforant pathways (MPP and LPP), respectively (). Activity-dependent changes of these inputs, and their dendritic integration by GCs are critical to dentate gyrus information processing (, , , , ). MPP and LPP inputs onto GCs express robust homosynaptic (, , ) and heterosynaptic (, ) long-term plasticity of AMPAR-mediated transmission. Furthermore, early studies showed that high-frequency stimulation can also trigger homosynaptic NMDAR plasticity in perforant path inputs (, ). Using physiologically relevant patterns of presynaptic and postsynaptic burst activity, a protocol known as burst-timing-dependent plasticity (BTDP), we have recently shown that MPP-GC synapses can undergo robust NMDAR LTP but not NMDAR-LTD (). Whether LPP-GC synapses can express homo- or heterosynaptic NMDAR plasticity is unknown.
Here, we examined whether BTDP induction protocols induce NMDAR plasticity at LPP inputs in acute hippocampal slices from rats and mice. We found that LPP-GC synapses do not express homosynaptic NMDAR plasticity but can undergo heterosynaptic plasticity induced by MPP-GC synapses. Conversely, homosynaptic AMPAR-LTP at MPP-GC synapses was accompanied by heterosynaptic AMPAR-LTD at LPP-GC synapses. Heterosynaptic NMDAR-LTP increased LPP-driven GC firing and was blocked by GluN2D antagonism and Grin2d postsynaptic conditional knockout. To our knowledge, these findings provide the first evidence of heterosynaptic LTP of NMDAR-mediated transmission and suggest that BTDP-induced homosynaptic plasticity at MPP can shape the transfer of information from LPP to GCs, thereby contributing to dentate gyrus-dependent forms of memory.
Materials and methods
Experimental model and subject details
Postnatal day 19 (P19) to P30 Sprague-Dawley rats (Charles River), P30 to P60 C57BL/6 (Jackson Laboratory) and floxed Grin2d mice (Grin2d fl/fl), of either sex were used for electrophysiological experiments. The Grin2d fl/fl mice were generated by removing the reporter cassette by crossing Grin2dTM1a(EUCOMM)Wtsi mice with B6-SJL-Tg(ACTFLPe)9205Dym/J line. All animals were group-housed under a standard 12 h light/12 h dark cycle. Animal handling and use followed a protocol approved by the Animal Care and Use Committee of Albert Einstein College of Medicine, in accordance with the National Institutes of Health guidelines.
Hippocampal slice preparation
Acute dorsal and intermediate transverse hippocampal slices (300–400 μm thick) were prepared from Sprague–Dawley rats, C57BL/6 and Grin2d fl/fl mice (300 μm thick). Briefly, the hippocampi were isolated and cut using a VT1200s microslicer (Leica Microsystems Co.) in a solution containing (in mM): 215 sucrose, 2.5 KCl, 26 NaHCO3, 1.6 NaH2PO4, 1 CaCl2, 4 MgCl2, 4 MgSO4 and 20 D-glucose. At 30 min post-sectioning, the cutting medium was gradually switched to extracellular artificial cerebrospinal fluid (ACSF) recording solution containing (in mM): 124 NaCl, 2.5 KCl, 26 NaHCO3, 1 NaH2PO4, 2.5 CaCl2, 1.3 MgSO4 and 10 D-glucose. Mouse hippocampal slices were prepared using an NMDG-based cutting solution containing (in mM): 93 N-Methyl-d-glucamin, 2.5 KCl, 1.25 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 D-glucose, 2 Thiourea, 5 Na-Ascorbate, 3 Na-Pyruvate, 0.5 CaCl2, 10 MgCl2. The NMDG-based cutting solution has been shown to improve tissue quality when preparing slices from older mice (P > 30) (). Slices were incubated in ACSF at room temperature for at least 45 min before recording.
Electrophysiology
All experiments, unless otherwise stated, were performed at 28 ± 1 °C in a submersion-type recording chamber perfused at ∼2 mL min–1 with ACSF. Whole-cell patch-clamp recordings were made from GCs voltage clamped at −45 mV (unless otherwise stated) using a Multiclamp 700A amplifier (Molecular Devices) and patch-type pipette electrodes (∼3–4 MΩ) containing (in mM): 135 K-Gluconate, 5 KCl, 0.1 EGTA, 0.04 CaCl2, 5 NaOH, 5 NaCl, 10 HEPES, 5 MgATP, 0.4 Na3GTP, and 10 D-glucose, pH 7.2 (280–290 mOsm). Both series resistance (∼7–25 MΩ) and input resistance were monitored throughout all experiments with a −5 mV, 80 ms voltage step to ensure recording quality, and cells showing a significant change in series resistance (>20%) were excluded from analysis.
To stimulate MPP and LPP inputs to GCs, stimulating patch-type pipettes were placed in the middle third of the medial molecular layer and in the distal part of the outer molecular layer of the dentate gyrus, respectively. To elicit synaptic responses, monopolar square-wave voltage or current pulses (100–200 μs pulse width, 4–30 V or 20–100 μA) were delivered through a stimulus isolator (Isoflex, AMPI, or Digitimer DS2A-MKII) connected to a broken tip (∼10–20 μm) stimulating patch-type micropipette filled with ACSF. Typically, stimulation was adjusted to obtain comparable-magnitude synaptic responses across experiments; e.g., 40–100 pA NMDAR-EPSCs [holding potential (Vh) = −45 mV, Figures 1–4]. Input/output experiments in Figure 5 show NMDAR excitatory postsynaptic potentials (NMDAR-EPSPs; Vh ∼−60 mV) evoked at stimulation intensities of 10, 20, and 30 V. Burst-timing NMDAR plasticity was induced in current clamp mode (Vh ∼−60 to −70 mV) by pairing presynaptic bursts (6 pulses at 50 Hz) with a postsynaptic burst of action potentials (5 action potentials at 100 Hz) delivered at a 10 ms interval, repeated 100 times at 2 Hz. Averaged traces include 20 consecutive individual responses.
FIGURE 1
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FIGURE 5
Postsynaptic Grin2d conditional knockout
Grin2d fl/fl mice were injected with Cre-expressing (AAV5-CaMKII-Cre-mCherry, 5.8 × 1012 Virus Molecules/mL, UNC Vector) or control (AAV5-CaMKII-mCherry, 4.9 × 1012 Virus Molecules/mL, UNC Vector) adeno-associated viruses. 1 μL of virus was injected (flow rate of 0.1 μL/min) unilaterally into the DG (relative to bregma: 2.06 mm posterior, 1.5 mm lateral, 1.8 mm ventral) of 5–6-week-old Grin2d fl/fl mice. Animals were placed in a stereotaxic frame and anesthetized with isoflurane (up to 5% for induction and 1%–2% for maintenance). Slices for electrophysiology were prepared from injected animals 3–4 weeks after injection.
Data analysis
Electrophysiological data were acquired at 5 kHz, filtered at 2.4 kHz, and analyzed using custom IgorPro software (Wavemetrics Inc.). The magnitude of LTP was assessed by comparing 10-min baseline responses with those recorded 20–30 min after LTP induction. A minimum of three animals were used for each experimental group.
Reagents
Reagents were bath-applied after dilution into ACSF from stock solutions stored at −20 °C and prepared in water or DMSO, depending on the manufacturer’s recommendations. The final DMSO concentration was <0.01%. Cyclopiazonic acid, heparin, ryanodine, DQP-1105, QNZ46, SCH 50911, D-APV, and MPEP were purchased from Tocris Biosciences. NBQX was purchased from Cayman Chemical Co, while BAPTA, picrotoxin, and all salts for making ACSF and internal solutions were purchased from Sigma-Millipore.
Quantification and statistical analysis
Statistical analysis was performed using OriginPro software (OriginLab). Normality of distributions was assessed using the Shapiro–Wilk test. For normal distributions, unpaired and paired two-tailed Student’s t-tests were used to assess between-group and within-group differences, respectively. For non-normal distributions, the nonparametric paired-sample Wilcoxon signed-rank test and the Mann–Whitney U test were used. Statistical comparisons in Figure 5 were performed using two-way ANOVA with repeated measures (RM), and Greenhouse–Geisser was used for correction of degrees of freedom when sphericity was not assumed.
To compare changes in EPSC amplitudes between groups, effect sizes were calculated using Hedges’ g for independent samples, comparing control and experimental groups. Hedges’ g is a bias-corrected version of Cohen’s d that reduces the overestimation of effect sizes in small samples, a common limitation in electrophysiological studies. This approach is recommended for biomedical data integration and meta-analyses ().
To quantify changes in synaptic strength before and after application of LTP induction protocol, effect sizes were calculated using Hedges’ g for repeated-measures designs (gav). Uncorrected effect sizes were first computed using the average variance of the baseline and 20–30 min post-induction measurements, providing a standardized metric comparable across experiments and studies. To correct for the upward bias associated with small sample sizes, Cohen’s dav was multiplied by a small-sample correction factor based on the paired-samples degrees of freedom (df = n−1) ().
Statistical significance was set at p < 0.05. All values are reported as the mean ± SEM.
Results
LPP-GC synapses express robust heterosynaptic NMDAR LTP but heterosynaptic AMPAR LTD
We examined whether inducing burst timing-dependent NMDAR-LTP at MPP-GC synapses could trigger plasticity at distal LPP-GC synapses. To test for NMDAR heterosynaptic plasticity, NMDAR excitatory postsynaptic currents (NMDAR-EPSCs) were monitored in the same GC in response to MPP and LPP stimulation with patch-type micropipettes placed in the MML and at the most distal border of the outer molecular layer (OML) of the dentate gyrus, respectively (see “Materials and methods”). Both pipettes were positioned on the same side of the GC to activate similar dendritic branches (Figures 1A, B). To isolate NMDAR-mediated transmission, whole-cell patch-clamp recordings of GCs [holding potential (Vh) = −45 mV] were performed in continuous presence of 10 μM NBQX and 100 μM picrotoxin to block AMPAR- and GABAA receptor-mediated transmission, respectively. After a 10-min baseline, we applied a burst pairing protocol (Harnett et al., 2009, ) known to induce homosynaptic NMDAR-LTP at MPP-GC synapses (), consisting of presynaptic stimulation (6 pulses at 50 Hz, at MPP) paired with postsynaptic firing (5 pulses at 100 Hz) with a 10 ms interval between pre- and postsynaptic activity, repeated 100 times (see “Materials and methods”). While homosynaptic AMPAR-LTP often induces compensatory, heterosynaptic AMPAR-LTD (, , ), we found that homosynaptic MPP-GC NMDAR-LTP was accompanied by heterosynaptic NMDAR-LTP at LPP-GC synapses (Figures 1A, B). No significant changes in input resistance were observed after LTP induction (97.37% ± 3.25% of baseline, n = 11, p = 0.44, paired t-test), supporting the stability of the recordings. We further confirmed that both homosynaptic and heterosynaptic NMDAR-LTP can also be elicited at a more physiological temperature (32 ± 1°C) (Supplementary Figure 1). Remarkably, applying the pre-post burst stimulation protocol only to LPP-GC synapses did not trigger any form of homosynaptic or heterosynaptic NMDAR long-term plasticity (Figure 1C), indicating that heterosynaptic NMDAR-LTP was not caused by stimulation crosstalk between the MPP and LPP inputs.
Because single dendritic branches perform local computations and may act as fundamental functional units of a neuron (, ), we hypothesized that the postsynaptic signaling that mediates NMDAR heterosynaptic plasticity could be restricted to the dendritic branches expressing homosynaptic LTP. To test this possibility, we placed the LPP stimulation pipette on the opposite side of the recorded GC (at least 250 μm lateral from the MPP stimulation pipette), which is expected to activate LPP inputs onto different dendritic branches. We observed that these LPP inputs did not exhibit heterosynaptic plasticity (Figure 1D), supporting the idea that NMDAR heterosynaptic plasticity depends on signaling confined to dendritic branches where homosynaptic LTP was induced. Lastly, we tested whether heterosynaptic LPP-GC NMDAR-LTP could also be observed in mice and repeated the experiment described in Figures 1A, B in acute mouse hippocampal slices. Both homosynaptic and heterosynaptic NMDAR plasticity were present in mice (Figure 1E), indicating a conserved plasticity mechanism. Overall, these results demonstrate that MPP and LPP inputs onto GCs undergo distinct forms of NMDAR plasticity. While pre-post burst stimulation induced homosynaptic NMDAR-LTP at MPP inputs (), only heterosynaptic NMDAR-LTP was observed at LPP inputs.
We wondered whether the homosynaptic and heterosynaptic BTDP we observed were exclusive to NMDARs or if they could also be exhibited by AMPARs. While almost nothing is known about NMDAR heterosynaptic plasticity, several reports have indicated that AMPARs can undergo heterosynaptic changes in neighboring synapses (, , ). However, only a few in vivo studies, with mixed results, have explored heterosynaptic AMPAR plasticity in the dentate gyrus (, , ), and to our knowledge, no in vitro study has characterized any form of functional heterosynaptic AMPAR plasticity at LPP-GC synapses. To fill this knowledge gap, we monitored AMPAR-EPSCs at both MPP- and LPP-GC synapses within the same GC (Vh = −60 mV), in the presence of the GABAA receptor antagonist picrotoxin (100 μM). After establishing a baseline, we applied pre-post burst stimulation at MPP-GC synapses and observed homosynaptic AMPAR-LTP at MPP-GC synapses but heterosynaptic AMPAR-LTD at LPP-GC synapses (Figure 1F). These findings indicate that BTDP modifies the relative contribution of AMPA and NMDA receptors at MPP and LPP synaptic inputs.
Internal Ca2+ stores are required for heterosynaptic but not homosynaptic NMDAR-LTP
NMDAR-LTP at central nervous system synapses (), including MPP-GC synapses (, ), relies on a postsynaptic induction mechanism involving NMDAR and type 5 metabotropic glutamate receptor (mGluR5) co-activation and a rise in postsynaptic [Ca2+]. We found that bath application of the mGluR5 antagonist MPEP (4 μM) (Figures 2A, B) and intracellular loading of the Ca2+ chelating agent BAPTA (10 mM) abolished LTP (Figure 2C), indicating that both mGluR5 and postsynaptic Ca2+ are required for pre-post burst-induced NMDAR LTP at MPP-GC synapses. Additionally, these manipulations also prevented heterosynaptic NMDAR-LTP at LPP-GC synapses recorded from the same GC (Figures 2D, E), suggesting a shared induction mechanism.
Internal Ca2+ stores contribute to the expression of homosynaptic NMDAR-LTP at mossy fiber to CA3 pyramidal cell synapses (). We then tested the role of internal Ca2+ stores in both homosynaptic and heterosynaptic NMDAR-LTP at dentate GC synapses. To this end, we used the cell-permeable inhibitor of sarcoplasmic reticulum Ca2+-ATPase, cyclopiazonic acid (CPA 30 μM, 40–50 min pre-incubation, also included in the perfusion) to deplete internal Ca2+ stores (Figures 3A, B). We found that CPA abolished the induction of LPP-GC heterosynaptic plasticity (Figure 3A) but had no effect on MPP-GC homosynaptic plasticity (Figure 3B). Internal Ca2+ can be released from endoplasmic reticulum (ER) stores via IP3 signaling or ryanodine receptor (RyR) activation. To determine which of these mechanisms is involved in heterosynaptic NMDAR-LTP, we incubated (40–50 min) and perfused hippocampal slices with heparin (2 mg/ml) and ryanodine (100 μM) to block IP3-mediated and RyR-mediated Ca2+ release, respectively (Figures 3C, D). We found that induction of heterosynaptic LPP-GC NMDAR-LTP was abolished by ryanodine but normally induced in the presence of heparin (Figure 3C), indicating that heterosynaptic LTP requires RyRs but not IP3 receptors. In contrast, homosynaptic NMDAR-LTP at MPP-GC was normally induced in the presence of both ryanodine and heparin (Figure 3D), strongly suggesting that the rise in postsynaptic Ca2+ at MPP-GC synapses required for this form of plasticity is independent of internal Ca2+ stores. Collectively, these findings demonstrate that homosynaptic vs. heterosynaptic NMDAR-LTP have distinct Ca2+ requirements for induction, with internal Ca2+ release necessary only for heterosynaptic NMDAR-LTP at LPP-GC synapses.
Heterosynaptic NMDAR-LTP is likely mediated by the synaptic recruitment of GluN2D-containing receptors
Previous studies reported that homosynaptic NMDAR-LTP at MPP-GC synapses is mediated by the recruitment of GluN2D-containing NMDARs to the synapse (, ). To assess the role of GluN2D subunits in burst timing-dependent heterosynaptic NMDAR-LTP, we first used two different non-competitive, activity-dependent GluN2C/GluN2D selective antagonists, DQP-1105 and QNZ46 (, ). Although these antagonists may not distinguish between GluN2C- and GluN2D-containing NMDARs, GluN2C expression in the adult hippocampus () and forebrain neurons () is negligible. We first tested the effect of DQP-1105 on heterosynaptic LPP-GC NMDAR-LTP and found that bath application of 30 μM DQP-1105 abolished heterosynaptic plasticity (Figure 4A). This manipulation also blocks homosynaptic NMDAR-LTP at MPP-GC synapses (). Most evidence indicates that GluN2D-containing receptors are primarily extrasynaptic in adult animals (). Consistent with this, bath application of the antagonists DQP-1105 or QNZ46 (30 μM) had no effect on LPP-GC basal synaptic transmission, whereas the non-selective NMDAR antagonist D-APV (50 μM) abolished these responses (Figure 4B). Remarkably, application of the selective GluN2D antagonists (DQP-1105 or QNZ46, 30 μM) 30 min after the induction of NMDAR-LTP significantly reduced LPP-GC NMDAR-mediated transmission (Figure 4C). These data strongly suggest that, like homosynaptic NMDAR-LTP, heterosynaptic NMDAR-LTP in the dentate gyrus likely results from the recruitment of GluN2D-containing receptors to the synapse.
To demonstrate the role of postsynaptic GluN2D-containing NMDARs in heterosynaptic NMDAR-LTP, we employed a conditional knockout strategy to selectively remove Grin2d from GCs (Grin2d postsynaptic cKO) by stereotaxically injecting adeno-associated virus (AAV) expressing Cre recombinase (AAV5.CaMKII.Cre-mCherry) or mCherry alone (AAV5.CaMKII.mCherry) into the dentate gyrus of adult Grin2d-floxed mice (Figure 4D). We recorded from mCherry+ GCs and found that heterosynaptic NMDAR-LTP was abolished in Cre-mCherry+ GCs (Grin2d postsynaptic cKO), whereas it remained intact in mCherry+ GCs (control) (Figures 4D, E). Consistent with findings that Grin2d postsynaptic cKO prevents homosynaptic NMDAR potentiation at MPP-GC synapses, these results suggest that postsynaptic GluN2D-containing NMDARs are also necessary for heterosynaptic NMDAR-LTP at LPP-GC synapses.
Heterosynaptic NMDAR-LTP enhances synaptically-driven GC output
We next sought to determine the functional impact of heterosynaptic NMDAR-LTP and whether it enhances the ability of LPP inputs to drive action potential (AP) firing in dentate GCs. To address this, we performed whole-cell current-clamp recordings from GCs and measured the probability of AP firing in response to LPP burst stimulation, which consisted of five pulses delivered at 50 Hz with varying stimulus strengths. NMDAR excitatory postsynaptic potentials (NMDAR-EPSPs) were recorded in the presence of NBQX (10 μM), picrotoxin (100 μM), and SCH50911 (20 μM) to block AMPA/Kainate and GABAA and GABAB receptors, respectively. We monitored LPP-evoked burst firing before and 15–20 min after LTP induction. Our results showed that inducing heterosynaptic NMDAR-LTP was associated with a significant increase in the number of APs in GCs (Figure 5A). No significant changes were observed in membrane input resistance post-LTP induction (Rinput, pre-LTP: 249.1 ± 41.7 MΩ, post-LTP: 230.7 ± 52.6 MΩ, n = 7, p = 0.26, paired t-test; data not shown). To test whether the increase in firing was mediated by GluN2D-dependent plasticity, we repeated the experiment in the presence of the selective GluN2D antagonist DQP-1105 (30 μM), which abolished heterosynaptic NMDAR-LTP at LPP-GC synapses (Figure 4A). Under these conditions, application of the pre–post burst stimulation protocol at MPP–GC synapses did not produce a significant change in LPP-evoked AP firing (Figure 5B). Together, these findings strongly suggest that GluN2D-mediated heterosynaptic NMDAR LTP enhances GC output driven by LPP burst activity.
Discussion
In this study, we describe a form of activity-dependent plasticity at entorhinal-dentate granule cell synapses, characterized by heterosynaptic potentiation of NMDAR-mediated transmission. Using a physiologically relevant burst timing-dependent paradigm, we show that triggering homosynaptic NMDAR-LTP at MPP synapses also causes robust heterosynaptic NMDAR-LTP at distal, non-stimulated LPP synapses. In contrast, the same induction protocol that triggered homo- and heterosynaptic NMDAR-LTP induced homosynaptic AMPAR-LTP at MPP-GC synapses but heterosynaptic AMPAR-LTD at LPP-GC synapses. Heterosynaptic NMDAR-LTP depended on Ca2+ release from internal stores, likely implicating long-range signaling via the ER. Additionally, the increased sensitivity to GluN2D antagonists after the induction of both homosynaptic and heterosynaptic NMDAR-LTP suggests that these forms of plasticity involve the synaptic recruitment of GluN2D-containing NMDARs. NMDAR-mediated synaptic strengthening results in a sustained increase in synaptically-driven GC activity. Together, our findings revealed a new activity-dependent synaptic mechanism in which functionally distinct entorhinal inputs work together to control the output of the dentate gyrus.
MPP-GC but not LPP-GC synapses exhibit homosynaptic NMDAR-LTP
BTDP of NMDAR-mediated transmission has previously been reported in midbrain dopamine neurons (Harnett et al., 2009), GC-CA3 synapses (), and MPP-GC synapses (). In vivo recordings showed that neurons from the medial entorhinal cortex fire bursts of action potentials (, , ), and GCs, which normally fire sparsely, can generate high-frequency bursts of back-propagating action potentials (, , ). In the present study, we found that pairing pre and postsynaptic burst activity, which selectively induces homosynaptic NMDAR-LTP at MPP-GC (), did not induce homosynaptic plasticity at LPP-GC synapses under our experimental conditions. This finding might be explained by a strong dendritic attenuation of backpropagating action potentials as a function of distance from the soma (, ). Also, as in distal synapses of other pyramidal neurons (, ), backpropagating action potentials may not be sufficient to unblock NMDARs at LPP-GCs. In a recent study, a more standard pre-post pairing protocol (i.e., single action potentials and synaptic responses) also failed to elicit LPP-GC AMPAR plasticity, whereas this plasticity was induced by theta-burst stimulation of LPP inputs that generated local dendritic spikes (). Our findings on NMDAR-mediated transmission support the idea that synaptic plasticity rules at proximal and distal entorhinal inputs onto GCs differ.
MPP-GC burst activity triggers opposite heterosynaptic LPP-GC plasticity of NMDAR- and AMPAR-mediated transmission
In this study, we present evidence of heterosynaptic NMDAR plasticity. Due to the unique properties of NMDARs, which can cause signal amplification, temporal summation, and increased Ca2+ influx (), it is likely that NMDAR potentiation modifies the dendritic properties of GCs at distal LPP inputs during heterosynaptic NMDAR LTP. Unlike NMDAR-mediated transmission, AMPAR potentiation at MPP-GC synapses was accompanied by heterosynaptic AMPAR-LTD at LPP-GC synapses. Since its initial discovery in the CA1 area (), similar heterosynaptic LTD of AMPAR-mediated transmission has been reported at several other synapses (, , ). Our findings provide further evidence that NMDARs and AMPARs exhibit opposite heterosynaptic plasticity rules, which might affect dendritic integration in GCs. While heterosynaptic NMDAR-LTP may enhance information transfer from LPP inputs to GCs, likely by facilitating temporal summation (), heterosynaptic AMPAR-LTD (Figure 1F) is expected to reduce the LPP-mediated drive of GCs. Therefore, concurrent induction of both forms of BTDP might favor burst-only LPP inputs. The potential role of such winner-take-all dynamics in the dentate gyrus warrants further exploration.
Homosynaptic vs. heterosynaptic NMDAR LTP have unique Ca2+ requirements
Mechanistically, the induction and expression of NMDAR plasticity share common properties across synapses (, ), including postsynaptic NMDAR-mediated Ca2+ influx and Ca2+ release from internal stores. A pivotal finding of our study is the mechanistic divergence between homosynaptic and heterosynaptic NMDAR plasticity. While homosynaptic MPP-GC NMDAR-LTP is unaffected by the depletion of internal Ca2+ stores, heterosynaptic potentiation at LPP synapses is abolished by ryanodine. Although ryanodine exhibits a complex, biphasic dose-response profile, the concentration used selectively blocks intracellular calcium release (Wang et al., 1996). However, we cannot rule out potential off-target effects. This dependence on RyR-mediated Ca2+ release suggests a long-range signaling mechanism that compensates for the dendritic attenuation of back-propagating action potentials in GCs (, ). Homosynaptic NMDAR-LTP may alter the synaptic rules at distal LPP-GC synapses, by eliciting the release of internal ER Ca2+ stores, although the diffusion of other signaling molecules cannot be discarded (, ). While homosynaptic NMDAR-LTP at GC-CA3 synapses requires IP3-dependent Ca2+ release (, ), evidence in the amygdala shows that heterosynaptic, but not homosynaptic plasticity, requires RyR-dependent Ca2+-induced Ca2+ release from internal stores (). A role for NMDAR-dependent Ca2+-induced Ca2+ release from the ER in heterosynaptic plasticity of AMPAR-mediated transmission has also been demonstrated in CA1 pyramidal neurons (, , ) and neocortex (). Furthermore, in mesolimbic dopamine neurons exposed to alcohol, increased susceptibility to induce NMDAR-LTP relies on Ca2+ release from ER stores (). Electron microscopy studies of pyramidal hippocampal neurons show that the smooth ER extends into distal dendrites and dendritic spines (). The coincident increase in ER-Ca2+ sources, along with increased GluN2D recruitment to potentially ease Mg2+ block, might provide an ideal scenario for the induction and expression of heterosynaptic NMDAR LTP at distal synapses. Future in vivo Ca2+ imaging studies combined with genetic strategies could clarify how these two events converge to mediate heterosynaptic plasticity and potentially contribute to dentate gyrus-dependent learning.
Heterosynaptic NMDAR-LTP is mediated by GluN2D recruitment
GluN2D expression in the adult hippocampus is generally believed to be primarily restricted to interneurons (, , , von Engelhardt et al., 2015). However, GluN2D is robustly expressed in the dendrites and axons of adult glutamatergic excitatory neurons in the subthalamic nucleus (), and GluN2D-containing NMDARs have also been reported in rat postnatal dentate gyrus GCs (). As for homosynaptic MPP-GC synapses (, ), the maintenance of heterosynaptic LPP-GC NMDAR-LTP was also significantly reduced by both GluN2D-subunit antagonists and postsynaptic Grin2d cKO, suggesting that both forms of plasticity require recruitment of GluN2D-containing receptors to the synapse, presumably forming triheteromers (GluN1/2B/2D) or diheteromers (GluN1/2D) (, Hansen et al., 2017, , ). GluN1/2B/2D triheteromers have functional properties intermediate to those of GluN1/2B and GluN1/2D diheteromers (Yi et al., 2019). The structural identification of functional triheteromeric GluN1-2B-2D receptors in the adult brain () confirms that GluN2D is a stable and functional component of the mature NMDAR landscape. While the recruitment of GluN1/2D diheteromers could result in NMDAR-EPSCs with a slower decay (, , ), no differences in NMDAR-EPSC decay were observed before and after homosynaptic LTP (), suggesting that GluN2D-containing NMDARs are primarily triheteromers. Importantly, a key property of GluN1/2D diheteromers is the low sensitivity to Mg2+ blockade relative to GluN2A or GluN2B-containing receptors (). Although backpropagating APs at distal synapses may not be sufficient to remove the NMDAR block (, ), expression of GluN2D-containing NMDARs after NMDAR LTP induction may be one factor that helps explain why NMDARs exhibit different synaptic plasticity learning rules than AMPARs.
By increasing the synaptically driven activity of GCs (Figure 5), heterosynaptic NMDAR-LTP could facilitate the transfer of information from the LPP to the dentate GCs, thereby dynamically regulating dentate gyrus computations and memory processes. Consistent with this idea, GC Grin2d cKO, which abolished heterosynaptic NMDAR LTP (Figure 4D), can also impair DG-dependent forms of memory (). Our finding that GluN2D antagonists can block the LPP-evoked GC firing after NMDAR-LTP induction aligns with previous work showing that spike firing rate was increased by the GluN2C/D-positive allosteric modulator CIQ, and decreased by the GluN2C/D antagonist DQP-1105 in subthalamic neurons in vivo (). The low sensitivity of GluN2D to voltage-dependent Mg2+ block allows these receptors to pass current even at or near resting membrane potentials (, ). Notably, heterosynaptic changes in NMDAR subunit composition have been previously reported in CA1 neurons (). GluN2D-mediated synaptic transmission has been reported in hippocampal interneurons of adult mice (von Engelhardt et al., 2015, , Yi et al., 2019), but it remains unclear whether activity modulates the expression of synaptic GluN2D-containing NMDARs in these neurons.
A potential limitation of the present study is that sex-specific differences in NMDAR heterosynaptic plasticity were not fully addressed. Although data from male and female subjects were pooled for analysis because no major sex differences were observed, we cannot exclude the possibility of more subtle sexually dimorphic effects. Furthermore, fluctuations in ovarian hormones during the estrous cycle may influence synaptic plasticity. Future studies with larger sample sizes and systematic assessment of estrous cycle stage will be required to provide sufficient statistical power to detect potential sex-dependent differences and to determine whether the mechanisms underlying NMDAR-dependent heterosynaptic plasticity differ between males and females.
In summary, BTDP of NMDAR-mediated transmission at MPP synapses can exert long-term, powerful control over NMDAR transmission at distal LPP-GC synapses. The coexistence of NMDAR heterosynaptic LTP and AMPAR heterosynaptic LTD at distal LPP synapses suggests a sophisticated regulatory mechanism for GC integration. Dynamic changes in NMDAR transmission could shift the induction threshold of NMDAR-dependent forms of plasticity, as seen in CA3 neurons, where NMDARs can selectively adjust synapses in a heterosynaptic manner (Tsukamoto et al., 2003), serve as a metaplastic switch for AMPAR-mediated plasticity (, ), and contribute to the metaplasticity of AMPAR LTP (). Behavioral deficits in GC Grin2d cKO mice () suggest a potential role for homo- and heterosynaptic plasticity in hippocampal-dependent behavior, but further work is required to determine the precise contribution of homo- and heterosynaptic NMDAR plasticity in behaving animals.
Statements
Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
Ethics statement
The animal study was approved by the Animal Care and Use Committee of Albert Einstein College of Medicine. The study was conducted in accordance with the National Institutes of Health guidelines.
Author contributions
KN: Methodology, Writing – original draft, Visualization, Validation, Conceptualization, Data curation, Writing – review & editing, Investigation. MC: Data curation, Visualization, Investigation, Formal analysis, Writing – review & editing. SL: Writing – review & editing, Formal analysis, Methodology, Investigation, Writing – original draft. PC: Methodology, Investigation, Data curation, Supervision, Conceptualization, Resources, Validation, Writing – original draft, Project administration, Writing – review & editing, Funding acquisition. AR-R: Conceptualization, Investigation, Methodology, Validation, Writing – review & editing, Supervision, Visualization, Formal analysis, Data curation, Writing – original draft.
Funding
The author(s) declared that financial support was received for this work and/or its publication. This research was supported by the National Institutes of Health: R01-MH125772, R01-MH116673, R01-NS113600, and R01-MH081935 to PC. AR-R was partially supported by the Brain & Behavior Research Foundation Young Investigator Award (2014) and the Ford Foundation Postdoctoral Fellowship (2013). KN was partially supported by the American Epilepsy Society Postdoctoral Research Fellowship (2020). MC was supported by NIH R25GM104547.
Acknowledgments
We thank Dr. Dravid Shashank for sharing floxed Grin2d mice and we thank the Wellcome Trust Sanger Institute Mouse Genetics Project (Sanger MGP) and its funders for providing the mutant mouse line [Grin2dTM1a(EUCOMM)Wtsi]. Funding and associated primary phenotypic information may be found at www.sanger.ac.uk/mouseportal. We thank all Castillo Lab members for helpful discussions.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that Generative AI was not used in the creation of this manuscript.
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Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnsyn.2026.1825949/full#supplementary-material
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Summary
Keywords
AMPA and NMDA receptors, entorhinal cortex, hippocampus, long-term depression (LTD), long-term potentiation (LTP)
Citation
Nasrallah K, Castillo M, Lutzu S, Castillo PE and Rodenas-Ruano A (2026) Heterosynaptic NMDA receptor plasticity in hippocampal dentate granule cells. Front. Synaptic Neurosci. 18:1825949. doi: 10.3389/fnsyn.2026.1825949
Received
09 March 2026
Revised
19 June 2026
Accepted
26 June 2026
Published
22 July 2026
Volume
18 - 2026
Edited by
P. Jesper Sjöström, McGill University, Canada
Reviewed by
Gayane Grigoryan, University of Freiburg, Germany
Pooja Tiwary, Bharati Vidyapeeth Deemed University, India
Updates
Copyright
© 2026 Nasrallah, Castillo, Lutzu, Castillo and Rodenas-Ruano.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Alma Rodenas-Ruano, arodenasruano@fordham.eduPablo E. Castillo, pablo.castillo@einsteinmed.edu
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