The subunit composition of N-methyl-d-aspartate receptors (NMDARs) at synaptic inputs onto a neuron can either vary or be uniform depending on the type of neuron and/or brain region. Excitatory pyramidal neurons in the frontal and somatosensory cortices (L5), for example, show pathway-specific differences in NMDAR subunit composition in contrast with the entorhinal cortex (L3), where we now show colocalization of NMDARs with distinct subunit compositions at individual synaptic inputs onto these neurons. Subunit composition was deduced electrophysiologically based on alterations of current-voltage relationship ( I–V) profiles, amplitudes, and decay kinetics of minimally evoked, pharmacologically isolated, NMDAR-mediated excitatory postsynaptic currents by known subunit-preferring antagonists. The I–Vs were outwardly rectifying in a majority of neurons assayed (~80%), indicating expression of GluN1/GluN2/GluN3-containing triheteromeric NMDARs ( t-NMDARs) and of the conventional type, reversing close to 0 mV with prominent regions of negative slope, in the rest of the neurons sampled (~20%), indicating expression of GluN1/GluN2-containing diheteromeric NMDARs ( d-NMDARs). Blocking t-NMDARs in neurons with outwardly rectifying I–Vs pharmacologically unmasked d-NMDARs, with all responses antagonized using D-AP5. Coimmunoprecipitation assays of membrane-bound protein complexes isolated from the medial entorhinal area using subunit-selective antibodies corroborated stoichiometry and together suggested the coexpression of t- and d-NMDARs at these synapses. Colocalization of functionally distinct NMDAR subtypes at individual synaptic inputs likely enhances the repertoire of pyramidal neurons for information processing and plasticity within the entorhinal cortex. NEW & NOTEWORTHY The subunit composition of a N-methyl-d-aspartate (NMDA) receptor, which dictates most aspects of its function, can vary between neurons in different brain regions and/or between synaptic inputs onto single neurons. Here we demonstrate colocalization of tri- and diheteromeric-NMDA receptors at the same/single synaptic input onto excitatory neurons in the entorhinal cortex. Synaptic colocalization of distinct NMDAR subtypes might endow entorhinal cortical neurons with the ability to encode distinct patterns of neuronal activity through single synapses.

Highlights The GluN3 subunit is the least understood of all subunits that make up functional NMDARs in the brain. We show through ion substitution experiments that NMDARs containing GluN3 are more permeable to Ca 2+ than those containing just GluN1 and GluN2. We attribute these differences to their ability to screen for Ca 2+ over Na + . Subunit-dependent cation selectivity represents a hitherto unrealized mechanism for finer control of Ca 2+ influx enhancing the repertoire of synaptic NMDARs.

Temporal lobe epilepsy (TLE) is the most common type of drug-resistant epilepsy in adults, with an unknown etiology. A hallmark of TLE is the characteristic loss of layer 3 neurons in the medial entorhinal area (MEA) that underlies seizure development. One approach to intervention is preventing loss of these neurons through better understanding of underlying pathophysiological mechanisms. Here, we show that both neurons and glia together give rise to the pathology that is mitigated by the amino acid D-serine whose levels are potentially diminished under epileptic conditions. Focal administration of D-serine to the MEA attenuates neuronal loss in this region thereby preventing epileptogenesis in an animal model of TLE. Additionally, treatment with D-serine reduces astrocyte counts in the MEA, alters their reactive status, and attenuates proliferation and/or infiltration of microglia to the region thereby curtailing the deleterious consequences of neuroinflammation. Given the paucity of compounds that reduce hyperexcitability and neuron loss, have anti-inflammatory properties, and are well tolerated by the brain, D-serine, an endogenous amino acid, offers new hope as a therapeutic agent for refractory TLE.

Temporal lobe epilepsy (TLE) is the most common type of epilepsy in adults that is often refractory to anti-epileptic medication therapy. Neither the pathology nor the etiology of TLE are fully characterized, although recent studies have established that the two are causally related. TLE pathology entails a stereotypic pattern of neuron loss in hippocampal and parahippocampal regions, predominantly in CA1 subfield of the hippocampus and layer 3 of the medial entorhinal area (MEA), deemed hallmark pathological features of the disease. Through this work, we address the contribution of glutamatergic N-methyl-D-aspartate receptors (NMDARs) to the pathology (vulnerability and pattern of neuronal loss), and by extension to the pathophysiology (Ca2+ induced excitotoxicity), by assaying the spatial expression of their subunit proteins (GluN1, GluN2A, GluN2B and GluN3A) in these regions using ASTA (area specific tissue analysis), a novel methodology for harvesting brain chads from hard-to-reach regions within brain slices for Western blotting. Our data suggest gradient expression of the GluN3A subunit along the mid-lateral extent of layer 3 MEA and along the CA1-subicular axis in the hippocampus, unlike GluN1 or GluN2 subunits which are uniformly distributed. Incorporation of GluN3A in the subunit composition of conventional diheteromeric (d-) NMDARs yield triheteromeric (t-) NMDARs which by virtue of their increased selectivity for Ca2+ render neurons vulnerable to excitotoxic damage. Thus, the expression profile of this subunit sheds light on the spatial extent of the pathology observed in these regions and implicates the GluN3 subunit of NMDARs in hippocampal and entorhinal cortical pathology underlying TLE.

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