GluN3 subunit expression correlates with increased vulnerability of hippocampus and entorhinal cortex to neurodegeneration in a model of temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) is the most common type of epilepsy in adults that is often refractory to antiepileptic medication therapy. Neither the pathology nor the etiology of TLE is 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 area-specific tissue analysis (ASTA), 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 that 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.NEW & NOTEWORTHY The role of the GluN3 subunit in NMDAR-mediated pathophysiology underlying TLE is not known. Here, we demonstrate using ASTA (area-specific tissue analysis) that its expression in specific regions of the entorhinal cortex and the hippocampus is correlated with significant cell loss and neurodegeneration, hallmark features of the disease.

d-Serine Intervention In The Medial Entorhinal Area Alters TLE-Related Pathology In CA1 Hippocampus Via The Temporoammonic Pathway.

Entrainment of the hippocampus by the medial entorhinal area (MEA) in Temporal Lobe Epilepsy (TLE), the most common type of drug-resistant epilepsy in adults, is believed to be mediated primarily through the perforant pathway (PP), which connects stellate cells in layer (L) II of the MEA with granule cells of the dentate gyrus (DG) to drive the hippocampal tri-synaptic circuit. Using immunohistochemistry, high-resolution confocal microscopy and the rat pilocarpine model of TLE, we show here that the lesser known temporoammonic pathway (TAP) plays a significant role in transferring MEA pathology to the CA1 region of the hippocampus independently of the PP. The pathology observed was region-specific and restricted primarily to the CA1c subfield of the hippocampus. As shown previously, daily intracranial infusion of d-serine (100 µm), an antagonist of GluN3-containing triheteromeric N-Methyl d-aspartate receptors (t-NMDARs), into the MEA prevented loss of LIII neurons and epileptogenesis. This intervention in the MEA led to the rescue of hippocampal CA1 neurons that would have otherwise perished in the epileptic animals, and down regulation of the expression of astrocytes and microglia thereby mitigating the effects of neuroinflammation. Interestingly, these changes were not observed to a similar extent in other regions of vulnerability like the hilus, DG or CA3, suggesting that the pathology manifest in CA1 is driven predominantly through the TAP. This work highlights TAP's role in the entrainment of the hippocampus and identifies specific areas for therapeutic intervention in dealing with TLE.

D-serine mitigates cell loss associated with temporal lobe epilepsy.

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.

The GluN3 subunit regulates ion selectivity within native N-methyl-d-aspartate receptors.

Glutamatergic N-methyl-d-aspartate receptors (NMDARs) are heterotetrameric proteins whose subunits are derived from three gene families, GRIN1 (codes for GluN1), GRIN2 (GluN2) and GRIN3 (GluN3). In addition to providing binding sites for glutamate and the co-agonist glycine, these subunits in their di (d-) and tri (t-) heteromeric configurations regulate various aspects of receptor function in the brain. For example, the decay kinetics of NMDAR-mediated synaptic currents depend on the type of GluN2 subunit (GluN2A-GluN2D) in the receptor subunit composition. While much is known about the contributions of GluN1 and GluN2 to d-NMDAR function, we know comparatively little about how GluN3 influences the function of t-NMDARs composed of one or more subunits from each of the three gene families. We report here that in addition to altering kinetics and voltage-dependent properties, the GluN3 subunit endows these receptors with ion selectivity wherein influx of Ca2+ is preferred over Na+. This became apparent in the process of assessing Ca2+ permeability through these receptors and is of significance given that NMDARs are generally believed to be nonselective to cations and increased selectivity can lead to enhanced permeability. This was true of two independent brain regions where t-NMDARs are expressed, the somatosensory cortex, where both receptor subtypes are expressed at separate inputs onto single neurons, and the entorhinal cortex, where they are co-expressed at individual synaptic inputs. Based on this data and the sequence of amino acids lining selectivity filters within these subunits, we propose GluN3 to be a regulatory subunit for ion selectivity in t-NMDARs.

Functional Connectivity of the Parasubiculum and Its Role in Temporal Lobe Epilepsy.

Temporal lobe epilepsy (TLE) is the commonest of adult epilepsies, often refractory to antiepileptic medications, whose prevention and treatment rely on understanding basic pathophysiological mechanisms in interlinked structures of the temporal lobe. The medial entorhinal area (MEA) is affected in TLE but mechanisms underlying hyperexcitability of MEA neurons require further elucidation. Previous studies have examined the role of the presubiculum (PrS) in mediating MEA pathophysiology but not the juxtaposed parasubiculum (Par). Here, we report on an electrophysiological assessment of the cells and circuits of the Par, their excitability under normal and epileptic conditions, and alterations in functional connectivity with neighboring PrS and MEA using the rat pilocarpine model of TLE. We show that Par, unlike the cell heterogeneous PrS, has a single dominant neuronal population whose excitability under epileptic conditions is altered by changes in both intrinsic properties and synaptic drive. These neurons experience significant reductions in synaptic inhibition and perish under chronic epileptic conditions. Connectivity between brain regions was deduced through changes in excitatory and inhibitory synaptic drive to neurons recorded in one region upon focal application of glutamate followed by NBQX to neurons in another using a microfluidic technique called CESOP and TLE-related circuit reorganization was assessed using data from normal and epileptic animals. The region-specific changes in Par and neighboring PrS and MEA together with their unexpected interactions are of significance in identifying ictogenic cells and circuits within the parahippocampal region and in unraveling pathophysiological mechanisms underlying TLE.

Colocalization of distinct NMDA receptor subtypes at excitatory synapses in the entorhinal cortex.

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.