The t-N-methyl-d-aspartate receptor: Making the case for d-Serine to be considered its inverse co-agonist.

The N-methyl-d-aspartate receptor (NMDAR) is an enigmatic macromolecule that has garnered a good deal of attention on account of its involvement in the cellular processes that underlie learning and memory, following its discovery in the mid twentieth century (Baudry and Davis, 1991). Yet, despite advances in knowledge about its function, there remains much more to be uncovered regarding the receptor's biophysical properties, subunit composition, and role in CNS physiology and pathophysiology. The motivation for this review stems from the need for synthesizing new information gathered about these receptors that sheds light on their role in synaptic plasticity and their dichotomous relationship with the amino acid d-serine through which they influence the pathogenesis of neurodegenerative diseases like temporal lobe epilepsy (TLE), the most common type of adult epilepsies (Beesley et al., 2020a). This review will outline pertinent ideas relating structure and function of t-NMDARs (GluN3 subunit-containing triheteromeric NMDARs) for which d-serine might serve as an inverse co-agonist. We will explore how tracing d-serine's origins blends glutamate-receptor biology with glial biology to help provide fresh perspectives on how neurodegeneration might interlink with neuroinflammation to initiate and perpetuate the disease state. Taken together, we envisage the review to deepen our understanding of endogenous d-serine's new role in the brain while also recognizing its therapeutic potential in the treatment of TLE that is oftentimes refractory to medications.

Visualizing the triheteromeric N-methyl-D-aspartate receptor subunit composition.

N-methyl-D-aspartate receptors (NMDARs) are one of three ligand-gated ionotropic channels that transduce the effects of neurotransmitter glutamate at excitatory synapses within the central nervous system. Their ability to influx Ca2+ into cells, unlike mature AMPA or kainate receptors, implicates them in a variety of processes ranging from synaptic plasticity to cell death. Many of the receptor's capabilities, including binding glutamate and regulating Ca2+ influx, have been attributed to their subunit composition, determined putatively using cell biology, electrophysiology and/or pharmacology. Here, we show that subunit composition of synaptic NMDARs can also be readily visualized in acute brain slices (rat) using highly specific antibodies directed against extracellular epitopes of the subunit proteins and high-resolution confocal microscopy. This has helped confirm the expression of triheteromeric t-NMDARs (containing GluN1, GluN2, and GluN3 subunits) at synapses for the first time and reconcile functional differences with diheteromeric d-NMDARs (containing GluN1 and GluN2 subunits) described previously. Even though structural information about individual receptors is still diffraction limited, fluorescently tagged receptor subunit puncta coalesce with precision at various magnifications and/or with the postsynaptic density (PSD-95) but not the presynaptic active zone marker Bassoon. These data are particularly relevant for identifying GluN3A-containing t-NMDARs that are highly Ca2+ permeable and whose expression at excitatory synapses renders neurons vulnerable to excitotoxicity and cell death. Imaging NMDAR subunit proteins at synapses not only offers firsthand insights into subunit composition to correlate function but may also help identify zones of vulnerability within brain structures underlying neurodegenerative diseases like Temporal Lobe Epilepsy.

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.

A Model for Predicting Cation Selectivity and Permeability in AMPA and NMDA Receptors Based on Receptor Subunit Composition.

Glutamatergic AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) and NMDA (N-methyl-D-aspartate) receptors are implicated in diverse functions ranging from synaptic plasticity to cell death. They are heterotetrameric proteins whose subunits are derived from multiple distinct gene families. The subunit composition of these receptors determines their permeability to monovalent and/or divalent cations, but it is not entirely clear how this selectivity arises in native and recombinantly-expressed receptor populations. By analyzing the sequence of amino acids lining the selectivity filters within the pore forming membrane helices (M2) of these subunits and by correlating subunit stoichiometry of these receptors with their ability to permeate Na+ and/or Ca2+, we propose here a mathematical model for predicting cation selectivity and permeability in these receptors. The model proposed is based on principles of charge attractivity and charge neutralization within the pore forming region of these receptors; it accurately predicts and reconciles experimental data across various platforms including Ca2+ permeability of GluA2-lacking AMPARs and ion selectivity within GluN3-containing di- and tri-heteromeric NMDARs. Additionally, the model provides insights into biophysical mechanisms regulating cation selectivity and permeability of these receptors and the role of various subunits in these processes.

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.

Layer-specific modulation of entorhinal cortical excitability by presubiculum in a rat model of temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) is the most common form of epilepsy in adults and is often refractory to antiepileptic medications. The medial entorhinal area (MEA) is affected in TLE but mechanisms underlying hyperexcitability of MEA neurons require further elucidation. Previous studies suggest that inputs from the presubiculum (PrS) contribute to MEA pathophysiology. We assessed electrophysiologically how PrS influences MEA excitability using the rat pilocarpine model of TLE. PrS-MEA connectivity was confirmed by electrically stimulating PrS afferents while recording from neurons within superficial layers of MEA. Assessment of alterations in PrS-mediated synaptic drive to MEA neurons was made following focal application of either glutamate or NBQX to the PrS in control and epileptic animals. Here, we report that monosynaptic inputs to MEA from PrS neurons are conserved in epileptic rats, and that PrS modulation of MEA excitability is layer-specific. PrS contributes more to synaptic inhibition of LII stellate cells than excitation. Under epileptic conditions, stellate cell inhibition is significantly reduced while excitatory synaptic drive is maintained at levels similar to control. PrS contributes to both synaptic excitation and inhibition of LIII pyramidal cells in control animals. Under epileptic conditions, overall excitatory synaptic drive to these neurons is enhanced while inhibitory synaptic drive is maintained at control levels. Additionally, neither glutamate nor NBQX applied focally to PrS now affected EPSC and IPSC frequency of LIII pyramidal neurons. These layer-specific changes in PrS-MEA interactions are unexpected and of significance in unraveling pathophysiological mechanisms underlying TLE.

N-acetyl cysteine mitigates the acute effects of cocaine-induced toxicity in astroglia-like cells.

Cocaine has a short half-life of only about an hour but its effects, predominantly on the central nervous system (CNS), are fairly long-lasting. Of all cells within the CNS, astrocytes may be the first to display cocaine toxicity owing to their relative abundance in the brain. Cocaine entry could trigger several early response changes that adversely affect their survival, and inhibiting these changes could conversely increase their rate of survival. In order to identify these changes and the minimal concentrations of cocaine that can elicit them in vitro, rat C6 astroglia-like cells were treated with cocaine (2-4 mM for 1h) and assayed for alterations in gross cell morphology, cytoplasmic vacuolation, viability, reactive oxygen species (ROS) generation, glutathione (GSH) levels, cell membrane integrity, F-actin cytoskeleton, and histone methylation. We report here that all of the above identified features are significantly altered by cocaine, and may collectively represent the key pathology underlying acute toxicity-mediated death of astroglia-like cells. Pretreatment of the cells with the clinically available antioxidant N-acetyl cysteine (NAC, 5 mM for 30 min) inhibited these changes during subsequent application of cocaine and mitigated cocaine-induced toxicity. Despite repeated cocaine exposure, NAC pretreated cells remained highly viable and post NAC treatment also increased viability of cocaine treated cells to a smaller yet significant level. We show further that this alleviation by NAC is mediated through an increase in GSH levels in the cells. These findings, coupled with the fact that astrocytes maintain neuronal integrity, suggest that compounds which target and mitigate these early toxic changes in astrocytes could have a potentially broad therapeutic role in cocaine-induced CNS damage.

Regular-spiking cells in the presubiculum are hyperexcitable in a rat model of temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) is the most common form of adult epilepsy, characterized by recurrent seizures originating in the temporal lobes. Here, we examine TLE-related changes in the presubiculum (PrS), a less-studied parahippocampal structure that both receives inputs from and projects to regions affected by TLE. We assessed the state of PrS neurons in TLE electrophysiologically to determine which of the previously identified cell types were rendered hyperexcitable in epileptic rats and whether their intrinsic and/or synaptic properties were altered. Cell types were characterized based on action potential discharge profiles followed by unsupervised hierarchical clustering. PrS neurons in epileptic animals could be divided into three major groups comprising of regular-spiking (RS), irregular-spiking (IR), and fast-adapting (FA) cells. RS cells, the predominant cell type encountered in PrS, were the only cells that were hyperexcitable in TLE. These neurons were previously identified as sending long-range axonal projections to neighboring structures including medial entorhinal area (MEA), and alterations in intrinsic properties increased their propensity for sustained firing of action potentials. Frequency and amplitude of both spontaneous excitatory and inhibitory synaptic events were reduced. Further analysis of nonaction potential-dependent miniature currents (in tetrodotoxin) indicated that reduction in excitatory drive to these neurons was mediated by decreased activity of excitatory neurons that synapse with RS cells concomitant with reduced activity of inhibitory neurons. Alterations in physiological properties of PrS neurons and their ensuing hyperexcitability could entrain parahippocampal structures downstream of PrS, including the MEA, contributing to temporal lobe epileptogenesis.

Electrophysiological and morphological characterization of cells in superficial layers of rat presubiculum.

The presubiculum (PrS) plays critical roles in spatial information processing and memory consolidation and has also been implicated in temporal lobe epileptogenesis. Despite its involvement in these processes, a basic structure-function analysis of PrS cells remains far from complete. To this end, we performed whole-cell recording and biocytin labeling of PrS neurons in layer (L)II and LIII to examine their electrophysiological and morphological properties. We characterized the cell types based on electrophysiological criteria, correlated their gross morphology, and classified them into distinct categories using unsupervised hierarchical cluster analysis. We identified seven distinct cell types: regular-spiking (RS), irregular-spiking (IR), initially bursting (IB), stuttering (Stu), single-spiking (SS), fast-adapting (FA), and late-spiking (LS) cells, of which RS and IB cells were common to LII and LIII, LS cells were specific to LIII, and the remaining types were identified exclusively in LII. Recorded neurons were either pyramidal or nonpyramidal and, except for Stu cells, displayed spine-rich dendrites. The RS, IB, and IR cells appeared to be projection neurons based on extension of their axons into LIII of the medial entorhinal area (MEA) and/or angular bundle. We conclude that LII and LIII of PrS are distinct in their neuronal populations and together constitute a more diverse population of neurons than previously suggested. PrS neurons serve as major drivers of circuits in superficial (LII-III) entorhinal cortex (ERC) and couple neighboring structures through robust afferentation, thereby substantiating the PrS's critical role in the parahippocampal region.

Diversity and excitability of deep-layer entorhinal cortical neurons in a model of temporal lobe epilepsy.

The entorhinal cortex (ERC) is critically implicated in temporal lobe epileptogenesis--the most common type of adult epilepsy. Previous studies have suggested that epileptiform discharges likely initiate in seizure-sensitive deep layers (V-VI) of the medial entorhinal area (MEA) and propagate into seizure-resistant superficial layers (II-III) and hippocampus, establishing a lamina-specific distinction between activities of deep- versus superficial-layer neurons and their seizure susceptibilities. While layer II stellate cells in MEA have been shown to be hyperexcitable and hypersynchronous in patients and animal models of temporal lobe epilepsy (TLE), the fate of neurons in the deep layers under epileptic conditions and their overall contribution to epileptogenicity of this region have remained unclear. We used whole cell recordings from slices of the ERC in normal and pilocarpine-treated epileptic rats to characterize the electrophysiological properties of neurons in this region and directly assess changes in their excitatory and inhibitory synaptic drive under epileptic conditions. We found a surprising heterogeneity with at least three major types and two subtypes of functionally distinct excitatory neurons. However, contrary to expectation, none of the major neuron types characterized showed any significant changes in their excitability, barring loss of excitatory and inhibitory inputs in a subtype of neurons whose dendrite extended into layer III, where neurons are preferentially lost during TLE. We confirmed hyperexcitability of layer II neurons in the same slices, suggesting minimal influence of deep-layer input on superficial-layer neuron excitability under epileptic conditions. These data show that deep layers of ERC contain a more diverse population of excitatory neurons than previously envisaged that appear to belie their seizure-sensitive reputation.

Neuron-specific nuclear antigen NeuN is not detectable in gerbil subtantia nigra pars reticulata.

NeuN (Neuronal Nuclei), the neuron-specific marker of nuclear protein is used extensively in histological procedures to identify major cell-types in adult vertebrate nervous systems of a variety of species including rodents and humans. Some notable exceptions (i.e., NeuN-negative neurons) include Purkinje cells in cerebellum, mitral cells in olfactory bulb, and photoreceptors in retina. Here we report that neurons in gerbil (Meriones unguiculatus) substantia nigra pars reticulata (SNr), whose "neuronal" phenotype was confirmed via electrophysiology, biocytin-labeling, histology, and in situ hybridization, are also devoid of NeuN-immunoreactivity as assayed with the widely used monoclonal antibody A60. Immunohistochemistry of rat SNr using the same antibody yielded robust staining. These data suggest lack of NeuN-immonoreactivity observed in certain cell-types and brain regions can be species-specific.

Recurrent circuits in layer II of medial entorhinal cortex in a model of temporal lobe epilepsy.

Patients and laboratory animal models of temporal lobe epilepsy display loss of layer III pyramidal neurons in medial entorhinal cortex and hyperexcitability and hypersynchrony of less vulnerable layer II stellate cells. We sought to test the hypothesis that loss of layer III pyramidal neurons triggers synaptic reorganization and formation of recurrent, excitatory synapses among layer II stellate cells in epileptic pilocarpine-treated rats. Laser-scanning photo-uncaging of glutamate focally activated neurons in layer II while excitatory synaptic responses were recorded in stellate cells. Photostimulation revealed previously unidentified, functional, recurrent, excitatory synapses between layer II stellate cells in control animals. Contrary to the hypothesis, however, control and epileptic rats displayed similar levels of recurrent excitation. Recently, hyperexcitability of layer II stellate cells has been attributed, at least in part, to loss of GABAergic interneurons and inhibitory synaptic input. To evaluate recurrent inhibitory circuits in layer II, we focally photostimulated interneurons while recording inhibitory synaptic responses in stellate cells. IPSCs were evoked more than five times more frequently in slices from control versus epileptic animals. These findings suggest that in this model of temporal lobe epilepsy, reduced recurrent inhibition contributes to layer II stellate cell hyperexcitability and hypersynchrony, but increased recurrent excitation does not.

Hyperexcitability, interneurons, and loss of GABAergic synapses in entorhinal cortex in a model of temporal lobe epilepsy.

Temporal lobe epilepsy is the most common type of epilepsy in adults, and its pathophysiology remains unclear. Layer II stellate cells of the entorhinal cortex, which are hyperexcitable in animal models of temporal lobe epilepsy, provide the predominant synaptic input to the hippocampal dentate gyrus. Previous studies have ascribed the hyperexcitability of layer II stellate cells to GABAergic interneurons becoming "dormant" after disconnection from their excitatory synaptic inputs, which has been reported to occur during preferential loss of layer III pyramidal cells. We used whole-cell recording from slices of entorhinal cortex in pilocarpine-treated epileptic rats to test the dormant interneuron hypothesis. Hyperexcitability appeared as multiple action potentials and prolonged depolarizations evoked in layer II stellate cells of epileptic rats but not controls. However, blockade of glutamatergic synaptic transmission caused similar percentage reductions in the frequency of spontaneous IPSCs in layer II stellate cells of control and epileptic rats, suggesting similar levels of excitatory synaptic input to GABAergic interneurons. Direct recordings and biocytin labeling revealed two major types of interneurons in layer III whose excitatory synaptic drive in epileptic animals was undiminished. Interneurons in layer III did not appear to be dormant; therefore, we tested whether loss of GABAergic synapses might underlie hyperexcitability of layer II stellate cells. Stereological evidence of fewer GABAergic interneurons, fewer gephyrin-immunoreactive punctae, and reduced frequency of spontaneous IPSCs and miniature IPSCs (recorded in tetrodotoxin) confirmed that layer II stellate cell hyperexcitability is attributable, at least in part, to reduced inhibitory synaptic input.

GABAA receptor-mediated IPSCs and alpha1 subunit expression are not reduced in the substantia nigra pars reticulata of gerbils with inherited epilepsy.

Domestic Mongolian gerbils, a model of inherited epilepsy, begin having spontaneous seizures at approximately 1.5 mo of age, making it possible to evaluate them during epileptic and pre-epileptic stages. Previous studies have shown that GABA binding is reduced in the substantia nigra pars reticulata (SNr) of both epileptic and pre-epileptic gerbils compared with controls, suggesting that reduced expression of GABAA receptors in SNr might be epileptogenic in this model. To test this hypothesis, we measured the expression of the GABAA receptor alpha1 subunit, the dominant alpha subunit expressed in the SNr, and evaluated GABAA receptor-mediated postsynaptic currents in SNr neurons. GABA(A) alpha1 subunit mRNA levels in substantia nigra-rich tissue from pre-epileptic animals were similar to controls, and immunocytochemistry for the alpha1 subunit showed similar strong expression in the SNr in both groups. Western analysis confirmed that expression of the alpha1 subunit protein was similar in substantia nigra-rich tissue from pre-epileptic and control gerbils. The frequency and amplitude of spontaneous inhibitory postsynaptic currents (IPSCs) and the frequency of miniature (m)IPSCs in SNr neurons of pre-epileptic gerbil were similar to those of controls. The amplitude of mIPSCs in the pre-epileptics was significantly larger than controls. Zolpidem, an alpha1 subunit-specific modulator of the GABAA receptor, was equally efficacious in prolonging the decay time of mIPSCs in both groups. Hence, contrary to the predictions of the hypothesis, mRNA and protein expression levels of the major GABAA receptor alpha subunit were normal, and neurons of the SNr in pre-epileptic gerbils displayed normal or enhanced IPSC frequencies and amplitudes. Therefore reduced expression of GABAA receptors in SNr is not likely to be an epileptogenic mechanism in this model.

Pathway-specific differences in subunit composition of synaptic NMDA receptors on pyramidal neurons in neocortex.

Heterogeneity of synaptic inputs onto neocortical layer 5 pyramidal neurons could result from differences in the underlying receptors, yet previous work has shown that functional attributes of AMPA receptors are uniform among synaptic connections onto these neurons. To determine whether NMDA receptors (NMDARs) would be similarly uniform, we compared in the same pyramidal neurons pharmacologically isolated NMDAR-mediated EPSCs evoked by stimulation of two anatomically distinguishable pathways, callosal or intracortical. Based on differences in voltage dependence, decay kinetics, apparent Mg2+sensitivity, and subunit-specific (NR2A, NR2B, and NR2C/D) pharmacology, we found NMDARs at these inputs to be distinct. Furthermore, NMDARs activated by the intracortical pathway were more efficient at integrating EPSPs and bringing the neuron closer to the spike-firing threshold than the callosal pathway. These results suggest that pyramidal neurons encode information differentially depending on the origin of their neocortical inputs and that NMDAR-dependent synaptic plasticity may be pathway specific.

A developmental switch of AMPA receptor subunits in neocortical pyramidal neurons.

AMPA receptors mediate most of the fast excitatory neurotransmission in the brain, and those lacking the glutamate receptor 2 (GluR2) subunit are Ca(2+)-permeable and expressed in cortical structures primarily by inhibitory interneurons. Here we report that synaptic AMPA receptors of excitatory layer 5 pyramidal neurons in the rat neocortex are deficient in GluR2 in early development, approximately before postnatal day 16, as evidenced by their inwardly rectifying current-voltage relationship, blockade of AMPA receptor-mediated EPSCs by external and internal polyamines, permeability to Ca(2+), and GluR2 immunoreactivity. Overall, these results indicate that neocortical pyramidal neurons undergo a developmental switch in the Ca(2+) permeability of their AMPA receptors through an alteration of their subunit composition. This has important implications for plasticity and neurotoxicity.