Endogenous PTEN-Induced Kinase 1 Regulates Dendritic Architecture and Spinogenesis.

Mutations in PTEN-induced kinase 1 (PINK1) contribute to autosomal recessive Parkinson's disease with cognitive and neuropsychiatric comorbidities. Disturbances in dendritic and spine architecture are hallmarks of neurodegenerative and neuropsychiatric conditions, but little is known of the impact of PINK1 on these structures. We used Pink1 -/- mice to study the role of endogenous PINK1 in regulating dendritic architecture, spine density, and spine maturation. Pink1 -/- cortical neurons of unknown sex showed decreased dendritic arborization, affecting both apical and basal arbors. Dendritic simplification in Pink1 -/- neurons was primarily driven by diminished branching with smaller effects on branch lengths. Pink1 -/- neurons showed reduced spine density with a shift in morphology to favor filopodia at the expense of mushroom spines. Electrophysiology revealed significant reductions in miniature EPSC (mEPSC) frequency in Pink1 -/- neurons, consistent with the observation of decreased spine numbers. Transfecting with human PINK1 rescued changes in dendritic architecture, in thin, stubby, and mushroom spine densities, and in mEPSC frequency. Diminished spine density was also observed in Golgi-Cox stained adult male Pink1 -/- brains. Western blot study of Pink1 -/- brains of either sex revealed reduced phosphorylation of NSFL1 cofactor p47, an indirect target of PINK1. Transfection of Pink1 -/- neurons with a phosphomimetic p47 plasmid rescued dendritic branching and thin/stubby spine density with a partial rescue of mushroom spines, implicating a role for PINK1-regulated p47 phosphorylation in dendrite and spine development. These findings suggest that PINK1-dependent synaptodendritic alterations may contribute to the risk of cognitive and/or neuropsychiatric pathologies observed in PINK1-mutated families.SIGNIFICANCE STATEMENT Loss of PINK1 function has been implicated in both familial and sporadic neurodegenerative diseases. Yet surprisingly little is known of the impact of PINK1 loss on the fine structure of neurons. Neurons receive excitatory synaptic signals along a complex network of projections that form the dendritic tree, largely at tiny protrusions called dendritic spines. We studied cortical neurons and brain tissues from mice lacking PINK1. We discovered that PINK1 deficiency causes striking simplification of dendritic architecture associated with reduced synaptic input and decreased spine density and maturation. These changes are reversed by reintroducing human PINK1 or one of its downstream mediators into PINK1-deficient mouse neurons, indicating a conserved function, whose loss may contribute to neurodegenerative processes.

Inhibitory and excitatory synaptic neuroadaptations in the diazepam tolerant brain.

Benzodiazepine (BZ) drugs treat seizures, anxiety, insomnia, and alcohol withdrawal by potentiating γ2 subunit containing GABA type A receptors (GABAARs). BZ clinical use is hampered by tolerance and withdrawal symptoms including heightened seizure susceptibility, panic, and sleep disturbances. Here, we investigated inhibitory GABAergic and excitatory glutamatergic plasticity in mice tolerant to benzodiazepine sedation. Repeated diazepam (DZP) treatment diminished sedative effects and decreased DZP potentiation of GABAAR synaptic currents without impacting overall synaptic inhibition. While DZP did not alter γ2-GABAAR subunit composition, there was a redistribution of extrasynaptic GABAARs to synapses, resulting in higher levels of synaptic BZ-insensitive α4-containing GABAARs and a concomitant reduction in tonic inhibition. Conversely, excitatory glutamatergic synaptic transmission was increased, and NMDAR subunits were upregulated at synaptic and total protein levels. Quantitative proteomics further revealed cortex neuroadaptations of key pro-excitatory mediators and synaptic plasticity pathways highlighted by Ca2+/calmodulin-dependent protein kinase II (CAMKII), MAPK, and PKC signaling. Thus, reduced inhibitory GABAergic tone and elevated glutamatergic neurotransmission contribute to disrupted excitation/inhibition balance and reduced BZ therapeutic power with benzodiazepine tolerance.

A versatile optical tool for studying synaptic GABAA receptor trafficking.

Live-cell imaging methods can provide critical real-time receptor trafficking measurements. Here, we describe an optical tool to study synaptic γ-aminobutyric acid (GABA) type A receptor (GABAAR) dynamics through adaptable fluorescent-tracking capabilities. A fluorogen-activating peptide (FAP) was genetically inserted into a GABAAR γ2 subunit tagged with pH-sensitive green fluorescent protein (γ2pHFAP). The FAP selectively binds and activates Malachite Green (MG) dyes that are otherwise non-fluorescent in solution. γ2pHFAP GABAARs are expressed at the cell surface in transfected cortical neurons, form synaptic clusters and do not perturb neuronal development. Electrophysiological studies show γ2pHFAP GABAARs respond to GABA and exhibit positive modulation upon stimulation with the benzodiazepine diazepam. Imaging studies using γ2pHFAP-transfected neurons and MG dyes show time-dependent receptor accumulation into intracellular vesicles, revealing constitutive endosomal and lysosomal trafficking. Simultaneous analysis of synaptic, surface and lysosomal receptors using the γ2pHFAP-MG dye approach reveals enhanced GABAAR turnover following a bicucculine-induced seizure paradigm, a finding not detected by standard surface receptor measurements. To our knowledge, this is the first application of the FAP-MG dye system in neurons, demonstrating the versatility to study nearly all phases of GABAAR trafficking.

Memantine and Ketamine Differentially Alter NMDA Receptor Desensitization.

Memantine and ketamine are clinically useful NMDA receptor (NMDAR) open channel blockers that inhibit NMDARs with similar potency and kinetics, but display vastly different clinical profiles. This discrepancy has been hypothesized to result from inhibition by memantine and ketamine of overlapping but distinct NMDAR subpopulations. For example, memantine but not ketamine may inhibit extrasynaptic NMDARs more effectively than synaptic NMDARs. However, the basis for preferential NMDAR inhibition depending on subcellular location has not been investigated systematically. We integrated recordings from heterologously expressed single NMDAR subtypes, kinetic modeling, and recordings of synaptically evoked NMDAR responses in acute brain slices to investigate mechanisms by which channel blockers may distinguish NMDAR subpopulations. We found that memantine and ketamine differentially alter NMDAR desensitization and that memantine stabilizes a Ca2+-dependent desensitized state. As a result, inhibition by memantine of GluN1/2A receptors in tsA201 cells and of native synaptic NMDARs in cortical pyramidal neurons from mice of either sex increased in conditions that enhanced intracellular Ca2+ accumulation. Therefore, differential inhibition by memantine and ketamine based on NMDAR location is likely to result from location dependence of the intensity and duration of NMDAR activation. Modulation of Ca2+-dependent NMDAR desensitization is an unexplored mechanism of inhibitory action with the potential to endow drugs with NMDAR selectivity that leads to superior clinical profiles. Our results suggest that designing compounds to target specific receptor states, rather than specific receptor types, may be a viable strategy for future drug development.SIGNIFICANCE STATEMENT Memantine and ketamine are NMDA receptor (NMDAR) channel-blocking drugs with divergent clinical effects. Understanding mechanistically their differential actions may advance our understanding of nervous system disorders and suggest strategies for the design of more effective drugs. Here, we show that memantine and ketamine have contrasting effects on NMDAR desensitization. Ketamine binding decreases occupancy of desensitized states of the GluN1/2B NMDAR subtype. In contrast, memantine binding increases occupancy of GluN1/2A and native NMDAR desensitized states entered after accumulation of intracellular Ca2+, a novel inhibitory mechanism. These properties may contribute to inhibition of distinct NMDAR subpopulations by memantine and ketamine and help to explain their differential clinical effects. Our results suggest stabilization of Ca2+-dependent desensitized states as a new strategy for pharmaceutical neuroprotection.

The Role of GluN2C-Containing NMDA Receptors in Ketamine's Psychotogenic Action and in Schizophrenia Models.

The NMDA receptor (NMDAR) hypofunction hypothesis of schizophrenia is supported by multiple lines of evidence. Notably, administration of the NMDAR antagonist, ketamine, to healthy human subjects has psychotogenic action, producing both positive and negative symptoms associated with schizophrenia. NMDARs have multiple subtypes, but the subtypes through which ketamine produces its psychotogenic effects are not known. Here we address this question using quantitative data that characterize ketamine's ability to block different NMDAR subtypes. Our calculations indicate that, at a concentration that has psychotogenic action in humans, ketamine blocks a substantial fraction of GluN2C subunit-containing receptors but has less effect on GluN2A-, GluN2B-, and GluN2D-containing receptors. Thus, GluN2C-containing receptors may have preferential involvement in psychotic states produced by ketamine. A separate line of experiments also points to a special role for GluN2C. That work demonstrates the ability of NMDAR antagonists to mimic the elevation in the awake-state δ frequency EEG power that occurs in schizophrenia. Physiological experiments in rodents show that NMDAR antagonists generate δ oscillations by their action on the GluN2C-containing NMDARs that are prevalent in the thalamus. Optogenetic experiments suggest that such oscillations could contribute to symptoms of schizophrenia.

Effects of memantine on the excitation-inhibition balance in prefrontal cortex.

Memantine is one of the few drugs currently approved for treatment of Alzheimer's disease (AD). The clinical effects of memantine are thought to be associated with inhibition of NMDA receptors (NMDARs). Surprisingly, other open-channel NMDAR blockers have unacceptable side effects that prevent their consideration for AD treatment. One of the mechanisms proposed to explain the therapeutic benefits of memantine involves preferential decrease of excitatory drive to inhibitory neurons in the cortical circuitry and consequent changes in balance between excitation and inhibition (E/I). In this study we addressed effects of memantine on E/I balance in the prefrontal cortex (PFC). We found that a moderate concentration of memantine shifted E/I balance away from inhibition in the PFC circuitry. Indeed, memantine decreased the frequency and amplitude of spontaneous inhibitory postsynaptic currents in pyramidal neurons while leaving spontaneous excitatory postsynaptic currents unaffected. These circuitry effects of memantine were occluded by the competitive NMDAR inhibitor AP-5, and thus are associated with NMDAR inhibition. We also found that memantine decreased feed-forward disynaptic inhibitory input to pyramidal neurons, which is thought to be mediated by parvalbumin (PV)-positive interneurons. Accordingly, memantine caused a greater decrease of the amplitude of NMDAR-mediated synaptic responses in PV-positive interneurons than in pyramidal neurons. Finally, memantine reduced firing activity in PV-positive interneurons while increasing firing in pyramidal neurons. This study elucidates a novel mechanism of action of memantine associated with shifting of the E/I balance away from inhibition in neocortical circuitry, and provides important insights for AD drug development.

Molecular bases of NMDA receptor subtype-dependent properties.

NMDA receptors (NMDARs) are a class of ionotropic glutamate receptors (iGluRs) that are essential for neuronal development, synaptic plasticity, learning and cell survival. Several features distinguish NMDARs from other iGluRs and underlie the crucial roles NMDARs play in nervous system physiology. NMDARs display slow deactivation kinetics, are highly Ca(2+) permeable, and require depolarization to relieve channel block by external Mg(2+) ions, thereby making them effective coincidence detectors. These properties and others differ among NMDAR subtypes, which are defined by the subunits that compose the receptor. NMDARs, which are heterotetrameric, commonly are composed of two GluN1 subunits and two GluN2 subunits, of which there are four types, GluN2A-D. 'Diheteromeric' NMDARs contain two identical GluN2 subunits. Gating and ligand-binding properties (e.g. deactivation kinetics) and channel properties (e.g. channel block by Mg(2+)) depend strongly on the GluN2 subunit contained in diheteromeric NMDARs. Recent work shows that two distinct regions of GluN2 subunits control most diheteromeric NMDAR subtype-dependent properties: the N-terminal domain is responsible for most subtype dependence of gating and ligand-binding properties; a single residue difference between GluN2 subunits at a site termed the GluN2 S/L site is responsible for most subtype dependence of channel properties. Thus, two structurally and functionally distinct regions underlie the majority of subtype dependence of NMDAR properties. This topical review highlights recent studies of recombinant diheteromeric NMDARs that uncovered the involvement of the N-terminal domain and of the GluN2 S/L site in the subtype dependence of NMDAR properties.

Mutant LRRK2 enhances glutamatergic synapse activity and evokes excitotoxic dendrite degeneration.

Mutations in leucine-rich repeat kinase 2 (LRRK2), which are associated with autosomal dominant Parkinson's disease, elicit progressive dendrite degeneration in neurons. We hypothesized that synaptic dysregulation contributes to mutant LRRK2-induced dendritic injury. We performed in vitro whole-cell voltage clamp studies of glutamatergic receptor agonist responses and glutamatergic synaptic activity in cultured rat cortical neurons expressing full-length wild-type and mutant forms of LRRK2. Expression of the pathogenic G2019S or R1441C LRRK2 mutants resulted in larger whole-cell current responses to direct application of AMPA and NMDA receptor agonists. In addition, mutant LRRK2-expressing neurons exhibited an increased frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs) in conjunction with increased excitatory synapse density as assessed by immunofluorescence for PSD95 and VGLUT1. Mutant LRRK2-expressing neurons showed enhanced vulnerability to acute synaptic glutamate stress. Furthermore, treatment with the NMDA receptor antagonist memantine significantly protected against subsequent losses in dendrite length and branching complexity. These data demonstrate an early association between mutant LRRK2 and increased excitatory synapse activity, implicating an excitotoxic contribution to mutant LRRK2 induced dendrite degeneration.

Mechanism of differential control of NMDA receptor activity by NR2 subunits.

N-methyl-d-aspartate (NMDA) receptors (NMDARs) are a major class of excitatory neurotransmitter receptors in the central nervous system. They form glutamate-gated ion channels that are highly permeable to calcium and mediate activity-dependent synaptic plasticity. NMDAR dysfunction is implicated in multiple brain disorders, including stroke, chronic pain and schizophrenia. NMDARs exist as multiple subtypes with distinct pharmacological and biophysical properties that are largely determined by the type of NR2 subunit (NR2A to NR2D) incorporated in the heteromeric NR1/NR2 complex. A fundamental difference between NMDAR subtypes is their channel maximal open probability (P(o)), which spans a 50-fold range from about 0.5 for NR2A-containing receptors to about 0.01 for receptors containing NR2C and NR2D; NR2B-containing receptors have an intermediate value (about 0.1). These differences in P(o) confer unique charge transfer capacities and signalling properties on each receptor subtype. The molecular basis for this profound difference in activity between NMDAR subtypes is unknown. Here we show that the subunit-specific gating of NMDARs is controlled by the region formed by the NR2 amino-terminal domain (NTD), an extracellular clamshell-like domain previously shown to bind allosteric inhibitors, and the short linker connecting the NTD to the agonist-binding domain (ABD). The subtype specificity of NMDAR P(o) largely reflects differences in the spontaneous (ligand-independent) equilibrium between open-cleft and closed-cleft conformations of the NR2-NTD. This NTD-driven gating control also affects pharmacological properties by setting the sensitivity to the endogenous inhibitors zinc and protons. Our results provide a proof of concept for a drug-based bidirectional control of NMDAR activity by using molecules acting either as NR2-NTD 'closers' or 'openers' promoting receptor inhibition or potentiation, respectively.

Mechanistic and structural determinants of NMDA receptor voltage-dependent gating and slow Mg2+ unblock.

NMDA receptor (NMDAR)-mediated currents depend on membrane depolarization to relieve powerful voltage-dependent NMDAR channel block by external magnesium (Mg(o)(2+)). Mg(o)(2+) unblock from native NMDARs exhibits a fast component that is consistent with rapid Mg(o)(2+) -unbinding kinetics and also a slower, millisecond time scale component (slow Mg(o)(2+) unblock). In recombinant NMDARs, slow Mg(o)(2+) unblock is prominent in GluN1/2A (an NMDAR subtype composed of GluN1 and GluN2A subunits) and GluN1/2B receptors, with slower kinetics observed for GluN1/2B receptors, but absent from GluN1/2C and GluN1/2D receptors. Slow Mg(o)(2+) unblock from GluN1/2B receptors results from inherent voltage-dependent gating, which increases channel open probability with depolarization. Here we examine the mechanisms responsible for NMDAR subtype dependence of slow Mg(o)(2+) unblock. We demonstrate that slow Mg(o)(2+) unblock from GluN1/2A receptors, like GluN1/2B receptors, results from inherent voltage-dependent gating. Surprisingly, GluN1/2A and GluN1/2B receptors exhibited equal inherent voltage dependence; faster Mg(o)(2+) unblock from GluN1/2A receptors can be explained by voltage-independent differences in gating kinetics. To investigate the absence of slow Mg(o)(2+) unblock in GluN1/2C and GluN1/2D receptors, we examined the GluN2 S/L site, a site responsible for several NMDAR subtype-dependent channel properties. Mutating the GluN2 S/L site of GluN2A subunits from serine (found in GluN2A and GluN2B subunits) to leucine (found in GluN2C and GluN2D) greatly diminished both voltage-dependent gating and slow Mg(o)(2+) unblock. Therefore, the residue at the GluN2 S/L site governs the expression of both slow Mg(o)(2+) unblock and inherent voltage dependence.

Structural rearrangements of NR1/NR2A NMDA receptors during allosteric inhibition.

Ionotropic glutamate receptor (iGluR) subunits contain a large N-terminal domain (NTD) that precedes the agonist-binding domain (ABD) and participates in subunit oligomerization. In NMDA receptors (NMDARs), the NTDs of NR2A and NR2B subunits also form binding sites for the endogenous inhibitor Zn(2+) ion. Although these allosteric sites have been characterized in detail, the molecular mechanisms by which the NTDs communicate with the rest of the receptor to promote its inhibition remain unknown. Here, we identify the ABD dimer interface as a major structural determinant that permits coupling between the NTDs and the channel gate. The strength of this interface also controls proton inhibition, another form of allosteric modulation of NMDARs. Conformational rearrangements at the ABD dimer interface thus appear to be a key mechanism conserved in all iGluR subfamilies, but have evolved to fulfill different functions: fast desensitization at AMPA and kainate receptors, allosteric inhibition at NMDARs.

Tonic NMDA receptor-mediated current in prefrontal cortical pyramidal cells and fast-spiking interneurons.

Tonically activated neuronal currents mediated by N-methyl-d-aspartate receptors (NMDARs) have been hypothesized to contribute to normal neuronal function as well as to neuronal pathology resulting from excessive activation of glutamate receptors (e.g., excitotoxicity). Whereas cortical excitatory cells are very vulnerable to excitotoxic insult, the data regarding resistance of inhibitory cells (or interneurons) are inconsistent. Types of neurons with more pronounced tonic NMDAR current potentially associated with the activation of extrasynaptic NMDARs could be expected to be more vulnerable to excessive activation by glutamate. In this study, we compared tonic activation of NMDARs in excitatory pyramidal cells and inhibitory fast-spiking interneurons in prefrontal cortical slices. We assessed tonic NMDAR current by measuring holding current shift as well as noise reduction following NMDAR blockade after removal of spontaneous glutamate release. In addition, we compared NMDAR miniature excitatory postsynaptic currents (EPSCs) in both cell types. We have demonstrated for the first time that tonic NMDAR currents are present in inhibitory fast-spiking interneurons. We found that the magnitude of tonic NMDAR current is similar in pyramidal cells and fast-spiking interneurons, and that quantal release of glutamate does not significantly impact tonic NMDAR current.

HSV delivery of a ligand-regulated endogenous ion channel gene to sensory neurons results in pain control following channel activation.

Persistent pain remains a tremendous health problem due to both its prevalence and dearth of effective therapeutic interventions. To maximize pain relief while minimizing side effects, current gene therapy-based approaches have mostly exploited the expression of pain inhibitory products or interfered with pronociceptive ion channels. These methods do not enable control over the timing or duration of analgesia, nor titration to analgesic efficacy. Here, we describe a gene therapy strategy that potentially overcomes these limitations by providing exquisite control over therapy with efficacy in clinically relevant models of inflammatory pain. We utilize a herpes simplex viral (HSV) vector (vHGlyRα1) to express a ligand-regulated chloride ion channel, the glycine receptor (GlyR) in targeted sensory afferents; the subsequent exogenous addition of glycine provides the means for temporal and spatial control of afferent activity, and therefore pain. Use of an endogenous inhibitory receptor not normally present on sensory neurons both minimizes immunogenicity and maximizes therapeutic selectivity.

Inhibition of NMDA receptors through a membrane-to-channel path.

N-methyl-D-aspartate receptors (NMDARs) are transmembrane proteins that are activated by the neurotransmitter glutamate and are found at most excitatory vertebrate synapses. NMDAR channel blockers, an antagonist class of broad pharmacological and clinical significance, inhibit by occluding the NMDAR ion channel. A vast literature demonstrates that NMDAR channel blockers, including MK-801, phencyclidine, ketamine, and the Alzheimer's disease drug memantine, can bind and unbind only when the NMDAR channel is open. Here we use electrophysiological recordings from transfected tsA201 cells and cultured neurons, NMDAR structural modeling, and custom-synthesized compounds to show that NMDAR channel blockers can enter the channel through two routes: the well-known hydrophilic path from extracellular solution to channel through the open channel gate, and also a hydrophobic path from plasma membrane to channel through a gated fenestration ("membrane-to-channel inhibition" (MCI)). Our demonstration that ligand-gated channels are subject to MCI, as are voltage-gated channels, highlights the broad expression of this inhibitory mechanism.

Design, synthesis, and in vitro and in vivo characterization of new memantine analogs for Alzheimer's disease.

Currently, of the few accessible symptomatic therapies for Alzheimer's disease (AD), memantine is the only N-methyl-d-aspartate receptor (NMDAR) blocker approved by the FDA. This work further explores a series of memantine analogs featuring a benzohomoadamantane scaffold. Most of the newly synthesized compounds block NMDARs in the micromolar range, but with lower potency than previously reported hit IIc, results that were supported by molecular dynamics simulations. Subsequently, electrophysiological studies with the more potent compounds allowed classification of IIc, a low micromolar, uncompetitive, voltage-dependent, NMDAR blocker, as a memantine-like compound. The excellent in vitro DMPK properties of IIc made it a promising candidate for in vivo studies in Caenorhabditis elegans (C. elegans) and in the 5XFAD mouse model of AD. Administration of IIc or memantine improved locomotion and rescues chemotaxis behavior in C. elegans. Furthermore, both compounds enhanced working memory in 5XFAD mice and modified NMDAR and CREB signaling, which may prevent synaptic dysfunction and modulate neurodegenerative progression.

Mg2+ imparts NMDA receptor subtype selectivity to the Alzheimer's drug memantine.

N-methyl-D-aspartate receptors (NMDARs) mediate interneuronal communication and are broadly involved in nervous system physiology and pathology (Dingledine et al., 1999). Memantine, a drug that blocks the ion channel formed by NMDARs, is a widely prescribed treatment of Alzheimer's disease (Schmitt, 2005; Lipton, 2006; Parsons et al., 2007). Research on memantine's mechanism of action has focused on the NMDAR subtypes most highly expressed in adult cerebral cortex, NR1/2A and NR1/2B receptors (Cull-Candy and Leszkiewicz, 2004), and has largely ignored interactions with extracellular Mg(2+) (Mg(2+)(o)). Mg(2+)(o) is an endogenous NMDAR channel blocker that binds near memantine's binding site (Kashiwagi et al., 2002; Chen and Lipton, 2005). We report that a physiological concentration (1 mM) of Mg(2+)(o) decreased memantine inhibition of NR1/2A and NR1/2B receptors nearly 20-fold at a membrane voltage near rest. In contrast, memantine inhibition of the other principal NMDAR subtypes, NR1/2C and NR1/2D receptors, was decreased only approximately 3-fold. As a result, therapeutic memantine concentrations should have negligible effects on NR1/2A or NR1/2B receptor activity but pronounced effects on NR1/2C and NR1/2D receptors. Quantitative modeling showed that the voltage dependence of memantine inhibition also is altered by 1 mM Mg(2+)(o). We report similar results with the NMDAR channel blocker ketamine, a drug used to model schizophrenia (Krystal et al., 2003). These results suggest that currently hypothesized mechanisms of memantine and ketamine action should be reconsidered and that NR1/2C and/or NR1/2D receptors play a more important role in cortical physiology and pathology than previously appreciated.

Interplay between Gating and Block of Ligand-Gated Ion Channels.

Drugs that inhibit ion channel function by binding in the channel and preventing current flow, known as channel blockers, can be used as powerful tools for analysis of channel properties. Channel blockers are used to probe both the sophisticated structure and basic biophysical properties of ion channels. Gating, the mechanism that controls the opening and closing of ion channels, can be profoundly influenced by channel blocking drugs. Channel block and gating are reciprocally connected; gating controls access of channel blockers to their binding sites, and channel-blocking drugs can have profound and diverse effects on the rates of gating transitions and on the stability of channel open and closed states. This review synthesizes knowledge of the inherent intertwining of block and gating of excitatory ligand-gated ion channels, with a focus on the utility of channel blockers as analytic probes of ionotropic glutamate receptor channel function.

Synaptic zinc inhibition of NMDA receptors depends on the association of GluN2A with the zinc transporter ZnT1.

The NMDA receptor (NMDAR) is inhibited by synaptically released zinc. This inhibition is thought to be the result of zinc diffusion across the synaptic cleft and subsequent binding to the extracellular domain of the NMDAR. However, this model fails to incorporate the observed association of the highly zinc-sensitive NMDAR subunit GluN2A with the postsynaptic zinc transporter ZnT1, which moves intracellular zinc to the extracellular space. Here, we report that disruption of ZnT1-GluN2A association by a cell-permeant peptide strongly reduced NMDAR inhibition by synaptic zinc in mouse dorsal cochlear nucleus synapses. Moreover, synaptic zinc inhibition of NMDARs required postsynaptic intracellular zinc, suggesting that cytoplasmic zinc is transported by ZnT1 to the extracellular space in close proximity to the NMDAR. These results challenge a decades-old dogma on how zinc inhibits synaptic NMDARs and demonstrate that presynaptic release and a postsynaptic transporter organize zinc into distinct microdomains to modulate NMDAR neurotransmission.

Memantine binding to a superficial site on NMDA receptors contributes to partial trapping.

Although many nervous system disorders are associated with N-methyl-D-aspartate (NMDA) receptor overactivation, pharmacological inhibition of NMDA receptors has typically demonstrated limited clinical value due to debilitating psychotomimetic side-effects. Memantine, however, induces far fewer behavioural side-effects than other NMDA receptor channel blockers such as ketamine, and slows the progressive cognitive decline associated with Alzheimer's disease. Memantine and ketamine inhibit NMDA receptors with similar affinity and kinetics. A prominent mechanistic difference between memantine and ketamine is the degree to which they are 'trapped' within the closed channel of NMDA receptors following removal of agonist: ketamine becomes trapped in nearly all NMDA receptors to which it was bound before agonist removal, whereas some bound memantine molecules dissociate after agonist removal, a phenomenon called partial trapping. Here we investigated the mechanism underlying partial trapping of memantine by recombinant NR1/2A NMDA receptors. We found that memantine dissociation from NR1/2A receptors after agonist removal (the process that results in partial trapping) followed an exponential time course with tau = 0.79 +/- 0.32 s. Neither membrane voltage depolarization nor maintained presence of memantine after agonist removal affected partial trapping, suggesting that partial trapping does not result from memantine escape through open channels. We tested the hypothesis that partial trapping results from binding of memantine to two sites, a superficial 'non-trapping' site and a deep 'trapping' site, which cannot be occupied simultaneously. This hypothesis was supported by the lack of ketamine binding to the superficial site, the voltage dependence of partial trapping, and the effect on partial trapping of a mutation near the deep site. The superficial binding site for memantine may, by causing partial trapping, contribute to memantine's unique therapeutic utility.

Oxygen-Glucose Deprivation Differentially Affects Neocortical Pyramidal Neurons and Parvalbumin-Positive Interneurons.

Stroke is a devastating brain disorder. The pathophysiology of stroke is associated with an impaired excitation-inhibition balance in the area that surrounds the infarct core after the insult, the peri-infarct zone. Here we exposed slices from adult mouse prefrontal cortex to oxygen-glucose deprivation and reoxygenation (OGD-RO) to study ischemia-induced changes in the activity of excitatory pyramidal neurons and inhibitory parvalbumin (PV)-positive interneurons. We found that during current-clamp recordings, PV-positive interneurons were more vulnerable to OGD-RO than pyramidal neurons as indicated by the lower percentage of recovery of PV-positive interneurons. However, neither the amplitude of OGD-induced depolarization observed in current-clamp mode nor the OGD-associated current observed in voltage-clamp mode differed between the two cell types. Large amplitude, presumably action-potential dependent, spontaneous postsynaptic inhibitory currents recorded from pyramidal neurons were less frequent after OGD-RO than in control condition. Disynaptic inhibitory postsynaptic currents (dIPSCs) in pyramidal neurons produced predominantly by PV-positive interneurons were reduced by OGD-RO. Following OGD-RO, dendrites of PV-positive interneurons exhibited more pathological beading than those of pyramidal neurons. Our data support the hypothesis that the differential vulnerability to ischemia-like conditions of excitatory and inhibitory neurons leads to the altered excitation-inhibition balance associated with stroke pathophysiology.