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Molecular and functional diversity among synapses is generated, in part, by differential expression of neurotransmitter receptors and their associated protein complexes. N -methyl- D -aspartate receptors (NMDARs) are tetrameric ionotropic glutamate receptors that most often comprise two GluN1 and two GluN2 subunits. NMDARs generate functionally diverse synapses across neuron populations through cell-type-specific expression patterns of GluN2 subunits (GluN2A - 2D), which have vastly different functional properties and distinct downstream signaling. Diverse NMDAR function has also been observed at anatomically distinct inputs to a single neuron population. However, the mechanisms that generate input-specific NMDAR function remain unknown as few studies have investigated subcellular GluN2 subunit localization in native brain tissue. We investigated NMDAR synaptic localization in thalamocortical (TC) neurons expressing all four GluN2 subunits. Utilizing super resolution imaging and knockout-validated antibodies, we revealed subtype- and input-specific GluN2 localization at corticothalamic (CT) versus sensory inputs to TC neurons in 4-week-old male and female C57Bl/6J mice. GluN2B was the most abundant postsynaptic subunit across all glutamatergic synapses followed by GluN2A and GluN2C, and GluN2D was localized to the fewest synapses. GluN2B was preferentially localized to CT synapses over sensory synapses, while GluN2A and GluN2C were more abundant at sensory inputs compared to CT inputs. Furthermore, postsynaptic scaffolding proteins PSD95 and SAP102 were preferentially localized with specific GluN2 subunits, and SAP102 was more abundant at sensory synapses than PSD95. This work indicates that TC neurons exhibit subtype- and input-specific localization of diverse NMDARs and associated scaffolding proteins that likely contribute to functional differences between CT and sensory synapses.

Localization of mRNAs to dendrites is a fundamental mechanism by which neurons achieve spatiotemporal control of gene expression. Translationally repressed neuronal mRNA transport granules, also referred to as ribonucleoprotein particles (RNPs), have been shown to be trafficked as single or low copy number RNPs and as larger complexes with multiple copies and/or species of mRNAs. However, there is little evidence of either population in intact neuronal circuits. Using single molecule fluorescence in situ hybridization studies in the dendrites of adult rat and mouse hippocampus, we provide evidence that supports the existence of multi-transcript RNPs with the constituents varying in amounts for each RNA species. By competing-off fluorescently labeled probe with serial increases of unlabeled probe, we detected stepwise decreases in Arc RNP number and fluorescence intensity, suggesting Arc RNAs localize to dendrites in both low- and multiple-copy number RNPs. When probing for multiple mRNAs, we find that localized RNPs are heterogeneous in size and colocalization patterns that vary per RNA. Further, localized RNAs that are targeted by the same trans-acting element (FMRP) display greater levels of colocalization compared to an RNA not targeted by FMRP. Simultaneous visualization of a dozen FMRP-targeted mRNA species using highly multiplexed imaging demonstrates that dendritic RNAs are mostly trafficked as heteromeric cargoes of multiple types of RNAs (at least one or more RNAs). Moreover, the composition of these RNA cargoes, as assessed by colocalization, correlates with the abundance of the transcripts even after accounting for the expected differences in colocalization based on expression. Collectively, these results suggest that dendritic RNPs are packaged as heterogeneous co-assemblies of different mRNAs and that RNP contents may be driven, at least partially, by highly abundant dendritic RNAs; a model that favors efficiency over fine-tuned control for sustaining long-distance trafficking of thousands of messenger molecules., Competing Interests: Conflict of interest The authors declare that they have no competing interests.

Background: Viral cardiac infection represents a significant clinical challenge encompassing several etiological agents, disease stages, complex presentation, and a resulting lack of mechanistic understanding. Myocarditis is a major cause of sudden cardiac death in young adults, where current knowledge in the field is dominated by later disease phases and pathological immune responses. However, little is known regarding how infection can acutely induce an arrhythmogenic substrate before significant immune responses. Adenovirus is a leading cause of myocarditis, but due to species specificity, models of infection are lacking, and it is not understood how adenoviral infection may underlie sudden cardiac arrest. Mouse adenovirus type-3 was previously reported as cardiotropic, yet it has not been utilized to understand the mechanisms of cardiac infection and pathology., Methods: We have developed mouse adenovirus type-3 infection as a model to investigate acute cardiac infection and molecular alterations to the infected heart before an appreciable immune response or gross cardiomyopathy., Results: Optical mapping of infected hearts exposes decreases in conduction velocity concomitant with increased Cx43 Ser368 phosphorylation, a residue known to regulate gap junction function. Hearts from animals harboring a phospho-null mutation at Cx43 Ser368 are protected against mouse adenovirus type-3-induced conduction velocity slowing. Additional to gap junction alterations, patch clamping of mouse adenovirus type-3-infected adult mouse ventricular cardiomyocytes reveals prolonged action potential duration as a result of decreased I K1 and I Ks current density. Turning to human systems, we find human adenovirus type-5 increases phosphorylation of Cx43 Ser368 and disrupts synchrony in human induced pluripotent stem cell-derived cardiomyocytes, indicating common mechanisms with our mouse whole heart and adult cardiomyocyte data., Conclusions: Together, these findings demonstrate that adenoviral infection creates an arrhythmogenic substrate through direct targeting of gap junction and ion channel function in the heart. Such alterations are known to precipitate arrhythmias and likely contribute to sudden cardiac death in acutely infected patients., Competing Interests: Disclosures None.

Somatosensory information is propagated from the periphery to the cerebral cortex by two parallel pathways through the ventral posterolateral (VPL) and ventral posteromedial (VPM) thalamus. VPL and VPM neurons receive somatosensory signals from the body and head, respectively. VPL and VPM neurons may also receive cell type-specific GABAergic input from the reticular nucleus of the thalamus. Although VPL and VPM neurons have distinct connectivity and physiological roles, differences in their functional properties remain unclear as they are often studied as one ventrobasal thalamus neuron population. Here, we directly compared synaptic and intrinsic properties of VPL and VPM neurons in C57Bl/6J mice of both sexes aged P25-P32. VPL neurons showed greater depolarization-induced spike firing and spike frequency adaptation than VPM neurons. VPL and VPM neurons fired similar numbers of spikes during hyperpolarization rebound bursts, but VPM neurons exhibited shorter burst latency compared with VPL neurons, which correlated with larger sag potential. VPM neurons had larger membrane capacitance and more complex dendritic arbors. Recordings of spontaneous and evoked synaptic transmission suggested that VPL neurons receive stronger excitatory synaptic input, whereas inhibitory synapse strength was stronger in VPM neurons. This work indicates that VPL and VPM thalamocortical neurons have distinct intrinsic and synaptic properties. The observed functional differences could have important implications for their specific physiological and pathophysiological roles within the somatosensory thalamocortical network. NEW & NOTEWORTHY This study revealed that somatosensory thalamocortical neurons in the VPL and VPM have substantial differences in excitatory synaptic input and intrinsic firing properties. The distinct properties suggest that VPL and VPM neurons could process somatosensory information differently and have selective vulnerability to disease. This work improves our understanding of nucleus-specific neuron function in the thalamus and demonstrates the critical importance of studying these parallel somatosensory pathways separately.

Subunit-selective inhibition of N -methyl-d-aspartate receptors (NMDARs) is a promising therapeutic strategy for several neurological disorders, including epilepsy, Alzheimer's and Parkinson's disease, depression, and acute brain injury. We previously described the dihydroquinoline-pyrazoline (DQP) analogue 2a ( DQP-26 ) as a potent NMDAR negative allosteric modulator with selectivity for GluN2C/D over GluN2A/B. However, moderate (<100-fold) subunit selectivity, inadequate cell-membrane permeability, and poor brain penetration complicated the use of 2a as an in vivo probe. In an effort to improve selectivity and the pharmacokinetic profile of the series, we performed additional structure-activity relationship studies of the succinate side chain and investigated the use of prodrugs to mask the pendant carboxylic acid. These efforts led to discovery of the analogue ( S )-(-)- 2i , also referred to as ( S )-(-)- DQP-997 - 74 , which exhibits >100- and >300-fold selectivity for GluN2C- and GluN2D-containing NMDARs (IC 50 0.069 and 0.035 μM, respectively) compared to GluN2A- and GluN2B-containing receptors (IC 50 5.2 and 16 μM, respectively) and has no effects on AMPA, kainate, or GluN1/GluN3 receptors. Compound ( S )-(-)- 2i is 5-fold more potent than ( S )- 2a . In addition, compound 2i shows a time-dependent enhancement of inhibitory actions at GluN2C- and GluN2D-containing NMDARs in the presence of the agonist glutamate, which could attenuate hypersynchronous activity driven by high-frequency excitatory synaptic transmission. Consistent with this finding, compound 2i significantly reduced the number of epileptic events in a murine model of tuberous sclerosis complex (TSC)-induced epilepsy that is associated with upregulation of the GluN2C subunit. Thus, 2i represents a robust tool for the GluN2C/D target validation. Esterification of the succinate carboxylate improved brain penetration, suggesting a strategy for therapeutic development of this series for NMDAR-associated neurological conditions.

Thalamocortical network dysfunction contributes to seizures and sleep deficits in Dravet syndrome (DS), an infantile epileptic encephalopathy, but the underlying molecular and cellular mechanisms remain elusive. DS is primarily caused by mutations in the SCN1A gene encoding the voltage-gated sodium channel Na V 1.1, which is highly expressed in GABAergic reticular thalamus (nRT) neurons as well as glutamatergic thalamocortical neurons. We hypothesized that Na V 1.1 haploinsufficiency alters somatosensory corticothalamic circuit function through both intrinsic and synaptic mechanisms in nRT and thalamocortical neurons. Using Scn1a heterozygous mice of both sexes aged P25-P30, we discovered reduced excitability of nRT neurons and thalamocortical neurons in the ventral posterolateral (VPL) thalamus, while thalamocortical ventral posteromedial (VPM) neurons exhibited enhanced excitability. Na V 1.1 haploinsufficiency enhanced GABAergic synaptic input and reduced glutamatergic input to VPL neurons, but not VPM neurons. In addition, glutamatergic input to nRT neurons was reduced in Scn1a heterozygous mice. These findings introduce alterations in glutamatergic synapse function and aberrant glutamatergic neuron excitability in the thalamus as disease mechanisms in DS, which has been widely considered a disease of GABAergic neurons. This work reveals additional complexity that expands current models of thalamic dysfunction in DS and identifies new components of corticothalamic circuitry as potential therapeutic targets., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Many physiologic effects of l-glutamate, the major excitatory neurotransmitter in the mammalian central nervous system, are mediated via signaling by ionotropic glutamate receptors (iGluRs). These ligand-gated ion channels are critical to brain function and are centrally implicated in numerous psychiatric and neurologic disorders. There are different classes of iGluRs with a variety of receptor subtypes in each class that play distinct roles in neuronal functions. The diversity in iGluR subtypes, with their unique functional properties and physiologic roles, has motivated a large number of studies. Our understanding of receptor subtypes has advanced considerably since the first iGluR subunit gene was cloned in 1989, and the research focus has expanded to encompass facets of biology that have been recently discovered and to exploit experimental paradigms made possible by technological advances. Here, we review insights from more than 3 decades of iGluR studies with an emphasis on the progress that has occurred in the past decade. We cover structure, function, pharmacology, roles in neurophysiology, and therapeutic implications for all classes of receptors assembled from the subunits encoded by the 18 ionotropic glutamate receptor genes. SIGNIFICANCE STATEMENT: Glutamate receptors play important roles in virtually all aspects of brain function and are either involved in mediating some clinical features of neurological disease or represent a therapeutic target for treatment. Therefore, understanding the structure, function, and pharmacology of this class of receptors will advance our understanding of many aspects of brain function at molecular, cellular, and system levels and provide new opportunities to treat patients., (U.S. Government work not protected by U.S. copyright.)

Allosteric modulators of ion channels typically alter the transitions rates between conformational states without changing the properties of the open pore. Here we describe a new class of positive allosteric modulators of N-methyl D-aspartate receptors (NMDARs) that mediate a calcium-permeable component of glutamatergic synaptic transmission and play essential roles in learning, memory and cognition, as well as neurological disease. EU1622-14 increases agonist potency and channel-open probability, slows receptor deactivation and decreases both single-channel conductance and calcium permeability. The unique functional selectivity of this chemical probe reveals a mechanism for enhancing NMDAR function while limiting excess calcium influx, and shows that allosteric modulators can act as biased modulators of ion-channel permeation.

N-methyl-D-aspartate receptors (NMDARs) mediate slow excitatory postsynaptic transmission in the central nervous system, thereby exerting a critical role in neuronal development and brain function. Rare genetic variants in the GRIN genes encoding NMDAR subunits segregated with neurological disorders. Here, we summarize the clinical presentations for 18 patients harboring 12 de novo missense variants in GRIN1, GRIN2A, and GRIN2B that alter residues in the M2 re-entrant loop, a region that lines the pore and is intolerant to missense variation. These de novo variants were identified in children with a set of neurological and neuropsychiatric conditions. Evaluation of the receptor cell surface expression, pharmacological properties, and biophysical characteristics show that these variants can have modest changes in agonist potency, proton inhibition, and surface expression. However, voltage-dependent magnesium inhibition is significantly reduced in all variants. The NMDARs hosting a single copy of a mutant subunit showed a dominant reduction in magnesium inhibition for some variants. These variant NMDARs also show reduced calcium permeability and single-channel conductance, as well as altered open probability. The data suggest that M2 missense variants increase NMDAR charge transfer in addition to varied and complex influences on NMDAR functional properties, which may underlie the patients' phenotypes., (© 2019 Wiley Periodicals, Inc.)

Developing cortical GABAergic interneurons rely on genetic programs, neuronal activity, and environmental cues to construct inhibitory circuits during early postnatal development. Disruption of these events can cause long-term changes in cortical inhibition and may be involved in neurological disorders associated with inhibitory circuit dysfunction. We hypothesized that tonic glutamate signaling in the neonatal cortex contributes to, and is necessary for, the maturation of cortical interneurons. To test this hypothesis, we used mice of both sexes to quantify extracellular glutamate concentrations in the cortex during development, measure ambient glutamate-mediated activation of developing cortical interneurons, and manipulate tonic glutamate signaling using subtype-specific NMDA receptor antagonists in vitro and in vivo We report that ambient glutamate levels are high (≈100 nm) in the neonatal cortex and decrease (to ≈50 nm) during the first weeks of life, coincident with increases in astrocytic glutamate uptake. Consistent with elevated ambient glutamate, putative parvalbumin-positive interneurons in the cortex (identified using G42:GAD1-eGFP reporter mice) exhibit a transient, tonic NMDA current at the end of the first postnatal week. GluN2C/GluN2D-containing NMDA receptors mediate the majority of this current and contribute to the resting membrane potential and intrinsic properties of developing putative parvalbumin interneurons. Pharmacological blockade of GluN2C/GluN2D-containing NMDA receptors in vivo during the period of tonic interneuron activation, but not later, leads to lasting decreases in interneuron morphological complexity and causes deficits in cortical inhibition later in life. These results demonstrate that dynamic ambient glutamate signaling contributes to cortical interneuron maturation via tonic activation of GluN2C/GluN2D-containing NMDA receptors. SIGNIFICANCE STATEMENT Inhibitory GABAergic interneurons make up 20% of cortical neurons and are critical to controlling cortical network activity. Dysfunction of cortical inhibition is associated with multiple neurological disorders, including epilepsy. Establishing inhibitory cortical networks requires in utero proliferation, differentiation, and migration of immature GABAergic interneurons, and subsequent postnatal morphological maturation and circuit integration. Here, we demonstrate that ambient glutamate provides tonic activation of immature, putative parvalbumin-positive GABAergic interneurons in the neonatal cortex via high-affinity NMDA receptors. When this activation is blocked, GABAergic interneuron maturation is disrupted, and cortical networks exhibit lasting abnormal hyperexcitability. We conclude that temporally precise activation of developing cortical interneurons by ambient glutamate is critically important for establishing normal cortical inhibition., (Copyright © 2019 the authors.)

In their 1994 paper, Monyer et al. described unique functional properties and cell type-specific expression of NMDA receptor (NMDAR) GluN2 subunits, suggesting that the roles of NMDAR vary across brain regions and throughout development. This influential work not only provided insight into how molecular diversity impacts synapse function, but also continues to inform new approaches for modulating brain circuits., (Copyright © 2018. Published by Elsevier Ltd.)

NMDA-type glutamate receptors (NMDARs) are ligand-gated ion channels that mediate excitatory neurotransmission in the CNS. Here we describe functional and single-channel properties of triheteromeric GluN1/GluN2A/GluN2C receptors, which contain two GluN1, one GluN2A, and one GluN2C subunits. This NMDAR has three conductance levels and opens in bursts similar to GluN1/GluN2A receptors but with a single-channel open time and open probability reminiscent of GluN1/GluN2C receptors. The deactivation time course of GluN1/GluN2A/GluN2C receptors is intermediate to GluN1/GluN2A and GluN1/GluN2C receptors and is not dominated by GluN2A or GluN2C. We show that triheteromeric GluN1/GluN2A/GluN2C receptors are the predominant NMDARs in cerebellar granule cells and propose that co-expression of GluN2A and GluN2C in cerebellar granule cells occludes cell surface expression of diheteromeric GluN1/GluN2C receptors. This new insight into neuronal GluN1/GluN2A/GluN2C receptors highlights the complexity of NMDAR signaling in the CNS., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Polymicrogyria is a malformation of cortical development. The aetiology of polymicrogyria remains poorly understood. Using whole-exome sequencing we found de novo heterozygous missense GRIN1 mutations in 2 of 57 parent-offspring trios with polymicrogyria. We found nine further de novo missense GRIN1 mutations in additional cortical malformation patients. Shared features in the patients were extensive bilateral polymicrogyria associated with severe developmental delay, postnatal microcephaly, cortical visual impairment and intractable epilepsy. GRIN1 encodes GluN1, the essential subunit of the N-methyl-d-aspartate receptor. The polymicrogyria-associated GRIN1 mutations tended to cluster in the S2 region (part of the ligand-binding domain of GluN1) or the adjacent M3 helix. These regions are rarely mutated in the normal population or in GRIN1 patients without polymicrogyria. Using two-electrode and whole-cell voltage-clamp analysis, we showed that the polymicrogyria-associated GRIN1 mutations significantly alter the in vitro activity of the receptor. Three of the mutations increased agonist potency while one reduced proton inhibition of the receptor. These results are striking because previous GRIN1 mutations have generally caused loss of function, and because N-methyl-d-aspartate receptor agonists have been used for many years to generate animal models of polymicrogyria. Overall, our results expand the phenotypic spectrum associated with GRIN1 mutations and highlight the important role of N-methyl-d-aspartate receptor signalling in the pathogenesis of polymicrogyria.

N-Methyl-d-aspartate receptors (NMDARs) are ionotropic glutamate receptors that mediate excitatory synaptic transmission and have been implicated in numerous neurological disorders. NMDARs typically comprise two GluN1 and two GluN2 subunits. The four GluN2 subtypes (GluN2A-GluN2D) have distinct functional properties and gene expression patterns, which contribute to diverse functional roles for NMDARs in the brain. Here, we present a series of GluN2C/2D-selective negative allosteric modulators built around a N-aryl benzamide (NAB) core. The prototypical compound, NAB-14, is >800-fold selective for recombinant GluN2C/GluN2D over GluN2A/GluN2B in Xenopus oocytes and has an IC 50 value of 580 nM at recombinant GluN2D-containing receptors expressed in mammalian cells. NAB-14 inhibits triheteromeric (GluN1/GluN2A/GluN2C) NMDARs with modestly reduced potency and efficacy compared to diheteromeric (GluN1/GluN2C/GluN2C) receptors. Site-directed mutagenesis suggests that structural determinants for NAB-14 inhibition reside in the GluN2D M1 transmembrane helix. NAB-14 inhibits GluN2D-mediated synaptic currents in rat subthalamic neurons and mouse hippocampal interneurons, but has no effect on synaptic transmission in hippocampal pyramidal neurons, which do not express GluN2C or GluN2D. This series possesses some druglike physical properties and modest brain permeability in rat and mouse. Altogether, this work identifies a new series of negative allosteric modulators that are valuable tools for studying GluN2C- and GluN2D-containing NMDAR function in brain circuits, and suggests that the series has the potential to be developed into therapies for selectively modulating brain circuits involving the GluN2C and GluN2D subunits.

N-methyl-d-aspartate receptors (NMDARs) play important roles in brain development and neurological disease. We report two individuals with similar dominant de novo GRIN1 mutations (c.1858 G>A and c.1858 G>C; both p.G620R). Both individuals presented at birth with developmental delay and hypotonia associated with behavioral abnormalities and stereotypical movements. Recombinant NMDARs containing the mutant GluN1-G620R together with either GluN2A or GluN2B were evaluated for changes in their trafficking to the plasma membrane and their electrophysiological properties. GluN1-G620R/GluN2A complexes showed a mild reduction in trafficking, a ~2-fold decrease in glutamate and glycine potency, a strong decrease in sensitivity to Mg 2+ block, and a significant reduction of current responses to a maximal effective concentration of agonists. GluN1-G620R/GluN2B complexes showed significantly reduced delivery of protein to the cell surface associated with similarly altered electrophysiology. These results indicate these individuals may have suffered neurodevelopmental deficits as a result of the decreased presence of GluN1-G620R/GluN2B complexes on the neuronal surface during embryonic brain development and reduced current responses of GluN1-G620R-containing NMDARs after birth. These cases emphasize the importance of comprehensive functional characterization of de novo mutations and illustrates how a combination of several distinct features of NMDAR expression, trafficking and function can be present and influence phenotype.

N-methyl-D-aspartate receptors (NMDARs), ligand-gated ionotropic glutamate receptors, play key roles in normal brain development and various neurological disorders. Here we use standing variation data from the human population to assess which protein domains within NMDAR GluN1, GluN2A and GluN2B subunits show the strongest signal for being depleted of missense variants. We find that this includes the GluN2 pre-M1 helix and linker between the agonist-binding domain (ABD) and first transmembrane domain (M1). We then evaluate the functional changes of multiple missense mutations in the NMDAR pre-M1 helix found in children with epilepsy and developmental delay. We find mutant GluN1/GluN2A receptors exhibit prolonged glutamate response time course for channels containing 1 or 2 GluN2A-P552R subunits, and a slow rise time only for receptors with 2 mutant subunits, suggesting rearrangement of one GluN2A pre-M1 helix is sufficient for rapid activation. GluN2A-P552R and analogous mutations in other GluN subunits increased the agonist potency and slowed response time course, suggesting a functionally conserved role for this residue. Although there is no detectable change in surface expression or open probability for GluN2A-P552R, the prolonged response time course for receptors that contained GluN2A-P552R increased charge transfer for synaptic-like activation, which should promote excitotoxic damage. Transfection of cultured neurons with GluN2A-P552R prolonged EPSPs, and triggered pronounced dendritic swelling in addition to excitotoxicity, which were both attenuated by memantine. These data implicate the pre-M1 region in gating, provide insight into how different subunits contribute to gating, and suggest that mutations in the pre-M1 helix can compromise neuronal health. Evaluation of FDA-approved NMDAR inhibitors on the mutant NMDAR-mediated current response and neuronal damage provides a potential clinical path to treat individuals harboring similar mutations in NMDARs., Competing Interests: SFT is a consultant of Janssen Pharmaceuticals, Boehringer Ingelheim Pharma GmbH, and Pfizer Inc, and co-founder of NeurOp Inc. SP serves on the advisory board and is an equity holder of Pairnomix. AEC is a founder of Q-State Biosciences.

Epilepsy and intellectual disability are associated with rare variants in the GluN2A and GluN2B (encoded by GRIN2A and GRIN2B) subunits of the N-methyl-D-aspartate receptor (NMDAR), a ligand-gated ion channel with essential roles in brain development and function. By assessing genetic variation across GluN2 domains, we determined that the agonist binding domain, transmembrane domain, and the linker regions between these domains were particularly intolerant to functional variation. Notably, the agonist binding domain of GluN2B exhibited significantly more variation intolerance than that of GluN2A. To understand the ramifications of missense variation in the agonist binding domain, we investigated the mechanisms by which 25 rare variants in the GluN2A and GluN2B agonist binding domains dysregulated NMDAR activity. When introduced into recombinant human NMDARs, these rare variants identified in individuals with neurologic disease had complex, and sometimes opposing, consequences on agonist binding, channel gating, receptor biogenesis, and forward trafficking. Our approach combined quantitative assessments of these effects to estimate the overall impact on synaptic and non-synaptic NMDAR function. Interestingly, similar neurologic diseases were associated with both gain- and loss-of-function variants in the same gene. Most rare variants in GluN2A were associated with epilepsy, whereas GluN2B variants were associated with intellectual disability with or without seizures. Finally, discerning the mechanisms underlying NMDAR dysregulation by these rare variants allowed investigations of pharmacologic strategies to correct NMDAR function., (Copyright © 2016 American Society of Human Genetics. All rights reserved.)

Alzheimer's disease (AD), the most common form of dementia in the elderly, is characterized by the presence of extracellular plaques composed of amyloid β (Aβ) peptides and intracellular tau aggregates. The plaques are surrounded by microglia, the brain's resident immune cells, which likely participate in the clearance of Aβ by phagocytosis. The microglia that are associated with plaques display an abnormal ameboid morphology and do not respond to tissue damage, in contrast to microglia in healthy brains. Here, we used time lapse confocal microscopy to perform a detailed real-time examination of microglial motility in acute hippocampal brain slices from the 5xFAD mouse model of AD, which was crossed to Cx3cr1(GFP/GFP) mice to achieve microglia-specific GFP expression for visualization. During baseline conditions, microglia around plaques appeared hypermotile, moving the processes that were pointing away from plaques at higher speed than microglia not associated with plaques. Yet, neither plaque-associated, nor plaque-free microglia were able to extend processes toward sites of modest mechanical damage. Application of the selective adenosine A2A receptor antagonist preladenant, which restores microglial response to cellular damage in a mouse model of Parkinson's disease, reduced the hypermotility of plaque-associated microglia, but did not restore motility toward damaged cells in slices from 5xFAD mice. Our results suggest that process hypermotility and resistance to A2A antagonism during response to tissue damage may represent unique functional phenotypes of plaque-associated microglia that impair their ability to function properly in the AD brain., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)

Communication among neurons is mediated through synaptic connections between axons and dendrites, and most excitatory synapses occur on actin-rich protrusions along dendrites called dendritic spines. Dendritic spines are structurally dynamic, and synapse strength is closely correlated with spine morphology. Abnormalities in the size, shape, and number of dendritic spines are prevalent in neurologic diseases, including autism spectrum disorders, schizophrenia, and Alzheimer disease. However, therapeutic targets that influence spine morphology are lacking. Rho-associated coiled-coil containing protein kinases (ROCK) 1 and ROCK2 are potent regulators of the actin cytoskeleton and highly promising drug targets for central nervous system disorders. In this report, we addressed how pharmacologic inhibition of ROCK1 and ROCK2 affects dendritic spine morphology. Hippocampal neurons were transfected with plasmids expressing fluorescently labeled Lifeact, a small actin binding peptide, and then incubated with or without Y-27632, an established pan-ROCK small molecule inhibitor. Using an automated 3D spine morphometry analysis method, we showed that inhibition of ROCK1 and ROCK2 significantly increased the mean protrusion density and significantly reduced the mean protrusion width. A trending increase in mean protrusion length was observed following Y-27632 treatment, and novel effects were observed among spine classes. Exposure to Y-27632 significantly increased the number of filopodia and thin spines, while the numbers of stubby and mushroom spines were similar to mock-treated samples. These findings support the hypothesis that pharmacologic inhibition of ROCK1 and ROCK2 may convey therapeutic benefit for neurologic disorders that feature dendritic spine loss or aberrant structural plasticity.

The GluN2D subunit of the NMDA receptor is prominently expressed in the basal ganglia and associated brainstem nuclei, including the subthalamic nucleus (STN), globus pallidus, striatum, and substantia nigra. However, little is known about how GluN2D-containing NMDA receptors contribute to synaptic activity in these regions. Using Western blotting of STN tissue punches, we demonstrated that GluN2D is expressed in the rat STN throughout development [age postnatal day 7 (P7)-P60] and in the adult (age P120). Immunoelectron microscopy of the adult rat brain showed that GluN2D is predominantly expressed in dendrites, unmyelinated axons, and axon terminals within the STN. Using subunit-selective allosteric modulators of NMDA receptors (TCN-201, ifenprodil, CIQ, and DQP-1105), we provide evidence that receptors containing the GluN2B and GluN2D subunits mediate responses to exogenously applied NMDA and glycine, as well as synaptic NMDA receptor activation in the STN of rat brain slices. EPSCs in the STN were mediated primarily by AMPA and NMDA receptors and GluN2D-containing NMDA receptors controlled the slow deactivation time course of EPSCs in the STN. In vivo recordings from the STN of anesthetized adult rats demonstrated that the spike firing rate was increased by the GluN2C/D potentiator CIQ and decreased by the GluN2C/D antagonist DQP-1105, suggesting that NMDA receptor activity can influence STN output. These data indicate that the GluN2B and GluN2D NMDA receptor subunits contribute to synaptic activity in the STN and may represent potential therapeutic targets for modulating subthalamic neuron activity in neurological disorders such as Parkinson's disease., (Copyright © 2015 the authors 0270-6474/15/3515971-13$15.00/0.)

Stroke remains a significant problem despite decades of work on neuroprotective strategies. NMDA receptor (NMDAR) antagonists are neuroprotective in preclinical models, but have been clinically unsuccessful, in part due to side effects. Here we describe a prototypical GluN2B-selective antagonist with an IC50 value that is 10-fold more potent at acidic pH 6.9 associated with ischemic tissue compared to pH 7.6, a value close to the pH in healthy brain tissue. This should maximize neuroprotection in ischemic tissue while minimizing on-target side effects associated with NMDAR blockade in noninjured brain regions. We have determined the mechanism underlying pH-dependent inhibition and demonstrate the utility of this approach in vivo. We also identify dicarboxylate dimers as a novel proton sensor in proteins. These results provide insight into the molecular basis of pH-dependent neuroprotective NMDAR block, which could be beneficial in a wide range of neurological insults associated with tissue acidification., (Copyright © 2015 Elsevier Inc. All rights reserved.)

NMDA receptors are tetrameric complexes of GluN1, GluN2A-D, and GluN3A-B subunits and are involved in normal brain function and neurologic disorders. We identified a novel class of stereoselective pyrrolidinone (PYD) positive allosteric modulators for GluN2C-containing NMDA receptors, exemplified by methyl 4-(3-acetyl-4-hydroxy-1-[2-(2-methyl-1H-indol-3-yl)ethyl]-5-oxo-2,5-dihydro-1H-pyrrol-2-yl)benzoate. Here we explore the site and mechanism of action of a prototypical analog, PYD-106, which at 30 μM does not alter responses of NMDA receptors containing GluN2A, GluN2B, and GluN2D and has no effect on AMPA [α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid] and kainate receptors. Coapplication of 50 μM PYD-106 with a maximally effective concentration of glutamate and glycine increases the response of GluN1/GluN2C NMDA receptors in HEK-293 cells to 221% of that obtained in the absence of PYD (taken as 100%). Evaluation of the concentration dependence of this enhancement revealed an EC50 value for PYD of 13 μM. PYD-106 increased opening frequency and open time of single channel currents activated by maximally effective concentrations of agonist but only had modest effects on glutamate and glycine EC50. PYD-106 selectively enhanced the responses of diheteromeric GluN1/GluN2C receptors but not triheteromeric GluN1/GluN2A/GluN2C receptors. Inclusion of residues encoded by GluN1-exon 5 attenuated the effects of PYD. Three GluN2C residues (Arg194, Ser470, Lys470), at which mutagenesis virtually eliminated PYD function, line a cavity at the interface of the ligand binding and the amino terminal domains in a homology model of GluN1/GluN2C built from crystallographic data on GluN1/GluN2B. We propose that this domain interface constitutes a new allosteric modulatory site on the NMDA receptor., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Microglia, the resident immune cells of the central nervous system, exist in either a "resting" state associated with physiological tissue surveillance or an "activated" state in neuroinflammation. We recently showed that ATP is the primary chemoattractor to tissue damage in vivo and elicits opposite effects on the motility of activated microglia in vitro through activation of adenosine A2A receptors. However, whether systemic inflammation affects microglial responses to tissue damage in vivo remains largely unknown. Using in vivo two-photon imaging of mice, we show that injection of lipopolysaccharide (LPS) at levels that can produce both clear neuroinflammation and some features of sepsis significantly reduced the rate of microglial response to laser-induced ablation injury in vivo. Under proinflammatory conditions, microglial processes initially retracted from the ablation site, but subsequently moved toward and engulfed the damaged area. Analyzing the process dynamics in 3D cultures of primary microglia indicated that only A2A , but not A1 or A3 receptors, mediate process retraction in LPS-activated microglia. The A2A receptor antagonists caffeine and preladenant reduced adenosine-mediated process retraction in activated microglia in vitro. Finally, administration of preladenant before induction of laser ablation in vivo accelerated the microglial response to injury following systemic inflammation. The regulation of rapid microglial responses to sites of injury by A2A receptors could have implications for their ability to respond to the neuronal death occurring under conditions of neuroinflammation in neurodegenerative disorders., (© 2014 Wiley Periodicals, Inc.)

Long-term synaptic plasticity involves changes in the expression and membrane insertion of cell-surface proteins. Interestingly, the mRNAs encoding many cell-surface proteins are localized to dendrites, but whether dendritic protein synthesis is required for activity-induced surface expression of specific proteins is unknown. Herein, we used microfluidic devices to demonstrate that dendritic protein synthesis is necessary for activity-induced insertion of GluN2A-containing NMDA receptors in rat hippocampal neurons. Furthermore, visualization of activity-induced local translation of GluN2A mRNA and membrane insertion of GluN2A protein in dendrites was directly observed and shown to depend on a 3' untranslated region cytoplasmic polyadenylation element and its associated translation complex. These findings uncover a novel mechanism for cytoplasmic polyadenylation element-mediated posttranscriptional regulation of GluN2A mRNA to control NMDA receptor surface expression during synaptic plasticity.

Synaptic activity is a spatially limited process that requires a precise, yet dynamic, complement of proteins within the synaptic micro-domain. The maintenance and regulation of these synaptic proteins is regulated, in part, by local mRNA translation in dendrites. Protein synthesis within the postsynaptic compartment allows neurons tight spatial and temporal control of synaptic protein expression, which is critical for proper functioning of synapses and neural circuits. In this review, we discuss the identity of proteins synthesized within dendrites, the receptor-mediated mechanisms regulating their synthesis, and the possible roles for these locally synthesized proteins. We also explore how our current understanding of dendritic protein synthesis in the hippocampus can be applied to new brain regions and to understanding the pathological mechanisms underlying varied neurological diseases., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Translational control of mRNAs in dendrites is essential for certain forms of synaptic plasticity and learning and memory. CPEB is an RNA-binding protein that regulates local translation in dendrites. Here, we identify poly(A) polymerase Gld2, deadenylase PARN, and translation inhibitory factor neuroguidin (Ngd) as components of a dendritic CPEB-associated polyadenylation apparatus. Synaptic stimulation induces phosphorylation of CPEB, PARN expulsion from the ribonucleoprotein complex, and polyadenylation in dendrites. A screen for mRNAs whose polyadenylation is altered by Gld2 depletion identified >100 transcripts including one encoding NR2A, an NMDA receptor subunit. shRNA depletion studies demonstrate that Gld2 promotes and Ngd inhibits dendritic NR2A expression. Finally, shRNA-mediated depletion of Gld2 in vivo attenuates protein synthesis-dependent long-term potentiation (LTP) at hippocampal dentate gyrus synapses; conversely, Ngd depletion enhances LTP. These results identify a pivotal role for polyadenylation and the opposing effects of Gld2 and Ngd in hippocampal synaptic plasticity., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Uncovering the mechanisms that regulate dendritic spine morphology has been limited, in part, by the lack of efficient and unbiased methods for analyzing spines. Here, we describe an automated 3D spine morphometry method and its application to spine remodeling in live neurons and spine abnormalities in a disease model. We anticipate that this approach will advance studies of synapse structure and function in brain development, plasticity, and disease.

Neurons are exquisitely polarized cells that extend intricate axonal and dendritic arbors. Developmental cues guide axons and dendrites into circuits by inducing rapid changes in local protein expression and cytoskeletal structure. Neurons can transduce these signals through local mRNA regulation. Here, we review the latest insights regarding post-transcriptional control of gene expression through mRNA transport and local protein synthesis in developing neurons. We focus on local mRNA regulation during axon growth and guidance, dendrite morphogenesis, and synapse formation and refinement. Dysregulated mRNA transport and translation in neurological disorders are also discussed. The collection of molecules and mechanisms reviewed includes sequence-specific RNA binding proteins, microtubule motors and adaptors, microRNAs, translation initiation factors, and the receptor-mediated signaling that modulates these molecules., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The localization of specific mRNAs into dendrites and/or axons is an important mechanism to enrich -proteins at their sites of function and influence neuronal development, plasticity, and repair. The fluorescence in situ hybridization (FISH) methods described here have provided high sensitivity and resolution enabling investigation into the mechanism, regulation, and function of mRNA localization in vitro and in vivo. Two methods are described in detail. The first method employs digoxigenin- or fluorophore-conjugated oligonucleotide probes for the detection of localized mRNAs in dendrites, spines, axons, and growth cones of cultured neurons. The second method employs digoxigenin-labeled RNA probes and fluorescence tyramide amplification for the detection of less abundant mRNAs localized to dendrites in vivo. Both methods enable the visualization and quantification of mRNA granules, and changes in their localization in response to various stimuli. The high-resolution FISH technology described here has broader applications beyond the study of mRNA localization. It enables the quantitative analyses of developmental and cell type-specific patterns of gene expression, and how these are modified by physiological signals or during disease states.

The RNA binding protein CPEB (cytoplasmic polyadenylation element binding) regulates cytoplasmic polyadenylation and translation in germ cells and the brain. In neurons, CPEB is detected at postsynaptic sites, as well as in the cell body. The related CPEB3 protein also regulates translation in neurons, albeit probably not through polyadenylation; it, as well as CPEB4, is present in dendrites and the cell body. Here, we show that treatment of neurons with ionotropic glutamate receptor agonists causes CPEB4 to accumulate in the nucleus. All CPEB proteins are nucleus-cytoplasm shuttling proteins that are retained in the nucleus in response to calcium-mediated signaling and alpha-calcium/calmodulin-dependent kinase protein II (CaMKII) activity. CPEB2, -3, and -4 have conserved nuclear export signals that are not present in CPEB. CPEB4 is necessary for cell survival and becomes nuclear in response to focal ischemia in vivo and when cultured neurons are deprived of oxygen and glucose. Further analysis indicates that nuclear accumulation of CPEB4 is controlled by the depletion of calcium from the ER, specifically, through the inositol-1,4,5-triphosphate (IP3) receptor, indicating a communication between these organelles in redistributing proteins between subcellular compartments.

Objective: Bone marrow stromal cells (MSC) are multipotent adult stem cells that have emerged as promising candidates for cell therapy in disorders including cardiac infarction, stroke, and spinal cord injury. While harvesting methods used by different laboratories are relatively standard, MSC culturing protocols vary widely. This study is aimed at evaluating the effects of initial plating density and total time in culture on proliferation, cell morphology, and differentiation potential of heterogeneous MSC cultures and more homogeneous cloned subpopulations., Materials and Methods: Rat MSC were plated at 20, 200, and 2000 cells/cm(2) and grown to 50% confluency. The numbers of population doublings and doubling times were determined within and across multiple passages. Changes in cell morphology and differentiation potential to adipogenic, chondrogenic, and osteogenic lineages were evaluated and compared among early, intermediate, and late passages, as well as between heterogeneous and cloned MSC populations., Results: We found optimal cell growth at a plating density of 200 cells/cm(2). Cultures derived from all plating densities developed increased proportions of flat cells over time. Assays for chondrogenesis, osteogenesis, and adipogenesis showed that heterogeneous MSC plated at all densities sustained the potential for all three mesenchymal phenotypes through at least passage 5; the flat subpopulation lost adipogenic and chondrogenic potential., Conclusion: Our findings suggest that the initial plating density is not critical for maintaining a well-defined, multipotent MSC population. Time in culture, however, affects cell characteristics, suggesting that cell expansion should be limited, especially until the specific characteristics of different MSC subpopulations are better understood.

The function of local protein synthesis in synaptic plasticity and its dysregulation in fragile X syndrome (FXS) is well studied, however the contribution of regulated mRNA transport to this function remains unclear. We report a function for the fragile X mental retardation protein (FMRP) in the rapid, activity-regulated transport of mRNAs important for synaptogenesis and plasticity. mRNAs were deficient in glutamatergic signaling-induced dendritic localization in neurons from Fmr1 KO mice, and single mRNA particle dynamics in live neurons revealed diminished kinesis. Motor-dependent translocation of FMRP and cognate mRNAs involved the C terminus of FMRP and kinesin light chain, and KO brain showed reduced kinesin-associated mRNAs. Acute suppression of FMRP and target mRNA transport in WT neurons resulted in altered filopodia-spine morphology that mimicked the FXS phenotype. These findings highlight a mechanism for stimulus-induced dendritic mRNA transport and link its impairment in a mouse model of FXS to altered developmental morphologic plasticity.

This study evaluates functional recovery after transplanting human bone marrow-derived stromal cells (BMSCs) into contusion models of spinal cord injury (SCI). The authors used a high-throughput process to expand BMSCs and characterized them by flow cytometry, ELISA, and gene expression. They found that BMSCs secrete neurotrophic factors and cytokines with therapeutic potential for cell survival and axon growth. In adult immune-suppressed rats, mild, moderate, or severe contusions were generated using the MASCIS impactor. One week following injury, 0.5 to 1 x 106 BMSCs were injected into the lesioned spinal cord; control animals received vehicle injection. Biweekly behavioral tests included the Basso, Beattie, and Bresnahan Locomotor Rating Scale (BBB), exploratory rearing, grid walking, and thermal sensitivity. Animals receiving moderate contusions followed by BMSC grafts showed significant behavioral recovery in BBB and rearing tests when compared to controls. Animals receiving BMSC grafts after mild or severe contusion showed trends toward improved recovery. Immunocytochemistry identified numerous axons passing through the injury in animals with BMSC grafts but few in controls. BMSCS were detected at 2 weeks after transplantation; however, at 11 weeks very few grafted cells remained. The authors conclude that BMSCs show potential for repairing SCI. However, the use of carefully characterized BMSCs improved transplantation protocols ensuring BMSC, survival, and systematic motor and sensory behavioral testing to identify robust recovery is imperative for further improvement.

Cell transplantation as a treatment for spinal cord injury is a promising therapeutic strategy whose effective clinical application would be facilitated by non-invasive delivery protocols. Cells derived from the bone marrow are particularly attractive because they can be obtained easily, expanded to large numbers and potentially used for autologous as well as allogeneic transplantation. In this study we tested the feasibility of a novel minimally invasive method--lumbar puncture (LP)--for transplanting bone marrow stromal stem cells (MSC) into a clinically relevant spinal cord contusion model. We further sought to determine optimal protocols for performing such minimally invasive cell transplantation. Sprague-Dawley rats received a moderate contusion injury at the midthoracic level followed by LP transplantation of MSC derived from transgenic rats that express the human placental alkaline phosphatase (AP) reporter gene. The recipients were analyzed histologically to evaluate the extent of cell delivery and survival at the injury site. We found that MSC delivered by LP reached the contused spinal cord tissues and exerted a significant beneficial effect by reducing cyst and injury size. Transplantation within 14 days of injury provided significantly greater grafting efficiency than more delayed delivery, and increasing MSC dosage improved cell engraftment. The techniques described here can easily be translated to patients, thus accelerating clinical application of stem cell therapies.

Neural precursor cells (NPCs) are promising grafts for treatment of traumatic CNS injury and neurodegenerative disorders because of their potential to differentiate into neurons and glial cells. When designing clinical protocols for NPC transplantation, it is important to develop alternatives to direct parenchymal injection, particularly at the injury site. We reasoned that since it is minimally invasive, intrathecal delivery of NPCs at lumbar spinal cord (lumbar puncture) represents an important and clinically applicable strategy. We tested this proposition by examining whether NPCs can be delivered to the injured cervical spinal cord via lumbar puncture using a mixed population of neuronal-restricted precursors (NRPs) and glial-restricted precursors (GRPs). For reliable tracking, the NPCs were derived from the embryonic spinal cord of transgenic donor rats that express the marker gene, human placental alkaline phosphatase, under the control of the ubiquitous Rosa 26 promoter. We found that mixed NRP/GRP grafts can be efficiently delivered to a cervical hemisection injury site by intrathecal delivery at the lumbar cord. Similar to direct parenchymal injections, transplanted NRP/GRP cells survive at the injury cavity for at least 5 weeks post-engraftment, migrate into intact spinal cord along white matter tracts and differentiate into all three mature CNS cell types, neurons, astrocytes, and oligodendrocytes. Furthermore, very few graft-derived cells localize to areas outside the injury site, including intact spinal cord and brain. These results demonstrate the potential of delivering lineage-restricted NPCs using the minimally invasive lumbar puncture method for the treatment of spinal cord injury.

Bone marrow stromal cells (MSC) are attractive candidates for developing cell therapies for central nervous system (CNS) disorders. They can be easily obtained, expanded in culture, and promote modest functional recovery following transplantation into animal models of injured or degenerative CNS. While syngeneic MSC grafts can be used efficiently, achieving long-term survival of allogeneic MSC grafts has been a challenge. We hypothesize that improved graft survival will enhance the functional recovery promoted by MSC. To improve MSC graft survival, we tested two dosages of the immune suppressant cyclosporin A (CsA) in an allogeneic model. Syngeneic transplantation of MSC where cells survive well without immune suppression was used as a control. Sprague-Dawley rats treated with standard dose (n = 12) or high-dose (n = 12) CsA served as allogeneic hosts; Fisher 344 rats (n = 12) served as syngeneic hosts. MSC were derived from transgenic Fisher 344 rats expressing human placental alkaline phosphatase and were grafted into cervical spinal cord. Animals treated with standard dose CsA showed significant decreases in allograft size 4 weeks posttransplantation; high CsA doses yielded significantly better graft survival 4 and 8 weeks posttransplantation compared to standard CsA. As expected, syngeneic MSC transplants showed good graft survival after 4 and 8 weeks. To investigate MSC graft elimination, we analyzed immune cell infiltration and cell death. Macrophage infiltration was high after 1 week in all groups. After 4 weeks, high-dose CsA and syngeneic animals showed significant reductions in macrophages at the graft site. Few T lymphocytes were detected in any group at each time point. Cell death occurred throughout the study; however, little apoptotic activity was detected. Histochemical analysis revealed no evidence of neural differentiation. These results indicate that allogeneic transplantation with appropriate immune suppression permits long-term survival of MSC; thus, both allogeneic and syngeneic strategies could be utilized in devising novel therapies for CNS injury.