Ionotropic glutamate receptors (iGluRs) mediate neuronal communication at synapses throughout vertebrate and invertebrate nervous systems. We have characterized a family of iGluR-related genes in Drosophila, which we name ionotropic receptors (IRs). These receptors do not belong to the well-described kainate, AMPA, or NMDA classes of iGluRs, and they have divergent ligand-binding domains that lack their characteristic glutamate-interacting residues. IRs are expressed in a combinatorial fashion in sensory neurons that respond to many distinct odors but do not express either insect odorant receptors (ORs) or gustatory receptors (GRs). IR proteins accumulate in sensory dendrites and not at synapses. Misexpression of IRs in different olfactory neurons is sufficient to confer ectopic odor responsiveness. Together, these results lead us to propose that the IRs comprise a novel family of chemosensory receptors. Conservation of IR/iGluR-related proteins in bacteria, plants, and animals suggests that this receptor family represents an evolutionarily ancient mechanism for sensing both internal and external chemical cues.

Pregnenolone sulfate (PS) is an abundant neurosteroid that can potentiate or inhibit ligand gated ion channel activity and thereby alter neuronal excitability. Whereas PS is known to inhibit kainate and AMPA responses while potentiating NMDA responses, the dependence of modulation on receptor subunit composition remains to be determined. Toward this end, the effect of PS on recombinant kainate (GluR6), AMPA (GluR1 or GluR3), and NMDA (NR1(100)+NR2A) receptors was characterized electrophysiologically with respect to efficacy and potency of modulation. With Xenopus oocytes expressing GluR1, GluR3 or GluR6 receptors, PS reduces the efficacy of kainate without affecting its potency, indicative of a noncompetitive mechanism of action. Conversely, with oocytes expressing NR1(100)+NR2A subunits, PS enhances the efficacy of NMDA without affecting its potency. Whereas the modulatory efficacy, but not the potency, of PS is increased two-fold by co-injection of NR1(100)+NR2A cRNAs as compared with NR1(100) cRNA alone, there is little or no effect of the NR2A subunit on efficacy or potency of pregnanolone (or epipregnanolone) sulfate as an inhibitor of the NMDA response. This suggests that the NR2A subunit controls the efficacy of neurosteroid enhancement, but not inhibition, which is consistent with our previous finding that potentiating and inhibitory steroids act at distinct sites on the NMDA receptor. This represents a first step towards understanding the role of subunit composition in determining neurosteroid modulation of ionotropic glutamate receptor function.

The past fifteen years has seen a revolution in our understanding of ionotropic glutamate receptor (iGluR) structure, starting with the first view of the ligand binding domain (LBD) published in 1998, and in many ways culminating in the publication of the full-length structure of GluA2 in 2009. These reports have revealed not only the central role played by subunit interfaces in iGluR function, but also myriad binding sites within interfaces for endogenous and exogenous factors. Changes in the conformation of inter-subunit interfaces are central to transmission of ligand gating into pore opening (itself a rearrangement of interfaces), and subsequent closure through desensitization. With the exception of the agonist binding site, which is located entirely within individual subunits, almost all modulatory factors affecting iGluRs appear to bind to sites in subunit interfaces. This review seeks to summarize what we currently understand about the diverse roles interfaces play in iGluR function, and to highlight questions for future research.

AMPA subtype ionotropic glutamate receptors (iGluRs) mediate the majority of fast neurotransmission across excitatory synapses in the central nervous system. Each AMPA receptor is composed of four multi-domain subunits that are organized into layers of two amino-terminal domain (ATD) dimers, two ligand-binding domain (LBD) dimers, transmembrane domains and carboxy-terminal domains. We introduced cysteine substitutions at the intersubunit interfaces of AMPA receptor subunit GluA2 and confirmed substituted cysteine crosslink formation by SDS-PAGE. The functional consequence of intersubunit crosslinks was assessed by recording GluA2-mediated currents in reducing and non-reducing conditions. Strong redox-dependent changes in GluA2-mediated currents were observed for cysteine substitutions at the LBD dimer-dimer interface but not at the ATD dimer-dimer interface. We conclude that during gating, LBD dimers undergo significant relative displacement, while ATD dimers either maintain their relative positioning, or their relative displacement has no appreciable effect on AMPA receptor function.

Neurological glutamate receptors are among the most important and intensely studied protein ligand binding systems in humans. They are crucial for the functioning of the central nervous system and involved in a variety of pathologies. Apart from the neurotransmitter glutamate, several artificial, agonistic and antagonistic ligands are known. Of particular interest here are novel photoswitchable agonists that would open the field of optogenetics to glutamate receptors. The receptor proteins are complex, membrane-bound multidomain oligomers that undergo large scale functional conformational changes, making detailed studies of their atomic structure challenging. Therefore, a thorough understanding of the microscopic details of ligand binding and receptor activation remains elusive in many cases. This topic has been successfully addressed by theoretical studies in the past and in this paper, we present extensive molecular dynamics simulation and free energy calculation results on the binding of AMPA and an AMPA derivative, which is the basis for designing light-sensitive ligands. We provide a two-step model for ligand binding domain activation and predict binding free energies for novel compounds in good agreement to experimental observations.

Mast cells are emerging as players in the communication between peripheral nerve endings and cells of the immune system. However, it is not clear the mechanism by which mast cells communicate with peripheral nerves. We previously found that mast cells located within healing tendons can express glutamate receptors, raising the possibility that mast cells may be sensitive to glutamate signaling. To evaluate this hypothesis, we stimulated primary mast cells with glutamate and showed that glutamate induced the profound upregulation of a panel of glutamate receptors of both the ionotropic type (NMDAR1, NMDAR2A, and NMDAR2B) and the metabotropic type (mGluR2 and mGluR7) at both the mRNA and protein levels. The binding of glutamate to glutamate receptors on the mast cell surface was confirmed. Further, glutamate had extensive effects on gene expression in the mast cells, including the upregulation of pro-inflammatory components such as IL-6 and CCL2. Glutamate also induced the upregulation of transcription factors, including Egr2, Egr3 and, in particular, FosB. The extensive induction of FosB was confirmed by immunofluorescence assessment. Glutamate receptor antagonists abrogated the responses of the mast cells to glutamate, supporting the supposition of a functional glutamate-glutamate receptor axis in mast cells. Finally, we provide in vivo evidence supporting a functional glutamate-glutamate receptor axis in the mast cells of injured tendons. Together, these findings establish glutamate as an effector of mast cell function, thereby introducing a novel principle for how cells in the immune system can communicate with nerve cells.

Glutamate is the major neurotransmitter in the vertebrate retina. Neurons involved in the glutamate pathway express α-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid (AMPA), kainic acid (KA) and N-methyl-D-aspartate (NMDA) receptors. Functional characterization of these ionotropic glutamate receptors can be achieved by using a cation channel permeating probe named agmatine (1-amino-4-guanidobutane; AGB). Retinal mapping using this guanidinium analog has certain advantages including the immunocytochemical identification of a whole population of neurons expressing functional glutamate gated receptor channels. We have extended AGB studies into the functionality of ionotropic receptors in peripheral aged human retina to serve as a comparison for functional analysis of retinopathies such as retinal detachment. We probed the human retina with AGB after activation with AMPA, KA and NMDA. The results showed patterns of AGB entry into neurons consistent with those previously observed in subunit localization studies in adult mammalian retinae including primates. Application of 30 μM AMPA activated receptors in virtually all calretinin immunoreactive AII amacrine cells in the mid-peripheral human retina. About half of the AII amacrine cells showed AGB permeation after incubation with 50 μM KA. Some bipolar cells including DB3 OFF bipolar cells displayed functional KA receptors. Colocalization of AGB with parvalbumin labeled horizontal cells revealed functional KA and AMPA receptors with no responsiveness to NMDA activation. NMDA activation resulted in AGB labeling of ganglion cells and amacrine cells. The present study provides a description of functional ionotropic glutamate receptors in the aged mid-peripheral human retina.

Ionotropic glutamate receptors (iGluRs) mediate excitatory signals between cells by binding neurotransmitters and conducting cations across the cell membrane. In the mammalian brain, most of these signals are mediated by two types of iGluRs: AMPA and NMDA (i.e. iGluRs sensitive to 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid and N-methyl-D-aspartic acid, respectively). Delta-type iGluRs of mammals also form neurotransmitter-binding channels in the cell membrane, but in contrast, their channel is not activated by neurotransmitter binding, raising biophysical questions about iGluR activation and biological questions about the role of delta iGluRs. We therefore investigated the divergence of delta iGluRs from their iGluR cousins using molecular phylogenetics, electrophysiology, and site-directed mutagenesis. We find that delta iGluRs are found in numerous bilaterian animals (e.g., worms, starfish, and vertebrates) and are closely related to AMPA receptors, both genetically and functionally. Surprisingly, we observe that many iGluRs of the delta family are activated by the classical inhibitory neurotransmitter, γ-aminobutyric acid (GABA). Finally, we identify nine amino acid substitutions that likely gave rise to the inactivity of today's mammalian delta iGluRs, and these mutations abolish activity when engineered into active invertebrate delta iGluRs, partly by inducing receptor desensitization. These results offer biophysical insight into iGluR activity and point to a role for GABA in excitatory signaling in invertebrates.

The cloning of the glutamate-gated ion channels of the brain revealed an unexpected level of complexity: there are many different genes that encode distinct subunits of the receptor/channel complex and an even larger number of possible receptor subunit combinations. Many--nearly all--of these gene products are expressed in the retina, and the questions that we face today are: how are they used and why are there so many? Answers to these questions will be found at several levels. At the level of transcription, we have learned that different sets of subunits are expressed by different retinal neurons. Little is known about the transcriptional control of these genes, so it remains to be determined whether these patterns of expression reflect the need for different gene products in different retinal neurons or whether these patterns of expression reflect the functional constraints of gene expression. Another level of complexity is caused by alternative splicing, and here we report that at least four and possibly all eight of the different NMDAR1 transcripts are present in the mouse retina. The consequences of this alternative splicing are poorly understood, but antibodies directed against the two different possible C-termini of NMDAR1 label many of the same cell types. It is possible that these different splice variants are combined to generate the channels. While immunohistochemistry provides us with a glimpse of the subunit proteins, much remains to be learned about their half-life within a retinal cell, their intracellular trafficking, their regulation at the synapse, and the proteins associated with their cytoplasmic domains. An approach we have taken towards studying the dynamic properties of receptor subunits has been to fuse them to the cDNA encoding the jellyfish Green Fluorescent Protein. This makes it possible to follow functional subunits in transfected cells over time and to begin to measure the mobility of the protein.

Ionotropic glutamate receptors have been associated with early development of the visual process by regulating cell differentiation, cell motility, and synaptic contacts. We determined the expression of functional ionotropic glutamate receptors during development of the mouse retina by assessing 1-amino-4-guanidobutane (agmatine; AGB) immunolabelling after application of a range of glutamate analogs. Colocalization of AGB with calretinin and islet-1 allowed the identification of functional receptors in neurochemically defined neurons. Activation with kainate (KA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and N-methyl-D-aspartate (NMDA) resulted in AGB entry into neurons consistent with that found previous receptor subunit localization studies in the developing retina. Temporal analysis revealed that application of 50 microM KA activated receptors as early as embryonic day 18 in the ventricular zone and in the ganglion cell layer, whereas 30 muM AMPA activated cells predominantly in the ganglion cell layer. Cholinergic amacrine cells showed functional KA and AMPA receptors upon their insertion into the conventional amacrine cell layer from postnatal day 1 (P1). OFF cone bipolar cells showed functional KA receptors from P6, at a developmental age when they are known to make contact with ganglion cells. NMDA activation led to diffuse AGB labeling at birth among cells in the ganglion cell layer, whereas, at P1, regularly spaced cholinergic amacrine cells in the conventional amacrine cell layer started to be responsive to NMDA. Non-NMDA receptors were first to show functional activation in the developing retina, and cholinergic amacrine cells displayed functional ionotropic glutamate receptors after reaching their final destination.

Previous studies have suggested that photoreceptor synaptic inputs to depolarizing bipolar cells (DBCs or ON bipolar cells) are mediated by mGluR6 receptors and those to hyperpolarizing bipolar cells (HBCs or OFF bipolar cells) are mediated by AMPA/kainate receptors. Here we show that in addition to mGluR6 receptors which mediate the sign-inverting, depolarizing light responses, subpopulations of cone-dominated and rod/cone mixed DBCs use GluR4 AMPA receptors to generate a transient sign-preserving OFF response under light adapted conditions. These AMPA receptors are located at the basal junctions postsynaptic to rods and they are silent under dark-adapted conditions, as tonic glutamate release in darkness desensitizes these receptors. Light adaptation enhances rod-cone coupling and thus allows cone photocurrents with an abrupt OFF depolarization to enter the rods. The abrupt rod depolarization triggers glutamate activation of unoccupied AMPA receptors, resulting in a transient OFF response in DBCs. It has been widely accepted that the DNQX-sensitive, OFF transient responses in retinal amacrine cells and ganglion cells are mediated exclusively by HBCs. Our results suggests that this view needs revision as AMPA receptors in subpopulations of DBCs are likely to significantly contribute to the DNQX-sensitive OFF transient responses in light-adapted third- and higher-order visual neurons.

Phenotypic plasticity is the ability held in many organisms to produce different phenotypes with a given genome in response to environmental stimuli, such as temperature, nutrition and various biological interactions. It seems likely that environmental signals induce a variety of mechanistic responses that influence ontogenetic processes. Inducible defenses, in which prey animals alter their morphology, behavior and/or other traits to help protect against direct or latent predation threats, are among the most striking examples of phenotypic plasticity. The freshwater microcrustacean Daphnia pulex forms tooth-like defensive structures, "neckteeth," in response to chemical cues or signals, referred to as "kairomones," in this case released from phantom midge larvae, a predator of D. pulex. To identify factors involved in the reception and/or transmission of a kairomone, we used microarray analysis to identify genes up-regulated following a short period of exposure to the midge kairomone. In addition to identifying differentially expressed genes of unknown function, we also found significant up-regulation of genes encoding ionotropic glutamate receptors, which are known to be involved in neurotransmission in many animal species. Specific antagonists of these receptors strongly inhibit the formation of neckteeth in D. pulex, although agonists did not induce neckteeth by themselves, indicating that ionotropic glutamate receptors are necessary but not sufficient for early steps of neckteeth formation in D. pulex. Moreover, using co-exposure of D. pulex to antagonists and juvenile hormone (JH), which physiologically mediates neckteeth formation, we found evidence suggesting that the inhibitory effect of antagonists is not due to direct inhibition of JH synthesis/secretion. Our findings not only provide a candidate molecule required for the inducible defense response in D. pulex, but also will contribute to the understanding of complex mechanisms underlying the recognition of environmental changes, which form the basis of phenotypic plasticity.

Ionotropic glutamate receptors (iGluRs) are responsible for extracellular signaling in the central nervous system. However, the relationship between the overall structure of the protein and its function has yet to be resolved. Atomic force microscopy (AFM) is an important technique that allows nano-scale imaging in liquid. In the present work we have succeeded in imaging by AFM of the external features of the most common iGluR, AMPA-R (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor), in a physiological environment.

A multitude of 18 iGluR receptor subunits, many of which are diversified by splicing and RNA editing, localize to >20 excitatory and inhibitory neocortical neuron types defined by physiology, morphology, and transcriptome in addition to various types of glial, endothelial, and blood cells. Here we have compiled the published expression of iGluR subunits in the areas and cell types of developing and adult cortex of rat, mouse, carnivore, bovine, monkey, and human as determined with antibody- and mRNA-based techniques. iGluRs are differentially expressed in the cortical areas and in the species, and all have a unique developmental pattern. Differences are quantitative rather than a mere absence/presence of expression. iGluR are too ubiquitously expressed and of limited use as markers for areas or layers. A focus has been the iGluR profile of cortical interneuron types. For instance, GluK1 and GluN3A are enriched in, but not specific for, interneurons; moreover, the interneurons expressing these subunits belong to different types. Adressing the types is still a major hurdle because type-specific markers are lacking, and the frequently used neuropeptide/CaBP signatures are subject to regulation by age and activity and vary as well between species and areas. RNA-seq reveals almost all subunits in the two morphofunctionally characterized interneuron types of adult cortical layer I, suggesting a fairly broad expression at the RNA level. It remains to be determined whether all proteins are synthesized, to which pre- or postsynaptic subdomains in a given neuron type they localize, and whether all are involved in synaptic transmission. J. Comp. Neurol. 525:976-1033, 2017. © 2016 Wiley Periodicals, Inc.

Diabetic retinopathy (DR) is a leading cause of vision loss and blindness among adults between the age 20 to 74. Changes in ionotropic glutamate receptor subunit composition can affect retinal glutamatergic neurotransmission and, therefore, contribute to visual impairment. The purpose of this study was to investigate whether diabetes leads to changes in ionotropic glutamate receptor subunit expression at the protein and mRNA level in the rat retina.

NMDA receptors (NMDAR) are key molecules involved in physiological and pathophysiological brain processes such as plasticity and excitotoxicity. Neuronal activity regulates NMDA receptor levels in the cell membrane. However, little is known on which time scale this regulation occurs and whether the two main diheteromeric NMDA receptor subtypes in forebrain, NR1/NR2A and NR1/NR2B, are regulated in a similar fashion. As these differ considerably in their electrophysiological properties, the NR2A/NR2B ratio affects the neurons' reaction to NMDA receptor activation. Here we provide evidence that the basal turnover rate in the cell membrane of NR2A- and NR2B-containing receptors is comparable. However, the level of the NR2A subtype in the cell membrane is highly regulated by NMDA receptor activity, resulting in a several-fold increased insertion of new receptors after blocking NMDAR for 8 h. Blocking AMPA receptors also increases the delivery of NR2A-containing receptors to the cell membrane. In contrast, the amount of NR2B-containing receptors in the cell membrane is not affected by ionotropic glutamate receptor block. Moreover, electrophysiological analysis of synaptic currents in hippocampal cultures and CA1 neurons of hippocampal slices revealed that after 8 h of NMDA receptor blockade the NMDA EPSCs increase as a result of augmented NMDA receptor-mediated currents. In conclusion, synaptic NR2A- but not NR2B-containing receptors are dynamically regulated, enabling neurons to change their NR2A/NR2B ratio within a time scale of hours.

The purpose of the present study was to elucidate the mechanisms that may underlie the sensitization of trigeminal spinal subnucleus caudalis (Vc) and upper cervical spinal cord (C1-C2) neurons to heat or cold stimulation of the orofacial region following glutamate (Glu) injection.

Glutamate is an excitatory neurotransmitter in the vertebrate retina. A previous study found vesicular glutamate transporter 2 (vGluT2) mRNA in the pigeon retina, suggesting that bipolar and ganglion cells are glutamatergic. The present study examined the localization of ionotropic glutamate receptors to identify receptor cells in the pigeon retina using in situ hybridization histochemistry. Nine subunits of AMPA receptor (GluA1, GluA2, GluA3, and GluA4), kainate receptor (GluK1, GluK2, and GluK4), and NMDA receptor (GluN1 and GluN2A) were found to be expressed in the inner nuclear layer (INL) and ganglion cell layers. GluA1, GluA2, GluA3, and GluA4 were primarily expressed in the inner half of INL, and the signal intensity was strong for GluA2, GluA3, and GluA4. GluK1 was intensely expressed in the outer half of INL, whereas GluK2 and GluK4 were mainly localized in the inner half of INL. GluN1 and GluN2A were moderately expressed in the inner half of INL. Horizontal cells expressed GluA3 and GluA4, and ganglion cells expressed all subunits examined. These results suggest that the glutamatergic neurotransmission in the pigeon retina is similar to that in mammals.

The expression of retinal CaMKII is up-regulated in the retina of the rdta mouse in which rod photoreceptors are genetically ablated. As ionotropic glutamate receptors are known substrates of CAMKII, this study set out to determine if the protein levels of ionotropic glutamate receptors in the rdta mouse retina are also affected.

The ability to optically manipulate specific neuronal signaling proteins with genetic precision paves the way for the dissection of their roles in brain function, behavior, and disease. Chemical optogenetic control with photoswitchable tethered ligands (PTLs) enables rapid, reversible and reproducible activation or block of specific neurotransmitter-gated receptors and ion channels in specific cells. In this study, we further engineered and characterized the light-activated GluK2 kainate receptor, LiGluR, to develop a toolbox of LiGluR variants. Low-affinity LiGluRs allow for efficient optical control of GluK2 while removing activation by native glutamate, whereas variant RNA edited versions enable the synaptic role of receptors with high and low Ca(2+) permeability to be assessed and spectral variant photoswitches provide flexibility in illumination. Importantly, we establish that LiGluR works efficiently in the cortex of awake, adult mice using standard optogenetic techniques, thus opening the door to probing the role of specific synaptic receptors and cellular signals in the neural circuit operations of the mammalian brain in normal conditions and in disease. The principals developed in this study are widely relevant to the engineering and in vivo use of optically controllable proteins, including other neurotransmitter receptors.