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Metabotropic glutamate receptors are class C G-protein-coupled receptors which respond to the neurotransmitter glutamate. Structural studies have been restricted to the amino-terminal extracellular domain, providing little understanding of the membrane-spanning signal transduction domain. Metabotropic glutamate receptor 5 is of considerable interest as a drug target in the treatment of fragile X syndrome, autism, depression, anxiety, addiction and movement disorders. Here we report the crystal structure of the transmembrane domain of the human receptor in complex with the negative allosteric modulator, mavoglurant. The structure provides detailed insight into the architecture of the transmembrane domain of class C receptors including the precise location of the allosteric binding site within the transmembrane domain and key micro-switches which regulate receptor signalling. This structure also provides a model for all class C G-protein-coupled receptors and may aid in the design of new small-molecule drugs for the treatment of brain disorders., Glutamate is the major excitatory neurotransmitter in the central nervous system. It mediates its activity through ionotropic channels and eight metabotropic G-protein-coupled receptors (GPCRs) which are expressed in neuronal and [...]

Mice lose their appetite in response to activation of a brain area that is involved in emotion and cognition. Eating is prompted, in part, by brain regions that help to [...]

Prolonged blockade of AMPA-type glutamate receptors in hippocampal neuron cultures leads to homeostatic enhancements of preand postsynaptic function that appear correlated at individual synapses, suggesting some form of transsynaptic coordination. The respective modifications are important for overall synaptic strength but their interrelationship, dynamics, and molecular underpinnings are unclear. Here we demonstrate that adaptation begins postsynaptically but is ultimately communicated to presynaptic terminals and expressed as an accelerated turnover of synaptic vesicles. Critical postsynaptic modifications occur over hours, but enable retrograde communication within minutes once AMPA receptor (AMPAR) blockade is removed, causing elevation of both spontaneous and evoked vesicle fusion. The retrograde signaling does not require spiking activity and can be interrupted by NBQX, philanthotoxin, postsynaptic BAPTA, or external sequestration of BDNF, consistent with the acute release of retrograde messenger, triggered by postsynaptic [Ca.sup.2+] elevation via [Ca.sup.2+]-permeable AMPARs. homeostasis | synaptic scaling | calcium signaling | miniature excitatory postsynaptic currents doi: 10.1073/pnas.1016399107

AMPA ([alpha]-amino-3-hydroxy-5-methyl-4-isoxazole-propionate) receptors desensitize rapidly and completely in the continued presence of their endogenous ligand glutamate; however, it is not clear what role AMPA receptor desensitization plays in the brain. We generated a knock-in mouse in which a single amino acid residue, which controls desensitization, was mutated in the GluA2 (GluR2) receptor subunit ([GluA2.sup.L483Y]). This mutation was homozygous lethal. However, mice carrying a single mutated allele, [GluA2.sup.L483Y/wt], survived past birth, but displayed severe and progressive neurological deficits including seizures and, ultimately, increased mortality. The expression of the AMPA receptor subunits GluA1 and GluA2 was decreased, whereas NMDA receptor protein expression was increased in [GluA2.sup.L483Y/wt] mice. Despite this, basal synaptic transmission and plasticity in the hippocampus were largely unaffected, suggesting that neurons preferentially target receptors to synapses to normalize synaptic weight. We found no gross neuroanatomical alterations in [GluA2.sup.L483Y/wt] mice. Moreover, there was no accumulation of AMPA receptor subunits in intracellular compartments, suggesting that folding and assembly of AMPA receptors are not affected by this mutation. Interestingly, EPSC paired pulse ratios in the CA1 were enhanced without a change in synaptic release probability, demonstrating that postsynaptic receptor properties can contribute to facilitation. The dramatic phenotype observed in this study by the introduction of a single amino acid change demonstrates an essential role in vivo for AMPA receptor desensitization. GluA2 (GluR2) | hippocampus | seizures | knock-in mouse doi: 10.1073/pnas.0908206107

Although pyramidal cells are the main excitatory neurons in the cerebral cortex, it has recently been reported that they can evoke inhibitory postsynaptic currents in neighboring pyramidal neurons. These inhibitory effects were proposed to be mediated by putative axo-axonic excitatory synapses between the axon terminals of pyramidal cells and perisomatic inhibitory axon terminals [Ren M, Yoshimura Y, Takada N, Horibe S, Komatsu Y (2007) Science 316:758-761]. However, the existence of this type of axo-axonic synapse was not found using serial section electron microscopy. Instead, we observed that inhibitory axon terminals synapsing on pyramidal cell bodies were frequently apposed by terminals that established excitatory synapses with neighbouring dendrites. We propose that a spillover of glutamate from these excitatory synapses can activate the adjacent inhibitory axo-somatic terminals. axo-axonic synapses | glutamate spillover | interpyramidal inhibition | perisomatic innervation | serial electron microscopy

In dopaminoceptive neurons, dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) plays a central role in integrating the effects of dopamine and other neurotransmitters. Phosphorylation of DARPP-32 at Thr-34 by protein kinase A results in inhibition of protein phosphatase 1 (PP1), and phosphorylation at Thr-75 by Cdk5 (cyclin-dependent kinase 5) results in inhibition of protein kinase A. Dephosphorylation at Thr-34 involves primarily the [Ca.sup.2+]-dependent protein phosphatase, PP2B (calcineurin), whereas dephosphorylation of Thr-75 involves primarily PP2A, the latter being subject to control by both cAMP- and [Ca.sup.2+]-dependent regulatory mechanisms. In the present study, we have investigated the mechanism of [Ca.sup.2+]-dependent regulation of Thr-75 by PP2A. We show that the PR72 (or B' or PPP2R3A) regulatory subunit of PP2A is highly expressed in striatum. Through the use of over-expression and down-regulation by using RNAi, we show that PP2A, in a heterotrimeric complex with the PR72 subunit, mediates [Ca.sup.2+]-dependent dephosphorylation at Thr-75 of DARPP-32. The PR72 subunit contains two [Ca.sup.2+] binding sites formed by E and F helices (EF-hands 1 and 2), and we show that the former is necessary for the ability of PP2A activity to be regulated by [Ca.sup.2+], both in vitro and in vivo. Our studies also indicate that the PR72-containing form of PP2A is necessary for the ability of glutamate acting at [alpha]-amino-3-hydroxy-5-methylisoxazole-4-propionic acid and NMDA receptors to regulate Thr-75 dephosphorylation. These studies further our understanding of the complex signal transduction pathways that regulate DARPP-32. In addition, our studies reveal an alternative intracellular mechanism whereby [Ca.sup.2+] can activate serine/threonine phosphatase activity. calcium | protein phosphorylation | dopamine | glutamate

The central nitrogen metabolic circuit in enteric bacteria consists of three enzymes: glutamine synthetase, glutamate synthase (GOGAT), and glutamate dehydrogenase (GDH). With the carbon skeleton provided by 2-oxoglutarate, ammonia/ammonium ([NH.sub.4].sup.+]) is assimilated into two central nitrogen intermediates, glutamate and glutamine. Although both serve as nitrogen donors for all biosynthetic needs, glutamate and glutamine play different roles. Internal glutamine serves as a sensor of external nitrogen availability, and its pool concentration decreases upon nitrogen limitation. A high glutamate pool concentration is required to maintain the internal [K.sup.+] pool. The configuration of high glutamate and low glutamine pools was disrupted in GOGAT-- mutants under low [NH.sub.4].sup.+] conditions: the glutamate pool was low, the difference between glutamate and glutamine was diminished, and growth was defective. When a GOGAT-- mutant was cultured in an [NH.sub.4].sup.+]limited chemostat, two sequential spontaneous mutations occurred. Each resulted in a suppressor mutant that outgrew its predecessor in the chemostat. The first suppressor overexpressed GDH, and the second also had a partially impaired glutamine synthetase. The result was a triple mutant in which [NH.sub.4].sup.+] was assimilated by two enzymes instead of the normal three and yet glutamate and glutamine pools and growth were essentially normal. The results indicate preference for the usual ratio of glutamate and glutamine and the resilient and compensatory nature of the circuit on pool control. Analysis of other suppressor mutants selected on solid medium suggests that increased GDH expression is the key for rescue of the growth defect of GOGAT- mutants under low [NH.sub.4].sup.+] conditions. ammonium assimilation | glutamine | metabolic circuit | suppressor

Metabotropic glutamate receptors (mGluRs) and Homer proteins play critical roles in neuronal functions including plasticity, nociception, epilepsy, and drug addiction. Furthermore, Homer proteins regulate mGluR1/5 function by acting as adapters and facilitating coupling to effectors such as the inositol triphosphate receptor. However, although Homer proteins and their interaction with mGluRs have been the subject of intense study, direct measurements of Homer-induced changes in postsynaptic mGluReffector coupling have not been reported. This question was addressed here by examining glutamatergic excitatory postsynaptic currents (EPSCs) in rat autaptic hippocampal cultures. In most neurons, the group I mGluR agonist (S)-3,5-dihydroxyphenylglycine strongly inhibited the EPSC acutely. This modulation occurred postsynaptically, was mediated primarily by mGluR5, and was inositol triphosphate receptor-dependent. Expression of the dominant negative, immediate early form of Homer, Homer la, strongly reduced EPSC modulation, but the W24A mutant of Homer la, which cannot bind mGluRs, had no effect. (S)-3,5-dihydroxyphenylglycine-mediated intracellular calcium responses in the processes of Homer 1a-expressing neurons were reduced compared with those in Homer la W24A-expressing cells. However, neither the distribution of mGluR5 nor the modulation of somatic calcium channels was altered by Homer la expression. These data demonstrate that Homer la can reduce mGluR5 coupling to postsynaptic effectors without relying on large changes in the subcellular distribution of the receptor. Thus, alteration of mGluR signaling by changes in Homer protein expression may represent a viable mechanism for fine-tuning synaptic strength in neurons. autapse | calcium channel | excitatory postsynaptic current | hippocampal

Metabotropic glutamate receptors play major roles in the activation of excitatory synapses in the central nerve system. We determined the crystal structure of the entire extracellular region of the group II receptor and that of the ligand-binding region of the group III receptor. A comparison among groups I, II, and III provides the structural basis that could account for the discrimination of group-specific agonists. Furthermore, the structure of group II includes the cysteine-rich domain, which is tightly linked to the ligand-binding domain by a disulfide bridge, suggesting a potential role in transmitting a ligand-induced conformational change into the downstream transmembrane region. The structure also reveals the lateral interaction between the two cysteine-rich domains, which could stimulate clustering of the dimeric receptors on the cell surface. We propose a general activation mechanism of the dimeric receptor coupled with both ligand-binding and interprotomer rearrangements. crystallization | cysteine-rich region | ligand binding | G protein-coupled receptor

[alpha]-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors mediate the majority of excitatory synaptic transmission in the brain. Recent studies have shown that activation of PKA regulates the membrane trafficking of the AMPA receptor Glu receptor 1 (GluR1) subunit, but the role of direct phosphorylation of GluR1 in regulating receptor redistribution is not clear. Here we show that phosphorylation of the GluR1 subunit on serine 845 by PKA is required for PKA-induced increases in AMPA receptor cell-surface expression because it promotes receptor insertion and decreases receptor endocytosis. Furthermore, dephosphorylation of GluR1 serine 845 triggers NMDA-induced AMPA receptor internalization. These findings strongly suggest that dynamic changes in direct phosphorylation of GluR1 by PKA are crucial in the modulation of AMPA receptor trafficking and synaptic plasticity. glutamate receptors | long-term potentiation | synaptic plasticity

HIV infection of the central nervous system can result in neurologic dysfunction with devastating consequences in AIDS patients. NeuroAIDS is characterized by neuronal injury and loss, yet there is no evidence that HIV can infect neurons. Here we show that the HIV-encoded protein tat triggers formation of a macromolecular complex involving the low-density lipoprotein receptor-related protein (LRP), postsynaptic density protein-95 (PSD-95), N-methyl-D-aspartic acid (NMDA) receptors, and neuronal nitric oxide synthase (nNOS) at the neuronal plasma membrane, and that this complex leads to apoptosis in neurons negative as well as positive for NMDA receptors and also in astrocytes. Blockade of LRP-mediated tat uptake, NMDA receptor activation, or neuronal nitric oxide synthase significantly reduces ensuing neuronal apoptosis, suggesting that formation of this complex is an early step in tat toxicity. We also show that the inflammatory chemokine, CCL2, protects against tat toxicity and inhibits formation of the complex. These findings implicate the complex in HIV-induced neuronal apoptosis and suggest therapeutic targets for intervention in the pathogenesis of NeuroAIDS. glutamate | dementia | HIV-1 | NeuroAIDS | excitotoxicity

Glutamyl-tRNA (Glu-tRNA), formed by Glu-tRNA synthetase (GluRS), is a substrate for protein biosynthesis and tetrapyrrole formation by the Cs pathway, in this route Glu-tRNA is transformed to [delta]-aminolevulinic acid, the universal precursor of tetrapyrroles (e.g., heme and chlorophyll) by the action of Glu-tRNA reductase (GluTR) and glutamate semialdehyde aminotransferase. GluTR is a target of feedback regulation by heme. In Acidithiobacillus ferrooxidans, an acidophilic bacterium that expresses two GluRSs (GluRS1 and GluRS2) with different tRNA specificity, the intracellular heme level varies depending on growth conditions. Under high heme requirement for respiration increased levels of GluRS and GluTR are observed. Strikingly, when intracellular heme is in excess, the cells respond by a dramatic decrease of GluRS activity and the level of GluTR. The recombinant GluRS1 enzyme is inhibited in vitro by hemin, but NADPH restores its activity. These results suggest that GluRS plays a major role in regulating the cellular level of heme. gene expression | protein biosynthesis | tetrapyrrole biosynthesis

It is known that Alzheimer's disease (AD) is a synaptic disease that involves various neurotransmitter systems, particularly those where synaptic transmission is mediated by acetylcholine or glutamate (Glu). Nevertheless, very little is known about the properties of neurotransmitter receptors of the AD human brain. We have shown previously that cell membranes, carrying neurotransmitter receptors from the human postmortem brain, can be transplanted to frog oocytes, and their receptors will still be functional. Taking advantage of this fact, we have now studied the properties of Glu receptors (GluRs) from the cerebral cortices of AD and non-AD brains and found that oocytes injected with AD membranes acquired GluRs that have essentially the same functional properties as those of oocytes injected with membranes from non-AD brains. However, the amplitudes of the currents elicited by Glu were always smaller in the oocytes injected with membranes from AD brains. Western blot analyses of the same membrane preparations used for the electrophysiological studies showed that AD membranes contained significantly fewer GluR2/3 subunit proteins. Furthermore, the corresponding mRNAs were also diminished in the AD brain. Therefore, the smaller amplitude of membrane currents elicited by Glu in oocytes injected with membranes from an AD brain is a consequence of a reduced number of GluRs in cell membranes transplanted from the AD brain. Thus, using the comparatively simple method of microtransplantation of receptors, it is now possible to determine the properties of neurotransmitter receptors of normal and diseased human brains. That knowledge may help to decipher the etiology of the diseases and also to develop new treatments. neurotransmitter receptors | postmortem brain | Xenopus oocytes

Serine racemase (SR) generates D-serine, a coagonist with glutamate at NMDA receptors. We show that SR is physiologically S-nitrosylated leading to marked inhibition of enzyme activity. Inhibition involves interactions with the cofactor ATP reflecting juxtaposition of the ATP-binding site and cysteine-113 (C113), the site for physiological S-nitrosylation. NMDA receptor physiologically enhances SR S-nitrosylation by activating neuronal nitricoxide synthase (nNOS). These findings support a model whereby postsynaptic stimulation of nitric-oxide (NO) formation feeds back to presynaptic cells to S-nitrosylate SR and decrease o-serine availability to postsynaptic NMDA receptors. neuronal nitric-oxide synthase | NMDA receptor | S-nitrosylation

Fragile X syndrome is a common heritable form of mental retardation in humans. Recent neuroanatomical studies indicate an apparent immature appearance of neurons in fragile X syndrome patients and fragile X mental retardation protein (FMRP)-knockout mice, an animal model of this condition. In this work, we investigated possible alterations in synaptic plasticity in the neocortex of FMRP-knockout mice. Extracellular field potentials were recorded from the deep-layer visual neocortex. Long-term potentiation (LTP) was severely attenuated in brain slices from knockout mice relative to that observed in slices from wild-type mice. Considering that neocortical LTP can involve both NMDA receptor-dependent and -independent mechanisms, we attempted to distinguish the nature of LTP attenuated in the knockout condition. In slices from wild-type mice, LTP was partially attenuated by the NMDA receptor antagonist 3-[([+ or -])-2-carboxypiperazin-4-yl]-propyl-1-phosphate (CPP); however, the general metabotropic glutamate receptor (mGluR) antagonist [alpha]-methyl-4-carboxyphenylglycine (MCPG) strongly attenuated LTP, resulting in a response indistinguishable from that observed in slices from knockout mice. The selective mGluRs antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP) attenuated LTP to a similar degree as did MCPG in wild-type slices, but MPEP did not alter the reduced potentiation in knockout slices. Our results suggest that LTP in layer V visual neocortex depends primarily on mGlu[R.sub.5] activation. Our data also indicate that mGlu[R.sub.5]mediated synaptic plasticity is absent in the neocortex of FMRP-knockout mice. Such an alteration may contribute to the cognitive and learning deficits exhibited in these mice as well as in fragile X syndrome. fragile X mental retardation protein | long-term potentiation | synaptic plasticity | visual cortex

Signal integration in the brain is determined by the size and kinetics of rapid synaptic responses. The latter, in turn, depends on the concentration profile of neurotransmitter in the synaptic cleft. According to a traditional view, narrower clefts should correspond to higher intracleft concentrations of neurotransmitter, and therefore to the enhanced activation of synaptic receptors. Here, we argue that narrowing the cleft also increases electrical resistance of the intracleft medium and therefore reduces local receptor currents. We employ detailed theoretical analyses and Monte Carlo simulations to propose that these two contrasting phenomena result in a relatively narrow range of cleft heights at which the synaptic receptor current reaches its maximum. Over a physiological range of synaptic parameters, the 'optimum' height falls between [approximately or equal to] 12 and 20 nm. This range is consistent with the structure of central synapses reported by electron microscopy. Therefore, our results suggest that a simple fundamental principle may underlie the synaptic cleft architecture: to maximize synaptic strength. AMPA receptor | diffusion | glutamate | Monte Carlo | neural communication

The NR2 subunit composition of NMDA receptors (NMDARs) varies during development, and this change is important in NMDAR-dependent signaling. In particular, synaptic NMDAR switch from containing mostly NR2B subunit to a mixture of NR2B and NR2A subunits. The pathways by which neurons differentially traffic NR2A- and NR2B-containing NMDARs are poorly understood. Using single-particle and -molecule approaches and specific antibodies directed against NR2A and NR2B extracellular epitopes, we investigated the surface mobility of native NR2A and NR2B subunits at the surface of cultured neurons. The surface mobility of NMDARs depends on the NR2 subunit subtype, with NR2A-containing NMDARs being more stable than NR2B-containing ones, and NR2A subunit overexpression stabilizes surface NR2B-containing NMDARs. The developmental change in the synaptic surface content of NR2A and NR2B subunits was correlated with a developmental change in the time spent by the subunits within synapses. This suggests that the switch in synaptic NM DAR subtypes depends on the regulation of the receptor surface trafficking. development | glutamate receptor | lateral mobility

In mammalian cortex, most excitatory inputs occur on dendritic spines, avoiding dendritic shafts. Although spines biochemically isolate inputs, nonspiny neurons can also implement biochemical compartmentalization; so, it is possible that spines have an additional function. We have recently shown that the spine neck can filter membrane potentials going into and out of the spine. To investigate the potential function of this electrical filtering, we used two-photon uncaging of glutamate and compared the integration of electrical signals in spines vs. dendritic shafts from basal dendrites of mouse layer 5 pyramidal neurons. Uncaging potentials onto spines summed linearly, whereas potentials on dendritic shafts reduced each other's effect. Linear integration of spines was maintained regardless of the amplitude of the response, distance between spines (as close as second harmonic | pyramidal cell | glutamate uncaging | two-photon

The retinal L-type [Ca.sup.2+] channel Cav1.4 is distinguished from all other members of the high voltage-activated (HVA) [Ca.sup.2+] channel family by lacking [Ca.sup.2+]-calmodulin-dependent inactivation. In synaptic terminals of photoreceptors and bipolar cells, this feature is essential to translate graded membrane depolarizations into sustained [Ca.sup.2+] influx and tonic glutamate release. The sequences conferring [Ca.sup.2+]-dependent inactivation (CDI) are conserved throughout the HVA calcium channel family, raising the question of how Cav1.4 manages to switch off CDI. Here, we identify an autoinhibitory domain in the distal C terminus of Cav1.4 that serves to abolish CDI. We show that this domain (ICDI, inhibitor of CDI) uncouples the molecular machinery conferring CDI from the inactivation gate by binding to the EF hand motif in the proximal C terminus. Deletion of ICDI completely restores [Ca.sup.2+]-calmodulin-mediated CDI in Cav1.4. CDI can be switched off again in the truncated Cav1.4 channel by coexpression of ICDI, indicating that ICDI works as an autonomous unit. Furthermore, we show that in the Cav1.2 L-type [Ca.sup.2+]-channel replacement of the distal C terminus by the corresponding sequence of Cav1.4 is sufficient to block CDI. This finding suggests that autoinhibition of CDI can be introduced principally into other [Ca.sup.2+] channel types. Our data provide a previously undescribed perspective on the regulation of HVA calcium channels by [Ca.sup.2+]. calmodulin | Cav1.4 | retina | Ca channels

The metabotropic glutamate receptors (mGluRs) within the Family C subclass of G protein-coupled receptors are crucial modulators of synaptic transmission. However, their closest relatives include a diverse group of sensory receptors whose biological functions are not associated with neurotransmission, raising the question of the evolutionary origin of amino acid-binding Family C receptors. A common feature of most, if not all, functional Family C receptors is the presence of an amino acid-binding site localized within the large extracellular Venus flytrap domain. Here, we used maximum likelihood methods to infer the ancestral state of key residues in the amino acid-binding pocket of a primordial Family C receptor. These residues were reconstructed in the background of the fish 5.24 chemosensory receptor, a broad-spectrum amino acid-activated receptor. Unlike the WT 5.24 receptor, which was not activated by mGluR agonists and displayed low sensitivity toward L-glutamate, the reconstructed ancestral receptor possessed a pharmacological profile characterized by high affinity for both L-glutamate and selective Group I mGluR agonists. This pharmacological phenotype could be largely recapitulated by mutating only two residues in the 5.24 receptor-binding pocket. Our results suggest that this primordial Family C receptor may have arisen early in metazoan evolution and that it already was preadapted as a glutamate receptor for its later use at excitatory synapses in glutamate-mediated neurotransmission. metabotropic glutamate receptor | primordial receptor | olfactory receptor | calcium-sensing receptor | taste receptor

Changes in synaptic strength that underlie memory formation in the CNS are initiated by pulses of [Ca.sup.2+] flowing through NMDA-type glutamate receptors into postsynaptic spines. Differences in the duration and size of the pulses determine whether a synapse is potentiated or depressed after repetitive synaptic activity. Calmodulin (CAM) is a major [Ca.sup.2+] effector protein that binds up to four [Ca.sup.2+] ions. CaM with bound [Ca.sup.2+] can activate at least six signaling enzymes in the spine. In fluctuating cytosolic [Ca.sup.2+], a large fraction of free CaM is bound to fewer than four [Ca.sup.2+] ions. Binding to targets increases the affinity of CaM's remaining [Ca.sup.2+]-binding sites. Thus, initial binding of CaM to a target may depend on the target's affinity for CaM with only one or two bound [Ca.sup.2+] ions. To study CaM-dependent signaling in the spine, we designed mutant CaMs that bind [Ca.sup.2+] only at the two N-terminal or two C-terminal sites by using computationally designed mutations to stabilize the inactivated [Ca.sup.2+]-binding domains in the 'closed' [Ca.sup.2+]-free conformation. We have measured their interactions with CaMKII, a major [Ca.sup.2+]/CaM target that mediates initiation of long-term potentiation. We show that CaM with two [Ca.sup.2+] ions bound in its C-terminal lobe not only binds to CaMKII with low micromolar affinity but also partially activates kinase activity. Our results support the idea that competition for binding of CaM with two bound [Ca.sup.2+] ions may influence significantly the outcome of local [Ca.sup.2+] signaling in spines and, perhaps, in other signaling pathways. postsynaptic | protein design | synaptic plasticity | microdomains

The neurotransmitter glutamate mediates excitatory synaptic transmission by activating ionotropic glutamate receptors (iGluRs). In Caenorhabditis elegans, the GLR-1 receptor subunit is required for glutamate-gated current in a subset of interneurons that control avoidance behaviors. Current mediated by GLR-1-containing iGluRs depends on SOL-1, a transmembrane CUB-domain protein that immunoprecipitates with GLR-1. We have found that reconstitution of glutamate-gated current in heterologous cells depends on three proteins, STG-1 (a C. elegans stargazin-like protein), SOL-1, and GLR-1. Here, we use genetic and pharmacological perturbations along with rapid perfusion electrophysiological techniques to demonstrate that SOL-1 functions to slow the rate and limit the extent of receptor desensitization as well as to enhance the recovery from desensitization. We have also identified a SOL-1 homologue from Drosophila and show that Dro SOL1 has a conserved function in promoting C. elegans glutamate-gated currents. SOL-1 homologues may play critical roles in regulating glutamatergic neurotransmission in more complex nervous systems. AMPA receptor | Caenorhabditis elegans | TARPs | stargazin | GLR-1

[alpha]-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs) are a major subtype of ionotropic glutamate receptors (iGluRs) that mediate rapid excitatory synaptic transmission in the vertebrate brain. Putative AMPARs are also expressed in the nervous system of invertebrates. In Caenorhabditis elegans, the GLR-1 receptor subunit is expressed in neural circuits that mediate avoidance behaviors and is required for glutamate-gated current in the AVA and AVD interneurons. Glutamate-gated currents can be recorded from heterologous cells that express vertebrate AMPARs; however, when C. elegans GLR-1 is expressed in heterologous cells, little or no glutamate-gated current is detected. This finding suggests that other receptor subunits or auxiliary proteins are required for function. Here, we identify Ce STG-1, a C. elegans stargazin-like protein, and show that expression of Ce STG-1 together with GLR-1 and the CUB-domain protein SOL-1 reconstitutes glutamate-gated currents in Xenopus oocytes. Ce STG-1 and homologues cloned from Drosophila (Dro STG1) and Apis mellifera (Apis STG1) have evolutionarily conserved functions and can partially substitute for one another to reconstitute glutamate-gated currents from rat, Drosophila, and C. elegans. Furthermore, we show that Ce STG-1 and Apis STG1 are primarily required for function independent of possible roles in promoting the surface expression of invertebrate AMPARs. C. elegans | SOL-1 | transmembrane AMPA receptor regulatory protein (TARP) | GLR-1

[alpha]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are the primary mediators of fast excitatory synaptic transmission in the mammalian CNS. Structures of the extracellular ligand-binding domain suggest that the extent of cleft closure in the ligand-binding domain controls the extent of activation of the receptor. Here we have developed a fluorescence resonance energy transfer-based probe that allows us to study the extent of cleft closure in the isolated ligand-binding domain in solution. These investigations show that the wild-type protein exhibits a graded cleft closure that correlates to the extent of activation, which is in qualitative agreement with the crystal structures. However, the changes in extent of cleft closure between the apo and agonist-bound states are smaller than that observed in the crystal structures. We have also used this method to study the L650T mutant and show that in solution the [alpha]-amino-5-methyl-3-hydroxy-4-isoxazole propionate-bound form of this mutant exists primarily in a conformation that is more closed than predicted based on the activity, indicating that the degree of cleft closure alone cannot be used as a measure of extent of activation of the receptor, and there are possibly other mechanisms in addition to cleft closure that mediate the subtleties in extent of activation by a given agonist. fluorescence | glutamate | ion channel

Glutamate is the major excitatory neurotransmitter in brain, and [alpha]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)type glutamate receptors (AMPARs) mediate the majority of postsynaptic depolarization. AMPAR ion channels display rapid gating, and their deactivation and desensitization determine the timing of synaptic transmission. AMPAR potentiators slow channel deactivation and desensitization, and these compounds represent exciting therapies for mental and neurodegenerative diseases. Previous studies showed that the AMPAR potentiators cyclothiazide and 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluorophenoxyacetamide display a preference for flip and flop alternatively spliced versions of glutamate receptor subunits, respectively. Here, we find that the AMPAR auxiliary subunit stargazin changes this pharmacology and makes both spliced forms of glutamate receptor subunit 1 sensitive to both classes of potentiator. Stargazin also enhances the effect of AMPAR potentiators on channel deactivation. This work demonstrates that stargazin controls AMPAR potentiator pharmacology, which has important implications for development of AMPAR potentiators as therapeutic agents. transmembrane AMPA receptor regulatory protein | (TARP) glutamate | synapse | psychiatry | cognition

Interaction between astrocytes and neurons enriches the behavior of brain circuits. By releasing glutamate and ATP, astrocytes can directly excite neurons and modulate synaptic transmission. In the rat olfactory bulb, we demonstrate that the release of GABA by astrocytes causes long-lasting and synchronous inhibition of mitral and granule cells. In addition, astrocytes release glutamate, leading to a selective activation of granule-cell NMDA receptors. Thus, by releasing excitatory and inhibitory neurotransmitters, astrocytes exert a complex modulatory control on the olfactory network. glutamate | GABA | inhibition | olfactory bulb | synchronization

The level of arousal in mammals is correlated with metabolic state and specific patterns of cortical neuronal responsivity. In particular, rhythmic transitions between periods of high activity (up phases) and low activity (down phases) vary between wakefulness and deep sleep/anesthesia. Current opinion about changes in cortical response state between sleep and wakefulness is split between neuronal network-mediated mechanisms and neuronal metabolism-related mechanisms. Here, we demonstrate that slow oscillations in network state are a consequence of interactions between both mechanisms. Specifically, recurrent networks of excitatory neurons, whose membrane potential is partly governed by ATP--modulated potassium ([K.sub.ATP]) channels, mediate response-state oscillations via the interaction between excitatory network activity involving slow, kainate receptor-mediated events and the resulting activation of ATP-dependent homeostatic mechanisms. These findings suggest that [K.sub.ATP] channels function as an interface between neuronal metabolic state and network responsivity in mammalian cortex. glutamate | slow-wave oscillation | potassium channel | rhythm

[Ca.sup.2+] channels play critical roles in the regulation of synaptic activity. In contrast to the weil established function of voltage-activated [Ca.sub.2+] channels in the presynaptic membrane for neurotransmitter release, some studies are just beginning to elucidate the functions of the [Ca.sub.2+] channels in the postsynaptic membrane. In this study, we demonstrated the functional association of [alpha]-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors with the neuronal [Ca.sub.2+] channels. A series of biochemical studies showed the specific association of [Ca.sub.v]2.1 ([[alpha].sub.1A]-class) and [Ca.sub.v]2.2 ([[alpha].sub.1B]-class) with AMPA receptors in the postsynaptic membrane. Our electrophysiological and [Ca.sup.2+] imaging analyses of recombinant [Ca.sub.v]2.1 and AMPA receptors also showed functional coupling of the two channels. Considering the critical roles of postsynaptic intracellular concentration of [Ca.sup.2+] ([[Ca.sup.2+].sub.i]) increase and AMPA receptor trafficking for long-term potentiation (LTP) and long-term depression (LTD), the functional association of [Ca.sup.2+] channels with the AMPA receptors may provide new insights into the mechanism of synaptic plasticity. voltage-activated [Ca.sup.2+] channel | stargazin | glutamate receptor | synaptic plasticity | postsynaptic density

It is widely thought that Alzheimer's disease (AD) begins as a malfunction of synapses, eventually leading to cognitive impairment and dementia. Homeostatic synaptic scaling is a mechanism that could be crucial at the onset of AD but has not been examined experimentally. In this process, the synaptic strength of a neuron is modified so that the overall excitability of the cell is maintained. Here, we investigate whether synaptic scaling mediated by L-[alpha]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) contributes to pathology in double knockin (2 x KI) mice carrying human mutations in the genes for amyloid precursor protein and presenilin-1. By using whole-cell recordings, we show that 2xKI mice exhibit age-related downscaling of AMPAR-mediated evoked currents and spontaneous, miniature currents. Electron microscopic analysis further corroborates the synaptic AMPAR decrease. Additionally, 2xKI mice show age-related deficits in bidirectional plasticity (long-term potentiation and long-term depression) and memory flexibility. These results suggest that AMPARs are important synaptic targets for AD and provide evidence that cognitive impairment may involve downscaling of postsynaptic AMPAR function. amyloid precursor protein | glutamate | presenilin

Intracellular calcium mobilization and signaling mechanisms triggered by activation of synaptic glutamate receptors in the striatum are important modulators of neurotransmission in striatal circuits. However, the expression and functions of scaffolding proteins anchoring glutamate receptors at striatal synapses have not been addressed so far. The long-form Homer1 proteins, Homer1b/c, assemble group I metabotropic glutamate receptors (mGluR1/5) in large macromolecular complexes with sources of calcium influx and release at synapses as well as with components of the NMDA receptor complex at the neuronal cell membrane. Homer1a, the short, activity-dependent splice variant of Homer1b/c, lacks the ability of linking mGluR1/5 to synaptic proteins and functions as an endogenous negative modulator of the mGluR1/5 inositol 1,4,5-trisphosphate receptor signaling complex. We have generated transgenic mice, which overexpress Homer1a in striatal medium spiny neurons either homogenously throughout the extrastriosomal matrix (Homer1a-matrix line) or predominantly in striosomal patches (Homer1a-striosome line). Homer1a-expressing mice demonstrated normal development of striatal structure and afferent-efferent connectivity. However, motor performance in behavioral tasks and striatal responses to the psychomotor stimulant amphetamine were significantly altered in the Homer1a-striosome line. Thus, glutamate receptor scaffolding proteins of the Homer1 family critically regulate the functions of striatal medium spiny neurons in complex motor tasks and its modulation by psychomotor stimulant drugs. metabotropic glutamate | motor stereotypy | striosome

The adrenal hormone corticosterone transcriptionally regulates responsive genes in the rodent hippocampus through nuclear mineralocorticoid and glucocorticoid receptors. Via this genomic pathway the hormone alters properties of hippocampal cells slowly and for a prolonged period. Here we report that corticosterone also rapidly and reversibly changes hippocampal signaling. Stress levels of the hormone enhance the frequency of miniature excitatory postsynaptic potentials in CA1 pyramidal neurons and reduce paired-pulse facilitation, pointing to a hormone-dependent enhancement of glutamate-release probability. The rapid effect by corticosterone is accomplished through a nongenomic pathway involving membrane-located receptors. Unexpectedly, the rapid effect critically depends on the classical mineralocorticoid receptor, as evidenced by the effectiveness of agonists, antagonists, and brain-specific inactivation of the mineralocorticoid but not the glucocorticoid receptor gene. Rapid actions by corticosterone would allow the brain to change its function within minutes after stress-induced elevations of corticosteroid levels, in addition to responding later through gene-mediated signaling pathways. [alpha]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor | CA1 hippocampus | glucocorticoid receptor knockout | miniature excitatory postsynaptic current | mineralocorticoid receptor knockout

Before synaptogenesis, early excitability implicating voltage-dependent and transmitter-activated channels is known to be crucial for neuronal development. We previously showed that preplate (PP) neurons of the mouse neocortex express functional [Na.sup.+] channels as early as embryonic day 12. In this study, we investigated the role of these [Na.sup.+] channels in signaling during early development. In the neocortex of embryonic-day-13 mice, activation of Nat channels with veratridine induced a large [Ca.sup.2+] response throughout the neocortex, even in cell populations that lack the [Na.sup.+] channel. This [Na.sup.+]-dependent [Ca.sup.2+] activity requires external [Ca.sup.2+] and is completely blocked by inhibitors of [Na.sup.+]/[Ca.sup.2+] exchangers. Moreover, veratridine-induced [Ca.sup.2+] increase coincides with a burst of exocytosis in the PP. In parallel, we show that Nat channel stimulation enhances glutamate secretion in the neocortical wall. Released glutamate triggers further [Ca.sup.2+] response in PP and ventricular zone, as indicated by the decreased response to veratridine in the presence of [alpha]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor and NMDA-receptor inhibitors. Therefore, the combined activation of the Nat channel and the [Na.sup.+]/[Ca.sup.2+] exchanger triggers [Ca.sup.2+] signaling in the PP neurons, leading to glutamate secretion, which amplifies the signal and serves as an autocrine/paracrine transmitter before functional synapses are formed in the neocortex. Membrane depolarization induced by glycine receptors activation could be one physiological activator of this [Na.sup.+] channel-dependent pathway. [Ca.sup.2+] signaling | [Na.sup.+]/[Ca.sup.2+] exchanger | exocytosis | neurogenesis

Metabotropic glutamate receptor (mGluR) subtypes (mGluR1 to mGluR8) act as important pre- and postsynaptic regulators of neurotransmission in the CNS. These receptors consist of two domains, an extracellular region containing the orthosteric agonist site and a transmembrane heptahelical domain involved in G protein activation and recognition of several recently synthesized pharmacological modulators. The presynaptic receptor mGluR7 shows the highest evolutionary conservation within the family, but no selective pharmacological tool was known. Here we characterize an mGluR7-selective agonist, N,N'-dibenzhydrylethane-1,2-diamine dihydrochloride (AMN082), which directly activates receptor signaling via an allosteric site in the transmembrane domain. At transfected mammalian cells expressing mGluR7, AMN082 potently inhibits cAMP accumulation and stimulates GTP[gamma]S binding (E[C.sub.50]-values, 64-290 nM) with agonist efficacies comparable with those of L-2-amino-4-phosphonobutyrate (L-A[P.sub.4]) and superior to those of L-glutamate. AMN082 ([less than or equal to]10 [micro]M) failed to show appreciable activating or inhibitory effects at other mGluR subtypes and selected ionotropic GluRs. Chimeric receptor studies position the binding site of AMN082 in the transmembrane region of mGluR7, and we demonstrate that this allosteric agonist has little, if any, effect on the potency of orthosteric ligands. Here we provide evidence for full agonist activity mediated by the heptahelical domain of family 3 G protein-coupled receptors (which have mGluR-like structure) that may lead to drug development opportunities. Further, AMN082 is orally active, penetrates the blood-brain barrier, and elevates the plasma stress hormones corticosterone and corticotropin in an mGluR7-dependent fashion. Therefore, AMN082 is a valuable tool for unraveling the role of mGluR7 in stress-related CNS disorders. G protein | mGluR

Several lines of work have shown that astrocytes release glutamate in response to receptor activation, which results in a modulation of local synaptic activity. Astrocytic glutamate release is [Ca.sup.2+]-dependent and occurs in conjunction with exocytosis of glutamate containing vesicles. However, astrocytes contain a millimolar concentration of cytosolic glutamate and express channels permeable to small anions, such as glutamate. Here, we tested the idea that astrocytes respond to receptor stimulation by dynamic changes in cell volume, resulting in volume-sensitive channel activation, and efflux of cytosolic glutamate. Confocal imaging and whole-cell recordings demonstrated that astrocytes exhibited a transient [Ca.sup.2+]-dependent cell volume increase, which activated glutamate permeable channels. HPLC analysis revealed that glutamate was released in conjunction with other amino acid osmolytes. Our observations indicate that volume-sensitive channel may constitute a previously uncharacterized target for modulation of astrocyte-neuronal interactions. electrophysiology | exocytosis | neurotransmitters | osmolarity | synapses

Glutamate receptors (GluRs) are the most abundant mediators of the fast excitatory neurotransmission in the human brain. Agonists will, after activation of the receptors, induce different degrees of desensitization. The efficacy of agonists strongly correlates with the agonist-induced closure of the ligand-binding domain. However, the differences in desensitization properties are less well understood. By using high-resolution x-ray structure of the GluR2 flop (GluR2o) ligand-binding core protein in complex with the partial glutamate receptor agonist (S)-2-amino-3-(3-hydroxy-5-tert-butyl-4-isothiazolyl)propionic acid [(S)-thio-ATPA], we show that (S)-thio-ATPA induces an 18[degrees] closure of the binding core similar to another partial agonist, (S)-2-amino-3-(4-bromo-3-hydroxy-5-isoxazolyl)propionic acid [(S)-Br-HIBO]. Despite the similar closure of the ligand-binding domain, we find in electrophysiological studies that (S)-thio-ATPA induced a 6.4-fold larger steady-state current than (RS)-Br-HIBO, and rapid agonist applications show that (S)-thio-ATPA induces a 3.6-fold higher steady-state/peak ratio and a 2.2-fold slower desensitization time constant than (RS)-Br-HIBO. Structural comparisons reveal that (S)-Br-HIBO, but not (S)-thio-ATPA, induces a twist of the ligand-binding core compared with the apostructure, and the agonist-specific conformation of Leu-650 correlates with the different kinetic profiles pointing at a key role in defining the desensitization kinetics. We conclude that, especially for intermediate efficacious agonists, the desensitization properties are influenced by additional ligand-induced factors beyond domain closure. 2-amino-3-(3-hydroxy-S-tert-butyl-4-isoxazolyl)propionic acid (thio-ATPA) | desensitization | domain closure | crystallization

Polyglutamylation of tubulin has been implicated in several functions of microtubules, but the identification of the responsible enzyme(s) has been challenging. We found that the neuronal tubulin polyglutamylase is a protein complex containing a tubulin tyrosine ligase-like (TTLL) protein, TTLL1. TTLL1 is a member of a large family of proteins with a TTL homology domain, whose members could catalyze ligations of diverse amino acids to tubulins or other substrates. In the model protist Tetrahymena thermophila, two conserved types of polyglutamylases were characterized that differ in substrate preference and subcellular localization., Polyglutamylation is an uncommon type of post-translational modification that adds multiple glutamic acids to a γ-carboxyl group of a glutamate residue of target proteins, including tubulin and nucleosome assembly proteins [...]

Glutamate is the predominant excitatory neurotransmitter in the mammalian brain. Once released, its rapid removal from the synaptic cleft is critical for preventing excitotoxicity and spillover to neighboring synapses. Despite consensus on the role of glutamate in normal and disease physiology, technical issues limit our understanding of its metabolism in intact cells. To monitor glutamate levels inside and at the surface of living cells, genetically encoded nanosensors were developed, The fluorescent indicator protein for glutamate (FLIPE) consists of the glutamate/aspartate binding protein ybeJ from Escherichia coil fused to two variants of the green fluorescent protein. Three sensors with lower affinities for glutamate were created by mutation of residues peristeric to the ybeJ binding pocket. In the presence of ligands, FLIPEs show a concentration-dependent decrease in FRET efficiency. When expressed on the surface of rat hippocampal neurons or PC12 cells, the sensors respond to extracellular glutamate with a reversible concentration-dependent decrease in FRET efficiency. Depolarization of neurons leads to a reduction in FRET efficiency corresponding to 300 nM glutamate at the cell surface. No change in FRET was observed when cells expressing sensors in the cytosol were superfused with up to 20 mM glutamate, consistent with a minimal contribution of glutamate uptake to cytosolic glutamate levels. The results demonstrate that FLIPE sensors can be used for real-time monitoring of glutamate metabolism in living cells, in tissues, or in intact organisms, providing tools for studying metabolism or for drug discovery. aspartate | hippocampal neuron | neurotransmitter | secretion | transport