Functional architecture of olfactory ionotropic glutamate receptors

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Electrophysiological demonstration of independent olfactory receptor types and associated neuronal responses in the trout olfactory bulb

The present study attempts to highlight the principles by which peripheral olfactory information of across- and within-class odorant signals is transformed into bulbar neuron responses. For this purpose, we performed electro-olfactogram cross-adaptation and mixture experiments as well as single unit recording of olfactory bulb neurons using amino acid, bile acid and F-prostaglandin stimulants in brown and rainbow trout. The results show that amino acids, a bile acid and a F-prostaglandin activate independent receptor types. However, within the class of amino acids, different receptor types are only partially independent. Neurons responsive to bile acid and amino acids were segregated to the mid-dorsal and latero-posterior olfactory bulb, respectively. Of the 43 responsive olfactory bulb neurons studied in brown trout, 41 showed specificity for one odorant class. Olfactory bulb neurons gained responsiveness to new amino acids with increasing stimulant concentration. We conclude that different odorant classes activate specific neurons located in different regions of the trout olfactory bulb, and that information distinguishing related amino acids can be represented in a limited number of bulbar neurons with distinct response profiles under the conditions investigated.     

Structural dynamics of an ionotropic glutamate receptor

Ionotropic glutamate receptors (iGluRs) are postsynaptic ion channels involved in excitatory neurotransmission. iGluRs play important roles in development and in forms of synaptic plasticity that underlie higher order processes such as learning and memory. Neurobiological and biochemical studies have long characterized iGluRs in detail. However, the structural basis for the function of iGluRs has not yet been investigated, because there is insufficient information about their three-dimensional structures. In 1998, a crystal structure called S1S2 lobes was first solved for the extracellular bilobed ligand-binding domain of the GluR2 subunit. Since then, the crystal structures for the S1S2 lobes both in the apo and in various liganded states have been reported, and recent biophysical studies have further elucidated the dynamic aspects of the structure of the S1S2 lobes. In this review, the dynamic structures of the S1S2 lobes and their ligands are summarized, and the importance of their structural flexibility and fluctuation is discussed in light of the mechanisms of ligand recognition, activation, and desensitization of the receptor.     

Pharmacological analysis of slow potentials recorded in forg olfactory bulb during natural stimulation

Pharmacological agents (strychnine, picrotoxin, pentobarbital, chloralose, GABA, penicillin, morphine) were used to investigate the nature of the slow potential recorded in the frog olfactory bulb in response to natural stimulation. Three possible hypotheses were tested: 1) The slow potential is neuroglial in nature; 2) it is the analog of the dorsal-root potential of the spinal cord and reflects depolarization of primary afferents arising in the terminals of the olfactory nerve and responsible for presynaptic inhibition in the frog olfactory bulb; 3) the slow potential reflects postsynaptic processes. The results showed great similarity between changes in the slow and dorsal-root potentials of the spinal cord in response to the action of pharmacological agents. However, the slow potential is evidently a complex response and incorporates at least one other component — depolarization of the dendrites of unknown nature.     

Ligand-specific conformations of an ionotropic glutamate receptor

A simple in silico procedure is proposed with a view to predict the agonist or antagonist character of new, AMPA-type Glu receptor channel ligands. Based on the experimental binding domain structures, the orientation of a single Lys residue close to the ligand binding core was found to be diagnostic of ligand-induced conformational changes. Acting as a switch, the position of the Lys residue indicates the agonist or antagonist character of AMPA receptor ligands, known to bind to the receptor. Stability centre analysis substantiated the key role this switch might play in ligand-induced conformational changes.     

Neuronal circuits and computations: Pattern decorrelation in the olfactory bulb

Neuronal circuits in the olfactory bulb transform odor-evoked activity patterns across the input channels, the olfactory glomeruli, into distributed activity patterns across the output neurons, the mitral cells. One computation associated with this transformation is a decorrelation of activity patterns representing similar odors. Such a decorrelation has various benefits for the classification and storage of information by associative networks in higher brain areas. Experimental results from adult zebrafish show that pattern decorrelation involves a redistribution of activity across the population of mitral cells. These observations imply that pattern decorrelation cannot be explained by a global scaling mechanism but that it depends on interactions between distinct subsets of neurons in the network. This article reviews insights into the network mechanism underlying pattern decorrelation and discusses recent results that link pattern decorrelation in the olfactory bulb to odor discrimination behavior.     

Roles of the N-terminal domain on the function and quaternary structure of the ionotropic glutamate receptor

The alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of ionotropic glutamate receptors (iGluRs) mediates fast excitatory neurotransmission in the mammalian brain. Although the most N-terminal leucine/isoleucine/valine-binding protein (LIVBP) domain is suggested to play a role in the initial assembly of iGluR subunits, it is unclear how this domain is arranged and functions in intact iGluRs. Similarly, although recent crystallographic analyses indicate that the isolated ligand-binding lysine/arginine/ornithine-binding protein domain forms a 2-fold symmetric dimer, the subunit stoichiometry of intact iGluRs remains elusive. Here, we developed a new approach to address these issues. The LIVBP domain of the GluR1 subunit of AMPA receptors was replaced by leucine-zipper peptides designed to form stable symmetric dimers, trimers, tetramers, or pentamers. All these mutant GluR1s were expressed in human embryonic kidney 293 cells and were transported to the cell surface as well as wild type GluR1. Functional and biochemical analyses indicated that these oligomerizing peptides specifically controlled the formation of the expected number of subunits in a channel complex. However, the channel function was only restored by the tetramer-forming peptide. Although the purified LIVBP domain of GluR1 formed a dimmer in solution, a dimer-forming peptide could not restore the function of GluR1. Moreover, a cross-linking assay indicated that four LIVBP domains are located in proximity to each other. These results suggest that the function of the LIVBP domain is not simply to form initial dimers but to adopt a conformation compatible with the overall tetrameric arrangement of subunits in intact AMPA receptors.     

Computer modeling of mechanisms of the information processing in the olfactory bulb. I. Model of the structure-functional organizations of neuronal elements in the olfactory bulb and receptor epithelium

A computer model of the olfactory bulb was constructed. The paper describes: 1) the general architecture of a model neuron network that reflects the neurophysiological experimental and theoretical data on the structural and functional organization of the peripheral part of the olfactory system, the olfactory bulb with inputs from olfactory receptor neurons; 2) the organization of each of three levels of the model: receptors, olfactory glomeruli, and basic neurons; and 3) a scenario of the computer model work. In some aspects, in particular, in the principle of information presentation, the treatment of the role of basic neurons (mitral and tufted cells), and their interrelations in modules, the model favorably differs from the available olfactory bulb models. The model is basic and provides further refinement of the architecture, an increase in the number of modules, and the modeling of the learning process.     

Guidepost neurons for the lateral olfactory tract: Expression of metabotropic glutamate receptor 1 and innervation by glutamatergic olfactory bulb axons

The guidepost neurons for the lateral olfactory tract, which are called lot cells, are the earliest‐generated neurons in the neocortex. They migrate tangentially and ventrally further down this tract, and provide scaffolding for the olfactory bulb axons projecting into this pathway. The molecular profiles of the lot cells are largely uncharacterized. We found that lot cells specifically express metabotropic glutamate receptor subtype‐1 at a very early stage of development. This receptor is functionally competent and responds to a metabotropic glutamate receptor agonist with a transient increase in the intracellular calcium ion concentration. When the glutamatergic olfactory bulb axons were electrically stimulated, lot cells responded to the stimulation with a calcium increase mainly via ionotropic glutamate receptors, suggesting potential neurotransmission between the axons and lot cells during early development. Together with the finding that lot cells themselves are glutamatergic excitatory neurons, our results provide another notable example of precocious interactions between the projecting axons and their intermediate targets. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

Thermodynamics and structural analysis of positive allosteric modulation of the ionotropic glutamate receptor GluA2

Positive allosteric modulators of the ionotropic glutamate receptor-2 (GluA2) are promising compounds for the treatment of cognitive disorders, e.g. Alzheimer's disease. These modulators bind within the dimer interface of the LBD (ligand-binding domain) and stabilize the agonist-bound conformation slowing receptor desensitization and/or deactivation. In the present study, we employ isothermal titration calorimetry to determine binding affinities and thermodynamic details of binding of modulators of GluA2. A mutant of the LBD of GluA2 (LBD-L483Y-N754S) that forms a stable dimer in solution was used. The potent GluA2 modulator BPAM-97 was used as a reference compound. Evidence that BPAM-97 binds in the same pocket as the well-known GluA2 modulator cyclothiazide was obtained from X-ray structures. The LBD-L483Y-N754S:BPAM-97 complex has a Kd of 5.6?μM (ΔH=-4.9 kcal/mol, -TΔS=-2.3 kcal/mol; where 1?kcal≈4.187?kJ). BPAM-97 was used in a displacement assay to determine a Kd of 0.46?mM (ΔH=-1.2 kcal/mol, -TΔS=-3.3 kcal/mol) for the LBD-L483Y-N754S:IDRA-21 complex. The major structural factors increasing the potency of BPAM-97 over IDRA-21 are the increased van der Waals contacts to, primarily, Met496 in GluA2 imposed by the ethyl substituent of BPAM-97. These results add important information on binding affinities and thermodynamic details, and provide a new tool in the development of drugs against cognitive disorders.     

Chemotopy of amino acids on the olfactory bulb predicts olfactory discrimination capabilities of zebrafish Danio rerio

Amino acids reliably evoke strong responses in fish olfactory system. The molecular olfactory receptors (ORs) are located in the membrane of cilia and microvilli of the olfactory receptor neurons (ORNs). Axons of ORNs converge on specific olfactory bulb (OB) glomeruli and the neural responses of ORNs expressing single Ors activate glomerular activity patterns typical for each amino acid. Chemically similar amino acids activate more similar glomerular activity patterns then chemically different amino acids. Differential glomerular activity patterns are the structural basis for amino acid perception and discrimination. We studied olfactory discrimination in zebrafish Danio rerio (Hamilton 1822) by conditioning them to respond to each of the following amino acids: L-Ala, L-Val, L-Leu, L-Arg, and L-Phe. Subsequently, zebrafish were tested for food searching activities with 18 nonconditioned amino acids. The food searching activity during 90 s of the test period was significantly greater after stimulation with the conditioned stimulus than with the nonconditioned amino acid. Zebrafish were able to discriminate all the tested amino acids except L-Ile from L-Val and L-Phe from L-Tyr. We conclude that zebrafish have difficulties discriminating amino acid odorants that evoke highly similar chemotopic patterns of activity in the OB.     

Conformational changes in the ligand-binding domain of a functional ionotropic glutamate receptor

Fluorescence resonance energy transfer was used to determine the structural changes in the extracellular ligand-binding segment in a functional glutamate receptor that contains the ligand-binding, transmembrane, and C-terminal segments. These studies indicate that the structural changes previously reported for the isolated ligand-binding domain due to the binding of partial and full agonists are also observed in this functional receptor, thus validating the detailed structure-function relationships that have been previously developed based on the structure of the isolated ligand-binding domain. Additionally, these studies provide the first evidence that there are no significant changes in the extent of cleft closure between the activated and desensitized states of the glutamate bound form of the receptor consistent with the previous functional investigations, which suggest that desensitization is mediated primarily by changes in the interactions between subunits composing the receptor.     

Insights into structure and function of ionotropic glutamate receptor channels: Starting from channel block

Ionotropic glutamate receptors (iGluRs) are modular proteins that contain ion channel permeable to different cations including calcium. The physiological role of iGluRs is mainly defined by the fact that currents conducted by their channels underlie communication between neurons in the majority of synapses in our brain. Knowing the structure and function of iGluR channel will not only give us a clue to how our brain works but also may help us to develop drugs for the treatment of multiple neurological disorders. Here I give a brief historical overview of the progress made in studies of iGluR channel structure and function that started more than two decades ago with studies of ion channel block. The article is published in the original.     

EEG analysis gives model of neuronal template-matching mechanism for sensory search with olfactory bulb

The spatial pattern of EEG activity at the surface of the olfactory bulb tends to be invariant with respect to input and to change to a new pattern whenever an animal is trained to expect or search for a particular odor. It is postulated here that the spatial EEG pattern is dependent on a neural template for that odor that is formed during training. This hypothesis is expressed in the form of a model consisting of an array of interconnected elements (1x10 or 6x6). Each element represents 2 excitatory and 2 inhibitory subsets of neurons with 3 types of internal feedback: negative, mutually excitatory, and mutually inhibitory. The elements are interconnected only by mutual excitation and mutual inhibition. Each neural subset is represented by a nonlinear differential equation; the connections are represented by modifiable coupling coefficients. With appropriate values of the time, coupling, and gain coefficients, and with input that is modelled on olfactory input, the set of 40 or 144 equations gives output that simulates the time and space patterns of the EEG.In the naive state the coefficients are uniform. A template is formed by giving input to selected elements, cross-correlating the outputs, and weighting the mutually excitatory coupling coefficient between each pair of elements by the corresponding correlation coefficient. When a template has been formed, input to nontemplate elements is treated as noise. Optionally a matched filter is made to simulate habituation by reducing the synaptic gain coefficients of those excitatory subsets that receive the noise. The model is tested by giving input to nontemplate elements and to none, part or all of the template elements.There are two outputs of the model. One is the spatial pattern V _j of the root mean square (rms) amplitudes of the individual outputs v(j,t) of the elements. The other output is the rms amplitude E _rms of the ensemble average E(t) over v(j, t).The results show that V _j depends on the template and is relatively insensitive to input, whether or not input is given to template elements. However, E _rms increases in proportion to the number of hits on the template. If the number of elements receiving noise does not exceed the number of elements in a template, or if the noise is matched with a habituation filter, then E _rms rises above the noise level for a hit on any one or more template elements irrespective of location or combination. V _j conforms to the performance of the surface EEG. E _rms is not yet accessible to physiological measurement.Supported by a grant MH 06686 from the National Institute of Mental Health and by a Research Professorship from the Miller Institute, Berkeley.     

Optical and genetic approaches toward understanding neuronal circuits in zebrafish

Optical and genetic tools are beginning to revolutionize thestudies of neuronal circuits. Neurons can now be labeled withconventional or genetically encoded indicators that allow theiractivity to be monitored during behavior in intact animals.Laser ablations and genetic inactivation offer ways to perturbactivity of specific cells to test their contributions to behavior.These approaches promise to speed progress in the understandingof vertebrate networks in genetic model systems such as miceand zebrafish. Here we review some of the progress in applyingthese tools, with an emphasis on our work to develop and applythese approaches in the zebrafish model.     

Analysis of the neuronal dendrite system of the olfactory bulb in the dog brain

A technique was worked out for quantitative analysis of accurate neuron drawings obtained from the preparations impregnated after Golgi. The analysis (according to 13 parameters) was performed to study the dendritic system within one main class of long-axonal neurons in layer II in areas having heterogenous structures of the polyfunctional formation in the dog cerebral olfactory tubercle. The most stable parameters were demonstrated to be the linear dimension of the cell body, branching of the apical and basal dendritic systems, as well as branching of the whole neuronal dendritic system. Values of other parameters change with statistical significance depending on the fact to what areas with heterogenous structures of the olfactory tubercle the neurons belong. The data obtained demonstrate a greater pyramidization of the neural class studied in the rostral portion and their similarity in the median and caudal portions of the olfactory tubercle and the neurons of highly differentiated subcortical nuclei.