Variability of calcium responses to agonists of glutamate receptors in hippocampal neurons

The subject of this work was to study the reasons of the variability of the calcium response amplitudes in individual neurons of the hippocampal cell culture to agonists of ionotropic glutamate receptors and the regularities of the calcium response amplitude distribution. Changes of [Ca²⁺] _i in the neurons in response to the NMDA-, AMPA-, and KA-receptor agonists were recorded using fluorescence probe Fura-2. The calcium response amplitudes (expressed as the ratio of fluorescence intensities of Fura-2 upon excitation at wave-lengths 340 and 380 nm) to short-term application of glutamate receptor agonists N-methyl-D-aspartate (NMDA), domoic acid (DA), α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), and (S)-(−)-5-fluorowillardiine (FW) were measured. Calcium responses of individual cells differed in shape and amplitude but always reproduced upon the second application of the agonist. To elucidate the nature of calcium response variability, we compared distributions of calcium response amplitudes to the NMDA-, KA-, and AMPA-receptor agonists in cultures of various ages in the presence of receptor desensitization inhibitors and different agonist concentrations. An even increase from 0.05 to 1.6 was characteristic for distributions of calcium response amplitudes. Nevertheless, in 1–3% neurons of the cell culture, calcium response amplitudes reached much higher values. The efficiency of the ligands usually increased in the following order: FW ≈ NMDA > DA. However, this regularity varied with age and depended on the presence of the receptor desensitization inhibitor. In the process of growth and differentiation of neurons in culture from 1 to 14 day in vitro, calcium response amplitude to AMPA- and KA-receptor agonists increased. Desensitization inhibitors transformed the response from pulse-like with a sharp peak into stepwise and increased the amplitude of calcium responses but did not abolish the character of even amplitude distribution. The effect of AMPA- and KA-receptor desensitization inhibitor decreased with calcium response amplitude growth in the control and approached zero in neurons with initially maximal amplitude. KA- and AMPA-receptor agonists at high concentrations possessed a property of desensitization inhibitors and transformed a transient response into a continuous one that lasted throughout the application time. Thus, the amplitude and shape of the calcium response to glutamate receptor agonists is a characteristic parameter of an individual cell.     

Glycine and serine as tentative agonists for metabotropic glutamate receptors

In experiments on slices of the rat hippocampus, glycine (Gly) and serine (Ser) in concentrations of 100 μM to I mM were found to reversible increase the amplitudes of population EPSP (pEPSP) in pyramidal neurons of theCA1 hippocampal area, evoked by single electrical stimuli applied to Schaffer collaterals (SchC). This potentiation was not affected by 100 μM of a non-competetive antagonist of NMDA glutamate receptors (GR), ketamine, but was considerably weakened by 500 μM of a competitive antagonist of metabotropic GR (mGR), (±)-4-carboxyphenylglycine (CFG). The effects of Gly and Ser were not observed in the presence of 50 μM of a blocker of protein kinase C (PKC) catalytic subunit, polymixin B, but were not modified by preliminary action on the slices of 10 μM of a calmodulin inhibitor, substance W-7. Gly and Ser also enhanced long-term post-tetanic potentiation (LTPP) of synaptic transmission caused by high-frequency rhythmic stimulation of SchC. Low-frequency (1/sec, 15 min) SchC stimulation abolished the potentiation of synaptic transmission evoked either by high-frequency SchC stimulation or by the actions of Gly and Ser. The data allow us to suggest that Gly and Ser in millimolar concentrations activate mGR, enhance relay functions of the synapses of pyramidal neurons in theCA1 hippocampal region, and facilitate plastic modifications in these synapses.     

The role of electrostatic interaction in the molecular recognition of selective agonists to metabotropic glutamate receptors

The influence of electrostatic interactions in determining selectivity for individual subtypes of metabotropic glutamate receptors (mGluRs) is evaluated for a small set of agonists by using the program Delphi and the information thus obtained is compared with docking experiments carried out with AutoDock. The evaluation of the electrostatic component of the free energy of binding for L-Glu, L-AP4, or S-PPG to mGluR1, mGluR2, and mGluR4 subtypes allowed for the detection of subtle differences in the electronic properties of the three subtypes, differences that can account for the observed agonist selectivity.     

Selective agonists for receptors of substance P and related neurokinins

Neurokinins and their receptors are a complex system consisting of at least three endogenous agents--substance P (SP), neurokinin A (NKA), and neurokinin B (NKB)--and their corresponding receptor types, respectively, NK-1, NK-2, and NK-3. Investigations on receptors have been made using sensitive and fairly selective pharmacological preparations (the dog carotid artery for the NK-1, the rabbit pulmonary artery devoid of endothelium for the NK-2, and the rat portal vein for the NK-3 receptor), and some natural peptides of mammalian and nonmammalian origin. Because of the nonselectivity of the natural peptides, analogues of the neurokinins have been found that act on one receptor only and show therefore high selectivity. The selective agonists [Sar9,Met(O2)11]SP, [Nle10]NKA (4-10), and [MePhe7]-NKB have been used successfully for (a) characterizing the three neurokinin receptors, (b) identifying isolated organs whose responses to neurokinins depend on the activation of a single (monoreceptor systems) or of more than one (multireceptor systems) receptor, and (c) elucidating some of the physiological function of the three receptor types. It is suggested that NK-1 mediate peripheral vasodilatation and exocrine secretions, NK-2 stimulate bronchial muscles and facilitate the release of catecholamines, and NK-3 promote the release of acetylcholine in peripheral organs.     

Stereostructure-activity studies on agonists at the AMPA and kainate subtypes of ionotropic glutamate receptors

(S)-Glutamic acid (Glu), the major excitatory neurotransmitter in the central nervous system, operates through ionotropic as well as metabotropic receptors and is considered to be involved in certain neurological disorders and degenerative brain diseases that are currently without any satisfactory therapeutic treatment. Until recently, development of selective Glu receptor agonists had mainly been based on lead compounds, which were frequently naturally occurring excitants structurally related to Glu. These Glu receptor agonists generally contain heterocyclic acidic moieties, which has stimulated the use of bioisosteric replacement approaches for the design of subtype-selective agonists. Furthermore, most of these leads are conformationally restricted and stereochemically well-defined Glu analogs. Crystallization of the agonist binding domain of the GluR2 subunit of the (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor subtype of ionotropic Glu receptors in the presence or absence of an agonist has provided important information about ligand-receptor interaction mechanisms. The availability of these binding domain crystal structures has formed the basis for rational design of ligands, especially for the AMPA and kainate subtypes of ionotropic Glu receptors. This mini-review will focus on structure-activity relationships on AMPA and kainate receptor agonists with special emphasis on stereochemical and three-dimensional aspects.     

Differential responsiveness of metabotropic glutamate receptors coupled to phosphoinositide hydrolysis to agonists in various brain areas of the adult rat

The effects of metabotropic glutamate receptor (mGluR) agonists on inositol phosphates (IP) accumulation were investigated in slices of the cerebral cortex, hippocampus, striatum and cerebellum of adult Sprague-Dawley rats. EC₅₀ values for 1S, 3R-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) did not differ significantly between various brain areas (range 10⁻⁵ M), quisqualate was the most potent in all the brain areas (range 10⁻⁷−10⁻⁶ M), except the cerebellum (10⁻⁵ M), ibotenate was the most potent in the striatum (range 10⁻⁶ M) and the least potent in the cerebral cortex and hippocampus (range 10⁻⁴ M). The efficacy in the four brain areas showed the following trend of ranking order for ACPD and quisqualate: hippocampus > striatum > cerebral cortex > cerebellum, and for ibotenate: hippocampus > cerebral cortex > striatum > cerebellum, although the observed differences reached the level of statistical significance only in the case of ACPD (hippocampus and striatum vs cerebellum) and ibotenate (hippocampus vs cerebellum). Co-incubation of the agonists at maximally effective concentrations in any pairwise combination resulted in no substantial additivity of IP accumulation. D,L-1-amino-3-phosphonopropionic acid (AP3) and D,L-2-amino-4-phosphonobutyric acid (AP4) at 0.5 mM concentration antagonized ACPD-induced IP accumulation by about 70 and 45%, respectively, without differences between brain areas. On the other hand, the antagonistic effects ofl-serine-o-phosphate (SOP) at 1 mM concentration were the highest in the hippocampus (75%) and the lowest in the cerebellum (25%). The comparative data indicate considerable regional receptor heterogeneity, in terms of different ratios of response to the agonists (but not antagonists, except SOP). There is a robust responsiveness of mGluRs not only in the hippocampus and cerebral cortex, but also in the striatum which exhibits the highest affinity to both quisqualate and ibotenate.     

Some interactions of L-DOPA and its related compounds with glutamate receptors

L-DOPA is probably a transmitter and/or modulator in the central nervous system (1). L-DOPA methyl ester (DOPA ME) is a competitive L-DOPA antagonist. However, it remains to be clarified whether there exist L-DOPAergic receptors. In Xenopus laevis oocytes injected with rat brain poly(A)+ RNA, L-DOPA induced small inward currents with ED50 of 2.2 mM at a holding potential of -70 mV. The currents were abolished by kynurenic acid or CNQX. Similar L-DOPA-currents were seen in oocytes co-injected with AMPA receptors, GluRs1,2,3 and 4. In brain membrane preparations, L-DOPA inhibited specific binding of [3H]-AMPA with IC50 of 260 microM. This inhibition was not modified by 200 microM ascorbic acid, an antioxidant. L-DOPA did not inhibit binding of [3H]-ligands of MK-801, kainate, DCKA and CGP39653. DOPA ME and L-DOPA cyclohexyl ester, a novel, potent and competitive antagonist (2), inhibited specific binding of [3H]-MK-801 with respective IC50 of 1 and 0.68 mM, but elicited no effect on that of the other [3H]-ligands. With low affinities, L-DOPA acts on AMPA receptors, while competitive antagonists act on NMDA ion channel domain. L-DOPAergic agonist and antagonist may not interact on ionotropic glutamate receptors. DOPA ME-sensitive L-DOPA recognition sites (1) seem to differ from glutamate receptors.     

Generalization mediates sensitivity to complex odor features in the honeybee

Animals use odors as signals for mate, kin, and food recognition, a strategy which appears ubiquitous and successful despite the high intrinsic variability of naturally-occurring odor quantities. Stimulus generalization, or the ability to decide that two objects, though readily distinguishable, are similar enough to afford the same consequence, could help animals adjust to variation in odor signals without losing sensitivity to key inter-stimulus differences. The present study was designed to investigate whether an animal's ability to generalize learned associations to novel odors can be influenced by the nature of the associated outcome. We use a classical conditioning paradigm for studying olfactory learning in honeybees to show that honeybees conditioned on either a fixed- or variable-proportion binary odor mixture generalize learned responses to novel proportions of the same mixture even when inter-odor differences are substantial. We also show that the resulting olfactory generalization gradients depend critically on both the nature of the stimulus-reward paradigm and the intrinsic variability of the conditioned stimulus. The reward dependency we observe must be cognitive rather than perceptual in nature, and we argue that outcome-dependent generalization is necessary for maintaining sensitivity to inter-odor differences in complex olfactory scenes.     

Binding of agonists and antagonists to muscarinic receptors

The binding of one irreversible and two reversible radioactive antagonists to muscarinic receptors in synaptosome preparations of rat cerebral cortex has been studied. The ligands all bind to the same receptor pool and directly and competitively yield self-consistent binding constants closely similar to those obtained by pharmacological methods on intact smooth muscle. The binding process for antagonists seems to be a simple mass action-determined process with a Hill slope of 1.0. The quantitative correlations strongly support the view that the receptor studied by ligand binding corresponds to the receptor studied by pharmacological methods. Inhibition of antagonist binding by most agonists shows a reduced Hill slope which also applies to direct binding studies of [³H] acetylcholine. Mechanisms that might account for the behavior of agonists are discussed but do not conclusively point to any single mechanism.     

Ionotropic glutamate receptors

In the brain, most fast excitatory synaptic transmission is mediated through L-glutamate acting on postsynaptic ionotropic glutamate receptors. These receptors are of two kinds—the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate (non-NMDA) and theN-methyl-D-aspartate (NMDA) receptors, which are thought to be colocalized onto the same postsynaptic elements. This excitatory transmission can be modulated both upward and downward, long-term potentiation (LTP) and long-term depression (LTD), respectively. Whether the expression of LTP/LTD is pre-or postsynaptically located (or both) remains an enigma. This article will focus on what postsynaptic modifications of the ionotropic glutamate receptors may possibly underly long-term potentiation/depression. It will discuss the character of LTP/LTD with respect to the temporal characteristics and to the type of changes that appears in the non-NMDA and NMDA receptor-mediated synaptic currents, and what constraints these findings put on the possible expression mechanism(s) for LTP/LTD. It will be submitted that if a modification of the glutamate receptors does underly LTP/LTD, an increase/decrease in the number of functional receptors is the most plausible alternative. This change in receptor number will have to include a coordinated change of both the non-NMDA and the NMDA receptors.     

Metabotropic glutamate receptors

Metabotropic glutamate receptors (mGlus) are a family of G-protein-coupled receptors activated by the neurotransmitter glutamate. Molecular cloning has revealed eight different subtypes (mGlu1-8) with distinct molecular and pharmacological properties. Multiplicity in this receptor family is further generated through alternative splicing. mGlus activate a multitude of signalling pathways important for modulating neuronal excitability, synaptic plasticity and feedback regulation of neurotransmitter release. In this review, we summarize anatomical findings (from our work and that of other laboratories) describing their distribution in the central nervous system. Recent evidence regarding the localization of these receptors in peripheral tissues will also be examined. The distinct regional, cellular and subcellular distribution of mGlus in the brain will be discussed in view of their relationship to neurotransmitter release sites and of possible functional implications.This work was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan (R.S.) and by the Austrian Science Fund FWF (grant no. P16720 to F.F.).     

Linking tricyclic antidepressants to ionotropic glutamate receptors

Although tricyclic antidepressants have been in existence since the 1940s when they were discovered upon screening iminodibenzyl derivatives for other potential therapeutic uses, their mechanism of action has remained unclear [A. Goodman Gilman, T.W. Rall, A.S. Nies, P. Taylor, Goodman and Gilman's The Pharmacological Basis of Therapeutics, eighth ed., Pergamon Press, New York, 1990]. In addition to their ability to hinder the reuptake of biogenic amines, there is mounting evidence that the tricyclic antidepressants inhibit glutamate transmission. Here, intrinsic tryptophan fluorescence spectroscopy is used to document the binding of desipramine, a member of the tricyclic antidepressant family, to a well-defined extracellular glutamate binding domain (S1S2) of the GluR2 subunit of the amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor. The binding is distinct from those of other known effectors of the receptor, including the endogenous sulfated neurosteroids pregnenolone sulfate and 3alpha-hydroxy-5beta-pregnan-20-one sulfate, and is consistent with a conformational change upon binding that is allosterically transmitted to the channel region of the receptor.     

Reduced sensitivity to beta-adrenergic agonists in Basenji-Greyhound dogs

To investigate the inhibitory effects of beta-adrenergic agonists and aminophylline on pulmonary responsiveness, we evaluated the ability of albuterol and aminophylline to attenuate pulmonary responses to aerosol challenge with methacholine and histamine in intact Basenji-Greyhound (BG) and selected mongrel dogs. Pulmonary responses were measured in untreated dogs and in dogs pretreated with albuterol (1 and 2.5 micrograms/kg) or aminophylline. Before aerosol challenge, baseline pulmonary resistance (RL) and dynamic compliance (Cdyn) were not significantly different between the BGs and the mongrels. In the untreated dogs, pulmonary responses to methacholine and histamine aerosols were not different between the BGs and the mongrels. Pretreatment with albuterol (1 microgram/kg) or aminophylline significantly attenuated the pulmonary response to methacholine in the mongrels but was without effect in the BGs. Albuterol (2.5 micrograms/kg) significantly attenuated the pulmonary response to methacholine in the BGs and the mongrels; however, this attenuation was significantly greater (P less than 0.05) in the mongrels than in the BGs. In response to histamine challenge, no differences were seen between the BGs and the mongrels in the control state (no pretreatment) or after pretreatment with albuterol or aminophylline. This study demonstrates that in BGs pulmonary responsiveness to methacholine but not histamine is resistant to inhibition by beta-adrenergic agonists. This may result from a qualitative or quantitative defect in either the cholinergic or beta-adrenergic receptor or to an abnormality distal to the receptors in the signal transduction mechanism at a site where the two signals interact.