Block of native Ca<SUP>2+</SUP>-permeable AMPA receptors in rat brain by intracellular polyamines generates double rectification

1. The influence of intracellular factors on current rectification of different subtypes of native alpha-amino-3-hydroxy-5-methyl-4- isoxazoleproprionate receptors (AMPARs) was studied in rat brain slices by combining fast application of glutamate with patch pipette perfusion. 2. The peak current-voltage (I-V) relation of the AMPARs expressed in Bergmann glial cells of cerebellum and dentate gyrus (DG) basket cells of hippocampus was weakly rectifying in outside-out patches and nystatin-perforated vesicles, but showed a doubly rectifying shape with a region of reduced slope between 0 and +40 mV in nucleated patches. The I-V relation of AMPARs expressed in hippocampal CA3 pyramidal neurones was linear in all recording configurations. 3. Intracellular application of 25 microM spermine, a naturally occurring polyamine, blocked outward currents in outside-out patches from Bergmann glial cells and DG basket cells in a voltage-dependent manner, generating I-V relations with a doubly rectifying shape which were similar to those recorded in nucleated patches. AMPARs in CA3 pyramidal cell patches were unaffected by 25 microM spermine. 4. The half-maximal blocking concentration of spermine at +40 mV was 0.3 microM in Bergmann glial cell patches and 1.5 microM in DG basket cell patches, whereas it was much higher (>> 100 microM) for CA3 pyramidal cell patches. Spermidine also affected current rectification, but with lower affinity. The block of outward current by polyamines following voltage jumps developed within < 0.5 ms. 5. We conclude that current rectification, rather than being an intrinsic property of the Ca(2+)-permeable AMPAR channel, is generated by polyamine block

Glycine receptors in CNS neurons as a target for nonretrograde action of cannabinoids

At many central synapses, endocannabinoids released by postsynaptic cells act retrogradely on presynaptic G-protein-coupled cannabinoid receptors to inhibit neurotransmitter release. Here, we demonstrate that cannabinoids may directly affect the functioning of inhibitory glycine receptor (GlyR) channels. In isolated hippocampal pyramidal and Purkinje cerebellar neurons, endogenous cannabinoids anandamide and 2-arachidonylglycerol, applied at physiological concentrations, inhibited the amplitude and altered the kinetics of rise time, desensitization, and deactivation of the glycine-activated current (

Fast interaction between AMPA and NMDA receptors by intracellular calcium

© 2016 Elsevier LtdSuppression of NMDA receptor (NMDAR)-mediated currents by intracellular Ca2+ has been described as a negative feedback loop in NMDAR modulation. In the time scale of tenths of milliseconds the depth of the suppression does not depend on the Ca2+ source. It may be caused by Ca2+ influx through voltage-gated calcium channels, NMDAR channels or release from intracellular stores. However, NMDARs are often co-expressed in synapses with Ca2+-permeable AMPA receptors (AMPARs). Due to significant differences in activation kinetics between these two types of glutamate receptors (GluRs), Ca2+ entry through AMPARs precedes full activation of NMDARs, and therefore, might have an impact on the amplitude of NMDAR-mediated currents. The study of Ca2+-mediated crosstalk between AMPAR and NMDAR in native synapses is challenging due to high NMDAR Ca2+ permeability. Therefore, recombinant Ca2+-permeable AMPAR and Ca2+-impermeable NMDAR mutant channels were co-expressed in HEK 293 cells to examine their interaction. An AMPAR-mediated increase in intracellular Ca2+ concentration ([Ca2+]i) reversibly reduced the size of NMDAR-mediated whole-cell currents. The time course of the NMDAR channel inactivation and recovery from inactivation followed the time course of the [Ca2+]i transient. When brief (1 ms) pulses of glutamate were applied to outside-out patches, the degree of NMDAR inactivation increased with the increase in charge carried by the currents through co-activated AMPARs. However, AMPAR-mediated NMDAR inactivation was abolished in the presence of intracellular fast Ca2+ buffer BAPTA or in Ca2+-free extracellular solution. We conclude that Ca2+ entering through AMPARs inactivates co-localized NMDARs in the time range of excitatory postsynaptic currents

Functional properties of human NMDA receptors associated with epilepsy-related mutations of GluN2A subunit

© 2017 Sibarov, Bruneau, Antonov, Szepetowski, Burnashev and Giniatullin. Genetic variants of the glutamate activated N-methyl-D-aspartate (NMDA) receptor (NMDAR) subunit GluN2A are associated with the hyperexcitable states manifested by epileptic seizures and interictal discharges in patients with disorders of the epilepsy-aphasia spectrum (EAS). The variants found in sporadic cases and families are of different types and include microdeletions encompassing the corresponding GRIN2A gene as well as nonsense, splice-site and missense GRIN2A defects. They are located at different functional domains of GluN2A and no clear genotype-phenotype correlation has emerged yet. Moreover, GluN2A variants may be associated with phenotypic pleiotropy. Deciphering the consequences of pathogenic GRIN2A variants would surely help in better understanding of the underlying mechanisms. This emphasizes the need for functional studies to unravel the basic functional properties of each specific NMDAR variant. In the present study, we have used patch-clamp recordings to evaluate kinetic changes of mutant NMDARs reconstituted after co-transfection of cultured cells with the appropriate expression vectors. Three previously identified missense variants found in patients or families with disorders of the EAS and situated in the N-terminal domain (p.Ile184Ser) or in the ligand-binding domain (p.Arg518His and p.Ala716Thr) of GluN2A were studied in both the homozygous and heterozygous conditions. Relative surface expression and current amplitude were significantly reduced for NMDARs composed of mutant p.Ile184Ser and p.Arg518His, but not p.Ala716His, as compared with wild-type (WT) NMDARs. Amplitude of whole-cell currents was still drastically decreased when WT and mutant p.Arg518His-GluN2A subunits were co-expressed, suggesting a dominant-negative mechanism. Activation times were significantly decreased in both homozygous and heterozygous conditions for the two p.Ile184Ser and p.Arg518His variants, but not for p.Ala716His. Deactivation also significantly increased for p.Ile184Ser variant in the homozygous but not the heterozygous state while it was increased for p.Arg518His in both states. Our data indicate that p.Ile184Ser and p.Arg518His GluN2A variants both impacted on NMDAR function, albeit differently, whereas p.Ala716His did not significantly influence NMDAR kinetics, hence partly questioning its direct and strong pathogenic role. This study brings new insights into the functional impact that GRIN2A variants might have on NMDAR kinetics, and provides a mechanistic explanation for the neurological manifestations seen in the corresponding human spectrum of disorders

The relative contribution of nmdars to excitatory postsynaptic currents is controlled by ca2+-induced inactivation

© 2016 Valiullina, Zakharova, Mukhtarov, Draguhn, Burnashev and Rozov. NMDA receptors (NMDARs) are important mediators of excitatory synaptic transmission and plasticity. A hallmark of these channels is their high permeability to Ca2+. At the same time, they are themselves inhibited by the elevation of intracellular Ca2+ concentration. It is unclear however, whether the Ca2+ entry associated with single NMDAR mediated synaptic events is sufficient to self-inhibit their activation. Such auto-regulation would have important effects on the dynamics of synaptic excitation in several central neuronal networks. Therefore, we studied NMDAR-mediated synaptic currents in mouse hippocampal CA1 pyramidal neurons. Postsynaptic responses to subthreshold Schaffer collateral stimulation depended strongly on the absence or presence of intracellular Ca2+ buffers. Loading of pyramidal cells with exogenous Ca2+ buffers increased the amplitude and decay time of NMDAR mediated EPSCs (EPSPs) and prolonged the time window for action potential (AP) generation. Our data indicate that the Ca2+ influx mediated by unitary synaptic events is sufficient to produce detectable self-inhibition of NMDARs even at a physiological Mg2+ concentration. Therefore, the contribution of NMDARs to synaptic excitation is strongly controlled by both previous synaptic activity as well as by the Ca2+ buffer capacity of postsynaptic neurons

Transient microstructural brain anomalies and epileptiform discharges in mice defective for epilepsy and language-related NMDA receptor subunit gene Grin2a

Wiley Periodicals, Inc. © 2018 International League Against Epilepsy Objective: The epilepsy-aphasia spectrum (EAS) is a heterogeneous group of age-dependent childhood disorders characterized by sleep-activated discharges associated with infrequent seizures and language, cognitive, and behavioral deficits. Defects in the GRIN2A gene, encoding a subunit of glutamate-gated N-methyl-d-aspartate (NMDA) receptors, represent the most important cause of EAS identified so far. Neocortical or thalamic lesions were detected in a subset of severe EAS disorders, and more subtle anomalies were reported in patients with so-called “benign” phenotypes. However, whether brain structural alterations exist in the context of GRIN2A defects is unknown. Methods: Magnetic resonance diffusion tensor imaging (MR-DTI) was used to perform longitudinal analysis of the brain at 3 developmental timepoints in living mice genetically knocked out (KO) for Grin2a. In addition, electroencephalography (EEG) was recorded using multisite extracellular electrodes to characterize the neocortical activity in vivo. Results: Microstructural alterations were detected in the neocortex, the corpus callosum, the hippocampus, and the thalamus of Grin2a KO mice. Most MR-DTI alterations were detected at a specific developmental stage when mice were aged 30 days, but not at earlier (15 days) or later (2 months) ages. EEG analysis detected epileptiform discharges in Grin2a KO mice in the third postnatal week. Significance: Grin2a KO mice replicated several anomalies found in patients with EAS disorders. Transient structural alterations detected by MR-DTI recalled the age-dependent course of EAS disorders, which in humans start during childhood and show variable outcome at the onset of adolescence. Together with the epileptiform discharges detected in young Grin2a KO mice, our data suggested the existence of early anomalies in the maturation of the neocortical and thalamocortical systems. Whereas the possible relationship of those anomalies with sleep warrants further investigations, our data suggest that Grin2a KO mice may serve as an animal model to study the neuronal mechanisms of EAS disorders and to design new therapeutic strategies

The relative contribution of nmdars to excitatory postsynaptic currents is controlled by ca²⁺-induced inactivation

© 2016 Valiullina, Zakharova, Mukhtarov, Draguhn, Burnashev and Rozov.NMDA receptors (NMDARs) are important mediators of excitatory synaptic transmission and plasticity. A hallmark of these channels is their high permeability to Ca2+. At the same time, they are themselves inhibited by the elevation of intracellular Ca2+ concentration. It is unclear however, whether the Ca2+ entry associated with single NMDAR mediated synaptic events is sufficient to self-inhibit their activation. Such auto-regulation would have important effects on the dynamics of synaptic excitation in several central neuronal networks. Therefore, we studied NMDAR-mediated synaptic currents in mouse hippocampal CA1 pyramidal neurons. Postsynaptic responses to subthreshold Schaffer collateral stimulation depended strongly on the absence or presence of intracellular Ca2+ buffers. Loading of pyramidal cells with exogenous Ca2+ buffers increased the amplitude and decay time of NMDAR mediated EPSCs (EPSPs) and prolonged the time window for action potential (AP) generation. Our data indicate that the Ca2+ influx mediated by unitary synaptic events is sufficient to produce detectable self-inhibition of NMDARs even at a physiological Mg2+ concentration. Therefore, the contribution of NMDARs to synaptic excitation is strongly controlled by both previous synaptic activity as well as by the Ca2+ buffer capacity of postsynaptic neurons

Formation of a disk-structure and jets in the symbiotic prototype Z And during its 2006-2010 active phase

We present an analysis of spectrophotometric observations of the latest cycle of activity of the symbiotic binary Z And from 2006 to 2010. We estimate the temperature of the hot component of Z And to be \approx 150000 - 170000 K at minimum brightness, decreasing to \approx 90000 K at the brightness maximum. Our estimate of the electron density in the gaseous nebula is N_{e}=10^{10}-10^{12} cm^{-3} in the region of formation of lines of neutral helium and 10^6-10^7 cm^{-3} in the region of formation of the [OIII] and [NeIII] nebular lines. A trend for the gas density derived from helium lines to increase and the gas density derived from [OIII] and [NeIII] lines to simultaneously decrease with increasing brightness of the system was observed. Our estimates show that the ratios of the theoretical and observed fluxes in the [OIII] and [NeIII] lines agree best when the O/Ne ratio is similar to its value for planetary nebulae. The model spectral energy distribution showed that, in addition to a cool component and gaseous nebula, a relatively cool pseudophotosphere (5250-11 500 K) is present in the system. The simultaneous presence of a relatively cool pseudophotosphere and high-ionization spectral lines is probably related to a disk-like structure of the pseudophotosphere. The pseudophotosphere formed very rapidly, over several weeks, during a period of increasing brightness of Z And. We infer that in 2009, as in 2006, the activity of the system was accompanied by a collimated bipolar ejection of matter. In contrast to the situation in 2006, the jets were detected even before the system reached its maximum brightness. Moreover, components with velocities close to 1200 km/s disappeared at the maximum, while those with velocities close to 1800 km/s appeared.Comment: 18 pages, 19 figures, Accepted for publication in Astronomy Report

Quantitative spectroscopy of Deneb

Quantitative spectroscopy of luminous BA-type supergiants offers a high potential for modern astrophysics. The degree to which we can rely on quantitative studies of this class of stars as a whole depends on the quality of the analyses for benchmark objects. We constrain the basic atmospheric parameters and fundamental stellar parameters as well as chemical abundances of the prototype A-type supergiant Deneb to unprecedented accuracy (Teff = 8525 +/- 75 K, log(g) = 1.10 +/- 0.05 dex, M_spec = 19 +/- 3 M_sun, L = 1.96 +/- 0.32 *10^5 L_sun, R = 203 +/- 17 R_sun, enrichment with CN-processed matter) by applying a sophisticated hybrid NLTE spectrum synthesis technique which has recently been developed and tested. The study is based on a high-resolution and high-S/N spectrum obtained with the Echelle spectrograph FOCES on the Calar Alto 2.2m telescope. Practically all inconsistencies reported in earlier studies are resolved. Multiple metal ionization equilibria and numerous hydrogen lines from the Balmer, Paschen, Brackett and Pfund series are brought into match simultaneously for the stellar parameter determination. Stellar wind properties are derived from H_alpha line-profile fitting using line-blanketed hydrodynamic non-LTE models. A self-consistent view of Deneb is thus obtained, allowing us to discuss its evolutionary state in detail by comparison with the most recent generation of evolution models for massive stars. (abridged)Comment: 17 pages, 12 figures. Accepted for publication in A&