N-methyl-d-aspartate (NMDA) receptors (NMDARs) are highly expressed in the CNS and mediate the slow component of excitatory transmission. The present study was aimed at characterizing the temperature dependence of the kinetic properties of native NMDARs, with special emphasis on the deactivation of synaptic NMDARs. We used patch-clamp recordings to study synaptic NMDARs at layer II/III pyramidal neurons of the rat cortex, recombinant GluN1/GluN2B receptors expressed in human embryonic kidney (HEK293) cells, and NMDARs in cultured hippocampal neurons. We found that time constants characterizing the deactivation of NMDAR-mediated excitatory postsynaptic currents (EPSCs) were similar to those of the deactivation of responses to a brief application of glutamate recorded under conditions of low NMDAR desensitization (whole-cell recording from cultured hippocampal neurons). In contrast, the deactivation of NMDAR-mediated responses exhibiting a high degree of desensitization (outside-out recording) was substantially faster than that of synaptic NMDA receptors. The time constants characterizing the deactivation of synaptic NMDARs and native NMDARs activated by exogenous glutamate application were only weakly temperature sensitive (Q(10)=1.7-2.2), in contrast to those of recombinant GluN1/GluN2B receptors, which are highly temperature sensitive (Q(10)=2.7-3.7). Ifenprodil reduced the amplitude of NMDAR-mediated EPSCs by approximately 50% but had no effect on the time course of deactivation. Analysis of GluN1/GluN2B responses indicated that the double exponential time course of deactivation reflects mainly agonist dissociation and receptor desensitization. We conclude that the temperature dependences of native and recombinant NMDAR are different; in addition, we contribute to a better understanding of the molecular mechanism that controls the time course of NMDAR-mediated EPSCs. Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

• MeSH
• antagonisté excitačních aminokyselin farmakologie   MeSH
• buněčné linie MeSH
• excitační postsynaptické potenciály fyziologie   účinky léků   MeSH
• hipokampus fyziologie   účinky léků   MeSH
• kinetika MeSH
• krysa rodu rattus MeSH
• kultivované buňky MeSH
• kyselina glutamová metabolismus   MeSH
• lidé MeSH
• mozková kůra fyziologie   účinky léků   MeSH
• nervový přenos fyziologie   účinky léků   MeSH
• piperidiny farmakologie   MeSH
• potkani Wistar MeSH
• pyramidové buňky fyziologie   účinky léků   MeSH
• receptory N-methyl-D-aspartátu metabolismus   MeSH
• synapse fyziologie   účinky léků   MeSH
• teplota MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH
• techniky in vitro MeSH

Pregnenolone sulfate (PS), an endogenously occurring neurosteroid, has been shown to modulate the activity of several neurotransmitter-gated channels, including the N-methyl-D-aspartate receptor (NMDAR). NMDARs are glutamate-gated ion channels involved in excitatory synaptic transmission, synaptic plasticity, and excitotoxicity. To determine the mechanism that controls PS sensitivity of NMDARs, we measured NMDAR responses induced by exogenous agonist application in voltage-clamped HEK293 cells expressing NR1/NR2B NMDARs and cultured rat hippocampal neurons. We report that PS potentiates the amplitude of whole-cell recorded NMDAR responses in cultured hippocampal neurons and HEK293 cells; however, the potentiating effect of PS on NMDAR in outside-out patches isolated from cultured hippocampal neurons and HEK293 cells was lost within 2 min after patch isolation in a neurosteroid-specific manner. The rate of diminution of the PS potentiating effect was slowed by protein phosphatase inhibitors. Treatment of cultured hippocampal neurons with a nonspecific protein kinase inhibitor and a specific protein kinase A (PKA) inhibitor diminished PS-induced potentiation, which was recovered by adding a PKA, but not a protein kinase C (PKC), activator. These results suggest that the effect of PS on NMDARs is controlled by cellular mechanisms that are mediated by dephosphorylation/phosphorylation pathways.

• MeSH
• buněčné linie MeSH
• fosforylace MeSH
• hipokampus fyziologie   MeSH
• krysa rodu rattus MeSH
• kultivované buňky MeSH
• lidé MeSH
• membránové potenciály MeSH
• metoda terčíkového zámku MeSH
• neurony fyziologie   MeSH
• pregnenolon metabolismus   MeSH
• proteinfosfatasy antagonisté a inhibitory   MeSH
• proteinkinasa C metabolismus   MeSH
• proteinkinasy závislé na cyklickém AMP metabolismus   MeSH
• receptory N-methyl-D-aspartátu agonisté   metabolismus   MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

Ionotropic glutamate receptors function can be affected by neurosteroids, both positively and negatively. N-methyl-D-aspartate (NMDA) receptor responses to exogenously applied glutamate are potentiated or inhibited (depending on the receptor subunit composition) by pregnenolone sulphate (PS) and inhibited by pregnanolone sulphate (3alpha5betaS). While PS effect is most pronounced when its application precedes that of glutamate, 3alpha5betaS only binds to receptors already activated. Synaptically activated NMDA receptors are inhibited by 3alpha5betaS, though to a lesser extent than those tonically activated by exogenous glutamate. PS, on the other hand, shows virtually no effect on any of the models of synaptically activated NMDA receptors. The site of neurosteroid action at the receptor molecule has not yet been identified, however, the experiments indicate that there are at least two distinct extracellularly located binding sites for PS mediating its potentiating and inhibitory effects respectively. Experiments with chimeric receptors revealed the importance of the extracellular loop connecting the third and the fourth transmembrane domain of the receptor NR2 subunit for the neurosteroid action. alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptors are inhibited by both PS and 3alpha5betaS. These neurosteroids also affect AMPA receptors-mediated synaptic transmission, however, in a rather indirect way, through presynaptically located targets of action.

• MeSH
• AMPA receptory metabolismus   MeSH
• excitační postsynaptické potenciály účinky léků   MeSH
• glutamátové receptory metabolismus   MeSH
• lidé MeSH
• mozek metabolismus   MeSH
• nervový přenos účinky léků   MeSH
• pregnenolon chemie   farmakologie   MeSH
• receptory N-methyl-D-aspartátu chemie   metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

• Publikační typ
• abstrakty MeSH

N-methyl-D-aspartate (NMDA) receptors are highly expressed in the CNS, mediate the slow component of excitatory transmission and play key roles in synaptic plasticity and excitotoxicity. These ligand-gated ion channels are heteromultimers composed of NR1 and NR2 subunits activated by glycine and glutamate. In this study, patch-clamp recordings were used to study the temperature sensitivity of recombinant NR1/NR2B receptors expressed in human embryonic kidney (HEK) 293 cells. Rate constants were assessed by fitting a six-state kinetic scheme to time courses of transient macroscopic currents induced by glutamate at 21.9-46.5 degrees C. Arrhenius transformation of the rate constants characterizing NMDA receptor channel activity indicates that the most sensitive were the rate constants of desensitization (temperature coefficient Q(10)=10.3), resensitization (Q(10)=4.6) and unbinding (Q(10)=3.6). Other rate constants and the amplitude of single-channel currents were less temperature sensitive. Deactivation of responses mediated by NR1/NR2B receptors after a brief application of glutamate was best fit by a double exponential function (tau(fast): Q(10)=3.7; tau(slow): Q(10)=2.7). From these data, we conclude that desensitization/resensitization of the NMDA receptor and glutamate unbinding are especially temperature sensitive and imply that at physiological temperatures the channel kinetics play an important role in determining amplitude and time course of NMDA receptor-mediated postsynaptic currents and these receptors mediated synaptic plasticity.

• MeSH
• algoritmy MeSH
• buněčné linie MeSH
• elektrofyziologie MeSH
• financování organizované MeSH
• gating iontového kanálu fyziologie   MeSH
• kinetika MeSH
• kyselina glutamová metabolismus   MeSH
• ledviny cytologie   metabolismus   MeSH
• lidé MeSH
• metoda terčíkového zámku MeSH
• receptory N-methyl-D-aspartátu fyziologie   genetika   účinky léků   MeSH
• rekombinantní proteiny MeSH
• statistické modely MeSH
• teplota MeSH
• transfekce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• techniky in vitro MeSH

• MeSH
• finanční podpora výzkumu jako téma MeSH
• Publikační typ
• abstrakty MeSH

It has been suggested that in mammals, trigeminal lamina I neurons play a role in the processing and transmission of sensory information from the orofacial region. We investigated the physiological and morphological properties of trigeminal subnucleus caudalis (Sp5C) lamina I neurons in slices prepared from the medulla oblongata of 13- to 15-day-old postnatal rats using patch-clamp recordings and subsequent biocytin-streptavidin-Alexa labeling. Twenty-five neurons were recorded and immunohistochemically stained. The Sp5C lamina I consisted of several types of neurons which, on the basis of their responses to somatic current injection, can be classified into four groups: tonic neurons, which fired throughout the depolarizing pulse; phasic neurons, which expressed an initial burst of action potentials; delayed onset neurons, which showed a significant delay of the first action potential; and single spike neurons, characterized by only one to five action potentials at the very beginning of the depolarizing pulse even at high levels of stimulation intensity. Electrical stimulation of the spinal trigeminal tract evoked AMPA receptor-mediated excitatory postsynaptic currents (EPSC) exhibiting a strong polysynaptic component. AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSC) were characterized by a 10-90% rise time of 0.50+/-0.06 ms and a decay time constant of 2.5+/-0.5 ms. The kinetic properties of NMDA receptor-mediated EPSCs were measured at +40 mV. The 10-90% rise time was 8+/-2 ms and the deactivation time constants were 94+/-31 and 339+/-72 ms, respectively. Intracellular staining and morphological analysis revealed three groups of neurons: fusiform, pyramidal, and multipolar. Statistical analysis indicated that the electrophysiological properties and morphological characteristics are correlated. Tonic and phasic neurons were fusiform or pyramidal and delayed onset and single spike neurons were multipolar. Our results show that both the physiological and morphological properties of Sp5C lamina I neurons exhibit significant differences, indicating their specific integration in the processing and transmission of sensory information from the orofacial region.

• MeSH
• algoritmy MeSH
• AMPA receptory fyziologie   MeSH
• elektrofyziologie MeSH
• excitační postsynaptické potenciály fyziologie   MeSH
• financování organizované MeSH
• imunohistochemie MeSH
• interpretace statistických dat MeSH
• krysa rodu rattus MeSH
• membránové potenciály fyziologie   MeSH
• metoda terčíkového zámku MeSH
• nuclei trigemini anatomie a histologie   fyziologie   MeSH
• polarita buněk fyziologie   MeSH
• potkani Wistar MeSH
• pyramidové buňky fyziologie   MeSH
• receptory N-methyl-D-aspartátu fyziologie   MeSH
• synapse fyziologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH

NMDA receptors are ligand-gated ion channels permeable to calcium and play a critical role in excitatory synaptic transmission, synaptic plasticity, and excitotoxicity. They are heteromeric complexes of NR1 combined with NR2A-D and/or NR3A-B subunits that are activated by glutamate and glycine and whose activity is modulated by allosteric modulators. In this study, patch-clamp recordings from human embryonic kidney 293 cells expressing NR1/NR2 receptors were used to study the molecular mechanism of the endogenous neurosteroid 20-oxo-5beta-pregnan-3alpha-yl sulfate (3alpha5betaS) action at NMDA receptors. 3alpha5betaS was a twofold more potent inhibitor of responses mediated by NR1/NR2C-D receptors than those mediated by NR1/NR2A-B receptors. The structure of the extracellular loop between the third and fourth transmembrane domains of the NR2 subunit was found to be critical for the neurosteroid inhibitory effect. The degree of 3alpha5betaS-induced inhibition of responses to glutamate was voltage independent, with recovery lasting several seconds. In contrast, application of 3alpha5betaS in the absence of agonist had no effect on the subsequent response to glutamate made in the absence of the neurosteroid. A kinetic model was developed to explain the use-dependent action of 3alpha5betaS at NMDA receptors. In accordance with the model, 3alpha5betaS was a less potent inhibitor of NMDA receptor-mediated EPSCs and responses induced by a short application of 1 mm glutamate than of those induced by a long application of glutamate. These results suggest that 3alpha5betaS is a use-dependent but voltage-independent inhibitor of NMDA receptors, with more potent action at tonically than at phasically activated receptors. This may be important in the treatment of excitotoxicity-induced neurodegeneration.

• MeSH
• buněčné linie MeSH
• financování organizované MeSH
• hipokampus cytologie   fyziologie   MeSH
• krysa rodu rattus MeSH
• kultivované buňky MeSH
• lidé MeSH
• membránové potenciály fyziologie   účinky léků   MeSH
• metoda terčíkového zámku MeSH
• modely neurologické MeSH
• neokortex fyziologie   MeSH
• novorozená zvířata MeSH
• pregnany farmakologie   MeSH
• receptory N-methyl-D-aspartátu antagonisté a inhibitory   MeSH
• transfekce MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• zvířata MeSH

Ligandem aktivované iontové kanály tvoří skupinu transmembránových proteinových struktur, které hrají klíčovou úlohu při vzniku a přenosu elektrických signálů. Pro zjištění mechanizmů, které se podílejí na vazbě ligandů, změnách pH a vysoké teploty vedoucích ke strukturálním změnám iontových kanálů, bylo v nedávné době využito nových technik. Cílem tohoto přehledu je sumarizovat komplexní mechanizmy podílející se na aktivaci glutamátových (NMDA, AMPA, kainátových) a vaniloidních (TRPV1) receptorů spojených s příslušnými iontovými kanály a rovněž shrnout výsledky studií provedených v nedávné době v Oddělení buněčné neurofyziologie Fyziologického ústavu AV ČR (http://www2.biomed.cas.cz /d331/index.html), které je součástí Centra neuropsychiatrických studií; http://www.pcp.lf3.cuni.cz/cns/.

Ligand-gated ion channels form a family of transmembrane proteins that play key role in generation and propagation of electrical signals. Recently, new techniques have become available for identification of the mechanisms involved in ligand binding, pH changes and elevated temperatures into structural rearrangements leading to channel gating. The aim of this article is to review the complexity of the gating mechanisms involved in the activation of glutamate (NMDA, AMPA, kainate) and vanilloid (TRPV1) receptor channels and summarize the recent results from studies performed in the Department of Cellular Neurophysiology of the Institute of Physiology AS CR (http://www2.biomed.cas.cz/d331/index.html) a member of the Centre of Neuropsychiatric Studies http://www.pcp.lf3.cuni.cz/cns/.

• MeSH
• finanční podpora výzkumu jako téma MeSH
• iontové kanály analýza   antagonisté a inhibitory   fyziologie   MeSH
• membránové proteiny fyziologie   MeSH
• neurofyziologie MeSH
• neurony fyziologie   MeSH
• receptory N-methyl-D-aspartátu fyziologie   MeSH
• synapse fyziologie   MeSH
• teplota MeSH
• Publikační typ
• přehledy MeSH

N-metyl-D-aspartátové (NMDA) receptory patří do skupiny ionotropních glutamátových receptorů, které společně s dalším podtypem glutamátových receptorů - AMPA/kainátovým receptorem zprostředkovávají rychlý synaptický přenos na většině excitačních synapsí v centrální nervové soustavě. NMDA receptory hrají zásadní úlohu pro zajištění komplexních funkcí nervové soustavy jako celku. Jejich aktivace je nutná pro navození dlouhodobé potenciace synaptického přenosu (LTP), o níž se předpokládá, že představuje buněčný mechanizmus, který se ve specifických oblastech nervové soustavy uplatňuje na vytváření paměťových stop. Nadměrná aktivace NMDA receptorů vede k patologickým procesům, které mohou vyústit až ve specifickou formu smrti neuronů (excitotoxicita). Existují experimentální důkazy o tom, že se excitotoxicita podílí na vzniku celé řady neurodegenerativních onemocnění a že antagonisté NMDA receptorů mohou představovat terapeutický potenciál pro léčbu onemocnění vykazující jak akutní, tak chronické formy neurodegenerace včetně Alzheimerovy demence. Memantin je látkou, která akompetitivním a napěťově závislým způsobem blokuje iontové kanály NMDA receptorů a existují důkazy o tom, že zlepšuje kognitivní funkce a zpomaluje jejich zhoršování u Alzheimerovy demence. Cílem tohoto přehledu je shrnout současné poznatky o mechanizmech působení memantinu na NMDA receptorech a představy o jeho možném využití při léčbě některých klinických syndromů, s nimiž se setkáváme u neurodegenerativních chorob, především Alzheimerovy demence.

N-methyl-D-aspartate (NMDA) receptors belong to the group of ionotropic glutamate receptors and together with AMPA/kainate receptors mediate fast excitatory synaptic transmission in the brain and spinal cord and play a key role in maintaining complex functions of the central nervous system. Their activation is an important step in the induction of long term changes in the efficacy of synaptic transmission (LTP) that is considered to represent the cellular mechanism underlying memory acquisition in specific brain regions. Excessive activation of NMDA receptors results in pathological states that may lead to a specific form of neuronal cell death (excitotoxicity). Experimental evidence suggests that excitotoxicity participates in the pathogenesis of many neurodegenerative disorders of the central nervous system. Therefore, NMDA receptor antagonists may act as potential therapeutic agents in both, the acute and chronic forms of neurodegeneration. Memantine is an uncompetitive and voltage-dependent blocker of NMDA receptor channels and was shown beneficial in improving cognition and slowing the progression of Alzheimer-s disease. The aim of this review is to summarize the present knowledge on the mechanisms involved in the effects of memantine on NMDA receptors and the ideas on its use as a potential remedy in the treatment of clinical symptoms that accompany Alzheimer-s dementia.

• MeSH
• Alzheimerova nemoc farmakoterapie   MeSH
• finanční podpora výzkumu jako téma MeSH
• glutamátové receptory účinky léků   MeSH
• lidé MeSH
• memantin aplikace a dávkování   farmakologie   terapeutické užití   MeSH
• neurodegenerativní nemoci farmakoterapie   MeSH
• receptory N-methyl-D-aspartátu antagonisté a inhibitory   fyziologie   terapeutické užití   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• přehledy MeSH