In this work we report on the implementation of methods for data processing signals from microelectrode arrays (MEA) and the application of these methods for signals originated from two types of MEAs to detect putative neurons and sort them into subpopulations. We recorded electrical signals from firing neurons using titanium nitride (TiN) and boron doped diamond (BDD) MEAs. In previous research, we have shown that these methods have the capacity to detect neurons using commercially-available TiN-MEAs. We have managed to cultivate and record hippocampal neurons for the first time using a newly developed custom-made multichannel BDD-MEA with 20 recording sites. We have analysed the signals with the algorithms developed and employed them to inspect firing bursts and enable spike sorting. We did not observe any significant difference between BDD- and TiN-MEAs over the parameters, which estimated spike shape variability per each detected neuron. This result supports the hypothesis that we have detected real neurons, rather than noise, in the BDD-MEA signal. BDD materials with suitable mechanical, electrical and biocompatibility properties have a large potential in novel therapies for treatments of neural pathologies, such as deep brain stimulation in Parkinson's disease.

• MeSH
• akční potenciály MeSH
• algoritmy MeSH
• bor chemie   MeSH
• diamant chemie   MeSH
• hipokampus fyziologie   MeSH
• krysa rodu Rattus MeSH
• mikroelektrody MeSH
• neurony fyziologie   MeSH
• potkani Wistar MeSH
• titan chemie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bor MeSH
• diamant MeSH
• titan MeSH
• titanium nitride MeSH Prohlížeč

Gain-of-function (GOF) mutations in ion channels are rare events, which lead to increased agonist sensitivity or altered gating properties, and may render the channel constitutively active. Uncovering and following characterization of such mutants contribute substantially to the understanding of the molecular basis of ion channel functioning. Here we give an overview of some GOF mutants in polymodal ion channels specifically involved in transduction of painful stimuli--TRPV1 and TRPA1, which are scrutinized by scientists due to their important role in development of some pathological pain states. Remarkably, a substitution of single amino acid in the S4-S5 region of TRPA1 (N855S) has been recently associated with familial episodic pain syndrome. This mutation increases chemical sensitivity of TRPA1, but leaves the voltage sensitivity unchanged. On the other hand, mutations in the analogous region of TRPV1 (R557K and G563S) severely affect all aspects of channel activation and lead to spontaneous activity. Comparison of the effects induced by mutations in homologous positions in different TRP receptors (or more generally in other distantly related ion channels) may elucidate the gating mechanisms conserved during evolution.

• MeSH
• bolest patofyziologie   MeSH
• kationtové kanály TRP chemie   genetika   metabolismus   MeSH
• kationtové kanály TRPV chemie   genetika   metabolismus   MeSH
• kationtový kanál TRPA1 MeSH
• lidé MeSH
• mícha patofyziologie   MeSH
• mutace genetika   MeSH
• percepce bolesti MeSH
• proteiny nervové tkáně chemie   genetika   metabolismus   MeSH
• vápníkové kanály chemie   genetika   metabolismus   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• kationtové kanály TRP MeSH
• kationtové kanály TRPV MeSH
• kationtový kanál TRPA1 MeSH
• proteiny nervové tkáně MeSH
• TRPA1 protein, human MeSH Prohlížeč
• TRPV1 protein, human MeSH Prohlížeč
• vápníkové kanály MeSH

NMDA receptors have received much attention over the last few decades, due to their role in many types of neural plasticity on the one hand, and their involvement in excitotoxicity on the other hand. There is great interest in developing clinically relevant NMDA receptor antagonists that would block excitotoxic NMDA receptor activation, without interfering with NMDA receptor function needed for normal synaptic transmission and plasticity. This review summarizes current understanding of the structure of NMDA receptors and the mechanisms of NMDA receptor activation and modulation, with special attention given to data describing the properties of various types of NMDA receptor inhibition. Our recent analyses point to certain neurosteroids as NMDA receptor inhibitors with desirable properties. Specifically, these compounds show use-dependent but voltage-independent block, that is predicted to preferentially target excessive tonic NMDA receptor activation. Importantly, neurosteroids are also characterized by use-independent unblock, compatible with minimal disruption of normal synaptic transmission. Thus, neurosteroids are a promising class of NMDA receptor modulators that may lead to the development of neuroprotective drugs with optimal therapeutic profiles.

• MeSH
• gating iontového kanálu účinky léků   MeSH
• konformace proteinů MeSH
• lidé MeSH
• mozek účinky léků   metabolismus   MeSH
• nemoci mozku farmakoterapie   metabolismus   MeSH
• nervový přenos účinky léků   MeSH
• neurony účinky léků   metabolismus   MeSH
• neuroprotektivní látky terapeutické užití   MeSH
• receptory N-methyl-D-aspartátu chemie   účinky léků   metabolismus   ultrastruktura   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• neuroprotektivní látky MeSH
• receptory N-methyl-D-aspartátu MeSH

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.

• MeSH
• antagonisté excitačních aminokyselin farmakologie   MeSH
• buněčné linie MeSH
• excitační postsynaptické potenciály účinky léků   fyziologie   MeSH
• hipokampus účinky léků   fyziologie   MeSH
• kinetika MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• kyselina glutamová metabolismus   MeSH
• lidé MeSH
• mozková kůra účinky léků   fyziologie   MeSH
• nervový přenos účinky léků   fyziologie   MeSH
• piperidiny farmakologie   MeSH
• potkani Wistar MeSH
• pyramidové buňky účinky léků   fyziologie   MeSH
• receptory N-methyl-D-aspartátu metabolismus   MeSH
• synapse účinky léků   fyziologie   MeSH
• techniky in vitro MeSH
• teplota * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antagonisté excitačních aminokyselin MeSH
• ifenprodil MeSH Prohlížeč
• kyselina glutamová MeSH
• piperidiny MeSH
• receptory N-methyl-D-aspartátu 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
• 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 účinky léků   genetika   fyziologie   MeSH
• rekombinantní proteiny MeSH
• statistické modely MeSH
• techniky in vitro MeSH
• teplota MeSH
• transfekce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kyselina glutamová MeSH
• NR1 NMDA receptor MeSH Prohlížeč
• NR2B NMDA receptor MeSH Prohlížeč
• receptory N-methyl-D-aspartátu MeSH
• rekombinantní proteiny MeSH

Ligand-gated ionic channels are integral membrane proteins that enable rapid and selective ion fluxes across biological membranes. In excitable cells, their role is crucial for generation and propagation of electrical signals. This survey describes recent results from studies performed in the Department of Cellular Neurophysiology, Institute of Physiology ASCR, aimed at exploring the conformational dynamics of the acetylcholine, glutamate and vanilloid receptors during their activation, inactivation and desensitization. Distinct families of ion channels were selected to illustrate a rich complexity of the functional states and conformational transitions these proteins undergo. Particular attention is focused on structure-function studies and allosteric modulation of their activity. Comprehension of the fundamental principles of mechanisms involved in the operation of ligand-gated ion channels at the cellular and molecular level is an essential prerequisite for gaining an insight into the pathogenesis of many psychiatric and neurological disorders and for efficient development of novel specifically targeted drugs.

• MeSH
• alosterická regulace MeSH
• elektrofyziologie MeSH
• gating iontového kanálu fyziologie   MeSH
• glutamátové receptory metabolismus   MeSH
• iontové kanály fyziologie   MeSH
• lidé MeSH
• ligandy MeSH
• molekulární sekvence - údaje MeSH
• receptory buněčného povrchu metabolismus   MeSH
• receptory cholinergní metabolismus   MeSH
• receptory léků metabolismus   MeSH
• sekvence aminokyselin MeSH
• signální transdukce MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• glutamátové receptory MeSH
• iontové kanály MeSH
• ligandy MeSH
• receptory buněčného povrchu MeSH
• receptory cholinergní MeSH
• receptory léků MeSH

The effects of high temperature (53-61 degrees C) on membrane currents (I(heat)) or depolarization (V(heat)) induced by noxious heat were studied in cultured dorsal root ganglia neurons from neonatal rats using the whole cell patch clamp technique. I(heat) or V(heat) produced by 3 s ramps of increasing temperature between 43 and 50 degrees C exhibited a fast slope (Q10>10) that was similar both during rising and falling temperature (n=85). Temperatures exceeding 52 degrees C resulted in slowdown in the recovery of I(heat), and the threshold for inducing I(heat) was shifted to lower temperatures in successive trials. These high temperatures (54-60 degrees C) caused a linear and incomplete recovery of I(heat) (Q10 decreased to <5; 4.5 +/- 0.4; n=17) and in successive trials the threshold of I(heat) decreased to temperatures close to that in the bath. The neurons, however, remained sensitive to capsaicin and to decreased extracellular pH. It is suggested that exposure of nociceptive neurons to excessive noxious heat results in an irreversible decrease of the energy barrier between the resting and activated state of the protein structures responsible for generation of I(heat). This may explain the sensitization of nociceptors after heat injury.

• MeSH
• cholin farmakologie   MeSH
• kapsaicin farmakologie   MeSH
• kinetika MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• membránové potenciály účinky léků   fyziologie   MeSH
• metoda terčíkového zámku MeSH
• neurony aferentní cytologie   fyziologie   MeSH
• nociceptory účinky léků   fyziologie   MeSH
• nootropní látky farmakologie   MeSH
• novorozená zvířata MeSH
• potkani Wistar MeSH
• spinální ganglia cytologie   MeSH
• vápník farmakologie   MeSH
• vysoká teplota MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cholin MeSH
• kapsaicin MeSH
• nootropní látky MeSH
• vápník MeSH

1. Membrane currents induced by noxious heat (Iheat) were studied in cultured dorsal root ganglion (DRG) neurones from newborn rats using ramps of increasing temperature of superfusing solutions. 2. Iheat was observed in about 70 % of small (< 25 microm) DRG neurones. At -60 mV, Iheat exhibited a threshold at about 43 C and reached its maximum, sometimes exceeding 1 nA, at 52 C (716 +/- 121 pA; n = 39). 3. Iheat exhibited a strong temperature sensitivity (temperature coefficient over a 10 C temperature range (Q10) = 17.8 +/- 2.1, mean +/- s.d., in the range 47-51 C; n = 41), distinguishing it from the currents induced by capsaicin (1 microM), bradykinin (5 microM) and weak acid (pH 6.1 or 6.3), which exhibited Q10 values of 1.6-2.8 over the whole temperature range (23-52 C). Repeated heat ramps resulted in a decrease of the maximum Iheat and the current was evoked at lower temperatures. 4. A single ramp exceeding 57 C resulted in an irreversible change in Iheat. In a subsequent trial, maximum Iheat was decreased to less than 50 %, its threshold was lowered to a temperature just above that in the bath and its maximum Q10 was markedly lower (5.6 +/- 0.8; n = 8). 5. DRG neurones that exhibited Iheat were sensitive to capsaicin. However, four capsaicin-sensitive neurones out of 41 were insensitive to noxious heat. There was no correlation between the amplitude of capsaicin-induced responses and Iheat. 6. In the absence of extracellular Ca2+, Q10 for Iheat was lowered from 25.3 +/- 7.5 to 4. 2 +/- 0.4 (n = 7) in the range 41-50 C. The tachyphylaxis, however, was still observed. 7. A high Q10 of Iheat suggests a profound, rapid and reversible change in a protein structure in the plasma membrane of heat-sensitive nociceptors. It is hypothesized that this protein complex possesses a high net free energy of stabilization (possibly due to ionic bonds) and undergoes disassembly when exposed to noxious heat. The liberated components activate distinct cationic channels to generate Iheat. Their affinity to form the complex at low temperatures irreversibly decreases after one exposure to excessive heat.

• MeSH
• bradykinin farmakologie   MeSH
• iontové kanály metabolismus   MeSH
• kapsaicin farmakologie   MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• membránové potenciály účinky léků   MeSH
• neurony aferentní účinky léků   metabolismus   MeSH
• receptory léků metabolismus   MeSH
• spinální ganglia cytologie   účinky léků   metabolismus   MeSH
• tachyfylaxe fyziologie   MeSH
• teplota MeSH
• vápník metabolismus   MeSH
• vysoká teplota * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• bradykinin MeSH
• iontové kanály MeSH
• kapsaicin MeSH
• receptory léků MeSH
• vápník MeSH

A technique is described that allows the application of fast temperature changes (time constant approximately 300 ms) of solutions superfusing cultured neurones under whole-cell mode of membrane current recording. Its principle is in heating the common outlet of the manifold which consists of 12 tubes connected to barrels containing test solutions of different composition. The outlet is made from a glass capillary (25 mm length, 620/350 microns outer/inner diameter) coated on the outside wall with platinum for a length of 12 mm. The heating element, a platinum layer, is electrically connected to the probe fixed to the micromanipulator used for positioning the manifold. The solutions, driven by gravity, are applied by opening electronic valves controlled either manually or in programmed sequences. The DC current for heating is controlled either manually or by external voltage command. The advantage of the technique is that the same temperature pattern can be applied to 12 different solutions. The technique is used for classifying sensory neurones in culture with respect to their sensitivity to heat and algogens; however, it is applicable to any study of the effects of increased temperature on the activity of ion channels in cultured cells.

• MeSH
• buněčná membrána fyziologie   MeSH
• cytologické techniky * MeSH
• kultivační média MeSH
• kultivované buňky MeSH
• lékové roztoky MeSH
• neurony účinky léků   fyziologie   MeSH
• vysoká teplota MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• kultivační média MeSH
• lékové roztoky MeSH

• MeSH
• elektrofyziologie přístrojové vybavení   metody   MeSH
• hipokampus metabolismus   MeSH
• ionty * MeSH
• mikroelektrody * MeSH
• morčata MeSH
• počítačové zpracování signálu * MeSH
• zvířata MeSH
• Check Tag
• morčata MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ionty * MeSH