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1

Wplyw podloza na stan zdrowotny konczyn prosiat ssacych

100%
Pasturczak E., Szczot M., Winnicki S.
Przegląd Hodowlany
|
1992
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tom 60
|
nr 11
26-28
2

Culture conditions severely alter the proportions between GABA-and glutamate-evoked currents in hippocampal pyramidal neurons

100%
Szczot M., Wojtowicz T., Mozrzymas J.
Acta Neurobiologiae Experimentalis
|
2009
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tom 69
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nr 3
Cultured hippocampal cells are commonly used as a convenient model but non-physiological conditions (impaired homeostasis, lack of glial cells etc.) persisting over long period of time raise concerns. Acute brain slices are believed to better maintain the physiological features but are often problematic for technical reasons (e.g. complicated access to neurons, attenuated spread of pharmaceutics). Non-physiological conditions in cell cultures during maturation of GABAergic and glutamatergic systems may alter expression of GABAA and glutamate receptors affecting thus the excitation to inhibition balance. Proportion between GABA and glutamate receptors may be evaluated by determining ratio between amplitudes of current responses to saturating agonist concentrations. We have recorded current responses to ultrafast applications of saturating GABA and glutamate concentrations in hippocampal cell cultures (9–15DIV) and in brain slices from CA1 pyramidal neurons of P19–P23 rats. For cell cultures, GABAergic and glutamergic currents ratio was 2.34 ± 0.45 (n=12) while for the brain slices it was only 0.24 ± 0.02 (n=5). These results provide evidence that non-physiological conditions in cell cultures may cause a dramatic change in expression pattern of GABAA and glutamate receptors. The underlying mechanisms are not known, but we may hypothesize that a homeostatic modulation due accumulation of neurotransmitters in culture medium could be involved. Support: grant no. 070231/Z/03/Z.
3

Mutation in F64 position of GABAAR alpha subunit affects the receptor gating

100%
Czyzewska M. M., Szczot M., Kisiel M., Mozrzymas J. W.
Acta Neurobiologiae Experimentalis
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2013
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tom 73
|
nr Suppl.1
GABAA receptor (GABAAR) is a pentamer, formed by 2α, 2β and γ subunit. GABA binding site is localized at the interface between α and β subunits. Our aim was to characterize how mutation localized at the binding pocket (α1F64) influences agonist binding and conformational transitions between bound receptor states (gating). We used patchclamp technique with ultrafast perfusion system and HEK 293 cells expressing native or mutated GABAARs. All mutations (α1F64C/L/A) right-shifted the dose-dependent curve and accelerated current deactivation, indicating impairment of binding. Reduction of fast desensitization, which in the case of α1F64C was complete, indicates changes in gating. Moreover, the mutation decreased the maximum open channel probability, a key feature of receptor gating. Experiments performed with different agonists confirmed mutation-induced changes in the channel’s opening/closing transitions (gating). Quantitative analysis based on model simulations indicated that this mutation mostly affected the channel state which precedes opening and is interpreted as a macromolecule destabilization (“priming” or “flipping”) following agonist binding, whereas desensitization or efficacy are affected to a smaller extent. Our data thus suggest that mutation of α1F64 residue affects the “transition wave” from binding sites to the channel gate.
4
Content available remote

GABAergic and glutamatergic currents in hippocampal slices and neuronal cultures show profound differences: a clue to a potent homeostatic modulation

100%
Szczot M., Wojtowicz T., Mozrzymas J. W.
Journal of Physiology and Pharmacology
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2010
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tom 61
|
nr 4
501-506
Acute hippocampal slices and primary neuronal cultures are often used with a tacit assumption that basic characteristics of the two models closely resemble each other. The use of the cell cultures, however, may raise controversies because of non-physiological conditions resulting from e.g. glial cells deficit, random neuronal sprouting, lack of specificity in the synaptic connections, impaired homeostasis, etc. Importantly, alteration in neuronal environment, especially when occurring over a prolonged period of time, may give rise to a profound homeostatic modulation. In the present study we have compared the properties of GABAergic and glutamatergic (non-NMDA) currents in pyramidal neurons from hippocampal slices and neuronal cell culture. We show that, most strikingly, amplitude ratio of currents elicited by ultrafast applications of saturating GABA and glutamate was nearly one order of magnitude larger in cultured neurons than that in slices. Miniature IPSCs and EPSCs also showed substantial differences between these two models. In particular, mEPSC amplitudes were larger and more frequent in cultured neurons but their time duration was longer in slices. Miniature IPSCs did not show differences in amplitude when comparing slices and cultures but their time duration was faster and occurrence more frequent in slices. In conclusion, we provide evidence that expression pattern of GABAA and glutamate receptors as well as synaptic current properties in the neuronal cell culture show profound differences with respect to that in the physiological conditions.
5

Hydrophobic residue in GABAA receptor ligand binding site strongly influences receptor gating

100%
Szczot M., Kisiel M., Czyzewska M. M., Mozrzymas J. W.
Acta Neurobiologiae Experimentalis
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2012
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tom 72
|
nr 2
Despite broad knowledge on GABAA receptors (GABAARs) structure, the mechanism of ligand induced conformational transitions remains poorly understood. To address this issue we have examined the activity of a recombinant α1/β1/γ2 GABAAR with point mutation introduced at the ligand binding site. Currents were elicited by ultrafast GABA applications and measured using patch-clamp technique. We show that cystein mutation of single hydrophobic residue not only weakened the agonist binding but also abolished fast desensitization and slowed the onset of currents evoked by saturating GABA. Non-stationary variance analysis showed that the mutation does not affect single channel conductance, but reduces maximal open probability, further indicating a change in gating properties. Ratio of current amplitudes elicited by pentobarbital (which activates GABAAR by different pathway than GABA), and by GABA was higher for mutant receptors, supporting interference with receptor gating. Our data show that the considered residue may strongly influence conformational transitions of GABAARs thus indicating this residue as a key element in transduction of free energy supplied by agonist binding to the conformational transitions.
6

Alpha1F64 influences GABAAR gating through flipping mechanism

88%
Kisiel M., Szczot M., Czyzewska M. M., Jatczak M., Mozrzymas J. W.
Acta Neurobiologiae Experimentalis
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2014
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tom 74
|
nr 3
GABAARs are crucial for neuronal inhibition. Using patch-clamp technique with ultrafast perfusion we found that mutations of hydrophobic residue at GABA-binding site affected not only binding affinity but also kinetics of macroscopic desensitization. Nonstationary variance analysis indicated that α1F64C mutation reduces maximum open probability. To obtain further information about the role of α1F64 we used two different agonists. Experiments with a partial agonist, P4S, suggested an impact of α1F64C mutation on the channel gating efficacy. Application of muscimol (with higher affinity than GABA) entailed a partial rescue of rapid desensitization in α1F64Lβ1γ2 receptors but in cysteine mutants – did not. Model simulations show that observed effects result from changes in flipping mechanism which links binding and gating. We conclude that α1F64 plays a crucial role in signal transduction from binding site to the channel gate. Supported by NCN Grant 350/B/P01/2011/40 to JWM
7

Mutation at GABA binding site (alpha1F64) affects both binding and gating properties of GABAA receptors

88%
Mozrzymas J. W., Kisiel M., Czyzewska M. M., Jatczak M., Szczot M.
Acta Neurobiologiae Experimentalis
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2014
|
tom 74
|
nr 3
In spite of broad knowledge of GABAAR pharmacokinetics, molecular mechanisms of conformation transitions remain elusive. Intriguingly, GABA binding site is distant from the channel gate (ca. 5 nm). In this study we searched for residues at GABA binding site involved in conveying binding energy to the channel gate of α1β2γ2 GABAARs. Mutation at α1F64 decreased the receptor affinity (shifted dose-response and reduced binding rate in racing protocol) and, in addition, strongly influenced onset, deactivation and desensitization of currents elicited by saturating agonist, indicating gating modification. Non-stationary variance analysis of GABA- or pentobarbital-evoked currents showed that the cysteine mutation strongly decreased the open channel probability confirming its impact on receptor gating. We conclude that α1F64 residue, although located at the binding site, strongly affects gating properties of GABAA receptors. Supported by NCN Grant 350/B/P01/2011/40 to JWM.
8

Absinthe ingredient monoterpenoid alfa-thujone is a modulator of neuronal GABAA receptors

88%
Czyzewska M. M., Szczot M., Pollastro F., Appendino G., Mozrzymas J. W.
Acta Neurobiologiae Experimentalis
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2011
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tom 71
|
nr S
Monoterpenoid α-thujone is a compound found in absinthe, alcoholic beverage commonly abused (often by famous artists) in late XIX and early XX century. It has been long speculated that α-thujone is responsible for some adverse effects of this liquor including seizures. It has been investigated that the effect of α-thujone is related to its action on GABAA receptors but a precise pharmacological analysis is lacking. In the present work we investigated mechanism of α-thujone action on GABAergic currents (current responses to exogenous GABA and miniature synaptic currents) in cultured hippocampal neurons. We found that high concentrations (100 - 300 μM) of α-thujone have only modest effect on amplitude of responses elicited by low (3 μM) [GABA], but it prolonged the current rise-time by nearly fivefold and significantly decreased the current fading during prolonged GABA application. At saturating [GABA] (10 mM), the amplitude of current response, elicited by rapid agonist applications, was significantly reduced by 300 µM α-thujone and current onset was also slowed down almost three times but this effect was markedly smaller than for currents evoked by low [GABA]. To assess the impact of α-thujone on desensitization, the time course of currents elicited by prolonged applications of saturating [GABA] were analyzed. In the presence of α-thujone the steady-state to peak value was markedly increased indicating a decrease in the extent of macroscopic desensitization. To check for the effect of α-thujone on synaptic transmission, we measured miniature inhibitory currents (mIPSC) and found that at 300 μM of α-thujone, amplitude and frequency of mIPSC were significantly reduced. In conclusion, our results suggest that α-thujone may act as a low potency allosteric modulator of GABAARs. Preliminary kinetic analysis suggests that this compound affects both binding and gating of neuronal GABAARs. This study was funded by the FNP award Mistrz (contract No 7/2008).
9
Content available remote

Impact of matrix metalloproteinase-9 overexpression on synaptic excitatory transmission and its plasticity in rat Ca3-Ca1 hippocampal pathway

76%
Wiera G., Szczot M., Wojtowicz T., Lebida K., Koza P., Mozrzymas J. W.
Journal of Physiology and Pharmacology
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2015
|
tom 66
|
nr 2
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