| 1. |
- Alrifaiy, Ahmed, et al.
(författare)
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Hypoxia on a chip - a novel approach for patch-clamp studies in a microfluidic system with full oxygen control
- 2012
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Ingår i: World Congress on Medical Physics and Biomedical Engineering, May 26-31, 2012, Beijing, China. - Berlin : Encyclopedia of Global Archaeology/Springer Verlag. - 9783642293047 - 9783642293054 ; , s. 313-316
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Konferensbidrag (refereegranskat)abstract
- A new approach to perform patch-clamp experiments on living cells under controlled anoxic and normoxic conditions was developed and tested. To provide an optimal control over the oxygen content and the biochemical environment a patch-clamp recording micropipette was integrated within an oxygen tight poly-methyl methacrylate (PMMA) based microchip. The oxygen content within the microfluidic chamber surrounding patch-clamp micropipette was maintained at 0.5-1.5 % by a continuous flow of artificial extracellular solution purged with nitrogen. The nerve and glial cells acutely obtained from the male rat brain were trapped by the optical tweezers and steered towards the patch-clamp micropipette through the channels of the microchip in order to achieve a close contact between the pipette and the cellular membrane. The patch-clamp recordings revealed that optical tweezers did not affect the electrophysiological properties of the tested cells suggesting that optical trapping is a safe and non-traumatizing method to manipulate living cells in the microfluidic system. Thus, our approach of combining optical tweezers and a gas-tight microfluidic chamber may be applied in various electrophysiological investigations of single cells were optimal control of the experimental conditions and the sample in a closed environment are necessary.
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| 2. |
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| 3. |
- Bitaraf, Nazanin, et al.
(författare)
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Development of a multifunctional microfluidic system for studies of nerve cell activity during hypoxic and anoxic conditions
- 2009
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Ingår i: World Congress on Medical Physics and Biomedical Engineering. - Berlin : Springer Science+Business Media B.V.. - 9783642038976 ; , s. 176-179, s. 176-179
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Konferensbidrag (refereegranskat)abstract
- Hemoproteins usually supply cells and tissue with oxygen. A new hemoprotein mainly present in nerve cells called Neuroglobin was recently discovered. Enhanced expression of the protein has been shown to reduce hypoxic neural injury but the mechanism behind this function remains unknown. Methods enabling investigation of the protein in single functional neurons need to be developed. Here, we have studied how the electrical signaling capacity of a neuron was affected by hypoxic environments. Preliminary results show a trend of higher noise-level when a neuron is exposed to hypoxic compared to normoxic surroundings, which implies increased ion-channel activity. The setup used today shows shortages such as reduced control over the oxygen content due to leakage. Therefore, a gas-tight, multifunctional microfluidic system is under development which enables us to study influences of Neuroglobin concentrations on neuronal activity during hypoxia and anoxia. For electrophysiological recordings a patch-clamp micro pipette will be molded into the walls of the microfluidic system. A single biological cell is steered towards the pipette and attached there by means of optical tweezers. The Neuroglobin oxygen binding state will be studied using optical spectroscopy and the neuron environment will be manipulated by applying flows of varying oxygen content through the microfluidic system. This system will constitute a powerful tool in the investigation of the Neuroglobin mechanism of action.
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| 4. |
- Chizhov, Anton V, et al.
(författare)
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Firing clamp : a novel method for single-trial estimation of excitatory and inhibitory synaptic neuronal conductances.
- 2014
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Ingår i: Frontiers in Cellular Neuroscience. - : Frontiers Media SA. - 1662-5102. ; 8:86, s. 86-
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Tidskriftsartikel (refereegranskat)abstract
- Understanding non-stationary neuronal activity as seen in vivo requires estimation of both excitatory and inhibitory synaptic conductances from a single trial of recording. For this purpose, we propose a new intracellular recording method, called "firing clamp." Synaptic conductances are estimated from the characteristics of artificially evoked probe spikes, namely the spike amplitude and the mean subthreshold potential, which are sensitive to both excitatory and inhibitory synaptic input signals. The probe spikes, timed at a fixed rate, are evoked in the dynamic-clamp mode by injected meander-like current steps, with the step duration depending on neuronal membrane voltage. We test the method with perforated-patch recordings from isolated cells stimulated by external application or synaptic release of transmitter, and validate the method with simulations of a biophysically-detailed neuron model. The results are compared with the conductance estimates based on conventional current-clamp recordings.
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| 5. |
- Das, Roshni, et al.
(författare)
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Medroxyprogesterone acetate positively modulates specific GABAA-receptor subtypes - affecting memory and cognition
- 2022
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Ingår i: Psychoneuroendocrinology. - : Elsevier. - 0306-4530 .- 1873-3360. ; 141
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Tidskriftsartikel (refereegranskat)abstract
- Medroxyprogesterone acetate (MPA) is a progestin widely used in humans as hormone replacement therapy and at other indications. Many progestin metabolites, as the progesterone metabolite allopregnanolone, have GABAA-receptor modulatory effects and are known to affect memory, learning, appetite, and mood. In women, 4 years chronic treatment with MPA doubles the frequency of dementia and in rats, MPA causes cognitive impairment related to the GABAergic system. Activation of the membrane bound GABAA receptor results in a chloride ion flux that can be studied by whole-cell patch-clamp electrophysiological recordings. The purpose of this study was to clarify the modulatory effects of MPA and specific MPA metabolites, with structures like known GABAA-receptor modulators, on different GABAA-receptor subtypes. An additional aim was to verify the results as steroid effects on GABA response in single cells taken from rat hypothalamus. HEK-293 cell-lines permanently expressing the recombinant human GABAA-receptor subtype α1β2γ2L or α5β3γ2L or α2β3γ2S were created. The MPA metabolites 3α5α-MPA,3β5α-MPA and 3β5β-MPA were synthesised and purified for electrophysiological patch-clamp measurements with a Dynaflow system. The effects of MPA and tetrahydrodeoxycorticosterone were also studied. None of the studied MPA metabolites affected the responses mediated by α1β2γ2L or α5β3γ2L GABAA receptors. Contrary, MPA clearly acted both as a positive modulator and as a direct activator of the α5β3γ2L and α2β3γ2S GABAA receptors. However, in concentrations up to 10 μM, MPA was inactive at the α1β2γ2L GABAA receptor. In the patch-clamp recordings from dissociated cells of the preoptic area in rats, MPA increased the amplitude of responses to GABA. In addition, MPA alone without added GABA, evoked a current response. In conclusion, MPA acts as a positive modulator of specific GABAA receptor subtypes expressed in HEK cells and at native GABA receptors in single cells from the hypothalamic preoptic area.
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| 6. |
- Druzin, Michael, et al.
(författare)
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2-aminoethyl diphenylborinate blocks GABAA-receptor-mediated currents in rat medial preoptic neurons
- 2016
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Ingår i: Opera Medica Et Physiologica. - Nizhni Novgorod : Lobachevsky State University of Nizhny Novgorod. - 2500-2295 .- 2500-2287. ; 2:1, s. 63-68
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Tidskriftsartikel (refereegranskat)abstract
- The effect of 2-aminoethyl diphenylborinate (2-APB), a commonly used drug to modulate inositol-1,4,5-triphosphate (IP3) receptors and transient receptor potential (TRP) channels, on GABAA receptor-mediatedcurrents was studied in neurons from the medial preoptic nucleus (MPN) of rat. 2-APB gradually and reversibly reducedthe currents evoked by GABA but had no effect on the currents evoked by glycine. The blocking effect was not mediatedby alterations in intracellular calcium concentration and showed a concentration dependence with half maximal effect at~50 μM 2-APB, for currents evoked by 100 μM, as well as by 1.0 mM GABA, suggesting that 2-APB is not competing withGABA for its binding site at the GABAA receptor. Thus, the present study describes a novel pharmacological property of2-APB as a non-competitive blocker of GABAA receptors and calls for caution in the interpretation of the results where2-APB is used to affect IP3 receptors or TRP channels.
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| 7. |
- Druzin, Michael, et al.
(författare)
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Bicuculline free base blocks voltage-activated K+ currents in rat medial preoptic neurons.
- 2004
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Ingår i: Neuropharmacology. - 0028-3908. ; 46:2, s. 285-95
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Tidskriftsartikel (refereegranskat)abstract
- The effects of the well-known GABA(A)-receptor blocker bicuculline on voltage-gated K(+) currents were studied in neurons from the medial preoptic nucleus (MPN) of rat. Whole-cell currents were recorded using the perforated-patch technique. Voltage steps from -54 to +6 mV resulted in tetraethylammonium-sensitive K(+) currents of delayed rectifier type. The total K(+) current (at 300 ms), including Ca(2+)-dependent and Ca(2+)-independent components, was reversibly reduced (17 +/- 4%) by 100 microM bicuculline methiodide and (37 +/- 5%) by 100 microM bicuculline as free base. The Ca(2+)-independent fraction (77 +/- 2%) of K(+) current evoked by a voltage step was, however, reduced (54 +/- 6%) only by bicuculline free base, but was not affected by bicuculline methiodide. The half-saturating concentration of bicuculline free base for blocking this purely voltage-gated K(+) current was 113 microM, whereas for blocking a steady Ca(2+)-dependent K(+) current it was 36 microM. The bicuculline-sensitive voltage-gated K(+) current was composed of 4-AP-sensitive and 4-AP-resistant components with different kinetic properties. No component of the purely voltage-gated K(+) current was affected neither by 100 nM alpha-dendrotoxin nor by 100 nM I-dendrotoxin. The possible K(+)-channel subtypes mediating the bicuculline-sensitive current in MPN neurons are discussed.
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| 8. |
- Druzin, Michael, et al.
(författare)
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Dual and opposing roles of presynaptic Ca2+ influx for spontaneous GABA release from rat medial preoptic nerve terminals.
- 2002
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Ingår i: Journal of Physiology. - : Wiley. - 0022-3751 .- 1469-7793. ; 542:Pt 1, s. 131-46
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Tidskriftsartikel (refereegranskat)abstract
- Calcium influx into the presynaptic nerve terminal is well established as a trigger signal for transmitter release by exocytosis. By studying dissociated preoptic neurons with functional adhering nerve terminals, we here show that presynaptic Ca2+ influx plays dual and opposing roles in the control of spontaneous transmitter release. Thus, application of various Ca2+ channel blockers paradoxically increased the frequency of spontaneous (miniature) inhibitory GABA-mediated postsynaptic currents (mIPSCs). Similar effects on mIPSC frequency were recorded upon washout of Cd2+ or EGTA from the external solution. The results are explained by a model with parallel Ca2+ influx through channels coupled to the exocytotic machinery and through channels coupled to Ca2+-activated K+ channels at a distance from the release site.
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| 9. |
- Druzin, Michael, et al.
(författare)
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Mechanism of estradiol-induced block of voltage-gated K+ currents in rat medial preoptic neurons.
- 2011
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Ingår i: PLOS ONE. - San Francisco : Public Library of Science. - 1932-6203. ; 6:5, s. e20213-
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Tidskriftsartikel (refereegranskat)abstract
- The present study was conducted to characterize possible rapid effects of 17-β-estradiol on voltage-gated K(+) channels in preoptic neurons and, in particular, to identify the mechanisms by which 17-β-estradiol affects the K(+) channels. Whole-cell currents from dissociated rat preoptic neurons were studied by perforated-patch recording. 17-β-Estradiol rapidly (within seconds) and reversibly reduced the K(+) currents, showing an EC(50) value of 9.7 µM. The effect was slightly voltage dependent, but independent of external Ca(2+), and not sensitive to an estrogen-receptor blocker. Although 17-α-estradiol also significantly reduced the K(+) currents, membrane-impermeant forms of estradiol did not reduce the K(+) currents and other estrogens, testosterone and cholesterol were considerably less effective. The reduction induced by estradiol was overlapping with that of the K(V)-2-channel blocker r-stromatoxin-1. The time course of K(+) current in 17-β-estradiol, with a time-dependent inhibition and a slight dependence on external K(+), suggested an open-channel block mechanism. The properties of block were predicted from a computational model where 17-β-estradiol binds to open K(+) channels. It was concluded that 17-β-estradiol rapidly reduces voltage-gated K(+) currents in a way consistent with an open-channel block mechanism. This suggests a new mechanism for steroid action on ion channels.
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| 10. |
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