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Yu Zhang - One of the best experts on this subject based on the ideXlab platform.

  • a method of marking neurons on brain slices using Patch Clamp Technique
    Chinese journal of applied physiology, 2021
    Co-Authors: Jie Gao, Yan Yun Huang, Yu Tong Zhang, Li Na Zhang, Na Hao, Xia Guo, Yu Zhang
    Abstract:

    Objective: To introduce a method of marking neurons using Patch Clamp Technique. Methods: The brain slices of the target area was cut with a vibrating microtome. The glass microelectrode was perfused with the electrode liquid containing NeurobiotinTM Tracer, and the whole-cell Patch-Clamp recording was performed. After recording, the brain slices were fixed and rinsed with 4% paraformaldehyde. After stained in phosphate buffer with Streptavidin-Texas Red and Triton X-100 for at least 2 hours, the neurons can be observed under a fluorescence microscope. Results: The cell membrane voltage was Clamped at -70 mV, and the neuron showed a gradually increasing membrane current after step stimulation. When recording in the current Clamp mode, the step stimulus caused the neuron to depolarize to the threshold potential and then burst into action potentials. The morphology of intact neurons with clear cell body and protrusions of a neuron could be observed under a fluorescence microscope. Conclusion: This method is suitable for observing the morphological features of the recorded neuron after Patch Clamp experiments, which is easy to operate, and the image is intuitive and clear.

  • Spontaneous firing properties of rat medial vestibular nucleus neurons in brain slices by infrared visual Patch Clamp Technique
    Frontiers of Medicine in China, 2008
    Co-Authors: Weijia Kong, Yan Zhou, Yu Zhang
    Abstract:

    Domestic application of infrared Patch Clamp Techniques on brain slices is limited. The key of the Technique is to prepare high-quality brain slices. The present paper describes the preparation procedure of brainstem slices and the spontaneous firing properties of rat medial vestibular nucleus (MVN) neurons. By infrared differential interference contrast Technique, neurons of rat MVN were visualized directly at the depth of 50–100 μm underneath the surface of slices. Firing activities of MVN neurons were recorded by the whole-cell Patch Clamp Technique in artificial cerebrospinal fluid (ACSF) and low Ca2+-high Mg2+ fluid. The firing mode was more irregular and depressive in low Ca2+-high Mg2+ fluid than in ACSF. According to the averaged waveform of action potentials, cells were classified as the neurons with monophasic after-hyperpolarization potential (AHP), and the neurons with biphasic AHP. The resting membrane potential (RMP), input resistance (Rin) and membrane capacitance (Cm) of neurons were recorded and compared between groups. With infrared videomicroscopy, Patch Clamp recordings could be made under direct observation in freshly prepared brainstem slices. The discharge activities of MVN neurons were spontaneous and the firing mode was modulated by extracellular calcium concentration. The basic membrane properties of two types of neurons were not significantly different, while the differences in waveform might play a role in the segregation between tonic and kinetic cells.

  • electrophysiological characteristics of muscarinic cholinergic receptor in rat medial vestibular nucleus neurons by visual Patch Clamp Technique
    Chinese journal of otorhinolaryngology head and neck surgery, 2007
    Co-Authors: Yu Zhang, Weijia Kong, Banghua Liu, Changkai Guo, Dawei Sun, Jiao Xia, Yun Zhu, Jian Zhang
    Abstract:

    Objective To establish the visual Patch Clamp whole-cell recording Technique and study the properties and functional significance of muscarinic receptor-mediated currents in rat medial vestibular nucleus neurons （MVNn）. Methods Brain slices containing the MVN were prepared from fifteen Wistar rats. By combining infrared differential interference contrast （IR-DIC） Technique and CCD-Camera system with visual Patch Clamp whole-cell recording Technique, twenty healthy neurons were located and muscarinic receptor-mediated currents in rat MVNn were observed and analyzed. Results Visual Patch Clamp Technique can be used to make direct localization and to make sure of active neuron. In MVNn, a comparison of the current-voltage relationships before and during the application of muscarine, which revealed an increase in the slope of the Ⅰ-Ⅴ curve and the reversal potential for this response lay at （ - 88. 4 ± 4. 9） mV （x ± s） , indicates that the activation of muscarinic cholinergic receptors leads to a decrease in potassium current. The test in the voltage sensitivity of the muscarine-induced response, which showed that the effect had a linear current-vohage relationship and reversed at （ - 86. 7 ± 3. 5 ） mV, indicates that the potassium current blocked by muscarine corresponds to the voltage-insensitive leak potassium current. Conclusions Visual Patch Clamp Technique, which was considered better than blind Patch Clamp Technique, can improve the success of sealing process. By the analysis of muscarinic receptor-mediated currents, the data provide support that muscarinic cholinergic mechanisms play a prominent role in the modulation of the excitability of MVNn and also offer a new idea for the efficacy of anticholinergic drugs. Key words: Vestibular nucleus; Receptors, muscarine; Patch Clamp Technique

M Watanabe - One of the best experts on this subject based on the ideXlab platform.

  • Ca-dependent K channels in smooth muscle cells permeabilized by beta-escin recorded using the cell-attached Patch-Clamp Technique.
    Pfl�gers Archiv European Journal of Physiology, 1992
    Co-Authors: K Muraki, Y Imaizumi, M Watanabe
    Abstract:

    Using the cell-attached Patch-Clamp Technique, the activity of single, Ca-dependent K channels was recorded in single smooth muscle cells permeabilized by beta-escin. The conductance and the relationship between the open probability of the channels and pCa recorded in permeabilized cells were very similar to those obtained in excised inside-out Patches. At pCa 7, application of 30 microM acetylcholine (ACh) or 0.1 microM substance P (SP) together with 1 mM guanosine 5'-trisphosphate to permeabilized cells elicited transient bursts of channel openings similar to those which occur in intact cells. Transient activation was also observed when 2-30 microM inositol trisphosphate (IP3) was applied to permeabilized cells. This single channel activity was inhibited by pretreatment with low-molecular-weight heparin at 50-100 micrograms/ml. Channel activity at pCa 7.0 was greatly enhanced by 200 microM cyclic adenosine monophosphate. These results provide direct evidence that single Ca-dependent K channel activity is regulated by the transmitters ACh and SP, as well as a second messenger, IP3, via the release of intracellular Ca from intracellular sites which are blocked by heparin. This novel approach is valuable in elucidating second messenger mechanisms involved in the regulation of single channel activity by transmitters and autocoids, since permeabilization by beta-escin preserves the entire system of receptor-operated signal transduction and allows intracellular application of second messengers at fixed concentrations.

Norio Akaike - One of the best experts on this subject based on the ideXlab platform.

  • gramicidin perforated Patch Clamp Technique reveals glycine gated outward chloride current in dissociated nucleus solitarii neurons of the rat
    Journal of Neurophysiology, 1994
    Co-Authors: Jeongseop Rhee, S Ebihara, Norio Akaike
    Abstract:

    1. The inhibitory response of exogenously applied glycine was investigated in freshly dissociated rat nucleus tractus solitarii neurons under whole cell configuration using new perforated Patch-Clamp Technique termed "gramicidin perforated Patch Technique," which maintains intact intracellular Cl- concentrations. 2. Using the gramicidin perforated Patch Technique, at a holding potential (VH) of -45 mV, glycine induced outward currents in a concentration-dependent manner with a EC50 of 4.0 x 10(-5) M and at a Hill coefficient of 1.5. In contrast, using the nystatin perforated Patch Technique, glycine induced inward currents at the same VH in a concentration-dependent manner with an EC50 of 4.9 x 10(-5) M and at a Hill coefficient of 1.2. 3. The glycine-induced outward currents were blocked by strychnine in a concentration dependent manner with an IC50 of 2.2 x 10(-8) M. The blockade was competitive. 4. The current-voltage relationship for the 10(-5) M glycine response showed a clear outward rectification. 5. Ten-fold change of extracellular Cl- with a large impermeable anion resulted in a 65 mV shift of the reversal potential of glycine-induced currents (EGly), indicating that the membrane behaves like a Cl- electrode in the presence of glycine. 6. The intracellular Cl- activity calculated from the EGly ranged from 7.3 to 18.2 mM, with a mean value of 13.3 mM. 7. The values of EGly in the individual neurons were significantly negative to the resting membrane potentials, suggesting the existence of active transport of Cl-.

  • Patch-Clamp Technique
    Yakubutsu seishin kodo = Japanese journal of psychopharmacology, 1992
    Co-Authors: Norio Akaike, Tetsuya Shirasaki
    Abstract:

    We first describe how to set up the system for Patch-Clamp recording and carry out the experiments with 4 different modes of conventional Patch-Clamp Technique: cell-attached, whole-cell, inside-out and outside-out configurations. Thereafter, we mention about an improved 'nystatin perforated' Patch-Clamp Technique which dissolves the fault of conventional Patch-Clamp Techniques except the cell-attached mode.

Michel De Waard - One of the best experts on this subject based on the ideXlab platform.

  • computer modeling of whole cell voltage Clamp analyses to delineate guidelines for good practice of manual and automated Patch Clamp
    Scientific Reports, 2021
    Co-Authors: Jerome Montnach, Maxime Lorenzini, Adrien Lesage, Isabelle Simon, Sebastien Nicolas, Eleonore Moreau, Celine Marionneau, Isabelle Baro, Michel De Waard
    Abstract:

    The Patch-Clamp Technique and more recently the high throughput Patch-Clamp Technique have contributed to major advances in the characterization of ion channels. However, the whole-cell voltage-Clamp Technique presents certain limits that need to be considered for robust data generation. One major caveat is that increasing current amplitude profoundly impacts the accuracy of the biophysical analyses of macroscopic ion currents under study. Using mathematical kinetic models of a cardiac voltage-gated sodium channel and a cardiac voltage-gated potassium channel, we demonstrated how large current amplitude and series resistance artefacts induce an undetected alteration in the actual membrane potential and affect the characterization of voltage-dependent activation and inactivation processes. We also computed how dose-response curves are hindered by high current amplitudes. This is of high interest since stable cell lines frequently demonstrating high current amplitudes are used for safety pharmacology using the high throughput Patch-Clamp Technique. It is therefore critical to set experimental limits for current amplitude recordings to prevent inaccuracy in the characterization of channel properties or drug activity, such limits being different from one channel type to another. Based on the predictions generated by the kinetic models, we draw simple guidelines for good practice of whole-cell voltage-Clamp recordings.

Eleonore Moreau - One of the best experts on this subject based on the ideXlab platform.

  • computer modeling of whole cell voltage Clamp analyses to delineate guidelines for good practice of manual and automated Patch Clamp
    Scientific Reports, 2021
    Co-Authors: Jerome Montnach, Maxime Lorenzini, Adrien Lesage, Isabelle Simon, Sebastien Nicolas, Eleonore Moreau, Celine Marionneau, Isabelle Baro, Michel De Waard
    Abstract:

    The Patch-Clamp Technique and more recently the high throughput Patch-Clamp Technique have contributed to major advances in the characterization of ion channels. However, the whole-cell voltage-Clamp Technique presents certain limits that need to be considered for robust data generation. One major caveat is that increasing current amplitude profoundly impacts the accuracy of the biophysical analyses of macroscopic ion currents under study. Using mathematical kinetic models of a cardiac voltage-gated sodium channel and a cardiac voltage-gated potassium channel, we demonstrated how large current amplitude and series resistance artefacts induce an undetected alteration in the actual membrane potential and affect the characterization of voltage-dependent activation and inactivation processes. We also computed how dose-response curves are hindered by high current amplitudes. This is of high interest since stable cell lines frequently demonstrating high current amplitudes are used for safety pharmacology using the high throughput Patch-Clamp Technique. It is therefore critical to set experimental limits for current amplitude recordings to prevent inaccuracy in the characterization of channel properties or drug activity, such limits being different from one channel type to another. Based on the predictions generated by the kinetic models, we draw simple guidelines for good practice of whole-cell voltage-Clamp recordings.