scholarly journals Mural propagation of descending vasa recta responses to mechanical stimulation

2013 ◽  
Vol 305 (3) ◽  
pp. F286-F294 ◽  
Author(s):  
Zhong Zhang ◽  
Kristie Payne ◽  
Chunhua Cao ◽  
Thomas L. Pallone
Keyword(s):  
Membrane Potential ◽  
Patch Clamp ◽  
Mechanical Stimulation ◽  
Channel Blockade ◽  
Patch Clamp Recording ◽  
Automated Method ◽  
Whole Cell Patch Clamp ◽  
Vasa Recta ◽  
Gated Channel ◽  
Phosphoinositide 3 Kinase

To investigate the responses of descending vasa recta (DVR) to deformation of the abluminal surface, we devised an automated method that controls duration and frequency of stimulation by utilizing a stream of buffer from a micropipette. During stimulation at one end of the vessel, fluorescent responses from fluo4 or bis[1,3-dibutylbarbituric acid-(5)] trimethineoxonol [DiBAC4(3)], indicating cytoplasmic calcium ([Ca2+]CYT) or membrane potential, respectively, were recorded from distant cells. Alternately, membrane potential was recorded from DVR pericytes by nystatin whole cell patch-clamp. Mechanical stimulation elicited reversible [Ca2+]CYT responses that increased with frequency. Individual pericyte responses along the vessel were initiated within a fraction of a second of one another. Those responses were inhibited by gap junction blockade with 18 β-glycyrrhetinic acid (100 μM) or phosphoinositide 3 kinase inhibition with 2-morpholin-4-yl-8-phenylchromen-4-one (50 μM). [Ca2+]CYT responses were blocked by removal of extracellular Ca2+ or L-type voltage-gated channel blockade with nifedipine (10 μM). At concentrations selective for the T-type channel blockade, mibefradil (100 nM) was ineffective. During mechanostimulation, pericytes rapidly depolarized, as documented with either DiBAC4(3) fluorescence or patch-clamp recording. Single stimuli yielded depolarizations of 22.5 ± 2.2 mV while repetitive stimuli at 0.1 Hz depolarized pericytes by 44.2 ± 4.0 mV. We conclude that DVR are mechanosensitive and that rapid transmission of signals along the vessel axis requires participation of gap junctions, L-type Ca2+ channels, and pericyte depolarization.

Effects of Methylphenidate on the Membrane Potential and Current in Neurons of the Rat Locus Coeruleus

2002 ◽  
Vol 87 (3) ◽  
pp. 1206-1212 ◽  
Author(s):  
Masaru Ishimatsu ◽  
Yuri Kidani ◽  
Akira Tsuda ◽  
Takashi Akasu
Keyword(s):  
Membrane Potential ◽  
Patch Clamp ◽  
Locus Coeruleus ◽  
Outward Current ◽  
Inward Rectification ◽  
Spontaneous Firing ◽  
Patch Clamp Recording ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp

Effects of methylphenidate (MPH), a therapeutic agent used in children presenting the attention deficit hyperactivity disorder (ADHD), on the membrane potential and current in neurons of the rat locus coeruleus (LC) were examined using intracellular and whole cell patch-clamp recording techniques. Application of MPH (30 μM) to artificial cerebrospinal fluid (ACSF) produced a hyperpolarizing response with amplitude of 12 ± 1 mV ( n = 29). Spontaneous firing of LC neurons was blocked during the MPH-induced hyperpolarization. Superfusion of LC neurons with ACSF containing 0 mM Ca2+ and 11 mM Mg2+ (Ca2+-free ACSF) produced a depolarizing response associated with an increase in spontaneous firing of the action potential. The MPH-induced hyperpolarization was blocked in Ca2+-free ACSF. Yohimbine (1 μM) and prazosin (10 μM), antagonists for α2 and α2B/2Creceptors, respectively, blocked the MPH-induced hyperpolarization in LC neurons. Tetrodotoxin (TTX, 1 μM) produced a partial depression of the MPH-induced hyperpolarization in LC neurons. Under the whole cell patch-clamp condition, MPH (30–300 μM) produced an outward current ( I MPH) with amplitude of 110 ± 6 pA ( n = 17) in LC neurons. The I MPH was blocked by Co2+ (1 mM). During prolonged application of MPH (300 μM for 45 min), the hyperpolarization gradually decreased in the amplitude and eventually disappeared, possibly because of depression of norepinephrine (NE) release from noradrenergic nerve terminals. At a low concentration (1 μM), MPH produced no outward current but consistently enhanced the outward current induced by NE. These results suggest that the MPH-induced response is mediated by NE via α2B/2C-adrenoceptors in LC neurons. I MPH was associated with an increase in the membrane conductance of LC neurons. The I MPH reversed its polarity at −102 ± 6 mV ( n = 8) in the ACSF. The reversal potential of I MPH was changed by 54 mV per decade change in the external K+ concentration. Current-voltage relationship showed that the I MPH exhibited inward rectification. Ba2+ (100 μM) suppressed the amplitude and the inward rectification of the I MPH.These results suggest that the I MPH is produced by activation of inward rectifier K+channels in LC neurons.

scholarly journals A Novel Membrane Potential-Sensitive Fluorescent Dye Improves Cell-Based Assays for Ion Channels

2002 ◽  
Vol 7 (1) ◽  
pp. 79-85 ◽  
Author(s):  
Deborah F. Baxter ◽  
Martin Kirk ◽  
Amy F. Garcia ◽  
Alejandra Raimondi ◽  
Mats H. Holmqvist ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Ion Channels ◽  
Patch Clamp ◽  
High Throughput Screening ◽  
Fluorescent Dye ◽  
Patch Clamp Recording ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp ◽  
Electrophysiological Studies

The study of ion channel-mediated changes in membrane potential using the conventional bisoxonol fluorescent dye DiBAC4(3) has several limitations, including a slow onset of response and multistep preparation, that limit both the fidelity of the results and the throughput of membrane potential assays. Here, we report the characterization of the FLIPR Membrane Potential Assay Kit (FMP) in cells expressing voltage- and ligand-gated ion channels. The steady-state and kinetics fluorescence properties of FMP were compared with those of DiBAC4(3), using both FLIPR and whole-cell patch-clamp recording. Our experiments with the voltage-gated K+ channel, hElk-1, revealed that FMP was 14-fold faster than DiBAC4(3) in response to depolarization. On addition of 60 mM KCl, the kinetics of fluorescence changes of FMP using FLIPR were identical to those observed in the electrophysiological studies using whole-cell current clamp. In addition, KCl concentration-dependent increases in FMP fluorescence correlated with the changes of membrane potential recorded in whole-cell patch clamp. In studies examining vanilloid receptor-1, a ligand-gated nonselective cation channel, FMP was superior to DiBAC4(3) with respect to both kinetics and amplitude of capsaicin-induced fluorescence changes. FMP has also been used to measure the activation of KATP1 and hERG.2 Thus this novel membrane potential dye represents a powerful tool for developing high-throughput screening assays for ion channels.

Measurement of spontaneous neuronal membrane activity by time lapse confocal microscopy

1995 ◽  
Vol 53 ◽  
pp. 972-973
Author(s):  
R H. Selinfreund ◽  
A. H. Cornell-Bell
Keyword(s):  
Membrane Potential ◽  
Confocal Microscopy ◽  
Patch Clamp ◽  
Electrical Activity ◽  
Time Lapse ◽  
Neuronal Firing ◽  
Neuronal Membrane ◽  
Pituitary Cells ◽  
Patch Clamp Recording ◽  
Spontaneous Electrical Activity

Cellular electrophysiological properties are normally monitored by standard patch clamp techniques . The combination of membrane potential dyes with time-lapse laser confocal microscopy provides a more direct, least destructive rapid method for monitoring changes in neuronal electrical activity. Using membrane potential dyes we found that spontaneous action potential firing can be detected using time-lapse confocal microscopy. Initially, patch clamp recording techniques were used to verify spontaneous electrical activity in GH4\C1 pituitary cells. It was found that serum depleted cells had reduced spontaneous electrical activity. Brief exposure to the serum derived growth factor, IGF-1, reconstituted electrical activity. We have examined the possibility of developing a rapid fluorescent assay to measure neuronal activity using membrane potential dyes. This neuronal regeneration assay has been adapted to run on a confocal microscope. Quantitative fluorescence is then used to measure a compounds ability to regenerate neuronal firing.The membrane potential dye di-8-ANEPPS was selected for these experiments. Di-8- ANEPPS is internalized slowly, has a high signal to noise ratio (40:1), has a linear fluorescent response to change in voltage.

Z f-and-H sys-based C m measurement under the whole-cell patch-clamp recording

2008 ◽  
Vol 457 (6) ◽  
pp. 1423-1434 ◽  
Author(s):  
Hao Zhang ◽  
Jie Luo ◽  
Jun Xiong ◽  
Xian-Guang Lin ◽  
Zheng-Xing Wu ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Patch Clamp Recording ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp

scholarly journals Descending vasa recta endothelial cells and pericytes form mural syncytia

2014 ◽  
Vol 306 (7) ◽  
pp. F751-F763 ◽  
Author(s):  
Zhong Zhang ◽  
Hai Lin ◽  
Chunhua Cao ◽  
Kristie Payne ◽  
Thomas L. Pallone
Keyword(s):  
Endothelial Cells ◽  
Membrane Potential ◽  
Patch Clamp ◽  
Gap Junction ◽  
Lucifer Yellow ◽  
Sampling Rate ◽  
Input Resistance ◽  
Contralateral Side ◽  
Vasa Recta ◽  
Endothelial Response

Using patch clamp, we induced depolarization of descending vasa recta (DVR) pericytes or endothelia and tested whether it was conducted to distant cells. Membrane potential was measured with the fluorescent voltage dye di-8-ANEPPS or with a second patch-clamp electrode. Depolarization of an endothelial cell induced responses in other endothelia within a millisecond and was slowed by gap junction blockade with heptanol. Endothelial response to pericyte depolarization was poor, implying high-resistance myo-endothelial coupling. In contrast, dual patch clamp of neighboring pericytes revealed syncytial coupling. At high sampling rate, the spread of depolarization between pericytes and endothelia occurred in 9 ± 2 or 12 ± 2 μs, respectively. Heptanol (2 mM) increased the overall input resistance of the pericyte layer to current flow and prevented transmission of depolarization between neighboring cells. The fluorescent tracer Lucifer yellow (LY), when introduced through ruptured patches, spread between neighboring endothelia in 1 to 7 s, depending on location of the flanking cell. LY diffused to endothelial cells on the ipsilateral but not contralateral side of the DVR wall and minimally between pericytes. We conclude that both DVR pericytes and endothelia are part of individual syncytia. The rate of conduction of membrane potential exceeds that for diffusion of hydrophilic molecules by orders of magnitude. Gap junction coupling of adjacent endothelial cells may be spatially oriented to favor longitudinal transmission along the DVR axis.

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