Periodic Current Injection (PCI) -A New Method to Image Steady-State Membrane Potential of Single Neurons in situ Using Extracellular Voltage-Sensitive Dyes

Abstract A new method is described which allows to image the steady-state distribution of m em brane potential of single neurons in situ. The method consists of staining the tissue with an extracellular voltage-sensitive dye (Di-4-ANEPPS) and impaling a single neuron with a microelectrode. After focusing the imaging system onto the cell a large series of images are taken with a CCD camera at the appropriate excitation wavelength of the voltage-sensitive dye while the neuron’s membrane potential is shifted by a periodic current injection (PCI). Afterwards two groups of images are averaged separately: those images while the cell was at rest and those images while the cell was hyperpolarized. After subtraction of these averaged images, the resulting difference image shows only the membrane potential of the cell which was altered periodically. The success of the method is demonstrated on leech cells in intact ganglia. If applied to cells with a basically two-dimensional arborization pattern, the decrease of activity in the difference image in areas further away from the injection site should relate to a decrease in membrane potential according to the passive electrotonic properties of the cell under study.

Download Full-text

The in situ Langendorff perfusion system: A new method for viral mediated gene transfer to porcine hearts prior transplantation

The Thoracic and Cardiovascular Surgeon ◽

10.1055/s-2008-1037868 ◽

2008 ◽

Vol 56 (S 1) ◽

Author(s):

L Burdorf ◽

N Schuhmann ◽

A Schulz ◽

M Köhn ◽

E Thein ◽

...

Keyword(s):

Gene Transfer ◽

New Method ◽

Perfusion System ◽

System A ◽

Langendorff Perfusion

Download Full-text

Faculty Opinions recommendation of Submillisecond optical reporting of membrane potential in situ using a neuronal tracer dye.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1163943.624582 ◽

2009 ◽

Author(s):

Naoum Issa ◽

Ari Rosenberg

Keyword(s):

Membrane Potential

Download Full-text

Acidification of rat liver lysosomes: quantitation and comparison with endosomes

AJP Cell Physiology ◽

10.1152/ajpcell.1993.265.4.c901 ◽

1993 ◽

Vol 265 (4) ◽

pp. C901-C917 ◽

Cited By ~ 42

Author(s):

R. W. Van Dyke

Keyword(s):

Steady State ◽

Membrane Potential ◽

Rat Liver ◽

Proton Pumps ◽

Intact Cells ◽

Early Endosomes ◽

Medium Permeability ◽

Electrogenic Proton Pump ◽

Secondary Lysosomes ◽

Permeability Studies

Both lysosomes and endosomes are acidified by an electrogenic proton pump, although studies in intact cells indicate that the steady-state internal pH (pHi) of lysosomes is more acid than that of endosomes. We undertook the present study to examine in detail the acidification mechanism of purified rat liver secondary lysosomes and to compare it with that of a population of early endosomes. Both endosomes and lysosomes exhibited ATP-dependent acidification, but proton influx rates were 2.4- to 2.7-fold greater for endosomes than for lysosomes because of differences in both buffering capacity and acidification rates, suggesting that endosomes exhibited greater numbers or rates of proton pumps. Lysosomes, however, exhibited a more acidic steady-state pHi due in part to a slower proton leak rate. Changes in medium Cl- increased acidification rates of endosomes more than lysosomes, and the lysosome ATP-dependent interior-positive membrane potential was only partially eliminated by high-Cl- medium. Permeability studies suggested that lysosomes were less permeable to Na+, Li+, and Cl- and more permeable to K+ and PO4(2-) than endosomes. Na-K-adenosine-triphosphatase did not appear to regulate acidification of either vesicle type. Endosome and lysosome acidification displayed similar inhibition profiles to N-ethylmaleimide, dicyclohexyl-carbodiimide, and vanadate, although lysosomes were somewhat more sensitive [concentration producing 50% maximal inhibition (IC50) 1 nM] to bafilomycin A1 than endosomes (IC50 7.6 nM). Oligomycin (1.5-3 microM) stimulated lysosome acidification due to shunting of membrane potential. Overall, acidification of endosomes and lysosomes was qualitatively similar but quantitatively somewhat different, possibly related to differences in the density or rate of proton pumps as well as vesicle permeability to protons, anions, and other cations.

Download Full-text

A new method for the in situ assay of α-glucosidase activity and inhibitor screening through an enzyme substrate-mediated DNA hybridization chain reaction

New Journal of Chemistry ◽

10.1039/c9nj01868a ◽

2019 ◽

Vol 43 (24) ◽

pp. 9458-9465

Author(s):

Xiquan Yue ◽

Lihong Su ◽

Xu Chen ◽

Junfeng Liu ◽

Longpo Zheng ◽

...

Keyword(s):

Small Molecule ◽

Dna Hybridization ◽

New Method ◽

Hybridization Chain Reaction ◽

Chain Reaction ◽

Inhibitor Screening ◽

Glucosidase Activity ◽

Enzyme Substrate ◽

In Situ Assay

The strategy is based on small molecule-mediated hybridization chain reaction.

Download Full-text

In situ measurement of the surface area during catalyst preparation: development of a new method

Applied Catalysis A General ◽

10.1016/s0926-860x(02)00035-2 ◽

2002 ◽

Vol 229 (1-2) ◽

pp. 273-281 ◽

Cited By ~ 8

Author(s):

A. Frennet ◽

V. Chitry ◽

N. Kruse

Keyword(s):

Surface Area ◽

Catalyst Preparation ◽

In Situ Measurement ◽

New Method

Download Full-text

Effects of hydrogen peroxide and hypochlorite on membrane potential of mitochondria in situ in rat heart cells

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y91-253 ◽

1991 ◽

Vol 69 (11) ◽

pp. 1705-1712 ◽

Cited By ~ 10

Author(s):

Noburu Konno ◽

K. J. Kako

Keyword(s):

Hydrogen Peroxide ◽

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Rat Heart ◽

Mitochondrial Membrane ◽

Isolated Rat Heart ◽

Heart Mitochondria ◽

High Concentrations ◽

Isolated Rat

Hydrogen peroxide (H2O2) and hypochlorite (HOCl) cause a variety of cellular dysfunctions. In this study we examined the effects of these agents on the electrical potential gradient across the inner membrane of mitochondria in situ in isolated rat heart myocytes. Myocytes were prepared by collagenase digestion and incubated in the presence of H2O2 or HOCl. Transmembrane electrical gradients were measured by distribution of [3H]triphenylmethylphosphonium+, a lipophilic cation. The particulate fraction was separated from the cytosolic compartment first by permeabilization using digitonin, followed by rapid centrifugal sedimentation through a bromododecane layer. We found that the mitochondrial membrane potential (161 ± 7 mV, negative inside) was relatively well maintained under oxidant stress, i.e., the potential was decreased only at high concentrations of HOCl and H2O2 and gradually with time. The membrane potential of isolated rat heart mitochondria was affected similarly by H2O2 and HOCl in a concentration- and time-dependent manner. High concentrations of oxidants also reduced the cellular ATP level but did not significantly change the matrix volume. When the extra-mitochondrial free calcium concentration was increased in permeabilized myocytes, the transmembrane potential was decreased proportionally, and this decrease was potentiated further by H2O2. These results support the view that heart mitochondria are equipped with well-developed defense mechanisms against oxidants, but the action of H2O2 on the transmembrane electrical gradient is exacerbated by an increase in cytosolic calcium. Keywords: ATP, calcium, cardiomyocyte, cell defense, mitochondrial membrane potential, oxidant, triphenylmethylphosphonium.

Download Full-text