Skip to main content
Journal cover image

Fluorescence imaging of changes in intracellular chloride in living brain slices.

Publication ,  Journal Article
Inglefield, JR; Schwartz-Bloom, RD
Published in: Methods
June 1999

In brain slice preparations, chloride movements across the cell membrane of living cells are measured traditionally with 36Cl- tracer methods, Cl--selective microelectrodes, or whole-cell recording using patch clamp analysis. We have developed an alternative, noninvasive technique that uses the fluorescent Cl- ion indicator, 6-methoxy-N-ethylquinolinium iodide (MEQ), to study changes in intracellular Cl- by epifluorescence or UV laser scanning confocal microscopy. In brain slices taken from rodents younger than 22 days of age, excellent cellular loading is achieved with the membrane-permeable form of the dye, dihydro-MEQ. Subsequent intracellular oxidation of dihydro-MEQ to the Cl--sensitive MEQ traps the polar form of the dye inside the neurons. Because MEQ is a single-excitation and single-emission dye, changes in intracellular Cl- concentrations can be calibrated from the Stern-Volmer relationship, determined in separate experiments. Using MEQ as the fluorescent indicator for Cl-, Cl- flux through the gamma-aminobutyric acid (GABA)-gated Cl- channel (GABAA receptor) can be studied by dynamic video imaging and either nonconfocal (epifluorescence) or confocal microscopy in the acute brain slice preparation. Increases in intracellular Cl- quench MEQ fluorescence, thereby reflecting GABAA receptor activation. GABAA receptor functional activity can be measured in discrete cells located in neuroanatomically defined populations within areas such as the neocortex and hippocampus. Changes in intracellular Cl- can also be studied under various conditions such as oxygen/glucose deprivation ("in vitro ischemia") and excitotoxicity. In such cases, changes in cell volume may also occur due to the dependence of cell volume regulation on Na+, K+, and Cl- flux. Because changes in cell volume can affect optical fluorescence measurements, we assess cell volume changes in the brain slice using the fluorescent indicator calcein-AM. Determination of changes in MEQ fluorescence versus calcein fluorescence allows one to distinguish between an increase in intracellular Cl- and an increase in cell volume.

Duke Scholars

Published In

Methods

DOI

ISSN

1046-2023

Publication Date

June 1999

Volume

18

Issue

2

Start / End Page

197 / 203

Location

United States

Related Subject Headings

  • Somatosensory Cortex
  • Receptors, GABA-A
  • Rats, Sprague-Dawley
  • Rats
  • Quinolinium Compounds
  • Microscopy, Fluorescence
  • Microscopy, Confocal
  • In Vitro Techniques
  • Hippocampus
  • Fluorescent Dyes
 

Citation

APA
Chicago
ICMJE
MLA
NLM
Inglefield, J. R., & Schwartz-Bloom, R. D. (1999). Fluorescence imaging of changes in intracellular chloride in living brain slices. Methods, 18(2), 197–203. https://doi.org/10.1006/meth.1999.0772
Inglefield, J. R., and R. D. Schwartz-Bloom. “Fluorescence imaging of changes in intracellular chloride in living brain slices.Methods 18, no. 2 (June 1999): 197–203. https://doi.org/10.1006/meth.1999.0772.
Inglefield JR, Schwartz-Bloom RD. Fluorescence imaging of changes in intracellular chloride in living brain slices. Methods. 1999 Jun;18(2):197–203.
Inglefield, J. R., and R. D. Schwartz-Bloom. “Fluorescence imaging of changes in intracellular chloride in living brain slices.Methods, vol. 18, no. 2, June 1999, pp. 197–203. Pubmed, doi:10.1006/meth.1999.0772.
Inglefield JR, Schwartz-Bloom RD. Fluorescence imaging of changes in intracellular chloride in living brain slices. Methods. 1999 Jun;18(2):197–203.
Journal cover image

Published In

Methods

DOI

ISSN

1046-2023

Publication Date

June 1999

Volume

18

Issue

2

Start / End Page

197 / 203

Location

United States

Related Subject Headings

  • Somatosensory Cortex
  • Receptors, GABA-A
  • Rats, Sprague-Dawley
  • Rats
  • Quinolinium Compounds
  • Microscopy, Fluorescence
  • Microscopy, Confocal
  • In Vitro Techniques
  • Hippocampus
  • Fluorescent Dyes