Measurement of chloride movement in neuronal preparations.
In this unit, protocols are described for biochemical and optical techniques that have been used by investigators to measure ligand-gated chloride movement in vesicular structures called synaptoneurosomes (also referred to as microsacs), in cultured neurons, and in the acute brain slice. These techniques can be applied to other ions as well. The measurement of uptake and efflux of radioisotopic chloride in synaptoneurosomes is used to study the responses of gamma-aminobutyric acid (GABA) receptors, which are coupled to chloride channels. Similar chloride flux assays for primary neuronal cultures are also presented. Alternatively, the efflux of chloride from synaptoneurosomes and primary neuronal cultures can be studied using fluorescent dyes and photometry. Finally, the measurement of chloride uptake can be studied in individual neurons in brain slices using fluorescent dyes and optical imaging by nonconfocal and confocal microscopy. Several support protocols are provided as well, outlining the preparation of synaptoneurosomes from specific brain regions, and the preparation, loading, and calibration of chloride-sensitive fluorescent dyes.
Schwartz-Bloom
R D
RD
Duke University Medical Center, Durham, North Carolina, USA.
Engblom
A C
AC
Akerman
K E
KE
Inglefield
J R
JR
D000818:Animals; D001921:Brain / Q000378:metabolism*; D018118:Chloride Channels / Q000378:metabolism; D002712:Chlorides / Q000032:analysis / Q000378:metabolism*; D002713:Chlorine / Q000032:analysis; D005456:Fluorescent Dyes / Q000032:analysis; D018755:GABA Agonists / Q000494:pharmacology; D015640:Ion Channel Gating; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009474:Neurons / Q000378:metabolism*; D011806:Quinolinium Compounds / Q000032:analysis; D011868:Radioisotopes / Q000032:analysis; D051381:Rats; D018079:Receptors, GABA / Q000187:drug effects / Q000502:physiology; D013574:Synaptosomes / Q000378:metabolism
D018118:Chloride Channels; D002712:Chlorides; D005456:Fluorescent Dyes; D018755:GABA Agonists; D011806:Quinolinium Compounds; D011868:Radioisotopes; D018079:Receptors, GABA; C070311:6-methoxy-N-ethylquinolinium; D002713:Chlorine