Simultaneous optical imaging of intracellular Cl − in neurons in different layers of rat neocortical slices: advantages and limitations

Abstract

Simultaneous recording of changes in intracellular Cl − concentration ([Cl −] i) in individual neurons situated in different layers (e.g. II/III–VI) of neocortical slices was found to be feasible by means of optical fluorescence measurements using 6-methoxy- N -ethylquinolinium iodide (MEQ). γ-Aminobutyric acid (GABA) caused a measurable increase in [Cl −] i in adult neocortical neurons, but a decrease in immature neurons. Developmental changes in the function of the Cl − pump and cation–Cl − co-transporters were evaluated using inhibitors such as furosemide (FURO), ethacrynic acid (ETA), and bumetanide (BMT). However, it was found that these inhibitors absorb and/or emit light of the wavelength that is used for the optical imaging of MEQ. In addition, quenching of MEQ fluorescence by Cl − and leakage of loaded MEQ was significantly enhanced at a higher temperature, which will limit experimentation at>30°C. Estimation of [Cl −] i in individual neurons in slices was made possible by calibrating intracellular MEQ fluorescence signals at known Cl − concentrations ([Cl −]) in the presence of tributyltin, a Cl −–OH − antiporter, nigericin, a K +–H +antiporter, and KSCN. This enables comparison of [Cl −] i between neurons in different slices. Thus, optical imaging of [Cl −] i in brain slices can provide valuable spatial information about [Cl −] i dynamics and homeostasis, although it should be emphasized that the technique does have some limitations.