Confocal imaging of intracellular chloride in living brain slices: measurement of GABAA receptor activity
Introduction
The responses of individual cells to various stimuli have been examined using fluorescent tracers for bio-active molecules (Tsien, 1988; Biwersi and Verkman, 1991; Hayashi and Miyata, 1994). For example, in cultured neurons and in brain slices, fluorescent indicators have been used to measure increases in intracellular calcium ([Ca2+]i), both in the somata (Yuste and Katz, 1991; Dailey and Smith, 1994; Porter and McCarthy, 1995) and in dendrites (Regehr et al., 1989; Guthrie et al., 1991; Llano et al., 1991; Frenguelli et al., 1993; Jaffe et al., 1994) during or after neuronal excitation. In contrast, changes in intracellular chloride ([Cl−]i) following GABAA receptor activation have been studied traditionally with electrophysiological measures (Krnjevic and Schwartz, 1967; Alger and Nicoll, 1979) and 36Cl− flux techniques (Schwartz et al., 1985; Harris and Allan, 1985). With the advent of Cl−-sensitive fluorescent dyes such as 6-methoxy-N-[3-sulfopropyl] quinolinium (SPQ) and 6-methoxy-N-ethyl-quinolinium chloride (MEQ), changes in [Cl−]i have been measured in non-neuronal cells (Biwersi and Verkman, 1991; Verkman, 1990for review) and engineered cell lines (Wong et al., 1992) with fluorescence photometry. In addition, Cl−-sensitive fluorescent indicators and fluorescence photometry have been applied to the study of GABA-induced Cl− transport within both synaptoneurosomes (Engblom and Akerman, 1991) and cultured neurons (Engblom et al., 1991; Hara et al., 1992). Recently, we developed an optical imaging technique to measure GABA-mediated changes in [Cl−]i within living brain slices using conventional epifluorescence microscopy and MEQ (Schwartz and Yu, 1995).
Optical imaging in living cell preparations has become a widely used technique with the aid of a confocal microscope. Imaging with laser-scanning confocal microscopy greatly improves the resolution (both temporal and spatial) of dynamic changes in intracellular processes. It is especially useful in brain slice preparations, where cells can be imaged deep in the slice, away from cut surfaces and potentially injured cells. Thus, we took advantage of better spatial and temporal resolution using ultraviolet (UV)-laser-scanning confocal microscopy to image optically the responses to GABAA receptor activation in various types of individual neurons, simultaneously, within their native environment. The purposes of this study were to: (1) characterize the measurement of GABAA receptor responses using confocal microscopy and the Cl−-sensitive dye, MEQ; (2) calibrate the change in MEQ fluorescence intracellularly in a brain slice preparation; and (3) demonstrate rapid changes in [Cl−]i following GABAA activation. These results have been presented in preliminary form (Inglefield and Schwartz-Bloom, 1996).
Section snippets
Synthesis of dihydro-MEQ
The reduction of MEQ (Molecular Probes, Eugene, OR) to a cell- permeable form, 6-methoxy-N-ethyl-1,2-dihydroquinoline (dihydro-MEQ) using sodium borohydride was described previously (Biwersi and Verkman, 1991), with modifications by our laboratory (Schwartz and Yu, 1995). Prior to each experiment, the dried dihydro-MEQ extract (under N2 at -20°C) was re-suspended in 15 μl of ethyl acetate and used immediately. For optimal use with confocal imaging, the dried dihydro-MEQ extract should be used
Biophysics of confocal imaging of MEQ-loaded cells
MEQ fluorescence has been characterized previously in a variety of biological preparations (cultured cells and slices) using fluorescence photometry and epifluorescence microscopy (Biwersi and Verkman, 1991; Schwartz and Yu, 1995). This dye has high Cl− sensitivity, relatively low toxicity, slow leakage rate (<10%/h), and resists photobleaching when exposed to incoherent light (Biwersi and Verkman, 1991). To determine the extent of photochemical bleaching (photo-bleaching) by the UV laser
Discussion
We have developed a confocal imaging method to measure Cl−- sensitive MEQ fluorescence within individual neurons in living brain slices. With confocal imaging of bath-loaded slices, we could measure, simultaneously, the GABAergic responses of several neurons within the field of view. Direct, independent evidence that changes in MEQ fluorescence were a measure of changes in [Cl−]i was provided by the calibration procedure. The Kq of 16 M−1 is in agreement with values calculated from other cell
Acknowledgements
This work was supported by a Fellowship from the American Heart Association, North Carolina Affiliate, Inc. to J.R.I., and NIH grant NS 28791 to R.D.S. We wish to thank Drs George Augustine, Victor Nadler, and Elizabeth Finch for helpful discussions relating to the development of the technique.
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