USE OF BCECF AND PROPIDIUM IODIDE TO ASSESS MEMBRANE INTEGRITY OF ACUTELY ISOLATED CA1 NEURONS FROM RAT HIPPOCAMPUS

We used 2 fluorescent dyes, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluore scein (BCECF) and propidium iodide (PI), to assess the membrane integr ity of neurons acutely isolated from the CA1 region of the rat hippoca mpus. Exciting BCECF at a relatively pH-insensitive wavelength (440 nm ), or exciting PI at 490 nm, we quantitatively recorded, in real time and in single cells, the rate constants for BCECF loss (-k(440)) and P I uptake (k(490)). We found that approximately 98% of intracellular BC ECF is rapidly released by applying 0.01% saponin. In neurons not trea ted with saponin, rate constants for BCECF loss and PI uptake typicall y were 1% min(-1) or less under control conditions, in the presence of NH3/NH4+ and in the absence of Na+. However, in a small number of neu rons, the rate constant for BCECF loss increased markedly (-k(440) > 5 % min(-1)), while pH(i) approached pH(o), suggesting that the plasma m embrane spontaneously became leaky. When neurons were progressively sw ollen in hypotonic solutions, rates constants for BCECF loss and PI up take generally were affected minimally unless osmolality was decreased to similar to 75 mOsmol/kg. Treating neurons with 0.001% saponin caus ed an increase in PI uptake rate only in a minority of neurons, wherea s in most experiments a similar treatment caused - k(440) for BCECF to exceed 5% min(-1), and led to a rapid deterioration of the pH gradien t across the cell membrane. At even lower saponin levels (0.0005-0.000 7%), we observed a much slower deterioration of pH(i), which occurred at low rates of BCECF loss (-k(440) = similar to 3% min(-1)). We concl ude that computing rate constants for BCECF loss and PI uptake may be useful for assessing neuronal health, and that BCECF loss may be more sensitive to cell damage than PI uptake.