Intracellular calcium measurements were performed by loading the cells with the fluorescent indicator, Fura-2AM, concomitantly with the application of high a KCl solution.
In the case of cultured PC12 and primary hippocampal cells, the vitality and responsiveness of neurons was assessed at the end of the imaging protocol by application of 54 mM KCl. KCl-mediated depolarization causes large, rapid and reversible Ca2+ transients in vital neurons.
In addition to orthoclase dissolution, excessive application of KCl as a fertilizer may have contributed to K+ and Cl− loadings in the groundwater (Lee et al. 2005).
Repetitive CSDs were induced in vivo by topical application of solid KCl.
However, due to the shortage of storage vesicles, no apparent release of stored tracers could be observed after the application of high KCl buffer compared to controls (Fig. 1b).
This is consistent with a two-step depolarization: an incremental depolarization by Conk-S1 and an additional depolarization on application of KCl, each associated with an increment in Ca2+ influx.
This amplitude was then compared to the current amplitude of an acute application of 30 mM KCl in the same cell (Figure 4A and 4B).
Topical application of KCl to the cortex triggered a series of DC shifts and cortical hyperemias characteristic of CSD.
For comparison with fluorescence approaches, we began these studies by using similar protocols as those described previously [4], [5], and Ca2+-responses were recorded with whole brain bioluminescence imaging after a short bath application of KCl (70 mM) (Figure 1B-D, movie S1).
A neuron was included in the analysis if the peak of the response from a 30 s reference application of 30 mM KCl was larger than threshold, defined as six S.D. of the baseline noise.
