To assess nuclear (spindle) movement, images of yeast cells expressing GFP-Tub1p were taken at 30 sec intervals for a total of 60 min, with an exposure time of 500 msec at each focal plane.
To examine nucleation and/or elongation activity, images of yeast cells expressing Bik1p-GFP were taken at 10 sec intervals for a total of 15 min, with an exposure time of 500 msec at each focal plane.
To measure parameters of dynamic instability, images of yeast cells with integrated GFP-TUB1 and BIK1-3GFP were taken at 3.5 sec intervals for a total of 5 10 min, with an exposure time of 600 msec at each focal plane.
Tweezer electrodes (BTX Tweezertrodes Model #522) connected to an electrostimulator (ElectroCellManipulator™, ECM® 830, BTX, a division of Genentronics), were placed on opposite sides of the kidney and 20 bipolar electrical pulses administered, 200 volts, each lasting 20 msec, at 1000 Hz after both injections.
After an additional 5 min of dark adaptation, rod response was measured by averaging 4 dim light flashes of 80 cd.m−2×0.12 msec at >2 sec interval.
Finally, after 10min light adaptation (25 cd.m−2), average single-flash cone response was determined from 4 flashes of 6,000 cd.m−2×0.5 msec at >15 sec interval.
The 360 nm excitation scan was repeated for another 500 msec at the end of the protocol and qualitative changes in intracellular Ca2+ concentration ([Ca2+]i) were inferred from the ratio of the fluorescence intensity at two wavelengths (360/380).
Then mice were further dark adapted for 3 min and maximal combined (rod and cone) response and oscillatory potential were determined from the average of responses to four flashes of 6,000 cd.m−2×0.5 msec at >10 sec inter-stimulus interval.
Maximum mean changes were 7.6 msec at 4 h, 3.1 msec at 2 h, and 4.5 msec at 4 h for 8, 10, or 12 mg/m, respectively.
Spike duration (measured at half-width of the action potential (AP), became shorter with prolonged differentiation, from 3.31±0.16 msec at day 23, to 2.57±0.10 msec at day 30, to a mean duration of 1.95±0.20 msec at 37 days.
Thus, 24 mg ondansetron likely results in a QTc effect of <5 msec at 4 h.
