Quantitative and Statistical Analysis of the Shape of Amperometric Spikes Recorded from Two Populations of Cells

Abstract

Previously used methods of comparing amperometric spike characteristics from two separate groups of cells have entailed pooling all the values for a spike characteristic from each group of cells and then statistically comparing the two samples. Although this approach has indicated that there are significant differences between the spike characteristics from coloboma and control mouse chromaffin cells, the results are not consistent between experiments. We have reexamined the assumptions of the statistical tests used as well as the variability inherent in amperometric data measured from two groups of cells. Our findings indicate that when comparing amperometric spike characteristics between groups of cells, it is more appropriate to compare samples of mean spike values. This method consistently indicates that there is no difference between coloboma and control amperometric spikes. These results have been validated by using samples of mean spike characteristics to detect changes in the shape of amperometric spikes from both mouse chromaffin cells at 37°C and PC12 cells previously exposed to 50 M L-3,4-dihydroxyphenylalanine and by the use of an additional analysis method, the nested ANOVA. Together, these results indicate that pooled samples of amperometric spike characteristics can give results that may confound the interpretation of amperometric data.

VMAT-Mediated Changes in Quantal Size and Vesicular Volume

“…The same electrode was used to measure release from a cell before and after drug treatment. This approach minimizes cell-to-cell and electrode variability and, as a result, should be more sensitive to changes in spike characteristics as compared with measuring release from separate groups of cells (Pothos et al, 1998a;Colliver et al, 2000). Additionally, with this technique it is possible to record from a cell within a fairly consistent time after treatment.…”

“…Because means from normal distributions should be less biased by outliers, we have created mean spike ratios from log-transformed values (Pothos et al, 1998b;Colliver et al, 2000). To create spike characteristic ratios, we determined the area, t 1/2 , and I max values at a cell before (pre) and after (post) the incubation period.…”

“…To ensure that cells with a large number of vesicles would not be over-represented within a treatment group, we calculated mean values for vesicle structures from each cell, and we statistically compared samples of mean values for different treatment groups, instead of pooled samples (Colliver et al, 2000). Amperometric spike ratios and means of vesicle structures were tested for significant differences by using the Student's t test (SigmaStat Version 2.03, SPSS).…”

Estradiol Inhibits Depolarization-Evoked Exocytosis in PC12 Cells via N-Type Voltage-Gated Calcium Channels

“…Peaks were counted and characterized with the MiniAnalysis software detection algorithm (Synaptosoft, Decatur, GA). Peaks were detected if both the amplitude of local maxima and the area under the curve exceeded a threshold of five times the root-meansquared noise for a flat, 2-s recording acquired at the beginning of each experiment (Colliver et al 2000). Peaks were visually inspected to confirm that electrical noise was not included and to include peaks manually that were not detected because of their proximity in the current trace.…”

“…Because the number of evoked peaks per cell can vary from 20 to 400 events, cells are best represented by averaging ratios from each cell rather than pooling pre-and posttreatment peaks from all cells prior to averaging (Colliver et al 2000;Westerink et al 2000), and were thus analyzed in this manner. Two-tailed t tests were used to compare all treatment groups, and Dunnett's t test was used to compare a range of E2 concentrations to control; P-values \0.05 were considered significant.…”