Fiber photometry analysis of spontaneous dopamine signals: The z-scored data are not the data

Fluorescent sensors have revolutionized the measurement of molecules in the brain, and the dLight dopamine sensor has been used extensively to examine reward- and cue-evoked dopamine release, but only recently has the field turned its attention to spontaneous release events. Analysis of spontaneous events typically requires evaluation of hundreds of events over minutes to hours, and the most common method of analysis, z-scoring, was not designed for this purpose. Here, we compare the accuracy and reliability of three different analysis methods to identify pharmacologically induced changes in dopamine release and uptake in freely moving C57BL/6J mice. The D1-like receptor antagonist SCH23390 was used to prevent dLight sensors from interacting with dopamine in the extracellular space, while cocaine was used to inhibit uptake and raclopride to increase release of dopamine in the nucleus accumbens. We examined peak-to-peak frequency, peak amplitude, and width, the time spent above an established cutoff. The three methods were 1) the widely-used "Z-Score Method", which automatically smooths baseline drift and normalizes recordings using signal-to-noise ratios, 2) a "Manual Method", in which local baselines were adjusted manually and individual cutoffs were determined for each subject, and 3) the "Prominence Method" that combines z-scoring with prominence assessment to tag individual peaks, then returns to the preprocessed data for kinetic analysis. First, SCH23390 drastically reduced the number of signals detected as expected, but only when the Manual Method was used. Z-scoring failed to identify any changes, due to its amplification of noise when signals were diminished. Cocaine increased signal width as expected using the Manual and Prominence Methods, but not the Z-Score Method. Finally, raclopride-induced increases in amplitude were correctly identified by the Manual and Prominence Methods. The Z-Score Method failed to identify any of the changes in dopamine release and uptake kinetics. Thus, analysis of spontaneous dopamine signals requires assessment of the %ΔF/F values, ideally using the Manual Method, and the use of z-scoring is not appropriate.