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Thus the total number of flies used for the curve is 50�C100). The standard PER assay was performed at room temperature (23��C �� 2��C). Mean acceptance thresholds (MATs) (Long et?al., 1986) were calculated as described below in the section ��Calculation of Mat. For TrpA1 experiments, flies were raised at 21��C. Flies were mounted at 23��C, and PER assays performed at either 23��C or 30��C.?The PER test was performed immediately after transferring the flies to different temperature and finished within 10?min. Th-GAL4;UAS-dTRPA1 flies showed partial proboscis extensions without sugar stimulation at 30��C but rarely showed full extensions without the stimulation. Only full extensions were scored as positive responses. In order to calculate MAT, sugar concentration where 50% of flies [http://www.selleckchem.com/products/pd-0332991-palbociclib-isethionate.html PD0332991 datasheet] show PER, sigmoid interpolation was performed (PER response curves were fitted into sigmoid curves). The sigmoid curve can be described as a logistic [http://www.selleckchem.com/products/Trichostatin-A.html TSA HDAC mw] function: Ss=11+e(?��slog2SconMAT). Ss: fraction of flies showing PER on different sugar concentration. Scon: sugar concentration (mM). MAT: sugar concentration where 50% of flies show PER. ��s: slope of the sigmoid curve. For all experimental data, polynomial curve fitting, which finds the coefficients that fit the data by the least-squares method, was done with Matlab (MathWorks). Goodness-of-fit was tested by two-way ANOVA between the actual PER response curves, and the sigmoid curve with the calculated coefficients (Figure?S3B). All wild-type sucrose response data were well-fit by a sigmoid curve (two-way ANOVA). The value of MAT decreased with starvation time, while ��s shows a statistical significant decrease after 2?days of starvation or L-dopa feeding (p?[http://en.wikipedia.org/wiki/PTEN_(gene) PTEN] indicates the increase in behavioral sugar sensitivity, and decrease in ��s implies (1) increase in distinguishability between two sugar concentration, (2) increase in sugar sensitivity at lower sugar concentration, or (3) increase in variation in sugar sensitivity among flies. Because we are interested in the changes in behavioral sugar sensitivity, MAT was used for data analysis. It should be noted that the use of MAT as a metric for comparison does not require that the PER response be titrated to saturation for each experimental or control condition, because MAT is a probabilistic measure (i.e., it measures the sucrose concentration at which 50% of the flies in a population are likely to show a PER response), whose value by definition therefore ranges between 0 and 1 ( Long et?al., 1986). The values of MAT are normally distributed among the data obtained from wild-type flies under the same condition of food deprivation (Lillifors test and Jarque-BARA test, and also checked by linearity in a probability plot). Thus parametric statistical tests were used for analysis of MAT data.