We used linear mixed models,53 which are extensions of linear regression models for data that are summarized in groups, to describe the relationship between the pupil response during the stimulation and independent variables, namely the baseline pupil size, the number of licks in each bout, the locomotor speed during stimulation, the duration of the stimulation and the interactions between the stimulation and the other three variables. Separate models were run with bouts from SERT-Cre and WT mice (in Figure 3) or with bouts from different protocols of SERT-Cre mice (in Figure 4), with coefficients that varied with respect to the different sessions (i.e., the random effect). Specifically, in these models, the parameters to estimate were the fixed-effects coefficients β applied to the predictors and the variance components of the random-effect associated with individual sessions, using maximum likelihood. The models were implemented using the linear mixed-effects model function in MATLAB (fitlme). In Figure 3, the analysis was restricted to bouts where the stimulation was at least 2.5 s, which is approximately the minimum stimulation duration that yields a significant acute effect (see Figure 2G). Predictors were z-scored before fitting the data and an intercept was included in the model. To check whether the distributions of the predictors’ values were different between SERT-Cre and WT animals, we used those values in a generalized linear model (GLM) to predict whether each trial belonged to a session from a SERT-Cre or a WT mouse. We estimated the predictive power of the model using the area under the curve (AUC) of the receiver operating characteristic (ROC) estimated on testing data (10-fold for cross-validation). We then compared the value we obtained with AUC estimated shuffling mouse identity (SERT-Cre or WT) session by session. We found that the non-shuffled AUC was not statistically different from the shuffled ones (p = 0.08), suggesting that the values of the predictors alone were not informative about the genotype of the animals.

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