Supplementary Figure 4: Habituation of old, young and juvenile animals to the chamber and the backpack

Old (n = 6) and young (n = 3) animals were habituated to the sound proof individual isolation chambers until stationary amounts of movement and singing were observed. Then, the backpacks were attached (day zero), backpacks were attached to old animals after 5 days and in young animals after 9 days. (a) Shown is the daily amount of movement measured by infrared video camera (motion, expressed in percent of the baseline motion (black dashed line) in old animals achieved on days [-3 -1]). The baseline value (2.27%) is an output of “Motion Detector” (range [0 100], %) averaged over light phases of days. For young animals the baseline motion (red dashed line) was given by the average motion on days [-6 -1] (5.63%). Error bars indicate s.e.m. Asterisks label days when motion significantly deviated from the corresponding baseline level (paired two-sided t-test, P < 0.05). Locomotor activity of young animals exceeded activity of old animals on all days (P < 0.05, non-paired two-sided t-test). (b) Number of song motifs produced per day. Baselines were computed by averaging number of motifs on days [-2 -1] in old birds and on days [-4 -2] in young birds. Asterisks mark significant deviations from baselines as before. Number of motifs produced by old and young animals differed on some days. (c) Number of calls per day. Number of calls in young group increased monotonically during 9 days of habituation to the chamber and did not reach saturation. For this reason we took the value on day -1 as estimate of baseline in young animals. Baseline calling in old animals was estimated as average on days [-2 -1]. Red horizontal bars along time axis indicates significant difference between old and young animals (P < 0.05, non-paired two-sided t-test). Notably, the rate of calling increased in young birds during habituation to the backpacks (days [1 9]) in roughly similar manner as during habituation to the chambers (days [-9 -1]). This means that the severity of calls disturbance in young birds caused by the backpacks is similar to the disturbance caused by the isolation chambers. (d) On two juveniles we attached 2.0 g backpacks on post-hatch-days 39 and 46 (time zero on x-axis). In the first juvenile (black) the backpack hardly decreased singing rate; in the second juvenile (red) the singing rate was transiently suppressed and picked up two days later. Both birds consistently produced more than 10’000 song syllables and introductory notes per day just 3 days after backpack attachment, suggesting that our recording technique is permissive of song learning studies in juveniles.