Seventeen mice (11 male and six female wild-type Thy1-GCaMP6, C57BL/6 background, 2-3 months) were single-housed. Each animal’s baseline weight was recorded on five consecutive days, while they had free access to plain water in their home-cage. They were then divided into three groups (with males and females randomly assigned), each with its own water regime. The first group (n = 8) received measured amounts of water each weekday. This was either 600 μl, or 40 μl of water per gram of body weight as measured right before water administration (range 600–1150 μl). On Saturday they received double this amount, while no water was administered on Sundays. The second group (n = 5) had access to free water in their home-cage, in which CA (1% or 2%) was dissolved. The last group (n = 4) remained on free water in their home-cage and served as a control. No animals showed signs of dehydration, or were dropped from the study.

The home-cage bottles with regular or 2% CA water were weighed daily, and the weight change used as a proxy for the volume of water consumed. To measure thirst, each animal was placed in a cage with 2 ml of plain water for 5 min on days 16, 24, and 29. For the animals on measured water, these 5 min replaced their daily measured water administration.

Mice in this study were on average nine weeks old on the first day of the experimental intervention. Expected weight curves were computed from weighings of age-matched animals not undergoing fluid restriction, from The Jackson Laboratory (2015).

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