Unexpected water rewards. Mice were water restricted for at least 3 days (1 ml water per day, making sure their weight always stays above 75% of their pre-water-restricted weight but below 85%). Then mice received unexpected water rewards while head fixed through a waterspout placed directly in front of their mouth but not touching it, though close enough for their tongue to reach comfortably. The spout was connected to a syringe filled with water and placed ~1 meter above the spout for gravity to push water through the tubbing, and was gated electronically through a solenoid valve, which was accompanied by a short ‘click’ noise. The water spout was connected to a sensor to detect when the mouse licked.
Mice were first trained to associate the spout with water by delivering a drop of water and pushing the spout closer to the mouse's mouth until they touched the water. This was done a couple times until the mice started to lick spontaneously.
Then mice were trained to associate the click noise with the water reward by delivering small rewards (4 μl) at random (10-30s intervals). Mice were considered trained when they licked consistently after the reward delivery times with a very short delay (reaction time). Usually a single 45 min session is enough for mice to learn.
This solenoid's click noise can be silenced by placing the solenoid inside a noise cancelling container. However, this prevents the mice from immediately realizing a reward is available, and will result in a delay in reward consumption that must be accounted for during analysis.
Large rewards - small 4 μl rewards were used for training, but during recording sessions 1/3 rewards were larger (16 μl). This was used for testing whether neuronal responses scaled for larger unexpected rewards.