Thursday, March 22, 2012

Notes on mouse licking

We recently got two things I've been waiting for: transgenic mice, and a lickometer. In anticipation of recording from taste cortex, I've started training mice to do a simple licking task: lick 9+ times following a short tone. (An FR5 lick schedule, borrowed from Sid Simon's lab.) (Doing this has made me realize the unique nuisance of investigating taste: unlike, other sensory systems, like olfaction or whisking, you need to train your mice before every experiment.)

Since the task is so simple, I designed a short, straightforward training protocol (see below). The first goal (Stage 1) was to get the mouse to lick following a tone. To reinforce this behaviour, every time the mouse licks within the "lick window," they get a water reward of 1-2uL (the mice are, of course, water deprived). Once the mice learn to lick after the tone, I gradually decrease the number of rewarded licks, until they only get water for a single lick in the middle of the window (Stage 2). To ensure they lick the whole time, the mice get a water reward after 9 licks. Finally, once the mice have completed Stage 2, we are ready to try (potentially aversive) tastants in the middle of the task (Stage 3). Once the mice perform Stage 3 correctly, we can record.
Learning protocol. (Top) Tone timecourse. (Middle) Liquid dispensed during licks. H = water; T = tastants. (Bottom) Example lick history. In Stage 1, each lick during the "lick window" is rewarded with water. In Stage 2, we gradually reduce the number of rewarded licks until only a single lick in the middle is rewarded. After nine licks, the mice get a water reward as well. In Stage 3, we give tastants in the middle of the lick window, instead of water.

Since we just got the lickometer equipment, I ran a pilot study with two mice, for two weeks. At the end of the two weeks, both mice were performing Stage 2.2 fairly well. Here is the lick-o-gram for the last 200 trials of training for Mouse 2 (M2):

Raster plot of licks during task. Mice were cued by a 200ms, 4kHz tone. Following that, they had a 2s lick window (blue line). In the lick window, they received water (1-2uL) on the fifth lick, and every lick from #10 onward.
Some observations:

1. Individual mice behave differently. The mice we're using, C57/Bl6, have been inbred for over twenty generations, and are practically clones. Despite the genetic similarity, the two mice I trained had different behaviour. Mouse 1 (M1) didn't hide in the behaviour tube, and refused to lick the water spout at first. In contrast, M2 hid in the behaviour tube, and had to be coaxed out; and quickly started licking the water spout. I don't know whether these differences are due to epigenetics, genetic drift, small differences in how I handled them, or something inscrutable. But it did surprise me.

2. The best way to get a mouse to move forward is to (gently) pull his tail.

3. Mice may solve your task in unexpected ways. The goal of training was for the mice to lick following the tone. M2 didn't quite understand this, but did know that if he licked a lot, he would get water. So he just licked continuously, ignoring the tone, and got his water. While I don't mind this in practice - since I just want them to lick - I wanted to see how well they could learn the complete task, so I extended the inter-trial interval, and M2 stopped licking constantly.

Continuing this theme, I do not think the mice quite understood that the tone preceded the lick window. In the lick-o-gram above the mouse stopped licking after 4 seconds, and restarted after 7 seconds. This makes me believe the mouse knew there was water every ~10s, rather than being cued by the sound. Also notice, the mouse did not really perform the task for the first 5 or so trials, then did well. MICE, WHAT ARE YOU THINKING!?!?

4. Motivation happens in bouts. The mice were water deprived before the experiment, and performed quite eagerly for the first 100 or so trials. However, after a while they would stop, only to restart the task for 5-10 trials. Two hypotheses: licking thousands of times gets tiring after a while; or a few hundred uL of water slakes their thirst for a bit.

5. When shaping mouse behaviour, you must move step-wise. In moving from Stage 1 to Stage 2, I started by removing the reward for licks 6-9, and the mice continued to perform pretty well (in general, they seem to lick for a few seconds after free-licking time). When removing the reward for the initial licks, the mice continued to perform when I dropped the first lick, but if I stopped rewarding the first two licks, they gave up, and simply stopped licking. So I will have to be patient when removing the initial reward licks.

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