Supplementing water rewards with sucrose increases the number of trials performed by mice.

<p>A. Example experiment, with water (black circles) and sucrose (red circles) rewards provided on alternating sessions. B. The number of trials is 23% larger with sucrose (p<0.001 in two mice; n.s. in the third). C. The number of rewards per session is larger (p<0.001 in two mice; n.s. in the third). D. The discriminability index is unchanged.</p

Performance of the lick-left/lick-right object location discrimination task with a delay epoch (data from Figure S1 [7]).

<p>A. Schematic of time-course of experiments. B. Learning curves showing the performance. Thin lines correspond to individual mice. Thick lines, average. Colors correspond to whisker trimming. Vertical dashed line indicates when the delay epoch was introduced. The four mice were from the same litter (2 males and 2 females). Same as Figure S1B in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088678#pone.0088678-Guo1" target="_blank">[7]</a>. C. Learning curves showing the discriminability index, d'. D. Bias: performance of lick-right trials minus performance of lick-left trials. Same as Figure S1C <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088678#pone.0088678-Guo1" target="_blank">[7]</a>. E. The fraction of trials with licking responses during the sample or delay epoch. Same as Figure S1D <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088678#pone.0088678-Guo1" target="_blank">[7]</a>. F. Water consumed. G. Trials per session. H. Health score. A health score larger than 3 (dashed line) triggers more detailed evaluation and possibly water supplements. I. Health score for four mice that were under water restriction for four months. A health score larger than 3 (dashed line) triggers more detailed evaluation and possibly water supplements.</p

A lick/no-lick object location discrimination task for head-fixed mice [23].

<p>A. Block-diagram of the possible events in a single trial. B. Schematic representation of event timing during a single lick trial. C. Schematic representation of the behavioral contingency. Mice had to lick for a water reward when the pole was in a posterior position and hold their tongue when the pole was in an anterior position. In some experiments, the contingency of the pole positions was reversed. D. Behavioral data from one session. The abscissa shows the time from trial start. Lick and no-lick trials are randomly interleaved. The pink ticks indicate licks. The red ticks indicate the first licks after the grace period. The blue bars correspond to the open times of the reward water valve. The horizontal green and red bars indicate whether each trial is correct or incorrect, respectively. The dark gray shading indicates that the pole is fully descended and in reach of the whiskers.</p

Normalized weight of 5 female mice after the initial water restriction (left) and after one day of free access to water (dotted line, day 0).

<p>Normalized weight of 5 female mice after the initial water restriction (left) and after one day of free access to water (dotted line, day 0).</p

Apparatus for head-fixation.

<p>A. Left, two types of titanium head plates. Right, stainless steel head bar holder and clamp (only one of two sides is shown). The head plate is inserted into notches in the holder and fastened with the clamp (right, top) and a thumbscrew (not shown). The simple head bar (left, top) is used when access to large parts of the brain is necessary. The larger head plate (left, middle) provides better stability. The simple head bar was cemented to the skull of the mouse (left, bottom). The head of the mouse (top view) was pointing downward. The skull was outfitted with a clear skull cap <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088678#pone.0088678-Guo1" target="_blank">[7]</a>. The head bar was aligned at the lambda sutures. The red dot indicates the location of bregma. B. Plexiglass body tube used for head-fixed mice. Mice rest their front paws on the front ledge. The bottom of the tube is coated with aluminum foil to produce electrical contact for electric lickports. The aluminum foil is connected to the red banana socket which will be connected to electric lickports for detecting licking events. C. Example caddy used in training apparatus, assembled from standard optomechanical components (Thorlabs). The head bar holder is mounted towards the left. D. A head-fixed mouse in the caddy.</p

Mice with one or more indicators of stress or pain are placed on detailed health assessment.

<p>Activity levels, grooming, and indicators of eating and drinking are scored daily in a health assessment sheet. The total aggregate health score determines if mice are supplied with additional water (see flowchart in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088678#pone-0088678-g002" target="_blank">Figure 2</a>).</p

Mouse weight and health during water restriction.

<p>All mice were trained in a lick/no-lick object location discrimination task using a single whisker (same mice as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088678#pone-0088678-g002" target="_blank">Figures 2</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088678#pone-0088678-g003" target="_blank">3</a> of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088678#pone.0088678-OConnor2" target="_blank">[18]</a>). Rewards consisted of approximately 8 µl of water per trial. A. Experimental time-course for one example mouse, from the beginning of water restriction to the end of electrophysiological recordings. An 85 day old mouse (25.4 g) was put on water restriction for eight days, followed by training (starting on day 9) and recording (starting on day 28). B. Body weight as a function of time. Same mouse as in A. The dashed line indicates 30% weight loss. C. Water consumed per day. After start of training mice mostly received their water during the training session. A larger number of correct trials will lead to more consumed water. Same mouse as in A. D. Health score as a function of time. A health score larger than 3 (dashed line) triggers more detailed evaluation and possibly water supplements. Same mouse as in A. E. Experimental time-course for a group of 5 mice. Same format as A. F. Average body weight of 5 mice (black line) and 2 mice with free access to water (grey line). Shading indicates standard deviation. Experimental time-course for all mice was similar, but not identical to A. G. Average water consumed. H. Average health score.</p

Performance as a function of normalized body weight.

<p>A. Performance as a function of normalized body weight. Each circle corresponds to one behavioral session. Different colors correspond to different mice (7–8 sessions per mouse). The sessions included are the first seven to eight sessions of discrimination training (corresponding to the training phase shown by open symbols in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088678#pone-0088678-g003" target="_blank">Figure 3a</a> of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088678#pone.0088678-OConnor4" target="_blank">[23]</a>. Multiple factors can compromise performance in behavioral experiments. In this experiment mice were trained in serial with individualized attention to reduce variability due to uncontrolled factors. The correlation coefficient is R² = 0.52 (<i>p</i><0.001). B. Number of trials as a function of normalized body weight. Mice usually perform less trials in the first few sessions of training. Same sessions as in (A). The correlation coefficient is R² = 0.24 (<i>p</i><0.001).</p

Performance of the lick/no-lick object location discrimination task.

<p>A. Time-course of experiments. B. Learning curves showing the discriminability index, d'. Thin lines correspond to individual mice. Thick lines, average. Red, recording sessions. C. Learning curves showing the fraction of correct trials. D. Water consumed. E. Health score. A health score larger than 3 (dashed line) triggers more detailed evaluation and possibly water supplements.</p

Key stages in mouse handling.

<p>A. Mouse eating a sunflower seed on the experimenter's hand. The pins emanating from the top of the mouse head correspond to ground and reference electrodes for extracellular recordings. B. Mouse being familiarized with the body tube. C. Mouse receiving a water reward in the body tube.</p