Characterizing the anticipatory activity in the home cage of male mice with one hour of restricted access to a novel female daily (“Experiment #1”).

<p>(<b>A</b>) The number of seconds in each hourly bin of high activity behaviors (walking, jumping, rearing, and hanging) that occurred on day 14 of restricted female access. Thin weight green lines represent data from individual mice with restricted female access while blue lines represent the controls. Median values are shown in thick green and blue lines for experimental and control mice, respectively. The gray area depicts the 11 hours of lights off. (<b>B</b>) An enlarged presentation of ZT 5 to 9, the four hours preceding female access, in panel A, to show the variance in the anticipatory behavior. (<b>C</b>) The number of seconds in each hourly bin of high activity behaviors that occurred on day 28 of the experiment. (<b>D</b>) An enlarged representation of bins 6 to 9 (which includes data from ZT 5-9) in C. (<b>E</b>) The sum of the number of seconds spent in any high activity behavior (walking, jumping, rearing, and hanging) during the last four hours prior to restricted female access. (<b>F</b>) Fraction of frames during the entire duration of the recording (∼23 hrs) during which the mice exhibited any high activity behavior. (<b>G</b>) Normalized high activity data in the four hours preceding restricted female access. The number of seconds spent in high activity behavior during the last four hours prior to female access divided by the total number of seconds of high activity across the entire 23 hour recording. (<b>H</b>) Table illustrating the relative strength of anticipatory activity versus nighttime activity for each individual mouse at every measurement in the experiment. For each animal, the maximum number of seconds per hour the mouse exhibits between ZT 5 and 9 is divided by the maximum number of seconds per hour the mouse exhibits during the lights off period. A ratio of greater than 1.1 is highlighted in red, between 1.0 and 1.1 in orange, between 0.9 and 1.0 in yellow, between 0.8 and 0.9 in light green, 0.7 and 0.8 in blue, 0.6 and 0.7 in purple, and less than 0.6 is white. All bar graphs represent median and the upper quartile of the interquartile range. N = 6 at all time points. Mann-Whitney Test, * = p<0.05, ** = p<0.01, *** = p<0.001.</p

Characterizing the anticipatory activity in the home cage of male mice with one hour of restricted access to a female partner daily (the same female for seven consecutive days, “Experiment #2”).

<p>(<b>A</b>) The number of seconds in each hourly bin of high activity behaviors (walking, jumping, rearing, and hanging) that occurred on day 14 of restricted female access. Thin weight green lines represent data from individual mice with restricted female access while blue lines represent the controls. Median values are shown in thick green and blue lines for experimental and control mice, respectively. The gray area depicts the 11 hours of lights off. (<b>B</b>) An enlarged presentation of ZT 5 to 9, the four hours preceding female access, in panel A, to show the variance in the anticipatory behavior. (<b>C</b>) The number of seconds in each hourly bin of high activity behaviors that occurred on day 21 of the experiment. (<b>D</b>) An enlarged representation of bins 6 to 9 (which includes data from ZT 5-9) in C. (<b>E</b>) The number of seconds in each hourly bin of high activity behaviors (walking, jumping, rearing, and hanging) during the last four hours prior to restricted female access. (<b>F</b>) Fraction of frames during the entire duration of the video recording (∼23 hrs) during which the mice exhibited any high activity behavior. (<b>G</b>) Normalized high activity data in the four hours preceding restricted female access. The number of seconds spent in high activity behavior during the last four hours prior to female access divided by the total number of seconds of high activity across the entire 23 hour recording. Quantifying the social interaction during the restricted female access period. The number of bouts in which each male mouse interacted with its respective female by sniffing the body, chasing, mounting, or thrusting on (<b>H</b>) day 15 is displayed as a scatter plot, with the solid line indicating the median value. (<b>I</b>) Sexual/social interaction on day 28 with the same female partner for the previous six days. (<b>J</b>) Interaction on day 29 with a novel female. (<b>K</b>) Table illustrating the relative strength of anticipatory activity versus nighttime activity for each individual mouse at every measurement in the experiment. For each animal, the maximum number of seconds per hour the mouse exhibits between ZT 5 and 9 is divided by the maximum number of seconds per hour the mouse exhibits during the lights off period. A ratio of greater than 1.1 is highlighted in red, between 1.0 and 1.1 in orange, between 0.9 and 1.0 in yellow, between 0.8 and 0.9 in light green, 0.7 and 0.8 in blue, 0.6 and 0.7 in purple, and less than 0.6 is white. n = 8 at all time points. All bar graphs represent median and the upper quartile of the interquartile range. * = p<0.05, ** = p<0.01, *** = p<0.001, Mann-Whitney Test.</p

Testing the effect of restricted running wheel on anticipatory activity.

<p>(<b>A</b>) Mice have access to a “low profile” running wheel or a (<b>B</b>) a shelter dome or have their bedding slightly disturbed as another control group (not shown). (<b>C</b>) The total number of seconds of high activity during the four hours prior to receiving running wheel access. (<b>D</b>) The fraction of frames during the entire twenty-two hours of recording during which the mice exhibited the high activity behaviors. (<b>E</b>) Normalized anticipatory activity. The number of seconds during which the mice exhibited high activity behaviors in the four hours preceding wheel or dome access or bedding disturbance is divided by the total number of seconds of high activity observed in the entire twenty-two hour recording period. (<b>F</b>) Food bin entry during the four hours preceding running wheel access. (<b>G</b>) The amount of time the mouse was awake during running wheel access, dome access, or after bedding disturbance. For wheel access mice the sum of the time it spent running on the wheel, interacting with the dome or wheel, or awake but not interaction is equivalent to time awake (Kruskal-Wallis Test, * = p<0.05, ** = p<0.01) * = p<0.05, Kruskal-Wallis Test.</p

Tracking the positions of two mice during social interaction.

<p>(<b>A</b>) The tracking software is able to locate the center of masses of both mice while at different positions in the cage. Representative images of the tracker are shown, showing various social interactions between male-female pairs in the top panels and female-female pairs in the bottom panels. (<b>B</b>) The fraction of frames of a video during which the male and female mouse are within a certain number of mouse widths away from each other. The dark solid line represents the median fraction of frames the mice were within a certain distance and the shaded boundaries represent the upper and lower quartile across the individual mouse pairs for that day. Day 15 (interaction with a novel female) is shown in dark blue, day 28 (interaction with a female for the seventh day in a row) in cyan, and Day 29 (interaction with another novel female) in olive. (<b>C</b>) The fraction of frames of a video during which the two female mice are within a certain number of mouse widths away from each other. Day 0 is shown in dark blue, day 7 in cyan, day 14 in pale green, day 21 in olive, and day 28 in orange. (<b>D</b>) The fraction of frames during which the male-female mice are within two mouse widths (approximately equal to one mouse length). Each point in the scatter plot represents the value for one video (one mouse interaction at one time point). (<b>E</b>) The fraction of frames during which the female-female mice are within two mouse widths.</p

Characterizing the anticipatory activity of female mice with one hour of restricted access to a stable female partner daily (“Experiment #3”).

<p>(<b>A</b>) The number of seconds in each hourly bin of high activity behaviors (walking, jumping, rearing, and hanging) that occurred on day 14 of restricted female access. Thin weight green lines represent data from individual mice with restricted female access while blue lines represent the controls. Median values are shown in thick green and blue lines for experimental and control mice, respectively. The gray area depicts the 11 hours of lights off. (<b>B</b>) An enlarged presentation of ZT 5 to 9, the four hours preceding female access, in panel A, to show the variance in the anticipatory behavior. (<b>C</b>) The number of seconds in each hourly bin of high activity behaviors that occurred on day 21 of the experiment. (<b>D</b>) An enlarged representation of bins 6 to 9 (which includes data from ZT 5-9) in C. (<b>E</b>) The number of seconds in each hourly bin of high activity behaviors (walking, jumping, rearing, and hanging) during the last four hours prior to restricted female access. (<b>F</b>) Fraction of frames during the entire duration of the video recording (∼23 hrs) during which the mice exhibited any high activity behavior. (<b>G</b>) Normalized high activity data in the four hours preceding restricted female access. The number of seconds spent in high activity behavior during the last four hours prior to female access divided by the total number of seconds of high activity across the entire 23 hour recording. Quantifying the social interaction during the restricted female access period. The number of bouts in which each female mouse interacted with its respective female partner by grooming, being groomed, sniffing the body, being sniffed, chasing, repeated pawing, or wrestling on day 0 (<b>H</b>), day 7 (<b>I</b>), day 14 (<b>J</b>), day 21 (<b>K</b>), and day 28 (<b>L</b>). Each point on the scatter plot represents the number of instances in which an individual mouse exhibited that behavior. The solid horizontal line represents the median value.</p

Diagrammatic representation of experiments.

<p>“Experiment #1” comprises conditions used at Simon Fraser University measuring activity mainly with running wheels and “Experiment #2” comprises conditions used at Caltech using computer vision to assess activity. Days of treatments are indicated as numbers below the colored bars, red represented calorie restriction, blue for fruit crunchies, brown for chocolate, white for no treatment, and yellow for high fat diet. For experiment #1, mice were fed the palatable snack from ZT4-6. For experiment #2, male mice were provided with the palatable snack at ZT10 and the snack was not removed; for female mice the snack was presented at ZT7. RF, restricted feeding; CR, calorie restriction.</p

Home cage activity and food bin entry of female mice in Experiment 2 during palatable meal access and withdrawal.

<p>(A) The fraction of all recorded frames within a 24 hour period during which the female mice were walking, hanging, jumping, or rearing. There were no significant differences in the fraction of frames during which the female mice exhibited high intensity activity. (B) The total seconds of walking, hanging, jumping, and rearing during the 2 h before the palatable meal is received. There is no significant difference between the female mice receiving a once-daily palatable meal and controls. (C) For each individual mouse the sum of the total seconds of walking, hanging, jumping, and rearing during the 2 h before the palatable meal is delivered divided by the total seconds of walking, hanging, jumping, and rearing during both recording periods within a 24 hour window. There is no significant difference between the female mice receiving a once-daily palatable meal and those that are disturbed by a pellet of normal chow. (D) The fraction of all recorded frames within a 24 h period during which the female mice inserted their nose into the food bin. The mice spent significantly less time with their nose in the food bin on all recorded days (day 0, 7, 14, 21, and 28) with the exception of the second day of withdrawal of the palatable meal. (E) The total seconds of food bin entry during the 2 h before the palatable meal is received. There is no significant difference between the female mice receiving a once-daily palatable meal and those that are disturbed by a pellet of normal chow. (F) For each individual mouse the sum of the total seconds of food bin entry during the 2 h before the palatable meal is divided by the total seconds of food bin entry during both recording periods within a 24 h window. There is only a significant difference between the female mice receiving a once-daily palatable meal and those that are disturbed by a pellet of normal chow on day 21 of the special feeding regimen. (G) The total sum of seconds of walking, hanging, jumping, or rearing observed during each hour of the 48 hour high fat withdrawal. Shaded boxes represent lights off; arrows represent expected meal time. (H) Sum of high intensity activity during the two hours following expected palatable treat access on day 1 and 2 of withdrawal. High fat entrained mice show more high intensity activity than ad libitum controls on both days of the withdrawal. (I) Total sum of seconds of food bin entry observed during each hour of the 48 hour high fat withdrawal. Shaded boxes represent lights off; arrows represent expected meal time. (J) Sum of food bin entry during the two hours following expected palatable treat access on day 1 and 2 of withdrawal. High fat entrained mice show more food bin entry than ad libitum controls on both days of the withdrawal, although it is only significantly greater on the second day. Arrows indicate the bin in which palatable meal would have been delivered. Bars show medians and interquartile ranges; the statistical test used was Mann-Whitney where * denotes p<0.05, ** denotes p<0.01, and *** denotes p<0.001. Note that no palatable meals were administered on day 28 and 29 (equivalent to withdrawal days 1 and 2), but all mice retained ad libitum access to food and water.</p

Home cage activity of male mice in Experiment 2.

<p>(A) The fraction of frames during the entire 23.5 to 24 h recording period during which the mice were walking, hanging, jumping, and rearing. Male mice receiving daily chocolate are significantly more active than <i>ad libitum</i> controls on days 7, 14, and 21. Mice receiving daily high fat are significantly more active than ad libitum controls on day 14. (B) The total seconds of walking, hanging, jumping, and rearing during the 2 h before the palatable meal is received. (C) For each individual mouse the sum of the total seconds of walking, hanging, jumping, and rearing during the 2 h before the palatable meal is divided by the total seconds spent walking, hanging, jumping, and rearing during the 23.5 to 24 h recording period. The mice fed high fat show a significantly greater difference in the fraction of high activity during this 5 h period than the mice fed chocolate. (D) The maximum number of seconds per hour of walking, hanging, jumping, and rearing in the 5 h preceding feeding is divided by the maximum number of seconds per hour of walking, hanging, jumping, and rearing at night. Ratios greater than 1 are shown in red, between 0.7 and 1 in orange, between 0.5 and 0.7 in yellow, and between 0.4 and 0.5 in green. Bars show medians and interquartile ranges. Statistical test was Kruskal-Wallis test with Dunn's post-test, * denotes p<0.05, ** denotes p<0.01, and *** denotes p<0.001.</p

Calorie restriction of ghrelin knockout mice.

<p>(A) Body weights of Ghrelin KO and WT mice on AL and CR feeding conditions. (B) Percent gain and loss of weight relative to day 0. There is no measurable difference in (C) High activity behaviors of Ghrelin KO and WT on day -7. (D) Food bin entry (E) Hang, (F) Jump, (G) Rear, (H) Walk, and (I) Sleep behaviors are not different among WT and KO groups on day -7. n = 16 for panels C–I. (J) Normalized high activity behaviors in the 3 hours preceding feeding for WT AL and WT CR. (K) Normalized high activity behaviors in the 3 hrs preceding feeding for KO AL and KO CR mice. Statistics were performed using the Mann-Whitney Test with post-test, *  =  p<0.05; **  =  p<0.01; and ***  =  p<0.001. Error bars represent IQR. n = 8–9 for panels J and K.</p

Calorie restriction and baseline home cage behavior of leptin knockout mice.

<p>(A) Leptin KO and WT mice on AL feeding conditions gain weight while Leptin KO and WT mice on CR lose weight. (B) Leptin KO mice on AL and CR feeding conditions have similar percent gain and loss of weight relative to percent changes in weight of WT controls on AL and CR feeding conditions. Leptin KO mice on CR are more resistant to weight loss (p<0.01 for Leptin KO CR vs. WT CR). (C) High activity behaviors of WT mice are much higher than Leptin KO mice. WT and Leptin KO mice have comparable (D) food bin entry on AL feeding. WT mice (E) hang, (F) jump, (G) rear and (H) walk more than Leptin KO mice. Statistics were performed using the Mann-Whitney Test *  =  p<0.05; **  =  p<0.01; ***  =  p<0.001. Error bars represent IQR for behavioral data and SEM for body weight data. n = 16 WT AL and n = 15 KO AL.</p