Sex differences in thermogenesis structure behavior and contact within huddles of infant mice.

Brown adipose tissue (BAT) is a thermogenic effector abundant in most mammalian infants. For multiparous species such as rats and mice, the interscapular BAT deposit provides both an emergency "thermal blanket" and a target for nestmates seeking warmth, thereby increasing the cohesiveness of huddling groups. Sex differences in BAT regulation and thermogenesis have been documented in a number of species, including mice (Mus musculus)--with females generally exhibiting relative upregulation of BAT. It is nonetheless unknown whether this difference affects the behavioral dynamics occurring within huddles of infant rodents. We investigated sex differences in BAT thermogenesis and its relation to contact while huddling in eight-day-old C57BL/6 mouse pups using infrared thermography, scoring of contact, and causal modeling of the relation between interscapular temperature relative to other pups in the huddle (T IS (rel)) and contacts while huddling. We found that females were warmer than their male siblings during cold challenge, under conditions both in which pups were isolated and in which pups could actively huddle in groups of six (3 male, 3 female). This difference garnered females significantly more contacts from other pups than males during cold-induced huddling. Granger analyses revealed a significant negative feedback relationship between contacts with males and T IS (rel) for females, and positive feedback between contacts with females and T IS (rel) for males, indicating that male pups drained heat from female siblings while huddling. Significant sex assortment nonetheless occurred, such that females made more contacts with other females than expected by chance, apparently outcompeting males for access to each other. These results provide further evidence of enhanced BAT thermogenesis in female mice. Slight differences in BAT can significantly structure the behavioral dynamics occurring in huddles, resulting in differences in the quantity and quality of contacts obtained by the individuals therein, creating sex differences in behavioral interactions beginning in early infancy

Granger (causal) analyses of relative T_IS and contact during cold challenge.

<p>The results of Granger analyses on relative T_IS (T_IS^rel) and contact with males and females. Asterisks indicate significant Granger causality, evaluated at <i>α</i> = .05/6 = .0083. Non-significant results (p<.05) are also shown to depict trends in the data. Arrows indicate that a change in one variable at time <i>t_n</i> predicts a change in another variable at <i>t_n+lag</i>. For example, in all but the Female Lag 0 models, T_IS^rel is a stronger predictor of contacts with females than the reverse. Arrows in both directions indicate a feedback relationship between the two variables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087405#pone.0087405-Granger3" target="_blank">[65]</a>; for example, our results indicate negative feedback between female T_IS^rel and female contacts with males and positive feedback between the male T_IS^rel and male contacts with females. The coloration of arrows indicates significant lagged Pearson product moment correlations between the two variables (<i>p</i><.001), with positive and negative correlations indicated by green and red, respectively. Tests between T_IS^rel and total contacts were run, but are not shown given that none were statistically significant. Models for Lag 3 were constructed but are not shown. For females, the model was null (no significant Granger causality detected), whereas for males the only significant effect was that male T_IS^rel Granger caused male contacts with males (F = 4.94, <i>p</i><.003).</p

Contactogon distributions for warm and cool phases.

<p>Distribution of contactogons (contact types) during the warm and cool phases of Experiment 2 (upper and lower panels, respectively). Values on the <i>y</i>-axis indicate total number of occurrences of each contactogon observed and expected by chance for males and females during the 50 minute trial. As can be seen, the distribution is skewed leftward, to more dispersed contactogons during the warm phase (which did not differ significantly from the chance distribution; <i>G</i> = 6.3, <i>p</i> = .705), and rightward, toward greater amounts of contact during the cool phase (which differed significantly from the distribution expected by chance; <i>G</i> = 39.7, <i>p</i><.00001). Asterisks indicate significant deviation from chance responding in specific contactogons using Fisher’s exact tests (<i>p</i><.005).</p

Granger (causal) analyses of relative T_IS and contact during the warm phase.

<p>The results of Granger analyses on relative T_IS (T_IS^rel) and contact with males and females. Asterisks indicate significant Granger causality, evaluated at <i>α</i> = .05/6 = .0083. Non-significant results (p<.05) are also shown to depict trends in the data. The coloration of arrows indicates significant lagged Pearson product moment correlations between the two variables (<i>p</i><.05), with positive and negative correlations indicated by green and red, respectively. Tests between T_IS^rel and total contacts were run, but are not shown.</p

Consistency of Sex Differences in Thermal and Contact Measures in Experiment 2.

<p><i>Note</i>. Results for Sign tests on thermal and contact measures obtained under identical T_a for the two sexes. <i>Asterisks</i> indicate a significant difference. In all cases of a significant difference this indicated higher female over male values and greater same- versus opposite-sex and female-female over male-male contacts.</p

Infrared thermography.

<p>(<b>A</b>) Sample thermograph from Experiment 1, showing 2 male (top and left) and 2 female (bottom and right) pups during cold challenge. (<b>B–C</b>) Sample thermographs of litters during cold challenge in Experiment 2. In (<b>C</b>) the zones used for measuring interscapular (<b>D</b>) and rump (<b>E</b>) temperatures (T_IS and T_rump, respectively) are shown for a pup that has separated from its huddle.</p

Correlation between relative T_IS and contact.

<p>Linear regressions, coefficients of determination (<i>R²</i>), and <i>p</i>-values for Pearson product moment correlations on T_IS relative to huddlemates (T_IS^rel) and (a) total contacts, (b) contacts with females, and (c) contacts with males, during the warm and cool phases of Experiment 2 (left and right, respectively). Asterisks indicate significant correlation, using a criterion of <i>α</i> = .05/6 = 0083. As can be seen, there was no relationship between T_IS^rel and contact during the warm phase, and a significant correlation between T_IS^rel and both total contacts and contacts with females, but not contacts with males, during the cool phase.</p

Thermal measurements for Experiment 2.

<p>Average temperatures for interscapular (T_IS) and rump (T_rump) regions ± SEM for male (blue lines) and female (red/pink lines) PND8 mouse pups.</p

Scoring of contact behavior.

<p>A depiction of the system used for scoring contact behavior, adapted from that employed by Sokoloff and Blumberg <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087405#pone.0087405-Sokoloff1" target="_blank">[28]</a>. Each pup in the litter was scored each minute for how many males (blue pups) and females (pink pups) they were in contact with, excluding contacts via tails and outstretched paws. Each combination of contacts was assigned a unique identifier or “contactogon”. For example, 0 M 2 F designates contact with zero males and two females. Contactogons possible for only a single sex (e.g., 3 M 0 F) are not shown, and were collapsed into a single category for the purposes of statistical analysis.</p

Thermal measurements for Experiment 1.

<p>Average temperatures for interscapular (T_IS) and rump (T_rump) regions ± SEM for male (blue lines) and female (red/pink lines) PND8 mouse pups.</p